Human chorionic gonadotropin (hCG) in the male reproductive tract.

Normal hypothalamic-pituitary testicular and prostatic functions are essential for maintenance of male fertility, whereby glycoprotein hormones (GPH) as well as androgens are major endocrine and local regulators. We have investigated whether the GPH human chorionic gonadotropin (hCG) and the free alpha and beta subunits thereof are produced in the target organs themselves and potentially act as auto/paracrine modulators of fertility. Immunofluorometric assays (IFMAs) based on our panel of highly selective monoclonal antibodies, immunohistochemistry (IHC), confocal laser scanning microscopy (CLSM) and 1- and 2D gel electrophoreses with subsequent western blotting have been utilized for the detection of hCGalpha, hCGbeta and its metabolite hCGbeta core fragment (cf) in human testis, prostate and seminal plasma. Both organs synthesize hCGalpha and hCGbeta, which are subsequently detectable at high concentrations in seminal plasma of healthy probands (n=17): hCGalpha 2630+/-520 ng/mL (mean+/-S.E.M.), hCGbeta 2+/-0.28 ng/mL, hCGbetacf and hCG 0.19+/-0.039 ng/mL. These parameters significantly exceed physiological values, e.g. ten thousand-fold in the case of hCGalpha, in serum of young men (n=20): hCGalpha 0.142+/-0.054 ng/mL (mean+/-S.E.M.), hCGbeta 0.05 ng/mL and hCG 0.004+/-0.003 ng/mL. Levels of these markers were not correlated with sperm counts. Of all body fluids including those of pregnant women seminal plasma is the richest physiological source for genuine free i.e. non-dissociated GPHalpha (M(r,app) 23k) which may even appear as di- or tetramers. Its concentration is similar to that observed in maternal serum (weeks 10-12 of gestation) and in extra-embryonic coelomic fluid. In contrast to those fluids where ratios of free subunits to hCG are in the range of 1:100 highly inverse ratios in the range of 10.000:1.000:1 were observed for hCGalpha:hCGbeta:hCG in seminal plasma. hCGalpha is not derived from heterodimeric GPH suggesting hCG-independent functions of hCGalpha and hCGbeta in male and female fertility.

[Hormone replacement therapy and the risk of developing breast cancer]. / Risiken der Hormonersatztherapie. Wie sind aktuelle Studien zu bewerten?

The Million Women Study (MWS) and the Women's Health Initiative (WHI) Study reveal an increased breast cancer risk and a higher relative risk for cardiovascular diseases in women on hormone replacement therapy (HRT) in comparison with control groups. For this reason, the WHI study was terminated prematurely. From the point of view of the internist, it would appear that, on the basis of the results of these studies, prophylactic treatment with estrogens and progesterone is no longer indicated. It must, however, be noted that numerous aspects are still unclear. This therefore means that in patients with severe menopausal symptoms giving rise to a high level of distress, HRT continues to be justified over the short term after a current cardiovascular risk or relevant tumor disease has been excluded.

The ERK signaling cascade inhibits gonadotropin-stimulated steroidogenesis.

The response of granulosa cells to luteinizing hormone (LH) and follicle-stimulating hormone (FSH) is mediated mainly by cAMP/protein kinase A (PKA) signaling. Notably, the activity of the extracellular signal-regulated kinase (ERK) signaling cascade is elevated in response to these stimuli as well. We studied the involvement of the ERK cascade in LH- and FSH-induced steroidogenesis in two granulosa-derived cell lines, rLHR-4 and rFSHR-17, respectively. We found that stimulation of these cells with the appropriate gonadotropin induced ERK activation as well as progesterone production downstream of PKA. Inhibition of ERK activity enhanced gonadotropin-stimulated progesterone production, which was correlated with increased expression of the steroidogenic acute regulatory protein (StAR), a key regulator of progesterone synthesis. Therefore, it is likely that gonadotropin-stimulated progesterone formation is regulated by a pathway that includes PKA and StAR, and this process is down-regulated by ERK, due to attenuation of StAR expression. Our results suggest that activation of PKA signaling by gonadotropins not only induces steroidogenesis but also activates down-regulation machinery involving the ERK cascade. The activation of ERK by gonadotropins as well as by other agents may be a key mechanism for the modulation of gonadotropin-induced steroidogenesis.

