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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.

Antibodies↗

[Fractionation of proteins of chorionic villi and chorionic membranes by two-dimensional electrophoresis].

Human chorionic villi and choriomembrane proteins were analyzed by two-dimensional electrophoresis. The samples were obtained during the first trimester of pregnancy and were solubilized in buffer containing Triton X-100 and CHAPS. High molecular weight proteins with Mr above 200 KDa and proteins with basic pI were found to be better resolved in this buffer system that in solution containing Triton X-100 only. Nearly 200 protein spots could be visualized on the silver stained gels.

Chorion↗

Cytogenetic analysis of chorionic villi for prenatal diagnosis: an ACC collaborative study of U.K. data. Association of Clinical Cytogeneticists Working Party on Chorionic Villi in Prenatal Diagnosis.

A prospective 3-year collaborative study was undertaken in 1987 to collect cytogenetic data from diagnostic chorionic villus samples (CVS) in the U.K. in order to determine the predictive value of the chromosome abnormalities encountered. Twenty-seven laboratories contributed a total number of 7595 cases, of which 97.6 per cent were successful. Excluding single cell anomalies, a total of 480 cytogenetic abnormalities were reported, of which 137 were familial structural rearrangements and 343 were de novo problems. Non-mosaic trisomies of chromosomes 13, 18, and 21 (n = 157), non-mosaic sex chromosome abnormalities (n = 33), and triploidy (n = 6) were all confirmed in cells of fetal origin where follow-up information was available. Of the nine remaining non-mosaics including tetraploidy, trisomies of other autosomes, and extra markers, only a trisomy 16 and a case of a supernumerary marker proved genuine. Eighty-eight cases of mosaicism were reported to the study, of which only nine were confirmed as genuine: two cases involving chromosome 13, one trisomy 18, two examples of extra marker chromosomes, three 45,X, and one 47,XXX. There were no reports of false-negative findings. Presumptive maternal cell contamination was encountered in 39 cases, a detected incidence of 0.5 per cent. Four cases of presumptive 'vanishing twin' were recorded: in three of these, direct preparations showed a female karyotype, whereas culture indicated a male (with male fetuses in two cases). The fourth case was of a female fetus with male and female cells in the CVS cultures. Subtle structural chromosome abnormalities were missed in three instances. Accurate prediction of the fetal karyotype was shown to require detailed knowledge of both the nature and the distribution of abnormal cells in the extra-embryonic tissues. In many cases, this could only be made where results from direct preparations and cultured cells were available. A number of conclusions were reached from these and similar data in the literature regarding the reliability of chromosome findings in CVS.

Chorionic Villi Sampling↗

Detection of a small molecular species of human chorionic gonadotropin in the urine of patients with carcinoma of the cervix and cervical intraepithelial neoplasia: comparison with other assays for human chorionic gonadotropin and its fragments.

A low-molecular-weight glycoprotein containing sequences of the beta subunit of human chorionic gonadotropin (hCG) has been found in the urine of patients with carcinoma of the cervix using an immunoradiometric assay. This fragment has chromatographic and immunological identity with hCG beta core. This molecule was present in 52 to 77% of all patients with invasive disease, while between 11 and 27% of patients with cervical intraepithelial neoplasia (CIN) also exhibited significant hCG beta-core immunoactivity. Few patients had either a positive assay for intact hCG or a positive assay directed at an epitope on the beta subunit (beta-hCG radioimmunoassay) in serum. However, between 17 and 40% of patients with invasive disease were positive for free beta-subunit immunoactivity in the blood. The origin of the beta-core immunoactivity in the urine is uncertain; while tumor production cannot be excluded, it is possible that the molecule originates from renal metabolism of small quantities of the beta subunit of hCG. Regardless of the source of the molecule, hCG beta core is a far more sensitive marker of hCG production by tumors than is serum hCG.

Carcinoma↗

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)

Adenylyl Cyclase Inhibitors↗

Desialylated and deglycosylated human chorionic gonadotropin are superagonists of native human chorionic gonadotropin in human thyroid follicles.

