AMER. ZOOL., 15:279-284 (1975).
In vitro Demonstration of Lactogenic Activity in the Mammalian Placenta
FRANK TALMANTES J R .
Department of Zoology and Cancer Research Laboratory, University of California,
Berkeley, California 94720
SYNOPSIS. As part of a comparative study of the occurrence of placental prolactins among
mammals, studies were done to see if the placentae of a variety of mammals produce a
prolactin, and if it is produced, during what period of gestation it is demonstrable. The
lactogenic activity of baboon, sheep, chinchilla, hamster, mouse, rat, and rabbit placentae at
different stages of pregnancy were examined by organ co-culture of placental explants with
prelactating mammary tissue, as well as by the addition of placental extracts to mammary
organ cultures. The lactogenic activity of human placental lactogen (hPL) was also
examined. Mammary tissue from nulliparous (day 11 or 12) pregnant BALB/c Crgl mice
were cultured in Waymouth's synthetic medium supplimented with insulin (5 /ttg/ml) and
aldosterone (1 /ig/ml). The lactogenic activity of the baboon, sheep, chinchilla, hamster, rat,
and mouse placentae was clearly demonstrable. In the rabbit, the lactogenic activity was not
detectable.
INTRODUCTION
The several aspects of mammary gland
development and function (ductal growth,
alveolar development, initiation and
maintenance of lactation) are influenced by
a variety of hormonal factors. Whereas
studies such as those of Lyons (1958) and of
Nandi (1959) have delineated the minimal
hormonal requirements for mammary
growth in the rat and mouse, it is likely that
during pregnancy, other hormones elaborated in or released from the placenta, certainly proteins and probably steroids, contribute importantly to normal mammogenesis. During gestation in the mouse,
the alveoli of the mammary gland proliferate and become organized into lobules. At
the beginning of the third week of pregnancy (day 13 or 14), the lobules gradually
acquire several indicators of active secreI am indebted to Professor Howard A. Bern for his
advice in the preparation of this paper, to Ann Mos
and Walter Jackson for excellent technical assistance,
to Paul Tibbetts, Sue Handa, and David Cohen for
their help with the mouse colony, and to John Underhill for the photographic work. The research was supported by N.I.H. grants CA-05388 and CA-05045 to
H. A. Bern and fellowships from the John Hay Whitney and Ford Foundations.
Present address of author: Biology Board and
Oakes College, Thimann Laboratories, University of
California, Santa Cruz, California 95064.
tion: stainable secretory material in the
lumina, distention owing to the accumulation of secretory product, and appearance
of intracellular and luminal lipid evident as
vacuoles in ordinary histological preparations. The growth and differentiation of
the mammary gland during pregnancy are
consequences of the action and interation
of several hormones (Cowie and Tindal,
1971).
It is known that the hormonal activity of
the placenta differs considerably in different species. In general, differences have
been reported in the ability of the placenta
to assume ovarian, adenohypophysial, and
adrenocortical functions. The hormones
from the placenta of mammals may differ
in several respects: site of production, time
of production, chemical nature, and biological activity. The placenta elaborates
gonadotropins similar to, although
physiologically and chemically different
from, those elaborated by the adenohypophysis. In the pregnant human female,
human chorionic gonadotropin (hCG) is
produced by the cytotrophoblast and syncytiotrophoblast cells, appears in the blood
and urine, and has the principal biological
property of causing luteinization. In the
pregnant mare, pregnant mare serum
gonadotropin (PMSG) is elaborated by the
tissues comprising the endometrial cups,
279
280
FRANK TALAMANTES JR.
appears in the blood and lymph, and has
the principal biological property of ovarian
follicle stimulation.
One of the hormones secreted by the
human placenta is a prolactin (human placental lactogen— hPL or human chorionic
somatomammotropin— HCS), a factor
which has attracted considerable interest in
the past few years (Josimovich and MacLaren, 1962; Grumbach and Kaplan, 1964;
Kaplan and Grumbach, 1965; Josimovich,
1966; Friesen, 1968; Sherwood etal., 1971;
Li, 1971). A comparative approach to the
study of placenta! prolactins is dictated by
the fact the endocrinology of gestation is
characterized by marked species differences (Amoroso, 1955). We do not know
whether the placentae of all mammals produce a prolactin, and if it is produced, during what period of gestation it is demonstrable. In vitro techniques have been successfully used to obtain information on
these issues (Kohmoto and Bern, 1970;
Buttle et al., 1972; Talamantes, 1973).
THE MOUSE MAMMARY GLAND ORGAN-CULTURE
SYSTEM AS A MEANS OF ANALYZING PLACENTAL
PROLACTINS
The mouse mammary gland has been
employed extensively as a target tissue for
detecting the presence of prolactin and for
studying the action of prolactin. In recent
years, the organ-culture technique based
on the Petri dish-watch glass method of Fell
and Robison (1929), or its modification for
liquid media by Chen (1954), has been increasingly applied in mammary gland
studies.