Disulfide bond formation is not required for human chorionic gonadotropin subunit association. Studies with dithiothreitol in JEG-3 cells.

To study the influence of disulfide bridge formation on the assembly of the subunits of human chorionic gonadotropin in JEG-3 choriocarcinoma cells, dithiothreitol (DTT) was used to create a reducing milieu in the endoplasmic reticulum (ER) in vivo. In the presence of 5 mM DTT during pulse-chase experiments all of the beta-subunit precursors observed in unperturbed cells (pbeta(0), pbeta(1), pbeta(2), and beta(*)) collapsed into the pbeta(0) form. The reducing milieu of the ER was reoxidized in less than 5 min after removal of DTT from the medium. DTT markedly increased the half-life of the pbeta(0) precursor from 8.8 to 65.2 min. Under reoxidation conditions, the beta-subunit precursors folded back from pbeta(0) in less than 5 min. In unperturbed JEG-3 cells, the alpha-subunit was present in both fully glycosylated and monoglycosylated precursor (pre-alpha) forms. The attachment of the second N-linked glycan residue of the alpha-subunit was accelerated in the presence of DTT, and consequently pre-alpha-subunit was missing from the DTT-treated cultures. The formation of alphabeta-dimers appeared to be at least partially independent of the oxidation state in the ER. The alphabeta-dimer was present under conditions in which disulfide bridge formation was prevented by exposure to 5 mM DTT before and during the pulse period. This clearly suggests that the human chorionic gonadotropin subunits may acquire association-competent conformations even when no disulfide bridge formation has taken place.

Long-term effect of cyclic AMP on N-glycosylation is caused by an increase in the activity of the cis-prenyltransferase.

Previously we have shown that long-term pretreatment of JEG-3 choriocarcinoma cells with 8-bromo-cAMP increases the capacity for N-glycosylation that was caused by an 8-10-fold enlargement of the dolichol pyrophosphoryl oligosaccharide (Dol-PP-oligosaccharide) pool [Konrad and Merz (1994) J. Biol. Chem. 269, 8659-8666]. The factors involved in the effect of cAMP on synthesis of Dol-PP-oligosaccharide are investigated here. The GlcNAc transfer to dolichol phosphate (Dol-P) was found to be unaffected by pretreatment with 8-bromo-cAMP. By measuring the uptake of [3H]mevalonate, a 20-fold increase in the incorporation of the label into Dol-P was observed in the cells treated with 8-bromo-cAMP. Under the same conditions, the synthesis of dolichol was enhanced 60-fold. However, the incorporation of the radioactivity into cholesterol was not increased in the JEG-3 cells pretreated with 8-bromo-cAMP, which suggests a specific stimulation of the dolichol/Dol-P pathway by cAMP. The cis-prenyltransferase activity was found to be increased 10-fold in cells pretreated with 8-bromo-cAMP. Dolichol kinase activity was unaffected by stimulation with 8-bromo-cAMP. The present study suggests that the larger glycosylation capacity in JEG-3 cells treated with 8-bromo-cAMP is caused by an increase in the microsomal cis-prenyltransferase activity.

The relative importance of the oligosaccharide units in human chorionic gonadotropin (CG) for LH/CG receptor activation in rat Leydig cells and mouse Leydig tumor cells.

The biological properties of deglycosylated human chorionic gonadotropin (DhCG), obtained by hydrogen fluoride treatment (HF-DhCG) of intact hCG or by oligonucleotide-directed mutagenesis (CHO-DhCG), and that of their fully glycosylated counterparts, were tested in terms of cAMP and steroid production in rat Leydig cells and in mouse Leydig tumor cells (MA-10 cells). In both cell types, HF-DhCG and CHO-DhCG possessed comparable biological activities. The maximum for DhCG-induced cAMP production was approximately 12% of that of intact hCG when tested in rat Leydig cells, and only 2% when tested in MA-10 cells. DhCG possessed significant steroidogenic activity in both cell types. In MA-10 cells the maximum for DhCG-induced steroidogenesis was 30-50% of that of intact hCG, while in rat Leydig cells DhCG and hCG induced similar steroidogenic maxima. Based on its ED50, DhCG possessed 10-17% of the steroidogenic potency of intact hCG in rat Leydig cells, while in MA-10 cells DhCG was only 2-fold less potent than hCG. When accurate hormone-receptor binding data are absent, the intrinsic receptor-stimulating activity of a ligand can still be estimated at full receptor occupancy, provided that over the whole dose range the biological response is proportional to receptor stimulation. The present data show that in transfected MA-10(P+29) cells which over-express rat phosphodiesterase, the hormone-induced stimulation of cAMP and steroid production is directly coupled to receptor activation up to maximal occupation of the LH/CG receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

