Citation
Bovine placental lactogen

Material Information

Title:
Bovine placental lactogen
Creator:
Wallace, Charles Ralph, 1955- ( Dissertant )
Collier, Robert J. ( Thesis advisor )
Bazer, Fuller W. ( Reviewer )
Wilcox, Charles J. ( Reviewer )
Caton, Donald ( Reviewer )
Buhi, William ( Reviewer )
Place of Publication:
Gainesville, Fla.
Publisher:
University of Florida
Publication Date:
Copyright Date:
1986
Language:
English

Subjects

Subjects / Keywords:
Cattle ( jstor )
Hormones ( jstor )
Human growth ( jstor )
Mammary glands ( jstor )
Placenta ( jstor )
Pregnancy ( jstor )
Rabbits ( jstor )
Rats ( jstor )
Secretion ( jstor )
Sheep ( jstor )
Animal Science thesis, Ph.D.
Dissertations, Academic -- Animal Science -- UF
Placental lactogen
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Abstract:
Studies were conducted to isolate and purify bovine placental lactogen (bPL) and to develop a radioimmunoassay to this protein. Bovine placental lactogen was isolated from culture medium after a 24 hr culture of fetal cotyledonary tissue. Cotyledonary explants were stimulated to secrete bPL by either addition of bovine growth hormone (NIH-B8) to the medium or co-culture of cotyledon and caruncular tissue. Production of bPL was greatly affected by explant size and 70%6 of that produced in a 48 hr culture was released in the first 12 hr. Purification of bPL was accomplished using a column chromatographic scheme that involved gel filtration, ion exchange and chromatofocusing chromatography. The bPL molecule was purified 600 fold with two forms at 30,000 MW and pi's of 4.95 and 5.15. The purified protein was utilized to develop antibodies in rabbits. A radioimmunoassay to bPL was developed using an antibody raised at the USDA Beltsville (F56). Approximately 20% specific binding was achieved with a 1:40,000 final working dilution of the antibody. Assay sensitivity was 300 ng/ml and the standard curve ranged from .1 8 ng. The antibody crossreacted with ovine placental lactogen at .2%. Dose response curves of amniotic or allantoic fluid or fetal and maternal serum were parallel to the standard curve and bPL was quantitatively recovered at from 82 125%. Using the radioimmunoassay, samples of amniotic and allantoic fluids and fetal and maternal serum were measured for bPL. Concentrations of bPL ranged from undetectable to 50 ng/ml, with fetal blood having the highest concentrations and amniotic fluid the lowest. Concentrations of bPL were measured in plasma samples from 12 cows collected three times a week from day 150 to 250 of gestation and then daily until term. Peak concentrations of bPL were at days 210 and 230 of gestation which may correspond to peak fetal growth periods. Concentrations of bPL in blood samples collected at 30 min. intervals for a period of 12 hours were quite similar both within and between cows, however two of the four animals sampled exhibited a spike of bPL secretion that was three to four times greater than baseline.
Thesis:
Thesis (Ph.D.)--University of Florida, 1986.
Bibliography:
Includes bibliographic references (leaves 147-163).
General Note:
Vita.

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Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
AEK5670 ( LTUF )
15525228 ( OCLC )
0029829037 ( ALEPH )

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BOVINE PLACENTAL LACTOGEN: ISOLATION, PURIFICATION
AND MFARTTRFMFNT IN TjT,,OOT(4(AT. FT,TTTTID





By

CHARLES RALPH WALLACE


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


1986















ACKNOWLEDGEMENTS

At this time I would like to acknowledge those people that have made

my PhD program at the University of Florida a memorable experience.

In the time that my family and I have been in Gainesville we have met and

interacted with so many interesting people that I regret that I cannot mention

them all.

Special thanks go to Dr. Robert J. Collier, chairman of the supervisory

committee, for his patience and support throughout this program. Dr. William

W. Thatcher is acknowledged for his ability to stimulate enthusiasm about

seemingly insignificant data in the author's perspective. Thanks are expressed

to Drs. Fuller W. Bazer and R. Michael Roberts for allowing the author to

work in their laboratories and gain the insight to begin to dissect animal

science problems into their biochemical root. Thanks are due to Dr. Charles

J. Wilcox for his statistical prowess and the ability to convey that prowess

to a novice. Dr. Donald Caton is acknowledged for his ability to remind

the author of the whole picture instead of just the part. Dr. William Buhi

is acknowledged for his excellent substitution near the end of this program.

Thanks are extended to Drs. David Beede and H.H. Head for the time

spent discussing research and daily events.

The technical assistance and friendship of Gail Knight, Annette 'Bee'

Leinart, Sergio Quintana, Catherine Ketchum and Carol Underwood were

greatly appreciated.

To the students and postdoctoral fellows that have enriched my program

with their presence, thanks are due to Marlin Dehoff, Lokenga Badinga,









Mark Maguire, Fran Romero and to Drs. Ron Kensinger, Jeff Knickerbocker,

Louis Guilbault, Rodney Geisert, Jeff Moffat, Randy Renegar, Jim Godkin,

George Baumbach, Asgi Fazlebas, Paul Schneider, John McNamara, Quim

Moya and Skip Bartol.

Without the assistance of Austin Green, Kent Bundy and Tom Bruce

the author would not have had animals to work with, thank you for your

help.

Last but not least I would like to thank my family for always being

there. To June, my wife, and Katherine and Steven, our children, you make

life worthwhile. To my parents, Ralph and Emily Wallace, your confidence

and moral support during this program was genuinely given and deeply felt.
















TABLE OF CONTENTS


Page


ACKNOWLEDGEMENTS . . .

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

LIST OF FIGURES . . .. .. ..

ABSTRACT . . . . .

CHAPTER

I REVIEW OF LITERATURE . .

History of Placental Lactogen .
Placental Type ............. *
Endocrine Control of Mammary Development
Secretion . . .
Function . . .
Nutrient Partitioning . .
Purification . . .


II COTYLEDON CULTURE EXPERIMENTS


. . ii

. .. vi

. . viii

. . X


. . 42


Introduction . .
Materials and Methods . *
Results and Discussion .. *


III PURIFICATION OF BOVINE PLACENTAL LACTOGEN 74

Introduction .................... 74
Materials and Methods . ............... 74
Results and Discussion ............... 77

IV DEVELOPMENT OF A RADIOIMMUNOASSAY TO BOVINE
PLACENTAL LACTOGEN . . . 96

Introduction . ................... 96
Materials and Methods . ............... 97
Results and Discussion ............... 103










V BOVINE PLACENTAL LACTOGEN CONCENTRATIONS
IN MATERNAL AND FETAL FLUIDS .. . 119

Introduction . . . . . 119
Materials and Methods ... . . . 119
Results and Discussion ... . . . 121

VI GENERAL DISCUSSION . .... . 140

LITERATURE CITED ................... 147

BIOGRAPHICAL SKETCH ............... .. .164















LIST OF TABLES


Table Page

2.1 Lactogenic Activity Produced by Various Tissues. 47

2.2 Analysis of Variance for Cotyledon Culture (Exp. #2). 48

2.3 Duncan's Multiple Range Test for Treatment in Exp. #2. 58

2.4 Analysis of Variance for Culture Exp. #3. 59

2.5 Analysis of Variance for Culture Exp. #4 60

2.6 Production of Lactogenic Activity by Different Mass 62
of Cotyledon Tissue.

2.7 Lactogenic Activity in Response to Arachidonic Acid. 68

2.8 Dose Response of Lactogenic Activity Produced by 70
Arachidonic Acid.

2.9 Lactogenic Activity Produced by Cotyledonary Tissue 70
Cultured in 0, 75, 150 or 300 -tM Arachidonic Acid
for 24 hr.

2.10 Analysis of Variance for Trials Two and Three, Exp. #6. 71

3.1 Purification of Bovine Placental Lactogen. 79

3.2 x nmoles 2-14-C-acetate Incorporated/100 mg Tissue/3 Hr. 94

4.1 Heterogeneity of Regression for Parallelism. 109

4.2 % Recovery of bPL From Maternal and Fetal Fluids. 113

4.3 Analysis of Variance for Assay Comparison Samples. 114

4.4 Determination of bPL or Lactogenic Activity (x SE) 115
in Cotyledon Culture Samples Tested with Estrogen (E)
or Growth Hormone (GH).

5.1 Bovine Placental Lactogen Concentrations in Maternal 122
and Fetal Fluids.

5.2 Estimated Fluid Volumes and Total bPL Concentrations. 126










5.3 Analysis of Variance for Maternal and Fetal Samples. 129

5.4 Analysis of Variance for bPL Concentrations Across 132
Gestation (Exp. #2).

5.5 Analysis of Variance for bPL Concentrations Across 133
Gestation (Exp. #2) Heterogeneity.















LIST OF FIGURES


Figure Page

2.1 Lactogenic activity (ng/ml) produced over time. 50

2.2 Lactogenic activity (ng/ml) produced by various 52
size tissue explants.

2.3 Lactogenic activity (ng/mg tissue) produced by 54
various size tissue explants.

2.4 Lactogenic activity (ng/mg tissue) produced by 56
various treatments.

2.5 Lactogenic activity (ng/ml) produced by various doses 64
of Estrogen or Growth Hormone.

2.6 Lactogenic activity (ng/ml) produced by increasing 67
Growth Hormone concentrations for individual animals.

3.1 Elution profile of lactogenic and somatotropic 81
activities and protein from a Sephacryl S-200 column.

3.2 Elution profile of lactogenic and somatotropic 83
activities and protein from a diethylaminoethyl
cellulose (DEAE) column.

3.3 Elution profile of lactogenic and somatotropic 86
activities and protein from a Chromatofocusing column.

3.4 Elution profile of lactogenic and somatotropic 88
activities of bPL p II from a Sephadex G-75 column.

3.5 Elution profile of lactogenic and somatotropic 90
activities of bPL p III from a Sephadex G-75 column.

3.6 Two dimensional polyacrylamide gels of crude culture 92
media (A) and purified bPL p III (B).

4.1 Lactogenic activity (ng/ml) eluted from native poly- 99
acrylamide gel slices.

4.2 Flurograph of immunoprecipitation of bPL by 50, 100, 106
or 200 jl Florida a bPL or 100, 100 or 200 4l USDA
a bPL.










4.3 Crossreactivity of anti-bPL with various protein 108
hormones.

4.4 Parallelism of 50, 100 or 200 jl of amniotic and 111
allantoic fluid and fetal and maternal serum com-
pared to the standard curve.

4.5 Immunohistochemical localization of bPL in bovine 117
placental tissue.

5.1 Concentrations of bPL (ng/ml) in amniotic and 124
allantoic fluids and maternal venous and fetal um-
bilical arterial and venous blood.

5.2 Linear regression of bPL concentrations in maternal 128
serum, fetal umbilical arterial and venous serum,
allantoic fluid and amniotic fluid from cows at
various gestational ages.

5.3 Least square regression (5th order) of bPL con- 135
centrations of Holstein heifers serviced with either
Holstein (1), Angus (2) or Brahman (3) semen.

5.4 Bovine placental lactogen concentrations from four 138
cows sampled at 30 min. intervals for a period of
12 hr.













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


BOVINE PLACENTAL LACTOGEN: ISOLATION, PURIFICATION
AND MEASUREMENT IN BIOLOGICAL FLUIDS

By

Charles Ralph Wallace

December, 1986

Chairman: Robert J. Collier
Major Department: Animal Science


Studies were conducted to isolate and purify bovine placental lactogen

(bPL) and to develop a radioimmunoassay to this protein. Bovine placental

lactogen was isolated from culture medium after a 24 hr culture of fetal

cotyledonary tissue. Cotyledonary explants were stimulated to secrete

bPL by either addition of bovine growth hormone (NIH-B8) to the medium

or co-culture of cotyledon and caruncular tissue. Production of bPL was

greatly affected by explant size and 70%6 of that produced in a 48 hr culture

was released in the first 12 hr.

Purification of bPL was accomplished using a column chromatographic

scheme that involved gel filtration, ion exchange and chromatofocusing

chromatography. The bPL molecule was purified 600 fold with two forms

at 30,000 MW and pi's of 4.95 and 5.15. The purified protein was utilized

to develop antibodies in rabbits.

A radioimmunoassay to bPL was developed using an antibody raised

at the USDA Beltsville (F56). Approximately 20% specific binding was








achieved with a 1:40,000 final working dilution of the antibody. Assay

sensitivity was 300 ng/ml and the standard curve ranged from .1 8 ng.

The antibody crossreacted with ovine placental lactogen at .2%. Dose

response curves of amniotic or allantoic fluid or fetal and maternal serum

were parallel to the standard curve and bPL was quantitatively recovered

at from 82 125%.

Using the radioimmunoassay, samples of amniotic and allantoic fluids

and fetal and maternal serum were measured for bPL. Concentrations of

bPL ranged from undetectable to 50 ng/ml, with fetal blood having the highest

concentrations and amniotic fluid the lowest. Concentrations of bPL were

measured in plasma samples from 12 cows collected three times a week

from day 150 to 250 of gestation and then daily until term. Peak concen-

trations of bPL were at days 210 and 230 of gestation which may correspond

to peak fetal growth periods. Concentrations of bPL in blood samples

collected at 30 min. intervals for a period of 12 hours were quite similar

both within and between cows, however two of the four animals sampled

exhibited a spike of bPL secretion that was three to four times greater than

baseline.
















CHAPTER I
REVIEW OF LITERATURE

History of Placental Lactogen

Placental lactogen is a protein hormone secreted by the fetal portion

of the placenta in several species (Talamantes, 1975). It was first 'discovered'

and named in 1962 (Josimovich and MacLaren, 1962) in the human. However,

workers had postulated the presence of a placental mammotropin in the

early 1900s. Bouchacourt (1902) suggested that the placenta was responsible

for 'witches-milk' in newborn infants and reported that the placental extract

from a sow, called chorionine, was galactopoietic. Halban (1905) proposed

that mammary gland development in pregnancy was controlled by substances

secreted by the placenta. He based his views on clinical cases of lactation

after ovariectomy or fetal death. At about the same time, Starling (1905)

concluded from experiments in rabbits that fetal extracts contained a

substance that stimulated mammary development. Hammond (1917) concurred

with Starlings findings when removal of the fetuses from rabbits at days

13-15 of gestation arrested mammary growth and was followed by secretion

of milk.

Hypophysectomy became a routine experimental procedure in the

early 1920s. Bell (1917) reported that hypophysectomy in the bitch resulted

in mammary atrophy. This gave rise to the hypothesis that the pituitary

gland was responsible for mammary gland function. Stricker and Grueter

(1928) strengthen this hypothesis by inducing milk secretion in castrated

virgin rabbits with a pituitary extract. To determine the effect of









hypophysectomy on parturition and mammary development Selye et al.

(1933) reported that rats hypophysectomized on days 10-14 of gestation

had normal parturition; however, pregnancy was prolonged. Milk secretion

was also normal at birth but stopped within the first 24 hours. These findings

suggested that a substance separate from the pituitary extract of Stricker

and Grueter (1928) was involved in mammary development. Selye et al.

(1935b) ovariectomized rats at midgestation and removed the fetuses while

leaving the placenta intact. This treatment had no adverse effect on the

mammary glands, which were well developed and contained milk. They

suggested that the placenta may produce a corpus luteum hormone because

the uterus showed distinct progestational changes. Newton and Lits (1938)

reported that ovariectomy on day 12-14 in the mouse also had no effect

on mammary development. Further investigation (Newton and Beck, 1939)

yielded the first suggestion that the placenta may produce a substance other

than an ovarian hormone. Mice were hypophysectomized at day 12 of

gestation and fetuses were removed leaving the placenta intact. Mice that

either aborted the placentas or had the placentas removed showed profound

mammary gland involution. However, the involution was prevented by the

presence of placental tissue. Another interesting result was that mice that

aborted lost 20% of their bodyweight by day 18 while those retaining placental

tissue lost 3%, suggesting that the placenta may act along with the ovaries

to prevent body weight loss in hypophysectomized rats. They hypothesized

that because the mammary gland can attain full development in the absence

of the pituitary, either the mammary gland is sensitive to the same placental

influences as the ovaries or it is susceptible to some different and independent

placental activity. More evidence supporting the hypothesis of a placental

substance was put forth when Lyons (1944) reported that rats








hypophysectomized and ovariectomized on days 7-8 of gestation and given

replacement therapy of either estrone + progesterone or progesterone alone

had normal lobulo-alveolar development. He stated that the placenta secretes

substances comparable to the anterior pituitary which synergize with estrone

and progesterone in their stimulation of lobulo-alveolar mammary growth

and cause lactation. He called this substance the placental mammotropin.

Mayer and Canivenc (1950) demonstrated that rat placental autografts placed

into the abdominal cavity of females had prominent large trophoblastic

cells and were responsible for luteotropic, mammotropic and lactogenic

effects. To further study the different roles of the placenta, Ray et al.

(1955) described a series of experiments that attempted to ascertain the

crop-sac stimulating, luteotropic, mammotropic and lactogenic functions

attributed to the placenta, plus the site of origin of the agent. They reported

that two, day 12, rat placentas (saline suspension) were adequate to stimulate

the pigeon crop-sac, or when injected into normal rats (for 10 days) would

inhibit the estrous cycle and stimulate lobulo-alveolar development. In

the hypophysectomized and ovariectomized virgin rat treatment with estrone,

progesterone and day 12 placenta (either extract or fresh tissue) for 6 or

10 days stimulated lobulo-alveolar mammary development. Placental extracts

were lactogenic when administered in combination with hydrocortisone

acetate, whereas, placental extract alone caused no gross lactation. To

localize the tissue that secreted or stored the mammotropic activity, the

placenta was separated into maternal and fetal regions. A homogenate

of the separate regions was injected into hypophysectomized-ovariectomized

rats treated with estrone and progesterone. All rats injected with placenta,

estrone and progesterone showed lobulo-alveolar development. A significant








result of this experiment was that homogenates from the decidua capsularis

region gave the same stimulating activity as whole placenta homogenates

having 10 times the original mass. In 1958 Lyons reviewed the literature

on mammary development. He concluded that because placental extracts

synergize with estrone and progesterone in the hypophysectomized-ovar-

iectomized rat to induce lobulo-alveolar mammary growth, the rat placenta

must produce a substance that imitates pituitary mammotropin. The evidence

also suggests that a placental somatotropin is present because of the marked

hyperplastic reaction in mammary development. Evidence for a placental

somatotropin was reported by Josimovich and MacLaren (1962). They

described a protein present in human term placenta and pregnancy sera

that reacted with antibodies to human growth hormone. This protein, which

they named human placental lactogen, was highly lactogenic in both the

pigeon crop-sac assay and in promoting milk synthesis in pseudopregnant

rabbit; however it had little growth promoting activity in the rat tibial plate

growth assay. Sciarra et al. (1963) used fluorescein labelled human growth

hormone (hGH) to localize the growth hormone like protein in the syncytial

cytoplasm of the villous trophoblast as early as the 12th week of gestation.

The discovery of a protein produced by the placenta that is immunologically

similar to human growth hormone with prolactin-like effects stimulated

other researchers in this area. Kaplan and Grumbach (1964) reported the

isolation of a protein from both human and simian term placentas that did

have growth hormone and prolactin like activities. They coined the name

chorionic growth hormone prolactin (CGP) and suggested that it was

responsible for metabolic changes in the mother in the last trimester of

pregnancy. They stated that CGP may serve as the metabolic hormone





5


of pregnancy ensuring as one function a maternal store of nitrogen and

minerals and the mobilization of fat to meet the requirements for fetal

growth during pregnancy. The growth hormone-like activity of the placental

protein was confirmed by Josimovich and Atwood (1964) but, they suggested

that human placental lactogen (hPL) potentiated the effect of human growth

hormone in the hypophysectomized rat tibial growth assay. In their assay

it would require four to five times the amount of hGH used alone as it did

when used in conjunction with hPL. Thus the controversy of whether human

placental lactogen had somatotropic activities was solved. Josimovich (1966)

fortified his position by reporting that four different hPL preparations

potentiated the effect of hGH in both the rat tibial growth assay and in

protection against insulin induced hypoglycemia. In that same year a new

name was given to the placental protein. Florini et al. (1966) purified a

placental protein and called it Purified Placental Protein (Human) (PPP[HI).

They agreed with previous reports on lactogenic and somatotropic activities

of their protein. Administration of placental lactogen to hypopituitary dwarfs

gave conflicting results. Grumbach et al. (1966) compared free fatty acid

concentrations in four children given either 400 mg CGP or 4 mg hGH.

In each case free fatty acid concentrations increased over resting values

however, hGH stimulated a larger increase than did CGP. In contrast, Schultz

and Blizzard (1966) reported that in two male patients with idiopathic

hypopituitarism, administration of 200 mg hPL had either no effect or a

negative effect on nitrogen retention. They also found that hPL did not

potentiate the effect of hGH on nitrogen retention. The differences in these

two reports may be explained by different dosages of placental lactogen

and different ages of patients.








The similarities between hGH and hPL in immunological and biological

properties stimulated further research in their chemical similarities. Catt

et al. (1967) demonstrated that hPL has a molecular weight of 18,000 21,000.

They also compared the first 17 amino acid residues from the amino terminus

of hPL and hGH. Eleven of the seventeen residues were identical between

the molecules which suggested that they were similar in chemical structure.

Sherwood (1967) extended the findings of Catt et al. (1967) by using trypsin

digestion of both hPL and hGH. The tryptic peptides generated from hPL

appeared to be identical or very similar to peptides from hGH. This suggested

that pituitary growth hormone and placental lactogen were closely related

with a common ancestor in the course of evolution.

In the literature, four different laboratories purified the same placental

factor and each has called it by a different name (Josimovich and MacLaren,

1962, Kaplan and Grumbach, 1964, Florini et al. 1966, and Friesen, 1965).

In 1968 a group of these researchers met and proposed to name the placental

factor chorionic somatomammotropin based on reported functions and site

of synthesis (Li et al., 1968). In spite of this proposed terminology, to avoid

confusion in the rest of this review, the placental protein will be called

placental lactogen.

Placental lactogens have been demonstrated in the human and monkey

(Kaplan and Grumbach, 1964) and suggested to be present in the rat (Lyons,

1944) and mouse (Newton and Beck, 1939). Gusdon et al. (1970) examined

placental extracts from several species to determine if a protein similar

to hPL was produced. They used a hemagglutination-inhibition assay and

cross-reaction with anti-hPL antibodies to determine the presence of a

hPL-like molecule in the monkey, rat, dog, pig, horse, sheep, rabbit or cow.








The monkey produced a large quantity of the lactogenic substance, which

had been previously reported (Kaplan and Grumbach, 1964), but the other

species produced only small amounts. Monkey placental lactogen was purified

by Shome and Friesen (1971) who demonstrated that it was similar to hGH

and hPL in molecular weight and amino acid composition. However, they

found two forms of the protein and the quantities recovered were lower

than in the purification of hPL. The presence of a goat placental lactogen

was suggested by Buttle et al. (1972). Plasma samples from five goats were

taken throughout gestation and were assayed for prolactin by

radioimmunoassay and for lactogenic activity by a rabbit mammary gland

organ culture method. The difference in lactogenic activity and prolactin

concentration was attributed to a placental lactogen. They demonstrated

a second lactogenic substance between the 9th and 15th weeks of gestation

in the goat and concluded that this lactogenic substance was goat placental

lactogen. In 1973 Shiu et al. developed a radioreceptor assay for prolactin

using the mammary cell membranes from midpregnant rabbits. They

demonstrated that prolactin ovinee, human, monkey and rat), hGH and hPL

inhibited the binding of 1251 human prolactin. They suggested that this

technique could be used to detect lactogenic hormones secreted during

pregnancy. Using this method, Fellows et al. (1974) reported the purification

of ovine placental lactogen. Ovine placental lactogen was similar to hPL

in its ability to bind to prolactin and growth hormone membrane receptors,

but it had higher somatotropic activity (Handwerger et al., 1974). They

suggested that because of the similarities between ovine placental lactogen

and hPL, the sheep may provide an excellent model for the study of placental

lactogen. Talamantes (1975) examined nine species of mammals for the








occurrence of a placental lactogen. He examined placental production of

lactogenic activity using either explants or placental extracts from baboon,

sheep, chinchilla, hampster, rat, mouse, guinea pig, rabbit and dog. Lactogenic

activity in a mouse mammary gland co-culture assay as compared to prolactin

was evident in all animals except the rabbit and dog, but, variation in

production was noted. Robertson and Friesen (1975) reported the purification

of rat placental lactogen. In the purified product they found two major

bands and two minor bands on polyacrylamide gel electrophoresis. This

heterogeneity is similar to that reported for growth hormone (Chrambach

et al., 1973).

The presence of a bovine placental lactogen was demonstrated by

Buttle and Forsyth (1976). They determined plasma lactogenic activity

in seven heifers across gestation using the method of Buttle et al. (1972)

and placental production of lactogenic activity using co-culture of

cotyledonary tissue and mouse mammary gland explants. There was no

lactogenic activity attributed to a placental lactogen in any of the 78 plasma

samples examined. However, the co-culture technique showed that the

cotyledon of the cow placenta from day 36, 180 and 270 of gestation did

produce placental lactogen. They suggested that bovine placental lactogen

may not be present in maternal plasma because of either a low secretion

rate or a rapid clearance rate. Becka et al. (1977) reported the purification

of goat placental lactogen from culture medium after tissue incubation.

Explants of placental tissue were cultured for 4 days with a fresh change

of medium every 24 hours. They demonstrated that placental proteins were

secreted into the medium, but only .1% of the proteins were associated

with lactogenic activity. They suggested that goat placental lactogen was








similar to ovine placental lactogen on the basis of lactogenic activity and

electrophoretic mobility. Bovine placental lactogen was purified (Roy et

al., 1977) and antibodies were raised in rabbits against the purified protein.

In the radioimmunoassay there was no crossreactivity with ovine placental

lactogen, bovine prolactin or growth hormone and hGH. Serum samples

from late pregnancy were assayed using both the radioimmunoassay and

a radioreceptor assay and had concentrations of less than 100 ng/ml. Similar

findings were reported by Hayden and Forsyth (1979).

The assays utilized to detect the presence of placental lactogens have

included the pigeon crop-sac assay (Nicoll, 1967), organ culture assays using

mammary explants from either the mouse or rabbit (Forsyth and Myres,

1971, Turkington, 1971), radioreceptor assays (Shiu et al., 1973) and in a

few cases, the radioimmunoassay (Roy et al., 1977). The problem with these

assays is that they may not be sensitive enough or specific enough to

determine small concentrations of placental lactogen. Tanaka et al. (1980)

developed a bioassay using the Nb2 lymphoma cell to determine the presence

of lactogenic hormones. Lymphoma cell replication was stimulated in a

dose dependent manner by prolactins (human, ovine, bovine and rat) and

placental lactogens (human, bovine and ovine) between 10 pg/ml and 4 ng/ml.

This assay could be used in conjunction with a specific radioimmuhoassay

for prolactin to determine concentrations of placental lactogen in serum

samples.

Beckers et al. (1980) reported in detail the purification of bovine

placental lactogen. They utilized a purification scheme that enriched the

placental lactogen 1,500-fold over the original cotyledonary extract. These

findings were confirmed by Murthy et al. (1982) and Eakle et al. (1982).








These laboratories reported a molecular weight of 32,000 which is appreciably

heavier than any of the other placental lactogens reported. Mouse placental

lactogen was first suggested in 1939 (Newton and Beck, 1939), but the protein

was not purified until 1982 (Colosi et al., 1982). They reported that mouse

placental lactogen had a similar molecular weight to human and ovine

placental lactogens. As more knowledge is attained about the placental

lactogens more complex interrelationships are involved. Robertson et al.

(1982) described the characterization of two forms of rat placental lactogen.

They demonstrated that the two forms differed in molecular weight,

isoelectric point and immunological properties. The forms also differed

in lactogenic and somatotropic activities with the early form being more

somatotropic and the late form being more lactogenic. The hypothesis was

proposed that the early form may correspond to the placental luteotropin

reported by Astwood and Greep (1938) whereas the late form was principally

mammotropic. Servely et al. (1983) reported that high concentrations of

antiprolactin receptor antibodies completely abolished the accumulation

of B-casein mRNA induced by ovine placental lactogen in rabbit mammary

gland explants and in coculture of ewe placenta and mammary tissue. This

indicated that the placenta secreted a lactogenic factor which acted via

the prolactin receptors. Voogt (1984) demonstrated that coincubation of

day 11 rat placenta and rat pituitary gland significantly decreased the

concentration of prolactin in the medium. This suggested that rat placental

lactogen had a direct inhibitory effect on prolactin secretion in vitro. The

prolactin like effect of rat placental lactogen was confirmed by Bussman

and Deis (1984). They reported that Y-glutamyltransferase activity was

maintained in mammary glands of ovariectomized rats treated with CB-154








while ovariectomized-hysterectomized rats lost Y -glutamyltransferase

activity. These findings suggest that rat placental lactogen can replace

rat prolactin in stimulating mammary enzyme activity during pregnancy.

Placental Type

Grosser (1909) classified placentas based on the number of layers of

tissue which, based upon the light microscopy, appeared to separate fetal

from maternal bloodstreams. The epitheliochorial placenta, which is

considered to represent the simplest form, has six layers: 1) endothelium

of fetal capillaries, 2) fetal connective tissue or mesenchyme, 3) fetal

chorionic epithelium, 4) maternal uterine epithelium, 5) maternal connective

tissue and 6) maternal endothelium. It was thought that the number of tissue

layers was directly related to the permeability of the placental barrier

(Steven, 1975). Barcroft (1946) disagreed with this concept stating that

with few exceptions the greater the number of tissue layers the more fully

developed the animal was at birth. Both pigs and horses have epitheliochorial

placenta. Cattle and sheep were classified as having syndesmochorial type

placenta because of the apparent absence, under light microscopic

examination, of the maternal epithelial layer. Recently, sheep and cattle

placenta have been examined using electron microscopy which indicated

that the correct placental type was epitheliochorial (Bjorkman, 1968). Grosser

(1909) classified other placental types as the endotheliochorial having five

layers as in dogs and cats and the hemochorial having four layers as in the

rodents and most primates. Enders (1965) demonstrated using electron

microscopy, that some capillaries of the hemochorial-type placenta are

covered by one or more layers of attenuated chorion. This finding gave

rise to three hemochorial subgroups: 1) hemomonochorial of man, 2)

hemodichorial of the rabbit, and 3) hemotrichorial of the rat and mouse.

The placentas within one classification (Grossner, 1909) may have









widely different functional characteristics (Steven, 1975). Placental shape

may act to group placental types more efficiently (Steven, 1975). For the

diffuse placenta of the mare and pig, the outer surface of the chorion is

covered by small villi or folds which lay in intimate contact with the uterine

epithelium. The majority of ruminants have a cotyledonary placenta where

chorionic villi are restricted to a well defined area of the chorionic sac.

The cow, sheep and goat have this type placenta (Amoroso, 1952). The zonary

placenta of the dog and cat has a equatorial girdle of chorionic villi, which

may be complete as in the dog or incomplete as in the bear (Young, 1968).

A discoid placenta is found in man, rodents and rabbits. In this placental

type the chorionic villi are restricted to a single disc shaped area. Wimsatt

(1962) stated that the trophoblast is probably the most important tissue

in the placenta of higher mammals. The trophoblast arises before

implantation, mediates attachment of the blastocyst, serves as the nutritive

front of the concepts and develops important secretary and regulatory

functions. The trophoblast in the allantoic placenta has three major cytologic

configurations, which may indicate physiologic specialization. The

cytotrophoblast, which may be the most common type, has distinct cellular

boundaries which are in an epithelial arrangement. In the syncytiotrophoblast

cell membranes are lacking and the nuclei are scattered at random or in

clumps throughout the cytoplasm. The third form consists of independent

structures, the trophoblastic giant cells, which can be uninucleate or

binucleate (Wimsatt, 1962).

The ultrastructure of the various placentas may vary even within a

group. The placenta of the pig, which is a diffuse placenta in the

epitheliochorial classification, has a band between maternal epithelium









and trophoblast of mutually interdigitating maternal and fetal microvilli

(Bjorkman, 1965). Over the mouths of the uterine glands the trophoblast

is not attached to the uterine epithelium, but forms regular or irregular

areolae for histotrophic nutrition (Amoroso, 1952). The trophoblast cells

contain numerous mitochondria in the apical portion and globular dense

granules in the basal part (Bjorkman, 1970). The horse placenta is also of

the diffuse type, but tufts of chorionic villi dip into maternal crypts to form

microcotyledons. Areolae, which form between the microcotyledons, are

associated with uterine glands, which are numerous within the endometrial

stroma. The trophoblast is cellular and uninucleate (Bjorkman, 1970).

The placenta of the cow is a cotyledonary placenta with binucleate

giant cells present in the trophoblast. Wimsatt (1951) considered the

binucleate cell to be homologous to the syncytical trophoblast in the deciduate

placenta. Bjorkman (1954) described PAS-positive staining within the

binucleate cells and suggested that a chorionic gonadotropin may be secreted.

Wooding and Wathes (1980) reported that fetal binucleate cells migrated

between the chorionic and uterine epithelia throughout pregnancy. They

suggested that binucleate cell migration may be required to transfer large

molecules across the microvillus junction. Binucleate cell migration was

studied more intensively by Wooding (1983), who reported that 15 to 20%

of the trophectodermal epithelial cells were binucleate. Of these cells,

20% were found to be migrating up to and across the microvillus junction

at all stages of pregnancy examined. The sheep placenta, which is also

cotyledonary, differs from the cow placenta in the shape of the placentome

as well as in the fact that a uterine syncytium is present (Bjorkman, 1970).

The trophoblast of the sheep placenta is similar to that of the cow in that









fetal binucleate cells are present. Wimsatt (1962) suggested that the

binucleate cells fuse to form a functional syncytialtrophoblast in the sheep.

However, other workers (Bjorkman, 1970, Boshier and Holloway, 1977)

determined that the syncytial layer was derived from maternal uterine

epithelium. Recently, Wooding (1980) reported that sheep binucleate cells

migrate across the microvillus junction to form the syncytium. The binucleate

cells of the sheep have been implicated in producing ovine placental lactogen

(Martal et al., 1977). This and the migration pattern of the binucleate cells

(Wooding, 1983) indicated how high concentrations of ovine placental lactogen

are secreted into the maternal system.

The endothelial placenta is characteristic of most carnivores. In the

dog the placenta is zonary or labyrinthine. The labyrinthine type placenta

are those in which the maternal vessels are surrounded by invading trophoblast

so that the vessels come to lie partially or exclusively within the boundaries

of the trophoblast (Steven, 1975). The ultrastructure of the dog placenta

is made up of maternal capillaries that are surrounded first by a dark

syncytium with well developed rough endoplasmic reticulum and numerous

mitochondria and second by a light cytotrophoblast (Bjorkman, 1970).

Anderson (1969) described the presence of decidual giant cells within the

syncytium. He stated that the cells were connective tissue of maternal

origin which had been surrounded by the syncytium in the same manner as

the maternal capillary. The cat placenta is the same type as the dog placenta.

The cat lamellae seem to run at right angles to the uterine surface. As

in the dog maternal capillaries are surrounded by a dark syncytium and light

cytotrophoblast. Decidual giant cells are apparent within the syncytium

(Dempsey and Wislocki, 1956) and are derived from fibroblasts which are









transformed into giant cells when the fetal trophoblast invades the uterine

endometrium.

The hemochorial placental morphology varies tremendously between

species, many of which are entirely unrelated except for the hemochorial

placenta. The hemomonochorial type placenta is seen in the human, some

monkeys and the nine-banded armadillo (Bjorkman, 1970). In the human

placenta the fetal capillary is surrounded by a light cytotrophoblast which

is surrounded by a syncytiotrophoblast layer. The syncytium is in direct

contact with the maternal blood space and has free microvilli for absorbing

nutrients (Bjorkman, 1970). The cytoplasm of the syncytium is electron

dense with free ribosomes and a very well developed rough endoplasmic

reticulum. This may suggest high synthetic activity (Bjorkman, 1970). Pierce

and Midgley (1963) suggested that cytotrophoblast cells were undifferentiated

cell types that matured into syncytiotrophoblastic giant cells. They also

demonstrated that the syncytial giant cell secretes human chorionic

gonadotropin (hCG) using fluorescein labelled antibody against hCG. Sciarra

et al. (1963) reported that human syncytial cytoplasm contained human

placental lactogen. They used flourescein labelled hGH to identify the

immunologically similar protein. The hemodichorial placenta of the rabbit,

also a labyrinthine type placenta, was described by Larsen (1962). He stated

that the trophoblast consisted of a cellular and syncytial component. The

cytotrophoblast contained mononuclear cells with oval nuclei and a light

cytoplasm. The syncytial trophoblast was found as a sheet covering the

cytotrophoblastic cells and surrounding a space containing maternal blood.

Larsen (1962) reported the presence of multinucleated giant cells in the

intermediate zone of the placenta. The intermediate zone was found









separating the syncytium from the maternal tissue. He suggested that the

multinucleated giant cells were fetal in origin and may be responsible for

transfer of nutrients. The hemotrichorial placenta of the rat and mouse

are similar in ultrastructure and labyrinthine in type (Jollie, 1964a, Kirby

and Bradbury, 1965). The placenta consist- of four cytoplasmic layers from

the maternal blood sinus to fetal capillary lumen. These layers are called,

1) trophoblast I, 2) trophoblast II, 3) element III, and 4) endothelium. Large

clusters of trophoblastic giant cells can be seen within the labyrinth

(Bjorkman, 1970). The giant cells lie within the junctional zone which is

adjacent to the decidua basalis (Jollie, 1964a).

