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The role of prolactin in establishment of pregnancy in pigs : studies on endometrial prolactin receptor regulation and uterine secretory physiology

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The role of prolactin in establishment of pregnancy in pigs : studies on endometrial prolactin receptor regulation and uterine secretory physiology
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Young, Kathleen Hart, 1960-
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English
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xv, 215 leaves : ill., photos ; 29 cm.

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Estrogens ( jstor )
Gilts ( jstor )
Hormones ( jstor )
Liver ( jstor )
Pregnancy ( jstor )
Prolactin receptors ( jstor )
Rats ( jstor )
Receptors ( jstor )
Secretion ( jstor )
Swine ( jstor )
Animal Science thesis Ph. D
Dissertations, Academic -- Animal Science -- UF
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1989.
Bibliography:
Includes bibliographical references (leaves 185-214).
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Also available online.
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Typescript.
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Vita.
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by Kathleen Hart Young.

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University of Florida
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THE ROLE OF PROLACTIN IN ESTABLISHMENT OF PREGNANCY IN PIGS:
STUDIES ON ENDOMETRIAL PROLACTIN RECEPTOR REGULATION
AND UTERINE SECRETARY PHYSIOLOGY



















By

KATHLEEN HART YOUNG


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1989




































"Nothing is impossible to the willing mind"



The Book of Hans Dynasty




































To those who believe in me,
"no matter what..."



WAH, RGY, JHN, LK, BHV, IJM and JMH















ACKNOWLEDGEMENTS

I would like to thank the members of my committee, Drs. Bazer,

Buhi, Shiverick, Simpkins and Thatcher, for time and knowledge they put

forth toward the completion of my research. I thank these individuals

who, through their conversations, actions, examples, and generosity,

were an integral part in my development as a scientist.

I am thankful to several people, behind the scenes, that

stimulated my pursuit of this degree, including Mr. Jerry Metzler, Fr.

Timothy Healy, Dr. Phil Senger, Dr. Brad Vaughn and my father, the late

William Hart.

Along the way, much appreciated examples and support were provided

by Drs. Mary Murray, Cheryl Ashworth-Stott, Saundra Tenbroeck and Susan

Ogilve.

I am indebted to Hironori Ohtsuka, founder of Wado-ryu Karate, and

his students, which ultimately led to the supportive friendships of

Mike McCoy, Mike Sawyer, Larry and Ellen Bellack, Cindy Silverstre and

Terry Minard.

The day-to-day events in the lab, barn and surgery room were

shared by several graduate students and post docs, including Dr. Randy

Renegar, Marlin DeHoff, Sue Chaichimansour, Dr. Wendy Campbell, Dr.

Jeff Vallet, Dan Dubois, Troy Ott, Jake Harney, Dr. Mark Mirando,

Saskia Beers and Matt Davis; I am grateful for their help and humour.

Special thanks go to Dan Dubois for lending me his computer for the

final stretch of dissertation polishing.

iv











I thank Dr. Bazer for the many opportunities to participate in

scientific meetings. I am much richer for the experiences. Also, I

thank him for the insight that social comfort doesn't always expand

your knowledge. My solo journey to the Prolactin Gordon Conference

allowed development of friendships, scientific collaboration and a

greater appreciation for prolactin.

Thanks are extended to Dr. Douglas Bolt for generously supplying

the porcine prolactin and other hormones used in these studies; to

Bennet Johnson, who patiently assayed serum samples for prolactin; to

Fil Fliss, for prostaglandin assays of uterine flushings; to Jenny

Davis for Nb2 lymphoma cell assays; and to Dr. Nelson Horseman for

pigeon crop sac assays.

Sincere thanks are extended to Dr. Wang, Dr. Shiverick and

personnel in her lab for their help and generous use of equipment,

computer time and borrowed keys during the period I was conducting

Scatchard analyses of the binding data.

Loving thanks go to Greg, my husband, friend and keeper of my

sanity; needless to say, but nonetheless essential to mention, is that

I am certainly more human because of you. Also, thanks go to my

sister, Judi Norton, who kept me going with letters, support, laughter

and love throughout my graduate program.

Lastly, although I'll be the person with the extra letters behind

by name, my success is truly because all of you were beside me. Thank

you!
















TABLE OF CONTENTS

PAGE

ACKNOWLEDGEMENTS............................................... iv

LIST OF TABLES ................................................. ix

LIST OF FIGURES................................................ x

ABSTRACT ....................................................... xiv

CHAPTERS

1 INTRODUCTION............................................. 1

2 REVIEW OF LITERATURE..................................... 3

History, Evolution and Structure of Prolactin............ 3
Microheterogeneity of Prolactin.......................... 6
Sources of Prolactin..................................... 9
Prolactin in the Circulation.............................12
Regulation of Pituitary Secretion of Prolactin...........13
Receptor Theory .......................................... 17
Analysis of Receptors.................................... 18
Prolactin Receptors...................................... 21
Signal Transduction Systems for Prolactin................30
Regulation of Prolactin Receptors........................35
Functions of Prolactin in the Uterus.....................40
Porcine Conceptus Development and Uterine Secretion......41

3 EFFECTS OF HYPOPROLACTINEMIA ON ESTABLISHMENT
OF PREGNANCY AND UTERINE SECRETARY FUNCTION IN PIGS......44

Introduction............................................. 44
Materials and Methods.................................... 45
Results .................................................. 51
Discussion............................................... 54

4 EFFECTS OF CYSTEAMINE ON CIRCULATING
PROLACTIN LEVELS IN PIGS.................................. 57

Introduction ..............................................57
Materials and Methods..................................... 57
Results ................................................... 58
Discussion................................................ 61











5 ESTABLISHMENT OF HYPERPROLACTINEMIA BY ADMINISTRATION
OF EXOGENOUS PORCINE PROLACTIN TO PIGS.................... 63

Introduction.............................................. 63
Materials and Methods ..................................... 63
Results ................................................... 65
Discussion ................................................ 65

6 EFFECT OF HYPERPROLACTINEMIA ON PROGESTERONE
AND ESTROGEN INDUCED UTERINE SECRETARY RESPONSE
IN PIGS ................................................... 67

Introduction.............................................. 67
Materials and Methods.....................................68
Results ................................................... 70
Discussion................................................ 77

7 DEVELOPMENT OF A HOMOLOGOUS RADIORECEPTOR ASSAY
FOR PORCINE ENDOMETRIAL PROLACTIN RECEPTORS............... 81

Introduction ..............................................81
Materials and Methods..................................... 83
Results ................................................... 89
Discussion................................................ 102

8 AFFINITY LABELLING OF PROLACTIN RECEPTORS
IN DAY 75 PRGENANT PORCINE ENDOMETRIUM WITH
PORCINE [125sI]-PROLACTIN.................................. 107

Introduction.............................................. 107
Materials and Methods..................................... 108
Results ................................................... 109
Discussion ................................................112

9 ENDOMETRIAL PROLACTIN RECEPTORS DETECTED BY
HOMOLOGOUS RADIORECPETOR ASSAY DURING THE ESTROUS
CYCLE AND EARLY PREGNANCY IN PIGS.........................114

Introduction .............................................. 114
Materials and Methods.....................................116
Results ................................................... 117
Discussion................................................ 119

10 EFFECTS OF ACUTE ESTRADIOL VALERATE ADMINISTRATION
ON ENDOMTRIAL PROLACTIN RECEPTORS DETECTED BY
HOMOLOGOUS RADIORECEPTOR ASSAY AND UTERINE
SECRETARY RESPONSE IN DAY 11 CYCLIC PIGS.................. 124

Introduction .............................................. 124
Materials and Methods..................................... 125
Results ................................................... 127
Discussion ................................................ 134











11 EFFECTS OF CHRONIC OVARIAN STEROID ADMINISTRATION
ON ENDOMETRIAL PROLACTIN RECEPTORS AS DETECTED BY
HOMOLOGOUS RADIORECEPTOR ASSAY AND UTERINE PROTEIN
SECRETARY RESPONSE IN OVARIECTOMIZED PIGS................. 139

Introduction.............................................. 139
Material and Methods...................................... 140
Results ................................................... 142
Discussion................................................ 144

12 STUDIES ON MITOGENICITY, LACTOGENICITY,
IMMUNOREACTIVITY AND RECEPTOR BINDING CHARACTERISTICS
OF NONGLYCOSYLATED AND GLYCOSYLATED PORCINE PROLACTIN ..... 150

Introduction............................................ 150
Materials and Methods..................................... 152
Results ................................................... 156
Discussion................................................ 163

13 GENERAL DISCUSSION........................................168


APPENDICES

A IODINATION OF PORCINE PROLACTIN AND
DETERMINATION OF SPECIFIC ACTIVITY........................ 178

B HOMOLOGOUS RADIORECEPTOR ASSAY FOR
PORCINE ENDOMETRIAL PROLACTIN RECEPTORS................... 181

C ACID PHOSPHATASE ASSAY FOR MEASUREMENT OF UTEROFERRIN......183


REFERENCES..................................................... 185

BIOGRAPHICAL SKETCH............................................ 215


viii















LIST OF TABLES


Tables Page

4-1: Effects on interestrous interval and cytoxicity
following cysteamine (CSH) and ethanolamine (control)
administration to cyclic gilts....................... 60

6-1: Composition of Day 15 uterine flushings from
ovariectomized gilts treated with daily injections
of progesterone and saline or porcine prolactin from
Days 4 through 14 (x+SEM) ............................. 71

11-1: Endometrial membrane prolactin receptors and total
protein and uteroferrin in uterine flushings from
ovariectomized gilts following steroid
administration for 11 days........................... 143















LIST OF FIGURES


Figure Page

3-I: Concentrations of total recoverable (A)
calcium, (B) chloride, (C) sodium and
(D) potassium in Day 12 uterine flushings
from cyclic gilts (Experiment 2) treated with
CB154 (100 mg/day) or vehicle (VHC, 4 ml/day)
on Days 10 and 11 and estradiol valerate
on Day 11............................................. 53

4-1: Mean concentrations of prolactin (ng/ml) in
serum of cyclic gilts treated with cysteamine
(solid line) or ethanolamine (dashed line) from
Days 10-16 (denoted by arrows)........................ 59

4-2: Reproductive tracts from gilts treated with
either (A) ethanolamine or (B) cysteamine.............62

5-1: Concentrations of immunoreactive prolactin in
serum during administration of 1 mg porcine
prolactin (circles), or 1 ml saline (squares),
at 0800 and 2000 h (denoted by arrows) on
Days 10 through 13 of the estrous cycle...............66

6-1: Concentrations of total recoverable (A) protein,
(B) uteroferrin, (C) glucose and (D) leucine
peptidase activity (LAP) in Day 12 uterine
flushings from cyclic gilts (Experiment 2)
treated with 1 ml saline (SAL) or 1 mg porcine
prolactin (PRL) at 0800 and 2000 h on Days 6-11
and 0.5 ml corn oil (OIL) or 5 mg estradiol
valerate (E2V) on Day 11 of the estrous cycle.........73

6-2: Concentrations of total recoverable (A) calcium,
(B) chloride, (C) sodium and (D) potassium in
Day 12 uterine flushings from cyclic gilts
(Experiment 2) treated with 1 ml saline (SAL) or
1 mg porcine prolactin (PRL) on Days 6-11 and
0.5 ml corn oil (OIL) or 5 mg estradiol valerate
(E2V) on Day 11 of the estrous cycle..................75













6-3: Concentrations of (A) PGF and (B) PGE in Day 12
uterine flushings from cyclic gilts (Experiment 2)
treated with 1 ml saline (SAL) or 1 mg porcine
prolactin (PRL) on Days 6-11 and 0.5 ml corn oil
(OIL) or 5 mg estradiol valerate (E2V) on Day 11
of the estrous cycle........................... ... .. 76

7-1: Effects of increasing magnesium chloride molarity
on binding of porcine [125I]-prolactin by
membranes from Day 75 porcine endometrium, amnion,
chorion, as well as post-parturient pig and rabbit
mammary gland (300 ug) ............................... 90

7-2: Effects of increasing protein concentrations of
Day 75 porcine endometrial membranes on
binding of porcine [(251]-prolactin...................91

7-3: Binding of porcine [125I]-prolactin by magnesium
chloride treated Day 75 porcine endometrial
(circles) or Day 20 rat liver (squares) membranes
at 4 C (dashed line) or 25 C (solid line).............93

7-4: Dissociation kinetics assay for magnesium
chloride treated (A) Day 75 porcine endometrial
or (B) Day 20 rat liver membranes.................... 95

7-5: Binding and displacement of ovine and porcine
prolactin from magnesium chloride treated Day 75
porcine endometrial membranes........................ 98

7-6: Crossreactivity of unlabelled porcine prolactin
(squares; pPRL), porcine growth hormone (triangles;
pGH),porcine luteinizing hormone (circles; pLH) and
porcine follicle stimulating hormone (diamonds;pFSH)
to porcine [125I]-prolactin with magnesium chloride
treated Day 75 porcine endometrial membranes..........99

7-7: Crossreactivity between unlabelled porcine growth
hormone (dashed line), or porcine prolactin (solid
line) and porcine [12SI]-prolactin with magnesium
chloride treated Day 75 porcine endometrial (circles)
and Day 20 rat liver (squares) membranes..............100

7-8: Scatchard analysis of porcine ['25I]-prolactin
displaced by unlabelled porcine prolactin using
magnesium chloride treated Day 75 porcine
endometrial membranes................................ 101

8-la: Autoradiography of affinity labelled, cross-linked
porcine endometrial membrane preparation prolactin
receptors ............................................. 111


Figure


Page











Figure


8-lb: Autoradiography of affinity labelled, cross-linked
rat liver membrane prolactin receptors................ ill

9-1: Prolactin receptors in endometrial membranes
of cyclic (squares) and pregnant (circles) gilts
over days of the estrous cycle and gestation..........118

10-1: Prolactin receptor numbers in endometrial membranes
at 1, 6, 12, and 24 h after administration (i.m.) of
estradiol valerate (0.5 mg, hatched bars) or 12 h
after corn oil (0.5 ml, solid bar) administration.....129

10-2: Total recoverable (A) calcium, (B) sodium and (C)
potassium in uterine flushings at 1, 6, 12 and
24 h following administration (i.m.) of estradiol
valerate (0.5 mg, hatched bars) or 12 h after
corn oil (0.5 ml, solid bar) administration...........131

10-3: Total recoverable (A) protein, (B) uteroferrin,
(C) leucine aminopeptidase (LAP) and (D) glucose
in uterine flushings at 1, 6, 12 and 24 h
following administration (i.m.) of estradiol
valerate (0.5 mg, hatched bars) or 12 h after
corn oil (0.5 ml, solid bar) administration...........133

11-1: Effect of chronic ovarian steroid administration on
uterine secretary response of ovariectomized gilts;
possible interaction with endogenous prolactin
to result in low and high uterine sector response .... 149

12-1: Separation of nonglycosylated and glycosylated
forms of porcine prolactin by Concanavalin A-
Sepharose 6B column chromatography................... 153

12-2: Evaluation of the purity of nonglycosylated and
glycosylated forms of porcine prolactin isolated
by Concanavalin-A Sepaharose 6B column chromatography
using 12.5% sodium dodecylsulphate one-dimensional
polyacrylamide gel electrophoresis .................... 155

12-3: Uptake of 3H-thymidine into Nb2 lymphoma cells
expressed as percent of Nb2 control (dashed line)
when cells were stimulated by total (circles),
nonglycosylated (squares) and glycosylated
(triangles) forms of porcine prolactin................158


xii


Page











Figure


12-4: Immunoaffinity of total (open and closed circles),
nonglycosylated (open and closed squares) and
glycosylated (open and closed triangles) forms of
porcine prolactin expressed as (A) percent bound
versus log concentration, (A inset) transformed to
log versus logit plot and (B) percent of Bo
(normalized to 100%) versus log concentration.........161

12-5: Scatchard analysis of competitive inhibtion curves
using magnesium chloride treated Day 75 pregnant
porcine endometrial membranes and [1251]-prolactin
(total) versus unlabelled total prolactin
(triangles) and [125I]-nonglycosylated:glycosylated
(2:1) prolactin versus unlabelled
nonglycosylated:glycosylated (2:1) (circles)
prolactin ............................................. 162

12-6: Scatchard analysis of porcine [125I]-(total)
prolactin binding to magnesium chloride treated
Day 75 pregnant porcine endometrial membranes
inhibited by unlabelled total (circles),
nonglycosylated (squares) or glycoslyated
(triangles) forms of porcine prolactin................164

13-1: Effects of hypoprolactinemia and hyperprolactinemia
on the composition of uterine flushings of Day 12
gilts at 24 h after a single administration of
estradiol valerate.................................... 171

13-2: Possible mechanisms) involving prolactin
during maternal recognition of pregnancy in pigs......174


xiii


Page
















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

THE ROLE OF PROLACTIN IN ESTABLISHMENT OF PREGNANCY IN PIGS:
STUDIES ON ENDOMETRIAL PROLACTIN RECEPTOR REGULATION
AND UTERINE SECRETARY PHYSIOLOGY

By

Kathleen Hart Young

May 1989

Chairman: Fuller W. Bazer
Major Department: Animal Science

Conceptus estrogens cause biphasic endometrial responses not

wholly explained by steroid receptor mechanism. Studies reported herein

support prolactin involvement in porcine uterine secretary function.

Estrogen-induced uterine secretary function was decreased in response

to hypoprolactinemia in pigs. Exogenous prolactin interacted with

estrogen, but not progesterone, to increase secretion of uteroferrin,

prostaglandin F2a and leucine-aminopeptidase. Prolactin alone

increased glucose in uterine flushings. Prolactin modulates quantity

of electrolytes released in response to estrogen and enhances secretion

of protein to nourish preimplantation porcine conceptuses.

A homologous porcine prolactin radioreceptor assay was developed,

using pig endometrial membrane preparations, which specifically

detected high affinity (0.3 x 10" M-1), prolactin receptors and was

used to detect changes in endometrial prolactin receptors during the

estrous cycle and early pregnancy. Endometrial prolactin receptors

were similar for pregnant and cyclic gilts on Day 8, then decreased

xiv










during the cycle. Receptors numbers increased following concepts

estrogen secretion (Day 12). Administration of estrogen to cyclic

gilts on Day 11 resulted in uterine secretary response similar to those

detected during pregnancy. Endometrial prolactin receptors increased

within 6h, decreased at 12h and recovered to basal values at 24h

following a single estrogen injection. Changes in prolactin receptors

were associated temporally with changes in uterine ion and protein

secretion. Endometrial prolactin receptor numbers decreased in

ovariectomized pigs treated with estrogen, or estrogen and

progesterone, while corn oil or progesterone alone had no affect. Low

prolactin receptor numbers were associated with low and high uterine

secretary responses. Estrogen stimulation of pituitary prolactin

release could down-regulate endometrial prolactin receptors and

increase steroid receptors. Availablity of exogenous steroids could

therefore influence uterine secretary response.

Porcine prolactin is microheterogeneic. Glycosylated porcine

prolactin has lower mitogenicity, immunoreactivity and binds to fewer

receptors, but higher lactogenicity and receptor binding affinity than

nonglycosylated or total prolactin.

Results of this study indicate that prolactin affects porcine

uterine physiology. Regulation and specificity of endometrial response

to prolactin appears to be controlled locally by regulation of receptor

number, but not affinity. Microheterogeneity of prolactin results in

different affinities for endometrial receptors and may account for

prolactin's diverse effects.















CHAPTER 1
INTRODUCTION

The existence of a lactogenic hormone was proposed in 1928, but

research on prolactin (PRL) did not flourish until the 1970's,

following its purification from several species, including the human.

Prolactin's diverse functions combined research from several scientific

disciplines, such as endocrinology, physiology, comparative

endocrinology, neuropharmacology and anatomy. From the early studies

on crop sac and mammary gland development, as well as lactogenesis, PRL

is now credited with over 100 functions (Nicoll and Bern, 1972).

Prolactin has osmoregulatory functions in amphibians, fish and mammals

and lactogenic functions in birds and mammals. Prolactin affects

growth of various organ systems in fish, amphibians, birds and mammals.

In mammals, PRL is associated primarily with reproductive processes,

e.g., mammogenesis, lactogenesis and luteotropic functions.

Previously, PRL was associated with uterine function, transport of

water, protein and prostaglandin secretion, and steroid uptake and

metabolism. In 1985, Chilton and Daniels, proposed a fourth function

for PRL in uterine physiology; endometriotrophism. Thus, PRL affects

the female reproductive system at all levels; ovaries, mammary gland

and uterus.

The uterus is regulated primarily by steroid hormones. Estrogen

induced biphasic responses, however, could not be explained by

classical steroid mechanisms, rapid signal transduction systems or

novel estrogen mechanisms. Therefore, estrogen's interaction with, or










modification by, another (peptide) hormone, to induce rapid uterine

responses was investigated.

Porcine conceptuses establish pregnancy through estrogen secretion

and cause direct physiological, biochemical and secretary changes in

uterine endometrium. Therefore, PRL interaction with estrogen can be

studied within the context of uterine secretary function. The pig is

an interesting model for reproductive physiology studies, particularly

those investigating effects of PRL. Porcine conceptuses undergo

noninvasive implantation, which does not decidualize or produce PRL

from the endometrium, and the placenta does not produce placental

lactogen. Therefore, the pig can be used for investigations of PRL

function in uterine physiology, without interference by decidial PRL or

placental lactogen. Lacking these placental lactogenic hormones, it

was intriguing to delineate the mechanisms) by which effects of PRL on

uterine physiology are induced in pigs.

This dissertation reports investigation of the role of PRL in

uterine physiology in pigs following induction of hypoprolactinemia and

hyperprolactinemia. Additionally, endometrial PRL receptors and their

regulation by ovarian steroids were investigated using a homologous

radioreceptor assay developed for porine PRL and porcine endometrial

PRL receptors. Homologous assays are advantagous since inconsistencies

associated with the use of heterologous hormones are avoided.

The literature review that follows is to familiarize the reader

with various aspects of PRL and its receptor. These areas are reviewed

with respect to basic function, characteristics and physiology that

must be considered in understanding the role of PRL in (porcine)

uterine physiology.















CHAPTER 2
REVIEW OF LITERATURE



History, Evolution and Structure of Prolactin

A lactogenic hormone was first suggested by experiments of John

Hunter (1788-1840) in which he described proliferation and secretion of

the pigeon crop sac. Hunter alleged that the pigeon crop sac was

analogous in its development to the mammary gland during gestation.

Interest in anterior pituitary secretions continues today from the

initial work of Hunter in the 1780's and characterization of the six

anterior pituitary hormones in the late 1930's. Research with

prolactin (PRL) was not very active until the 1920's. Injection of

anterior pituitary extracts into rats (Evans and Simpson, 1929) and

intact (Strickler and Grueter, 1928) and ovariectomized (Corner, 1930)

rabbits resulted in mammary distention due to milk synthesis. Yet, it

was the industrious work of Oscar Riddle and coworkers (1933) that

involved isolating and purifying the lactogenic substance which was

named "prolactin." However, it was development of the pigeon crop sac

bioassay, based on John Hunter's early work, that allowed confirmation

of PRL activity during its purification. Great controversy prevailed

as to whether a separate growth promoting hormone existed. Riddle et

al. (1933) demonstrated that PRL was different from growth hormone and

established PRL as an independent hormone. Despite a desire for

complete separatation of functions for the new known hormones, PRL and










growth hormone, overlapping functions are observed in several species

(Nicoll, 1975; 1982).

Since its discovery, PRL from several species has been sequenced.

Prolactin is a simple polypeptide containing 199 amino acids. Three

disulphide bonds, located between cysteines 4-11, 58-174 and 191-199,

are observed for PRL in most mammalian species, except horse (Li and

Chung, 1983) and fish (Farmer et al., 1977) which lack the NH2-terminal

loop. Biological activity is affected by breaking the disulfide bonds

indicating that function is dependent on the three dimensional

structure of PRL. Microheterogeneity has been established for PRL;

with 25,000 Mr glycosoylated and 60,000 Mr dimer forms in addition to

the well characterized 23,000 Mr form of PRL. These forms are present

in the pituitary as well as the circulation. Prolactin has been

sequenced for humans (Shome and Parlow, 1977; Cooke et al., 1981),

sheep (Li et al., 1970), cows (Wallis et al., 1974; Sasavage et al.,

1982), rats (Parlow and Schome, 1976; Gubbins et al., 1980), mice

(Kohmoto et al., 1984), whales (partial (Kawachi and Tubokawa, 1979),

elephants (Li et al., 1987) and pigs (Li, 1976). Porcine PRL has

typtophan residues at amino acids 91 and 150 and an isoelectric point

of 5.85 (Brewly and Li, 1975). Species specificity is considerable,

with 20% of the residues dissimilar between pig/whale and ruminants,

and a 40% difference between rat and other mammalian PRLs suggesting

that PRL has evolved differently, even within the mammalian species,

from an ancestral molecule it shared with growth hormone (Wallis,

1981).

Prolactin appears to have undergone three evolutionary

accelerations. Rat PRL evolved the fastest, showing 44 point










mutations/100 residues/108 years, followed by human (19 point

mutations), and ovine and bovine (16 point mutations) PRLs. Porcine

and cetacea PRL show only 5 point mutations/108 years, which is much

slower than for other species. Differential regulation of PRL

development between species may suggest specified functions. Porcine

PRL has evolved only as fast as cytochrome C, while rat PRL has evolved

so quickly that is has only a 40% difference in its structure compared

to human PRL. This is a smaller difference than that between human and

nonprimate PRLs. The reasons for the differential evolutionary rates

of PRL are unknown; as are the resulting changes in function or

specificity. Most evolutionary change is neutral (King and Jukes,

1969); however, increased rates of change may be due to new selection

pressures or loss of specific function (Wallis, 1981).

These points, suggest that species specificity is an important

attribute of PRL to further our understanding of its biological

functions. Previous views were that a hormone, regardless of species

source, would function similarly in each species into which it was

injected. This fallacy is discussed by Nicoll (1982), who noted that

early research on PRL was with ovine PRL. By chance, the majority of

ovine PRL's functions are "PRL-like" in nature. Had the early work

been conducted with human PRL, the literature may have described a very

different set of functions. Hormones are named usually for their

suggested functions, thus restricted by man's attempt to organize and

understand himself. Although first thought to be primarily involved in

lactation, PRL is now cited with over 100 functions (Riddle, 1963;

Nicoll and Bern, 1972), most of which are categorized by 1)

osmoregulation and electrolyte balance, 2) growth and development, 3)










mammalian reproduction, 4) parental behaviour, 5) intergumentary

structures and 6) interaction with steroid hormones (Nicoll et al.,

1985). Based on PRL's diversity of actions, the chronobiologist,

Hablan (1980), has suggested its renaming to 'versatilin' (as cited by

Nicoll, 1979).



Microheterogeneity of Prolactin

Glycosylated Prolactin

The anterior pituitary produces several glycosylated hormones.

Therefore the discovery of a glycosylated form of PRL was novel, yet

not entirely unexpected. First reported in 1984, by Lewis and

coworkers, glycosylated PRL has received increasing attention. The

diversity of PRL's action may be a function of its microheterogeneity.

Presently, several molecular weight forms of PRL (23,000, 25,000 and

50-60,000 Mr) have been isolated from pituitaries of sheep (Lewis et

al., 1984), pigs (Pankov and Butnev, 1986), rats (Wallis, 1980), mice

(Sinha and Jacobsen, 1987) and humans (Lewis et al., 1985; Markoff and

Lee, 1987), suggesting that PRL is actually a 'family' of hormones

(Lewis et al., 1984). Glycosylated PRL is produced also in uterine

decidua (Lee and Markoff, 1986) and found in amniotic fluid (Meuris et

al., 1985) of humans. The carbohydrate moiety of glycosylated porcine

PRL resembles that of ovine LH (Bedi et al., 1982). and is N-linked at

asparigine 31 to the polypeptide (Pankov and Butnev, 1986).

The glycosylated form of PRL is less mitogenic and immunoreactive

in Nb2 lymphoma cell assays and polyclonal RIAs, respectively

(Pellegini et al., 1988; Scott et al., 1988). Lactogenic activity, as

measured by the pigeon crop sac or mouse mammary explant casein










synthesis assays, is lower for human and sheep glycosylacted PRL (Lewis

et al., 1984, 1985), but higher for porcine glycosylated PRL (Pankov

and Butnev, 1986). Porcine glycosylated PRL had decreased binding to

rabbit or porcine mammary gland membrane preparations than ovine PRL

(Pellegini et al., 1988; Seely et al., 1988). However, these assays

were conducted in heterologous systems and results may be inconsistent

with the true activity of glycosylated PRL.

The function of the carbohydrate moiety of PRL is unknown.

Speculation suggests that this form of PRL could be reserve source and

deglycosylated as needed or have a longer half-life than the 23,000 Mr

nonglycosylated PRL. The ratio of nonglycosylated to glycosylated PRL

may be important in physiological effects of PRL. The ratio changes in

late pregnancy of humans (Markoff and Lee, 1987) and during the first

year of life for pigs (Sinha et al., 1988), but a functional change

associated with shifting ratios has not been elucidated. Further work

on the role of the forms of PRL, individually or combined, is

necessary to understand the diversity of PRL's functions.

Additionally, investigation of these forms at the receptor level may

provide insight into interactions with PRL receptor(s) and subsequent

biological actions.



Cleaved and Clipped Prolactin

Another variation of PRL is the "cleaved" form in which the large

loop formed by the disulfide bond between amino acids 54 and 174 is

severed at amino acids 148-149. A polypeptide containing two strands

results, joined by the disulfide bridge which previously caused a large

loop in PRL. Cleaved PRL contains 16,000 Mr (amino acids 1-148) and











8,000 Mr (149-199) peptides. Cleavage of the large loop of PRL does

not inhibit its binding to PRL receptors in mammary gland or ventral

prostate of rats (Clapp, 1987; Vick et al., 1987). Additionally,

cleaved PRL has full potency in Nb2 lymphoma cell mitogenic assays.

However, only 50-60% of cleaved PRL is detected by RIA (Vick et al.,

1987), suggesting that the biological and antigenic determinents are at

different sites, as suggested by Amit et al. (1985).

Cleaved PRL is found in pituitaries of mice (Sinha and Gillian,

1981), rats (Mittra, 1980) and humans (Sinha et al., 1985) and is

produced by ventral prostate of male rats. Mammary tissue of lactating

rats has higher enzyme activity, thus more cleaved PRL is produced by

this tissue (Vick et al., 1987; Clapp, 1987). Target tissues of PRL

may be expected to contain cleavage enzymes (Nolin, 1982), but it is

uncertain if these enzymes are regulated physiologically. Cleavage of

PRL is by nonspecific multipurpose proteases influenced by

configuration of the PRL molecule (Wong et al., 1986). Cleavage also

depends on pH, with enzymatic activity increasing at pH 5 to 3.6

(Clapp, 1987).

Reduction of the disulfide bond joining the chains generates a

16,000 Mr and 8,000 Mr fragments. The 16,000 Mr fragment maintains PRL

activity on mammary epithelial cells, despite decreased receptor

binding, mitogenic activity and immunoreactivity. However, functions

of the cleaved, 16,000 Mr and 8,000 Mr fragments of PRL await further

investigation.










Sources of Prolactin

Anterior Pituitary Prolactin

The anterior pituitary is derived from an evagination of embryonic

ectodermal tissue, Rathke's pouch, and attaches to neural tissue from

the brain to form the pituitary. The cells of the anterior pituitary

can be classified as acidophils, basophils and chromophobes by

microscopy and perferential uptake of certain dyes. Acidophils are

further defined as alpha or epsilon cells, being oranophils or

carminophils, respectively. Carminophils, but not orangophils,

fluctuate during pregnancy. Both cell types are located in the lateral

aspects of the anterior pituitary.

Lactotrophs and somatotrophs, which secrete PRL and growth

hormone, respectively, comprise the acidophilic cells of the anterior

pituitary. Somatotrophs, which comprise 50% of anterior pituitary

cells, were described by Kurasumi (1968) as round or oval polygonal

cells with short clubbed mitochondria and round dense secretary

granules of approximately 200-350 mm. Lactotrophs, comprise 15-20% of

the anterior pituitary cells, and were initially described by Farquhar

and Rinehart (1954). These PRL secreting cells have large nuclei and

relatively small amount of cytoplasm which contains well developed

golgi complex and abundant rough endoplamic reticulum. Secretory

granules of the lactotroph are elongated (600-900 mm) although small

(200 mm) secretary vesicles are also observed.

Unlike other pituitary hormones, PRL is synthesized with a signal

peptide. Newly synthesized PRL is preferenially released, independent

of TSH, while secretion of stored PRL is dependent on TSH stimulation

(Walker and Farquhar, 1980). Crinophagy, another unique feature of










lactotrophs, occurs when secretary vesicles fuse with lysosomes instead

of plasma membranes when PRL secretion is inhibited (Farquhar, 1985).

Dopamine receptors are present on lactotrophs. These receptors, along

with dopamine, are internalized to secretary granules however the role

of interalized neurotransmitter has not been fully elucidated.

Lactotrophs and somatotrophs were thought to be from

morphologically distint cell lines (Farquhar, 1985). However, recent

findings suggest that PRL secreting lactotrophs can be specific cells,

or another class of PRL secreting cells, derived from somatotrophs, and

termed mammosomatotrophs (Stattman, 1974).



Mammosomatotrophs

The anterior pituitary is comprised of different cell types, each

responsible for synthesis and secretion a hormone. The 'one cell/one

hormone' theory is supported by anatomical and microscopy studies of

the anterior pituitary. Growth hormone and PRL are secreted from

somatotrophs and lactotrophs, respectively; however, distinction

between these cell types is fading. Early work by Stattman (1974)

suggested that some cells in the anterior pituitary, termed

mammosomatotrophs, secrete both PRL and growth hormone, or exhibit a

functional shift from growth hormone to PRL secretion.

Mammosomatotrophs have thus far been recorded only in rats.

Mammosomatotrophs constitute one-half of PRL secreting cells in the

male pituitary; however, percentages are not available for female

pituitaries (Brookfor et al., 1986). Estrogen stimulation of male

anterior pituitary cells in culture did not cause cell mitosis,

contrary to results by Corenblum et al. (1980), but shifted cell










secretion from growth hormone to dual secretion with PRL (Brookfor et

al., 1986), suggesting a functional 'plasticity' for conversion. It is

unknown if mammosomatotrophs serve as a stem cell or are a normal stage

within the cell cycle (Frawley et al., 1985).



Decidual Prolactin

Prolactin is found in the amniotic fluid during pregnancy. Yet,

PRL in the maternal circulation does not fluctuate greatly during

gestation and, therefore, could not account for the high concentrations

of PRL in the amniotic fluid of rhesus monkeys (Josimovich et al.,

1974). Additionally, PRL levels in amniotic fluid of anecephalic

fetuses or fetus that have died in utero are similar to normal fetuses

(Walsh et al., 1977). Placental membranes do not produce PRL, but

decidua attached to placental membranes stains positively for PRL

(Golander et al., 1978; Healy et al., 1979). Studies of decidual

tissue of pregnancy determined that PRL was produced from these

tissues. In 1977, the suggestion that a pituitary hormone could be

produced in a nonpituitary tissue was considered novel. However, many

hormones previously associated only with brain tissue are now found in

other tissues and vice versa (vasoactive intestinal peptide). Riddick

and Kusmik (1977) confirmed that PRL was produced during normal

pregnancy and by secretary endometrium from Day 22 of the menstrual

cycle (McRae et al., 1986).

Decidual PRL is identical in structure and biological function

(Riddick et al., 1978) to pituitary PRL. Prolactin secretion is 1000-

fold less from decidual (400 ng/g tissue) when compared to pituitary

(400 ug/g tissue) (Tomita et al., 1982). However, the weight of the










decidua is far greater than that for the anterior pituitary, which

weights approximately 0.6 g. The two sources of PRL are regulated

differently; TRH and dopamine, which stimulate and inhibit pituitary

PRL release, respectively, do not affect PRL production by decidual

tissue. Release of PRL by decidual tissue is inhibited by arachadonic

acid and stimulated by calcium and progesterone (Healy and Hodgen,

1983). Differential regulation of PRL from its two sources may be due

to storage properties of PRL in the tissues (Harkoff et al., 1983). In

addition, placental peptides are secreted that stimulate (23,500 Mr;

Handwerger et al., 1983) and inhibit (Markoff et al., 1983) decidual

PRL. Local production of PRL in species that decidualize at

implantation, including humans, rabbits and rats, further support PRL's

role in uterine physiology. However, in species with noninvasive

placentae, such as pigs, other mechanisms) may exist, i.e., receptor

regulation, to allow similar effects of PRL on uterine function.



Prolactin in the Circulation

Circulating levels of PRL are relatively constant during pregnancy

(Dusza and Krzymowska, 1981; Kensinger et al., 1986; DeHoff et al.,

1986) in pigs; elevated slightly on Day 10 (20 ng/ml), then declining

by Day 20 and remaining constant (5-10 ng/ml) until parturition

(Kraeling et al., 1982). In cyclic pigs, PRL levels are similar to

those for pregnant pigs (5-10 ng/ml) except that PRL is elevated (15-20

ng/ml) on Days 0 to 2 and 16 to 17 (Brinkley et al., 1973; Dusza and

Krzymowska, 1979; Foxcroft and Van der Weil, 1982) when concentrations

of circulating estrogens increase.











In rats, PRL levels increase 10-fold with diunrnal and nocturnal

increases (de Greef et al., 1977; Neill, 1980), following mating or

cervical stimulation to approximately Day 12 for intact, or Day 6 for

ovariectomized rats. Studies with ovariectomized rats suggest that

progesterone is associated with the noctural increase in PRL levels,

while estrogen accentuates the diurnal rise and inhibits the nocturnal

increase (Freeman and Sterman, 1978). For cyclic rats, PRL levels

increase at proestrus, in association with increases in circulating

estrogens (Butcher et al., 1974; Kelly et al., 1975).



Regulation of Pituitary Secretion of Prolactin

Hypothalamic Factors

The mechanisms responsible for regulation of secretion of PRL by

the anterior pituitary are currently under intense investigation.

Hypothalamic dopamine tonically inhibits PRL secretion, a mechanism

which is unique among the anterior pituitary cells. However, the

posterior pituitary may also be a source of dopamine (Ben-Johnathen and

Peters, 1982) and may be transported to the anterior pituitary through

the blood (Page et al., 1982) as for other substances (Baertschi,

1980). Gamma butyric acid (GABA) also contributes to the inhibition of

PRL secretion (Duvilanski et al., 1986) especially in pigs (Schally et

al., 1977). Other factors are PRL releasing factors; opiods (Bero and

Kuhn, 1987; Rauhala et al., 1987), serotonin (Bero and Kuhn, 1987;

Thomas et al., 1988), Vasoactive intestinal peptide (VIP; Nagy et al.,

1988), peptide histidine isoleucine (PHI; Abe et al., 1985), oxytocin,

(Samson et al., 1987; Lumpkin et al., 1983) and posterior pituitary

factor (Murai et al., 1988). Complete control of PRL secretion in pigs










may be due to a combination of these factors and not under the sole

regulation of the tuberoinfindibular dopaminergic system (Moore, 1988).



Estrogen

Lactotrophs are unique among anterior pituitary cells since they

are generally inhibited by tonic hypothalamic dopamine. Several

factors can override dopamine inhibition, but estrogen is most

effective. The mechanisms) responsible for estrogenic stimulation of

PRL secretion are relatively unknown; however, following an increase in

estrogen, circulating PRL increases 10-fold, on the afternoon of

proestrus in rats (Butcher et al., 1974). Administration of exogenous

estrogen to ovariectomized rats increased PRL over 2-3 days (Maurer and

Gorski, 1977); however, Yamamoto et al. (1975) demonstrated increases

in PRL within 12 h following injection of a single dose of estrogen

into ovariectomized rats.

Despite chronic inhibition of lactotrophs by dopamine, estrogen

increases the synthesis and secretion (Thorner and MacLeod, 1980) of

pituitary PRL, as well as the number of lactotrophs (Neill and Frawley,

1983). Estrogens' effects may be due to 1) direct stimulation of

pituitary lactotrophs, 2) modulation of hypothalamic regulation of the

pituitary or 3) effects on the physiological responsiveness of

lactotrophs to other regulatory mechanisms.

Estrogen stimulates increases in PRL in pituitary cells when added

to in vitro culture medium (Nicoll and Meites, 1972) or in vivo when

implanted into anterior pituitaries of rabbits (Kanematsu and Sawyer,

1963) and sheep (Vivian et al., 1979). Hypothalamic control,

especially by dopamine, of pituitary PRL is dampened due to estrogen











(Shull and Gorski, 1984). Dopamine released into the portal veins is

decreased (Ben-Johnathan et al., 1977) during proestrus and in

ovariectomized rats treated with estrogen (Cramer et al., 1979).

However, chronic estrogen treatment increases dopamine turnover (Fuxe

et al., 1969) and release (Gudelsky et al., 1981), possibly through

short-loop feedback mechanisms of hyperprolactinemia (Moore, 1988).

Estrogen may also stimulate release of TRH, a known PRL releasing

factor (Shull and Gorski, 1984). The number of TRH receptors on

lactotrophs are increased in pituitaries of estrogen treated rats

(DeLean et al., 1977).

Estrogen effects lactotrophs through both protein dependent and

independent mechanisms. Estrogen acts directly on the genome of the

lactotrophs to increase transcription and PRL mRNA and then a 5-fold

increase in PRL within 24h. This mechanism of estrogen action is

thyroid and hypothalamus independent since estrogen increases PRL


in thyroidectomized


rats or rats with pituitaries


transplanted to the kidney capsule. In ovariectomized rats, both rapid

and prolonged effects of estrogen on PRL secretion occurs. Estrogen

does not affect the growth hormone gene in rats. Estrogen receptors

reach a peak in the nucleus at 1 hour, then decrease, but PRL synthesis

and secretion is stimulated for 48 to 72h following estrogen

stimulation. Shull and Gorski (1986) suggested that estrogen affects

stable nuclear components, such as chromatin proteins, DNA sequences

around the PRL gene, a second regulatory factor (unknown or pituitary

transcriptional activator; PIT-1) or a combination of these effects.


secretion











Estrogen Receptors

The hydrophobic similarities between the pentanophenanthrene ring

of steroid hormones and membrane lipids allow steroids to enter a cell

through simple diffusion. A 'two-step' theory for expression of

steroid hormones was proposed by Jensen et al. (1968) whereby the

unbound estrogen receptor resided in the cytoplasm. Activation by

estrogen binding shifted the sedimentation coefficent from 4S to 5S

(O'Malley and Schrader, 1979) and translocation to the nucleus allowed

interaction with acceptor nonhistone chromatin proteins (see review

Grody et al., 1982). Estrogen receptor mechanisms have undergone

substantial revision. Williams and Gorski (1972) proposed an

equilibrium theory where all estrogen receptors are nuclear, but their

affinity is dictated by binding status. Receptors not bound by

estrogen have a lower affinity and move to the cytoplasm during tissue

processing. Martin and Sheridan (1982), Welshons et al. (1984) and

King and Green (1984) agree with Williams and Gorski (1972) on the

artifactual nature of cytoplasmic receptors. Buffer volumes affect

recovery of cytoplasmic and nuclear estrogen receptors. Monoclonal

antibodies and immunoperoxidase staining localized estrogen receptors

in nuclei of human breast tumour and rabbit uterine cells.

Steroid receptors are a family of ligand regulated positive

transcription factors with a common structural organization. There is

a central DNA binding domain, hinged to a carboxy terminus that is

common to all steroid receptors. This also contains zinc finger

proteins (Miller et al., 1985) of 2 pairs of four consecutive

cysteines, that act as ligands for zinc atoms.










Steroid interaction at the PRL genome DNA is thought to stimulate

PRL transcription though involvement of pituitary transcriptional

activator, PIT-1. This factor, PIT-1, must bind DNA in conjunction

with estrogen binding to its DNA domain to stimulate PRL gene

transcription (Adler et al., 1988).



Receptor Theory

Peptide hormones usually interact with cells through receptors.

Although the concept of 'receptors' seems commonplace, it was

introduced by J.N. Langley (1852-1926) following his observations of

mutual antagonism between curare and nicotine. The drugs interacted

with a "receptive substance" during autonomic transmission in

neuromuscular communication of the frog leg (Langley, 1909).

Qualitative aspects of receptor saturation and selectivity were

transformed to quantitative analysis by A.J. Clark (1885-1941).

Studying acetylcholine and atropine, he recognized that the rate at

which drugs combined with receptors was dependent on the concentration

of drugs and receptors and that the dissociation rate was proportional

to the number of complexes formed. These properties were similar to

mass action isotherms used by Langmuir (1881-1957) and, therefore,

drug-receptor interactions were found to obey the laws of mass action.

However, not all drug-receptor binding phenomena was explained by

Clark's observations and mass action equations. Stephanson (1956)

further refined drug-receptor interactions with the role of efficacy in

biological responses, noting that agonist response curves for tissues

were often steeper than dose-response curves. He postualated that 1)

maximal effects are produced when an agonist occupies only a small










proportion of the receptors; 2) biological response is not linear in

proportion to the number of receptors; and 3) equal biological

responses can be produced by drugs of different capacity for receptor

occupancy; that is, increased efficacy. The concept of spare receptors

was developed by Paton (1941), from studies on effects of antagonism.

He noted that agonists can elicit maximal biological responses even

when only a small fraction of the total receptors are occupied.

Receptor occupancy was not rate limiting for tissue activation.

Nickerson (1956) observed that only 1% of guinea pig ileum histamine

receptors needed to be occupied for maximal contraction, confirming

Paton's (1961) theory.



Analysis of Receptors

With developments in in vitro techniques, quantification of ligand

binding to membranes and defining receptors required evidence of

saturation, specificity, and kinetics realistic for the time course of

biological action. Receptor binding data are obtained through

saturation or competitive inhibition studies, and generate curvilinear

results. Linear transformation of data to obtain binding parameters is

achieved through Scatchard (1949) (Rosenthal; 1969) interpretation.

Scatchard analysis is based on equilibrium kinetics resulting in a

linear plot of data where bound/free ratio and free hormone data are

ploted on the abcissa and the ordinate, respectively. This results in

linear interpretation of data where the negative slope of the generated

line defines the affinity constant (Ka) or its reciprocal defines the

dissociation constant (1/Ka or KD). Maximal binding and density of

binding sites are estimated by the y- and x-intercepts, respectively.










The linear plot is an algebraic derivation from original theory that

numbers of complexes formed are dependent on the concentration of

receptors and hormone available as well as the rates of association and

dissociation.


ki
[H] RI [HR] (1)


[H] [R] = [HR]/(KD + [H])


with [HR] = amount bound (B); [R] = maximal binding (Bmax) and

[H] = free hormone (F), the equation is restated to


B = [(F)(Bmax)]/KD + F


rearrangment generates


(B)(Ko) + (B)(F) = (F)(B.ax)


division by Free hormone


[(B)(Ko)/F] + B = B.ax


rearranged to


B/F = (B.ax B)/KD


and transformed to the linear (y = mx + b) expression


B/F = (-1/KD)(B) + B.aX/KD











Thus, the mathematical model and equations for binding data

resemble those for enzyme kinetics (Eadie, 1942; Hofstee, 1952).

Scatchard analysis assumes a known number of receptor sites. This is

not often the case during membrane receptor investigation. Therefore,

the Rosenthal (1969) analysis is used, since in theory, it is not based

on known receptor numbers. However, the two data transformations are

mathematically equivalent and receptor data are analyzed through

Scatchard analysis and interpretation.



Dissociation Rate Constants

Hormone receptor complexes are, in part, dependent on the rate at

which these complexes form (ki) and dissociate (k-i) at equlibrium.


ki
CH] [R] [HRJ (8)
k-i


Dissociation rate constants are determinable when rebinding of

labeled hormone is prevented and kinetics are reduced to simple

first order reactions.


dB/dt = (-k-,)(B) (9)


If bound (B) is equal to bound at time = 0 (Bo), then integration

results in


ln B/Bo = (-k-,)(t) (10)


and a plot of ln B/Bo versus time generates a slope of -k-i;

i.e. the dissociation rate constant. At equilibrium, the rates










of association and dissociation are equal (Baxter and Funder,

1979) to


k[H] [R] = k-i [HR] (11)

or redefined as


[H][R]/[HR] = k-i/ki = KD (12)


Thus, the ratio of ligand 'off' to 'on' a receptor is the equilibrium

dissociation constant (KD) and is equal to the reciprocal of the

association constant (K.) generated from the negative slope of

Scatchard analysis as previously mentioned. The association constant

depicts the affinity and tightness of binding between a hormone or

ligand and receptor (Limbird, 1986).

Scatchard analysis can generate a linear relationship between B/F

and F, describing a single class of hormone receptors. Curvilinear

Scatchard can be due to two separate binding sites with different

affinities or negative cooperativity (DeMeyts et al., 1976)



Prolactin Receptors

General Characteristics

Although PRL was discovered in the 1930's (Riddle et al., 1933),

binding of PRL to tissues was not demonstrated until the 1960's.

Specific high affinity binding of PRL and its relation to biological

response characteristics of hormone-receptor interaction was first

investigated in mammary glands of mice (Frantz and Turkington, 1972)

and rabbits (Shiu et al., 1973). Investigation of the PRL receptor

structure has been with rat liver. Binding of hormone to membrane does











not necessarily induce a biological response. However, binding sites

in the mammary gland (Shiu et al., 1976) and liver (Chen et al., 1972)

have been correlated with cellular changes in mRNA and enzyme activity

and therefore, suggest that binding sites serve as receptors essential

for biological responses. Target cell membranes having saturable PRL

binding sites include mammary epithelial cells, hepatocytes, renal

tubules, adrenal cortex, prostate, seminal vesicles, and brain (Hughes

et al., 1985). Characteristics of PRL receptors include high affinity

and saturability. The high affinity constant, as determined by

heterologous ovine PRL assay (Ka = 109 M-1; Shiu and Freisen, 1974a),

is correlated with hormone biopotency. Homologous detection of PRL

receptors generates an affinity constant 109 (Haro and Talamantes,

1985; this study), only slightly lower than that reported for rat liver

(Dave and Knazek, 1980; Dave et al., 1981; Liscia et al., 1982; and

Liscia and Vonderhaar, 1982); cow mammary gland (Ashkenazi et al.,

1987) and mouse liver (Posner et al., 1974b; Knazek et al., 1977;

1978). However, the reversability of PRL bound to its receptor has

been questioned, as observed in vitro which often inefficiently

replicates the in vivo binding environment. Prolactin receptors have

been noted in several other tissues and are associated with other

physiological effects. Prolactin stimulates steroid production in ovary

and Leydig cells (Barkte and Dalterio, 1976; Husto et al., 1972),

uptake of testosterone by prostate (Farnsworth and Gonder, 1977),

increased uteroglobin mRNA and steroid receptors in the uterus (Chilton

and Daniels, 1985) and water and ion regulation in fetal membranes

(Rabee and McCoshen, 1986; Kensinger et al., 1986). Prolactin binds to

kidney membranes, but a clear function has not been established. These











kidney binding sites for PRL appear to be specific and therefore are

considered to be PRL receptors (Hughes et al., 1985).



Binding Characteristics

Most studies conducted of PRL receptors utilize heterologous assay

systems, combining tracer, competing hormone and tissue source of

membrane receptors from various species. Although a great deal of

information regarding in vitro binding of PRL receptors has been

obtained, these systems do not mimic the in vivo environment. Thus,

questions remain as to whether heterologous hormone interactions are

identical to those for homologous hormones. Heterologous hormones can

generate binding results that may not characterize in vivo homologous

binding kinetics and properties or produce immunological artifacts

(Hughes et al., 1982; Amit et al., 1983). Additionally, other

hormones, placental lactogens or proliferin and the microheterogeneity

of PRL and growth hormones contribute to the difficulties in

interpreting results from heterologous radioreceptor assays. To date,

results from only one homologous assay has described interactions

between mouse PRL and its liver receptors (Haro and Talamantes, 1985a).

Binding characteristics are similar, but the affinity constant is

slightly lower than obtained from heterologous assays. This would

results in a more rapid hormone-receptor dissociation, as speculated to

occur in vivo (van der Gugten et al., 1980). Thus it appears

advantageous, although more difficult, to utilize homologous hormones

for studying receptors.

Scatchard (1949) analysis of binding studies assumes that

receptors are saturable, specific and freely reversible at equilibrium.











Studies with PRL receptors (in vitro), whether by heterologous or

homologous assay systems, fulfill the constrains of saturability and

specificity; however, complete reversibility has been questioned. Slow

or difficult dissociation has been observed for some peptide hormones

including PRL (Kelly et al., 1983; van der Gugten et al., 1980), growth

hormone (Donner et al., 1980), TSH (Powell-Jones et al., 1979), LH

(Katitineni et al., 1980) and insulin (Donner and Corin, 1980).

Increases in association time from lh to 10h (in vitro) are directly

correlated with increases in dissociation time, as well as incomplete

dissociation, after 48 hours (Kelly et al., 1983). Longer association

times may allow tighter binding and decreased ability for hormone

dissociation. Internalization of PRL and other hormones is often

preceded by tightening of the hormone-receptor linkage (Catt et al.,

1979).

Prolactin receptors within different cell membranes have different

affinity constants and dissociation times (Kelly et al., 1983).

Therefore, affinity and dissociation could depend on location of the

receptor within cellular membranes. The proportion of subcellular

membranes within microsomes commonly used during in vitro receptor

studies is not known. Differences in affinity or dissociation

constants may be a function of the ratio of PRL receptors within plasma

or golgi membranes in the microsomal pellet.

Dissociation of ovine PRL from rat liver (Kelly et al., 1983) and

porcine corpora luteua (Brambly and Menzies, 1985) membranes is 60%

complete after 48h. This slow dissociation rate in not likely due to

damaged hormone or receptors and must be more efficient in vivo than in

vitro (van der Gugten et al., 1980). Therefore, in vivo, energy











dependent factors or other factors may account for rapid reversibility

that does not yet occur in vitro. Additionally, intrinsic factors

which affect dissociation may differ between tissues since it appears

that PRL receptors are differentially regulated. A high apparent

activiation energy be involved in slow dissociation for PRL binding and

as reported for CL of pigs (64.8 kJ, Brambly and Menzies, 1987), and

liver of mice (43.6 kJ/mole; Haro and Talmantes, 1985b) and rats (34

kJ/mole; Rae-Ventner and Dao, 1982). Extensive hydrophobic

interactions may be involved in PRL interaction with its receptor since

monovalent anions, acetate and phosphate stablize homologous binding

(Haro and Talamantes, 1985a). Amino acids at position 20 through 36

form such a hydrophobic region with histidines located at positions 27

and 30 in cow (Wallis, 1974), sheep (Li et al., 1970), pig (Li et al.,

1976) and human (Cooke et al., 1981) and at positions 25 and 28 in rat

(Cooke et al., 1980) and mouse (Kohomoto et al., 1984) PRLs.



Prolactin Receptor Turnover

Once PRL binds to its receptor, internalization is rapid and

biphasic. Internalization of bound PRL occurs in as little as 5 min.

At 5 min, radiolabeled PRL was associated with the low density membrane

fraction having high galactosyl transferase activity characteristic of

golgi membranes. At 10 min, radiolabeled PRL was associated with the

high density membrane fraction with high acid phosphatase activity,

characteristic of lysosomes. This internalization and trafficking is

similar to that for insulin (Posner, et al., 1981). The golgi contains

twice the amount of PRL, and processes PRL slower than insulin. The

golgi network has been redefined as the trans golgi network (TRN) or










golgi saccule endoplasmic reticulum lysosome (GERL) which functions to

transport proteins to the plasmalemma or to lysosomes (see review,

Griffiths and Simon, 1986).



Molecular Weight of Prolactin Receptor

Prolactin receptor structure has been investigated through various

biochemical methods including gel chromatography, solubilization,

affinity labelling and cross-linking. Prolactin receptors are

hydrophobic glycoproteins, since solubilized receptors bind to

Concanavalin-A, a lectin which binds mannose and glucose residues.

Triton X-100 solubilized PRL receptors aggregate to form larger

molecular weight (220,000 Mr) complexes while purification of receptors

with 3-[(3-cholamidopropyo)-dimethylamimonio]-l-propane sulfonate

(CHAPS), a nondetergent zwitterionic solution, results in a single

electrophoretic band of 32,000-37,000 Mr (Djiane et al., 1987).

Molecular weight estimates of PRL receptors from rabbit mammary gland

suggest a Mr of 35,000 to 42,000. Rabbit mammary gland membrane

homogenates that are cross-linked with radiolabeled ovine PRL and

analyzed by SDS-PAGE and autoradiography indicated a 58,000-60,000 Mr

band. Subtraction of the Mr of PRL, yields a 35,000-37,000 Mr estimate

for PRL receptor from rabbit mammary gland (Djiane et al., 1987).

Identical results were obtained for PRL receptors in ovary, kidney and

adrenal gland from rabbits, but an additional protein band of 63,000 Mr

for ovarian and adrenal tissues was detected. (Djiane et al., 1987).

Affinity labelling results in a lower Mr estimate for prolactin

receptor subunit while gel electrophoresis suggests a higher (99,800-

340,000) Mr estimate. Berthon et al. (1987a) used hormone affinity










and monoclonal antibody detection and showed PRL receptors for sow

mammary gland at 42,000 with faint bands at 31,000 and 53,000 Mr.

Djiane and coworkers (1987) suggest that the holo-PRL receptor contains

2 or more of the 32,000-40,000 Mr units that are not linked by

disulphide bonds, but may be noncovalently associated.

Ovarian lactogenic receptors in rats (Dufau and Kusuda, 1988) have

two active subunits, 88,000 and 40,000 Mr, as purified by sequential

affinity chromatography. The 40,000 Mr subunit is part of the 80,000

Mr receptor form and is similar to the 35,000-44,000 Mr PRL receptor

subunit from rabbit, rat and mouse liver and mammary gland (Haeuptle et

al., 1983; Hughes et al., 1983; Liscia and Vonderhaar, 1982; Liscia et

al., 1982). The higher Mr species was also observed in ovarian,

testicular, kidney and mammary gland tissues following cross-linking of

receptor subunits. Different molecular species are observed in various

rat tissues including the ovary (88,000 and 40,000; Bonifacino, 1985),

Leydig cells (91,000; 81,000; 37,000 and 31,000), mammary gland

(93,000; 83,000; 30,000 and 28,000) and kidney (65,000 and 30,000)

(Bonifianco et al., 1985). Additionally, ovarian and Leydig cells

contained the 37,000 Mr form within the 81,000 form. The female rat

liver 87,000 Mr form contains subunits of 40,000 and 35,000 which may

or may not be linked by disulfide bonds (Haldosen and Gustafsson,

1987). Lactogenic receptors from mammary gland from pigs and rabbits

range in molecular weight from 28,000 to 69,000 (Haeuptle et al., 1983;

Hughes et al., 1983; Sakai et al., 1985; Katoh et al., 1985).










Molecular Structure of Cloned Prolactin Receptor

The prolactin receptor from rat liver has been cloned (Boutin et

al., 1988). Association with other proteins is not required for the

40,000 Mr structure which supports results of Liscia and Vonderhaar,

(1982); Haeputle et al. (1983) and Necessary et al. (1984) but not

those of Dufau and Kusuda (1987). The PRL receptor (40,000 Mr) subunit

contains a 19 amino acid signal sequence, an extracellular domain (210

amino acids), speculated to bind PRL, a single transmembrane section

(24 amino acids) and a short cytoplasmic domain (54 amino acids). This

PRL receptor has 30% overall homology to growth hormone receptor (Leung

et al., 1987) following removal of 293 cytoplasmic amino acids from the

growth hormone receptor structure. The two receptors share 67%

homology between the first and second, and third and fourth cysteine

residues. A 40-60% homology exists in three other extracellular

regions. A 19 amino acid series in the cytoplasmic domain has 68%

structural identity to growth hormone. These two receptors do not

share sequence homology with other proteins. The short cytoplasmic

domain of the PRL receptor does not possess tyrosine kinase activity or

phosphorylation sites as seen in other growth factor receptors (Hunter,

1987). However, the short cytoplasmic domain is similar to other

protein receptors which transport various compounds; such as

transferring receptor transport of transferring (Schneider et al., 1984),

LDL receptor transport of cholesterol (Yamamoto et al., 1984) and IGF-

II receptor transport of mannose-6-phosphate (Morgan et al., 1987).

Prolactin receptors cloned from other rat tissues (ovary, adrenal and

mammary gland) are more similar to growth hormone receptor than liver

PRL receptor by their longer cytoplamic domains (Kelly et al., 1989).










Structural differences for PRL receptors between mammary gland and

liver have been suggested previously (Sakai et al., 1985).

During sequencing of purified growth hormone receptor, 20-50% of

the receptor was actually sequenced as ubiquitin (Leung et al., 1987).

Ubiquitin has been associated with the intracellular domain of the

growth hormone receptor and may play a functional role in receptor

activation and cellular response (Leung et al., 1988). A peptide bond

forms between the epsilon-amino group associated with the cytoplasmic

domain of the receptor and the carboxy terminal end of ubiquitin

(Goldknopf and Busch, 1977) is suggested. This feature is also present

in lymphocyte homing receptor (Siegelman et al., 1986) and platelet

derived growth factor (Yardin et al., 1986) and may extend to ovarian

and mammary gland PRL receptors with long cytoplasmic domains (Kelly et

al., 1989).



Water Soluble Prolactin Receptors

Water soluble PRL receptors have been described for pig mammary

glands (Berthon et al., 1987b) and rat liver (Amit et al., 1984). This

receptor is not precipitable by polyetheylene glycol as are cytosolic

steroid receptors (Kelly et al., 1983). Water soluble receptors have

been reported for follicle stimulating hormone (Dufau et al., 1977),

human chorionic gonadotropin (Pahnke and LeidenBerger, 1978) and human

growth hormone (McGuffin et al., 1976; Herrington 1981), suggesting

that water soluble receptors are generally associated with polypeptide

hormones (Berthon et al., 1987b). The water soluble PRL receptor from

porcine mammary gland has similar specificty and affinity as membrane

associated PRL receptors and binding was higher with ovine PRL. Porcine










and rabbit PRLs were not able to compete with ovine PRL and exhibited

only 3% binding. These results also question the true validity of PRL

binding receptor estimates when using a heterologous system.

Antibodies to rabbit mammary gland PRL receptors recognize water

soluble receptors and block their binding of ovine PRL (Berthon et al.,

1987b). Water soluble and membrane associated PRL receptors probably

share antigenic determinants and hormone binding sites with rabbit

mammary membrane PRL receptors. It is unclear, however, whether water

soluble receptors are involved in signal tranduction or transport of

PRL from mammary cells into blood or milk.



Signal Transduction Systems for Prolactin

Intracellular Mediators

Peptide hormone-receptor interaction is mediated through a

transduction system to stimulate changes in cell physiology and overall

tissue response. Transduction systems involve either 1) ligand

modulated ion channel activity, 2) ligand regulating enzyme activity or

3)ligand regulation of cryptic mediators through interactions between

intracellular receptor domains or other submembranous constituents

similar to G and N regulatory components. Transduction systems have

been classified for several hormones (see review, Hollenburg, 1986;

Cockcroft and Stutchfeld, 1988). Mobility of peptide receptors is

paramount to some transduction systems, i.e., adenylate cyclase, and

functions due to the fluid mosaic properties of the cell membrane

(Singer and Nicholson, 1979) with possible involvement of microtubules

and microfilaments.










Prolactin, however, possesses a receptor leading to biological

changes within target cells, but without a known transduction system

between hormone binding and cellular response. Prolactin generates

such diverse physiological affects within a vast array of tissues that

several concomitent transduction systems are feasible. Witorsch et al.

(1987) and Hughes et al. (1985) review trannduction systems that have

been investigated for PRL. Additionally, the source of tissue for

receptor studies may bias transduction system results since PRL's

different responses may be achieved through different pathways.

Mammary gland is a complex tissue requiring support from insulin,

glucocorticoid and estrogen, in addition to PRL, for maintainence and

milk systhesis. Other hormones or factors, such as thyroxine or growth

hormone, may be essential to mammary cell function. Explant cultures of

mammary tissue have been studied to elucidate the mechanisms) of PRL

action. A recent model for PRL effects on casein biosynthesis was

proposed by Rillema (1980). Components included decreased cAMP as a

stimulatory component, calcium as an obligatory factor, phospholipase C

to generate diacylglycerol to stimulate protein kinase C and increases

ornithine decarboxylase (ODC) activity. Phospholipase A2, ODC and

prostaglandins appear to be involved in prolactin stimulation of casein

biosynthesis in mammary gland explants from mice (Cameron and Rillema,

1983). Polyamines (Rillema, 1979) and Na4-K+-ATPase (Falconer and

Rowe, 1977) may mediate PRLs action, as well as changes in

phosphatidylcholine, as PRL stimulation is associated with increased

choline uptake and decrease phosphatidylcholine turnover (Ko et al.,

1986).











1980) or transduction mechaniams of cellular hardware. Cells may not

possess identical intracellular machinery, e.g., hepatocytes versus

epithelial cells, and are inherently different and programmed to

respond differently from each other, but characteristically for each

cell type.



Extracellular Mediators

Actions of growth hormone are acheived indirectly through

stimulation of liver to release somatomedin C (Laron, 1982), now

recognized as insulin-like growth factors (IGF). This mechanisms of

action is extended to PRL (Anderson et al., 1983), which stimulates

release of a hepatic factor (Nicoll et al., 1983) that mediates PRL's

effect on mammary cells or pigeon crop sac cells. This factor, termed

"synlactin" (Nicoll et al., 1983), has no ettect alone. It is secreted

when circulating PRL increases, such as during lactation, but is not

detected in male or virgin female rats. Pigeon crop sac proliferation

increases (in vivo) when ovine PRL is injected in the hepatic, but not


jugular vein


of pigeons;


suggesting that this mitogen is produced by


liver of pigeons (Hick and Nicoll, 1985).

Prolactin stimulation of somatomedin production by rat liver is

20-fold greater than that of growth hormone. However, PRL does not

stimulate growth in male rats. Synlactin may be an IGF-like molecule

that is detected during sulphate determination of IGFs (Mick and

Nicoll, 1985). Whether synlactin is a fragment of PRL or similar to

IGF awaits determination of its amino acid structure.


Another extracellular


mediator of PRL effects is liver lactogenic


factor (LLF) (Hoeffler and Frawley 1987). Both synlactin and LLF are










secreted from the liver is response to high circulating levels of PRL.

However, Hoeffler and Frawley (1987) suggest that the two compounds are

different. The LLF is lactogenic, exerts potent biological activity

individually, and acts additively with PRL when tested in the reverse

hemolytic plaque assay (Neill and Frawley, 1983) using mammary cells.

Synlactin is mitogenic, devoid of activity alone and acts

synergistically with PRL when tested in pigeon crop sac assays.

Neither synlactin or LLF have been sequenced, nor tested in reverse

hemolytic plaque or pigeon crop sac assays, respectively.

A role for a PRL stimulated liver mitogenic factor is suggested

since liver receptors for PRL increase during pregnancy in rats (Sasaki

et al., 1982a), mice (Sasaki et al., 1982b) and rabbits (Kelly et al.,

1974; Fix et al., 1981) and may be associated with increases in

synlactin secretion and possibly mammary growth and development during

gestation (Hick and Nicoll, 1985).



Internalization of Prolactin and its Receptor

A clearly defined transduction mechanism has eluded researchers

studying PRL-receptor interaction; therefore the role of

internalization in PRL function was investigated. Prolactin has been

detected in the subcellular fraction of the plasmalemma (Posner et al.,

1981), and golgi regions of cells from rat ventral prostate and liver

(Bergeron et al., 1978). Receptor internalization through coated pits

was proposed for LDL receptors (Goldstein et al., 1979). Following

internalization, the hormone is degraded while the receptor can be

degraded or recycled. Proteolysis of internalized hormones allows

epidermal growth factor and insulin to affect target tissues (Goldfine










et al., 1987; Rosen et al., 1987; Walaas and Walaas, 1988).

Internalization and processing of PRL by lysosomes in mammary and

ovarian tissues may be involved in PRL stimulation of these cells

(Nolin and Bagdonanov 1980; Mittra, 1980; Nolin, 1982). Proteolytic

fragments of 8,000 and 16,000 Mr of PRL may be involved in PRL action

(Clapp, 1987) suggesting that 23,000 Mr prolactin is a prohormone prior

to being internalized (Nolin, 1982).

Hormone-receptor internalization to mediate PRL action suggests

involvement of cellular microtubules and microfiliments. Chloroquine,

which binds tubulin and destabilizes microtubular structures, inhibits

downregulation of PRL receptors and stimulation of casein synthesis

(Houdebine and Djiane, 1980). Subsequent results indicate that

chloroquine binds tubulin at the plasma and golgi membranes. Another

microtubular destabilizer, griseofulvin, did not affect PRL stimulation

casein synthesis of mammary cells; suggesting PRL affects are at the

cell surface (Houdebine et al., 1982).

Advances in receptor purification enabled development of

antibodies to PRL receptors. Administration of high doses of anti-PRL

receptor serum to rats blocked PRL stimulation whereas low doses

actually mimiced PRL effects, suggesting that PRL effects are through

the receptor at the cell membrane and not following internalization and

processing to cleaved or clipped forms (Witorsch et al., 1987). Whole

anti-PRL receptor serum, bivalent F(ab)z or monovalent F(ab.) fragments

demonstrated similar abilities to inhibit PRL binding to mammary gland

(Djiane et al., 1987). In mammary gland explant cultures, similar

effects on PRL receptor downregulation were observed for all forms of

anti-PRL receptor, as well as PRL itself. Low doses of bivalent F(ab)2











fragments stimulated casein DNA synthesis, while whole antibody serum

stimulated casein DNA 50-60%. Monovalent F(ab) fragment had no activity

in stimulation of casein biosynthesis. Interactions between two PRL

receptor molecules is, therefore suggested to be involved in PRL

stimulation; similar to the mechanism of insulin (Rosen, 1988). Under

these conditions, the half-life for PRL in the plasmalemma is three

times greater and movement of PRL receptors within the cell membrane

could constitute an additional regulatory mechanism for cell

receptivity. Microaggregation of receptors following hormone binding

is essential for induction of biological effects of several hormones

(Brown and Goldstein, 1983).



Regulation of Prolactin Receptors

Regulation by Ovarian Steroids

Prolactin receptor concentrations are sex specific (Sherman,

1977; Waters et al., 1978). Changes in PRL receptor numbers within

several target tissues occur during puberty (Kelly et al., 1974), the

estrous cycle (Kelly et al., 1975), pregnancy (DeHoff et al. 1984;

Grissom and Littleton, 1988), lactation (Kelly et al., 1975; Sherman et

al., 1977; Shiu et al., 1981), and in response to ovariectomy (Posner

et al., 1974a; Kelly et al., 1979; Marshall et al., 1979; Daniels et

al., 1984), orchotomy (Kelly et al., 1976; Bohnet et al., 1977) and

following administration of exogenous steroids (Waters et al., 1978;

Shiu et al., 1982). However, alterations in PRL receptor

concentrations are not necessarily similar between tissues or within

the same tissue of different species.










Steroid regulation of hepatic PRL receptors has been extensively

investigated. Hepatic PRL receptors decrease in ovariectomized rats

(Posner et al., 1974a; Kelly et al., 1979), but increase following 8 to

12 days of chronic estrogen administration (Posner et al., 1974a).

Increases in PRL receptors following estrogen treatment are thought to

be mediated indirectly, through stimulation of pituitary PRL release

and auto-upregulation of receptors. Hypophysectomy (Posner et al.,

1978), but not CB154 administration (Kelly et al., 1976), blocked the

estrogen-induced increase in rat hepatic PRL receptors, suggesting

pituitary involvement, but not exclusively an effect of PRL.

Involvement of growth hormone (Knazek et al., 1975), ACTH and TSH

(Bhattacharya and Vonderhaar, 1979) has been implicated.

Contrary to results in rats, ovariectomy increases hepatic PRL

receptors in mice (Marshall et al., 1979) and exogenous estrogen

reverses effects of ovariectomy. Estrogen administration also

decreases PRL receptors in prostate (Kledzik et al., 1976; Amit et al.,

1983) adrenal, kidney, (Monkemeyer et al., 1974) and mammary gland of

mice (Marshall et al., 1979) and rats (Bohnet et al., 1977; Smith et

al., 1976).

The increase in PRL receptors in the mammary gland following

parturition (Holcomb et al., 1975; Djiane et al., 1977) is thought to

result from autoregulation due to increases in concentrations of serum

PRL since administration of CB154 at parturition, decreases mammary

gland PRL receptors (Bohnet et al., 1977). Administration of PRL to

pseudopregnant rabbits increases mammary gland PRL binding sites; an

effect blocked by administration of exogenous progesterone (Djiane and

Durand, 1977). Sakai et al. (1978, 1979) suggest that progesterone











indirectly decreases or supresses PRL receptors by competing with

glucocorticoid receptors to block stimulation of PRL receptors.

Progesterone retards PRL auto-upregulation of PRL receptors in mammary

glands of rabbits (Djiane and Durand, 1977). However, progesterone

appears to have no effect on PRL receptors in mammary gland (Sherman et

al., 1977) or liver (Posner et al., 1974a) of rats.



Regulation by Peptides

Prolactin regulates its own receptor in target tissues. However,

unlike other peptide hormones, PRL both increases and decreases its

receptors. Auto-downregulation of PRL receptors has been detected in

vitro (Djiane et al., 1979a) and in vivo (Djiane et al., 1979b) for

rabbit mammary gland. Downregulation of PRL receptors is rapid and

transient, usually following an acute, physiological stimulus by PRL,

whereas, for other hormones, downregulation is much longer (Posner et

al., 1978).

Up-regulation of PRL receptors by physiological levels of

circulating PRL occurs in mouse and rat hepatic (Costlow et al., 1975;

Dave et al., 1981, 1982; Amit et al., 1985 and Rui et al., 1987), and

prostatic (Dave and Witorsch, 1985) membranes. Sustained high

circulating levels of PRL increased PRL receptors in rat liver (Posner

et al., 1975), rat (Holcomb et al., 1976) and rabbit (Djiane et al.,

1977) mammary gland and pigeon crop sac (Klediz et al., 1975). Auto-

upregulation of rabbit mammary gland PRL receptors have a slower onset,

requiring several days of sustained PRL levels, and is more stable

(Djiane and Durand, 1977). Therefore, Djiane and coworkers (1979)










suggested that PRL regulation of its receptor, with up and down

regulation, is through two non-antagonistic mechanisms.

Increases in ventral prostate PRL receptors occur 6-12 h following

ovine PRL administration in vitro. Increases are dose dependent (Rui

et al., 1986) and not mimiced by estrogen, androgen, hCG, insulin,

calcium, prostaglandins or cAMP. Positive regulation of ventral

prostate PRL receptors by estrogen is confirmed by Dave and Witorsch

(1985) and Blankenstein et al. (1985). Similarly PRL auto-upregulates

its receptors in testes (Amodor et al., 1985), liver (Amit et al.,

1985), lung (Amit et al., 1985), adrenal gland (Calvo et al., 1981) and

mammary gland (Djiane and Durand, 1977; Djiane et al., 1979).

Studies with hypophysectomized rats suggest that hepatic PRL

receptors may be regulated by anterior pituitary hormones, other than

PRL. Hypophysectomized rats bearing pituitary implants have increased

hepatic PRL receptor numbers which is not mimiced by adminsitration of

ovine PRL (Posner et al., 1975). Additionally, adminsitration of CB154

to reduce PRL levels had no effect (Norstedt et al., 1981). Continuous

infusion of rat growth hormone, but not rat PRL, to male rats resulted

in feminization of hepatic PRL receptor profiles (Norstedt et al.,

1987). Growth hormone may induce PRL hepatic receptors differently

between sexes since its secretary pattern differs between males and

females (Eden, 1979). Induction of hepatic PRL receptors in rats was

achieved by exogenous administration of human, bovine and rat growth

hormones, but not by PRL or human placental lactogen. Hepatic PRL

receptors of prepubertal (17 day old) female rats increased to levels

typical of adult females following 7 days of human growth hormone

infusion. These studies support growth hormone regulation of PRL










receptors. However, growth hormone may regulate PRL receptors during

growth and development while PRL may regulate its receptors to meet

adult physiological demands.



Regulation by Membrane Fluidity

The fluid mosaic membrane (Singer and Nicholson, 1979) allows

lateral and vertical movement of receptors and other proteins.

Additionally, proteins and receptors may assemble in different

configurations (Koch et al., 1979) or states of availability. The rat

liver PRL receptor is a glycoprotein with a single transmembrane domain

(Boutin et al., 1988). Therefore the microenvironment as well as the

physical status of the membrane may influence PRL receptor binding and

tissue receptivity. Changes in membrane fluidity and PRL receptor

binding were investigated in rat hepatic tissue.

Composition of unsaturated fatty acids can influence membrane

viscosity. Rats fed a diet deficient in essential fatty acids had

increased membrane viscosity that resulted in a progressive decrease in

hepatic PRL binding, not reversable by exogenous administration of PRL

(Knazek and Liu, 1979). Phospholipase Az generates arachadonic acid

from membrane phospholipids and results in a biphasic increase and

decrease in PRL binding to hepatocytes in vitro (Dave et al., 1981).

Prostacyclin treatment in vitro also increases PRL binding to hepatic

cells (Dave and Knazek, 1980) which was blocked by in vivo

administration of indomethacin (Knazek, et al., 1981). These compounds

share the ability to increase hepatic membrane fluidity and increase

PRL receptor availability. As summarized by Witorsch et al. (1987)

membrane fluidity is correlated with increases in PRL receptor numbers.










Modulation of membrane viscosity and PRL receptors by phospholipase A2

is through generation of prostaglandins. Prostaglandins may also

mediate PRL receptor auto-upregulation by decreasing membrane

viscosity. Prolactin may also change the phospholipid:cholesterol

ratio resulting in membrane fluidity changes that increase the

availability of cryptic receptors.

Subcellular localization of PRL receptors may be affected by

membrane fluidity since PRL receptors are preferentially located within

the cell. Seventy percent of mammary gland (rabbit) and liver (rat)

cells' lactogenic receptors are located in the plasmalemma (Bergeron et

al., 1978) The golgi rich fraction of cells contain 4 to 6 times more

PRL receptors and are 2.5 times more fluid than plasma membranes from

preparations from prostate and (male and female) liver cells (Dave and

Witorsch, 1986). Additionally, only plasma membranes increased PRL

receptor numbers in respond to increases in membrane fluidity.



Prolactin Functions in the Uterus

Prolactin is credited with over 100 functions in several species

(Riddle, 1963; Nicoll and Bern, 1972; Nicoll et al., 1986). However,

for the scope of this review, PRL's functions in uterine physiology

will be reviewed. For more complete discussion of PRL functions, the

following reviews are suggested: Riddle, 1963; Bern, 1975; Nicoll,

1982; and Nicoll et al., 1986. The function of PRL within uterine

physiology is suggested by the evidence of endometrial PRL receptors in

humans (Healy, 1984), sheep (Posner et al., 1974b), pigs (DeHoff et

al., 1984) rabbits (Daniels et al., 1984; Chilton and Daniels, 1985;

Grissom and Littleton, 1988), rats (Williams et al., 1978), mice (Sinha










et al., 1983), and mink (Rose et al., 1983); and by production of PRL

by decidual endometrium (Riddick et al., 1978). Exogenous PRL

administered to longterm ovariectomized rabbits stimulates uterine

proliferation and uteroglobin secretion to levels similar to that

detected in estrous does (Chilton and Daniels, 1985). Prolactin

increases the concentration of estrogen and progesterone receptors in

endometrium of rabbits (Daniels et al., 1984) and increases endometrial

uptake of estrogen in rats (Leung and Sasaki, 1973). Armstrong and

King, (1977) detected increases in progesterone metabolism by the rat

uterus following administration of exogenous PRL. Prolactin modifies

the accumulation of uterine lumen fluid in rats, possibly though

synergistic actions with estrogen (Kennedy and Armstrong, 1972).

Additionally, PRL may function during conceptus-endometrial

interactions since PRL affects blastocyst growth and terminates delayed

implantation in the mink (Martinet et al., 1981). Prolactin may also

regulate the synthesis or direction of secretion of uterine

prostaglandins in pigs (Mirando et al., 1988) and humans (Healy, 1984).

Therefore, PRL appears to affect the uterine environment through

modification of secretary functions, or effects on endometrial

proliferation or conceptus-endometrial interactions.


Porcine Conceptus Development and Uterine Secretory Response

The porcine concepts develops from a spherical form on Day 10 of

gestation to a tubular form on Days 10.5-11 and reaches a filimentous

(200 mm) form late on Day 11 (Geisert et al., 1982a). The porcine

concepts continues to elongate, initially through cellular

rearrangment and then through cellular hypertrophy and hyperplasia to











reach a lenght of 900 mm (Geisert et al., 1982a). During the initial

elongation phase, porcine conceptuses secrete estrogens (Heap et al.,

1979) which signals the maternal physiology to change the uterine

environment from cyclicity to that of pregnancy. During the

preimplantation period, the concepts relies on secretion of protein,

sugars, ions, and other compounds, collectively termed histotroph. for

nutritional support until placentation is established around Day 18.

Therefore, prior to any physical attachment to the uterine endometrium,

conceptuses must insure luteostasis and histotroph secretion and thus

establish a viable pregnancy (Bazer et al., 1982). Luetostasis is

thought to occur through the redirection of secretion of prostaglandin

secretion from an endocrine mode toward the uterine vasculature to an

endocrine mode, into the uterine lumen (Bazer and Thatcher, 1977).

Redirection of the secretion of prostaglandins is well documented both

in vivo (Frank et al., 1977) and in vitro (Gross et al., 1988),

although, the mechanism is unknown, increasing evidence suggests that

prolactin may be involved (Young and Bazer, 1988; Mirando et al, 1988).

Secretion of histotroph is a well characterized series of events

during the time of maternal recognition of pregnancy (Geisert et al.,

1982b; 1982c; Bazer et al, 1987; Roberts and Bazer, 1988).