[Fragments of human chorionic gonadotropin (hCG): diagnosis of pregnancy and tumors]. / Fragmente des humanen Choriongonadotropins (hCG): Schwangerschafts- und Tumordiagnostik.

The pregnancy and tumor marker human chorionic gonadotropin (hCG) belongs to the family of the glycoprotein hormones. Information on epitope forming sequences of hCG and its subunits hCG alpha and hcg beta has significant impact on the examination of intra- and extracellular metabolism and the standardization of diagnostic assay systems. Variants of hCG appear in biological fluids with variable modifications on different parts of the molecule. These changes may influence the binding patterns of monoclonal antibodies (MCA), thereby causing erroneous results in hCG immunoassays. The aim of the present work was to investigate the influence of peptide bond cleavages and the loss of certain segments of the molecule, which were induced by proteases on the expression of the seven hCG alpha-(alpha 1-alpha 7), nine hCG beta- (beta 1-beta 9) and four hCG beta-core-fragment-epitopes (beta 10-beta 13), previously identified by us [1-10]. To this end, we digested hCG alpha and hCG beta with chymotrypsin. Hormone fragments were separated by high performance liquid chromatography (HPLC) and subsequently immunochemically examined by direct binding radioimmunoassay (DB-RIA), competitive RIA and immunoenzymometric assays (IEMA). Fractions containing hCG-like immunoreactivity were sequenced by Edman and carboxypeptidase-Y degradation. It appeared that: (I) Amino acids (AA) alpha 41-47 and the peptide bonds between AA alpha 40/41, alpha 47/48 and alpha 29/30 do not influence the expression of the 7 alpha-epitopes, (II) The absence of the hCG beta N-terminus plays a crucial role for the formation of epitopes beta 10 and beta 13. (III) Neither the presence nor the absence of the C-terminal peptide of hCG beta (hCG beta CTP, AA beta 114-145) has any importance for the expression of epitopes beta 1-beta 7 and beta 10-beta 13 (IV).(ABSTRACT TRUNCATED AT 250 WORDS)

Lack of self-regulation of human chorionic gonadotropin biosynthesis in human choriocarcinoma cells.

Human gestational trophoblastic neoplasms overexpress hCG/LH receptors. Whether this overexpression is a reflection of a loss of self-regulation of hCG biosynthesis was investigated using JAR human choriocarcinoma cells. The results show that exogenous hCG did not affect steady state hCG alpha and hCG beta mRNA or dimer hCG protein levels in JAR cells. The JAR cells, however, responded to 8-bromo-cAMP with an increase in hCG alpha mRNA levels, suggesting that cAMP-mediated regulation of the hCG subunit genes was intact in the cells. Disruption of receptor function by a receptor antibody, which resulted in an increase in hCG alpha mRNA levels and hCG secretion in normal trophoblasts, had no effect on JAR cells. Unlike normal trophoblasts, which contain a predominant receptor transcript of 1.8 kilobases (kb), with minor higher molecular size (7.5 and 5.4 kb) transcripts occasionally seen, JAR cells contain a higher abundance of multiple transcripts (7.5, 5.4, 3.5, and 1.8 kb), with the predominant transcript being 5.4 kb. In addition, although normal trophoblasts contain an 80-kilodalton receptor protein, JAR cells contain only a 50-kilodalton hCG/LH receptor isoform. In contrast to the effects of exogenous hCG on normal placental tissue in vitro, it was unable to down-regulate receptor transcripts or receptor protein in JAR cells. In summary, JAR cells lack the ability to self-regulate hCG biosynthesis. This loss could explain how hCG can reach very high levels in gestational trophoblastic disease compared to those in normal pregnancy.

Regulation of N-glycosylation. Long term effect of cyclic AMP mediates enhanced synthesis of the dolichol pyrophosphate core oligosaccharide.