Highly purified human chorionic gonadotropin (hCG) interacts with the thyrotropin (TSH) receptor and stimulates triiodothyronine (T3) secretion, iodide uptake and organification, and cyclic adenosine monophosphate (cAMP) formation in human thyroid follicles. Because of interest in the role of the carbohydrate component in the structure-function relationships of hCG we undertook to deplete hCG of its sialic acid or carbohydrate residues and assess the thyrotropic activity of the carbohydrate-modified forms. For this purpose, we used our assay system consisting of human thyroid follicles cultured and suspended in collagen gel in serum-free medium. Under these conditions, the cells are organized as follicular three-dimensional structures with normal polarity, enabling enhanced responsiveness to hormonal stimulation, and T3 secretion can be measured as a response parameter. Desialylated (ds)-hCG and deglycosylated (dg)-hCG dose-dependently stimulated T3 secretion, iodide uptake and organification, and in each case did so with about twice the intrinsic activity of native hCG. Indeed, removal of the sialic acid or carbohydrate residues from native hCG transformed it into a thyroid stimulator that elicited a maximal response in terms of iodide uptake, organification and T3 secretion by human thyroid follicles as high as TSH and almost twice as high as native hCG. Not only were ds-hCG and dg-hCG more intrinsically active than hCG, they were more than five times as potent. As with hCG, both ds-hCG and dg-hCG managed to elicit such responses in human thyrocytes while evoking minimal amounts of cAMP, illustrating the concept of cAMP superfluity and highlighting the potential pitfalls of using cAMP as a measure of hormonal bioactivity. hCG, and to a greater extent ds-hCG and dg-hCG, inhibited, as did TSH, gamma-interferon-induced human leukocyte antigen-DR (HLA-DR) expression in human thyrocytes, again reflecting the intrinsic thyrotropic activity of native hCG and its variants depleted of sialic acid or carbohydrate residues. In conclusion, this is the first report on the thyrotropic activity of ds-hCG and dg-hCG using the physiologically relevant hormonal end-point response, thyroid hormone secretion. The study was conducted in a serum-free culture system of human thyroid follicles and shows that removal of the sialic acid or carbohydrate residues from native hCG transform hCG variants into thyroid stimulating superagonists. The hCG variants inhibited, as did TSH, gamma-interferon-induced HLA-DR expression.

Asialoglycoproteins↗

The effect of human chorionic gonadotropin, dibutyryl cyclic adenosine 3',5'-monophosphate, prostaglandins, and 25-hydroxycholesterol on acute progesterone secretion by dissociated rabbit luteal cells in vitro: evidence for independent effect of human chorionic gonadotropins and lipoproteins.

Although estradiol is the established luteotropic hormone in the rabbit, the corpus luteum also contains a luteinizing hormone (LH)-activated adenylate cyclase system and cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase, which suggests that LH and cAMP may play a physiological role in regulating luteal progesterone production. The present study examined whether human chorionic gonadotropin (hCG) and cAMP derivatives stimulate progesterone production by dispersed rabbit luteal cells in static and perifusion incubations. Results of this study show that progesterone production by rabbit luteal cells is significantly stimulated (p less than 0.05) by hCG concentrations at or greater than 0.1 IU/ml or by dibutyryl cAMP concentrations at or greater than 5 mM. Both agents produce maximal stimulations of approximately 4-fold. However, neither prostaglandin E2 or F2 alpha at concentrations of 0.1-3.0 micrograms/ml altered progesterone secretion. When luteal cells were incubated with maximal concentrations of hCG and lipoproteins together, the resultant progesterone secretion was additive. This suggests that the effects of hCG and lipoprotein are independent. Both responses could be blocked completely by cycloheximide (10(-4) M), and thus appear to be dependent on protein synthesis. The cholesterol derivative 25-hydroxycholesterol (20 micrograms/ml) partially overcame the steroidogenic block by cycloheximide, suggesting that transport of cholesterol, regardless of its origin, into mitochondria was an essential protein-mediated event in these cells. Inhibition of the side-chain-cleavage enzyme by aminoglutethamide blocked progesterone production by rabbit luteal cells in vitro. Although estradiol may dominate in the regulation of luteal progesterone production physiologically, this study clearly demonstrates that potential mechanisms do exist in the rabbit corpus luteum for cAMP-mediated stimulation of progesterone production in the rabbit.

Animals↗

Luteinizing hormone/chorionic gonadotropin bioactivity in the common marmoset (Callithrix jacchus) is due to a chorionic gonadotropin molecule with a structure intermediate between human chorionic gonadotropin and human luteinizing hormone.

