BOVINE PLACENTAL LACTOGEN: ISOLATION, PURIFICATION
AND MFARTTRFMFNT IN TjT,,OOT(4(AT. FT,TTTTID
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
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
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
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
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
ACKNOWLEDGEMENTS . . . . .
LIST OF TABLES ..... .... ........
LIST OF FIGURES . . . . . .. .. ..
ABSTRACT . . . . . . . . . .
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
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
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
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
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
4.3 Crossreactivity of anti-bPL with various protein 108
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
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
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
Charles Ralph Wallace
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
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
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
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 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
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.
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
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
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
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.
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.
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.
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,
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
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.
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
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,
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.
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
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.
COTYLEDON CULTURE EXPERIMENTS
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
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
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
Table 2.2. Analysis of Variance for Cotyledon Culture (Exp # 2)
+Tisswt was tested using Type I Sums of Squares.
'Cow, Trt, CowxTrt, Time, CowxTime, TrtxTime and CowxTrtxTime were
tested using Type III SS.
1 I - - -
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N AIIAIIH NH
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II I I I'
5 5 5 5 5 5
a Q l m N S
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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.
Acetate + Glucose
*Means with same letter are not significantly different (P<.05).
Table 2.4. Analysis of Variance for Culture Exp. #3.
+Grid was tested using cow (Grid) as the error term.
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.
Pooled SEM = 80.34
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.
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
+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.
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
C 1W/INJ AiIA l^11 I N3aEOL1D,
-- a hi ~
-- EJ hi
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.
Activity (LA) jg LA/g
6.67 .184 1.464
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.
ng/ml 24 hr
Table 2.9. Lactogenic Activity Produced by Cotyledonary Tissue Cultured
in 0, 75, 150 or 300 uM Arachidonic Acid for 24 hr.
(ng LA/mg Tissue)
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
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.
PURIFICATION OF BOVINE PLACENTAL LACTOGEN
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,
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
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
o c -
d o w a f W W(
(IW/Bu) AIAIJOV OldOIILOLVYlOS JO OIN3D01OOV
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'C S_ f
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I I I oI
o o o o
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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).
0 0 *- 0)
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