Daily immunoactive and bioactive human chorionic gonadotropin profiles in periimplantation urine samples

A need exists for broadly applicable biomarkers of pregnancy outcome in population-based studies that assess environmental hazards to human reproduction. Previous studies have demonstrated that during the periimplantation period, measures of the circulating levels of immunoreactive hCG (IhCG) are not predictive of pregnancy outcome, whereas measurements of the circulating levels of bioactive hCG (BhCG) provide information relating to pregnancy outcome and might provide the basis for an early biomarker of pregnancy outcome. However, for this biomarker to have broad application in population-based studies, it must be adapted to urinary hCG metabolites. The principle objective of the present study was to characterize the periimplantation excretion patterns of urinary hCG metabolites of pregnancies that resulted in live birth (LB), early pregnancy loss (EPL), and recognized clinical abortion (CAB) with an immunoenzymometric assay specific to intact hCG and an LH/chorionic gonadotropin cellular bioassay as the basis for a preliminary comparison between successful (LB) and failing (EPL and CAB) outcome groups. Automated immunoassays for FSH and hCG were used to define each conceptive cycle's implantation window. The timing of first hCG detection was significantly later for the EPL group. Pregnancies that resulted in LB had consistently rising average daily IhCG and BhCG levels, with no significant differences when average daily IhCG and BhCG measurements were compared (Student t-test, P>0.05), whereas pregnancies that resulted in CAB and EFL had lower average daily IhCG and BhCG levels that increased inconsistently. These findings demonstrate that critical information related to pregnancy outcome may be present when multiple urinary hCG isoforms are measured. Further data suggest that the rate of change for the ratio of daily BhCG over IhCG levels might be useful as the basis of a broadly applicable early biomarker for pregnancy outcome.     

The synthesis and secretion of human chorionic gonadotropin by tissue slices from first trimester placentas

Placental tissue slices from first trimester placentas synthesize and secrete proteins labeled by radioactive glucosamine are preferentially secreted as compared to proteins in general. One of the proteins synthesized and secreted is hCG. Processing and secretion of proteins, including hCG, by the tissue slices need a two-hour period. Both secretion and glycosylation of the protein can take place independently of protein synthesis. A method was developed for the specific determination of newly synthesized radioactive hCG in placental tissue.Department of Reproductive Biology, Case Western Reserve University, Cleveland, Ohio, USA     

Protein-secretory patterns of normal and abnormal human placentas with special reference to human chorionic gonadotropin

[35S]Methionine-labeled protein-secretory patterns resolved by two-dimensional polyacrylamide gel electrophoresis in abnormal hydatidiform-mole placentas were compared with those in normal full-term placentas with special reference to human chorionic gonadotropin (hCG) by means of immunoblotting and immunoelectron-microscopic techniques. Although basic protein-secretory patterns of both placentas were similar to each other, four polypeptide spots appeared and one spot disappeared in the hydatidiform-mole samples. Among four newly synthesized and secreted spots, three were immunoreacted with anti-hCG serum by an immunoblotting experiment. Ultrastructural localization of hCG showed that the labeling intensity of anti-hCG serum in hydatidiform-mole placentas was much heavier than that in full-terms ones. Particularly, the Golgi apparatus, middle-sized granules and large bodies were highly immunoreactive. The present study reveals that hydatidiform-mole placentas have different protein-secretory functions especially in hCG synthesis and secretion from those of normal pregnancy.     

Biological actions of monoclonal luteinizing hormone/human chorionic gonadotropin receptor antibodies.

