Human fetal nongonadal tissues contain human chorionic gonadotropin/luteinizing hormone receptors

Human chorionic gonadotropin (hCG), a heterodimeric glycoprotein hormone produced in abundance by placental syncytiotrophoblasts, is preferentially secreted into maternal circulation. Fetal circulation also contains low levels of hCG that are probably derived from fetal kidney, liver, anterior pituitary gland, etc. In addition, the fetus has access to hCG present in exocoelomic and amniotic fluids. hCG has been found in a number of fetal tissues known to stimulate fetal adrenal and testicular steroidogenesis and is also thought to play a role in growth and differentiation of fetal tissues. This led us to test the hypothesis that fetal nongonadal tissues, as in the adult, may also contain hCG/LH receptors. This hypothesis was tested by immunocytochemistry, Western blotting, in situ hybridization, and RT-PCR. The results demonstrate that kidney, liver, pancreas, lung, small and large intestines, and adrenals contained hCG/LH receptors. Although the role of fetal nongonadal hCG/LH receptors is not known, they may mediate the pleiotropic actions of hCG in the growing human fetus.     

Cellular localization of chorionic gonadotropin in human fetal kidney and liver

Earlier, we reported that second trimester human fetal kidney and, to a much lesser extent, human fetal liver were capable of synthesizing and secreting the beta-subunit of hCG. Recently, we also have shown that these tissues, likewise, synthesize and secrete the alpha-subunit of hCG. The hCG produced is biologically active. To determine the cellular localization of these peptides, immunocytochemical studies were performed on human fetal tissues using antibodies against beta hCG, alpha hCG, and the intact hormone. Placental syncytiotrophoblast served as an immunopositive control. In the human fetal kidney, the ascending (thick) limb of the loop of Henle, distal convoluted tubule, and occasional cells in the collecting ducts were distinctly immunopositive for both beta hCG and the alpha-subunit. Small amounts of light positive staining occurred in only a few hepatocytes. Placental syncytiotrophoblast was routinely positive for both subunits, but fetal lung and striated muscle were negative. These immunocytochemical results indicate that immunoreactive beta hCG as well as the alpha-subunit are present in placental syncytiotrophoblast, in the distal renal nephron, and in a limited population of hepatocytes. The qualitative number and intensity of immunopositive cells closely correlate with the quantitative amounts of their hCG subunit synthesis. Taken together with our previous biosynthetic data, the immunocytochemical localization reported here indicates the probable cellular sites of alpha- and beta hCG synthesis in these tissues. The presence of comparable alpha- and beta-subunit staining in identical cell populations suggests that both hCG subunits and, therefore, perhaps intact hCG are produced at these same cellular sites during fetal life.     

Radioreceptor assay for human chorionic gonadotropin.

A sensitive radioreceptor assay capable of detecting 100 pg of human chorionic gonadotropin has been developed using ovarian receptors and (125I)hCG. The dose-response curves of a series of urine and serum samples from pregnant women were linear and parallel to standard curves of 2nd International Standard hCG and purified hCG when logit of percent bound was plotted against log dose. Potency estimates of pregnancy sera, measured by radioreceptor assays, were approximately 2 times greater than those determined by radioimmunoassays. Examination of hCG concentrations in the urine of pregnant and nonpregnant women, using a receptor assay, showed good correlation with the result obtained with a standard agglutination pregnancy test. This readioreceptor assay, which can be made as sensitive as radioimmunoassay, provides a rapid and simple method for the measurement of biologically active hCG and LH.     

Presence of luteinizing hormone/human chorionic gonadotropin receptors in male breast tissues

Receptors for LH/human chorionic gonadotropin (hCG) have been found in a variety of nongonadal tissues including the female breast. Using in situ hybridization and immunohistochemistry, we demonstrated the presence of LH/hCG receptor mRNA and protein in normal male breast tissue obtained at autopsy (n = 4) and archival samples of benign gynecomastia (n = 14) and male breast carcinoma (n = 5). Although the function of these receptors remains to be determined, the findings suggest the possibility that LH and hCG may play a role in the pathogenesis of male breast disorders.     

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.     

