Reduction of testicular human chorionic gonadotropin receptors by human chorionic gonadotropin in vivo and in vitro

Changes in rat and human testicular human chorionic gonadotropin (hCG) binding sites induced by hCG were estimated in vivo and in vitro. After a single administration of hCG, the specific 125I-hCG bindings were significantly reduced for 7 and 5 days in rat and human testes, respectively. Thereafter, 125I-hCG bindings had recovered to pretreatment values by the 14th day after the administration. Occupied hCG bindings accounted for about half of the reduced bindings on the day after administration of hCG. After this time, however, the occupancy did not contribute so much to the reduction of the bindings. In experiments in vitro using the organ culture technique, an exposure to hCG for 24 h induced a dose-related significant loss of the specific 125I-hCG bindings for 7 and 5 days in rat and human testes, respectively. Thereafter, the loss was gradually recovered. These patterns of changes in 125I-hCG bindings in vitro were similar to those in vivo. These findings suggest that the reduction in hCG binding sites by hCG is due to not only occupancy but also downregulation of the binding sites and that the testicular organ culture method used in the present study is useful to study hormonal regulation of testicular function, especially in human testes.     

The secretory patterns of relaxin and human chorionic gonadotropin in human pregnancy

Relaxin and human chorionic gonadotropin (hCG) were simultaneously determined in the same serum samples obtained from pregnant women. Although the secretory pattern of relaxin, in general, appeared to parallel that of hCG during human pregnancy, several discrepancies were discerned in the secretory patterns of the two hormones. The mean hCG concentration significantly differed between weeks 4-7 and 8-11 of pregnancy, but the mean relaxin concentration did not. The mean relaxin concentration began to decrease at weeks 16-19 whereas that of hCG did so at weeks 12-15. The mean relaxin concentration at weeks 4-7 was significantly higher than that at weeks 24-27, though there was no significant difference between the mean hCG concentrations in the two periods. These differences in the secretory pattern of relaxin from that of hCG indicate that relaxin secretion in pregnancy is not determined only by the circulating level of hCG. The responsiveness of the corpus luteum of pregnancy to hCG stimulation of relaxin secretion may vary as a function of the age of the corpus luteum, and this may partially account for the differences between the secretory pattern of relaxin and that of hCG observed in the present study.     

Overexpression of human chorionic gonadotropin causes multiple reproductive defects in transgenic mice

Human CG is a pregnancy marker secreted by the placenta, and it utilizes the same receptors as does LH. Human CG is a heterodimer, and its subunits are expressed in tissues other than placenta. Similarly, LH/hCG receptors are also expressed in multiple tissues; however, the physiological significance of this expression is unknown. Free hCGbeta is efficiently secreted in vitro in transfected cells and is highly expressed in many human cancers; however, the biological effects of free hCGbeta in vivo are unknown. To study in vivo consequences of elevated levels of free hCGbeta and hCG dimer in both male and female reproductive physiology, we used mouse metallothionein 1 promoter to generate multiple lines of transgenic mice that overexpressed either one or both subunits of hCG. Although mice expressing the glycoprotein hormone alpha subunit are normal and fertile, both male and female transgenic mice overexpressing only the hormone-specific hCGbeta subunit are infertile. The hCGbeta subunit-expressing transgenic female mice progressively develop cystic ovaries, whereas the male transgenic mice are infertile but otherwise are not phenotypically discernible. In contrast, both the male and female transgenic mice coexpressing high levels of the hCG subunits (i.e., the hCG dimer) demonstrate multiple reproductive defects. The male transgenic mice have Leydig cell hyperplasia, very high levels of serum testosterone, reduced testis size, and dramatically enlarged seminal vesicles and are infertile and display overly aggressive behavior when caged with females. The female transgenic mice are also infertile, have elevated levels of serum estradiol, and progressively develop hemorrhagic and cystic ovaries with thecal layer enlargement and stromal cell proliferation and degenerating kidneys. These results suggest that the in vivo biological effects of ectopically expressed free hCGbeta subunit are distinct from those of the hCG dimer and are gender specific. These transgenic mice are useful models for studying the biology of free hCGbeta subunit, for further analyzing the gain of function effects of hCG during early Leydig cell development, and for studying the roles of hCG in ovarian and kidney pathophysiology and function.     