Additionally, these uterine secretary events can be mimiced by a single

exogenous dose of estradiol valerate on Day 11 (Geisert et al., 1982c;

Young et al., 1987). Pseudopregnancy can be established in pigs

following injection of estradiol valerate on Days 11-15 or by

injections on Days 11 and 15-16 (Geisert et al., 1987). Major events

of porcine uterine secretary activity following concepts estrogen

secretion on Day 10-11 are as follows: (1) calcium is rapidly released










into the uterine lumen on Day 12 and its reuptake occurs by Day 14; (2)

sodium and potassium increase in the uterine lumen, as calcium levels

decrease, with greatest increases between Days 14 and 16; (3) protein

and uteroferrin concentrations in the uterine flushings increase on

about Days 14 to 16 and (4) glucose concentrations in uterine flushings

of pregnant pigs increase between Days 14 and 16 of gestation. In

cyclic pigs: (1) calcium increases on Day 12, but maximal levels are

lower than for pregnant pigs, and declines gradually between Days 14

to 16; (2) sodium and potassium increase as calcium decreases but to

amounts lower than for pregnant pigs; (3) protein and uteroferrin

increase on Days 15 to 16 and (4) glucose concentrations are low and

affected by day of the cycle.

Nonpregnant pigs injected with estradiol valerate on Day 11 of the

estrous cycle have uterine flushings on Day 12 that are similar in

composition to those collected on Day 12 of pregnancy (Geisert et al.,

1982c). Thus, estrogen induced uterine secretary function can be

investigated in regard to pregnancy without confounding effects of

other concepts secretary products (Geisert et al., 1982c, Young et

al., 1987).















CHAPTER 3
EFFECTS OF HYPOPROLACTINEMIA ON ESTABLISHMENT
OF PREGNANCY AND UTERINE SECRETARY FUNCTION IN PIGS



Introduction

Endocrinological studies to establish a hormone's involvement in a

physiological system is often through manipulation of the endogenous

concentration of circulating hormone followed by observation of

physiological changes in the target tissue of interest. Bromocryptine,

(CB154) a dopamine agonist, has been used in rats (Kelly et al., 1979),

pigs (Kraeling et al., 1982) and humans (see review, Barbieri and Ryan,

1983) to lower the circulating prolactin (PRL) levels through tonic

inhibition of PRL by stimulating dopamine receptors on the pituitary.

Prolactin affects proliferation (Chilton and Daniels, 1985),

protein secretion (Chilton and Daniels, 1985; Young and Bazer, 1988),

steroid receptor concentration (Daniels et al., 1984), estrogen uptake

(Leung and Sasakai, 1973) and progesterone metabolism (Kennedy and

Armstrong, 1977) in the uterine endometrium, as well as water and ion

concentrations in fetal membranes (Rabee and McCoshen, 1986; Goldstein,

1980), and fetal growth (Nicoll et al., 1986). Prolactin involvement

in uterine physiology within several species is well established.

However, very few studies utilized pigs and those studied effects of

PRL during the latter part of gestation. Studies conducted with

rabbits, (Daniels et al., 1984; Chilton and Daniels, 1985) support PRL

involvement in endometrial proliferation and secretion of uteroglobin.

44










Rabbits, like pigs, lack placental lactogen. Therefore, PRL may also

be important for endometrial function in pigs. Experiments were

conducted to lower circulating PRL in pregnant and cyclic gilts and to

observe effects on fetal survival and uterine secretary function.



Materials and Methods

Animals

Crossbred gilts of similar age (7 to 9 months) and weight (110 to

120 kg) were used in all studies after experiencing at least two

estrous cycles of normal length (18-22 days). Using intact boars,

gilts were observed for estrus daily and the first day of behavioral

estrus was designated Day 0. Gilts were mated when detected in estrus

(Day 0) and 12 and 24h later in studies using pregnant pigs.



Surgical Procedures

Uterine flushings were collected in 20 ml of double distilled

water per uterine horn as described previously (Bazer et al., 1978).

Flushing volumes were recorded and flushings were centrifuged at 10,000

x g for 15 min at 4 C. Supernatants were collected and stored at -20 C

until analyzed.



Bromocryptine

Bromocryptine (CB154; was a gift of SANDOZ Pharmaceutical, East

Hanover, NJ), a dopamine agonist, was solubilized in absolute ethanol

and mixed with saline (1:1, v/v) to a concentration of 25 mg/ml.

Treated gilts received 100 mg CB154/day (4 ml) subcutaneously while











control gilts received 4 ml of vehicle solution on the basis of

results of Kraeling et al. (1982).



Protein

Total recoverable protein concentrations in uterine flushings were

determined by the method of Lowry et al. (1951) using bovine serum

albumin as standard.


Uteroferrin

Concentrations of uteroferrin in uterine flushings were

by measurement of acid phosphatase activity (Basha et

Scholsnagle et al., 1974). Values are expressed

paranitrophenol phosphate (pNP) released per ml per 10 min

and 37 C.


determined

al., 1979;

as umoles

at pH 4.9


Calcium

The Calcette 4009 (Precision Systems, Inc., Sudbury, NA) was used

to determine calcium concentrations using ethylene glycol-bis-N-

N'tetraacetic acid (EGTA) for fluorometric titration of calcium in an

aqueous solution (Alexander, 1971).



Chloride

Concentrations of chloride in uterine flushings were determined by

a colorometric assay adapted from Hamilton (1966). Uterine flushings

were diluted 1:10 (v/v) with the mercurous reagent in 12 x 75 mm glass

tubes, covered with parafilm and inverted several times. Sodium

chloride was used for the standard curve and yielded a linear










relationship (correlation coefficient=0.99; y = intercept +6.3x)

between absorbance (510 nm) and concentration of chloride (mEq/L). The

assay was sensitive at 60 mEq/L.



Sodium and Potassium

A flame photometer (Perkin Elmer 51Ca; Coleman Instruments

Division, Oak Brook, IL) was used to determine concentrations of sodium

and potassium as described previously (Young et al., 1987).



Glucose

The Beckman Glucose Analyzer 2 (Beckman Instruments, Columbia, MD)

was used to determine glucose concentrations as a direct proportion to

oxygen consumption (Bazer et al., 1984).



Leucine-acyl Aminopeptidase (LAP)

This membrane marker protein was used as an index of secretary

activity and its concentration was determined using a colorometric

assay (Zavy et al., 1984). One Sigma Unit (SU) will release 1 umole

(143 ug) of 0-naphthylamine from L-leucyl-o-nahpthylamine per hour at

37 C and pH 7.1.


Prostaglandin (PG) F

Uterine flushings were analyzed for PGF by radioimmunoassay (RIA)

as described by Knickerbocker et al. (1986) using the antibody

characterized by Kennedy (1985) and tritiated PGF2a

([5,6,8,9,11,12,14,15-3H]:PGF2a; specific activity 160-180 Ci/mmole;

Amersham Corporation, Arlington Heights, IL). Standard curves were










prepared in charcoal-stripped uterine flushings with known amounts of

radioinert PGF2Q (0, 10, 25, 50, 100, 250, 1000 and 2500 pg). A 1:5000

dilution of antiserum enabled detection of 10 pg PGF per tube. Cross-

reactivities of PGF2a antiserum with other prostaglandins were: 94% for

PGFla; 2.4% for PGE2 and <0.1% for 13,14, dihydro-15-keto-PGF2a. PGE

and arachadonic acid. Unextracted uterine flushings (0.2 ml) were

assayed for PGF in duplicate. A pool of uterine flushings

(approximately 3 ng PGF/ml) assayed in serial dilutions (0.01, 0.025,

0.05, 0.1 and 0.2 ml with a final volume of 0.2 ml in charcoal stripped

uterine flush) resulted in an inhibition curve that was parallel to,

and not different from the standard curve when tested for heterogeneity

of regression. Further characterization of the assay involved

measurement of known amounts (10, 25, 50, 100, 250, 500, 1000 and 2500

pg) of PGF in chacrcoal-stripped uterine flushings ([y=-9.6 + 1.11x];

where y=amount of PGF measured (pg/0.2 ml) and x=amount of PGF added

(pg/0.2 ml); R2=0.947). Inter- and intra- assay coefficients of

variation were 14.1% and 15.7%, respectively.



Prostaglandin E

Concentrations of PGE2 in uterine flushings were determined using

an assay similar to that described for PGF with a modification (Lewis

et al., 1978) using tritiated PGE2 ([5,6,8,11,12,14,15-3H]:PGE;

specific activity 140-170 Ci/mmole; Amersham Corporation, Arlington

Heights, IL). A 1:6000 dilution of antiserum (Eli Lilly,

Indiannapolis, IN) enabled detection of 5 pg PGE2/tube as different

from zero. Cross-reactivities of PGE2 antiserum with other

prostaglandins were; 24% for PGEi; 1.7% for PGF2 ; 0.1% for 13,14-










dihydro-15-keto-PGFza, PGF, and arachidonic acid. A pool of charcoal-

stripped uterine flushing (approximately 3 ng PGE/ml) was serially

diluted and assayed as described previously. The inhibition curve was

parallel to, and not different from, the standard curve when tested for

heterogeneity of regression. The assay was further characterized by

measurement of known amounts of PGE2 added to charcoal-stripped uterine

flush ([y=-6.9 + 1.09x]; where y= the amount of PGE2 measured

(pg/0.2ml) and x=the amount of PGE2 added (pg/0.2ml); R2=0.928)].

Inter- and intra- assay coefficients of variation were 9.7 and 12.4%,

respectively.



Prolactin

Concentrations of PRL in serum of gilts were measured by a

radioimmunoassay (RIA) sensitive to 1 ng/ml (Kraeling et al., 1982).

The inter- and intra- assay coefficients of variation were 15.2 and

16.3%, respectively (Kraeling et al., 1982).



Statistics

Data were analyzed by least squares analysis of variance using the

General Linear Models procedures of the Statistical Analysis System

(SAS) (Barr et al., 1979) to detect effects of treatment.



Experiment 1

This experiment determined effects of hypoprolactinemia on

concepts survival. Ten gilts were mated on Days 0 and 1 of the

estrous cycle and assigned randomly, 5 per treatment group, to receive

either CB154 (100 mg/day) or vehicle (4ml/day) once daily on Days 10










through 16 of gestation. Jugular vein blood samples were collected on

Days 10 (preinjection), 15 (during injection) and 20 (4 days

postinjection) using a vacutainer single sample collection method to

avoid prolonged stress. Serum was assayed for concentrations of PRL to

determine effectiveness of CB154. On Day 25 of gestation, gilts were

injected with sodium thiamylal (ig, i.v.) to induce anesthesia, which

was then maintained with halothane using a closed-circuit gas

anesthetic unit, and subjected to midventral laparotomy. The uterus was

exposed and examined for evidence of normal pregnancy. Each gilt

received 15 mg of PGFz2a (Lutalyse, Upjohn Company, Kalamazoo, MI) to

terminate the pregnancy. Gilts experienced two normal estrous cycles

before being used in Experiment 2. This protocol was necessary because

of the requirement that CB154-treated gilts be euthanized.



Experiment 2

This experiment was to determine effects of hypoprolactinemia on

uterine secretary activity. The protocol was to mimic effects of

estrogens from conceptuses on endometrial secretary activity during the

time of maternal recognition of pregnancy without interactions with

other concepts products (Geisert et al., 1982b). The 10 gilts used in

Experiment 1 were assigned randomly, 5 per treatment group, to receive

either CB154 (100 mg/day) or vehicle (4ml/day) on Days 10 and 11 of the

estrous cycle. All gilts then received 5 mg of estradiol valerate

(i.m.) on Day 11. On Day 12, uterine flushings and a jugular vein

blood sample were collected.










Results

Effects of Hypoprolactinemia

Administration of CB154 to pregnant gilts (Experiment 1) decreased

circulating concentrations of PRL by 40% (P<0.06) on Day 15. Prolactin

levels (+0.33 ng/ml) were 5.0, 3.3, and 4.4 ng/ml and 5.2, 5.0, and

4.4 ng/ml on Days 10, 15 and 20 of gestation for gilts that received

CB154 and vehicle only, respectively. The number of corpora lutea

(CL) was not different on Day 25 of gestation between gilts that

received CB154 (13.6 +0.9) and vehicle (14.0 +0.9) and litter size was

similar between gilts that received CB154 (7.3 +1.75) and vehicle (8.4

+1.75).

Administration of CB154 to cyclic gilts (Experiment 2) decreased

circulating concentrations of PRL by 50% (2.7 +0.33 ng/ml vs 5.3 +0.33

ng/ml; P<0.06) on Day 12 to levels that were similar to those reported

for CB154-treated pigs by Kraeling et al. (1982), Whitacre et al.

(1981) and Smith and Wagner (1986). The inability to totally supress

circulating PRL in pigs by CB154 administration may be due to PRL

regulation by factors other than dopamine. Previously reported dosages

of 480 mg/day did not affect PRL support of corpus luteum function in

mid-gestational gilts (R.R. Kraeling as cited by Bazer and First,

1983). Total recoverable protein (mg), uteroferrin (umoles), and

glucose (mg) concentrations, respectively, were not different in

uterine flushings from gilts that received CB154 (17.6+2.4 mg; 541+92

umoles and 2.23+0.13 mg) compared to those that received vehicle

(19.2+2.4 mg; 608+92 umoles and 2.35+0.13 mg). However, concentrations

of leucine aminopeptidase were lower (P<0.025) in uterine flushings of

CB154 (140+18 SU) compared to vehicle (230+18 SU) treated gilts.

































Figure 3-1: Concentrations of total recoverable (A) calcium,
(B) chloride, (C) sodium and (D) potassium in Day 12 uterine
flushings from cyclic gilts (Experiment 2) treated with CB154
(100 mg/day) or vehicle (VHC; 4 ml) on Days 10 and 11 and
estradiol valerate on Day 11. Treatment effects were
detected for calcium and potassium (P<0.05), as well as
sodium and chloride (P<0.01). The overall SEM was +0.14 for
calcium, +0.12 for chloride, +12 for sodium and +8 for
potassium.



























CB154 VHC




c


CB154 VHC


150

hO
v
E
<" 7s --
f 75
6

!-


HA


B













CB154 VHC




D











I4
CB154 VHC


300-


hO

E
150-


I-


1 -










Concentrations of PGF (30+5 vs 17 +5 ng/horn) and PGE (14+2.2 vs 11

+2.2 ng/horn) in uterine flushings were not affected by treatment.

Gilts that received CB154 had lower concentrations of calcium (P<0.03),

sodium (P(0.02), potassium (P<0.01) and chloride (P<0.01) in uterine

flushings (Figure 3-1).



Discussion

Bromocryptine, effectively lowers PRL to near undecteable levels

in rats. In this study, PRL was decreased 40-50% in pigs. Fetal

survival was not affected despite decreased circulating PRL. The

remaining concentrations of circulating PRL may have been adequate for

fetal survival. Additionally, porcine conceptuses may have compensated

through other physiological mechanism, which at this time are unknown,

to maintain the pregnancy. Prolactin modulates endometrial physiology

of rabbits (Daniels et al., 1984; Chilton and Dainels, 1985), but

adminsitration of CB154 to pigs on Days 10-15 of gestation may have

been unable to reverse prior affects of basal PRL on endometrium.

Therefore, although PRL levels were lowered, the endometrium was

already stimulated to respond to concepts signals. Lowered PRL levels

following concepts estrogen signal may have blunted the uterine

secretary response, but PRL concentrations were adequate for concepts

survival. Fetal survival may have been affected if CB154

administration began earlier in gestation to block possible effects or

modulation of PRL on uterine physiology.

However, lowered PRL affected estrogen-stimulated ionic changes in

the uterine lumen, suggesting that PRL may affect the ionic environment

of the developing concepts. In this study, slight hypoprolactinemia










significantly decreased concentrations of calcium, sodium, potassium

and chloride ions in uterine flushings, but did not affect total

protein. The inability of lowered PRL levels to alter secretion of

proteins may be due to lack of effects on ion ratios despite lowered

individual ion levels. The effects of decreasing concentrations of

ions may not have occurred in a temporal pattern necessary to alter in

the secretion of proteins. The aminopeptidase activity in uterine

flushings was decreased in response to hypoprolactinemia suggesting a

decrease in secretary activity (membrane processing) of endometrial

epithelium (Zavy et al., 1984).

The decrease in circulating PRL levels in the present study may

have been compensated for physiologically through other mechanisms,

e.g., an increase in PRL receptor numbers, has been previously

demonstrated for rabbit mammary tissue (Dijane et al., 1977) and rat

liver (Kelly et al., 1979). Hypoprolactinemia may also affect hormonal

regulation at the central nervous system, receptor levels in the

hypothalamus and target tissue (Muldoon, 1985) or intrapituitary

communication (Murai et al., 1988). Additionally, regulation of PRL

secretion by gamma amino butyric acid (Schally et al., 1978; Duvalinski

et al., 1987) in pigs suggests a different control mechanism for which

CB154 may be less effective.

In summary, circulating PRL levels were decreased 40-50% in pigs

following administration of CB154, but fetal survival at Day 25 of

gestation was not affected. Ions and LAP concentrations, but not

secreted proteins, in uterine flushings of estrogen stimulated Day 11

cyclic gilts were decreased. Hypoprolactinemia had begun to affect

uterine secretary function, but changes in ions may not have occurred







56


in the proper ratio or temporal pattern to secretion of proteins.

Additionally, decreases in circulating PRL may have been compensated

for physiologically through other mechanisms.














CHAPTER 4
EFFECTS OF CYSTEAMINE ON CIRCULATING
PROLACTIN LEVELS IN PIGS



Introduction

Cysteamine (CSH; SH2-CH2-CH2-NHz) disturbs the tertiary structure

of prolactin (PRL) and thereby depletes circulating levels of bioactive

PRL (Millard et al., 1982). Several short-term studies of effects of

CSH on PRL levels in rats have been reported (Millard et al., 1982;

McComb et al., 1985; Sagar et al., 1985). Dose response tests

determined that rats administered 90 mg/kg CSH (subcutaneously) had

decreased PRL levels in both the anterior pituitary (2.0 vs 0.57 ug/mg

protein) and serum (3.7 vs 0.44 ng/ml) 4h post-administration. No

adverse effects were reported (Millard et al., 1982). Cysteamine can,

therefore, lower circulating levels of bioactive PRL without

involvement of dopamine or other receptors at the anterior pituitary.

This study investigated the use of CSH for inhibition of circulating

PRL levels in pigs as an alternate method to bromocryptine

administration.



Materials and Methods

Experimental Design

On Day 4 of the estrous cycle, gilts were anesthetized with

thiamylal sodium (1 g, i.v.) and maintained under surgical anesthesia

using a closed circuit anesthesia machine. Gilts were fitted with

57










jugular catheters (Ford and Maurer, 1978), allowed 4 days rest and

housed individually to recover from surgery through Day 7. On Day 8,

7-10 ml blood samples were collected at 0800, 1300 and 1930 h. Gilts

were assigned randomly to receive either CSH (100 mg/kg/day) or an

isoosmotic control of ethanolamine (100 mg/kg/day). Administration of

treatment began on Day 10 at 0830h and was scheduled to continue until

Day 16 of the estrous cycle. Blood samples were taken two days prior

to, and following, the treatment period.



Results

Due to unexpected cytotoxic effects of CSH at the injection sites,

(tissue necrosis and gangrenous leasions), this project was cancelled

after four gilts received treatments for various lenghts of time.

Blood samples were analyzed for PRL concentrations and results are

summarized in Figure 4-1. Due to the inconsistent number of blood

samples and limited data, statistical analyses were not conducted.

Administration of CSH lowered serum PRL levels about 30% (1.97 vs 2.7

ng/ml) of controls after three days and about 70% (1.33 vs 4.32 ng/ml)

after five days of treatment. Comparison of PRL levels from CSH- and

CB154-treated gilts (Chapter 3) on a similar "day post-injection" basis

revealed that CSH lowered serum PRL levels 67% more than CB154.

Interestrous intervals and (arbitary) cytotoxic reactions to the

treatments are summarized in Table 4-1. A few comments on these

results are in order: Gilt 328 started the experiment in which 100

mg/kg dose was given twice daily to prevent possible increases in PRL

in the evening. This dosage was very cytotoxic, therefore CSH was

administered to gilt 305 at the same dose but only once a day in the





















A
/
I


p
/ N
I
/


I
I-
*'*~--~


10 11 12 13 14 15 16 17 18


Day of estrous cycle





Figure 4-1: Mean concentrations of prolactin (ng/ml) in
serum of cyclic gilts treated with cysteamine (solid line) or
ethanoloamine (dashed line) from Days 10-16 (denoted by
arrows).





























Table 4-1: Effects on interestrus interval and cytotoxicity
reaction to cysteamine (CSH) or ethanoloamine (control)
administration to cyclic gilts.

Gilts Group Dose #Inj #B1 Smpl Cytotoxicity Cycle length
mg/day score days

328 CSH 200 9 19 10 28+

326 Control 100 7 33 5 30

305 CSH 100 7 33 7 45+

12 Control 100 3 33 3 23










AM. The other gilts also had cytotoxic reactions to the injections and

were rated arbitarily on a 1 to 10 scale. The cycle lengths are

reported, although of limited value since CSH-treated gilts did not

return to estrus before being euthanized.



Discussion

These data suggest that CSH may be effective in decreasing

circulating levels of bioactive PRL and interestrous intervals may have

been increased in gilts that received CSH. Additionally, the gross

morphological appearance of the reproductive tract included atrophy and

regression (see Figure 4-2). Although CSH appeared to be effective in

lowering PRL levels and changing uterine appearance, use of this

compound in gilts in not warranted due to the severe cytotoxicity it

induced when administered subcutaneously at the dosage used for rats.












































Figure 4-2: Reproductive tracts from gilts treated with
either (A) ethanolamine or (B) cysteamine.











CHAPTER 5
ESTABLISHMENT OF HYPERPROLACTINEMIA
BY ADMINISTRATION OF EXOGENOUS PORCINE PROLACTIN TO PIGS



Introduction

Establishment of function for a hormone can be acheived through

administration of that hormone followed by studies of the physiological

changes it induces. Circulating prolactin (PRL), however, is subject to

increases due to stress such as those that may be experienced during

frequent blood sampling and confinement. Therefore, this study was

conducted to establish that hyperprolactinemia could be acheived by

injecting porcine PRL twice daily. This was done as a separate

experiment to avoid compromising uterine secretary responses in

subsequent experiments (Chapter 6) due to confinement and chronic

bleeding of the gilts which would likely cause stress-induced release

of PRL.



Material and Methods


Animals

Crossbred gilts of similar age

120 kg) were used in all studies

estrous cycles of normal length

gilts were observed for estrus daily

estrus was designated Day 0.


(7 to 9 months) and weight (110 to

after experiencing at least two

(18-22 days). Using intact boars,

and the first day of behavioral











Catheterizations

On the assigned day, gilts were anesthetized and fitted with

indwelling jugular catheters (Ford and Mauer, 1978) which were

maintained with 200 lU/ml heparin:saline solution. Following surgery,

gilts were housed individually until after collection of the final

blood sample and removal of the catheters.



Hormones

Exogenous porcine prolactin (PRL; USDA-B-1; gift from Dr. Douglas

Bolt, National Animal Hormone Program Director) was diluted in

phosphate buffered saline (PBS; 1 mg/ml; pH 7.2), aliquoted into 1.2 ml

volumes and stored at 4 C until injected subcutaneously.



Experimental Design

Six nonpregnant gilts, 3 per treatment group, were assigned

randomly to receive either porcine PRL (1 mg) or vehicle (SAL; 1 ml

saline). On Day 7, gilts were anesthetized and fitted with indwelling

jugular vein catheters. Jugular vein blood (7 ml) samples were

collected on Days 10 through 13 of the estrous cycle at 0730, 1000,

1200, 1930 and 2400h. Prolactin or saline was administered

subcutaneously at 0800 and 2000h, 30 min after the morning and evening

blood samples, on Days 10 through 14. Blood samples were assayed for

concentrations of PRL in serum.



Prolactin

Concentrations of PRL in serum of gilts were measured by a

radioimmunoassay (RIA) sensitive to 1 ng/ml (Kraeling et al., 1982).










The inter- and intra- assay coefficients of variation were 15.2 and

16.3%, respectively.



Statistics

Data were analyzed by least squares analysis of variance using the

General Linear Models procedures of the Statistical Analysis System

(SAS) (Barr et al., 1979). Included in the model were the effects of

pig, treatment, time and treatment by time interaction.



Results

Effects of administration of exogenous porcine PRL are presented

in Figure 5-1. Concentrations of PRL in serum increased within 2h in

gilts that received exogenous porcine PRL. Over the 4 days of

administration, concentrations of PRL were 4.5-fold higher (P<0.O01)

for gilts that received exogenous porcine PRL (19.6+1.24 ng/ml)

compared to control gilts (4.3 +0.13 ng/ml).



Discussion

Administration of exogenous porcine PRL at 12 h intervals was

effective in elevating the circulating levels of PRL. Prolactin levels

were increased at 2 h post-administration and remained elevated

throughout the treatment period. Additionally, the increase in PRL

levels were not pharmacological since concentrations of PRL in pigs

during estrus and the late luteal phase are 15 to 20 ng/ml (Brinkley et

al., 1973; Dusza and Kryzmowska, 1979).















24
22- -
| 20-
C

18-

16-
bo 14-
0- 12-
E 10-

8-
6-
4!
4 ..........^ - H-^h^ N^

10 11 12 13
Day of Estrous Cycle





Figure 5-1: Concentrations of immunoreactive prolactin in
serum during administration of 1 mg porcine prolactin,
(circles), or 1 ml saline (squares), at 0800 and 2000h
(denoted by arrows) on Days 10 through 13 of the estrous
cycle. Blood samples were collected at 0730, 1000, 1200,
1930 and 2400 h. The SEM were +1.24 ng/ml for prolactin
treated and +0.12 ng/ml for saline treated gilts.















CHAPTER 6
EFFECTS OF HYPERPROLACTINEMIA ON PROGESTERONE AND ESTROGEN
INDUCED UTERINE SECRETARY RESPONSE IN PIGS



Introduction

As mentioned in Chapter 3, early endocrinological studies involved

manipulation of endogenous hormones to establish physiological roles at

the tissue of interest. Converse to lowering endogenous hormones,

manipulations which increase endogenous hormone levels followed by

observation of physiological changes can also allow insight into a

hormones potential involvement in function. Several mechanisms have

been used, ranging from injection of crude tissue homogenates to

sophisticated gene manipulations. Each technique acheived a similar

endpoint, increased or supplemented endogenous hormone levels to allow

to investigation of resulting physiological changes.

Exogenous prolactin (PRL) results in increased endometrial

proliferation and protein secretion (Chilton and Daniels, 1985) in long

term (12 week) ovariectomized rabbits. Prolactin also modulates ion

channels (Falconer and Rowe, 1977), gap junction formation (Sorenson et

al., 1987) auto up-regulates its own receptor (Djaine et al., 1977;

1987) and increases steroid receptors (Daniels et al., 1984). Through

these mechanisms, PRL may affect the uterine environment of pigs.

Uterine secretary function is critical for nourishment of

preimplantation porcine conceptuses. Investigation of PRL interactions

with ovarian steroid hormones, especially estrogen, may explain the

67










biphasic responses of the uterus that occur following estrogen

administration (Szego et al., 1978; Geisert et al., 1982c; Young et

al., 1987) possibly through modification of the uterine environment or

through more rapid effects of the peptide hormones. Therefore,

interactions between PRL and progesterone, in the absence of estrogens

of ovarian origin, were investigated in pigs. Knight et al. (1973)

demonstrated that ovariectomized pigs treated with progesterone alone,

secrete the same proteins after 11 days of treatment as ovarian intact

gilts on Day 15 of the estrous cycle or pregnancy. In a second

experiment, the interaction between estrogen and PRL was investigated

following administration of exogenous estrogen. Administration of

exogenous estrogen on Day 11 of the cycle mimics the maternal

recognition of pregnancy factor, porcine concepts estrogens, without

confounding effects of other concepts secretary products (Geisert et

al., 1982c).



Materials and Methods

Animals

Crossbred gilts of similar weight (100-120 kg) and age (7-9

months) were used in this study after they experienced at least two

normal estrous cycles (18-22 days). In the presence of intact boars,

gilts were observed daily for behavioral estrus. The first day of

behavioral estrous was designated Day 0.



Exogenous Hormone Administration

For chronic steroid treatment, gilts received 200 mg progesterone

(P4; Sigma, St. Louis, MO) in 4 ml corn oil:ethanol solution (90:10,











v/v). Control gilts received 4 ml vehicle only. Acute steroid

treatment consisted of 0.5 ml (10 mg/ml) estradiol valerate (E2V;

Squibb, Raritan, NJ) or 0.5 ml corn oil. Exogenous porcine prolactin

(PRL; USDA-B-i; gift from Dr. Douglas Bolt, National Animal Hormone

Program Director) was diluted in phosphate buffered saline (PBS; 1

mg/ml; pH 7.2), aliquoted into 1.2 ml volumes and stored at 4 C until

injected subcutaneously.



Surgical Procedures

Gilts used in studies of PRL effects on P4-induced uterine

secretary components in Experiment 1 were ovariectomized on Day 4 of

the estrous cycle. Gilts were anesthetized, subjected to midventral

laparotomy, the ovaries were exteriorized, all ovarian vessels were

ligated and the ovaries removed with minimal trauma to the uterus.

Uterine flushings were collected as described in Chapter 3.



Experiment 1

Seven nonpregnant gilts were ovariectomized on Day 4 of the

estrous cycle and injected with 200 mg P4 once daily from Days 4

through 14. Gilts were assigned randomly to receive either porcine PRL

(n=4; 1 mg) or vehicle (n=3; 1 ml saline) daily at 0800 and 2000h on

Days 4 through 14. Uterine flushings were collected on Day 15.



Experiment 2

Twelve nonpregnant gilts, 4 per treatment group, were assigned

randomly to receive one of three treatments. Gilts in the negative

control group (corn oil only on Day 11) and positive control group (5










mg E2V in corn oil on Day 11) received 1 ml saline twice daily at 0730

and 1930h on Days 6 through 11. Gilts in the treatment group received 1

mg porcine PRL in saline twice daily at 0730 and 1930h and 5 mg E2V on

Day 11. Uterine flushings were collected on Day 12, approximately 24h

after treatment with corn oil or E2V. A PRL and corn oil treatment

group was not included because results of Experiment 1 indicated no

interaction between PRL and progesterone on uterine secretary activity.



Uterine Flushings

Uterine flushings were processed and evaluated for total

recoverable protein, uteroferrin, leucine aminopeptidase (LAP),

glucose, calcium, sodium, potassium, chloride, PGF, and PGE as

described in Chapter 3.



Results

Effects of PRL on P4-induced uterine secretary responses are

summarized in Table 6-1. Administration of exogenous PRL did not

affect any of the P4-induced uterine secretary components examined in

the uterine flushings.

Effects of exogenous PRL on estrogen-induced uterine secretary

activity are summarized in Figures 6-1, 6-2, and 6-3. Treatment

effects were detected for (see Figure 6-1A, B and C) total recoverable

protein and total uteroferrin (P<0.001), total glucose (P<0.01), and

total leucine aminopeptidase (P<0.05); (see Figure 6-2A, B and D),

total calcium (P<0.03), total chloride (P<0.02), total potassium

(P<0.01); and (see Figure 6-3A) total PGF (P<0.02) in uterine

flushings. There was no effect of treatment on sodium (Figure 6-2C) or
















Table 6-1: Composition of Day 15 uterine flushings from
ovariectomized gilts treated with daily injections of
progesterone and saline or porcine prolactin from Days
4 through 14 (x + SEM).

ITEM8 SALINE PROLACTIN


Total Protein (mg) 74.3 +19.3 74.0 +16.7

Total Uteroferrinb 4138 +1903 4230 +1648
(umoles/uterine horn)

Uteroferrin/mg Protein 45.7 +14.9 51.7 +13.0

Total Calcium (mag) 0.5 +0.2 0.4 +0.2

Total Cloride (mg) 1.7 +0.3 1.5 +0.2

Total Sodium (ug) 125.3 +32.1 93.5 +27.9

Total Potassium (ug) 170.2 +46.7 131.8 +40.4

Total Glucose (mg) 2.5 +0.3 2.8 +0.3

Total L-acyl aminopeptidasec 655 +112 604 +92

Total PGF (ng/uterine horn) 235 +22 219 +19

Total PGE (ng/uterine horn) 38.6 +6.7 34.6 +5.8


*Treatment effects were not detected (P>0.05).

bAcid phosphatase activity; umoles p-nitrophenol
released/ml/10 min at 37 C.

cSigma Units: One Sigma Unit will release 1 umole (143 ug)
of B-napthylamine from L-leucine-B-nalpthylamine per hour
at 37 C, pH 7.1.































Figure 6-1: Concentrations of total recoverable (A) protein,
(B) uteroferrin, (C) glucose, and (D) leucine aminopeptidase
(LAP) activity in Day 12 uterine flushings from cylic gilts
(Experiment 2) treated with 1 ml saline (SAL) or 1 mg porcine
prolactin (PRL) at 0800 and 2000h on Days 6-11 and 0.5 ml
corn oil (OIL) or 5 mg estradiol valerate (E2V) on Day 11 of
the estrous cycle. Overall treatment effects were detected
for protein (P<0.O01), uteroferrin (P<0.01) and leucine
aminopeptidase activity (P letters are different (P<0.05). The overall SEM was +2.72
for protein, +272 for uteroferrin, +0.16 for glucose and
+52.7 for LAP.



















4000-4


2000--


Sal PRL Sal
Oil l-E2V-j


400-


Sal PRL Sal
Oil -E2V--


D


200-I-


Sal PRL Sal
Oil -E2V--


Sal PRL Sal
Oil I-E2V-j


0
E
c
'S

-20-


I-


3.0-4-


1.5-+


4A
40-1-
































Figure 6-2: Concentrations of total recoverable (A) calcium,
(B) chloride, (C) sodium and (D) potassium in Day 12 uterine
flushings from cyclic gilts (Experiment 2) treated with 1 ml
saline (SAL) or 1 mg porcine prolactin (PRL) on Days 6-11 and
0.5 ml corn oil (OIL) or 5 mg estradiol valerate (E2V) on Day
11 of the estrous cycle. Overall treatment effects were
detected for calcium and chloride (P<0.05) as well as
potassium (P different (P<0.05). The overall SEM was +0.3 for calcium,
+0.22 for chloride, +10.7 for sodium and +9.23 for potassium.






















E


60
E
3n
5 1
m
@


2-


oi -E2V-I


80-4 c


70-+


60-+


50-+


01-

U


- -
0
J1-


0I1 rnL 2 I1
Oil ^2-


100-






250-
E


-o

03
0-
C6


D


2-_



















A B



50
50-- 50--




I11
SOa
25 25 .. ....


eUU


III

Sal PRL Sal Sal PRL Sal
Oil [-E2V-j Oil I-E2V--




Figure 6-3: Concentrations of (A) PGF and (B) PGE in Day 12
uterine flushings from cyclic gilts (Experiment 2) treated
with 1 ml saline (SAL) or 1 mg porcine prolactin (PRL) on
Days 6-11 and 0.5 ml corn oil (OIL) or 5 mg estradiol
valerate (E2V) on Day 11 of the estrous cycle. Overall
treatment effects were detected for PGF (P(<0.01). Values
with different letters are different (P<0.05). The overall
SEM was +10.2 for PGF and +6.7 for PGE.










PGE (Figure 6-3B) in uterine flushings. Additionally, gilts treated

with PRL+E2V had greater amounts of glucose (P<0.01) and PGF (P<0.01)

in uterine flushings than gilts receiving E2V alone. But, values for

glucose and PGF were not different for gilts treated on Day 11 with E2V

or corn oil. The specific activity of uteroferrin (umoles/mg protein)

was higher (P<0.01) in uterine flushings from gilts treated with

PRL+E2V (106.5 +9.3) compared to gilts receiving SAL+E2V alone (56.7

+9.3) or SAL+corn oil (8.4 +9.3). Gilts receiving PRL+E2V had greater

uterine secretary responses for total protein, potassium and leucine

aminopeptidase compared to gilts receiving SAL+E2V, but differences

were not statistically significant.



Discussion

Exogenous PRL interacted with estrogen, but not progesterone, to

cause significant effects on uterine epithelial secretary activity.

Shifts in concentrations of ions could account for the earlier release

of several uterine secretary components; although such shifts could not

be not accounted for by static measurements on Day 12 in the present

study. Shifts in ions occur prior to protein secretion during early

pregnancy (Geisert et al., 1982b; Bazer et al., 1984) and in response

to administration of exogenous estradiol on Day 11. With respect to

the secretary profiles of uterine components in the experiments

reported here, exogenous PRL may have advanced the rapid release and

reuptake of calcium which characteristically follows estrogen

stimulation and precedes accumulation of proteins in the uterine lumen.

Luminal calcium flux may have occurred early on Day 11 in

hyperprolactinemic gilts. Therefore, calcium measured in uterine










flushings on Day 12 could be influenced by advanced calcium reuptake

that does not normally occur until Days 13-14 of pregnancy. This may

account for the increase in glucose and uteroferrin on Day 12 for PRL

treated pigs compared to previous reports that glucose and uteroferrin

concentrations do not increase until Days 14 to 16 during a normal

pregnancy (Bazer et al., 1984). Hyperprolactinemia resulted in

increased concentrations and specific activity of uteroferrin in (Day

12) uterine flushings. Increases in this protein usually occur on Day

14 of gestation (Zavy et al., 1982). Since there was not a concomitant

increase in aminopeptidase, a membrane marker protein indicative of

cell membrane processing and secretary activity, PRL may have altered

uteroferrin synthesis resulting in an increase in the releaseable pool

stored within intracellular secretary granules and available for

release following estrogen stimulation. Additionally,

hyperprolactinemia resulted in increased total glucose in uterine

flushings on Day 12 which normally does not occur until Days 14 to 16

of pregnancy (Zavy et al., 1984). Administration of PRL appeared to

advance the ability of uterine epithelium to respond to estrogen-

induced secretary events.

Stimulation of secretion of prostaglandins of the F, but not the

E, series suggests that effects of PRL may be on the uterine

epithelium. Previous reports have localized PGF and PGE secretion to

the endometrial epithelium and stroma, respectively, of cows (Grasso et

al., 1987). Prolactin exerts luteostatic effects in several species

(Murphy and Rajkumar, 1985), primarily through stimulation of ovarian

steroid production. Prolactin appears to advance the ability of the

uterus to respond to estradiol since an increase in luminal PGF










occurred 48 to 72 h following a single injection of estradiol valerate

on Day 11 to cyclic gilts (Geisert et al., 1982b). During

establishment of pregnancy in the pig, PGF secretion must be redirected

from an endocrine to an exocrine direction (Bazer and Thatcher, 1977)

to protect the corpus luteum from regression. The ability of PRL to

stimulate secretion of PGF and enhance its secretion into the uterine

lumen (Mirando et al., 1988) is a novel finding suggesting that PRL

plays a luteostatic role, in conjunction with estrogen, in early

pregnancy of pigs.

The present findings in the pig, support the hypothesis that PRL

modulates uterine secretary activity during establishment of pregnancy

as previously described for long-term ovariectomized rabbits (Chilton

and Daniels, 1985). Prolactin increased uterine secretary function and

caused differential changes in ions, increased secretion of proteins,

PGF and glucose. Although the mechanisms) by which PRL influences the

endometrial secretary profile is not known, several mechanisms may be

involved. These include 1) activation of ion channels to facilitate

transport and secretion of cellular components (Petersen and Maruyama,

1985) or stimulate membrane cycling of calcium (Alkon and Rasmussin,

1988); 2) increased estrogen binding by cells of rat liver (Chamness

et al., 1975) and uterus (Leung and Sasaki, 1973); 3) facilitation of

formation of gap junctions to increase intercellular communication

(Sorenson et al., 1987), and 4) up-regulation of PRL receptors to

increase membrane fluidity (Dave and Witorsch, 1985) and increase the

availability of cryptic hormone receptors. Dave et al. (1983) observed

an increase in PRL binding during early pregnancy which they attributed

to alterations in membrane fluidity.







80


In summary, during the time of maternal recognition of pregnancy

in the pig, PRL interacts with estrogen, rather than progesterone, to

influence uterine endometrial secretary activity. This selective

interaction with estrogen may allow the uterine endometrium to respond

maximally to the estrogen signal from the porcine concepts which

allows establishment of pregnancy.















CHAPTER 7
DEVELOPMENT OF A HOMOLOGOUS RADIORECEPTOR ASSAY FOR
PORCINE ENDOMETRIAL PROLACTIN RECEPTORS



Introduction

Prolactin (PRL) binds to high affinity, low capacity receptors

(Shiu et al., 1973; Sakai et al., 1985) in target tissues and induces

responses that are biologically important to the reproductive system.

Prolactin binding sites have been reported in reproductive tissues of

sheep (Posner et al., 1974b), rats (Williams et al., 1974), humans

(Healy, 1984), mink (Rose et al., 1983), rabbits (Grissom and

Littleton, 1988) cow (Posner et al., 1974b) and pig (Posner et al.,

1974b; DeHoff et al., 1984; Bramley and Menzies, 1987). Prolactin

exerts effects on steroidogenesis in corpora lutea (CL) (Veldhuis et

al., 1980; Brambley and Menzies, 1987), transport of water by placental

membranes (Goldstein et al., 1980), uterine endometrial proliferation,

protein synthesis and secretion (Chilton and Daniels, 1985; Young and

Bazer, 1988), enhanced secretion of prostaglandin F2a into the uterine

lumen (Mirando et al., 1988), as well as mammary growth and lactation

(Vonderhaar et al., 1984).

Endometrial PRL binding sites have been detected in several

species. Local production of PRL from uterine decidual tissue may

affect uterine physiology in an autocrine or paracrine manner (Healy,

1984; Jayatilak and Gibori, 1986). However, in species with

noninvasive implantation, such as the pig, changes in the number of PRL

81










receptors in the endometrium may determine effects of PRL on the

physiology of the uterus. While these receptors may respond to

decidual PRL and/or placental lactogen, the pig has neither and

circulating levels of immunoreactive PRL are relatively constant

throughout pregnancy (Dusza and Krzymowska, 1981). Therefore, PRL

effects are probably regulated by changes in the PRL receptor

population or receptor characteristics.

Heterologous ovinee) PRL radioreceptor assays (RRA) enabled

detection of PRL receptors (fmoles/g wet weight; fm/gww) in porcine

endometrium as early as Day 15 (75 fm/gww), increasing receptor numbers

to Days 45 and 75 (575 and 700 fm/gww, respectively) and decreased

receptor numbers by Day 90 (65 fm/gww) of gestation (DeHoff et al.,

1984). These changes in PRL receptor numbers are temporally associated

with increases in concentration of circulating estrogens (DeHoff et

al., 1986), PRL-stimulated endometrial proliferation, induction of

steroid receptors, protein secretion (Chilton and Daniels, 1985),

epithelial ion transport (Rabee and McCoshen, 1986), transport of

placental water (Goldstein et al., 1986) and fetal growth (Nicoll et

al., 1985).

Several heterologous RRAs have been described for PRL and are used

routinely (Shiu et al., 1973; Waters et al., 1974; Kelly et al., 1979)

but may be complicated by indiscriminant binding due to effects of

heterologous hormones (Nicoll, 1982). However, PRL, a member of the

growth hormone-prolactin-placental lactogen family, can crossreact with

other hormones in that family in RRAs. Prolactin from one species

often has biochemical homology and physiological actions related to

growth hormone of another species. Additionally, high variablity in










structural properties between PRL of different species (Nicoll et al.,

1985) could contribute to inaccurate measurement of receptors within a

species when a heterologus RRA is used for receptor quantification. A

homologus RRA has the advantage of insuring that a receptor is specific

for the hormone under investigation and that the results can be

accurately correlated to the in vivo hormonal environment and provide a

method for investigating changes in in vivo PRL receptor populations

that are of physiological interest. Additionally, development of a

homologous RRA for porcine tissues may indicate the feasibility of

developing and using homologous RRAs for other species or hormone

systems.



Materials and Methods

Hormones

Porcine PRL, growth hormone (GH), follicle stimulating hormone

(FSH), luteinizing hormone (LH) and ovine PRL were from the USDA (grade

USDA-B-1) and were generously supplyed by Dr. Douglas Bolt (Director of

Animal Hormone Program).



iodination of Hormones

Porcine PRL (USDA-B-1) was iodinated using the lodo-gen procedure

adapted from Markwell and Fox (1978). Radioactivity in the eluant from

a gel filtration column separating protein bound iodine from free

iodine was monitored by counting 10 ul aliqouts from each fraction to

detect labelled PRL. The peak (approximately 750,000-1,000,000 cpm/10

ul) and the descending 2-3 tubes were tested for PRL receptor binding

activity. Specific activity (approximately 83 uCi/ug) was determined











by trichloro acetic acid (TCA) precipitation prior to column elution

and calculating the incorporation of 125I into PRL (see appendix C).

Ovine PRL (USDA-B-1) was iodinated as described for porcine PRL with a

specific activity of 97 uCi/ug. The [125I]-PRL fractions were diluted

1:1 in assay buffer, stored at 4 C and used within 10 days.



Animals

Pregnant gilts, anesthetized on Day 75 using sodium thiamylal (Ig)

and maintained under surgical anesthesia on a closed circuit anesthetic

machine using Halothane, were subjected to a midventral laparotomy and

hysterectomized. Endometrium was separated from placental and

myometrial tissue layers and placed on ice. Liver and mammary gland

tissue was collected from Day 75 pregnant gilts at slaughter and

processed as described below. Day 20 pregnant rats were euthanized

with an overdose of sodium pentobarbital and livers were excised and

processed. Rabbit mammary glands were prepared by the method of Shiu

et al. (1973) and obtained following euthanasia with an overdose with

sodium pentobarbital.



Membrane Preparation

Endometrium from Day 75 pregnant gilts and liver from Day 20

pregnant rats were collected and placed on ice, rinsed three times in

ice-cold 0.9% (w/v) saline, rinsed in ice-cold homogenization buffer

(100 mM Tris, 150 mM sodium chloride, 50 mM ethyleneglycol-bis[B-

aminoethyl ether] N,N'-tetraacetic acid [EGTA], 50 mM ethylenediamine

tetraacetic acid [EDTA], 300 mM sucrose, 1 mM

phenylmethylsulfonylfluoride (PMSF) and Aprotinin (400 Kallikrin










inhibitory units/mi; pH 9.0), and then frozen at -70 C within 15 min

following collection. Tissue was thawed in ice-cold homogenization

buffer (4 ml/g tissue) on ice, processed using a Polytron homogenizer

(3 x 10 sec bursts at full speed), and homogenates centrifuged at

15,000 x g for 20 min at 4 C. The resulting supernatant was centrifuged

at 100,000 x g for 120 min at 4 C. The 100,000 x g pellet was

resuspended in buffer [100 mM sodium phosphate, 150 mM NaCI, 10 mM

EDTA, 0.1% (w/v) NaN3, pH 7.6], aliquoted and stored at -70 C until

assayed.



Protein Determination

Membrane protein concentrations were determined by the method of

Lowry et al., (1951) using bovine serum albumin as standard.



Chaotropic Treatment of Membranes

To ensure removal of endogenous hormone from endometrial prolactin

receptors, membranes were treated with magnesium chloride (MgCl2) as

described by Kelly et al. (1979). A 75 ul aliqout containing 150 ug

protein was added to 500 ul 4 M MgC2lz, vortexed, and incubated for 5

min. The reaction was stopped by addition of 3 ml ice-cold assay

buffer [10 mM sodium phosphate, 150 mM NaCI, 10 mM EDTA, 0.1% (w/v) BSA

and 0.1% sodium azide, pH 7.6]. Samples were centrifuged at 1800 x g

for 15 min at 4 C, decanted, and placed in an ice bath. Ice-cold assay

buffer (300 ul) was added immediately and tubes were vortexed

extensively to ensure pellet resuspension. All assays were conducted

using polypropylene 12 x 75 mm tubes (Sarstedt, Princeton, NJ) which

reduced nonspecific binding when compared to borosilicate glass tubes.




Full Text
82
receptors in the endometrium may determine effects of PRL on the
physiology of the uterus. While these receptors may respond to
decidual PRL and/or placental lactogen, the pig has neither and
circulating levels of immunoreactive PRL are relatively constant
throughout pregnancy (Dusza and Krzymowska, 1981). Therefore, PRL
effects are probably regulated by changes in the PRL receptor
population or receptor characteristics.
Heterologous (ovine) PRL radioreceptor assays (RRA) enabled
detection of PRL receptors (fmoles/g wet weight; fm/gww) in porcine
endometrium as early as Day 15 (75 fm/gww), increasing receptor numbers
to Days 45 and 75 (575 and 700 fm/gww, respectively) and decreased
receptor numbers by Day 90 (65 fm/gww) of gestation (DeHoff et al.,
1984). These changes in PRL receptor numbers are temporally associated
with increases in concentration of circulating estrogens (DeHoff et
al., 1986), PRL-stimulated endometrial proliferation, induction of
steroid receptors, protein secretion (Chilton and Daniels, 1985),
epithelial ion transport (Rabee and McCoshen, 1986), transport of
placental water (Goldstein et al., 1986) and fetal growth (Nicoll et
al., 1985).
Several heterologous RRAs have been described for PRL and are used
routinely (Shiu et al., 1973; Waters et al., 1974; Kelly et al., 1979)
but may be complicated by indiscriminant binding due to effects of
heterologous hormones (Nicoll, 1982). However, PRL, a member of the
growth hormone-prolactin-placental lactogen family, can crossreact with
other hormones in that family in RRAs. Prolactin from one species
often has biochemical homology and physiological actions related to
growth hormone of another species. Additionally, high variablity in


152
mitogenic activities by pigeon crop sac and Nb2 lymphoma cell assays,
respectively. Additionally, immunoaffinity and binding characteristics
of total, nonglycosylated and glycosylated forms of PRL were
investigated using homologous radioimmuno- (RIA) and radioreceptor
(RRA) assay systems, respectively.
Materials and Methods
Separation of Nonglycosylated and Glycosylated Forms of Prolactin
The glycosylated and nonglycosylated forms of porcine PRL were
separated using a Concanavalin A-Sepharose CL-6B (Con-A) column. The
column (1 x 2 cm; 4 C) was equilibrated with 10 mM phosphate buffered
saline (PBS, pH 7.5) followed by 0.2M alpha methyl mannoside in PBS to
remove Con-A that had detached from the Sepharose matrix. The column
was extensively rinsed with PBS prior to addition of porcine PRL.
Porcine PRL (USDA-1, from Dr. Douglas Bolt, Animal Hormone Program
Director, USDA, Beltsville, MD) was solubilized in PBS (3.5 mg/ml) and
slowly loaded onto the Con-A column. The buffer flow was stopped to
allow for maximal binding of glycosylated PRL. After 30 min, the
unretained nonglycosylated PRL was washed through the column with PBS
(flow rate of approximately 30 ml/hour) and collected as 1 ml
fractions. Protein was followed by absorbance at 280 nm. The retained
glycosylated porcine PRL was eluted from the column with 0.2 M alpha
methyl mannoside in PBS and collected in a similar manner as
nonglycosylated PRL.
Separation of the two forms of PRL yielded a 2:1 ratio of
nonglycosylated to glycosylated forms, respectively (Figure 12-1). The
1 ml fractions from each peak representing each form were pooled to


194
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Percent Nb2 lyphoma cell control
158
Hormone per well (ng)


186
Aragena, C., Bohnet, H.G., Fang, V.S. and Friesen, H.G. (1976)
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112
linked radiolabelled porcine PRL to either porcine endometrium (Figure
8-la, lane 7) or rat liver (Figure 8-2b, lane 6) membrane preparations
not treated with MgCli and in the absence of unlabelled hormone.
Additionally, cross-linking performed on concentrations of porcine
endometrial membrane protein used in homologous RRA (chapter 7) showed
electrophoretic bands similar to these previously described for porcine
endometrium (Figure 8-la, lane 13). Radiolabelled bands were not
present when membranes were incubated in the presence (Figure 8-la,
lane 12) of unlabeled porcine PRL or when not pretreated with MgCl2
(Figure 8-la, lanes 11 and 12).
Discussion
Affinity labeling of PRL receptors with iodinated porcine PRL
resulted in four (65,000, 55,000, 39,500 and 22,000) and two (39,000
and 23,000) Mr estimates of porcine endometrial and rat liver membrane
preparations, respectively. The 39,500 Mr protein from pig endometrium
and 39,000 Mr protein from rat liver are similar to PRL receptors
described previously for rat liver (Kelly et al., 1983), mammary gland
from sows treated with CB154 (Berthon et al., 1987a) and untreated sows
(Sasaki et al., 1985; Katoh et al., 1983) as well as rabbits (Sasaki
and Ike, 1985) mammary gland. However, the detection of multiple bands
in crude homogenates of pig endometrial and rat liver membranes may
have resulted from protein degradation or dimer formation during the
incubation or cross-linking procedures.
Several molecular weight bands (79, 58, 53, 42, 31 and 18K) are
detected when solubilized PRL receptors are hormone-affinity purified
and radiolabeled (not cross-linked; Berthon et al., 1987a). Further
¡


CHAPTER 10
EFFECTS OF ACUTE OF ESTRADIOL VALERATE ADMINISTRATION
ON ENDOMETRIAL PROLACTIN RECEPTORS DETECTED BY HOMOLOGOUS
RADIORECEPTOR ASSAY AND UTERINE SECRETORY
RESPONSE IN DAY 11 CYCLIC PIGS
Introduction
Estrogen involvement in prolactin (PRL) receptor regulation is
suggested since ovariectomy (Kelly et al., 1975), stage of reproductive
cycle (Kelly et al., 1974) and administration of exogenous estrogen
(Posner et al., 1974a; Kelly et al., 1975) affects PRL receptor numbers
in various target tissues. Endometrial membrane PRL receptors in pigs
increase initially in association with secretion of estrogens by
conceptuses and establishment of pregnancy between Days 11 to 12
(Chapter 9) and then vary throughout gestation (DeHoff et al., 1984).
During the time of maternal recognition of pregnancy, a succession of
endometrial secretory events are essential for nourishment and support
of preimplantation porcine conceptuses (Geisert et al., 1982b; 1982c;
Bazer and Roberts, 1983; Young et al., 1987). Prolactin affects
uterine secretory physiology of pigs (Young and Bazer, 1988), rabbits
(Chilton and Daniels, 1985) and rats (Kennedy and Armstrong, 1972).
Estrogen appears to increase endometrial PRL receptors to increase
uterine responsiveness to rather constant circulating levels of PRL
(Dusza and Krzymowska, 1979). Therefore, porcine endometrial PRL
receptors were studied using a homologous RRA to correlate changes in
124


CHAPTER 3
EFFECTS OF HYPOPROLACTINEMIA ON ESTABLISHMENT
OF PREGNANCY AND UTERINE SECRETORY FUNCTION IN PIGS
Introduction
Endocrinological studies to establish a hormone's involvement in a
physiological system is often through manipulation of the endogenous
concentration of circulating hormone followed by observation of
physiological changes in the target tissue of interest. Bromocryptine,
(CB154) a dopamine agonist, has been used in rats (Kelly et al., 1979),
pigs (Kraeling et al., 1982) and humans (see review, Barbieri and Ryan,
1983) to lower the circulating prolactin (PRL) levels through tonic
inhibition of PRL by stimulating dopamine receptors on the pituitary.
Prolactin affects proliferation (Chilton and Daniels, 1985),
protein secretion (Chilton and Daniels, 1985; Young and Bazer, 1988),
steroid receptor concentration (Daniels et al., 1984), estrogen uptake
(Leung and Sasakai, 1973) and progesterone metabolism (Kennedy and
Armstrong, 1977) in the uterine endometrium, as well as water and ion
concentrations in fetal membranes (Rabee and McCoshen, 1986; Goldstein,
1980), and fetal growth (Nicoll et al., 1986). Prolactin involvement
in uterine physiology within several species is well established.
However, very few studies utilized pigs and those studied effects of
PRL during the latter part of gestation. Studies conducted with
rabbits, (Daniels et al., 1984; Chilton and Daniels, 1985) support PRL
involvement in endometrial proliferation and secretion of uteroglobin.
44


92
Unlabeled Hormone (Hog M)


180
Test binding of labelled hormone The peak tube and the descending 2 to
3 tubes are tested for specificity of binding. Set up binding assay
with total count tube (TCT), Maximal binding (Bo) and nonspecific
binding (NSB) of 1000 or 2500 ng of unlabelled porcine prolactin using
Day 75 pregnant pig endometrial membranes (see assay protocol, appendix
B). The NSB should be approximately 4-7% of TCT and Bo should be
approximately 20-30% of TCT.
Clean up Properly dispose of ALL radioactive solutions and materials.
Storage of hormone Radiolabelled porcine prolactin for use in RRA
should be kept at 4 C for approximately 10-15 days. Store tubes in
lead-lined container. As radiolabelled hormone deteriorates, the NSB
will increase.
Determination of specific activity Dilute 5 ul radiolabelled porcine
prolactin into 1000 ul assay buffer. Aliquot 10 ul of diluted solution
and add to this 350 ul assay buffer and 100 ul 10% trichloroacetic acid
(TCA) solution. Incubate on ice for 30 min. Centrifuge (2500 x g) for
20 min at 4 C. Decant and count pellets. Calculate percent
incorporation of the 10 ul labelled hormone from cpm before and after
TCA precipitation based on the amount of hormone in 5 ul taken from the
reaction vessel.


167
glycosylated PRL may differ from that of nonglycosylated PRL, but those
functions may include those previously attributed to 'total' PRL.
Glycosylated PRL may function as a reserve or long half-life form while
the deglycosylated hormone is active at the tissue level. Further
investigation of microheterogeneity and molecular weight forms of PRL
is needed to increase our understanding of how these forms of PRL
account for the complexities of function and diversity of physiological
responses to PRL.


2
modification by, another (peptide) hormone, to induce rapid uterine
responses was investigated.
Porcine conceptuses establish pregnancy through estrogen secretion
and cause direct physiological, biochemical and secretory changes in
uterine endometrium. Therefore, PRL interaction with estrogen can be
studied within the context of uterine secretory function. The pig is
an interesting model for reproductive physiology studies, particularly
those investigating effects of PRL. Porcine conceptuses undergo
noninvasive implantation, which does not decidualize or produce PRL
from the endometrium, and the placenta does not produce placental
lactogen. Therefore, the pig can be used for investigations of PRL
function in uterine physiology, without interference by decidial PRL or
placental lactogen. Lacking these placental lactogenic hormones, it
was intriguing to delineate the mechanism(s) by which effects of PRL on
uterine physiology are induced in pigs.
This dissertation reports investigation of the role of PRL in
uterine physiology in pigs following induction of hypoprolactinemia and
hyperprolactinemia. Additionally, endometrial PRL receptors and their
regulation by ovarian steroids were investigated using a homologous
radioreceptor assay developed for porine PRL and porcine endometrial
PRL receptors. Homologous assays are advantagous since inconsistencies
associated with the use of heterologous hormones are avoided.
The literature review that follows is to familiarize the reader
with various aspects of PRL and its receptor. These areas are reviewed
with respect to basic function, characteristics and physiology that
must be considered in understanding the role of PRL in (porcine)
uterine physiology.


61
AM. The other gilts also had cytotoxic reactions to the injections and
were rated arbitarily on a 1 to 10 scale. The cycle lengths are
reported, although of limited value since CSH-treated gilts did not
return to estrus before being euthanized.
Discussion
These data suggest that CSH may be effective in decreasing
circulating levels of bioactive PRL and interestrous intervals may have
been increased in gilts that received CSH. Additionally, the gross
morphological appearance of the reproductive tract included atrophy and
regression (see Figure 4-2). Although CSH appeared to be effective in
lowering PRL levels and changing uterine appearance, use of this
compound in gilts in not warranted due to the severe cytotoxicity it
induced when administered subcutaneously at the dosage used for rats.


134
was similar at 1 and 6h (186 and 132 SU, respectively), however,
concentrations increased (P<0.05) at 12 and 24h (223 and 230 SU,
respectively); post E2V administration. Total LAP measured at 12h post
injection was greater (P<0.05) for gilts that received E2V versus corn
oil (223 vs 135 SU). Total glucose (mg + 0.17; Figure 10-4d) increased
(P<0.01) between 1 and 6h (2.0 and 2.9 mg, respectively), remained
constant through 12h (2.7 mg) and then increased further (P<0.03) at
24h (3.3 mg). Total glucose at 12h post injection was greater (P<0.01)
for gilts that received E2V rather than corn oil (3.3 vs 2.1 mg).
Changes in PRL receptor numbers were negatively correlated with changes
in calcium (-.88); sodium (-.80) and potassium (-.77), as well as the
membrane marker protein, leucine aminopeptidase (-.9). But positive
correlations also were detected between some components of uterine
flushings, i.e., sodium and potassium (0.99), sodium and glucose
(0.80), potassium and glucose (0.82), protein and LAP (0.83) and
protein and uteroferrin (0.88).
Discussion
The uterine secretory response to administration of E2V was
similar to that previously reported (Geisert et al., 1982b; Bazer and
Roberts; 1983; Bazer et al., 1986; Young et al., 1987). Uterine
flushings collected at 6h had increases in glucose, while at 12h
calcium, sodium, potassium and LAP were increased. Additionally, total
protein and uteroferrin increased by 24h. Prolactin may modulate
changes in ions in porcine uterine fluids since ion concentrations are
decreased in hypoprolactinemic pigs (Young and Bazer, 1988). Ion
shifts affect secretory processes (Bazer et al., 1984) as detected at


Percent Specific Binding
100
0 2.5 10 40 100 200 500
Unlabeled Hormone (ng)
Figure 7-7: Crossreactivity between unlabelled porcine
growth hormone (dashed line) or porcine prolactin (solid
line) and porcine t123I]-prolactin with magnesium chloride
treated (circles) Day 75 porcine endometrial and (squares)
Day 20 rat liver membranes.


I certify that I have read this study and that in my opinion it
conforms to acceptable standard of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
William W. Thatcher
Graduate Research Professor
of Dairy Science
This dissertation was submitted to the Graduate Faculty of the
College of Agriculture and to the Graduate School and was accepted as
partial fulfillment of the requirements for the degree of Doctor of
Philosophy.
May, 1989
Dean, College of Agriculture
Dean, Graduate School


137
stimulation and cellular response may be due to estrogen's interaction
at the lipid bilayer of the target cell plasma membrane; potentially
affecting PRL receptor microenvironment or PRL receptor recycling.
Increases in membrane fluidity increase available PRL receptors in rat
and mouse liver (Dave et al., 1983). A high estrogenrprogesterone
ratio increases membrane fluidity of human monocytes (Bagdade and
Sabbaiah, 1988). Estrogen may affect different membranes in cells.
Prolactin receptors increase more rapidily (<1 day) in golgi membranes
than plasma membranes (>3 days) in response to estrogen (Posner et al.,
1979). Seventy percent of PRL receptors are associated with the golgi
membranes. However, it is not known whether golgi receptors are newly
synthesized or membrane receptors being processed for degradation or
recycling.
Estrogen acutely affects the microtubular and microvillar
apparatus of endometrial epithelial cell membranes (Szego et al.,
1988). Early effects of estrogen on target tissue may be mediated
through changes in membrane integrity or fluidity or through effects on
other membrane components such as receptors and ion channels. The PRL
receptor cloned from rat ovaries (Kelly et al., 1989) has a long
cytoplasmic domain; increases in membrane fluidity would increase
extracellular domain exposure. Yet, PRL receptors cloned from rat
kidney (Boutin et al., 1988) has a short cytoplasmic domain and thus,
increases in membrane fluidity would reduce its extracellular domain
exposure (Shinitzky, 1984) By causing similar effects on membrane
fluidity, estrogen could cause different PRL receptor responses in
different tissues. Interactions between estrogen and the plasma


121
increased amounts of hormones in the uterine lumen. Estrone levels are
high in uterine flushings of pregnant gilts on Day 8, followed by
increased estradiol levels on Day 12 (Zavy et al.r 1980). Estrone was
found to be equally potent to estradiol in stimulation of hepatic
lactogenic receptors in adult male or immature rats (Posner et al.,
1974a). Although, estradiol is considered more uteropotent than
estrone; estrone may also modulate endometrial PRL receptors. Changes
in uterine function around Day 12 are of interest due to temporal
associations with critical stages of development by porcine conceptuses
(Geisert et al., 1982b; Bazer and Roberts, 1983).
Porcine conceptuses undergo drastic morphological changes during
the period when they initiate estrogen secretion on Days 11-12. The
conceptus rapidly elongates, intially by cellular rearrangement, and
then by hypertrophy and hyperplasia, from tubular (20-40 mm) and
filimentous forms (100-200 mm), to reach lenghts of 900 mm by Day 14
(Geisert et al., 1982b). In mink, PRL accelerates blastocyst growth,
possibly through effects on the CL and increases in progesterone
secretion (Martinet et al., 1981). Growth of the uterus and elongation
of the porcine conceptus is synchronous (Bazer et al., 1982), thus
fetal-placental units do not overlap. The underlying mechanism is
unknown, but PRL has been associated with growth promoting affects
(Nicoll, 1982). The increase in PRL receptors only in pregnant
endometrial membranes (Day 12), may be necessary for uterine growth
during conceptus elongation.
Correlated with secretion of estrogens by pig conceptuses, the
maternal recognition of pregnancy factor (Heap et al., 1979), are
changes in ion fluxes, specifically calcium, followed by release


30
and rabbit PRLs were not able to compete with ovine PRL and exhibited
only 3% binding. These results also question the true validity of PRL
binding receptor estimates when using a heterologous system.
Antibodies to rabbit mammary gland PRL receptors recognize water
soluble receptors and block their binding of ovine PRL (Berthon et al.,
1987b). Water soluble and membrane associated PRL receptors probably
share antigenic determinants and hormone binding sites with rabbit
mammary membrane PRL receptors. It is unclear, however, whether water
soluble receptors are involved in signal tranduction or transport of
PRL from mammary cells into blood or milk.
Signal Transduction Systems for Prolactin
Intracellular Mediators
Peptide hormone-receptor interaction is mediated through a
transduction system to stimulate changes in cell physiology and overall
tissue response. Transduction systems involve either 1) ligand
modulated ion channel activity, 2) ligand regulating enzyme activity or
3)ligand regulation of cryptic mediators through interactions between
intracellular receptor domains or other submembranous constituents
similar to G and N regulatory components. Transduction systems have
been classified for several hormones (see review, Hollenburg, 1986;
Cockcroft and Stutchfeld, 1988). Mobility of peptide receptors is
paramount to some transduction systems, i.e., adenylate cyclase, and
functions due to the fluid mosaic properties of the cell membrane
(Singer and Nicholson, 1979) with possible involvement of microtubules
and microfilaments.


149
ESTROGEN
Figure 11-1: Effect of chronic ovarian steroid adminsitration on
uterine secretory response of ovariectomized gilts; possible
interaction with endogenous prolactin to result in low and high
uterine secretory response.


142
Measurement of Endometrial Prolactin Receptors
Endometrial PRL receptors were detected using the homologous RRA
described in Chapter 7. Endometrial preparation, homogenization,
protein determination, iodination, specific activity determination (83
uCi/ug), chaotropic treatment of endometrial membrane preparations,
radioreceptor assay conditions, inhibition curves and analysis of
binding data were as described in Chapter 7.
Statistics
Data were analyzed by least squares analysis of variance using the
General Linear Methods of the Statistical Analysis System (SAS) (Barr
et al., 1979). Orthogonal contrasts were used to determine differences
between means.
Results
The affinity constants generated from LIGAND analysis of
inhibition curves from homologous RRA were not affected by steroid
treatment. The average Ka was .20 + .03 x 108 M-1, (data not shown)
and was similar to values previously determined using the homologous
porcine PRL RRA in Chapters 7, 9 and 10. Effects of ovarian steroid
administration on porcine endometrial membrane PRL receptor numbers are
summarized in Table 11-1. Gilts treated with CO or P4 had similar
endometrial membrane PRL receptor numbers (60 vs 58 + 7 pmoles/mg
protein, respectively). Gilts that received E2V alone (45 pmoles/mg
protein) or E2V+P4 (44 pmoles/mg protein), had lower (P<0.06)
endometrial PRL receptors than gilts that received either P4 alone or
corn oil. When PRL receptor numbers were calculated per gram wet


115
F2 secretion into the uterine lumen (Young and Bazer, 1988; Mirando et
al., 1988).
The period of maternal recognition of pregnancy is of great
interest due to the morphological, physiological and endocrinological
changes that must occur for preimplantation porcine conceptuses to
survive. Estrogens of conceptus origin affect uterine secretory
function and may modulate the population of endometrial PRL receptors.
However, conceptus estrogens act locally, since they are metabolized to
a biologically inactive form, estrone sulfate (Heap et al., 1979),
before entering the porcine maternal circulation (Stoner et al., 1980).
Changes in endometrial PRL receptors throughout pregnancy (DeHoff
et al., 1984) have been associated with other gestational events
(Kensinger et al., 1986, Rabee and McCoshen, 1986) such as fetal growth
(Nicoll, 1982), endometrial synthesis and secretion of protein (Chilton
and Daniels, 1985; Young and Bazer, 1988), ion and water movement
across the placenta (Goldstein et al., 1980) and changes in circulating
levels of estrogen (DeHoff et al., 1986). Biological effects occur
through either increases in circulating concentrations of a hormone
with receptor numbers remaining constant, or hormone levels may remain
constant with receptor numbers increasing. The objectives of this
study were to compare endometrial PRL receptors during early pregnancy
and the estrous cycle and determine whether changes in receptor numbers
were associated with PRL biological activity during early pregnancy.
J


40
Modulation of membrane viscosity and PRL receptors by phospholipase A2
is through generation of prostaglandins. Prostaglandins may also
mediate PRL receptor auto-upregulation by decreasing membrane
viscosity. Prolactin may also change the phospholipid:cholesterol
ratio resulting in membrane fluidity changes that increase the
availability of cryptic receptors.
Subcellular localization of PRL receptors may be affected by
membrane fluidity since PRL receptors are preferentially located within
the cell. Seventy percent of mammary gland (rabbit) and liver (rat)
cells' lactogenic receptors are located in the plasmalemma (Bergeron et
al., 1978) The golgi rich fraction of cells contain 4 to 6 times more
PRL receptors and are 2.5 times more fluid than plasma membranes from
preparations from prostate and (male and female) liver cells (Dave and
Vitorsch, 1986). Additionally, only plasma membranes increased PRL
receptor numbers in respond to increases in membrane fluidity.
Prolactin Functions in the Uterus
Prolactin is credited with over 100 functions in several species
(Riddle, 1963; Nicoll and Bern, 1972; Nicoll et al., 1986). However,
for the scope of this review, PRL's functions in uterine physiology
will be reviewed. For more complete discussion of PRL functions, the
following reviews are suggested: Riddle, 1963; Bern, 1975; Nicoll,
1982; and Nicoll et al., 1986. The function of PRL within uterine
physiology is suggested by the evidence of endometrial PRL receptors in
humans (Healy, 1984), sheep (Posner et al., 1974b), pigs (DeHoff et
al., 1984) rabbits (Daniels et al., 1984; Chilton and Daniels, 1985;
Grissom and Littleton, 1988), rats (Williams et al., 1978), mice (Sinha


78
flushings on Day 12 could be influenced by advanced calcium reuptake
that does not normally occur until Days 13-14 of pregnancy. This may
account for the increase in glucose and uteroferrin on Day 12 for PRL
treated pigs compared to previous reports that glucose and uteroferrin
concetrations do not increase until Days 14 to 16 during a normal
pregnancy (Bazer et al., 1984). Hyperprolactinemia resulted in
increased concentrations and specific activity of uteroferrin in (Day
12) uterine flushings. Increases in this protein usually occur on Day
14 of gestation (Zavy et al., 1982). Since there was not a concomitant
increase in aminopeptidase, a membrane marker protein indicative of
cell membrane processing and secretory activity, PRL may have altered
uteroferrin synthesis resulting in an increase in the releaseable pool
stored within intracellular secretory granules and available for
release following estrogen stimulation. Additionally,
hyperprolactinemia resulted in increased total glucose in uterine
flushings on Day 12 which normally does not occur until Days 14 to 16
of pregnancy (Zavy et al., 1984). Administration of PRL appeared to
advance the ability of uterine epithelium to respond to estrogen-
induced secretory events.
Stimulation of secretion of prostaglandins of the F, but not the
E, series suggests that effects of PRL may be on the uterine
epithelium. Previous reports have localized PGF and PGE secretion to
the endometrial epithelium and stroma, respectively, of cows (Grasso et
al., 1987). Prolactin exerts luteostatic effects in several species
(Murphy and Rajkumar, 1985), primarily through stimulation of ovarian
steroid production. Prolactin appears to advance the ability of the
uterus to respond to estradiol since an increase in luminal PGF


70
mg E2V in corn oil on Day 11) received 1 ml saline twice daily at 0730
and 1930h on Days 6 through 11. Gilts in the treatment group received 1
mg porcine PRL in saline twice daily at 0730 and 1930h and 5 mg E2V on
Day 11. Uterine flushings were collected on Day 12, approximately 24h
after treatment with corn oil or E2V. A PRL and corn oil treatment
group was not included because results of Experiment 1 indicated no
interaction between PRL and progesterone on uterine secretory activity.
Uterine Flushings
Uterine flushings were processed and evaluated for total
recoverable protein, uteroferrin, leucine aminopeptidase (LAP),
glucose, calcium, sodium, potassium, chloride, PGF, and PGE as
described in Chapter 3.
Results
Effects of PRL on P4-induced uterine secretory responses are
summarized in Table 6-1. Administration of exogenous PRL did not
affect any of the P4-induced uterine secretory components examined in
the uterine flushings.
Effects of exogenous PRL on estrogen-induced uterine secretory
activity are summarized in Figures 6-1, 6-2, and 6-3. Treatment
effects were detected for (see Figure 6-1A, B and C) total recoverable
protein and total uteroferrin (PCO.OOl), total glucose (P<0.01), and
total leucine aminopeptidase (P<0.05); (see Figure 6-2A, B and D),
total calcium (P<0.03), total chloride (P<0.02), total potassium
(P<0.01); and (see Figure 6-3A) total PGF (P<0.02) in uterine
flushings. There was no effect of treatment on sodium (Figure 6-20 or


Figure 6-2: Concentrations of total recoverable (A) calcium,
(B) chloride, (C) sodium and (D) potassium in Day 12 uterine
flushings from cyclic gilts (Experiment 2) treated with 1 ml
saline (SAL) or 1 mg porcine prolactin (PRL) on Days 6-11 and
0.5 ml corn oil (OIL) or 5 mg estradiol valerate (E2V) on Day
11 of the estrous cycle. Overall treatment effects were
detected for calcium and chloride (P<0.05) as well as
potassium (P<0.01). Values with different letters are
different (P<0.05). The overall SEM was +0.3 for calcium,
+0.22 for chloride, +10.7 for sodium and +9.23 for potassium.


62
Figure 4-2: Reproductive tracts from gilts treated with
either (A) ethanolamine or (B) cysteamine.


9
Sources of Prolactin
Anterior Pituitary Prolactin
The anterior pituitary is derived from an evagination of embryonic
ectodermal tissue, Rathke's pouch, and attaches to neural tissue from
the brain to form the pituitary. The cells of the anterior pituitary
can be classified as acidophils, basophils and chromophobes by
microscopy and perferential uptake of certain dyes. Acidophils are
further defined as alpha or epsilon cells, being oranophils or
carminophils, respectively. Carminophils, but not orangophils,
fluctuate during pregnancy. Both cell types are located in the lateral
aspects of the anterior pituitary.
Lactotrophs and somatotrophs, which secrete PRL and growth
hormone, respectively, comprise the acidophilic cells of the anterior
pituitary. Somatotrophs, which comprise 50% of anterior pituitary
cells, were described by Kurasumi (1968) as round or oval polygonal
cells with short clubbed mitochondria and round dense secretory
granules of approximately 200-350 mm. Lactotrophs, comprise 15-20% of
the anterior pituitary cells, and were intitially described by Farquhar
and Rinehart (1954). These PRL secreting cells have large nuclei and
relatively small amount of cytoplasm which contains well developed
golgi complex and abundant rough endoplamic reticulum. Secretory
granules of the lactotroph are elongated (600-900 mm) although small
(200 mm) secretory vesicles are also observed.
Unlike other pituitary hormones, PRL is synthesized with a signal
peptide. Newly synthesized PRL is preferenially released, independent
of TSH, while secretion of stored PRL is dependent on TSH stimulation
(Walker and Farquhar, 1980). Crinophagy, another unique feature of


5 ESTABLISHMENT OF HYPERPROLACTINEMIA BY ADMINISTRATION
OF EXOGENOUS PORCINE PROLACTIN TO PIGS 63
Introduction 63
Materials and Methods 63
Results 65
Discussion 65
6 EFFECT OF HYPERPROLACTINEMIA ON PROGESTERONE
AND ESTROGEN INDUCED UTERINE SECRETORY RESPONSE
IN PIGS 67
Introduction 67
Materials and Methods 68
Results 70
Discussion 77
7 DEVELOPMENT OF A HOMOLOGOUS RADIORECEPTOR ASSAY
FOR PORCINE ENDOMETRIAL PROLACTIN RECEPTORS 81
Introduction 81
Materials and Methods 83
Results 89
Discussion 102
8 AFFINITY LABELLING OF PROLACTIN RECEPTORS
IN DAY 75 PRGENANT PORCINE ENDOMETRIUM WITH
PORCINE [i231]-PROLACTIN 107
Introduction 107
Materials and Methods 108
Results 109
Discussion 112
9 ENDOMETRIAL PROLACTIN RECEPTORS DETECTED BY
HOMOLOGOUS RADIORECPETOR ASSAY DURING THE ESTROUS
CYCLE AND EARLY PREGNANCY IN PIGS 114
Introduction 114
Materials and Methods 116
Results 117
Discussion 119
10EFFECTS OF ACUTE ESTRADIOL VALERATE ADMINISTRATION
ON ENDOMTRIAL PROLACTIN RECEPTORS DETECTED BY
HOMOLOGOUS RADIORECEPTOR ASSAY AND UTERINE
SECRETORY RESPONSE IN DAY 11 CYCLIC PIGS 124
Introduction 124
Materials and Methods 125
Results 127
Discussion 134
vii


165
porcine PRL (Pankov and Butnev, 1986; Seely et al.f 1988). Pankov and
Butnev (1986) suggested that the carbohydrate moiety on PRL could mask
antigenic determinants. Pellegini and coworkers (1988) found similar
detection of the two forms of human PRL using a monoclonal antibody.
The ratio between the two forms of PRL may vary during different
physiological states (Markoff and Lee, 1987) and change the overall
activity of PRL without altering the amount detected by RIA (Sinha et
al., 1984).
The bioactivity of the two forms of PRL exhibit different
potencies when mitogenic and lactogenic effects were measured.
Mitogenicity, as determined in the Nb2 lymphoma cell assay, indicated
that nonglycosylated and glycosylated forms of PRL were 10- and 100-
fold less potent, respectively, than total PRL. These results are
similar to those of Markoff and Lee (1987), Scott et al. (1988) and
Pellegini et al. (1988). Lactogenic bioactivity, however, was
enhanced for glycosylated, compared to nonglycosylated and porcine
total, PRL which is consistent with previous results (Pankov and
Butnev, 1986). But glycosylated ovine PRL had decreased lactogenic
activity in the pigeon crop sac assay (Lewis et al., 1984) and in the
mouse mammary casein bioassay (Seely et al., 1988).
Results from the homologous RRA suggest that glycosylated porcine
PRL may have higher affinity for PRL binding sites in porcine
endometrial membranes. Prolactin receptors for different tissues
within the same animal may be differentially regulated since several
studies have shown differential changes in receptor numbers in tissues
under the same physiological conditions (Posner et al., 1974b; Kelly et
al., 1979; Shiu et al., 1981; DeHoff et al., 1984). Data from the


CHAPTER 11
EFFECT OF CHRONIC OVARIAN STEROID ADMINISTRATION ON
ENDOMETRIAL PROLACTIN RECEPTORS AS DETECTED BY HOMOLOGOUS
RADIORECEPTOR ASSAY AND UTERINE PROTEIN SECRETORY
RESPONSE IN OVARIECTOMIZED PIGS
Introduction
The direct involvement of ovarian steroids in uterine functions is
well established; however, these steroids can also modulate, or be
modulated by, peptide hormones. Although several protein hormones have
been implicated, recent reports suggest that prolactin (PRL), in
addition to its mammotrophic, lactogenic and luteotrophic roles,
influences uterine physiology. Endometrial PRL receptors have been
detected for humans (Healy, 1984), rabbits (Ohno, 1982; Grissom and
Littleton, 1988), sheep and cows (Posner et al., 1974b), rats (Williams
et al., 1978), mink (Rose et al., 1983) and pigs (DeHoff et al., 1984;
Young and Bazer, 1987). Effects of PRL on uterine physiology include
water and ion movement (Rabee and McCoshen, 1986; Goldstein et al.,
1980), fetal growth (Nicoll et al., 1975), protein secretion (Daniels
et al., 1984; Chilton and Daniels, 1985; Young and Bazer; 1988),
endometrial proliferation (Chilton and Daniels, 1985), steroid receptor
concentrations (Chilton and Daniels, 1985; Muldoon, 1981; 1987),
prostaglandin secretion (Mirando et al., 1988; Young and Bazer, 1988),
progesterone metabolism (Armstrong and King, 1977) and estrogen uptake
(Leung and Sasakai, 1973).
139


39
receptors. However, growth hormone may regulate PRL receptors during
growth and development while PRL may regulate its receptors to meet
adult physiological demands.
Regulation by Membrane Fluidity
The fluid mosaic membrane (Singer and Nicholson, 1979) allows
lateral and vertical movement of receptors and other proteins.
Additionally, proteins and receptors may assemble in different
configurations (Koch et al., 1979) or states of availablity. The rat
liver PRL receptor is a glycoprotein with a single transmembrane domain
(Boutin et al., 1988). Therefore the microenvironment as well as the
physical status of the membrane may influence PRL receptor binding and
tissue receptivity. Changes in membrane fluidity and PRL receptor
binding were investigated in rat hepatic tissue.
Composition of unsaturated fatty acids can influence membrane
viscosity. Rats fed a diet deficient in essential fatty acids had
increased membrane viscosity that resulted in a progressive decrease in
hepatic PRL binding, not reversable by exogenous administration of PRL
(Knazek and Liu, 1979). Phospholipase A2 generates arachadonic acid
from membrane phospholipids and results in a biphasic increase and
decrease in PRL binding to hepatocytes in vitro (Dave et al., 1981).
Prostacyclin treatment in vitro also increases PRL binding to hepatic
cells (Dave and Knazek, 1980) which was blocked by in vivo
administration of indomethacin (Knazek, et al., 1981). These compounds
share the ability to increase hepatic membrane fluidity and increase
PRL receptor availablity. As summarized by Witorsch et al. (1987)
membrane fluidity is correlated with increases in PRL receptor numbers.