Effect of human chorionic gonadotropin on human thyroid tissue in vitro.

Thyroid stimulating substances other than TSH have been found in certain disease states associated with hyperthyroidism. The thyroid stimulator associated with the thyrotoxicosis of trophoblastic disease is uncertain; however, recent evidence suggests a role for hCG. To explore the thyroid stimulating properties of hCG further, we examined the ability of hCG to displace [1252]TSH from receptors on human thyroid membrane and to generate cyclic-AMP (c-AMP) from human thyroid slices. Human chorionic gonadotropin at a concentration of 40 IU/ml displaced labeled TSH from human thyroid membranes and, at a concentration of 69 IU/ml, hCG caused the generation of c-AMP in thyroid slices. These results suggest that hCG can bind to the TSH receptor on thyroid cells and can stimulate them to produce c-AMP at concentrations of hCG within the range that is found in trophoblastic disease.     

The thyrotropin in hydatidiform moles is human chorionic gonadotropin.

Thyrotropic activity (TSH), measured by the McKenzie mouse bioassay, has been correlated with human chorionic gonadotropin (hCG) activity, measured by radioimmunoassay, in serum and tissue samples from 11 patients with hydatidiform mole and in partially and highly purified preparations of urinary hCG. Serum samples, taken at various times before and after removal of the moles, gave a ratio of 0.42 plus or minus 0.24 muU TSH/U hCG (mean plus or minus SD) (N)=43). In all cases where hCG activity fell below 150-175 U/ml (n=49), thyroid stimulating activity was undetectable (smaller than 40 muU/ml). We extracted lyophylized molar tissue by a modification of the Bates alcohol-saline method and purified the resultant extract by a combination of gel chromatography, affinity chromatography using Concanavalin A coupled to Sepharose, and isoelectrofocusing. Following extraction, an approximately 20-fold purification was achieved without significant alteration of the ratio of the two activities. Using results from all phases of purification the ratio of muU TSH/U hCG was 0.51 plus or minus 0.35(n = 23).Both activities were in the same position on disc gel electrophoresis. Activity ratios were less constant when partially purified preparations of urinary hCG were assayed for both thyrotropic and hCG activities. The presence of an hCG immunoreactive species, presumably hCG-beta subunit, which contains no thyrotropic activity but has an approximately 10-fold greater activity on a weight basis than intact hCG, may be a partial explanation for this observation. Isoelectrofocusing of a urinary hCG preparation showed that all hCG immunoreactive species with pl's between 3. 5 and 5.0 contained thyrotropic activity in proportion to their hCG content. Seven highly purified hCG preparations had thyrotropic activity with a ratio of 0.48 plus or minus 0.18 muU TSH/U hCG. These results indicate that hCG has intrinsic thyrotropic activity. On a molecular basis it is calculated that hCG contains approximately 1/4000 the thyrotropic activity of human pituitary TSH. In conditions of grossly elevated serum hCG levels, such as hydatidiform mole, this thyrotropic activity can be sufficient to produce hyperthyroidism.     

Human chorionic gonadotropin binding to human fetal testes as a function of gestational age

The characteristics of binding of hCG to testicular tissue obtained from human abortuses of 10--24 weeks gestational age were studied. Specific, saturable binding of [125I]hCG was demonstrated using homogenates of human fetal testicular tissue. The equilibrium dissociation constant ranged from 0.4 x 10(-10) M to 5.5 x 10(-10) M, a finding that is indicative of a high affinity receptor. The capacity to bind hCG was low, but varied strikingly with gestational age. The binding capacity for hCG of tissues from abortuses of gestational age less than 15 weeks and greater than 22 weeks was consistently less than 10.0 pg x mg-1 tissue (2.2 fmol x mg-1 tissue). The binding capacity for hCG of tissues from abortuses of gestational age between 15--20 weeks ranged from 2.4--29.8 pg x mg-1 tissue (0.5--6.5 fmol x mg-1 tissue) with the majority of values being greater than 10 pg x mg-1 tissue (2.2 fmol x mg-1 tissue). On the other hand, receptors for hCG in human fetal ovarian tissue were undetectable, irrespective of gestational age. It is concluded that specific high affinity binding sites for hCG are present in human fetal testes and that the binding capacity is maximum between gestational ages of 15--20 weeks. This increase in binding capacity parallels the surge in testosterone production known to occur during the same period of development. These results suggest that the increase in fetal plasma levels of testosterone during this time in gestation is the result of an increase in the sensitivity of the fetal testis to hCG caused by an increase in the number of hCG receptors, and that hCG most likely is responsible for stimulation of fetal testicular steroidogenesis in utero at this time of gestation.     