Several recent studies have elucidated the structure of the mammalian LH/hCG receptor; as reported in the present work, we have developed a series of monoclonal antibodies (mAbs) against the rat ovarian LH/hCG receptor using highly purified receptor as immunogen and by screening hybridomas with purified LH/hCG receptors. The mAbs were able to specifically immunoprecipitate LH/hCG receptors from solubilized preparations of rat ovarian membranes as well as from partially purified preparations. Western blotting with mAb P1B4 detected a probable receptor dimer and a receptor fragment in rat and porcine ovarian tissue but not in other tissues. This mAb also partially inhibited hCG binding to rat and porcine ovarian tissues. The receptor mAbs were able to inhibit hCG-induced progesterone synthesis in cultured human and porcine granulosa cells without affecting cAMP- and FSH-induced progesterone synthesis. The mAb P1B4 was used to demonstrate that the majority of ovarian receptors are internalized after hCG treatment and that in pseudopregnant rats receptors are present in the rough endoplasmic reticulum and in microvesicles. Bovine corpus luteal cells also contained P1B4 binding sites, as detected by immunohistochemical technique. Taken together, these results suggest that the mAbs are specific for the LH/hCG receptor, mAb P1B4 recognizes an epitope that is highly conserved among mammals, and this epitope is probably in the extracellular domain.     

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.     

Biological and immunological properties of zebra pituitary gonadotropins: comparison with horse and donkey gonadotropins

Previous studies from this laboratory have described the properties of purified luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from horse and donkey anterior pituitary glands. The present study afforded the opportunity to further characterize these previously purified hormone preparations and to compare them with enriched gonadotropin fractions from zebra pituitary glands. Although a single LH and FSH fraction was usually obtained for each pool of pituitaries, two separate zebra LH and two donkey FSH preparations were generated. Purified hormone preparations from the horse were designated eLH and eFSH. Preparations zLH-A, zLH-B, and zFSH were obtained from zebra pituitaries, and fractions dLH, dFSH-A, and dFSH-B were prepared from donkey pituitary glands. These preparations were analyzed by LH and FSH radioimmunoassays (RIAs), radioreceptor assays (RRAs), LH bioassay, and chromatofocusing. Clear immunological differences were observed between equid gonadotropins. Homologous RIAs for eLH and eFSH did not cross-react similarly, or in a parallel fashion, with gonadotropins from the donkey and zebra. In contrast, RIAs capable of assessing LH or FSH in a wide number of species showed all equid gonadotropin preparations to have considerable activity and to produce parallel dilution curves. Relative to eLH (1.00), zLH-A was found to have higher LH bioactivity:LH RIA (2.50), LH RRA:LH RIA (1.42), and LH bioactivity: LH RRA (2.21) activity ratios. The dLH and zLH-B fractions only differed from eLH in LH RRA:LH RIA activity (0.69 and 0.62, respectively). Only LH from the horse possessed clear intrinsic FSH-receptor-binding activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Evidence that constitutively active luteinizing hormone/human chorionic gonadotropin receptors are rapidly internalized.

The luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor, which belongs to the family of G-protein coupled receptors, plays an important role in gonadal steroidogenesis. Substitution of aspartic acid 556 of the LH/hCG receptor with glycine (D556G) creates a constitutively active receptor that activates adenylyl cyclase in the absence of hormone. To examine receptor internalization, human embryonic kidney cells (293 T) expressing wild type (WT) or D556G mutant receptors were incubated with [125I]hCG and subsequently analyzed for cell surface bound and internalized radioactivity. Comparison of the rate constants of internalization of the D556G mutant and WT receptors revealed that the rate of internalization of the D556G mutant was five times greater than that of the WT receptor. Although the D556G receptor internalizes [125I]hCG rapidly, a corresponding increase in [125I]hCG degradation was not seen. The internalization of another constitutively active LH/hCG receptor (aspartic acid 556 to tyrosine) was also greater than that of the WT receptor. Internalization of receptor bound [125I]hCG was inhibited by a hypertonic sucrose solution, confirming that the ligand enters the cell by receptor-mediated endocytosis. Furthermore, the constitutively active D556G and D556Y LH/hCG receptors utilize the arrestin dependent internalization pathway. These results suggest that the active state conformation of the constitutively active receptor is conducive to rapid internalization.     

Autoradiographic analysis of follicle-stimulating hormone and human chorionic gonadotropin receptors in the ovary of immature rats treated with equine chorionic gonadotropin.