43
into the uterine lumen on Day 12 and its reuptake occurs by Day 14; (2)
sodium and potassium increase in the uterine lumen, as calcium levels
decrease, with greatest increases between Days 14 and 16; (3) protein
and uteroferrin concentrations in the uterine flushings increase on
about Days 14 to 16 and (4) glucose concentrations in uterine flushings
of pregnant pigs increase between Days 14 and 16 of gestation. In
cyclic pigs: (1) calcium increases on Day 12, but maximal levels are
lower than for pregnant pigs, and declines gradually between Days 14
to 16; (2) sodium and potassium increase as calcium decreases but to
amounts lower than for pregnant pigs; (3) protein and uteroferrin
increase on Days 15 to 16 and (4) glucose concentrations are low and
affectd by day of the cycle.
Nonpregnant pigs injected with estradiol valerate on Day 11 of the
estrous cycle have uterine flushings on Day 12 that are similar in
composition to those collected on Day 12 of pregnancy (Geisert et al.,
1982c). Thus, estrogen induced uterine secretory function can be
investigated in regard to pregnancy without confounding effects of
other conceptus secretory products (Geisert et al., 1982c, Young et
al., 1987).


159
Antibody Affinity of the Forms of Porcine Prolactin
Results from RIA trials are depicted as percent bound versus log
concentration of hormone (Figure 12-4a), normalized percent bound
versus log concentration of hormone (Figure 12-4b) and as data
transformed to log (concentration) versus logit (cpm) (Figure 12-4a,
inset). Transformation of data (log/logit) resulted in generation of
straight lines with similar slopes, but different (P<0.02) intercepts.
When results were normalized and expressed as percent of Bo, the
polyclonal antibody had slightly higher affinity for total, than
nonglycosylated, PRL; followed by glycosylated PRL (Figure 12-4b).
Results from the log-logit plot suggest that 30 and 45% more total PRL
(1.44 ng) was detected than nonglycosylated (0.9743 ng) or glycosylated
PRL (0.798 ng), respectively, at 50% bound (P<0.01).
Receptor Affinity as Measured in a Homologous Radioreceptor Assay
Evaluation of inhibition curves by Scatchard analysis resulted in
an average Ka of 0.21 x 108 M*1, similar to that previously reported
for a homologous porcine PRL RRA (Chapters 7, 9, 10 and 11). The
number of PRL receptors detected in MgCU-treated Day 75 pregnant pig
endometrial memebranes were greatest when [,23I]-PRL was displaced with
unlabelled total PRL followed by unlabelled nonglycosylated and then
glycosylated PRL. When the separated forms of nonglycosylated and
glycosylated PRL were remixed in a 2:1 ratio, the displacement curve
had a similar slope, but estimates of receptor numbers were lower (58
vs 15 pmoles/mg protein; Figure 12-5) In a separate assay, the
nonglycosylated and glycosylated forms of porcine PRL were competed
against radiolabelled total PRL. There were no differences in the Kas


46
control gilts received 4 ml of vehicle solution on the basis of
results of Kraeling et al. (1982) .
Protein
Total recoverable protein concentrations in uterine flushings were
determined by the method of Lowry et al. (1951) using bovine serum
albumin as standard.
Uteroferrin
Concentrations of uteroferrin in uterine flushings were determined
by measurement of acid phosphatase activity (Basha et al., 1979;
Scholsnagle et al., 1974). Values are expressed as umoles
paranitrophenol phosphate (pNP) released per ml per 10 min at pH 4.9
and 37 C.
Calcium
The Calcette 4009 (Precision Systems, Inc., Sudbury, MA) was used
to determine calcium concentrations using ethylene glycol-bis-N-
N'tetraacetic acid (EGTA) for fluorometric titration of calcium in an
aqueous solution (Alexander, 1971).
Chloride
Concentrations of chloride in uterine flushings were determined by
a colorometric assay adapted from Hamilton (1966). Uterine flushings
were diluted 1:10 (v/v) with the mercurous reagent in 12 x 75 mm glass
tubes, covered with parafilm and inverted several times. Sodium
chloride was used for the standard curve and yielded a linear


105
complete for either membrane (Kelly et al., 1983). The difficulty in
removing endogenous PRL from membranes in vitro is also indicative of
incomplete reversal of PRL binding. Dissociation of PRL from pig
endometrial receptor was 40% complete after 48h, with the majority of
this dissociation occuring within the first lOh. In the present study,
60% of bound porcine t125I]-PRL could be dissociated from rat liver
membranes which is consistent with findings for ovine PRL (Kelly et
al., 1983). Bramley and Menzies (1985) reported similar results for
pig CL and attributed the the slow dissociation rate to the high (64.8
kJ/mol) apparant activation energy for PRL binding, which was reported
also for mouse (43.6 kJ/mol; Haro and Talamantes, 1985b) and rat
(34kJ/mol; Rae-Ventner and Dao, 1982) liver. This may be due to
extensive hydrophobic interactions. Amino acids at positions 20
through 30 are known to be hydrophobic with histidines located at
positions 27 and 30 in cow (Wallis, 1974), sheep (Li et al., 1970), pig
(Li, 1976) and human (Cooke et al., 1981) and at positions 25 and 28 in
rat (Cooke et al., 1980) and mouse (Kohmoto et al., 1984) PRL.
Characteristics of PRL receptors determined with this homologous
RRA have many similarities to those characterized using heterologous
RRAs. Additionally, this homologous RRA, which more closely
approximate in vivo conditions, provide additional support to confirm
previous reports. Receptors are nearly saturated and binding data are
not easily obtainable without treatment of membranes with chaotropic
agents. This was also the case for other binding assays for sheep,
rabbit and pig. Therefore, the homologous RRA, permits evaluation of
specific PRL receptors within reproductive (and other) tissues without
confounding effects associated with heterologous RRAs. Homologous


187
Bazer, F.W. and Roberts, R.M. (1983) Biochemical aspects of conceptus
endometrial interactions. J. Exp. Zool. 228, 373-391.
Bazer, F.W., Roberts, R.M., Mejia, A.M., Clark, W.R. and Vallet, J.V.
(1984) Protein, electrolytes and glucose in uterine flushings of
pregnant and nonpregnant gilts. Biol. Reprod. (Supp 1) 30, 192
(Abstract).
Bazer, F.V., Roberts, R.M. and Sharp, D.C. (1978) Collection and
analysis of female genital tract sectretions. In: Methods in Mammalian
Reproduction, pp. 503-527. (ed. J.C. Daniels) Academic Press, New
York.
Bazer, F.W. and Thatcher, W.W. (1977). Theory of maternal recognition
of pregnancy in swine based on estrogen controlled endocrine versus
exocrine secretion of prostaglandin F2C1 by the uterine endometrium.
Prostaglandins. 14, 397-400.
Bedi, G.S., French, W.C. and Bahl, O.P. (1982) Structure of
carbohydrate units of ovine luteinizing hormone. J. Biol. Chem. 257,
4345-4355.
Ben-Johnathan, N., Oliver, C., Weiner, H.J., Mical, R.A. and Porter,
J.C. (1977) Dopamine in hypophysial portal plasma of the rat during the
estrous cycle and throughout pregnancy. Endocrinology. 100, 452-458.
Ben-Johnathan, N. and Peters, L. (1982) Posterior pituitary lobectomy:
differential elevation of plasma prolactin and luteinizing hormone in
estrous and lactating rats. Endocrinology. 110, 861-865.
Bergeron, J.J.M., Posner, B.I., Josefsberg, A. and Sisktrom, R. (1978)
Intracellular polypeptide hormone receptors: the demonstration of
specific sites for insulin and human growth hormone in golgi fractions
isolated from the livers of female rats. J. Biol. Chem. 253, 4058-
4062.
Bern, H.A. (1975) Prolactin and osmoregulation. Am. Zool. 15, 937-
948.
Bero, L.A. and Kuhn, C.M. (1987) Differential ontogeny of opiod,
dopaminergic and serotonergic regulation of prolactin. J. Pharm. Exp.
Therapeutics. 240, 825-830.
Berthois, Y., Pourreau-Schneider, H., Gandilon, P., Mittre, H.,
Tubiana, N. and Martin, P.M. (1986) Estradiol membrane binding sites
on human breast cancer cell lines. Use of fluorescent estradiol
conjugate to demonstrate plasma membrane binding systems. J. Steroid
Biochem. 25, 963-972.
Berthon, P., Katoh, M., Dusanter-Fourt, I., Kelly, P.A. and Djiane, J.
(1987a) Purification of prolactin receptors from sow mammary gland and
polyclonal antibody production. Mol. Cell. Endocrinol. 51, 71-81.


140
Concentrations of PRL in blood of pigs remains rather constant
throughout most of gestation (Dusza and Krzymowska, 1981; Foxcroft and
van der Weil, 1982; DeHoff et al., 1986). Control of tissue response
by local changes in PRL receptors may alter tissue responsiveness to a
circulating hormone. Ovarian steroids affect PRL receptors in liver,
kidney, adrenal, mammary and ovarian tissues (Waters et al., 1978; Shiu
et al., 1982 ). Considering the importance of ovarian steroids in
uterine physiology, their effects on porcine endometrial PRL receptors
were studied using a homologous RRA. Relationships between changes in
porcine endometrial PRL receptors and endometrial secretion of total
uterine protein and uteroferrin were also evaluated.
Materials and Methods
Animals
Crossbred gilts of similar weight (100-120 kg) and age (7-9
months) were used in this study following completion of one normal (18
22 days) estrous cycle. Gilts were checked daily for estrus in the
presence of intact boars. The morning when gilts were observed in
behavioural estrus was designated Day 0.
Hormones
Estradiol valerate, progesterone and corn oil were obtained from
Sigma (St. Louis, MO). Estradiol valerate was solubilized in 10/90
(v/v) ethanolrcorn oil to a concentration of 200 ug/ml. Progesterone
was solubilized in a similar manner to a concentration of 50 mg/ml.
Ethanol:corn oil (10:90, v/v) was the hormone vehicle and control
solution.


34
et al., 1987; Rosen et al., 1987; Walaas and Walaas, 1988).
Internalization and processing of PRL by lysosomes in mammary and
ovarian tissues may be involved in PRL stimulation of these cells
(Nolin and Bagdonanov 1980; Mittra, 1980; Nolin, 1982). Proteolytic
fragments of 8,000 and 16,000 Mr of PRL may be involved in PRL action
(Clapp, 1987) suggesting that 23,000 Mr prolactin is a prohormone prior
to being internalized (Nolin, 1982).
Hormone-receptor internalization to mediate PRL action suggests
involvement of cellular microtubules and microfiliments. Chloroquine,
which binds tubulin and destabilizes microtubular structures, inhibits
downregulation of PRL receptors and stimulation of casein synthesis
(Houdebine and Djiane, 1980). Subsequent results indicate that
chloroquine binds tubulin at the plasma and golgi membranes. Another
microtubular destabilizer, griseofulvin, did not affect PRL stimulation
casein synthesis of mammary cells; suggesting PRL affects are at the
cell surface (Houdebine et al., 1982).
Advances in receptor purification enabled development of
antibodies to PRL receptors. Adminstration of high doses of anti-PRL
receptor serum to rats blocked PRL stimulation whereas low doses
actually mimiced PRL effects, suggesting that PRL effects are through
the receptor at the cell membrane and not following internalization and
processing to cleaved or clipped forms (Witorsch et al., 1987). Whole
anti-PRL receptor serum, bivalent F(ab)2 or monovalent F(ab) fragments
demonstrated similar abilities to inhibit PRL binding to mammary gland
(Djiane et al., 1987). In mammary gland explant cultures, similar
effects on PRL receptor downregulation were observed for all forms of
anti-PRL receptor, as well as PRL itself. Low doses of bivalent F(ab)2


69
v/v). Control gilts received 4 ml vehicle only. Acute steroid
treatment consisted of 0.5 ml (10 mg/ml) estradiol valerate (E2V;
Squibb, Raritan, NJ) or 0.5 ml corn oil. Exogenous porcine prolactin
(PRL; USDA-B-1; gift from Dr. Douglas Bolt, National Animal Hormone
Program Director) was diluted in phosphate buffered saline (PBS; 1
mg/ml; pH 7.2), aliquoted into 1.2 ml volumes and stored at 4 C until
injected subcutaneously.
Surgical Procedures
Gilts used in studies of PRL effects on P4-induced uterine
secretory components in Experiment 1 were ovariectomized on Day 4 of
the estrous cycle. Gilts were anesthetized, subjected to midventral
laparotomy, the ovaries were exteriorized, all ovarian vessels were
ligated and the ovaries removed with minimal trauma to the uterus.
Uterine flushings were collected as descibed in Chapter 3.
Experiment 1
Seven nonpregnant gilts were ovariectomized on Day 4 of the
estrous cycle and injected with 200 mg P4 once daily from Days 4
through 14. Gilts were assigned randomly to receive either porcine PRL
(n=4; 1 mg) or vehicle (n=3; 1 ml saline) daily at 0800 and 2000h on
Days 4 through 14. Uterine flushings were collected on Day 15.
Experiment 2
Twelve nonpregnant gilts, 4 per treatment group, were assigned
randomly to receive one of three treatments. Gilts in the negative
control group (corn oil only on Day 11) and positive control group (5


ACKNOWLEDGEMENTS
I would like to thank the members of my committee, Drs. Bazer,
Buhi, Shiverick, Simpkins and Thatcher, for time and knowledge they put
forth toward the completion of my research. I thank these individuals
who, through their conversations, actions, examples, and generosity,
were an integral part in my development as a scientist.
I am thankful to several people, behind the scenes, that
stimulated my pursuit of this degree, including Mr. Jerry Metzler, Fr.
Timothy Healy, Dr. Phil Senger, Dr. Brad Vaughn and my father, the late
William Hart.
Along the way, much appreciated examples and support were provided
by Drs. Mary Murray, Cheryl Ashworth-Stott, Saundra Tenbroeck and Susan
Ogilve.
I am indebted to Hironori Ohtsuka, founder of Wado-ryu Karate, and
his students, which ultimately led to the supportive friendships of
Mike McCoy, Mike Sawyer, Larry and Ellen Bellack, Cindy Silverstre and
Terry Minard.
The day-to-day events in the lab, barn and surgery room were
shared by several graduate students and post docs, including Dr. Randy
Renegar, Marlin DeHoff, Sue Chaichimansour, Dr. Wendy Campbell, Dr.
Jeff Vallet, Dan Dubois, Troy Ott, Jake Harney, Dr. Mark Mirando,
Saskia Beers and Matt Davis; I am grateful for their help and humour.
Special thanks go to Dan Dubois for lending me his computer for the
final stretch of dissertation polishing.
iv


To those who believe in me,
"no matter what..
VAH, RGY, JHN, LK, BHV, IJM and JMH


106
RRAs may be used to measure receptors during different physiological
states, and to compare receptor levels in different tissues, since
receptors appear to change differentially in tissues within an animal
being maintained under steady-state physiological conditions.
Additionally, homologous and heterologous assays could be used
comparatively, to elucidate the intricate ligand-receptor-membrane
interactions between members of hormonal families and their receptors.


85
inhibitory units/ml; pH 9.0), and then frozen at -70 C within 15 min
following collection. Tissue was thawed in ice-cold homogenization
buffer (4 ml/g tissue) on ice, processed using a Polytron homogenizer
(3 x 10 sec bursts at full speed), and homogenates centrifuged at
15,000 x g for 20 min at 4 C. The resulting supernatant was centrifuged
at 100,000 x g for 120 min at 4 C. The 100,000 x g pellet was
resuspended in buffer [100 mM sodium phosphate, 150 mM NaCl, 10 mM
EDTA, 0.1% (w/v) NaN3, pH 7.6], aliquoted and stored at -70 C until
assayed.
Protein Determination
Membrane protein concentrations were determined by the method of
Lowry et al., (1951) using bovine serum albumin as standard.
Chaotropic Treatment of Membranes
To ensure removal of endogenous hormone from endometrial prolactin
receptors, membranes were treated with magnesium chloride (MgCU) as
described by Kelly et al. (1979). A 75 ul aliqout containing 150 ug
protein was added to 500 ul 4 M MgCl2, vortexed, and incubated for 5
min. The reaction was stopped by addition of 3 ml ice-cold assay
buffer [10 mM sodium phosphate, 150 mM NaCl, 10 mM EDTA, 0.1% (w/v) BSA
and 0.1% sodium azide, pH 7.6]. Samples were centrifuged at 1800 x g
for 15 min at 4 C, decanted, and placed in an ice bath. Ice-cold assay
buffer (300 ul) was added immediately and tubes were vortexed
extensively to ensure pellet resuspension. All assays were conducted
using polypropylene 12 x 75 mm tubes (Sarstedt, Princeton, NJ) which
reduced nonspecific binding when compared to borosilicate glass tubes.


Figure Page
8-lb: Autoradiography of affinity labelled, cross-linked
rat liver membrane prolactin receptors Ill
9-1: Prolactin receptors in endometrial membranes
of cyclic (squares) and pregnant (circles) gilts
over days of the estrous cycle and gestation 118
10-1: Prolactin receptor numbers in endometrial membranes
at 1, 6, 12, and 24 h after administration (i.m.) of
estradiol valerate (0.5 mg, hatched bars) or 12 h
after corn oil (0.5 ml, solid bar) administration 129
10-2: Total recoverable (A) calcium, (B) sodium and (C)
potassium in uterine flushings at 1, 6, 12 and
24 h following administration (i.m.) of estradiol
valerate (0.5 mg, hatched bars) or 12 h after
corn oil (0.5 ml, solid bar) administration 131
10-3: Total recoverable (A) protein, (B) uteroferrin,
(C) leucine aminopeptidase (LAP) and (D) glucose
in uterine flushings at 1, 6, 12 and 24 h
following administration (i.m.) of estradiol
valerate (0.5 mg, hatched bars) or 12 h after
corn oil (0.5 ml, solid bar) administration 133
11-1: Effect of chronic ovarian steroid administration on
uterine secretory response of ovariectomized gilts;
possible interaction with endogenous prolactin
to result in low and high uterine sectory response. 149
12-1: Separation of nonglycosylated and glycosylated
forms of porcine prolactin by Concanavalin A-
Sepharose 6B column chromatography 153
12-2: Evaluation of the purity of nonglycosylated and
glycosylated forms of porcine prolactin isolated
by Concanavalin-A Sepaharose 6B column chromatography
using 12.5% sodium dodecylsulphate one-dimensional
polyacrylamide gel electrophoresis 155
12-3: Uptake of 3H-thymidine into Nb2 lymphoma cells
expressed as percent of Nb2 control (dashed line)
when cells were stimulated by total (circles),
nonglycosylated (squares) and glycosylated
(triangles) forms of porcine prolactin 158
xii


191
deGreef, V.J., Dullaart, J. and Zeilmaker, G.H. (1977) Serum
concentrations of progesterone, lutenizing hormone, follicle
stimulating hormone and prolactin in pseudopregnant rats: effect of
decidualization. Endocrinology. 101, 1054-1063.
DeHoff, M.H., Bazer, F.W. and Collier, R.J. (1984) Ontogeny of
prolactin receptors in porcine uterine endometrium during pregnancy.
Proc. 4th Int. Prolactin Congr. 95 (Abstract).
DeHoff, M.H., Stoner, C.S., Bazer, F.W., Collier, R.J., Kraeling, R.R.
and Buonomo, F.C. (1986) Temporal changes in steroids prolactin and
growth hormone in pregnant and pseudopregnant gilts during mammogenesis
and lactogeneis. Dorn. Anim. Res. 3, 95-105.
DeLean, A., Ferland, L., Drouin, J., Kelly, P.A. and Labne, F. (1977)
Modulation of pituitary thyrotropin releasing hormone receptor levels
by estrogens and thyroid hormones. Endocrinology. 100, 1496-1504.
DeMeyts, P., Bianco, A. and Roth, J. (1976) Site-site interactions
among insulin receptors. Characterization of the negative
cooperativity. J. Biol. Chem. 251, 1877-1888.
Djiane, J., Clauser, H. and Kelly, P.A. (1979a) Rapid down regulation
of prolactin receptors in the mammary gland and liver. Biochem.
Biophys. Res. Comm. 90, 1371-1378.
Djiane, J. Delouis, C. and Kelly, P.A. (1979b) Prolactin receptors on
organ culture of rabbit mammary gland: effects of cylcoheximide and
prolactin. Proc. Soc. Exp. Biol. Med. 162, 342-345.
Djiane, J. and Durand, P. (1977) Prolactin-progesterone antagonism in
self-regulation of prolactin receptors in the mammary gland. Nature.
266, 641-643.
Djiane, J., Durand, P. and Kelly, P.A. (1977) Evolution of prolactin
receptors in rabbit mammary gland during pregnancy and lactation.
Endocrinology. 100, 1348-1356.
Djiane, J., Kelly, P.A., Katoh, M., Dusanter-Fourt, I., and Berthon,
P. (1987) The prolactin receptor. In: The Molecular Biology of
Receptors: Techniques and Applications of Receptor Research, (ed. A.D.
Stosberg) pp. 92-127. VCH Publishers, New York.
Donner, D.B., Casadei, J., Hartstein, L., Martin, D. and Sonenberg, M.
(1980) Characterization of the slowly dissociable human growth
hormone binding component of isolated rat hepatocytes. Biochemistry.
19, 3293-3300.
Donner, D.B. and Corin, R.E. (1980) Formation of a receptor state from
which insulin dissociates slowly in hepatic cells and plasma membrane.
J. Biol. Chem. 255, 9005-9008.


6
mammalian reproduction, 4) parental behaviour, 5) intergumentary
structures and 6) interaction with steroid hormones (Nicoll et al.,
1985). Based on PRL's diversity of actions, the chronobiologist,
Hablan (1980), has suggested its renaming to 'versatilin' (as cited by
Nicoll, 1979).
Microheterogeneity of Prolactin
Glycosylated Prolactin
The anterior pituitary produces several glycosylated hormones.
Therefore the discovery of a glycosylated form of PRL was novel, yet
not entirely unexpected. First reported in 1984, by Lewis and
coworkers, glycosylated PRL has received increasing attention. The
diversity of PRL's action may be a function of its microheterogeneity.
Presently, several molecular weight forms of PRL (23,000, 25,000 and
50-60,000 Mr) have been isolated from pituitaries of sheep (Lewis et
al., 1984), pigs (Pankov and Butnev, 1986), rats (Wallis, 1980), mice
(Sinha and Jacobsen, 1987) and humans (Lewis et al., 1985; Markoff and
Lee, 1987), suggesting that PRL is actually a 'family' of hormones
(Lewis et al., 1984). Glycosylated PRL is produced also in uterine
decidua (Lee and Markoff, 1986) and found in amniotic fluid (Meuris et
al., 1985) of humans. The carbohydrate moiety of glycosylated porcine
PRL resembles that of ovine LH (Bedi et al., 1982). and is N-linked at
asparigine 31 to the polypeptide (Pankov and Butnev, 1986).
The glycosylated form of PRL is less mitogenic and immunoreactive
in Nb2 lymphoma cell assays and polyclonal RIAs, respectively
(Pellegini et al., 1988; Scott et al., 1988). Lactogenic activity, as
measured by the pigeon crop sac or mouse mammary explant casein


138
membrane as well as its receptor may cause the biphasic effects of
estrogen on target tissues.
Previous results indicate PRL involvement in uterine endometrial
physiology (Williams et al.f 1978; Rose et al., 1983; Chilton and
Daniels, 1985; Young and Bazer, 1988; Mirando et al., 1988). Results
from the present studies indicate that estrogen affects PRL receptor
numbers within 12h. Considering the rapid successsion of uterine
secretory events essential to the preimplantation porcine conceptus,
one effect of estrogen appears to be modulation of endometrial PRL
receptors that are temporally associated with uterine secretory
processes. Rapid induction of endometrial PRL receptors may be caused
by estrogens interaction with membrane receptors, changes in membrane
dynamics and fluidity or unmasking of receptors, rather than PRL
receptor synthesis. Additionally, stimulation of pituitary PRL release
by exogenous estrogen is likely to affect receptor binding and
internalization, but not auto-upregulate of PRL receptors within 24h.
The involvement of PRL in estrogen-induced uterine secretory activity
is well documented. Further investigations of both acute and chronic
effects of steroid-PRL interactions are necessary to fully elucidate
the mechanisms whereby they affect uterine physiology.


208
Rosenthal, H.E. (1967) Graphic method for the determination and
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Gluccocorticoid regulation of prolactin receptors in mammary cells in
culture. Endocrinology. 104, 1447-1449.
Sakai, S., Enami, J., Nandi, S. and Banerjee, M.R. (1978) Prolactin
receptor in dissociated mammary epithelial cells at different stages of
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Sakai, S. and Ike, F. (1987) Two separate receptors for prolactin in
the rabbit mammary gland. Endocrinol. Jpn. 34, 863-870.
Sakai, S., Katoh, M., Berthon, P. and Kelly, P.A. (1984)
Characterization of prolactin receptors in porcine mammary gland.
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Sakai, S., Katoh, M., Berthon, P. and Kelly, P.A. (1985)
Characterization of prolactin receptors in pig mammary gland. Biochem.
J. 224, 991-922.
Samson, V.K., Lumpkin, M.D. and McCann, S.M. (1986) Evidence for a
physiological role for oxytocin in the control of prolactin secretion.
Endocrinology. 119, 554-560.
Sasakai, N., Imai, Y., Tsushima, T., Matsuzaki, F. (1982a) Regulation
of somatotropic and lactogenic binding sites in mouse liver membranes.
Acta Endocrinol. 101, 574-579.
Sasakai, N., Tanaka, Y., Imai, Y., Tsushima, T. and Matsuzaki, F.
(1982b) Different characteristics of solubilized liver of pregnant and
nonpregnant female rats. Biochem. J. 203, 653-656.
Sasavage, N.L., Nilson, J.H., Horowitz, S. and Rottman, F.M. (1982)
Nucleotide sequence for bovine prolactin messenger RNA. Evidence for
sequence polymorphism. J. Biol. Chem. 257, 678-681.
Scatchard, G. (1949) The attraction of proteins for small molecules
and ions. Ann. N.Y. Acad. Sci. 51, 660-672.
Schally, A.V., Redding, T.W., Arimura, A., Dupont, A. and Linthicum,
G.L. (1977) Isolation of gamma-amino butyric acid from pig hypothalami
and demonstation of its prolactin release-inhibiting (PIF) activity in
vivo and in vitro. Endocrinology. 100, 681-691.
Schneider, C., Owen, M.J., Banville, D. and Williams, J.G. (1984)
Primary structure of human transferrin receptor deduced from the mRNA
sequence. Nature. 311, 675-678.


CHAPTER 1
INTRODUCTION
The exsistence of a lactogenic hormone was proposed in 1928, but
research on prolactin (PRL) did not flourish until the 1970's,
following its purification from several species, including the human.
Prolactin's diverse functions combined research from several scientific
disciplines, such as endocrinology, physiology, comparative
endocrinology, neuropharmacology and anatomy. From the early studies
on crop sac and mammary gland development, as well as lactogenesis, PRL
is now credited with over 100 functions (Nicoll and Bern, 1972).
Prolactin has osmoregulatory functions in amphibians, fish and mammals
and lactogenic functions in birds and mammals. Prolactin affects
growth of various organ systems in fish, amphibians, birds and mammals.
In mammals, PRL is associated primarily with reproductive processes,
e.g., mammogenesis, lactogenesis and luteotropic funtions.
Previously, PRL was associated with uterine function, transport of
water, protein and prostaglandin secretion, and steroid uptake and
metabolism. In 1985, Chilton and Daniels, proposed a fourth function
for PRL in uterine physiology; endometriotrophism. Thus, PRL affects
the female reproductive system at all levels; ovaries, mammary gland
and uterus.
The uterus is regulated primarily by steroid hormones. Estrogen
induced biphasic responses, however, could not be explained by
classical steroid mechanisms, rapid signal transduction systems or
novel estrogen mechanisms. Therefore, estrogen's interaction with, or
1


54
Concentrations of PGF (30+5 vs 17 +5 ng/horn) and PGE (14+2.2 vs 11
+2.2 ng/horn) in uterine flushings were not affected by treatment.
Gilts that received CB154 had lower concentrations of calcium (P<0.03),
sodium (P<0.02), potassium (P<0.01) and chloride (P<0.01) in uterine
flushings (Figure 3-1).
Discussion
Bromocryptine, effectively lowers PRL to near undecteable levels
in rats. In this study, PRL was decreased 40-50% in pigs. Fetal
survival was not affected despite decreased circulating PRL. The
remaining concentrations of circulating PRL may have been adequate for
fetal survival. Additionally, porcine conceptuses may have compensated
through other physiological mechanism, which at this time are unknown,
to maintain the pregnancy. Prolactin modulates endometrial physiology
of rabbits (Daniels et al., 1984; Chilton and Dainels, 1985), but
adminsitration of CB154 to pigs on Days 10-15 of gestation may have
been unable to reverse prior affects of basal PRL on endometrium.
Therefore, although PRL levels were lowered, the endometrium was
already stimulated to respond to conceptus signals. Lowered PRL levels
following conceptus estrogen signal may have blunted the uterine
secretory response, but PRL concentrations were adequate for conceptus
survival. Fetal survival may have been affected if CB154
administration began earlier in gestation to block possible effects or
modulation of PRL on uterine physiology.
However, lowered PRL affected estrogen-stimulated ionic changes in
the uterine lumen, suggesting that PRL may affect the ionic environment
of the developing conceptus. In this study, slight hypoprolactinemia


35
fragments stimulated casein DNA synthesis, while whole antibody serum
stimulated casein DNA 50-60%. Monovalent F(ab) fragment had no activity
in stimulation of casein biosynthesis. Interactions between two PRL
receptor molecules is, therefore suggested to be involved in PRL
stimulation; similar to the mechanism of insulin (Rosen, 1988). Under
these conditions, the half-life for PRL in the plasmalemma is three
times greater and movement of PRL receptors within the cell membrane
could constitute an additional regulatory mechanism for cell
receptivity. Microaggregation of receptors following hormone binding
is essential for induction of biological effects of several hormomes
(Brown and Goldstein, 1983).
Regulation of Prolactin Receptors
Regulation by Ovarian Steroids
Prolactin receptor concentrations are sex specific (Sherman,
1977; Waters et al., 1978). Changes in PRL receptor numbers within
several target tissues occur during puberty (Kelly et al., 1974), the
estrous cycle (Kelly et al., 1975), pregnancy (DeHoff et al. 1984;
Grissom and Littleton, 1988), lactation (Kelly et al., 1975; Sherman et
al., 1977; Shiu et al., 1981), and in response to ovariectomy (Posner
et al., 1974a; Kelly et al., 1979; Marshall et al., 1979; Daniels et
al., 1984), orchotomy (Kelly et al., 1976; Bohnet et al., 1977) and
following administration of exogenous steroids (Waters et al., 1978;
Shiu et al., 1982). However, alterations in PRL receptor
concentrations are not necessarily similar between tissues or within
the same tissue of different species.


123
diapause and delayed implantation; possibly through alterations in the
metabolism of progesterone, as suggested for the rat uterus (Armstrong
and King, 1971). Porcine CL have PRL receptors which fluctuate during
the estrous cycle or early gestation, but are highest in the mid-luteal
phase of the cycle (Bramley and Menzies, 1987) and are higher for CL of
pregnancy (Rolland et al., 1978).
In summary, endometrial membrane PRL receptor numbers fluctuate
during early pregnancy which may explain enhanced biological effects of
PRL in the uterine environment, despite rather constant levels in
plasma during early pregnancy. Additionally, increases in PRL receptor
numbers are temporally associated with reproductively important events,
such as conceptus elongation, uterine growth, luteostasis,
establishment of pregnancy and uterine secretory function. These
results support PRL's involvement in uterine physiology. Although,
regulation of hepatic PRL receptors is well investigated, further work
is needed to establish if similar mechanisms are responsible for tissue
specific changes in PRL receptors within endometrium and other
reproductive tissues.


53
E
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144
weight (+41 pmoles/gww) of endometrial tissues, gilts treated with CO
(233 pmoles/gww) were greater than for E2V alone and P4 alone (176 and
185 pmoles/gww, respectively) which were higher than for E2V+P4 (157
pmoles/gww).
Treatment of gilts with ovarian steroids affected (P<0.001) total
recoverable protein and uteroferrin in uterine flushings. Total
protein (mg + 5) was similar in gilts that received CO or E2V alone
(1.7 and 7.1 mg, respectively). Progesterone administration increased
(P<0.001) protein secretion (36.8 mg) as did administration of E2V+P4
(83.8 mg, P<0.001). Uteroferrin (umoles + 524) was secreted in a
similar pattern as protein. Administration of E2V or CO resulted in
low levels of uteroferrin in uterine flushings (8.1 vs 1.7 umoles,
respectively), which increased (P<0.001) in response to P4 alone (4227
umoles) and E2V+P4 (8405 umoles) (see Table 11-1) .
Discussion
Prolactin (PRL) receptor concentrations are sex specific
(Sherman, 1977; Waters et al., 1978). Changes in PRL receptor numbers
within several target tissues occur during puberty (Kelly et al.,
1974), the estrous cycle (Kelly et al., 1975; Chapter 9), pregnancy
(DeHoff et al. 1984; Grissom and Littleton, 1988; Chapter 9), lactation
(Shiu et al., 1974; Kelly et al., 1975; Sherman et al., 1977), and in
response to ovariectomy (Posner et al., 1974a; Kelly et al., 1979;
Daniels et al., 1984), orchotomy (Kelly et al., 1976; Bohnet et al.,
1977) and following administration of exogenous steroids (Waters et
al., 1978; Shiu et al., 1982). However, alterations in PRL receptor


APPENDIX C
ACID PHOSPHATASE ASSAY FOR MEASUREMENT OF UTEROFERRIN
Solutions
1) Tris buffer: 0.5 M pH 7.0
2) Sodium Acetate: 1M pH 4.9
3) Sodium Hydroxide: 1M
Make fresh prior to assay:
4) Assay Buffer:
5) pNPP Substrate:
6.005 g/L dHzO
13.68 g/100 ml dH20
20.0 g/150 ml dH20 (stir)
0.1M B-Me in 0.05M Tris buffer (780 ul/100ml)
10 ml tris buffer 11 + 90 ml dH20
FV=371.1 14.88 gm/L in 0.01M NaAC
500 ul 1M NaAc (#2) + 49.5 ml dH20
Calculations for Proper Volumes
t tubes in assay +5 x 400 ul
" x 100 ul
" x 500 ul
" x 1.5 ml
vol assay buffer needed
vol of 1 M NaAc needed
vol pNPP substrate needed
vol 1M NaOH needed
Standard Curve
umoles dil
pNPP
pNPP
dH2 0
OD
0
1:10
Oul
100
ul
calb
0.01
1.10
10 ul
90
ul
0.02
1:10
20 ul
80
ul
0.04
1:10
40 ul
60
ul
0.08
1:10
80 ul
20
ul
0.15
none
15 ul
85
ul
0.20
none
20 ul
80
ul
Protocol for Acid Phosphatase Assay
Note: Since this is a timed assay, only run about 30 to 40 tubes,
including the standard curve, in one assay.
1) Label tubes. Run each sample in duplicate.
Turn on water bath and set to 37 C, and set the spectrophotometer
at 410 nM (visible light).
183


11
secretion from growth hormone to dual secretion with PRL (Brookfor et
al., 1986), suggesting a functional 'plasticity' for conversion. It is
unknown if mammosomatotrophs serve as a stem cell or are a normal stage
within the cell cycle (Frawley et al., 1985).
Decidual Prolactin
Prolactin is found in the amniotic fluid during pregnancy. Yet,
PRL in the maternal circulation does not fluctuate greatly during
gestation and, therefore, could not account for the high concentrations
of PRL in the amniotic fluid of rhesus monkeys (Josimovich et al.,
1974). Additionally, PRL levels in amniotic fluid of anecephalic
fetuses or fetus that have died in tero are similar to normal fetuses
(Walsh et al., 1977). Placental membranes do not produce PRL, but
decidua attached to placental membranes stains positively for PRL
(Golander et al., 1978; Healy et al., 1979). Studies of decidual
tissue of pregnancy determined that PRL was produced from these
tissues. In 1977, the suggestion that a pituitary hormone could be
produced in a nonpituitary tissue was considered novel. However, many
hormones previously associated only with brain tissue are now found in
other tissues and vice versa (vasoactive intestinal peptide). Riddick
and Kusmik (1977) confirmed that PRL was produced during normal
pregnancy and by secretory endometrium from Day 22 of the menstrual
cycle (McRae et al., 1986).
Decidual PRL is identical in structure and biological function
(Riddick et al., 1978) to pituitary PRL. Prolactin secretion is 1000-
fold less from decidual (400 ng/g tissue) when compared to pituitary
(400 ug/g tissue) (Tomita et al., 1982). However, the weight of the


179
3) Dry iodo-gen under a gentle nitrogen stream. Rinse with buffer #1
(NO BSA). After air drying, a faint white ring should be visible on the
bottom of the tube.
Make Column Use a new, disposable 10 ml pipette with the top cut off.
Rinse inside of 10 ml pipette with buffer 12 (BSA). Pour sephadex
column quickly to avoid air bubbles. (NOTE: if buffers are not at room
temperature then bubbles form in the column, start over) Equilibrate
with buffer 12 (BSA) and finish with at least 1 volume of buffer 11 (NO
BSA). Stop Column.
Prepare prolactin Weigh as small amount of prolactin as accurately
possible (50-100 ug) on the Cahn balance. Dilute with 0.1 M NaHC03, pH
8.3 to a concentration of 20 ug/10 ul. Dilute 1:1 with buffer #1 (NO
BSA; use correct buffer otherwise you will iodinate BSA as well as the
hormone).
Iodination proceedure Set up iodination hood with the following:
Sephadex column, 10 ul Hamilton (stored in hood), fraction collector
(stored under hood), 20 ul pipette (remove metal automatic tip
remover), pasteur pipette and bulb, 50 tubes (13 x 100 mm borosilicate)
each containing 500 ul buffer 12 (BSA), iodo-gen tube, hormone, pipette
tips, excess buffer fl (NO BSA) and extra borosilicate tube for 5 ul
aliquot. Log out 10 ul Na-[125I].
Add 1 mCi (10 ul) Na129I to iodo-gen tube.
Add 10 ul buffer II (NO BSA)
Add 20 ul hormone (10 ug)
Add 20 ul buffer fl (NA BSA)
Hand vortex 5 sec every min for 15 min.
Following the 15 min reaction, take a 5 ul aliquot and put it into an
extra (empty) tube and set it aside (use for calculation of specific
activity).
Using pastuer pipette, load remaining 55 ul onto Sephadex column. DO
NOT scratch iodo-gen from the tube. Start column flow. After the
reactants have entered the column, add a small amount of buffer II (NO
BSA) to the column. DO NOT disturb the gel surface. DO NOT let the
gel run dry.
Collect 20 drops into the first tube and 10 drops into the remaining 49
tubes. Stop column.
Determination of peak Aliquot 10 ul from each tube into another set of
12 x 75 mm borosilicate tubes and count for either 0.5 or 1 min.
Identify hormone label peak (around tube 120). Hormone peak should
contain approximately 500,000 to 1,000,000 cpm/10 ul.


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Figure 10-1: Prolactin receptor numbers in endometrial
membranes at 1, 6, 12, and 24 h after administration (i.m.)
of estradiol valerate (0.5 mg, hatched bars) or 12 h after
corn oil (0.5 ml, solid bar) administration. The solid line
denotes the mean value prior to injection (time zero) of Day
11 cyclic gilts. Values with different letters are different
(P<0.02). The SEM was + 11.8 pmoles/mg protein.


LIST OF TABLES
Tables Page
4-1: Effects on interestrous interval and cytoxicity
following cysteamine (CSH) and ethanolamine (control)
administration to cyclic gilts 60
6-1: Composition of Day 16 uterine flushings from
ovariectomized gilts treated with daily injections
of progesterone and saline or porcine prolactin from
Days 4 through 14 (x+SEM) 71
11-1: Endometrial membrane prolactin receptors and total
protein and uteroferrin in uterine flushings from
ovariectomized gilts following steroid
administration for 11 days 143
ix


96
Radioinert ovine PRL
was
less effective
than
porcine PRL in
displacing [123I]-PRL of
either
species
from
porcine endometrial
membranes (Figure 7-5).
The
homologous
RRA
system
resulted in 75%
displacement when radioinert porcine PRL competed with radiolabelled
porcine PRL. Additionally, maximum specific binding was higher for
radiolabeled porcine PRL (25%) compared to radiolabelled ovine PRL
(18%). The percent displacement for the four hormone combinations
(labelled/unlabelled) were as follows: 40% for 123I-ovine/ovine; 30%
i23I-porcine/ovine; 60% 125I-ovine/porcine; and 75% for 125I-
porcine/porcine. Thus, the homologous (label vs unlabel)
hormone/membrane combination resulted in a more effective displacement
of PRL from Day 75 porcine endometrial membranes.
Hormonal specificity of the porcine endometrial PRL receptor is
depicted in Figure 7-6. Porcine [125I]-PRL is not displaced by porcine
LH or FSH and only slight displacement was detected for porcine GH when
compared to the displacement observed with radioinert porcine PRL
(Figure 7-7); approximately 100-fold more porcine GH than porcine PRL
was needed to displace a comparable amount of radiolabelled porcine PRL
from the endometrial receptor. Binding constants for GH could not be
determined when crossreactivity assays were analyzed using the LIGAND
program.
Homologous competitive displacement assays were conducted with Day
75 pregnant endometrial membranes. Scatchard analysis of binding data
(Figure 7-8) indicated an equilibrium dissociation constant (Kd) of
3.06 x 10'8 M (Ka = .326+0.011 x 10 M-1). Pretreatment of membranes
with MgCl2 did not alter the Kd but did increase maximum binding 4- to


166
present study suggested that glycosylated porcine PRL interacts
differently with PRL receptors. Neither form alone, nor when remixed,
displaced [125I]-PRL (total) as effectivly as unlabelled total PRL.
This may result from 1) the need for both forms to be present for
effective receptor binding or activation, 2) detrimental effects of
separation of the two forms on binding ability or 3) targeting of the
different forms of PRL to different parts of the receptor(s) or
membrane. Discrepancies between biological activity and RIAs have been
reported previously (Asawaroengchai et al., 1978; Leung et al., 1978;
Owens et al., 1986) which may extend to results for binding data
(Nicoll, 1975). Uncertainty remains regarding the number of PRL
receptors necessary to elicite a biological response (Bohnet et al.,
1977).
These results suggest that glycosylated porcine PRL has greater
lactogenic activity, but lower mitogenic activity than the
nonglycosylated form. Lower immunoaffinity of glycosylated PRL
suggests that the RIA may not accurately measure biopotency of
circulating PRL. Additionally, the ratio of the two forms may be
important physiologically (Sinha et al., 1988). Glycosylated porcine
PRL may possess different receptor binding characteristics than
nonglycosylated or total PRL. Additionally, the presence of both forms
of PRL, without manipulation, is necessary to obtain binding
characteristics similar to those previously reported for the porcine
endometrial membrane PRL receptor (DeHoff et al., 1984; Chapters 7, 9,
10 and 11). Prolactin may be present in the circulation as a higher Mr
(50-60,000; Suh and Frantz, 1974; Whitaker et al., 1983) form which
may be disrupted during separation. Thus, the physiological roles of


47
relationship (correlation coefficient=0.99; y = intercept +6.3x)
between absorbance (510 nm) and concentration of chloride (mEq/L). The
assay was sensitive at 60 mEq/L.
Sodium and Potassium
A flame photometer (Perkin Elmer 51Ca; Coleman Instruments
Division, Oak Brook, IL) was used to determine concentrations of sodium
and potassium as described previously (Young et al., 1987).
Glucose
The Beckman Glucose Analyzer 2 (Beckman Instruments, Columbia, MD)
was used to determine glucose concentrations as a direct proportion to
oxygen consumption (Bazer et al., 1984).
Leucine-acyl Aminopeptidase (LAP)
This membrane marker protein was used as an index of secretory
activity and its concentration was determined using a colorometric
assay (Zavy et al., 1984). One Sigma Unit (SU) will release 1 umole
(143 ug) of 5-naphthylamine from L-leucyl-p-nahpthylamine per hour at
37 C and pH 7.1.
Prostaglandin (PG) F
Uterine flushings were analyzed for PGF by radioimmunoassay (RIA)
as described by Knickerbocker et al. (1986) using the antibody
characterized by Kennedy (1985) and tritiated PGF2C1
((5,6,8,9,11,12,14,15-3H]:PGF2 Amersham Corporation, Arlington Heights, IL). Standard curves were


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
THE ROLE OF PROLACTIN IN ESTABLISHMENT OF PREGNANCY IN PIGS:
STUDIES ON ENDOMETRIAL PROLACTIN RECEPTOR REGULATION
AND UTERINE SECRETORY PHYSIOLOGY
By
Kathleen Hart Young
May 1989
Chairman: Fuller W. Bazer
Major Department: Animal Science
Conceptus estrogens cause biphasic endometrial responses not
wholly explained by steroid receptor mechanism. Studies reported herein
support prolactin involvement in porcine uterine secretory function.
Estrogen-induced uterine secretory function was decreased in response
to hypoprolactinemia in pigs. Exogenous prolactin interacted with
estrogen, but not progesterone, to increase secretion of uteroferrin,
prostaglandin F2CL and leucine-aminopeptidase. Prolactin alone
increased glucose in uterine flushings. Prolactin modulates quantity
of electrolytes released in reponse to estrogen and enhances secretion
of protein to nourish preimplantation porcine conceptuses.
A homologous porcine prolactin radioreceptor assay was developed,
using pig endometrial membrane preparations, which specifically
detected high affinity (0.3 x 108 M-1), prolactin receptors and was
used to detect changes in endometrial prolactin receptors during the
estrous cycle and early pregnancy. Endometrial prolactin receptors
were similar for pregnant and cyclic gilts on Day 8, then decreased
xiv


Figure
Page
12-4: Immunoaffinity of total (open and closed circles),
nonglycosylated (open and closed squares) and
glycosylated (open and closed triangles) forms of
porcine prolactin expressed as (A) percent bound
versus log concentration, (A inset) transformed to
log versus logit plot and (B) percent of Bo
(normalized to 100%) versus log concentration 161
12-5: Scatchard analysis of competitive inhibtion curves
using magnesium chloride treated Day 75 pregnant
porcine endometrial membranes and t125I]-prolactin
(total) versus unlabelled total prolactin
(triangles) and t125I]-nonglycosylatedrglycosylated
(2:1) prolactin versus unlabelled
nonglycosylated:glycosylated (2:1) (circles)
prolactin 162
12-6: Scatchard analysis of porcine t1251]-(total)
prolactin binding to magnesium chloride treated
Day 75 pregnant porcine endometrial membranes
inhibited by unlabelled total (circles),
nonglycosylated (squares) or glycoslyated
(triangles) forms of porcine prolactin 164
13-1: Effects of hypoprolactinemia and hyperprolactinemia
on the composition of uterine flushings of Day 12
gilts at 24 h after a single administration of
estradiol valerate 171
13-2: Possible mechanism(s) involving prolactin
during maternal recognition of pregnancy in pigs 174
xiii


212
Veldhuis, J.D., Klase, P. and Hammond, J.M. (1980) Divergent effects of
prolactin on steroidogenesis by porcine granulosa cells in vitro:
influence of cytodifferentiation. Endocrinology. 107, 42-46.
Vician, L., Shupnik, M.A. and Gorski, J. (1982) Effect of estrogen on
primary ovine pituitary cell culture. Stimulation of prolactin
secretion, synthesis and preprolactin messenger ribonucleaic acid
activity. Endocrinology. 104, 736-743.
Vick, R.S., Wong, V.L.K. and Witorsch, R.J. (1987) Biological,
immunological and biochemical characterization of cleaved prolactin
generated by lactating mammary gland. Biochem. Biophys. Acta. 931,
196-204.
Vonderhaar, B.K., Bhattacharya, A., Alhadi, T., Liscia, D.S., Andrew,
E.M., Young, J.K., Ginsberg, R., Bhattacharjee, M. and Horn, T.M.
(1984) Production symposium: Prolactin effects on mammary epithelial
cell. Isolation characterization and regulation of the prolactin
receptor. J. Dairy Sci. 68, 466-488.
Walaas, 0. and Walaas, S.I. (1988) Second messengers identified for
insulin. TIPS. 9, 151-152.
Walker, A.M. and Farquhar, M.G. (1980) Preferential release of newly
synthesized prolactin granules is the result of functional
heterogeneity among mammotrophs. Endocrinology. 107, 1095-1104.
Wallis, M. (1974) The primary structure of bovine prolactin. Febs.
Lett. 44, 205-208.
Wallis, M. (1981) The molecular evolution of pituitary growth hormone
prolactin and placental lactogen: a protein family showing variable
rates of evolution. J. Mol. Evol. 17, 10-18.
Wallis, M., Daniels, M. and Ellis, S.A. (1980) Size heterogeneity of
rat pituitary prolactin. Biochem. J. 189, 605-614.
Walsh, S.W., Meyer, R.K., Wolf, R.C. and Friesen, H.G. (1977) Corpus
luteum and fetoplacental functions in monkey hypophysectomized during
late pregnancy. Endocrinology. 100, 845-850.
Waters, M.J., Friesen, H.G. and Bohnet, H.G. (1978) Regulation of
prolactin receptors by steroid hormones and use of radioligand assays
in endocrine research. In: Receptors and Hormone Action. Vol III, pp.
457-477. (eds. L. Birnbaumer and B.W. O'Malley) Academic Press, New
York.
Waters M.J., Lusins, S. and Friesen, H.G. (1974) Immunology and
physiochemical evidence for tissue specific prolactin-receptors in the
rabbit. Endocrinology. 115, 1-10.


88
Dissociation of Porcine [123I]-PRL
Porcine [123]-PRL (45,000 cpm; 0.24 ng) was incubated with 150 ug
Day 75 pregnant pig endometrial or Day 20 pregnant rat liver membrane
at 25 C in 500 ul of assay buffer in the presence (1 ug) and absence of
unlabelled porcine PRL for 24 hours. After incubation, the membranes
were washed in 3 ml ice-cold assay buffer, centrifuged (2300 x g; 30
min 4 C), and decanted to remove any free hormone. Pellets were
resuspended in fresh assay buffer (500 ul) containing 5 ug unlabelled
porcine PRL. Dissociation of previously bound porcine f1 231]-PRL was
determined at various time points (0, 1, 2.5, 5, 7.5, 10, 12, 24, 32
and 48h). Labeled porcine PRL specifically bound to membranes was
expressed as a fraction of the amount bound at equilibrium.
Hormonal Specificity of Porcine [*231]-PRL
Porcine [123I]-PRL was incubated with 150 ug of Day 75 pregnant
pig endometrial or Day 20 pregnant rat liver membranes for 24 hours at
25 C in the presence and absence of increasing concentrations of
unlabeled porcine PRL (0-5120), porcine growth hormone (GH), porcine
luteinizing hormone (LH) and porcine follicle stimulating hormone (FSH)
(0-10240 ng). Binding assays were conducted as described previously.
Heterologous versus Homologous Binding and Displacement of Ovine
and Porcine PRL
The effects of heterologous (ovine) and homologous (porcine)
PRL were investigated using radiolabelled and radioinert forms of each
hormone. Assays were conducted using Day 75 pregnant pig endometrial
membranes in the following combinations: 1) ovine [231]-PRL versus


66
10 11 12 13
Day of Estrous Cycle
Figure 5-1: Concentrations of immunoreactive prolactin in
serum during administration of 1 mg porcine prolactin,
(circles), or 1 ml saline (squares), at 0800 and 2000h
(denoted by arrows) on Days 10 through 13 of the estrous
cycle. Blood samples were collected at 0730, 1000, 1200,
1930 and 2400 h. The SEM were +1.24 ng/ml for prolactin
treated and +0.12 ng/ml for saline treated gilts.


APPENDIX A
IODINATION OF PORCINE PROLACTIN
AND DETERMINATION OF SPECIFIC ACTIVITY
Buffer #1
pH 7.2
Buffer |2
pH 7.6
Reagents
25 mM Tris-HCl
3.94 g/L
10 mM CaCl2
1.109 g/L
0.01% Sodium Azide
0.1 g/L
0.1% PMSF
2 ml stock
solution/L
0.1% BSA 0.5 g
into 500 ml
of Buffer
PMSF stock 100 mM phenylmethylsulfonylflouride in absolute
alcohol. Store at -20 C.
Porcine Prolactin (USDA-B-1)
Na-[129I], 100 oCi (Amersham Corp. Arlington Heights, IL)
Iodo-gen powder (Pierce Chemical Corp., Rockford, IL)
0.01 M NaCHOa (pH 8.3) 0.8401 g/100 ml
Chloroform
Sephadex G75 (Pharmacia Fine Chemicals, Piscataway, NJ)
Swell in 25 mM Tris-HCl; 0.01% Thimasol for 24 h,
pour off fines and degas. Use at 25 C.
The day before
1) Acid wash (Glacial acetic acid) 12 x 75 borosilicate tubes for
iodo-gen.
2) Bring buffers and G75 to 25 C.
3) Start assembly of separation column (see below).
Morning of Iodination
1) Turn on Cahn Balance and allow 20 min warm-up. Weigh iodo-gen
(approximately 500 ug) using Cahn balance (in Dr. Hansen's lab) and
dilute to 0.1 mg/ml in chloroform. Stir gently in small beaker with
micro stir bar.
2) Carefully aliquot 20 ul of iodo-gen solution into bottom of acid
washed tubes. Add 40 ul of chloroform to each tube.
178


29
Structural differences for PRL receptors between mammary gland and
liver have been suggested previously (Sakai et al., 1985).
During sequencing of purified growth hormone receptor, 20-50% of
the receptor was actually sequenced as ubiquitin (Leung et al., 1987).
Ubiquitin has been associated with the intracellular domain of the
growth hormone receptor and may play a functional role in receptor
activation and cellular response (Leung et al., 1988). A peptide bond
forms between the epsilon-amino group associated with the cytoplasmic
domain of the receptor and the carboxy terminal end of ubiquitin
(Goldknopf and Busch, 1977) is suggested. This feature is also present
in lymphocyte homing receptor (Siegelman et al., 1986) and platelet
derived growth factor (Yardin et al., 1986) and may extend to ovarian
and mammary gland PRL receptors with long cytoplasmic domains (Kelly et
al., 1989).
Water Soluble Prolactin Receptors
Water soluble PRL receptors have been described for pig mammary
glands (Berthon et al., 1987b) and rat liver (Amit et al., 1984). This
receptor is not precipitable by polyetheylene glycol as are cytosolic
steroid receptors (Kelly et al., 1983). Water soluble receptors have
been reported for follicle stimulating hormone (Dufau et al., 1977),
human chorionic gonadotropin (Pahnke and LeidenBerger, 1978) and human
growth hormone (McGuffin et al., 1976; Herrington 1981), suggesting
that water soluble receptors are generally associated with polypeptide
hormones (Berthon et al., 1987b). The water soluble PRL receptor from
porcine mammary gland has similar specificty and affinity as membrane
associated PRL receptors and binding was higher with ovine PRL. Porcine


122
(secretion) of uteroferrin and other proteins into the uterine lumen.
Signals from the porcine conceptus must prevent CL regression and
facilitate redirection of prostaglandin F2a secretion from the uterine
vasculature (endocrine) toward the uterine lumen (exocrine) (Bazer and
Thatcher, 1977). Concentrations of prostaglandin F2C1 are higher in
uterine flushings from pregnant than cyclic gilts between Days 12 to 14
(Zavy et al., 1980). Prolactin interacts with estrogen in vivo (Young
and Bazer, 1988) and in vitro (Mirando et al., 1988) to stimulate
release of PGF toward the uterine lumen. In the present study,
increased numbers of PRL receptors in endometrial membranes on Day 12
of gestation may enhance biological effects of PRL which include
facilitation of the redirection of prostaglandin F2C1 into the uterine
lumen to allow luteostasis and maternal recognition of pregnancy as
proposed by Bazer and Thatcher (1977) .
Uterine PRL receptors have been measured throughout gestation in
pigs (DeHoff et al., 1984); mid and late-gestation in rabbits (Grissom
and Littleton, 1988) and at a single time point in pregnant rats
(Williams et al., 1978), sheep (Posner et al., 1974b) and anestrous
mink (Rose et al., 1983). During gestation in the pig, endometrial PRL
receptors increase between days 30-45, decrease to Day 60, increase to
maximum values on Day 75 and then decrease to low levels by Day 90
(DeHoff et al., 1984). Uterine PRL receptors increase 25-fold between
Days 5 and 20 of gestation in rabbits (Grissom and Littleton, 1988)
with the majority of receptors in the endometrium. Prolactin receptor
numbers are comparable for the pregnant uterus and artificially induced
decidual tissue of rats (Williams et al., 1978). Prolactin binding
sites in the uterus of mink (Rose et al., 1983) may stop embryonic


LIST OF FIGURES
Figure Page
3-1: Concentrations of total recoverable (A)
calcium, (B) chloride, (C) sodium and
(D) potassium in Day 12 uterine flushings
from cyclic gilts (Experiment 2) treated with
CB154 (100 mg/day) or vehicle (VHC, 4 ml/day)
on Days 10 and 11 and estradiol valerate
on Day 11 53
4-1: Mean concentrations of prolactin (ng/ml) in
serum of cyclic gilts treated with cysteamine
(solid line) or ethanolamine (dashed line) from
Days 10-16 (denoted by arrows) 59
4-2: Reproductive tracts from gilts treated with
either (A) ethanolamine or (B) cysteamine 62
5-1: Concentrations of immunoreactive prolactin in
serum during administration of 1 mg porcine
prolactin (circles), or 1 ml saline (squares),
at 0800 and 2000 h (denoted by arrows) on
Days 10 through 13 of the estrous cycle 66
6-1: Concentrations of total recoverable (A) protein,
(B) uteroferrin, (C) glucose and (D) leucine
peptidase activity (LAP) in Day 12 uterine
flushings from cyclic gilts (Experiment 2)
treated with 1 ml saline (SAL) or 1 mg porcine
prolactin (PRL) at 0800 and 2000 h on Days 6-11
and 0.5 ml corn oil (OIL) or 5 mg estradiol
valerate (E2V) on Day 11 of the estrous cycle 73
6-2: Concentrations of total recoverable (A) calcium,
(B) chloride, (C) sodium and (D) potassium in
Day 12 uterine flushings from cyclic gilts
(Experiment 2) treated with 1 ml saline (SAL) or
1 mg porcine prolactin (PRL) on Days 6-11 and
0.5 ml corn oil (OIL) or 5 mg estradiol valerate
(E2V) on Day 11 of the estrous cycle 75
x


4
growth hormone, overlapping functions are observed in several species
(Nicoll, 1975; 1982).
Since its discovery, PRL from several species has been sequenced.
Prolactin is a simple polypeptide containing 199 amino acids. Three
disulphide bonds, located between cysteines 4-11, 58-174 and 191-199,
are observed for PRL in most mammalian species, except horse (Li and
Chung, 1983) and fish (Farmer et al., 1977) which lack the NH2-terminal
loop. Biological activity is affected by breaking the disulfide bonds
indicating that function is dependent on the three dimensional
structure of PRL. Microheterogeneity has been established for PRL;
with 25,000 Mr glycosoylated and 60,000 Mr dimer forms in addition to
the well characterized 23,000 Mr form of PRL. These forms are present
in the pituitary as well as the circulation. Prolactin has been
sequenced for humans (Shome and Parlow, 1977; Cooke et al., 1981),
sheep (Li et al., 1970), cows (Wallis et al., 1974; Sasavage et al.,
1982), rats (Parlow and Schome, 1976; Gubbins et al., 1980), mice
(Kohmoto et al., 1984), whales (partial (Kawachi and Tubokawa, 1979),
elephants (Li et al., 1987) and pigs (Li, 1976). Porcine PRL has
typtophan residues at amino acids 91 and 150 and an isoelectric point
of 5.85 (Brewly and Li, 1975). Species specificity is considerable,
with 20% of the residues dissimilar between pig/whale and ruminants,
and a 40% difference between rat and other mammalian PRLs suggesting
that PRL has evolved differently, even within the mammalian species,
from an ancestral molecule it shared with growth hormone (Wallis,
1981) .
Prolactin appears to have undergone three evolutionary
accelerations. Rat PRL evolved the fastest, showing 44 point


192
Donner, D.B., Martin, D.V. and Sonnenberg, M. (1978) Accumulation of a
slowly dissociable peptide hormone binding components by isolated rat
target cells. Proc. Nat. Acad. Sci. USA. 75, 672-676.
Dufau, M.L. and Kusuda, S. (1987) Purification and characterization of
ovarian LH/hCG and prolactin receptors. J. Receptor Res. 7, 167-193.
Dufau, M.L., Ryan, D.W. and Catt, K.J. (1977) Soluble FSH receptors
from rat testis. FEBS Lett. 81, 359-362.
Dunand, M., Kraehenbuhl, J.P., Rossier, B.C. and Aubert, M. (1988)
Purification of PRL receptors from toad kidney: comparisons with rabbit
mammary PRL receptors. Am. Physiol. Soc. 254, C372-382.
Dusza, L. and Krzymowska, H. (1979) Plasma prolactin concentrations
during the oestrous cycle of sows. J. Reprod. Fert. 57, 511-514.
Dusza, L. and Krzymowska, H. (1981) Plasma prolactin levels in sows
during pregnancy, parturiation and early lactation. J. Reprod. Fert.
61, 131-134.
Duvilanski, B.H., Seilicovich, A., Diaz, M., Kasaga, M. and Debeljuk,
L. (1986) GABA-Prolactin interactions. In: GABA and Endocrine
Functions. pp. 119-130. (eds. G. Racagni and A.O. Donoso) Raven
Press, New York.
Eadie, G.S. (1942) The inhibition of cholinesterase by physostigmine
and prostigmine. J. Biol. Chem. 146, 85-93.
Eden, S. (1979) Age- and sex-related differences in episodic growth
hormone secretion in the rat. Endocrinology. 105, 555-580.
Evans, H.M. and Simpson, M.E. (1929) Hyperplasia of the mammary
apparatus of adult virgin females induced by anterior hypophyseal
hormones. Proc. Soc. Exp. Biol. Med. 26, 598-601.
Falconer, I.R. and Rowe, J.M. (1977) Effect of prolactin on sodium and
potassium concentrations in mammary alveolar tissue. Endocrinology.
101, 181-186.
Farmer, S., Papkoff, H., Brewley, T.A., Hayasida, T., Nishioka, R.S.
and Bern, H.A. (1977) Isolation and properties of teleost prolactin.
Gen. Comp. Endocrinol. 31, 60-71.
Farnsworth, V.E. and Gonder, M.J. (1977) Prolactin and prostate cancer.
Urology. 10, 33-34.
Farquhar, M.G. (1985) Membrane traffic in prolactin and other secretory
cells. In: Prolactin, Basic and Clinical Correlates, pp. 3-16. (eds.
R.M. MacLeod, M.O. Thorner and U. Scapagini) Fidia Research Series,
Springer-Verlag, Berlin.


11EFFECTS OF CHRONIC OVARIAN STEROID ADMINISTRATION
ON ENDOMETRIAL PROLACTIN RECEPTORS AS DETECTED BY
HOMOLOGOUS RADIORECEPTOR ASSAY AND UTERINE PROTEIN
SECRETORY RESPONSE IN OVARIECTOMIZED PIGS 139
Introduction 139
Material and Methods 140
Results 142
Discussion 144
12 STUDIES ON MITOGENICITY, LACTOGENICITY,
IMMUNOREACTIVITY AND RECEPTOR BINDING CHARACTERISTICS
OF NONGLYCOSYLATED AND GLYCOSYLATED PORCINE PROLACTIN 150
Introduction 150
Materials and Methods 152
Results 156
Discussion 163
13 GENERAL DISCUSSION 168
APPENDICES
A IODINATION OF PORCINE PROLACTIN AND
DETERMINATION OF SPECIFIC ACTIVITY 178
B HOMOLOGOUS RADIORECEPTOR ASSAY FOR
PORCINE ENDOMETRIAL PROLACTIN RECEPTORS 181
C ACID PHOSPHATASE ASSAY FOR MEASURMENT OF UTEROFERRIN 183
REFERENCES 185
BIOGRAPHICAL SKETCH 215
viii


83
structural properties between PRL of different species (Nicoll et al.(
1985) could contribute to inaccurate measurement of receptors within a
species when a heterologus RRA is used for receptor quantification. A
homologus RRA has the advantage of insuring that a receptor is specific
for the hormone under investigation and that the results can be
accurately correlated to the in vivo hormonal environment and provide a
method for investigating changes in in vivo PRL receptor populations
that are of physiological interest. Additionally, development of a
homologous RRA for porcine tissues may indicate the feasibility of
developing and using homologous RRAs for other species or hormone
systems.
Materials and Methods
Hormones
Porcine PRL, growth hormone (GH), follicle stimulating hormone
(FSH), luteinizing hormone (LH) and ovine PRL were from the USDA (grade
USDA-B-1) and were generously supplyed by Dr. Douglas Bolt (Director of
Animal Hormone Program).
Iodination of Hormones
Porcine PRL (USDA-B-1) was iodinated using the Iodo-gen procedure
adapted from Markwell and Fox (1978). Radioactivity in the eluant from
a gel filtration column separating protein bound iodine from free
iodine was monitored by counting 10 ul aliqouts from each fraction to
detect labelled PRL. The peak (approximately 750,000-1,000,000 cpm/10
ul) and the descending 2-3 tubes were tested for PRL receptor binding
activity. Specific activity (approximately 83 uCi/ug) was determined


103
concentrations of membrane protein. Optimal binding conditions were
similar to those reported for heterologous RRAs and were adapted from a
homologous RRA for mouse hepatic PRL receptors (Haro and Talamantes,
1985a) Binding of labelled porcine PRL to endometrium is enhanced 4-
to 6-fold when membranes are pretreated with 4 M MgCl2. The labelled
porcine PRL was specifically displaced (80%) over a range (0-2560 ug)
of unlabelled (porcine) PRL commonly used in heterologous RRAs. The
MgCli treatment did not affect Ka, but did increase the number of
receptors. This weak chaotropic agent is thought to remove endogenous
PRL not displaced during mechanical processing of the tissue (Kelly et
al., 1979) by destabilizing the membrane's water structure and
disrupting the hydrophobic and electrostatic forces involved during
protein-receptor-membrane interactions (Hafeti and Hanstein, 1974). It
is not known whether altering the microenvironment surrounding the PRL
receptor changes availability of other PRL receptors (cryptic or
golgi). Changes in the lipid microenvironment; however, are thought to
play a role in binding of hormone to its receptor (Dave et al., 1983)
and may affect PRL binding (Dave and Witorsch, 1985).
The Ka of the porcine endometrial PRL receptor (0.326 x 108 M_1)
is similar to that reported for solubilized bovine mammary gland
(Ashkenazi et al., 1987) and is slightly lower than that reported by
Haro and Talamantes (1985a) using a homologous mouse PRL RRA and by
Posner et al. (1974b) using a heterologous RRA with mouse liver tissue.
This Ka is also slightly lower than those reported for ovine PRL with
porcine endometrium (DeHoff et al., 1984), porcine ovary (Rolland et
al., 1976; Bramley and Menzies, 1987) and porcine mammary tissue (Shiu
et al., 1973; Berthon et al., 1987b), and ovine PRL with uterine tissue


CHAPTER 7
DEVELOPMENT OF A HOMOLOGOUS RADIORECEPTOR ASSAY FOR
PORCINE ENDOMETRIAL PROLACTIN RECEPTORS
Introduction
Prolactin (PRL) binds to high affinity, low capacity receptors
(Shiu et al., 1973; Sakai et al., 1985) in target tissues and induces
responses that are biologically important to the reproductive system.
Prolactin binding sites have been reported in reproductive tissues of
sheep (Posner et al., 1974b), rats (Williams et al., 1974), humans
(Healy, 1984), mink (Rose et al., 1983), rabbits (Grissom and
Littleton, 1988) cow (Posner et al., 1974b) and pig (Posner et al.,
1974b; DeHoff et al., 1984; Bramley and Menzies, 1987). Prolactin
exerts effects on steroidogenesis in corpora ltea (CL) (Veldhuis et
al., 1980; Brambley and Menzies, 1987), transport of water by placental
membranes (Goldstein et al., 1980), uterine endometrial proliferation,
protein synthesis and secretion (Chilton and Daniels, 1985; Young and
Bazer, 1988), enhanced secretion of prostaglandin F2d into the uterine
lumen (Mirando et al., 1988), as well as mammary growth and lactation
(Vonderhaar et al., 1984).
Endometrial PRL binding sites have been detected in several
species. Local production of PRL from uterine decidual tissue may
affect uterine physiology in an autocrine or paracrine manner (Healy,
1984; Jayatilak and Gibori, 1986). However, in species with
noninvasive implantation, such as the pig, changes in the number of PRL
81


2b
golgi saccule endoplasmic reticulum lysosome (GERL) which functions to
transport proteins to the plasmalemma or to lysosomes (see review,
Griffiths and Simon, 1986).
Molecular Weight of Prolactin Receptor
Prolactin receptor structure has been investigated through various
biochemical methods including gel chromatography, solubilization,
affinity labelling and cross-linking. Prolactin receptors are
hydrophobic glycoproteins, since solubilized receptors bind to
Concanavalin-A, a lectin which binds mannose and glucose residues.
Triton X-100 solubilized PRL receptors aggregate to form larger
molecular weight (220,000 Mr) complexes while purification of receptors
with 3-[(3-cholamidopropyo)-dimethylammonio]-1-propane sulfonate
(CHAPS), a nondetergent zwitterionic solution, results in a single
electrophoretic band of 32,000-37,000 Mr (Djiane et al., 1987).
Molecular weight estimates of PRL receptors from rabbit mammary gland
suggest a Mr of 35,000 to 42,000. Rabbit mammary gland membrane
homogenates that are cross-linked with radiolabeled ovine PRL and
analyzed by SDS-PAGE and autoradiography indicated a 58,000-60,000 Mr
band. Subtraction of the Mr of PRL, yields a 35,000-37,000 Mr estimate
for PRL receptor from rabbit mammary gland (Djiane et al., 1987).
Identical results were obtained for PRL receptors in ovary, kidney and
adrenal gland from rabbits, but an additional protein band of 63,000 Mr
for ovarian and adrenal tissues was detected. (Djiane et al., 1987).
Affinity labelling results in a lower Mr estimate for prolactin
receptor subunit while gel electrophoresis suggests a higher (99,800-
340,000) Mr estimate. Berthon et al. (1987a) used hormone affinity


APPENDIX B
HOMOLOGOUS RADIORECEPTOR ASSAY FOR PORCINE
ENDOMETRIAL PROLACTIN RECEPTORS
Materials
Iodinated porcine prolactin
Unlabelled porcine prolactin
Plastic 12 x 75 mm tubes (Sarstedt, Princeton, NJ)
Assay Buffer pH 7.6, stored at 4 C.
150 mM NaCl
10 mM NaPOz
10 mM EDTA
0.1% BSA
0.1% NaAzide
4M MgClz
Wheaton hand homogenizers (5 or 15 ml)
Vortex
Repeat dispenser 3 ml
Centrifuge capable of 3500 x g at 4 C
Homologous RRA
1) Homogenize tissue and determine protein concentrations.
Store at -70 C in small aliquots (750 ul), only thaw once.
2) Figure dilution for final concentration of 150 ug
protein/75 ul assay buffer. Prepare enough membrane protein
to run 45 tubes for each sample. Homgenize and resuspend
membrane, by hand, using a wheaton homogenizer on ice.
2) Chaotropic treatment of membranes
Label tubes 1-15; fl,2 and #15 in triplicate, #3-14
in duplicate.
Add 500 ul 4M magnesium chloride to tubes #2-15
To each tube, (#2-15) add 75 ul membrane preparation containing
150 ug protein (diluted in assay buffer)
Vortex quickly
Incubate 5 min at 25 C
Wash with 3 ml ice-cold assay buffer
Centrifuge (2300 x g) for 15 min at 4 C.
Decant and blot
Add fresh ice-cold assay buffer (300 ul) and immediately
vortex extensively to resuspend pellet
181


Total LAP (sigma units) Total Protein (mg)
133
A
1 6 12 24 12
Hours post injection
(PC0.05)
c
240-
Hours post injection
(PC0.05)
B
co
i
o
V)
jl>
O
E
3
4.6-
4.4-
4.2-
4.0-
!
Hours post injection
(P<0.01)
D
Hours post injection
(PC0.01)


177
receptors, about which much is known, but so much remains to be
discovered.


202
McGuffin, W.L., Gavin, J.R., Lesmak, M.A., Gorden, P. and Roth, J.
(1976) Water-soluble specific growth hormone binding sites from
cultured human lymphocytes: prepartation and partial characterization.
Endocrinology. 98, 1401-1407.
McRae, M.A., Newman, G.R., Walker, S.M. and Jasani, B. (1986)
Immunohistochemical identification of prolactin and 24K protein in
secretory endometrium. Frtil. Steril. 45, 643-648.
Meites, J. and Clemens, J.A. (1972) Hypothalamic control of prolactin
secretion. Vit. Horm. 30, 165-216.
Heuns, S., Sroboda, M., Chnstophe, J. and Robyn, C. (1985) Evidence
for a glycosylated prolactin variant in human pituitary and amniotic
fluid. In: Prolactin, Basic and Clinical Correlates, pp. 487-493.
(eds. R.M. Macleod, M.O. Thorner, U. Scapagnini) Fidia Research Series,
Springer-Verlag, Berlin.
Mick, C.C.W. and Nicoll, C.S. (1985) Prolactin directly stimulates the
liver in vivo to secrete a factor (Synlactin) which acts
synergistically with the hormone. Endocrinology. 116, 2049-2053.
Millard, W.J., Sagar, S.M., Landis, D.M.D. and Martin, J.B. (1982)
Cysteamine: a potent and specific depleator of pituitary prolactin.
Science. 271, 452-459.
Miller, J., McLaclan, A.D. and Klug, A. (1985) Repetitive zinc
binding domains in the protein transcription factor IIIA from Xenopus
oocyte. EMBO J. 4, 1609-1614.
Mirando, M.A., Gross, T.S., Young, K.H. and Bazer, F.W. (1988).
Reorientation of prostaglandin F2C1 (PGF) secretion by calcium
ionophore, oestradiol and prolactin in perifused porcine endometrium.
J. Reprod. Fert. Abstract Series 1, 58 (Abstract).
Mittra, I. (1980) A novel "cleaved prolactin" in the rat pituitary.
Part 2. In vivo mitogenic activity of its N-terminal 16 K moiety.
Biochem. Biophys. Res. Commun. 95, 1760-1767.
Monkemeyer, H., Kelly, P.A. and Friesen, H.G. (1974) A simplified
procedure for studying tissue receptors for protein hormones. Clin.
Res. 22, 733 (Abstract).
Moore, K.E. (1987) Interactions between prolactin and dopaminergic
neurons. Biol. Reprod. 36, 47-58.
Morgan, D.O., Edman, J.C., Strandring, F.N., Fried, V.A., Smith, M.C.,
Roth, R.A. and Rutter, W.J. (1987) Insulin-like growth factor II
receptor as a multifunctional binding protein. Nature. 239, 301-307.
Muldoon, T. (1981) Interplay between estradiol and prolactin: the
regulation of steroid hormone receptor levels, nature and functionality
in normal mouse mammary tissue. Endocrinology. 109, 1339-1346.


148
al., 1975); however, estrogen acts synergistically with progesterone to
further increase secretion of uteroferrin. Differences in the
secretory response may be due to 1) estrogen stimulation of pituitary
PRL (Chen and Meites, 1970); 2) PRL stimulation of progesterone
receptors (Chamness et al., 1975; Chilton and Daniels, 1985; 1987;
Muldoon, 1987) or 3) availability of steroids, i.e. exogenous
progesterone available to bind to increased progesterone receptors
stimulated by effects of estrogen on pituitary PRL release. Prolactin
increases steroid receptors in mammary tissue of mice, however, the
steroid must be available for steroid receptor translocation to the
nucleus (Muldoon et al., 1987). Although the number of PRL receptors
measured in E2V and E2V+P4 treatment groups were low, this may not be
rate limiting for the secretory response. Greater uterine secretory
responses in gilts receiving E2V and P4 may reflect the proper hormonal
milieu necessary to utilize enhanced steroid receptors stimulated by
binding of PRL to its receptor in endometrial cells (see Figure 11-1).
Following chronic steroid administration, PRL affects include
modulation of steroid receptor concentrations in addition to direct
effects through PRL receptors. Prolactin receptor numbers change
during early pregnancy (Chapter 9) and throughout gestation (DeHoff et
al., 1984). Uterine secretory function is reduced by hypoprolactinemia
and increased by exogenous PRL in vivo (Young and Bazer, 1988) and in
vitro (Mirando et al., 1988). Therefore, it appears that PRL
influences the complexities of porcine uterine physiology during the
cycle, e.g., endometrial activity during early pregnancy when
conceptus-endometrial interactions are necessary for the establishment
and maintenance of gestation.


Figure 6-1: Concentrations of total recoverable (A) protein,
(B) uteroferrin, (C) glucose, and (D) leucine aminopeptidase
(LAP) activity in Day 12 uterine flushings from cylic gilts
(Experiment 2) treated with 1 ml saline (SAL) or 1 mg porcine
prolactin (PRL) at 0800 and 2000h on Days 6-11 and 0.5 ml
corn oil (OIL) or 5 mg estradiol valerate (E2V) on Day 11 of
the estrous cycle. Overall treatment effects were detected
for protein (P<0.001), uteroferrin (P<0.01) and leucine
aminopeptidase activity (P<0.05). Values with different
letters are different (P<0.05). The overall SEM was +2.72
for protein, +272 for uteroferrin, +0.16 for glucose and
+52.7 for LAP.


184
2) 100 ul sample, previously diluted if necessary
3) Add 400 ul tris buffer vortex
4) Incubate at 37 C for 20 min
5) Add 100 ul 1M NaAc vortex
Add 500 ul pNPP substrate vortex gently
6) Incubate at 37 C for 10 min.
7) Add 1.5 ml 1M NaOH Vortex.
8) Read OD at 410 nM
Calculations
1) Plot the standard curve and obtain correlation coefficient
2) Average OD and obtain umole/100 ul from standard curve
3) umole pNPP/100 ul x dilution factor x 10 =pNPP/ml/10 min
Note: Only accept values that are on the standard curve. The assay
usually needs to be run several times before the proper dilution is
attained.