The influence of 8-bromo-cAMP on N-glycosylation in JEG-3 choriocarcinoma cells was investigated using the octanoyl-tripeptide (OTP; N-octanoyl-asparagyl-125I-tyrosyl-threonine amide) as glycosyl acceptor. In cells pretreated with 8-bromo-cAMP (2.5 nM to 1 mM), the amount of glycosylated OTP released into the culture medium was increased up to 35-fold. Under the same conditions, a 23-fold higher quantity of the glycoprotein hormone human chorionic gonadotropin was secreted. Preincubation of 10-90 min with 250 microM 8-bromo-cAMP caused only a 2-fold increase of the total amount of glycosylated OTP, whereas it was approximately 20-fold higher when the pretreatment was extended to 40 h. This strongly suggests involvement of gene activation rather than cAMP-mediated phosphorylation. The specific activity of the oligosaccharyltransferase, as well as the mRNA levels of ribophorin I and II (presumptive subunits of the enzyme), remained unchanged. In pulse-chase experiments, [3H]mannose incorporation into dolichol-linked Glc3Man9(GlcNAc)2 was up to 20-fold higher in cells pretreated with 8-bromo-cAMP (250 microM, 40 h). The radioactivity was chased from the lipid-linked oligosaccharide pool and shifted to the glycoprotein fraction 10 times more rapidly in the pretreated cells. The flux of [3H]mannose through the dolichol phosphate mannose pool was only slightly affected by the 8-bromo-cAMP pretreatment. Our investigations show that the oligosaccharyltransferase activity in JEG-3 cells is not rate-limiting. N-Glycosylation seems to be controlled by the amount of lipid-linked core oligosaccharide. The size of the lipid-linked core oligosaccharide pool, as well as the flux through, is markedly increased by pretreatment with 8-bromo-cAMP.

The primate placenta and human chorionic gonadotropin.

In the primate placenta various peptide and proteohormones are synthesized which control growth and development of the fetus as well as the exchange of nutrients and metabolic products between the mother and the fetus. In humans, maintenance of pregnancy in the first trimester depends on the synthesis of the bioactive glycoprotein hormone human chorionic gonadotropin (hCG). It is expressed in placenta by the syncytiothrophoblast of early pregnancy. In cell culture, hCG production seems to mark a certain step in the process of differentiation of cytotrophoblasts and choriocarcinoma cells. It is neither understood how hCG synthesis is initiated and maintained at the beginning of gestation nor what control mechanisms are responsible for the down-regulation of the synthesis at the end of the first trimester. Besides a long list of various other substances which have been described to act as intrinsic placental stimulators of hCG biosynthesis, gonadoliberin and gamma-aminobutyric acid seem to play an important role. This establishes to some extent an analogy to the regulation of gonadotropin synthesis in the central nervous system. Recently, a full-length form of functional LH/hCG receptors of approximately 80 kD has been found in term placenta suggesting autoregulation as a regulatory principle of hCG biosynthesis. In the first trimester placenta as well as in choriocarcinoma cells a truncated form (50 kd) of LH/hCG receptors seems to exist. In these cases, exogenous hCG was unable to down-regulate its own synthesis. The carbohydrate moiety of hCG influences folding, subunit assembly, circulatory half-life, receptor interaction and biological response. A surplus of glycosylation may prevent subunit assembly. Experimental deglycosylation induces a different conformation of hCG, which partly acquires antagonistic properties. Recent results indicate that cAMP, which increases transcription and mRNA stability, also expands the N-glycosylation capacity and thus may accomplish an over-all coordination of hCG biosynthesis including post-translational events.

Novel self-regulation of human chorionic gonadotropin biosynthesis in term pregnancy human placenta.