Embryonic and immature mouse mammary glands have been cultured in complex
media supplemented by hormones
(Blainsky, 1950; Lasfargues and Murray,
1959; Prop, 1959, 1960, 1961). Owing to
the complexity of these incompletely
defined media, a question exists as to
whether unknown factors may have contributed to the observed response. With the
use of defined media, the importance of
insulin in maintaining alveolar structure
was recognized (Elias, 1957, 1959; Trowell,
1959; Elias and Rivera, 1959; Lasfargues,
1960; Rivera and Bern, 1961). The mouse
mammary gland organ-culture system was
used by Nicoll et al. (1966) for examining
various vertebrate pituitaries for the presence of lactogenic activity and has been developed into a sensitive assay for prolactin
(Franz and Kleinberg, 1970). The coculture of mouse mammary gland explants
and placental explants has provided information on the mammotropic influence of
the placenta (Kohmoto and Bern, 1970;
Forsyth, 1972).
I have developed further the technique
of Kohmoto and Bern in order to test not
only placental explants but also placental
extracts for lactogenic activity. Mammary
gland explants from nulliparous (day 11 or
12) pregnant B ALB/cCrgl mice are allowed
to float freely instead of being placed on top
of a stainless steel-dacron raft. Extracts
from a variety of mammalian placentae
were prepared for assay by KC1 extraction
(Talamantes, 1973, and unpublished); lactogenic activity of both placental explants
and placental extracts are evaluated according to the criteria given in Table 1.
The advantages of the in vitro technique
lie in the many disadvantages of the in vivo
methods. In vivo methods involve several
problems: the need for large quantities of
the test material to produce an observable
response, the possibility of systemic inactivation or modification of the hormone
being tested, and the possible activation of
other endocrine systems which may modify
any direct action of the hormone. Organculture methods overcome these difficulties. On the other hand, the in vitro system
has some inherent disadvantages: cultured
explants lack the functional blood circulation of the whole animal; thus, delivery of
oxygen and nutrients to the cultured
explants may not always be adequate and
uniform. The explants located between the
TABLE 1. Mammary gland explant histological grading system.
No colloidal secretion
Trace of secretion without distention
of alveoli
Less than 50% of alveoli with secretion
More than 50% of alveoli with secretion
More than 50% of alveoli have secretion
and width of alveoli more than
5 times that of alveolar wall
±
+
++
+++
LACTOGENIC ACTIVITY IN THE MAMMALIAN PLACENTA
281
fluid and gas phase may have unequal access to oxygen and nutrients; the portion of
the explant surrounded by the gas phase
receives adequate oxygenation while the
lower part of the explant may become
hypoxic and undergo partial necrosis. The
upper part of the explant may also suffer
from a lack of nutrients since their availability is dependent upon capillary action. As a
consequence, when the hormonal response
of the explants is graded, it may occur that
the explants do not respond homogeneously to the hormone.
tion for a positive lactogenic response of the
concentrations tested was 0.01 /ig/ml.
Among the nonhuman primates, extracts
from term Hamadryas baboon placenta
produced a positive lactogenic response in
the mouse mammary gland organ-culture
system. Positive responses were also obtained from extracts of sheep placenta (day
90 of gestation) and of chinchilla placenta
(days 85 and 95). Hamster placental extracts and co-cultured placental explants
from days 12, 14, and 16 of gestation have a
positive response. Both mouse and rat placental extracts and explants from days 10,
12, 14, 16, 18, and 20 of pregnancy were
OCCURRENCE OF LACTOGENIC ACTIVITY IN MAM- also positive. On the other hand, rabbit plaMALIAN PLACENTAE
cental extracts from days 15, 17, and 28, as
well as placental explants from day 15 of
Table 2 summarizes the biological evi- gestation, were negative.
dence for placental prolactin from a variety
of mammalian species. Using the in vitro
system described above and judging secreDISCUSSION
tory activity by histological criteria, mammary gland explants from BALB/cCrgl
Although some effort has recently been
mice were observed to give a lactogenic re- applied to comparative studies of placental
sponse to human placental lactogen (hPL) prolactins, the field has hardly been
at all concentrations tested (0.01-5 /ttg/ml) touched. Studies on placental prolactins
(Figs. 1, 2). The minimum hPL concentra- have been reported for only 16 species
TABLE 2. Biological evidence for placenta prolactins
Species
Assay System
Man
Man
Man
Pigeon crop sac
In vivo rabbit assay
Mouse mammary gland
organ culture
Pigeon crop sac
Mouse mammary gland
organ culture
Mouse mammary gland
organ culture
Mouse mammary gland
organ culture
Mouse mammary gland
organ culture
Mouse mammary gland
organ culture
In vivo mouse assay
Mouse mammary gland
organ culture
In vivo rat assay
In vivo rat assay
Pigeon crop sac
and in vivo rat assay
Mouse mammary gland
organ culture
Mouse mammary gland
organ culture
Rhesus Monkey
Hamadryas Baboon
Goat
Sheep
Chinchilla
Hamster
Mouse
Mouse
Rat
Rat
Rat
Rat
Rabbit
Response
Reference
Josimovich and MacLaren (1962)
Friesen (1966)
Turkington and Topper (1966);
Talamantes (unpublished)
Kaplan and Grumbach (1964)
Talamantes (unpublished)
Forsyth (1972)
Talamantes (unpublished)
Talamantes (1973)
Talamantes (1973)
Cerruti and Lyons (1960)
Kohmoto and Bern (1970);
Talamantes (unpublished)
Matthies (1967, 1968)
Cohen and Gala (1969)
Shani et al. (1970)
Talamantes (1973)
Talamantes (unpublished)
282
FRANK TALAMANTES JR.
