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.     

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.     

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.     

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.     

Gonadotropin-releasing hormone effects on placental hormones during gestation: I. Alpha-human chorionic gonadotropin, human chorionic gonadotropin and human chorionic somatomammotropin

The release of alpha-human chorionic gonadotropin (alpha hCG), gonadotropin human chorionic gonadotropin (hCG) and human chorionic somatomammotropin (hCS) in vitro from placentas of different gestational ages was studied. In addition, the effect of gonadotropin-releasing hormone (GnRH) on these hormonal releases, as related to the gestational age of the placenta cultured and the dose of GnRH, was determined. The basal release of alpha hCG and hCG was greatest at 9-13 wk of gestation (1000-1500 ng/mg and 250-350 ng/mg, respectively). Lowest release rates were at term (28 ng/mg and 20 ng/mg, respectively). Hormonal release declined with extended culture, except from the cultures of 13- and 15-wk placentas, in which the initially high release continued throughout the 8 days of culture. The initial release of hCS was low at 6 wk, increased to maximum rates by 15 wk, and was similar to the initial rate of release at term. Gonadotropin-releasing hormone stimulated the release of alpha hCG and hCG most dramatically in cultures of 16-wk and 17-wk placentas, where as much as a 400- and 250-fold increase, respectively, on Day 6 of culture was observed (p less than 0.0001). In term placenta cultures after 6 days in vitro, a 20-fold stimulation of alpha hCG and a 10-fold increase of hCG was effected by GnRH (p less than 0.001). The largest responses of alpha hCG and hCG to GnRH were observed when estrogen levels were low. Dose-related responses were observed in some placentas, yet in some instances, maximal effects were attained with all doses utilized in these studies (0.2 to 50 micrograms/ml). These data demonstrate that human placentas of different gestational ages have varying hormonogenic capabilities in vitro. The data also establish that synthetic GnRH is capable of stimulating alpha hCG and hCG production, but the degree and pattern of response to GnRH stimulation are related to the gestational age of the placental tissue and its time in culture. The most responsive period to exogenous GnRH stimulation of alpha hCG and hCG release was on Days 5 and 6 of culture, when basal estrogen release was very low. These data support the hypothesis that hCG release might be controlled by a chorionic GnRH stimulation and suggest that local steroid levels may modulate the hCG response to GnRH stimulation.     

Preliminary studies on the indium slide immunoassay for estimation of human chorionic gonadotropin and antihuman chorionic gonadotropin antibody

Human chorionic gonadotropin (hCG) is synthesized and secreted as early as 170 hr after fertilization and has been used as an index for pregnancy. Neutralization of hCG with a beta-subunit hCG vaccine(s) has been proposed as a contraceptive technique. To monitor the duration of effectiveness of the vaccine, it will be necessary to monitor the anti-hCG antibodies, especially those responsible for inhibiting the hCG bioactivity. We report a simple, rapid technique using an indium slide immunoassay for the qualitative estimation of hCG and to monitor a bioeffective anti-hCG antibody. The sensitivity of the indium slide assay to measure hCG ranged from 1 microgram/ml to 1 ng/ml, depending on the format of the assay. The indium slide assay also detected anti-hCG antibodies generated against a specific determinant on hCG recognized by a neutralizing monoclonal antibody (P3W80) in women immunized with a contraceptive vaccine.     

Preliminary X-ray diffraction analysis of human chorionic gonadotropin

Hexagonal bipyramidal crystals of deglycosylated human chorionic gonadotropin have been grown using the method of vapor diffusion against ammonium sulfate. These crystals grow to nearly 0.4 mm along each axis, diffract to better than 3.5-A resolution and are relatively stable to irradiation. The crystals belong to the hexagonal space group P6(1)22 or enantiomer, and have unit cell parameters a = b = 88.7 A and c = 177.3 A.     

Production of monoclonal antibodies to human chorionic gonadotropin

Production of monoclonal antibodies against human chorionic gonadotropin (hCG) has been studied using hCG as an immunogen. Spleen cells of BALB/c mice immunized with hCG were fused with NS-1 mouse myeloma cells. This study reports the successful isolation of a hybrid clone secreting a monoclonal antibody specific for hCG. By using PEG 4,000 as a fusion agent, the fusion rates were between 42.0 and 50.2%. In total 842 hybridomas were produced. Among them, 403 hybridomas had hCG antibody production. After cloning twice by limiting dilution and alternately screening by enzyme immunoassay and by radioimmunoassay, there were 39 cell lines having specific antibody production. Among them, the No. 57-42-2 had the highest reactivity. By Ouchterlony test, the monoclonal antibody was shown to be IgG1. The affinity constant of the antibody to hCG was 0.6 x 10(9) 1/mole. In radioimmunoassay, the cross reactivity of the antibody to human luteinizing hormone (LH) and human follicle-stimulating hormone (FSH) was 1.5% and 0.7%, respectively. There was no cross reaction with human thyrotropin-stimulating hormone (TSH).     