Term pregnancy human placenta contains hCG/LH receptor mRNA transcripts and immunoreactive receptor protein. Both the receptor transcripts and receptor proteins are present only in trophoblasts. These findings led us to investigate whether hCG can regulate its own synthesis in term pregnancy human placenta. Treatment of placental tissue in static cultures or in a dynamic superfusion system with increasing concentrations of highly purified hCG provoked a biphasic effect on the steady state hCG subunit mRNA levels. Although low concentrations of hCG (< 200 mIU/ml) were not effective, moderate concentrations (200-1000 mIU/ml) increased, and high concentrations (> or = 5000 mIU/ml) either had no effect or actually decreased mRNA levels relative to the control values. This response was specific, because none of the hCG concentrations tested had any effect on glyceraldehyde-3-phosphate dehydrogenase or beta-actin mRNA levels. The effects of hCG on steady state hCG subunit mRNA levels were paralleled by corresponding changes in tissue hCG protein levels. Endogenous hCG appears to down-regulate alpha-subunit mRNA levels and hCG secretion. The hCG effect is probably receptor mediated, because a receptor antagonist, deglycosylated hCG, partially antagonized the hCG action. Treatment with exogenous hCG also down-regulated its own receptor mRNA and receptor protein levels. hCG regulation of its alpha-subunit and receptor levels involved both transcriptional as well as posttranscriptional mechanisms. In summary, this is the first demonstration of hCG regulating its own synthesis in term pregnancy human placenta. The findings of this study could offer a potential molecular explanation for the profile of hCG levels in normal pregnant women.

Functional and immunological relevance of the COOH-terminal extension of human chorionic gonadotropin beta: implications for the WHO birth control vaccine.

The World Health Organisation (WHO) Task Force on Birth Control Vaccines has selected the pregnancy hormone human chorionic gonadotropin (hCG) as a target molecule for a contraceptive vaccine. A synthetic peptide antigen corresponding to the amino acid sequence 109-145 of the carboxyl-terminal portion of the hCG beta-subunit (hCG beta CTP), which is supposed to elicit hCG-immunoneutralizing antibodies, has been submitted to clinical trials. Recent findings suggest that hCG beta CTP does not play a role in the biological activity of hCG. This raises the question concerning the assumed mechanism of action of the hCG beta CTP-based birth control vaccine. We therefore investigated the immunoneutralizing capacity of antibodies directed against hCG beta CTP. Although it is possible to generate specific monoclonal and polyclonal antibodies for hCG by using hCG beta CTP as an immunogen, it appeared that the biological response to hCG was not affected by such antibodies. The reason for this is that the hCG-antibody-complex is still able to bind to target cell receptors and therefore the intended contraceptive effect should not occur. In addition there is a risk of hazardous possible side effects such as an autoimmune reaction against the ovary because we found that at least one epitope is still accessible for antibody binding on receptor-bound hCG. We conclude from our results that both the efficacy and safety of the WHO vaccine are not yet ensured.

Significance of the carbohydrate moiety of the rat ovarian luteinizing-hormone/chorionic-gonadotropin receptor for ligand-binding specificity and signal transduction.

The contribution of the carbohydrate moiety of the rat ovarian luteinizing-hormone (LH)/chorionic-gonadotropin (CG) receptor to ligand-binding specificity and signal transduction was investigated by using glycosidases. Purified membranes from pseudo-pregnant rat ovaries were treated with neuraminidase or peptide N-glycosidase F, to remove terminal sialic acids and N-linked oligosaccharides of the receptor, respectively. Ligand blotting and densitometric scanning of the autoradiograms showed that 90-95% of the receptors were deglycosylated, and that desialylation was virtually complete. Neither the desialylated nor the deglycosylated receptors were able to bind human follicle-stimulating hormone or bovine thyroid-stimulating hormone, as revealed by competition binding experiments. The 50% effective dose of hCG for adenylate cyclase activation, as determined by measuring the formation of cyclic [32P]AMP from [alpha-32P]ATP for 15 min at 30 degrees C, was similar in the control and deglycosylated membranes: 10.2 +/- 3.3 nM and 12.2 +/- 3.8 nM respectively. The same was true for the time course of the basal, hCG- and forskolin-stimulated enzyme activity. In addition, removal of oligosaccharides from the receptor did not restore the ability of desialylated hCG, nor of the deglycosylated hormone, to stimulate adenylate cyclase. In conclusion, the carbohydrate moiety of the native membrane-inserted rat ovarian LH/CG receptor does not contribute to the ligand-binding specificity, and it is not required for the functional coupling of the occupied receptor and the adenylate cyclase system. These functions are associated with the polypeptide portion of the receptor.

Evidence for a modulation of human chorionic gonadotropin (hCG) subunit messenger ribonucleic acid levels and hCG secretion by gamma-aminobutyric acid in human first trimester placenta in vitro.