The gonadotropin-primed immature rat has become the most common model for the study of follicular development and ovulation. In this study, prepubertal female rats, 23 and 24 days old, were injected s. c. with 5 IU eCG, and ovaries were collected for topical autoradiography of FSH and hCG receptors at 48 or 24 h post-eCG, respectively (i.e., Day 25). In a baseline group, on Day 25 (before eCG), even the smallest preantral follicles with 1 layer of granulosa cells (GCs; primary follicles) possessed FSH receptors, but hCG receptors were found only on the theca of follicles with 2 or more layers of GCs. Human CG receptors were especially prominent in the interstitium that intimately surrounds preantral follicles without any distinction between theca and interstitial cells. There was a discrete theca surrounding antral follicles. Occasionally antral follicles had hCG receptors in the interstitium, but the adjacent theca was negative, suggesting that these follicles might be destined for atresia. By 24 h post-eCG, a now-discrete theca layer with hCG receptors surrounded all preantral follicles except for the primary follicles, which never responded to eCG. The interstitium was hypertrophied and epithelioid, as was the theca surrounding nonatretic preantral and antral follicles. Increased mitotic activity characterized the growing preantral follicle, and for the first time, FSH binding in GCs of antral follicles was greater than in the preantral population. By 48 h post-eCG, the primary follicles were still unresponsive to eCG. FSH receptors were even more pronounced in the GCs of large antral follicles, although hCG receptors were present in the GCs of only one third of the antral follicles, reflecting the small dose of eCG administered. By 48 h post-eCG, receptors in the interstitium were barely detectable. Using this model, the following study considers the functional in vitro changes in steroidogenesis in follicles from the smallest preantral follicles to the largest antral follicles.     

Testicular steroidogenesis after human chorionic gonadotropin desensitization in rats.

When a single injection of 500 I.U. of human chorionic gonadotropin (hCG) is given to rats there is an initial acute rise of plasma testosterone and of testicular content for both cyclic AMP and testosterone. This response correlates with an increase in both lyase and 17 alpha-hydroxylase activities. Thereafter both plasma and testicular testosterone decline and do not increase after a second injection of hCG. During this period of desensitization, isolated Leydig cells were insensitive to the steroidogenic stimulatory effect of both hCG and dibutyryl cyclic AMP. The post-cyclic AMP block is not due to an alteration of the cyclic AMP-dependent protein kinase but it is correlated with a decrease in both lyase and 17 alpha-hydroxylase activities of the Leydig cell's microsomes. This decrease is not caused by the absence of the recently described cytosol activator of this enzyme because its addition did not restore the enzymatic activity. Within 60 to 96 h after hCG injection there was a spontaneous increase of both plasma and testicular testosterone and this parallels the recovery of lyase and 17 alpha-hydroxylase activities. These results suggest that both enzymatic activities are regulated, directly or indirectly, by hCG, and that this is partly responsible for the hCG-induced steroidogenic refractoriness of Leydig cells.     

Human chorionic gonadotropin, its receptor and mechanism of action.

Human chorionic gonadotropin (HCG) is a glycoprotein hormone consisting of two noncovalently bonded subunits, alpha and beta. The hormone can be dissociated and reassociated. Whereas the individual subunits do not show any receptor binding activity, the reconstituted molecule is almost fully active. The amino acid and carbohydrate sequences in hCG-alpha and hCG-beta are described. There are in all seven carbohydrate units, four complex asparagine-linked and three serine-linked short oligosaccharide chains. The sequential removal of monosaccharides from the carbohydrate moiety of the hormone results in derivatives that bind to the cell surface receptors but inhibit the hCG-induced accumulation of cAMP. The derivatives, however, still are able to produce steroidogenesis maximally. The data raise the possibility of other mediator(s) of the hormone action in addition to cAMP. The hCG/LH (luteinizing hormone) receptor has been labeled by the incorporation of N-acetyl-D-1-[14C] glucosamine and also by the selective incorporation of 125I or 131I. Using 131I-labeled bovine corpus luteal plasma membranes, a method for the purification of the receptor to homogeneity has been developed. The purified receptor has properties similar to the membrane-bound receptor. Availability of the purified receptor offers newer approaches to the study of molecular mechanisms of polypeptide hormone action.     