In the placental types described previously, a common theme is seen.

With the possible exception of the rabbit, if trophoblastic giant cells are

present, the placenta secretes the hormone placental lactogen. As previously

stated, binucleate giant cells have been implicated in producing placental

lactogen (sheep, Martal et al., 1977, cattle, Wallace, 1985, and human, Sciarra

et al., 1963). Placental lactogen is present in the mouse and rat, but tissue

localization studies have not been reported. The pig and horse have no

trophoblastic giant cells and the dog and cat have giant cells but it is decidual

in origin. A placental lactogen may be present in the rabbit, but, reports

are conflicting.

Endocrine Control of Mammary Development

Mammary growth is a continuous process from embryonic life through

reproductive senescence. The mammary gland is an integral part of the

reproductive process in all mammals. Just as the placenta delivers nutrients

to the developing fetus during intrauterine life, the mammary glands contain

nutrients for the extrauterine survival of the young. The endocrine control









of the mammary gland, to synchronize its development and secretion with

the needs of the offspring, has intrigued researchers for centuries.

Lane-Claypon and Starling (1906) were the first to examine the stimulus

for mammary growth using experimental methods. They administered aqueous

extracts of placenta, fetuses or ovaries to virgin rabbits and concluded that

the fetus contained a product that stimulated mammary growth.

Estrogen. The isolation of estrogen (Allen and Doisy, 1923) was followed

by reports of its stimulation of mammary growth in mice and rats. Allen

et al. (1924) demonstrated that estrogen injection in ovariectomized rats

and mice stimulated mammary gland duct growth. Species variation in

response to estrogen administration is quite marked. Duct development

was stimulated by estrogen in the mouse (Bradbury, 1932), cat (Turner and

DeMoss, 1934), dog (Turner and Gomez, 1934) and rat (Turner and Schultze,

1931). Although in the rabbit (Frazier and Mu, 1935) and guinea pig (Nelson

and Smelser, 1933) estrogen also stimulated lobulo-alveolar development.

Turner and Allen (1933) demonstrated that estrogen administered over a

period of 65 days induced lobulo-alveolar development in male rhesus monkeys.

Progesterone. Turner and Schultze (1931) demonstrated that

administration of progesterone to rats and rabbits had no effect on mammary

development. However, these workers were able to induce lobule formation

by injection of progesterone and estrogen in animals that had been pretreated

with estrogen. The importance of estrogen-progesterone synergism in

lobulo-alveolar mammary development was clearly demonstrated (Turner

and Frank, 1931). They reported that estrogen stimulated ductal development

in castrated male or female rabbits. However, greatly increasing the dosage

had no increased effect. Injection of a crude corpus luteum extract into









estrogen primed rabbits had no stimulatory effect. However, the injection

of estrogen plus the crude corpus luteum extract stimulated lobulo-alveolar

development.

Pituitary Hormones. The interest of mammary physiologists in hormonal

control of mammary development was temporarily diverted to the pituitary

when Stricker and Grueter (1928) induced milk secretion in ovariectomized

virgin rabbits with a pituitary extract.

In the absence of the pituitary, estrogen and progesterone have no

effect on mammary gland development (Selye et al., 1935a). Gomez et

al. (1937) reported that hypophysectomized male guinea pigs treated with

pituitaries from estrogen primed rats had extensive alveolar development.

These findings suggested that estrogens stimulated mammary development

by working through the pituitary gland. Gomez and Turner (1938) proposed

the hypothesis that there were two pituitary factors involved in

mammogenesis; the duct growth factor, which was stimulated by estrogen

secretion and the lobulo-alveolar growth factor, which was stimulated by

estrogen plus progesterone. This hypothesis was supported by the work of

Nathanson et al. (1939) who demonstrated ductal development in

hypophysectomized rats treated with estrogen plus pituitary extracts, which

they called growth complex. Lyons (1943) reported that in the

hypophysectomized-ovariectomized rat, estrogen, progesterone and prolactin

provided the minimal hormonal requirements for stimulating lobulo-alveolar

development. This finding was expanded (Lyons et al., 1953) when full

lobulo-alveolar development was attained in hypophysectomized-ovariecto-

mized rats treated with the above hormones plus purified growth hormone.

The importance of the pituitary hormones was slightly diminished when








Ahren and Jacobsohn (1956) demonstrated that long acting insulin plus estrogen

and progesterone stimulated mammary development in the absence of pituitary

hormones. They suggested that any hormone with powerful metabolic actions

can play a role in mammary gland growth.

Placental Hormones. Placental involvement in mammmogenesis was

first suggested for mice (Newton and Beck, 1939) and rats (Lyons, 1944).

They suggested that the placenta produced a mammotropin that could replace

pituitary prolactin in stimulation of the mammary gland. Ray et al. (1955)

compared the placental factors of the rat and pituitary hormones in their

ability to stimulate mammary development and reported that the action

of rat placental mammotropin was similar to that of pituitary prolactin.

Hypophysectomy of rats had no effect on mammary growth at days 12 and

20 of gestation using the DNA method of quantitation (Anderson and Turner,

1969). Positive significant correlations were obtained between both fetal

number and weight of placentas and mammary development in mice measured

by RNA, DNA or RNA/DNA ratio (Nagasawa and Yanai, 1971). They suggested

that the placental mammotropic hormone may play an important role in

mammary development during pregnancy. Anderson (1975) demonstrated

that increasing the number of fetal placental units in either intact or

hypophysectomized rats increased mammary DNA concentration. Removal

of the fetuses in hypophysectomized rats while leaving 0-5 placentas intact

indicated that as little as two placentas were required to allow mammary

DNA values to reach control values. Schams et al. (1984) examined the

effects of bromocryptine on mammary gland development in ewes and heifers.

Administration of bromocryptine to ewes markedly decreased prolactin

concentrations, but had no effect on serum placental lactogen concentrations.









Mammary glands of bromocryptine treated ewes were similar to controls.

In some lobules the secretion of lipid droplets was diminished. In heifers,

bromocryptine treatment depressed serum prolactin concentrations, but

placental lactogen was not measured. Mammary development in heifers

was similar to that for ewes. These findings suggest that placental lactogen

may be able to replace pituitary prolactin in the stimulation of mammary

development. As stated previously, the hormones involved in mammary

development are estrogens, progesterone and pituitary hormones (prolactin

and growth hormone) or placental lactogen. The placenta of many mammals

secrete steroids as well as protein hormones. Progesterone secretion by

the placenta has been reported for the human (Diczfalusy and Borell, 1961),

sheep (Linzell and Heap, 1968), guinea pig (Heap and Deanesly, 1966), cow

(Erb et al., 1967) and rat (Csapo and Wiest, 1969). In the ovariectomized

guinea pig plasma progesterone was shown to be correlated with placental

weight (Heap and Deanesly, 1966). Linzell and Heap (1968) reported that

sheep placenta secrete up to 14 mg progesterone per day, which is about

five times greater than ovarian secretion. Placental hypertrophy was reported

in ovariectomized rats that maintained pregnancies (Csapo and Wiest, 1969).

Estrogen secretion by the placenta of mammals is even more widespread

than progesterone. There is no known species with a gestation length longer

than 70 days that doesn't have placental estrogen synthesis (Davies and Ryan,

1972). Estrogen synthesis was demonstrated in vitro for the sheep, cow,

horse and sow placenta (Ainsworth and Ryan, 1966). Tissue preparations

were incubated in the presence of androstenedione and dehydroepiandrosterone

for 1 hr at 37 C. Tissue preparations incubated with either pregnenolone

or progesterone failed to synthesize estrogens. These findings supported

the concept of the fetoplacental unit (Diczfalusy, 1964). According to this








concept the fetus and placenta together carry out steroid biosynthetic

pathways which neither could do alone. In the case of estrogen secretion

the placenta secretes progesterone to the fetus which converts it to

androstenedione in the adrenal. The androstenedione then is converted by

the placenta into estrogen.

In Vitro Studies. To remove the effects of possible hormonal inter-

actions within the animal, Lasfargues and Murray (1959) suggested that

the ideal approach would be to study mammary development in a physio-

logically defined environment. They recalled the work of Hardy (1950),

who reported differentiation of mouse mammary ducts in vitro. Organ culture

of explants of 10-15 day old mouse abdominal wall was utilized to determine

the effects of hormonal stimulation of mammary differentiation (Lasfargues

and Murray, 1959). The results indicated that estradiol and progesterone

inhibit growth of the mammary epithelium while growth hormone and prolactin

promote active growth and cortisol induced distension of ducts to form alveoli.

The influence of cortisol on explants from prelactating mice was studied

by Rivera and Bern (1961) who demonstrated that cortisol plus insulin could

maintain alveolar structure, but only in the presence of prolactin or growth

hormone was secretary activity maintained or stimulated. In mice pretreated

with either estrogen and progesterone for 9 days or estrogen, progesterone,

prolactin and growth hormone for 7 days, lobulo-alveolar differentiation

was attained only when explants were cultured in the presence of estrogen,

progesterone, aldosterone, prolactin, growth hormone and insulin (Ichinose

and Nandi, 1966). To determine the steroidal requirements explants from

pretreated mice were cultured in the presence of prolactin, growth hormone

and insulin plus various steroid hormones. Lobulo-alveolar differentiation

was stimulated when aldosterone was in the medium either alone or in








combination with estrogen or progesterone. In fetal mouse mammary tissue

insulin is required for prolactin to stimulate duct growth while aldosterone

and progesterone enhance ductal branching and lobulo-alveolar differentiation

in the presence of insulin and prolactin (Ceriani, 1970). Turkington and

Topper (1966) reported that hPL in combination with insulin and hydrocortisone

stimulated midpregnant mouse mammary tissue to approximate the alveolar

development of full term pregnancy. This combination of hormones also

stimulated casein synthesis, which indicated that hPL may have an important

role in mammary gland development during pregnancy. Recently the effect

of epidermal growth factor on mammary development has been reported.

Tonelli and Sorof (1980) indicated that cultured mouse mammary glands

that had undergone development in the presence of prolactin, insulin,

aldosterone and hydrocortisol and then regression by removing the hormonal

stimulation could be stimulated to develop a second time when epidermal

growth factor was added to the medium along with the above stimulatory

hormones. Epidermal growth factor had no effect on the first cycle of

development. They suggested that endogenous epidermal growth factor

was available in the first cycle. The addition of epidermal growth factor

to cultured mouse mammary epithelial cells stimulated cell proliferation

(Taketani and Oka, 1983). Casein production by mammary epithelial cells

was inhibited by epidermal growth factor. One possible explanation for

the inhibition of casein synthesis was that binding of epidermal growth factor

may block the prolactin receptors and thus inhibit prolactin binding.

The organ culture method has indicated that hormones directly affect

the mammary gland to stimulate differentiation. The presence of a receptor

for these stimulatory hormones has been postulated. Shiu et al. (1973)

identified a specific receptor site for prolactin or lactogenic hormones in









membrane fractions obtained from pregnant or lactating rabbits. Purification

of the prolactin receptor from rabbit mammary glands was reported (Shiu

and Friesen, 1974). Djiane and Durand (1977) described the regulation of

prolactin receptor numbers in the rabbit mammary gland. They demonstrated

that receptor numbers increased in mammary tissue from rabbits treated

with 100 I.U. ovine prolactin, whereas progesterone plus prolactin treatment

had no effect. The self-regulation of prolactin receptors by prolactin was

confirmed in the rat (Bohnet et al., 1977). They demonstrated that a peak

in receptor numbers coincided with the peak in prolactin after parturition.

Injection of either estradiol valerate, 17 hydroxyprogesterone caproate or

bromocryptine reduced prolactin receptor numbers compared to controls.

Injection of anti-prolactin receptor serum into lactating rats resulted in

increased serum prolactin and decreased litter weight gains (Bohnet et al.,

1978). They suggested that the antiserum may inhibit some of the effects

of endogenous prolactin. Haslam and Shyamala (1979) reported that

progesterone receptor numbers in mice were inversely proportional to the

secretary activity of the gland.

Secretion

Placental lactogens are secreted by the trophoblastic portion of the

placenta (Sciarra et al., 1963, Martal et al., 1977). However, the regulation

of that secretion still is unclear. To determine the regulatory mechanisms

involved in secretion of placental lactogens, researchers have developed

two methods. The first was to manipulate the whole animal (or human)

by increasing or decreasing metabolites thought to be important in placental

lactogen secretion, and second to culture placental tissue (explants, dispersed

cells or whole placenta) in presence or absence of hormones or metabolites.









Changes in Concentrations. Radioimmunoassays were first developed

for hPL (Kaplan and Grumbach, 1965, Beck et al., 1965) to determine the

pattern of secretion. Placental lactogen concentrations of .534g/ml in

pregnant women were detectable at 8 weeks of gestation and increased

to 10-40 ug/ml at term (Kaplan and Grumbach, 1965). Human placental

lactogen concentrations fell drastically after parturition. The presence

of hPL in the placenta was detectable at day 12 (Beck, 1970). At term,

concentrations of hPL in umbilical plasma were reported as undetectable

(Beck et al., 1965) or 50 to 200 times lower than maternal concentrations

(Kaplan and Grumbach, 1965) and amniotic fluid hPL concentrations ranged

from 2-11 ig/ml. The estimated halflife of hPL was 21-23 min. Taking

into account maternal concentrations and the reported halflife, a production

rate of 3-12g hPL per day was proposed (Beck et al., 1965). Infusions of

hPL into men or nonpregnant women at 93-373 jg/min resulted in plasma

hPL concentrations of 2-3 jg/ml after 90 min (Beck and Daughaday, 1967).

Spellacy et al. (1966) stated that hPL concentrations were not related to

placental or fetal weight. However, Sciarra et al. (1968) reported a low

positive correlation between hPL concentrations and placental weight in

patients between 38 and 42 weeks of gestation. Concentrations of hPL vary

randomly over a 24 hr period (Pavlou et al., 1972) with no apparent diurnal

rhythm. Vigneri et al. (1975) suggested that hPL concentrations fluctuate

irregularly, therefore, a frequent sampling procedure is required to correctly

determine the secretary activity of hPL.

Ovine placental lactogen, as measured by radioimmunoassay (Handwerger

et al., 1977, Chan et al., 1978b) was first detectable at day 40 of gestation

and reached a peak of 2,400 ng/ml at day 130. Concentrations of ovine

placental lactogen in umbilical cord plasma and allantoic fluid were









approximately one-tenth and one-hundredth of maternal concentrations,

respectively (Handwerger et al., 1977). While amniotic fluid had

concentrations of 5-90 ng/ml of ovine placental lactogen at day 50, levels

were undetectable thereafter (Chan et al., 1978b). Chronic catheterization

of animals is an ideal method for examining the secretion of ovine placental

lactogen (Gluckman et al., 1979, Taylor et al., 1980). Fetal concentrations

of ovine placental lactogen peaked at days 120-124 while maternal

concentrations peaked at days 130-139 and were 10 times that for the fetus

(Taylor et al., 1980). Fetal concentrations were unaffected by fetal numbers;

however, maternal concentrations increased significantly with increasing

fetal numbers (Taylor et al., 1980).

Kelly et al. (1976) reported lactogenic and somatotropic activities

in serum or plasma samples throughout pregnancy of nine species. Two

peaks of lactogenic activity were detected in mice and rats, one at

midgestation and one near term. Peaks of somatotropic activities were

coincident with lactogenic peaks; however, the first peak was much lower

than the lactogenic peak and the second much higher. Peaks of lactogenic

and somatotropic activities were similar in the hampster, guinea pig, goat,

sheep, monkey and human. Somatotropic activities were much lower in

the hampster, guinea pig, sheep and human and higher in the goat. Activities

were low throughout gestation in the cow. Concentrations of placental

lactogen in the goat increases with fetal number (Hayden et al., 1979) and

plateaued from 16 weeks of gestation until term. Bovine placental lactogen

concentrations were measured using the Nb2 lymphoma assay (Schellenberg

and Friesen, 1982). Placental lactogen concentrations in maternal plasma

were below the sensitivity of the assay while fetal samples from day 180









of gestation revealed concentrations between 5 and 22 ng/ml. They suggested

that bovine placental lactogen acts to stimulate fetal growth.

The presence of placental lactogen in rodents has been postulated

since Newton and Beck (1939); but not until recently has a mouse or rat

placental lactogen been purified and a radioimmunoassay developed.

Robertson and Friesen (1981) compared rat placental lactogen concentrations

measured in either a radioreceptor assay or radioimmunoassay. The

radioreceptor assay detected two similar peaks of 1,000 ng/ml at days 11-13

and 13-21 whereas the radioimmunoassay detected a peak at day 11-13 which

was minor and a major peak of 1,000 ng/ml at days 13-21. Plasma samples

collected at 5-10 min intervals from rats at day 19 of gestation were used

to demonstrate that rat placental lactogen concentrations vary greatly over

short periods of time (Klindt et al. 1982). A radioimmunoassay for mouse

placental lactogen (Soares et al., 1982) was used to measure placental lactogen

concentrations. Using both a radioreceptor assay and radioimmunoassay,

concentrations of mouse placental lactogen ranged from 1 ng/ml at day

9 to greater than 250 ng/ml at day 18 and were similar as measured by the

two assays. However, the radioreceptor assay detected a large peak at

day 10 while the radioimmunoassay did not. Profiles of mouse placental

lactogen were different between different strains of mice. Markoff and

Talamantes (1981) indicated that mouse placental lactogen concentrations

increased with fetal numbers.

In summary, concentrations of placental lactogen in the maternal

system vary greatly between species. Concentrations of hPL peaked at

10-40 jg/ml (Handwerger et al., 1977), while bovine placental lactogen

concentrations were below the sensitivity of the assay utilized (Schellenberg

and Friesen, 1982). The pattern of placental lactogen secretion was similar








among the species reported, with placental lactogen being first detected

at low concentrations and increasing almost linearly to just before term.

Concentrations of placental lactogen in the fetal circulation, as well as,

amniotic and allantoic fluids, were lower than those for maternal blood.

The only apparent regulatory mechanism was the association between

increasing placental mass or fetal numbers and placental lactogen

concentrations in maternal blood.

Nutritional Effects on Secretion. Reports on the effects of alteration

of hormonal or metabolic factors on hPL concentrations have been conflicting.

Kaplan and Grumbach (1964) suggested that human placental lactogen may

serve as the metabolic hormone of pregnancy. Possible functions included

increased nitrogen retention, increasing free fatty acids, increasing circulating

insulin, resistance to exogenous insulin, increased transfer of amino acids

across the placenta and fetal growth. Spellacy et al. (1966) demonstrated

that hPL concentrations were unaffected by hyper- or hypoglycemia or time

of day. Subsequent reports indicated that glucose administration to pregnant

women after a 15 hr fast significantly reduced hPL concentrations during

the first 30 min (Burt et al., 1970), while fasting pregnant women for 84

hr to induce hypoglycemia significantly elevated hPL concentrations (Kim

and Felig, 1971). Prieto et al. (1976) reported that hPL concentrations were

not affected by glucose administered either orally or as a continuous infusion.

They did state that an acute pulse of glucose transiently suppressed hPL

concentrations. Variations in goat placental lactogen concentrations were

not correlated with changes in blood glucose (Hayden et al., 1980), while

culture of human placental tissue in the presence of 2x glucose inhibited

hPL release (Belleville et al., 1979). Plasma hPL concentrations were

unaffected by free fatty acid concentrations (Gaspard et al., 1975) which







is similar to results in the goat (Hayden et al., 1980). Arginine infusion

had no effect on hPL concentrations (Tyson et al., 1969) while there was

a dramatic increase in ovine placental lactogen concentrations 2-3 hr after

the start of infusion of arginine (Handwerger et al., 1978). Culture of bovine

placental tissue in the presence of arginine stimulated the release of bovine

placental lactogen (Forsyth and Hayden, 1980).

Infusion of either alanine or glycine slightly increased ovine placental

lactogen concentrations while glutamic acid had no effect (Handwerger

et al., 1978). Subsequent reports demonstrated that infusion of ornithine

stimulated ovine placental lactogen secretion while citrulline had no effect

(Handwerger et al., 1981c). In summary, nutritional metabolites such as

glucose or free fatty acids had either a transient or non-existant effect

on maternal placental lactogen concentrations in the species studied.

Manipulation of the glucose concentration in medium used for culture of

human placental tissue demonstrated that glucose was required for hPL

secretion, but increasing or decreasing the minimum glucose concentration

inhibited hPL release (Belleville et al., 1979). Hypoglycemia induced by

fasting pregnant women stimulated hPL concentrations, but the mechanism

is not clear. Infusion of amino acids into sheep and possibly cows may affect

the release of placental lactogens, but again the mechanism was not studied.

Placental lactogens may be the metabolic hormone of pregnancy as Kaplan

and Grumbach (1964) theorized; however, extensive and well designed research

will be required to determine the exact function.

Hormonal Effects on Secretion. Culture of placental tissue to determine

the regulatory mechanisms involved in secretion of placental lactogen was

first reported for the human (Suwa and Friesen, 1969). They cultured explants

from human term placentas in the presence of 3H-leucine. Eighty percent








of the hPL released into the medium was released in the first 24 hr. The

addition of hPL, progesterone, insulin, cortisol or dibutyryl cyclic AMP had

no effect on the release of hPL. Placental explants cultured for 96 hr

demonstrated that during the first 72 hr hPL constituted approximately

10% of the proteins secreted into the medium; however, between 72 and

96 hr there was a dramatic increase in secretion of hPL so that it constituted

approximately 50% of the proteins secreted into the medium (Friesen et

al., 1969). Stimulation of hPL release in culture in response to addition

of pimozide (Macaron et al., 1978), estradiol (Belleville et al., 1978),

arachidonic acid (Handwerger et al., 1981a), EDTA, EGTA or

methoxyverapamil (Handwerger et al., 1981) and insulin (Perlman et al.,

1978) has been reported. Addition of adrenalin, noradrenalin or progesterone

(Belleville et al., 1978), as well as, dopamine (Macaron et al., 1978) inhibited

hPL release and prostaglandins E2 or F2a (Belleville et al., 1978) or

somatostatin (Macaron et al., 1978) had no effect on hPL secretion in vitro.

Cyclohexamide and dopamine had no effect on bovine placental explants

in secretion of bovine placental lactogen (Forsyth and Hayden, 1980). Macaron

et al. (1978) suggested that hPL release may be modulated by dopaminergic

receptors while Handwprger et al. (1981b) suggested that calcium flux may

mediate hPL release. Administration of either prostaglandin E2 or F2a

(Keller et al., 1972) or thyroid releasing hormone (Hershman et al., 1973)

to pregnant women had no effect on hPL concentrations. Ylikorkala and

Pennanen (1973) reported that induction of abortion with PGF2a by either

intra or extra amniotic routes decreased hPL secretion. The decrease was

greatest in patients given PGF2a by the extra amniotic route while saline

had no effect on hPL concentrations. Manipulation of progesterone

concentrations in pregnant sheep had no effect on ovine placental lactogen









concentrations (Taylor et al., 1982). However, Moore et al. (1984) recently

demonstrated that infusion of epidermal growth factor for 24-28 hr

significantly increased ovine placental lactogen concentrations in pregnant

sheep. Thorburn et al. (1981) suggested that epidermal growth factor may

affect transplacental migration of binucleate cells which stimulates the

secretion of ovine placental lactogen. A second theory is that epidermal

growth factor stimulates the production of binucleate cells from mononucleate

cells to increase the number of migrating binucleate cells.

In summary, hormonal manipulation either in vivo or in vitro has been

shown to affect placental lactogen secretion, but the mechanism of action

was not identified. The proposed hypotheses of either dopaminergic receptors

(Macaron et al., 1978) or calcium flux (Handwerger et al., 1981b) mediating

hPL release gave an indication of the complexity of the regulation of placental

lactogen secretion. Placental lactogen secretion does not appear to be

autonomous as suggested for the human (Spellacy et al., 1966). In vivo and

in vitro studies have demonstrated that manipulation of hormonal or metabolic

factors can effect the secretion rate of placental lactogen. Fasted pregnant

women had elevated hPL concentrations (Kim and Felig, 1971); however,

the direct cause of the increase was not addressed. The need for elucidation

of the mechanism involved in regulation of placental lactogen remains.

Function

Since the discovery of human placental lactogen in 1962 (Josimovich

and MacLaren, 1962) the search for the role of this placental peptide has

continued. The exact function of placental lactogen remains unclear; however,

the original hypothesis of Kaplan and Grumbach (1964) still stimulates further

research. Potential activities of placental lactogen that have received major

attention are: 1) lactogenic activity, 2) somatotropic activity, 3) luteotropic

activity, and 4) miscellaneous function.








Lactogenic Activity. Josimovich and MacLaren (1962) reported that

hPL was lactogenic in the pigeon crop sac and rabbit intraductal assay and

was 50% as potent as NIH ovine prolactin. This lactogenic activity was

responsible for the name placental lactogen. Lactogenic activity was

confirmed for hPL (Kaplan and Grumbach, 1964) and demonstrated for ovine

placental lactogen (Handwerger et al., 1974). Talamantes (1975) exploited

the lactogenic activity of placental lactogen to determine the presence

of this hormone in nine different species. Mouse mammary tissue cultured

in the presence of placental extracts demonstrated lactogenic activity in

the baboon, sheep, chinchilla, hampster, rat, mouse and guinea pig, but not

the rabbit and dog. Forsyth (1974) used a mammary and placental co-culture

technique to demonstrate the presence of placental lactogen in the goat,

cow, sheep and fallow deer, but not the pig. Shiu et al. (1973) developed

a radioreceptor assay utilizing the prolactin receptor from rabbit mammary

gland membranes. This technique has been utilized to expand the knowledge

of placental lactogens in several species. Reddy and Watkins (1975) injected

1251 hPL into rats to determine tissue distribution. Labelled hPL was found

mainly in the kidney and mammary gland. Immunohistochemical localization

indicated that hPL bound to the proximal tubule of the kidney and the alveolar

cell membrane of the mammary gland.

Turkington (1968) demonstrated that addition of hPL to mouse mammary

explants in the presence of insulin induced production of casein, a-lactalbumin

and B-lactoglobulin. The addition of colchicine or actinomycin D to the

culture media demonstrated that hPL stimulated differentiated cells formed

in vitro through a DNA directed RNA synthesis. Mammary development

in hypophysectomized pregnant animals has suggested a role for placental

lactogen in the mouse (Selye et al., 1933), rat (Lyons, 1944), guinea pig








(Pencharz and Lyons, 1934), ewe (Denamur and Martinet, 1961), rhesus monkey

(Agate, 1952), goat (Buttle et al., 1978) and woman (Kaplan, 1961). To further

implicate placental lactogen in mammary development, ovine placental

lactogen has been evaluated for lactogenic activity using both rabbit and

ewe mammary explants and membrane (Servely et al., 1983). Ovine placental

lactogen inhibited binding of 125I human growth hormone to rabbit mammary

membranes, but was only slightly inhibitory in ewe mammary membranes.

In mammary explant cultures, ovine placental lactogen stimulated S-casein

synthesis in rabbit mammary tissue but this effect was inconsistent for ewe

mammary tissue. Co-culture of ovine placenta and mammary tissue resulted

in increased B-casein mRNA accumulation. Analysis of the culture media

by radioreceptor assay indicated ovine placental lactogen concentrations

of 70 ug/ml. This suggested that ovine placental lactogen was lactogenic

in the ewe, but was required at high concentrations. Treatment of pregnant

ewes and heifers with bromocryptine indicated that a substance other than

prolactin was present that could stimulate mammary development and

lactation (Schams et al., 1984). Chomczynski and Topper (1974) demonstrated

that hPL stimulated RNA synthesis by isolated rat and mouse mammary

epithelial nuclei. They suggested that hPL and prolactin nray act by binding

directly to the nucleus.

Somatotropic Activity. Placental lactogen was originally identified

because of the cross reaction with antibodies to hGH (Josimovich and

MacLaren, 1962), but final purification steps removed the somatotropic

activity of the protein. Kaplan and Grumbach (1964) reported that their

preparation of human placental lactogen stimulated radioactive sulfate

uptake by hypophysectomized rat tibia. Another growth hormone like activity

was reported when hPL was demonstrated to increase weight gain in hypophy-








sectomized rats (Friesen, 1964) and stimulate 3H thymidine incorporation

into DNA of cartilage from hypophysectomized rats (Breuer, 1969). The

similarity between hPL and hGH was further fortified when Niall et al. (1971)

reported that comparison of the amino acid sequence of hPL and hGH showed

a homology of over 80%. The hypothesis that hPL was an important hormone

of pregnancy that regulated the metabolism of pregnant woman stimulated

research in that area.

The growth hormone-like activity of hPL was demonstrated when hPL

was administered to hypopituitary dwarfs (Grumbach et al., 1966). Plasma

free fatty acids were increased after hPL, but the magnitude of the increase

was less than that after hGH. Turtle et al. (1966) reported that hPL

stimulated lipolysis in rat epididymal fat cells in vitro. They concluded

that hPL 1) stimulates lipolysis through a DNA-RNA mediated process, 2)

potentiated the lipolytic effect of physiological levels of growth hormone,

and 3) may account for the progressive rise in plasma free fatty acids during

pregnancy. The lipolytic action of hPL was confirmed when women that

were fasted for up to 72 hr had increased plasma hPL and free fatty acid

concentrations (Tyson et al., 1971). The relationship between hPL and free

fatty acid concentrations was disputed when altered free fatty acid

concentrations were found to have no effect on circulating hPL concentrations

(Gaspard et al., 1977). Handwerger et al. (1976) demonstrated that adminis-

tration of ovine placental lactogen decreased free fatty acids, glucose and

amino nitrogen, but increased insulin.

The insulin like activity of placental lactogen had been previously

reported. The administration of 400 mg of hPL per day to two hypopituitary

dwarfs caused increased nitrogen and potassium retention, increased insulin

response to glucose and decreased rate of glucose disappearance from the









plasma (Grumbach et al., 1968). Malaisse et al. (1969) demonstrated that

treatment of hypophysectomized rats with hPL caused a reduction in plasma

sugar, but increased both content and output of insulin by pancreatic tissue

in vitro. Insulin induced hypoglycemia stimulated an increase in hPL

concentrations while glucose loading suppressed hPL concentrations transiently

in pregnant women (Gaspard et al., 1974). Brinsmead et al. (1981) conducted

a series of experiments to determine the effects of hyper- or hypoglycemia

or fasting on maternal and fetal ovine placental lactogen concentrations.

Insulin induced hypoglycemia in either the ewe or fetus had no effect on

fetal ovine placental lactogen concentrations while maternal concentrations

of ovine placental lactogen decreased after 120 min, which is directly opposite

the response of hPL. Infusion of glucose to the fetus had no effect on ovine

placental lactogen concentrations in either the ewe or fetus, while fasting

the ewe for 72 hr increased ovine placental lactogen concentrations in both

the ewe and fetus. Ovine placental lactogen stimulated 14C glucose

incorporation into glycogen in fetal rat hepatocytes (Freemark and

Handwerger, 1984) and the action was potentiated by insulin. The two

hormones acted synergistically to promote liver glycogen synthesis. They

observed that ovine placental lactogen was more potent than ovine growth

hormone, suggesting that ovine placental lactogen may have metabolic

functions in the fetus that are subsequently controlled by growth hormone

in the postnatal period. Chan et al. (1978a) reported specific binding of

1251 ovine placental lactogen to nonpregnant ewe liver, adipose tissue, ovary,

corpus luteum, uterus and fetal liver. The binding was inhibited by ovine

growth hormone, but not by ovine prolactin suggesting that ovine placental

lactogen may act more like growth hormone than prolactin.






35

Several reports have indicated that ovine placental lactogen may be

important in fetal growth. Hurley et al. (1977a) demonstrated that admin-

istration of ovine placental lactogen to hypophysectomized rats stimulated

release of somatomedin. Subsequently, Adams et al. (1983) reported that

ovine placental lactogen stimulated insulin-like growth factor II (IGF II)

production by fetal rat fibroblasts while in adult rat fibroblasts either hGH

or ovine placental lactogen stimulated production of IGF I. Ovine placental

lactogen and growth hormone stimulated ornithine decarboxylase activity

in neonatal rat liver (Butler et al., 1978) while only ovine placental lactogen

stimulated ornithine decarboxylase activity in fetal rat liver (Hurley et

al., 1980). To add further support that ovine placental lactogen replaces

growth hormone in the fetus, Freemark and Handwerger (1982) reported

that ovine placental lactogen stimulated alpha amino isobutyric acid transport

into weanling rat diaphragm cells with equal potency to ovine growth hormone.

A year later the same researchers indicated that in fetal rat diaphragm

cells ovine placental lactogen stimulates alpha amino isobutyric acid transport

while ovine growth hormone was without effect (Freemark and Handwerger,

1983).

Luteotropic Activity. Ray et al. (1955) implicated placental lactogen

as a luteotropic substance when they reported that injection of two day

12 rat placentas inhibited the estrous cycle in normal rats. Human placental

lactogen maintained an induced decidual reaction in hypophysectomized

pseudopregnant rats (Josimovich et al., 1963). This action was abolished

when hPL was preincubated with antibodies to hGH. Josimovich and Atwood

(1964) proposed the hypothesis that hPL synergized with human chorionic

gonadotorpin to stimulate the corpus luteum of pregnancy and this was








confirmed when human chorionic gonadotropin and hPL maintained a decidual

reaction for the normal duration (Josimovich, 1968).

Additional reports have not only supported the luteotropic activity

of hPL but also raised questions to the function of hPL in fetal development.

El Tomi et al. (1971) reported that immunization of rabbits with hPL caused

either total or partial fetal resorption during pregnancy. The ovaries and

uterus of hPL immunized rabbits were significantly smaller than controls.

In rats injected with antibodies to hPL implantation was normal, but no

births occurred (Gusdon, 1972). The immunized rats resumed normal estrous

cycles and were rebred but over a period of 11 months none of the rats

delivered a litter. Monkeys immunized with either hPL or placental extracts

had decreased fertility (Gusdon and Witherow, 1976). However, the titer

raised against hPL did not seem to be related to whether the monkeys became

pregnant.

In contrast to reports supporting the luteotropic activity of placental

lactogen, Martal and Djiane (1977) stated that ovine placental lactogen

infused into the uterus of a ewe on day 12 of the estrous cycle did not extend

the lifespan of the corpus luteum. This may indicate that placental lactogens

are luteotropic in species that rely on prolactin for luteal maintenance.

Miscellaneous Function. Spellacy et al. (1971) described the use of

hPL concentrations as a placental function test. They examined hPL

concentrations across gestation in approximately 1400 pregnancies. After

examining the data they described a fetal distress zone, where after 30

weeks of gestation hPL concentrations were below 4 ~g/ml. Of patients

with hPL concentrations within the fetal distress zone, 2406 of the infants

died. Subsequent reports have suggested that hPL concentrations were not

the best indicator of fetal distress. Nielsen et al. (1981) stated that








monitoring hPL concentrations was not recommended as a routine procedure

in all pregnancies, but may be beneficial in some complicated pregnancies.

In 1964, Kaplan and Grumbach suggested that human placental lactogen

(hPL) may regulate metabolic functions in the maternal system. They

implicated hPL in increasing: free fatty acid concentrations, resistance

to exogenous insulin, levels of circulating insulin, lean body mass and changes

in body fluid compartments during pregnancy. They also suggested that

hPL stimulates transfer of amino acids across the placenta which would

affect fetal metabolism and growth. Few of these proposed functions have

been confirmed. Freemark and Handwerger (1982) demonstrated that ovine

placental lactogen stimulated alpha amino isobutyric acid transport into

fetal rat diaphragm cells. However, further evidence for a role of placental

lactogens is lacking.

Nutrient Partitioning

The control mechanism involved in nutrient partitioning are complex

and incompletely understood at present. The partitioning of nutrients to

various body tissues involves two types of regulation, homeostasis and

homeorhesis (Bauman and Currie, 1980). Homeostasis is involved in

maintaining a physiological equilibrium in the animal such as body

temperature, while homeorhesis is involved with coordinated changes in

metabolism of body tissues necessary to support a physiological state such

as lactation or pregnancy.

Total nutrient requirements throughout pregnancy are about 75% greater

than for a nonpregnant animal of the same weight (Moe and Tyrrell, 1972).

The efficiency of utilization of metabolizable energy during pregnancy in

sheep and cattle was estimated as 16.1% (Rattray et al., 1974) and 25%,

(Moe and Tyrrell, 1972) respectively. However, this efficiency may increase

if a maintenance requirement for the fetus is taken into account (Rattray

et al., 1974).









Fetal metabolism has recently been reviewed by Jones and Rolph (1985).

They stated that the fetus utilizes glucose, lactate, amino acids, acetate,

glycerol and fatty acids for the energy required for growth. The fetus receives

glucose from the maternal circulation via the placenta (Battaglia and Meschia,

1978). Hay et al. (1983) reported partitioning of glucose in the pregnant

ewe during both normal and hypoglycemic states. In this experiment, the

fetus consumed 10% of the available glucose in both conditions. Assessing

fetal glucose metabolism is difficult because fetal concentrations can be

influenced by fetal hepatic or placental glycogenolysis (Jones et al., 1983).

