Journal o/Steroid Pcrgamon Press
Ltd 1979. Rinted in Great Britain
HUMAN CHORJONIC GONADOTROPIN AND OVARIAN AND PLACENTAL STEROIDOGENESIS G. P. Department
of Biochemistry, All India Institute of Medical Sciences, New Delhi-l 10016, India
,!%hCG distinctive from @hLH, as read by the immune system [ 10,111.
Human chorionic gonadotrophin (hCG) is a unique and fascinating molecule. It was considered till lately to be produced primarily in pregnancy and its determination in urine or blood served as a diagnostic index of pregnancy. Recently however, reports have appeared on the presence of hCG like material in a variety of normal tissues and cancers [l-S]. While in some studies the evidence is highly suggestive, in others inferences have been based mostly on competitive reactions in radioi~un~s~y systems. In view of the observation  that fractions apparently giving reactions like hCG (such as those obtained from crab’s stomach) were in fact not hCG and their reactivity in radioimmunoassay was abolished by inhibitors of proteolytic enzymes, caution must be exercized in drawing tirm conclusions for the presence of hCG in every type of tissue. It is possible that small amounts of this substance are made by normal human tissues, but there is little doubt that it is synthesized in appreciable amounts only in pregnancy and in some cancers. The smalf amount, if truly found to be hCG in normal tissues, may either be a m~if~~tion of the leaky genes or a product of a handful. of undifferentiated stem cells with active embryonic genome. In either case the biological function of the low amounts of hCG are not discernible with certainty, it does not seem to disturb the menstrual cycle and may have no physiological relevance. The biological actions of this hormone are manifest when it is secreted in large amounts in pregnancy. hCG is active as a gonadotrophin in almost all mammalian species ranging from mouse to man. Some essential structural features of the molecule have been conserved during evolution. The hormone is composed of two non-identi~l subunits (a and /l). The g-subunit of hCG is nearly identical to that in FSH, TSH and LH, the p-subunits ascribe in each case specificity to the hormone . The amino acid sequences of j?-hCG and b-hLH reveal large areas of homologies, but fl-hCG differs from fi-hLH at 51 amino acid residues including the 30 amino acid C-terminal piece unique to hCG. hCG can thus be considered to have some individuality apart from its similarity to hLH. Its distinctive character is further exemplified by the following characteristics: (1) hCG has some FSH and TSH like activities besides that of hLH [8,9]. (2) Antisera raised against @-hCG can be highly specific, and non-reactive or very poorty reactive with hLH, indicating conformations in
LCG SYNTHESIS AND ITS REGULATION
Isolated syncytiotrophoblasts retain capability for synthesis of hCG over prolonged periods of time in uirro (Table if. These observations of my coworker Sudhir Paul using a homogenous in vitro system provide evidence for the ability of these cells to synthesize the hormone without the essential coparticipation of other tissues. The amount of the hormone produced by cells prepared from early pregnancy placenta was larger than that produced by cells prepared by a similar procedure from term placenta. The capacity for synthesis and secretion differs quite markedly (Table 2). Another interesting feature of these studies is the remarkable consistency of the amounts produced by the cells over a finite period. A given cell preparation produces and secretes nearly the same amount of hCG every 72 h over 15 days to 6 months of maintenance in culture. A balancing mechanism seems to be operative. The signals for the synthesis and secretion of hCG are not defined A recent paper [12) describes the rapid fall in hCG synthesis by choriocarcinoma cell lines upon exclusion of serum from the medium. hCG synthesis was sustained on readdition of the serum to the culture medium. Serum thus seems to contain
Table 1. hCG synthesized and secreted by the human syncytio~ophobl~ts in culture Day of cuIture
: 9 12 15
hCG secreted (pg/pg DNA) (Mean 2 SD) 0.20 0.23 0.19 0.23 0.19
i 0.04 +_0.03 _+0.04 & 0.02 * 0.03
Syncytiotrophoblasts isolated from placenta of 9.5 weeks were cultured in triplicate in M 199 containing 10% fetal calf serum as described elsewhere. Every three days before change of medium, 1 mI aliquots of the o&suspension was withdrawn for analysis. hCG was measured in the ceil-free su~matants by radioimm~oas~y and the DNA in the pellet according to Burton. gestation
G. P. TALWAR
Table 2. Capacity for synthesis and secretion of hCG in vitro by syncytiotrophoblasts from first and third trimester pregnancy Gestation age of syncytiotrophoblasts 4-12 weeks 24 weeks-Full-term
Number of placentae as source of the cells 9 12
143.90 f 28.57 0.23 f 0.11
Syncytiotrophoblasts were prepared from early and late placentae and cultivated according to Paul et at.[26,37]. Medium was changed every 24 h and hCG content determined by radioimmunoas~ys.