Effect of super-ovulatory doses of pregnant mare serum gonadotropin and human chorionic gonadotropin in blastocyst implantation in golden hamsters

The effect of super-ovulatory dose of pregnant mare serum gonadotropin and human chorionic gonadotropin on ovulation, advancement of ovulation, subsequent embryo development and implantation were studied in the hamster. Groups of hamsters received pregnant mare serum gonadotropin injection on day 1 of the estrous cycle followed by human chorionic gonadotropin injection either at 56 or 76 h later, pregnant mare serum gonadotropin alone on day 1 or human chorionic gonadotropin alone on day 3.The combination therapy (pregnant mare serum gonadotropin and human chorionic gonadotropin) resulted in super-ovulation (an average of 40 mature ova/animal) while human chorionic gonadotropin alone yielded an average of 10 mature ova/animal. Ovulation was advanced by 24 h by giving human chorionic gonadotropin at 56 h instead of 76 h after pregnant mare serum gonadotropin. Subsequent embryo development and implantation occurring under different hormonal regimens were studied. The ova obtained by giving human chorionic gonadotropin injection at 56 h were poorly fertilizablein vivo and hence the pregnancy rate was low (6 %). These ova however, were fertilizablein vitro, suggesting that the low fertilization rate and developmental failure may be due to inhibition of sperm capacitation/transport because of premature human chorionic gonadotropin administration. In the group receiving human chorionic gonadotropin alone on day 3 there was fertilization and cleavage, but no implantation occurred due to failure of functional corpora lutea. However, administration of progesterone and estrone from day 2 of gestation resulted in 80% implantation and sustenance of pregnancy. On the other hand, the pregnant mare serum gonadotropin and human chorionic gonadotropin combination therapy resulted in super-pregnancy. The number of fetuses present at term was higher in the group receiving pregnant mare serum gonadotropin alone than in the group receiving the combination therapy. Embryo resorption however was higher (37%) in the latter group compared with the former (9.5%). However, preimplantation embryos were found to be viable as evidenced by fluorescein diacetate staining.     

Nucleotides do not modulate rat luteocyte human chorionic gonadotropin responsiveness by inhibiting human chorionic gonadotropin binding

Nucleotide inhibition of ¹²⁵I-labeled human chorionic gonadotropin binding to luteocyte receptor was studied by investigating effects of nucleotides on the apparent equilibrium association constant (K_a) and number of binding sites (B_max), and on rate constants for association (k₊₁) and dissociation (k_?1, k_?2). K_aandB_max were determined by various analyses of equilibrium binding data using washed 2000g pellet of an ovarian homogenate from rats 7 days after pregnant mare's serum gonadotropin-human chorionic gonadotropin priming. Adenyl and guanyl nucleotides, as well as other nucleotides, lowered the K_a of ¹²⁵I-labeled human chorionic gonadotropin binding to luteocyte receptor without affecting B_max. The degree of inhibition was dose related at nucleotide concentrations greater than 10^?3 m. GTP and guanyl-5′-ylimidodiphosphate inhibitions were similar in the presence or absence of EDTA (1.25 × 10^?3 m). ATP and GTP lowered K_a by slowing the rate of association. Inhibition of binding could not be demonstrated at lower nucleotide concentrations even when luteocyte membranes were purified partially by sucrose density gradient ultracentrifugation. In light of the high nucleotide concentrations required to inhibit ¹²⁵I-labeled human chorionic gonadotropin binding and the inhibition by Mg²⁺ and PP₁ at similar concentrations, the effect appears to be a nonspecific ionic effect. Therefore, in contrast to the glucagon-hepatocyte system, luteocyte human chorionic gonadotropin responsiveness does not appear to be modulated by nucleotide inhibition of human chorionic gonadotropin-receptor interaction.     