Total Glucose (mg)
Total LAP (SU)
to
o
o
400
Total Uteroferrin (umoles)
to
o

o
4-
o
o
l
u>


Figure 8-la: Autoradiography of affinity labelled, cross-
linked porcine endometrial membrane PRL receptors. Lanes 1
through 8 were loaded with 750 ug endometrial protein and
250,000 cpm iodinated porcine PRL. Lanes 10 through 13 were
loaded with 150 ug endomterial protein and 45,000 cpm
labelled porcine PRL.
Lane designations are as follows:
1) MgCl treated, 10 ug unlabelled porcine PRL
2) MgCl treated, 20 ug unlabelled porcine PRL
3) MgCl treated, 0 ug unlabelled hormone (Bo)
4) MgCl treated, 10 ug unlabelled porcine GH
5) Untreated, 10 ug unlabelled porcine PRL
6) Untreated, 20 ug unlabelled porcine PRL
7) Untreated, 0 ug unlabelled hormone (Bo)
8) Untreated, 10 ug unlabelled porcine PRL
9) labeled porcine PRL
10) untreated, 2.5 ug unlabelled porcine PRL
11) untreated, 0 ug unlabelled hormone (Bo)
12) MgCl treated, 2.5 ug unlabelled porcine PRL
13) MgCl treated, 0 ug hormone (Bo)
Figure 8-lb: Autoradiography of affinity labelled, cross-
linked rat liver membrane PRL receptors. Lanes 1 through 8
were loaded with 750 ug membrane protein and 250,000 cpm
iodinated porcine PRL. Lane designations are as follows:
1) MgCl treated, 10 ug unlabelled porcine PRL
2) MgCl treated, 0 ug unlabelled hormone (Bo)
3) MgCl treated, 10 ug unlabelled porcine GH
4) MgCl treated, 0 ug unlabelled hormone (Bo)
5) Untreated, 10 ug unlabelled porcine PRL
6) Untreated, 0 ug unlabelled hormone (Bo)
7) Untreated, 10 ug unlabelled porcine GH
8) Untreated, 0 ug unlabelled hormone (Bo)


THE ROLE OF
STUDIES
PROLACTIN IN ESTABLISHMENT OF PREGNANCY IN PIGS
ON ENDOMETRIAL PROLACTIN RECEPTOR REGULATION
AND UTERINE SECRETORY PHYSIOLOGY
By
KATHLEEN HART YOUNG
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1989

"Nothing is impossible to the willing mind"
The Book of Hans Dynasty

To those who believe in me,
"no matter what..
VAH, RGY, JHN, LK, BHV, IJM and JMH

ACKNOWLEDGEMENTS
I would like to thank the members of my committee, Drs. Bazer,
Buhi, Shiverick, Simpkins and Thatcher, for time and knowledge they put
forth toward the completion of my research. I thank these individuals
who, through their conversations, actions, examples, and generosity,
were an integral part in my development as a scientist.
I am thankful to several people, behind the scenes, that
stimulated my pursuit of this degree, including Mr. Jerry Metzler, Fr.
Timothy Healy, Dr. Phil Senger, Dr. Brad Vaughn and my father, the late
William Hart.
Along the way, much appreciated examples and support were provided
by Drs. Mary Murray, Cheryl Ashworth-Stott, Saundra Tenbroeck and Susan
Ogilve.
I am indebted to Hironori Ohtsuka, founder of Wado-ryu Karate, and
his students, which ultimately led to the supportive friendships of
Mike McCoy, Mike Sawyer, Larry and Ellen Bellack, Cindy Silverstre and
Terry Minard.
The day-to-day events in the lab, barn and surgery room were
shared by several graduate students and post docs, including Dr. Randy
Renegar, Marlin DeHoff, Sue Chaichimansour, Dr. Wendy Campbell, Dr.
Jeff Vallet, Dan Dubois, Troy Ott, Jake Harney, Dr. Mark Mirando,
Saskia Beers and Matt Davis; I am grateful for their help and humour.
Special thanks go to Dan Dubois for lending me his computer for the
final stretch of dissertation polishing.
iv

I thank Dr. Bazer for the many opportunities to participate in
scientific meetings. I am much richer for the experiences. Also, I
thank him for the insight that social comfort doesn't always expand
your knowledge. My solo journey to the Prolactin Gordon Conference
allowed development of friendships, scientific collaboration and a
greater appreciation for prolactin.
Thanks are extended to Dr. Douglas Bolt for generously supplying
the porcine prolactin and other hormones used in these studies; to
Bennet Johnson, who patiently assayed serum samples for prolactin; to
Fil Fliss, for prostaglandin assays of uterine flushings; to Jenny
Davis for Nb2 lymphoma cell assays; and to Dr. Nelson Horseman for
pigeon crop sac assays.
Sincere thanks are extended to Dr. Wang, Dr. Shiverick and
personnel in her lab for their help and generous use of equipment,
computer time and borrowed keys during the period I was conducting
Scatchard analyses of the binding data.
Loving thanks go to Greg, my husband, friend and keeper of my
sanity; needless to say, but nonetheless essential to mention, is that
I am certainly more human because of you. Also, thanks go to my
sister, Judi Norton, who kept me going with letters, support, laughter
and love throughout my graduate program.
Lastly, although Ill be the person with the extra letters behind
by name, my success is truly because all of you were beside me. Thank
you!
v

TABLE OF CONTENTS
PAGE
ACKNOWLEDGEMENTS iv
LIST OF TABLES ix
LIST OF FIGURES X
ABSTRACT xiv
CHAPTERS
1 INTRODUCTION 1
2 REVIEW OF LITERATURE 3
History, Evolution and Structure of Prolactin 3
Microheterogeneity of Prolactin 6
Sources of Prolactin 9
Prolactin in the Circulation 12
Regulation of Pituitary Secretion of Prolactin 13
Receptor Theory 17
Analysis of Receptors 18
Prolactin Receptors 21
Signal Transduction Systems for Prolactin 30
Regulation of Prolactin Receptors 35
Functions of Prolactin in the Uterus 40
Porcine Conceptus Development and Uterine Secretion 41
3 EFFECTS OF HYPOPROLACTINEMIA ON ESTABLISHMENT
OF PREGNANCY AND UTERINE SECRETORY FUNCTION IN PIGS 44
Introduction 44
Materials and Methods 45
Results 51
Discussion 54
4EFFECTS OF CYSTEAMINE ON CIRCULATING
PROLACTIN LEVELS IN PIGS 57
Introduction 57
Materials and Methods 57
Results 58
Discussion 61
vi

5 ESTABLISHMENT OF HYPERPROLACTINEMIA BY ADMINISTRATION
OF EXOGENOUS PORCINE PROLACTIN TO PIGS 63
Introduction 63
Materials and Methods 63
Results 65
Discussion 65
6 EFFECT OF HYPERPROLACTINEMIA ON PROGESTERONE
AND ESTROGEN INDUCED UTERINE SECRETORY RESPONSE
IN PIGS 67
Introduction 67
Materials and Methods 68
Results 70
Discussion 77
7 DEVELOPMENT OF A HOMOLOGOUS RADIORECEPTOR ASSAY
FOR PORCINE ENDOMETRIAL PROLACTIN RECEPTORS 81
Introduction 81
Materials and Methods 83
Results 89
Discussion 102
8 AFFINITY LABELLING OF PROLACTIN RECEPTORS
IN DAY 75 PRGENANT PORCINE ENDOMETRIUM WITH
PORCINE [i231]-PROLACTIN 107
Introduction 107
Materials and Methods 108
Results 109
Discussion 112
9 ENDOMETRIAL PROLACTIN RECEPTORS DETECTED BY
HOMOLOGOUS RADIORECPETOR ASSAY DURING THE ESTROUS
CYCLE AND EARLY PREGNANCY IN PIGS 114
Introduction 114
Materials and Methods 116
Results 117
Discussion 119
10EFFECTS OF ACUTE ESTRADIOL VALERATE ADMINISTRATION
ON ENDOMTRIAL PROLACTIN RECEPTORS DETECTED BY
HOMOLOGOUS RADIORECEPTOR ASSAY AND UTERINE
SECRETORY RESPONSE IN DAY 11 CYCLIC PIGS 124
Introduction 124
Materials and Methods 125
Results 127
Discussion 134
vii

11EFFECTS OF CHRONIC OVARIAN STEROID ADMINISTRATION
ON ENDOMETRIAL PROLACTIN RECEPTORS AS DETECTED BY
HOMOLOGOUS RADIORECEPTOR ASSAY AND UTERINE PROTEIN
SECRETORY RESPONSE IN OVARIECTOMIZED PIGS 139
Introduction 139
Material and Methods 140
Results 142
Discussion 144
12 STUDIES ON MITOGENICITY, LACTOGENICITY,
IMMUNOREACTIVITY AND RECEPTOR BINDING CHARACTERISTICS
OF NONGLYCOSYLATED AND GLYCOSYLATED PORCINE PROLACTIN 150
Introduction 150
Materials and Methods 152
Results 156
Discussion 163
13 GENERAL DISCUSSION 168
APPENDICES
A IODINATION OF PORCINE PROLACTIN AND
DETERMINATION OF SPECIFIC ACTIVITY 178
B HOMOLOGOUS RADIORECEPTOR ASSAY FOR
PORCINE ENDOMETRIAL PROLACTIN RECEPTORS 181
C ACID PHOSPHATASE ASSAY FOR MEASURMENT OF UTEROFERRIN 183
REFERENCES 185
BIOGRAPHICAL SKETCH 215
viii

LIST OF TABLES
Tables Page
4-1: Effects on interestrous interval and cytoxicity
following cysteamine (CSH) and ethanolamine (control)
administration to cyclic gilts 60
6-1: Composition of Day 16 uterine flushings from
ovariectomized gilts treated with daily injections
of progesterone and saline or porcine prolactin from
Days 4 through 14 (x+SEM) 71
11-1: Endometrial membrane prolactin receptors and total
protein and uteroferrin in uterine flushings from
ovariectomized gilts following steroid
administration for 11 days 143
ix

LIST OF FIGURES
Figure Page
3-1: Concentrations of total recoverable (A)
calcium, (B) chloride, (C) sodium and
(D) potassium in Day 12 uterine flushings
from cyclic gilts (Experiment 2) treated with
CB154 (100 mg/day) or vehicle (VHC, 4 ml/day)
on Days 10 and 11 and estradiol valerate
on Day 11 53
4-1: Mean concentrations of prolactin (ng/ml) in
serum of cyclic gilts treated with cysteamine
(solid line) or ethanolamine (dashed line) from
Days 10-16 (denoted by arrows) 59
4-2: Reproductive tracts from gilts treated with
either (A) ethanolamine or (B) cysteamine 62
5-1: Concentrations of immunoreactive prolactin in
serum during administration of 1 mg porcine
prolactin (circles), or 1 ml saline (squares),
at 0800 and 2000 h (denoted by arrows) on
Days 10 through 13 of the estrous cycle 66
6-1: Concentrations of total recoverable (A) protein,
(B) uteroferrin, (C) glucose and (D) leucine
peptidase activity (LAP) in Day 12 uterine
flushings from cyclic gilts (Experiment 2)
treated with 1 ml saline (SAL) or 1 mg porcine
prolactin (PRL) at 0800 and 2000 h on Days 6-11
and 0.5 ml corn oil (OIL) or 5 mg estradiol
valerate (E2V) on Day 11 of the estrous cycle 73
6-2: Concentrations of total recoverable (A) calcium,
(B) chloride, (C) sodium and (D) potassium in
Day 12 uterine flushings from cyclic gilts
(Experiment 2) treated with 1 ml saline (SAL) or
1 mg porcine prolactin (PRL) on Days 6-11 and
0.5 ml corn oil (OIL) or 5 mg estradiol valerate
(E2V) on Day 11 of the estrous cycle 75
x

Figure
Page
6-3: Concentrations of (A) PGF and (B) PGE in Day 12
uterine flushings from cyclic gilts (Experiment 2)
treated with 1 ml saline (SAL) or 1 mg porcine
prolactin (PRL) on Days 6-11 and 0.5 ml corn oil
(OIL) or 5 mg estradiol valerate (E2V) on Day 11
of the estrous cycle 76
7-1: Effects of increasing magnesium chloride molarity
on binding of porcine [*23I]-prolactin by
membranes from Day 75 porcine endometrium, amnion,
chorion, as well as post-parturient pig and rabbit
mammary gland (300 ug) 90
7-2: Effects of increasing protein concentrations of
Day 75 porcine endometrial membranes on
binding of porcine [123I]-prolactin 91
7-3: Binding of porcine t1251]-prolactin by magnesium
chloride treated Day 75 porcine endometrial
(circles) or Day 20 rat liver (squares) membranes
at 4 C (dashed line) or 25 C (solid line) 93
7-4: Dissociation kinetics assay for magnesium
chloride treated (A) Day 75 porcine endometrial
or (B) Day 20 rat liver membranes 95
7-5: Binding and displacement of ovine and porcine
prolactin from magnesium chloride treated Day 75
porcine endometrial membranes 98
7-6: Crossreactivity of unlabelled porcine prolactin
(squares; pPRL), porcine growth hormone (triangles;
pGH),porcine luteinizing hormone (circles; pLH) and
porcine follicle stimulating hormone (diamonds;pFSH)
to porcine t123I]-prolactin with magnesium chloride
treated Day 75 porcine endometrial membranes 99
7-7: Crossreactivity between unlabelled porcine growth
hormone (dashed line), or porcine prolactin (solid
line) and porcine f123I]-prolactin with magnesium
chloride treated Day 75 porcine endometrial (circles)
and Day 20 rat liver (squares) membranes 100
7-8: Scatchard analysis of porcine [123I]-prolactin
displaced by unlabelled porcine prolactin using
magnesium chloride treated Day 75 porcine
endometrial membranes 101
8-la: Autoradiography of affinity labelled, cross-linked
porcine endometrial membrane preparation prolactin
receptors Ill
xi

Figure Page
8-lb: Autoradiography of affinity labelled, cross-linked
rat liver membrane prolactin receptors Ill
9-1: Prolactin receptors in endometrial membranes
of cyclic (squares) and pregnant (circles) gilts
over days of the estrous cycle and gestation 118
10-1: Prolactin receptor numbers in endometrial membranes
at 1, 6, 12, and 24 h after administration (i.m.) of
estradiol valerate (0.5 mg, hatched bars) or 12 h
after corn oil (0.5 ml, solid bar) administration 129
10-2: Total recoverable (A) calcium, (B) sodium and (C)
potassium in uterine flushings at 1, 6, 12 and
24 h following administration (i.m.) of estradiol
valerate (0.5 mg, hatched bars) or 12 h after
corn oil (0.5 ml, solid bar) administration 131
10-3: Total recoverable (A) protein, (B) uteroferrin,
(C) leucine aminopeptidase (LAP) and (D) glucose
in uterine flushings at 1, 6, 12 and 24 h
following administration (i.m.) of estradiol
valerate (0.5 mg, hatched bars) or 12 h after
corn oil (0.5 ml, solid bar) administration 133
11-1: Effect of chronic ovarian steroid administration on
uterine secretory response of ovariectomized gilts;
possible interaction with endogenous prolactin
to result in low and high uterine sectory response. 149
12-1: Separation of nonglycosylated and glycosylated
forms of porcine prolactin by Concanavalin A-
Sepharose 6B column chromatography 153
12-2: Evaluation of the purity of nonglycosylated and
glycosylated forms of porcine prolactin isolated
by Concanavalin-A Sepaharose 6B column chromatography
using 12.5% sodium dodecylsulphate one-dimensional
polyacrylamide gel electrophoresis 155
12-3: Uptake of 3H-thymidine into Nb2 lymphoma cells
expressed as percent of Nb2 control (dashed line)
when cells were stimulated by total (circles),
nonglycosylated (squares) and glycosylated
(triangles) forms of porcine prolactin 158
xii

Figure
Page
12-4: Immunoaffinity of total (open and closed circles),
nonglycosylated (open and closed squares) and
glycosylated (open and closed triangles) forms of
porcine prolactin expressed as (A) percent bound
versus log concentration, (A inset) transformed to
log versus logit plot and (B) percent of Bo
(normalized to 100%) versus log concentration 161
12-5: Scatchard analysis of competitive inhibtion curves
using magnesium chloride treated Day 75 pregnant
porcine endometrial membranes and t125I]-prolactin
(total) versus unlabelled total prolactin
(triangles) and t125I]-nonglycosylatedrglycosylated
(2:1) prolactin versus unlabelled
nonglycosylated:glycosylated (2:1) (circles)
prolactin 162
12-6: Scatchard analysis of porcine t1251]-(total)
prolactin binding to magnesium chloride treated
Day 75 pregnant porcine endometrial membranes
inhibited by unlabelled total (circles),
nonglycosylated (squares) or glycoslyated
(triangles) forms of porcine prolactin 164
13-1: Effects of hypoprolactinemia and hyperprolactinemia
on the composition of uterine flushings of Day 12
gilts at 24 h after a single administration of
estradiol valerate 171
13-2: Possible mechanism(s) involving prolactin
during maternal recognition of pregnancy in pigs 174
xiii

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
THE ROLE OF PROLACTIN IN ESTABLISHMENT OF PREGNANCY IN PIGS:
STUDIES ON ENDOMETRIAL PROLACTIN RECEPTOR REGULATION
AND UTERINE SECRETORY PHYSIOLOGY
By
Kathleen Hart Young
May 1989
Chairman: Fuller W. Bazer
Major Department: Animal Science
Conceptus estrogens cause biphasic endometrial responses not
wholly explained by steroid receptor mechanism. Studies reported herein
support prolactin involvement in porcine uterine secretory function.
Estrogen-induced uterine secretory function was decreased in response
to hypoprolactinemia in pigs. Exogenous prolactin interacted with
estrogen, but not progesterone, to increase secretion of uteroferrin,
prostaglandin F2CL and leucine-aminopeptidase. Prolactin alone
increased glucose in uterine flushings. Prolactin modulates quantity
of electrolytes released in reponse to estrogen and enhances secretion
of protein to nourish preimplantation porcine conceptuses.
A homologous porcine prolactin radioreceptor assay was developed,
using pig endometrial membrane preparations, which specifically
detected high affinity (0.3 x 108 M-1), prolactin receptors and was
used to detect changes in endometrial prolactin receptors during the
estrous cycle and early pregnancy. Endometrial prolactin receptors
were similar for pregnant and cyclic gilts on Day 8, then decreased
xiv

during the cycle. Receptors numbers increased following conceptus
estrogen secretion (Day 12). Administration of estrogen to cyclic
gilts on Day 11 resulted in uterine secretory response similar to those
detected during pregnancy. Endometrial prolactin receptors increased
within 6h, decreased at 12h and recovered to basal values at 24h
following a single estrogen injection. Changes in prolactin receptors
were associated temporally with changes in uterine ion and protein
secretion. Endometrial prolactin receptor numbers decreased in
ovariectomized pigs treated with estrogen, or estrogen and
progesterone, while corn oil or progesterone alone had no affect. Low
prolactin receptor numbers were associated with low and high uterine
secretory responses. Estrogen stimulation of pituitary prolactin
release could down-regulate endometrial prolactin receptors and
increase steroid receptors. Availablity of exogenous steroids could
therefore influence uterine secretory response.
Porcine prolactin is microheterogeneic. Glycosylated porcine
prolactin has lower mitogenicity, immunoreactivity and binds to fewer
receptors, but higher lactogenicity and receptor binding affinity than
nonglycosylated or total prolactin.
Results of this study indicate that prolactin affects porcine
uterine physiology. Regulation and specificity of endometrial response
to prolactin appears to be controlled locally by regulation of receptor
number, but not affinity. Microheterogeneity of prolactin results in
different affinities for endometrial receptors and may account for
prolactin's diverse effects.
XV

CHAPTER 1
INTRODUCTION
The exsistence of a lactogenic hormone was proposed in 1928, but
research on prolactin (PRL) did not flourish until the 1970's,
following its purification from several species, including the human.
Prolactin's diverse functions combined research from several scientific
disciplines, such as endocrinology, physiology, comparative
endocrinology, neuropharmacology and anatomy. From the early studies
on crop sac and mammary gland development, as well as lactogenesis, PRL
is now credited with over 100 functions (Nicoll and Bern, 1972).
Prolactin has osmoregulatory functions in amphibians, fish and mammals
and lactogenic functions in birds and mammals. Prolactin affects
growth of various organ systems in fish, amphibians, birds and mammals.
In mammals, PRL is associated primarily with reproductive processes,
e.g., mammogenesis, lactogenesis and luteotropic funtions.
Previously, PRL was associated with uterine function, transport of
water, protein and prostaglandin secretion, and steroid uptake and
metabolism. In 1985, Chilton and Daniels, proposed a fourth function
for PRL in uterine physiology; endometriotrophism. Thus, PRL affects
the female reproductive system at all levels; ovaries, mammary gland
and uterus.
The uterus is regulated primarily by steroid hormones. Estrogen
induced biphasic responses, however, could not be explained by
classical steroid mechanisms, rapid signal transduction systems or
novel estrogen mechanisms. Therefore, estrogen's interaction with, or
1

2
modification by, another (peptide) hormone, to induce rapid uterine
responses was investigated.
Porcine conceptuses establish pregnancy through estrogen secretion
and cause direct physiological, biochemical and secretory changes in
uterine endometrium. Therefore, PRL interaction with estrogen can be
studied within the context of uterine secretory function. The pig is
an interesting model for reproductive physiology studies, particularly
those investigating effects of PRL. Porcine conceptuses undergo
noninvasive implantation, which does not decidualize or produce PRL
from the endometrium, and the placenta does not produce placental
lactogen. Therefore, the pig can be used for investigations of PRL
function in uterine physiology, without interference by decidial PRL or
placental lactogen. Lacking these placental lactogenic hormones, it
was intriguing to delineate the mechanism(s) by which effects of PRL on
uterine physiology are induced in pigs.
This dissertation reports investigation of the role of PRL in
uterine physiology in pigs following induction of hypoprolactinemia and
hyperprolactinemia. Additionally, endometrial PRL receptors and their
regulation by ovarian steroids were investigated using a homologous
radioreceptor assay developed for porine PRL and porcine endometrial
PRL receptors. Homologous assays are advantagous since inconsistencies
associated with the use of heterologous hormones are avoided.
The literature review that follows is to familiarize the reader
with various aspects of PRL and its receptor. These areas are reviewed
with respect to basic function, characteristics and physiology that
must be considered in understanding the role of PRL in (porcine)
uterine physiology.

CHAPTER 2
REVIEW OF LITERATURE
History, Evolution and Structure of Prolactin
A lactogenic hormone was first suggested by experiments of John
Hunter (1788-1840) in which he described proliferation and secretion of
the pigeon crop sac. Hunter alleged that the pigeon crop sac was
analogous in its development to the mammary gland during gestation.
Interest in anterior pituitary secretions continues today from the
initial work of Hunter in the 1780's and characterization of the six
anterior pituitary hormones in the late 1930s. Research with
prolactin (PRL) was not very active until the 1920's. Injection of
anterior pituitary extracts into rats (Evans and Simpson, 1929) and
intact (Strickler and Grueter, 1928) and ovariectomized (Corner, 1930)
rabbits resulted in mammary distention due to milk synthesis. Yet, it
was the industrious work of Oscar Riddle and coworkers (1933) that
involved isolating and purifying the lactogenic substance which was
named "prolactin." However, it was development of the pigeon crop sac
bioassay, based on John Hunter's early work, that allowed confirmation
of PRL activity during its purification. Great controversy prevailed
as to whether a separate growth promoting hormone exsisted. Riddle et
al. (1933) demonstrated that PRL was different from growth hormone and
established PRL as an independent hormone. Despite a desire for
complete separatation of functions for the new known hormones, PRL and
3

4
growth hormone, overlapping functions are observed in several species
(Nicoll, 1975; 1982).
Since its discovery, PRL from several species has been sequenced.
Prolactin is a simple polypeptide containing 199 amino acids. Three
disulphide bonds, located between cysteines 4-11, 58-174 and 191-199,
are observed for PRL in most mammalian species, except horse (Li and
Chung, 1983) and fish (Farmer et al., 1977) which lack the NH2-terminal
loop. Biological activity is affected by breaking the disulfide bonds
indicating that function is dependent on the three dimensional
structure of PRL. Microheterogeneity has been established for PRL;
with 25,000 Mr glycosoylated and 60,000 Mr dimer forms in addition to
the well characterized 23,000 Mr form of PRL. These forms are present
in the pituitary as well as the circulation. Prolactin has been
sequenced for humans (Shome and Parlow, 1977; Cooke et al., 1981),
sheep (Li et al., 1970), cows (Wallis et al., 1974; Sasavage et al.,
1982), rats (Parlow and Schome, 1976; Gubbins et al., 1980), mice
(Kohmoto et al., 1984), whales (partial (Kawachi and Tubokawa, 1979),
elephants (Li et al., 1987) and pigs (Li, 1976). Porcine PRL has
typtophan residues at amino acids 91 and 150 and an isoelectric point
of 5.85 (Brewly and Li, 1975). Species specificity is considerable,
with 20% of the residues dissimilar between pig/whale and ruminants,
and a 40% difference between rat and other mammalian PRLs suggesting
that PRL has evolved differently, even within the mammalian species,
from an ancestral molecule it shared with growth hormone (Wallis,
1981) .
Prolactin appears to have undergone three evolutionary
accelerations. Rat PRL evolved the fastest, showing 44 point

5
mutations/100 residues/108 years, followed by human (19 point
mutations), and ovine and bovine (16 point mutations) PRLs. Porcine
and cetacea PRL show only 5 point mutations/108 years, which is much
slower than for other species. Differential regulation of PRL
development between species may suggest specified functions. Porcine
PRL has evolved only as fast as cytochrome C, while rat PRL has evolved
so quickly that is has only a 40% difference in its structure compared
to human PRL. This is a smaller difference than that between human and
nonprimate PRLs. The reasons for the differential evolutionary rates
of PRL are unknown; as are the resulting changes in function or
specificity. Most evolutionary change is neutral (King and Jukes,
1969); however, increased rates of change may be due to new selection
pressures or loss of specific function (Wallis, 1981).
These points, suggest that species specificity is an important
attribute of PRL to further our understanding of its biological
functions. Previous views were that a hormone, regardless of species
source, would function similarly in each species into which it was
injected. This fallacy is discussed by Nicoll (1982), who noted that
early research on PRL was with ovine PRL. By chance, the majority of
ovine PRL's functions are "PRL-like" in nature. Had the early work
been conducted with human PRL, the literature may have described a very
different set of functions. Hormones are named usually for their
suggested functions, thus restricted by man's attempt to organize and
understand himself. Although first thought to be primarily involved in
lactation, PRL is now cited with over 100 functions (Riddle, 1963;
Nicoll and Bern, 1972), most of which are categorized by 1)
osmoregulation and electrolyte balance, 2) growth and development, 3)

6
mammalian reproduction, 4) parental behaviour, 5) intergumentary
structures and 6) interaction with steroid hormones (Nicoll et al.,
1985). Based on PRL's diversity of actions, the chronobiologist,
Hablan (1980), has suggested its renaming to 'versatilin' (as cited by
Nicoll, 1979).
Microheterogeneity of Prolactin
Glycosylated Prolactin
The anterior pituitary produces several glycosylated hormones.
Therefore the discovery of a glycosylated form of PRL was novel, yet
not entirely unexpected. First reported in 1984, by Lewis and
coworkers, glycosylated PRL has received increasing attention. The
diversity of PRL's action may be a function of its microheterogeneity.
Presently, several molecular weight forms of PRL (23,000, 25,000 and
50-60,000 Mr) have been isolated from pituitaries of sheep (Lewis et
al., 1984), pigs (Pankov and Butnev, 1986), rats (Wallis, 1980), mice
(Sinha and Jacobsen, 1987) and humans (Lewis et al., 1985; Markoff and
Lee, 1987), suggesting that PRL is actually a 'family' of hormones
(Lewis et al., 1984). Glycosylated PRL is produced also in uterine
decidua (Lee and Markoff, 1986) and found in amniotic fluid (Meuris et
al., 1985) of humans. The carbohydrate moiety of glycosylated porcine
PRL resembles that of ovine LH (Bedi et al., 1982). and is N-linked at
asparigine 31 to the polypeptide (Pankov and Butnev, 1986).
The glycosylated form of PRL is less mitogenic and immunoreactive
in Nb2 lymphoma cell assays and polyclonal RIAs, respectively
(Pellegini et al., 1988; Scott et al., 1988). Lactogenic activity, as
measured by the pigeon crop sac or mouse mammary explant casein

7
synthesis assays, is lower for human and sheep glycosylacted PRL (Lewis
et al., 1984, 1985), but higher for porcine glycosylated PRL (Pankov
and Butnev, 1986). Porcine glycosylated PRL had decreased binding to
rabbit or porcine mammary gland membrane preparations than ovine PRL
(Pellegini et al., 1988; Seely et al., 1988). However, these assays
were conducted in heterologous systems and results may be inconsistent
with the true activity of glycosylated PRL.
The function of the carbohydrate moiety of PRL is unknown.
Speculation suggests that this form of PRL could be reserve source and
deglycosylated as needed or have a longer half-life than the 23,000 Mr
nonglycosylated PRL. The ratio of nonglycosylated to glycosylated PRL
may be important in physiological effects of PRL. The ratio changes in
late pregnancy of humans (Markoff and Lee, 1987) and during the first
year of life for pigs (Sinha et al., 1988), but a functional change
associated with shifting ratios has not been elucidated. Further work
on the role of the forms of PRL, individually or combinded, is
necessary to understand the diversity of PRL's functions.
Additionally, investigation of these forms at the receptor level may
provide insight into interactions with PRL receptor(s) and subsequent
biological actions.
Cleaved and Clipped Prolactin
Another variation of PRL is the "cleaved" form in which the large
loop formed by the disulfide bond between amino acids 54 and 174 is
severed at amino acids 148-149. A polypeptide containing two strands
results, joined by the disulfide bridge which previously caused a large
loop in PRL. Cleaved PRL contains 16,000 Mr (amino acids 1-148) and

8
8,000 Mr (149-199) peptides. Cleavage of the large loop of PRL does
not inhibit its binding to PRL receptors in mammary gland or ventral
prostate of rats (Clapp, 1987; Vick et al., 1987). Additionally,
cleaved PRL has full potency in Nb2 lymphoma cell mitogenic assays.
However, only 50-60% of cleaved PRL is detected by RIA (Vick et al.,
1987), suggesting that the biological and antigenic determinents are at
different sites, as suggested by Amit et al. (1985).
Cleaved PRL is found in pituitaries of mice (Sinha and Gillian,
1981), rats (Mittra, 1980) and humans (Sinha et al., 1985) and is
produced by ventral prostate of male rats. Mammary tissue of lactating
rats has higher enzyme activity, thus more cleaved PRL is produced by
this tissue (Vick et al., 1987; Clapp, 1987). Target tissues of PRL
may be expected to contain cleavage enzymes (Nolin, 1982), but it is
uncertain if these enzymes are regulated physiologically. Cleavage of
PRL is by nonspecific multipurpose proteases influenced by
configuration of the PRL molecule (Wong et al., 1986). Cleavage also
depends on pH, with enzymatic activity increasing at pH 5 to 3.6
(Clapp, 1987).
Reduction of the disulfide bond joining the chains generates a
16,000 Mr and 8,000 Mr fragments. The 16,000 Mr fragment maintains PRL
activity on mammary epithelial cells, despite decreased receptor
binding, mitogenic activity and immunoreactivity. However, functions
of the cleaved, 16,000 Mr and 8,000 Mr fragments of PRL await further
investigation.

9
Sources of Prolactin
Anterior Pituitary Prolactin
The anterior pituitary is derived from an evagination of embryonic
ectodermal tissue, Rathke's pouch, and attaches to neural tissue from
the brain to form the pituitary. The cells of the anterior pituitary
can be classified as acidophils, basophils and chromophobes by
microscopy and perferential uptake of certain dyes. Acidophils are
further defined as alpha or epsilon cells, being oranophils or
carminophils, respectively. Carminophils, but not orangophils,
fluctuate during pregnancy. Both cell types are located in the lateral
aspects of the anterior pituitary.
Lactotrophs and somatotrophs, which secrete PRL and growth
hormone, respectively, comprise the acidophilic cells of the anterior
pituitary. Somatotrophs, which comprise 50% of anterior pituitary
cells, were described by Kurasumi (1968) as round or oval polygonal
cells with short clubbed mitochondria and round dense secretory
granules of approximately 200-350 mm. Lactotrophs, comprise 15-20% of
the anterior pituitary cells, and were intitially described by Farquhar
and Rinehart (1954). These PRL secreting cells have large nuclei and
relatively small amount of cytoplasm which contains well developed
golgi complex and abundant rough endoplamic reticulum. Secretory
granules of the lactotroph are elongated (600-900 mm) although small
(200 mm) secretory vesicles are also observed.
Unlike other pituitary hormones, PRL is synthesized with a signal
peptide. Newly synthesized PRL is preferenially released, independent
of TSH, while secretion of stored PRL is dependent on TSH stimulation
(Walker and Farquhar, 1980). Crinophagy, another unique feature of

10
lactotrophs, occurs when secretory vesicles fuse with lysosomes instead
of plasma membranes when PRL secretion is inhibited (Farquhar, 1985).
Dopamine receptors are present on lactotrophs. These receptors, along
with dopamine, are internalized to secretory granules however the role
of interalized neurotranmitter has not been fully elucidated.
Lactotrophs and somatotrophs were thought to be from
morphologically distint cell lines (Farquhar, 1985). However, recent
findings suggest that PRL secreting lactotrophs can be specific cells,
or another class of PRL secreting cells, derived from somatotrophs, and
termed mammosomatotrophs (Stattman, 1974).
Mammosomatotrophs
The anterior pituitary is comprised of different cell types, each
responsible for synthesis and secretion a hormone. The 'one cell/one
hormone theory is supported by anatomical and microscopy studies of
the anterior pituitary. Growth hormone and PRL are secreted from
somatotrophs and lactotrophs, respectively; however, distinction
between these cell types is fading. Early work by Stattman (1974)
suggested that some cells in the anterior pituitary, termed
mammosomatotrophs, secrete both PRL and growth hormone, or exhibit a
functional shift from growth hormone to PRL secretion.
Mammosomatotrophs have thus far been recorded only in rats.
Mammosomatotrophs constitute one-half of PRL secreting cells in the
male pituitary; however, percentages are not available for female
pituitaries (Brookfor et al., 1986). Estrogen stimulation of male
anterior pituitary cells in culture did not cause cell mitosis,
contrary to results by Corenblum et al. (1980), but shifted cell

11
secretion from growth hormone to dual secretion with PRL (Brookfor et
al., 1986), suggesting a functional 'plasticity' for conversion. It is
unknown if mammosomatotrophs serve as a stem cell or are a normal stage
within the cell cycle (Frawley et al., 1985).
Decidual Prolactin
Prolactin is found in the amniotic fluid during pregnancy. Yet,
PRL in the maternal circulation does not fluctuate greatly during
gestation and, therefore, could not account for the high concentrations
of PRL in the amniotic fluid of rhesus monkeys (Josimovich et al.,
1974). Additionally, PRL levels in amniotic fluid of anecephalic
fetuses or fetus that have died in tero are similar to normal fetuses
(Walsh et al., 1977). Placental membranes do not produce PRL, but
decidua attached to placental membranes stains positively for PRL
(Golander et al., 1978; Healy et al., 1979). Studies of decidual
tissue of pregnancy determined that PRL was produced from these
tissues. In 1977, the suggestion that a pituitary hormone could be
produced in a nonpituitary tissue was considered novel. However, many
hormones previously associated only with brain tissue are now found in
other tissues and vice versa (vasoactive intestinal peptide). Riddick
and Kusmik (1977) confirmed that PRL was produced during normal
pregnancy and by secretory endometrium from Day 22 of the menstrual
cycle (McRae et al., 1986).
Decidual PRL is identical in structure and biological function
(Riddick et al., 1978) to pituitary PRL. Prolactin secretion is 1000-
fold less from decidual (400 ng/g tissue) when compared to pituitary
(400 ug/g tissue) (Tomita et al., 1982). However, the weight of the

12
decidua is far greater than that for the anterior pituitary, which
weights approximately 0.6 g. The two sources of PRL are regulated
differently; TRH and dopamine, which stimulate and inhibit pituitary
PRL release, respectively, do not affect PRL production by decidual
tissue. Release of PRL by decidual tissue is inhibited by arachadonic
acid and stimulated by calcium and progesterone (Healy and Hodgen,
1983). Differential regulation of PRL from its two sources may be due
to storage properties of PRL in the tissues (Markoff et al., 1983). In
addition, placental peptides are secreted that stimulate (23,500 Mr;
Handwerger et al., 1983) and inhibit (Markoff et al., 1983) decidual
PRL. Local production of PRL in species that decidualize at
implantation, including humans, rabbits and rats, further support PRL's
role in uterine physiology. However, in species with noninvasive
placentae, such as pigs, other mechanism(s) may exist, i.e., receptor
regulation, to allow similar effects of PRL on uterine function.
Prolactin in the Circulation
Circulating levels of PRL are relatively constant during pregnancy
(Dusza and Krzymowska, 1981; Kensinger et al., 1986; DeHoff et al.,
1986) in pigs; elevated slightly on Day 10 (20 ng/ml), then declining
by Day 20 and remaining constant (5-10 ng/ml) until parturition
(Kraeling et al., 1982). In cyclic pigs, PRL levels are similar to
those for pregnant pigs (5-10 ng/ml) except that PRL is elevated (15-20
ng/ml) on Days 0 to 2 and 16 to 17 (Brinkley et al., 1973; Dusza and
Krzymowska, 1979; Foxcroft and Van der Weil, 1982) when concentrations
of circulating estrogens increase.

13
In rats, PRL levels increase 10-fold with diunrnal and nocturnal
increases (de Greef et al.r 1977; Neill, 1980), following mating or
cervical stimulation to approximately Day 12 for intact, or Day 6 for
ovariectomized rats. Studies with ovariectomized rats suggest that
progesterone is associated with the noctural increase in PRL levels,
while estrogen accentuates the diurnal rise and inhibits the nocturnal
increase (Freeman and Sterman, 1978). For cyclic rats, PRL levels
increase at proestrus, in association with increases in circulating
estrogens (Butcher et al., 1974; Kelly et al., 1975).
Regulation of Pituitary Secretion of Prolactin
Hypothalamic Factors
The mechanisms responsible for regulation of secretion of PRL by
the anterior pituitary are currently under intense invesigation.
Hypothalamic dopamine tonically inhibits PRL secretion, a mechanism
which is unique among the anterior pituitary cells. However, the
posterior pituitary may also be a source of dopamine (Ben-Johnathen and
Peters, 1982) and may be transported to the anterior pituitary through
the blood (Page et al., 1982) as for other substances (Baertschi,
1980). Gamma butyric acid (GABA) also contributes to the inhibition of
PRL secretion (Duvilanski et al., 1986) especially in pigs (Schally et
al., 1977). Other factors are PRL releasing factors; opiods (Bero and
Kuhn, 1987; Rauhala et al., 1987), serotonin (Bero and Kuhn, 1987;
Thomas et al., 1988), Vasoactive intestinal peptide (VIP; Nagy et al.,
1988), peptide histidine isoleucine (PHI; Abe et al., 1985), oxytocin,
(Samson et al., 1987; Lumpkin et al., 1983) and posterior pituitary
factor (Murai et al., 1988). Complete control of PRL secretion in pigs

14
may be due to a combination of these factors and not under the sole
regulation of the tuberoinfindibular dopaminergic system (Moore, 1988).
Estrogen
Lactotrophs are unique among anterior pituitary cells since they
are generally inhibited by tonic hypothalamic dopamine. Several
factors can override dopamine inhibition, but estrogen is most
effective. The mechanism(s) responsible for estrogenic stimulation of
PRL secretion are relatively unknown; however, following an increase in
estrogen, circulating PRL increases 10-fold, on the afternoon of
proestrus in rats (Butcher et al., 1974). Administration of exogenous
estrogen to ovariectomized rats increased PRL over 2-3 days (Maurer and
Gorski, 1977); however, Yamamoto et al. (1975) demonstrated increases
in PRL within 12 h following injection of a single dose of estrogen
into ovariectomized rats.
Despite chronic inhibition of lactotrophs by dopamine, estrogen
increases the synthesis and secretion (Thorner and MacLeod, 1980) of
pituitary PRL, as well as the number of lactotrophs (Neill and Frawley,
1983). Estrogens' effects may be due to 1) direct stimulation of
pituitary lactotrophs, 2) modulation of hypothalamic regulation of the
pituitary or 3) effects on the physiological responsiveness of
lactotrophs to other regulatory mechanisms.
Estrogen stimulates increases in PRL in pituitary cells when added
to in vitro culture medium (Nicoll and Meites, 1972) or in vivo when
implanted into anterior pituitaries of rabbits (Kanematsu and Sawyer,
1963) and sheep (Vivian et al., 1979). Hypothalamic control,
especially by dopamine, of pituitary PRL is dampened due to estrogen

15
(Shull and Gorski, 1984). Dopamine released into the portal veins is
decreased (Ben-Johnathan et al., 1977) during proestrus and in
ovariectomized rats treated with estrogen (Cramer et al., 1979).
However, chronic estrogen treatment increases dopamine turnover (Fuxe
et al., 1969) and release (Gudelsky et al., 1981), possibly through
short-loop feedback mechanisms of hyperprolactinemia (Moore, 1988).
Estrogen may also stimulate release of TRH, a known PRL releasing
factor (Shull and Gorski, 1984). The number of TRH receptors on
lactotrophs are increased in pituitaries of estrogen treated rats
(DeLean et al., 1977).
Estrogen effects lactotrophs through both protein dependent and
independent mechanisms. Estrogen acts directly on the genome of the
lactotrophs to increase transcription and PRL mRNA and then a 5-fold
increase in PRL within 24h. This mechanism of estrogen action is
thyroid and hypothalamus independent since estrogen increses PRL
secretion in thyroidectomized rats or rats with pituitaries
transplanted to the kidney capsule. In ovariectomized rats, both rapid
and prolonged effects of estrogen on PRL secretion occurs. Estrogen
does not affect the growth hormone gene in rats. Estrogen receptors
reach a peak in the nucleus at 1 hour, then decrease, but PRL synthesis
and secretion is stimulated for 48 to 72h following estrogen
stimulation. Shull and Gorski (1986) suggested that estrogen affects
stable nuclear components, such as chromatin proteins, DNA sequences
around the PRL gene, a second regulatory factor (unknown or pituitary
transcriptional activator; PIT-1) or a combination of these effects.

16
Estrogen Receptors
The hydrophobic similarities between the pentanophenanthrene ring
of steroid hormones and membrane lipids allow steroids to enter a cell
through simple diffusion. A 'two-step' theory for expression of
steroid hormones was proposed by Jensen et al. (1968) whereby the
unbound estrogen receptor resided in the cytoplasm. Activation by
estrogen binding shifted the sedimentation coefficent from 4S to 5S
(O'Malley and Schrader, 1979) and translocation to the nucleus allowed
interaction with acceptor nonhistone chromatin proteins (see review
Grody et al., 1982). Estrogen receptor mechanisms have undergone
substanial revision. Williams and Gorski (1972) proposed an
equilibrium theory where all estrogen receptors are nuclear, but their
affinity is dictated by binding status. Receptors not bound by
estrogen have a lower affinity and move to the cytoplasm during tissue
processing. Martin and Sheridan (1982), Welshons et al. (1984) and
King and Green (1984) agree with Williams and Gorski (1972) on the
artifactual nature of cytoplasmic receptors. Buffer volumes affect
recovery of cytoplasmic and nuclear estrogen receptors. Monoclonal
antibodies and immunoperoxidase staining localized estrogen receptors
in nuclei of human breast tumour and rabbit uterine cells.
Steroid receptors are a family of ligand regulated positive
transcription factors with a common structural organization. There is
a central DNA binding domain, hinged to a carboxy terminus that is
common to all steroid receptors. This also contains zinc finger
proteins (Miller et al., 1985) of 2 pairs of four consecutive
cysteines, that act as ligands for zinc atoms.

Steroid interaction at the PRL genome DNA is thought to stimulate
PRL transcription though involvement of pituitary transcriptional
activator, PIT-1. This factor, PIT-1, must bind DNA in conjunction
with estrogen binding to its DNA domain to stimulate PRL gene
transcription (Adler et al., 1988).
Receptor Theory
Peptide hormones usually interact with cells through receptors.
Although the concept of 'receptors' seems commonplace, it was
introduced by J.N. Langley (1852-1926) following his observations of
mutual antagonism between curare and nicotine. The drugs interacted
with a "receptive substance" during autonomic transmission in
neuromuscular communication of the frog leg (Langley, 1909).
Qualitative aspects of receptor saturation and selectivity were
transformed to quantitative analysis by A.J. Clark (1885-1941).
Studying acetylcholine and atropine, he recognized that the rate at
which drugs combined with receptors was dependent on the concentration
of drugs and receptors and that the dissociation rate was proportional
to the number of complexes formed. These properties were similar to
mass action isotherms used by Langmuir (1881-1957) and, therefore,
drug-receptor interactions were found to obey the laws of mass action.
However, not all drug-receptor binding phenomena was explained by
Clarks observations and mass action equations. Stephanson (1956)
further refined drug-receptor interactions with the role of efficacy in
biological responses, noting that agonist response curves for tissues
were often steeper than dose-response curves. He postualated that 1)
maximal effects are produced when an agonist occupies only a small

18
proportion of the receptors; 2) biological response is not linear in
proportion to the number of receptors; and 3) equal biological
responses can be produced by drugs of different capacity for receptor
occupancy; that is, increased efficacy. The concept of spare receptors
was developed by Patn (1941), from studies on effects of antagonism.
He noted that agonists can elicit maximal biological responses even
when only a small fraction of the total receptors are occupied.
Receptor occupancy was not rate limiting for tissue activation.
Nickerson (1956) observed that only 1% of guinea pig ileum histamine
receptors needed to be occupied for maximal contraction, confirming
Paton's (1961) theory.
Analysis of Receptors
With developments in in vitro techniques, quantification of ligand
binding to membranes and defining receptors required evidence of
saturation, specificity, and kinetics realistic for the time course of
biological action. Receptor binding data are obtained through
saturation or competitive inhibition studies, and generate curvilinear
results. Linear transformation of data to obtain binding parameters is
achieved through Scatchard (1949) (Rosenthal; 1969) interpretation.
Scatchard analysis is based on equilibrium kinetics resulting in a
linear plot of data where bound/free ratio and free hormone data are
ploted on the abcissa and the ordinate, respectively. This results in
linear interpretation of data where the negative slope of the generated
line defines the affinity constant (Ka) or its reciprocal defines the
dissociation constant (1/Ka or Kd). Maximal binding and density of
binding sites are estimated by the y- and x-intercepts, respectively.

19
The linear plot is an algebraic derivation from original theory that
numbers of complexes formed are dependent on the concentration of
receptors and hormone available as well as the rates of association and
dissociation.
ki
[H] [R]-*~- [HR] (1)
k-,
and
[H][R] = [HR]/(Kd + [H]) (2)
with [HR] = amount bound (B); [R] = maximal binding (Bmax) and
[H] = free hormone (F), the equation is restated to
B = [(F) (Boa x ) ] /Kd + F (3)
rearrangment generates
(B) (Kd) + (B) (F) = (F) (B.ax)
division by Free hormone
[(B) (Kd ) /F] + B = B.ax
rearranged to
B/F (Bmax B)/Kd
and transformed to the linear (y = mx + b) expression
(4)
(5)
(6)
B/F = (-1/Kd)(B) + B.ax/Ko
(7)

20
Thus, the mathematical model and equations for binding data
resemble those for enzyme kinetics (Eadie, 1942; Hofstee, 1952).
Scatchard analysis assumes a known number of receptor sites. This is
not often the case during membrane receptor investigation. Therefore,
the Rosenthal (1969) analysis is used, since in theory, it is not based
on known receptor numbers. However, the two data transformations are
mathematically equivalent and receptor data are analyzed through
Scatchard analysis and interpretation.
Dissociation Rate Constants
Hormone receptor complexes are, in part, dependent on the rate at
which these complexes form (ki) and dissociate (k-i) at equlibrium.
ki
[H] [R]^ [HR] (8)
k-i
Dissociation rate constants are determinable when rebinding of
labeled hormone is prevented and kinetics are reduced to simple
first order reactions.
dB/dt = (-k-i)(B) (9)
If bound (B) is equal to bound at time = 0 (Bo), then integration
results in
In B/Bo = (-k-1) (t) (10)
and a plot of In B/Bo versus time generates a slope of -k-x;
i.e. the dissociation rate constant. At equilibrium, the rates

21
of association and dissociation are equal (Baxter and Funder,
1979) to
ki [H] [R] = k-i [HR] (11)
or redefined as
[H][R]/[HR] = k-i/ki = Kd (12)
Thus, the ratio of ligand 'off' to 'on' a receptor is the equilibrium
dissociation constant (Kd) and is equal to the reciprocal of the
association constant (Ka) generated from the negative slope of
Scatchard analysis as previously mentioned. The association constant
depicts the affinity and tightness of binding between a hormone or
ligand and receptor (Limbird, 1986).
Scatchard analysis can generate a linear relationship between B/F
and F, describing a single class of hormone receptors. Curvilinear
Scatchard can be due to two separate binding sites with different
affinities or negative cooperativity (DeMeyts et al., 1976)
Prolactin Receptors
General Characteristics
Although PRL was discovered in the 1930s (Riddle et al., 1933),
binding of PRL to tissues was not demonstrated until the 1960s.
Specific high affinity binding of PRL and its relation to biological
response characteristics of hormone-receptor interaction was first
investigated in mammary glands of mice (Frantz and Turkington, 1972)
and rabbits (Shiu et al., 1973). Investigation of the PRL receptor
structure has been with rat liver. Binding of hormone to membrane does

22
not necessarily induce a biological response. However, binding sites
in the mammary gland (Shiu et al., 1976) and liver (Chen et al., 1972)
have been correlated with cellular changes in mRNA and enzyme activity
and therefore, suggest that binding sites serve as receptors essential
for biological responses. Target cell membranes having saturable PRL
binding sites include mammary epithelial cells, hepatocytes, renal
tubules, adrenal cortex, prostate, seminal vesicles, and brain (Hughes
et al., 1985). Characteristics of PRL receptors include high affinity
and saturability. The high affinity constant, as determined by
heterologous ovine PRL assay (Ka = 109 M_1; Shiu and Freisen, 1974a),
is correlated with hormone biopotency. Homologous detection of PRL
receptors generates an affinity constant 108 (Haro and Talamantes,
1985; this study), only slightly lower than that reported for rat liver
(Dave and Knazek, 1980; Dave et al., 1981; Liscia et al., 1982; and
Liscia and Vonderhaar, 1982); cow mammary gland (Ashkenazi et al.,
1987) and mouse liver (Posner et al., 1974b; Knazek et al., 1977;
1978). However, the reversability of PRL bound to its receptor has
been questioned, as observed in vitro which often inefficiently
replicates the in vivo binding environment. Prolactin receptors have
been noted in several other tissues and are associated with other
physiological effects. Prolactin stimulates steroid production in ovary
and Leydig cells (Barkte and Dalterio, 1976; Musto et al., 1972),
uptake of testosterone by prostate (Farnsworth and Gonder, 1977),
increased uteroglobin mRNA and steroid receptors in the uterus (Chilton
and Daniels, 1985) and water and ion regulation in fetal membranes
(Rabee and McCoshen, 1986; Kensinger et al., 1986). Prolactin binds to
kidney membranes, but a clear function has not been established. These

23
kidney binding sites for PRL appear to be specific and therefore are
considered to be PRL receptors (Hughes et al., 1985).
Binding Characteristics
Most studies conducted of PRL receptors utilize heterologous assay
systems, combining tracer, competing hormone and tissue source of
membrane receptors from various species. Although a great deal of
information regarding in vitro binding of PRL receptors has been
obtained, these systems do not mimic the in vivo environment. Thus,
questions remain as to whether heterologous hormone interactions are
identical to those for homologous hormones. Heterologous hormones can
generate binding results that may not characterize in vivo homologous
binding kinetics and properties or produce immunological artifacts
(Hughes et al., 1982; Amit et al., 1983). Additionally, other
hormones, placental lactogens or proliferin and the microheterogeneity
of PRL and growth hormones contribute to the difficulties in
interpreting results from heterologous radioreceptor assays. To date,
results from only one homologous assay has descibed interactions
between mouse PRL and its liver receptors (Haro and Talamantes, 1985a).
Binding characteristics are similar, but the affinity constant is
slightly lower than obtained from heterologous assays. This would
results in a more rapid hormone-receptor dissociation, as speculated to
occur in vivo (van der Gugten et al., 1980). Thus it appears
advantageous, although more difficult, to utilize homologous hormones
for studying receptors.
Scatchard (1949) analysis of binding studies assumes that
receptors are saturable, specific and freely reversible at equilibrium.

24
Studies with PRL receptors (in vitro), whether by heterologous or
homologous assay systems, fulfill the constrains of saturability and
specificity; however, complete reversibility has been questioned. Slow
or difficult dissociation has been observed for some peptide hormones
including PRL (Kelly et al., 1983; van der Gugten et al., 1980), growth
hormone (Donner et al., 1980), TSH (Powell-Jones et al., 1979), LH
(Katitineni et al., 1980) and insulin (Donner and Corin, 1980).
Increases in association time from lh to lOh (in vitro) are directly
correlated with increases in dissociation time, as well as incomplete
dissociation, after 48 hours (Kelly et al., 1983). Longer association
times may allow tighter binding and decreased ability for hormone
dissociation. Internalization of PRL and other hormones is often
preceded by tightening of the hormone-receptor linkage (Catt et al.,
1979) .
Prolactin receptors within different cell membranes have different
affinity constants and dissociation times (Kelly et al., 1983).
Therefore, affinity and dissociation could depend on location of the
receptor within cellular membranes. The proportion of subcellular
membranes within microsomes commonly used during in vitro receptor
studies is not known. Differences in affinity or dissociation
constants may be a function of the ratio of PRL receptors within plasma
or golgi membranes in the microsomal pellet.
Dissociation of ovine PRL from rat liver (Kelly et al., 1983) and
porcine corpora luteua (Brambly and Menzies, 1985) membranes is 60%
complete after 48h. This slow dissociation rate in not likely due to
damaged hormone or receptors and must be more efficient in vivo than in
vitro (van der Gugten et al., 1980). Therefore, in vivo, energy

25
dependent factors or other factors may account for rapid reversibility
that does not yet occur in vitro. Additionally, intrinsic factors
which affect dissociation may differ between tissues since it appears
that PRL receptors are differentially regulated. A high apparant
activiation energy be involved in slow dissociation for PRL binding and
as reported for CL of pigs (64.8 kJ, Brambly and Menzies, 1987), and
liver of mice (43.6 kJ/mole; Haro and Taimantes, 1985b) and rats (34
kJ/mole; Rae-Ventner and Dao, 1982). Extensive hydrophobic
interactions may be involved in PRL interaction with its receptor since
monovalent anions, acetate and phosphate stablize homologous binding
(Haro and Talamantes, 1985a). Amino acids at position 20 through 36
form such a hydrophobic region with histidines located at positions 27
and 30 in cow (Wallis, 1974), sheep (Li et al., 1970), pig (Li et al.,
1976) and human (Cooke et al., 1981) and at positions 25 and 28 in rat
(Cooke et al., 1980) and mouse (Kohomoto et al., 1984) PRLs.
Prolactin Receptor Turnover
Once PRL binds to its receptor, internalization is rapid and
biphasic. Internalization of bound PRL occurs in as little as 5 min.
At 5 min, radiolabeled PRL was associated with the low density membrane
fraction having high galactosyl transferase activity characteristic of
golgi membranes. At 10 min, radiolabeled PRL was associated with the
high density membrane fraction with high acid phosphatase activity,
characteristic of lysosomes. This internalization and trafficing is
similar to that for insulin (Posner, et al., 1981). The golgi contains
twice the amount of PRL, and processes PRL slower than insulin. The
golgi network has been redefined as the trans golgi network (TRN) or

2b
golgi saccule endoplasmic reticulum lysosome (GERL) which functions to
transport proteins to the plasmalemma or to lysosomes (see review,
Griffiths and Simon, 1986).
Molecular Weight of Prolactin Receptor
Prolactin receptor structure has been investigated through various
biochemical methods including gel chromatography, solubilization,
affinity labelling and cross-linking. Prolactin receptors are
hydrophobic glycoproteins, since solubilized receptors bind to
Concanavalin-A, a lectin which binds mannose and glucose residues.
Triton X-100 solubilized PRL receptors aggregate to form larger
molecular weight (220,000 Mr) complexes while purification of receptors
with 3-[(3-cholamidopropyo)-dimethylammonio]-1-propane sulfonate
(CHAPS), a nondetergent zwitterionic solution, results in a single
electrophoretic band of 32,000-37,000 Mr (Djiane et al., 1987).
Molecular weight estimates of PRL receptors from rabbit mammary gland
suggest a Mr of 35,000 to 42,000. Rabbit mammary gland membrane
homogenates that are cross-linked with radiolabeled ovine PRL and
analyzed by SDS-PAGE and autoradiography indicated a 58,000-60,000 Mr
band. Subtraction of the Mr of PRL, yields a 35,000-37,000 Mr estimate
for PRL receptor from rabbit mammary gland (Djiane et al., 1987).
Identical results were obtained for PRL receptors in ovary, kidney and
adrenal gland from rabbits, but an additional protein band of 63,000 Mr
for ovarian and adrenal tissues was detected. (Djiane et al., 1987).
Affinity labelling results in a lower Mr estimate for prolactin
receptor subunit while gel electrophoresis suggests a higher (99,800-
340,000) Mr estimate. Berthon et al. (1987a) used hormone affinity

27
and monoclonal antibody detection and showed PRL receptors for sow
mammary gland at 42,000 with faint bands at 31,000 and 53,000 Mr.
Djiane and coworkers (1987) suggest that the holo-PRL receptor contains
2 or more of the 32,000-40,000 Mr units that are not linked by
disulphide bonds, but may be noncovalently associated.
Ovarian lactogenic receptors in rats (Dufau and Kusuda, 1988) have
two active subunits, 88,000 and 40,000 Mr, as purified by sequential
affinity chromatography. The 40,000 Mr subunit is part of the 80,000
Mr receptor form and is similar to the 35,000-44,000 Mr PRL receptor
subunit from rabbit, rat and mouse liver and mammary gland (Haeuptle et
al., 1983; Hughes et al., 1983; Liscia and Vonderhaar, 1982; Liscia et
al., 1982). The higher Mr species was also observed in ovarian,
testicular, kidney and mammary gland tissues following cross-linking of
receptor subunits. Different molecular species are observed in various
rat tissues including the ovary (88,000 and 40,000; Bonifacino, 1985),
Leydig cells (91,000; 81,000; 37,000 and 31,000), mammary gland
(93,000; 83,000; 30,000 and 28,000) and kidney (65,000 and 30,000)
(Bonifianco et al., 1985). Additionally, ovarian and Leydig cells
contained the 37,000 Mr form within the 81,000 form. The female rat
liver 87,000 Mr form contains subunits of 40,000 and 35,000 which may
or may not be linked by disulfide bonds (Haldosen and Gustafsson,
1987) Lactogenic receptors from mammary gland from pigs and rabbits
range in molecular weight from 28,000 to 69,000 (Haeuptle et al., 1983;
Hughes et al., 1983; Sakai et al., 1985; Katoh et al., 1985).

28
Molecular Structure of Cloned Prolactin Receptor
The prolactin receptor from rat liver has been cloned (Boutin et
al.f 1988). Association with other proteins is not required for the
40,000 Mr structure which supports results of Liscia and Vonderhaar,
(1982); Haeputle et al. (1983) and Necessary et al. (1984) but not
those of Dufau and Kusuda (1987). The PRL receptor (40,000 Mr) subunit
contains a 19 amino acid signal sequence, an extracellular domain (210
amino acids), speculated to bind PRL, a single transmembrane section
(24 amino acids) and a short cytoplasmic domain (54 amino acids). This
PRL receptor has 30% overall homology to growth hormone receptor (Leung
et al., 1987) following removal of 293 cytoplasmic amino acids from the
growth hormone receptor structure. The two receptors share 67%
homology between the first and second, and third and fourth cysteine
residues. A 40-60% homology exists in three other extracellular
regions. A 19 amino acid series in the cytoplasmic domain has 68%
structural identity to growth hormone. These two receptors do not
share sequence homology with other proteins. The short cytoplasmic
domain of the PRL receptor does not possess tyrosine kinase activity or
phosphorylation sites as seen in other growth factor receptors (Hunter,
1987). However, the short cytoplasmic domain is similar to other
protein receptors which transport various compounds; such as
transferrin receptor transport of transferrin (Schneider et al., 1984),
LDL receptor transport of cholesterol (Yamamoto et al., 1984) and IGF-
II receptor transport of mannose-6-phosphate (Morgan et al., 1987).
Prolactin receptors cloned from other rat tissues (ovary, adrenal and
mammary gland) are more similar to growth hormone receptor than liver
PRL receptor by their longer cytoplamic domains (Kelly et al., 1989).

29
Structural differences for PRL receptors between mammary gland and
liver have been suggested previously (Sakai et al., 1985).
During sequencing of purified growth hormone receptor, 20-50% of
the receptor was actually sequenced as ubiquitin (Leung et al., 1987).
Ubiquitin has been associated with the intracellular domain of the
growth hormone receptor and may play a functional role in receptor
activation and cellular response (Leung et al., 1988). A peptide bond
forms between the epsilon-amino group associated with the cytoplasmic
domain of the receptor and the carboxy terminal end of ubiquitin
(Goldknopf and Busch, 1977) is suggested. This feature is also present
in lymphocyte homing receptor (Siegelman et al., 1986) and platelet
derived growth factor (Yardin et al., 1986) and may extend to ovarian
and mammary gland PRL receptors with long cytoplasmic domains (Kelly et
al., 1989).
Water Soluble Prolactin Receptors
Water soluble PRL receptors have been described for pig mammary
glands (Berthon et al., 1987b) and rat liver (Amit et al., 1984). This
receptor is not precipitable by polyetheylene glycol as are cytosolic
steroid receptors (Kelly et al., 1983). Water soluble receptors have
been reported for follicle stimulating hormone (Dufau et al., 1977),
human chorionic gonadotropin (Pahnke and LeidenBerger, 1978) and human
growth hormone (McGuffin et al., 1976; Herrington 1981), suggesting
that water soluble receptors are generally associated with polypeptide
hormones (Berthon et al., 1987b). The water soluble PRL receptor from
porcine mammary gland has similar specificty and affinity as membrane
associated PRL receptors and binding was higher with ovine PRL. Porcine

30
and rabbit PRLs were not able to compete with ovine PRL and exhibited
only 3% binding. These results also question the true validity of PRL
binding receptor estimates when using a heterologous system.
Antibodies to rabbit mammary gland PRL receptors recognize water
soluble receptors and block their binding of ovine PRL (Berthon et al.,
1987b). Water soluble and membrane associated PRL receptors probably
share antigenic determinants and hormone binding sites with rabbit
mammary membrane PRL receptors. It is unclear, however, whether water
soluble receptors are involved in signal tranduction or transport of
PRL from mammary cells into blood or milk.
Signal Transduction Systems for Prolactin
Intracellular Mediators
Peptide hormone-receptor interaction is mediated through a
transduction system to stimulate changes in cell physiology and overall
tissue response. Transduction systems involve either 1) ligand
modulated ion channel activity, 2) ligand regulating enzyme activity or
3)ligand regulation of cryptic mediators through interactions between
intracellular receptor domains or other submembranous constituents
similar to G and N regulatory components. Transduction systems have
been classified for several hormones (see review, Hollenburg, 1986;
Cockcroft and Stutchfeld, 1988). Mobility of peptide receptors is
paramount to some transduction systems, i.e., adenylate cyclase, and
functions due to the fluid mosaic properties of the cell membrane
(Singer and Nicholson, 1979) with possible involvement of microtubules
and microfilaments.

31
Prolactin, however, possesses a receptor leading to biological
changes within target cells, but without a known transduction system
between hormone binding and cellular response. Prolactin generates
such diverse physiological affects within a vast array of tissues that
several concomitent transduction systems are feasible. Witorsch et al.
(1987) and Hughes et al. (1985) review trannduction systems that have
been investigated for PRL. Additionally, the source of tissue for
receptor studies may bias transduction system results since PRL's
different responses may be achieved through different pathways.
Mammary gland is a complex tissue requiring support from insulin,
glucocorticoid and estrogen, in addition to PRL, for maintainence and
milk systhesis. Other hormones or factors, such as thyroxine or growth
hormone, may be essential to mammary cell function. Explant cultures of
mammary tissue have been studied to elucidate the mechanism(s) of PRL
action. A recent model for PRL effects on casein biosynthesis was
proposed by Rillema (1980). Components included decreased cAMP as a
stimulatory component, calcium as an obligatory factor, phospholipase C
to generate diacylglycerol to stimulate protein kinase C and increases
ornithine decarboxylase (ODC) activity. Phospholipase A2, ODC and
prostaglandins appear to be involved in prolactin stimulation of casein
biosynthesis in mammary gland explants from mice (Cameron and Rillema,
1983). Polyamines (Rillema, 1979) and Na*-K*-ATPase (Falconer and
Rowe, 1977) may mediate PRLs action, as well as changes in
phosphatidylcholine, as PRL stimulation is associated with increased
choline uptake and decrease phosphatidylcholine turnover (Ko et al.,
1986).

32
1980) or transduction mechaniams of cellular hardware. Cells may not
possess identical intracellular machinery, e.g., hepatocytes versus
epithelial cells, and are inherently different and programmed to
respond differently from each other, but characteristically for each
cell type.
Extracellular Mediators
Actions of growth hormone are acheived indirectly through
stimulation of liver to release somatomedin C (Laron, 1982), now
recognized as insulin-like growth factors (IGF). This mechanisms of
action is extended to PRL (Anderson et al., 1983), which stimulates
release of a hepatic factor (Nicoll et al., 1983) that mediates PRL's
effect on mammary cells or pigeon crop sac cells. This factor, termed
"synlactin" (Nicoll et al., 1983), has no effect alone. It is secreted
when circulating PRL increases, such as during lactation, but is not
detected in male or virgin female rats. Pigeon crop sac proliferation
increases (in vivo) when ovine PRL is injected in the hepatic, but not
jugular vein of pigeons; suggesting that this mitogen is produced by
liver of pigeons (Mick and Nicoll, 1985).
Prolactin stimulation of somatomedin production by rat liver is
20-fold greater than that of growth hormone. However, PRL does not
stimulate growth in male rats. Synlactin may be an IGF-like molecule
that is detected during sulphate determination of IGFs (Mick and
Nicoll, 1985). Whether synlactin is a fragment of PRL or similar to
IGF awaits determination of its amino acid structure.
Another extracellular mediator of PRL effects is liver lactogenic
factor (LLF) (Hoeffler and Frawley 1987). Both synlactin and LLF are

33
secreted from the liver is response to high circulating levels of PRL.
However, Hoeffler and Frawley (1987) suggest that the two compounds are
different. The LLF is lactogenic, exerts potent biological activity
individually, and acts additively with PRL when tested in the reverse
hemolytic plaque assay (Neill and Frawley, 1983) using mammary cells.
Synlactin is mitogenic, devoid of activity alone and acts
synergistically with PRL when tested in pigeon crop sac assays.
Neither synlactin or LLF have been sequenced, nor tested in reverse
hemolytic plaque or pigeon crop sac assays, respectively.
A role for a PRL stimulated liver mitogenic factor is suggested
since liver receptors for PRL increase during pregnancy in rats (Sasaki
et al., 1982a), mice (Sasaki et al., 1982b) and rabbits (Kelly et al.,
1974; Fix et al., 1981) and may be associated with increases in
synlactin secretion and possibly mammary growth and development during
gestation (Mick and Nicoll, 1985).
Internalization of Prolactin and its Receptor
A clearly definded transduction mechanism has eluded researchers
studying PRL-receptor interaction; therefore the role of
internalization in PRL function was investigated. Prolactin has been
detected in the subcellular fraction of the plasmalemma (Posner et al.,
1981) and golgi regions of cells from rat ventral prostate and liver
(Bergeron et al., 1978). Receptor internalization through coated pits
was proposed for LDL receptors (Goldstein et al., 1979). Following
internalization, the hormone is degraded while the receptor can be
degraded or recycled. Proteolysis of internalized hormones allows
epidermal growth factor and insulin to affect target tissues (Goldfine

34
et al., 1987; Rosen et al., 1987; Walaas and Walaas, 1988).
Internalization and processing of PRL by lysosomes in mammary and
ovarian tissues may be involved in PRL stimulation of these cells
(Nolin and Bagdonanov 1980; Mittra, 1980; Nolin, 1982). Proteolytic
fragments of 8,000 and 16,000 Mr of PRL may be involved in PRL action
(Clapp, 1987) suggesting that 23,000 Mr prolactin is a prohormone prior
to being internalized (Nolin, 1982).
Hormone-receptor internalization to mediate PRL action suggests
involvement of cellular microtubules and microfiliments. Chloroquine,
which binds tubulin and destabilizes microtubular structures, inhibits
downregulation of PRL receptors and stimulation of casein synthesis
(Houdebine and Djiane, 1980). Subsequent results indicate that
chloroquine binds tubulin at the plasma and golgi membranes. Another
microtubular destabilizer, griseofulvin, did not affect PRL stimulation
casein synthesis of mammary cells; suggesting PRL affects are at the
cell surface (Houdebine et al., 1982).
Advances in receptor purification enabled development of
antibodies to PRL receptors. Adminstration of high doses of anti-PRL
receptor serum to rats blocked PRL stimulation whereas low doses
actually mimiced PRL effects, suggesting that PRL effects are through
the receptor at the cell membrane and not following internalization and
processing to cleaved or clipped forms (Witorsch et al., 1987). Whole
anti-PRL receptor serum, bivalent F(ab)2 or monovalent F(ab) fragments
demonstrated similar abilities to inhibit PRL binding to mammary gland
(Djiane et al., 1987). In mammary gland explant cultures, similar
effects on PRL receptor downregulation were observed for all forms of
anti-PRL receptor, as well as PRL itself. Low doses of bivalent F(ab)2

35
fragments stimulated casein DNA synthesis, while whole antibody serum
stimulated casein DNA 50-60%. Monovalent F(ab) fragment had no activity
in stimulation of casein biosynthesis. Interactions between two PRL
receptor molecules is, therefore suggested to be involved in PRL
stimulation; similar to the mechanism of insulin (Rosen, 1988). Under
these conditions, the half-life for PRL in the plasmalemma is three
times greater and movement of PRL receptors within the cell membrane
could constitute an additional regulatory mechanism for cell
receptivity. Microaggregation of receptors following hormone binding
is essential for induction of biological effects of several hormomes
(Brown and Goldstein, 1983).
Regulation of Prolactin Receptors
Regulation by Ovarian Steroids
Prolactin receptor concentrations are sex specific (Sherman,
1977; Waters et al., 1978). Changes in PRL receptor numbers within
several target tissues occur during puberty (Kelly et al., 1974), the
estrous cycle (Kelly et al., 1975), pregnancy (DeHoff et al. 1984;
Grissom and Littleton, 1988), lactation (Kelly et al., 1975; Sherman et
al., 1977; Shiu et al., 1981), and in response to ovariectomy (Posner
et al., 1974a; Kelly et al., 1979; Marshall et al., 1979; Daniels et
al., 1984), orchotomy (Kelly et al., 1976; Bohnet et al., 1977) and
following administration of exogenous steroids (Waters et al., 1978;
Shiu et al., 1982). However, alterations in PRL receptor
concentrations are not necessarily similar between tissues or within
the same tissue of different species.

36
Steroid regulation of hepatic PRL receptors has been extensively
investigated. Hepatic PRL receptors decrease in ovariectomized rats
(Posner et al., 1974a; Kelly et al., 1979), but increase following 8 to
12 days of chronic estrogen administration (Posner et al., 1974a).
Increases in PRL receptors following estrogen treatment are thought to
be mediated indirectly, through stimulation of pituitary PRL release
and auto-upregulation of receptors. Hypophysectomy (Posner et al.,
1978), but not CB154 administration (Kelly et al., 1976), blocked the
estrogen-induced increase in rat hepatic PRL receptors, suggesting
pituitary involvement, but not exclusively an effect of PRL.
Involvement of growth hormone (Knazek et al., 1975), ACTH and TSH
(Bhattacharya and Vonderhaar, 1979) has been implicated.
Contrary to results in rats, ovariectomy increases hepatic PRL
receptors in mice (Marshall et al., 1979) and exogenous estrogen
reverses effects of ovariectomy. Estrogen administration also
decreases PRL receptors in prostate (Kledzik et al., 1976; Amit et al.,
1983) adrenal, kidney, (Monkemeyer et al., 1974) and mammary gland of
mice (Marshall et al., 1979) and rats (Bohnet et al., 1977; Smith et
al., 1976).
The increase in PRL receptors in the mammary gland following
parturition (Holcomb et al., 1975; Djiane et al., 1977) is thought to
result from autoregulation due to increases in concentrations of serum
PRL since administration of CB154 at parturition, decreases mammary
gland PRL receptors (Bohnet et al., 1977). Administration of PRL to
pseudopregnant rabbits increases mammary gland PRL binding sites; an
effect blocked by administration of exogenous progesterone (Djiane and
Durand, 1977). Sakai et al. (1978, 1979) suggest that progesterone

37
indirectly decreases or supresses PRL receptors by competing with
glucocorticoid receptors to block stimulation of PRL receptors.
Progesterone retards PRL auto-upregulation of PRL receptors in mammary
glands of rabbits (Djiane and Durand, 1977). However, progesterone
appears to have no effect on PRL receptors in mammary gland (Sherman et
al., 1977) or liver (Posner et al., 1974a) of rats.
Regulation by Peptides
Prolactin regulates its own receptor in target tissues. However,
unlike other peptide hormones, PRL both increases and decreases its
receptors. Auto-downregulation of PRL receptors has been detected in
vitro (Djiane et al., 1979a) and in vivo (Djiane et al., 1979b) for
rabbit mammary gland. Downregulation of PRL receptors is rapid and
transient, usually following an acute, physiological stimulus by PRL,
whereas, for other hormones, downregulation is much longer (Posner et
al., 1978).
Up-regulation of PRL receptors by physiological levels of
circulating PRL occurs in mouse and rat hepatic (Costlow et al., 1975;
Dave et al., 1981, 1982; Amit et al., 1985 and Rui et al., 1987), and
prostatic (Dave and Witorsch, 1985) membranes. Sustained high
circulating levels of PRL increased PRL receptors in rat liver (Posner
et al., 1975), rat (Holcomb et al., 1976) and rabbit (Djiane et al.,
1977) mammary gland and pigeon crop sac (Klediz et al., 1975). Auto-
upregulation of rabbit mammary gland PRL receptors have a slower onset,
requiring several days of sustained PRL levels, and is more stable
(Djiane and Durand, 1977). Therefore, Djiane and coworkers (1979)

38
suggested that PRL regulation of its receptor, with up and down
regulation, is through two non-antagonistic mechanisms.
Increases in ventral prostate PRL receptors occur 6-12 h following
ovine PRL administration in vitro. Increases are dose dependent (Rui
et al., 1986) and not mimiced by estrogen, androgen, hCG, insulin,
calcium, prostaglandins or cAMP. Positive regulation of ventral
prostate PRL receptors by estrogen is confirmed by Dave and Witorsch
(1985) and Blankenstein et al. (1985). Similarly PRL auto-upregulates
its receptors in testes (Amodor et al., 1985), liver (Amit et al.,
1985), lung (Amit et al., 1985), adrenal gland (Calvo et al., 1981) and
mammary gland (Djiane and Durand, 1977; Djiane et al., 1979).
Studies with hypophysectomized rats suggest that hepatic PRL
receptors may be regulated by anterior pituitary hormones, other than
PRL. Hypophysectomized rats bearing pituitary implants have increased
hepatic PRL receptor numbers which is not mimiced by adminsitration of
ovine PRL (Posner et al., 1975). Additionally, adminsitration of CB154
to reduce PRL levels had no effect (Norstedt et al., 1981). Continuous
infusion of rat growth hormone, but not rat PRL, to male rats resulted
in feminization of hepatic PRL receptor profiles (Norstedt et al.,
1987). Growth hormone may induce PRL hepatic receptors differently
between sexes since its secretory pattern differs between males and
females (Eden, 1979). Induction of hepatic PRL receptors in rats was
achieved by exogenous administration of human, bovine and rat growth
hormones, but net by PRL or human placental lactogen. Hepatic PRL
receptors of prepubertal (17 day old) female rats increased to levels
typical of adult females following 7 days of human growth hormone
infusion. These studies support growth hormone regulation of PRL

39
receptors. However, growth hormone may regulate PRL receptors during
growth and development while PRL may regulate its receptors to meet
adult physiological demands.
Regulation by Membrane Fluidity
The fluid mosaic membrane (Singer and Nicholson, 1979) allows
lateral and vertical movement of receptors and other proteins.
Additionally, proteins and receptors may assemble in different
configurations (Koch et al., 1979) or states of availablity. The rat
liver PRL receptor is a glycoprotein with a single transmembrane domain
(Boutin et al., 1988). Therefore the microenvironment as well as the
physical status of the membrane may influence PRL receptor binding and
tissue receptivity. Changes in membrane fluidity and PRL receptor
binding were investigated in rat hepatic tissue.
Composition of unsaturated fatty acids can influence membrane
viscosity. Rats fed a diet deficient in essential fatty acids had
increased membrane viscosity that resulted in a progressive decrease in
hepatic PRL binding, not reversable by exogenous administration of PRL
(Knazek and Liu, 1979). Phospholipase A2 generates arachadonic acid
from membrane phospholipids and results in a biphasic increase and
decrease in PRL binding to hepatocytes in vitro (Dave et al., 1981).
Prostacyclin treatment in vitro also increases PRL binding to hepatic
cells (Dave and Knazek, 1980) which was blocked by in vivo
administration of indomethacin (Knazek, et al., 1981). These compounds
share the ability to increase hepatic membrane fluidity and increase
PRL receptor availablity. As summarized by Witorsch et al. (1987)
membrane fluidity is correlated with increases in PRL receptor numbers.

40
Modulation of membrane viscosity and PRL receptors by phospholipase A2
is through generation of prostaglandins. Prostaglandins may also
mediate PRL receptor auto-upregulation by decreasing membrane
viscosity. Prolactin may also change the phospholipid:cholesterol
ratio resulting in membrane fluidity changes that increase the
availability of cryptic receptors.
Subcellular localization of PRL receptors may be affected by
membrane fluidity since PRL receptors are preferentially located within
the cell. Seventy percent of mammary gland (rabbit) and liver (rat)
cells' lactogenic receptors are located in the plasmalemma (Bergeron et
al., 1978) The golgi rich fraction of cells contain 4 to 6 times more
PRL receptors and are 2.5 times more fluid than plasma membranes from
preparations from prostate and (male and female) liver cells (Dave and
Vitorsch, 1986). Additionally, only plasma membranes increased PRL
receptor numbers in respond to increases in membrane fluidity.
Prolactin Functions in the Uterus
Prolactin is credited with over 100 functions in several species
(Riddle, 1963; Nicoll and Bern, 1972; Nicoll et al., 1986). However,
for the scope of this review, PRL's functions in uterine physiology
will be reviewed. For more complete discussion of PRL functions, the
following reviews are suggested: Riddle, 1963; Bern, 1975; Nicoll,
1982; and Nicoll et al., 1986. The function of PRL within uterine
physiology is suggested by the evidence of endometrial PRL receptors in
humans (Healy, 1984), sheep (Posner et al., 1974b), pigs (DeHoff et
al., 1984) rabbits (Daniels et al., 1984; Chilton and Daniels, 1985;
Grissom and Littleton, 1988), rats (Williams et al., 1978), mice (Sinha

41
et al., 1983), and mink (Rose et al., 1983); and by production of PRL
by decidual endometrium (Riddick et al., 1978). Exogenous PRL
administered to longterm ovariectomized rabbits stimulates uterine
proliferation and uteroglobin secretion to levels similar to that
detected in estrous does (Chilton and Daniels, 1985). Prolactin
increases the concentration of estrogen and progesterone receptors in
endometrium of rabbits (Daniels et al., 1984) and increases endometrial
uptake of estrogen in rats (Leung and Sasaki, 1973). Armstrong and
King, (1977) detected increases in progesterone metabolism by the rat
uterus following administration of exogenous PRL. Prolactin modifies
the accumulation of uterine lumen fluid in rats, possibly though
synergistic actions with estrogen (Kennedy and Armstrong, 1972).
Additionally, PRL may function during conceptus-endometrial
interactions since PRL affects blastocyst growth and terminates delayed
implantation in the mink (Martinet et al., 1981). Prolactin may also
regulate the synthesis or direction of secretion of uterine
prostaglandins in pigs (Mirando et al., 1988) and humans (Healy, 1984).
Therefore, PRL appears to affect the uterine environment through
modification of secretory functions, or effects on endometrial
proliferation or conceptus-endometrial interactions.
Porcine Conceptus Development and Uterine Secretory Response
The porcine conceptus develops from a spherical form on Day 10 of
gestation to a tubular form on Days 10.5-11 and reaches a filimentous
(200 mm) form late on Day 11 (Geisert et al., 1982a). The porcine
conceptus continues to elongate, initially through cellular
rearrangment and then through cellular hypertrophy and hyperplasia to

42
reach a lenght of 900 mm (Geisert et al., 1982a). During the initial
elongation phase, porcine conceptuses secrete estrogens (Heap et al.,
1979) which signals the maternal physiology to change the uterine
environment from cyclicity to that of pregnancy. During the
preimplantation period, the conceptus relies on secretion of protein,
sugars, ions, and other compounds, collectively termed histotroph, for
nutritional support until placentation is established around Day 18.
Therefore, prior to any physical attachment to the uterine endometrium,
conceptuses must insure luteostasis and histotroph secretion and thus
establish a viable pregnancy (Bazer et al., 1982). Luetostasis is
thought to occur through the redirection of secretion of prostaglandin
secretion from an endocrine mode toward the uterine vasculature to an
endocrine mode, into the uterine lumen (Bazer and Thatcher, 1977).
Redirection of the secretion of prostaglandins is well documented both
in vivo (Frank et al., 1977) and in vitro (Gross et al., 1988),
although, the mechanism is unknown, increasing evidence suggests that
prolactin may be involved (Young and Bazer, 1988; Mirando et al, 1988).
Secretion of histotroph is a well characterized series of events
during the time of maternal recognition of pregnancy (Geisert et al.,
1982b; 1982c; Bazer et al, 1987; Roberts and Bazer, 1988).
Additionally, these uterine secretory events can be mimiced by a single
exogenous dose of estradiol valerate on Day 11 (Geisert et al., 1982c;
Young et al., 1987). Pseudopregnancy can be established in pigs
following injection of estradiol valerate on Days 11-15 or by
injections on Days 11 and 15-16 (Geisert et al., 1987). Major events
of porcine uterine secretory activity following conceptus estrogen
secretion on Day 10-11 are as follows: (1) calcium is rapidly released

43
into the uterine lumen on Day 12 and its reuptake occurs by Day 14; (2)
sodium and potassium increase in the uterine lumen, as calcium levels
decrease, with greatest increases between Days 14 and 16; (3) protein
and uteroferrin concentrations in the uterine flushings increase on
about Days 14 to 16 and (4) glucose concentrations in uterine flushings
of pregnant pigs increase between Days 14 and 16 of gestation. In
cyclic pigs: (1) calcium increases on Day 12, but maximal levels are
lower than for pregnant pigs, and declines gradually between Days 14
to 16; (2) sodium and potassium increase as calcium decreases but to
amounts lower than for pregnant pigs; (3) protein and uteroferrin
increase on Days 15 to 16 and (4) glucose concentrations are low and
affectd by day of the cycle.
Nonpregnant pigs injected with estradiol valerate on Day 11 of the
estrous cycle have uterine flushings on Day 12 that are similar in
composition to those collected on Day 12 of pregnancy (Geisert et al.,
1982c). Thus, estrogen induced uterine secretory function can be
investigated in regard to pregnancy without confounding effects of
other conceptus secretory products (Geisert et al., 1982c, Young et
al., 1987).