Lactate is supplied to the fetus by the placenta (Burd et al., 1972) and is

used either directly for energy metabolism by various fetal organs or in

gluconeogenesis.

Up to 50 percent of all fatty acids required by the fetus have been

estimated to be supplied from placental transfer (Alling et al., 1972) and

maternal diet markedly affects fetal lipid composition (Thomas and Lowry,

1984).

The fetus has a high requirement for nitrogen, which is met by reincorpo-

ration of amino acids produced by protein degredation (Lewis et al., 1984).

Lemons et al. (1976) measured the venoarterial concentration differences

of 22 amino acids across the umbilical circulation of the fetal lamb and

stated that neutral and basic amino acids are transported from the maternal

to fetal system while acidic amino acids are not. In fact, glutamic acid

is delivered from the fetus to the placenta in large amounts.

Fetal oxidative metabolism was reviewed by Battaglia and Meschia

(1978). Oxygen consumption by fetal sheep, goats and cattle ranged from

7 to 9 ml/min/kg fetal body weight. In the fetal lamb oxidative metabolism

consumes primarily carbohydrates and amino acids, therefore, approximately








56 kcal/day is consumed. Comparable figures for cattle in late pregnancy

was estimated at 2.3 Meal/day while about 1 Meal/day is accumulated in

the fetus (Bauman and Currie, 1980). The metabolic cost of maintaining

the fetus is high.

Purification

Placental lactogens have been isolated and purified in the human

(Josimovich and MacLaren, 1962), monkey (Shome and Friesen, 1971), sheep

(Martal and Djiane, 1975), rat (Robertson and Friesen, 1975), goat (Becka

et al., 1977), cow (Beckers et al., 1980) and mouse (Colosi et al., 1982).

The purification procedures reported for the various species are quite similar.

In all the above reports, except in the goat (Becka et al., 1977) placental

tissue was extracted and the placental lactogen was precipitated with

ammonium sulfate. Column chromatography by gel filtration and ion exchange

was used in all cases. Preparative isoelectric-focusing was an additional

step in purification of goat (Becka et al., 1977) and rat (Robertson and Friesen,

1975) placental lactogens. Purification of bovine placental lactogen required

a more rigorous purification scheme. Gel filtration and ion exchange

chromatography was enhanced by hydroxyapatite (Murthy et al., 1982, Eakle

Et al., 1982) and chromatofocusing (Eakle et al., 1982) columns. The

hydroxyapatite column separates proteins by hydrophobic interactions while

the chromatofocusing column separates proteins on the basis of their

isoelectric points. Affinity chromatography was introduced (Beckers et al.,

1980) to remove bovine serum albumin from the placental lactogen

preparation. Originally column fractions were monitored for lactogenic

or somatotropic activities using the pigeon crop sac or rabbit intraductal

assays for lactogenic acitivy (Josimovich and MacLaren, 1962) or rat tibial

growth assay for somatotropic activity (Josimovich and MacLaren, 1962,







Shome and Friesen, 1971). However, after the report of Shiu et al. (1973)

researchers used the radioreceptor assay to detect the presence of lactogenic

or somatotropic proteins. The membranes utilized in the radioreceptor

assays were either rabbit mammary gland (Robertson and Friesen, 1975)

or rabbit (Arima and Bremel, 1983) or rat liver (Murthy et al., 1982) for

lactogenic activity and rabbit liver membrane for somatotropic activity

(Robertson and Friesen, 1975).

The reported purification procedures yielded from 2% (Murthy et al.,

1982) to 29% (Colosi et al., 1982) of the original somatotropic or lactogenic

activity in the purified form. The molecular weights of the various placental

lactogens were estimated at 22,000 for the human (Friesen, 1965), goat

(Becka et al., 1977), mouse (Colosi et al., 1982), rat (Robertson and Friesen,

1975), monkey (Shome and Friesen, 1971) and sheep (Hurley et al., 1977b,

Martal and Djiane, 1975) and approximately 30,000 for the cow (Beckers

et al., 1980, Murthy et al., 1982, Eakle et al., 1982, Arima and Bremel, 1983).

The isoelectric point of placental lactogen ranged from 5.5 in the cow (Murthy

et al., 1982, Arima and Bremel, 1983), 6.0 in the rat (Robertson and Friesen,

1975), 6.8 or 7.2 in the sheep (Hurley et al., 1977, Martal and Djiane, 1975,

respectively), 7.1 in the mouse (Colosi et al., 1982), to 8.8 in the goat (Becka

et al., 1977).

Multiple forms of placental lactogen have been demonstrated in the

human (Suwa and Friesen, 1969), rat (Robertson et al., 1982), mouse (Soares

et al., 1982), monkey (Shome and Friesen, 1971), and cow (Arima and Bremel,

1983). Suwa and Friesen (1969) reported that two peaks of hPL were detected

after gel filtration. The molecular weight of the proteins was 100,000 and

20,000, respectively. They suggested that the large molecular weight protein

was an aggregate of hPL while the smaller molecular weight protein was

native hPL. In the mouse, two forms of placental lactogen are secreted








at different times of gestation (Soares et al., 1982). Two peaks of lactogenic

activity were present at days 10 and 18 in the mouse, but only the second

peak crossreacted with a specific antibody to mouse placental lactogen.

Similar findings were reported for the rat (Robertson et al., 1982). Two

peaks of lactogenic activity were detected in a radioreceptor assay at days

11 13 and 12 21. The peak at days 11 13 was not detected using a specific

radioimmunoassay. The two forms of rat placental lactogen had molecular

weights of 40,000 and 20,000, respectively with isoelectric points of 4.5

and 6.2. They suggested that the early form (days 11 13) of rat placental

lactogen may be responsible for the luteotropic activity reported by Astwood

and Greep (1938) and the late form (day 12 21) was primarily mammotropic.

Monkey placental lactogen had two forms with similar molecular weights,

but different electrophoretic mobilities (Shome and Friesen, 1971). They

suggested that deamidation may be responsible for this difference. Arima

and Bremel (1983) reported three forms of bovine placental lactogen with

similar molecular weights, but isoelectric points of 5.85, 5.52 and 5.39.

They suggested that genetic variability may be responsible for the different

forms.

In conclusion, placental lactogen has been purified for a number of

different species. The purification procedure utilized was similar between

species, but the resulting protein was quite different. The molecular weight

of placental lactogen was reported as 22,000 for each species except the

cow (30,000). Multiple forms of the molecule were reported.
















CHAPTER II
COTYLEDON CULTURE EXPERIMENTS


Introduction

Placental lactogen production by placental tissue in culture was first

reported in the human by Suwa and Friesen (1969). They indicated that

80% of the human placental lactogen (hPL) released into the medium was

secreted in the first 24 hr. The release of hPL may be stimulated by the

addition of pimozide (Macaron et al., 1978), estradiol (Belleville, et al.,

1978), arachidonic acid (Handwerger et al., 1981a), EDTA, EGTA or

methoxyverapamil (Handwerger et al., 1981b) and insulin (Perlman et al.,

1985) to the medium. Bovine placental tissue also secretes placental lactogen

in vitro (Buttle and Forsyth, 1976) and this secretion was stimulated by the

addition of arginine to the media (Forsyth and Hayden, 1980). The regulatory

factors involved in placental lactogen secretion in culture remain unknown,

however hypotheses include the stimulation of secretion by inhibiting the

calcium flux (Handwerger et al., 1981b) or modulating dopaminergic receptors

(Macaron et al., 1978). The purpose of the studies described in this chapter

was to evaluate the factors which may be involved in regulation of bovine

placental lactogen (bPL) secretion in vitro.

Materials and Methods

Six experiments were conducted to determine the role of substrate

or hormone supplementation on secretion of bPL. Whole uteri were collected

at slaughter from cows at approximately day 200 of gestation. The uteri

were transported to the laboratory where placentomes were removed







aseptically. Placentomes were separated into maternal caruncle and fetal

cotyledon. Cotyledons were placed in cold minimum essential media (MEM)

(Gibco, Grand Island, NY) on ice and taken to the processing laboratory.

In a laminar flow hood cotyledonary villi were removed with scissors and

minced with scalpel blades. Explants weighing approximately 1 mg were

placed on a stainless steel grid in either a falcon culture dish (Falcon Plastics

Co., Oxnard, CA) or 24 well culture plate (Costar Rochester Scientific,

Rochester, NY) in the presence of 1 ml MEM plus the appropriate treatment.

Tissue explants were cultured on a rocker table (Bellco Glass, Vineland,

NJ) in an incubator at 37C (National, Portland, OR) in the presence of 50%

N2:45%02:5%CO2. After incubation culture medium was assayed for

lactogenic activity in a Prolactin radioreceptor (Prl-RRA) assay by the method

of Shiu et al. (1973).

Experiment 1. To determine the site of production of the lactogenic

activity by the bovine placenta, six explants from cotyledon, caruncle or

inter-cotyledonary tissue were placed into culture for 24 hr.

Experiment 2. To determine the role of either energy substrate or

hormonal supplementation on production of lactogenic activity, cotyledonary

explants were cultured in the presence of either additional substrate or

hormones. Treatments consisted of

1 ml MEM + 0 Insulin + 50 ig/ml Acetate + 1 mg/ml Glucose

1 ml MEM + .2 u Insulin + 50 ug/ml Acetate + 1 mg/ml Glucose

1 ml MEM + .2 u Insulin + 50 ug/ml Acetate + 5 mg Glucose

1 ml MEM + .2 j Insulin + 100 jg Acetate + 1 mg Glucose

1 ml MEM + .2 4 Insulin + 100 ug Acetate + 5 mg Glucose

1 ml MEM + .2 j Insulin + 50 ig Acetate + 1 mg Glucose + 50 ng L-T4 (Sigma)

1 ml MEM + .2 p Insulin + 50 pg Acetate + 1 mg Glucose + 10 ng Cortisol

1 ml MEM + .2 u Insulin + 50 ug Acetate + 1 mg Glucose + 1 ng Estrone









1 ml MEM + .2 p Insulin + 50 Pg Acetate + 1 mg Glucose + 1 ng Estradiol

1 ml MEM + .2 4 Insulin + 50 4g Acetate + 1 mg Glucose + 10 ng Progesterone

1 ml MEM + .2 p Insulin + 50 Pg Acetate + 1 mg Glucose + 100 ng GH (NIH B8)

1 ml MEM + .2 p Insulin + 50 Pg Acetate + 1 mg Glucose + coculture with caruncle

Each culture was in duplicate and the cultures were terminated at 12, 24,

36 or 48 hr. The tissue was blotted dry and weighed and medium was stored

at -200C until analyzed. The experiment was replicated three times,

using three cows (two Angus and one Brown Swiss) ranging from 230 to 250

days of gestation.

Experiment 3. To further evaluate hormonal regulation of secretion

of lactogenic activity, placental tissue from four cows at approximately

day 230 of gestation was cultured in the presence of nine different hormones

at three different concentrations. The treatments consisted of

Dose

Growth Hormone (NIH B8), ng/ml 100, 10, 1

Estradiol, pg/ml 1000, 100, 10

Seratonin, pg/ml 1000, 100, 10

Dopamine, pg/ml 1000, 100, 10

Norepinephrine, pg/ml 1000, 100, 10

Epinephrine, pg/ml 1000, 100, 10

Ergocryptine, pg/ml 1000, 100, 10

Thyroid releasing hormone, pg/ml 1000, 100, 10

Somatostatin, pg/ml 1000, 100, 10

Explants were cultured for 24, 48, or 72 hr. In the first two replicates the

explants were immersed in 1 ml culture media; however, because of irregular

results the third and fourth replicates were conducted with explants placed


on grids.










Experiment 4. Placental explants from three cows, at approximately

day 186 of gestation, were cultured for 24 hr in the presence of estradiol

or growth hormone using a 4 x 4 latin square design. The treatments consisted

of bovine Growth Hormone (NIH B8) at 0, 10, 100 and 1000 ng and estradiol-

17B at 0, .1, 1 and 10 ng. Treatments were conducted in triplicate.

Experiment 5. To determine the optimum amount of tissue to culture

in spinner flasks (Bellco Glass, Vineland, NJ) to maximize production of

lactogenic activity, cotyledonary tissue at 6.3, 12.8, 18.4, 25.9, 30.9 or 36.2

g was cultured in 500 ml MEM for 24 hr. Culture medium was analyzed

for lactogenic activity and protein concentration.

Experiment 6. To determine the effect of arachidonic acid on lactogenic

activity production, cotyledonary tissue was cultured in medium containing

concentrations of arachidonic acid ranging from 0 to 318 pM. Arachidonic

acid (Fluka Chemical Corp., Hauppauge, NY) in methylene chloride CHCl2

was dried under N2 gas before addition to culture media. Three trials were

conducted to determine the effect of arachidonic acid on production of

lactogenic activity. In the first trial, four petri dishes, each containing

1 g cotyledonary tissue, were cultured for 24 hr. At the end of the incubation,

the MEM was removed and replaced. In two of the dishes 300 pM arachidonic

acid was added and the tissue was incubated for an additional 24 hr. In the

second and third trials cotyledonary tissue was cultured in the presence

of 0, 75, 150 or 300 uM arachidonic acid for 24 hr. At the end of incubation

media was measured for lactogenic activity by a Prl RRA.

Statistical Analysis. Experiments 2, 3, 4 and 6 were analyzed by the

General Linear Models procedure on the Statistical Analysis System (SAS).

In Exp. 2 means were calculated for each treatment and Duncan's Multiple

Range test was conducted to detect treatment effects. Experiment 1 and

5 were not analyzed.







Results and Discussion

Experiment 1. Six explants of either cotyledon, caruncle or

intercaruncular area were cultured for 24 hr. Highest concentrations of

lactogenic activity measured either as ng/ml or ng lactogenic activity/mg

tissue was detected in medium from culture of cotyledonary tissue (187.66

14.67 and 136.28 27.12, respectively) (table 2.1). The caruncular tissue

did produce lactogenic activity, but the variability was great. No lactogenic

activity was produced by the intercaruncular area and the lactogenic activity

produced by the caruncular tissue probably resulted from cotyledonary

contamination. During the placentome separation, pieces of cotyledon can

be trapped in the crypts of the caruncle.

Experiment 2. The effect of additional substrate or hormones on

production of lactogenic activity was examined in this experiment. Statistical

analysis indicated that cow, trt, cow x trt, trt x time, cow x trt x time and

tissue weight to the 4th order were significant (table 2.2). The cow x trt

interaction indicated that cows reacted differently to treatments.

Cotyledonary tissue produced approximately 500 ng lactogenic activity/ml

in the first 12 hr of culture with only an additional 200 ng/ml in the subsequent

36 hr (fig. 2.1). Production of lactogenic activity was affected (P<.001)

by tissue weight (mg) (fig. 2.2). Tissue weights ranged from .17-8.91 mg.

To normalize data on production of lactogenic activity by different size

explants, lactogenic activity (ng) per mg of tissue was plotted versus tissue

weight (fig. 2.3). Production of lactogenic activity expressed in this manner

over a range of tissue weights from 1 8 mg was similar; however, production

by explants weighing less than 1 mg was higher, possibly because values

were multiplied. Addition of substrate or thyroxine, cortisol, estrone,

estradiol-17B or progesterone had no effect on production of lactogenic

activity (fig. 2.4). The absence of insulin from the culture medium resulted














Table 2.1. Lactogenic Activity Produced by Cotyledonary, Caruncular
and InterCaruncular Tissues From a Cow at Approximately Day
200 of Gestation.


Total Lactogenic Activity


Tissue


Cotyledon

Carcuncle


ng/ml
(x SE)

187.6614.67

36.7737.20


ng/mg
(x SE)

136.2827.12

21.9118.09


InterCaruncular
Area


.050.02


.010.01











Table 2.2. Analysis of Variance for Cotyledon Culture (Exp # 2)


Source

Cow'

Trt

CowxTrt

Time

CowxTime

TrtxTime

CowxTrtxTime

Tisswt+

Tisswt2

Tisswt3

Tisswt4

Residual


SS

95580822

261924951

282405564

33808993

72618595

253183425

459610224

58973143

54460193

37514058

5974688

725929234


F Value

8.82***

4.40***

2.48***

2.08

2.23*

1.42*

1.35*

10.89**

10.05**

6.92**

11.03**


*P<.1

**P<.01

***P<.001

+Tisswt was tested using Type I Sums of Squares.

'Cow, Trt, CowxTrt, Time, CowxTime, TrtxTime and CowxTrtxTime were

tested using Type III SS.


















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in lower concentrations of lactogenic activity. Duncan's Multiple Range

test indicated that addition of growth hormone or co-culture of cotyledon

and caruncular tissue stimulated (P<.05) production of lactogenic activity

when measured on a per mg of tissue basis (table 2.3). These results suggest

that the majority of bPL is secreted into the medium in the first 12 hr of

culture. This is in agreement with the findings of Suwa and Friesen (1969).

Tissue explants did not require additional acetate or glucose to produce

more lactogenic activity. Other nutritional factors such as amino acids

may affect production. Hormonal stimulation of lactogenic activity by

growth hormone was not affected by crossreactivity of GH in the Prl RRA

(4.3%) but is unclear at this time. Similarly, the mechanism for stimulation

of production of lactogenic activity by co-culture of cotyledon and caruncle

is unclear. The lactogenic activity/mg tissue did not take into account weight

of caruncular tissue, so that cotyledonary tissue contamination as

demonstrated in Exp. 1 could have been a factor. Another explanation is

that the caruncular tissue secretes a substance that stimulates release of

lactogenic activity from the cotyledon. The identification of this substance

was not attempted.

Experiment 3. In this experiment the analysis of variance (table 2.4)

indicated that grid, whether tissue was incubated on stainless steel grids

or immersed in culture medium, was not a main effect, but there was a

grid x time interaction. Hormonal treatment and dose of hormone also had

no effect on production of lactogenic activity (table 2.5). There was a

significant time effect, and time interactions with grid and cow (grid) were

significant. Bovine growth hormone (NIH-B8) did not affect production

of lactogenic activity which was in contrast to Exp. 2. The significant time

effect was also in contrast to the results in Exp. 2. This effect may be












Table 2.3. Duncan's Multiple Range Test for Treatment in Exp. #2.


Duncan Grouping*

A
A
A

B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B


x Lactogenic
Activity
(ng/mg Tissue)

645.0

568.7

378.6

369.7

366.9

349.3

348.4

309.3

280.1

270.4

264.5

253.9


Treatment

Growth Hormone

CoCulture

Progesterone

Estradiol-17B

Cortisol

Control

Estrone

Acetate

Glucose

Thyroxine

Acetate + Glucose

-Insulin


*Means with same letter are not significantly different (P<.05).














Table 2.4. Analysis of Variance for Culture Exp. #3.


Source


Grid+

Cow (Grid)

Trt

Dose

Time

GridxTime

Cow (Grid)xTime

TrtxTime

DosexTime

TrtxDose

GridxTrtxTime

Cow (Grid)xTrtxTime

Residual


SS

21139032

823850054

51936936

31575667

133253219

43799379

125011255

106651824

49560746

54768973

199656550

340298401

1057785899


*P<.1

**P<.01

***P<.001

+Grid was tested using cow (Grid) as the error term.


F Value

.51

73.21***

1.15

1.87

11.84***

3.89*

5.55**

1.18

1.47

.65

1.31

1.14










Table 2.5. Mean (x) Production of Lactogenic Activity (ng/ml) by Cotyledonary
Explants In Vitro. Effect of Three Doses of Growth Hormone (GH), Estradiol
(E2), Seratonin, Dopamine, Norepinephrin, Epinephrin, Ergocryptine, Thyroid
Releasing Hormone (TRH) and Somatostatin.


Time (hr)


Hormone
Control
GH




E2




Seratonin




Dopamine




Norepinephrin




Epinephrin




Ergocyptine




TRH




Somatostatin


Dose (ng)
0.00
10.00
100.00
1000.00
.01
.10
1.00
.01
.10
1.00
.01
.10
1.00
.01
.10
1.00
.01
.10
1.00
.01
.10
1.00
.01
.10
1.00
.01
.10
1.00


Pooled SEM = 80.34


24
300.0
187.2
277.3
288.0
175.4
226.8
276.5
221.0
283.2
376.2
257.8
260.7
211.8
273.5
237.2
246.1
229.3
261.8
284.3
239.0
244.7
255.6
380.3
785.6
269.8
200.3
226.6
652.7


48
785.2
535.0
439.7
402.9
581.9
426.6
493.6
381.6
470.9
610.1
389.9
404.4
680.0
367.9
446.1
302.8
326.1
430.5
291.5
407.1
400.2
530.2
374.1
376.4
755.7
433.0
544.2
411.7


72
412.0
374.0
1030.0
477.7
273.7
376.8
486.8
457.0
381.4
391.1
489.3
384.9
490.6
402.1
394.4
742.3
314.4
422.1
504.2
628.7
394.0
345.3
438.9
602.8
352.3
304.0
328.6
343.3









explained by the different patterns of production of lactogenic activity

over time as depicted in table 2.5. The cows utilized in this experiment

were at a similar stage of gestation to the cows in Exp. 2, but the culture

times were different. These results agree with previous reports that thyroid

releasing hormone (Hershman et al., 1973), somatostatin (Macaron et al.,

1978) and dopamine (Forsyth and Hayden, 1980) had no effect on placental

lactogen production. However, hPL secretion was stimulated by the addition

of estradiol (Belleville et al., 1978) and inhibited by the addition of

epinephrine, norepinephrine (Belleville et al., 1978) and dopamine (Macaron

et al., 1978). The results of this experiment have not increased our

understanding of the regulatory mechanisms involved in the secretion of

bovine placental lactogen (as measured by lactogenic activity) in vitro.

Experiment 4. In Exp. 2 preliminary analysis suggested that the

hormones GH (NIH B8) and estradiol-17B enhanced the production of

lactogenic activity. Therefore, the purpose of this experiment was to

determine if there was a dose response in production of lactogenic activity

and if hormonal interactions could affect production. Statistical analysis

(table 2.6) indicated that cow, estradiol (E), GH and cow x GH effects were

significant (P<.1). The least squares means plotted by hormone and dose

(fig. 2.5) suggested that estradiol at doses of .1 and 1 ng inhibited the

production of lactogenic activity, but the 10 ng dose had no effect. Increasing

the concentration of growth hormone stimulated the production of lactogenic

activity by a mean of 85 ng/ml for the 1,000 ng dose. This may be partially

explained by the 4.3% crossreactivity of GH in the Prl RRA, which would

add 43 ng of lactogenic activity to the 1,000 ng GH concentration. When

least squares means for each cow for growth hormone were plotted (fig.





62





Table 2.6. Analysis of Variance for Culture Exp. #4.



Source df SS+ F Value

Cow 2 26873472 25.97***

Estradiol 3 3396251 2.19*

Cow x Estradiol 6 1851052 .60

Growth Hormone 3 3710058 2.39*

Cow x Growth Hormone 6 7842747 2.53*

Estradiol 9 2359430 .51

Tissue Weight 1 16158946 31.24*

Residual 110 56905808



*P<.1

**P<.01

***P<.001

+Tissue weight was tested using the Type I SS and all other factors

were tested using the Type III SS.













Fig. 2.5. Lactogenic activity (ng/ml) produced by cotyledonary tissue in a
4x4 Latin Square design using 0, .1, 1 and 10 ng Estradiol-17B and
0, 10, 100 and 1000 ng Growth Hormone.





























I


DOSE CNEI


I 5EM


150



IBO
loo


ili I -un









2.6) the significant GH effect and cow x GH may be explained by the result

from the 1,000 ng GH concentration for cow 7.

Experiment 5. A preliminary experiment was conducted to determine

the amount of cotyledonary tissue to culture in 500 ml MEM (table 2.7).

As tissue weight increased so did lactogcnic activity (pg/ml) and protein

(mg/ml), although when put on a per gram tissue basis the 6.3 g culture flask

was the most efficient in producing lactogenic activity with the least amount

of protein. The specific activity (ug lactogenic activity/mg protein) was

highest for the 6.3 g flask and decreased with increasing tissue weight.

Thus to maximize the production of lactogenic activity with the least amount

of protein in the medium 10 15 g of cotyledonary tissue was chosen as

the optimal amount of tissue to produce bovine placental lactogen (as

measured by lactogenic activity) for purification of the molecule.

Experiment 6. Three preliminary experiments were conducted to

determine the effect of arachidonic acid on production of lactogenic activity.

Results are depicted in table 2.8 for the first trial.

Production of lactogenic activity was similar in the four petri dishes

after 24 hr of culture. Addition of 300 jM arachidonic acid to dishes one

and two may have attenuated the decrease of production of lactogenic activity

(51.8 vs. 64.8% for dishes one and two versus three and four) by cotyledonary

tissue in vitro.

In the second and third trials, tissue exposed to 0 300 pM arachidonic

acid (table 2.9) was cultured for 24 hr. Statistical analysis (table 2.10) using

the model cow explant (cow) dose x cow x dose indicated that dose of

arachidonic acid was not significantly different. This is in direct contrast

to the reports of Handwerger et al. (1981a) in humans and Huyler et al. (1985)

in sheep. Using explant (cow) as the error term for cow demonstrated that
















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0
*-


c









E









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0



0



a
o
















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i-






e-
Ec


Cb


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ca

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^ h
^c

























s4








































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- M


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68





Table 2.7. Production of Lactogen Activity by Different Mass of
Cotyledonary Tissue in Spinner Culture Flasks with 500 ml Minimum
Essential Medium. Tissue was Cultured at 37 C for 24 hr.


Tissue
Weight (g)


Lactogenic
Activity (LA) jg LA/g
ug/ml Tissue


Protein
mg/g
mg/ml Tissue


Specific
Activity
4g LA/mg
Protein


.457

.346

.290


.385

.803

.920


.212 1.041

.194 1.380


6.67 .184 1.464


.061 7.48

.063 5.52

.050 5.80

.039 5.28

.045 4.35

.040 4.57


6.3


12.8

18.4

25.9

30.9

36.2


2.88

4.43

5.34

5.50

5.98






69





Table 2.8. Lactogenic Activity Produced by Cotyledonary Tissue
During a 24 hr Culture. Medium was changed after the initial 24
hr. Petri Dish 1 and 2 Contained 300 uM Arachidonic Acid.


Lactogenic Activity
ng/ml 24 hr


1886.1

1818.2

2494.3


48 hr

770.4

1008.7

678.5


1715.8 740.7


% Decrease


59.1

44.5

72.8

56.8


Petri
Dish











Table 2.9. Lactogenic Activity Produced by Cotyledonary Tissue Cultured
in 0, 75, 150 or 300 uM Arachidonic Acid for 24 hr.


Dose Trial

0 2

2

3

3

75 2

2

3

3

150 2

2

3

3

300 2

2

3

3


Tissue
Weight (mg)

364

458

60

126

522

472

106

180

556

406

170

213

418

460

175

181


Lactogenic Activity
(LA) (ng/ml)

745.0

1028.7

731.0

849.0

692.8

855.9

803.0

1028.0

1195.0

788.2

1004.0

1192.0

1026.6

894.0

1220.5

1066.5


Specific Activity
(ng LA/mg Tissue)

2.04

2.25

12.17

6.74

1.33

1.81

7.58

5.71

2.15

1.89

5.91

5.60

2.46

1.94

7.00

5.90














Table 2.10. Analysis of Variance for Trials Two and Three, Exp. #6.



Source df SS+ F Value

Cow* 1 8.86 1.56

Explant (cow) 2 11.39 5.59

Dose 3 1.97 .65

Cow x Dose 3 .32 .11

Residual 6 6.11



+Type III Sum of Squares.

*Cow was treated using Explant (cow) as the error term.










the cows were not different. Production of lactogenic activity by

cotyledonary explants in the two trials was similar (table 2.9), but the specific

activity (ng lactogenic activity/mg tissue) was higher in trial three because

of the smaller explants utilized, which is in agreement with Exp. 2.

In summary, bovine placental lactogen, as measured by lactogenic

activity, was produced by fetal cotyledonary tissue in vitro and 70% of the

lactogenic activity was produced in the first 12 hr of a 48 hr culture. These

results are similar to the findings of Suwa and Friesen (1969) in the human.

Production of lactogenic activity was affected by amount of tissue therefore,

production/mg tissue was utilized to account for differences in weight of

explants. The increased production of lactogenic activity by explants weighing

less than one milligram may be due to increased surface area for transport

of nutrients into the tissue or lactogenic substances out of the tissue. This

possibility was not examined further, but tissue was minced finely in

subsequent studies.

Addition of acetate, glucose, thyroxine, cortisol, estrone, estradiol-17B,

progesterone, seratonin, dopamine, norepinephrin, epinephrin, ergocryptine,

thyroid releasing hormone, somatostatin or arachidonic acid had no effect

on production of lactogenic activity by cotyledonary explants. This is in

contrast to reports in the human (Bellville et al., 1978; Handwerger et al.,

1981a) and sheep (Huyler et al., 1985) but in agreement with a report in

the cow (Forsyth and Hayden, 1980). Growth hormone significantly increased

production of lactogenic activity in Exp. 2 and 4, but the response in Exp.

4 may be due to a single animal. Increasing doses of growth hormone (Exp.

3) did not stimulate production of lactogenic activity over control, which

may have been affected by very high control values.










Production of lactogenic activity by spinner culture increased as amount

of tissue increased, however; the concentration of protein in the media also

increased. The specific activity of the lactogenic activity (ng lactogenic

activity/mg protein) decreased as tissue weight increased, which would hinder

the purification process. Thus the optimum amount of cotyledonary tissue

to produce lactogenic activity with the least amount of protein was

determined to be 10 15 g in 500 ml MEM. The experiments in this chapter

failed to elucidate the regulatory factors involved in secretion of bPL in

vitro. A possible explanation for this is that culture conditions may not

have been optimal. Lactogenic activity was produced for eventual purifi-

cation of bPL, therefore factors such as, fetal calf serum, were omitted

from the culture media. Fetal calf serum has been proven to be a requirement

in several culture systems. The time between slaughter and incubation of

the tissue may be a factor in viability of tissue, which ranged from 30-120

minutes. Finally, production of lactogenic activity by cotyledonary tissue

may be an autonomous process as has been suggested in the human.














CHAPTER III
PURIFICATION OF BOVINE PLACENTAL LACTOGEN

Introduction

Bovine placental lactogen (bPL) has been isolated and purified (Beckers

et al., 1980, Murthy et al., 1982, Arima and Bremel, 1983). The molecular

weight and isoelectric point reported for the molecule was 32,000 (Murthy

et al., 1982, Arima and Bremel, 1983) and 5.5 (Murthy et al., 1982, Arima

and Bremel, 1983), respectively. Bovine placental lactogen was purified

from fetal cotyledons following homogenization, extraction in ammonium

bicarbonate buffer, ammonium sulfate precipitation and column chroma-

tography. All three groups used gel filtration and ion exchange chroma-

tography; however, Beckers et al. (1980) utilized an affinity column to remove

bovine serum albumin, while Murthy et al. (1982) and Arima and Bremel

(1983) also added a hydroxyapatite column and Arima and Bremel (1983)

utilized a chromatofocusing column. Approach of the present study was

to purify bPL from material secreted into medium by cotyledonary explants

as described by R. Kensinger (unpublished).

Materials and Methods

Whole uteri were collected at slaughter and transported to the

laboratory. Placentomes were removed aseptically and separated into

maternal caruncles and fetal cotyledons. Cotyledonary tissue was placed

in sterile minimum essential medium (MEM) on ice. Villi were removed

and minced with scissors in a laminar flow hood. Forty grams minced










cotyledonary tissue was placed in a spinner flask (Bellco, Vineland, NJ) in

1 L MEM. The flask was aerated with 50%N2:45%02:5%C02, placed on a

magnetic stir plate and slowly stirred for 24 hr in a 37 C incubator. At

the end of incubation, culture medium was centrifuged at 10,000 x g for

10 min. Supernatant was saved for bPL purification by a method similar

to Arima and Bremel (1983). Columns consisted of Sephacryl 200 (S-200),

diethylaminoethyl cellulose (DEAE), chromatofocusing and Sephadex G-75.

Column fractions were monitored for protein using the BioRad protein

assay (BioRad Laboratories, Richmond, CA), for lactogenic activity using

a prolactin radioreceptor assay (Prl-RRA) by the method of Shiu et al. (1973)

and for somatotrophic activity using a homologous growth hormone radio-

receptor assay (GH-RRA) by the method of Haro et al. (1984). Prolactin

(NIAMDD-oPrl-14) and growth hormone (recombinant bovine growth hormone

[rbGH], Monsanto, St. Louis, MO) were iodinated using lodo-Gen reagent

(Pierce Chemical Co., Rockford, IL). Prolactin was diluted to 5 ug/25 ul

in 25 mM Tris buffer, pH 7.6 while rbGH was weighed and diluted in .1 M

NaHC03 buffer, pH 9.0. Hormone and 1 mCi of Na1251 (1 mCi/10 pl)

(Amersham, Arlington Heights, IL) were added to a 12 x 75 borosilicate

tube which was coated with 2 pg lodo-Gen (50 ul reaction volume) and the

reaction was allowed to procede for 15 min for rbGH and 5 min for Prl.

Iodinated hormone was separated from free 1251 on a .7 x 25 cm Biogel P-60

column (BioRad Laboratories, Richmond, CA).

The procedure utilized for the radioreceptor assay was similar for

both Prl and GH-RRA's. Approximately 50,000 cpm of iodinated hormone

was combined with either 100 ul rabbit mammary membrane (8 mg/ml) (diluted

1:3 in assay buffer) for the Prl-RRA or 400 Il steer liver membrane (1-7

mg/ml) for the GH-RRA.










Two dimensional polyacrylamide gel electrophoresis (2D-PAGE) was

conducted on peak fractions from the G-75 column and the crude culture

medium by the method of Roberts et al. (1984). Approximately 1.3 Pg of

protein was dissolved in 100 ul of a solution containing 5 mM K2C03, 2%

(v/v) Nonidet P-40, .5% dithiothreitol, 2% Ampholines and 9.3 M urea. This

solution was loaded on to a 4.3% acrylamide isoelectric focusing gel containing

N'N'-diallytartardiamide, 2% Nonidet P-40 and 9.3 M urea. After isoelectric

focusing the gels were equilibrated in 65 mM Tris, .1% sodium dodecyl sulfate,

1% 2-mercaptoethanol, pH 6.8. The gels were then overlaid on 10% (w/v)

acrylamide slab gels and electrophoresis conducted toward the anode. After

completion of electrophoresis, slabs were fixed in acetic acid:ethanol (7:40).

Slabs then were equilibrated in acetic acid methanol (5:10) and stained with

BioRad silver stain kit (BioRad Laboratories, Richmond, CA). We obtained

K2C03 and Nonidet P-40 from Sigma Chemical Co. (St. Louis, MO),

dithiothreitol, acrylamide, N'N'-diallytartardiamide, sodium dodecyl sulfate

and 2-mercaptoethanol from BioRad Laboratories (Richmond, CA), urea

from Schwarz Mann (Cambridge, MA) and ampholines from LKB (Gethersberg,

MD).

To test the biological activity of purified bPL, a bovine mammary

gland explant culture was performed. Four cows (two Holstein and two

Jersey) at approximately day 240 of gestation were utilized. Rate of two

14C-acetate (New England Nuclear, Boston, MA) incorporation into fatty

acids was examined. Mammary biopsies were performed by Dr. E.L. Bliss,

at the University of Florida Large Animal Veterinary Clinic. Cows were

anesthetized locally with Lidocaine plus epinephrine (Tech America, Elwood,

KS). An incision was made in the left front quarter near the body wall.

A 30 gm explant of mammary tissue was removed and placed in sterile 25










mM Tris, 200 mM sucrose, pH 7.2. Two explants per day were then taken

to the laboratory for processing. Tissue was sliced using a Stadie-Riggs

hand microtome (Stadie and Riggs, 1944). Tissue slices were minced with

scissors and three explants were placed on a stainless steel grid in a 24 well

culture dish (Costar, Rochester Scientific, Rochester, NY) with 1 ml medium.

The medium used was Tissue Culture Media 199 (Difco Laboratories, Detroit,

MI) which contained 10 mM acetate, 10 mM glucose, non-essential amino

acids (Gibco, Grand Island, NY), cortisol (4-pregnen-11B, 17a, 21-triol-3,

20 dione, Steraloids, Inc., Pawling, NY), insulin (Sigma Chemical Co., St.

Louis, MO), antimycotic-antibiotic (Gibco, Grand Island, NY) and either

0, 1, 10, 100, 250, 500 or 1,000 ng prolactin (NIAMDD-oPrl-14) or 1, 10,

100, 250, 500, 1,000 or 5,000 ng bPL (peak II or III). Explants were incubated

for 48 hr (in triplicate) at 37 C in an atmosphere of 50%N2:45%02:5%C02.

At the end of incubation, the tissue was placed in a 25 ml Erlenmeyer flask

with 3 ml of Krebs-Ringer bicarbonate buffer, pH 7.3 containing 10 mM

acetate, 10 mM glucose, 133 mU insulin and 2-14C-acetate (1 ICi). Tissues

were incubated for 3 hr in a Dubnoff metabolic water bath at 37 C. Incubation

was terminated by the addition of 100 pl 1N sulfuric acid and tissue was

blotted dry and weighed. Tissues were saponified and fatty acids were

extracted and quantitated by the method of Bauman et al. (1970).