Chorionic gonadotropin (CG), a pregnancy-specific heterodimeric hormone found in primates, is responsible for CL rescue with pregnancy maintenance. Of the primates, the human and baboon gene sequences are the only structures so far determined. In order to study the structure and function of CG in other primates, we have isolated and sequenced the coding regions for the two subunits of marmoset CG (mCG) by the reverse transcription/polymerase chain reaction method. Study of multiple clones confirmed a high degree of homology with the human sequences (88% and 80% for the alpha and beta nucleotide sequences, respectively). Marmoset CG alpha has an extra four amino acids compared to hCG alpha, whereas the mCG beta sequence has a 3-bp deletion that maintains the reading frame and C-terminal amino acid sequence. Most of the differences between hCG beta and mCG beta peptides occur in the C-terminal region, which includes the loss of two of the O-linked glycosylation consensus sequences and the presence of an N-linked glycosylation consensus sequence. When mCG alpha and beta were co-expressed in CHO cells, assembly of biologically active hormone was confirmed by induced steroid secretion by MA10 cells. Partially purified mCG beta was used to raise anti-mCG antibodies. To date, an antibody has been obtained that is capable of detecting recombinant mCG beta, recombinant mCG dimer, and mCG dimer secreted by cultured marmoset trophoblast. Marmoset CG alpha and beta were also detectable at the transcriptional level in cultured trophoblast by in situ hybridization. This suggests that the LH/CG bioactivity reported from marmoset placentae and embryos is due to a molecule with structural features common to hLH (glycosylation pattern) and hCG (CG beta C-terminal structure).

Amino Acid Sequence↗

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.

Animals↗

Asialoagalacto-human chorionic gonadotropin, a carbohydrate-modified variant of human chorionic gonadotropin, antagonizes the stimulatory actions of bovine thyroid-stimulating hormone on thyroid function and HLA-DR expression in human thyroid in vitro and in vivo.

The concept of using thyroid-stimulating hormone (TSH) receptor antagonists in the management of Graves' disease is intriguing. Therefore, we investigated a TSH receptor antagonist derived from human chorionic gonadotropin (hCG) with respect to TSH receptor binding, adenylate cyclase activity, thyroid hormone release, and HLA class II antigen expression in vitro and in an in vivo model. A variant of hCG, asialoagalacto-hCG, like asialo-hCG and unlike hCG itself, inhibited both 125I-bTSH binding and cAMP response to bTSH in human thyroid membranes. However, like intact or deglycosylated hCG and unlike asialo-hCG, asialoagalacto-hCG displayed a limited affinity for hepatic asialoglycoprotein receptors, a likely marker for its in vivo turnover rate. It proved capable of inhibiting bTSH-stimulated thyroid hormone release in human thyroid slices as well as in the nude mouse bearing human thyroid transplants. It also prevented bTSH induced hypertrophy of transplanted thyrocytes. Further, HLA-DR expression induced by bTSH in the presence of gamma-interferon on human thyrocytes was inhibited. In conclusion, we present evidence that asialogalacto-hCG antagonizes bTSH actions on thyroid function and HLA-DR expression in human thyroid in vitro and, more importantly, in an in vivo model. Hence, the hCG variant described here or similar agents should warrant further exploration in the study and treatment of Graves' disease.

Adenylyl Cyclases↗

Failure of somatostatin to affect human chorionic somatomammotropin and human chorionic gonadotropin secretion in vitro.

The effect of somatostatin (SRIF) on human chorionic somatomammotropin (hCS) secretion was studied in human placental explants cultured in vitro. In the experimental flasks, SRIF was added in a concentration of 10, 100, and 1000 ng/ml media; hCS levels measured by RIA were not different from those found in the control flasks. In separate experiments, we investigated the action of SRIF on hCG secretion by a human malignant choriocarcinoma cell line maintained in tissue culture. SRIF (1000 ng/ml) did not inhibit basal or dibutyryl cAMP-induced stimulation of hCG secretion. These results suggest that somatostatin does not suppress hCS or hCG release in vitro from normal or malignant trophoblast, respectively.

Bucladesine↗

Human chorionic gonadotropin secretion and protein phosphorylation in chorionic tissue.