Immunobiology of human chorionic gonadotropin

A comprehensive review of the immunobiology of human chorionic gonadotropin (hCG), including the structure of both alpha and beta chains, immunogenicity of various segments and epitopes of each, secretion and function of the hormone, determinants of receptor recognition, and finally, clinical studies of possible contraceptive beta-hCG-based vaccines, is presented. hCG is composed of 2 glycosylated peptides. The alpha subunit is identical to that found in hLH, hFSH and hTSH. The beta subunit, which is limiting in the sense that it is secreted in smaller amounts, defines the biological activity of hCG. hCG is secreted throughout pregnancy from 170 hours after fertilization to a peak at 8-10 weeks of and is essential for maintenance of early pregnancy by progesterone secreted by the corpus luteum. Although native hCG evokes antibodies, they cross react with LH, so such a vaccine would not be useful for contraception. Beta-hCG has been purified and also produced by monoclonal antibodies, and shown to produce antibodies and infertility in baboons. Phase I clinical trials of immunologically purified beta-hCG complexed to tetanus toxoid were conducted on 63 women in an international study in the mid-1970s, but results were mixed in terms of antibody titer and duration. New vaccines have been designed based on more sophisticated adjuvants, beta- hCG-terminal peptides, and polyvalent vaccines and are being tested in 4 Phase I trials currently, sponsored by the Population Council, the Indian government-sponsored program, and the WHO.     

A nicked beta-subunit of human chorionic gonadotropin purified from pregnancy urine.

Human chorionic gonadotropin (hCG) is a glycoprotein hormone composed of two dissimilar subunits (alpha and beta) and normally excreted in urine of pregnant women. An uncommon beta-subunit of hCG was purified from fresh early normal pregnancy urine by Sepralyte C8, resin adsorption. Sephadex G-100 column chromatography, and reverse-phase HPLC. SDS-PAGE under non-reducing conditions showed that the apparent molecular weight (39,000) of this beta-subunit was extremely similar to that of the native beta-subunit, which is known to consist of 145 amino acid residues and carbohydrates. However, SDS-PAGE, under reducing conditions, resulted in two bands with apparent molecular weights of 22,000 and 18,000, indicating that it consisted of two peptide fragments connected with disulfide bridge(s). These two peptide fragments, separated and purified from the reduced and carboxymethylated protein, were subjected to amino acid and N-terminal sequence analyses. It was found that this beta-subunit consisted of two polypeptide chains composed of residues 1-47 disulfide-bridged to residues 48-145 of the beta-subunit, which may be produced by nicking of the beta-subunit at the one site (Gly47-Val48). This beta-subunit was termed a nicked beta-subunit of hCG (N-hCG beta). It was also found that N-hCG beta was present in urine as an alpha beta dimer, indicating that an intrachain nicking of this site in the beta-subunit does not inhibit alpha beta dimer formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carboxypeptidase digestion of human chorionic gonadotropin

Native human chorionic gonadotropin (hCG) was resistant to carboxypeptidase digestion even in the presence of urea. Isolated alpha subunit of the hormone (hCG-alpha), though unreactive to enzyme treatment in the absence of denaturant, released up to four amino acid residues from the C-terminus on incubation with a mixture of carboxypeptidases A and B in urea. While an hCG-alpha product which lacked Ser-92 recombined completely with intact hCG-beta, hCG-alpha from which Ser-92 recombined completely with intact hCG-beta, hCG-alpha from which Ser-92 and Lys-91 were removed showed only partial recombination. The two recombinants were devoid of any in vivo biologic activity, but retained some of the immunologic activity of the native recombinant. These findings indicate that the integrity of the C-terminal residue of serine in hCG-alpha is essential for the expression of in vivo biologic activity of the native hormone.     

Inhibition of adenylyl cyclase activity in rat corpora luteal tissue by glycopeptides of human chorionic gonadotropin and the alpha-subunit of human chorionic gonadotropin

Indirect evidence has indicated that the carbohydrate moieties of the glycoprotein hormones are involved in the activation of the receptor-adenylyl cyclase system of reproductive tissues. In the present study, we have isolated the glycopeptides (GP) from human chorionic gonadotropin (hCG), the alpha-subunit of hCG, fetuin, and bovine gamma-globulin (b gamma G). These along with a number of synthetic oligosaccharides were tested for their ability to inhibit adenylyl cyclase (AC). There was less than 0.001% cross-reactivity of the GP from hCG, hCG alpha, fetuin, and b gamma G when tested in a double-antibody hCG radioimmunoassay or rat corpora lutea radioreceptor assay. The GP of fetuin, b gamma G, and the synthetic oligosaccharides did not inhibit AC activity of 2000 g corpora lutea membranes when coincubated with 100 ng of hCG/mL (ED50). However, when the GP of hCG and hCG alpha were included with intact hCG, there was a dose-related inhibition. Inhibition of cyclase activity was enhanced when the hCG GP were desialylated. This occurred without a change in the lag time of hCG activation which was calculated to be 1-1.5 min. Changing the concentration of ATP and Mg2+ did not affect the inhibitory effects of the hCG alpha GP on hCG-stimulated AC activity. Inhibition by hCG GP followed uncompetitive kinetics. The inhibition by the GP of hCG seems to be restricted to the LH/hCG-stimulatable AC system because the same dosage of hCG GP which inhibited the rat luteal AC system did not have any effect on the rat hepatocyte AC system when coincubated with glucagon or on NaF-stimulated activity in luteal membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