Results and Discussion

Tissue culture incubation was halted 24 hr after initiation. Culture

medium became very acidic and incidence of contamination was increased

with longer incubations. The culture medium was lyophilized and stored

at -20 C until chromatography.

S-200. The lyophilized culture medium was reequilibrated with 25

mM Tris buffer, pH 6.2 and contained 2315.5 mg/ml lactogenic activity,










1545.0 ng/ml somatotrophic activity and 871.2 ug/ml protein (table 3.1).

The protein was loaded on a 3.2 x 85 cm Sephacryl 200 column and eluted

with 25 mM Tris, 200 mM NaCI, pH 6.2. Fractions (6 ml) were collected

on a Gilson fraction collector (Middleton, WI), and monitored for protein,

lactogenic and somatotropic activity. Protein in the peak fractions was

reduced 3-fold; however, both lactogenic and somatotropic activities were

also reduced to result in no overall increase in purification (table 3.1). The

elution profile from the S-200 column (fig. 3.1) indicates that somatotropic

activity is eluted as a symmetrical peak with peak height greater than that

for lactogenic activity. The lactogenic activity peak was broader and both

peaks were associated with the right hand shoulder of the protein profile.

DEAE. Peak fractions (tubes 48 57) were dialysed against 25 mm

Tris, pH 6.2 and loaded on a 1.25 x 17.5 cm DEAE column. The column was

eluted with a 0 .3 M NaC1 gradient and 6 ml fractions were collected.

The elution profile (fig. 3.2) indicated that both the lactogenic and

somatotropic activity peaks preceded the protein peak, but the somatotrophic

activity was attenuated. This could be explained by a conformational change

in the molecule or a loss of the protein associated with the somatotropic

activity. The lactogenic peak was again much broader than the somatotropic

peak and may be attributed to several proteins. The overall specific activity

increased 10-fold for lactogenic activity, but only 3-fold for somatotropic

activity.

Chromatofocusing. Peak fractions (tubes 51 70) from the DEAE

column were pooled and dialysed against 25 mM Imidazole buffer, pH 6.2.

The pooled fraction was then loaded on a .75 x 50 cm chromatofocusing

column and eluted with Poly buffer PBE 94 (Pharmacia Fine Chemicals,










Table 3.1. Purification of Bovine Placental Lactogen


Column

Culture Media

S-200

DEAE

Chromatofocusing


II

III

G-75 II

III


Lactogenic
Activity
(ng/ml)

2315.5

613.1

777.6


217.7

136.0

125.0

213.0


Somatotropic
Activity Protein
(ng/ml) (Pg/ml)

1545.0 871.2

299.3 273.5

87.4 23.5


10.7

51.8

10.0

12.8


7.64

4.20

.13

.13


Specific
Activity
ng Activity
/pg Protein
LA SA

2.66 1.77

2.24 1.09

33.1 3.72


28.5

32.4

961.5

1638.5


1.4

12.3

76.9

98.5











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pH gradient. To inhibit protein aggregation, fractions (6 ml) were collected

in .6 ml 1 M sucrose to make a final concentration of .1 M. Two peaks of

lactogenic and somatotropic activity are evident in the chromatofocusing

column elution profile. A third peak (peak I) was present when high

concentrations of bPL were purified. Peak II (p II) was eluted at a pH of

5.3 while peak III (p III) was eluted at a pH of 4.9 (fig. 3.3). This is in

agreement with the report of Arima and Bremel (1983). Total lactogenic

activity was greater than somatotropic activity and the ratio of lactogenic

to somatotropic activity was greater in p II than in p III (fig. 3.3). Fractions

associated with peaks (II and III) were pooled separately and each was purified

on a 2.5 x 62 cm Sephadex G-75 column.

G-75. Pooled p II and p III were loaded on the G-75 column and eluted

with 25 mM Tris, 200 mM NaCI, pH 6.2. Fractions (5 ml) were collected

in .5 ml of 1 M sucrose. The elution profiles of p II and p III from the G-75

column had protein concentrations which were <1 pg/ml which is the

sensitivity of the BioRad protein assay (Richmond, CA) (fig. 3.4 and 3.5),

therefore, elution profiles were not plotted. Concentrations of protein were

determined in pooled peak samples after concentration by lyophilization

(table 3.1). Somatotropic activities were further diminished in both p II and

p III, while lactogenic activity remained unchanged (table 3.1). Peak activities

were shifted five fractions to the left for p III over p II which would suggest

that p III contains proteins of slightly larger molecular weight than p II.

Results of 2D-PAGE of a) crude culture medium, and b) pooled (p III) fractions

from G-75 are depicted in fig. 3.6. The crude culture medium (fig. 3.6A)

contained a large array of proteins. The pooled p III G-75 fractions (fig.

3.6B) is a graphic illustration of data from the purification table (table 3.1).













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Full Text

PAGE 1

BOVINE PLACENTAL LACTOGEN: ISOLATION, PURIFICATION A. Nn MF. A STTR FMFN'T' TN i:nnT OGTr A. T PT TTTDS By CHARLES RALPH WALLACE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSPHY UNIVERSITY OF FLORIDA 1986

PAGE 2

ACKNOWLEDGEMENTS At this time I would like to acknowledge those people that have made my PhD program at the University of Florida a memorable experience. In the time that my family and I have been in Gainesville we have met and interacted with so many interesting people that I regret that I cannot mention them all. Special thanks go to Dr. Robert J. Collier, chairman of the supervisory committee, for his patience and support throughout this program. Dr. William W. Thatcher is acknowledged for his ability to stimulate enthusiasm about seemingly insignificant data in the author's perspective. Thanks are expressed to Drs. Fuller W. Bazer and R. Michael Roberts for allowing the author to work in their laboratories and gain the insight to begin to dissect animal science problems into their biochemical root. Thanks are due to Dr. Charles J. Wilcox for his statistical prowess and the ability to convey that prowess to a novice. Dr. Donald Caton is acknowledged for his ability to remind the author of the whole picture instead of just the part. Dr. William Buhi is acknowledged for his excellent substitution near the end of this program. Thanks are extended to Drs. David Beede and H.H. Head for the time spent discussing research and daily events. The technical assistance and friendship of Gail Knight, Annette 'Bee' Leinart, Sergio Quintana, Catherine Ketchum and Carol Underwood were greatly appreciated. To the students and postdoctoral fellows that have enriched my program with their presence, thanks are due to Marlin Dehoff, Lokenga Badinga ii

PAGE 3

Mark Maguire, Fran Romero and to Drs. Ron Kensinger, Jeff Knickerbocker, Louis Guilbault, Rodney Geisert, Jeff Moffat, Randy Renegar, Jim Godkin, George Baumbach, Asgi Fazlebas, Paul Schneider, John McNamara, Quim Moya and Skip Bartol. Without the assistance of Austin Green, Kent Bundy and Tom Bruce the author would not have had animals to work with, thank you for your help. Last but not least I would like to thank my family for always being there. To June, my wife, and Katherine and Steven, our children, you make life worthwhile. To my parents, Ralph and Emily Wallace, your confidence and moral support during this program was genuinely given and deeply felt. iii

PAGE 4

TABLE OF CONTENTS ACKNOWLEDGEMENTS LIST OF TABLES LIST OF FIGURES ABSTRACT CHAPTER II III REVIEW OF LITERATURE History of Placental Lactogen Placental Type Endocrine Control of Mammary Development Secretion Function Nutrient Partitioning Purification COTYLEDON CULTURE EXPERIMENTS Introduction Materials and Methods Results and Discussion PURIFICATION OF BOVINE PLACENTAL LACTOGEN Introduction Materials and Methods Results and Discussion IV DEVELOPMENT OF A RADIOIMMUNOASSA Y TO BOVINE ii vi viii X 1 1 11 16 23 30 37 39 42 42 42 46 74 74 74 77 PLACENTAL LACTOGEN 96 Introduction Materials and Methods Results and Discussion iv 96 97 103

PAGE 5

V BOVINE PLACENTAL LACTOGEN CONCENTRATIONS IN MATERNAL AND FETAL FLUIDS , 119 VI Introduction . . Materials and Methods Results and Discussion GENERAL DISCUSSION LITERATURE CITED BIOGRAPHICAL SKETCH V 119 119 121 140 147 164

PAGE 6

LIST OF TABLES Table 2. 1 Lactogenic Activity Produced by Various Tissues. 47 2.2 Analysis of Variance for Cotyledon Culture (Exp. #2). 48 2. 3 Duncan I s Multiple Range Test for Treatment in Exp. # 2. 58 2.4 A nal ys is of Variance for Culture Exp. #3. 59 2.5 Analysis of Variance for Culture Exp. #4 60 2. 6 Production of Lactogenic Activity b y Different Mass 62 of Cotyledon Tissue. 2 7 Lactogenic Activity in Re s ponse to Arac hidonic Acid. 68 2. 8 Dose Response of Lactogenic Ac tivit y Produced b y 70 Arachidonic Acid. 2 9 Lactogenic Activity Produced b y Cotyledonar y Ti ss ue 70 Cultured in O, 75, 150 or 300 it M A rachidonic Ac id for 24 hr. 2.10 Analysis of Variance for Trial s Two a nd Three Exp. #6. 71 3 .1 Purification of Bovine Placental Lactogen. 79 3. 2 x nmole s 21 4-c-acetate Incorporated/100 mg Ti ss ue /3 Hr. 94 4 .1 Heterogeneit y of Regres s ion for P ara 11 e 1 i s m. 109 4. 2 % Recover y of bPL From Maternal a nd Fetal Fluids. 113 4.3 A nal ys is of Variance for Assay C ompari s on Samples. 114 4.4 Determination of bPL or Lactogenic Activity (x SE) 115 in Cotyledon Culture Sample s Tested w ith E s tro ge n (E) or Growth Hormone ( G H) 5 .1 Bovine Placental Lactogen C oncentrations in Ma ternal 122 and Fetal Fluids. 5.2 E s timated Fluid Volume s and Total bPL C on ce ntrations. 126 vi

PAGE 7

5.3 5.4 5.5 Analysis of Variance for Maternal and Fetal Samples. Analysis of Variance for bPL Concentrations Across Gestation ( Exp. # 2) Ana 1 ysis of Variance for bPL Concentrations Across Gestation ( Exp. # 2) Heterogeneity. vii 129 132 133

PAGE 8

LIST OF FIGURES Figure 2 .1 Lactogenic activity (ng/m 1) produced over time. 50 2. 2 Lactogenic activity ( ng/m I) produced by various 5 2 size tissue explants. 2. 3 Lactogenic activity (ng/mg tissue) produced by 54 various size tissue exp 1 an ts. 2.4 Lactogenic activity (ng/mg tissue) produced by 56 various treatments. 2. 5 Lactogenic activity ( ng/m 1) produced by various doses 64 of Estrogen or Growth Hormone. 2.6 Lactogenic activity (ng/ml) produced by increasing 67 Growth Hormone concentrations for individual animals. 3 .1 Elution profile of lactogenic and somatotropic 81 activities and protein from a Sephacryl S-200 column. 3.2 Elution profile of lactogenic and somatotropic 83 activities and protein from a diethylaminoethyl cellulose (DEAE) column. 3.3 Elution profile of lactogenic and somatotropic 86 activities and protein from a Chromatofocusing column. 3.4 Elution profile of lactogenic and somatotropic 88 activities of bPL p II from a Sephadex G-75 column. 3. 5 Elution profile of lactogenic and somatotropic 90 activities of bPL p III from a Sephadex G-75 column. 3. 6 Two dimensional polyacrylamide gels of crude culture 92 media (A) and purified bPL p III ( B). 4.1 Lactogenic activity (ng/ml) eluted from native poly99 acrylamide gel slices. 4.2 Flurograph of immunoprecipitation of bPL by 50, 100, 106 or 200 l Florida a bPL or 100, 100 or 200 l USDA a bPL. viii

PAGE 9

4.3 4.4 5 .1 5.2 5.3 5.4 Crossreactivity of anti-bPL with various protein hormones. Parallelism of 50, 100 or 200 l of amniotic and allantoic fluid and fetal and maternal serum com pared to the standard curve. Immunohistochemical localizati0n of bPL ~n b0vin':' p 1 acenta 1 tissue. Concentrations of bPL ( ng/m 1) in amniotic and allantoic fluids and maternal venous and fetal um bilical arterial and venous blood. Linear regression of bPL concentrations in maternal serum, fetal umbilical arterial and venous serum, allantoic fluid and amniotic fluid from cows at various gestational ages. Least square regression ( 5th order) of bPL con centrations of Holstein heifers serviced with either Holstein ( 1), Angus ( 2) or Brahman ( 3) semen. Bovine placental lactogen concentrations from four cows sampled at 30 min. intervals for a period of 12 hr. ix 108 111 117 124 128 135 138

PAGE 10

Abstract of Dissert a ti o n Pre s ent e d to the Graduate School of the U niver s it y of Flori da in P a rt ia l Fulfillment of the Requirements for the De g ree o f Doctor of P h ilo s oph y BO V INE PLACENTAL LACTOGEN: ISOLATIO N, P U RIFIC A TION A ND M EASURE I\ 'IE N T I N BIOL O GICAL FLUIDS B y Charles Ralph Wa llac e D ec e mber 198 6 Chairm a n: Robert J. C ollier M ajor Department: A nimal S ci ence Studie s w ere c onduct e d ta i s ol a te a n d p ur if y b ovine p l ace nt a l l ac to g e n ( bPL) a nd to develop a r a dioimmun oassa y t o t hi s p rot e i n. B ovine pl ace n ~ a l lactogen w as isolated from c ulture me d ium a f ter a 24 h r c u lt u r e of f et a l cot y ledonar y tis s ue. C ot y ledonar y e xpl a nt s w er e s timul a te d t o s e c r ete bPL b y either addition of bovine g ro w th h o rm o ne ( N IHB8) t o the med i um or co-culture of c ot y ledon a nd ca run cu l ar t i ss u e. P rod u cti on o f bPL 1 .,v as g reatl y affe c ted b y explant s ize a nd 70 % o f tha t p ro d uc ed in a -+ 8 h r c ult u r e was relea s ed in the first 12 hr. Purification of bPL w as a ccompli sh e d us in g a c olumn c hr o m a to g r a phi c s cheme that involved gel filtration i on ex cha n ge a nd c h rom a tofocusing chromatograph y The bPL rnole c ule w a s purif i e d 60 0 fo ld w ith t w o forms at 3 0 000 M W a nd pI' s of 4.9 5 a nd 5.15. The pur ifi e d p r o tein w a s utilized to develop a ntibodies in r a bbits. A radioimmunoass ay to bPL w as developed usin g a n a ntibod y rai s ed at the U SD A Belt s ville (F56). Approximatel y 2 0 % s pe c ific bindin g was X

PAGE 11

achieved with a 1:40,000 final working dilution of the antibody. Assay sensitivity was 300 ng/ml and the standard curve ranged from .1 8 ng. The antibody crossreacted with ovine placental lactogen at .2 96 Dose response curves of amniotic or allantoic fluid or fetal and maternal serum were parallel to the standard curve and bPL was quantitatively recovered at from 82 125 96 Using the radioimmunoassay, samples of amniotic and allantoic fluids and fetal and maternal serum were measured for bPL. Concentrations of bPL ranged from undetectable to 50 ng / ml with fetal blood having the highest concentrations and amniotic fluid the lowest. Concentrations of bPL were measured in plasma samples from 12 cows collected three times a week from day 150 to 250 of gestation and then daily until term. Peak concen trations of bPL were at days 210 and 230 of gestation which ma y correspond to peak fetal growth periods. Concentrations of bPL in blood s ample s collected at 30 min. intervals for a period of 1 2 hours were quite s imilar both within and between cows however two of the four animals sampled exhibited a spike of bPL secretion that was three to four times greater than baseline. XI

PAGE 12

CHAPTER I REVIEW OF LITERATURE History of Placental Lactogen Placental lactogen is a protein hormone secreted by the fetal portion of the placenta in several species (Talamantes, 197 5). It was first 'discovered' and named in 1962 (Josimovich and MacLaren, 1962) in the human. However, workers had postulated the presence of a placental mammotropin in the early 1900s. Bouchacourt (190 2) suggested that the placenta was responsible for 'witches-milk' in newborn infants and reported that the placental extract from a sow, called chorionine, was galactopoietic. Halban (1905) proposed that mammary gland development in pregnancy was controlled by substances secreted by the placenta. He based his views on clinical cases of lactation after ovariectomy or fetal death. At about the same time, Starling (1905) concluded from experiments in rabbi ts that fetal extracts contained a substance that stimulated mammary development. Hammond (1917) concurred with Starlings findings when removal of the fetuses from rabbits at days 13-15 of gestation arrested mammary growth and was followed by secretion of milk. Hypophysectomy became a routine experimental procedure in the early 1920s. Bell (1917) reported that hypophysectomy in the bitch resulted in mammary atrophy. This gave rise to the hypothesis that the pituitary gland was responsible for mammary gland function. Stricker and Grueter (192 8) strenghthen this hypothesis by inducing milk secretion in castrated virgin rabbits with a pituitary extract. To determine the effect of 1

PAGE 13

2 hypophysectomy on parturition and mammary development Selye et al. (1933) reported that rats hypophysectomized on days 1 0-14 of gestation had normal parturition; however, pregnancy was prolonged. Milk secretion was also normal at birth but stopped within the first 24 hours. These finding s suggested that a substance separate from the pituitary extract of Stricker and Grueter (1928) was involved in mammar y development. Sel y e et al. (1935b) ovariectomized rats at midgestation and removed the fetuses while leaving the placenta intact. This treatment had no adverse effect on the mammary glands, which were well developed and contained milk. They suggested that the placenta may produce a corpus luteum hormone because the uterus s howed distinct progestational changes. Newton and Lits (1938) reported that ovariectomy on day 12-14 in the mouse also had no effect on mammary development. Further investigation (Newton and Beck, 1939) yielded the first sugge s tion that the placenta ma y produce a substance other than an ovarian hormone. Mice were h y pophysectomized at day 12 of gestation and fetuses were removed leaving the placenta intact. M ice that either aborted the placentas or had the placentas removed s howed profound mammary gland involution. However, the involution was prevented by the pre s ence of placental ti s sue. Another intere s ting result was that mice that aborted lo s t 20 96 of their bodyweight b y day 18 while tho s e retaining placental tis s ue lost 3 96, suggesting that the placenta ma y act along with the ovarie s to prevent body weight loss in h y pophysectomized rat s The y hypothesized that because the mammary gland can attain full development in the absence of the pituitar y, either the mammar y gland is s ensitive to the s ame placental influences as the ovaries or it is s usceptible to s ome different and independent placental activity. More evidence s upporting the h y pothesis of a placental substance was put forth when Lyons (1944) reported that rats

PAGE 14

3 hypophysectomized and ovariectomized on days 7-8 of gestation and given replacement therapy of either estrone + progesterone or progesterone alone had normal lobulo-alveolar development. He stated that the placenta secretes substances comparable to the anterior pituitary which synergize with estrone and progesterone in their stimulation of lobulo-alveolar mammary growth and cause lactation. He called this substance the placental mammotropin. Mayer and Canivenc (1950) demonstrated that rat placental autografts placed into the abdominal cavity of females had prominent large trophoblastic cells and were responsible for luteotropic mammotropic and lactogenic effects. To further study the different roles of the placenta, Ray et al. (1955) described a series of experiments that attempted to ascer tain the crop-sac stimulating, luteotropic mammotropic and lactogenic functions attributed to the placenta, plus the site of origin of the agent. The y reported that two, day 12 rat placentas ( sa line sus pension) were adequate to s timulate the pigeon crop-sac, or when injected into normal rats (for 10 days) would inhibit the estrous cycle and stimulate lobule-alveolar development. In the hypoph ys ectomized and ovariectomized virgin rat treatment with estrone, proge s terone and day 12 placenta (either extract or fresh tissue) for 6 or 10 days stimulated lobulo-alveolar mammar y development. Placental extracts were lactogenic when a dministered in combination with hydrocortisone acetate, whereas, placental extract alone caused no gross lactation. To localize the tis su e that s ecreted or s tored the mammotropic activity, the placenta was separated into maternal and fetal regions. A homogenate of the separate regions was injected into h y poph ysecto mized-ovariectomized rats treated with estrone and progesterone. All rats injected with placenta estrone and progesterone s howed lobulo-alveolar development. A significant

PAGE 15

4 result of this experiment was that homogenates from the decidua capsularis region gave the same stimulating activity as whole placenta homogenates having 10 times the original mass. In 1958 Lyons reviewed the literature on mammary development. He concluded that because placental extracts synergize with estrone and progesterone in the hypophysectomized-ovar iectomized rat to induce lobulo-alveolar mammary growth, the rat placenta must produce a substance that imitates pituitary mammotropin. The evidence also suggests that a placental somatotropin is present because of the marked hyperplastic reaction in mammary development. Evidence for a placental somatotropin was reported by Josimovich and MacLaren (196 2). They described a protein present in human term placenta and pregnancy sera that reacted with antibodies to human growth hormone. This protein, which they named human placental lactogen, was highly lactogenic in both the pigeon crop-sac assay and in promoting milk synthesis in pseudopregnant rabbit; however it had little growth promoting activity in the rat tibial plate growth assay. Sciarra et al. (1963) used fluorescein labelled human growth hormone (hG H) to localize the growth hormone like protein in the syncytial cytoplasm of the villous trophoblast as early as the 12th week of gestation. The discovery of a protein produced by the placenta that is immunologically similar to human growth hormone with prolactin-like effects stimulated other researchers in this area. Kaplan and Grumbach (1964) reported the isolation of a protein from both human and simian term placentas that did have growth hormone and prolactin like activities. They coined the name chorionic growth hormone prolactin (CGP) and suggested that it was responsible for metabolic changes in the mother in the last trimester of pregnancy. They stated that CGP may serve as the metabolic hormone

PAGE 16

5 of pregnancy ensuring as one function a maternal store of nitrogen and minerals and the mobilization of fat to meet the requirements for fetal growth during pregnancy. The growth hormone-like activity of the placental protein was confirmed by Josimovich and Atwood (1964) but, they suggested that human placental lactogen (hPL) potentiated the effect of human growth hormone m the hypophysectomized rat tibial growth assay. In their assay it would require four to five times the amount of hGH used alone as it did when used in conjunction with hPL. Thus the controversy of whether human placental lactogen had somatotropic activities was solved. Josimovich (1966) fortified his position by reporting that four different hPL preparations potentiated the effect of hG H in both the rat tibial growth assay and in protection against insulin induced hypoglycemia. In that same year a new name was given to the placental protein. Florini et al. (1966) purified a placental protein and called it Purified Placental Protein (Human) (PPP[H]). They agreed with previous reports on lactogenic J nd somatotropic activities of their protein. Administration of placental lactogen to hypopituitary dwarfs gave conflicting results. Grumbach et al. (1966) compared free fatty acid concentrations in four children given either 400 mg CGP or 4 mg hGH. In each case free fatty acid concentrations increased over resting values however, hGH stimulated a larger increase than did CGP. In contrast, Schultz and Blizzard (1966) reported that in two male patients with idiopathic hypopituitarism, administration of 200 mg hPL had either no effect or a negative effect on nitrogen retention. They also found that hPL did not potentiate the effect of hGH on nitrogen retention. The differences in these two reports may be explained by different dosages of placental lactogen and different ages of patients.

PAGE 17

6 The similarities between hG H and hPL in immunological and biological properties stimulated further research in their chemical similarities. Catt et al. (1967) demonstrated that hPL has a molecular weight of 18,000 21,000. They also compared the first 17 amino acid residues from the amino terminus of hPL and hG H. Eleven of the seventeen residues were identical between the molecules which suggested that they were similiar in chemical structure. Sherwood (1967) extended the findings of Catt et al. (1967) by using trypsin digestion of both hPL and hGH. The tryptic peptides generated from hPL appeared to be identical or very s imilar to peptides from hGH. This suggested that pituitary growth hormone and placental lactogen were closely related with a common ancestor in the course of evolution. In the literature, four different laboratories purified the same placental factor and each has called it by a different name (Josimovich and MacLaren, 1962, Kaplan and Grumbach, 1964, Florini et al. 1966, and Friesen, 1965). In 1968 a group of these researchers met and roposed to name the placental factor chorionic somatomammotropin based on reported functions and site of synthesis (Li et al., 1968). In spite of this proposed terminology, to avoid confusion in the rest of this review, the placental protein will be called placental lactogen. Placental lactogens have been demonstrated in the human and monke y (Kaplan and Grumbach, 1964) and suggested to be present in the rat (Lyons, 1944) and mouse (Newton and Beck, 1939). Gusdon et al. (1970) examined placental extracts from several species to determine if a protein similar to hPL was produced. They used a hemagglutination-inhibition assay and cross-reaction with anti-hPL antibodies to determine the presence of a hPL-like molecule in the monkey, rat, dog, pig, horse, sheep, rabbit or cow.

PAGE 18

7 The monkey produced a large quantity of the lactogenic substance, which had been previously reported (Kaplan and Grumbach, 1964), but the other species produced only small amounts. Monkey placental lactogen was purified by Shome and Friesen (1971) who demonstrated that it was similar to hGH and hPL in molecular weight and amino acid composition. However, they found two forms of the protein and the quantities recovered were lower than in the purification of hPL. The presence of a goat placental lactogen was suggested by Buttle et al. (1972). Plasma samples from five goats were taken throughout gestation and were assayed for prolactin by radioimmunoassay and for lactogenic activity by a rabbit mammary gland organ culture method. The difference in lactogenic activity and prolactin concentration was attributed to a placental lactogen. They demonstrated a second lactogenic substance between the 9th and 15th weeks of gestation in the goat and concluded that this lactogenic substance was goat placental lactogen. In 1973 Shiu et al. developed a radioreceptor assay for prolactin using the mammary cell membranes from midpregnant rabbits. They demonstrated that prolactin (ovine, human, monkey and rat), hGH and hPL inhibited the binding of 1 25 r human prolactin. They suggested that this technique could be used to detect lactogenic hormones secreted during pregnancy. Using this method, Fellows et al. (197 4) reported the purification of ovine placental lactogen. Ovine placental lactogen was similar to hPL in its ability to bind to prolactin and growth hormone membrane receptors, but it had higher somatotropic activity (Handwerger et al., 197 4). They suggested that because of the similarities between ovine placental lactogen and hPL, the sheep may provide an excellent model for the study of placental lactogen. Talamantes (1975) examined nine species of mammals for the

PAGE 19

8 occurrence of a placental lactogen. He examined placental production of lactogenic activity using either explants or placental extracts from baboon, sheep, chinchilla, hampster, rat, mouse, guinea pig, rabbit and dog. Lactogenic activity in a mouse mammary gland co-culture assay as compared to prolactin was evident in all animals except the rabbit and dog, but, variation in production was noted. Robertson and Friesen (1975) reported the purification of rat placental lactogen. In the purified product they found two major bands and two minor bands on polyacrylamide gel electrophoresis. This heterogeneity is similar to that reported for growth hormone (Chrambach et al., 1973). The presence of a bovine placental lactogen was demonstrated by Buttle and Forsyth (1976). They determined plasma lactogenic activity in seven heifers across gestation using the method of Buttle et al. (1972) and placental production of lactogenic activity using co-culture of cotyledonary tissue and mouse mammary gland explants. There was no lactogenic activity attributed to a placental lactogen in an y of the 78 plasma samples examined. However, the co-culture technique showed that the cotyledon of the cow placenta from day 36, 180 and 270 of gestation did produce placental lactogen. They suggested that bovine placental lactogen may not be present in maternal plasma because of either a low secretion rate or a rapid clearance rate. Becka et al. (1977) reported the purification of goat placental lactogen from culture medium after tissue incubation. Explants of placental tissue were cultured for 4 days with a fresh change of medium every 24 hours. They demonstrated that placental proteins were secreted into the medium, but only .1 % of the proteins were associated with lactogenic activity. They suggested that goat placental lactogen was

PAGE 20

similar to ovine placental lactogen on the basis of lactogenic activity and electrophoretic mobilit y. Bovine placental lactogen was purified (Roy et al., 1977) and antibodies were raised in rabbits against the purified protein. In the radioimmunoassay there was no crossreactivity with ovine placental lactogen, bovine prolactin or growth hormone and hG H. Serum samples from late pregnancy were assayed using both the radioimmunoassay and a radioreceptor assay and had concentrations of less than 100 ng/ml. Similar findings were reported by Hayden and Forsyth (197 9). The assays utilized to detect the presence of placental lactogens have included the pigeon crop-sac a ss ay (Nicoll, 1967), organ culture assays using mammary explants from either the mouse or rabbit (Fors y th and Myres, 1971, Turkington 1971), radioreceptor assays (Shiu et al., 1973) and in a few cases, the radioimmunoassay (Roy et al., 1977). The problem with these assays is that they may not be s ensitive enough or s pecific enough to determine s mall concentrations of placental lactogen. Tanaka et al. (1980) developed a bioassay using the Nb2 lymphoma cell to determine the presence of lactogenic hormones. Lymphoma cell replication was stimulated in a dose dependent manner by prolactins (human, ovine, bovine and r at) ana placental lactogens (human, bovine and ovine) between 10 pg / ml and 4 ng / ml. This assay could be used in conjunction with a specific radioimmu'noassay for prolactin to determine concentrations of placental lactogen in seruni sa mples. Beckers et al. (1980) reported in detail the purification of bovine placental lactogen. They utilized a purification scheme that enriched the placental lactogen 1,500-fold over the original cotyledonary extract. These findings were confirmed by Murthy et al. (1982) and Eakle et al. (1982).

PAGE 21

10 These laboratories reported a molecular weight of 32,000 which is appreciably heavier than any of the other placental lactogens reported. Mouse placental lactogen was first suggested in 1939 (Newton and Beck, 1939), but the protein was not purified until 1982 (Colosi et al., 1982). They reported that mouse placental lactogen had a similar molecular weight to human and ovine placental lactogens. As more knowledge is attained about the placental lactogens more complex interrelationships are involved. Robertson et al. (1982) described the characterization of two forms of rat placental lactogen. They demonstrated that the two forms differed in molecular weight, isoelectric point and immunological properties. The forms also differed in lactogenic and somatotropic activities with the early form being more somatotropic and the late form being more lactogenic. The hypothesis was proposed that the early form may correspond to the placental luteotropin reported by Astwood and Greep (1938) whereas the late form was principally mammotropic. Servely et al. (1983) reported that high concentrations of antiprolactin receptor antibodies completely abolished the accumulation of 8 -casein mRNA induced by ovine placental lactogen in rabbit mammary gland explants and in coculture of ewe placenta and mammary tissue. This indicated that the placenta secreted a lactogenic factor which acted via the prolactin receptors. Voogt (1984) demonstrated that coincubation of day 11 rat placenta and rat pituitary gland significantly decreased the concentration of prolactin in the medium. This suggested that rat placental lactogen had a direct inhibitory effect on prolactin secretion in vitro. The prolactin like effect of rat placental lactogen was confirmed by Bussman and Deis (1984). They reported that Y -glutamyltransferase activity was maintained in mammary glands of ovariectomized rats treated with CB-154

PAGE 22

11 while ovariectomized-hysterectomized rats lost Y-glutamyltransferase activity. These findings suggest that rat placental lactogen can replace rat prolactin in stimulating mammary enzyme activity during pregnancy. Placental Type Grosser (1909) classified placentas based on the number of layers of tissue which, based upon the light microscopy, appeared to separate fetal from maternal bloodstreams. The epitheliochorial placenta, which is considered to represent the simplest form, has six layers: 1) endothelium of fetal capillaries, 2) fetal connective tissue or mesenchyme, 3) fetal chorionic epithelium, 4) maternal uterine epithelium 5 ) maternal connective tissue and 6) maternal endothelium. It was thought that the number of tissue layers was directly related to the permeabilit y of the placental barrier (Steven, 1975). Barcroft (1946) disagreed with this concept s tating that with few exceptions the greater the number of tissue la y ers the more fully developed the animal was at birth. Both pigs and horses have epitheliochorial placenta. Cattle and sheep were classified as having syndesmochorial t y pe placenta because of the apparent absence, under light microscopic examination, of the maternal epithelial la y er. Recentl y, s heep and cattle placenta have been examined using electron microscopy which indicated that the correct placental type was epitheliochorial (Bjorkman, 1968). Grosser (1909) classified other placental types as the endotheliochorial having five layers as in dogs and cats and the hemochorial having four la y ers as in the rodents and most primates. Enders (1965) demonstrated using electron microscopy that some capillaries of the hemochorial-t y pe placenta are covered by one or more layers of attenuated chorion. This finding gave rise to three hemochorial subgroups: 1) hemomonochorial of man, 2) hemodichorial of the rabbit, and 3) hemotrichorial of the rat and mouse. The placentas within one classification (Grossner, 1909) ma y have

PAGE 23

12 widely different functional characteristics (Steven, 1975). Placental shape may act to group placental types more efficiently (Steven, 1975). For the diffuse placenta of the mare and pig, the outer surface of the chorion is covered by small villi or folds which lay in intimate contact with the uterine epithelium. The majority of ruminants have a cotyledonary placenta where chorionic villi are restricted to a well defined area of the chorionic sac. The cow, sheep and goat have this type placenta (Amoroso, 1952). The zonary placenta of the dog and cat has a equatorial girdle of chorionic villi, which may be complete as in the dog or incomplete as in the bear (Young, 1968). A discoid placenta is found in man, rodents and rabbits. In this placental type the chorionic villi are restricted to a s ingle disc shaped area. Wimsatt (1962) stated that the trophoblast is probably the most important tissue m the placenta of higher mammals. The trophoblast arises before implantation, mediates attachment of the blastocyst, serves as the nutritive front of the conceptus and develops important s ecretory and regulatory functions. The trophoblast in the allantoic placenta has three major cytologic configurations, which may indicate physiologic specialization. The cytotrophoblast, which may be the most common t y pe, has distinct cellular boundaries which are in an epithelial arrangement. In the syncytiotrophoblast cell membranes are lacking and the nuclei are scattered at random or in clumps throughout the cytoplasm. The third form consists of independent structures, the trophoblastic giant cells, which can be uninucleate or binucleate (Wimsatt, 1962). The ultrastructure of the various placentas may var y even within a group. The placenta of the pig, which is a diffuse placenta in the epitheliochorial classification, has a band between maternal epithelium

PAGE 24

13 and trophoblast of mutually interdigitating maternal and fetal microvilli (Bjorkman, 1965). Over the mouths of the uterine glands the trophoblast is not attached to the uterine epithelium, but forms regular or irregular areolae for his to trophic nutrition (A morose, 19 5 2). The trophoblast cells contain numerous mitochondria in the apical portion and globular dense granules in the basal part (Bjorkman, 1970). The horse placenta is also of the diffuse type, but tufts of chorionic villi dip into maternal crypts to form microcotyledons. Areolae, which form between the microcotyledons, are associated with uterine glands, which are numerous within the endometrial stroma. The trophoblast is cellular and uninucleate (Bjorkman, 1970). The placenta of the cow is a cotyledonary placenta with binucleate giant cells present in the trophoblast. Wimsatt (1951) considered the binucleate cell to be homologous to the syncytical trophoblast in the deciduate placenta. Bjorkman (1954) described PAS-positive staining within the binucleate cells and suggested that a chorionic gonadotropin may be secreted. Wooding and Wathes (1980) reported that fetal binucleate cells migrated between the chorionic and uterine epithelia throughout pregnancy. They suggested that binucleate cell migration may be required to transfer large molecules across the microvillus junction. Binucleate cell migration was studied more intensively by Wooding (1983), who reported that 15 to 20 % of the trophectodermal epithelial cells were binucleate. Of these cells, 20 % were found to be migrating up to and across the microvillus junction at all stages of pregnancy examined. The sheep placenta, which is also cotyledonary, differs from the cow placenta in the shape of the placentome as well as in the fact that a uterine syncytium is present (Bjorkman, 1970). The trophoblast of the sheep placenta is similar to that of the cow in that

PAGE 25

14 fetal binucleate cells are present. Wimsatt (1962) suggested that the binucleate cells fuse to form a functional syncytialtrophoblast in the sheep. However, other workers (Bjorkman, 1970, Boshier and Holloway, 1977) determined that the syncytial layer was derived from maternal uterine epithelium. Recently, Wooding (1980) reported that sheep binucleate cells migrate across the microvillus junction to form the syncytium. The binucleate cells of the sheep have been implicated in producing ovine placental lactogen (Martal et al., 1977). This and the migration pattern of the binucleate cells (Wooding, 1983) indicated how high cncentrations of ovine placental lactogen are secreted in to the maternal system. The endothelial placenta is characteristic of most carnivores. In the dog the placenta is zonary or labyrinthine. The labyrinthine type placenta are those in which the maternal vessels are surrounded by invading trophoblast so that the vessels come to lie partially or exclusively within the boundaries of the trophoblast (Steven, 1975). The ultrastructure of the dog placenta is made up of maternal capillaries that are surrounded first by a dark syncytium with well developed rough endoplasmic reticulum and numerous mitochondria and second by a light cytotrophoblast (Bjorkman, 1970). Anderson (1969) described the presence of decidual giant cells within the syncytium. He stated that the cells were connective tissue of maternal origin which had been surrounded by the syncytium in the same manner as the maternal capillary. The cat placenta is the same type as the dog placenta. The cat lamellae seem to run at right angles to the uterine surface. As in the dog maternal capillaries are surrounded by a dark syncytium and light cytotrophoblast. Decidual giant cells are apparent within the syncytium (Dempsey and Wislocki, 1956) and are derived from fibroblasts which are