factors regulating hCG synthesis and secretion. Epithelial growth factor (EGF) has been hinted to be one
BIOLOGICAL ROLE OF HCG IN PREGNANCY
To begin with, it may be stated that all is not known about the biological functions of hCG. In this lecture I will confine discussion to the role of hCG in (a) early pregnancy, (b) midpregnancy and (c) as a molecular interphase contributing to protection against immunological rejection of the conceptus.
lity of hCG in vitro and in uiuo to stimulate progesterone production by the corpus luteum. (ii) The ability of hCG to sustain blood progesterone levels in women undergoing medical termination of pregnancy during the first trimester . The source of progesterone during this phase of pregnancy is the ovaries, as ov~iectomy leads to a decline in progesterone levels and interruption of pregnancy. The tropic hormone for steroidogenesis is not of pituitary origin, since hypophysectomy does not arrest steroidogenesis, nor interferes with pregnancy [ 181.
Is hCG the only luteotropic factor EARLY PREGNANCY
hCG is detectable in circulation by 8 to 10 days post-fertilization [13, 143. Whether the hormone is synthesized by the preimpl~~tion blastocyst is debated, but its presence is certainly recognizable around the time of implantation. The hormone levels rise steeply in the early period of gestation and a plateau is reached around 8 to 10 weeks of pregnancy, after which a decline is noticed. It may be emphasized that hCG secretion does not shut off in later periods of pregnancy and is maintained throughout gestation. The blood hCG comes down to sub-detection range only after expulsion of placenta and trophoblastic elements [l.Sj.
A number of clinical observations indicate the essential requirement of hCG in the first few weeks of pregnancy. Declining levels or even stationary levels of hCG over a period of 48 h in the early phase of pregnancy are usually associated with imminent abortion [ 161.
Possible role of hCG in early pregnancy
Rising levels of hCG in blood are temporally coincident with a pick up of progesterone after the falling trend of the luteal phase of the cycle, suggesting strongly the luteotropic action of hCG. Other evidences in favour of this role of hCG are: (if The abi-
While the above mentioned evidence points strongly to the luteotropic role of hCG in early pregnancy, there may also be a requirement of additional components for maintenance of ovarian steroidogenesis. An indication of the insufficiency of hCG alone for prolonged ma~tenan~ of corpus luteum function is furnished by the inability of hCG to prevent the onset of menstruation in nonpregnant cycling women, in spite of repeated administration of the hormone . The first injection of hCG in the late luteal phase of the cycle, no doubt, leads to a spurt in progesterone production but this is not sustained by continuing administration of hCG, presumably due to the downgrading action and loss of receptors. The menstrual cycle is lengthened by a few days but not indefinitely. These observations elicit a serious enquiry as to whether hCG is indeed the only luteotropic factor in pregnancy. Dr. Chandana Das in our laboratory recently carried out a series of experiments to see whether there was additional luteotropic activity in early pregnancy sera, besides hCG. The highly sensitive Leydig cell bioassay was employed. The sera were tested before and after incubation with specific anti-hCG sera. Antisera raised against total hCG and against p-hCG almost entirely eliminated the stimulating factor in early pregnancy sera for production of testosterone by the Leydig cells (Table 3). The receptors on Leydig cells have similar characteristics to those present in the corpus luteum [ZO] and both are equally sensitive to the gonadotropins. It is therefore apparent that the steroidogenic stimulator in early pregnancy is
Human chorionic gonadotropin Table 3. Neutralization
by anti-hCG sera of the gonadotropic
activity in early pregnancy serum Residual gonadotropic activity after incubation with antisera (per cent of control)
Serum sample from:
Day of pregnancy
Testosterone production by pregnancy plasma + normal monkey plasma
+ Anti-/l hCG
+ Anti- hCG
MEE PAR DAL JAY KAM KEL
52 45 45 43 43 31
1.2 ng 5.9 ng 6.1 ng 6.3 ng 5.0 ng 5.4 ng
7 I 1 9 5 4
8 6 9 9 9 3
Aliquots of pregnancy plasma (1:100 dilution) were incubated with immunoglobulin preparation from monkey antisera against /I-hCG or total hCG for 2 h at 31°C and overnight at 4°C. The steroidogenie potency of the pregnancy plasma samples was determined in mouse Leydig cell assay system before and after incubation with antibodies. Pregnancy plasma incubated with immunoglobulins from normal monkey serum under identical conditions served as control and was taken as 100%. The residual gonadotropic activity after incubation with antisera is given as percent of the respective control. Each value is a mean of quadruplicate determinations.