CHAPTER 3
EFFECTS OF HYPOPROLACTINEMIA ON ESTABLISHMENT
OF PREGNANCY AND UTERINE SECRETORY FUNCTION IN PIGS
Introduction
Endocrinological studies to establish a hormone's involvement in a
physiological system is often through manipulation of the endogenous
concentration of circulating hormone followed by observation of
physiological changes in the target tissue of interest. Bromocryptine,
(CB154) a dopamine agonist, has been used in rats (Kelly et al., 1979),
pigs (Kraeling et al., 1982) and humans (see review, Barbieri and Ryan,
1983) to lower the circulating prolactin (PRL) levels through tonic
inhibition of PRL by stimulating dopamine receptors on the pituitary.
Prolactin affects proliferation (Chilton and Daniels, 1985),
protein secretion (Chilton and Daniels, 1985; Young and Bazer, 1988),
steroid receptor concentration (Daniels et al., 1984), estrogen uptake
(Leung and Sasakai, 1973) and progesterone metabolism (Kennedy and
Armstrong, 1977) in the uterine endometrium, as well as water and ion
concentrations in fetal membranes (Rabee and McCoshen, 1986; Goldstein,
1980), and fetal growth (Nicoll et al., 1986). Prolactin involvement
in uterine physiology within several species is well established.
However, very few studies utilized pigs and those studied effects of
PRL during the latter part of gestation. Studies conducted with
rabbits, (Daniels et al., 1984; Chilton and Daniels, 1985) support PRL
involvement in endometrial proliferation and secretion of uteroglobin.
44

45
Rabbits, like pigs, lack placental lactogen. Therefore, PRL may also
be important for endometrial function in pigs. Experiments were
conducted to lower circulating PRL in pregnant and cyclic gilts and to
observe effects on fetal survival and uterine secretory function.
Materials and Methods
Animals
Crossbred gilts of similar age (7 to 9 months) and weight (110 to
120 kg) were used in all studies after experiencing at least two
estrous cycles of normal length (18-22 days). Using intact boars,
gilts were observed for estrus daily and the first day of behavioral
estrus was designated Day 0. Gilts were mated when detected in estrus
(Day 0) and 12 and 24h later in studies using pregnant pigs.
Surgical Procedures
Uterine flushings were collected in 20 ml of double distilled
water per uterine horn as described previously (Bazer et al., 1978).
Flushing volumes were recorded and flushings were centrifuged at 10,000
x g for 15 min at 4 C. Supernatants were collected and stored at -20 C
until analyzed.
Bromocryptine
Bromocryptine (CB154; was a gift of SANDOZ Pharmaceutical, East
Hanover, NJ), a dopamine agonist, was solubilized in absolute ethanol
and mixed with saline (1:1, v/v) to a concentration of 25 mg/ml.
Treated gilts received 100 mg CB154/day (4 ml) subcutaneously while

46
control gilts received 4 ml of vehicle solution on the basis of
results of Kraeling et al. (1982) .
Protein
Total recoverable protein concentrations in uterine flushings were
determined by the method of Lowry et al. (1951) using bovine serum
albumin as standard.
Uteroferrin
Concentrations of uteroferrin in uterine flushings were determined
by measurement of acid phosphatase activity (Basha et al., 1979;
Scholsnagle et al., 1974). Values are expressed as umoles
paranitrophenol phosphate (pNP) released per ml per 10 min at pH 4.9
and 37 C.
Calcium
The Calcette 4009 (Precision Systems, Inc., Sudbury, MA) was used
to determine calcium concentrations using ethylene glycol-bis-N-
N'tetraacetic acid (EGTA) for fluorometric titration of calcium in an
aqueous solution (Alexander, 1971).
Chloride
Concentrations of chloride in uterine flushings were determined by
a colorometric assay adapted from Hamilton (1966). Uterine flushings
were diluted 1:10 (v/v) with the mercurous reagent in 12 x 75 mm glass
tubes, covered with parafilm and inverted several times. Sodium
chloride was used for the standard curve and yielded a linear

47
relationship (correlation coefficient=0.99; y = intercept +6.3x)
between absorbance (510 nm) and concentration of chloride (mEq/L). The
assay was sensitive at 60 mEq/L.
Sodium and Potassium
A flame photometer (Perkin Elmer 51Ca; Coleman Instruments
Division, Oak Brook, IL) was used to determine concentrations of sodium
and potassium as described previously (Young et al., 1987).
Glucose
The Beckman Glucose Analyzer 2 (Beckman Instruments, Columbia, MD)
was used to determine glucose concentrations as a direct proportion to
oxygen consumption (Bazer et al., 1984).
Leucine-acyl Aminopeptidase (LAP)
This membrane marker protein was used as an index of secretory
activity and its concentration was determined using a colorometric
assay (Zavy et al., 1984). One Sigma Unit (SU) will release 1 umole
(143 ug) of 5-naphthylamine from L-leucyl-p-nahpthylamine per hour at
37 C and pH 7.1.
Prostaglandin (PG) F
Uterine flushings were analyzed for PGF by radioimmunoassay (RIA)
as described by Knickerbocker et al. (1986) using the antibody
characterized by Kennedy (1985) and tritiated PGF2C1
((5,6,8,9,11,12,14,15-3H]:PGF2 Amersham Corporation, Arlington Heights, IL). Standard curves were

48
prepared in charcoal-stripped uterine flushings with known amounts of
radioinert PGF2a (0, 10, 25, 50, 100, 250, 1000 and 2500 pg). A 1:5000
dilution of antiserum enabled detection of 10 pg PGF per tube. Cross
reactivities of PGF2a antiserum with other prostaglandins were: 94% for
PGFla; 2.4% for PGE2 and <0.1% for 13,14, dihydro-15-keto-PGF2a. PGE
and arachadonic acid. Unextracted uterine flushings (0.2 ml) were
assayed for PGF in duplicate. A pool of uterine flushings
(approximately 3 ng PGF/ml) assayed in serial dilutions (0.01, 0.025,
0.05, 0.1 and 0.2 ml with a final volume of 0.2 ml in charcoal stripped
uterine flush) resulted in an inhibition curve that was parallel to,
and not different from the standard curve when tested for heterogeneity
of regression. Further characterization of the assay involved
measurement of known amounts (10, 25, 50, 100, 250, 500, 1000 and 2500
pg) of PGF in charcoal-stripped uterine flushings ([y=-9.6 + l.llx];
where y=amount of PGF measured (pg/0.2 ml) and x=amount of PGF added
(pg/0.2 ml); R2=0.947). Inter- and intra- assay coefficients of
variation were 14.1% and 15.7%, respectively.
Prostaglandin E
Concentrations of PGE2 in uterine flushings were determined using
an assay similar to that described for PGF with a modification (Lewis
et al., 1978) using tritiated PGEZ ([5,6,8,11,12,14,15-3H]:PGE;
specific activity 140-170 Ci/mmole; Amersham Corporation, Arlington
Heights, IL). A 1:6000 dilution of antiserum (Eli Lilly,
Indiannapolis, IN) enabled detection of 5 pg PGE2/tube as different
from zero. Cross-reactivities of PGE2 antiserum with other
prostaglandins were; 24% for PGEi; 1.7% for PGF2 ; 0.1% for 13,14-

dihydro-15-keto-PGF2a, PGFi and arachidonic acid. A pool of charcoal-
stripped uterine flushing (approximately 3 ng PGE/ml) was serially
diluted and assayed as described previously. The inhibition curve was
parallel to, and not different from, the standard curve when tested for
heterogeneity of regression. The assay was further characterized by
measurement of known amounts of PGE2 added to charcoal-stripped uterine
flush ([y=-6.9 + 1.09x]; where y= the amount of PGE2 measured
(pg/0.2ml) and x=the amount of PGE2 added (pg/0.2ml); R2=0.928)].
Inter- and intra- assay coefficients of variation were 9.7 and 12.4%,
respectively.
Prolactin
Concentrations of PRL in serum of gilts were measured by a
radioimmunoassay (RIA) sensitive to 1 ng/ml (Kraeling et al., 1982).
The inter- and intra- assay coefficients of variation were 15.2 and
16.3%, respectively (Kraeling et al., 1982).
Statistics
Data were analyzed by least squares analysis of variance using the
General Linear Models procedures of the Statistical Analysis System
(SAS) (Barr et al., 1979) to detect effects of treatment.
Experiment 1
This experiment determined effects of hypoprolactinemia on
conceptus survival. Ten gilts were mated on Days 0 and 1 of the
estrous cycle and assigned randomly, 5 per treatment group, to receive
either CB154 (100 mg/day) or vehicle (4ml/day) once daily on Days 10

50
through 16 of gestation. Jugular vein blood samples were collected on
Days 10 (preinjection), 15 (during injection) and 20 (4 days
postinjection) using a vacutainer single sample collection method to
avoid prolonged stress. Serum was assayed for concentrations of PP.L to
determine effectiveness of CB154. On Day 25 of gestation, gilts were
injected with sodium thiamylal (lg, i.v.) to induce anesthesia, which
was then maintained with halothane using a closed-circuit gas
anesthetic unit, and subjected to midventral laparotomy. The uterus was
exposed and examined for evidence of normal pregnancy. Each gilt
received 15 mg of PGF2a (Lutalyse, Upjohn Company, Kalamazoo, MI) to
terminate the pregnancy. Gilts experienced two normal estrous cycles
before being used in Experiment 2. This protocol was necessary because
of the requirement that CB154-treated gilts be euthanized.
Experiment 2
This experiment was to determine effects of hypoprolactinemia on
uterine secretory activity. The protocol was to mimic effects of
estrogens from conceptuses on endometrial secretory activity during the
time of maternal recognition of pregnancy without interactions with
other conceptus products (Geisert et al., 1982b). The 10 gilts used in
Experiment 1 were assigned randomly, 5 per treatment group, to receive
either CB154 (100 mg/day) or vehicle (4ml/day) on Days 10 and 11 of the
estrous cycle. All gilts then received 5 mg of estradiol valerate
(i.m.) on Day 11. On Day 12, uterine flushings and a jugular vein
blood sample were collected.

51
Results
Effects of Hypoprolactinemia
Administration of CB154 to pregnant gilts (Experiment 1) decreased
circulating concentrations of PRL by 40% (P<0.06) on Day 15. Prolactin
levels (+0.33 ng/ml) were 5.0, 3.3, and 4.4 ng/ml and 5.2, 5.0, and
4.4 ng/ml on Days 10, 15 and 20 of gestation for gilts that received
CB154 and vehicle only, respectively. The number of corpora ltea
(CL) was not different on Day 25 of gestation between gilts that
received CB154 (13.6 +0.9) and vehicle (14.0 +0.9) and litter size was
similar between gilts that received CB154 (7.3 +1.75) and vehicle (8.4
+1.75).
Administration of CB154 to cyclic gilts (Experiment 2) decreased
circulating concentrations of PRL by 50% (2.7 +0.33 ng/ml vs 5.3 +0.33
ng/ml; P<0.06) on Day 12 to levels that were similar to those reported
for CB154-treated pigs by Kraeling et al. (1982), Whitacre et al.
(1981) and Smith and Wagner (1986). The inability to totally supress
circulating PRL in pigs by CB154 administration may be due to PRL
regulation by factors other than dopamine. Previously reported dosages
of 480 mg/day did not affect PRL support of corpus luteum function in
mid-gestational gilts (R.R. Kraeling as cited by Bazer and First,
1983). Total recoverable protein (mg), uteroferrin (umoles), and
glucose (mg) concentrations, respectively, were not different in
uterine flushings from gilts that received CB154 (17.6+2.4 mg; 541+92
umoles and 2.23+0.13 mg) compared to those that received vehicle
(19.2+2.4 mg; 608+92 umoles and 2.35+0.13 mg). However, concentrations
of leucine aminopeptidase were lower (P<0.025) in uterine flushings of
CB154 (140+18 SU) compared to vehicle (230+18 SU) treated gilts.

Figure 3-1: Concentrations of total recoverable (A) calcium,
(B) chloride, (C) sodium and (D) potassium in Day 12 uterine
flushings from cyclic gilts (Experiment 2) treated with CB154
(100 mg/day) or vehicle (VHC; 4 ml) on Days 10 and 11 and
estradiol valerate on Day 11. Treatment effects were
detected for calcium and potassium (P<0.05), as well as
sodium and chloride (P<0.01). The overall SEM was +0.14 for
calcium, +0.12 for chloride, +12 for sodium and +8 for
potassium.

53
E
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T3
k.
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U
(9
o

54
Concentrations of PGF (30+5 vs 17 +5 ng/horn) and PGE (14+2.2 vs 11
+2.2 ng/horn) in uterine flushings were not affected by treatment.
Gilts that received CB154 had lower concentrations of calcium (P<0.03),
sodium (P<0.02), potassium (P<0.01) and chloride (P<0.01) in uterine
flushings (Figure 3-1).
Discussion
Bromocryptine, effectively lowers PRL to near undecteable levels
in rats. In this study, PRL was decreased 40-50% in pigs. Fetal
survival was not affected despite decreased circulating PRL. The
remaining concentrations of circulating PRL may have been adequate for
fetal survival. Additionally, porcine conceptuses may have compensated
through other physiological mechanism, which at this time are unknown,
to maintain the pregnancy. Prolactin modulates endometrial physiology
of rabbits (Daniels et al., 1984; Chilton and Dainels, 1985), but
adminsitration of CB154 to pigs on Days 10-15 of gestation may have
been unable to reverse prior affects of basal PRL on endometrium.
Therefore, although PRL levels were lowered, the endometrium was
already stimulated to respond to conceptus signals. Lowered PRL levels
following conceptus estrogen signal may have blunted the uterine
secretory response, but PRL concentrations were adequate for conceptus
survival. Fetal survival may have been affected if CB154
administration began earlier in gestation to block possible effects or
modulation of PRL on uterine physiology.
However, lowered PRL affected estrogen-stimulated ionic changes in
the uterine lumen, suggesting that PRL may affect the ionic environment
of the developing conceptus. In this study, slight hypoprolactinemia

55
significantly decreased concentrations of calcium, sodium, potassium
and chloride ions in uterine flushings, but did not affect total
protein. The inability of lowered PRL levels to alter secretion of
proteins may be due to lack of effects on ion ratios despite lowered
individual ion levels. The effects of decreasing concentrations of
ions may not have occurred in a temporal pattern necessary to alter in
the secretion of proteins. The aminopeptidase activity in uterine
flushings was decreased in response to hypoprolactinemia suggesting a
decrease in secretory activity (membrane processing) of endometrial
epithelium (Zavy et al., 1984).
The decrease in circulating PRL levels in the present study may
have been compensated for physiologically through other mechanisms,
e.g., an increase in PRL receptor numbers, has been previously
demonstrated for rabbit mammary tissue (Dijane et al., 1977) and rat
liver (Kelly et al., 1979). Hypoprolactinemia may also affect hormonal
regulation at the central nervous system, receptor levels in the
hypothalamus and target tissue (Muldoon, 1985) or intrapituitary
communication (Murai et al., 1988). Additionally, regulation of PRL
secretion by gamma amino butyric acid (Schally et al., 1978; Duvalinski
et al., 1987) in pigs suggests a different control mechanism for which
CB154 may be less effective.
In summary, circulating PRL levels were decreased 40-50% in pigs
following administration of CB154, but fetal survival at Day 25 of
gestation was not affected. Ions and LAP concentrations, but not
secreted proteins, in uterine flushings of estrogen stimulated Day 11
cyclic gilts were decreased. Hypoprolactinemia had begun to affect
uterine secretory function, but changes in ions may not have occurred

56
in the proper ratio or temporal pattern to secretion of proteins.
Additionally, decreases in circulating PRL may have been compensated
for physiologically through other mechanisms.

CHAPTER 4
EFFECTS OF CYSTEAMINE ON CIRCULATING
PROLACTIN LEVELS IN PIGS
Introduction
Cysteamine (CSH; SHz-CH2-CH2-NH2) disturbs the tertiary structure
of prolactin (PRL) and thereby depletes circulating levels of bioactive
PRL (Millard et al., 1982). Several short-term studies of effects of
CSH on PRL levels in rats have been reported (Millard et al., 1982;
McComb et al., 1985; Sagar et al., 1985). Dose response tests
determined that rats administered 90 mg/kg CSH (subcutaneously) had
decreased PRL levels in both the anterior pituitary (2.0 vs 0.57 ug/mg
protein) and serum (3.7 vs 0.44 ng/ml) 4h post-administration. No
adverse effects were reported (Millard et al., 1982). Cysteamine can,
therefore, lower circulating levels of bioactive PRL without
involvement of dopamine or other receptors at the anterior pituitary.
This study investigated the use of CSH for inhibition of circulating
PRL levels in pigs as an alternate method to bromocryptine
administration.
Materials and Methods
Experimental Design
On Day 4 of the estrous cycle, gilts were anesthetized with
thiamylal sodium (1 g, i.v.) and maintained under surgical anesthesia
using a closed circuit anesthesia machine. Gilts were fitted with
57

58
jugular catheters (Ford and Maurer, 1978), allowed 4 days rest and
housed individually to recover from surgery through Day 7. On Day 8,
7-10 ml blood samples were collected at 0800, 1300 and 1930 h. Gilts
were assigned randomly to receive either CSH (100 mg/kg/day) or an
isoosmotic control of ethanolamine (100 mg/kg/day). Administration of
treatment began on Day 10 at 0830h and was scheduled to continue until
Day 16 of the estrous cycle. Blood samples were taken two days prior
to, and following, the treatment period.
Results
Due to unexpected cytotoxic effects of CSH at the injection sites,
(tissue necrosis and gangrenous leasions), this project was cancelled
after four gilts received treatments for various lenghts of time.
Blood samples were analyzed for PRL concentrations and results are
summarized in Figure 4-1. Due to the inconsistent number of blood
samples and limited data, statistical analyses were not conducted.
Administration of CSH lowered serum PRL levels about 30% (1.97 vs 2.7
ng/ml) of controls after three days and about 70% (1.33 vs 4.32 ng/ml)
after five days of treatment. Comparison of PRL levels from CSH- and
CB154-treated gilts (Chapter 3) on a similar "day post-injection" basis
revealed that CSH lowered serum PRL levels 67% more than CB154.
Interestrous intervals and (arbitary) cytotoxic reactions to the
treatments are summarized in Table 4-1. A few comments on these
results are in order: Gilt 328 started the experiment in which 100
mg/kg dose was given twice daily to prevent possible increases in PRL
in the evening. This dosage was very cytotoxic, therefore CSH was
administered to gilt 305 at the same dose but only once a day in the

Figure 4-1: Mean concentrations of prolactin (ng/ml) in
serum of cyclic gilts treated with cysteamine (solid line) or
ethanoloamine (dashed line) from Days 10-16 (denoted by
arrows).
59

60
Table 4-1: Effects on interestrus interval and cytotoxicity
reaction to cysteamine (CSH) or ethanoloamine (control)
administration to cyclic gilts.
Gilts
Group
Dose
mg/day
#Inj
IB1 Smpl
Cytotoxicity
score
Cycle length
days
328
CSH
200
9
19
10
28+
326
Control
100
7
33
5
30
305
CSH
100
7
33
7
45+
12
Control
100
3
33
3
23

61
AM. The other gilts also had cytotoxic reactions to the injections and
were rated arbitarily on a 1 to 10 scale. The cycle lengths are
reported, although of limited value since CSH-treated gilts did not
return to estrus before being euthanized.
Discussion
These data suggest that CSH may be effective in decreasing
circulating levels of bioactive PRL and interestrous intervals may have
been increased in gilts that received CSH. Additionally, the gross
morphological appearance of the reproductive tract included atrophy and
regression (see Figure 4-2). Although CSH appeared to be effective in
lowering PRL levels and changing uterine appearance, use of this
compound in gilts in not warranted due to the severe cytotoxicity it
induced when administered subcutaneously at the dosage used for rats.

62
Figure 4-2: Reproductive tracts from gilts treated with
either (A) ethanolamine or (B) cysteamine.

CHAPTER 5
ESTABLISHMENT OF HYPERPROLACTINEMIA
BY ADMINISTRATION OF EXOGENOUS PORCINE PROLACTIN TO PIGS
Introduction
Establishment of function for a hormone can be acheived through
administration of that hormone followed by studies of the physiological
changes it induces. Circulating prolactin (PRL), however, is subject to
increases due to stress such as those that may be experienced during
frequent blood sampling and confinement. Therefore, this study was
conducted to establish that hyperprolactinemia could be acheived by
injecting porcine PRL twice daily. This was done as a separate
experiment to avoid compromising uterine secretory responses in
subsequent experiments (Chapter 6) due to confinement and chronic
bleeding of the gilts which would likely cause stress-induced release
of PRL.
Material and Methods
Animals
Crossbred gilts of similar age (7 to 9 months) and weight (110 to
120 kg) were used in all studies after experiencing at least two
estrous cycles of normal length (18-22 days). Using intact boars,
gilts were observed for estrus daily and the first day of behavioral
estrus was designated Day 0.
63

64
Catheterizations
On the assigned day, gilts were anesthetized and fitted with
indwelling jugular catheters (Ford and Mauer, 1978) which were
maintained with 200 IU/ml heparin:saline solution. Following surgery,
gilts were housed individually until after collection of the final
blood sample and removal of the catheters.
Hormones
Exogenous porcine prolactin (PRL; USDA-B-1; gift from Dr. Douglas
Bolt, National Animal Hormone Program Director) was diluted in
phosphate buffered saline (PBS; 1 mg/ml; pH 7.2), aliquoted into 1.2 ml
volumes and stored at 4 C until injected subcutaneously.
Experimental Design
Six nonpregnant gilts, 3 per treatment group, were assigned
randomly to receive either porcine PRL (1 mg) or vehicle (SAL; 1 ml
saline). On Day 7, gilts were anesthetized and fitted with indwelling
jugular vein catheters. Jugular vein blood (7 ml) samples were
collected on Days 10 through 13 of the estrous cycle at 0730, 1000,
1200, 1930 and 2400h. Prolactin or saline was administered
subcutaneously at 0800 and 2000h, 30 min after the morning and evening
blood samples, on Days 10 through 14. Blood samples were assayed for
concentrations of PRL in serum.
Prolactin
Concentrations of PRL in serum of gilts were measured by a
radioimmunoassay (RIA) sensitive to 1 ng/ml (Kraeling et al., 1982).

65
The inter- and intra- assay coefficients of variation were 15.2 and
16.3%, respectively.
Statistics
Data were analyzed by least squares analysis of variance using the
General Linear Models procedures of the Statistical Analysis System
(SAS) (Barr et al.f 1979). Included in the model were the effects of
pig, treatment, time and treatment by time interaction.
Results
Effects of administration of exogenous porcine PRL are presented
in Figure 5-1. Concentrations of PRL in serum increased within 2h in
gilts that received exogenous porcine PRL. Over the 4 days of
administration, concentrations of PRL were 4.5-fold higher (P<0.001)
for gilts that received exogenous porcine PRL (19.6+1.24 ng/ml)
compared to control gilts (4.3 +0.13 ng/ml).
Discussion
Administration of exogenous porcine PRL at 12 h intervals was
effective in elevating the circulating levels of PRL. Prolactin levels
were increased at 2 h post-administration and remained elevated
throughout the treatment period. Additionally, the increase in PRL
levels were not pharmacological since concentrations of PRL in pigs
during estrus and the late luteal phase are 15 to 20 ng/ml (Brinkley et
al., 1973; Dusza and Kryzmowska, 1979).

66
10 11 12 13
Day of Estrous Cycle
Figure 5-1: Concentrations of immunoreactive prolactin in
serum during administration of 1 mg porcine prolactin,
(circles), or 1 ml saline (squares), at 0800 and 2000h
(denoted by arrows) on Days 10 through 13 of the estrous
cycle. Blood samples were collected at 0730, 1000, 1200,
1930 and 2400 h. The SEM were +1.24 ng/ml for prolactin
treated and +0.12 ng/ml for saline treated gilts.

CHAPTER 6
EFFECTS OF HYPERPROLACTINEMIA ON PROGESTERONE AND ESTROGEN
INDUCED UTERINE SECRETORY RESPONSE IN PIGS
Introduction
As mentioned in Chapter 3, early endocrinological studies involved
manipulation of endogenous hormones to establish physiological roles at
the tissue of interest. Converse to lowering endogenous hormones,
manipulations which increase endogenous hormone levels followed by
observation of physiological changes can also allow insight into a
hormones potential involvement in function. Several mechanisms have
been used, ranging from injection of crude tissue homogenates to
sophisticated gene manipulations. Each technique acheived a similar
endpoint, increased or supplemented endogenous hormone levels to allow
to investigation of resulting physiological changes.
Exogenous prolactin (PRL) results in increased endometrial
proliferation and protein secretion (Chilton and Daniels, 1985) in long
term (12 week) ovariectomized rabbits. Prolactin also modulates ion
channels (Falconer and Rowe, 1977), gap junction formation (Sorenson et
al., 1987) auto up-regulates its own receptor (Djaine et al., 1977;
1987) and increases steroid receptors (Daniels et al., 1984). Through
these mechanisms, PRL may affect the uterine environment of pigs.
Uterine secretory function is critical for nourishment of
preimplantation porcine conceptuses. Investigation of PRL interactions
with ovarian steroid hormones, especially estrogen, may explain the
67

68
biphasic responses of the uterus that occur following estrogen
administration (Szego et al., 1978; Geisert et al.( 1982c; Young et
al., 1987) possibly through modification of the uterine environment or
through more rapid effects of the peptide hormones. Therefore,
interactions between PRL and progesterone, in the absence of estrogens
of ovarian origin, were investigated in pigs. Knight et al. (1973)
demonstrated that ovariectomized pigs treated with progesterone alone,
secrete the same proteins after 11 days of treatment as ovarian intact
gilts on Day 15 of the estrous cycle or pregnancy. In a second
experiment, the interaction between estrogen and PRL was investigated
following administration of exogenous estrogen. Administration of
exogenous estrogen on Day 11 of the cycle mimics the maternal
recognition of pregnancy factor, porcine conceptus estrogens, without
confounding effects of other conceptus secretory products (Geisert et
al., 1982c) .
Materials and Methods
Animals
Crossbred gilts of similar weight (100-120 kg) and age (7-9
months) were used in this study after they experienced at least two
normal estrous cycles (18-22 days). In the presence of intact boars,
gilts were observed daily for behavioural estrus. The first day of
behavioural estrous was designated Day 0.
Exogenous Hormone Administration
For chronic steroid treatment, gilts received 200 mg progesterone
(P4; Sigma, St. Louis, MO) in 4 ml corn oil:ethanol solution (90:10,

69
v/v). Control gilts received 4 ml vehicle only. Acute steroid
treatment consisted of 0.5 ml (10 mg/ml) estradiol valerate (E2V;
Squibb, Raritan, NJ) or 0.5 ml corn oil. Exogenous porcine prolactin
(PRL; USDA-B-1; gift from Dr. Douglas Bolt, National Animal Hormone
Program Director) was diluted in phosphate buffered saline (PBS; 1
mg/ml; pH 7.2), aliquoted into 1.2 ml volumes and stored at 4 C until
injected subcutaneously.
Surgical Procedures
Gilts used in studies of PRL effects on P4-induced uterine
secretory components in Experiment 1 were ovariectomized on Day 4 of
the estrous cycle. Gilts were anesthetized, subjected to midventral
laparotomy, the ovaries were exteriorized, all ovarian vessels were
ligated and the ovaries removed with minimal trauma to the uterus.
Uterine flushings were collected as descibed in Chapter 3.
Experiment 1
Seven nonpregnant gilts were ovariectomized on Day 4 of the
estrous cycle and injected with 200 mg P4 once daily from Days 4
through 14. Gilts were assigned randomly to receive either porcine PRL
(n=4; 1 mg) or vehicle (n=3; 1 ml saline) daily at 0800 and 2000h on
Days 4 through 14. Uterine flushings were collected on Day 15.
Experiment 2
Twelve nonpregnant gilts, 4 per treatment group, were assigned
randomly to receive one of three treatments. Gilts in the negative
control group (corn oil only on Day 11) and positive control group (5

70
mg E2V in corn oil on Day 11) received 1 ml saline twice daily at 0730
and 1930h on Days 6 through 11. Gilts in the treatment group received 1
mg porcine PRL in saline twice daily at 0730 and 1930h and 5 mg E2V on
Day 11. Uterine flushings were collected on Day 12, approximately 24h
after treatment with corn oil or E2V. A PRL and corn oil treatment
group was not included because results of Experiment 1 indicated no
interaction between PRL and progesterone on uterine secretory activity.
Uterine Flushings
Uterine flushings were processed and evaluated for total
recoverable protein, uteroferrin, leucine aminopeptidase (LAP),
glucose, calcium, sodium, potassium, chloride, PGF, and PGE as
described in Chapter 3.
Results
Effects of PRL on P4-induced uterine secretory responses are
summarized in Table 6-1. Administration of exogenous PRL did not
affect any of the P4-induced uterine secretory components examined in
the uterine flushings.
Effects of exogenous PRL on estrogen-induced uterine secretory
activity are summarized in Figures 6-1, 6-2, and 6-3. Treatment
effects were detected for (see Figure 6-1A, B and C) total recoverable
protein and total uteroferrin (PCO.OOl), total glucose (P<0.01), and
total leucine aminopeptidase (P<0.05); (see Figure 6-2A, B and D),
total calcium (P<0.03), total chloride (P<0.02), total potassium
(P<0.01); and (see Figure 6-3A) total PGF (P<0.02) in uterine
flushings. There was no effect of treatment on sodium (Figure 6-20 or

71
Table 6-1: Composition of Day 15 uterine flushings from
ovariectomized gilts treated with daily injections of
progesterone and saline or porcine prolactin from Days
4 through 14 (x + SEM).
ITEM
SALINE
PROLACTIN
Total
Protein (mg)
74.3
+19.3
74.0
+16.7
Total Uteroferrinb
(umoles/uterine horn)
4138
+1903
4230
+ 1648
Uteroferrin/mg Protein
45.7
+14.9
51.7
+13.0
Total
Calcium (mg)
0.5
+0.2
0.4
+0.2
Total
Cloride (mg)
1.7
+0.3
1.5
+0.2
Total
Sodium (ug)
125.3
+32.1
93.5
+27.9
Total
Potassium (ug)
170.2
+46.7
131.8
+40.4
Total
Glucose (mg)
2.5
+0.3
2.8
+0.3
Total
L-acyl aminopeptidasec
655
+112
604
+92
Total
PGF (ng/uterine horn)
235
+22
219
+19
Total
PGE (ng/uterine horn)
38.6
+6.7
34.6
+ 5.8
treatment effects were not detected (P>0.05).
bAcid phosphatase activity; umoles p-nitrophenol
released/ml/10 min at 37 C.
cSigma Units: One Sigma Unit will release 1 umole (143 ug)
of B-napthylamine from L-leucine-B-nalpthylamine per hour
at 37 C, pH 7.1.

Figure 6-1: Concentrations of total recoverable (A) protein,
(B) uteroferrin, (C) glucose, and (D) leucine aminopeptidase
(LAP) activity in Day 12 uterine flushings from cylic gilts
(Experiment 2) treated with 1 ml saline (SAL) or 1 mg porcine
prolactin (PRL) at 0800 and 2000h on Days 6-11 and 0.5 ml
corn oil (OIL) or 5 mg estradiol valerate (E2V) on Day 11 of
the estrous cycle. Overall treatment effects were detected
for protein (P<0.001), uteroferrin (P<0.01) and leucine
aminopeptidase activity (P<0.05). Values with different
letters are different (P<0.05). The overall SEM was +2.72
for protein, +272 for uteroferrin, +0.16 for glucose and
+52.7 for LAP.

Total Glucose (mg)
Total LAP (SU)
to
o
o
400
Total Uteroferrin (umoles)
to
o

o
4-
o
o
l
u>

Figure 6-2: Concentrations of total recoverable (A) calcium,
(B) chloride, (C) sodium and (D) potassium in Day 12 uterine
flushings from cyclic gilts (Experiment 2) treated with 1 ml
saline (SAL) or 1 mg porcine prolactin (PRL) on Days 6-11 and
0.5 ml corn oil (OIL) or 5 mg estradiol valerate (E2V) on Day
11 of the estrous cycle. Overall treatment effects were
detected for calcium and chloride (P<0.05) as well as
potassium (P<0.01). Values with different letters are
different (P<0.05). The overall SEM was +0.3 for calcium,
+0.22 for chloride, +10.7 for sodium and +9.23 for potassium.

Total Sodium (ug)
W ffS V|
o o o
Total Potassium (ug)
Ul
o
100
'-J
Total Chloride (mg)
ro
09

76
50 --
\
60
25 -
Sal PRL Sal
Oil |E2VJ
B
50 --
o
JC
\
bo
e
25
LU
u
CL
o
Sal PRL Sal
Oil |E2V|
Figure 6-3: Concentrations of (A) PGF and (B) PGE in Day 12
uterine flushings from cyclic gilts (Experiment 2) treated
with 1 ml saline (SAL) or 1 mg porcine prolactin (PRL) on
Days 6-11 and 0.5 ml corn oil (OIL) or 5 mg estradiol
valerate (E2V) on Day 11 of the estrous cycle. Overall
treatment effects were detected for PGF (P<0.01). Values
with different letters are different (P<0.05). The overall
SEM was +10.2 for PGF and +6.7 for PGE.

77
PGE (Figure 6-3B) in uterine flushings. Additionally, gilts treated
with PRL+E2V had greater amounts of glucose (P<0.01) and PGF (P<0.01)
in uterine flushings than gilts receiving E2V alone. But, values for
glucose and PGF were not different for gilts treated on Day 11 with E2V
or corn oil. The specific activity of uteroferrin (umoles/mg protein)
was higher (P<0.01) in uterine flushings from gilts treated with
PRL+E2V (106.5 +9.3) compared to gilts receiving SAL+E2V alone (56.7
+9.3) or SAL+corn oil (8.4 +9.3). Gilts receiving PRL+E2V had greater
uterine secretory responses for total protein, potassium and leucine
aminopeptidase compared to gilts receiveing SAL+E2V, but differences
were not statistically significant.
Discussion
Exogenous PRL interacted with estrogen, but not progesterone, to
cause significant effects on uterine epithelial secretory activity.
Shifts in concentrations of ions could account for the earlier release
of several uterine secretory components; although such shifts could not
be not accounted for by static measurments on Day 12 in the present
study. Shifts in ions occur prior to protein secretion during early
pregnancy (Geisert et al., 1982b; Bazer et al., 1984) and in response
to administration of exogenous estradiol on Day 11. With respect to
the secretory profiles of uterine components in the experiments
reported here, exogenous PRL may have advanced the rapid release and
reuptake of calcium which characteristically follows estrogen
stimulation and precedes accumulation of proteins in the uterine lumen.
Luminal calcium flux may have occurred early on Day 11 in
hyperprolactinemic gilts. Therefore, calcium measured in uterine

78
flushings on Day 12 could be influenced by advanced calcium reuptake
that does not normally occur until Days 13-14 of pregnancy. This may
account for the increase in glucose and uteroferrin on Day 12 for PRL
treated pigs compared to previous reports that glucose and uteroferrin
concetrations do not increase until Days 14 to 16 during a normal
pregnancy (Bazer et al., 1984). Hyperprolactinemia resulted in
increased concentrations and specific activity of uteroferrin in (Day
12) uterine flushings. Increases in this protein usually occur on Day
14 of gestation (Zavy et al., 1982). Since there was not a concomitant
increase in aminopeptidase, a membrane marker protein indicative of
cell membrane processing and secretory activity, PRL may have altered
uteroferrin synthesis resulting in an increase in the releaseable pool
stored within intracellular secretory granules and available for
release following estrogen stimulation. Additionally,
hyperprolactinemia resulted in increased total glucose in uterine
flushings on Day 12 which normally does not occur until Days 14 to 16
of pregnancy (Zavy et al., 1984). Administration of PRL appeared to
advance the ability of uterine epithelium to respond to estrogen-
induced secretory events.
Stimulation of secretion of prostaglandins of the F, but not the
E, series suggests that effects of PRL may be on the uterine
epithelium. Previous reports have localized PGF and PGE secretion to
the endometrial epithelium and stroma, respectively, of cows (Grasso et
al., 1987). Prolactin exerts luteostatic effects in several species
(Murphy and Rajkumar, 1985), primarily through stimulation of ovarian
steroid production. Prolactin appears to advance the ability of the
uterus to respond to estradiol since an increase in luminal PGF

79
occurred 48 to 72 h following a single injection of estradiol valerate
on Day 11 to cyclic gilts (Geisert et al., 1982b). During
establishment of pregnancy in the pig, PGF secretion must be redirected
from an endocrine to an exocrine direction (Bazer and Thatcher, 1977)
to protect the corpus luteum from regression. The ability of PRL to
stimulate secretion of PGF and enhance its secretion into the uterine
lumen (Mirando et al., 1988) is a novel finding suggesting that PRL
plays a luteostatic role, in conjunction with estrogen, in early
pregnancy of pigs.
The present findings in the pig, support the hypothesis that PRL
modulates uterine secretory activity during establishment of pregnancy
as previously described for long-term ovariectomized rabbits (Chilton
and Daniels, 1985). Prolactin increased uterine secretory function and
caused differential changes in ions, increased secretion of proteins,
PGF and glucose. Although the mechanism(s) by which PRL influences the
endometrial secretory profile is not known, several mechanisms may be
involved. These include 1) activation of ion channels to facilitate
transport and secretion of cellular components (Petersen and Maruyama,
1985) or stimulate membrane cycling of calcium (Alkon and Rasmussin,
1988); 2) increased estrogen binding by cells of rat liver (Chamness
et al., 1975) and uterus (Leung and Sasaki, 1973); 3) facilitation of
formation of gap junctions to increase intercellular communication
(Sorenson et al., 1987), and 4) up-regulation of PRL receptors to
increase membrane fluidity (Dave and Witorsch, 1985) and increase the
availablity of cryptic hormone receptors. Dave et al. (1983) observed
an increase in PRL binding during early pregnancy which they attributed
to alterations in membrane fluidity.

80
In summary, during the time of maternal recognition of pregnancy
in the pig, PRL interacts with estrogen, rather than progesterone, to
influence uterine endometrial secretory activity. This selective
interaction with estrogen may allow the uterine endometrium to respond
maximally to the estrogen signal from the porcine conceptus which
allows establishment of pregnancy.

CHAPTER 7
DEVELOPMENT OF A HOMOLOGOUS RADIORECEPTOR ASSAY FOR
PORCINE ENDOMETRIAL PROLACTIN RECEPTORS
Introduction
Prolactin (PRL) binds to high affinity, low capacity receptors
(Shiu et al., 1973; Sakai et al., 1985) in target tissues and induces
responses that are biologically important to the reproductive system.
Prolactin binding sites have been reported in reproductive tissues of
sheep (Posner et al., 1974b), rats (Williams et al., 1974), humans
(Healy, 1984), mink (Rose et al., 1983), rabbits (Grissom and
Littleton, 1988) cow (Posner et al., 1974b) and pig (Posner et al.,
1974b; DeHoff et al., 1984; Bramley and Menzies, 1987). Prolactin
exerts effects on steroidogenesis in corpora ltea (CL) (Veldhuis et
al., 1980; Brambley and Menzies, 1987), transport of water by placental
membranes (Goldstein et al., 1980), uterine endometrial proliferation,
protein synthesis and secretion (Chilton and Daniels, 1985; Young and
Bazer, 1988), enhanced secretion of prostaglandin F2d into the uterine
lumen (Mirando et al., 1988), as well as mammary growth and lactation
(Vonderhaar et al., 1984).
Endometrial PRL binding sites have been detected in several
species. Local production of PRL from uterine decidual tissue may
affect uterine physiology in an autocrine or paracrine manner (Healy,
1984; Jayatilak and Gibori, 1986). However, in species with
noninvasive implantation, such as the pig, changes in the number of PRL
81

82
receptors in the endometrium may determine effects of PRL on the
physiology of the uterus. While these receptors may respond to
decidual PRL and/or placental lactogen, the pig has neither and
circulating levels of immunoreactive PRL are relatively constant
throughout pregnancy (Dusza and Krzymowska, 1981). Therefore, PRL
effects are probably regulated by changes in the PRL receptor
population or receptor characteristics.
Heterologous (ovine) PRL radioreceptor assays (RRA) enabled
detection of PRL receptors (fmoles/g wet weight; fm/gww) in porcine
endometrium as early as Day 15 (75 fm/gww), increasing receptor numbers
to Days 45 and 75 (575 and 700 fm/gww, respectively) and decreased
receptor numbers by Day 90 (65 fm/gww) of gestation (DeHoff et al.,
1984). These changes in PRL receptor numbers are temporally associated
with increases in concentration of circulating estrogens (DeHoff et
al., 1986), PRL-stimulated endometrial proliferation, induction of
steroid receptors, protein secretion (Chilton and Daniels, 1985),
epithelial ion transport (Rabee and McCoshen, 1986), transport of
placental water (Goldstein et al., 1986) and fetal growth (Nicoll et
al., 1985).
Several heterologous RRAs have been described for PRL and are used
routinely (Shiu et al., 1973; Waters et al., 1974; Kelly et al., 1979)
but may be complicated by indiscriminant binding due to effects of
heterologous hormones (Nicoll, 1982). However, PRL, a member of the
growth hormone-prolactin-placental lactogen family, can crossreact with
other hormones in that family in RRAs. Prolactin from one species
often has biochemical homology and physiological actions related to
growth hormone of another species. Additionally, high variablity in

83
structural properties between PRL of different species (Nicoll et al.(
1985) could contribute to inaccurate measurement of receptors within a
species when a heterologus RRA is used for receptor quantification. A
homologus RRA has the advantage of insuring that a receptor is specific
for the hormone under investigation and that the results can be
accurately correlated to the in vivo hormonal environment and provide a
method for investigating changes in in vivo PRL receptor populations
that are of physiological interest. Additionally, development of a
homologous RRA for porcine tissues may indicate the feasibility of
developing and using homologous RRAs for other species or hormone
systems.
Materials and Methods
Hormones
Porcine PRL, growth hormone (GH), follicle stimulating hormone
(FSH), luteinizing hormone (LH) and ovine PRL were from the USDA (grade
USDA-B-1) and were generously supplyed by Dr. Douglas Bolt (Director of
Animal Hormone Program).
Iodination of Hormones
Porcine PRL (USDA-B-1) was iodinated using the Iodo-gen procedure
adapted from Markwell and Fox (1978). Radioactivity in the eluant from
a gel filtration column separating protein bound iodine from free
iodine was monitored by counting 10 ul aliqouts from each fraction to
detect labelled PRL. The peak (approximately 750,000-1,000,000 cpm/10
ul) and the descending 2-3 tubes were tested for PRL receptor binding
activity. Specific activity (approximately 83 uCi/ug) was determined

84
by trichloro acetic acid (TCA) precipitation prior to column elution
and calculating the incorporation of 125I into PRL (see appendix C).
Ovine PRL (USDA-B-1) was iodinated as described for porcine PRL with a
specific activity of 97 uCi/ug. The t1231]-PRL fractions were diluted
1:1 in assay buffer, stored at 4 C and used within 10 days.
Animals
Pregnant gilts, anesthetized on Day 75 using sodium thiamylal (lg)
and maintained under surgical anesthesia on a closed circuit anesthetic
machine using Halothane, were subjected to a midventral laparotomy and
hysterectomized. Endometrium was separated from placental and
myometrial tissue layers and placed on ice. Liver and mammary gland
tissue was collected from Day 75 pregnant gilts at slaughter and
processed as described below. Day 20 pregnant rats were euthanized
with an overdose of sodium pentobarbital and livers were excised and
processed. Rabbit mammary glands were prepared by the method of Shiu
et al. (1973) and obtained following euthanasia with an overdose with
sodium pentobarbital.
Membrane Preparation
Endometrium from Day 75 pregnant gilts and liver from Day 20
pregnant rats were collected and placed on ice, rinsed three times in
ice-cold 0.9% (w/v) saline, rinsed in ice-cold homogenization buffer
(100 mM Tris, 150 mM sodium chloride, 50 mM ethyleneglycol-bis[B-
aminoethyl ether] N,N'-tetraacetic acid [EGTA], 50 mM ethylenediamine
tetraacetic acid [EDTA], 300 mM sucrose, 1 mM
phenylmethylsulfonylfluoride (PMSF) and Aprotinin (400 Kallikrin

85
inhibitory units/ml; pH 9.0), and then frozen at -70 C within 15 min
following collection. Tissue was thawed in ice-cold homogenization
buffer (4 ml/g tissue) on ice, processed using a Polytron homogenizer
(3 x 10 sec bursts at full speed), and homogenates centrifuged at
15,000 x g for 20 min at 4 C. The resulting supernatant was centrifuged
at 100,000 x g for 120 min at 4 C. The 100,000 x g pellet was
resuspended in buffer [100 mM sodium phosphate, 150 mM NaCl, 10 mM
EDTA, 0.1% (w/v) NaN3, pH 7.6], aliquoted and stored at -70 C until
assayed.
Protein Determination
Membrane protein concentrations were determined by the method of
Lowry et al., (1951) using bovine serum albumin as standard.
Chaotropic Treatment of Membranes
To ensure removal of endogenous hormone from endometrial prolactin
receptors, membranes were treated with magnesium chloride (MgCU) as
described by Kelly et al. (1979). A 75 ul aliqout containing 150 ug
protein was added to 500 ul 4 M MgCl2, vortexed, and incubated for 5
min. The reaction was stopped by addition of 3 ml ice-cold assay
buffer [10 mM sodium phosphate, 150 mM NaCl, 10 mM EDTA, 0.1% (w/v) BSA
and 0.1% sodium azide, pH 7.6]. Samples were centrifuged at 1800 x g
for 15 min at 4 C, decanted, and placed in an ice bath. Ice-cold assay
buffer (300 ul) was added immediately and tubes were vortexed
extensively to ensure pellet resuspension. All assays were conducted
using polypropylene 12 x 75 mm tubes (Sarstedt, Princeton, NJ) which
reduced nonspecific binding when compared to borosilicate glass tubes.

86
Prolactin Radioreceptor Assay (RRA) Procedure
Following treatment of the membrane preparations with MgCl2,
binding assays were conducted as adapted from Haro and Talamantes
(1985a). Radioinert porcine PRL (100 ul) was added in serially
increasing concentrations (0-5120 ng). Radiolabeled porcine PRL (100
ul; 45,000 cpm; 0.24 ng) was added and tubes were vortexed extensively.
Total assay volume was 500 ul. The binding reaction was conducted at
room temperature (25 C) for 24 hours, terminated by addition of 3 ml
ice-cold assay buffer, and centrifuged at 3500 x g for 30 min. Samples
were decanted, inverted to drain, and pellets counted (1 min) to detect
gamma radioactivity (GammaTrac 1191, Nuclear Chicago Corp., Des Plains,
IL). Nonspecific binding, 7-4% of total binding, was determined by the
amount of radioactivity bound in the presence of either 2560 or 5120 ng
unlabelled hormone, respectively. Results are expressed as percent
bound [(total bound minus nonspecific binding/total counts) X 100].
Total bound binding represents radioactivity in the absence (Bo) or
presence (1-1000 ng) of radioinert hormone. In each experiment the
total and nonspecific binding was determined in triplicate. The intra-
and inter- assay coefficients of variation for receptor number were 9
and 10%, respectively, and for Ka, 18 and 23%, respectively.
Effects of Magnesium Chloride Molarity
Binding of porcine t1231]-PRL was tested as a function of MgCl2
molarity. Membrane preparations from Day 75 pregnant porcine
endometrium (300-500 ug), amnion, chorion and post-parturient (rabbit
or pig) mammary tissues were tested for binding of [123I]-PRL following

87
treatment of the membranes with increasing concentrations (0,1,2,4, and
6 M) of MgClz.
Binding of Porcine [12aI]-PRL as a Function of Increasing Membrane
Protein Concentration
Porcine [12SI]-PRL (45,000 cpm 0.24 ng) was incubated in 500 ul of
assay buffer containing increasing concentrations (0 to 500 ug) of Day
75 pregnant pig endometrial membrane protein in the presence (1 ug) or
absence of unlabelled porcine PRL. This assay was conducted in a
similar manner with identical protein concentrations of untreated
membranes and membranes pretreated with 4 M MgCl2 or distilled water.
Determination of Protein:Radiolabelled Ratio Concentration
Three levels of protein (50, 150, 250 ug) were assayed with
increasing amounts of radiolabelled PRL (15,000; 30,000; 45,000;
60,000; 75,000 and 90,000 cpm) in a total volume of 500 ul assay
buffer. Each protein:radiolabel concentration combination assay was
conducted in the presence (1 ug) and absence of unlabelled PRL to
determine maximal specific binding.
Analysis of Porcine [*23I]-PRL Association Kinetics
At time zero, [125I]-PRL (45,000 cpm) was incubated at 4 and 25 C
with 150 ug of Day 75 pregnant pig endometrial or Day 20 pregnant rat
liver membranes in 500 ul assay buffer either in the presence (1 ug) or
absence of unlabelled porcine PRL. Specific binding of porcine [12SI]-
PRL was determined after 1.5, 4, 8, 12 and 24h.

88
Dissociation of Porcine [123I]-PRL
Porcine [123]-PRL (45,000 cpm; 0.24 ng) was incubated with 150 ug
Day 75 pregnant pig endometrial or Day 20 pregnant rat liver membrane
at 25 C in 500 ul of assay buffer in the presence (1 ug) and absence of
unlabelled porcine PRL for 24 hours. After incubation, the membranes
were washed in 3 ml ice-cold assay buffer, centrifuged (2300 x g; 30
min 4 C), and decanted to remove any free hormone. Pellets were
resuspended in fresh assay buffer (500 ul) containing 5 ug unlabelled
porcine PRL. Dissociation of previously bound porcine f1 231]-PRL was
determined at various time points (0, 1, 2.5, 5, 7.5, 10, 12, 24, 32
and 48h). Labeled porcine PRL specifically bound to membranes was
expressed as a fraction of the amount bound at equilibrium.
Hormonal Specificity of Porcine [*231]-PRL
Porcine [123I]-PRL was incubated with 150 ug of Day 75 pregnant
pig endometrial or Day 20 pregnant rat liver membranes for 24 hours at
25 C in the presence and absence of increasing concentrations of
unlabeled porcine PRL (0-5120), porcine growth hormone (GH), porcine
luteinizing hormone (LH) and porcine follicle stimulating hormone (FSH)
(0-10240 ng). Binding assays were conducted as described previously.
Heterologous versus Homologous Binding and Displacement of Ovine
and Porcine PRL
The effects of heterologous (ovine) and homologous (porcine)
PRL were investigated using radiolabelled and radioinert forms of each
hormone. Assays were conducted using Day 75 pregnant pig endometrial
membranes in the following combinations: 1) ovine [231]-PRL versus

89
unlabelled ovine PRL; 2) ovine [12,I]-PRL versus unlabelled porcine
PRL; 3) porcine [1231]-PRL versus unlabelled ovine PRL and 4) porcine
[i25i]_prl versus unlabelled porcine PRL. This enabled investigation
of the combination of radiolabelled and unlabelled hormones that would
result in the most desirable displacement from porcine endometrial PRL
receptors. Assays were conducted as described previously.
Analysis of Binding Data
The amount of bound radiolabelled hormone obtained in the various
assay procedures was subjected to binding analysis using a LIGAND
program (Munson and Rodbard, 1985) adapted for the Macintosh Computer
(Apple Computers, Los Angeles, CA). This program analyzes data through
Scatchard (1949) computations to generate binding responses. Binding
data were tested for best fit assuming 1 and 2 binding sites.
Results
The effect of increasing MgCl2 molarity is depicted in Figure 7-1.
All tissues showed very low binding (<3%) when not pretreated with
MgCl2. Membranes from rabbit mammary gland showed highest binding when
pretreated with 2M MgCl2 whereas membranes from Day 75 pig mammary
gland, chorion and endometrium had the highest total binding when
pretreated with 4M MgCl2. Porcine anmiotic membranes had the greatest
binding when pretreated with 6M MgCl2; however, all other membranes had
similar or decreased binding at that molarity.
Increased binding of porcine [1231]~PRL occurred with increasing
concentrations of membrane protein only when membranes were pretreated
with 4M MgCl2 (Figure 7-2). Untreated membranes or membranes that were

Percentage 125l Porcine Prolactin Bound
90
Figure 7-1: Effects of increasing magnesium chloride molarity
on binding of porcine [125I]-prolactin by membranes from Day
75 porcine endometrium, amnion, chorion, as well as post
parturient pig and rabbit mammary gland (300 ug).

91
Micrograms Membrane Protein
Figure 7-2: Effects of increasing protein concentrations of
Day 75 porcine endometrial membranes on binding of porcine
[i23i]_prolactin. Membranes were treated with either 4 M
MgCl2; 4 M MgCl2 and incubated with 1 ug unlabelled porcine
prolactin (NSB); untreated membrane (Bo); or distilled water
(H20).

92
pretreated with 500 ul distilled water had low binding similar to that
obtained when MgCli-pretreated membranes were incubated in the presence
of unlabelled porcine PRL.
The optimal membrane proteinrradiolabel concentration ratio was
150 ug membrane using 45,000 cpm porcine [123I]-PRL. Maximal specific
binding of approximately 20% for Day 75 pig endometrial membranes was
slightly lower than the 25% specific binding observed for Day 20
pregnant rat liver membranes under similar assay conditions.
The Day 75 porcine endometrial and Day 20 rat liver membrane
preparations were tested for binding saturability at 4 and 25 C (Figure
7-3). Binding curves were similar for the two membrane sources at each
temperature. Porcine endometrial and rat liver membranes reached
saturation at 24h attained specific binding of approximately 20% and
25%, respectively, at 25 C. At the lower temperature, porcine
endometrial and rat liver membranes had specific binding values of 4
and 7%, respectively.
Assays to determine dissociation kinetics suggested that the
majority of the porcine t1291]-PRL displacement occurred within lOh.
The dissociation rate constant (Kd) for the porcine endometrial PRL
receptor (Figure 7-4a) was slightly lower than that for rat liver
receptor (Figure 7-4b) with Kd's of 3.79 x 10_6/s and 9.3 x 10_6/s,
respectively. Over the remaining 38h only slight displacement
occurred for the porcine endometrial PRL receptor (Kd=1.63 x 10-6/s).
Additionally, displacement of porcine [129I]-PRL from receptor by 5 ug
unlabeled porcine PRL was incomplete for both porcine endometrial (60%)
and rat liver (40%) membranes. This is consistent with previous
results by van der Gugten et al. (1980) and Kelly et al. (1983).

Percent Specific Binding
93
Time in Hours
Figure 7-3: Binding of porcine [12SI]-prolactin by magnesium
chloride treated Day 75 porcine endometrial (circles) or Day 20
rat liver (squares) membranes at 4 C (dashed line) or 25 C (solid
line).

Figure 7-4: Dissociation kinetics assay for magnesium
chloride treated (A) Day 75 porcine endometrial or (B) Day 20
rat liver membranes. Tissue was incubated with porcine
[i2 ai]-prolactin in the presence (1 ug) and absence of
unlabelled porcine prolactin for 24 h at 25 C, rinsed, and
fresh buffer containing 5 ug unlabelled porcine prolactin was
added. Specific binding was determined at each time point.

In (B/Bo)
NSB subtracted
i i i i I
o o o o o
00
Hours
In (B/Bo)
NSB subtracted
l I i i i i I
o o o o o o o

ro ro ro o o

96
Radioinert ovine PRL
was
less effective
than
porcine PRL in
displacing [123I]-PRL of
either
species
from
porcine endometrial
membranes (Figure 7-5).
The
homologous
RRA
system
resulted in 75%
displacement when radioinert porcine PRL competed with radiolabelled
porcine PRL. Additionally, maximum specific binding was higher for
radiolabeled porcine PRL (25%) compared to radiolabelled ovine PRL
(18%). The percent displacement for the four hormone combinations
(labelled/unlabelled) were as follows: 40% for 123I-ovine/ovine; 30%
i23I-porcine/ovine; 60% 125I-ovine/porcine; and 75% for 125I-
porcine/porcine. Thus, the homologous (label vs unlabel)
hormone/membrane combination resulted in a more effective displacement
of PRL from Day 75 porcine endometrial membranes.
Hormonal specificity of the porcine endometrial PRL receptor is
depicted in Figure 7-6. Porcine [125I]-PRL is not displaced by porcine
LH or FSH and only slight displacement was detected for porcine GH when
compared to the displacement observed with radioinert porcine PRL
(Figure 7-7); approximately 100-fold more porcine GH than porcine PRL
was needed to displace a comparable amount of radiolabelled porcine PRL
from the endometrial receptor. Binding constants for GH could not be
determined when crossreactivity assays were analyzed using the LIGAND
program.
Homologous competitive displacement assays were conducted with Day
75 pregnant endometrial membranes. Scatchard analysis of binding data
(Figure 7-8) indicated an equilibrium dissociation constant (Kd) of
3.06 x 10'8 M (Ka = .326+0.011 x 10 M-1). Pretreatment of membranes
with MgCl2 did not alter the Kd but did increase maximum binding 4- to

Figure 7-5: Binding and displacement of ovine and porcine
prolactin from magnesium cloride treated Day 75 porcine
endometrial membranes. Porcine [l23I]-prolactin vs unlabelled
porcine prolactin (P/p); porcine t123I]-prolactin vs
unlabelled ovine prolactin (P/o); ovine [123I]-prolactin vs
unlabelled ovine prolactin (O/o); and ovine t123I]-prolactin
vs unlabelled porcine prolactin (0/p).

92
Unlabeled Hormone (Hog M)

Percent Specific Binding
99
Figure 7-6: Crossreactivity of unlabelled porcine prolactin
(squares; pPRL), porcine growth hormone (triangles; pGH),
porcine luteinizing hormone (circles; pLH) and porcine
follicle stimulating hormone (diamonds; pFSH) to porcine
[125I]-prolactin with magnesium chloride treated Day 75
porcine endometrial membranes.

Percent Specific Binding
100
0 2.5 10 40 100 200 500
Unlabeled Hormone (ng)
Figure 7-7: Crossreactivity between unlabelled porcine
growth hormone (dashed line) or porcine prolactin (solid
line) and porcine t123I]-prolactin with magnesium chloride
treated (circles) Day 75 porcine endometrial and (squares)
Day 20 rat liver membranes.

101
Figure 7-8: Scatchard analysis of porcine C123I]-prolactin
displaced by unlabelled porcine prolactin using magnesium
chloride treated Day 75 porcine endometrial membranes.
Actual and 'best fit' data points are represented by open
squares and closed triangles, respectively.

102
6-fold (7% vs 29%) and resulted in an increase in the number of
available receptors (285 fmoles vs 34 pmoles/mg protein).
Conservative interpretation of Scatchard analysis of competitive
inhibition curves resulted in one binding site. However, two binding
sites were also determined, although not as consistently, through the
LIGAND analysis. These binding sites contained a higher affinity (109
or 1010 M-1) but low capacity component as well as a lower affinity
(107) but high capacity component than when data were analyzed and
displacement curves fitted for a single receptor site.
Discussion
Prolactin receptors are generally measured by RRAs employing ovine
PRL (Kelly et al., 1979, 1983; DeHoff et al., 1984; Bramley and
Menzies, 1987; Grissom and Littleton, 1988). These investigators have
reported high affinity, low capacity receptors for PRL between various
tissues and among species. Prolactin belongs to a family of hormones
including GH, placental lactogen and proliferin (Linzer and Nathans,
1984). Nicoll (1982) described complications inherent in RRAs due to
interplay between members of this hormone family, especially GH and
PRL, and their receptors. Ovine PRL may not be purely analogous when
employed in heterologous systems during in vitro binding analyses.
Therefore, the homologous RRA may provide an in vitro environment that
is most similar to the in vivo hormone-membrane milieu.
Results of the present study validate a homologous RRA for porcine
PRL which allows quantitation of PRL receptors in porcine endometrial
membrane. This assay demonstrated saturable binding sites and a direct
relationship between the amount of PRL bound and increasing

103
concentrations of membrane protein. Optimal binding conditions were
similar to those reported for heterologous RRAs and were adapted from a
homologous RRA for mouse hepatic PRL receptors (Haro and Talamantes,
1985a) Binding of labelled porcine PRL to endometrium is enhanced 4-
to 6-fold when membranes are pretreated with 4 M MgCl2. The labelled
porcine PRL was specifically displaced (80%) over a range (0-2560 ug)
of unlabelled (porcine) PRL commonly used in heterologous RRAs. The
MgCli treatment did not affect Ka, but did increase the number of
receptors. This weak chaotropic agent is thought to remove endogenous
PRL not displaced during mechanical processing of the tissue (Kelly et
al., 1979) by destabilizing the membrane's water structure and
disrupting the hydrophobic and electrostatic forces involved during
protein-receptor-membrane interactions (Hafeti and Hanstein, 1974). It
is not known whether altering the microenvironment surrounding the PRL
receptor changes availability of other PRL receptors (cryptic or
golgi). Changes in the lipid microenvironment; however, are thought to
play a role in binding of hormone to its receptor (Dave et al., 1983)
and may affect PRL binding (Dave and Witorsch, 1985).
The Ka of the porcine endometrial PRL receptor (0.326 x 108 M_1)
is similar to that reported for solubilized bovine mammary gland
(Ashkenazi et al., 1987) and is slightly lower than that reported by
Haro and Talamantes (1985a) using a homologous mouse PRL RRA and by
Posner et al. (1974b) using a heterologous RRA with mouse liver tissue.
This Ka is also slightly lower than those reported for ovine PRL with
porcine endometrium (DeHoff et al., 1984), porcine ovary (Rolland et
al., 1976; Bramley and Menzies, 1987) and porcine mammary tissue (Shiu
et al., 1973; Berthon et al., 1987b), and ovine PRL with uterine tissue

104
from rabbit (Grissom and Littleton, 1988), mink (Rose et al., 1983),
rat (Williams et al., 1978), and sheep (Posner et al., 1974b). The sow
mammary gland lactogenic receptor has a higher affinity for ovine than
porcine PRL. The binding of [123I]-PRL to homologous receptors was low
(<3%) when labelled porcine PRL was bound to porcine mammary tissue,
labelled rabbit PRL to rabbit mammary tissue and labelled sheep PRL to
sheep mammary tissue (Berthon et al., 1987b) none of which was
pretreated with MgCl2. Low binding was detected in the present study,
but homologous binding was increased following treatment of endometrial
membranes with MgCli.
Crossreactivity of GH for PRL receptors is not uncommon. Growth
hormome is often reported to have the same affinity as PRL for
receptors when tested in heterologous assay systems and has been
suggested to upregulate the hepatic prolactin receptor (Knazek et al.,
1974; Webb et al., 1986). It is unlikely, however, that GH bound to
the PRL receptor is biologically agonistic in pig endometrium since
crossreactivity between PRL and GH was not apparent, suggesting that
PRL is binding to its own receptor.
The reversibility of binding after PRL has bound to its receptor
has been questioned. An exhaustive study by Van der Gugten and
coworkers (1980) suggested that PRL may not freely dissociate from its
receptor in vitro. Additional work by Kelly et al. (1983) showed that
the dissociation rate of PRL was much slower following longer
association time. Prolactin receptors from different membrane
subpopulations also have differences in affinity and dissociation rate
constants. Golgi membrane receptors have faster dissociation rate
constants than receptors in plasma membrane. Yet, dissociation was not

105
complete for either membrane (Kelly et al., 1983). The difficulty in
removing endogenous PRL from membranes in vitro is also indicative of
incomplete reversal of PRL binding. Dissociation of PRL from pig
endometrial receptor was 40% complete after 48h, with the majority of
this dissociation occuring within the first lOh. In the present study,
60% of bound porcine t125I]-PRL could be dissociated from rat liver
membranes which is consistent with findings for ovine PRL (Kelly et
al., 1983). Bramley and Menzies (1985) reported similar results for
pig CL and attributed the the slow dissociation rate to the high (64.8
kJ/mol) apparant activation energy for PRL binding, which was reported
also for mouse (43.6 kJ/mol; Haro and Talamantes, 1985b) and rat
(34kJ/mol; Rae-Ventner and Dao, 1982) liver. This may be due to
extensive hydrophobic interactions. Amino acids at positions 20
through 30 are known to be hydrophobic with histidines located at
positions 27 and 30 in cow (Wallis, 1974), sheep (Li et al., 1970), pig
(Li, 1976) and human (Cooke et al., 1981) and at positions 25 and 28 in
rat (Cooke et al., 1980) and mouse (Kohmoto et al., 1984) PRL.
Characteristics of PRL receptors determined with this homologous
RRA have many similarities to those characterized using heterologous
RRAs. Additionally, this homologous RRA, which more closely
approximate in vivo conditions, provide additional support to confirm
previous reports. Receptors are nearly saturated and binding data are
not easily obtainable without treatment of membranes with chaotropic
agents. This was also the case for other binding assays for sheep,
rabbit and pig. Therefore, the homologous RRA, permits evaluation of
specific PRL receptors within reproductive (and other) tissues without
confounding effects associated with heterologous RRAs. Homologous

106
RRAs may be used to measure receptors during different physiological
states, and to compare receptor levels in different tissues, since
receptors appear to change differentially in tissues within an animal
being maintained under steady-state physiological conditions.
Additionally, homologous and heterologous assays could be used
comparatively, to elucidate the intricate ligand-receptor-membrane
interactions between members of hormonal families and their receptors.

CHAPTER 8
AFFINITY LABELLING OF PROLACTIN RECEPTORS
IN DAY 75 PREGNANT PORCINE ENDOMETRIUM
WITH PORCINE [l25I]-PROLACTIN
Introduction
The molecular weight of the prolactin (PRL) receptor has been
estimated through several biochemical techniques such as affinity
chromatography, antibody detection, and solubilization (see review,
Djiane et al., 1987). Through these methods, the PRL receptor has an
estimated molecular weight of 37,000 to 42,000. A higher relative
molecular weight (Mr) estimate (80-90,000) also exsists, but this may
be a dimer configuration of the lower molecular weight form. Treatment
of PRL receptors with dithiothreitol (DTT) prior to electrophoresis
does not produce multiple bands and, therefore, the receptor, if
containing multiple subunits, is not complexed by disulfide bonds.
Cross-linking of PRL to its receptor can elucidate the molecular weight
of receptors when the molecular weight of the hormone is subtracted
from the resulting molecular weight estimates of the electrophoretic
bands.
This study investigated the similarity between affinity labeling
of the PRL receptor in rat liver membrane preparations, a common and
well investigated source of PRL receptors, to that of porcine
endometrial membrane preparations.
107

108
Materials and Methods
Tissue Collection and Preparation
Day 75 pregnant pig endometrium and Day 20 pregnant rat liver were
collected and processed as described in Chapter 7.
Affinity Labeling of Prolactin Receptors
Affinity labeling of prolactin receptors from Day 75 pregnant
porcine endometrial, and Day 20 pregnant rat liver preparations were by
a cross-linking procedures adapted from Hughes et al. (1983), Wang et
al. (1987) and Berthon et al. (1987a). Iodination of porcine PRL and
homologous RRA for porcine PRL were conducted as described in Chapter
7. All volumes used for chaotropic treatment of membrane preparations
were proportionally increased to accommodate 750 ug membrane protein,
rather than the 150 ug normally used in binding assays. Additionally,
200,000-300,000 cpm of labeled porcine PRL was added in the presence or
absence of 10 or 20 ug unlabeled porcine PRL or 10 ug unlabeled porcine
GH. Following incubation, membranes were washed and centrifuged as
previously described and resuspended in 500 ul assay buffer. Then 5 ul
of ethylene glycol bis succininimidyl succinate (EGS) dissolved in
dimethylsulfoxide (DMSO) (22.81 mg EGS/1 ml DMSO; 50 mM solution) was
added to the resuspended pellet to give a final concentration of 500
uM. Samples were incubated for 30 min on ice. The reaction was
quenched with 3 ml of tris buffer (10 mM Tris-HCl, 1 mM EDTA pH 7.4),
centrifuged at 3000 x g for 20 min, decanted and counted. Pellets were
solubilized in 50 ul Laemelli buffer and prepared for 10% one
dimensional polyacrylamide gel electrophoresis (Roberts et al., 1984).

109
Gels were stained with comassie blue, dried and exposed to Kodak XRP
film for 4 weeks at -70 C.
Estimates of the molecular weights of cross-linked hormone
receptor complexes were estimated from semilog plots of protein band
migration (mm) from the top of gel versus its molecular weight. The
following proteins were used as molecular weight markers: 14,000,
lysozyme; 20,000, soybean trypsin inhibitor; 29,000, carbonic
anhydrase; 45,000, ovalbumin; 57,500, catalase; 69,000, bovine serum
albumin; 97,000, phosphoralase B and 335,000, thyroglobulin.
Results
Cross-linking of porcine [12SI]-PRL to magnesium chloride treated
porcine endometrial membrane preparations resulted in four
electrophoretic bands detected by autoradiography with molecular weight
estimates of 45,000, 62,500; 78,000 and 88,000 (Figure 8-la, lane 3).
Following subtraction of the molecular weight of PRL (23,000), the
porcine endometrial PRL receptor(s) molecular weights are estimated to
be 22,000; 39,500, 55,000 and 65,000. Two bands were observed from
autoradiography of cross-linked PRL to rat liver membrane prepations
(62,000 and 46,000 Mr; Figure 8-lb, lanes 2 and 4) resulting in
estimates of 39,000 and 23,000 Mr for the rat liver membrane PRL
receptor. The addition of 10 ug unlabelled porcine PRL blocked all of
these bands in both the pig endometrium (Figure 8-la, lanes 1 and 2)
and rat liver (Figure 8-lb, lane 1). Bands were faintly detectable
despite the addition of unlabelled porcine GH to porcine endometrium
(Figure 8-la, lane 4) and rat liver (Figure 8-lb, lane 3).
Electrophoretic bands at 60,000 Mr were barely detectable for cross-

Figure 8-la: Autoradiography of affinity labelled, cross-
linked porcine endometrial membrane PRL receptors. Lanes 1
through 8 were loaded with 750 ug endometrial protein and
250,000 cpm iodinated porcine PRL. Lanes 10 through 13 were
loaded with 150 ug endomterial protein and 45,000 cpm
labelled porcine PRL.
Lane designations are as follows:
1) MgCl treated, 10 ug unlabelled porcine PRL
2) MgCl treated, 20 ug unlabelled porcine PRL
3) MgCl treated, 0 ug unlabelled hormone (Bo)
4) MgCl treated, 10 ug unlabelled porcine GH
5) Untreated, 10 ug unlabelled porcine PRL
6) Untreated, 20 ug unlabelled porcine PRL
7) Untreated, 0 ug unlabelled hormone (Bo)
8) Untreated, 10 ug unlabelled porcine PRL
9) labeled porcine PRL
10) untreated, 2.5 ug unlabelled porcine PRL
11) untreated, 0 ug unlabelled hormone (Bo)
12) MgCl treated, 2.5 ug unlabelled porcine PRL
13) MgCl treated, 0 ug hormone (Bo)
Figure 8-lb: Autoradiography of affinity labelled, cross-
linked rat liver membrane PRL receptors. Lanes 1 through 8
were loaded with 750 ug membrane protein and 250,000 cpm
iodinated porcine PRL. Lane designations are as follows:
1) MgCl treated, 10 ug unlabelled porcine PRL
2) MgCl treated, 0 ug unlabelled hormone (Bo)
3) MgCl treated, 10 ug unlabelled porcine GH
4) MgCl treated, 0 ug unlabelled hormone (Bo)
5) Untreated, 10 ug unlabelled porcine PRL
6) Untreated, 0 ug unlabelled hormone (Bo)
7) Untreated, 10 ug unlabelled porcine GH
8) Untreated, 0 ug unlabelled hormone (Bo)

Ill
Pig Endometrium
4M MgCI NO MgCI
PRL PRL Bo GH PRL PRL Bo GH
o
RRA cone
ra 4M MgCI
J NSB Bo NSB Bo
Q Rat Liver
4M MgCI NO MgCI
PRL Bo GH Bo PRL Bo GH B^
1 2 3 4 5 6 7 8

112
linked radiolabelled porcine PRL to either porcine endometrium (Figure
8-la, lane 7) or rat liver (Figure 8-2b, lane 6) membrane preparations
not treated with MgCli and in the absence of unlabelled hormone.
Additionally, cross-linking performed on concentrations of porcine
endometrial membrane protein used in homologous RRA (chapter 7) showed
electrophoretic bands similar to these previously described for porcine
endometrium (Figure 8-la, lane 13). Radiolabelled bands were not
present when membranes were incubated in the presence (Figure 8-la,
lane 12) of unlabeled porcine PRL or when not pretreated with MgCl2
(Figure 8-la, lanes 11 and 12).
Discussion
Affinity labeling of PRL receptors with iodinated porcine PRL
resulted in four (65,000, 55,000, 39,500 and 22,000) and two (39,000
and 23,000) Mr estimates of porcine endometrial and rat liver membrane
preparations, respectively. The 39,500 Mr protein from pig endometrium
and 39,000 Mr protein from rat liver are similar to PRL receptors
described previously for rat liver (Kelly et al., 1983), mammary gland
from sows treated with CB154 (Berthon et al., 1987a) and untreated sows
(Sasaki et al., 1985; Katoh et al., 1983) as well as rabbits (Sasaki
and Ike, 1985) mammary gland. However, the detection of multiple bands
in crude homogenates of pig endometrial and rat liver membranes may
have resulted from protein degradation or dimer formation during the
incubation or cross-linking procedures.
Several molecular weight bands (79, 58, 53, 42, 31 and 18K) are
detected when solubilized PRL receptors are hormone-affinity purified
and radiolabeled (not cross-linked; Berthon et al., 1987a). Further
¡

113
purification of membrane preparation by Affigel-10 ovine PRL affinity
chromtography and electrophorisis resulted in a major band of 42,000 Mr
and faint bands of 53,000 and 31,000. Additionally, monoclonal
antibody detection of purified PRL receptor resulted in three bands of
66,000; 45,000 and 31,000.
Therefore, the PRL receptor in porcine endometrial membranes,
following affinity labeling, cross-linking and electrophoresis, has an
estimated molecular weight that is similar to that for rat liver PRL
receptors. However, other molecular weight proteins were detected and
agree with results of other estimates of Mr for porcine mammary gland
PRL receptors.

CHAPTER 9
ENDOMETRIAL PROLACTIN RECEPTORS DETECTED
BY HOMOLOGOUS RADIORECEPTOR ASSAY DURING THE
ESTROUS CYCLE AND EARLY PREGNANCY IN PIGS
Introduction
Circulating levels of prolactin (PRL) are relatively constant
during pregnancy (Dusza and Krzymowska, 1981; Kensinger et al., 1986;
DeHoff et al., 1986). Prolactin is slightly elevated on Day 10 of
gestation, declines by Day 20 and remains constant (approximately 10
ng/ml) until parturition. The PRL levels are similar for cyclic gilts
except that PRL is elevated on Days 0-2 and 16-17 (Brinkley et al.,
1973; Dusza and Krzymowkska, 1979; Foxcroft and Van der Veil, 1982)
when concentration of circulating estrogens increase.
Although PRL is associated primarily with mammary growth and
development (Shiu et al., 1973; Costlow et al., 1974; Shiu, 1980),
recent data support a role for PRL in uterine physiology. Prolactin
interacts with ovarian steroids to enhance uteroglobin secretion and
endometrial proliferation in rabbits (Chilton and Daniels, 1985), as
well as increased uteroferrin, prostaglandin Fa, and glucose in
uterine secretions (Young and Bazer, 1988) of pigs. Prolactin may
affect ion transport and calcium cycling across the porcine endometrial
epithelium (Mirando et al., 1988), thus modulating uterine secretory
function (Young and Bazer, 1988). Prolactin may also exert
antiluteolytic effects through redirection of endometrial prostaglandin
114


115
F2 secretion into the uterine lumen (Young and Bazer, 1988; Mirando et
al., 1988).
The period of maternal recognition of pregnancy is of great
interest due to the morphological, physiological and endocrinological
changes that must occur for preimplantation porcine conceptuses to
survive. Estrogens of conceptus origin affect uterine secretory
function and may modulate the population of endometrial PRL receptors.
However, conceptus estrogens act locally, since they are metabolized to
a biologically inactive form, estrone sulfate (Heap et al., 1979),
before entering the porcine maternal circulation (Stoner et al., 1980).
Changes in endometrial PRL receptors throughout pregnancy (DeHoff
et al., 1984) have been associated with other gestational events
(Kensinger et al., 1986, Rabee and McCoshen, 1986) such as fetal growth
(Nicoll, 1982), endometrial synthesis and secretion of protein (Chilton
and Daniels, 1985; Young and Bazer, 1988), ion and water movement
across the placenta (Goldstein et al., 1980) and changes in circulating
levels of estrogen (DeHoff et al., 1986). Biological effects occur
through either increases in circulating concentrations of a hormone
with receptor numbers remaining constant, or hormone levels may remain
constant with receptor numbers increasing. The objectives of this
study were to compare endometrial PRL receptors during early pregnancy
and the estrous cycle and determine whether changes in receptor numbers
were associated with PRL biological activity during early pregnancy.
J

116
Materials and Methods
Animals
Crossbred gilts of similar weight (100-120 kg) and age (7 to 9
months) were used in this study following completion of two normal (18
to 22 days) estrous cycles. Thrirty-nine gilts were assigned randomly
to either the pregnant (n=21) or cyclic (n=18) reproductive status.
Within status, gilts (three per day) were assigned to Days 8, 10, 11,
12, 14, and 15 of the estrous cycle and pregnancy, as well as Day 30 of
gestation. Gilts were observed daily for estrus in the presence of
intact boars. The first day of behavioural estrus was designated Day
0. Gilts assigned to the pregnant status group were mated when
detected in estrus and 12 and 24 h later.
Surgical Procedures
On the appropriate day of the estrous cycle or gestation, gilts
were anesthetized with thiamylal sodium (1 g; i.v.) and maintained
under surgical anesthesia on a closed circuit anesthesia machine using
halothane. Gilts were subjected to midventral laparotomy and
hysterectomized. Endometrial tissue was separated from myometrium and
and processed as descibed in Chapter 7. Tissue was maintained at -70 C
from within 15 min of collection until receptor assays were conducted.
Mesurement of Endometrial Prolactin Receptors
The homologous RRA used for dectection of porcine endometrial PRL
receptors was conducted as described in Chapter 7. This includes
membrane preparation, protein determination, iodination of hormone,
detection of specific activity (83 uCi/ug), chaotropic treatment of

117
membrane preparations, RRA conditions, inhibition curves and analysis
of binding data.
Binding Analysis
Inhibition binding assays were also conducted on Day 75 pregnant
porcine endomtrial membranes to serve as a positive control. Analysis
of binding data was as described in Chapter 7.
Statistics
Data were analyzed by least squares analysis of variance using the
General Linear Models procedure of the Statistical Analysis System
(SAS) (Barr et al., 1979) to detect effects of reproductive status
(pregnant or cyclic), day and their interaction. Within reproductive
status, orthogonal contrasts were used to detect differences between
means on different days. Student's t-test was used to detect
differences between reproductive status on individual days.
Results
Results from analysis of porcine endometrial membrane PRL
receptors throughout the estrous cycle and early pregnancy are depicted
in Figure 9-1. The affinity of the porcine endometrial membrane PRL
receptor (Ka = 0.21 + 0.05 x 108 M_1) was not affected by day, status
or their interaction. However, receptor numbers were affected (P<0.01)
by reproductive status since endometrial PRL receptors (pmole/mg
protein) were higher for endometrial membranes from pregnant (31.2
+1.7) than cyclic (24.3+1.7) gilts. Changes in endometrial PRL
receptor numbers were affected by a day by status interaction (P<0.06)

Total Prolactin Receptors (pmoles/mg protein)
118
Day
Figure 9-1: Prolactin receptors in endometrial membranes of
cyclic (squares) and pregnant (circles) gilts over days of the
estrous cycle and gestation. Values with different letters are
significantly different (P<0.05) within reproductive status and
between status on Day 12. Values were obtained from three gilts
per day in each reproductive status. The SEM for prolactin
receptor numbers is + 4 pmoles/mg protein.