Biochemical evidence that human placental lactogen and human chorionic gonadotropin are not stored in cytoplasmic secretion granules

The intracellular storage sites for the human placental hormones placental lactogen (hPL) and chorionic gonadotropin (hCG) are unknown. To determine whether hPL and hCG are stored in cytoplasmic secretion granules, we have compared the localization of hPL and hCG in placental homogenates following differential and density-gradient centrifugations to those of prolactin (PRL) and luteinizing hormone (LH) in human and rat pituitary homogenates. In the differential centrifugation studies, 93.1 +/- 4.1% (mean +/- SE) of the hPL and 79.4 +/- 6.0% of the hCG were detected in the postmicrosomal supernatant of placental homogenates. In contrast, 95-98% of the hPRL and hLH in the pituitary homogenates were detected in particulate fractions. Following centrifugation on sucrose-density gradients, particulate hPL and hCG were distributed diffusely throughout the gradients, while greater than 90% of the pituitary hormones sedimented as single peaks with densities of 1.22 g/cm3. When human placental and rat pituitary tissues were homogenized together prior to differential and density-gradient centrifugations, similar marked differences were observed between the distribution of the placental and pituitary hormones. These results strongly suggest that the placental hormones hPL and hCG, unlike pituitary PRL and LH, are not stored in large secretory granules. Differences in the intracellular storage sites of the hormones may explain, in part, differences in the regulation of peptide hormone secretion by placental and pituitary tissues.     

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.     

Urinary chorionic gonadotropin levels in pregnant golden lion tamarins. Preliminary observations.

We have established the interval over which urinary chorionic gonadotropin can be detected by a radioimmunoassay during pregnancy in the golden lion tamarin. Preliminary findings indicate the potential value of this radioimmunoassay system for (1) diagnosis of pregnancy at about four weeks after fertilization; (2) estimation of the expected time of delivery; and (3) identification of individual monkeys having an apparent high risk of spontaneous abortion.     

Deglycosylated human chorionic gonadotropin. An antagonist to desensitization and down-regulation of the gonadotropin receptor-adenylate cyclase system

Human chorionic gonadotropin (hCG) was deglycosylated with anhydrous HF and compared with native hCG for binding and biological activity. The deglycosylated hormone (DG-hCG) had the same affinity as hCG for gonadotropin receptors in murine Leydig tumor cells (MLTC-1) but was less than 1% as potent as hCG in stimulating cyclic AMP production in these cells. Exposure of MLTC-1 cells for 30 min to hCG caused a desensitization of hCG-stimulated adenylate cyclase activity, whereas DG-hCG did not induce desensitization even after 4 h. hCG induced down-regulation of hCG receptors; by 4 h, 40% of the receptors had disappeared, whereas there was no receptor loss in cells exposed to DG-hCG for the same time. By 6 h, receptor down-regulation began to occur in the DG-hCG-treated cells and could be mimicked by exposing the cells to dibutyryl cyclic AMP or cholera toxin. Thus, the small increase in cyclic AMP generated by DG-hCG appears to result in some loss of receptors. Cells were incubated with iodinated hCG or DG-hCG for 30 min, washed, and incubated in fresh medium. Both bound ligands were degraded as measured by disappearance of cell-associated radioactivity and appearance of trichloroacetic acid-soluble label in the medium. The half-lives were 3 and 6 h for hCG and DG-hCG, respectively. Our results indicate that DG-hCG in contrast to hCG does not cause either rapid desensitization of hCG-stimulated adenylated cyclase or rapid down-regulation of hCG receptors. Therefore, receptor occupancy alone is insufficient to induce these phenomena.