Title: Bovine placental lactogen
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Title: Bovine placental lactogen
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Creator: Wallace, Charles Ralph, 1955-
Copyright Date: 1986
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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









C.-


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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--aaa


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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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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 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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