either hCG or a protein closely resembling hCG and cross-reacting with it immunologically. There remains however, a gap in our understanding of the continuous stimulation of steroidogenesis by hCG in early pregnancy. It is tempting to speculate that a factor is also present in early pregnancy which enables the continued responsiveness of the ovarian cells to hCG. Mechanism of stimulation of steroidogenesis by hCG
(a) Receptors. The hormone sensitive cells bear receptors which bind this hormone with high affinity. The receptors increase in number with maturation of the follicle and are maximal in the corpus luteum (Table 4). FSH is believed to induce receptors for LHjhCG. (b) What do the receptors recognize? hCG is composed of two non-identical subunits. Optimal hormonal activity is expressed by the subunits in an associated state, and it is this conformation which is best recognized by the tissue receptors. Dissociation of the subunits results in a steep fall in the hormonal activity . The beta-subunit is not totally devoid of biological activity. Using highly sensitive assay systems, Ramakrishnan et a/.[221 were able to show that chemically and immunochemically purified /I-hCG
devoid of contamination with trace amounts of hCG, gives a dose dependent rise in testosterone synthesis by mouse Leydig cells (Fig. 1). The activity was 400 fold lower than that of the intact hormone. Studies with synthetic fragments conforming to various parts of the B-hCG, further showed that the carboxyl-terminal portion of the beta subunit was not biologically active, and the peptides did not stimulate steroidogenesis by the Leydig cells (Fig. 2). They did not also compete with hCG for the binding sites in tissue receptors (Fig. 3). The fragment conforming to the sequence 39-71 induced testosterone synthesis in the Leydig cells, whereas the fragment 39-56 failed to do so. It is thus likely that a part of the determinants which are recognized by the tissue receptors and which trigger the synthesis of steroids by the cells are located between the amino-acids 57 to 71 of the fi-hCG. Nevertheless, for effective transmission of the hormonal message, the rest of the molecule has an important role in assuming the most appropriate conformation. (c) Mechanism of action. Hormone-receptor interaction leads to activation of adenyl cyclase with the production of cyclic AMP which in turn activates protein kinases phosphorylating a variety of cellular proteins and enzymes. Phosphorylation results in a shift of the catalytic activities of proteins and enzymes
Table 4. Binding of [ ‘251]-hCGmto particumctions
from developing follicles and corpora lutea
Incubation conditions Tissue
Corpora lutea Mature follicle Immature follicle
7411 f 633 1466 f 248 698 a 45
[‘251]-hCG + 2 pg hCG 628 + 25 581 f 36 431 f 53
(c.p.mJ0.6mg protein) Net [“‘I]-hCG
6789 1 633 885 f 251 261 k 69
6OCMl sr pellets were prepared from homogenates of immature and mature follicles and corpora lutea pooled from ovaries of 20 goats. Results are mean + S.E.M. of six assays of two different pooled preparations .
