The role of calcium in luteinizing hormone/human chorionic gonadotrophin stimulation of Leydig cell immunoactive inhibin secretion in vitro.

The mechanism by which luteinizing hormone (LH) stimulates Leydig cell immunoactive inhibin (I-inhibin) secretion was investigated using Percoll-purified adult rat Leydig cells. Using a maximally stimulating dose of LH (16 ng/ml). Leydig cell I-inhibin secretion was non-detectable at 1-2 h of incubation, but subsequently increased at all time points during a 25 h incubation period. LH stimulated both Leydig cell content and release of I-inhibin. Increasing concentrations of LH stimulated both inhibin and testosterone immunoactivity in the incubation media over a similar dose-response range, with a 2- to 4-fold rise in I-inhibin secretion at maximal doses of LH. Dibutyryl cAMP stimulated testosterone secretion in a manner similar to that of LH, but I-inhibin secretion was less sensitive than testosterone and a significant stimulation was observed only at the highest doses (200-1000 micrograms/ml). LH-stimulated I-inhibin secretion was significantly decreased when Leydig cells were incubated in calcium-depleted (0.15 mM Ca2+ + 1 mM EGTA) or low [Ca2+] media (0.15 mM) as compared to normal (1.15 mM) or high [Ca2+] (2-5 mM) media. In contrast, LH-stimulated testosterone secretion remained unchanged by altering extracellular [Ca2+], and although decreased in the presence of EGTA, testosterone secretion remained significantly greater than basal levels. Furthermore both diltiazem and verapamil completely blocked the LH and dibutyryl cAMP-stimulated increase in Leydig cell I-inhibin, but did not reduce either LH or dibutyryl cAMP-stimulated testosterone production to basal levels. We conclude that LH stimulates both I-inhibin synthesis and release by adult rat Leydig cells in culture, by mechanisms involving calcium.     

Proliferation and differentiation of possible Leydig cell precursors after destruction of the existing Leydig cells with ethane dimethyl sulphonate: the role of LH/human chorionic gonadotrophin

The influence of LH levels on the proliferation and differentiation of possible Leydig cell precursors was investigated in adult rats, after the destruction of the existing Leydig cells with the cytotoxic drug ethane dimethyl sulphonate (EDS). In rats bearing a testosterone implant which prevented the rise in plasma LH levels and kept them within the normal range after the destruction of the Leydig cells, the proliferative activity of possible Leydig cell precursors still increased seven- to eightfold 2 days after EDS administration. Apparently, in this situation, locally produced factors, and not LH, may play a role in the stimulation of proliferation. The proliferative activity of the possible precursor cells could be further stimulated by treating rats with daily injections of human chorionic gonadotrophin (hCG) following EDS administration. It was concluded that the proliferative activity of possible Leydig cell precursors is probably regulated by both paracrine and endocrine factors. Almost no Leydig cells were formed in the rats bearing a testosterone implant during the first 4 weeks after EDS administration. When these rats were treated with hCG, starting 28 days after administration of EDS, a substantial number of Leydig cells was found after 2 days, and these cells also showed 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and alpha-naphtyl esterase (alpha-NE) activity. When hCG treatment was started at 14 or 21 days after EDS administration, some cells with the nuclear characteristics of Leydig cells were present after 2 days, but no 3 beta-HSD or alpha-NE activity could be detected.(ABSTRACT TRUNCATED AT 250 WORDS)     

The response of inhibin to human chorionic gonadotrophin is decreased in senescent men compared with young men

Inhibin and testosterone were measured in the serum of young and old men with proven fertility before and after stimulation with human chorionic gonadotrophin (hCG) in order to characterize endocrinological changes in senescence further. While there was a significant increase of both hormones in all young men, there was a decreased response of serum testosterone and an insignificant increase in inhibin in the older men. Although basal hormone levels and ejaculate parameters were not different, hCG stimulation revealed that there were decreased secretory capacities of Leydig as well as of Sertoli cells in old age.     

Effects of hypophysectomy and human chorionic gonadotrophin on Leydig cell function in mature rats

The biochemical activities involved in the maintenance of Leydig cell functions, and the effects of hypophysectomy and human chorionic gonadotrophin (hCG) on these functions are largely unknown. In the present study, adult hypophysectomized rats were used as a model to determine the effects of these treatments on a number of biochemical and morphological parameters. After 33 days of hypophysectomy, the morphology of the Leydig cells had been drastically altered. In addition, alpha-naphthol and beta-naphthol esterase activity as well as the steroidogenic capacity of the Leydig cells were greatly reduced at this time. In contrast, the level of sterol carrier protein 2 (SCP2), a Leydig cell-specific protein, was affected by hypophysectomy much less than the other parameters measured. Two daily injections of hCG to rats hypophysectomized for 31 days resulted in no change in the morphology of the Leydig cells, or in their proliferative activity. Non-specific esterase activities were also unaffected by 2 days of treatment with hCG. However, two injections of hCG to rats hypophysectomized for 31 days resulted in nearly complete restoration of steroidogenic capacity, and a 3.5-fold increase in the level of SCP2. These findings indicate that hypophysectomy results in significant morphological and biochemical changes in Leydig cells, and that hCG is capable of restoring some of these capacities within a short time.     