119
and were best described separately. Endometrial membrane receptor
numbers (pmoles/mg protein) were not different between pregnant and
cyclic gilts on Day 8 (27.6 vs 30.4 +4, respectively). After that,
endometrial membranes from cyclic gilts had decreased PRL receptor
numbers on Days 10 (21.1+4) to Day 14 (21 to 23+4) which then increased
slightly on Day 15 (28.4 +4). Prolactin receptors in endometrial
membranes from pregnant gilts remained relatively constant for Days 8,
10 and 11 (27.6, 26.9 and 28.0 +4 pmoles/mg protein, respectively),
then increased (P<0.05) on Day 12 (37.0 +4) and remained elevated
through Days 14 (33.5 +4), 15 (31.6 +4) and 30 (34.5+4) of gestation.
Endometrial PRL receptor numbers on Day 12 were greater (P<0.05) for
pregnant than cyclic gilts.
Discussion
Histotroph, contains proteins, sugars and other constituents
essential to preimplantation porcine conceptuses and must be secreted
into the uterine lumen at the appropriate time to support and nourish
the porcine conceptus until placentation is established. Prolactin
enhances effects of ovarian steroids on uterine physiology.
Interactions between PRL and progesterone enhance endometrial
proliferation and uteroglobin secretion in the long-term ovariectomized
rabbit (Chilton and Daniels, 1985). In the pig, however, PRL interacts
with estrogen to enhance secretion of uteroferrin, prostaglandin F2C1
and glucose into the uterine lumen (Young and Bazer, 1988). Prolactin
concentrations in plasma of pigs are relatively constant throughout
gestation (Dusza and Krzymowska, 1981; Kensinger et al., 1986; DeHoff
et al., 1986); yet PRL exerts an effect on uterine secretory

120
physiology in pigs (Young and Bazer, 1988; Mirando et al.( 1988).
Changes in the biological effects of a hormone may occur through either
1) increasing hormone concentrations in the presence of constant
receptor numbers or 2) increasing receptor numbers, but constant
concentrations of hormone. Results of this study support the latter
mechanism since PRL receptor numbers remained relatively constant in
pregnant gilts until Day 12, then increased and remained elevated
through Day 30. Despite similar numbers of receptors in endometrial
membranes on Day 8 for cyclic and pregnant pigs, PRL receptors in
cyclic endometrial membranes decreased by Day 10 and remained
consistently low until Day 15.
The Kd detected for endometrial PRL receptors in regard to
circulating PRL levels would suggest that only a small proportion
receptors may need to be bound to elicit a response, as previously
mentioned for histamine receptors (Nickerson, 1956). Scatchard
analysis of homologous competitive inhibition curves for endometrial
PRL suggest the presence of two binding sites, having high (109 or 1010
M-1) and low (107) affinities (see Chapter 7). Two binding sites were
detected in endometrial membranes of gilts, most notably on Day 11 and
12 of pregnancy, however, results were not conclusive. A higher
affinity binding site for PRL, if temporally associated with porcine
conceptus estrogen secretion, would allow an additional regulatory
mechanism for affects of PRL on target tissues. Therefore, although
multiple binding sites may be present in porcine endometrial membranes,
additional investigation is necessary.
The increase in PRL receptors detected for pregnant endometrial
membranes in the present study may have resulted from effects of

121
increased amounts of hormones in the uterine lumen. Estrone levels are
high in uterine flushings of pregnant gilts on Day 8, followed by
increased estradiol levels on Day 12 (Zavy et al.r 1980). Estrone was
found to be equally potent to estradiol in stimulation of hepatic
lactogenic receptors in adult male or immature rats (Posner et al.,
1974a). Although, estradiol is considered more uteropotent than
estrone; estrone may also modulate endometrial PRL receptors. Changes
in uterine function around Day 12 are of interest due to temporal
associations with critical stages of development by porcine conceptuses
(Geisert et al., 1982b; Bazer and Roberts, 1983).
Porcine conceptuses undergo drastic morphological changes during
the period when they initiate estrogen secretion on Days 11-12. The
conceptus rapidly elongates, intially by cellular rearrangement, and
then by hypertrophy and hyperplasia, from tubular (20-40 mm) and
filimentous forms (100-200 mm), to reach lenghts of 900 mm by Day 14
(Geisert et al., 1982b). In mink, PRL accelerates blastocyst growth,
possibly through effects on the CL and increases in progesterone
secretion (Martinet et al., 1981). Growth of the uterus and elongation
of the porcine conceptus is synchronous (Bazer et al., 1982), thus
fetal-placental units do not overlap. The underlying mechanism is
unknown, but PRL has been associated with growth promoting affects
(Nicoll, 1982). The increase in PRL receptors only in pregnant
endometrial membranes (Day 12), may be necessary for uterine growth
during conceptus elongation.
Correlated with secretion of estrogens by pig conceptuses, the
maternal recognition of pregnancy factor (Heap et al., 1979), are
changes in ion fluxes, specifically calcium, followed by release

122
(secretion) of uteroferrin and other proteins into the uterine lumen.
Signals from the porcine conceptus must prevent CL regression and
facilitate redirection of prostaglandin F2a secretion from the uterine
vasculature (endocrine) toward the uterine lumen (exocrine) (Bazer and
Thatcher, 1977). Concentrations of prostaglandin F2C1 are higher in
uterine flushings from pregnant than cyclic gilts between Days 12 to 14
(Zavy et al., 1980). Prolactin interacts with estrogen in vivo (Young
and Bazer, 1988) and in vitro (Mirando et al., 1988) to stimulate
release of PGF toward the uterine lumen. In the present study,
increased numbers of PRL receptors in endometrial membranes on Day 12
of gestation may enhance biological effects of PRL which include
facilitation of the redirection of prostaglandin F2C1 into the uterine
lumen to allow luteostasis and maternal recognition of pregnancy as
proposed by Bazer and Thatcher (1977) .
Uterine PRL receptors have been measured throughout gestation in
pigs (DeHoff et al., 1984); mid and late-gestation in rabbits (Grissom
and Littleton, 1988) and at a single time point in pregnant rats
(Williams et al., 1978), sheep (Posner et al., 1974b) and anestrous
mink (Rose et al., 1983). During gestation in the pig, endometrial PRL
receptors increase between days 30-45, decrease to Day 60, increase to
maximum values on Day 75 and then decrease to low levels by Day 90
(DeHoff et al., 1984). Uterine PRL receptors increase 25-fold between
Days 5 and 20 of gestation in rabbits (Grissom and Littleton, 1988)
with the majority of receptors in the endometrium. Prolactin receptor
numbers are comparable for the pregnant uterus and artificially induced
decidual tissue of rats (Williams et al., 1978). Prolactin binding
sites in the uterus of mink (Rose et al., 1983) may stop embryonic

123
diapause and delayed implantation; possibly through alterations in the
metabolism of progesterone, as suggested for the rat uterus (Armstrong
and King, 1971). Porcine CL have PRL receptors which fluctuate during
the estrous cycle or early gestation, but are highest in the mid-luteal
phase of the cycle (Bramley and Menzies, 1987) and are higher for CL of
pregnancy (Rolland et al., 1978).
In summary, endometrial membrane PRL receptor numbers fluctuate
during early pregnancy which may explain enhanced biological effects of
PRL in the uterine environment, despite rather constant levels in
plasma during early pregnancy. Additionally, increases in PRL receptor
numbers are temporally associated with reproductively important events,
such as conceptus elongation, uterine growth, luteostasis,
establishment of pregnancy and uterine secretory function. These
results support PRL's involvement in uterine physiology. Although,
regulation of hepatic PRL receptors is well investigated, further work
is needed to establish if similar mechanisms are responsible for tissue
specific changes in PRL receptors within endometrium and other
reproductive tissues.

CHAPTER 10
EFFECTS OF ACUTE OF ESTRADIOL VALERATE ADMINISTRATION
ON ENDOMETRIAL PROLACTIN RECEPTORS DETECTED BY HOMOLOGOUS
RADIORECEPTOR ASSAY AND UTERINE SECRETORY
RESPONSE IN DAY 11 CYCLIC PIGS
Introduction
Estrogen involvement in prolactin (PRL) receptor regulation is
suggested since ovariectomy (Kelly et al., 1975), stage of reproductive
cycle (Kelly et al., 1974) and administration of exogenous estrogen
(Posner et al., 1974a; Kelly et al., 1975) affects PRL receptor numbers
in various target tissues. Endometrial membrane PRL receptors in pigs
increase initially in association with secretion of estrogens by
conceptuses and establishment of pregnancy between Days 11 to 12
(Chapter 9) and then vary throughout gestation (DeHoff et al., 1984).
During the time of maternal recognition of pregnancy, a succession of
endometrial secretory events are essential for nourishment and support
of preimplantation porcine conceptuses (Geisert et al., 1982b; 1982c;
Bazer and Roberts, 1983; Young et al., 1987). Prolactin affects
uterine secretory physiology of pigs (Young and Bazer, 1988), rabbits
(Chilton and Daniels, 1985) and rats (Kennedy and Armstrong, 1972).
Estrogen appears to increase endometrial PRL receptors to increase
uterine responsiveness to rather constant circulating levels of PRL
(Dusza and Krzymowska, 1979). Therefore, porcine endometrial PRL
receptors were studied using a homologous RRA to correlate changes in
124

125
uterine secretory activity at selected intervals following
administration of exogenous estrogen.
Materials and Methods
Animals
Crossbred gilts of similar weight (100-120 kg) and age (7 to 9
months) were used in this study after they experienced at least two
normal estrous cycles (18 to 22 days). In the presence of intact
boars, gilts were observed daily for behavioral estrus. The first day
of behavioral estrus was designated Day 0.
Experimental Design
On Day 11 of the estrous cycle, 15 gilts were anesthetized with
thiamylal sodium (1 g, i.v.) and maintained under surgical anesthesia
using a closed curcuit anesthesia machine. Gilts were subjected to
midventral laparotomy and one uterine horn was exposed. Uterine
flushings were collected in 20 ml double distilled water (Bazer et al.,
1978) and endometrium was separated from myometrium and placed on ice.
These samples provided intragilt control data (time zero). Twelve
gilts then received estradiol valerate (E2V; 5 mg) and at either 1, 6,
12 and 24h post-E2V injection (three gilts per group), uterine
flushings and endometrium were collected in an identical manner from
the second uterine horn. Three gilts were injected with corn oil
(control group, 0.5 ml) and were assigned to 12h post-injection since
estrogen effects on the uterine environment are readily detectable at
12h (Geisert et al., 1982c). Gilts assigned for collection of time 0
and lh samples, remained under anesthesia whereas the 6, 12 and 24h

126
post-injection samples were obtained after gilts were reanesthetized
just before the time of the second sample collection. This procedure
avoided prolonged exposure to general anesthesia, and is not
detrimental to uterine secretory physiology (Young et al., 1987).
Tissue Preparation
Uterine flushings were prepared as described in Chapter 3.
Endometrial tissue was collected and prepared for homologous PRL RRA as
described in Chapter 7.
Protein Determination
Protein concentrations of endometrial membrane preparations and
uterine flushings were determined by the method of Lowry et al. (1961)
using bovine serum albumin as standard.
Measurement of Endomtrial Prolactin Receptors
Prolactin receptors were measured using the homologous RRA
described in Chapter 7. Tissue processing, protein determination,
iodination, determination of specific activity (83 uCi/ug) chaotropic
treatment of endometrial homogenates, RRA conditions, inhibition curves
and binding data analysis were all conducted as described in Chapter 7.
Binding analysis
Binding assays were also conducted using Day 75 pregnant pig
endometrial membranes as a positive homologous control (Young and
Bazer, 1987) since this tissue had high PRL receptor concentrations
(DeHoff et al., 1984).

127
Analysis of Uterine Flushings
Total recoverable protein, uteroferrin, calcium, sodium,
potassium, leucine aminopeptidase (LAP) and glucose in uterine
flushings were queantitated as described in Chapter 3.
Statistics
Data were analyzed by Least Squares Analysis of Covariance using
the Statistical Analysis System (SAS) (Barr et al., 1979). Included in
the model were effects of treatment (E2V vs corn oil), time (1, 6, 12
and 24h post-injection), treatment by time interaction and the
covariate. Each gilt served as its own control, i.e., the covariate
(time zero values for uterine flushings and endometrium), provided data
on basal levels of these constituents and was used to determine whether
changes due to treatment, time or their interaction were significant.
Orthogonal contrasts were used to detect differences between means.
Results
Evaluation of receptor data indicated that the affinity constant
(Ka=0.32 + 0.12 x 108 M-1) was not affected by treatment over the time
periods studied. However, PRL receptor numbers in the porcine
endometrial membranes did change after administration of exogenous E2V
as depicted in Figure 10-1. Receptor numbers (pmoles/mg protein +
11.8) were higher at 1 and 6h after E2V (43 and 55 pmoles/mg,
respectively), but then decreased (P<0.02) by 12h (9.6 pmoles/mg) and
24 h (23.4 pmoles/mg) after E2V administration to values that were not
different from values measured at 12h for gilts treated with corn oil
(26 pmoles/mg).

Figure 10-1: Prolactin receptor numbers in endometrial
membranes at 1, 6, 12, and 24 h after administration (i.m.)
of estradiol valerate (0.5 mg, hatched bars) or 12 h after
corn oil (0.5 ml, solid bar) administration. The solid line
denotes the mean value prior to injection (time zero) of Day
11 cyclic gilts. Values with different letters are different
(P<0.02). The SEM was + 11.8 pmoles/mg protein.

129
W
L.
o
*->
Cl
CD
O
CD
C
O
jg
o
Q_
50-
1 6 12 24
Hours post injection
(P< 0.02)

130
Analysis of ions in uterine flushings are summarized in Figure 10-
2. Total recoverable calcium (mg + 0.18; Figure 10-2a) was not
different between 1 and 6h (0.3-0.4 mg), increased (P<0.03) at 12h (1.0
mg) and then decreased at 24h (0.7 mg) following E2V administration.
Calcium in uterine flushings at 12h post injection was greater (P<0.05)
for gilts that received E2V compared to those that received corn oil
(1.0 vs 0.47 mg). Total recoverable sodium (ug + 17; Figure 10-2b)
increased between 1 and 6h (28 and 57 ug, respectively), increased
(P<0.01) again at 12h and remained elevated at 24h (96 and 106 ug,
respectively) following administration of E2V. Potassium (ug + 17;
Figure 10-2c) changed in a pattern similar to that for sodium. Values
were 48 ug and 57 ug at 1 and 6h, respectively, then increased (PC0.05)
at 12h (93 ug) and remained elevated at 24h (101 ug) post-E2V
administration. However, sodium and potassium values at 12 h post
injection were not different between gilts that received E2V or corn
oil.
Total recoverable protein (mg + 4; Figure 10-3a) was not different
at 1, 6, or 12h (13, 5, and 11 mg, respectively), but increased
(P<0.05) between 12 and 24h (11 vs 21 mg) following E2V administration.
Total protein in uterine flushing collected at 12h post-injection was
not different for gilts that received E2V or corn oil (11 vs 7.6 mg).
Total recoverable uteroferrin (umoles+168; Figure 10-3b) remained
stable through 12h, (285, 92, and 437, umoles respectively), but then
increased (P<0.001) at 24h (4690 umoles) post-administration of E2V.
Total uteroferrin in uterine flushings at 12h post-injection was
similar for gilts that received E2V or corn oil (437 vs 150 umoles).
Total leucine aminopeptidase (LAP; sigma units (SU)+30; Figure 10-3c)

Total mg / uterine horn
131
A Calcium
B Sodium
C Potassium
1.0-
0.8-
0.6-
0.4-
0.2-
1 6 12 24 12
Hours post injection
(P<0.05)
1 6 12 24 12 1 6 12 24 12
Hours post injection ^p<
(P<0.01)
Figure 10-2: Total recoverable (A) calcium, (B) sodium and (C)
potassium in uterine flushings at 1, 6, 12 and 24h following
administration (i.m.) of estradiol valerate (0.5 mg, hatched bars) or
12 h after corn oil (0.5 ml, solid bar) administration. The solid line
denotes the mean value prior to injection (time zero) of Day 11 cyclic
gilts. Values with different letters are different for calicum and
potassium (P<0.05) and for sodium (P<0.01). The SEM was +0.18 for
calcium; +17 for sodium and potassium.

Figure 10-3: Total recoverable (A) protein, (B) uteroferrin,
(C) leucine aminopeptidase (LAP) and (D) glucose in uterine
flushings at 1, 6, 12 and 24h following administration (i.m.)
of estradiol valerate (0.5 mg, hatched bars) or 12 h after
corn oil (0.5 ml, solid bar) administration. The soild line
denotes the mean value prior to injection (time zero) of Day
11 cyclic gilts. Values with different letters are different
for protein and LAP (P<0.05); glucose (P <0.01) and
uteroferrin (P<0.01). The SEM are +4 for protein; +168 for
uteroferrin; +30 for LAP and +0.17 for glucose.

Total LAP (sigma units) Total Protein (mg)
133
A
1 6 12 24 12
Hours post injection
(PC0.05)
c
240-
Hours post injection
(PC0.05)
B
co
i
o
V)
jl>
O
E
3
4.6-
4.4-
4.2-
4.0-
!
Hours post injection
(P<0.01)
D
Hours post injection
(PC0.01)

134
was similar at 1 and 6h (186 and 132 SU, respectively), however,
concentrations increased (P<0.05) at 12 and 24h (223 and 230 SU,
respectively); post E2V administration. Total LAP measured at 12h post
injection was greater (P<0.05) for gilts that received E2V versus corn
oil (223 vs 135 SU). Total glucose (mg + 0.17; Figure 10-4d) increased
(P<0.01) between 1 and 6h (2.0 and 2.9 mg, respectively), remained
constant through 12h (2.7 mg) and then increased further (P<0.03) at
24h (3.3 mg). Total glucose at 12h post injection was greater (P<0.01)
for gilts that received E2V rather than corn oil (3.3 vs 2.1 mg).
Changes in PRL receptor numbers were negatively correlated with changes
in calcium (-.88); sodium (-.80) and potassium (-.77), as well as the
membrane marker protein, leucine aminopeptidase (-.9). But positive
correlations also were detected between some components of uterine
flushings, i.e., sodium and potassium (0.99), sodium and glucose
(0.80), potassium and glucose (0.82), protein and LAP (0.83) and
protein and uteroferrin (0.88).
Discussion
The uterine secretory response to administration of E2V was
similar to that previously reported (Geisert et al., 1982b; Bazer and
Roberts; 1983; Bazer et al., 1986; Young et al., 1987). Uterine
flushings collected at 6h had increases in glucose, while at 12h
calcium, sodium, potassium and LAP were increased. Additionally, total
protein and uteroferrin increased by 24h. Prolactin may modulate
changes in ions in porcine uterine fluids since ion concentrations are
decreased in hypoprolactinemic pigs (Young and Bazer, 1988). Ion
shifts affect secretory processes (Bazer et al., 1984) as detected at

135
12h by increased LAP concentrations which were followed by increases in
other components. Estrogen, either alone or in conjunction with PRL,
could affect the synthesis of uterine secretory components. Total
protein and uteroferrin in uterine flushings increased at 24h without a
further increase in LAP; suggesting an increase in rate of synthesis,
and secretion of proteins without a detectable change in membrane
processing as measured by LAP. These data agree with previous reports
wherein estrogen-PRL interaction increased porcine endometrial
uteroferrin secretion (Young and Bazer, 1988). The temporal changes
observed following administration of E2V suggest that estrogen
increases PRL receptor numbers in porcine endometrial membranes prior
to observed changes in secretory products. An effect of estrogen on
PRL receptor numbers was suggested previously since endometrial
membrane PRL receptors increase following conceptus estrogen secretion
during early pregnancy. Endometrial membrane PRL receptors also
flucutate throughout gestation in association with changes in
concentrations of estrogens (DeHoff et al., 1984). Changes in PRL
receptor numbers may be necessary for estrogen stimulation of changes
in ion concentrations, or ratios, as occurred in this and previous
studies (Geisert et al., 1982c; Bazer et al., 1984; Young et al.,
1987). Additionally, estrogen increases pituitary secretion of PRL
(Chen and Meites, 1970; Baxter, 1985) which would increase available
PRL for receptor binding. This may increase internalization of
endometrial receptors since receptor numbers decreased at 12h. Maximal
occupation of rabbit mammary gland PRL receptors is at 15 min and
returns to normal levels by 12h (Djiane et al., 1979a). The half-life
of rabbit mammary gland PRL receptors is approximately 40-50 min

136
(Djiane et al., 1979a). A similar half-life was determined for rat
hepatic PRL receptors (Baxter, 1985). Downregulation of PRL receptors
is more pronounced and of longer duration than for other receptors
(Djiane et al., 1979a). Rapid turnover of PRL receptors is suggested
since receptors must recognize pulastile release of the hormone; unlike
insulin in which the receptor half-life is greater than 2h. Although,
the half-life of endometrial PRL receptors is not known, receptor
numbers increased between 12 and 24h following estrogen administration
to levels similar to those detected prior to treatment. Additionally,
the decrease in PRL receptors as 12h following estrogen may be due to
changes in the secretion or half-life of PRL or changes in receptor
processing.
Although estrogen increases pituitary release of PRL, it is
unlikely that endometrial receptors are auto-upregulated as this is a
delayed process (Djiane et al., 1979a). Additionally, increases in rat
hepatic PRL receptors in response to exogenous estrogen are not
affected by bromocryptine adminstration (hypoprolactinemia) (Kelly et
al., 1976).
The rapid increases in PRL receptor numbers at 1 to 6h post-E2V
are not easily explained by classical steroid-receptor interactions
(Jensen et al., 1968) and suggest that estrogen may affect mechanisms
other than receptor synthesis. Short response intervals to estrogen
stimulation could occur through steroid interaction with plasma
membrane receptors (Peitras and Szego, 1977; 1979; Parrikh et al.,
1980; Nenci et al., 1981; Towle and Sze, 1983 and Berthois et al.,
1988); suggesting a mechanism more similar to that for peptide
hormones. Additionally, short response intervals to estrogenic

137
stimulation and cellular response may be due to estrogen's interaction
at the lipid bilayer of the target cell plasma membrane; potentially
affecting PRL receptor microenvironment or PRL receptor recycling.
Increases in membrane fluidity increase available PRL receptors in rat
and mouse liver (Dave et al., 1983). A high estrogenrprogesterone
ratio increases membrane fluidity of human monocytes (Bagdade and
Sabbaiah, 1988). Estrogen may affect different membranes in cells.
Prolactin receptors increase more rapidily (<1 day) in golgi membranes
than plasma membranes (>3 days) in response to estrogen (Posner et al.,
1979). Seventy percent of PRL receptors are associated with the golgi
membranes. However, it is not known whether golgi receptors are newly
synthesized or membrane receptors being processed for degradation or
recycling.
Estrogen acutely affects the microtubular and microvillar
apparatus of endometrial epithelial cell membranes (Szego et al.,
1988). Early effects of estrogen on target tissue may be mediated
through changes in membrane integrity or fluidity or through effects on
other membrane components such as receptors and ion channels. The PRL
receptor cloned from rat ovaries (Kelly et al., 1989) has a long
cytoplasmic domain; increases in membrane fluidity would increase
extracellular domain exposure. Yet, PRL receptors cloned from rat
kidney (Boutin et al., 1988) has a short cytoplasmic domain and thus,
increases in membrane fluidity would reduce its extracellular domain
exposure (Shinitzky, 1984) By causing similar effects on membrane
fluidity, estrogen could cause different PRL receptor responses in
different tissues. Interactions between estrogen and the plasma

138
membrane as well as its receptor may cause the biphasic effects of
estrogen on target tissues.
Previous results indicate PRL involvement in uterine endometrial
physiology (Williams et al.f 1978; Rose et al., 1983; Chilton and
Daniels, 1985; Young and Bazer, 1988; Mirando et al., 1988). Results
from the present studies indicate that estrogen affects PRL receptor
numbers within 12h. Considering the rapid successsion of uterine
secretory events essential to the preimplantation porcine conceptus,
one effect of estrogen appears to be modulation of endometrial PRL
receptors that are temporally associated with uterine secretory
processes. Rapid induction of endometrial PRL receptors may be caused
by estrogens interaction with membrane receptors, changes in membrane
dynamics and fluidity or unmasking of receptors, rather than PRL
receptor synthesis. Additionally, stimulation of pituitary PRL release
by exogenous estrogen is likely to affect receptor binding and
internalization, but not auto-upregulate of PRL receptors within 24h.
The involvement of PRL in estrogen-induced uterine secretory activity
is well documented. Further investigations of both acute and chronic
effects of steroid-PRL interactions are necessary to fully elucidate
the mechanisms whereby they affect uterine physiology.

CHAPTER 11
EFFECT OF CHRONIC OVARIAN STEROID ADMINISTRATION ON
ENDOMETRIAL PROLACTIN RECEPTORS AS DETECTED BY HOMOLOGOUS
RADIORECEPTOR ASSAY AND UTERINE PROTEIN SECRETORY
RESPONSE IN OVARIECTOMIZED PIGS
Introduction
The direct involvement of ovarian steroids in uterine functions is
well established; however, these steroids can also modulate, or be
modulated by, peptide hormones. Although several protein hormones have
been implicated, recent reports suggest that prolactin (PRL), in
addition to its mammotrophic, lactogenic and luteotrophic roles,
influences uterine physiology. Endometrial PRL receptors have been
detected for humans (Healy, 1984), rabbits (Ohno, 1982; Grissom and
Littleton, 1988), sheep and cows (Posner et al., 1974b), rats (Williams
et al., 1978), mink (Rose et al., 1983) and pigs (DeHoff et al., 1984;
Young and Bazer, 1987). Effects of PRL on uterine physiology include
water and ion movement (Rabee and McCoshen, 1986; Goldstein et al.,
1980), fetal growth (Nicoll et al., 1975), protein secretion (Daniels
et al., 1984; Chilton and Daniels, 1985; Young and Bazer; 1988),
endometrial proliferation (Chilton and Daniels, 1985), steroid receptor
concentrations (Chilton and Daniels, 1985; Muldoon, 1981; 1987),
prostaglandin secretion (Mirando et al., 1988; Young and Bazer, 1988),
progesterone metabolism (Armstrong and King, 1977) and estrogen uptake
(Leung and Sasakai, 1973).
139

140
Concentrations of PRL in blood of pigs remains rather constant
throughout most of gestation (Dusza and Krzymowska, 1981; Foxcroft and
van der Weil, 1982; DeHoff et al., 1986). Control of tissue response
by local changes in PRL receptors may alter tissue responsiveness to a
circulating hormone. Ovarian steroids affect PRL receptors in liver,
kidney, adrenal, mammary and ovarian tissues (Waters et al., 1978; Shiu
et al., 1982 ). Considering the importance of ovarian steroids in
uterine physiology, their effects on porcine endometrial PRL receptors
were studied using a homologous RRA. Relationships between changes in
porcine endometrial PRL receptors and endometrial secretion of total
uterine protein and uteroferrin were also evaluated.
Materials and Methods
Animals
Crossbred gilts of similar weight (100-120 kg) and age (7-9
months) were used in this study following completion of one normal (18
22 days) estrous cycle. Gilts were checked daily for estrus in the
presence of intact boars. The morning when gilts were observed in
behavioural estrus was designated Day 0.
Hormones
Estradiol valerate, progesterone and corn oil were obtained from
Sigma (St. Louis, MO). Estradiol valerate was solubilized in 10/90
(v/v) ethanolrcorn oil to a concentration of 200 ug/ml. Progesterone
was solubilized in a similar manner to a concentration of 50 mg/ml.
Ethanol:corn oil (10:90, v/v) was the hormone vehicle and control
solution.

141
Experimental Design
On Day 4 of the estrous cycle, 12 gilts were ovariectomized and
assigned randomly (3 per group) to one of four hormone treatments:
estradiol valerate (E2V; 100 ug/0.5 ml/day); progesterone (P4; 200 mg/4
ml/day); combined treatments of estradiol valerate and progesterone
(E2V+P4); or corn oil (CO; 4 ml/day). Treatments were administered
from Days 4 to 14. Gilts were hysterectomized and uterine flushings
{Bazer et al., 1978) and endometrium were collected on Day 15.
Surgical Procedure
On Day 4 of the estrous cycle, gilts were anesthetized with
thiamylal sodium (1 g, i.v.) and maintained under surgical anesthesia
using a closed circuit anesthesia machine. Gilts were subjected to
midventral laparotomy, the ovaries were exteroirized, blood vessels
tied, and the ovaries removed. The uterus was not exteriorized to
minimize manipulation of the reproductive tract. On Day 15, the gilts
were subjected to a second midventral laparotomy. The uterus was
exteriorized, each uterine horn was flushed with 20 ml sterile saline
(Bazer et al., 1978) and endometrium was obtained immediately following
hysterectomy.
Preparation and Analysis of Uterine Flushings
Uterine flushings were collected and determination of total
recoverable protein and uteroferrin were conducted as described in
Chapter 3.

142
Measurement of Endometrial Prolactin Receptors
Endometrial PRL receptors were detected using the homologous RRA
described in Chapter 7. Endometrial preparation, homogenization,
protein determination, iodination, specific activity determination (83
uCi/ug), chaotropic treatment of endometrial membrane preparations,
radioreceptor assay conditions, inhibition curves and analysis of
binding data were as described in Chapter 7.
Statistics
Data were analyzed by least squares analysis of variance using the
General Linear Methods of the Statistical Analysis System (SAS) (Barr
et al., 1979). Orthogonal contrasts were used to determine differences
between means.
Results
The affinity constants generated from LIGAND analysis of
inhibition curves from homologous RRA were not affected by steroid
treatment. The average Ka was .20 + .03 x 108 M-1, (data not shown)
and was similar to values previously determined using the homologous
porcine PRL RRA in Chapters 7, 9 and 10. Effects of ovarian steroid
administration on porcine endometrial membrane PRL receptor numbers are
summarized in Table 11-1. Gilts treated with CO or P4 had similar
endometrial membrane PRL receptor numbers (60 vs 58 + 7 pmoles/mg
protein, respectively). Gilts that received E2V alone (45 pmoles/mg
protein) or E2V+P4 (44 pmoles/mg protein), had lower (P<0.06)
endometrial PRL receptors than gilts that received either P4 alone or
corn oil. When PRL receptor numbers were calculated per gram wet

143
Table 11-1: Endometrial membrane prolactin receptors and
total protein and uteroferrin in uterine flushings
from ovariectomized gilts following steroid administration
for 11 days.
Treatment
Component
CO
E2V
P4
E2V+P4
(SEM)d
Endometrial
PRL receptors
(pmoles/mg protein)
60a
45
58a
43b
(+7)
Protein
(total mg/uterine horn)
2a
7a
37b
OO
n
(+5)
Uteroferrin
2a
8a
4227b
8408c
(+524)
(total umoles/uterine horn)
abc Within rows, values with different letters are different
for PRL receptors (P<0.06), protein and (P<0.01) and
uteroferrin (P<0.001).
d Three gilts/treatment

144
weight (+41 pmoles/gww) of endometrial tissues, gilts treated with CO
(233 pmoles/gww) were greater than for E2V alone and P4 alone (176 and
185 pmoles/gww, respectively) which were higher than for E2V+P4 (157
pmoles/gww).
Treatment of gilts with ovarian steroids affected (P<0.001) total
recoverable protein and uteroferrin in uterine flushings. Total
protein (mg + 5) was similar in gilts that received CO or E2V alone
(1.7 and 7.1 mg, respectively). Progesterone administration increased
(P<0.001) protein secretion (36.8 mg) as did administration of E2V+P4
(83.8 mg, P<0.001). Uteroferrin (umoles + 524) was secreted in a
similar pattern as protein. Administration of E2V or CO resulted in
low levels of uteroferrin in uterine flushings (8.1 vs 1.7 umoles,
respectively), which increased (P<0.001) in response to P4 alone (4227
umoles) and E2V+P4 (8405 umoles) (see Table 11-1) .
Discussion
Prolactin (PRL) receptor concentrations are sex specific
(Sherman, 1977; Waters et al., 1978). Changes in PRL receptor numbers
within several target tissues occur during puberty (Kelly et al.,
1974), the estrous cycle (Kelly et al., 1975; Chapter 9), pregnancy
(DeHoff et al. 1984; Grissom and Littleton, 1988; Chapter 9), lactation
(Shiu et al., 1974; Kelly et al., 1975; Sherman et al., 1977), and in
response to ovariectomy (Posner et al., 1974a; Kelly et al., 1979;
Daniels et al., 1984), orchotomy (Kelly et al., 1976; Bohnet et al.,
1977) and following administration of exogenous steroids (Waters et
al., 1978; Shiu et al., 1982). However, alterations in PRL receptor

145
concentrations are not necessarily similar between tissues or within
the same tissue of different species.
Steroid regulation of hepatic PRL receptors has been extensively
investigated. Hepatic PRL receptors decrease in ovariectomized rats
(Posner et al., 1974a; Kelly et al., 1979), but increase following 8 to
12 days of chronic estrogen administration (Posner et al., 1974a).
Increases in PRL receptors following estrogen treatment may be
mediated, indirectly, through stimulating release of pituitary PRL and
auto-upregulation of PRL receptors. Hypophesectomy (Posner et al.,
1974a), but not CB154 administration (Kelly et al., 1976), blocked the
estrogen stimulated increase in rat hepatic PRL receptors, suggesting
pituitary involvement, but not exclusively an effect of PRL.
Involvement of growth hormone (Knazek et al., 1975), ACTH and TSH
(Bhattacharya and Vonderhaar, 1979) is implicated (Waters et al.,
1978). Contrary to results in rats, ovariectomy increases hepatic PRL
receptors in mice (Marshall et al., 1979) and exogenous estrogen
reverses the effect of ovariectomy. Estrogen administration also
decreases PRL receptors in prostate (Kledzik et al., 1976; Amit et al.,
1983) adrenal, kidney, (Monkemeyer et al., 1974) and mammary gland of
mice (Marshall et al., 1979) and rats (Smith et al., 1976; Bohnet et
al., 1977).
The increase in PRL receptors in the mammary gland following
parturition (Holcomb et al., 1975; Djiane et al., 1977) is thought to
be due to autoregulation by increases in serum PRL associated with
parturition since administration of CB154 at parturition, decreases
mammary gland PRL receptors (Bohnet et al., 1977). Admistration of PRL
to pseudopregnant rabbits increases mammary gland PRL binding sites; an

146
effect blocked by progesterone administration (Djiane and Durand,
1977). Sakai et al. (1978, 1979) suggest that progesterone indirectly
decreases or supresses PRL receptors by competing with glucocorticoid
receptors to block stimulation of PRL receptors. Progesterone retards
PRL auto-upregulation of PRL receptors in mammary glands of rabbits
(Djiane and Durand, 1977). However, progesterone appears to have no
effect on PRL receptors in mammary gland (Sherman et al., 1977) or
liver (Posner et al., 1974a) of rats. In the present study, porcine
endometrial membrane PRL receptors were not affected by progesterone
since receptor numbers were similar to those of corn oil-treated
controls. Previous studies confirm that PRL receptors in different
tissues do not respond similarly to identical hormonal regimes (Posner
et al., 1974a; Kelly et al., 1976; Grissom and Littleton, 1988). The
mammary gland PRL receptor is upregulated by PRL, similar to liver, but
decreased by estrogen administration, unlike the liver in which the PRL
receptor is upregulated. Additionally, changes in PRL receptors are
not similar between tissues associated with reproductive functions,
i.e., uterine and mammary tissue (DeHoff et al., 1984).
Changes in PRL receptors in porcine endometrial membranes in the
present study agree with previous reports indicating steroid regulation
of PRL receptors in mammary gland, liver, (except rat), adrenal and
kidney. However, relationships between uterine secretory responses and
endometrial PRL receptor numbers must be interpreted with respect to
interactions between steroids and PRL and regulation of their
individual receptors. In addition to regulation of PRL receptors by
ovarian steroids, PRL regulates steroid receptors in reproductive
tissues (Chilton and Daniels, 1985; Muldoon et al., 1987) and liver

147
(Chamness et al., 1975). Estrogen stimulates pituitary PRL release
which may not result in auto-upregulation of PRL receptors, but
increased concentrations of PRL in serum may influence steroid receptor
numbers.
With regard to uterine secretory responses, interpretation of PRL
affects is not readily evident since lower PRL receptor numbers were
associated with both low (E2 alone) and high (E2V+P4) uterine secretory
responses. There is uncertainty regarding the number of PRL receptors
necessary to induce a biological response (Bohnet et al., 1977; van der
Gugten et al., 1980). Prolactin receptor numbers in the present study
were slightly higher than those detected on Day 15 of the estrous cycle
or pregnancy (Chapter 9). Following inititation of lactation, PRL
receptors in the mammary gland continually decrease, despite constant
milk production (Bohnet et al., 1977) suggesting that the number of PRL
receptors is not rate limiting. Additionally, lower numbers of PRL
receptors in gilts that received estrogen may be due to increases in
circulating PRL since increased hormone may be available for receptor
binding. This may mask endmoetrial PRL receptors or cause receptors to
become cryptic. Additionally, estrogen may cause changes in
subcellular trafficing of PRL receptors into lysosomes to be degraded.
Differences in secretory response could be due to availablity (or
unavailablity) of other (exogenous) hormones needed for complete
uterine secretory response.
Although the number of PRL receptors were not different between
gilts that received estrogen alone and E2V+P4, the uterine secretory
response was greater for the latter group. Uteroferrin is synthesized
and secreted in response to progesterone (Knight et al., 1973; Chen et

148
al., 1975); however, estrogen acts synergistically with progesterone to
further increase secretion of uteroferrin. Differences in the
secretory response may be due to 1) estrogen stimulation of pituitary
PRL (Chen and Meites, 1970); 2) PRL stimulation of progesterone
receptors (Chamness et al., 1975; Chilton and Daniels, 1985; 1987;
Muldoon, 1987) or 3) availability of steroids, i.e. exogenous
progesterone available to bind to increased progesterone receptors
stimulated by effects of estrogen on pituitary PRL release. Prolactin
increases steroid receptors in mammary tissue of mice, however, the
steroid must be available for steroid receptor translocation to the
nucleus (Muldoon et al., 1987). Although the number of PRL receptors
measured in E2V and E2V+P4 treatment groups were low, this may not be
rate limiting for the secretory response. Greater uterine secretory
responses in gilts receiving E2V and P4 may reflect the proper hormonal
milieu necessary to utilize enhanced steroid receptors stimulated by
binding of PRL to its receptor in endometrial cells (see Figure 11-1).
Following chronic steroid administration, PRL affects include
modulation of steroid receptor concentrations in addition to direct
effects through PRL receptors. Prolactin receptor numbers change
during early pregnancy (Chapter 9) and throughout gestation (DeHoff et
al., 1984). Uterine secretory function is reduced by hypoprolactinemia
and increased by exogenous PRL in vivo (Young and Bazer, 1988) and in
vitro (Mirando et al., 1988). Therefore, it appears that PRL
influences the complexities of porcine uterine physiology during the
cycle, e.g., endometrial activity during early pregnancy when
conceptus-endometrial interactions are necessary for the establishment
and maintenance of gestation.

149
ESTROGEN
Figure 11-1: Effect of chronic ovarian steroid adminsitration on
uterine secretory response of ovariectomized gilts; possible
interaction with endogenous prolactin to result in low and high
uterine secretory response.

CHAPTER 12
STUDIES ON MITOGENICITY, LACTOGENICITY,
IMMUNOREACTIVITY AND RECEPTOR BINDING CHARACTERISTICS
OF NONGLYCOSYLATED AND GLYCOSYLATED PORCINE PROLACTIN
Introduction
Prolactin (PRL) exerts a diversity of physiological effects on
several target tissues in many vertebrate species (Riddle, 1963; Nicoll
and Bern, 1972). Prolactin affects osmoregulation in fish and
amphibians (Bern, 1975), ion and water regulation across the intestine
and kidneys, mammary development and lactation, as well as several
reproductive processes in mammals (Shiu et al., 1981). The ability of a
single hormone to exert such diverse actions is intriguing. Recent
data suggest that prolactin, itself, is composed of a "family" of
hormones (Lewis, 1984). Several molecular weight forms of PRL (23,000,
25,000 and 50-60,000) have been isolated from pituitary extracts. The
23,000 Mr form is a simple peptide structure consisting of 199 amino
acids with three disulfide bridges (Li et al., 1960). The 25,000 Mr
variant is identical in primary structure but contains a N-linked
carbohydrate chain on asparigine 31 (Parkov and Butnev, 1986) while
the 50-60,000 Mr form (Suh and Frantz, 1974; Whitaker et al., 1983) is
thought to be a dimer of the lower Mr forms (Shoupe et al., 1985).
The glycosylated form of PRL has been detected in pituitaries of
sheep (Lewis et al., 1984), humans (pituitary and decidual sources,
Lewis et al., 1985; Markoff et al., 1987), pigs (Pankov and Butnev,
1986), rats (Wallis et al., 1980) and mice (Sinha et al., 1987; Sinha
150

151
and Baxter, 1979). The glycosylated form accounts for approximately
10-30% of pituitary PRL (Lewis et al., 1984; Pankov and Butnev, 1986),
and 30-40% of circulating PRL (Markoff and Lee, 1987). Glycosylated
PRL is secreted from near-term decidua in humans (Lee and Markoff,
1986) and is found in human amniotic fluid (Meuris et al., 1985). The
carbohydrate structure of porcine PRL (25,000 Mr) has been identified
(Pankov and Butnev, 1986), and is similar to the carbohydrate moiety of
ovine LH (Bedi et al., 1982). Several studies have compared biological
activities and immunoaffinities of glycosylated and nonglycosylated
PRL. The glycosylated form has been reported to have both higher
(Pankov and Butnev, 1986) and lower (Lewis, 1984; 1985) activity in
lactogenic assays, lower mitogenic activity and immunoaffinity
(Pellegini et al., 1988; Scott et al., 1988). The decreased
immunoaffinity of glycosylated PRL is expected since the carbohydrate
moiety may mask the antigenic determinant (Pankov and Butnev, 1986;
Pellegini et al., 1988).
Comparisons of the affinities of glycosylated and nonglycosylated
forms of PRL for the lactogenic receptor have been conducted using
heterologous assays (Pellegini et al., 1988; Seely et al., 1988), but
are confounded by involvement of from two or three species through by
sources of tracer, unlabelled hormone and source of receptor.
Information from these studies suggest in vitro binding characteristics
which may not accurately reflect physiological hormone-receptor
interactions. Therefore, investigation of hormone binding
characteristics using an assay homologous to a species is important.
Porcine PRL was investigated to determine the presence of a
glycosylated form. Then, both forms were tested for lactogenic and

152
mitogenic activities by pigeon crop sac and Nb2 lymphoma cell assays,
respectively. Additionally, immunoaffinity and binding characteristics
of total, nonglycosylated and glycosylated forms of PRL were
investigated using homologous radioimmuno- (RIA) and radioreceptor
(RRA) assay systems, respectively.
Materials and Methods
Separation of Nonglycosylated and Glycosylated Forms of Prolactin
The glycosylated and nonglycosylated forms of porcine PRL were
separated using a Concanavalin A-Sepharose CL-6B (Con-A) column. The
column (1 x 2 cm; 4 C) was equilibrated with 10 mM phosphate buffered
saline (PBS, pH 7.5) followed by 0.2M alpha methyl mannoside in PBS to
remove Con-A that had detached from the Sepharose matrix. The column
was extensively rinsed with PBS prior to addition of porcine PRL.
Porcine PRL (USDA-1, from Dr. Douglas Bolt, Animal Hormone Program
Director, USDA, Beltsville, MD) was solubilized in PBS (3.5 mg/ml) and
slowly loaded onto the Con-A column. The buffer flow was stopped to
allow for maximal binding of glycosylated PRL. After 30 min, the
unretained nonglycosylated PRL was washed through the column with PBS
(flow rate of approximately 30 ml/hour) and collected as 1 ml
fractions. Protein was followed by absorbance at 280 nm. The retained
glycosylated porcine PRL was eluted from the column with 0.2 M alpha
methyl mannoside in PBS and collected in a similar manner as
nonglycosylated PRL.
Separation of the two forms of PRL yielded a 2:1 ratio of
nonglycosylated to glycosylated forms, respectively (Figure 12-1). The
1 ml fractions from each peak representing each form were pooled to

Absorbance at 280 nM
153
Figure 12-1: Separation of nonglycosylated and glycosylated forms
of porcine prolactin by Concanavalin A-Sepharose 6B column
chromatography. Protein was detected by its absorbtion at 280 nm.

154
provide the nonglycosylated and glycosylated forms. The proteins were
dialyzed against 10 mM sodium phosphate (pH 7.6) to provide forms of
PRL to be tested in mitogenic, immunoaffinity and receptor binding
assays. The forms of PRL to be tested for lactogenic activity were
dialyzed against distilled water. Protein concentration was determined
as described by Lowry et al. (1951) after dialysis. The purity of the
two forms of porcine PRL was determined by using 12.5% SDS one
dimensional polyacylaminde gel electrophoresis (Figure 12-2; Roberts et
al., 1984).
Nb2 Lymphoma Cell Assay
Cells were propagated in Fishers medium supplemented with 10%
horse serum, 10% fetal bovine serum, 10 units/ml each penicillin and
streptomycin and 100 urn (S-mercaptoethanol. Cultures were rendered
quiescent by transferring cells into medium with 1% fetal bovine serum
for 24h and then into medium completely deficient in fetal bovine
serum. Cells were plated at an initial cell density of 4 x 103
cells/ml into a 96-well plate (200 ul/well). Each test sample was
assayed in triplicate while "Nb2 alone" (no test sample) control was
assayed in quadruplicate. Nb2 cells were harvested 48 hours later
(Cambridge PhD Cell Harvester, Cambridge, MA) following a 4 hour DNA
labeling period with 3H-thymidine (0.5 uCi/well). The uptake of 3H-
thymidine was quantified by liquid scintillation counting.
Pigeon Crop Sac Assay
Pigeon crop sac assays were conducted as described by Nicoll et
al. (1985) and Pukac and Horseman (1984). Pigeons were injected for 3

155
1 2 3 4 5 6
Figure 12-2: Evaluation of the purity of nonglycosylated and
glycosylated forms of porcine prolactin isolated by column
chromoatography using 12.5% sodium dodecylsulphate one
dimensional polyacrylamide gel electrophoresis. Lanes 1 and
6 contain molecular weight markers (12,400, cytochrome C;
20,000, soybean trypsin inhibitor; 29,000, carbonic
anhydrase; 45,000, ovalbumin; 63,000-65,000, bovine serum
albumin). Lanes 2 and 5 contain porcine prolactin (total)
prior to separation. Lane 3 contains the nonglycosylated
form, while Lane 4 contains the glycosylated form of porcine
prolactin.

156
Statistics
Data were analyzed using the General Linear Models of Analysis of
Variance of the Statistical Analysis System (SAS) (Barr et al., 1979).
Data from Nb2 lymphoma cell assays were analyzed for heterogenity of
regression. Data from RIA curves were transformed to log-logit plots
to generate straight lines which were tested for parallelism.
Orthogonal contrasts were used to detect differences between slopes and
intercepts.
Results
Mitoqenicity of the Forms of Porcine Prolactin
Results of the Nb2 lymphoma cell assay comparing nonglycosylated,
glycosylated and total porcine PRL are depicted in Figure 12-3.
Nonglycosylated PRL had greater mitogenicity (P<0.01) than glycosylated
PRL. However, nonglycosylated and glycosylated PRL were 10- and 100-
fold less active (P<0.01), respectively, than total PRL in stimulating
uptake of 3H-thymidine by Nb2 lymphoma cells.
Lactogenic Activity of the Forms of Porcine Prolactin
At the lower dose (10 mg) of hormone administration, pigeons that
received glycosylated PRL had a 64% increase in crop sac mucosal dry
weight compared to pigeons that received nonglycosylated PRL (12.8 vs
7.8 mg, respectively). Crop sac mucosal dry weights were not different
between pigeons that received total or nonglycosylated PRL. However,
at a higher dose (50 mg) of the PRL forms, no differences in crop sac
mucosal dry weight were detected between pigeons that received
glycosylated and nonglycosylated (12.8 vs 11.0 mg, respectively) PRL.

Figure 12-3: Uptake of 3H-thymidine into Nb2 lymphoma cells
expressed as percent of Nb2 control (dashed line) when cells
were stimulated by total (circles), nonglycosylated (squares)
and glycosylated (triangles) forms of porcine prolactin.

Percent Nb2 lyphoma cell control
158
Hormone per well (ng)

159
Antibody Affinity of the Forms of Porcine Prolactin
Results from RIA trials are depicted as percent bound versus log
concentration of hormone (Figure 12-4a), normalized percent bound
versus log concentration of hormone (Figure 12-4b) and as data
transformed to log (concentration) versus logit (cpm) (Figure 12-4a,
inset). Transformation of data (log/logit) resulted in generation of
straight lines with similar slopes, but different (P<0.02) intercepts.
When results were normalized and expressed as percent of Bo, the
polyclonal antibody had slightly higher affinity for total, than
nonglycosylated, PRL; followed by glycosylated PRL (Figure 12-4b).
Results from the log-logit plot suggest that 30 and 45% more total PRL
(1.44 ng) was detected than nonglycosylated (0.9743 ng) or glycosylated
PRL (0.798 ng), respectively, at 50% bound (P<0.01).
Receptor Affinity as Measured in a Homologous Radioreceptor Assay
Evaluation of inhibition curves by Scatchard analysis resulted in
an average Ka of 0.21 x 108 M*1, similar to that previously reported
for a homologous porcine PRL RRA (Chapters 7, 9, 10 and 11). The
number of PRL receptors detected in MgCU-treated Day 75 pregnant pig
endometrial memebranes were greatest when [,23I]-PRL was displaced with
unlabelled total PRL followed by unlabelled nonglycosylated and then
glycosylated PRL. When the separated forms of nonglycosylated and
glycosylated PRL were remixed in a 2:1 ratio, the displacement curve
had a similar slope, but estimates of receptor numbers were lower (58
vs 15 pmoles/mg protein; Figure 12-5) In a separate assay, the
nonglycosylated and glycosylated forms of porcine PRL were competed
against radiolabelled total PRL. There were no differences in the Kas

Figure 12-4: Immunoaffinity of total (open and closed
circles), nonglycosylated (open and closed squares) and
glycosylated (open and closed triangles) forms of porcine
prolactin expressed as (A) percent bound versus log
concentration, (A inset) transformed to log versus logit plot
and (B) percent of Bo (normalized to 100%) versus log
concentration.

Percent Bound
161
Hormone (log concentration)
B

162
nmoles unlabeled hormone
T
20
Figure 12-5: Scatchard analysis of competitive inhibtion
curves using magnesium chloride treated Day 75 pregnant
porcine endometrial membranes and [!25I]-prolactin (total)
versus unlabelled total prolactin (triangles) and [125I]-
nonglycosylated:glycosylated (2:1) prolactin versus
unlabelled nonglycosylated:glycosylated (2:1) prolactin
(circles).

163
generated from Scatchard analyses of the inhibition curves (Figure 12-
6) using radioinert nonglycosylated or total PRL (0.129 vs 0.168 x 108
M*1) against radiolabelled total PRL, however, receptor numbers were
lower when radioinert nonglycosylated PRL than total PRL (14.64 vs
22.77 pmoles/mg protein, respectively). When radioinert glycosylated
PRL competed against radiolabelled total PRL for endometrial binding
sites, the Ka was higher (0.3 vs .17 x 10B M*1) but receptors were only
33% of those measured using total PRL (7.6 vs 22.77 pmoles/mg protein,
respectively). These data suggest the possibility of two PRL receptor
populations, one site being high affinity-low capacity and another
being low affinity-high capacity. Importantly, summation of receptors
measured using each form of PRL alone was approximately equal to that
measured using total PRL.
Discussion
Results from this study suggest that the two forms of PRL may
interact with various target tissues either individually or in
combination. Investigation of these forms of PRL; nonglycosylated
(23000); glycosylated (25000) and total PRL, by various techniques is
necessary to elucidate physiological effects of PRL. Results indicated
that the two forms caused different relative responses in different
assays.
Results from the RIA using a polyclonal antibody suggest that the
two forms of PRL, assayed alone, are detectable at higher
concentrations than for total PRL. These results are similar to those
for the glycosylated forms of ovine (Lewis et al., 1984) and human PRL
(Lewis et al., 1985; Pellegini et al., 1988) and previous results for

164
0.15-
0
0
i
Li.
T5
C
D
O
CD
0.10*
Total
Nongly
A
Glycos A
i r
1 2 3
T
4
T
5
T
6
nmoles unlabeled hormone
Figure 12-6: Scatchard analysis of porcine t1231]-(total)
prolactin binding to magnesium chloride treated Day 75
pregnant porcine endometrial membranes inhibited by unlabeled
total (circles), nonglycosylated (squares) or glycoslyated
(triangles) forms of porcine prolactin.

165
porcine PRL (Pankov and Butnev, 1986; Seely et al.f 1988). Pankov and
Butnev (1986) suggested that the carbohydrate moiety on PRL could mask
antigenic determinants. Pellegini and coworkers (1988) found similar
detection of the two forms of human PRL using a monoclonal antibody.
The ratio between the two forms of PRL may vary during different
physiological states (Markoff and Lee, 1987) and change the overall
activity of PRL without altering the amount detected by RIA (Sinha et
al., 1984).
The bioactivity of the two forms of PRL exhibit different
potencies when mitogenic and lactogenic effects were measured.
Mitogenicity, as determined in the Nb2 lymphoma cell assay, indicated
that nonglycosylated and glycosylated forms of PRL were 10- and 100-
fold less potent, respectively, than total PRL. These results are
similar to those of Markoff and Lee (1987), Scott et al. (1988) and
Pellegini et al. (1988). Lactogenic bioactivity, however, was
enhanced for glycosylated, compared to nonglycosylated and porcine
total, PRL which is consistent with previous results (Pankov and
Butnev, 1986). But glycosylated ovine PRL had decreased lactogenic
activity in the pigeon crop sac assay (Lewis et al., 1984) and in the
mouse mammary casein bioassay (Seely et al., 1988).
Results from the homologous RRA suggest that glycosylated porcine
PRL may have higher affinity for PRL binding sites in porcine
endometrial membranes. Prolactin receptors for different tissues
within the same animal may be differentially regulated since several
studies have shown differential changes in receptor numbers in tissues
under the same physiological conditions (Posner et al., 1974b; Kelly et
al., 1979; Shiu et al., 1981; DeHoff et al., 1984). Data from the

166
present study suggested that glycosylated porcine PRL interacts
differently with PRL receptors. Neither form alone, nor when remixed,
displaced [125I]-PRL (total) as effectivly as unlabelled total PRL.
This may result from 1) the need for both forms to be present for
effective receptor binding or activation, 2) detrimental effects of
separation of the two forms on binding ability or 3) targeting of the
different forms of PRL to different parts of the receptor(s) or
membrane. Discrepancies between biological activity and RIAs have been
reported previously (Asawaroengchai et al., 1978; Leung et al., 1978;
Owens et al., 1986) which may extend to results for binding data
(Nicoll, 1975). Uncertainty remains regarding the number of PRL
receptors necessary to elicite a biological response (Bohnet et al.,
1977).
These results suggest that glycosylated porcine PRL has greater
lactogenic activity, but lower mitogenic activity than the
nonglycosylated form. Lower immunoaffinity of glycosylated PRL
suggests that the RIA may not accurately measure biopotency of
circulating PRL. Additionally, the ratio of the two forms may be
important physiologically (Sinha et al., 1988). Glycosylated porcine
PRL may possess different receptor binding characteristics than
nonglycosylated or total PRL. Additionally, the presence of both forms
of PRL, without manipulation, is necessary to obtain binding
characteristics similar to those previously reported for the porcine
endometrial membrane PRL receptor (DeHoff et al., 1984; Chapters 7, 9,
10 and 11). Prolactin may be present in the circulation as a higher Mr
(50-60,000; Suh and Frantz, 1974; Whitaker et al., 1983) form which
may be disrupted during separation. Thus, the physiological roles of

167
glycosylated PRL may differ from that of nonglycosylated PRL, but those
functions may include those previously attributed to 'total' PRL.
Glycosylated PRL may function as a reserve or long half-life form while
the deglycosylated hormone is active at the tissue level. Further
investigation of microheterogeneity and molecular weight forms of PRL
is needed to increase our understanding of how these forms of PRL
account for the complexities of function and diversity of physiological
responses to PRL.

CHAPTER 13
GENERAL DISCUSSION
Prolactin (PRL) function in mammals is associated mainly with
reproduction. In females, effects of PRL on the ovary and mammary
gland are well established. However, previous reports and results from
this dissertation expand on these roles to include an effect of PRL on
endometrial function which affects the uteine environment. Thereby,
extending the functions of PRL to include all tissues of the female
reproductive system.
Prolactin's effects on uterine physiology are well documented.
Most species studied to date, however, undergo invasive implantation
and have production of PRL by uterine decidual tissue and/or placental
production of placental lactogen. The pig is similar to the rabbit in
some aspects of conceptus-uterine interactions. Both species have
conceptuses that secrete estrogen to establish pregnancy and have
placentae that do not produce placental lactogen. However, the rabbit
endometrium does decidualize during implantation and produces PRL
locally. The pig conceptus undergoes noninvasive implantation and has
no local source of endometrial PRL. However, PRL does affect uterine
secretory function in pigs (chapters 3 and 6; Mirando et al., 1988).
Administration of CB154 decreased circulating concentrations of
PRL by 40-50%, similar to results of Kraeling et al. (1982) and
Vhitacre et al., (1981). However, in these studies, PRL was not
decreased below chronic circulating levels, as observed for rats.
Bromocryptine may be effective in blocking large increases in
168

169
circulating PRL associated with parturition in pigs or mating in rats,
but CB154 may be less effective, at least in pigs, in decreasing tonic
release of PRL. Additionally, PRL secretion may be controlled
differently in pigs through a nondopaminergic mechanism. Treatment of
pregnant gilts with CB154 had no effect on fetal survival suggesting
that 1) treatment was not early enough to alter the uterine environment
prior to conceptus estrogen secretion; 2) basal levels of PRL were
adequate for reproduction despite CB154 treatment; 3) endometrial PRL
receptors increased to allow physiological responses despite lowered
concentrations of PRL (Kelly et al., 1979) and 4) physiological
compensation by the porcine conceptus by other, unknown, mechanisms.
Admmstration of CB154 decreased the quantity of electrolytes in
uterine flushings of cyclic gilts 24 h following a single dose of
exogenous estrogen on Day 11. Lowered circulating PRL levels
interfered with estrogen-induced uterine ion fluxes and membrane
turnover, suggesting detrimental effects of lower PRL on uterine
secretory function.
The preimplantation porcine conceptus must stimulate histotroph
secretion and provide an antiluteolytic signal to insure maintenance of
CL function and progesterone secretion. Both actions are essential to
the establishment of a viable pregnancy and both appear to be modulated
or affected by PRL. Administration of exogenous PRL enhanced secretion
of uteroferrin, prostaglandin Fa and glucose into uterine flushings
(Day 12) of gilts treated 24 h earlier with exogenous estradiol. These
components of uterine flushings do not usually increase to high
concentrations until Day 14 to 16 of gestation, but pretreatment of
pigs with PRL (5 days) allowed a more rapid or maximal response to

170
exogenous estrogen given on Day 11. Increases in uterine secretory
response were detected when there was an interaction with estrogen, but
not progesterone. By manipulation of endogenous PRL in vivo, uterine
secretory response was decreased due to hypoprolactinemia and increased
in conjunction with hyperprolactinemia (Figure 13-1), suggesting that
PRL affects uterine secretory responses in pigs, as previously observed
in rabbits (Chilton and Daniels, 1985) and rats (Leung and Sasaki,
1973).
Prolactin concentrations in the circulation of pigs are rather
constant during the estrous cycle (Dusza and Krzymowska, 1979) and
early pregnany (Dusza and Krzymowska, 1981), and increase in
association with increases in circulating estrogens (Brinkley et al.,
1973; DeHoff et al., 1986). Effects of PRL may be regulated through
changes in numbers of PRL receptors in target tissues. These changes
may account for differences in physiological affects associated with
reproductive status or following administration of exogenous estrogen.
Prolactin receptors, in general, have been detected by
heterologous assays using ovine PRL. In vitro quantification can be
affected by heterologous assays due to differences in crossreactivity
between PRL, receptors and immunological artifacts (Hughes et al.,
1982) as well as other problems discussed by Nicoll (1982). In the
present study, a homologous RRA for porcine PRL was developed to obtain
binding characteristics for porcine endometrial PRL receptors. Results
obtained were similar to those generated in homologous assays for mouse
liver and to results obtained using heterologous binding conditions for
PRL receptors in reproductive tissues of pigs (Rolland et al., 1978;
DeHoff et al., 1984; Bramley and Menzies, 1987) and other species

171
Calcium
Sodium
Potassium
Chloride
LAP
Uteroferrin
Glucose
PGF2a
Elevation of:
Potassium
LAP
Figure 13-1: Effects of hypoprolactinemia and
hyperprolactinemia on the composition of uterine flushings of
Day 12 gilts at 24h after a single administration of
estradiol valerate.

172
(Posner et al., 1974b; Grissom and Littleton, 1988). In the present
study, porcine endometrial membrane preparations had low binding for
porcine PRL unless pretreated with MgCl2 to remove endogenous hormone.
Difficulty in binding PRL to tissue from a homologous source has been
reported for rabbit, sheep and pigs (Berthon et al., 1987b), however,
the tissues were not treated with a chaotropic agent (MgClz).
Validation of the homologous RRA for porcine prolactin assures that PRL
is binding to its own receptor since crossreactivity with GH was very
low, an attribute not always acheivable in heterologous assays.
Additionally, investigation of competative inhibition curves for two
receptors sites suggested that the porcine endometrial membranes may
contain PRL receptors with high affinity, low capacity and low
affinity, high capacity binding characteristics (Chapters 7, 9 and 12).
Investigation of the molecular weight estimates of the porcine
endometrial PRL receptors agreed with previous estimates for PRL
receptors from rat liver and also detected the two molecular weight
variants reported for sow mammary gland (Berthon et al., 1987a), but
not for rat liver membranes. Heterogeneity between PRL receptors has
been suggested (Nicoll et al., 1980) since PRL receptors are
differentially regulated in tissues from animals under steady-state
physiological conditions.
The effects of pregnancy, as well as acute and chronic ovarian
steroid administation, on the ontogeny of endometrial prolactin
receptors were investigated using the homologous RRA for porcine PRL.
Circulating levels of PRL do not change during establishment of
pregnancy. Rather, physiological effects of PRL appear to be regulated
by changes in receptor numbers, but not affinity, in endometrial

173
membranes. During pregnancy, endometrial PRL receptors increased, only
in pregnant pigs on Day 12, and remained elevated to Days 15 and 30
(Chapter 9) This increase was temporally associated with increased
production of estrogens by the conceptus and agrees with the premise
that changes in endometrial PRL receptors occur throughout gestation in
pigs (DeHoff et al., 1984). Receptors for PRL increased between 1 and
6h following estrogen administration, then decreased at 12h, in
temporal association with increases in luminal concentrations of
calcium, but prior to increses in uterine secretion protein secretions.
The mechanism(s) whereby PRL modulates of the porcine uterine
environment to enable maximal response to estrogen secretion by
conceptuses are presented in Figure 13-2. Prior to Day 8, circulating
PRL and endomtrial PRL receptors are similar regardless of reproductive
status. Prolactin could then increase endometrial proliferation
(Chilton and Daneils, 1984), gap junction formation (Sorenson et al.,
1987), steroid receptor concentrations (Leung and Sasaki, 1973; Daniels
et al., 1984) and affect ion channels (Petersen and Maruyama, 1985).
After Day 8, PRL receptors decreased in cyclic gilts, but were
maintained in the endometrium of pregnant gilts. Estrogens secreted by
pig conceptuses binds to estrogen receptors, increased in number by
PRL, and has enhanced effects. Conceptus estrogens may affect
endometrial cell membranes directly, increasing membrane fluidity or
shifting subcellular localization of PRL receptors. This leads to
increased unoccupied PRL receptors in the endometrium, as observed in
most tissues except for rat liver. Thus, estrogen and progesterone can
stimulate the endometrium maximally since steroid receptor numbers were
increased by PRL. Prolactin can affect the uterus since receptor

174
SruUirin
Endometrial proliferation
Increase gap junctions
Ion channels
Increase E and P receptors "*CjL prOgCSlCiOfie
of pregnancy
Figure 13-2: Possible mechanism(s) involving prolactin during
maternal recognition of pregnancy in pigs.

175
numbers are increased by conceptus estrogens. Prolactin can then
interact with conceptus estrogens to enhance histotroph sectretion and
redirect prostaglandin secretion into the uterine lumen thereby
facilitating the mechanism of luteostasis. Therefore, PRL modulates
the uterus to increase its response to conceptus estrogens and enhance
histotroph secretion and luteostasis; both of which are essential for
establish pregnancy in pigs.
Chronic administration of ovarian steroids to ovariectomized gilts
resulted in decreased PRL receptor numbers in association with both low
(E2V alone) and high (E2V+P4) uterine secretory responses. Estrogen
administration was common to these two groups and may have increased
pituitary secretion of PRL. Higher circulating PRL may stimulate an
increase steroid receptors. But without the proper presence of
progesterone in the E2V (alone) treated group, uterine secretory
activity could not progress as observed during pregnancy (maintained
luteal function) or following administration of both exogenous estrogen
and progesterone. Increases in PRL secretion due to exogenous
administration of estrogen may have resulted in masking of endometrial
PRL receptors or differential regulation of subcellular trafficing of
PRL receptors to lysosomes (see Figure 11-2) .
Porcine PRL is microheterogeneic with glycosylated and
nonglycosylated forms. Glycosylated PRL has lower mitogenicity and
immunoaffinity, but higher lactogenicity when compared to
nonglycosylated or total porcine PRL. Additionally, unlabelled
glycosylated porcine PRL competes with radiolabelled total porcine PRL
for binding sites with higher affinity but lower capacity than usually
detected for unlabelled total pig PRL. Therefore, glycosylated PRL may

176
be important physiologically, especially in species which lack
placental lactogen.
Results of the present studies support the concept that PRL
affects the uterine physiology of pigs and suggests that effects of PRL
on uterine physiology are not limited to species with decidual or
placental sources of lactogenic hormones. Additionally, a homologous
radioreceptor assay for PRL was acheiveable and provided data for PRL
receptor characteristics which were similar to those obtained using a
heterologous assay. Meaurement of PRL receptors using a homologous RRA
has the advantage of increased similarity to in vivo hormone-receptor
environmnent. The endometrium of pigs apparently regulates PRL
receptor numbers, not affinity, to modify responses to relatively
constant concentrations of PRL in blood. These changes in hormone-
receptor-membrane interaction result in physiological changes similar
to those achieved by other hormones through increased in concentrations
of the hormone. Elucidation of the signal transduction system for PRL
will increase our understanding of different mechanisms of regulation
of the actions of PRL by target tissues and suggest other mechanisms of
hormone-tissue response regulation. This has been suggested by
receptor characterization studies, and by the microheterogeneity of PRL
between its cleaved, clipped, and glycosylated forms, for which little
is known regarding their physiological affects.
In conclusion, results from PRL physiology and receptor studies
described in this dissertation indicate that PRL affects porcine
uterine physiology. Further studies are required to elucidate the
functions of PRL microheterogeneity, and expand our knowledge of PRL

177
receptors, about which much is known, but so much remains to be
discovered.

APPENDIX A
IODINATION OF PORCINE PROLACTIN
AND DETERMINATION OF SPECIFIC ACTIVITY
Buffer #1
pH 7.2
Buffer |2
pH 7.6
Reagents
25 mM Tris-HCl
3.94 g/L
10 mM CaCl2
1.109 g/L
0.01% Sodium Azide
0.1 g/L
0.1% PMSF
2 ml stock
solution/L
0.1% BSA 0.5 g
into 500 ml
of Buffer
PMSF stock 100 mM phenylmethylsulfonylflouride in absolute
alcohol. Store at -20 C.
Porcine Prolactin (USDA-B-1)
Na-[129I], 100 oCi (Amersham Corp. Arlington Heights, IL)
Iodo-gen powder (Pierce Chemical Corp., Rockford, IL)
0.01 M NaCHOa (pH 8.3) 0.8401 g/100 ml
Chloroform
Sephadex G75 (Pharmacia Fine Chemicals, Piscataway, NJ)
Swell in 25 mM Tris-HCl; 0.01% Thimasol for 24 h,
pour off fines and degas. Use at 25 C.
The day before
1) Acid wash (Glacial acetic acid) 12 x 75 borosilicate tubes for
iodo-gen.
2) Bring buffers and G75 to 25 C.
3) Start assembly of separation column (see below).
Morning of Iodination
1) Turn on Cahn Balance and allow 20 min warm-up. Weigh iodo-gen
(approximately 500 ug) using Cahn balance (in Dr. Hansen's lab) and
dilute to 0.1 mg/ml in chloroform. Stir gently in small beaker with
micro stir bar.
2) Carefully aliquot 20 ul of iodo-gen solution into bottom of acid
washed tubes. Add 40 ul of chloroform to each tube.
178

179
3) Dry iodo-gen under a gentle nitrogen stream. Rinse with buffer #1
(NO BSA). After air drying, a faint white ring should be visible on the
bottom of the tube.
Make Column Use a new, disposable 10 ml pipette with the top cut off.
Rinse inside of 10 ml pipette with buffer 12 (BSA). Pour sephadex
column quickly to avoid air bubbles. (NOTE: if buffers are not at room
temperature then bubbles form in the column, start over) Equilibrate
with buffer 12 (BSA) and finish with at least 1 volume of buffer 11 (NO
BSA). Stop Column.
Prepare prolactin Weigh as small amount of prolactin as accurately
possible (50-100 ug) on the Cahn balance. Dilute with 0.1 M NaHC03, pH
8.3 to a concentration of 20 ug/10 ul. Dilute 1:1 with buffer #1 (NO
BSA; use correct buffer otherwise you will iodinate BSA as well as the
hormone).
Iodination proceedure Set up iodination hood with the following:
Sephadex column, 10 ul Hamilton (stored in hood), fraction collector
(stored under hood), 20 ul pipette (remove metal automatic tip
remover), pasteur pipette and bulb, 50 tubes (13 x 100 mm borosilicate)
each containing 500 ul buffer 12 (BSA), iodo-gen tube, hormone, pipette
tips, excess buffer fl (NO BSA) and extra borosilicate tube for 5 ul
aliquot. Log out 10 ul Na-[125I].
Add 1 mCi (10 ul) Na129I to iodo-gen tube.
Add 10 ul buffer II (NO BSA)
Add 20 ul hormone (10 ug)
Add 20 ul buffer fl (NA BSA)
Hand vortex 5 sec every min for 15 min.
Following the 15 min reaction, take a 5 ul aliquot and put it into an
extra (empty) tube and set it aside (use for calculation of specific
activity).
Using pastuer pipette, load remaining 55 ul onto Sephadex column. DO
NOT scratch iodo-gen from the tube. Start column flow. After the
reactants have entered the column, add a small amount of buffer II (NO
BSA) to the column. DO NOT disturb the gel surface. DO NOT let the
gel run dry.
Collect 20 drops into the first tube and 10 drops into the remaining 49
tubes. Stop column.
Determination of peak Aliquot 10 ul from each tube into another set of
12 x 75 mm borosilicate tubes and count for either 0.5 or 1 min.
Identify hormone label peak (around tube 120). Hormone peak should
contain approximately 500,000 to 1,000,000 cpm/10 ul.

180
Test binding of labelled hormone The peak tube and the descending 2 to
3 tubes are tested for specificity of binding. Set up binding assay
with total count tube (TCT), Maximal binding (Bo) and nonspecific
binding (NSB) of 1000 or 2500 ng of unlabelled porcine prolactin using
Day 75 pregnant pig endometrial membranes (see assay protocol, appendix
B). The NSB should be approximately 4-7% of TCT and Bo should be
approximately 20-30% of TCT.
Clean up Properly dispose of ALL radioactive solutions and materials.
Storage of hormone Radiolabelled porcine prolactin for use in RRA
should be kept at 4 C for approximately 10-15 days. Store tubes in
lead-lined container. As radiolabelled hormone deteriorates, the NSB
will increase.
Determination of specific activity Dilute 5 ul radiolabelled porcine
prolactin into 1000 ul assay buffer. Aliquot 10 ul of diluted solution
and add to this 350 ul assay buffer and 100 ul 10% trichloroacetic acid
(TCA) solution. Incubate on ice for 30 min. Centrifuge (2500 x g) for
20 min at 4 C. Decant and count pellets. Calculate percent
incorporation of the 10 ul labelled hormone from cpm before and after
TCA precipitation based on the amount of hormone in 5 ul taken from the
reaction vessel.

APPENDIX B
HOMOLOGOUS RADIORECEPTOR ASSAY FOR PORCINE
ENDOMETRIAL PROLACTIN RECEPTORS
Materials
Iodinated porcine prolactin
Unlabelled porcine prolactin
Plastic 12 x 75 mm tubes (Sarstedt, Princeton, NJ)
Assay Buffer pH 7.6, stored at 4 C.
150 mM NaCl
10 mM NaPOz
10 mM EDTA
0.1% BSA
0.1% NaAzide
4M MgClz
Wheaton hand homogenizers (5 or 15 ml)
Vortex
Repeat dispenser 3 ml
Centrifuge capable of 3500 x g at 4 C
Homologous RRA
1) Homogenize tissue and determine protein concentrations.
Store at -70 C in small aliquots (750 ul), only thaw once.
2) Figure dilution for final concentration of 150 ug
protein/75 ul assay buffer. Prepare enough membrane protein
to run 45 tubes for each sample. Homgenize and resuspend
membrane, by hand, using a wheaton homogenizer on ice.
2) Chaotropic treatment of membranes
Label tubes 1-15; fl,2 and #15 in triplicate, #3-14
in duplicate.
Add 500 ul 4M magnesium chloride to tubes #2-15
To each tube, (#2-15) add 75 ul membrane preparation containing
150 ug protein (diluted in assay buffer)
Vortex quickly
Incubate 5 min at 25 C
Wash with 3 ml ice-cold assay buffer
Centrifuge (2300 x g) for 15 min at 4 C.
Decant and blot
Add fresh ice-cold assay buffer (300 ul) and immediately
vortex extensively to resuspend pellet
181

182
3) Set up binding assay
Make serial dilutions of unlabelled porcine prolactin
ranging from 0-5120 ng/100 ul (Stock solution 50 ug/ml in 0.1
NaHC03 can be kept in Reveo (-70 C) for 10 days).
Add 100 ul unlabeled prolactin to each tube 2-14), (0, 1.25,
2.5, 5, 10, 20, 40, 80, 160, 320, 640, 1280, 2560, and
5120 ng cold)
(Note: fl is TCT, 12 is Bo, 114 and 15 are NSB at two levels)
Vortex
Add 100 ul labelled prolactin (45,000 cpm/100 ul,
approximately 0.24 ng) to all tubes 1-15
Vortex 2-15, cap #1 (TCT)
Incubate 24 h at 25 C
4) Take off binding assay
Add 3 ml ice-cold assay buffer to each tube except #1
Centrifuge (3500 x g) for 30 min at 4 C.
Decant and Blot
Count pellets for 1 min
Determine maximal binding (20-30%; Bo/TCT) and nonspecific
binding (4-5% TCT)
Analyze displacement curves by Scatchard analysis determine
affinity constant and receptor density.

APPENDIX C
ACID PHOSPHATASE ASSAY FOR MEASUREMENT OF UTEROFERRIN
Solutions
1) Tris buffer: 0.5 M pH 7.0
2) Sodium Acetate: 1M pH 4.9
3) Sodium Hydroxide: 1M
Make fresh prior to assay:
4) Assay Buffer:
5) pNPP Substrate:
6.005 g/L dHzO
13.68 g/100 ml dH20
20.0 g/150 ml dH20 (stir)
0.1M B-Me in 0.05M Tris buffer (780 ul/100ml)
10 ml tris buffer 11 + 90 ml dH20
FV=371.1 14.88 gm/L in 0.01M NaAC
500 ul 1M NaAc (#2) + 49.5 ml dH20
Calculations for Proper Volumes
t tubes in assay +5 x 400 ul
" x 100 ul
" x 500 ul
" x 1.5 ml
vol assay buffer needed
vol of 1 M NaAc needed
vol pNPP substrate needed
vol 1M NaOH needed
Standard Curve
umoles dil
pNPP
pNPP
dH2 0
OD
0
1:10
Oul
100
ul
calb
0.01
1.10
10 ul
90
ul
0.02
1:10
20 ul
80
ul
0.04
1:10
40 ul
60
ul
0.08
1:10
80 ul
20
ul
0.15
none
15 ul
85
ul
0.20
none
20 ul
80
ul
Protocol for Acid Phosphatase Assay
Note: Since this is a timed assay, only run about 30 to 40 tubes,
including the standard curve, in one assay.
1) Label tubes. Run each sample in duplicate.
Turn on water bath and set to 37 C, and set the spectrophotometer
at 410 nM (visible light).
183

184
2) 100 ul sample, previously diluted if necessary
3) Add 400 ul tris buffer vortex
4) Incubate at 37 C for 20 min
5) Add 100 ul 1M NaAc vortex
Add 500 ul pNPP substrate vortex gently
6) Incubate at 37 C for 10 min.
7) Add 1.5 ml 1M NaOH Vortex.
8) Read OD at 410 nM
Calculations
1) Plot the standard curve and obtain correlation coefficient
2) Average OD and obtain umole/100 ul from standard curve
3) umole pNPP/100 ul x dilution factor x 10 =pNPP/ml/10 min
Note: Only accept values that are on the standard curve. The assay
usually needs to be run several times before the proper dilution is
attained.

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S. (1985) Vasoactive intestinal peptide is a physiological mediator of
prolactin release in the rat. Endocrinology. 116, 1383-1386.
Adler, S., Waterman, M.L., He, X. and Rosenfeld, M.G. (1988) Steroid
receptor-mediated inhibition of rat prolactin gene expression does not
require the receptor DNA-binding domain. Cell. 52, 685-695.
Alexander, R.L. (1971) Evaluation of an automatic calcium titrator.
Clin. Chem. 17, 1171-1175.
Alkon, D.L. and Rasmussin, H. (1988) A spatial-temporal model of cell
activation. Science. 239, 998-1005.
Amador, A., Klemcke, H.G., Bartke, A., Soares, M.J., Siler-Kodhr, T.M.
and Talamantes, F. (1985) Effects of different numbers of etopic
pituitary transplants on regulation of testicular LH/hCH and prolactin
receptors in the hamster (Mesocriecetus auratus). J. Reprod. Fert.
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215
BIOGRAPHICAL SKETCH
Kathleen Mary Hart, third and youngest child of William Alan and
Jeanne Alyce Hart, was born Feburary 24, 1960, in Cleveland, Ohio. She
spent her childhood in Lakewood, Ohio and graduated from Lakewood High
School in 1978. She obtained a Bachelor of Science degree in animal
bioscience from The Pennsylvania State University, graduating with
honors in 1982. In 1984, she obtained her Master of Science degree in
animal science from the University of Florida. Under the direction of
Dr. Fuller Bazer, she continued with doctoral research at The
Univeristy of Florida. She married Robert Gregory Young in November,
1985. She obtained her second degree black belt in karate in 1988.
Upon completion of her doctoral studies, she will move to the Chicago
area to further her studies on prolactin under the direction of Dr.
Daniel Linzer, Department of Biochemistry, Molecular Biology and
Cellular Biology, at Northwestern University.

I certify that I have read
conforms to acceptable standard
adequate, in scope and quality,
Doctor of Philosophy.
this study and that in my opinion it
of scholarly presentation and is fully
as a dissertation for the degree of
'Fuller W. Bazer, Chair
Graduate Reseach Professor of
Animal Science
I certify that I have read
conforms to acceptable standard
adequate, in scope and quality,
Doctor of Philosophy.
this study and that in my opinion it
of scholarly presentation and is fully
as a dissertation for the degree of
'A C*-
L
William C. Buhi
Assistant Professor of
Biochemistry and Molecular
Biology
I certify that I have read this study and that in my opinion it
conforms to acceptable standard of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for
the degree of Doctor of Philosophy.
Kathleen T. Shiverick
Professor of Pharmacology
and Therapeutics
I certify that I have read this study and that in my opinion it
conforms to acceptable standard of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
fies W. Simpkins /
Professor of Pharmacodynamics

I certify that I have read this study and that in my opinion it
conforms to acceptable standard of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
William W. Thatcher
Graduate Research Professor
of Dairy Science
This dissertation was submitted to the Graduate Faculty of the
College of Agriculture and to the Graduate School and was accepted as
partial fulfillment of the requirements for the degree of Doctor of
Philosophy.
May, 1989
Dean, College of Agriculture
Dean, Graduate School



55
significantly decreased concentrations of calcium, sodium, potassium
and chloride ions in uterine flushings, but did not affect total
protein. The inability of lowered PRL levels to alter secretion of
proteins may be due to lack of effects on ion ratios despite lowered
individual ion levels. The effects of decreasing concentrations of
ions may not have occurred in a temporal pattern necessary to alter in
the secretion of proteins. The aminopeptidase activity in uterine
flushings was decreased in response to hypoprolactinemia suggesting a
decrease in secretory activity (membrane processing) of endometrial
epithelium (Zavy et al., 1984).
The decrease in circulating PRL levels in the present study may
have been compensated for physiologically through other mechanisms,
e.g., an increase in PRL receptor numbers, has been previously
demonstrated for rabbit mammary tissue (Dijane et al., 1977) and rat
liver (Kelly et al., 1979). Hypoprolactinemia may also affect hormonal
regulation at the central nervous system, receptor levels in the
hypothalamus and target tissue (Muldoon, 1985) or intrapituitary
communication (Murai et al., 1988). Additionally, regulation of PRL
secretion by gamma amino butyric acid (Schally et al., 1978; Duvalinski
et al., 1987) in pigs suggests a different control mechanism for which
CB154 may be less effective.
In summary, circulating PRL levels were decreased 40-50% in pigs
following administration of CB154, but fetal survival at Day 25 of
gestation was not affected. Ions and LAP concentrations, but not
secreted proteins, in uterine flushings of estrogen stimulated Day 11
cyclic gilts were decreased. Hypoprolactinemia had begun to affect
uterine secretory function, but changes in ions may not have occurred


147
(Chamness et al., 1975). Estrogen stimulates pituitary PRL release
which may not result in auto-upregulation of PRL receptors, but
increased concentrations of PRL in serum may influence steroid receptor
numbers.
With regard to uterine secretory responses, interpretation of PRL
affects is not readily evident since lower PRL receptor numbers were
associated with both low (E2 alone) and high (E2V+P4) uterine secretory
responses. There is uncertainty regarding the number of PRL receptors
necessary to induce a biological response (Bohnet et al., 1977; van der
Gugten et al., 1980). Prolactin receptor numbers in the present study
were slightly higher than those detected on Day 15 of the estrous cycle
or pregnancy (Chapter 9). Following inititation of lactation, PRL
receptors in the mammary gland continually decrease, despite constant
milk production (Bohnet et al., 1977) suggesting that the number of PRL
receptors is not rate limiting. Additionally, lower numbers of PRL
receptors in gilts that received estrogen may be due to increases in
circulating PRL since increased hormone may be available for receptor
binding. This may mask endmoetrial PRL receptors or cause receptors to
become cryptic. Additionally, estrogen may cause changes in
subcellular trafficing of PRL receptors into lysosomes to be degraded.
Differences in secretory response could be due to availablity (or
unavailablity) of other (exogenous) hormones needed for complete
uterine secretory response.
Although the number of PRL receptors were not different between
gilts that received estrogen alone and E2V+P4, the uterine secretory
response was greater for the latter group. Uteroferrin is synthesized
and secreted in response to progesterone (Knight et al., 1973; Chen et


182
3) Set up binding assay
Make serial dilutions of unlabelled porcine prolactin
ranging from 0-5120 ng/100 ul (Stock solution 50 ug/ml in 0.1
NaHC03 can be kept in Reveo (-70 C) for 10 days).
Add 100 ul unlabeled prolactin to each tube 2-14), (0, 1.25,
2.5, 5, 10, 20, 40, 80, 160, 320, 640, 1280, 2560, and
5120 ng cold)
(Note: fl is TCT, 12 is Bo, 114 and 15 are NSB at two levels)
Vortex
Add 100 ul labelled prolactin (45,000 cpm/100 ul,
approximately 0.24 ng) to all tubes 1-15
Vortex 2-15, cap #1 (TCT)
Incubate 24 h at 25 C
4) Take off binding assay
Add 3 ml ice-cold assay buffer to each tube except #1
Centrifuge (3500 x g) for 30 min at 4 C.
Decant and Blot
Count pellets for 1 min
Determine maximal binding (20-30%; Bo/TCT) and nonspecific
binding (4-5% TCT)
Analyze displacement curves by Scatchard analysis determine
affinity constant and receptor density.