G. P. TALWAR
_._______ ____ _______. ___.. -_,c ________________----_ - _____em x ARCM-RhCG
, 4s la
Fig. 1. Effect of human chorionic gonadotropin (0) and immunochemically purified /Z-hCG (0) on testosterone production by Leydig cells. The values are means for quadruplicate determinations. The broken line denotes the basal production of testosterone by the Leydig cells in the absence of hormone. The reduced carboxymethyl derivative of the asialo-beta-subunit of hCG (m) did not cause stimulation of steroidogenesis at the concentrations tested (data from Ramakrishnan et a1.). with consequent activation of discrete metabolic reactions. In stimulation of steroidogenesis, the points affected by phosphorylation reactions are believed to be: (i) release of free cholesterol from esterified form,
(ii) its transport to mitochondria, (iii) its cleavage to pregnenolone, (iv) improved transport of pregnenolone to the cytoplasmic compartment from the mitochondria. Fig. 4 summarizes these notions. PLACENTAL STEROIDOGENESIS During pregnancy the placenta produces substantial amounts of progesterone and oestrogens. Steroi-
dogenesis in the normal steroid producing glands, the adrenals, ovaries and testes, is regulated by tropic hormones. The question that arises is, whether placental steroidogenesis also requires a tropic hormone and if so, does hCG perform this function? Immunoenzymatic techniques demonstrate the location of anti-hCG reacting components on the membranes of trophoblasts in early pregnancy chorionic villi (Fig. 5). This may signify the presence of binding sites for hCG on membranes. This conclusion is however not warranted because trophoblasts are also the cells that synthesize the hormone. In other steroid sensitive tissues, the gonadotropins
moles / Tube
Fig. 2. Effect of synthetic peptides and enzyme-cleaved fragment of the b-hCG on testosterone production by mouse Leydig cells. 0, C-terminal peptide residues 115-145; W, C-terminal peptide residues 111-145; A, C-terminal peptide residues 101-145; A, Core 18-amino acid peptide residues 39-56; l , Core 33-amino acid peptide residues 39-71; 0, Thermolysin cleaved fragment of the asialo+?-hCG (fragment 140 linked to fragment 50-114). The broken line indicates the basal level of testosterone production. The values are means for quadruplicate determinations (data from Ramakrishnan et al.).
Human chorionic gonadotropin
Fig. 3. Competition of C-terminal synthetic peptide of j-hCG and [““I]-hCG for the binding sites in corpora luteal receptors. Sheep corpora luteal receptor preparation (20008) were used for the binding assay. hCG was radioidodinated by lactoperoxidase method (sp. S.A.: approx. [email protected]
/pg). (O), C-terminal peptides (115-145; 111-145; 101-145). interact with membrane associated receptors and one of the consequences of this interaction is the activation of adenyl cyclase. Menon et a/.[241 and Demers et aI. have reported an increase in cyclic AMP in the placental tissue by hCG. Prem Mohini and Shail Sharma in our laboratory have however obtained equivocal results. Further studies are required to establish whether hCG is indeed a stimulator of adenyl cyclase in the placental tissue. The effect of hCG on conversion of A-5 pregnenolone to progesterone
Chorionic villi from early pregnancy as well as syncytiotrophoblasts prepared in a fairly homogenous state by a method described elsewhere were employed. hCG did not stimulate the conversion of pregnenolone to progesterone at any of the concentrations tested up to 7OIU/ml (Fig. 6). In another set of experiments, the effect of anti-hCG serum on this reaction was studied on the premise that a prestimulated state may exist because of the endogenous hCG in trophoblasts, thus rendering the cells insensitive to exogenous hCG. The antibodies did not sup press the progesterone synthesis (Fig. 7). hCG thus does not appear to exercise an influence on the conversion of pregnenolone to progesterone. Its possible effects on other steps of steroidogenesis are however not excluded by this experiment.