The signal transduction system acts either monodirectionally (synergistically) or bidirectionally (antagonistically) in different tissues. We activated the signal transduction system in BeWo human choriocarcinoma cells and studied the effects on human chorionic gonadotropin (hCG) secretion, cell proliferation, and DNA synthesis. Protein phosphorylation in the cytosolic fraction was also studied by two-dimensional polyacrylamide gel electrophoresis. When phorbol 12-myristate 13-acetate (PMA) was added to cultures, hCG secretion was increased dose-dependently, but cell proliferation and 3H-thymidine uptake were not affected. When only cholera toxin was added, hCG secretion was also stimulated dose-dependently, but when both PMA and cholera toxin were added there was a synergistic potentiation of hCG production. In contrast, Ca ionophore A23187 had almost no effect on hCG secretion. Two-dimensional gel electrophoresis and autoradiography showed that phosphorylation of a 33 kd acidic protein was produced by cholera toxin, while phosphorylation of a 45 kd acidic protein and dephosphorylation of a 14 kd acidic protein were caused by the phorbol ester. These proteins may be specific substrates of protein kinase A and protein kinase C, respectively, in BeWo cells.

Cell Division↗

Binding of human chorionic gonadotrophin to testicular biopsy tissue from infertile men and the effect of prior treatment with human chorionic gonadotrophin.

The binding of 125I-labelled human chorionic gonadotrophin (HCG) to testicular tissue obtained by biopsy from 27 infertile men has been investigated. Fresh tissue cut into pieces was used for these studies as homogenization and/or freezing severely reduced the ability of testicular tissue to bind HCG. In five infertile men who had a normal endocrine profile and normal testicular histology, 10.3 +/- 1.4 (S.D.) pg 125I-labelled HCG were bound/mg testis, which was similar to binding (7.9 +/- 3.6 pg/mg) to testicular tissue obtained from three apparently normal men who underwent orchidectomy for prostatic carcinoma. However, in five infertile men with germ cell aplasia binding was increased to 16.6 +/- 5.7 pg/mg tissue. Fourteen infertile men were injected 24 h before biopsy with HCG, and this treatment consistently, and in most cases significantly, reduced the testicular binding of 125I-labelled HCG compared with that found in untreated, infertile men. It was concluded that measurement of HCG-binding in testicular biopsy specimens is feasible and may prove useful in assessing similarities between Leydig cell function in man and animals.

Adult↗

Precocious ovulation induced by human chorionic gonadotrophin in the immature female rat: comparison of follicle growth induced by treatment with human chorionic gonadotrophin and by electrical stimulation of the hypothalamus.

Precocious first ovulation, preceded by an endogenous preovulatory LH surge, could be predictably induced in immature female rats by administering repeated injections of human chorionic gonadotrophin (hCG). Administration of a dose of 0.05-0.075 i.u. hCG, four times a day from day 28 to day 31 of age resulted in a highly constant ovulatory response: at 4.0 +/- 0.0 days after the start of treatment 7.7 +/- 0.3 (n = 15) ova were found. Use of a higher dose of hCG (0.1 i.u.) resulted in lower numbers of ova (5.6 +/- 0.4, n = 7; P less than 0.005) whereas use of a lower dose of hCG (0.025-0.038 i.u.) resulted in a less constant timing of the induced ovulation at 5.4 +/- 0.2 days after the start of treatment (n = 7; P less than 0.0005). In animals treated with the dose of 0.05-0.075 i.u. hCG, a positive correlation was found between body weight at the start of treatment and the number of ova released (r = 0.75, n = 25; P less than 0.001). Ovarian follicle dynamics were studied on the various days of hCG treatment (dose 0.05-0.075 i.u.) and compared with the follicle changes that take place after electrical stimulation of the hypothalamus, performed on day 28, a treatment known to result in first ovulation 4-5 days later. In both groups a decrease in the number of the smallest and the middle-sized antral follicles as compared with their respective controls was seen, whereas numbers of follicles in the largest, 'ovulatable' size classes gradually increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Reduction of testicular human chorionic gonadotropin receptors by human chorionic gonadotropin in infertile men.