22
not necessarily induce a biological response. However, binding sites
in the mammary gland (Shiu et al., 1976) and liver (Chen et al., 1972)
have been correlated with cellular changes in mRNA and enzyme activity
and therefore, suggest that binding sites serve as receptors essential
for biological responses. Target cell membranes having saturable PRL
binding sites include mammary epithelial cells, hepatocytes, renal
tubules, adrenal cortex, prostate, seminal vesicles, and brain (Hughes
et al., 1985). Characteristics of PRL receptors include high affinity
and saturability. The high affinity constant, as determined by
heterologous ovine PRL assay (Ka = 109 M_1; Shiu and Freisen, 1974a),
is correlated with hormone biopotency. Homologous detection of PRL
receptors generates an affinity constant 108 (Haro and Talamantes,
1985; this study), only slightly lower than that reported for rat liver
(Dave and Knazek, 1980; Dave et al., 1981; Liscia et al., 1982; and
Liscia and Vonderhaar, 1982); cow mammary gland (Ashkenazi et al.,
1987) and mouse liver (Posner et al., 1974b; Knazek et al., 1977;
1978). However, the reversability of PRL bound to its receptor has
been questioned, as observed in vitro which often inefficiently
replicates the in vivo binding environment. Prolactin receptors have
been noted in several other tissues and are associated with other
physiological effects. Prolactin stimulates steroid production in ovary
and Leydig cells (Barkte and Dalterio, 1976; Musto et al., 1972),
uptake of testosterone by prostate (Farnsworth and Gonder, 1977),
increased uteroglobin mRNA and steroid receptors in the uterus (Chilton
and Daniels, 1985) and water and ion regulation in fetal membranes
(Rabee and McCoshen, 1986; Kensinger et al., 1986). Prolactin binds to
kidney membranes, but a clear function has not been established. These


Steroid interaction at the PRL genome DNA is thought to stimulate
PRL transcription though involvement of pituitary transcriptional
activator, PIT-1. This factor, PIT-1, must bind DNA in conjunction
with estrogen binding to its DNA domain to stimulate PRL gene
transcription (Adler et al., 1988).
Receptor Theory
Peptide hormones usually interact with cells through receptors.
Although the concept of 'receptors' seems commonplace, it was
introduced by J.N. Langley (1852-1926) following his observations of
mutual antagonism between curare and nicotine. The drugs interacted
with a "receptive substance" during autonomic transmission in
neuromuscular communication of the frog leg (Langley, 1909).
Qualitative aspects of receptor saturation and selectivity were
transformed to quantitative analysis by A.J. Clark (1885-1941).
Studying acetylcholine and atropine, he recognized that the rate at
which drugs combined with receptors was dependent on the concentration
of drugs and receptors and that the dissociation rate was proportional
to the number of complexes formed. These properties were similar to
mass action isotherms used by Langmuir (1881-1957) and, therefore,
drug-receptor interactions were found to obey the laws of mass action.
However, not all drug-receptor binding phenomena was explained by
Clarks observations and mass action equations. Stephanson (1956)
further refined drug-receptor interactions with the role of efficacy in
biological responses, noting that agonist response curves for tissues
were often steeper than dose-response curves. He postualated that 1)
maximal effects are produced when an agonist occupies only a small


18
proportion of the receptors; 2) biological response is not linear in
proportion to the number of receptors; and 3) equal biological
responses can be produced by drugs of different capacity for receptor
occupancy; that is, increased efficacy. The concept of spare receptors
was developed by Patn (1941), from studies on effects of antagonism.
He noted that agonists can elicit maximal biological responses even
when only a small fraction of the total receptors are occupied.
Receptor occupancy was not rate limiting for tissue activation.
Nickerson (1956) observed that only 1% of guinea pig ileum histamine
receptors needed to be occupied for maximal contraction, confirming
Paton's (1961) theory.
Analysis of Receptors
With developments in in vitro techniques, quantification of ligand
binding to membranes and defining receptors required evidence of
saturation, specificity, and kinetics realistic for the time course of
biological action. Receptor binding data are obtained through
saturation or competitive inhibition studies, and generate curvilinear
results. Linear transformation of data to obtain binding parameters is
achieved through Scatchard (1949) (Rosenthal; 1969) interpretation.
Scatchard analysis is based on equilibrium kinetics resulting in a
linear plot of data where bound/free ratio and free hormone data are
ploted on the abcissa and the ordinate, respectively. This results in
linear interpretation of data where the negative slope of the generated
line defines the affinity constant (Ka) or its reciprocal defines the
dissociation constant (1/Ka or Kd). Maximal binding and density of
binding sites are estimated by the y- and x-intercepts, respectively.


71
Table 6-1: Composition of Day 15 uterine flushings from
ovariectomized gilts treated with daily injections of
progesterone and saline or porcine prolactin from Days
4 through 14 (x + SEM).
ITEM
SALINE
PROLACTIN
Total
Protein (mg)
74.3
+19.3
74.0
+16.7
Total Uteroferrinb
(umoles/uterine horn)
4138
+1903
4230
+ 1648
Uteroferrin/mg Protein
45.7
+14.9
51.7
+13.0
Total
Calcium (mg)
0.5
+0.2
0.4
+0.2
Total
Cloride (mg)
1.7
+0.3
1.5
+0.2
Total
Sodium (ug)
125.3
+32.1
93.5
+27.9
Total
Potassium (ug)
170.2
+46.7
131.8
+40.4
Total
Glucose (mg)
2.5
+0.3
2.8
+0.3
Total
L-acyl aminopeptidasec
655
+112
604
+92
Total
PGF (ng/uterine horn)
235
+22
219
+19
Total
PGE (ng/uterine horn)
38.6
+6.7
34.6
+ 5.8
treatment effects were not detected (P>0.05).
bAcid phosphatase activity; umoles p-nitrophenol
released/ml/10 min at 37 C.
cSigma Units: One Sigma Unit will release 1 umole (143 ug)
of B-napthylamine from L-leucine-B-nalpthylamine per hour
at 37 C, pH 7.1.


Absorbance at 280 nM
153
Figure 12-1: Separation of nonglycosylated and glycosylated forms
of porcine prolactin by Concanavalin A-Sepharose 6B column
chromatography. Protein was detected by its absorbtion at 280 nm.


Figure 7-4: Dissociation kinetics assay for magnesium
chloride treated (A) Day 75 porcine endometrial or (B) Day 20
rat liver membranes. Tissue was incubated with porcine
[i2 ai]-prolactin in the presence (1 ug) and absence of
unlabelled porcine prolactin for 24 h at 25 C, rinsed, and
fresh buffer containing 5 ug unlabelled porcine prolactin was
added. Specific binding was determined at each time point.


12
decidua is far greater than that for the anterior pituitary, which
weights approximately 0.6 g. The two sources of PRL are regulated
differently; TRH and dopamine, which stimulate and inhibit pituitary
PRL release, respectively, do not affect PRL production by decidual
tissue. Release of PRL by decidual tissue is inhibited by arachadonic
acid and stimulated by calcium and progesterone (Healy and Hodgen,
1983). Differential regulation of PRL from its two sources may be due
to storage properties of PRL in the tissues (Markoff et al., 1983). In
addition, placental peptides are secreted that stimulate (23,500 Mr;
Handwerger et al., 1983) and inhibit (Markoff et al., 1983) decidual
PRL. Local production of PRL in species that decidualize at
implantation, including humans, rabbits and rats, further support PRL's
role in uterine physiology. However, in species with noninvasive
placentae, such as pigs, other mechanism(s) may exist, i.e., receptor
regulation, to allow similar effects of PRL on uterine function.
Prolactin in the Circulation
Circulating levels of PRL are relatively constant during pregnancy
(Dusza and Krzymowska, 1981; Kensinger et al., 1986; DeHoff et al.,
1986) in pigs; elevated slightly on Day 10 (20 ng/ml), then declining
by Day 20 and remaining constant (5-10 ng/ml) until parturition
(Kraeling et al., 1982). In cyclic pigs, PRL levels are similar to
those for pregnant pigs (5-10 ng/ml) except that PRL is elevated (15-20
ng/ml) on Days 0 to 2 and 16 to 17 (Brinkley et al., 1973; Dusza and
Krzymowska, 1979; Foxcroft and Van der Weil, 1982) when concentrations
of circulating estrogens increase.


65
The inter- and intra- assay coefficients of variation were 15.2 and
16.3%, respectively.
Statistics
Data were analyzed by least squares analysis of variance using the
General Linear Models procedures of the Statistical Analysis System
(SAS) (Barr et al.f 1979). Included in the model were the effects of
pig, treatment, time and treatment by time interaction.
Results
Effects of administration of exogenous porcine PRL are presented
in Figure 5-1. Concentrations of PRL in serum increased within 2h in
gilts that received exogenous porcine PRL. Over the 4 days of
administration, concentrations of PRL were 4.5-fold higher (P<0.001)
for gilts that received exogenous porcine PRL (19.6+1.24 ng/ml)
compared to control gilts (4.3 +0.13 ng/ml).
Discussion
Administration of exogenous porcine PRL at 12 h intervals was
effective in elevating the circulating levels of PRL. Prolactin levels
were increased at 2 h post-administration and remained elevated
throughout the treatment period. Additionally, the increase in PRL
levels were not pharmacological since concentrations of PRL in pigs
during estrus and the late luteal phase are 15 to 20 ng/ml (Brinkley et
al., 1973; Dusza and Kryzmowska, 1979).


104
from rabbit (Grissom and Littleton, 1988), mink (Rose et al., 1983),
rat (Williams et al., 1978), and sheep (Posner et al., 1974b). The sow
mammary gland lactogenic receptor has a higher affinity for ovine than
porcine PRL. The binding of [123I]-PRL to homologous receptors was low
(<3%) when labelled porcine PRL was bound to porcine mammary tissue,
labelled rabbit PRL to rabbit mammary tissue and labelled sheep PRL to
sheep mammary tissue (Berthon et al., 1987b) none of which was
pretreated with MgCl2. Low binding was detected in the present study,
but homologous binding was increased following treatment of endometrial
membranes with MgCli.
Crossreactivity of GH for PRL receptors is not uncommon. Growth
hormome is often reported to have the same affinity as PRL for
receptors when tested in heterologous assay systems and has been
suggested to upregulate the hepatic prolactin receptor (Knazek et al.,
1974; Webb et al., 1986). It is unlikely, however, that GH bound to
the PRL receptor is biologically agonistic in pig endometrium since
crossreactivity between PRL and GH was not apparent, suggesting that
PRL is binding to its own receptor.
The reversibility of binding after PRL has bound to its receptor
has been questioned. An exhaustive study by Van der Gugten and
coworkers (1980) suggested that PRL may not freely dissociate from its
receptor in vitro. Additional work by Kelly et al. (1983) showed that
the dissociation rate of PRL was much slower following longer
association time. Prolactin receptors from different membrane
subpopulations also have differences in affinity and dissociation rate
constants. Golgi membrane receptors have faster dissociation rate
constants than receptors in plasma membrane. Yet, dissociation was not


117
membrane preparations, RRA conditions, inhibition curves and analysis
of binding data.
Binding Analysis
Inhibition binding assays were also conducted on Day 75 pregnant
porcine endomtrial membranes to serve as a positive control. Analysis
of binding data was as described in Chapter 7.
Statistics
Data were analyzed by least squares analysis of variance using the
General Linear Models procedure of the Statistical Analysis System
(SAS) (Barr et al., 1979) to detect effects of reproductive status
(pregnant or cyclic), day and their interaction. Within reproductive
status, orthogonal contrasts were used to detect differences between
means on different days. Student's t-test was used to detect
differences between reproductive status on individual days.
Results
Results from analysis of porcine endometrial membrane PRL
receptors throughout the estrous cycle and early pregnancy are depicted
in Figure 9-1. The affinity of the porcine endometrial membrane PRL
receptor (Ka = 0.21 + 0.05 x 108 M_1) was not affected by day, status
or their interaction. However, receptor numbers were affected (P<0.01)
by reproductive status since endometrial PRL receptors (pmole/mg
protein) were higher for endometrial membranes from pregnant (31.2
+1.7) than cyclic (24.3+1.7) gilts. Changes in endometrial PRL
receptor numbers were affected by a day by status interaction (P<0.06)


5
mutations/100 residues/108 years, followed by human (19 point
mutations), and ovine and bovine (16 point mutations) PRLs. Porcine
and cetacea PRL show only 5 point mutations/108 years, which is much
slower than for other species. Differential regulation of PRL
development between species may suggest specified functions. Porcine
PRL has evolved only as fast as cytochrome C, while rat PRL has evolved
so quickly that is has only a 40% difference in its structure compared
to human PRL. This is a smaller difference than that between human and
nonprimate PRLs. The reasons for the differential evolutionary rates
of PRL are unknown; as are the resulting changes in function or
specificity. Most evolutionary change is neutral (King and Jukes,
1969); however, increased rates of change may be due to new selection
pressures or loss of specific function (Wallis, 1981).
These points, suggest that species specificity is an important
attribute of PRL to further our understanding of its biological
functions. Previous views were that a hormone, regardless of species
source, would function similarly in each species into which it was
injected. This fallacy is discussed by Nicoll (1982), who noted that
early research on PRL was with ovine PRL. By chance, the majority of
ovine PRL's functions are "PRL-like" in nature. Had the early work
been conducted with human PRL, the literature may have described a very
different set of functions. Hormones are named usually for their
suggested functions, thus restricted by man's attempt to organize and
understand himself. Although first thought to be primarily involved in
lactation, PRL is now cited with over 100 functions (Riddle, 1963;
Nicoll and Bern, 1972), most of which are categorized by 1)
osmoregulation and electrolyte balance, 2) growth and development, 3)


"Nothing is impossible to the willing mind"
The Book of Hans Dynasty


201
Liscia, D.S., Alhadi, T. and Vonderhaar, B.K. (1982) Solubilization of
active prolactin receptors by a non-denaturing swittlerionic detergent.
J. Biol. Chem. 257, 9401-9405.
Liscia, D.S. and Vonderhaar, B.K. (1982) Purification of a prolactin
receptor. Proc. Nat. Acad. Sci. USA. 79, 5930-5934.
Lowry, O.H., Rosenburgh, N.J., Farr, A.L. and Randell, R.J. (1951)
Protein measurment with the folin phenol reagent. J. Biol. Chem. 193,
265-275.
Lumpkin, M.D., Samson, V.K. and McCann, S.M. (1983) Hypothalamic and
pituitary sites of action of oxytocin to alter prolactin secretion in
the rat. Endocrinology. 112, 1711-1714.
Mainoya, J.R., Bern, H.A. and Regan. J.W. (1974) Influence of ovine
prolactin on transport of fluid and sodium chloride by mammalian
intestine and gall bladder. J. Endocrinol. 63, 311-317.
Markoff, E., Howell, S., Barry, S. and Handwerger, S. (1983) Local
regulation of decidual prolactin release in vitro. Proc. 65th Ann.
Meet. Endocrine Soc. 239 (Abstract).
Markoff, E and Lee, D.V. (1987) Glycosylated prolactin is a major
circulating variant in human serum. J. Clin. Endocrinol. Metab. 65,
1102-1106.
Markwell, M.K. and Fox, C.F. (1978) Surface specific iodination of
membrane proteins of viruses and eukaryotic cells using 1,3,4,6-
tetracholo-30, 60 diphenyl glycouride. Biochemistry. 17, 4807-4817.
Marshall, S., Bruni, J.F. and Meites, J. (1978) Prolactin receptors in
mouse liver: species differences in response to estrogenic stimulation.
Proc. Soc. Exp. Biol. Med. 159, 256-259.
Martinet, L., Aliis, C. and Allain, D. (1981). The role of prolactin
and LH in luteal function and blastocyst growth in mink (Mustela
vison). J. Reprod. Fert. Supp 29, 119-130.
Maurer, R.A. (1982) Estradiol regulates the transcription of the
prolactin gene. J. Biol. Chem. 257, 2133-2136.
Maurer, R.A. and Gorski, J. (1977) Effect of estradiol-176 and
pimozole on prolactin synthesis in male and female rats.
Endocrinology. 101, 76-84.
McComb, D.J., Caires, P.D., Kovacs, K. and Szabo, S. (1985) Effects of
cysteamine on the hypothalmc-pituitary axis in the rat. Fed. Proc. 44,
2551-2258.


CHAPTER 6
EFFECTS OF HYPERPROLACTINEMIA ON PROGESTERONE AND ESTROGEN
INDUCED UTERINE SECRETORY RESPONSE IN PIGS
Introduction
As mentioned in Chapter 3, early endocrinological studies involved
manipulation of endogenous hormones to establish physiological roles at
the tissue of interest. Converse to lowering endogenous hormones,
manipulations which increase endogenous hormone levels followed by
observation of physiological changes can also allow insight into a
hormones potential involvement in function. Several mechanisms have
been used, ranging from injection of crude tissue homogenates to
sophisticated gene manipulations. Each technique acheived a similar
endpoint, increased or supplemented endogenous hormone levels to allow
to investigation of resulting physiological changes.
Exogenous prolactin (PRL) results in increased endometrial
proliferation and protein secretion (Chilton and Daniels, 1985) in long
term (12 week) ovariectomized rabbits. Prolactin also modulates ion
channels (Falconer and Rowe, 1977), gap junction formation (Sorenson et
al., 1987) auto up-regulates its own receptor (Djaine et al., 1977;
1987) and increases steroid receptors (Daniels et al., 1984). Through
these mechanisms, PRL may affect the uterine environment of pigs.
Uterine secretory function is critical for nourishment of
preimplantation porcine conceptuses. Investigation of PRL interactions
with ovarian steroid hormones, especially estrogen, may explain the
67


188
Berthon, P., Kelly, P.A. and Djiane, J. (1987b) Water-soluble
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Bhattacharya, A. and Vonderhaar, B.K. (1979) Thyroid hormone
regulation of prolactin binding to mouse mammary gland. Biochem.
Biophys. Res. Comm. 88, 1405-1411.
Blankenstein, M.A., Bolt-se Vries, A., Coert, A., Nievelstein, H. and
Schroder, F.H. (1985) Effect of long-term hyperprolactinemia in the
prolactin receptor content of the rat ventral prostate. Prostate. 6,
277-283.
Bohnet, H.G., Gomez, F. and Friesen, H.G. (1977) Prolactin and
estrogen binding in the mammary gland of the lactating and non-
lactating rat. Endocrinology. 101, 1111-1121.
Bonifacino, J. and Dufau, M.L. (1984) Stucture of the ovarian lactogen
receptors analysis with cross-linking reagents. J. Biol. Chem. 259,
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Bonifacino, J. and Dufau, M.L. (1985) Lactogenic receptors in rat
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of the growth hormone/prolactin receptor gene family. Cell. 53, 69-77.
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concentrations. J. Endocrin. 113, 355-364.
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Brinkley, H.J., Wilfinger, W.W. and Young, E.P. (1973). Plasma
prolactin in the estrous cycle of the pig. J. Anim. Sci. 37, 303
(Abstract).
Brookfor, F.R., Hoeffler, J.P. and Frawley, L.S. (1986) Estradiol
induced a shift in cultured cells that release prolactin or growth
hormone. Am. J. Physiol. 250, E103-E105.
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Invest. 72, 743-747.


170
exogenous estrogen given on Day 11. Increases in uterine secretory
response were detected when there was an interaction with estrogen, but
not progesterone. By manipulation of endogenous PRL in vivo, uterine
secretory response was decreased due to hypoprolactinemia and increased
in conjunction with hyperprolactinemia (Figure 13-1), suggesting that
PRL affects uterine secretory responses in pigs, as previously observed
in rabbits (Chilton and Daniels, 1985) and rats (Leung and Sasaki,
1973).
Prolactin concentrations in the circulation of pigs are rather
constant during the estrous cycle (Dusza and Krzymowska, 1979) and
early pregnany (Dusza and Krzymowska, 1981), and increase in
association with increases in circulating estrogens (Brinkley et al.,
1973; DeHoff et al., 1986). Effects of PRL may be regulated through
changes in numbers of PRL receptors in target tissues. These changes
may account for differences in physiological affects associated with
reproductive status or following administration of exogenous estrogen.
Prolactin receptors, in general, have been detected by
heterologous assays using ovine PRL. In vitro quantification can be
affected by heterologous assays due to differences in crossreactivity
between PRL, receptors and immunological artifacts (Hughes et al.,
1982) as well as other problems discussed by Nicoll (1982). In the
present study, a homologous RRA for porcine PRL was developed to obtain
binding characteristics for porcine endometrial PRL receptors. Results
obtained were similar to those generated in homologous assays for mouse
liver and to results obtained using heterologous binding conditions for
PRL receptors in reproductive tissues of pigs (Rolland et al., 1978;
DeHoff et al., 1984; Bramley and Menzies, 1987) and other species


205
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Induction of prolactin receptors in rat liver after administration of
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experimental and physiological conditions. Mol. Cell. Endocrinol. 39,
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chorionic gonadotropin binding component obtained from Leydig cell
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Pankov, Y.A. and Butnev, V.Y. (1986) Multiple forms of pituitary
prolactin, a glycosylated form of porcine prolactin with enhanced
biological activity. Int. J. Pept. Prot. Res. 28, 113-115.
Parlow, A.F. and Shome, B. (1976) Rat prolactin: the entire linear
amino acid sequence. Fed. Proc. 35, 219 (Abstract).
Parnkh, I., Anderson, W.L. and Neame, P. (1980) Evidence for a
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Patn, W.D.M. (1961) A theory of drug action based on drug-receptor
combination. Proc. Royal Soc. B. 154, 21-69.
Pellegini, I., Gunz, G., Ronin, C., Fenouillet, E.,'Peyet, J.P.,
Delori, P. and Jaquet, P. (1988) Polymorphism of prolactin secretion
by human prolactinoma cells: Immunological, receptor binding and
biological properties of the glycosylated and nonglycosylated forms.
Endocrinology. 122, 2667-2674.


CHAPTER 13
GENERAL DISCUSSION
Prolactin (PRL) function in mammals is associated mainly with
reproduction. In females, effects of PRL on the ovary and mammary
gland are well established. However, previous reports and results from
this dissertation expand on these roles to include an effect of PRL on
endometrial function which affects the uteine environment. Thereby,
extending the functions of PRL to include all tissues of the female
reproductive system.
Prolactin's effects on uterine physiology are well documented.
Most species studied to date, however, undergo invasive implantation
and have production of PRL by uterine decidual tissue and/or placental
production of placental lactogen. The pig is similar to the rabbit in
some aspects of conceptus-uterine interactions. Both species have
conceptuses that secrete estrogen to establish pregnancy and have
placentae that do not produce placental lactogen. However, the rabbit
endometrium does decidualize during implantation and produces PRL
locally. The pig conceptus undergoes noninvasive implantation and has
no local source of endometrial PRL. However, PRL does affect uterine
secretory function in pigs (chapters 3 and 6; Mirando et al., 1988).
Administration of CB154 decreased circulating concentrations of
PRL by 40-50%, similar to results of Kraeling et al. (1982) and
Vhitacre et al., (1981). However, in these studies, PRL was not
decreased below chronic circulating levels, as observed for rats.
Bromocryptine may be effective in blocking large increases in
168


THE ROLE OF
STUDIES
PROLACTIN IN ESTABLISHMENT OF PREGNANCY IN PIGS
ON ENDOMETRIAL PROLACTIN RECEPTOR REGULATION
AND UTERINE SECRETORY PHYSIOLOGY
By
KATHLEEN HART YOUNG
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1989


154
provide the nonglycosylated and glycosylated forms. The proteins were
dialyzed against 10 mM sodium phosphate (pH 7.6) to provide forms of
PRL to be tested in mitogenic, immunoaffinity and receptor binding
assays. The forms of PRL to be tested for lactogenic activity were
dialyzed against distilled water. Protein concentration was determined
as described by Lowry et al. (1951) after dialysis. The purity of the
two forms of porcine PRL was determined by using 12.5% SDS one
dimensional polyacylaminde gel electrophoresis (Figure 12-2; Roberts et
al., 1984).
Nb2 Lymphoma Cell Assay
Cells were propagated in Fishers medium supplemented with 10%
horse serum, 10% fetal bovine serum, 10 units/ml each penicillin and
streptomycin and 100 urn (S-mercaptoethanol. Cultures were rendered
quiescent by transferring cells into medium with 1% fetal bovine serum
for 24h and then into medium completely deficient in fetal bovine
serum. Cells were plated at an initial cell density of 4 x 103
cells/ml into a 96-well plate (200 ul/well). Each test sample was
assayed in triplicate while "Nb2 alone" (no test sample) control was
assayed in quadruplicate. Nb2 cells were harvested 48 hours later
(Cambridge PhD Cell Harvester, Cambridge, MA) following a 4 hour DNA
labeling period with 3H-thymidine (0.5 uCi/well). The uptake of 3H-
thymidine was quantified by liquid scintillation counting.
Pigeon Crop Sac Assay
Pigeon crop sac assays were conducted as described by Nicoll et
al. (1985) and Pukac and Horseman (1984). Pigeons were injected for 3


135
12h by increased LAP concentrations which were followed by increases in
other components. Estrogen, either alone or in conjunction with PRL,
could affect the synthesis of uterine secretory components. Total
protein and uteroferrin in uterine flushings increased at 24h without a
further increase in LAP; suggesting an increase in rate of synthesis,
and secretion of proteins without a detectable change in membrane
processing as measured by LAP. These data agree with previous reports
wherein estrogen-PRL interaction increased porcine endometrial
uteroferrin secretion (Young and Bazer, 1988). The temporal changes
observed following administration of E2V suggest that estrogen
increases PRL receptor numbers in porcine endometrial membranes prior
to observed changes in secretory products. An effect of estrogen on
PRL receptor numbers was suggested previously since endometrial
membrane PRL receptors increase following conceptus estrogen secretion
during early pregnancy. Endometrial membrane PRL receptors also
flucutate throughout gestation in association with changes in
concentrations of estrogens (DeHoff et al., 1984). Changes in PRL
receptor numbers may be necessary for estrogen stimulation of changes
in ion concentrations, or ratios, as occurred in this and previous
studies (Geisert et al., 1982c; Bazer et al., 1984; Young et al.,
1987). Additionally, estrogen increases pituitary secretion of PRL
(Chen and Meites, 1970; Baxter, 1985) which would increase available
PRL for receptor binding. This may increase internalization of
endometrial receptors since receptor numbers decreased at 12h. Maximal
occupation of rabbit mammary gland PRL receptors is at 15 min and
returns to normal levels by 12h (Djiane et al., 1979a). The half-life
of rabbit mammary gland PRL receptors is approximately 40-50 min


8
8,000 Mr (149-199) peptides. Cleavage of the large loop of PRL does
not inhibit its binding to PRL receptors in mammary gland or ventral
prostate of rats (Clapp, 1987; Vick et al., 1987). Additionally,
cleaved PRL has full potency in Nb2 lymphoma cell mitogenic assays.
However, only 50-60% of cleaved PRL is detected by RIA (Vick et al.,
1987), suggesting that the biological and antigenic determinents are at
different sites, as suggested by Amit et al. (1985).
Cleaved PRL is found in pituitaries of mice (Sinha and Gillian,
1981), rats (Mittra, 1980) and humans (Sinha et al., 1985) and is
produced by ventral prostate of male rats. Mammary tissue of lactating
rats has higher enzyme activity, thus more cleaved PRL is produced by
this tissue (Vick et al., 1987; Clapp, 1987). Target tissues of PRL
may be expected to contain cleavage enzymes (Nolin, 1982), but it is
uncertain if these enzymes are regulated physiologically. Cleavage of
PRL is by nonspecific multipurpose proteases influenced by
configuration of the PRL molecule (Wong et al., 1986). Cleavage also
depends on pH, with enzymatic activity increasing at pH 5 to 3.6
(Clapp, 1987).
Reduction of the disulfide bond joining the chains generates a
16,000 Mr and 8,000 Mr fragments. The 16,000 Mr fragment maintains PRL
activity on mammary epithelial cells, despite decreased receptor
binding, mitogenic activity and immunoreactivity. However, functions
of the cleaved, 16,000 Mr and 8,000 Mr fragments of PRL await further
investigation.


Figure 4-1: Mean concentrations of prolactin (ng/ml) in
serum of cyclic gilts treated with cysteamine (solid line) or
ethanoloamine (dashed line) from Days 10-16 (denoted by
arrows).
59


56
in the proper ratio or temporal pattern to secretion of proteins.
Additionally, decreases in circulating PRL may have been compensated
for physiologically through other mechanisms.


174
SruUirin
Endometrial proliferation
Increase gap junctions
Ion channels
Increase E and P receptors "*CjL prOgCSlCiOfie
of pregnancy
Figure 13-2: Possible mechanism(s) involving prolactin during
maternal recognition of pregnancy in pigs.


38
suggested that PRL regulation of its receptor, with up and down
regulation, is through two non-antagonistic mechanisms.
Increases in ventral prostate PRL receptors occur 6-12 h following
ovine PRL administration in vitro. Increases are dose dependent (Rui
et al., 1986) and not mimiced by estrogen, androgen, hCG, insulin,
calcium, prostaglandins or cAMP. Positive regulation of ventral
prostate PRL receptors by estrogen is confirmed by Dave and Witorsch
(1985) and Blankenstein et al. (1985). Similarly PRL auto-upregulates
its receptors in testes (Amodor et al., 1985), liver (Amit et al.,
1985), lung (Amit et al., 1985), adrenal gland (Calvo et al., 1981) and
mammary gland (Djiane and Durand, 1977; Djiane et al., 1979).
Studies with hypophysectomized rats suggest that hepatic PRL
receptors may be regulated by anterior pituitary hormones, other than
PRL. Hypophysectomized rats bearing pituitary implants have increased
hepatic PRL receptor numbers which is not mimiced by adminsitration of
ovine PRL (Posner et al., 1975). Additionally, adminsitration of CB154
to reduce PRL levels had no effect (Norstedt et al., 1981). Continuous
infusion of rat growth hormone, but not rat PRL, to male rats resulted
in feminization of hepatic PRL receptor profiles (Norstedt et al.,
1987). Growth hormone may induce PRL hepatic receptors differently
between sexes since its secretory pattern differs between males and
females (Eden, 1979). Induction of hepatic PRL receptors in rats was
achieved by exogenous administration of human, bovine and rat growth
hormones, but net by PRL or human placental lactogen. Hepatic PRL
receptors of prepubertal (17 day old) female rats increased to levels
typical of adult females following 7 days of human growth hormone
infusion. These studies support growth hormone regulation of PRL


Percent Specific Binding
93
Time in Hours
Figure 7-3: Binding of porcine [12SI]-prolactin by magnesium
chloride treated Day 75 porcine endometrial (circles) or Day 20
rat liver (squares) membranes at 4 C (dashed line) or 25 C (solid
line).


68
biphasic responses of the uterus that occur following estrogen
administration (Szego et al., 1978; Geisert et al.( 1982c; Young et
al., 1987) possibly through modification of the uterine environment or
through more rapid effects of the peptide hormones. Therefore,
interactions between PRL and progesterone, in the absence of estrogens
of ovarian origin, were investigated in pigs. Knight et al. (1973)
demonstrated that ovariectomized pigs treated with progesterone alone,
secrete the same proteins after 11 days of treatment as ovarian intact
gilts on Day 15 of the estrous cycle or pregnancy. In a second
experiment, the interaction between estrogen and PRL was investigated
following administration of exogenous estrogen. Administration of
exogenous estrogen on Day 11 of the cycle mimics the maternal
recognition of pregnancy factor, porcine conceptus estrogens, without
confounding effects of other conceptus secretory products (Geisert et
al., 1982c) .
Materials and Methods
Animals
Crossbred gilts of similar weight (100-120 kg) and age (7-9
months) were used in this study after they experienced at least two
normal estrous cycles (18-22 days). In the presence of intact boars,
gilts were observed daily for behavioural estrus. The first day of
behavioural estrous was designated Day 0.
Exogenous Hormone Administration
For chronic steroid treatment, gilts received 200 mg progesterone
(P4; Sigma, St. Louis, MO) in 4 ml corn oil:ethanol solution (90:10,


92
pretreated with 500 ul distilled water had low binding similar to that
obtained when MgCli-pretreated membranes were incubated in the presence
of unlabelled porcine PRL.
The optimal membrane proteinrradiolabel concentration ratio was
150 ug membrane using 45,000 cpm porcine [123I]-PRL. Maximal specific
binding of approximately 20% for Day 75 pig endometrial membranes was
slightly lower than the 25% specific binding observed for Day 20
pregnant rat liver membranes under similar assay conditions.
The Day 75 porcine endometrial and Day 20 rat liver membrane
preparations were tested for binding saturability at 4 and 25 C (Figure
7-3). Binding curves were similar for the two membrane sources at each
temperature. Porcine endometrial and rat liver membranes reached
saturation at 24h attained specific binding of approximately 20% and
25%, respectively, at 25 C. At the lower temperature, porcine
endometrial and rat liver membranes had specific binding values of 4
and 7%, respectively.
Assays to determine dissociation kinetics suggested that the
majority of the porcine t1291]-PRL displacement occurred within lOh.
The dissociation rate constant (Kd) for the porcine endometrial PRL
receptor (Figure 7-4a) was slightly lower than that for rat liver
receptor (Figure 7-4b) with Kd's of 3.79 x 10_6/s and 9.3 x 10_6/s,
respectively. Over the remaining 38h only slight displacement
occurred for the porcine endometrial PRL receptor (Kd=1.63 x 10-6/s).
Additionally, displacement of porcine [129I]-PRL from receptor by 5 ug
unlabeled porcine PRL was incomplete for both porcine endometrial (60%)
and rat liver (40%) membranes. This is consistent with previous
results by van der Gugten et al. (1980) and Kelly et al. (1983).


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109
Gels were stained with comassie blue, dried and exposed to Kodak XRP
film for 4 weeks at -70 C.
Estimates of the molecular weights of cross-linked hormone
receptor complexes were estimated from semilog plots of protein band
migration (mm) from the top of gel versus its molecular weight. The
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Results
Cross-linking of porcine [12SI]-PRL to magnesium chloride treated
porcine endometrial membrane preparations resulted in four
electrophoretic bands detected by autoradiography with molecular weight
estimates of 45,000, 62,500; 78,000 and 88,000 (Figure 8-la, lane 3).
Following subtraction of the molecular weight of PRL (23,000), the
porcine endometrial PRL receptor(s) molecular weights are estimated to
be 22,000; 39,500, 55,000 and 65,000. Two bands were observed from
autoradiography of cross-linked PRL to rat liver membrane prepations
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estimates of 39,000 and 23,000 Mr for the rat liver membrane PRL
receptor. The addition of 10 ug unlabelled porcine PRL blocked all of
these bands in both the pig endometrium (Figure 8-la, lanes 1 and 2)
and rat liver (Figure 8-lb, lane 1). Bands were faintly detectable
despite the addition of unlabelled porcine GH to porcine endometrium
(Figure 8-la, lane 4) and rat liver (Figure 8-lb, lane 3).
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101
Figure 7-8: Scatchard analysis of porcine C123I]-prolactin
displaced by unlabelled porcine prolactin using magnesium
chloride treated Day 75 porcine endometrial membranes.
Actual and 'best fit' data points are represented by open
squares and closed triangles, respectively.


21
of association and dissociation are equal (Baxter and Funder,
1979) to
ki [H] [R] = k-i [HR] (11)
or redefined as
[H][R]/[HR] = k-i/ki = Kd (12)
Thus, the ratio of ligand 'off' to 'on' a receptor is the equilibrium
dissociation constant (Kd) and is equal to the reciprocal of the
association constant (Ka) generated from the negative slope of
Scatchard analysis as previously mentioned. The association constant
depicts the affinity and tightness of binding between a hormone or
ligand and receptor (Limbird, 1986).
Scatchard analysis can generate a linear relationship between B/F
and F, describing a single class of hormone receptors. Curvilinear
Scatchard can be due to two separate binding sites with different
affinities or negative cooperativity (DeMeyts et al., 1976)
Prolactin Receptors
General Characteristics
Although PRL was discovered in the 1930s (Riddle et al., 1933),
binding of PRL to tissues was not demonstrated until the 1960s.
Specific high affinity binding of PRL and its relation to biological
response characteristics of hormone-receptor interaction was first
investigated in mammary glands of mice (Frantz and Turkington, 1972)
and rabbits (Shiu et al., 1973). Investigation of the PRL receptor
structure has been with rat liver. Binding of hormone to membrane does


Figure 10-3: Total recoverable (A) protein, (B) uteroferrin,
(C) leucine aminopeptidase (LAP) and (D) glucose in uterine
flushings at 1, 6, 12 and 24h following administration (i.m.)
of estradiol valerate (0.5 mg, hatched bars) or 12 h after
corn oil (0.5 ml, solid bar) administration. The soild line
denotes the mean value prior to injection (time zero) of Day
11 cyclic gilts. Values with different letters are different
for protein and LAP (P<0.05); glucose (P <0.01) and
uteroferrin (P<0.01). The SEM are +4 for protein; +168 for
uteroferrin; +30 for LAP and +0.17 for glucose.


86
Prolactin Radioreceptor Assay (RRA) Procedure
Following treatment of the membrane preparations with MgCl2,
binding assays were conducted as adapted from Haro and Talamantes
(1985a). Radioinert porcine PRL (100 ul) was added in serially
increasing concentrations (0-5120 ng). Radiolabeled porcine PRL (100
ul; 45,000 cpm; 0.24 ng) was added and tubes were vortexed extensively.
Total assay volume was 500 ul. The binding reaction was conducted at
room temperature (25 C) for 24 hours, terminated by addition of 3 ml
ice-cold assay buffer, and centrifuged at 3500 x g for 30 min. Samples
were decanted, inverted to drain, and pellets counted (1 min) to detect
gamma radioactivity (GammaTrac 1191, Nuclear Chicago Corp., Des Plains,
IL). Nonspecific binding, 7-4% of total binding, was determined by the
amount of radioactivity bound in the presence of either 2560 or 5120 ng
unlabelled hormone, respectively. Results are expressed as percent
bound [(total bound minus nonspecific binding/total counts) X 100].
Total bound binding represents radioactivity in the absence (Bo) or
presence (1-1000 ng) of radioinert hormone. In each experiment the
total and nonspecific binding was determined in triplicate. The intra-
and inter- assay coefficients of variation for receptor number were 9
and 10%, respectively, and for Ka, 18 and 23%, respectively.
Effects of Magnesium Chloride Molarity
Binding of porcine t1231]-PRL was tested as a function of MgCl2
molarity. Membrane preparations from Day 75 pregnant porcine
endometrium (300-500 ug), amnion, chorion and post-parturient (rabbit
or pig) mammary tissues were tested for binding of [123I]-PRL following


CHAPTER 4
EFFECTS OF CYSTEAMINE ON CIRCULATING
PROLACTIN LEVELS IN PIGS
Introduction
Cysteamine (CSH; SHz-CH2-CH2-NH2) disturbs the tertiary structure
of prolactin (PRL) and thereby depletes circulating levels of bioactive
PRL (Millard et al., 1982). Several short-term studies of effects of
CSH on PRL levels in rats have been reported (Millard et al., 1982;
McComb et al., 1985; Sagar et al., 1985). Dose response tests
determined that rats administered 90 mg/kg CSH (subcutaneously) had
decreased PRL levels in both the anterior pituitary (2.0 vs 0.57 ug/mg
protein) and serum (3.7 vs 0.44 ng/ml) 4h post-administration. No
adverse effects were reported (Millard et al., 1982). Cysteamine can,
therefore, lower circulating levels of bioactive PRL without
involvement of dopamine or other receptors at the anterior pituitary.
This study investigated the use of CSH for inhibition of circulating
PRL levels in pigs as an alternate method to bromocryptine
administration.
Materials and Methods
Experimental Design
On Day 4 of the estrous cycle, gilts were anesthetized with
thiamylal sodium (1 g, i.v.) and maintained under surgical anesthesia
using a closed circuit anesthesia machine. Gilts were fitted with
57


189
Butcher, R.L., Collins, W.E. and Fugo, N.V. (1974) Plasma
concentrations of LH, FSH, prolactin, progesterone and estradiol-178
throughout the 4-day estrous cycle of the rat. Endocrinology 94,
1704-1708.
Calvo, J.C., Finocchiaro, L., Luthy, I., Charreau, E.H., Calandra,
R.S., Engstrom, B. and Hansson, V. (1981) Specific prolactin binding
in the rat ventral adrenal gland: its characterization and hormonal
regulation. J. Endocrinol. 6, 303-325.
Cameron, C.M. and Rillema, J.A. (1983) Effects of prostaglandins on the
prolactin stimulation of lipid biosynthesis in mouse mammary gland
explants. Prostaglandins Leukotrienes Med. 10, 433-441.
Catt, K.J., Harwood, J.P., Aguilera, G. and Dufau, M.L. (1979)
Hormonal regulation of peptide receptors and target cell responses.
Nature (London). 280, 109-116.
Chamness, G.C., Costlow, M.E. and McGuire, W.L. (1975) Estrogen
receptor in rat liver and its dependence on prolactin. Steroids. 3,
363-371.
Chen, C.L. and Meites, J. (1970) Effects of estrogen and progesterone
on serum and pituitary prolactin levels in ovariectomized rats.
Endocrinology. 86, 503-506.
Chen, H.W., Hamer, D.H., Heininger, H. and Meier, H. (1972)
Stimulation of hepatic RNA synthesis in drawf mice by ovine prolactin.
Biochem. Biophys. Acta. 287, 90-98.
Chen, T.T., Bazer, F.W. and Gebhardt, B.M. (1975) Uterine secretion in
mammals: synthesis and placental transport of a purple acid
phosphatase in pigs. Biol. Reprod. 13, 304-313.
Chilton, B.S. and Daniels, J.C. (1985) Influence of prolactin on DNA
synthesis and glandular differentiation in rabbit uterine endometrium.
In: Prolactin, Basic and Clinical Correlates, pp. 351-359. (Eds. R.M.
Macleod, M.O. Throner and U. Scapagni) Fidia Research Series,
Springer-Verlag, Berlin.
Chilton, B.S. and Daniels, J.C. (1987) Differences in the rabbit
uterine response to progesterone as influenced by growth hormone or
prolactin. J. Reprod. Fert. 79, 581-587.
Clapp, C. (1987) Analysis of the proteolytic cleavage of prolactin by
the mammary gland and liver of rat: characterization of the cleaved
and 16K forms. Endocrinology. 121, 2055-2063.
Clark, A.J. (1927) The reaction between acetyl choline and muscle
cells. Part II. J. Physiol. 64, 123-143.


Ill
Pig Endometrium
4M MgCI NO MgCI
PRL PRL Bo GH PRL PRL Bo GH
o
RRA cone
ra 4M MgCI
J NSB Bo NSB Bo
Q Rat Liver
4M MgCI NO MgCI
PRL Bo GH Bo PRL Bo GH B^
1 2 3 4 5 6 7 8


32
1980) or transduction mechaniams of cellular hardware. Cells may not
possess identical intracellular machinery, e.g., hepatocytes versus
epithelial cells, and are inherently different and programmed to
respond differently from each other, but characteristically for each
cell type.
Extracellular Mediators
Actions of growth hormone are acheived indirectly through
stimulation of liver to release somatomedin C (Laron, 1982), now
recognized as insulin-like growth factors (IGF). This mechanisms of
action is extended to PRL (Anderson et al., 1983), which stimulates
release of a hepatic factor (Nicoll et al., 1983) that mediates PRL's
effect on mammary cells or pigeon crop sac cells. This factor, termed
"synlactin" (Nicoll et al., 1983), has no effect alone. It is secreted
when circulating PRL increases, such as during lactation, but is not
detected in male or virgin female rats. Pigeon crop sac proliferation
increases (in vivo) when ovine PRL is injected in the hepatic, but not
jugular vein of pigeons; suggesting that this mitogen is produced by
liver of pigeons (Mick and Nicoll, 1985).
Prolactin stimulation of somatomedin production by rat liver is
20-fold greater than that of growth hormone. However, PRL does not
stimulate growth in male rats. Synlactin may be an IGF-like molecule
that is detected during sulphate determination of IGFs (Mick and
Nicoll, 1985). Whether synlactin is a fragment of PRL or similar to
IGF awaits determination of its amino acid structure.
Another extracellular mediator of PRL effects is liver lactogenic
factor (LLF) (Hoeffler and Frawley 1987). Both synlactin and LLF are


Figure 12-4: Immunoaffinity of total (open and closed
circles), nonglycosylated (open and closed squares) and
glycosylated (open and closed triangles) forms of porcine
prolactin expressed as (A) percent bound versus log
concentration, (A inset) transformed to log versus logit plot
and (B) percent of Bo (normalized to 100%) versus log
concentration.


151
and Baxter, 1979). The glycosylated form accounts for approximately
10-30% of pituitary PRL (Lewis et al., 1984; Pankov and Butnev, 1986),
and 30-40% of circulating PRL (Markoff and Lee, 1987). Glycosylated
PRL is secreted from near-term decidua in humans (Lee and Markoff,
1986) and is found in human amniotic fluid (Meuris et al., 1985). The
carbohydrate structure of porcine PRL (25,000 Mr) has been identified
(Pankov and Butnev, 1986), and is similar to the carbohydrate moiety of
ovine LH (Bedi et al., 1982). Several studies have compared biological
activities and immunoaffinities of glycosylated and nonglycosylated
PRL. The glycosylated form has been reported to have both higher
(Pankov and Butnev, 1986) and lower (Lewis, 1984; 1985) activity in
lactogenic assays, lower mitogenic activity and immunoaffinity
(Pellegini et al., 1988; Scott et al., 1988). The decreased
immunoaffinity of glycosylated PRL is expected since the carbohydrate
moiety may mask the antigenic determinant (Pankov and Butnev, 1986;
Pellegini et al., 1988).
Comparisons of the affinities of glycosylated and nonglycosylated
forms of PRL for the lactogenic receptor have been conducted using
heterologous assays (Pellegini et al., 1988; Seely et al., 1988), but
are confounded by involvement of from two or three species through by
sources of tracer, unlabelled hormone and source of receptor.
Information from these studies suggest in vitro binding characteristics
which may not accurately reflect physiological hormone-receptor
interactions. Therefore, investigation of hormone binding
characteristics using an assay homologous to a species is important.
Porcine PRL was investigated to determine the presence of a
glycosylated form. Then, both forms were tested for lactogenic and


126
post-injection samples were obtained after gilts were reanesthetized
just before the time of the second sample collection. This procedure
avoided prolonged exposure to general anesthesia, and is not
detrimental to uterine secretory physiology (Young et al., 1987).
Tissue Preparation
Uterine flushings were prepared as described in Chapter 3.
Endometrial tissue was collected and prepared for homologous PRL RRA as
described in Chapter 7.
Protein Determination
Protein concentrations of endometrial membrane preparations and
uterine flushings were determined by the method of Lowry et al. (1961)
using bovine serum albumin as standard.
Measurement of Endomtrial Prolactin Receptors
Prolactin receptors were measured using the homologous RRA
described in Chapter 7. Tissue processing, protein determination,
iodination, determination of specific activity (83 uCi/ug) chaotropic
treatment of endometrial homogenates, RRA conditions, inhibition curves
and binding data analysis were all conducted as described in Chapter 7.
Binding analysis
Binding assays were also conducted using Day 75 pregnant pig
endometrial membranes as a positive homologous control (Young and
Bazer, 1987) since this tissue had high PRL receptor concentrations
(DeHoff et al., 1984).


196
Hayden, T.J., Bourney, R.C. and Forsyth, I.A. (1979) Ontogeny and
control of prolactin receptors in the mammary gland and liver of
virgin, pregnant and lactating rats. J. Endocrinol. 80, 259-269.
Healy, D.L. (1984) The clinical significance of endometrial
prolactin. Aust. N.Z. J. Obstet. Gynecol. 24, 111-116.
Healy, D.L., and Hodgen, G.D. (1983) The endocrinology of human
endometrium. Obstet. Gynecol. Surv. 38, 509-530.
Healy, D.L., Kimpton, V.G., Muller, H.K. and Burger, H.G. (1978) The
synthesis of immunoreactive prolactin by decidua-chorion. Br. J.
Obstet Gynaecol. 86, 307-312.
Heap, R.B., Flint, A.P.F., Gadsby, J.E. and Rice, C. (1979). Hormones,
the early embryo and the uterine environment. J. Reprod. Fert. 55, 275-
287.
Herrington, A.C., Elson, D. and Ymer, S. (1981) Water soluble receptors
for human growth hormone from rabbit liver. J. Recept. Res. 2, 203-
220.
Hoeffler, J.P. and Frawley, L.S. (1987) Liver tissue produces a potent
lactogen that partially mimics the actions of prolactin.
Endocrinology. 120, 1679-1681.
Hofstee, B.H.J. (1952) On the evaluation of the constants V and Km in
enzyme reactions. Science. 116, 329-331.
Holcomb, H.H., Costlow, M.E., Bischow, R.A. and McGuire, V.L. (1977)
Prolactin binding in rat mammary gland during pregnancy and lactation.
Biochem. Biophys. Acta. 428, 104-112.
Hollenburg, M.D. (1986) Mechanisms of receptor-mediated transmembrane
signalling. Experientia. 42, 718-726.
Houdebine, L.M. and Djiane, J. (1980) Effects of lysomotropic agents
and of microfiliment and microtubule disrupting drugs on the activation
of casein gene expression by prolactin in the mammary gland. Mol.
Cell. Endocrinol. 17, 1-15.
Hughes, H.P., Elsholtz, H.P. and Friesen, H.G. (1982) Up-regulation of
lactogenic receptors an immunological artifact? Endocrinology. Ill,
702-704.
Hughes, J.P., Elsholtz. P. and Friesen, H.G. (1985) Growth hormone and
prolactin receptors. In: Polypeptide Hormone Receptors, pp. 157-199.
(ed. B.I. Posner) Marcel Dekker, Inc., New York.
Hughes, J.P., Simpson, J.S.A. and Friesen, H.G. (1983) Solubilization
of active prolactin receptor by a non-denaturing switterionic
detergent. Endocrinology. 112, 1980-1985.


58
jugular catheters (Ford and Maurer, 1978), allowed 4 days rest and
housed individually to recover from surgery through Day 7. On Day 8,
7-10 ml blood samples were collected at 0800, 1300 and 1930 h. Gilts
were assigned randomly to receive either CSH (100 mg/kg/day) or an
isoosmotic control of ethanolamine (100 mg/kg/day). Administration of
treatment began on Day 10 at 0830h and was scheduled to continue until
Day 16 of the estrous cycle. Blood samples were taken two days prior
to, and following, the treatment period.
Results
Due to unexpected cytotoxic effects of CSH at the injection sites,
(tissue necrosis and gangrenous leasions), this project was cancelled
after four gilts received treatments for various lenghts of time.
Blood samples were analyzed for PRL concentrations and results are
summarized in Figure 4-1. Due to the inconsistent number of blood
samples and limited data, statistical analyses were not conducted.
Administration of CSH lowered serum PRL levels about 30% (1.97 vs 2.7
ng/ml) of controls after three days and about 70% (1.33 vs 4.32 ng/ml)
after five days of treatment. Comparison of PRL levels from CSH- and
CB154-treated gilts (Chapter 3) on a similar "day post-injection" basis
revealed that CSH lowered serum PRL levels 67% more than CB154.
Interestrous intervals and (arbitary) cytotoxic reactions to the
treatments are summarized in Table 4-1. A few comments on these
results are in order: Gilt 328 started the experiment in which 100
mg/kg dose was given twice daily to prevent possible increases in PRL
in the evening. This dosage was very cytotoxic, therefore CSH was
administered to gilt 305 at the same dose but only once a day in the


Figure
Page
6-3: Concentrations of (A) PGF and (B) PGE in Day 12
uterine flushings from cyclic gilts (Experiment 2)
treated with 1 ml saline (SAL) or 1 mg porcine
prolactin (PRL) on Days 6-11 and 0.5 ml corn oil
(OIL) or 5 mg estradiol valerate (E2V) on Day 11
of the estrous cycle 76
7-1: Effects of increasing magnesium chloride molarity
on binding of porcine [*23I]-prolactin by
membranes from Day 75 porcine endometrium, amnion,
chorion, as well as post-parturient pig and rabbit
mammary gland (300 ug) 90
7-2: Effects of increasing protein concentrations of
Day 75 porcine endometrial membranes on
binding of porcine [123I]-prolactin 91
7-3: Binding of porcine t1251]-prolactin by magnesium
chloride treated Day 75 porcine endometrial
(circles) or Day 20 rat liver (squares) membranes
at 4 C (dashed line) or 25 C (solid line) 93
7-4: Dissociation kinetics assay for magnesium
chloride treated (A) Day 75 porcine endometrial
or (B) Day 20 rat liver membranes 95
7-5: Binding and displacement of ovine and porcine
prolactin from magnesium chloride treated Day 75
porcine endometrial membranes 98
7-6: Crossreactivity of unlabelled porcine prolactin
(squares; pPRL), porcine growth hormone (triangles;
pGH),porcine luteinizing hormone (circles; pLH) and
porcine follicle stimulating hormone (diamonds;pFSH)
to porcine t123I]-prolactin with magnesium chloride
treated Day 75 porcine endometrial membranes 99
7-7: Crossreactivity between unlabelled porcine growth
hormone (dashed line), or porcine prolactin (solid
line) and porcine f123I]-prolactin with magnesium
chloride treated Day 75 porcine endometrial (circles)
and Day 20 rat liver (squares) membranes 100
7-8: Scatchard analysis of porcine [123I]-prolactin
displaced by unlabelled porcine prolactin using
magnesium chloride treated Day 75 porcine
endometrial membranes 101
8-la: Autoradiography of affinity labelled, cross-linked
porcine endometrial membrane preparation prolactin
receptors Ill
xi


169
circulating PRL associated with parturition in pigs or mating in rats,
but CB154 may be less effective, at least in pigs, in decreasing tonic
release of PRL. Additionally, PRL secretion may be controlled
differently in pigs through a nondopaminergic mechanism. Treatment of
pregnant gilts with CB154 had no effect on fetal survival suggesting
that 1) treatment was not early enough to alter the uterine environment
prior to conceptus estrogen secretion; 2) basal levels of PRL were
adequate for reproduction despite CB154 treatment; 3) endometrial PRL
receptors increased to allow physiological responses despite lowered
concentrations of PRL (Kelly et al., 1979) and 4) physiological
compensation by the porcine conceptus by other, unknown, mechanisms.
Admmstration of CB154 decreased the quantity of electrolytes in
uterine flushings of cyclic gilts 24 h following a single dose of
exogenous estrogen on Day 11. Lowered circulating PRL levels
interfered with estrogen-induced uterine ion fluxes and membrane
turnover, suggesting detrimental effects of lower PRL on uterine
secretory function.
The preimplantation porcine conceptus must stimulate histotroph
secretion and provide an antiluteolytic signal to insure maintenance of
CL function and progesterone secretion. Both actions are essential to
the establishment of a viable pregnancy and both appear to be modulated
or affected by PRL. Administration of exogenous PRL enhanced secretion
of uteroferrin, prostaglandin Fa and glucose into uterine flushings
(Day 12) of gilts treated 24 h earlier with exogenous estradiol. These
components of uterine flushings do not usually increase to high
concentrations until Day 14 to 16 of gestation, but pretreatment of
pigs with PRL (5 days) allowed a more rapid or maximal response to


127
Analysis of Uterine Flushings
Total recoverable protein, uteroferrin, calcium, sodium,
potassium, leucine aminopeptidase (LAP) and glucose in uterine
flushings were queantitated as described in Chapter 3.
Statistics
Data were analyzed by Least Squares Analysis of Covariance using
the Statistical Analysis System (SAS) (Barr et al., 1979). Included in
the model were effects of treatment (E2V vs corn oil), time (1, 6, 12
and 24h post-injection), treatment by time interaction and the
covariate. Each gilt served as its own control, i.e., the covariate
(time zero values for uterine flushings and endometrium), provided data
on basal levels of these constituents and was used to determine whether
changes due to treatment, time or their interaction were significant.
Orthogonal contrasts were used to detect differences between means.
Results
Evaluation of receptor data indicated that the affinity constant
(Ka=0.32 + 0.12 x 108 M-1) was not affected by treatment over the time
periods studied. However, PRL receptor numbers in the porcine
endometrial membranes did change after administration of exogenous E2V
as depicted in Figure 10-1. Receptor numbers (pmoles/mg protein +
11.8) were higher at 1 and 6h after E2V (43 and 55 pmoles/mg,
respectively), but then decreased (P<0.02) by 12h (9.6 pmoles/mg) and
24 h (23.4 pmoles/mg) after E2V administration to values that were not
different from values measured at 12h for gilts treated with corn oil
(26 pmoles/mg).


Total Sodium (ug)
W ffS V|
o o o
Total Potassium (ug)
Ul
o
100
'-J
Total Chloride (mg)
ro
09


Percent Bound
161
Hormone (log concentration)
B


175
numbers are increased by conceptus estrogens. Prolactin can then
interact with conceptus estrogens to enhance histotroph sectretion and
redirect prostaglandin secretion into the uterine lumen thereby
facilitating the mechanism of luteostasis. Therefore, PRL modulates
the uterus to increase its response to conceptus estrogens and enhance
histotroph secretion and luteostasis; both of which are essential for
establish pregnancy in pigs.
Chronic administration of ovarian steroids to ovariectomized gilts
resulted in decreased PRL receptor numbers in association with both low
(E2V alone) and high (E2V+P4) uterine secretory responses. Estrogen
administration was common to these two groups and may have increased
pituitary secretion of PRL. Higher circulating PRL may stimulate an
increase steroid receptors. But without the proper presence of
progesterone in the E2V (alone) treated group, uterine secretory
activity could not progress as observed during pregnancy (maintained
luteal function) or following administration of both exogenous estrogen
and progesterone. Increases in PRL secretion due to exogenous
administration of estrogen may have resulted in masking of endometrial
PRL receptors or differential regulation of subcellular trafficing of
PRL receptors to lysosomes (see Figure 11-2) .
Porcine PRL is microheterogeneic with glycosylated and
nonglycosylated forms. Glycosylated PRL has lower mitogenicity and
immunoaffinity, but higher lactogenicity when compared to
nonglycosylated or total porcine PRL. Additionally, unlabelled
glycosylated porcine PRL competes with radiolabelled total porcine PRL
for binding sites with higher affinity but lower capacity than usually
detected for unlabelled total pig PRL. Therefore, glycosylated PRL may


130
Analysis of ions in uterine flushings are summarized in Figure 10-
2. Total recoverable calcium (mg + 0.18; Figure 10-2a) was not
different between 1 and 6h (0.3-0.4 mg), increased (P<0.03) at 12h (1.0
mg) and then decreased at 24h (0.7 mg) following E2V administration.
Calcium in uterine flushings at 12h post injection was greater (P<0.05)
for gilts that received E2V compared to those that received corn oil
(1.0 vs 0.47 mg). Total recoverable sodium (ug + 17; Figure 10-2b)
increased between 1 and 6h (28 and 57 ug, respectively), increased
(P<0.01) again at 12h and remained elevated at 24h (96 and 106 ug,
respectively) following administration of E2V. Potassium (ug + 17;
Figure 10-2c) changed in a pattern similar to that for sodium. Values
were 48 ug and 57 ug at 1 and 6h, respectively, then increased (PC0.05)
at 12h (93 ug) and remained elevated at 24h (101 ug) post-E2V
administration. However, sodium and potassium values at 12 h post
injection were not different between gilts that received E2V or corn
oil.
Total recoverable protein (mg + 4; Figure 10-3a) was not different
at 1, 6, or 12h (13, 5, and 11 mg, respectively), but increased
(P<0.05) between 12 and 24h (11 vs 21 mg) following E2V administration.
Total protein in uterine flushing collected at 12h post-injection was
not different for gilts that received E2V or corn oil (11 vs 7.6 mg).
Total recoverable uteroferrin (umoles+168; Figure 10-3b) remained
stable through 12h, (285, 92, and 437, umoles respectively), but then
increased (P<0.001) at 24h (4690 umoles) post-administration of E2V.
Total uteroferrin in uterine flushings at 12h post-injection was
similar for gilts that received E2V or corn oil (437 vs 150 umoles).
Total leucine aminopeptidase (LAP; sigma units (SU)+30; Figure 10-3c)


13
In rats, PRL levels increase 10-fold with diunrnal and nocturnal
increases (de Greef et al.r 1977; Neill, 1980), following mating or
cervical stimulation to approximately Day 12 for intact, or Day 6 for
ovariectomized rats. Studies with ovariectomized rats suggest that
progesterone is associated with the noctural increase in PRL levels,
while estrogen accentuates the diurnal rise and inhibits the nocturnal
increase (Freeman and Sterman, 1978). For cyclic rats, PRL levels
increase at proestrus, in association with increases in circulating
estrogens (Butcher et al., 1974; Kelly et al., 1975).
Regulation of Pituitary Secretion of Prolactin
Hypothalamic Factors
The mechanisms responsible for regulation of secretion of PRL by
the anterior pituitary are currently under intense invesigation.
Hypothalamic dopamine tonically inhibits PRL secretion, a mechanism
which is unique among the anterior pituitary cells. However, the
posterior pituitary may also be a source of dopamine (Ben-Johnathen and
Peters, 1982) and may be transported to the anterior pituitary through
the blood (Page et al., 1982) as for other substances (Baertschi,
1980). Gamma butyric acid (GABA) also contributes to the inhibition of
PRL secretion (Duvilanski et al., 1986) especially in pigs (Schally et
al., 1977). Other factors are PRL releasing factors; opiods (Bero and
Kuhn, 1987; Rauhala et al., 1987), serotonin (Bero and Kuhn, 1987;
Thomas et al., 1988), Vasoactive intestinal peptide (VIP; Nagy et al.,
1988), peptide histidine isoleucine (PHI; Abe et al., 1985), oxytocin,
(Samson et al., 1987; Lumpkin et al., 1983) and posterior pituitary
factor (Murai et al., 1988). Complete control of PRL secretion in pigs


CHAPTER 8
AFFINITY LABELLING OF PROLACTIN RECEPTORS
IN DAY 75 PREGNANT PORCINE ENDOMETRIUM
WITH PORCINE [l25I]-PROLACTIN
Introduction
The molecular weight of the prolactin (PRL) receptor has been
estimated through several biochemical techniques such as affinity
chromatography, antibody detection, and solubilization (see review,
Djiane et al., 1987). Through these methods, the PRL receptor has an
estimated molecular weight of 37,000 to 42,000. A higher relative
molecular weight (Mr) estimate (80-90,000) also exsists, but this may
be a dimer configuration of the lower molecular weight form. Treatment
of PRL receptors with dithiothreitol (DTT) prior to electrophoresis
does not produce multiple bands and, therefore, the receptor, if
containing multiple subunits, is not complexed by disulfide bonds.
Cross-linking of PRL to its receptor can elucidate the molecular weight
of receptors when the molecular weight of the hormone is subtracted
from the resulting molecular weight estimates of the electrophoretic
bands.
This study investigated the similarity between affinity labeling
of the PRL receptor in rat liver membrane preparations, a common and
well investigated source of PRL receptors, to that of porcine
endometrial membrane preparations.
107


15
(Shull and Gorski, 1984). Dopamine released into the portal veins is
decreased (Ben-Johnathan et al., 1977) during proestrus and in
ovariectomized rats treated with estrogen (Cramer et al., 1979).
However, chronic estrogen treatment increases dopamine turnover (Fuxe
et al., 1969) and release (Gudelsky et al., 1981), possibly through
short-loop feedback mechanisms of hyperprolactinemia (Moore, 1988).
Estrogen may also stimulate release of TRH, a known PRL releasing
factor (Shull and Gorski, 1984). The number of TRH receptors on
lactotrophs are increased in pituitaries of estrogen treated rats
(DeLean et al., 1977).
Estrogen effects lactotrophs through both protein dependent and
independent mechanisms. Estrogen acts directly on the genome of the
lactotrophs to increase transcription and PRL mRNA and then a 5-fold
increase in PRL within 24h. This mechanism of estrogen action is
thyroid and hypothalamus independent since estrogen increses PRL
secretion in thyroidectomized rats or rats with pituitaries
transplanted to the kidney capsule. In ovariectomized rats, both rapid
and prolonged effects of estrogen on PRL secretion occurs. Estrogen
does not affect the growth hormone gene in rats. Estrogen receptors
reach a peak in the nucleus at 1 hour, then decrease, but PRL synthesis
and secretion is stimulated for 48 to 72h following estrogen
stimulation. Shull and Gorski (1986) suggested that estrogen affects
stable nuclear components, such as chromatin proteins, DNA sequences
around the PRL gene, a second regulatory factor (unknown or pituitary
transcriptional activator; PIT-1) or a combination of these effects.


215
BIOGRAPHICAL SKETCH
Kathleen Mary Hart, third and youngest child of William Alan and
Jeanne Alyce Hart, was born Feburary 24, 1960, in Cleveland, Ohio. She
spent her childhood in Lakewood, Ohio and graduated from Lakewood High
School in 1978. She obtained a Bachelor of Science degree in animal
bioscience from The Pennsylvania State University, graduating with
honors in 1982. In 1984, she obtained her Master of Science degree in
animal science from the University of Florida. Under the direction of
Dr. Fuller Bazer, she continued with doctoral research at The
Univeristy of Florida. She married Robert Gregory Young in November,
1985. She obtained her second degree black belt in karate in 1988.
Upon completion of her doctoral studies, she will move to the Chicago
area to further her studies on prolactin under the direction of Dr.
Daniel Linzer, Department of Biochemistry, Molecular Biology and
Cellular Biology, at Northwestern University.


64
Catheterizations
On the assigned day, gilts were anesthetized and fitted with
indwelling jugular catheters (Ford and Mauer, 1978) which were
maintained with 200 IU/ml heparin:saline solution. Following surgery,
gilts were housed individually until after collection of the final
blood sample and removal of the catheters.
Hormones
Exogenous porcine prolactin (PRL; USDA-B-1; gift from Dr. Douglas
Bolt, National Animal Hormone Program Director) was diluted in
phosphate buffered saline (PBS; 1 mg/ml; pH 7.2), aliquoted into 1.2 ml
volumes and stored at 4 C until injected subcutaneously.
Experimental Design
Six nonpregnant gilts, 3 per treatment group, were assigned
randomly to receive either porcine PRL (1 mg) or vehicle (SAL; 1 ml
saline). On Day 7, gilts were anesthetized and fitted with indwelling
jugular vein catheters. Jugular vein blood (7 ml) samples were
collected on Days 10 through 13 of the estrous cycle at 0730, 1000,
1200, 1930 and 2400h. Prolactin or saline was administered
subcutaneously at 0800 and 2000h, 30 min after the morning and evening
blood samples, on Days 10 through 14. Blood samples were assayed for
concentrations of PRL in serum.
Prolactin
Concentrations of PRL in serum of gilts were measured by a
radioimmunoassay (RIA) sensitive to 1 ng/ml (Kraeling et al., 1982).


Total Prolactin Receptors (pmoles/mg protein)
118
Day
Figure 9-1: Prolactin receptors in endometrial membranes of
cyclic (squares) and pregnant (circles) gilts over days of the
estrous cycle and gestation. Values with different letters are
significantly different (P<0.05) within reproductive status and
between status on Day 12. Values were obtained from three gilts
per day in each reproductive status. The SEM for prolactin
receptor numbers is + 4 pmoles/mg protein.


TABLE OF CONTENTS
PAGE
ACKNOWLEDGEMENTS iv
LIST OF TABLES ix
LIST OF FIGURES X
ABSTRACT xiv
CHAPTERS
1 INTRODUCTION 1
2 REVIEW OF LITERATURE 3
History, Evolution and Structure of Prolactin 3
Microheterogeneity of Prolactin 6
Sources of Prolactin 9
Prolactin in the Circulation 12
Regulation of Pituitary Secretion of Prolactin 13
Receptor Theory 17
Analysis of Receptors 18
Prolactin Receptors 21
Signal Transduction Systems for Prolactin 30
Regulation of Prolactin Receptors 35
Functions of Prolactin in the Uterus 40
Porcine Conceptus Development and Uterine Secretion 41
3 EFFECTS OF HYPOPROLACTINEMIA ON ESTABLISHMENT
OF PREGNANCY AND UTERINE SECRETORY FUNCTION IN PIGS 44
Introduction 44
Materials and Methods 45
Results 51
Discussion 54
4EFFECTS OF CYSTEAMINE ON CIRCULATING
PROLACTIN LEVELS IN PIGS 57
Introduction 57
Materials and Methods 57
Results 58
Discussion 61
vi


206
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171
Calcium
Sodium
Potassium
Chloride
LAP
Uteroferrin
Glucose
PGF2a
Elevation of:
Potassium
LAP
Figure 13-1: Effects of hypoprolactinemia and
hyperprolactinemia on the composition of uterine flushings of
Day 12 gilts at 24h after a single administration of
estradiol valerate.


CHAPTER 12
STUDIES ON MITOGENICITY, LACTOGENICITY,
IMMUNOREACTIVITY AND RECEPTOR BINDING CHARACTERISTICS
OF NONGLYCOSYLATED AND GLYCOSYLATED PORCINE PROLACTIN
Introduction
Prolactin (PRL) exerts a diversity of physiological effects on
several target tissues in many vertebrate species (Riddle, 1963; Nicoll
and Bern, 1972). Prolactin affects osmoregulation in fish and
amphibians (Bern, 1975), ion and water regulation across the intestine
and kidneys, mammary development and lactation, as well as several
reproductive processes in mammals (Shiu et al., 1981). The ability of a
single hormone to exert such diverse actions is intriguing. Recent
data suggest that prolactin, itself, is composed of a "family" of
hormones (Lewis, 1984). Several molecular weight forms of PRL (23,000,
25,000 and 50-60,000) have been isolated from pituitary extracts. The
23,000 Mr form is a simple peptide structure consisting of 199 amino
acids with three disulfide bridges (Li et al., 1960). The 25,000 Mr
variant is identical in primary structure but contains a N-linked
carbohydrate chain on asparigine 31 (Parkov and Butnev, 1986) while
the 50-60,000 Mr form (Suh and Frantz, 1974; Whitaker et al., 1983) is
thought to be a dimer of the lower Mr forms (Shoupe et al., 1985).
The glycosylated form of PRL has been detected in pituitaries of
sheep (Lewis et al., 1984), humans (pituitary and decidual sources,
Lewis et al., 1985; Markoff et al., 1987), pigs (Pankov and Butnev,
1986), rats (Wallis et al., 1980) and mice (Sinha et al., 1987; Sinha
150


CHAPTER 9
ENDOMETRIAL PROLACTIN RECEPTORS DETECTED
BY HOMOLOGOUS RADIORECEPTOR ASSAY DURING THE
ESTROUS CYCLE AND EARLY PREGNANCY IN PIGS
Introduction
Circulating levels of prolactin (PRL) are relatively constant
during pregnancy (Dusza and Krzymowska, 1981; Kensinger et al., 1986;
DeHoff et al., 1986). Prolactin is slightly elevated on Day 10 of
gestation, declines by Day 20 and remains constant (approximately 10
ng/ml) until parturition. The PRL levels are similar for cyclic gilts
except that PRL is elevated on Days 0-2 and 16-17 (Brinkley et al.,
1973; Dusza and Krzymowkska, 1979; Foxcroft and Van der Veil, 1982)
when concentration of circulating estrogens increase.
Although PRL is associated primarily with mammary growth and
development (Shiu et al., 1973; Costlow et al., 1974; Shiu, 1980),
recent data support a role for PRL in uterine physiology. Prolactin
interacts with ovarian steroids to enhance uteroglobin secretion and
endometrial proliferation in rabbits (Chilton and Daniels, 1985), as
well as increased uteroferrin, prostaglandin Fa, and glucose in
uterine secretions (Young and Bazer, 1988) of pigs. Prolactin may
affect ion transport and calcium cycling across the porcine endometrial
epithelium (Mirando et al., 1988), thus modulating uterine secretory
function (Young and Bazer, 1988). Prolactin may also exert
antiluteolytic effects through redirection of endometrial prostaglandin
114



51
Results
Effects of Hypoprolactinemia
Administration of CB154 to pregnant gilts (Experiment 1) decreased
circulating concentrations of PRL by 40% (P<0.06) on Day 15. Prolactin
levels (+0.33 ng/ml) were 5.0, 3.3, and 4.4 ng/ml and 5.2, 5.0, and
4.4 ng/ml on Days 10, 15 and 20 of gestation for gilts that received
CB154 and vehicle only, respectively. The number of corpora ltea
(CL) was not different on Day 25 of gestation between gilts that
received CB154 (13.6 +0.9) and vehicle (14.0 +0.9) and litter size was
similar between gilts that received CB154 (7.3 +1.75) and vehicle (8.4
+1.75).
Administration of CB154 to cyclic gilts (Experiment 2) decreased
circulating concentrations of PRL by 50% (2.7 +0.33 ng/ml vs 5.3 +0.33
ng/ml; P<0.06) on Day 12 to levels that were similar to those reported
for CB154-treated pigs by Kraeling et al. (1982), Whitacre et al.
(1981) and Smith and Wagner (1986). The inability to totally supress
circulating PRL in pigs by CB154 administration may be due to PRL
regulation by factors other than dopamine. Previously reported dosages
of 480 mg/day did not affect PRL support of corpus luteum function in
mid-gestational gilts (R.R. Kraeling as cited by Bazer and First,
1983). Total recoverable protein (mg), uteroferrin (umoles), and
glucose (mg) concentrations, respectively, were not different in
uterine flushings from gilts that received CB154 (17.6+2.4 mg; 541+92
umoles and 2.23+0.13 mg) compared to those that received vehicle
(19.2+2.4 mg; 608+92 umoles and 2.35+0.13 mg). However, concentrations
of leucine aminopeptidase were lower (P<0.025) in uterine flushings of
CB154 (140+18 SU) compared to vehicle (230+18 SU) treated gilts.


163
generated from Scatchard analyses of the inhibition curves (Figure 12-
6) using radioinert nonglycosylated or total PRL (0.129 vs 0.168 x 108
M*1) against radiolabelled total PRL, however, receptor numbers were
lower when radioinert nonglycosylated PRL than total PRL (14.64 vs
22.77 pmoles/mg protein, respectively). When radioinert glycosylated
PRL competed against radiolabelled total PRL for endometrial binding
sites, the Ka was higher (0.3 vs .17 x 10B M*1) but receptors were only
33% of those measured using total PRL (7.6 vs 22.77 pmoles/mg protein,
respectively). These data suggest the possibility of two PRL receptor
populations, one site being high affinity-low capacity and another
being low affinity-high capacity. Importantly, summation of receptors
measured using each form of PRL alone was approximately equal to that
measured using total PRL.
Discussion
Results from this study suggest that the two forms of PRL may
interact with various target tissues either individually or in
combination. Investigation of these forms of PRL; nonglycosylated
(23000); glycosylated (25000) and total PRL, by various techniques is
necessary to elucidate physiological effects of PRL. Results indicated
that the two forms caused different relative responses in different
assays.
Results from the RIA using a polyclonal antibody suggest that the
two forms of PRL, assayed alone, are detectable at higher
concentrations than for total PRL. These results are similar to those
for the glycosylated forms of ovine (Lewis et al., 1984) and human PRL
(Lewis et al., 1985; Pellegini et al., 1988) and previous results for


197
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50
through 16 of gestation. Jugular vein blood samples were collected on
Days 10 (preinjection), 15 (during injection) and 20 (4 days
postinjection) using a vacutainer single sample collection method to
avoid prolonged stress. Serum was assayed for concentrations of PP.L to
determine effectiveness of CB154. On Day 25 of gestation, gilts were
injected with sodium thiamylal (lg, i.v.) to induce anesthesia, which
was then maintained with halothane using a closed-circuit gas
anesthetic unit, and subjected to midventral laparotomy. The uterus was
exposed and examined for evidence of normal pregnancy. Each gilt
received 15 mg of PGF2a (Lutalyse, Upjohn Company, Kalamazoo, MI) to
terminate the pregnancy. Gilts experienced two normal estrous cycles
before being used in Experiment 2. This protocol was necessary because
of the requirement that CB154-treated gilts be euthanized.
Experiment 2
This experiment was to determine effects of hypoprolactinemia on
uterine secretory activity. The protocol was to mimic effects of
estrogens from conceptuses on endometrial secretory activity during the
time of maternal recognition of pregnancy without interactions with
other conceptus products (Geisert et al., 1982b). The 10 gilts used in
Experiment 1 were assigned randomly, 5 per treatment group, to receive
either CB154 (100 mg/day) or vehicle (4ml/day) on Days 10 and 11 of the
estrous cycle. All gilts then received 5 mg of estradiol valerate
(i.m.) on Day 11. On Day 12, uterine flushings and a jugular vein
blood sample were collected.


209
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116
Materials and Methods
Animals
Crossbred gilts of similar weight (100-120 kg) and age (7 to 9
months) were used in this study following completion of two normal (18
to 22 days) estrous cycles. Thrirty-nine gilts were assigned randomly
to either the pregnant (n=21) or cyclic (n=18) reproductive status.
Within status, gilts (three per day) were assigned to Days 8, 10, 11,
12, 14, and 15 of the estrous cycle and pregnancy, as well as Day 30 of
gestation. Gilts were observed daily for estrus in the presence of
intact boars. The first day of behavioural estrus was designated Day
0. Gilts assigned to the pregnant status group were mated when
detected in estrus and 12 and 24 h later.
Surgical Procedures
On the appropriate day of the estrous cycle or gestation, gilts
were anesthetized with thiamylal sodium (1 g; i.v.) and maintained
under surgical anesthesia on a closed circuit anesthesia machine using
halothane. Gilts were subjected to midventral laparotomy and
hysterectomized. Endometrial tissue was separated from myometrium and
and processed as descibed in Chapter 7. Tissue was maintained at -70 C
from within 15 min of collection until receptor assays were conducted.
Mesurement of Endometrial Prolactin Receptors
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receptors was conducted as described in Chapter 7. This includes
membrane preparation, protein determination, iodination of hormone,
detection of specific activity (83 uCi/ug), chaotropic treatment of


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903.


I thank Dr. Bazer for the many opportunities to participate in
scientific meetings. I am much richer for the experiences. Also, I
thank him for the insight that social comfort doesn't always expand
your knowledge. My solo journey to the Prolactin Gordon Conference
allowed development of friendships, scientific collaboration and a
greater appreciation for prolactin.
Thanks are extended to Dr. Douglas Bolt for generously supplying
the porcine prolactin and other hormones used in these studies; to
Bennet Johnson, who patiently assayed serum samples for prolactin; to
Fil Fliss, for prostaglandin assays of uterine flushings; to Jenny
Davis for Nb2 lymphoma cell assays; and to Dr. Nelson Horseman for
pigeon crop sac assays.
Sincere thanks are extended to Dr. Wang, Dr. Shiverick and
personnel in her lab for their help and generous use of equipment,
computer time and borrowed keys during the period I was conducting
Scatchard analyses of the binding data.
Loving thanks go to Greg, my husband, friend and keeper of my
sanity; needless to say, but nonetheless essential to mention, is that
I am certainly more human because of you. Also, thanks go to my
sister, Judi Norton, who kept me going with letters, support, laughter
and love throughout my graduate program.
Lastly, although Ill be the person with the extra letters behind
by name, my success is truly because all of you were beside me. Thank
you!
v


89
unlabelled ovine PRL; 2) ovine [12,I]-PRL versus unlabelled porcine
PRL; 3) porcine [1231]-PRL versus unlabelled ovine PRL and 4) porcine
[i25i]_prl versus unlabelled porcine PRL. This enabled investigation
of the combination of radiolabelled and unlabelled hormones that would
result in the most desirable displacement from porcine endometrial PRL
receptors. Assays were conducted as described previously.
Analysis of Binding Data
The amount of bound radiolabelled hormone obtained in the various
assay procedures was subjected to binding analysis using a LIGAND
program (Munson and Rodbard, 1985) adapted for the Macintosh Computer
(Apple Computers, Los Angeles, CA). This program analyzes data through
Scatchard (1949) computations to generate binding responses. Binding
data were tested for best fit assuming 1 and 2 binding sites.
Results
The effect of increasing MgCl2 molarity is depicted in Figure 7-1.
All tissues showed very low binding (<3%) when not pretreated with
MgCl2. Membranes from rabbit mammary gland showed highest binding when
pretreated with 2M MgCl2 whereas membranes from Day 75 pig mammary
gland, chorion and endometrium had the highest total binding when
pretreated with 4M MgCl2. Porcine anmiotic membranes had the greatest
binding when pretreated with 6M MgCl2; however, all other membranes had
similar or decreased binding at that molarity.
Increased binding of porcine [1231]~PRL occurred with increasing
concentrations of membrane protein only when membranes were pretreated
with 4M MgCl2 (Figure 7-2). Untreated membranes or membranes that were


214
Young, K.H. and Bazer, F.W. (1987) Development of a homologous
radioreceptor assay (RRA) for porcine prolactin. Biol. Reprod. 36
(Supp 1), 74 (Abstract).
Young, K.H. and Bazer, F.W. (1988) The role of prolactin in the
establishment of pregnancy in the pig: effects on fetal survival and
uterine secretory function. Biol. Reprod. 38 (Supp 1), 105 (Abstract).
Young, K.H., Bazer, F.W., Simpkins, J.W. and Roberts, R.M. (1987)
Effects of early pregnancy and acute 17(3-estradiol administration on
porcine uterine secretion, cyclic nucleotides, and catecholamines.
Endocrinology. 120, 254-263.
Zavy, M.T., Clark, W.R., Sharp, D.C., Roberts, R.M. and Bazer, F.W.
(1982) Comparison of glucose, fructose, ascorbic acid and glucose
phosphate insomerase enzymatic activities in uterine flushings from
nonpregnant and pregnant gilts and pony mares. Biol. Reprod. 27, 1147-
1158.
Zavy, M.T., Roberts, R.M. and Bazer, F.W. (1984) Acid phosphatase and
leucine aminopeptidase activity in the uterine flushing of nonpregnant
and pregnant gilts. J. Reprod Fert. 72, 503-507.


