The role of prolactin in establishment of pregnancy in pigs : studies on endometrial prolactin receptor regulation and u...

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Title:
The role of prolactin in establishment of pregnancy in pigs : studies on endometrial prolactin receptor regulation and uterine secretory physiology
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xv, 215 leaves : ill., photos ; 29 cm.
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English
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Young, Kathleen Hart, 1960-
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Dissertations, Academic -- Animal Science -- UF
Animal Science thesis Ph. D
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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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oclc - 22714811
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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.