We measured testicular human chorionic gonadotropin (hCG) receptors in 30 infertile men either before or 1, 3, 5, 7, or 14 days after a single administration of 5000 IU of hCG. For 5 days the administration of hCG significantly reduced the testicular binding of 125I-hCG compared with that of the testes before administration. From the 7th day the binding capacity began to increase and returned to the preadministration level 14 days after the treatment. Occupied hCG binding sites accounted for about half of the reduction in binding sites on the day after administration of hCG. After this time, however, the occupancy did not contribute so much to the reduction in binding sites. These findings suggest that the reduction in testicular hCG binding sites after a single administration of 5000 IU of hCG is due to not only occupancy but also down-regulation of the binding sites.

Binding Sites↗

Ectopic human chorionic gonadotropin beta secretion by epithelial tumors and human chorionic gonadotropin beta-induced apoptosis in Kaposi's sarcoma: is there a connection?

Since it was first claimed in 1995 that human chorionic gonadotropin (hCG) inhibits HIV-associated Kaposi's sarcoma (KS), focus has been lost on establishing a credible hypothesis to explain a novel action ascribed to hCG, which is, after all, a pregnancy hormone. After 7 years, no real progress has been made, and there remains no consensus as to which component of hCG preparations is responsible for inducing apoptosis of HIV-associated KS. It would certainly seem apparent that many basic experiments on hCG species have been overlooked in the studies that have been published. Furthermore, the much wider literature concerning the association of hCG with promoting oncogenesis has been ignored. This review puts into context the numerous studies on hCG, HIV-associated KS, and oncogenesis.

Animals↗

Decreasing risk of pregnancy loss following chorionic villus sampling. Elimination of transabdominal chorionic villus sampling during the ninth week of pregnancy.

Chorionic villus sampling (CVS) is a method of obtaining fetal cells in the first trimester of pregnancy for genetic analysis. The transcervical (TC) approach was the first technique to be widely used. In the National Institute of Child Health and Human Development collaborative study the absolute loss rate following CVS (the total number of spontaneous abortions and neonatal deaths following CVS) was 4%. More recently the transabdominal (TA) approach has been introduced. This study compares the loss rates for the two approaches at various gestational ages for three 6-month periods following the addition of the TA approach with each other and with the loss rates prior to the introduction of TA CVS. We found that the percentage of pregnancy losses following TA CVS during the ninth week of gestation (63-69 days) was consistently higher than for TC CVS performed at the same gestational age. The loss rate for TC CVS has steadily decreased since the introduction of TA CVS after remaining the same for the two years prior to the introduction of the TA approach. After minimizing the number of TA CVS performed during the ninth week of gestation, the overall loss rate during the most recent 6-month period has been reduced to 0.94%. We conclude that the lowest loss rate following CVS can be obtained if both the TA and TC methods are available, and that the number of TA procedures performed during the ninth week of gestation is minimized.

Abortion, Spontaneous↗

Human chorionic gonadotropin. V. Tissue specificity of binding and partial characterization of soluble human chorionic gonadotropin-receptor complexes.

An in vivo human chorionic gonadotropin (hCG)-receptor complex was solubilized from the subcellular fraction of ovarian and testicular tissues of rats that had been injected with 125-I-labeled hCG. The soluble hCG-receptor complex was partially characterized by Sepharose 6B chromatography in the presence of the nonionic detergent, Emulphogene, and was shown to have a molecular size of about 65 A. By this method it was also shown that the in vivo uptake of radioactivity by rat gonadal tissues represents 125-I-hCG and not the dissociated subunits or degradation products of the hormone. A soluble hCG-receptor complex isolated in vitro in approximately the same yield from both rat testicular and ovarian homogenates was shown to be the same size. The hCG-receptor appears to be specifically located in gonadal tissue; a corresponding hCG-receptor complex was not obtained from liver or kidney that incorporated significant levels of 125-I-hCG administered in vivo. Furthermore, a desialyzed hCG-receptor complex was obtained from rat testis but not liver; desialyzed hCG, like other desialyzed glycoproteins, is nonspecifically bound by rat liver homogenates. The binding of hCG and luteinizing hormone (LH) by rat testis receptor exhibits a high degree of specificity. Other glycoprotein hormones without LH activity, such as follicle-stimulating hormone and thyroid-stimulating hormone, and glycoproteins such as fetuin or alpha1-acid glycoprotein do not bind to the hCG/LH receptors. Desialyzed hCG was 2 times more effective in competing for binding to rat testis receptors than "native" hCG, indicating that caution must be exercised when the radioligand receptor assay is utilized to assay hCG preparations varying in sialic acid content.

Animals↗