A test of maternal human chorionic gonadotropin during pregnancy as an adaptive filter of human gestations

The risk of abnormalities and morbidity among live births increases with advanced maternal age. Explanations for this elevated morbidity invoke several maternal mechanisms. The relaxed filter stringency (RFS) hypothesis asserts that mothers, nearing the end of their reproductive lifespan, reduce the stringency of a screen of offspring quality in utero based on life-history traits of parity and interbirth interval (IBI). A separate line of research implicates human chorionic gonadotropin (hCG) during pregnancy as a signal of offspring quality. We test the RFS hypothesis directly by examining whether the difference in gestational hCG across consecutive live births varies positively with the mother''s number of previous live births but inversely with her most recent IBI. We applied multivariable regression methods to a unique dataset of gestational hCG for over 500 000 live births from 2002 to 2007. The difference in gestational hCG across mothers'' consecutive live births varies positively with both mothers'' parity and IBI. These associations remain similar among older mothers (35+ years). Findings support the RFS hypothesis for the parity expectation but not for the IBI expectation. Further evidence for the RFS hypothesis among contemporary human gestations would have to invoke screening mechanisms other than hCG.     

Regulation by human chorionic gonadotropin of protein kinases in rabbit endometrium

The effect of human chorionic gonadotropin (CG) on protein kinase levels has been studied in rabbit endometrium. A good correlation was observed between alterations of total protein kinase activity and DNA content in the tissue. The level of type I cyclic AMP-dependent protein kinase had slightly increased by 6 h after human CG treatment, and then decreased later. In contrast, the type II enzyme gradually increased after 3 days. The increase of the activity at 7 days was mainly due to that of type II enzyme, and the ratio of type II to type I enzyme increased. The activity pattern at the later stage closely resembled that of treatment with progesterone.     

Slow internalization of human chorionic gonadotropin by cultured granulosa cells

Kinetic studies were performed on two-day cultures of rat ovarian granulosa cells to follow the fate of surface-bound 125I-labeled human chorionic gonadotropin (125I-hCG). Low pH was used to release hCG from its surface receptor, allowing us to distinguish between surface-bound and internalized hormone. Because our results indicated that hormone is lost from the cell surface by dissociation as well as internalization, equations were derived to determine independent rate constants for each process. We calculate that if hormone binding were irreversible, the t 1/2 for internalization would be 8.5 hour. Morphometric studies on the uptake of horseradish peroxidase indicate that the t 1/2 for internalization of bulk membrane in granulosa cells is 55 to 77 minutes. Thus, the rate of uptake of surface-bound hCG appears to be seven to nine times slower than the rate of uptake of bulk plasma membrane, which suggests that the LH/hCG receptor may be selectively excluded from the endocytic vesicles of granulosa cells.     

Upregulation of placental indoleamine 2,3-dioxygenase by human chorionic gonadotropin

We tested the hypothesis that hCG can upregulate human trophoblast indoleamine 2, 3-dioxygenase (INDO), which catalyzes the breakdown of tryptophan in villous circulation. The results revealed that it can. Treatment of human trophoblasts with hCG resulted in a time and dose dependent increase in INDO mRNA and protein levels and its enzyme activity. The hCG effect was hormone specific and required the dimer conformation of hCG. The hCG effect required its receptors and was mediated by a cAMP dependent, but protein kinase A independent, mitogen-activated protein kinase 3/1 (MAPK3/1) signaling mechanism. In summary, the present data demonstrate a novel hCG effect on human placental INDO, which probably plays a key role at maternal fetal interface in preventing fetal rejection.