The cytotrophoblasts are the site of production of liberins and statins in human placenta, whereas the syncytiotrophoblasts synthesize tropic hormones. These placental cell layers seem to interact like the hypothalamus and pituitary. In the central nervous system, gamma-aminobutyric acid (GABA)-ergic neurons represent one important control mechanism that seems to influence the lutropin biosynthesis indirectly (via gonadoliberin) and directly. It was the objective of the present study to find out whether GABA also may influence the biosynthesis and secretion of hCG by human first trimester placenta. Already one single pulse of GABA (1 h; 0.01-100 microM) stimulated hCG secretion significantly (P less than 0.0001). GABA also induced a marked increase in the mRNA levels of both subunits, with an optimum at 10 microM. The effect on hCG secretion was mimicked by the GABA-A receptor agonist muscimol (P less than 0.002), but under the experimental conditions used (multiple pulses; 1 microM), only the beta mRNA was increased. The GABA-A receptor antagonist bicuculline (two pulses; 10 microM) suppressed basal hCG secretion (P less than 0.001) and abolished the episodic secretion pattern observed in the control cultures. Applying a combination of equimolar amounts of GABA and bicuculline, hCG secretion and the episodic secretion pattern were similar as in control cultures. The data seem to suggest a regulation of hCG biosynthesis in human first trimester placenta in which GABA is involved, probably acting via GABA-A-like receptor sites.

Number and topography of epitopes of human chorionic gonadotropin (hCG) are shared by desialylated and deglycosylated hCG.

A previously established map of the surface epitopes of human chorionic gonadotropin (hCG) served as template for the present study in which we investigated the antigenic surfaces of two glycosylation variants of hCG, i.e. desialylated hCG (asialo-hCG) and deglycosylated hCG (degly-hCG). This map allocates five epitopes to the alpha subunit, five to the beta subunit and four alpha beta epitopes to structures formed only by the alpha/beta heterodimer holo-hCG (Schwarz et al. (1986) Endocrinology 118, 189-197; Berger et al. (1990) J. Endocrinol. 125, 301-309). Here it is described that both variants complied with this template: each of the 14 distinct monoclonal antibodies with which the epitopes of hCG were defined reacted with radiolabeled asialo-hCG and degly-hCG as well and generally bound degly-hCG with greater affinity than hCG. Moreover, every combination of capture and radiolabeled detection antibody that was either compatible or incompatible on unlabeled hCG was so also on unlabeled asialo-hCG and degly-hCG. It thus appears that alterations of the carbohydrate structure of hCG can be associated with a change in affinity between some antibodies and their respective epitopes but not with a loss of an epitope or with a change in the topographical relationships of the 14 epitopes.

Epitope mapping of the receptor-bound agonistic form of human chorionic gonadotropin (hCG) in comparison to the antagonistic form (deglycosylated hCG).

On the surface of free human chorionic gonadotropin (hCG), we can distinguish with our panel of monoclonal antibodies (MCA) 14 topographically distinct epitopes (designated alpha 1 - alpha 5, beta 1 - beta 5, alpha beta 1 - alpha beta 4, depending on the subunit they are attached to). Only 2, i.e. the adjacent beta 3 and beta 5 epitopes, of these 14 are accessible to 125I-labeled MCA binding, when hCG is first allowed to bind to the rat testis hCG receptor. This result indicates that the agonist hCG assumes a defined orientation in its receptor-bound state and that, except for that small area comprising the beta 3 and beta 5 epitopes, most of its surface is masked by the hCG receptor. We therefore asked whether the competitive antagonist deglycosylated hCG (degly-hCG), which, when free, is antigenically (as to number and topography of epitopes) indistinguishable from native hCG, would interact with the receptor differently, that is, in a way that can be discerned by this epitope accessibility paradigm. Here we describe that on receptor-bound degly-hCG the beta 3 and beta 5 epitopes were concealed as were all other epitopes. This observation, together with finding the receptor affinity of degly-hCG to be 4 times higher than that of native hCG, suggests that degly-hCG assumes a signal transduction-incompetent ligand orientation and at the same time interacts with the receptor more intensively, i.e. establishes additional ("antagonist accessory") protein-protein contacts besides those involved in agonist binding. It thus appears that the carbohydrate moieties function to prevent formation of such accessory contacts.