PAGE 26

15 transformed into giant cells when the fetal trophoblast invades the uterine endometrium. The hemochorial placental morphology varies tremendously between species, many of which are entirely unrelated except for the hemochorial placenta. The hemomonochorial type placenta is seen in the human, some monkeys and the nine-banded armadillo (Bjorkman, 1970). In the human placenta the fetal capillary is surrounded by a light cytotrophoblast which is surrounded by a syncytiotrophoblast layer. The syncytium is in direct contact with the maternal blood s pace and has free microvilli for absorbing nutrients (Bjorkman, 1970). The cytoplasm of the sy ncytium is electron dense with free ribosomes and a very well developed rough endoplasmic reticulum. This may suggest high synthetic activity (Bjorkman, 1970). Pierce and Midgley (1963) suggested that cytotrophoblast cells were undifferentiated cell types that matured into syncytiotrophoblastic giant cells. They also demonstrated that the syncytial giant cell s ecretes human chorionic gonadotropin (hCG) using fluorescein labelled antibody against hCG. Sciarra et al. (1963) reported that human syncytial cytoplasm contained human placental lactogen. They used flourescein labelled hG H to identify the immunologically similar protein. The hemodichorial placenta of the rabbit also a labyrinthine type placenta, was described by Larsen (1962). He stated that the trophoblast consisted of a cellular and syncytial component. The cytotrophoblast contained mononuclear cells with oval nuclei and a light cytoplasm. The syncytial trophoblast was found as a sheet covering the cytotrophoblastic cells and surrounding a s pace containing maternal blood. Larsen (1962) reported the presence of multinucleated giant cells in the intermediate zone of the placenta. The intermediate zone was found

PAGE 27

16 separating the syncytium from the maternal tissue. He suggested that the multinucleated giant cells were fetal in origin and ma y be responsible for transfer of nutrients. The hemotrichorial placenta of the rat and mouse are similar in ultrastructure and labyrinthine in type (Jollie, 1964a, Kirby ancl Rranta c0nsist<: of four cytopl::ismir. lay8rs from the maternal blood sinus to fetal capillary lumen. These layers are called, 1) trophoblast I, 2) trophoblast II, 3) element III, and 4) endothelium. Large clusters of trophoblastic giant cells can be seen within the labyrinth (Bjorkman, 1970). The giant cells lie within the junctional zone which is adjacent to the decidua basalis (Jollie, 1964a). In the placental types described previousl y, a c ommon theme is seen. With the possible exception of the rabbit, if trophoblastic giant cells are present, the placenta s ecretes the hormone placental lactogen. As previously s tated binucleate giant cells have been implicated in producing placental lactogen (sheep, Martal et al., 1977 cattle, Wallace, 1985, and human, Sciarra et al., 1963). Placental lactogen is present in the mou se and rat, but tissue localization studies have not been reported. The pig and horse have no trophoblastic giant cells and the dog a nd cat have giant cells but it is decidual in origin. placental lactogen may be pre se nt in the rabbit, but reports are conflicting. Endocrine Control of Mammary Development Mammary growth is a continuous process from embryonic life through reproductive senescence. The mammary gland is an integral part of the reproductive process in all mammals. Just as the placenta delivers nutrients to the developing fetus during intrauterine life, the mammary glands contain nutrients for the extrauterine survival of the young. The endocrine control

PAGE 28

17 of the mammary gland, to synchronize its development and secretion with the needs of the offspring, has intrigued researchers for centuries. Lane-Claypon and Starling (1906) were the first to examine the stimulus for mammary growth using experimental methods . They administered aqueous extracts of placenta, fetuses or ovaries to virgin rabbits and concluded that the fetus contained a product that stimulated mammary growth. Estrogen. The isolation of estrogen (Allen and Doisy, 1923) was followed by reports of its stimulation of mammary growth in mice and rats. Allen et al. (1924) demonstrated that estrogen injection in ovariectomized rats and mice stimulated mammary gland duct growth. Species variation in response to estrogen administration is quite marked. Duct development was stimulated by estrogen in the mouse (Bradbury, 1932), cat (Turner and De!Vloss, 1934), dog (Turner and Gomez, 1934) and rat (Turner and Schultze, 1931). Although in the rabbit (Frazier and Mu, 1935) and guinea pig (Nelson and Smelser, 1933) estrogen also stimulated lobulo-alveolar development. Turner and Allen (1933) demonstrated that estrogen administered over a period of 65 days induced lobulo-alveolar development in male rhesus monkeys. Progesterone. Turner and Schultze (1931) demonstrated that administration of progesterone to rats and rabbits had no effect on mammary development. However, these workers were able to induce lobule formation by injection of progesterone and estrogen in animals that had been pretreated with estrogen. The importance of estrogen-progesterone synergism in lobulo-alveolar mammary development was clearly demonstrated (Turner and Frank, 1931). They reported that estrogen stimulated ductal development in castrated male or female rabbits. However, greatly increasing the dosage had no increased effect. Injection of a crude corpus luteum extract into

PAGE 29

18 estrogen primed rabbits had no stimulator y effect. However, the injection of estrogen plus the crude corpus luteum extract stimulated lobulo-alveolar development. Pituitar y Hormone!3. The interest of mammar y ph y siologists in hormonal control of mammary development was temporarily diverted to the pituitar y when Stricker and Grueter (1928) induced milk secretion in ovariectomized virgin rabbits with a pituitary extract. In the absence of the pituitar y, estrogen and progesterone have no effect on mammary gland development ( Sel y e et al., 19 3 5a). Gomez et al. (1937) reported that hypoph y sectomized male guinea pigs treated w ith pituitaries from estrogen primed rats had extensive a lveolar development. These findings suggested that estrogens stimulated mamm a r y development b y working through the pituitar y gland. Gomez and Turner ( 19 3 8) proposed the hypothesis that there were two pituitar y fact ors involved m mammogenesis; the duct growth factor, which wa s s timul a t ed b y e s trogen s ecretion and the lobulo-alveolar growth fs1ctor, which was s timulated b y estrogen plus progesterone. Thi s h y pothesi s w as s upported b y the w ork of Nathanson et al. (1939) who demonstrated ductal d evelopment in hypophysectomized rats treated with estrogen plus pituitar y extra c t s, w hich the y called growth complex. L y ons (1 943 ) r eported that m the hypophysectomized-ovariectomized rat, estrogen proge s terone and prolactin provided the minimal hormonal requirement s fo r s timulatin g lobulo-alveolar development. This finding was expanded ( L y ons et al. i 953) w hen full lobulo-alveolar development was attained in hypoph ys e c tomized-ovariecto mized rats treated with the above hormones plus purified growth h ormone The importance of the pituitary hormones was s lightl y diminished when

PAGE 30

19 Ahren and Jacobsohn (1956) demonstrated that long acting insulin plus estrogen and progesterone stimulated mammary development in the absence of pituitary hormones. They suggested that any hormone with powerful metabolic actions can play a role in mammary gland growth. Placental Hormones. Placental involvement in mammmogenesis was first suggested for mice (Newton and Beck, 1939) and rats (Lyons, 1944). The y suggested that the placenta produced a mammotropin that could replace pituitary prolactin in stimulation of the mammary gland. Ra y et al. (1955) compared the placental factors of the rat and pituitar y hormones in their ability to stimulate mammary development and reported that the action of rat placental mammotropin was similar to that of pituitar y prolactin. Hypophysectomy of rats had no effect on mammar y growth at days 12 and 20 of gestation using the DNA method of quantitation (Anderson and Turneri 1969). Positive significant correlations were obtained between both fetal number and weight of placentas and mammar y development' in mice measured by RNA, DNA or RNA / DNA ratio ( Nagasawa a nd Yanai 1971). The y s uggested that the placental mammotropic hormone ma y pla y an important role in mammar y development during pregnanc y Anderson ( 1975) demonstrated that increasing the number of fetal placental units in either intact or hypophysectomized rats increased mammar y DNA co ncentration. Removal of the fetuses in hypoph y sectomized rats w hile leaving 0-5 placentas intact indicated that as little as two placentas were required to a llow mammar y DNA values to reach control values. Schams et al. (1984) examined the effects of bromocryptine on mammary gland development in ewes and heifers. Administration of bromocryptine to ewes markedl y decreased prolactin concentrations, but had no effect on serum placental lactogen concentrations.

PAGE 31

20 Mammary glands of bromocryptine treated ewes were similar to controls. In some lobules the secretion of lipid droplets was diminished. In heifers, bromocryptine treatment depressed serum prolactin concentrations, but placental lactogen was not measured. Mammary development in heifers was similar to that for ewes. These findings suggest that placental lactogen may be able to replace pituitary prolactin in the stimulation of mammary development. As stated previousl y, the hormones involved in mammary development are estrogens, progesterone and pituitary hormones (prolactin and growth hormone) or placental lactogen. The placenta of many mammals secrete steroids as well as protein hormones. Progesterone secretion by the placenta has been reported for the human (Diczfalusy and Borell, 1961 ), sheep (Linzell and Heap, 1968), guinea pig (Heap and Deanesl y, 1966), cow (Erb et al., 1967) and rat (Csapo and Wiest, 1969). In the ovariectomized guinea pig plasma progesterone was shown to be correlated with placental weight (Heap and Deanesly 1966). Linzell and Heap (1968) reported that sheep placenta secrete up to 14 mg progesterone per da y, which is about five times greater than ovarian secretion. Placental hypertrophy was reported in ovariectomized rats that maintained pregnancie s (Csapo and Wiest, 1969). Estrogen secretion by the placenta of mammals is even more widespread than progesterone. There is no known species wit h a gestation length longer than 70 da ys that doesn't have placental estrogen synthesis (Davies and Ryan, 197 2). Estrogen synthesis was demonstrated in vitro for the sheep, cow, horse and sow placenta (Ainsworth and R ya n, 1966). Tissue preparations were incubated in the presence of androstenedione and dehydroepiandrosterone for 1 hr at 37 C. Tissue preparations incubated with either pregnenolone or progesterone failed to synthesize estrogens. These findings supported the concept of the fetoplacental unit (Diczfalusy, 1964). According to this

PAGE 32

21 concept the fetus and placenta together carr y out steroid bios y nthetic path w a y s whi c h neither could do alone. In the case of estrogen secretion the placenta secretes progesterone to the fetus which converts it to androstenedione in the adrenal. The androstenedione then is converted b y the placenta into estrogen. In Vitro Studies. To remove the effects of possible hormonal interactions within the animal, Lasfargues and M urra y (1959) suggested that the ideal approach would be to stud y mammar y development in a ph y sio logically defined environment. The y re c alled the w ork of Hard y ( 1950) who reported differentiation of mouse mammar y ducts i n vitro. Organ c ulture of explants of 10-15 da y old mouse a bdominal w all wa s utilized t o determine the effects of hormonal stimulation of mammar y diff e rentiation ( Lasfargue s and M urra y, 1959). The results indicated that estradiol a nd pro g esterone inhibit growth of the mammar y e pithelium w hile g rowth hormone and prolactin promote active growth and c ortisol i nduced di s tension of d ucts to form a lveoli. The influence of c ortisol on explant s from prelact a ting mice w as s tudied b y Rivera and Bern (1961) w ho demonstrated that c orti s ol plus in s ulin c ould maintain alveolar s tructure but onl y in the presen c e o f prol ac tin or g rowth hormone was s ecretor y activit y maintained or s timulated. In mi c e pretreated with e ither estrogen a nd proge s terone fo r 9 d a y s o r es tro g en proge s t e rone prolactin and growth hormone for 7 d a ys, lobulea lve o lar differentiation w as attained onl y when explant s were c ultured in t he presence of estrogen progesterone aldosterone prolactin growth hormone a nd insulin ( Ichinose and Nandi 1966). To determine the s teroidal requirement s e xplants from pretreated mice were cultured in the presence of prola c tin growth hormone and insulin plus various steroid hormones. Lobule-alveolar differentiation was s timulated when aldosterone was in the medium either a lone or i n

PAGE 33

22 combination with estrogen or progesterone. In fetal mouse mammary tissue insulin is required for prolactin to stimulate duct growth while aldosterone and progesterone enhance ductal branching and lobule-alveolar differentiation in the presence of insulin and prolactin (Ceriani, 1970). Turkington and Topper (1966) reported that hPL in combination with insulin and hydrocortisone stimulated midpregnant mouse mammary tissue to approximate the alveolar development of full term pregnancy. This combination of hormones also stimulated casein synthesis, which indicated that hPL may have an important role in mammary gland development during pregnancy. Recently the effect of epidermal growth factor on mammary development has been reported. Tonelli and Sorof (1980) indicated that cultured mouse mammary glands that had undergone development in the presence of prolactin, insulin, aldosterone and hydrocortisol and then regression by removing the hormonal stimulation could be stimulated to develop a second time when epidermal growth factor was added to the medium along with the above stimulatory hormones. Epidermal growth factor had no effect on the first cycle of development. They suggested that endogenous epidermal growth factor was available in the first cycle. The addition of epidermal growth factor to cultured mouse mammary epithelial cells stimulated cell proliferation (Taketani and Oka, 1983). Casein production by mammary epithelial cells was inhibited by epidermal growth factor. One possible explanation for the inhibition of casein s y nthesis was that binding of epidermal growth factor may block the prolactin receptors and thus inhibit prolactin binding. The organ culture method has indicated that hormones directly affect the mammary gland to stimulate differentiation. The presence of a receptor for these stimulatory hormones has been postulated. Shiu et al. (1973) identified a specific receptor site for prolactin or lactogenic hormones in

PAGE 34

23 membrane fraction s obtained from pregnant or lactating rabbits. Purification of the prolactin receptor from rabbit mammar y glands was reported (Shiu and Friesen, 197 4). Djiane and Durand (1977) described the regulation of prolactin receptor numbers in the rabbit mammar y gland. The y demonstrated that receptor numbers increased in mammar y tissue from rabbit s treated with 100 I. U. ovine prolactin, whereas progesterone plu s prolactin treatment had no effect. The self-regulation of prolactin receptors b y prolactin was confirmed in the rat (Bohnet et al. 1977). The y demonstrated that a peak in receptor number s coincided with the peak in prolactin after parturition. Inje c tion of either e s tradiol valerate 17 h y drox y progesterone c aproate or bromocr y ptine reduced prolactin receptor numbers c ompared to control s Injection of anti-prolactin re c eptor s erum into lactating ::-ats resulted in increased serum prolactin a nd decreased litter weight g ains (Bohnet et al. 197 8). The y s uggested th a t the anti s erum ma y inhibit some of the effe c t s of endogenous prolactin. Ha s lam and Sh y amala (1979) reported that progesterone receptor numbers in mi c e were inver s ely proportional to the s ecretor y a ctivit y of the gland. Secretion Placental lactogen s are secreted by the trophoblastic portion of the placenta (Sciarra et al. 1963, Ma rtal et al. 1977). However the re g ulation of that s ecretion s till i s unclear. To determine the r e gulator y mechanisms involved in s ecretion of placental lactogens resear c hers have developed two methods The fir s t was to manipulate the w hole a nimal (or human) b y increasing or decreasing metabolite s thought to be important in placental lactogen secretion, and s econd to culture placental tissue (explants, di s per s ed cell s or whole placenta) in presence or absence of hormones or metabolites.

PAGE 35

24 Changes in Concentrations. Radioimmunoassays were first developed for hPL (Kaplan and Grumbach, 1965, Beck et al., 1965) to determine the pattern of secretion. Placental lactogen concentrations of .53g/ml in pregnant women were detectable at 8 weeks of gestation and increased to 10-40 g/ml at term (Kaplan and Grumbach, 1965). Human placental lactogen concentrations fell drastically after parturition. The presence of hPL in the placenta was detectable at day 12 (Beck, 197 0). At term, concentrations of hPL in umbilical plasma were reported as undetectable (Beck et al., 1965) or 50 to 200 times lower than maternal concentrations (Kaplan and Grumbach, 1965) and amniotic fluid hPL concentrations ranged from 2-11 / ml. The estimated halflife of hPL was 2 12 3 min. Taking into account maternal concentrations and the reported halflife, a production rate of 3-12g hPL per day was proposed (Beck et al., 196 5). Infusions of hPL into men or nonpregnant women at 93-373 / min resulted in plasma hPL concentrations of 2-3 g/ml after 90 min (Beck and Daughaday, 1967). Spellacy et al. (1966) stated that hPL concentrations were not related to placental or fetal weight. However, Sciarra et al. (1968) reported a low positive correlation between hPL concentrations and placental weight in patients between 38 and 42 weeks of gestation. Concentrations of hPL vary randomly over a 24 hr period (Pavlou et al., 1972) with no apparent diurnal rhythm. Vigneri et al. (1975) suggested that hPL concentrations fluctuate irregularly, therefore, a frequent sampling procedure is required to correctly determine the secretory activity of hPL. Ovine placental lactogen, as measured by radioimmunoassa y (Handwerger et al., 1977, Chan et al., 1978b) was first detectable at da y 40 of gestation and reached a peak of 2,400 ng/ml at day 130. Concentrations of ovine placental lactogen in umbilical cord plasma and allantoic fluid were

PAGE 36

25 approximately one-tenth and one-hundredth of maternal concentrations, respectively (Handwerger et al., 1977). While amniotic fluid had concentrations of 5-90 ng/ml of ovine placental lactogen at day 50, levels were undetectable thereafter (Chan et al., 1978b). Chronic catheterization of animals is an ideal method for examining the secretion of ovine placental lactogen (Gluckman et al., 1979, Taylor et al., 1980). Fetal concentrations of ovine placental lactogen peaked at days 120-124 while maternal concentrations peaked at days 130-139 and were 10 times that for the fetus (Taylor et al., 1980). Fetal concentrations were unaffected by fetal numbers; however maternal concentrations increased significantly with increasing fetal numbers (Taylor et al., 1980). Kelly et al. (1976) reported lactogenic and somatctropic activities in se rum or plasma samples throughout pregnanc y of nine species. Two pe aks of lactogenic activity were detected in mi ce and rats, one at mi dg estation and one near term. Peaks of somatotropic activities were coincident with lactogenic peaks; however, the first peak was much lower than the lactogenic peak and the second much higher. Peaks of lactogenic and s omatotropic activities were similar in the hampster, guinea pig, goat, sheep, monkey and human. Somatotropic activities were much lower in the hampster, guinea pig sheep and human and higher in the goat. Activities were low throughout gestation in the cow. Concentrations of placental lactogen in the goat increases with fetal number (Hayden et al., 1979) and plateaued from 16 weeks of gestation until term. Bovine placental lactogen concentrations were measured using the Nb2 lymphoma assay (Schellenberg and Friesen 1982). Placental la c togen concentrations in maternal plasma were below the sensitivity of the assay while fetal samples from day 180

PAGE 37

26 of gestation revealed concentrations between 5 and 22 ng/ml. They suggested that bovine placental lactogen acts to stimulate fetal growth. The presence of placental lactogen in rodents has been postulated since Newton and Beck (1939); but not until recently has a mouse or rat placental lactogen been purified and a radioimmunoassay developed. Robertson and Friesen (1981) compared rat placental lactogen concentrations measured in either a radioreceptor assay or radioimmunoassay. The radioreceptor assay detected two similar peaks of 1,000 ng/ml at days 11-13 and 13-21 whereas the radioimmunoassay detected a peak at day 11-13 which was minor and a major peak of 1,000 ng/ml at days 13-21. Plasma samples collected at 5-10 min intervals from rats at day 19 of gestation were used to demonstrate that rat placental lactogen concentrations vary greatly over short periods of time (Klindt et al. 1982). A radioimmunoassay for mouse placental lactogen (Soares et al., 1982) was used to measure placental lactogen concentrations. Using both a radioreceptor assay and radioimmunoassay, concentrations of mouse placental lactogen ranged from 1 ng/ml at day 9 to greater than 250 ng/ml at day 18 and were similar as measured by the two assays. However, the radioreceptor assay detected a large peak at day 10 while the radioimmunoassay did not. Profiles of mouse placental lactogen were different between different strains of mice. Markoff and Talamantes (1981) indicated that mouse placental lactogen concentrations increased with fetal numbers. In summary, concentrations of placental lactogen in the maternal system vary greatly between species. Concentrations of hPL peaked at 10-40 g/ml (Handwerger et al., 1977), while bovine placental lactogen concentrations were below the sensitivity of the assay utilized (Schellenberg and Friesen, 1982). The pattern of placental lactogen secretion was similar

PAGE 38

27 among the species reported, with placental lactogen being first detected at low concentrations and increasing almost linearl y to just before term. Concentrations of placental lactogen in the fetal circulation, as well as, amniotic and allantoic fluids, were lower than those for maternal blood. The onl y apparent regulatory mechanism was the association between increasing placental mass or fetal numbers and placental lactogen concentrations in maternal blood. Nutritional Effects on Secretion. Reports on the effects of alteration of hormonal or metabolic factors on hPL c oncentrations have been c onflicting. Kaplan and Grumbach ( 1964) suggested that human placental lactogen ma y s erve as the metabolic hormone of pregnanc y Possible f un c tion s i ncluded increased nitrogen retention increasing free fatt y acids increasing c irculating insulin, resistance to exogenous insulin increased tran s fer of a mino acids across the placenta and fetal growth. Spellac y et a l. ( 1 9 66) demonstrated that hPL c oncentrations were unaffected b y h y peror h y pogl y cemia or time of da y Subsequent reports indicated that g lucose admini s tration to pre g nant women after a 15 hr fast s ignificantl y reduced hPL concentrations during the first 3 0 min (Burt et al. 1970) w hile fa s ting pregnant w omen for 8 4 hr to induce h y pogl y cemia s ignific a ntl y elevated hPL c oncentr a tion s ( Kim and Felig, 1971). Prieto et al. (1976) reported that h PL c oncentr a tions w ere not affected b y glucose a dministered either orall y o r a s a c ontinuou s i nfusion. The y did state that an acute pulse of g lucose tran s ientl y s uppressed hPL c oncentrations. Variations in goat placental lactogen c oncentrations w ere not correlated with changes in blood g lucose ( Ha y den et a l. 1980) while culture of human placental tissue in the presence of 2 x g lu c ose inhibited hPL release (Belleville et al., 1979) Plasma hPL c oncentrations were unaffected by free fatty acid concentrations (Gaspard et al. 197 5) which

PAGE 39

28 is similar to results in the goat (Hayden et al., 1980). Arginine infusion had no effect on hPL concentrations (Tyson et al., 1969) while there was a dramatic increase in ovine placental lactogen concentrations 2-3 hr after the start of infusion of arginine (Handwerger et al., 197 8). Culture of bovine placental tissue in the presence of arginine stimulated the release of bovine placental lactogen (Forsyth and Ha y den, l 98UJ. Infusion of either alanine or glycine slightly increased ovine placental lactogen concentrations while glutamic acid had no effect (Handwerger et al., 1978). Subsequent reports demonstrated that infusion of ornithine stimulated ovine placental lactogen secretion while citrulline had no effect (Handwerger et al., 1981c). In summary, nutritional metabolites such as glucose or free fatty acids had either a transient or non-existant effect on maternal placental lactogen concentrations in the species studied. Manipulation of the glucose concentration in medium used for culture of human placental tissue demonstrated that glucose was required for hPL secretion, but increasing or decreasing the minimum glucose c oncentration inhibited hPL release (Belleville et al., 1979). Hypogl y cemia induced by fasting pregnant women s timulated hPL concentrations, but the mechanism is not clear. Infusion of amino acids into s heep and possibl y cows ma y affect the release of placental lactogens but again the mechanism was not s tudied. Placental lactogens ma y be the metabolic hormone of pregnanc y as Kaplan and Grumbach (1964) theorized; however, extensive and well designed research will be required to determine the exact function. Hormonal Effects on Secretior.. Culture of placental tissue to determine the regulator y mechanisms involved in secretion of placental lactogen was first reported for the human (Suwa and Friesen, 1969). The y c ultured explants from human term placentas in the presence of 3H-leucine. Eighty percent

PAGE 40

29 of the hPL released into the medium was released in the first 24 hr. The addition of hPL, progesterone, insulin, cortisol or dibutyryl cyclic AMP had no effect on the release of hPL. Placental explants cultured for 96 hr demonstrated that during the first 72 hr hPL constituted approximately 10 % of the proteins secreted into the medium; however between 72 and 96 hr there was a dramatic increase in secretion of hPL so that it constituted approximately 50% of the proteins secreted into the medium (Friesen et al., 1969). Stimulation of hPL release in culture in response to addition of pimozide (Macaron et al., 1978), estradiol (Belleville et al., 1978), arachidonic acid (Handwerger et al., 1981a), EDTA, EGTA or methoxyverapamil (Handwerger et al., 1981) and insulin (Perlman et al., 1978) has been reported. Addition of adrenalin, noradrenalin or progesterone (Belleville et al., 1978), as well as, dopamine (Macaron et al., 197 8) inhibited hPL release and prostaglandins E2 or F2a (Belleville et al., 1978) or s omatostatin (Macaron et al., 1978) had no e ffe ct on hPL s ecretion in vitro. Cyclohexamide and dopamine had no effect on bovine placental explants in secretion of bovine placental lactogen (Forsyth and Ha y den 1980). Maca ron et al. (1978) suggested that hPL release ma y be modul a ted by dopaminergi c I receptors while Handwfrger et al. (1981b) s ugge s ted that calcium flux ma y mediate hPL release. Administration of either prostaglandin E2 or F2a (Keller et al., 1972) or thyroid relea s ing hormone (Her s hman et al., 197 3) to pregnant women had no effe c t on hPL c oncentrations. Ylikorkala and Pennanen (1973) reported that induction of abortion with PGF2a by either intra or extra amniotic routes decreased hPL s ecretion. The decrease was greatest in patients given PGF2a by the extra a mniotic route while sa line had no effect on hPL concentrations. Manipulation of proge s terone concentrations in pregnant sheep had no effect on ovine placental lactogen

PAGE 41

30 concentrations (Taylor et al., 1982). However, Moore et al. (1984) recently demonstrated that infusion of epidermal growth factor for 24-28 hr significantly increased ovine placental lactogen concentrations in pregnant sheep. Thorburn et al. (1981) suggested that epidermal growth factor may affect transplacental migration of binucleate cells which stimulates the secretion of ovine placental lactogen. A second theory is that epidermal growth factor stimulates the production of binucleate cells from mononucleate cells to increase the number of migrating binucleate cells. In summary, hormonal manipulation either in vivo or in vitro has been shown to affect placental lactogen secretion, but the mechanism of action was not identified. The proposed hypotheses of either dopaminergic receptors (Macaron et al., 1978) or calcium flux (Handwerger et al., 1981b) mediating hPL release gave an indication of the complexity of the regulation of placental lactogen secretion. Placental lactogen secretion does not appear to be autonomous as suggested for the human (Spellacy et al., 1966). In vivo and in vitro studies have demonstrated that manipulation of hormonal or metabolic factors can effect the secretion rate of placental lactogen. Fasted pregnant women had elevated hPL concentrations (Kim and Felig, 1971); however, the direct cause of the increase was not addressed. The need for elucidation of the mechanism involved in regulation of placental lactogen remains. Function Since the discovery of human placental lactogen in 1962 (Josimovich and MacLaren, 1962) the search for the role of this placental peptide has continued. The exact function of placental lactogen remains unclear; however, the original hypothesis of Kaplan and Grumbach (1964) still stimulates further research. Potential activities of placental lactogen that have received major attention are: 1) lactogenic activity, 2) somatotropic activity, 3) luteotropic activity, and 4) miscellaneous function.

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31 Lactogenic Activity. Josimovich and MacLaren (1962) reported that hP L was lactogenic in the pigeon crop sac and rabbit intraductal assay and was 50% as potent as NIH ovine prolactin. This lactogenic activity was responsible for the name placental lactogen. Lactogenic activity was confirmed for hPL (Kaplan and Grumbach, 1964) and demonstrated for ovine placental lactogen (Handwerger et al., 197 4). Talamantes (197 5) exploited the lactogenic activity of placental lactogen to determine the presence of this hormone in nine different species. Mouse mammary tissue cultured in the presence of placental extracts demonstrated lactogenic activity in the baboon sheep, chi nchilla, hampster rat, mouse and guinea pig, but not the rabbit and dog. Forsyth (1974) used a mammar y and placental c o-culture technique to demonstrate the presence of placental lactogen in the goat, cow, sheep and fallow deer, but not the pig. Shiu et al. (1973) developed a radioreceptor assay utilizing the prolactin rece ptor from rabbit mammar y gland membranes. This technique llas been utilized to expand the knowledge of placental lactogens in s everal species. Reddy and Watkins ( 1975) injected 1 25 1 hPL into rats to determine tissue distribution. Labelled hPL was found mainly in the kidney and mammar y gland. Imm unohistochemical localization indicated that hPL bound to the proximal tubule of the kidne y and the a lveolar cell membrane of the mammar y gland. Turkington (1968) demonstrated that addition of hPL to mouse mammar y explants in the presence of insulin induced production of case in a -lactalbumin and B-lactoglobulin. The addition of colchicine or actinomycin D to the culture media demonstrated that hPL s timulated differentiated cells formed in vitro through a DNA directed RN A sy nthesis. Mammar y development in hypophysectomized pregnant animals has suggested a role for placental lactogen in the mouse (Selye et al., 1933), rat (Lyons, 1944), guinea pig

PAGE 43

32 (Pencharz and Lyons, 1934), ewe (Denamur and Vlartinet, 1961), rhesus monkey (Agate, 1952), goat (Buttle et al., 1978) and woman (Kaplan, 1961). To further implicate placental lactogen in mammary development, ovine placental lactogen has been evaluated for lactogenic activity using both rabbit and ewe mammar y explants and membrane (Servely et al., 1983). Ovine placental lactogen inhibited binding of 1251 human growth hormone to rabbit mammary membranes, but was only slightly inhibitory in ewe mammar y membranes. In mammary explant cultures, ovine placental lactogen stimulated B -casein synthesis in rabbit mammary tissue but this effect was inconsistent for ewe mammary tissue. Co-culture of ovine placenta and mammar y tissue resulted m increased B -casein mRN A accumulation. .--\nalysis of t he c ulture media by radioreceptor assay indicated ovine placental lactogen c oncentrations of 70 / ml. This suggested that ovine placental lactogen was lactogenic in the ewe, but was required at high concentrations. Treatment of pregnant ewes and heifers with bromocryptine indicated that a s ubstance other than prolactin was present that could s timulate mammar y development and lactation (Schams et al., 1984). Chomczynski and Topper (1974) demonstrated that hPL stimulated RNA sy nthesis by isolated ra t and : mouse mammar y epithelial nuclei. The y s uggested that hP L and prolactin rljla y ac t b y binding directly to the nucleus. Somatotropic Activity. Placent a l lactogen w as originall y identified because of the cr oss reaction with antibodies to hGH (Josimovich and MacLaren, 1962), but final purification s tep s removed the s omatotropic activity of the protein. Kaplan and Grumbach (1964) reported that their preparation of human placental lactogen s timulated radioactive s ulfate uptake by hypophysectomized rat tibia. Another growth hormone like activit y was reported when hPL was demonstrated to increase weight gain in h y poph y

PAGE 44

33 sectomized rats (Friesen, 1964) and stimulate 3 H thymidine incorporation into DNA of cartilage from hypophysectomized rats (Breuer, 1969). The similarity between hPL and hGH was further fortified when Niall et al. (1971) reported that comparison of the amino acid sequence of hPL and hGH showed a homology of over 80%. The hypothesis that hPL was an important hormone of pregnancy that regulated the metabolism of pregnant woman stimulated research in that area. The growth hormone-like activity of hPL was demonstrated when hPL was administered to hypopituitary dwarfs (Grumbach et al., 1966). Plasma free fatty acids were increased after hPL, but the magnitude of the increase was less than that after hGH. Turtle et al. (1966) reported that hPL stimulated lipolysis in rat epididymal fat cells in vitro. They concluded that hPL 1) stimulates lipolysis through a DNA-RNA mediated process, 2) potentia ted the lipolytic effect of physiological levels of growth hormone, and 3) may account for the progressive rise in plasma free fatty acids during pregnancy. The lipolytic action of hPL was confirmed when women that were fasted for up to 72 hr had increased plasma hPL and free fatty acid concentrations (Tyson et al., 1971). The relationship between hPL and free fatty acid concentrations was disputed when altered free fatty acid concentrations were found to have no effect on circulating hPL concentrations (Gaspard et al., 1977). Handwerger et al. (1976) demonstrated that adminis tration of ovine placental lactogen decreased free fatty acids, glucose and amino nitrogen, but increased insulin. The insulin like activity of placental lactogen had been previously reported. The administration of 400 mg of hPL per day to two hypopituitary dwarfs caused increased nitrogen and potassium retention, increased insulin response to glucose and decreased rate of glucose disappearance from the

PAGE 45

34 plasma (Grumbach et al., 1968). Malaisse et al. (1969) demonstrated that treatment of hypophysectomized rats with hPL caused a reduction in plasma sugar, but increased both content and output of insulin by pancreatic tissue in vitro. Insulin induced hypoglycemia stimulated an increase in hPL concentrations while glucose loading suppressed hPL concentrations transiently in pregnant women (Gaspard et al., 1974). Brinsmead et al. (1981) conducted a series of experiments to determine the effects of hyperor hypoglycemia or fasting on maternal and fetal ovine placental lactogen concentrations. Insulin induced hypoglycemia in either the ewe or fetus had no effect on fetal ovine placental lactogen concentrations while maternal concentrations of ovine placental lactogen decreased after 120 min, which is directly opposite the response of hPL. Infusion of gluco5-e to the fetus had no effect on ovine placental lactogen concentrations in either the ewe or fetus, while fasting the ewe for 72 hr increased ovine placental lactogen c oncentrations in both the ewe and fetus. Ovine placental lactogen s timulated 14c glucose incorporation into glycogen in fetal rat hepatoc y tes (Freemark and Handwerger, 1984) and the action was potentiated b y insulin. The two hormones acted synergistically to promote liver glycogen synthesis. They observed that ovine placental lactogen was more potent than ovine growth hormone, s uggesting that ovine placental lactogen ma y have metabolic functions in the fetus that are subsequentl y controlled by growth hormone in the postnatal period. Chan et al. (1978a) reported specific binding of 1251 ovine placental lactogen to nonpregnant ewe liver, adipose tissue, ovary, corpus luteum, uterus and fetal liver. The binding was inhibited by ovine growth hormone, but not by ovine prolactin suggesting that ovrne placental lactogen may act more like growth hormone than prolactin.

PAGE 46

35 Several reports have indicated that ovine placental lactogen may be important in fetal growth. Hurley et al. (1977a) demonstrated that admin istration of ovine placental lactogen to h y poph ys ectomized rats stimulated release of somatomedin. Subsequentl y, Adams et al. (1983) reported that ovine placental lactogen stimulated insulin-like growth factor II (IGF II) production by fetal rat fibroblasts while in adult rat fibroblasts either hGH or ovine placental lactogen stimulated production of IGF I. Ovine placental lactogen and growth hormone stimulated ornithine decarboxylase activity in neonatal rat liver (Butler et al., 1978) while only ovine placental lactogen stimulated ornithine decarboxylase activity in fetal rat liver (Hurley et al., 1980). To add further s upport that ovine placental lactogen replaces growth hormone in the fetus, Free mark and Handwerger (1982) reported that ovine placental lactogen s timulated alpha a mino isobutyric acid transport into weanling rat diaphragm cells with equal potenc y to ovine growth hormone. A y ear later the s ame r esearchers indicated that in fetal rat diaphragm cells ovine placental lac tog en stimulates alpha a mino isobut y ric acid transport while ovine growth hormone was without effect ( Freemark and Handwerger 1983). Luteotropic Activity. Ra y et a l. (195:')) implicated placental la c togen as a luteotropic s ubstance when they reported that injection of two day 12 rat placentas inhibited the estrous cycle in normal rats. Human placental lactogen maintained an induced decidual reaction in h y poph ysec tomized pseudopregnant rats (Josimovich et al., 1963). T hi;:; action was abolished when hPL was preincubated with antibodies to hGH. Josimovich and Atwood (1964) proposed tile h y pothesis that hPL sy nergized w ith human chorionic gonadotorpin to stimulate the corpus luteum of pregnanc y and this was

PAGE 47

36 confirmed when human chorionic gonadotropin and hPL maintained a decidual reaction for the normal duration (Josimovich, 1968). Additional reports have not onl y supported the luteotropic activity of hPL but also raised questions to the function of hPL in fetal development. El Tomi et al. (1971) reported that immunization of rabbits with hPL caused either total or partial fetal resorption during pregnancy. The ovaries and uterus of hPL immunized rabbits were significantly smaller than controls. In rats injected with antibodies to hPL implantation was normal, but no births occurred (Gusdon, 1972). The immunized rats resumed normal estrous cycles and were rebred but over a period of 11 months none of the rats delivered a litter. Monkeys immunized with either hPL or placental extracts had decreased fertility (Gusdon and Witherow 1976). However, the titer raised against hPL did not seem to be related to whether the monke ys became pregnant. In contrast to reports supporting the luteotropic activity of placental lactogen, Martal and Djiane (1977) s tated that ovine placental lactogen infused into the uterus of a ewe on day 12 of the estrous cyc le did not extend the lifespan of the corpus luteum. This ma y indicate that placental lactogens are luteotropic in species that rely on prolactin for luteal maintenance. Miscellaneous Function. Spellac y et al. ( 1971) described the use of hPL concentrations as a placental function test. They examined hPL concentrations across gestation in approximately 1400 pregnancies. After examining the data they described a fetal distress zone where after 30 weeks of gestation hPL concentrations were below 4 / ml. Of patients with hPL concentrations within the fetal distress zone 24 % of the infants died. Subsequent reports have suggested that hPL c oncentrations were not the best indicator of fetal distress. Nielsen et al. (1981) stated that

PAGE 48

37 monitoring hPL concentrations was not recommended as a routine procedure in all pregnancies, but may be beneficial in some complicated pregnancies. In 1964, Kaplan and Grumbach suggested that human placental lactogen (hPL) may regulate metabolic functions in the maternal system. They implicated hPL in increasing: free fatty acid concentrations, resistance to exogenous insulin, levels of circulating insulin, lean body mass and changes in body fluid compartments during pregnancy. They also suggested that hPL stimulates transfer of amino acids across the placenta which would affect fetal metabolism and growth. Few of these proposed functions have been confirmed. Freemark and Handwerger (1982) demonstrated that ovine placental lactogen stimulated alpha amino isobutyric acid transport into fetal rat diaphragm cells. However, further evidence for a role of placental lactogens is lacking. Nutrient Partitioning The control mechanism involved in nutrient partitioning are complex and incompletely understood at present. The partitioning of nutrients to various body tissues involves two types of regulation, homeostasis and homeorhesis (Bauman and Currie, 1980). Homeostasis is involved in maintaining a physiological equilibrium in the animal such as body temperature, while homeorhesis is involved with coordinated changes in metabolism of body tissues necessary to support a physiological state such as lactation or pregnancy. Total nutrient requirements throughout pregnancy are about 75 % greater than for a nonpregnant animal of the same weight (Moe and Tyrrell, 1972). The efficiency of utilization of metabolizable energy during pregnancy in sheep and cattle was estimated as 16.1 % (Rattray et al., 1974) and 25%, (Moe and Tyrrell, 1972) respectively. However, this efficiency may increase if a maintenance requirement for the fetus is taken into account (Rattray et al., 1974).