Gallbladder carcinoma producing human chorionic gonadotropin

A primary carcinoma of the gallbladder producing human chorionic gonadotropin (HCG) was encountered in an 83-yr-old Japanese woman, with elevation of HCG/beta-HCG in urine and serum. Remarkable elevation of serum estradiol was an associated finding, with increased HCG. At autopsy, we found that the primary carcinoma of the gallbladder extensively involved the liver. Histologically, the tumor revealed adenosquamous cell carcinoma in the primary site, and moderately to poorly differentiated adenocarcinoma in metastatic foci. Immunohistochemical staining for beta-HCG showed a positive reaction in adenocarcinoma components. This is an extremely rare case of an HCG-producing gallbladder carcinoma, which leads us to speculate that HCG-positive tumor cells may occur due to dedifferentiation.     

Highly sensitive radioimmunoassay for chorionic gonadotropin in human urine.

The value of RIAs that measure hCG levels in human urine has been limited principally because of cross-reactivity with human LH. Recently, antisera generated to antigenic determinants on the intact hCG beta subunit and its carboxyl-terminal peptide have been shown to exhibit substantially reduced human LH cross-reactivity. To take maximal advantage of these antisera and to minimize interference by nonspecific substances in urine, a procedure for extracting and concentrating hCG from 24-h urine samples was developed. The procedure involves preparation of a standard kaolin-acetone urine concentrate and adsorption of the hCG in the concentrate to Concanavalin A covalently linked to agarose for purification and subsequent RIA. In urine samples obtained from patients with gestational trophoblastic disease, there was a direct correlation between hCG levels measured by RIA and those estimated by mouse uterine weight bioassay. In individual subjects, hCG levels were determined in serum and urine obtained the same day. When hCG was clearly detectable in the serum at levels greater than 1 ng/ml, the quantity of hCG measured in the urine concentrate exceeded 500 ng/24 h. The concentrates prepared from the urine of normal persons contained an hCG-like glycoprotein substance with antigenic determinants similar to those of the carboxyl-terminal peptide of hCG beta. As the range of hCG immunoreactivity measured in the urine concentrates of normal subjects was 6-52 ng/24 h, specific and sensitive detection of urinary hCG could be accomplished in patients whose sera contained hCG undetectable by conventional RIA. Partial purification and concentration of urinary hCG by this procedure with subsequent RIA provides a sensitive and reliable method for detecting hCG in urine.     

Tumor necrosis factor-alpha inhibits human chorionic gonadotropin secretion.

The placenta is a major source of tumor necrosis factor-alpha (TNF alpha) and is rich in TNF alpha receptors. TNF alpha inhibited hCG secretion by normal chorionic villi at 6 weeks gestation, but chorionic villi contain the heterogenous cell population. To investigate the direct effects of TNF alpha on trophoblasts, the NUC1 choriocarcinoma cell line was used as an in vitro placental cell model. TNF alpha also inhibited hCG secretion by NUC1 cells at concentrations from 1-100 U/mL. TNF alpha at concentrations of 1, 10, and 100 U/mL significantly decreased hCG secretion for 24 h to 88%, 81%, and 71% of the control level, respectively. The expression of hCG beta mRNA was also decreased to 23% of the control level after 24 h of TNF alpha treatment (100 U/mL). Inhibition occurred after 12 h of TNF alpha treatment (100 U/mL). Two measurements, trypan blue dye exclusion and 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide reduction, revealed that these inhibitory effects were not due to the cytotoxic activity of TNF alpha on NUC1. In conclusion, TNF alpha reduces hCG secretion in vitro.