significantly the production of potentiates DHEA [27,28]. The stimulation caused by hCG up to 22 weeks of pregnancy is much more than that caused by ACTH in early time periods of gestation, after which ACTH is more effective though hCG continues to exercise stimulation. Cedard et aI. have also reported the influence of hCG on aromatization of A-4 androstenedione. An augmentation of free oestriol was obtained by Troen during perfusion of the placenta with hCG. hCG induced production of testosterone by the foetoplacental unit There appears to be some correlation between serum levels of hCG and foetal production of testosterone in midpregnancy . The foetal testes are
A symbotic relationship exists between the mother and the foeto-placental unit for supply of substrates and intermediates. The immediate precursor for the oestrogens is dehydroepiandrostenedione (DHEA) made either by maternal or foetal adrenals. This is converted to oestrogens by the reactions summarized in Fig. 8. The production of DHEA is apparently under the control of tropic hormones. In the fetal adrenals, hCG S.B.I l/IA-D
Fig. 4. Schematic model of the action of hCG/hLH on a target cell producing progesterone. The binding of the hormone with the receptors located on the plasma membrane is expressed in activation of adenyl cyclase with the production of cyclic AMP from ATP. CAMP activates phosphokinase (PK). The phosphorylation reactions influence the indicated steps in the formation of progesterone (P4) from cholesterol ester.
Fig. 5. Location of hCG in chorionic villi from first trimester human placenta. The villi were incubated with mono-specific anti-hCG serum followed by anti-globulins tagged with peroxidase. The reaction products are seen as electron dense material lining the trophoblast of the unstained preparation (16,400 x ). Photomicrograph is by Dr. P. D. Gupta.
highly sensitive to hCG at 12 to 13 weeks of gestation as shown by the in vivo data and by in vitro experi-
ments. hCG is presumably the important tropic stimulus for the production of testosterone by the foetal Leydig cells. To sum up, hCG does not appear to influence the conversion of pregnenolone to progesterone in the trophoblasts. It may have however, an important role
on oestrogen synthesis by the foeto-placental unit by stimulation of the production of DHEA by the adrenals and thereby increasing the supply of this precursor for oestrone and oestradiol synthesis in the placenta. hCG has also an important function in the mid-pregnancy production of testosterone by the male foetus, which in turn has an important role in differentiation of the foetus.
Fig. 6. Purified syncytiotrophoblasts obtained from placenta of 16 weeks gestation were cultured in M 199 medium supplemented with 10% foetal calf serum and 2OOng/ml of pregnenolone. Purified hCG (10,000 IU/mg) was added to the cultures at the indicated concentrations. Progesterone was determined in the culture media and in homogenate of the cells by radioimmunoassay  after Celite chromatography .
Human chorionic gonadotropin
“foreign” to the maternal immune system from immunological rejection . hCG has a fairly high content of carbohydrates. The high negative charge covering trophoblasts may repel lymphocytes from interacting with the foetal tissue antigens. Besides this, hCG is a protein to which the mother is immunologically tolerant and a coating of this protein could conceivably provide a protection to immunological reactions. It may be emphasized that while hCG may well be a contributory factor, it will be an over-simplification to state that this is the only or the main mechanism by which the foeto-placental unit survives. Many other mechanisms are recognized and have been discussed by Beer and Voisin.
would like to acknowledge the fruitful collaboration of my brilliant students and coworkers Sudhir Paul, S. Ramakrishnan, and C. Das. The photomicrographs were taken by Dr. P. D. Gupta. Research support was provided by grants from the IDRC, Canada, The Population Council Inc., New York and the Family Planning Foundation of India.
Fig. 7. Effect of anti-hCG antibodies on the conversion of pregnenolone to progesterone by human chorionic tissue in organ culture. Seven-week old villous tissue segments were cultured in M 199 containing 2OOng/ml A’-pregnenolone in either, the presence of 10% nonimmune control serum, or anti-hCG sera. Fresh guinea-pig serum was added to a final concentration of 10% as source of complement. The culture medium was changed after 24 h and the cultures were incubated for another 24 h. Progesterone was determined in the culture media and in the homogenates of the tissue by radioimmunoassay after Celite chromatography.
Role of hCG immunological
in the protection
of the concepfus
hCG forms a molecular interphase between the foeto-placental unit and the maternal tissues. Hypothesis have been advanced that it may also contribute to the many mechanisms which protect the foetus, with paternal genome-directed proteins intrinsically
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G. P. TALWAR
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