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38 Fetal metabolism has recently been reviewed by Jones and Rolph (1985). They stated that the fetus utilizes glucose, lactate, amino acids, acetate, glycerol and fatty acids for the energy required for growth. The fetus receives glucose from the maternal circulation via the placenta (Battaglia and Meschia, 1978). Hay et al. (1983) reported partitioning of glucose in the pregnant ewe during both normal and hypoglycemic states. In this experiment, the fetus consumed 10 % of the available glucose in both conditions. Assessing fetal glucose metabolism is difficult because fetal concentrations can be influenced by fetal hepatic or placental glycogenolysis (Jones et al., 1983). Lactate is supplied to the fetus by the placenta (Burd et al., 1972) and is used either directly for energy metabolism by various fetal organs or in gluconeogenesis. Up to 50 percent of all fatty acids required by the fetus have been estimated to be supplied from placental transfer (Alling et al., 1972) and maternal diet markedly affects fetal lipid composition (Thomas and Lowry, 1984). The fetus has a high requirement for nitrogen which is met b y reincorpo ration of amino acids produced by protein degredation (Lewis et al., 1984). Lemons et al. (1976) measured the venoarterial concentration differences of 22 amino acids across the umbilical circulation of the fetal lamb and stated that neutral and basic amino acids are transported from the maternal to fetal system while acidic amino acids are not. In fact, glutamic acid is delivered from the fetus to the placenta in large amounts. Fetal oxidative metabolism was reviewed by Battaglia and Meschia (1978). Oxygen consumption by fetal s heep, goats and cattle ranged from 7 to 9 ml/min/kg fetal body weight. In the fetal lamb oxidative metabolism consumes primarily carbohydrates and amino acids, therefore, approximately

PAGE 50

39 56 kcal/day is consumed. Comparable figures for cattle in late pregnancy was estimated at 2.3 Meal/day while about 1 Meal/day is accumulated in the fetus (Bauman and Currie, 1980). The metabolic cost of maintaining the fetus is high. Purification Placental lactogens have been isolated and purified in the human (Josimovich and MacLaren, 1962), monkey (Shome and Friesen, 1971), sheep (Martal and Djiane, 1975), rat (Robertson and Friesen, 1975), goat (Becka et al., 1977), cow (Beckers et al., 1980) and mouse (Colosi et al., 1982). The purification procedures reported for the various species are quite similar. In all the above reports, except in the goat (Becka et al. 1977) placental tissue was extracted and the placental lactogen was precipitated with ammonium sulfate. Column chromatography by gel filtration and ion exchange was used in all cases. Preparative isoelectric-focusing was an additional step in purification of goat (Becka et al., 1977) and rat (Robertson and Friesen, 1975) placental lactogens. Purification of bovine placental lactogen required a more rigorous purification scheme. Gel filtration and ion exchange c;:hroma tography was enhanced by hydroxyapatite (Murthy et al., 1982, Eakle s t al., 1982) and chromatofocusing (Eakle et al., 1982) columns. The I hydroxyapatite column separates proteins by hydrophobic interactions while I the chromatofocusing column separates proteins on the basis of their isoelectric points. Affinity chromatography was introduced (Beckers et al., 1980) to remove bovine serum albumin from the placental lactogen preparation. Originally column fractions were monitored for lactogenic or somatotropic activities using the pigeon crop sac or rabbit intraductal assays for lactogenic acitivy (Josimovich and MacLaren, 1962) or rat tibial growth assay for somatotropic activity (Josi movich and MacLaren, 196 2,

PAGE 51

40 Shome and Friesen, 1971). However, after the report of Shiu et al. (1973) researchers used the radioreceptor assay to detect the presence of lactogenic or somatotropic proteins. The membranes utilized in the radioreceptor assays were either rabbit mammary gland (Robertson and Friesen, 1975) or rabbit (Arima and Bremel, 1983) or rat liver (Murthy et al., 1982) for lactogenic activity and rabbit liver membrane for somntotropic activity (Robertson and Friesen, 197 5). The reported purification procedures yielded from 2% (Murthy et al., 1982) to 29 % (Colosi et al., 1982) of the original somatotropic or lactogenic activity in the purified form. The molecular weights of the various placental lactogens were estimated at 22,000 for the human (Friesen, 1965), goat (Becka et al., 1977), mouse (Colosi et al., 1982), rat (Robertson and Friesen, 1975), monkey (Shome and Friesen, 1971) and sheep (Hurley et al., 1977b, Martal and Djiane, 1975) and approximately 30,000 for the cow (Beckers et al., 1980, Murthy et al., 1982, Eakle et al., 1982, Arima and Bremel, 1983). The isoelectric point of placental lactogen ranged from 5.5 in the cow (Murthy et al., 1982, Arima and Bremel, 1983), 6.0 in the rat (Robertson and Friesen, 1975), 6.8 or 7 .2 in the sheep (Hurley et al., 1977, Martal and Djiane, 197 5, respectively), 7.1 in the mouse (Colosi et al., 1982), to 8.8 in the goat (Becka et al., 1977). Multiple forms of placental lactogen have been demonstrated in the human (Suwa and Friesen, 1969), rat (Robertson et al., 1982), mouse (Soares et al., 1982), monkey (Shame and Friesen, 1971), and cow (Arima and Bremel, 1983). Suwa and Friesen (1969) reported that two peaks of hPL were detected after gel filtration. The molecular weight of the proteins was 100,000 and 20,000, respectively. They suggested that the large molecular weight protein was an aggregate of hPL while the smaller molecular weight protein was native hPL. In the mouse, two forms of placental lactogen are secreted

PAGE 52

41 at different times of gestation (Soares et al., 1982). Two peaks of lactogenic activity were present at days 10 and 18 in the mou s e but only the second peak crossreacted with a specific antibody to mouse placental lactogen. Similar finding s were reported for the rat (Robertson et al., 1982). Two peaks of lactogenic activity were detected in a radioreceptor assay at days 11 13 and 12 21. The peak at days 11 13 was not detected using a specific radioimmunoassa y The two forms of rat placental lactogen had molecular weights of 40,000 and 20,000, respectively with isoelectric points of 4.5 and 6.2. The y s uggested that the early form (days 11 13) of rat placental lactogen may be responsible for the luteotropic activity reported by Astwood and Greep (1938) and the late form (day 12 21) was primaril y mammotropic. M onke y placental lactogen had two forms with s imilar molecular weights, but different electrophoretic mobilities (Shome and Friesen, 1 ~71) They suggested that deamidation may be responsible for thi s difference. Arima and Bremel (1983) reported three forms of bovin e placental lactogen with si milar molecular weights, but isoelectric point s of 5.85, 5.52 and 5.39. The y s uggested that genetic variabilit y may be responsible for the different forms. In conclusion, placental lactogen has been purified for a number of different species. The purification procedure utilized was s imilar between species, but the resulting protein wa s quite different. The molecular weight of placental lactogen was reported as 22,000 for each s pecies except the cow (30,000). Multiple form s of the molecule were reported.

PAGE 53

CHAPTER II COTYLEDON CULTURE EXPERIMENTS Introduction Placental lactogen production by placental tissue in culture was first reported in the human by Suwa and Friesen (1969). They indicated that 80% of the human placental lactogen (hPL) released into the medium was secreted in the first 24 hr. The release of hPL ma y be stimulated by the addition of pimozide (Macaron et al., 1978), estradiol (Belleville, et al., 1978), arachidonic acid (Handwerger et al., 1981a), EDTA, EGTA or methoxyverapamil (Handwerger et al., 1981b) and insulin (Perlman et al., 1985) to the medium. Bovine placental tissue also secretes placental lactogen in vitro (Buttle and Fors y th, 1976) and this sec retion was s timulated b y the addition of arginine to the media (Forsyth and Hayden, 1980). The regulatory factors involved in placental lactogen secr etion in culture remain unknown, however hypotheses include the stimulation of s ecretion b y inhibiting the calcium flux (Handwerger et al., 1981b) or modulating dopaminergic receptors (l\'lacaron et al., 1978). The purpose of the s tudies described in this chapter was to evaluate the factors which may be involved in regulation of bovine placental lactogen (bPL) s ecretion in vitro. Materials and Methods Six experiments were conducted to determine the role of s ubstrate or hormone supplementation on s ecretion of bPL. Whole uteri were collected at slaughter from cows at approximately day 200 of gestation. The uteri were transported to the laboratory where placentomes were removed 42

PAGE 54

43 aseptically. Placentomes were separated into maternal caruncle and fetal cotyledon. Cotyledons were placed in cold minimum essential media (MEM) (Gibco, Grand Island, NY) on ice and taken to the processing laboratory. In a laminar flow hood cotyledonary villi were removed with scissors and minced with scalpel blades. Explants weighing approximately 1 mg were placed on a stainless steel grid in either a falcon culture dish (Falcon Plastics Co., Oxnard CA) or 24 well culture plate (Costar Rochester Scientific Rochester, NY) in the presence of 1 ml M EM plus the appropriate treatment. Tissue explants were cultured on a rocker table ( Belko Glass Vineland, NJ) in an incubator at 3 7C (National, Portland OR) in the presence of 50% N 2 :45 % O2:5 %C O 2 After incubation c ulture medium w as ass a y ed for lactogenic activity in a Prolactin radioreceptor ( Prl-RR A) ass a y by the method of Shiu et al. ( 1 9 7 3 ) Experiment 1. To determine the s ite of production of the lactogenic activity b y the bovine placenta s ix explants from cotyledon, ca runcle or inter-cot y ledonary tissue were placed into culture for 24 hr. Experiment 2. To determine the role of either energy s ubstrate or hormonal s upplementation on production of lactogenic activity, cotyledonary explants were c ultured in the presence of either additional s ubstrate or hormones. Treatments consisted of 1 ml MEM + O Insulin + 50 / ml Acetate + l mg / ml Glucose 1 ml M EM + .2 Insulin + 50 ug / ml Ac etate + 1 mg / ml Glucose 1 ml MEM + .2 Insulin + 50 / ml Acetate + 5 mg Glucose 1 ml M EM + .2 Insulin + 100 g Acetate + 1 mg Gluco s e 1 ml MEM + .2 Insulin + 100 g Acetate+ 5 mg Glucose 1 ml MEM + .2 Insulin+ 50 g Acetate+ 1 mg Glucose+ 50 ng L-T4 ( Sigma) 1 ml MEM + 2 Insulin + 50 g Acetate + 1 mg Glucose + 10 ng Cortisol 1 ml MEM + .2 Insulin+ 50 g Acetate + 1 mg Glucose + 1 ng Estrone

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44 1 ml MEM + .2 Insulin + 50 g Acetate+ 1 mg Glucose+ 1 ng Estradiol 1 ml MEM + .2 Insulin+ 50 g Acetate+ 1 mg Glucose+ 10 ng Progesterone 1 ml MEM + 2 Insulin+ 50 g Acetate+ 1 mg Glucose+ 100 ng GH (NIH B8) 1 ml MEM + .2 Insulin + 50 g Acetate + 1 mg Glucose + coculture with caruncle Each culture was in duplicate and the cultures were terminated at 12, 24, 36 or 48 hr. The tissue was blotted dry and weighed and medium was stored at -20C until analyzed. The experiment was replicated three times, using three cows (two Angus and one Brown Swiss) ranging from 230 to 250 days of gestation. Experiment 3. To further evaluate hormonal regulation of secretion of lactogenic activity, placental tissue from four cows at approximately day 230 of gestation was cultured in the presence of nine different hormones at three different concentrations. The treatments consisted of Dose Growth Hormone ( NIH B8) ng/ m 1 100, 10, 1 Estradiol, pg/ml 1000, 100, 10 Seratonin, pg/ml 1000, 100, 10 Dopamine, pg/ml 1000, 100, 10 Norepinephrine, pg/ml 1000, 100, 10 Epinephrine, pg/m 1 1000, 100, 10 Ergocryptine, pg/ml 1000, 100, 10 Thyroid re 1 easing hormone, pg/ml 1000, 100, 10 Somatostatin, pg/ml 1000, 100, 10 Explants were cultured for 24, 48, or 72 hr. In the first two replicates the explants were immersed in 1 ml culture media; however, because of irregular results the third and fourth replicates were conducted with explants placed on grids.

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45 Experiment 4. Placental explants from three cows, at approximately day 186 of gestation, were cultured for 24 hr in the presence of estradiol or growth hormone using a 4 x 4 latin square design. The treatments consisted of bovine Growth Hormone (NIH B8) at 0, 10, 100 and 1000 ng and estradiol17B at 0, .1, 1 and 10 ng. Treatments were conducted in triplicate. Experiment 5. To determine the optimum amount of tissue to culture in spinner flasks (Bellco Glass, Vineland, NJ) to maximize production of lactogenic activity, cotyledonary tissue at 6.3, 12.8 18.4, 25.9, 30.9 or 36.2 g was cultured in 500 ml MEM for 24 hr. Culture medium was analyzed for lactogenic activity and protein concentration. Experiment 6. To determine the effect of arachidonic acid on lactogenic activity production cotyledonary tissue was c ultured in medium containing concentrations of arachidonic acid ranging from 0 to 3 18 M. Arachidonic acid (Fluka Chemical Corp., Hauppauge, NY) in meth y lene chloride CHCl2 was dried under N 2 gas before addition to culture media. Three trials were conducted to determine the effect of arachidonic acid on production of lactogenic activity. In the first trial, four petri dishes, each containing 1 1 g cotyledonary tissue, were cultured for 24 hr. At the end of the incub a tion, I the MEM was removed and replaced. In two of the dishes 300 IV! arachidonic acid was added and the tissue was incubated for an additional 24 hr. In the second and third trials cotyledonary tissue was cultured in the presence of 0, 75, 150 or 300 M arachidonic acid for 24 hr. At the end of incubation media was measured for lactogenic activity by a Prl RRA. Statistical Analysis. Experiments 2, 3, 4 and 6 were analyzed by the General Linear Models procedure on the Statistical Analysis System (SAS). In Exp. 2 means were calculated for each treatment and Duncan's M ultiple Range test was conducted to detect treatment effects. Experiment 1 and 5 were not analyzed.

PAGE 57

Results and Discussion Experiment 1. 46 Six explants of either cotyledon, caruncle or intercaruncular area were cultured for 24 hr. Highest concentrations of lactogenic activity measured either as ng / ml or ng lactogenic activity/mg tissue was detected in medium from culture of cotyledonary tissue (187 .66 14.67 and 136.28 27.12, respectively) (table 2.1). The caruncular tissue did produce lactogenic activity, but the variabilit y was great. No lactogenic activity was produced by the intercaruncular area and the lactogenic activity produced by the caruncular tissue probably resulted from cot y ledonary contamination. During the placentome separation pieces of cot y ledon can be trapped in the crypts of the caruncle. Experiment 2. The effect of additional substrate or hormones on production of lactogenic activity was examined in this experiment. Statistical analysis indicated that cow, trt, cow x trt, trt x time, cow x trt x time and tissue weight to the 4th order were s ignificant ( table 2. 2 ). The cow x trt interaction indicated that cows reacted differentl y to treatments. Cotyledonar y tissue produced approximatel y 500 ng lactogenic activity / ml in the first 12 hr of culture with only an additional 2 00 ng / ml in the s ubsequent 36 hr (fig. 2.1). Production of lactogenic activity was affected (P<.001) by tissue weight (mg) (fig. 2.2). Tis s ue weights ranged from .17-8.91 mg. To normalize data on production of lactogenic activit y b y different size explants, lactogenic activity (ng) per mg of tissue w as plotted versus tissue weight (fig. 2.3). Production of lactogenic activit y expressed in this manner over a range of tissue weights from 1 8 mg was similar ; however production by explants weighing less than 1 mg was higher, possibl y because values were multiplied. Addition of substrate or thyroxine, cortisol estrone estradiol-17B or progesterone had no effect on production of lactogenic activity (fig. 2.4). The absence of insulin from the culture medium resulted

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47 Table 2.1. Lactogenic Activity Produced by Cotyledonary, Caruncular and InterCaruncular Tissues From a Cow at Approximately Day 200 of Gestation. Total Lactogenic Activity ng/ml ng/mg Tissue (x SE) (x SE) N Cotyledon 187.66.67 136.28.12 6 Carcuncle 36.77 20 21.91.09 6 InterCaruncu 1 ar .05.02 .01.01 6 Area

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48 Ta b le 2.2. Analysis of Variance for Cotyledon Culture (Exp # 2) Source Cow Trt C o wxTrt Time CowxTime TrtxTime CowxTrtxTime Tisswt+ Tisswt 2 Tisswt3 Tisswt 4 Residual *P<.1 **P< 01 ***P<.001 df ss 2 95580822 11 261924951 21 282405564 3 33808993 6 72618595 33 253183425 63 459610224 1 58973143 1 54460193 1 37514058 1 5974688 134 725929234 +Tis:-;wt was tested usi n g T y pe I Sum s of Squares. F Value 8.82*** 4.4C*** 2.48*** 2.08 2.23* 1. 42* 1. 35 10.89** 10.05** 6.92** 11. 03** cow, Trt, CowxTrt, Time, CowxTime, TrtxTime and CowxTrtxTime were tested using Type III SS.

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Fig. 2 .1. Lactogenic activity (ng/ml) produced by cotyledonary tissue over time (P<.0001). The points plotted are the mean of all treatments for that time.

PAGE 61

ISi C11 J: w U1 lS2 1SJ UJ !SI iSl Lt1 lSl lSl T 50 [S1 iSl r, ts: m T ...... r-, ct I ..I w f'\1

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Fig. 2.2. Lactogenic activity (ng/ml) produced by cotyledonary tissue over a range of .2-9 mg (P<.04). The explants were placed into groups by weights which were: .1-.5, .5-1, 1-2, 2-4, 4-6 and 6-9 mg. Weight groups combined lactogenic activity produced by all treatment groups.

PAGE 63

tSI lS2 f\l tS1 rs. lSJ ts2 lS2 ill IS1 tsl U] 5 2 lS2 tsI J"' tS1 tS1 N lSI ISl Ul lSI Lit !SI T !SI fTJ tsI N ISl ,-, l.!J :E L.J II La w 3 w ::J tJt U1 r

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Fig. 2.3. Lactogenic activity (ng/mg ti ss ue) produced by c otyledonary explants over a range of weights from .17-9 mg (P<.0001). Explant weight group s were .1-.5, .5-1 1-2, 2-4 4 6 and 6-9 mg. Weight groups combined lactogenic activity produced by all treatment groups.

PAGE 65

54 w en H .,_ _,. __ ...., __ .__,_,...,_ _,. __ ..,_ __ ,.__....& 1Sl ls:! Ill lSl !SJ rn ls:! iS T ISl iI1 lS2 I" ISl 1D lSl i./1 lS2 T lS2 r, isi N ISl [SJ !SJ ., t!J :E w II Ill w 3: w ::J lJ1 U1 I

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Fig. 2.4. Lactogenic activity (ng/mg tissue) produced by cotyledonary tissue (x SE) exposed to either Control (C), No Insulin ( 1), Acetate (Ac), Glucose (G), Acetate + Glucose (Ac + G), Thyroxine (T 4), Cortisol (F), Estrone (E1 ), Estradiol (E2), Progesterone (P 4), Growth Hormone (G H) or Caruncular co-culture (Co) over all time periods (P<.0001 ).

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700 r, 600 SEM l!) 1: l!J z S00 . LJ )t> Li00 . ._ V a: 300 ... V z w l!) 200 a tV a: ... -I C A G C _J 100 ... 0 0 2 3 Lt A 1i F E C I G 6 7 8 TREATMENT E 2 9 I IZJ G H I I C 0 12 <:Jl Cf)

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57 in lower concentrations of lactogenic activity. Duncan's Multiple Range test indicated that addition of gro w th hormone or co-culture of cot y ledon and caruncular tissue stimulated (P<.05) production of lactogenic activity when measured on a per mg of tissue basis (table 2.3). These results suggest that the majorit y of bPL is secreted into the medium in the first 12 hr of culture. This is in agreement with the findings of Suwa and Friesen (1969). Tissue explants did not require additional acetate or glucose to produce more lactogenic activit y Other nutritional factors such as amino acids may affect production. Hormonal stimulation of lactogenic activit y by growth hormone was not affected by crossreactivit y of G H in the Prl RRA ( 4.3 % ) but is unclear at this time. Similarl y, the mechanism for s timulation of production of lactogenic activit y by co-culture of c ot y ledon and caruncle is unclear. The lactogenic activit y/ mg tis s ue did not take into account w eight of caruncular tissue s o that c ot y ledonar y tis s ue contamination as demonstrated in Exp. 1 could have b een a fa ctor Another explanation is that the caruncular tissue secretes a s ubstance that s timulates release of lactogenic activit y from the cot y ledon. The identification of this substance was not attempted. Experiment 3. In this experiment the a n a l ys is of variance (table 2.-1) indicated that grid w hether tis s ue was incub a ted on s tainles s s teel g rids or immersed in culture medium, was not a main e ffe c t, but there w as a grid x time interaction. Hormonal treatment a nd dose of hormone also had no effect on production of lactogenic activit y ( t a ble 2 5). There w as a significant time effect and time interactions w ith grid and c ow ( gTid) w ere significant. Bovine growth hormone (NIH-88) did not a ffect production of lactogenic activity which was in contrast to Exp 2 The s ignificant time effect was also in contrast to the results in Exp. 2 This effect ma y be

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58 Table 2.3. Duncan's Multi ple Range Test for Treatment in Exp. # 2. x Lactogenic Activity Duncan Grouping* ( ng/ mg Tissue) N Treatment A 645.0 24 Growth Hormone A A 568.7 16 CoCul ture B 378.6 24 Progesterone B B 369.7 23 Estradio 1-1 7 B B B 366.9 24 Cortisol B B 349.3 24 Control B B 348 4 24 Estrone B B 309.3 24 Acetate B B 280 .1 24 Glucose B B 270.4 24 Thyroxine B B 264.5 24 Acetate + Glucose B B 253 9 23 -Insulin *Means with same letter are not significantly different (P< .05).

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59 Table 2.4. Analysis of Variance for Culture Exp. # 3. Source Grid+ Cow (Grid) Trt Dose Time GridxTime Cow (Grid)xTime TrtxTime DosexTime TrtxDose GridxTrtxTime Cow (Grid)xTrtxTime Residual *P<.1 **P<.01 ***P<.001 df ss 1 21139032 2 823850054 8 51936936 3 31575667 2 133253219 2 43799379 4 125011255 16 106651824 6 49560746 15 54768973 27 199656550 53 340298401 188 1057785899 +Grid was tested using cow (Grid) as the error term. F Value .51 73.21*** 1.15 1. 87 11.84*** 3.89* 5.55** 1.18 1.47 .65 1. 31 1.14

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60 Table 2.5. Mean (x) Production of Lactogenic Activity (ng/ml) by Cotyledonary Explants In Vitro. Effect of Three Doses of Growth Hormone (GH), Estradiol (E2), Seratonin, Dopamine, Norepinephrin, Epinephrin, Ergocryptine, Thyroid Releasing Hormone (TRH) and Somatostatin. Time (hr) Hormone Dose (ng) 24 48 '7'2 --Control 0.00 300.0 785.2 412.0 GH 10.00 187.2 535.0 374.0 100.00 277.3 439.7 1030.0 1000.00 288.0 402.9 477.7 E2 .01 175.4 581.9 273.7 .10 226.8 426.6 376.8 1.00 276.5 493 6 486.8 Seratonin .01 221.0 381.6 457.0 .10 283.2 470 9 381.4 1.00 376.2 610.1 391.1 Dopamine .01 257.8 389.9 489.3 .10 260.7 404.4 384.9 1.00 211. 8 680.0 490.6 N orepinephrin .01 273.5 367.9 402.1 .10 237.2 446.1 394.4 1.00 246.1 302 8 742.3 Epinephrin .01 229.3 326 .1 314.4 .10 261. 8 430.5 422.1 1.00 284 3 291. 5 504.2 Ergocyptine .01 239 .0 407.1 628.7 .10 244 7 400.2 394.0 1.00 255.6 530.2 345.3 TRH .01 380 3 374.1 438.9 .10 785.6 376.4 602.8 1.00 269.8 755.7 352.3 Somatostatin .01 200.3 433.0 304.0 .10 226.6 544.2 328.6 1.00 652.7 411. 7 343.3 Pooled SEM = 80.34

PAGE 72

61 explained by the different patterns of production of lactogenic activity over time as depicted in table 2.5. The cows utilized in this experiment were at a similar stage of gestation to the cows in Exp. 2, but the culture times were different. These results agree with previous reports that thyroid releasing hormone (Hershman et al., 1973), somatostatin (Maca1 on et al., 1978) and dopamine (Forsyth and Hayden, 1980) had no effect on placental lactogen production. However, hPL secretion was stimulated by the addition of estradiol (Belleville et al., 1978) and inhibited by the addition of epinephrine, norepinephrine (Belleville et al., 1978) and dopamine (Macaron et al., 1978). The results of this experiment have not increased our understanding of the regulator y mechanisms involved in the secretion of bovine placental lactogen (as measured by lactogenic activity) in vitro. Experiment 4. In Exp. 2 preliminary analysis suggested that the hormones G H (NIH B8) and estradiol-1 7 B enhanced the production of lac to genic activity. Therefore, the purpose of this experiment was to determine if there was a dose response in production of lactogenic activity and if hormonal interactions could affect production. Statistical analysis (table 2.6) indicated that cow, estradiol (E), GH and cow x GH effects were significant (P<.1 ). The least squares means plotted by hormone and dose (fig. 2 .5) suggested that estradiol at doses of .1 and 1 ng inhibited the production of lactogenic activity, but the 10 ng dose had no effect. Increasing the concentration of growth hormone stimulated the production of lactogenic activity by a mean of 85 ng / ml for the 1,000 ng dose. This may be partially explained by the 4.3 % crossreactivity of GH in the Prl RRA, which would add 43 ng of lactogenic activity to the 1,000 ng GH concentration. When least squares means for each cow for growth hormone were plotted (fig.

PAGE 73

62 Table 2.6. Anal y sis of Variance for Culture Exp. #4. Source Cow Estradiol Cow x Estradiol Growth Hormone Cow x Growth E s tradiol Tissue Weight Residua 1 *P<.1 **P<.01 ***P<.001 Hormone df ss+ 2 26873472 3 3396251 6 1851052 3 3710058 6 7842747 9 2 359430 1 16158946 110 56905808 F Value 2 5.97*.;c* 2.19* .60 2.39* 2.53* 51 3 1.24* +Tissue weight was tested using the T y pe I SS and all other factors were tested using the Type III SS.

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Fig. 2.5. Lactogenic activit y (ng / ml) produced b y cotyledonar y tissue in a 4x4 Latin Square design using 0 .1 1 and 10 ng Estradiol-178 and 0 10, 100 and 1000 ng Growth Hormone.

PAGE 75

64 .. M I SEM ..J 200 l: l!J z '-' ,--ISB I,---. > IV a: V 10121 '9 z Lal l!l C tV SJa a: .J I 0 I I I .. a J I e 121 21 a 21 a 21 E GH DCSE CNGJ

PAGE 76

65 2.6) the significant GH effect and cow x GH ma y be explained by the result from the 1,000 ng G H concentration for cow 7. Experiment 5. A preliminary experiment was conducted to determine the amount of cotyledonary tissue to culture in 500 ml MEM (table 2.7). As tissue weight increased so did lactcgcnic activity (g/ml) &;id protein (mg / ml), although when put on a per gram tissue basis the 6.3 g culture flask was the most efficient in producing lactogenic activity with the least amount of protein. The specific activity (g lactogenic activity/mg protein) was highest for the 6 .3 g flask and decreased with increasing tissue weight. Thus to maximize the production of lactogenic activity with the least amount of protein in the medium 10 15 g of cotyledonary tissue was c hosen as the optimal amount of tissue to produce bovine placental lactogen (as measured by lactogenic activity) for purification of the molecule. Experiment 6. Three preliminar y experiments were conducted to determine the effect of arachidonic acid on production of lactogenic activity. Results are depicted in table 2 .8 for the fir s t trial. Production of lactogenic activity was s imilar in the four petri dishes after 24 hr of c ulture. 1 Addition of 300 M arac hidonic acid to dishes one and two ma y have attenu 1 ated the decrease of production of lactogenic activity (51.8 vs. 64.8 % for dishes one and two versus three and four) by cotyledonary tissue in vitro. In the second and third trials, tissue exposed to O 300 M arachidonic acid (table 2.9) was cultured for 24 hr. Statistical analysis (table 2.10) using the model cow explant (cow) dose x cow x dose indicated that dose of arachidonic acid was not significantly different. This is in direct contrast to the reports of Handwerger et al. (1981a) in humans and Hu y ler et al. (1985) in sheep. Using explant (cow) as the error term for cow demonstrated that

PAGE 77

Fig. 2.6. Lactogenic activity (ng/ml) produced by cotyledonary tissue of individual animals c 1 1ltured in either 0, 10, 100 or 1000 ng Growth Hormone (NIH-88).

PAGE 78

I ISi r, . l: w Ln .. ,.. .. iSl lSJ N I l 67 I I I . ~ 1 --ISi ISi ISi ISi ISi I ISi I IS1 ISi ISl ISi ISi ISi ISi ISi ISi ISi ISi ISi ISi ISi IS1 . ISi en l.t1 w in I: ::J z 3: V

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68 Table 2. 7. Production of Lactogen Activity b y Different Mass of Cotyledonary Tissue in Spinner Culture Flasks with 500 ml Minimum Essential Medium. Tissue was Cultured at 37 C for 24 hr. Specific Lactogenic Protein Activity Tissue Activity (LA) g LA/g mg/g g LA/mg Weight ( g) / ml Tissue mg/ml Tissue Protein 6.3 2.88 .457 .385 .061 7.48 12.8 4.43 .346 .803 .063 5 .52 18.4 5.34 .290 .920 .050 5.80 25.9 5.50 212 1.041 .0 39 5.28 30.9 5.98 .194 1.380 .045 4.35 36.2 6.67 .184 1. 464 .040 4.57

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69 Table 2.8. Lactogenic Activity Produced b y Cotyledonary Tissue During a 24 hr Culture Med ium was cha nged after the initial 24 hr. Petri Dish 1 and 2 Contained 300 M Arachidonic Acid. Petri Lactogenic Activity Dish ng/ml 24 hr 48 hr % Decrease -1 1886.1 770 4 59.1 2 1818.2 1008.7 44.5 3 2494 3 678 5 72.8 4 1715.8 740.7 56.8

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7 0 T a bl e 2 9 Lact o ge ni c Act i vi t y Pro duced b y C o tyledo n ary Tissue Cu l tured in 0, 75, 150 or 300 M A r achid on ic Acid for 24 hr. Tiss u e Lactogenic Act i vity S pe cific Ac t ivity D o se Trial We i gh t (mg) (LA) ( n g/ml) ( n g L A/mg T i ssue) 0 2 364 745.0 2.04 2 458 1028 7 2.25 3 60 731. 0 12.17 3 126 849 0 6.74 75 2 522 692.8 1. 33 2 472 855 9 1.81 3 106 803.0 7.58 3 180 1028.0 5.71 150 2 556 1195. 0 2.15 2 406 788.2 1. 89 3 170 1004. 0 5.91 3 213 1192 0 5.60 300 2 418 1026.6 2 46 2 460 894 0 1. 94 3 175 1220.5 7 00 3 181 1066.5 5.90

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71 Table 2.10. Analysis of Variance for Trials Two and Three, Exp. #6. Sau~ce df ss+ F Value Cow* 1 8.86 1. 56 Explant (cow) 2 11. 39 5.59 Dose 3 1. 97 .65 Cow X Dose 3 .3 2 .11 Residual 6 6 .11 +Ty pe III Sum of Squares. *Cow was treated using Explant (cow) as the error term.

PAGE 83

72 the cows were not different. Production of lactogenic activity by cotyledonary explants in the two trials was similar (table 2.9), but the specific activity (ng lactogenic activity/mg tissue) was higher in trial three because of the smaller explants utilized, which is in agreement with Exp. 2. In summary, bovine placental lactogen, as measured by lactogenic activity, was produced by fetal cotyledonary tissue in vitro and 70% of the lactogenic activity was produced in the first 12 hr of a 48 hr culture. These results are similar to the findings of Suwa and Friesen (1969) in the human. Production of lactogenic activity was affected b y amount of tissue therefore, production/mg tissue was utilized to account for differences in weight of explants. The increased production of lactogenic activity by explants weighing less than one milligram may be due to increased surface area for transport of nutrients into the tissue or lactogenic s ubstances out of the tissue. This possibility was not examined further, but tissue was minced finely in subsequent studies. Addition of acetate, glucose thyroxine, cortisol, estrone, estradiol-178, progesterone, s eratonin, dopamine, norepinephrin epinephrin, ergocryptine, thyroid releasing hormone, somatostatin or arachidonic acid had no effect on production of lactogenic activity by cotyledonary explants. This is in contrast to reports in the human (Bellville et al., 197 8; Handwerger et al., 1981a) and sheep (Huyler et al., 1985) but in agreement with a report in the cow (Forsyth and Hayden, 1980). Growth hormone significantly increased production of lactogenic activity in Exp. 2 and 4, but the response in Exp. 4 may be due to a single animal. Increasing doses of growth hormone (Exp. 3) did not stimulate production of lactogenic activity over control, which may have been affected by very high control values.

PAGE 84

73 Production of lactogenic activity b y spinner culture increased as amount of tissue increased, however; the concentration of protein in the media also increased. The specific activity of the lactogenic activity (ng lactogenic activity/mg protein) decreased as tissue weight increased, which would hinder the purification process. Thus the optimum amount of cotyledonary tissue to produce lactogenic activity with the least amount of protein was determined to be 10 15 g in 500 ml MEM. The experiments in this chapter failed to elucidate the regulatory factors involved in secretion of bPL in vitro. A possible explanation for this is that culture conditions may not have been optimal. Lactogenic activity was produced for eventual purifi cation of bPL, therefore factors such as, fetal calf s erum, were omitted from the culture media. Fetal calf s erum has been proven to be a requirement in several culture systems. The time between s laughter and incubation of the tissue may be a factor in viabilit y of tissue, which ranged from 30-120 minutes. Finally, production of lactogenic activity by cotyledonary tissue may be an autonomous process as has been s uggested in the human.