r .
represent only semi-quantitative estimates
at best, and thus essentially serve only to
indicate presence or absence of placental
prolactin at the times chosen. In general,
placental explants result in stronger lactogenic responses than those obtained from
extracts. The placenta may have limited
storage capacity for prolactin, although it
may secrete actively at a high rate. In addition, during the extraction procedure inactivation of the lactogenic activity may occur.
The present data on the lactogenic activity
of the human, rat, and mouse placenta are
in agreement with the results of other in
vivo and in vitro studies (Turkington and
Topper, 1966; Matthies, 1967, 1968;
Cohen and Gala, 1969; Kohmoto and Bern,
1970).
The isolation, purification, and chemical
nature of human placental lactogen are
FIG. 1. Midpregnant mouse mammary tissue cultured
in the presence of aldosterone (1/ig/ml), insulin (5
/u.g/ml), and hPL (0.01 /ng/ml).
from 5 mammalian orders (Primates, Carnivora, Perissodactyla, Artiodactyla,
Rodentia, and Lagomorpha), hardly representative of the total number of eutherian mammals. The results of my studies
clearly demonstrate the lactogenic
(secretion-stimulating) activity of a variety
of mammalian placentae. In the rat, mouse,
and hamster, lactogenic activity was demonstrable at several times during gestation. In the rabbit, lactogenic activity was
not detectable. Kelly et al. (1973), using a
radioreceptor assay, were able to quantify
placental lactogen levels in serum of the
following pregnant animals: hamster,
mouse, guinea pig, and sheep. However,
placental lactogen was not detectable in the
serum of the rabbit, cat, dog, pig, and cow.
My bioassay results, based on histological
responses of mammary gland explants,
FIG. 2. Midpregnant mouse mammary tissue cultured
in the presence of aldosterone (1 /xg/ml), insulin (5
/ig/ml), and hPL (3 ^g/ml).
LACTOGENIC ACTIVITY IN THE MAMMALIAN PLACENTA
well documented (Li, 1971; Sherwood et
al., 1971). Information on the chemical
structure of nonhuman placental lactogens
is preliminary. Initial attempts to isolate the
placental prolactin of BALB/cCrgl mice are
now under way (Talamantes and Dickhoff).
The detection of the active placental prolactin band(s) from polyacrylamide gel columns is being accomplished by elution of
segments of the column and testing them
for lactogenic activity using the mouse
mammary gland organ-culture system.
Once this identification is made, it should
be possible to estimate meaningfully the
prolactin content of placentae at different
days of gestation using the densitometric
measurement of the stained bands after gel
electrophoresis (Nicoll et al., 1969).
Molecular weight can also be estimated by
means of polyacrylamide electrophoresis in
the presence of sodium dodecyl sulfate
(Weber and Osborn, 1969).
Our understanding of the physiological
role of placental prolactins in pregnancy is
incomplete, although there has been considerable speculation on the functions of
human placental lactogen (hPL). By definition, placental lactogens are lactogenic in
mammary gland test systems; therefore, it
is likely that these hormones contribute to
maternal mammary gland development
during pregnancy and are associated with
the initiation of milk secretion at the termination of pregnancy. HPL has also been
shown to be luteotropic when administered
alone or in conjunction with human
chorionic gonadotropin in the rat
(Josimovich et al., 1963). Furthermore,
hPL may contribute to the regulation of
human growth hormone secretion during
pregnancy (Spellacy et al., 1970). A lipolytic
role for hPL, wherein free fatty acids become available to the mother during pregnancy thus sparing maternal glucose and
amino acids for utilization by the fetus and
the placenta, has also been postulated
(Grumbach et al., 1968). The anabolic and
somatotropic activity of hPL has been
documented in several studies (Josimovich
et al., 1964, 1966; Grumbach et al., 1968).
Little hPL seems to enter the fetal circulation; at most, the concentration is only
about 0.3% of that found in the maternal
283
circulation at term. We have no information on the availability of placental prolactins to the embryo and fetus in nonhuman
mammals, so that if only insignificant
amounts of hPL are available in human interauterine development, one should bear
in mind that in other mammalian species,
considerable prolactin of placental origin
may be having a major influence on the
fetus. Even small quantities of placental
prolactin to the fetus could aid in normal
growth and development and/or in osmoregulatory adjustments in utero (Bern,
1972).
In general, I would emphasize that the
role of placental prolactins in mammals as a
group presents a considerable challenge to
the comparative physiologist and to the developmental biologist, whose studies may
add further to our concept of what contributions hPL may be making in the
human species.
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