41
et al., 1983), and mink (Rose et al., 1983); and by production of PRL
by decidual endometrium (Riddick et al., 1978). Exogenous PRL
administered to longterm ovariectomized rabbits stimulates uterine
proliferation and uteroglobin secretion to levels similar to that
detected in estrous does (Chilton and Daniels, 1985). Prolactin
increases the concentration of estrogen and progesterone receptors in
endometrium of rabbits (Daniels et al., 1984) and increases endometrial
uptake of estrogen in rats (Leung and Sasaki, 1973). Armstrong and
King, (1977) detected increases in progesterone metabolism by the rat
uterus following administration of exogenous PRL. Prolactin modifies
the accumulation of uterine lumen fluid in rats, possibly though
synergistic actions with estrogen (Kennedy and Armstrong, 1972).
Additionally, PRL may function during conceptus-endometrial
interactions since PRL affects blastocyst growth and terminates delayed
implantation in the mink (Martinet et al., 1981). Prolactin may also
regulate the synthesis or direction of secretion of uterine
prostaglandins in pigs (Mirando et al., 1988) and humans (Healy, 1984).
Therefore, PRL appears to affect the uterine environment through
modification of secretory functions, or effects on endometrial
proliferation or conceptus-endometrial interactions.
Porcine Conceptus Development and Uterine Secretory Response
The porcine conceptus develops from a spherical form on Day 10 of
gestation to a tubular form on Days 10.5-11 and reaches a filimentous
(200 mm) form late on Day 11 (Geisert et al., 1982a). The porcine
conceptus continues to elongate, initially through cellular
rearrangment and then through cellular hypertrophy and hyperplasia to


87
treatment of the membranes with increasing concentrations (0,1,2,4, and
6 M) of MgClz.
Binding of Porcine [12aI]-PRL as a Function of Increasing Membrane
Protein Concentration
Porcine [12SI]-PRL (45,000 cpm 0.24 ng) was incubated in 500 ul of
assay buffer containing increasing concentrations (0 to 500 ug) of Day
75 pregnant pig endometrial membrane protein in the presence (1 ug) or
absence of unlabelled porcine PRL. This assay was conducted in a
similar manner with identical protein concentrations of untreated
membranes and membranes pretreated with 4 M MgCl2 or distilled water.
Determination of Protein:Radiolabelled Ratio Concentration
Three levels of protein (50, 150, 250 ug) were assayed with
increasing amounts of radiolabelled PRL (15,000; 30,000; 45,000;
60,000; 75,000 and 90,000 cpm) in a total volume of 500 ul assay
buffer. Each protein:radiolabel concentration combination assay was
conducted in the presence (1 ug) and absence of unlabelled PRL to
determine maximal specific binding.
Analysis of Porcine [*23I]-PRL Association Kinetics
At time zero, [125I]-PRL (45,000 cpm) was incubated at 4 and 25 C
with 150 ug of Day 75 pregnant pig endometrial or Day 20 pregnant rat
liver membranes in 500 ul assay buffer either in the presence (1 ug) or
absence of unlabelled porcine PRL. Specific binding of porcine [12SI]-
PRL was determined after 1.5, 4, 8, 12 and 24h.


27
and monoclonal antibody detection and showed PRL receptors for sow
mammary gland at 42,000 with faint bands at 31,000 and 53,000 Mr.
Djiane and coworkers (1987) suggest that the holo-PRL receptor contains
2 or more of the 32,000-40,000 Mr units that are not linked by
disulphide bonds, but may be noncovalently associated.
Ovarian lactogenic receptors in rats (Dufau and Kusuda, 1988) have
two active subunits, 88,000 and 40,000 Mr, as purified by sequential
affinity chromatography. The 40,000 Mr subunit is part of the 80,000
Mr receptor form and is similar to the 35,000-44,000 Mr PRL receptor
subunit from rabbit, rat and mouse liver and mammary gland (Haeuptle et
al., 1983; Hughes et al., 1983; Liscia and Vonderhaar, 1982; Liscia et
al., 1982). The higher Mr species was also observed in ovarian,
testicular, kidney and mammary gland tissues following cross-linking of
receptor subunits. Different molecular species are observed in various
rat tissues including the ovary (88,000 and 40,000; Bonifacino, 1985),
Leydig cells (91,000; 81,000; 37,000 and 31,000), mammary gland
(93,000; 83,000; 30,000 and 28,000) and kidney (65,000 and 30,000)
(Bonifianco et al., 1985). Additionally, ovarian and Leydig cells
contained the 37,000 Mr form within the 81,000 form. The female rat
liver 87,000 Mr form contains subunits of 40,000 and 35,000 which may
or may not be linked by disulfide bonds (Haldosen and Gustafsson,
1987) Lactogenic receptors from mammary gland from pigs and rabbits
range in molecular weight from 28,000 to 69,000 (Haeuptle et al., 1983;
Hughes et al., 1983; Sakai et al., 1985; Katoh et al., 1985).


91
Micrograms Membrane Protein
Figure 7-2: Effects of increasing protein concentrations of
Day 75 porcine endometrial membranes on binding of porcine
[i23i]_prolactin. Membranes were treated with either 4 M
MgCl2; 4 M MgCl2 and incubated with 1 ug unlabelled porcine
prolactin (NSB); untreated membrane (Bo); or distilled water
(H20).


136
(Djiane et al., 1979a). A similar half-life was determined for rat
hepatic PRL receptors (Baxter, 1985). Downregulation of PRL receptors
is more pronounced and of longer duration than for other receptors
(Djiane et al., 1979a). Rapid turnover of PRL receptors is suggested
since receptors must recognize pulastile release of the hormone; unlike
insulin in which the receptor half-life is greater than 2h. Although,
the half-life of endometrial PRL receptors is not known, receptor
numbers increased between 12 and 24h following estrogen administration
to levels similar to those detected prior to treatment. Additionally,
the decrease in PRL receptors as 12h following estrogen may be due to
changes in the secretion or half-life of PRL or changes in receptor
processing.
Although estrogen increases pituitary release of PRL, it is
unlikely that endometrial receptors are auto-upregulated as this is a
delayed process (Djiane et al., 1979a). Additionally, increases in rat
hepatic PRL receptors in response to exogenous estrogen are not
affected by bromocryptine adminstration (hypoprolactinemia) (Kelly et
al., 1976).
The rapid increases in PRL receptor numbers at 1 to 6h post-E2V
are not easily explained by classical steroid-receptor interactions
(Jensen et al., 1968) and suggest that estrogen may affect mechanisms
other than receptor synthesis. Short response intervals to estrogen
stimulation could occur through steroid interaction with plasma
membrane receptors (Peitras and Szego, 1977; 1979; Parrikh et al.,
1980; Nenci et al., 1981; Towle and Sze, 1983 and Berthois et al.,
1988); suggesting a mechanism more similar to that for peptide
hormones. Additionally, short response intervals to estrogenic


146
effect blocked by progesterone administration (Djiane and Durand,
1977). Sakai et al. (1978, 1979) suggest that progesterone indirectly
decreases or supresses PRL receptors by competing with glucocorticoid
receptors to block stimulation of PRL receptors. Progesterone retards
PRL auto-upregulation of PRL receptors in mammary glands of rabbits
(Djiane and Durand, 1977). However, progesterone appears to have no
effect on PRL receptors in mammary gland (Sherman et al., 1977) or
liver (Posner et al., 1974a) of rats. In the present study, porcine
endometrial membrane PRL receptors were not affected by progesterone
since receptor numbers were similar to those of corn oil-treated
controls. Previous studies confirm that PRL receptors in different
tissues do not respond similarly to identical hormonal regimes (Posner
et al., 1974a; Kelly et al., 1976; Grissom and Littleton, 1988). The
mammary gland PRL receptor is upregulated by PRL, similar to liver, but
decreased by estrogen administration, unlike the liver in which the PRL
receptor is upregulated. Additionally, changes in PRL receptors are
not similar between tissues associated with reproductive functions,
i.e., uterine and mammary tissue (DeHoff et al., 1984).
Changes in PRL receptors in porcine endometrial membranes in the
present study agree with previous reports indicating steroid regulation
of PRL receptors in mammary gland, liver, (except rat), adrenal and
kidney. However, relationships between uterine secretory responses and
endometrial PRL receptor numbers must be interpreted with respect to
interactions between steroids and PRL and regulation of their
individual receptors. In addition to regulation of PRL receptors by
ovarian steroids, PRL regulates steroid receptors in reproductive
tissues (Chilton and Daniels, 1985; Muldoon et al., 1987) and liver


Figure 7-5: Binding and displacement of ovine and porcine
prolactin from magnesium cloride treated Day 75 porcine
endometrial membranes. Porcine [l23I]-prolactin vs unlabelled
porcine prolactin (P/p); porcine t123I]-prolactin vs
unlabelled ovine prolactin (P/o); ovine [123I]-prolactin vs
unlabelled ovine prolactin (O/o); and ovine t123I]-prolactin
vs unlabelled porcine prolactin (0/p).


125
uterine secretory activity at selected intervals following
administration of exogenous estrogen.
Materials and Methods
Animals
Crossbred gilts of similar weight (100-120 kg) and age (7 to 9
months) were used in this study after they experienced at least two
normal estrous cycles (18 to 22 days). In the presence of intact
boars, gilts were observed daily for behavioral estrus. The first day
of behavioral estrus was designated Day 0.
Experimental Design
On Day 11 of the estrous cycle, 15 gilts were anesthetized with
thiamylal sodium (1 g, i.v.) and maintained under surgical anesthesia
using a closed curcuit anesthesia machine. Gilts were subjected to
midventral laparotomy and one uterine horn was exposed. Uterine
flushings were collected in 20 ml double distilled water (Bazer et al.,
1978) and endometrium was separated from myometrium and placed on ice.
These samples provided intragilt control data (time zero). Twelve
gilts then received estradiol valerate (E2V; 5 mg) and at either 1, 6,
12 and 24h post-E2V injection (three gilts per group), uterine
flushings and endometrium were collected in an identical manner from
the second uterine horn. Three gilts were injected with corn oil
(control group, 0.5 ml) and were assigned to 12h post-injection since
estrogen effects on the uterine environment are readily detectable at
12h (Geisert et al., 1982c). Gilts assigned for collection of time 0
and lh samples, remained under anesthesia whereas the 6, 12 and 24h


19
The linear plot is an algebraic derivation from original theory that
numbers of complexes formed are dependent on the concentration of
receptors and hormone available as well as the rates of association and
dissociation.
ki
[H] [R]-*~- [HR] (1)
k-,
and
[H][R] = [HR]/(Kd + [H]) (2)
with [HR] = amount bound (B); [R] = maximal binding (Bmax) and
[H] = free hormone (F), the equation is restated to
B = [(F) (Boa x ) ] /Kd + F (3)
rearrangment generates
(B) (Kd) + (B) (F) = (F) (B.ax)
division by Free hormone
[(B) (Kd ) /F] + B = B.ax
rearranged to
B/F (Bmax B)/Kd
and transformed to the linear (y = mx + b) expression
(4)
(5)
(6)
B/F = (-1/Kd)(B) + B.ax/Ko
(7)


129
W
L.
o
*->
Cl
CD
O
CD
C
O
jg
o
Q_
50-
1 6 12 24
Hours post injection
(P< 0.02)


28
Molecular Structure of Cloned Prolactin Receptor
The prolactin receptor from rat liver has been cloned (Boutin et
al.f 1988). Association with other proteins is not required for the
40,000 Mr structure which supports results of Liscia and Vonderhaar,
(1982); Haeputle et al. (1983) and Necessary et al. (1984) but not
those of Dufau and Kusuda (1987). The PRL receptor (40,000 Mr) subunit
contains a 19 amino acid signal sequence, an extracellular domain (210
amino acids), speculated to bind PRL, a single transmembrane section
(24 amino acids) and a short cytoplasmic domain (54 amino acids). This
PRL receptor has 30% overall homology to growth hormone receptor (Leung
et al., 1987) following removal of 293 cytoplasmic amino acids from the
growth hormone receptor structure. The two receptors share 67%
homology between the first and second, and third and fourth cysteine
residues. A 40-60% homology exists in three other extracellular
regions. A 19 amino acid series in the cytoplasmic domain has 68%
structural identity to growth hormone. These two receptors do not
share sequence homology with other proteins. The short cytoplasmic
domain of the PRL receptor does not possess tyrosine kinase activity or
phosphorylation sites as seen in other growth factor receptors (Hunter,
1987). However, the short cytoplasmic domain is similar to other
protein receptors which transport various compounds; such as
transferrin receptor transport of transferrin (Schneider et al., 1984),
LDL receptor transport of cholesterol (Yamamoto et al., 1984) and IGF-
II receptor transport of mannose-6-phosphate (Morgan et al., 1987).
Prolactin receptors cloned from other rat tissues (ovary, adrenal and
mammary gland) are more similar to growth hormone receptor than liver
PRL receptor by their longer cytoplamic domains (Kelly et al., 1989).


79
occurred 48 to 72 h following a single injection of estradiol valerate
on Day 11 to cyclic gilts (Geisert et al., 1982b). During
establishment of pregnancy in the pig, PGF secretion must be redirected
from an endocrine to an exocrine direction (Bazer and Thatcher, 1977)
to protect the corpus luteum from regression. The ability of PRL to
stimulate secretion of PGF and enhance its secretion into the uterine
lumen (Mirando et al., 1988) is a novel finding suggesting that PRL
plays a luteostatic role, in conjunction with estrogen, in early
pregnancy of pigs.
The present findings in the pig, support the hypothesis that PRL
modulates uterine secretory activity during establishment of pregnancy
as previously described for long-term ovariectomized rabbits (Chilton
and Daniels, 1985). Prolactin increased uterine secretory function and
caused differential changes in ions, increased secretion of proteins,
PGF and glucose. Although the mechanism(s) by which PRL influences the
endometrial secretory profile is not known, several mechanisms may be
involved. These include 1) activation of ion channels to facilitate
transport and secretion of cellular components (Petersen and Maruyama,
1985) or stimulate membrane cycling of calcium (Alkon and Rasmussin,
1988); 2) increased estrogen binding by cells of rat liver (Chamness
et al., 1975) and uterus (Leung and Sasaki, 1973); 3) facilitation of
formation of gap junctions to increase intercellular communication
(Sorenson et al., 1987), and 4) up-regulation of PRL receptors to
increase membrane fluidity (Dave and Witorsch, 1985) and increase the
availablity of cryptic hormone receptors. Dave et al. (1983) observed
an increase in PRL binding during early pregnancy which they attributed
to alterations in membrane fluidity.


REFERENCES
Abe, H., Elngler, D., Molitch, M.E., Bollinger-Gruber. J. and Reichler,
S. (1985) Vasoactive intestinal peptide is a physiological mediator of
prolactin release in the rat. Endocrinology. 116, 1383-1386.
Adler, S., Waterman, M.L., He, X. and Rosenfeld, M.G. (1988) Steroid
receptor-mediated inhibition of rat prolactin gene expression does not
require the receptor DNA-binding domain. Cell. 52, 685-695.
Alexander, R.L. (1971) Evaluation of an automatic calcium titrator.
Clin. Chem. 17, 1171-1175.
Alkon, D.L. and Rasmussin, H. (1988) A spatial-temporal model of cell
activation. Science. 239, 998-1005.
Amador, A., Klemcke, H.G., Bartke, A., Soares, M.J., Siler-Kodhr, T.M.
and Talamantes, F. (1985) Effects of different numbers of etopic
pituitary transplants on regulation of testicular LH/hCH and prolactin
receptors in the hamster (Mesocriecetus auratus). J. Reprod. Fert.
73, 483-489.
Amit, T., Barkey, R.J., Gavish, M. and Youdim, M.B.H. (1984) Induction
of prolactin (PRL) receptors by PRL in the rat lung and liver.
Demonstration and characterization of a soluble receptor.
Endocrinology. 114, 545-552.
Amit T, Barkey, R.J., Gavish, M. and Youdim, M.B.H. (1985) Induction
of prolactin receptors by prolactin in the rat lung and liver:
demonstration of separate receptor and antibody entities. Mol. Cell.
Endocrinol. 39, 21-29.
Amit, T., Barkey, R.J. and Youdim, M.B.H. (1983) Effect of prolactin,
testosterone and estrogen on prolactin binding in the rat testis,
prostate, seminal vesicle and liver. Mol. Cell. Endocrinol. 30, 179-
180.
Anderson, L.L., Boradinelli, J.C., Malven, P.V. and Ford, J.J., (1982)
Prolactin secretion after hypopheseal stalk transection in pigs.
Endocrinology. Ill, 380-384.
Anderson, T.R., Rodrigues, J., Nicoll, C.S., Spencer, E.M. (1983) The
synlactin hypothesis: prolactin's mitogenic action may involve
synergism with a somatomedin-like molecule. In: Insulin-like Growth
Factors/Somatomedins. p. 71-89, (ed. E.M. Spencer), de Gruyter, Berlin.
185


Total mg / uterine horn
131
A Calcium
B Sodium
C Potassium
1.0-
0.8-
0.6-
0.4-
0.2-
1 6 12 24 12
Hours post injection
(P<0.05)
1 6 12 24 12 1 6 12 24 12
Hours post injection ^p<
(P<0.01)
Figure 10-2: Total recoverable (A) calcium, (B) sodium and (C)
potassium in uterine flushings at 1, 6, 12 and 24h following
administration (i.m.) of estradiol valerate (0.5 mg, hatched bars) or
12 h after corn oil (0.5 ml, solid bar) administration. The solid line
denotes the mean value prior to injection (time zero) of Day 11 cyclic
gilts. Values with different letters are different for calicum and
potassium (P<0.05) and for sodium (P<0.01). The SEM was +0.18 for
calcium; +17 for sodium and potassium.


37
indirectly decreases or supresses PRL receptors by competing with
glucocorticoid receptors to block stimulation of PRL receptors.
Progesterone retards PRL auto-upregulation of PRL receptors in mammary
glands of rabbits (Djiane and Durand, 1977). However, progesterone
appears to have no effect on PRL receptors in mammary gland (Sherman et
al., 1977) or liver (Posner et al., 1974a) of rats.
Regulation by Peptides
Prolactin regulates its own receptor in target tissues. However,
unlike other peptide hormones, PRL both increases and decreases its
receptors. Auto-downregulation of PRL receptors has been detected in
vitro (Djiane et al., 1979a) and in vivo (Djiane et al., 1979b) for
rabbit mammary gland. Downregulation of PRL receptors is rapid and
transient, usually following an acute, physiological stimulus by PRL,
whereas, for other hormones, downregulation is much longer (Posner et
al., 1978).
Up-regulation of PRL receptors by physiological levels of
circulating PRL occurs in mouse and rat hepatic (Costlow et al., 1975;
Dave et al., 1981, 1982; Amit et al., 1985 and Rui et al., 1987), and
prostatic (Dave and Witorsch, 1985) membranes. Sustained high
circulating levels of PRL increased PRL receptors in rat liver (Posner
et al., 1975), rat (Holcomb et al., 1976) and rabbit (Djiane et al.,
1977) mammary gland and pigeon crop sac (Klediz et al., 1975). Auto-
upregulation of rabbit mammary gland PRL receptors have a slower onset,
requiring several days of sustained PRL levels, and is more stable
(Djiane and Durand, 1977). Therefore, Djiane and coworkers (1979)


25
dependent factors or other factors may account for rapid reversibility
that does not yet occur in vitro. Additionally, intrinsic factors
which affect dissociation may differ between tissues since it appears
that PRL receptors are differentially regulated. A high apparant
activiation energy be involved in slow dissociation for PRL binding and
as reported for CL of pigs (64.8 kJ, Brambly and Menzies, 1987), and
liver of mice (43.6 kJ/mole; Haro and Taimantes, 1985b) and rats (34
kJ/mole; Rae-Ventner and Dao, 1982). Extensive hydrophobic
interactions may be involved in PRL interaction with its receptor since
monovalent anions, acetate and phosphate stablize homologous binding
(Haro and Talamantes, 1985a). Amino acids at position 20 through 36
form such a hydrophobic region with histidines located at positions 27
and 30 in cow (Wallis, 1974), sheep (Li et al., 1970), pig (Li et al.,
1976) and human (Cooke et al., 1981) and at positions 25 and 28 in rat
(Cooke et al., 1980) and mouse (Kohomoto et al., 1984) PRLs.
Prolactin Receptor Turnover
Once PRL binds to its receptor, internalization is rapid and
biphasic. Internalization of bound PRL occurs in as little as 5 min.
At 5 min, radiolabeled PRL was associated with the low density membrane
fraction having high galactosyl transferase activity characteristic of
golgi membranes. At 10 min, radiolabeled PRL was associated with the
high density membrane fraction with high acid phosphatase activity,
characteristic of lysosomes. This internalization and trafficing is
similar to that for insulin (Posner, et al., 1981). The golgi contains
twice the amount of PRL, and processes PRL slower than insulin. The
golgi network has been redefined as the trans golgi network (TRN) or


195
Grody, W.W., Schrader, W.T. and O'Malley, B.V. (1982) Activation
transformation and subunit structure of steroid hormone receptors.
Endocrine Rev. 3, 141-163.
Gross, T.S., LaCroix, M.C., Bazer, F.W., Thatcher, V.V. and Harney,
J.P. (1988) Prostaglandin secretion by perifused porcine endometrium:
further evidence for an endocrine versus exocrine secretion of
prostaglandins. Prostaglandins. 35, 327-341.
Gubbins, E.J., Maurer, R.A., Lagrimini, M., Erwin, C.R. and Donelson,
J.W. (1980) Structure of the rat prolactin gene. J. Biol. Chem. 255,
8655-8662.
Gudelsky, G.A., Nansel, D.D. and Porter, J.C. (1981) Role of estrogen
in the dopaminergic control of prolactin secretion. Endocrinology.
108, 440-444.
Haeuptle, M.T., Aubert, M.L., Djiane, J. and Kraenhenbuhl. J.P. (1983)
Binding sites for lactogenic and somatogenic hormones from rabbit
mammary gland and liver. J. Biol. Chem. 258, 305-314.
Hafeti, Y. and Hanstein, V.G. (1974) Destabilization of membranes with
chaotropic ions. Meth. Enzymol. 31, 770-790.
Haldosen, L.A. and Gustafsson, J.A. (1987) Characterization of hepatic
lactogen receptor. Subunit composition and hydrodynamic properties.
J. Biol. Chem. 262, 7404-7411.
Hamilton, R.H. (1966) A direct photometric method for chloride in
biological fluids employing mercuric thiocyanate and perchloric acid.
Clin. Chem. 12, 1-17.
Handwerger, S., Capel, D., Korner, G. and Richards, R. (1987)
Purification of decidual prolactin-releasing factor, a placental
protein that stimulates prolactin release from human decidual tissue.
Biochem. Biophys. Res. Commun. 147, 452-459.
Handwerger, S., Barry, S., Markoff, E., Barrett, J. and Conn. P.M.
(1983) Stimulation of the synthesis and release of decidual prolactin
by a placental polypeptide. Endocrinology. 112, 1370-1374.
Haro, L.S. and Talamantes, F.J. (1985a) Interaction of mouse prolactin
with mouse hepatic receptors. Mol. Cell. Endocrinol. 41, 93-104.
Haro, L.S. and Talamantes, F.J. (1985b) Thermodynamics and kinetics
of mouse prolactin-hepatic receptor interaction. Mol. Cell.
Endocrinol. 43, 199-204.
Haro L.S. and Talamantes, F.J. (1986) Studies on the dissociation of
mouse prolactin from mouse prolactin hepatic receptors. Mol. Cell.
Endocrinol. 44, 159-164.


42
reach a lenght of 900 mm (Geisert et al., 1982a). During the initial
elongation phase, porcine conceptuses secrete estrogens (Heap et al.,
1979) which signals the maternal physiology to change the uterine
environment from cyclicity to that of pregnancy. During the
preimplantation period, the conceptus relies on secretion of protein,
sugars, ions, and other compounds, collectively termed histotroph, for
nutritional support until placentation is established around Day 18.
Therefore, prior to any physical attachment to the uterine endometrium,
conceptuses must insure luteostasis and histotroph secretion and thus
establish a viable pregnancy (Bazer et al., 1982). Luetostasis is
thought to occur through the redirection of secretion of prostaglandin
secretion from an endocrine mode toward the uterine vasculature to an
endocrine mode, into the uterine lumen (Bazer and Thatcher, 1977).
Redirection of the secretion of prostaglandins is well documented both
in vivo (Frank et al., 1977) and in vitro (Gross et al., 1988),
although, the mechanism is unknown, increasing evidence suggests that
prolactin may be involved (Young and Bazer, 1988; Mirando et al, 1988).
Secretion of histotroph is a well characterized series of events
during the time of maternal recognition of pregnancy (Geisert et al.,
1982b; 1982c; Bazer et al, 1987; Roberts and Bazer, 1988).
Additionally, these uterine secretory events can be mimiced by a single
exogenous dose of estradiol valerate on Day 11 (Geisert et al., 1982c;
Young et al., 1987). Pseudopregnancy can be established in pigs
following injection of estradiol valerate on Days 11-15 or by
injections on Days 11 and 15-16 (Geisert et al., 1987). Major events
of porcine uterine secretory activity following conceptus estrogen
secretion on Day 10-11 are as follows: (1) calcium is rapidly released


24
Studies with PRL receptors (in vitro), whether by heterologous or
homologous assay systems, fulfill the constrains of saturability and
specificity; however, complete reversibility has been questioned. Slow
or difficult dissociation has been observed for some peptide hormones
including PRL (Kelly et al., 1983; van der Gugten et al., 1980), growth
hormone (Donner et al., 1980), TSH (Powell-Jones et al., 1979), LH
(Katitineni et al., 1980) and insulin (Donner and Corin, 1980).
Increases in association time from lh to lOh (in vitro) are directly
correlated with increases in dissociation time, as well as incomplete
dissociation, after 48 hours (Kelly et al., 1983). Longer association
times may allow tighter binding and decreased ability for hormone
dissociation. Internalization of PRL and other hormones is often
preceded by tightening of the hormone-receptor linkage (Catt et al.,
1979) .
Prolactin receptors within different cell membranes have different
affinity constants and dissociation times (Kelly et al., 1983).
Therefore, affinity and dissociation could depend on location of the
receptor within cellular membranes. The proportion of subcellular
membranes within microsomes commonly used during in vitro receptor
studies is not known. Differences in affinity or dissociation
constants may be a function of the ratio of PRL receptors within plasma
or golgi membranes in the microsomal pellet.
Dissociation of ovine PRL from rat liver (Kelly et al., 1983) and
porcine corpora luteua (Brambly and Menzies, 1985) membranes is 60%
complete after 48h. This slow dissociation rate in not likely due to
damaged hormone or receptors and must be more efficient in vivo than in
vitro (van der Gugten et al., 1980). Therefore, in vivo, energy


16
Estrogen Receptors
The hydrophobic similarities between the pentanophenanthrene ring
of steroid hormones and membrane lipids allow steroids to enter a cell
through simple diffusion. A 'two-step' theory for expression of
steroid hormones was proposed by Jensen et al. (1968) whereby the
unbound estrogen receptor resided in the cytoplasm. Activation by
estrogen binding shifted the sedimentation coefficent from 4S to 5S
(O'Malley and Schrader, 1979) and translocation to the nucleus allowed
interaction with acceptor nonhistone chromatin proteins (see review
Grody et al., 1982). Estrogen receptor mechanisms have undergone
substanial revision. Williams and Gorski (1972) proposed an
equilibrium theory where all estrogen receptors are nuclear, but their
affinity is dictated by binding status. Receptors not bound by
estrogen have a lower affinity and move to the cytoplasm during tissue
processing. Martin and Sheridan (1982), Welshons et al. (1984) and
King and Green (1984) agree with Williams and Gorski (1972) on the
artifactual nature of cytoplasmic receptors. Buffer volumes affect
recovery of cytoplasmic and nuclear estrogen receptors. Monoclonal
antibodies and immunoperoxidase staining localized estrogen receptors
in nuclei of human breast tumour and rabbit uterine cells.
Steroid receptors are a family of ligand regulated positive
transcription factors with a common structural organization. There is
a central DNA binding domain, hinged to a carboxy terminus that is
common to all steroid receptors. This also contains zinc finger
proteins (Miller et al., 1985) of 2 pairs of four consecutive
cysteines, that act as ligands for zinc atoms.


31
Prolactin, however, possesses a receptor leading to biological
changes within target cells, but without a known transduction system
between hormone binding and cellular response. Prolactin generates
such diverse physiological affects within a vast array of tissues that
several concomitent transduction systems are feasible. Witorsch et al.
(1987) and Hughes et al. (1985) review trannduction systems that have
been investigated for PRL. Additionally, the source of tissue for
receptor studies may bias transduction system results since PRL's
different responses may be achieved through different pathways.
Mammary gland is a complex tissue requiring support from insulin,
glucocorticoid and estrogen, in addition to PRL, for maintainence and
milk systhesis. Other hormones or factors, such as thyroxine or growth
hormone, may be essential to mammary cell function. Explant cultures of
mammary tissue have been studied to elucidate the mechanism(s) of PRL
action. A recent model for PRL effects on casein biosynthesis was
proposed by Rillema (1980). Components included decreased cAMP as a
stimulatory component, calcium as an obligatory factor, phospholipase C
to generate diacylglycerol to stimulate protein kinase C and increases
ornithine decarboxylase (ODC) activity. Phospholipase A2, ODC and
prostaglandins appear to be involved in prolactin stimulation of casein
biosynthesis in mammary gland explants from mice (Cameron and Rillema,
1983). Polyamines (Rillema, 1979) and Na*-K*-ATPase (Falconer and
Rowe, 1977) may mediate PRLs action, as well as changes in
phosphatidylcholine, as PRL stimulation is associated with increased
choline uptake and decrease phosphatidylcholine turnover (Ko et al.,
1986).


I certify that I have read
conforms to acceptable standard
adequate, in scope and quality,
Doctor of Philosophy.
this study and that in my opinion it
of scholarly presentation and is fully
as a dissertation for the degree of
'Fuller W. Bazer, Chair
Graduate Reseach Professor of
Animal Science
I certify that I have read
conforms to acceptable standard
adequate, in scope and quality,
Doctor of Philosophy.
this study and that in my opinion it
of scholarly presentation and is fully
as a dissertation for the degree of
'A C*-
L
William C. Buhi
Assistant Professor of
Biochemistry and Molecular
Biology
I certify that I have read this study and that in my opinion it
conforms to acceptable standard of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for
the degree of Doctor of Philosophy.
Kathleen T. Shiverick
Professor of Pharmacology
and Therapeutics
I certify that I have read this study and that in my opinion it
conforms to acceptable standard of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
fies W. Simpkins /
Professor of Pharmacodynamics


143
Table 11-1: Endometrial membrane prolactin receptors and
total protein and uteroferrin in uterine flushings
from ovariectomized gilts following steroid administration
for 11 days.
Treatment
Component
CO
E2V
P4
E2V+P4
(SEM)d
Endometrial
PRL receptors
(pmoles/mg protein)
60a
45
58a
43b
(+7)
Protein
(total mg/uterine horn)
2a
7a
37b
OO
n
(+5)
Uteroferrin
2a
8a
4227b
8408c
(+524)
(total umoles/uterine horn)
abc Within rows, values with different letters are different
for PRL receptors (P<0.06), protein and (P<0.01) and
uteroferrin (P<0.001).
d Three gilts/treatment


20
Thus, the mathematical model and equations for binding data
resemble those for enzyme kinetics (Eadie, 1942; Hofstee, 1952).
Scatchard analysis assumes a known number of receptor sites. This is
not often the case during membrane receptor investigation. Therefore,
the Rosenthal (1969) analysis is used, since in theory, it is not based
on known receptor numbers. However, the two data transformations are
mathematically equivalent and receptor data are analyzed through
Scatchard analysis and interpretation.
Dissociation Rate Constants
Hormone receptor complexes are, in part, dependent on the rate at
which these complexes form (ki) and dissociate (k-i) at equlibrium.
ki
[H] [R]^ [HR] (8)
k-i
Dissociation rate constants are determinable when rebinding of
labeled hormone is prevented and kinetics are reduced to simple
first order reactions.
dB/dt = (-k-i)(B) (9)
If bound (B) is equal to bound at time = 0 (Bo), then integration
results in
In B/Bo = (-k-1) (t) (10)
and a plot of In B/Bo versus time generates a slope of -k-x;
i.e. the dissociation rate constant. At equilibrium, the rates


36
Steroid regulation of hepatic PRL receptors has been extensively
investigated. Hepatic PRL receptors decrease in ovariectomized rats
(Posner et al., 1974a; Kelly et al., 1979), but increase following 8 to
12 days of chronic estrogen administration (Posner et al., 1974a).
Increases in PRL receptors following estrogen treatment are thought to
be mediated indirectly, through stimulation of pituitary PRL release
and auto-upregulation of receptors. Hypophysectomy (Posner et al.,
1978), but not CB154 administration (Kelly et al., 1976), blocked the
estrogen-induced increase in rat hepatic PRL receptors, suggesting
pituitary involvement, but not exclusively an effect of PRL.
Involvement of growth hormone (Knazek et al., 1975), ACTH and TSH
(Bhattacharya and Vonderhaar, 1979) has been implicated.
Contrary to results in rats, ovariectomy increases hepatic PRL
receptors in mice (Marshall et al., 1979) and exogenous estrogen
reverses effects of ovariectomy. Estrogen administration also
decreases PRL receptors in prostate (Kledzik et al., 1976; Amit et al.,
1983) adrenal, kidney, (Monkemeyer et al., 1974) and mammary gland of
mice (Marshall et al., 1979) and rats (Bohnet et al., 1977; Smith et
al., 1976).
The increase in PRL receptors in the mammary gland following
parturition (Holcomb et al., 1975; Djiane et al., 1977) is thought to
result from autoregulation due to increases in concentrations of serum
PRL since administration of CB154 at parturition, decreases mammary
gland PRL receptors (Bohnet et al., 1977). Administration of PRL to
pseudopregnant rabbits increases mammary gland PRL binding sites; an
effect blocked by administration of exogenous progesterone (Djiane and
Durand, 1977). Sakai et al. (1978, 1979) suggest that progesterone


Percent Specific Binding
99
Figure 7-6: Crossreactivity of unlabelled porcine prolactin
(squares; pPRL), porcine growth hormone (triangles; pGH),
porcine luteinizing hormone (circles; pLH) and porcine
follicle stimulating hormone (diamonds; pFSH) to porcine
[125I]-prolactin with magnesium chloride treated Day 75
porcine endometrial membranes.


In (B/Bo)
NSB subtracted
i i i i I
o o o o o
00
Hours
In (B/Bo)
NSB subtracted
l I i i i i I
o o o o o o o

ro ro ro o o


23
kidney binding sites for PRL appear to be specific and therefore are
considered to be PRL receptors (Hughes et al., 1985).
Binding Characteristics
Most studies conducted of PRL receptors utilize heterologous assay
systems, combining tracer, competing hormone and tissue source of
membrane receptors from various species. Although a great deal of
information regarding in vitro binding of PRL receptors has been
obtained, these systems do not mimic the in vivo environment. Thus,
questions remain as to whether heterologous hormone interactions are
identical to those for homologous hormones. Heterologous hormones can
generate binding results that may not characterize in vivo homologous
binding kinetics and properties or produce immunological artifacts
(Hughes et al., 1982; Amit et al., 1983). Additionally, other
hormones, placental lactogens or proliferin and the microheterogeneity
of PRL and growth hormones contribute to the difficulties in
interpreting results from heterologous radioreceptor assays. To date,
results from only one homologous assay has descibed interactions
between mouse PRL and its liver receptors (Haro and Talamantes, 1985a).
Binding characteristics are similar, but the affinity constant is
slightly lower than obtained from heterologous assays. This would
results in a more rapid hormone-receptor dissociation, as speculated to
occur in vivo (van der Gugten et al., 1980). Thus it appears
advantageous, although more difficult, to utilize homologous hormones
for studying receptors.
Scatchard (1949) analysis of binding studies assumes that
receptors are saturable, specific and freely reversible at equilibrium.


155
1 2 3 4 5 6
Figure 12-2: Evaluation of the purity of nonglycosylated and
glycosylated forms of porcine prolactin isolated by column
chromoatography using 12.5% sodium dodecylsulphate one
dimensional polyacrylamide gel electrophoresis. Lanes 1 and
6 contain molecular weight markers (12,400, cytochrome C;
20,000, soybean trypsin inhibitor; 29,000, carbonic
anhydrase; 45,000, ovalbumin; 63,000-65,000, bovine serum
albumin). Lanes 2 and 5 contain porcine prolactin (total)
prior to separation. Lane 3 contains the nonglycosylated
form, while Lane 4 contains the glycosylated form of porcine
prolactin.


176
be important physiologically, especially in species which lack
placental lactogen.
Results of the present studies support the concept that PRL
affects the uterine physiology of pigs and suggests that effects of PRL
on uterine physiology are not limited to species with decidual or
placental sources of lactogenic hormones. Additionally, a homologous
radioreceptor assay for PRL was acheiveable and provided data for PRL
receptor characteristics which were similar to those obtained using a
heterologous assay. Meaurement of PRL receptors using a homologous RRA
has the advantage of increased similarity to in vivo hormone-receptor
environmnent. The endometrium of pigs apparently regulates PRL
receptor numbers, not affinity, to modify responses to relatively
constant concentrations of PRL in blood. These changes in hormone-
receptor-membrane interaction result in physiological changes similar
to those achieved by other hormones through increased in concentrations
of the hormone. Elucidation of the signal transduction system for PRL
will increase our understanding of different mechanisms of regulation
of the actions of PRL by target tissues and suggest other mechanisms of
hormone-tissue response regulation. This has been suggested by
receptor characterization studies, and by the microheterogeneity of PRL
between its cleaved, clipped, and glycosylated forms, for which little
is known regarding their physiological affects.
In conclusion, results from PRL physiology and receptor studies
described in this dissertation indicate that PRL affects porcine
uterine physiology. Further studies are required to elucidate the
functions of PRL microheterogeneity, and expand our knowledge of PRL


dihydro-15-keto-PGF2a, PGFi and arachidonic acid. A pool of charcoal-
stripped uterine flushing (approximately 3 ng PGE/ml) was serially
diluted and assayed as described previously. The inhibition curve was
parallel to, and not different from, the standard curve when tested for
heterogeneity of regression. The assay was further characterized by
measurement of known amounts of PGE2 added to charcoal-stripped uterine
flush ([y=-6.9 + 1.09x]; where y= the amount of PGE2 measured
(pg/0.2ml) and x=the amount of PGE2 added (pg/0.2ml); R2=0.928)].
Inter- and intra- assay coefficients of variation were 9.7 and 12.4%,
respectively.
Prolactin
Concentrations of PRL in serum of gilts were measured by a
radioimmunoassay (RIA) sensitive to 1 ng/ml (Kraeling et al., 1982).
The inter- and intra- assay coefficients of variation were 15.2 and
16.3%, respectively (Kraeling et al., 1982).
Statistics
Data were analyzed by least squares analysis of variance using the
General Linear Models procedures of the Statistical Analysis System
(SAS) (Barr et al., 1979) to detect effects of treatment.
Experiment 1
This experiment determined effects of hypoprolactinemia on
conceptus survival. Ten gilts were mated on Days 0 and 1 of the
estrous cycle and assigned randomly, 5 per treatment group, to receive
either CB154 (100 mg/day) or vehicle (4ml/day) once daily on Days 10


120
physiology in pigs (Young and Bazer, 1988; Mirando et al.( 1988).
Changes in the biological effects of a hormone may occur through either
1) increasing hormone concentrations in the presence of constant
receptor numbers or 2) increasing receptor numbers, but constant
concentrations of hormone. Results of this study support the latter
mechanism since PRL receptor numbers remained relatively constant in
pregnant gilts until Day 12, then increased and remained elevated
through Day 30. Despite similar numbers of receptors in endometrial
membranes on Day 8 for cyclic and pregnant pigs, PRL receptors in
cyclic endometrial membranes decreased by Day 10 and remained
consistently low until Day 15.
The Kd detected for endometrial PRL receptors in regard to
circulating PRL levels would suggest that only a small proportion
receptors may need to be bound to elicit a response, as previously
mentioned for histamine receptors (Nickerson, 1956). Scatchard
analysis of homologous competitive inhibition curves for endometrial
PRL suggest the presence of two binding sites, having high (109 or 1010
M-1) and low (107) affinities (see Chapter 7). Two binding sites were
detected in endometrial membranes of gilts, most notably on Day 11 and
12 of pregnancy, however, results were not conclusive. A higher
affinity binding site for PRL, if temporally associated with porcine
conceptus estrogen secretion, would allow an additional regulatory
mechanism for affects of PRL on target tissues. Therefore, although
multiple binding sites may be present in porcine endometrial membranes,
additional investigation is necessary.
The increase in PRL receptors detected for pregnant endometrial
membranes in the present study may have resulted from effects of


7
synthesis assays, is lower for human and sheep glycosylacted PRL (Lewis
et al., 1984, 1985), but higher for porcine glycosylated PRL (Pankov
and Butnev, 1986). Porcine glycosylated PRL had decreased binding to
rabbit or porcine mammary gland membrane preparations than ovine PRL
(Pellegini et al., 1988; Seely et al., 1988). However, these assays
were conducted in heterologous systems and results may be inconsistent
with the true activity of glycosylated PRL.
The function of the carbohydrate moiety of PRL is unknown.
Speculation suggests that this form of PRL could be reserve source and
deglycosylated as needed or have a longer half-life than the 23,000 Mr
nonglycosylated PRL. The ratio of nonglycosylated to glycosylated PRL
may be important in physiological effects of PRL. The ratio changes in
late pregnancy of humans (Markoff and Lee, 1987) and during the first
year of life for pigs (Sinha et al., 1988), but a functional change
associated with shifting ratios has not been elucidated. Further work
on the role of the forms of PRL, individually or combinded, is
necessary to understand the diversity of PRL's functions.
Additionally, investigation of these forms at the receptor level may
provide insight into interactions with PRL receptor(s) and subsequent
biological actions.
Cleaved and Clipped Prolactin
Another variation of PRL is the "cleaved" form in which the large
loop formed by the disulfide bond between amino acids 54 and 174 is
severed at amino acids 148-149. A polypeptide containing two strands
results, joined by the disulfide bridge which previously caused a large
loop in PRL. Cleaved PRL contains 16,000 Mr (amino acids 1-148) and


172
(Posner et al., 1974b; Grissom and Littleton, 1988). In the present
study, porcine endometrial membrane preparations had low binding for
porcine PRL unless pretreated with MgCl2 to remove endogenous hormone.
Difficulty in binding PRL to tissue from a homologous source has been
reported for rabbit, sheep and pigs (Berthon et al., 1987b), however,
the tissues were not treated with a chaotropic agent (MgClz).
Validation of the homologous RRA for porcine prolactin assures that PRL
is binding to its own receptor since crossreactivity with GH was very
low, an attribute not always acheivable in heterologous assays.
Additionally, investigation of competative inhibition curves for two
receptors sites suggested that the porcine endometrial membranes may
contain PRL receptors with high affinity, low capacity and low
affinity, high capacity binding characteristics (Chapters 7, 9 and 12).
Investigation of the molecular weight estimates of the porcine
endometrial PRL receptors agreed with previous estimates for PRL
receptors from rat liver and also detected the two molecular weight
variants reported for sow mammary gland (Berthon et al., 1987a), but
not for rat liver membranes. Heterogeneity between PRL receptors has
been suggested (Nicoll et al., 1980) since PRL receptors are
differentially regulated in tissues from animals under steady-state
physiological conditions.
The effects of pregnancy, as well as acute and chronic ovarian
steroid administation, on the ontogeny of endometrial prolactin
receptors were investigated using the homologous RRA for porcine PRL.
Circulating levels of PRL do not change during establishment of
pregnancy. Rather, physiological effects of PRL appear to be regulated
by changes in receptor numbers, but not affinity, in endometrial


during the cycle. Receptors numbers increased following conceptus
estrogen secretion (Day 12). Administration of estrogen to cyclic
gilts on Day 11 resulted in uterine secretory response similar to those
detected during pregnancy. Endometrial prolactin receptors increased
within 6h, decreased at 12h and recovered to basal values at 24h
following a single estrogen injection. Changes in prolactin receptors
were associated temporally with changes in uterine ion and protein
secretion. Endometrial prolactin receptor numbers decreased in
ovariectomized pigs treated with estrogen, or estrogen and
progesterone, while corn oil or progesterone alone had no affect. Low
prolactin receptor numbers were associated with low and high uterine
secretory responses. Estrogen stimulation of pituitary prolactin
release could down-regulate endometrial prolactin receptors and
increase steroid receptors. Availablity of exogenous steroids could
therefore influence uterine secretory response.
Porcine prolactin is microheterogeneic. Glycosylated porcine
prolactin has lower mitogenicity, immunoreactivity and binds to fewer
receptors, but higher lactogenicity and receptor binding affinity than
nonglycosylated or total prolactin.
Results of this study indicate that prolactin affects porcine
uterine physiology. Regulation and specificity of endometrial response
to prolactin appears to be controlled locally by regulation of receptor
number, but not affinity. Microheterogeneity of prolactin results in
different affinities for endometrial receptors and may account for
prolactin's diverse effects.
XV


84
by trichloro acetic acid (TCA) precipitation prior to column elution
and calculating the incorporation of 125I into PRL (see appendix C).
Ovine PRL (USDA-B-1) was iodinated as described for porcine PRL with a
specific activity of 97 uCi/ug. The t1231]-PRL fractions were diluted
1:1 in assay buffer, stored at 4 C and used within 10 days.
Animals
Pregnant gilts, anesthetized on Day 75 using sodium thiamylal (lg)
and maintained under surgical anesthesia on a closed circuit anesthetic
machine using Halothane, were subjected to a midventral laparotomy and
hysterectomized. Endometrium was separated from placental and
myometrial tissue layers and placed on ice. Liver and mammary gland
tissue was collected from Day 75 pregnant gilts at slaughter and
processed as described below. Day 20 pregnant rats were euthanized
with an overdose of sodium pentobarbital and livers were excised and
processed. Rabbit mammary glands were prepared by the method of Shiu
et al. (1973) and obtained following euthanasia with an overdose with
sodium pentobarbital.
Membrane Preparation
Endometrium from Day 75 pregnant gilts and liver from Day 20
pregnant rats were collected and placed on ice, rinsed three times in
ice-cold 0.9% (w/v) saline, rinsed in ice-cold homogenization buffer
(100 mM Tris, 150 mM sodium chloride, 50 mM ethyleneglycol-bis[B-
aminoethyl ether] N,N'-tetraacetic acid [EGTA], 50 mM ethylenediamine
tetraacetic acid [EDTA], 300 mM sucrose, 1 mM
phenylmethylsulfonylfluoride (PMSF) and Aprotinin (400 Kallikrin


108
Materials and Methods
Tissue Collection and Preparation
Day 75 pregnant pig endometrium and Day 20 pregnant rat liver were
collected and processed as described in Chapter 7.
Affinity Labeling of Prolactin Receptors
Affinity labeling of prolactin receptors from Day 75 pregnant
porcine endometrial, and Day 20 pregnant rat liver preparations were by
a cross-linking procedures adapted from Hughes et al. (1983), Wang et
al. (1987) and Berthon et al. (1987a). Iodination of porcine PRL and
homologous RRA for porcine PRL were conducted as described in Chapter
7. All volumes used for chaotropic treatment of membrane preparations
were proportionally increased to accommodate 750 ug membrane protein,
rather than the 150 ug normally used in binding assays. Additionally,
200,000-300,000 cpm of labeled porcine PRL was added in the presence or
absence of 10 or 20 ug unlabeled porcine PRL or 10 ug unlabeled porcine
GH. Following incubation, membranes were washed and centrifuged as
previously described and resuspended in 500 ul assay buffer. Then 5 ul
of ethylene glycol bis succininimidyl succinate (EGS) dissolved in
dimethylsulfoxide (DMSO) (22.81 mg EGS/1 ml DMSO; 50 mM solution) was
added to the resuspended pellet to give a final concentration of 500
uM. Samples were incubated for 30 min on ice. The reaction was
quenched with 3 ml of tris buffer (10 mM Tris-HCl, 1 mM EDTA pH 7.4),
centrifuged at 3000 x g for 20 min, decanted and counted. Pellets were
solubilized in 50 ul Laemelli buffer and prepared for 10% one
dimensional polyacrylamide gel electrophoresis (Roberts et al., 1984).


113
purification of membrane preparation by Affigel-10 ovine PRL affinity
chromtography and electrophorisis resulted in a major band of 42,000 Mr
and faint bands of 53,000 and 31,000. Additionally, monoclonal
antibody detection of purified PRL receptor resulted in three bands of
66,000; 45,000 and 31,000.
Therefore, the PRL receptor in porcine endometrial membranes,
following affinity labeling, cross-linking and electrophoresis, has an
estimated molecular weight that is similar to that for rat liver PRL
receptors. However, other molecular weight proteins were detected and
agree with results of other estimates of Mr for porcine mammary gland
PRL receptors.


173
membranes. During pregnancy, endometrial PRL receptors increased, only
in pregnant pigs on Day 12, and remained elevated to Days 15 and 30
(Chapter 9) This increase was temporally associated with increased
production of estrogens by the conceptus and agrees with the premise
that changes in endometrial PRL receptors occur throughout gestation in
pigs (DeHoff et al., 1984). Receptors for PRL increased between 1 and
6h following estrogen administration, then decreased at 12h, in
temporal association with increases in luminal concentrations of
calcium, but prior to increses in uterine secretion protein secretions.
The mechanism(s) whereby PRL modulates of the porcine uterine
environment to enable maximal response to estrogen secretion by
conceptuses are presented in Figure 13-2. Prior to Day 8, circulating
PRL and endomtrial PRL receptors are similar regardless of reproductive
status. Prolactin could then increase endometrial proliferation
(Chilton and Daneils, 1984), gap junction formation (Sorenson et al.,
1987), steroid receptor concentrations (Leung and Sasaki, 1973; Daniels
et al., 1984) and affect ion channels (Petersen and Maruyama, 1985).
After Day 8, PRL receptors decreased in cyclic gilts, but were
maintained in the endometrium of pregnant gilts. Estrogens secreted by
pig conceptuses binds to estrogen receptors, increased in number by
PRL, and has enhanced effects. Conceptus estrogens may affect
endometrial cell membranes directly, increasing membrane fluidity or
shifting subcellular localization of PRL receptors. This leads to
increased unoccupied PRL receptors in the endometrium, as observed in
most tissues except for rat liver. Thus, estrogen and progesterone can
stimulate the endometrium maximally since steroid receptor numbers were
increased by PRL. Prolactin can affect the uterus since receptor


204
Nicoll, C.S. (1979) Ontogeny and evolution of prolactin's functions.
Fed. Proc. 39, 2563-2566.
Nicoll, C.S. (1982). Prolactin and growth hormone: specialists on one
hand and mutual mimics on the other. Perspec. Biol. Med. 25, 369-381.
Nicoll, C.S., Anderson, T.R., Herbert, N.J. and Russell, S.M. (1985)
Comparative aspects of the growth-promoting actions of prolactin on its
target organs: evidence for synergism with an insulin-like grwoth
factor. In: Prolactin, Basic and Clinical Correlates, pp. 393-407.
(eds. R.M. MacLeod, M.O. Thorner and U. Scapagini) Fidia Reseach
Series, Spinger-Verlag, Berlin.
Nicoll, C.S. and Bern, H.A. (1972) On the actions of prolactin among
the vertebrates: is there a common denominator? In: Lactogenic
hormones, pp. 299-317. (eds. G.E. Wolstenholme, J. Knight) Churchill
Livingstone, London.
Nicoll, C.S., Herbert, N., Steiny, S. and Delidow, B. (1983) A
synergist of prolactin's mitogenic activity (synlactin) is secreted by
the liver. Am. Zool. 23, 899-905.
Nicoll, C.S. and Meites, J. (1962) Estrogen stimulation of prolactin
production by rat adenohypophysis in vitro. Endocrinology. 70, 272-
277.
Nicoll, C.S. Tarpey, J.F., Mayer, G.L. and Russell, S.M. (1986)
Similarites and differences among prolactins and growth hormones and
their receptors. Am. Zool. 26, 965-983.
Nicoll, C.S., White, B.A. and Leung, F.C. (1980) Evolution of
prolactin, its functions, and its receptors. In: Central and Periferal
Regulation of Prolactin Function, pp.11-24. (eds. R.M. Macleod and U.
Scapagini) Raven Press, New York.
Nolin, J.M. (1982) Profiles target-cell prolactin and
adrenocorticotropin during lactational diestrus. In: Reproductive
Processes and Contraception, pp.195-213. (ed. K.W. McKerns), Plenum
Press, New York.
Nolin, J.M. and Bogdanove, E.M. (1980) Effects of estrogen on
prolactin (PRL) incorporation by lutein and milk secretory cells and on
pituitary PRL secretion in the postpartum rat: correlations with target
cell reponsiveness to PRL. Biol. Reprod. 22, 393-416.
Norstedt, G., Husman, B., Mode, A., Eneroth, P., Lewis, U.J. and
Gustafsson, J. -A. (1987) Induction of prolactin receptors in the
liver is more closely related to the growth-promoting than to the
lactogenic potency of peptides. Acta Endocrinol. 114, 350-356.
Norstedt, G. and Mode, A. (1982) On the primary site of action of
estrogens and androgens in the regulation of hepatic prolactin
receptors. Endocrinology. Ill, 645-649.


Percentage 125l Porcine Prolactin Bound
90
Figure 7-1: Effects of increasing magnesium chloride molarity
on binding of porcine [125I]-prolactin by membranes from Day
75 porcine endometrium, amnion, chorion, as well as post
parturient pig and rabbit mammary gland (300 ug).


48
prepared in charcoal-stripped uterine flushings with known amounts of
radioinert PGF2a (0, 10, 25, 50, 100, 250, 1000 and 2500 pg). A 1:5000
dilution of antiserum enabled detection of 10 pg PGF per tube. Cross
reactivities of PGF2a antiserum with other prostaglandins were: 94% for
PGFla; 2.4% for PGE2 and <0.1% for 13,14, dihydro-15-keto-PGF2a. PGE
and arachadonic acid. Unextracted uterine flushings (0.2 ml) were
assayed for PGF in duplicate. A pool of uterine flushings
(approximately 3 ng PGF/ml) assayed in serial dilutions (0.01, 0.025,
0.05, 0.1 and 0.2 ml with a final volume of 0.2 ml in charcoal stripped
uterine flush) resulted in an inhibition curve that was parallel to,
and not different from the standard curve when tested for heterogeneity
of regression. Further characterization of the assay involved
measurement of known amounts (10, 25, 50, 100, 250, 500, 1000 and 2500
pg) of PGF in charcoal-stripped uterine flushings ([y=-9.6 + l.llx];
where y=amount of PGF measured (pg/0.2 ml) and x=amount of PGF added
(pg/0.2 ml); R2=0.947). Inter- and intra- assay coefficients of
variation were 14.1% and 15.7%, respectively.
Prostaglandin E
Concentrations of PGE2 in uterine flushings were determined using
an assay similar to that described for PGF with a modification (Lewis
et al., 1978) using tritiated PGEZ ([5,6,8,11,12,14,15-3H]:PGE;
specific activity 140-170 Ci/mmole; Amersham Corporation, Arlington
Heights, IL). A 1:6000 dilution of antiserum (Eli Lilly,
Indiannapolis, IN) enabled detection of 5 pg PGE2/tube as different
from zero. Cross-reactivities of PGE2 antiserum with other
prostaglandins were; 24% for PGEi; 1.7% for PGF2 ; 0.1% for 13,14-


33
secreted from the liver is response to high circulating levels of PRL.
However, Hoeffler and Frawley (1987) suggest that the two compounds are
different. The LLF is lactogenic, exerts potent biological activity
individually, and acts additively with PRL when tested in the reverse
hemolytic plaque assay (Neill and Frawley, 1983) using mammary cells.
Synlactin is mitogenic, devoid of activity alone and acts
synergistically with PRL when tested in pigeon crop sac assays.
Neither synlactin or LLF have been sequenced, nor tested in reverse
hemolytic plaque or pigeon crop sac assays, respectively.
A role for a PRL stimulated liver mitogenic factor is suggested
since liver receptors for PRL increase during pregnancy in rats (Sasaki
et al., 1982a), mice (Sasaki et al., 1982b) and rabbits (Kelly et al.,
1974; Fix et al., 1981) and may be associated with increases in
synlactin secretion and possibly mammary growth and development during
gestation (Mick and Nicoll, 1985).
Internalization of Prolactin and its Receptor
A clearly definded transduction mechanism has eluded researchers
studying PRL-receptor interaction; therefore the role of
internalization in PRL function was investigated. Prolactin has been
detected in the subcellular fraction of the plasmalemma (Posner et al.,
1981) and golgi regions of cells from rat ventral prostate and liver
(Bergeron et al., 1978). Receptor internalization through coated pits
was proposed for LDL receptors (Goldstein et al., 1979). Following
internalization, the hormone is degraded while the receptor can be
degraded or recycled. Proteolysis of internalized hormones allows
epidermal growth factor and insulin to affect target tissues (Goldfine


60
Table 4-1: Effects on interestrus interval and cytotoxicity
reaction to cysteamine (CSH) or ethanoloamine (control)
administration to cyclic gilts.
Gilts
Group
Dose
mg/day
#Inj
IB1 Smpl
Cytotoxicity
score
Cycle length
days
328
CSH
200
9
19
10
28+
326
Control
100
7
33
5
30
305
CSH
100
7
33
7
45+
12
Control
100
3
33
3
23


77
PGE (Figure 6-3B) in uterine flushings. Additionally, gilts treated
with PRL+E2V had greater amounts of glucose (P<0.01) and PGF (P<0.01)
in uterine flushings than gilts receiving E2V alone. But, values for
glucose and PGF were not different for gilts treated on Day 11 with E2V
or corn oil. The specific activity of uteroferrin (umoles/mg protein)
was higher (P<0.01) in uterine flushings from gilts treated with
PRL+E2V (106.5 +9.3) compared to gilts receiving SAL+E2V alone (56.7
+9.3) or SAL+corn oil (8.4 +9.3). Gilts receiving PRL+E2V had greater
uterine secretory responses for total protein, potassium and leucine
aminopeptidase compared to gilts receiveing SAL+E2V, but differences
were not statistically significant.
Discussion
Exogenous PRL interacted with estrogen, but not progesterone, to
cause significant effects on uterine epithelial secretory activity.
Shifts in concentrations of ions could account for the earlier release
of several uterine secretory components; although such shifts could not
be not accounted for by static measurments on Day 12 in the present
study. Shifts in ions occur prior to protein secretion during early
pregnancy (Geisert et al., 1982b; Bazer et al., 1984) and in response
to administration of exogenous estradiol on Day 11. With respect to
the secretory profiles of uterine components in the experiments
reported here, exogenous PRL may have advanced the rapid release and
reuptake of calcium which characteristically follows estrogen
stimulation and precedes accumulation of proteins in the uterine lumen.
Luminal calcium flux may have occurred early on Day 11 in
hyperprolactinemic gilts. Therefore, calcium measured in uterine


CHAPTER 5
ESTABLISHMENT OF HYPERPROLACTINEMIA
BY ADMINISTRATION OF EXOGENOUS PORCINE PROLACTIN TO PIGS
Introduction
Establishment of function for a hormone can be acheived through
administration of that hormone followed by studies of the physiological
changes it induces. Circulating prolactin (PRL), however, is subject to
increases due to stress such as those that may be experienced during
frequent blood sampling and confinement. Therefore, this study was
conducted to establish that hyperprolactinemia could be acheived by
injecting porcine PRL twice daily. This was done as a separate
experiment to avoid compromising uterine secretory responses in
subsequent experiments (Chapter 6) due to confinement and chronic
bleeding of the gilts which would likely cause stress-induced release
of PRL.
Material and Methods
Animals
Crossbred gilts of similar age (7 to 9 months) and weight (110 to
120 kg) were used in all studies after experiencing at least two
estrous cycles of normal length (18-22 days). Using intact boars,
gilts were observed for estrus daily and the first day of behavioral
estrus was designated Day 0.
63


76
50 --
\
60
25 -
Sal PRL Sal
Oil |E2VJ
B
50 --
o
JC
\
bo
e
25
LU
u
CL
o
Sal PRL Sal
Oil |E2V|
Figure 6-3: Concentrations of (A) PGF and (B) PGE in Day 12
uterine flushings from cyclic gilts (Experiment 2) treated
with 1 ml saline (SAL) or 1 mg porcine prolactin (PRL) on
Days 6-11 and 0.5 ml corn oil (OIL) or 5 mg estradiol
valerate (E2V) on Day 11 of the estrous cycle. Overall
treatment effects were detected for PGF (P<0.01). Values
with different letters are different (P<0.05). The overall
SEM was +10.2 for PGF and +6.7 for PGE.


156
Statistics
Data were analyzed using the General Linear Models of Analysis of
Variance of the Statistical Analysis System (SAS) (Barr et al., 1979).
Data from Nb2 lymphoma cell assays were analyzed for heterogenity of
regression. Data from RIA curves were transformed to log-logit plots
to generate straight lines which were tested for parallelism.
Orthogonal contrasts were used to detect differences between slopes and
intercepts.
Results
Mitoqenicity of the Forms of Porcine Prolactin
Results of the Nb2 lymphoma cell assay comparing nonglycosylated,
glycosylated and total porcine PRL are depicted in Figure 12-3.
Nonglycosylated PRL had greater mitogenicity (P<0.01) than glycosylated
PRL. However, nonglycosylated and glycosylated PRL were 10- and 100-
fold less active (P<0.01), respectively, than total PRL in stimulating
uptake of 3H-thymidine by Nb2 lymphoma cells.
Lactogenic Activity of the Forms of Porcine Prolactin
At the lower dose (10 mg) of hormone administration, pigeons that
received glycosylated PRL had a 64% increase in crop sac mucosal dry
weight compared to pigeons that received nonglycosylated PRL (12.8 vs
7.8 mg, respectively). Crop sac mucosal dry weights were not different
between pigeons that received total or nonglycosylated PRL. However,
at a higher dose (50 mg) of the PRL forms, no differences in crop sac
mucosal dry weight were detected between pigeons that received
glycosylated and nonglycosylated (12.8 vs 11.0 mg, respectively) PRL.


141
Experimental Design
On Day 4 of the estrous cycle, 12 gilts were ovariectomized and
assigned randomly (3 per group) to one of four hormone treatments:
estradiol valerate (E2V; 100 ug/0.5 ml/day); progesterone (P4; 200 mg/4
ml/day); combined treatments of estradiol valerate and progesterone
(E2V+P4); or corn oil (CO; 4 ml/day). Treatments were administered
from Days 4 to 14. Gilts were hysterectomized and uterine flushings
{Bazer et al., 1978) and endometrium were collected on Day 15.
Surgical Procedure
On Day 4 of the estrous cycle, gilts were anesthetized with
thiamylal sodium (1 g, i.v.) and maintained under surgical anesthesia
using a closed circuit anesthesia machine. Gilts were subjected to
midventral laparotomy, the ovaries were exteroirized, blood vessels
tied, and the ovaries removed. The uterus was not exteriorized to
minimize manipulation of the reproductive tract. On Day 15, the gilts
were subjected to a second midventral laparotomy. The uterus was
exteriorized, each uterine horn was flushed with 20 ml sterile saline
(Bazer et al., 1978) and endometrium was obtained immediately following
hysterectomy.
Preparation and Analysis of Uterine Flushings
Uterine flushings were collected and determination of total
recoverable protein and uteroferrin were conducted as described in
Chapter 3.


145
concentrations are not necessarily similar between tissues or within
the same tissue of different species.
Steroid regulation of hepatic PRL receptors has been extensively
investigated. Hepatic PRL receptors decrease in ovariectomized rats
(Posner et al., 1974a; Kelly et al., 1979), but increase following 8 to
12 days of chronic estrogen administration (Posner et al., 1974a).
Increases in PRL receptors following estrogen treatment may be
mediated, indirectly, through stimulating release of pituitary PRL and
auto-upregulation of PRL receptors. Hypophesectomy (Posner et al.,
1974a), but not CB154 administration (Kelly et al., 1976), blocked the
estrogen stimulated increase in rat hepatic PRL receptors, suggesting
pituitary involvement, but not exclusively an effect of PRL.
Involvement of growth hormone (Knazek et al., 1975), ACTH and TSH
(Bhattacharya and Vonderhaar, 1979) is implicated (Waters et al.,
1978). Contrary to results in rats, ovariectomy increases hepatic PRL
receptors in mice (Marshall et al., 1979) and exogenous estrogen
reverses the effect of ovariectomy. Estrogen administration also
decreases PRL receptors in prostate (Kledzik et al., 1976; Amit et al.,
1983) adrenal, kidney, (Monkemeyer et al., 1974) and mammary gland of
mice (Marshall et al., 1979) and rats (Smith et al., 1976; Bohnet et
al., 1977).
The increase in PRL receptors in the mammary gland following
parturition (Holcomb et al., 1975; Djiane et al., 1977) is thought to
be due to autoregulation by increases in serum PRL associated with
parturition since administration of CB154 at parturition, decreases
mammary gland PRL receptors (Bohnet et al., 1977). Admistration of PRL
to pseudopregnant rabbits increases mammary gland PRL binding sites; an


102
6-fold (7% vs 29%) and resulted in an increase in the number of
available receptors (285 fmoles vs 34 pmoles/mg protein).
Conservative interpretation of Scatchard analysis of competitive
inhibition curves resulted in one binding site. However, two binding
sites were also determined, although not as consistently, through the
LIGAND analysis. These binding sites contained a higher affinity (109
or 1010 M-1) but low capacity component as well as a lower affinity
(107) but high capacity component than when data were analyzed and
displacement curves fitted for a single receptor site.
Discussion
Prolactin receptors are generally measured by RRAs employing ovine
PRL (Kelly et al., 1979, 1983; DeHoff et al., 1984; Bramley and
Menzies, 1987; Grissom and Littleton, 1988). These investigators have
reported high affinity, low capacity receptors for PRL between various
tissues and among species. Prolactin belongs to a family of hormones
including GH, placental lactogen and proliferin (Linzer and Nathans,
1984). Nicoll (1982) described complications inherent in RRAs due to
interplay between members of this hormone family, especially GH and
PRL, and their receptors. Ovine PRL may not be purely analogous when
employed in heterologous systems during in vitro binding analyses.
Therefore, the homologous RRA may provide an in vitro environment that
is most similar to the in vivo hormone-membrane milieu.
Results of the present study validate a homologous RRA for porcine
PRL which allows quantitation of PRL receptors in porcine endometrial
membrane. This assay demonstrated saturable binding sites and a direct
relationship between the amount of PRL bound and increasing


CHAPTER 2
REVIEW OF LITERATURE
History, Evolution and Structure of Prolactin
A lactogenic hormone was first suggested by experiments of John
Hunter (1788-1840) in which he described proliferation and secretion of
the pigeon crop sac. Hunter alleged that the pigeon crop sac was
analogous in its development to the mammary gland during gestation.
Interest in anterior pituitary secretions continues today from the
initial work of Hunter in the 1780's and characterization of the six
anterior pituitary hormones in the late 1930s. Research with
prolactin (PRL) was not very active until the 1920's. Injection of
anterior pituitary extracts into rats (Evans and Simpson, 1929) and
intact (Strickler and Grueter, 1928) and ovariectomized (Corner, 1930)
rabbits resulted in mammary distention due to milk synthesis. Yet, it
was the industrious work of Oscar Riddle and coworkers (1933) that
involved isolating and purifying the lactogenic substance which was
named "prolactin." However, it was development of the pigeon crop sac
bioassay, based on John Hunter's early work, that allowed confirmation
of PRL activity during its purification. Great controversy prevailed
as to whether a separate growth promoting hormone exsisted. Riddle et
al. (1933) demonstrated that PRL was different from growth hormone and
established PRL as an independent hormone. Despite a desire for
complete separatation of functions for the new known hormones, PRL and
3


Figure 12-3: Uptake of 3H-thymidine into Nb2 lymphoma cells
expressed as percent of Nb2 control (dashed line) when cells
were stimulated by total (circles), nonglycosylated (squares)
and glycosylated (triangles) forms of porcine prolactin.


210
Shull, J.D. and Gorski, J. (1983) Prolactin gene transcription: two
stages of reglation by estrogen. Fed. Proc. Am. Soc. Exp. Biol. 42.
206 (Abstract).
Shull, J.D. and Gorski, J. (1984) Estrogen stimulates prolactin gene
transcription by a mechanism independent of pituitary protein
synthesis. Endocrinology. 114, 1550-1557.
Shull, J.D. and Gorski, J. (1986) The hormonal regulation of prolactin
gene expression: an examination of mechanisms controlling prolactin
synthesis and the possible relationship of estrogen to these
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Siegelman, M. Bond, M.W., Gallatin, W.H., St. John, T., Smith, H.,
Fried, M.H. and Veissman, I.L. (1986) Cell surface molecular
association with lymphocyte homing is a ubiquitinated branched-chain
glycoprotein. Science. 231, 823-829.
Singer, S.J. and Nicholson, G.L. (1979) The fluid mosaic model of the
structure of cell membranes. Science. 175, 720-731.
Sinha, Y.N. and Baxter, S.R. (1979) Metabolism of prolactin in mice
with a high incidence of mammary tumours: Evidence for greater
conversion into nonimmunoassayable form. J. Endocrinol. 81, 299-314.
Sinha, Y.N., Campion, D.R., Jacobson, B.P. and Lewis, U.J. (1988)
Glycosylated prolactin in porcine plasma: immunoblotic measurement from
birth to one year of age. Endocrinology. 123, 1728-1734.
Sinha, Y.N. and Gilligan, T.A. (1981) Identification of a less
immunoreactive form of prolactin in the rat pituitary. Endocrinology.
108, 1091-1094.
Sinha, Y.N., Gillian, T.A. and Lee, D.V. (1984) Detection of a high
molecular weight variant of prolactin in human plasma by a combination
of electrophoretic and immunologic techniques. J. Clin. Endocrin.
Metab. 58, 752-754.
Sinha, Y.N., Gillian, T.A., Lee, D.W., Hollingsworth, D. and Markoff,
E. (1985) Cleaved prolactin: evidence for its occurance in human
pituitary gland and plasma. J. Clin. Endocrinol. Metab. 60, 239-241.
Sinha, Y.N. and Jacobsen, B.P. (1987) Glycosylated prolactin in the
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Smith, B.C. and Wagner, W.C. (1985) Effect of dopamine agonist or
antagonists, TRH, stress and piglet removal in plasma prolactin
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207
Rae-Ventner, B. and Dao, T.L. (1982) Kinetic properties of rat
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Riddick, D.H. and Kusmik, W.F. (1977) Decidua: a possible source of
amniotic fluid prolactin. Am. J. Obstet. Gynecol. 127, 187-190.
Riddick, D.H., Luciano, A.A., Kusmik, W.F. and Maslar, I.A. (1978) De
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Riddle, 0. (1963) Prolactin invertebrate function and organization.
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Riddle, 0. Bates, R.W. and Dykshorn, S. (1933) The preparation,
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Roberts, R.M. and Bazer, F.W. (1988) The functions of uterine
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200
Lee, D.W. and Markoff, E. (1986) Synthesis and release of glycosylated
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Proc. Nat. Acad. Sci. USA. 81, 4255-4259.


119
and were best described separately. Endometrial membrane receptor
numbers (pmoles/mg protein) were not different between pregnant and
cyclic gilts on Day 8 (27.6 vs 30.4 +4, respectively). After that,
endometrial membranes from cyclic gilts had decreased PRL receptor
numbers on Days 10 (21.1+4) to Day 14 (21 to 23+4) which then increased
slightly on Day 15 (28.4 +4). Prolactin receptors in endometrial
membranes from pregnant gilts remained relatively constant for Days 8,
10 and 11 (27.6, 26.9 and 28.0 +4 pmoles/mg protein, respectively),
then increased (P<0.05) on Day 12 (37.0 +4) and remained elevated
through Days 14 (33.5 +4), 15 (31.6 +4) and 30 (34.5+4) of gestation.
Endometrial PRL receptor numbers on Day 12 were greater (P<0.05) for
pregnant than cyclic gilts.
Discussion
Histotroph, contains proteins, sugars and other constituents
essential to preimplantation porcine conceptuses and must be secreted
into the uterine lumen at the appropriate time to support and nourish
the porcine conceptus until placentation is established. Prolactin
enhances effects of ovarian steroids on uterine physiology.
Interactions between PRL and progesterone enhance endometrial
proliferation and uteroglobin secretion in the long-term ovariectomized
rabbit (Chilton and Daniels, 1985). In the pig, however, PRL interacts
with estrogen to enhance secretion of uteroferrin, prostaglandin F2C1
and glucose into the uterine lumen (Young and Bazer, 1988). Prolactin
concentrations in plasma of pigs are relatively constant throughout
gestation (Dusza and Krzymowska, 1981; Kensinger et al., 1986; DeHoff
et al., 1986); yet PRL exerts an effect on uterine secretory


45
Rabbits, like pigs, lack placental lactogen. Therefore, PRL may also
be important for endometrial function in pigs. Experiments were
conducted to lower circulating PRL in pregnant and cyclic gilts and to
observe effects on fetal survival and uterine secretory function.
Materials and Methods
Animals
Crossbred gilts of similar age (7 to 9 months) and weight (110 to
120 kg) were used in all studies after experiencing at least two
estrous cycles of normal length (18-22 days). Using intact boars,
gilts were observed for estrus daily and the first day of behavioral
estrus was designated Day 0. Gilts were mated when detected in estrus
(Day 0) and 12 and 24h later in studies using pregnant pigs.
Surgical Procedures
Uterine flushings were collected in 20 ml of double distilled
water per uterine horn as described previously (Bazer et al., 1978).
Flushing volumes were recorded and flushings were centrifuged at 10,000
x g for 15 min at 4 C. Supernatants were collected and stored at -20 C
until analyzed.
Bromocryptine
Bromocryptine (CB154; was a gift of SANDOZ Pharmaceutical, East
Hanover, NJ), a dopamine agonist, was solubilized in absolute ethanol
and mixed with saline (1:1, v/v) to a concentration of 25 mg/ml.
Treated gilts received 100 mg CB154/day (4 ml) subcutaneously while


193
Farquhar, M.G. and Rinehart, F.J. (1954) Cytological alteration in
anterior pituitary gland following tyroidectomy: An electron
microspical observation. Acta Endocrinol. (Cophen). Supp (100), 161
(Abstract) .
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prolactin binding to rabbit liver plasma membranes. Horm. Metab. Res.
13, 510-515.
Flint, A.P.F., Burton, R.D., Gadsby, J.D., Saunders, P.T.K. and
Heap, R.B. (1979) Blastocyst oestrogen synthesis and the maternal
recognition of pregnancy. In: Maternal Recognition of Pregnancy, pp.
209-208. (ed. J. Whelan). CIBA Foundation Symposium on Nutrition, New
Series 64, Excepta Medica, Amsterdam.
Ford, J.J. and Maurer, R.R. (1978) Simple technique for chronic venous
catheterization of swine. Lab. Anim. Sci. 28, 615-618.
Foxcroft, G.R. and Van der Wiel, D.F.M. (1982). Endocrine control of
the oestrous cycle. In: Control of Pig Reproduction, pp. 161-177. (eds.
D.J.A. Cole and G.R. Foxcroft). Butterworth Scienctific, Boston.
Frank, M., Bazer, F.W., Thatcher, W.W. and Wilcox, C.J. (1977) A study
of prostaglandin F2 as the luteolysin in swine: III. Effects of
estradiol valerate on prostaglandin F, progestins, estrone and
estradiol concentrations in the utero-ovarian vein of nonpregnant
gilts. Prostaglandins. 14, 1183-1196.
Franks, S. (1983) Regulation of prolactin secretion by oestrogens:
physiological and pathological significance. Clin. Sci. 65, 457-462.
Frantz, W.L. and Turkinton, R.W. (1972) Formation of biologically
active 125I-prolactin by enzymatic radioiodination. Endocrinology. 91,
1545-1548.
Frawley, L.S., Brookfor, F.R. and Hoeffler, J.P. (1985) Identification
by plaque assays of a pituitary cell type that secretes both growth
hormone and prolactin. Endocrinology. 116, 734-737.
Freeman, M.E. and Sterman, J.R. (1978) Ovarian steroid modulation of
prolactin surges in cervically stimulted ovariectomized rats.
Endocrinology. 94, 1915-1920.
Fuxe, K., Hokfelt, T. and Nilsson, 0. (1969) Castration, sex hormones
and tuberoinfindibular dopamine neuron. Neuroendocrinology. 5, 107-
113.
Geisert, R.D., Brookbank, J.W., Roberts, R.M. and Bazer, F.W. (1982a)
Establishment of pregnancy in the pig: II cellular remodeling of
porcine blastocyst during elongation on Day 12 of pregnancy. Biol.
Reprod. 27, 941-955.


162
nmoles unlabeled hormone
T
20
Figure 12-5: Scatchard analysis of competitive inhibtion
curves using magnesium chloride treated Day 75 pregnant
porcine endometrial membranes and [!25I]-prolactin (total)
versus unlabelled total prolactin (triangles) and [125I]-
nonglycosylated:glycosylated (2:1) prolactin versus
unlabelled nonglycosylated:glycosylated (2:1) prolactin
(circles).


10
lactotrophs, occurs when secretory vesicles fuse with lysosomes instead
of plasma membranes when PRL secretion is inhibited (Farquhar, 1985).
Dopamine receptors are present on lactotrophs. These receptors, along
with dopamine, are internalized to secretory granules however the role
of interalized neurotranmitter has not been fully elucidated.
Lactotrophs and somatotrophs were thought to be from
morphologically distint cell lines (Farquhar, 1985). However, recent
findings suggest that PRL secreting lactotrophs can be specific cells,
or another class of PRL secreting cells, derived from somatotrophs, and
termed mammosomatotrophs (Stattman, 1974).
Mammosomatotrophs
The anterior pituitary is comprised of different cell types, each
responsible for synthesis and secretion a hormone. The 'one cell/one
hormone theory is supported by anatomical and microscopy studies of
the anterior pituitary. Growth hormone and PRL are secreted from
somatotrophs and lactotrophs, respectively; however, distinction
between these cell types is fading. Early work by Stattman (1974)
suggested that some cells in the anterior pituitary, termed
mammosomatotrophs, secrete both PRL and growth hormone, or exhibit a
functional shift from growth hormone to PRL secretion.
Mammosomatotrophs have thus far been recorded only in rats.
Mammosomatotrophs constitute one-half of PRL secreting cells in the
male pituitary; however, percentages are not available for female
pituitaries (Brookfor et al., 1986). Estrogen stimulation of male
anterior pituitary cells in culture did not cause cell mitosis,
contrary to results by Corenblum et al. (1980), but shifted cell


80
In summary, during the time of maternal recognition of pregnancy
in the pig, PRL interacts with estrogen, rather than progesterone, to
influence uterine endometrial secretory activity. This selective
interaction with estrogen may allow the uterine endometrium to respond
maximally to the estrogen signal from the porcine conceptus which
allows establishment of pregnancy.


14
may be due to a combination of these factors and not under the sole
regulation of the tuberoinfindibular dopaminergic system (Moore, 1988).
Estrogen
Lactotrophs are unique among anterior pituitary cells since they
are generally inhibited by tonic hypothalamic dopamine. Several
factors can override dopamine inhibition, but estrogen is most
effective. The mechanism(s) responsible for estrogenic stimulation of
PRL secretion are relatively unknown; however, following an increase in
estrogen, circulating PRL increases 10-fold, on the afternoon of
proestrus in rats (Butcher et al., 1974). Administration of exogenous
estrogen to ovariectomized rats increased PRL over 2-3 days (Maurer and
Gorski, 1977); however, Yamamoto et al. (1975) demonstrated increases
in PRL within 12 h following injection of a single dose of estrogen
into ovariectomized rats.
Despite chronic inhibition of lactotrophs by dopamine, estrogen
increases the synthesis and secretion (Thorner and MacLeod, 1980) of
pituitary PRL, as well as the number of lactotrophs (Neill and Frawley,
1983). Estrogens' effects may be due to 1) direct stimulation of
pituitary lactotrophs, 2) modulation of hypothalamic regulation of the
pituitary or 3) effects on the physiological responsiveness of
lactotrophs to other regulatory mechanisms.
Estrogen stimulates increases in PRL in pituitary cells when added
to in vitro culture medium (Nicoll and Meites, 1972) or in vivo when
implanted into anterior pituitaries of rabbits (Kanematsu and Sawyer,
1963) and sheep (Vivian et al., 1979). Hypothalamic control,
especially by dopamine, of pituitary PRL is dampened due to estrogen


164
0.15-
0
0
i
Li.
T5
C
D
O
CD
0.10*
Total
Nongly
A
Glycos A
i r
1 2 3
T
4
T
5
T
6
nmoles unlabeled hormone
Figure 12-6: Scatchard analysis of porcine t1231]-(total)
prolactin binding to magnesium chloride treated Day 75
pregnant porcine endometrial membranes inhibited by unlabeled
total (circles), nonglycosylated (squares) or glycoslyated
(triangles) forms of porcine prolactin.


Figure 3-1: Concentrations of total recoverable (A) calcium,
(B) chloride, (C) sodium and (D) potassium in Day 12 uterine
flushings from cyclic gilts (Experiment 2) treated with CB154
(100 mg/day) or vehicle (VHC; 4 ml) on Days 10 and 11 and
estradiol valerate on Day 11. Treatment effects were
detected for calcium and potassium (P<0.05), as well as
sodium and chloride (P<0.01). The overall SEM was +0.14 for
calcium, +0.12 for chloride, +12 for sodium and +8 for
potassium.