The secretion of human chorionic gonadotropin as well as the alpha- and beta messenger ribonucleic acid levels are stimulated by exogenous gonadoliberin pulses applied to first trimester placenta in a superfusion culture system.

It is well documented that the hypothalamic decapeptide gonadoliberin (GnRH) controls the biosynthesis and secretion of pituitary gonadotropins; however, it is still unclear whether GnRH synthesized by the placenta plays the same role with respect to hCG. In the current study we have investigated the acute response of placenta tissue to a single GnRH pulse as well as the influence of GnRH pulses on the secretion of hCG and hCG mRNA concentrations elicited several hours after application of the peptide hormone. For this purpose we have used a superfusion culture model of first trimester placenta tissue (8-12 weeks of gestation). In the first hour after explantation of the tissue, hCG secretion was decreased, and increasing amounts of free subunits were released. Afterward, the original hCG secretion rates were recovered and maintained for several days, attended by decreased levels of free subunits in the culture medium. The superfusion model was superior to static incubations, since it showed approximately a 4-fold higher amount of hCG to be secreted within 24 h (day 3 of cultures). A single GnRH pulse (1 mumol/L; 30 min) caused a significantly increased transient release of hCG (P less than 0.0001). Two GnRH pulses (concentration range, 0.01-10 mumol/L; 30 min) applied 24 h after (first pulse) and in the interval between 36-48 h after (second pulse) the start of the superfusion culture elicited a long-lasting 2-fold increase in the hCG secretion rate, which rose approximately 6 h after the second GnRH pulse. This was correlated with increased mRNA concentrations measured by means of Northern blots of total RNA. At 0.02 mumol/L GnRH, 4-fold higher beta mRNA levels were observed. The alpha mRNA levels were 2.5-fold elevated. GnRH pulses of 0.01 and 10 mumol/L, respectively, were ineffective. A further effect of GnRH pulses was augmentation of the episodic character of hCG secretion. Our results suggest that GnRH causes different specific acute and late effects on the amount and pattern of hCG secretion as well as on hCG biosynthesis at the levels of both hCG subunit mRNAs.

Significance of the glycan moiety of the rat ovarian luteinizing hormone/chorionic gonadotropin (CG) receptor and human CG for receptor-hormone interaction.

The role of the glycan moiety of the rat ovarian LH/CG receptor and human CG (hCG) in high-affinity receptor-hormone interaction was investigated by cross-linking and quantitative binding experiments. hCG and its derivatives, desialylated hCG and deglycosylated hCG were labeled either to the alpha-subunit (125I) or the beta-subunit (3H). The ligands were attached to ovarian membrane particles, which were treated with neuraminidase or peptide-N-glycosidase F to remove terminal sialic acids or N-linked oligosaccharides of the receptor, respectively, and the complexes formed were solubilized, cross-linked with glutaraldehyde, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All of the ligands produced similar autoradiographic patterns with the native or glycosidase-treated receptor, and only the receptor-(alpha)hCG and receptor-(alpha, beta)hCG complexes were detected. Moreover, quantitative binding studies indicated that all of the hormone derivatives had similar affinities for the native or glycosidase-treated receptor. In addition, the orientation of the carbohydrate side chains on the receptor-hormone complex was studied by digesting the complex with the glycosidases. The molecular weight of the receptor, evidenced by ligand blotting, was reduced to the same extent, whether the membrane-bound free receptor or receptor-hormone complex was treated with the glycosidases, suggesting that the oligosaccharide side chains of the receptor are apart from the hormone binding region. As peptide-N-glycosidase F treatment reduced the size of the Mr 90,000 receptor first to about Mr 67,000 and finally to about Mr 62,000, there may possibly be 2 N-linked carbohydrate chains per receptor polypeptide. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the glycosidase-treated receptor-[125I]hCG complex also revealed that neuraminidase was able to remove the sialic acids from both subunits of the receptor-bound hormone. In conclusion, the results suggest that hCG interacts with the polypeptide backbone of its ovarian receptor mainly through the peptide core of its alpha-subunit. Moreover, the carbohydrate side chains of both subunits of hCG are positioned on the outward face of the receptor-hormone complex.