PAGE 85

CHAPTER III PURIFICATION OF BOVINE PLACENTAL LACTOGEN Introduction Bovine placental lactogen (bPL) has been isolated and purified (Beckers et al., 1980, Murthy et al., 1982 Arima and Bremel 1983). The molecular weight and isoelectric point reported for the molecule was 32,000 (Murthy et al. 1982 Arima and Bremel, 1983) and 5.5 (Murthy et al., 1982, Arima and Bremel, 1983), respectively. Bovine placental lactogen was purified from fetal cotyledons following homogenization, extraction in ammonium bicarbonate buffer, ammonium sulfate precipitation and column chroma tography. All three groups used gel filtration and ion exchange chroma tography; however, Beckers et al. (1980) utilized an affinity column to remove bovine serum a lbumin, while Murthy et al. (1982) and Arima and Bremel (1983) also added a hydroxyapatite column and Arima and Bremel (1983) utilized a chromatofocusing column. Approach of the present study was to purify bPL from material secreted into medium by cotyledonar y explants as described by R. Kensinger (unpublished). Materials and Methods Whole uteri were collected at s laughter and transported to the laboratory. Placentomes were removed aseptically and separated into maternal car uncles and fetal cotyledons. Cotyledonar y tissue was placed in sterile minimum essential medium (MEM) on ice. Villi were removed and minced with scissors in a laminar flow hood. Forty grams minced 74

PAGE 86

75 cotyledonary tissue was placed in a spinner flask (Bellco, Vineland, NJ) in 1 L MEM. The flask was aerated with 50 % N2:45 % 02:5 % C02, placed on a magnetic stir plate and slowly stirred for 24 hr in a 37 C incubator. At the end of incubation, culture medium was centrifuged at 10,000 x g for 10 min. Supernatant was saved for bPL purification by a method similar to Ari ma and Brem el (1983). Columns consisted of Sephacryl 200 (S-200), diethylaminoethyl cellulose (DEAE), chromatofocusing and Sephadex G-75. Column fractions were monitored for protein using the BioRad protein assay (BioRad Laboratories, Richmond, CA), for lactogenic activity using a prolactin radioreceptor assay (Prl-RRA) by the method of Shiu et al. (197 3) and for somatotrophic activity using a homologous growth hormone radio receptor assay (GH-RRA) by the method of Haro et al. (1984). Prolactin (NIAMDD-oPrl-14) and growth hormone (recombinant bovine growth hormone [rbGH], Monsanto, St. Louis, MO) were iodinated using Iodo-Gen reagent (Pierce Chemical Co., Rockford, IL). Prolactin was diluted to 5 /2 5 l in 25 mM Tris buffer pH 7.6 while rbGH was weighed and diluted in .1 l\1 NaHC03 buffer, pH 9.0. Hormone and 1 mCi of Na 1 2 51 (1 mCi /1 0 l) (Amersham, Arlington Heights, IL) were added to a 12 x 75 borosilicate tube which was coated with 2 g Iodo-Gen (50 l reaction volume) and the reaction was allowed to procede for 15 min for rbG H and 5 min for Prl. Iodinated hormone was separated from free 1251 on a .7 x 25 cm Biegel P-60 column (BioRad Laboratories, Richmond, CA). The procedure utilized for the radioreceptor assay was si milar for both Prl and GH-RRA's. Approximately 50,000 cpm of iodinated hormone was combined with either 100 l rabbit mammar y membrane (8 mg / ml) (diluted 1:3 in assay buffer) for the Prl-RRA or 400 l steer liver membrane (1-7 mg/ml) for the GH-RRA.

PAGE 87

76 Two dimensional polyacrylamide gel electrophoresis (2D-PAGE) was conducted on peak fractions from the G-7 5 column and the crude culture medium by the method of Roberts et al. (1984). Approximately 1.3 g of protein was dissolved in 100 l of a solution containing 5 mM K2C03, 2 96 (v/v) Nonidet P-40, 5 % dithiothreitol 2 96 Ampholines and 9.3 M urea. This solution was loaded on to a 4.3 % acrylamide isoelectric focusing gel containing N'N'-diallytartardiamide, 2 96 Nonidet P-40 and 9.3 M urea. After isoelectric focusing the gels were equilibrated in 65 mM Tris .1 % sodium dodecyl sulfate 1 % 2-mercaptoethanol, pH 6.8. The gels were then overlaid on 10 % (w / v) acr y lamide slab gels and electrophoresis conducted toward the anode. After completion of electrophoresis, s labs were fixed in acetic acid:ethanol (7:40). Slabs then were equilibrated in acetic acid methanol (5:10) and s tained with BioRad silver stain kit (BioRad Laboratories, Richmond CA) We obtained K2C03 and Nonidet P-40 from Sigma Chemical Co. (St. Louis MO), dithiothreitol, acrylamide, N'N'-diallytartardiamide, s odium dodec y l sulfate and 2-mercaptoethanol from BioRad Laboratories (Richmond, CA), urea from Schwarz Mann (Cambridge MA) and ampholines from LKB (Gethersberg, MD). To test the biological activit y of purified bPL a bovine mammary gland explant culture was performed. Four c ows (two Holstein and two Jersey) at approximately da y 240 of gestation were utilized. Rate of two 14c-acetate (New England Nuclear Boston M A) incorporation into fatty acids was examined. Mammar y biopsies were performed by Dr. E.L. Bliss, at the Universit y of Florida Large Animal Veterinar y Clinic. Cows were anesthetized locally with Lidocaine plus epinephrine (Tech A merica Elwood, KS). An incision was made in the left front quarter near the body wall. A 30 gm explant of mammary tissue was removed and placed in sterile 25

PAGE 88

77 mM Tris, 200 mM sucrose, pH 7.2. Two explants per day were then taken to the laboratory for processing. Tissue was sliced using a Stadie-Riggs hand microtome (Stadie and Riggs, 1944). Tissue slices were minced with scissors and three explants were placed on a stainless steel grid in a 24 well culture dish (Costar, Rochester Scientific, Rochester, NY) with 1 ml medium. The medium used was Tissue Culture Media 199 (Difeo Laboratories, Detroit, MI) which contained 10 mM acetate, 10 mM glucose, non-essential amino acids (Gibco, Grand Island, NY), cortisol (4-pregnen-11 B, 17 a 21-triol-3, 20 dione, Steraloids, Inc., Pawling, NY), insulin (Sigma Chemical Co., St. Louis, MO), antimycotic-antibiotic (Gibco, Grand Island, NY) and either 0, 1, 10, 100, 250, 500 or 1,000 ng prolactin (NIAMDD-oPrl-14) or 1, 10, 100, 250, 500, 1,000 or 5,000 ng bPL (peak II or III). Explants were incubated for 48 hr (in triplicate) at 37 C in an atmosphere of 50 % N 2:45 % 02:5 % C02. At the end of incubation, the tissue was placed in a 25 ml Erlenmeyer flask with 3 ml of Krebs-Ringer bicarbonate buffer, pH 7.3 containing 10 mM acetate, 10 mM glucose, 133 mu insulin and 214 c-acetate (1 Ci). Tissues were incubated for 3 hr in a Dubnoff metabolic water bath at 37 C. Incubation was terminated by the addition of 100 l 1 N s ulfuric acid and tissue was blotted dry and weighed. Tissues were saponified and fatty acids were extracted and quantitated by the method of Bauman et al. (1970). Results and Discussion Tissue culture incubation was halted 24 hr after initiation. Culture medium became very acidic and incidence of contamination was increased with longer incubations. The culture medium was lyophilized and stored at -20 C until chromatography. S-200. The lyophilized culture medium was reequilibrated with 25 mM Tris buffer, pH 6.2 and contained 2315.5 mg/ml lactogenic activity,

PAGE 89

78 1545.0 ng/ml somatotrophic activity and 871.2 g/ml protein (table 3.1). The protein was loaded on a 3.2 x 85 cm Sephacryl 200 column and eluted with 25 mM Tris, 200 mM NaCl, pH 6.2. Fractions (6 ml) were collected on a Gilson fraction collector (Middleton, WI), and monitored for protein, lactogenic and somatotropic activity. Protein in the peak fractions was reduced 3-fold; however, both lactogcnic and somatotropic activities were also reduced to result in no overall increase in purification (table 3.1). The elution profile from the S-200 column (fig. 3.1) indicates that somatotropic activity is eluted as a symmetrical peak with peak height greater than that for lactogenic activity. The lactogenic activity peak was broader and both peaks were associated with the right hand shoulder of the protein profile. DEAE. Peak fractions (tubes 48 57) were dialysed against 25 mm Tris, pH 6.2 and loaded on a 1.25 x 17 .5 cm DEAE column. The column was eluted with a 0 .3 M NaCl gradient and 6 ml fractions were collected. The elution profile (fig. 3.2) indicated that both the lactogenic and somatotropic activity peaks preceded the protein peak, but the somatotrophic activity was attenuated. This coulq be explained by a conformational change in the molecule or a loss of the activity. The lactogcnic peak was rotein associated with the somatotropic I again much broader than the somatotropic I peak and may be attributed to several proteins. The overall specific activity increased 10-fold for lactogenic activity, but only 3-fold for somatotropic activity. Chromatofocusing. Peak fractions (tubes 51 70) from the DEAE column were pooled and dialysed against 25 mM Imidazole buffer, pH 6.2. The pooled fraction was then loaded on a .75 x 50 cm chromatofocusing column and eluted with Poly buffer PBE 94 (Pharmacia Fine Chemicals,

PAGE 90

79 Table 3.1. Purification of Bovine Placental Lactogen Specific Activity Lactogenic Somatotropic ng Activity Activity Activity Protein /g Protein Column ( ng/ml) (ng / ml) (g/m 1) LA SA Culture Media 23 15.5 1545.0 871. 2 2.66 1. 77 S-200 613.1 299.3 273.5 2.24 1.09 DEAE 777.6 87.4 23.5 33.1 3.72 Chromatofocusing II 217 .7 10.7 7.64 28 .5 1. 4 III 136.0 51. 8 4.20 32.4 12.3 G-75 II 125.0 10.0 .13 961.5 76.9 III 213 .0 12.8 .13 1638.5 98.5

PAGE 91

Fig. 3.1. Elution profile of lactogenic and somatotropic activities and protein from a 3. 2 x 85 cm Sephacryl S-200 column equilibrated with 25 mM Tris-HCl, pH 6.2, containing .3 M NaCl. The dashed line indicates protein (g/ml), the bold line indicates lactogenic activity and the narrow line indicates somatotropic activity.

PAGE 92

0 0 0 .,.. 81 _ _ .,,,,,. . . _.0 0 0 0 0 0 0 0 Cl) C\I a:, ~ I : ,. . ( ,! < ' ( \ i 1 I (IW/6U) A.l.lAl.l.OV OldOl::l.l.O.l. Vr40S JO OIN3~0.l.OV1 (IW/6n) N13.l.Ol::ld ..l.. 0 0) 0 a:, 0 Cl) 1 0 10 0 0 C'? 0 "' 0 .,.. 0 ... z 0 0 < a: u.

PAGE 93

Fig. 3.2. Elution profile of lactogenic and somatotropic activities and protein from a 1.25 x 17 .5 cm diethylaminoethyl cellulose (DEA E) column. Column was equilibrated with 25 ml\1 Tris-HCI, pH 6.2 and eluted with a 300 ml gradient of 0 .3 M NaCl in 25 MM Tris-HCl, pH 6.2. The dashed line indicates protein (g/rnl), the bold line indicates lactogenic activity and the na1-row line indicates somatotropic activity.

PAGE 94

83 0 --I CD __. ,,,,,,,------_,--..s I' l ~--l r ----------I ----I ---40 "\. I CD l { c.. <. : ... < z l 0 I0 () < C: f ~. I i, 0 _,, .... I N t L 0 0 0 0 0 0 0 0 It) 0 10 (IW/OU) A11AU.::>V ::>ldOl:LLO! VriOS JO ::>IN3DO.l::>V1 0 0 0 0 10 0 IO (IW/On) Nl3lO~d

PAGE 95

84 Uppsala, Sweden) pH 4.0. The chromatofocusing column generates its own pH gradient. To inhibit protein aggregation, fractions (6 ml) were collected in .6 ml 1 M sucrose to make a final concentration of .1 M. Two peaks of lactogenic and somatotropic activity are evident in the chromatofocusing column elution profile. A third peak (peak I) was present when hrgh concentrations of bPL were purified. Peak II (p II) was eluted at a pH of 5.3 while peak III (p III) was eluted at a pH of 4.9 (fig. 3.3). This is in agreement with the report of Arima and Bremel (1983). Total lactogenic activity was greater than somatotropic activity and the ratio of lactogenic to s omatotropic activity was greater in p II than in p III (fig. 3.3). Fractions associated with peaks (II and III) were pooled separately and each was purified on a 2.5 x 62 cm Sephadex G-75 column. G-7 5. Pooled p II and p III were loaded on the G-7 5 column and eluted with 25 mM Tris, 200 mM NaCl, pH 6.2. Fractions (5 ml) were collected in .5 ml of 1 M s ucrose. The elution profiles of p II and p III from the G-7 5 column had protein concentrations which were <1 / ml which is the sensitivity of the BioRad protein assay (Richmond, CA) (fig. 3.4 and 3 .5), therefore, elution profiles were not plotted. Concentrations of protein were determined in pooled peak samples after concentration by lyophilization (table 3.1). Somatotropic activites were further diminished in both p II and p III, while lactogenic activity remained unchanged (table 3.1). Peak activities were shifted five fractions to the left for p III over p II which would sugg est that p III contains proteins of slightly larger molecular weight than p II. Results of 2D-PAGE of a) crude culture medium, and b) pooled (p III) fractions from G-7 5 are depicted in fig. 3.6. The crude culture medium (fig. 3.6A) contained a large array of proteins. The pooled p III G-75 fractions (fig. 3.6B) is a graphic illustration of data from the purification table (table 3.1).

PAGE 96

Fig. 3.3. Elution profile of lactogenic and somatotropic activities and protein from a .75 x 50 cm Chromato focusing column Column was equilibrated in 25 ml\1 lmidazole, pH 6.2 and eluted with Poly buffer PBE 94, pH 4.0. The bold dashed line indicates protein (g/ml), the bold solid line indicates lactogenic activity, the narrow solid line indicates somatotropic activity and the narrow da~hed line indicates the pH gTadient that was generated on the column.

PAGE 97

co 1---4 -+ o 0 0 0 (') 86 Hd IO .,,,, ~ _ .. I . .. ... .. .... ,. .. _,. ..,. ---+----'-----+-, 0 0 (\I 0 0 (IW/OU) A.ll,\l.lOV OldOt:t.10.l v~os JO OIN300.10Vl 0 .,.. .l .l. l. ... _;_ ... .l. .... 0 0 It) .,.. 0 0 .... 0 It) 0 .. z 0 t(J < a: ..

PAGE 98

Fig. 3.4. Elution profile of lactogenic and somatotropic activities of bPL p II from a 2.5 x 65 cm Sephadex G-75 column. Column was equilibrated and eluted in 25 mM Tris-HCl, pH 6.2, containing .2 M NaCl. Fractions were collected in .5 ml of 1 M sucrose. Bold solid line indicates lactogenic activity and narrow solid line indicates somatotropic activity.

PAGE 99

0 0 C\I 0 0 ... 88 (IW/Ou) A.llAll.OV OldOl::f.lO.lVriOS Jo OIN3DO.J.OV1 0 0 IX) 0 (I) 0 .... 0 C\I 0 ... z C """' (.) < C: u..

PAGE 100

Fig. 3.5. Elution profile of lactogenic and somatotropic activities of bPL p III from a 2.5 x 65 cm Sephadex G-75 column. Column was equilibrated and eluted in 25 mM Tris-HCl, pH 6.2, containing .2 M NaCl. Fractions were collected in .5 ml 1 M sucrose. Bold solid line indicates lactogenic activity and narrow solid line indicates somatotropic activity.

PAGE 101

0 0 0 0 (') 0 0 N 90 0 0 (IW/0U) All,\11:)V :)ldO~.lO.l.V~OS JO OIN300l.:)V1 I I I I t i I I I .L 0 0 a:> 0 a, ... z 0 .,_ u < C: I.I..

PAGE 102

Fig. 3.6. Two dimensional polyacrylamide gel electrophoresis of crude culture medium (A) from bovine cotyledonary culture and purified bPL p III (B). Isoelectric focusing was performed in the first dimension and sodium dodecyl sulfate electrophoresis in the second. Gel was save1 stained (Dio-RaJ La001 atories, Richmond, CA).

PAGE 103

94 67 43 30 20 14 43 30 20 14

PAGE 104

93 One band is present at approximately 30,000 MW at a isoelectric point of 5.1 which agrees with the report of Arima and Bremel (1983). The gel also shows a high molecular weight contaminant which may be an aggregation product because the molecular weight exclusion of the G-7 5 column is 75,000 1-"lnd twn ~orit~irnin!'.lnt<. ::it 60,000 IV!W /:lt a simila!' pl to the 30,000 MW protein. The purified bPL was tested for lactogenic activity in a bovine mammary gland explant culture. Increasing concentration of prolactin (NIADD-oPrl-14) were used as standard for bPL, p II and p III activity. Peak 14 c-acetate incorporation was stimulated with 250 ng prolactin and incorporation plateaued with increasing prolactin doses (table 3.2). Alteration of 14c-acetate incorporation by bPL, p II and p III was quite variable (table 3.2) with increasing doses actually inhibiting incorporation. The inhibition of l4c acetate incorporation was probably due to sucrose and NaCl present in the bPL preparation. Servely et al. (1983) demonstrated that ovine placental lactogen was not lactogenic in vitro but when placental and mammary tissues were co-cultured the mammary tissue was stimulated. They suggested that the placenta produced very high concentrations of ovine placental lactogen (70 g/ml) that was lactogenic in their culture system. Bovine placental tissue co-cultured with mouse mammary explants was lactogenic (Forsyth, 1974). If bPL is similar to ovine placental lactogen then concentrations used in this study would not affect bovine mammary tissue. However, concentrations of bPL of 70 g/ml would actually be pharmacological in vivo. In summary, bPL has been purified from bovine cotyledonary culture medium by a series of column chromatographic steps (S-200, DEAE, Chromate

PAGE 105

94 Ta bl e 3.2. Mean rates of 2 14c acetate i n corporated i n to fatty acids b y bovi n e m ammary tissue ex pl an t s in short ter m i n cu b atio n after O or 48 hr of cu lt ure i n medium con t ai n i n g P rl, b PL II o r b PL III. H834 H450 J155 Jl09 -Hormo n e No ( n g/ml) 53 3 61. 2 83.5 35.6 Prl 1 71. 6 98.7 65.5 41. 6 10 59 3 62 1 59.0 57.0 100 83 5 89.3 64.6 68.0 250 173.0 100 .1 111.1 86.5 500 187.4 119 1 112 7 146.7 1000 178 1 149.7 118 .6 96.6 bP L II 1 100.8 174.4 102 2 44.7 10 45 6 85.0 56 7 57 6 100 78.7 64.2 54.0 58.4 250 113. 5 68 7 56.1 55 3 500 84.5 115. 0 66.1 11.1 1000 39 0 54.6 23 0 74.9 5000 1. 7 b PL III 1 73.7 109.9 29.5 46.5 10 124.6 64 7 58.0 47 9 100 67.9 55 3 44.8 37 0 250 64 4 84.5 35 6 76 9 500 40.9 73 1 36.6 58.7 1000 29.9 39.5 14.3 10.2 5000 2.4

PAGE 106

95 focusing and G-75). The chromatographic scheme was similar to previous reports (Arima and Bremel, 1983). The final yield of bPL was low (14 % ) (both p II and p III), but the purity was increased 362 and 616-fold for p II and p III, respectively. The attenuation of the somatotropic activity after DEAE celluiose chromatography may be due to a conformational cha nge in the protein which ma y affect the homologous GH-RRA utilized, but not affecting the heterologous Prl-RRA. Two dimensional polyacr y lamide gel electrophoresis demonstrated a broad array of proteins in the cotyledonary culture medium. The major protein was presented at 67,000 MW, similar to bovine serum albumin. Purification of bPL resulted in a major band at 30,000 MW with contaminants at 60,000 and 90,000 MW, which were probably aggregation products. Purified bPL was tested for lactogenic activity in a bovine mammary gland explant culture sys tem. Alteration of 2 14 c-acetate incorporation into fatty acids was examined using increasing doses of either prolactin, bPL p II or bPL p III. Peak stimulation by prolactin was achieved with 250 ng, but bPL (either p II or p III) resulted in an inhibition. This inhibition may have been caused by sucrose contained m the bPL [_)reparations. An i interesting result was that the two Holstein cows utilized were more responsive to bPL stimulation than the two Jersey cows, but the results in both breeds were inconsistant.

PAGE 107

CHAPTER IV DEVELOPMENT OF A RADIOIMMUNOASSA Y TO BOVINE PLACENTALLACTOGEN Introduction In 1976, Bolander and Fellows reported purification of bovine placental lactogen (bPL) and development of a radioimmunoassay (RIA) to this protein. A subsequent report (Bolander et al., 1976) described changes in bPL concentrations in dairy and beef cows during gestation. They reported that bPL concentrations in dairy cows were twice as high as in beef cows, which suggested that bPL was related to subsequent milk production. Other laboratories have not been able to repeat these findings. Roy et al. (1977) developed a RIA to purified bPL and reported that concentrations of bPL in serum from late pregnant cows was below the sensitivity of the assay (<100 ng/ml). Beckers et al. (1980) reported low concentrations of bPL in pregnant cow serum across gestation. Schellenberg and Friesen (198 2) utilized the very sensitive Nb2 lymphoma assay, which measures lactogenic activity, to measure concentrations of bPL in maternal and fetal blood. Maternal concentrations of bPL were undetectable while fetal concentrations ranged from 5-22 ng/ml. Each of the above researchers purified bPL by different methods and developed RIAs to the resulting proteins. The different results indicate that either the proteins purified were different or the assays developed did not have required sensitivity. To clarify the situation, this study involved collaboration with Dr. R.D. Bremel at the University of Wisconsin, Madison to purify bPL by similar procedures and develop RIAs for bPL in both laboratories. 96

PAGE 108

97 Materials and Methods Antibody Development. Bovine placental lactogen purified as previously described, was further purified using a native polyacr y lamide gel system (fig. 4.1). Purified bPL was loaded on a 10 % polyacr y lamide slab gel in the absence of chemical denaturing agents such as sodium dodecyl sulfate or 2-mercaptoethanol. After electrophoresis the slab gel was divided into lanes. One lane was stained with Coomassie Brilliant Blue R-250 (BioRad Richmond, CA) and the other lane was cut into .5 cm pieces and placed in 200 l of 10 mM Tris, pH 8.2 for an overnight incubation. After the incubation the samples were centrifuged at 2,000 x g for 10 min and super natants were assayed for lactogenic activity in a Prl RRA. Slices with peak lactogenic activit y were then homogenized and injected into rabbits for antibody development (injected two times at three month intervals and bleed 15 days after injection). lmmunoprecipitation. Antibodies produced b y the native gel method at the University of Florida or by the method of Vaitukaitus et al. (1971) at the USDA at Beltsville by Dr. Douglas Bolt using bPL purified at the University of Florida, were tested for the abilit y to immunoprecipitate bPL from culture media. Fetal cotyledons were collected as previousl y described. Villi were removed with scissors and minced with s calpel blades. A pproxi matel y 700 mg of tissue was incubated in 15 ml M E M in the presence of 125 Ci35s Methionine (Amersham, Arlington Heights, IL) for 2 4 hr at 37 C. After incubation the medium was centrifuged and supernatant was dialyzed against two changes of 10 mM Tris, pH 8.2. Dialyzed medium (800 l) was then combined with either 50, 100 or 200 l of serum antibod y preparation or 100 l normal rabbit serum plus 200 l immunoprecipitation buffer (.3 M NaCl, .5 M Tris acetate, 1 mM PMSF 1 mM EDTA, .1 % BSA, 2 % Nonidet P-40 and .02 % Na Azide, pH 7 .5) and incub11.ted overnight at 4 C. The next

PAGE 109

Fig. 4.1. Lactogenic activity (ng/ml), as measured in a Prl RRA, eluted from .5 cm native polyacrylamide gel slices with 25 mM Tris HCl, pH 8.2. The native 10% polyacrylamide gel was electrophoresed in the absence of sodium dodecyl sulfate and was stained with Coomassie Brilliant Blue R-250.

PAGE 111

100 morning 100 l of a 10 % (v / v) s olution of Protein A Sepharose (Pharmacia, Pi s cata w a y, NJ) w as added and samples were incubated for 5 hr at room temperature on a tube turner. After this incubation the samples were centrifuged and the pellets were washed six times with immunoprecipitation wash buffer (50 m M Tris-acetate 3 [ VI NaCl .5 % Nonidet P4 0 and 1 % s odium dodec y l sulfate, pH 7.5). A fter the la s t wash 30 l of loading gel buffer containing 25 mM Tris, 192 mM gl y cine and .1 % sodium dodec y l s ulfate plus 2 l 6-mercaptoethanol wa s added and tubes were boiled for 3 min. Tubes were centrifuged and s upernatants loaded on a 10 % pol y acr y lamide s lab gel. Electrophoresi s was toward the a node. After electrophoresis the s lab gel was s tained with Cooma ss ie Brilli a nt Blue R2 50 (BioRad, Richmond CA) and then destained in a cetic acid ethanol (7:10) s olution. The gel was then washed in water for 3 0 mm laid o n a pie c e of blotter paper ( Whatman Clifton, NJ) a nd c overed w ith a s heet of Saran W rap (Dow Chemical M idland M I). This was placed on a g el d r y er ( Hoefer San Francisco C A ) and heat and v acc uum w as a pplied to remove w ater. The dried gel was then placed i n a f ilm c assette ( Hal s e y X -r ay Product s, Inc. Brookl y n, NY) with a piece of Kodak X RP x-ra y film ( E a stman Kodak Rochester NY) to visualize radioa c tivel y labelled protein s from the pol y acr y lamide g el. Pol y acr y lamide g el a nd x-ra y film w ere incub a ted for 7 da y s and then the x-ra y film was developed. Assa y development. Antibod y to bPL d eveloped a t U SDA Belt s ville (F56) was utilized to develop a RIA to bPL. Bovine placental lactogen (Dr. R.D. Bremel, M adison WI) was iodinated using the Iodo-G e n ( Pier c e C hemical) procedure. Iodo-Gen (2 g) dissolved in c hloroform wa s dried under nitrogen. The Iodo-Gen tube was rinsed with Tris buffer pH 7 .6 and allowed to dr y Bovine placental lactogen (5 g in 25 l NaHCO3, pH 9.0) and 1 mCi Na

PAGE 112

101 1251 (Amersham, Arlington Heights, IL) were combined in the Iodo-Gen tube for an incubation time of 4 min. Labelled bPL and free iodine were separated on a 7 x 17 cm Biogel P-6O (Bio Rad, Richmond, CA) column. Two hundred microliters rabbit anti-bPL antibody diluted 1:10,000 in 25 m M Tri s, 50 mM EDTA .1 % BSA pH 7.8 was combined with 100 I 1251 bPL (20.000 cpm) and incubated at 4 C for 24 hr. The next da y, 200 I s heep anti-rabbit IgG antibody (1 :80 Cappel, Ma lvern PA) and 500 I 6% pol y eth y lene glycol (PEG 8000) (Fisher Scientific Orlando, FL) were added. Tubes were vortexed and incubated for 30 min at 4 C. Tubes were then centrifuged for 30 min at 3,790 x g in a RC3B ce ntrifuge (DuPont Co., Newtown, CT) using a 6000A rotor. Supernatant was deca nted. tu bes were swa bbed and pellets were counted in a Gamma Trac 1191 ( Tm Analytic, Elk Grove Village, IL) gamma counter. Assay Validation. Because bPL c oncentrations m maternal and fetal fluids ma y have an important biological function, the bPL assay was validated in amniotic and allantoic fluids, maternal se rum a nd fetal umbilical veinous and arterial s era. Crossreactivity of anti-bPL antibody was tested against ovine, bovine and porcine, prolactin, growth hormone. luteinizing hormone and follicle s timulating hormone, bovine th yroid stimulating hormone (gifts from D. Bolt, USDA), bovine insulin ( Sigma Che mical, St. Louis. M O) and ovine placental lactogen (I. Fors y th Reading England). P ara lleli s m was tested using 50, 100 or 200 l of either allantoic or a mnioti c fluids or fetal or maternal serum. Addition of .25, .5, 1, 2 or -l ng bPL to a mniotic or allantoic fluid or fetal or maternal se rum was quantitativel y recovered. Biological Validation. To compare the biological activ it y of bPL as measured in the Prl radioreceptor assay (Prl-RRA) w ith the immunologic properties of the molecule as measured in the RIA, the sa me sa mples were

PAGE 113

102 quantified for bPL or lactogenic activity. Two replicates of culture Exp. 4 were utilized. The samples originally were measured for lactogenic activity in December 1983 and were stored at -20 C until analyzed for bPL via RIA (July 1985). The assay results were evaluated using General Linear Models analysis by the Statistical Analysis System (SAS). Immunohistochemistry. The antibody to bPL (USDA F56) also was utilized to determine immunohistochemically the site of origin of bPL. Whole placentomes were collected as previously described. Placentomes were cut through the midline to make a 1 cm section containing both maternal caruncle and fetal cotyledon. This section then was cut into 1 cm x 1 cm sections of placentome. Sections then were dehydrated in increasing <'oncentrations of EtOH (20-100 % ) and then embedded in paraffin. Seven micron sections were cut on an American Optical microtome and two sections were placed on each slide. Sections were deparaffinized and hydrated using toluene and decreasing concentrations of EtoH (100-50 % ). Immunohistochemical staining was conducted using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Sections were incubated with 3 96 H2O2 in methanol for 30 min to quench endogenous peroxide activity a11d then washed. Sections were then incubated in 3 % normal goat serum plus .1 % bovine serum albumin (Sigma Chemical Co., St. Louis, MO) for 20 min to inhibit nonspecific binding. Normal goat serum was removed and sections were incubated with either rabbit anti-bPL (USDA F56 1:20) or normal rabbit serum (1 :20) for 30 min. Slides were washed for 10 min and then incubated for 15 min with Vectastain ABC reagent at 37 C. This reagent contains A vidin DH and biotinylated horseradish peroxidase which complex and then bind to the biotinylated antibody complexed with the tissue section.

PAGE 114

103 The slides were washed for 10 min and then incubated for 5 min in peroxidase substrate solution, which activates the horseradish peroxidase. This was then washed for 5 min in tap water and then counterstained for 2 min in heamatox y lin to stain nuclei. The sections then were photographed using a Nikon 105 Phase Contrast microscope with the phase rings removed. Statistical Analysis. Parallelism and assay comparison studies were analyzed s tatisticall y using the General Linear Models procedure on the Statistical Analysis System (SAS) (Barr et al., 197 6). Regression analysis was conducted using heterogeneity of regression. Heterogeneity of regression was determined by comparing the error term from the statistical model used to determine main effects (called whole model Y = Fluid, Dose, Dose x Dose) and the model used to partition the Fluid x Do s e interaction into the individual components (called partial model Y = Fluid, Fluid x Dose, Fluid x Dose x Dose). The difference in the degrees of freedom and s ums of squares in the 2 models (whole partial) was used to calculate the mean square error and this was divided b y the mean square error of the whole model to determine s ignificance. A s ignifi ca nt result would indicate that the regression lines generated were not similar, but a non-significant result would indicate a failure to detect a difference. Results and Discussion Lactogenic activit y recovered from native pol yacry lamide gel slices is depicted in fig. 4.1. The peak lactogenic activity was associated with the top band of the Coomassie stained gel. The two s lices containing peak lactogenic activity (600 ng) were pooled, homogenized and injected into rabbits. This technique further purifies the bPL and the polyacr y lamide acts as an adjuvant in the rabbit.

PAGE 115

104 Proteins produced in cotyledon culture in the presence of 3 5 s Methionine that were radiolabelled are depicted on the flurograph (fig. 4.2). A large number of proteins from the culture medium were radiolabelled as depicted in the medium lane. Normal rabbit serum (NRS) nonspecifically immuno precipitated several of these proteins. The 45,000 MW protein that was nonspecifically precipitated may be a placental immuno-globulin that inter acted with the protein A sepharose. A specifically immunoprecipitated band was apparent in both antibody preparations with increasing intensity at higher antibody volumes (Fla 200, 100 and 50 l and USDA, 200, 100 and 100 I). The molecular weight of the specific band was estimated at 37,000 which was slightly heavier than the 32,000 molecular weight reported by Murthy et al. (1982) and Arima and Bremel (1983). Twenty percent specific binding was achieved with a 1 :40,000 final working dilution of anti-bPL antibody (USDA F 5 6) the antibody generated at Fla did not attain a sufficient titer to utilize. T h e standard curve ranged from .1 8 ng with an assay sensitivity of .1 ng. Eight nanograms of bPL displaced 77 % of the specifically bound counts. This assay was more sensitive than the assay reported by Roy et al. (1977) and s imilar to that reported by Beckers (1983). Crossreactivity of the anti-bPL antibody is depi c ted on fig. 4.3. The antibody to bPL (F56) did crossreact with ovine pla c ental lactogen, but only at .2 96 which is in contrast to Roy et al. (1977). There was no evidence of crossreactivity with any of the other ovine, bovine or porcine hormones tested. This indicates that the anti-bPL antibod y is specific for a bPL. Parallelism of either 50, 100 or 200 I of allantoic or amniotic fluid or fetal or maternal plasma was tested by heterogeneity of regression in the analysis of variance of SAS (table 4.1), which was unable to detect differences between the dose response curves and the standard curve (P>.9) (fig. 4.4), consequently,

PAGE 116

Fig. 4.2. Fluorograph of a 1 Dimension polyacrylamide gel electrophoresis gel (10 % acrylumide) depicting proteins secreted into minimum essential medium by cotyledonary explants in the presence of 100 uCi 3 5 s Methionine. Proteins were immunoprecipitated with 50, 100 or 200 l Fla anti-bPL antibody or 100 or 200 l USDA anti-bPL (F56) antibody.

PAGE 117

,.,, FLA. USDA MW NRS 200 100 50 200 100 media MW 3 20.G116.~ .97 .. +77.&. 45.9. 1 29.020.0 .. .,_

PAGE 118

Fig. 4.3. Crossreactivity of anti-bPL with 10 1000 ng of bovine prolactin (PRL), growth hormone (GH), follicle stimulating hormone (FSH), insulin (INS), luteinizing hormone (LH), thyroid stimulating hormone (TSH) and ovine pla c ental lactogen (oPL) compared with .1 8 ng bPL.

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100 ---------===================== SPRL GH ~=============~~~==================== gff~5LH +---BTSH 90 ao I \~\ 701' I \ 60 l\ "O I C: ::, ...... 0 00 \ 0 so I co '# 40 30 \BPL 20 10 0 ,._ ______ ..., ________ .... _______________ ,. 0. 1 1.0 10.0 100.0 1000.0 10000.0 Log Hormone (ng)

PAGE 120

109 Table 4.1. Heterogeneity of Regre ssi on for Paralleli s m. Whole Mode l: Source Fluid. Dose+ Dose x Do se+ Error Partial Model: Source Fluid. Fluid x Dose+ Fluid x Dose x Error *F<. 001 **F<.0001 Dose+ df ss F Value 4 .183998 46.25** 1 .509472 513.90** 1 .028002 28 25** 23 .022801 df ss F Value 4 .0051 23 1.55** 5 .516827 125.47** 5 .03109 2 7.55* 15 .012357 significance tested using Type III Sums of Squares +significance tested using Type I Sums of Squares

PAGE 121

Fig. 4.4. Parallelism of 50, 100 or 200 111 of amniotic (AM) and allantoic (AL) fluids and fetal (F) and maternal (M) serum compared to .1 8 ng bPL.

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IS1 IS1 + IS1 en IS1 llJ 111 + :JNl~NIE lN:3:>el:3d _J u.. a: 0 Iu, / + ts2 N t I I ISJ ts2 ts2 t:il ts2 ts2 ts2 ts2 ts2 ts2 ts2 w U1 C C) l!J C ...J

PAGE 123

112 there was no inhibition of binding other than bPL in the various fluids tested. Recovery of .25, .5, 1, 2 or 4 ng bPL from amniotic or allantoic fluids or fetal or maternal serum ranged from 82.5 125 % (table 4.2). Mean recovery of bPL was 116.3, 103.5, 100.5 and 108.7 % for maternal sera, fetal sera, amniotic fluid and allantoic fluid, respectively. Comparison of bPL concentrations in cultures of cotyledon by either RIA or RRA assay indicated that concentrations of either bPL or lactogenic activity were similar, but not identical. Statistical analysis using the model RIA RRA = Cow Estrogen Cow x Estrogen GH Cow x GH Estrogen x GH indicated that cow was significant using either assay (table 4.3); however, using the RIA there were significant cow x estrogen and estrogen x G H effects. This may be explained by the increased sensitivity of the RIA and the fact that the RIA was more variable between treatments. The means of both cows for treatment by each assay are depicted on table 4.4. No treatment effects were detected by either assay on bPL production in culture over values for controls. The RIA detected a suppression of bPL production by increasing estradiol concentrations, but the one and ten ng dose of estradiol in combination with GH increased bPL production over either GH or Estradiol alone. Immunohistochemical Localization. Tissue sections treated with either normal rabbit serum (A) or rabbit anti-bPL (USDA F56) (B) are depicted in fig. 4.5. The nuclei are stained with heamatoxylin. This stain accentuates the binucleate cells, which are further stained by the horseradish peroxidase in the Vectastain kit. This suggests the binucleate cells either produce or store bPL. Localization of placental lactogen in sheep binucleate cells has been reported (Martal et al., 1977) and human placental lactogen was localized in the syncytical cytoplasm (Sciarra et al., 1963). Wooding (1980) reported

PAGE 124

113 Table 4.2. % Recovery of bPL from Materna l and Fetal Fluids. Fluids ng Added Maternal Fetal Amniotic Al lantoic 25 98.2 94.4 112. 4 102.2 .50 124.5 114 .1 99.0 96.0 1.00 123.0 103.4 103.9 117 .4 2.00 111. 0 110. 7 104.6 119. 7 4 .00 125.0 94 8 82 5 108.3

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114 Table 4.3. Analysis of Variance For Assay Comparison Samples ANOVA Source Cow Estrogen Cow x Estrogen GH Cow x GH Estrogen x GH Residual *P< .1 **P<.01 ***P<.001 RIA df ss 1 235750 3 9506 3 206739 3 64450 3 37909 9 284648 71 1276818 RRA F Value df ss F Value 13.11** 1 413370 29.42*** .18 3 41584 .99 3.83* 3 11911 .28 1.19 3 6280 .15 .1 7 3 2 9932 71 1.76* 9 86461 .68 70 983669

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115 Table 4.4. Determination of bPL or Lactogenic Activity (xSE)* in Cotyledon Culture Samples Treated with Estrogen (E) or Growth Hormone ( G H). Treatment (ng) Assay (ng/ml) E GH RIA RRA 0 0 388 358 0 10 226 305 0 100 262 292 0 1000 250 295 .1 0 292 304 1 0 245 304 10 0 194 304 1 10 315 382 .1 100 275 333 .1 1000 291 287 1 10 265 282 1 100 306 259 1 1000 409 324 10 10 430 352 10 100 395 325 10 1000 377 304 xSE = 310.3.6

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Fig. 4.5. Immunohistochemical localization of bPL in bovine placental tissue. Placental tissue was embedded in parafin and stained using either normal rabbit serum (a) or anti-bPL (F56 1:50) (b) and the Vectastain ABC kit. Tissue was counterstained with heamatoxylin.