Immunological properties of the β-subunit of human chorionic gonadotropin

The β-subunit of human chorionic gonadotropin (hCG-β) has been modified to a varying degree by the cleavage of the disulfide bonds by reduction with dithioerythritol followed by S-carboxamidomethylation. The resulting derivatives of hCG-β show a preferential loss in their immunological cross-reactivity with human luteinizing hormone (hLH). The immunological specificity of the partially reduced and S-alkylated derivatives can be further enhanced by conjugation with tetanous toxoid using glutaraldehyde. Neither conjugation of hCG-β with the toxoid nor its treatment with anti-hLH immunoadsorbent affects its cross-reactivity with hLH.     

Solution structure of the alpha-subunit of human chorionic gonadotropin.

The three-dimensional solution structure of the alpha-subunit in the alpha, beta heterodimeric human chorionic gonadotropin (hCG), deglycosylated with endo-beta-N-acetylglucosaminidase-B (dg-alpha hCG), was determined using 2D homonuclear and 2D heteronuclear 1H, 13C NMR spectroscopy at natural abundance in conjunction with the program package XPLOR. The distance geometry/simulated annealing protocol was modified to allow for the efficient modelling of the cystine knot motif present in alpha hCG. The protein structure was modelled with 620 interproton distance restraints and the GlcNAc residue linked to Asn78 was modelled with 30 protein-carbohydrate and 3 intraresidual NOEs. The solution structure of dg-alpha hCG is represented by an ensemble of 27 structures. In comparison to the crystal structure of the dimer, the solution structure of free dg-alpha hCG exhibits: (a) an increased structural disorder (residues 33-57); (b) a different backbone conformation near Val76 and Glu77; and (c) a larger flexibility. These differences are caused by the absence of the interactions with the beta-subunit. Consequently, in free dg-alpha hCG, compared to the intact dimer, the two hairpin loops 20-23 and 70-74 are arranged differently with respect to each other. The beta-GlcNAc(78) is tightly associated with the hydrophobic protein-core in between the beta-hairpins. This conclusion is based on the NOEs from the axial H1, H3, H5 atoms and the N-acetyl protons of beta-GlcNAc(78) to the protein-core. The hydrophobic protein-core between the beta-hairpins is thereby shielded from the solvent.     

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)     

Monoclonal antibodies against the beta subunit of human chorionic gonadotropin

The aim of investigation was creation of hybridomas, which produce monoclonal antibodies to the beta-subunit of human chorionic gonadotropin (beta-hCG), characterization of monoclonal antibodies, which necessary for hCG immunoassay in biological fluids, as an immunological methods of detection of early stage of pregnancy and choriocarcinoma. 4 hybridomas, producing monoclonal antibodies to-hCG of IgG1 isotype, were created. On the base of monoclonal antibodies, which produced by D2 hybridoma cell line, test-systems for RIA of hCG in blood serum and urine were elaborated. These test-systems can be used in medical practice for diagnosis of early stages of pregnancy and choriocarcinoma.     

Amphiregulin regulates the production of human chorionic gonadotropin in trophoblasts

AimsThe aim of this study was to investigate the significance of epidermal growth factor receptor (EGFR) ligands produced in syncytiotrophoblasts during normal pregnancy.Main methodsWe examined the expression of EGFR ligands in human pregnancy by real-time PCR, and analyzed the relationship between EGFR ligands and human chorionic gonadotropin (hCG) or human placental lactogen in amniotic fluid by ELISA. In addition, we also examined the EGFR ligands in syncytiotrophoblasts and the amount of hCG secretion in JAR, JEG3 and BeWo cells in the presence of each EGFR ligand.Key findingsIn order to identify possible candidates among the EGFR ligands, we examined the predominant expression of an EGFR ligand in the chorionic villi and amniotic fluid during normal pregnancy, and analyzed the relationship between EGFR ligands and hCG in trophoblastic model cells. Amphiregulin was primarily expressed throughout human pregnancy and stimulated the secretion of hCG, indicating that amphiregulin is a key molecule among EGFR ligands.SignificanceAmphiregulin may play a pivotal role in the development or maturation of placenta.