PAGE 129

118 that sheep binucleate cells migrate to form the s y ncytium, which may indicate an evolutionar y adaptation. Thorburn et al. (1981) suggested that ovine placental lactogen was secreted when the binucleate cell was removed from the fetal environment. Wooding (1983) quantitated the frequency of binucleate cells in various ruminant placentas and demonstrated that 15 to 20 % of the trophectodermal cells were binucleate and, of those, 20 % were migrating across the microvillus junction into the maternal s ystem. A radioimmunoassa y was developed specific to bPL, which detected bPL in maternal and fetal blood and amniotic and allantoic fluids. The role of bPL in these fluids is unknown but it has been suggested that placental lactogens are involved in maternal nutrient mobilization and fetal growth. This assay ma y be utilized to determine the s ignificance of bPL s ecretion during pregnanc y in the cow. The high s pecificit y and s en s itivit y of the anti-bPL antibody can detect minute variations in bPL concentrations during pregnancy. Bovine placental lactogen was localized in the fetal binucleate cell using immunohistochemical techniques. Thi s indicates that the binucleate cell is important for the s ecretion and/or s torage of bPL.

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CHAPTER V BO V INE PLACENTAL LACTOGEN CONCENTRATIONS IN M ATERNAL AND FET A L FLUIDS. Introduction Concentrations of placental lactogen in maternal plasma have been measured b y radioimmunoassay (RIA) during gestation for the rat (Robertson and Friesen 1981), mouse (Soares et al. 1982), sheep (H a nd w erger et a l., 1977) a nd human (Kaplan and Grumbach, 1965). A ttempt s to measure bovine placental lactogen (bPL) b y either RI A (Ro y et a l. 1977) or bioas s a y (Schellenberg and Frie s en, 198 2) have y ielded c onfli c ting information a s to concentrations of bPL in the m a ternal sys tem. Fetal bPL concentrations ranged from 5 22 ng / ml (S c hellenberg a nd Frie s en 198 2 ). The RI A developed as de s cribed in the preceding c hapter w a s u s ed to mea s ure bPL in maternal a nd fetal fluids during ge s tation. M aterials and M ethods Three experiment s were conducted to examine bPL con c entration s in maternal a nd fetal fluids during g e s t a tion. Experiment 1. S a mple s of m a ternal venou s a nd fet a l umbili ca l a rterial a nd venou s blood a s w ell as allantoic a nd a mnioti c fluids were c ollected from cows a t s laughter. W hole uteri w ere c olle c ted and transported to the laborator y for s ampling. The uterine w all wa s c ut with a s calpel blade to reveal the c horio-allantoic membrane. The membrane w a s pierced with an 18 gauge needle (Becton-Dickinson, Rutherford NJ) and 10 ml of allantoic 119

PAGE 131

120 fluid was withdrawn. The same procedure was followed when the amniotic membrane was reached. Fetal umbilical blood was sampled through the umbilical cord and vessel determination was made by palpation. Maternal blood was collected when the animal was exsanguinated. Fetal crown rump length was measured and used to estimate gestational age, by the method of Rexroad et al. (197 4). Fluid samples were placed on ice and stored at 4 C overnight. The next day the samples were centrifuged at 1,100 x g for 10 min in an RC2-B centrifuge using an SS24 rotor. After centrifugation, supernatants were stored at -20 C until analyzed for bPL. Experiment 2. Twenty-one Holstein heifers were randomly assigned to be bred with either Holstein Angus or Brahman semen, to determine the effect of sire of fetus on maternal and feta! hormone production (Guilbault et al., 1985). Blood samples were collected by jugular venipuncture thrice weekly from day 150 260 of gestation and then dail y until term. Samples were collected in heparinized tubes on ice and immediately centrifuged at 5,000 x g for 15 min in an RC2-B centrifuge using a SS24 rotor. Plasma was collected and stored at -20 C until analyzed. Samples from 12 of the heifers (four from each breed group) were utilized for bPL analysis. Plasma samples from day 260 until term were previously used for quantitation of estrogen, progesterone and 13, 14-dihydro-15-keto PGF2 a Experiment 3. Four Holstein cows at approximately day 250 of gestation were utilized for this experiment. Cannula (V9 Bolab, Lake Haversan City, AZ) were placed nonsurgically in the jugular vein the day prior to sa mpling. Blood samples were collected at 30 min intervals for a period of 12 hr. Samples were placed on ice and allowed to clot overnight. Samples were then centrifuged at 1,100 x g for 10 min in an RC2-B centrifuge using an SS24 rotor. Serum was harvested and stored at -20 C until analyzed for bPL.

PAGE 132

121 Bovine Placental Lactogen Analysis. Samples were assa y ed in duplicate (volumes ranged from 100 300 l) as described previously with a slight modification. Samples with very low concentrations of bPL (maternal serum or plasma and amniotic fluid) were allowed to incubate an additional 24 hr to attain the sensitivity to detect low concentrations of hormone. In these fluids the assay procedure was: sample was combined with 200 l anti-bPL antibody and 100 l (20,000 cpm) 1251-labelled bPL. This was vortexed and incubated at 4 C for 24 hr. At the end of the first incubation 200 l sheep anti-rabbit IgG (Cappell Cooper Biomedical, Malvern, PA) was added and tubes were vortexed and incubated at 4 C for 24 hr. After the second incubation 500 l 6 % {w / v) PEG was added, tube s were vortexed and incubated for 30 min at 4 C. Tubes were then centrifuged at 3,790 x g for 30 min in an RC3-B centrifuge using a 6000A rotor. Supernatants were decanted and tubes were swabbed before pellets were counted for radioactivit y Statistical Analysis. Experiments 1 a nd 2 were analyzed s tatistically using the General Linear Models procedure on the Statistical Analysis S ys tem (Barr et al., 1976). Regression analysis was conducted using heterogeneit y of regression. Source was used to describe the different fluids tested and day was run as a continuous variable. Results and Discussion Experiment 1. Samples were collected from 28 cows between 123 and 268 days of gestation. Mean bPL c oncentrations for the various fluids are depicted in table 5.1. Fetal blood contained the highest concentration of bPL compared to the other fluid pools. Concentrations in the fetal circulation were similar between umbilical artery and vein ranging from 3.8 50. 7 ng/ml (fig. 5.1). The highest concentrations of bPL were detected at the earliest

PAGE 133

122 Table 5.1. Bovine Placental Lactogen Concentrations in Maternal and Fetal Fluids. Pool n bPL ( x SE) Amniotic 18 .9 1 Al lantoic 27 3.1 5 Fetal Umbilical Arterial 26 18.8 2.5 Fetal Umbilical Venous 27 19.7 2.7 Maternal 24 2.2 2

PAGE 134

Fig. 5.1: Concentrations of bPL (ng/ml) in amniotic and allantoic fluids and maternal venous and fetal umbilical arterial and venous blood. Samples were collected at slaughter and day of gestation was calculated u s ing the crown rump length (Rexroad et al., 197 4). 1 I

PAGE 135

12 4 I --: ;, > ..L [[ L.....U.l: + C < Y .. -lJ s !:SJ !SI Q iS2 Lrl Li, ...... fT1 f\l ..J [ 7W / C7N J 7d3 / Q m j\J UJ N rs! :r N -,. --rr [Sl r!Sl L 7 f\J LJ n C lSl 1 rn 1 l '. u: :I ISl J ...... .J J I [SJ N !Si

PAGE 136

125 days in gestation examined with peaks occurring at day 210 220 and 230. Fetal calf blood volumes have not been reported previously but blood volumes in the fetal lamb are about 18.1 % when fetal weights vary over a range of 1 6 kg (Assali, 1968). Assuming that this relationship holds true for the fetal calf, total bPL increases throughout gestation (table 5.2) along with fetal weight (Swett et al., 1948). Regression analysis (fig. 5. 2) indicated that concentrations decreased from day 123 to day 268. Sex of calf did not effect bPL concentrations (table 5.3). Ovine placental lactogen has been implicated in replacing growth hormone in the fetus and bPL may serve the same function. Placental growth and nutrient transfer may be stimulated directly by bPL or indirectly through an insulin-like growth factor intermediate. Allantoic fluid concentrations of bPL ranged from .3 6.8 ng/ml. Arthur (1957) measured fetal calf allantoic and amniotic fluid volumes and reported that allantoic fluid volumes increase dramatically from day 120 until term. Using the allantoic fluid volumes reported by Arthur (1957), total bPL was calculated (table 5.2) and increased throughout gestation. Peak concentrations in this study were determined at days 154 and 2 29. The high concentrations of bPL in allantoic fluid implies that bPL is removed from the fetus via the urine. The role of the peptide in allantoic fluid is unclear, but the lactogenic activit y that the protein displays may suggest an osmoregulatory role similar to prolactin. Amniotic fluid concentrations of bPL were the lowest of the fluids tested (table 5.1), and ranged from undetectable to 2.0 ng / ml. Peak bPL concentrations were determined at days 166 and 232 of gestation. Total bPL in amniotic fluid, calculated from reported amniotic fluid volumes

PAGE 137

Table 5.2. Est i mated Fluid Volumes and Tota l bPL Concentrat ion s Allantoic Amniotic Fetal Blood Maternal Blood Fetal A~ Vol (ml) bPL (ng) Vo l (ml) bPL (ng) Vol (ml) bPL (ng) Vol (ml) bPL (ng) ---140 920 5704 3325 5320 395 12845 38181 89727 ...... Nl 165 2950 2655 3285 6405 924 13593 38181 120272 en 226 4800 18720 3150 3780 2360 19119 38181 1833 7 2 264 9600 45120 2400 1680 5377 29308 38181 57272

PAGE 138

Fig. 5.2: Linear regression of bPL concentrations in maternal serum, fetal umbilical arterial and venous serum, allantoic fluid and amniotic fluid collected at slaughter from cows at b ~ tween 120-268 days of gestation.

PAGE 139

S:121 Y121 r, i 30 I!] z LJ _J n. tO 2121 10 0 ~. ~FR --....--.....____ f y AL M RM I I I C. ... . . I I 120 IY0 160 180 200 220 2Y0 260 DRY OF GESTATION >-' l--.J 00

PAGE 140

129 Table 5.3. Analysis of Variance for Maternal and Fetal Samples Source* Fluid Sex Day Residual df 4 1 1 85 ss 5757.36 43.92 524 .45 4854.60 F value 25 .20*** .77 9.18** *Fluid and sex were tested using Type III Sums of Squares (SS). Day was tested using Type I SS. **P < .01 ***P < .001

PAGE 141

130 (Arthur 1957) peak at day 165 of gestation and then decrease until term coincident with fluid volume (table 5. 2). Maternal bPL concentrations were similar to allantoic fluid bPL concentrations, ranging from .5 4.8 ng / ml with peak concentrations at da y s 205 and 213 of gestation. Total bPL in maternal blood was calculated assuming a maternal weight of 545.5 kg and that bleed volume was 7 % of the body weight (table 5.2), and found to increase to a peak at day 226 of gestation and then decrease to term (table 5.2). As the name implies bPL ma y s timulate mammar y development during gestation, but the evidence in ruminants does not support this h y pothesis. Kaplan and Grumbach (1965) s uggested using anecdotal type evidence t hat placental lactogen ma y be important in nutrient partitioning during gestation and this may be one of the functions of bPL. This is the first report of bPL c oncentrations rn maternal and fetal fluids. Ro y et al. (1977) attempted to measure maternal c oncentrations but the sensitivity of the RIA was inadequate (< 100 ng / ml). Schellenberg and Friesen (1982) reported fetal bPL concentrations ranging from 5 2 2 ng / ml in the Nbz lymphoma assay which a re c onsistant w ith values obtained in this study. The ratio of bPL concentrations between fluids in this s tud y 1s not m agreement with reports for other s pecie s Kaplan a nd Grumba c h (1965) reported that human fetal hPL c oncentrations w ere 5 0 to 2 00 time s lower than maternal concentrations (10 -lO / ml) a nd a mniotic fluid concentrations were only 20 % of maternal hPL con c entrations. Handwerger et al. (1977) demonstrated that sheep umbilical cord plasma and allantoic fluid concentrations of ovine placental lactogen w ere 10 % and 1 % respectively, of maternal concentrations (peak of 2400 ng / ml) while amniotic fluid concentrations ranged from 5 90 ng / ml at da y 50 but were undetectable

PAGE 142

131 thereafter (Chan et al., 1978). Fetal concentrations of bPL in this study were 10 times maternal concentrations indicating larger differences even between closel y related species like the sheep and cow. Total bPL, in the fluid pools studied was highest in maternal blood throughout the period examined (table 5 .2) which suggests that bPL is secreted preferrentiall y in the maternal s y stem as occurs in sheep (Handwerger et al., 197 7 ). Experiment 2 Statistical analysis indicated ( table 5.-D that breed was not a significant main effect but, Cow ( Breed) and Da y t o the 5th order were significant. Heterogeneit y of r egression w as c alculated a s previousl y described i n Chapter IV. The difference i n mean squares o f t he models i n tables 5A and 5.5 w as significantly d ifferent than t he mean s quare of t he model in table 5.4, which suggested that the regression lines of the Breed x Day interaction ( bPL ( ng / ml) vs da y of g estation) w ere d iffe r ent ( fig. 5. 3 ). This is depicted by differences in pattern o f c hange i n bPL indicated b y the regression lines tn fig. 5. 3 Concentrations of bPL r anged from undetectable to 3 .1 ng / ml, with peak c oncentrations bet w een d a y s 21 0 a nd 2 30 of gestation. This is in agreement with data f rom s ingle s amples o btained in Exp. 1. The concentration of bPL in maternal s amples w as much lo w er than placental lactogen concentrations reported for t he human ( Kaplan a nd Grumbach, 1965 ), sheep ( Handwerger e t a l.. 197 7 ) ra t ( Rober t son a nd Fri e s en. 1981) and mouse ( Soares et al. 198 2 ) a nd t he pattern o f s ecretion i s d istinct from that of the other species. The c oe f ficient o f var i ation of bPL c oncen trations in the -!64 blood samples in this experiment was -!1. 2 96 w ith a mean value of 1.22 ng / ml. This indicates that the s ample s ize f or breed was probably not large enough to detect statistical differences. Guilbault et al. (1985) detected breed differences in concentrations of estrogen proges terone and 13 14-dih y dro-15 keto-PGF 2 'J. using the s ame s amples a s w ere

PAGE 143

132 Table 5.4. Anal y sis of Variance for bPL Concentrations Across Gestation (Exp. #2). Source df ss F value Breed* 2 .069 .02 Cow (Breed) 8 2 9.011 24.60*** Da y 1 .133 90 Da y xDa y 1 .083 .57 Da y xDa y xDa y 1 .183 1. 2 4 Da y xDa y xDa y xDa y 1 3 76 2 5 5 Da y xDa y xDa y xDa y xDa y 1 6 1 9 4. 2 0** Residual 44 2 65. 15 5 *Breed was tested for significance using C: ) w ( Breed) a s the error term. **P < .1 ***P < 0 01 I I I I I I I I I I I I I I I I I I I

PAGE 144

133 Table 5.5. Analysis of Variance for bPL Concentrations across Gestation (Exp. # 2) Heterogeneity Source df ss F valuP. Breed* 2 393 .06 Cow (Breed) 8 26.343 25.30*** B,eedxDay 3 .423 1.08 BreedxDayxDay 3 .255 .65 BreedxDayxDayxDay 3 .278 71 BreedxDayxDayxDayxDa y 3 451 1. 16 BreedxDayxDayxDayxDayxDay 3 713 1. 83 Residua 1 432 56.232 *Breed was tested for significance using Cow (Breed) as the error term. ***P < .001

PAGE 145

Fig. 5.3: Least squares regression (5th Ol'<1el') of bPL concentrations of Holstein heifers : ,erviced by either Holstein (1 ), Angus (2) 01 Brahman (3) semen. Blood sumples weIe collecte<1 thrice weekly from day 150 to day 260 of gestation and then daily until pal'turition. lndivirlual data points are lhe arithmetic mean (n = 4).

PAGE 146

135 N + 1; + I CS2 l"'l I + z tS2 rl.t1 a:: I :::J ra:: a: a.. + ISi l' rI a:: a:: a.. tS2 tn en I a: C ISi .._ _______ ____ _., __ ....., __ ...,._ ____ ,.__.. r, iSl l.t1 tS2 l.t1 . N CS2 C 7W/:JN J 7dQ

PAGE 147

136 utilized in this study but they had seven animals in each breed group. They suggested that both fetal and maternal hormones were influenced by conceptus genotype. Experiment 3. Concentrations of bPL in samples collected from four cows at 30 min intervals for 12 hr ranged from .3-8.2 ng / ml. Concentrations 'Jf b?L w ere si::nil:J.::~oth .vithin ar.d between cows but two cows exhibited a pulsatile release of bP L during the 12 hr bleeding period ( fig. j_4 l. The rapid disappearance of the bP L from the blood suggests a short half-life for bPL. The variability tn bPL concentrations was low f or most of the 12 hr examined which is in contrast to the report b y Klindt et al. ( 1982) for the rat. However the y examined s amples co llected a t :5 1 0 min intervals while samples in this study were collected at 30 min i ntervals. The pulsatile release of bPL may be important to stimulate nutrient transfer ac ross the placenta, similar to the suggestion of Kaplan and Grumbach ( 1965) f or the human. Another possible explanation for the pulsa tile release of bP L is that binucleate cells may migrate across the placenta in groups and release bPL into the maternal system. In s ummary bPL was detected in maternal a nd f etal fl uids during gestation. Fetal blood contained the highest co n ce ntrations of bfL c ompared I with amniotic and allantoic fluids as well as maternal blood. However. calculation of total bPL rn each fluid pool i ndicated t hat large amounts of bPL are secreted into the maternal sys tem. The pattern of c hange in bP L in maternal plasma during gestation is s ugge s ted b y i ncreasing s ecretion of bPL from day 150 to day 250 of gestation and then decreasing s ecretion until term. Swett et al. (1948) demonstrated that fetal weight increases two-fold during each month beginning at approximatel y the fifth month of gestation and peak rate of weight gain occurred at day 23 0 ( Eley et al.,

PAGE 148

Fig. 5.4: Bovine placental lactogen co ncentra lions from four Holstein cows at approximately day 250 of gestation. Blood samples were collected at 30 min inte1vals for a period of 12 h1 during a single day.

PAGE 149

!SJ l: w lJl H 138 rn rn ill LIi T r, N lSl N lSl a, [D Ul ll] n:: LIi T r, N ::J I

PAGE 150

139 1978). Variabilit y in secretion of bPL in acute bleeding experiments with frequent samples is low; however, pulses of bPL do occur (3 % of samples). The physiological significance of bPL in maternal and fetal fluids is still unclear, but the presence of this protein in high concentrations in the fetal circulation suggests a role in fetal development.

PAGE 151

CHAPTER VI GENERAL DISCUSSION The experiments described in this dissertation indicated that bovine placental lactogen (bPL) was produced by the fetal cotyledon of the bovine placentome. The cell type that produced bPL was immunohistochemically identified as the binucleate cell. Duello et al. (1985) indicated that bPL was contained in secretory granules within the binucleate cell and Byatt et al. (1985) reported isolation of bPL by isolating these secretory granules. Wooding (1980) demonstrated that fetal sheep binucleate cells migrate across the placental barrier and fuse to form the maternal syncytium. This may explain the high concentrations of ovine placental lactogen in the maternal circulation. Thorburn et al. (1981) reported that epidermal growth factor infusion into pregnant ewes stimulated release of ovine placental lactogen into the maternal circulation. They further suggested that epidermal growth factor may be the placental mitogen that determines the rate of migration of fetal binucleate cells. They also observed that binucleate cells cultured in calf serum degranulate and when the medium was replaced with fetal calf serum the granules reappeared. They hypothesized that ovine placental lactogen stimulates release of somatomedins such as insulin-like growth factor II, which in turn enhances the transport of glucose and amino acids across the placenta to stimulate fetal growth. The role of placental lactogen in fetal growth has been suggested previously. Niall et al. (1971) reported that over 80 % of the amino acid sequences of human placental lactogen (hPL) and human growth hormone (hG H) were identical. Binding of 1 Z 51 ovine placental lactogen to membrane 140

PAGE 152

141 receptors was inhibited by growth hormone, but uneff ected by prolactin (Chan et al., 1978). Hurley et al. (1977) demonstrated that administration of ovine placental lactogen stimulated release of somatomedin. Several researchers have reported different responses to administration of ovine placental lactogen in vitro when fetal or neonatal tissues were utilized. Adams et al. (1983) reported that ovine placental lactogen stimulated insulin-like growth factor II production b y fetal r-a t fibroblasts while in adult rat fibroblasts either hGH or ovine placental lactogen stimulated production of insulin-like growth factor I. Ovine placental lactogen or growth hormone stimulate ornithine decarboxylase activit y in neonatal rat liver (Butler et al. 1978) and tr a nsport of alpha amino i s obut y ric a cid into w eanling rat diaphram cells (Freemark and Handwerger 1982). However in fetal tissues onl y ovine placental lactogen was effective in stimulating ornithine decarboxylase activity (Hurle y et al., 1980) or alpha amino isobutyric acid transport (Free mark and Handwerger 1983). These results suggest that ovine placental lactogen may replace growth hormone in the fetus. Attempts to stimulate the production of bPL b y explants of cotyledons in vitro indicated that bovine growth hormone or c o-culture of cot y ledonar y and caruncular tissue stimulated the relea s e of bPL a s mea s ured b y lactogenic activit y in a prolactin radioreceptor assa y (Prl RRA). The mechanism of this stimulator y action of either growth hormone or c aruncular co-culture on bPL secretion is unknown. The caruncular ti s sue ma y s ecrete a s timulatory substance such as epidermal growth fa c tors a s s uggested b y Thorburn et al. (1981), to stimulate bPL release into the medium. Fors y th and Ha y den (1980) stimulated the release of bPL from cot y ledonar y explants by the addition of arginine to the medium while addition of either c y clohexamide or dopamine had no effect. Release of hPL from placental explant culture was stimulated b y the addition of pimozide (Macaron et al., 1978), estradiol

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142 (Belleville et al., 1978), arachidonic acid (Handwerger et al., 1981a), EDTA, EGTA or methoxyverapamil (Handwerger et al., 1981 b) and insulin (Perlman et al., 1978). Neither dopamine, estradiol or arachidonic acid had an effect on bPL release. Macaron et al. (1978) suggested that hPL release may be modulated by dopaminergic receptors, which is in contrast to the results of these studies. A major factor in the production of lactogenic activity in these studies was quantity of tissue in the explant culture. The term lactogenic activity (ng/mg tissue) was used to correct for the variance in total activity due to tissue size. In doing so, hormonally stimulated responses may have been masked. Another variable in explant cultures was tissue heterogeneity. Histology of placental explants (fig. 4.6) demonstrated this variability. Explants with higher numbers of binucleate cells may produce more bPL, therefore, a binucleate cell culture system may be more valuable in determining the hormonal requirements for placental lactogen release. Bovine placental lactogen was purified from cotyledonary culture medium in this study. Using this method, culture medium contained bPL with a specific activity (ng bPL/g protein) of 2.66 which was from 3.6 10 times greater than the purity of the starting material used by Murthy et al. (1982), Beckers et al. (1980) and Arima and Bremel (1983). These workers used cotyledonary homogenates and ammonium sulfate precipitation as the initial step in purification. The purification scheme utilized in this study was similar to the method of Arima and Bremel (1983). However, with the advantage in initial purity of material from culture medium, the purification scheme wasn't as rigorous. Using the culture medium and this purification scheme bPL was purified by 362 and 616 fold (peaks II and III, respectively) (table 3.1) which is similar to previous reports. Two dimensional

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143 polyacrylamide gel electrophoresis (fig. 3.6) depicts the results summarized in table 3.1. The protein purified in this study was not a major protein in cotyledonary culture medium. However, evaluation of lactogenic and somato tropic activity in the column fractions allowed identification of a single protein after the chromatographic scheme. The dramatic decrease in somato tropic activity after DEAE ion exchange remains puzzling. This could result from either a conformational change in the molecule or elution of a second somatotropic molecule; however, there is no precedent in the literature for these explanations. Arima and Bremel (1983) and Beckers et al. (1980) reported both lactogenic and somatotropic activities for bPL, but used heterologous assay systems for both activities. Somatotropic activity of bPL in this study was detected b y using a homologous growth hormone radioreceptor assay (Haro et al., 1984). Comparison of the lactogenic and somatotropic activity elution profiles indicated that the homologous assay is much more specific than the heterologous prolactin radioreceptor assay, but because of the loss of somatotropic activity during purification, the lactogenic activity profile was followed to determine protein purit y Purified bPL was not lactogenic in a bovine mammary gland explant culture system over a range of 0 5,000 ng / ml. This range ma y not have been adequate to detect an effect. Servel y et al. (1983) reported that concentrations of ovine placental lactogen of 70 / ml were lactogenic; however these were not physiological. Placental co-culture with mammary explants has been used to identify the presence of placental lactogens in several species (Fors y th, 197 4), but the homologous system utilized in this study may be more specific. The purified bPL, peak III was utilized to raise antibodies in rabbits both at the USDA (Beltsville, MD) by Dr. Doug Bolt and at the Universit y

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144 of Florida. Different techniques were utilized to prepare the antigen for immunization. At Florida, a native gel method was used to further purify the molecule. By increasing the purity and injecting the bPL in polyacryl amide, approximately 1 g of protein was enough to stimulate antibody production. The antibodies produced at both locations immunoprecipated a single specific band of approximately 37,000 MW from culture medium (fig. 4.2). The USDA antibody (F56) immunohistochemically localized bPL in fetal binucleate cells and indicated that they are the site of production and/or storage of bPL; this supports data from sheep (Marta! et al., 1977) and humans (Sciarra et al., 1963). Wooding (1983) found that 15 % of the binucleate cell population was in the process of migrating from fetal into the maternal portion of the placenta at gestational stages included in this study. This would explain how a 32,000 MW molecule could cross the placenta into the maternal blood. A radioimmunoassay was developed using antibodies produced against purified bPL (USDA F56). A 1:40,000 final working dilution of antibody was utilized to achieve approximately 20 % specific binding of l 25r-bPL. The standard curve ranged from .1 8 ng with a sensitivity of .1 ng. The antibody failed to crossreact with porcine, ovine or bovine prolactin, growth hormone, luteinizing hormone and follicle stimulating hormone or bovine thyroid stimulating hormone and insulin at concentrations ranging from 10 to 1,000 ng/tube. It did crossreact with ovine placental lactogen at approximately .2 % Dose response curves of 50, 100 or 200 l of either amniotic fluid, allantoic fluid, fetal sera or maternal sera were parallel to the standard curve (P>.9), which indicates that molecules in these fluids did not inhibit binding. Bovine placental lactogen added at .25, .5, 1.0, 2.0 or 4.0 ng was recovered quantitatively from amniotic fluid, allantoic fluid,

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145 fetal sera or maternal sera at from 82 125 % Radioimmunoassays of bPL have been reported previously (Bolander et al., 197 6; Roy et al., 1977 and Beckers, 1983). The report by Bolander et al. (1976) has not been repeated. Roy et al. (1977) reported that bPL concentrations were below the sensitivity of the assa y (
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146 that the binucleate cells may secrete placental lactogen when it is removed from the fetal influence. The role that bPL plays in either the fetal or maternal s y stem was not elucidated by these studies, but several possibilities exist. Kaplan and Grumbach (1965) suggested that hPL was responsible for nutrient partitioning during gestation, but support for this h y pothesis has not been reported. Increased placental transport of nutrients may be stimulated by increased placental lactogen, which could directly affect the fetus. Placental lactogen may locall y stimulate placental growth to increase transport of nutrients. Stimulation of fetal growth has been suggested b y s everal researchers. The fact that ovine placental lactogen replaces growth hormone in fetal rat liver (Hurley et al., 1980), diaphram cells (Freemark and Handwerger, 1983) and fibroblasts (Adams et al., 1983) indicates that fetal tissues ma y recognize placental lactogens as the fetal growth hormone. As the name implies, placental lactogen ma y stimulate mammar y development or milk secretion. However, there are no reports that support this hypothesis. Placental lactogen may stimulate the mammar y gland indirectly, by increasing insulin-like growth factors or nutrients Milk secretion during a c oncurrent pregnanc y do~s not increa s e as bPL increases during gestation which suggests that the growth hormone-like a ctivit y of bPL is not enough to over-ride the nutrient drain o( the fetus. As bPL becomes available research exploring the affect of bPL on the developing mammar y gland may be beneficial to the understanding of this hormone.

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LITERATURE CITED Adams, S.O., S.P. Nissley S. Handwerger and M.M. Rechler. 1983. Develop mental pattern s of in:suli11-like grow ti 1 factor-i and -Ii synthesis and regulation in rat fibroblasts. Nature 302:150-153. Agate, F .J. 1952. The growth and secretory activity of the mammary glands of the pregnant rhesus monkey (macaca mulatta) following hypophy sectomy. Amer. J. Anat. 90:257-284. Ahren, K. and D. Jacobsohn. 1956. Mammary growth in hypoph ysec tomized rats injected with ovarian hormones and insulin. Acta Ph ys iol. Scand. 37:190-203. Ainsworth, L. and K.J. R y an. 1966. Steroid hormone transformations b y endocrine organs from pregnant mammal s I. Estrogen bios y nthesi s by mammalian placental preparations in vitro. Endocrinology 79:875-883. Allen, E. and E.A. Doi sy 1923. An ovarian hormone preliminar y report on its localization, extraction and partial purification and action in test animals. J. Amer. Med. Assoc. 81:819-821. Allen, E., 8.F. Francis, L.L. Robert so n C .E. Co lgate and C .G. Johnston. 1924. The hormone of the ovarian follicle: It s localization a nd action in test animals, and additional point s bearing upon the internal s ecretion of the ovar y Amer. J. Anat. 34:133-182. Alling, C., A. Bruce, I. Karlsson 0. Sapia and L. Svennerholm. 1972. Effect of maternal essential fatty acid supply on fatty acid composition of brain, liver, muscle and serum in 21-day-old rats. J. Nutr. 102:773-782. A moroso E.C. 1952. Placentation. PP. 127-311. In A .S. Park s (Ed) Mars hall 's Phyiology of Reproduction. Vol. 2 Longmans Green a nd Co Lt. London. Anderson, J. W. 1969. Ultrastructure of the placenta a nd fetal membranes of the dog. I. The placental labyrinth. Anat. Rec. 165:1 5-36. Anderson, R.R. 1975. Mammary gland growth in the h y poph ys ectomized pregnant rat. Proc. Soc. Exp. Biol. Med. 148:283-287. Anderson, R.R. and C. W. Turner. 1969. Ma intenance of pregnanc y and mammary gland growth in the hypoph ys ectomized rat. J. Anim. Sci. 29:183. 147

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152 Fellows 1 R.E., T. Hurley, W. Maurer, and S. Handwerger. 1974. Isolation and chemical characterization of ovine placental lactogen. Endocrinology 94:A113. Florini, J.R., G. Tonelli, C.B. Brewer, J. Coppola, I. Ringler, and P.H. Bell. 1966. Characterization and biological effects of purified placental protein (Human). Endocrinology 79:692-708. Forsyth, I.A. 197 4. The comparative study of placental lactogenic hormones: A review. PP. 49-67. In Lactogenic Hormones, Fetal Nutrition and Lactation. John Wiley and Sons, New York. Forsyth, I.A. and T.J. Hayden. 1980. Effects of dopamine and arginine on bovine placental lactogen production in vitro. J. Endocrinol. 85:31 P-32P. Forsyth, I.A. and R.P. Myres. 1971. Human prolactin. Evidence obtained by bioassay of human plasma. J. Endocrinol. 51:157-168. Frazier C.N. and J. W. Mu. 1935. Development of female characteristics in adult male rabbits following prolonged administration of estrogenic substance. Proc. Soc. Exp. Biol. Med. 32:997-1001. Freemark, M. and S. Handwerger. 1982. Ovine placental lactogen stimulate3 amino acid transport in rat diaphragm. Endocrinolog y 110:2201-2203. Freemark, M. and S. Handwerger. 1983. Ovine placental lactogen, but not growth hormone, stimulates ammo acid transport in fetal rat diaphragm. Endocrinology 112:402-404. Freemark, M. and S. Handwerger. 1984. S y nergistic effects of OPL and insulin on glycogen metabolism in fetal rat hepatoc y tes. Amer. J. Physiol. 247:-718. Friesen, H.G. 1964. Purification of a placental factor with immunological and biological similarity to human growth hormone. Endocrinolog y 74:1006. Friesen, H.G. 1965. Purification of a placental factor with immunological and chemical similarity to human growth hormone. Endocrinology 76:369-381. Friesen, H.G., S. Suwa, and P. Pare. 1969. S y nthesis and s ecretion of pla cental lactogen and other proteins by the placenta. Rec. Prog. Horm. Res. 25:161-205. Gaspard, U .J ., A.S. Luyckx, A.N. George. and P .J. Lefebure. 1977. Relation ship between plasma free fatty acid levels and human placental lactogen secretion in late pregnancy. J. Clin. Endocrinol. Metab. 45:246-254. Gaspard, U.J., H. Sandront, and A. Luyckx. 1974. Glucose-insulin interaction and the modulation of human placental lactogen (hPL) secretion during pregnancy. J. Obstet. Gynecol. Brit. Commw. 81:201209.

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BIOGRAPHICAL SKETCH Charles R. Wallace was born in Pontiac, Michigan on January 3, 1955, to Ralph J. and Emily C. Wallace. He attended primary and secondary schools in Birmingham, Michigan and graduated from Big Rapids High School, Big Rapids, Michigan, in 197 3. After receiving his Bachelor of Science in zoology from Michigan State University, East Lansing, Michigan (March 1977), he was employed as a Research Technician in the Dairy Science Department at Michigan State University. He married June A. Vallender on May 20, 1978 and they honeymooned on their trip to the University of Georgia, A thens, Georgia, to begin a Master of Science program in the Animal and Dairy Science Department under the direction of Terry E. Kiser. After the completion of his Master of Science program he journeyed south to begin a Doctor of Philosophy program in the Dairy Science Department at the University of Florida, Gainesville, Florida, under the direction of Robert J. Collier. While at Florida he added to his family with the births of Katherine June (August 8, 1982) and Steven Charles (September 3, 1984). Upon completion of his PhD program he has accepted an NIH Training Grant Postdoctoral Fellowship at Cornell University, Ithaca, New York, in the Animal Science Department. 164

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I certif y that I have read this study and that in my opm10n it conforms to acceptable standards of scholarly presentation and is full y adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. 7:Zyl~~ C,~t,L L L Robert J. Collier, Chairman Associate Professor of Animal Science I certify that I have read this study and that in m y opm10n it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and qualit y as a dissertation for the degree of Doctor of Philosoph y / Fuller W. Bazer _/ Professor of Animal Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is full y adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosoph y T Wdce!'/ Charles J. Wilcox Professor of Dairy Science

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I certify that I have read this study and that in m y opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and qualit y, as a dissertation for the degree of Doctor of Philosoph y DonM Caton Professor of Anesthesiology and Obstetrics and Gynecology I certify that I have read this study and that in m y opinion it conforms to acceptable standards of scholarly presentation and is fully adequatet in scope and quality as a dissertation for the degree of Doctor of Philosoph y ----( / ( ',. ( "'> [ I \j : -:) ( \..; William Buhi Ass istant Professor of Biochemistr y and M olecular Biolog y

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This dissertation was s ubmitted to the Graduate Facult y of the College of Agriculture and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. December, 1986 Dean, Graduate School

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