Bovine inhibin immediately inhibits the electrophysiological response to chorionic gonadotrophin in ovarian follicles of Xenopus laevis

Inhibin was originally isolated from ovarian tissue using, as a bioassay, its ability to diminish synthesis and secretion of gonadotrophins in pituitary cells after several days. Inhibin is known also to modify responses to gonadotrophins in the ovary after stimulation times of hours or days. Here we show that action for less than 4 min of purified bovine inhibin on ovarian follicles from Xenopus laevis causes a specific, dose-responsive (greater than 10 U/ml) inhibition of the membrane hyperpolarization produced by stimulation with chorionic gonadotrophin. The CG-induced change in follicle membrane potential has previously been shown to be caused by potassium efflux produced by rises in intracellular cAMP. Inhibin did not affect similar hyperpolarization stimulated by 10 microM adenosine. The apparent Ca(2+)-dependent Cl- currents which are often associated with CG action, were not inhibited by inhibin. At concentrations up to 2000 U/ml, inhibin did not significantly alter the timing of CG-induced germinal vesicle breakdown in the oocytes, which may suggest functional action on other developmental processes.     

Destruction of testicular Leydig cells reveals a role of endogenous inhibin in regulating follicle-stimulating hormone secretion in the adult male rat

It has previously been demonstrated that passive immunoneutralization of endogenous inhibin results in a dramatic elevation in follicle-stimulating hormone (FSH) secretion in the adult female rat but not in the adult male. The purpose of the present study was to investigate whether the effects of immunoneutralizing endogenous inhibin on FSH secretion in the adult male rat might be masked by the presence of additional, compensating, FSH-suppressing factors. This was determined by examining the individual and combined effects of removing the testicular influences provided by the Leydig cells using the selective toxicant, ethane dimethane sulfonate (EDS), and passive immunoneutralization of endogenous inhibin. Within 24 h of a single i.p. injection of EDS, plasma testosterone levels were lowered to near assay limits and by 3 days were undetectable. Plasma FSH levels were significantly elevated 3 and 7 days after EDS treatment, but not to the levels observed in rats castrated for similar periods of time. Castration of rats, treated 3 days earlier with EDS, resulted in a further significant increase in FSH secretion as compared with EDS-treated, sham-operated controls, indicating that the testes were providing an additional FSH-suppressing factor(s) other than those originating in the Leydig cells. Injection of anti-inhibin serum, into rats treated 3 or 7 days earlier with EDS, induced a further significant increase in FSH secretion that raised plasma FSH to a level comparable to that observed in male rats castrated for similar periods of time. Plasma LH secretion was also dramatically elevated by EDS treatment to levels that equaled or exceeded those observed in similarly timed castrates. Pituitary sensitivity, as tested by the injection of an exogenous challenge of luteinizing hormone-releasing hormone (LHRH), was significantly increased 3 or 7 days after either EDS treatment or castration in terms of LHRH-stimulated LH release, but not in terms of LHRH-stimulated FSH release. Immunoneutralization of endogenous inhibin induced no further observable changes in pituitary sensitivity to LHRH. These results demonstrate that in the absence of the Leydig cells a secondary role is revealed for endogenous inhibin in suppressing FSH secretion that, in combination with the Leydig cell influence(s), accounts for the postcastration increase in FSH. The need to remove the Leydig cell influence(s) to reveal an effect of endogenous inhibin on FSH secretion in the adult male rat may suggest that the inhibin effect is normally masked by the presence of the comparatively larger suppressive influence(s) derived from the Leydig cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Basal inhibin B and the testosterone response to human chorionic gonadotropin correlate in prepubertal boys

During childhood, the quiescent phase of testicular activity, the hCG stimulation test is widely used to evaluate testicular function. Inhibin B, a gonadal peptide regulating FSH secretion, is an established marker of Sertoli cell function and spermatogenesis in adults. In contrast to the other hormones of the hypothalamo-pituitary-gonadal axis, inhibin B is also secreted in detectable amounts during childhood. The aim of this study was to determine whether basal inhibin B levels are able to predict prepubertal testicular function, so as to avoid a stimulation test. Inhibin B and testosterone before and after hCG stimulation were measured in 54 male children with various testicular disorders by an immunoassay specific for inhibin B. Basal inhibin B was compared to the testosterone increase after hCG. Inhibin B and the hCG-induced testosterone increment correlated strongly (r = 0.84; P<0.0001). Patients with anorchia were clearly distinguishable from those with abdominal testes, having undetectable (inhibin B, <15 pg/mL) respective normal inhibin B levels for age. Inhibin B and the testosterone response to hCG were low in boys with testicular damage (delayed diagnosis of cryptorchidism; after testicular torsion) and in patients with gonadal dysgenesis, but were normal or increased in children with androgen insensitivity syndrome. We conclude that basal inhibin B predicts the testosterone response to hCG in boys and therefore gives reliable information about both the presence and function of the testes. The diagnostic procedure in cryptorchidism may be reduced to a single inhibin B measurement. Furthermore, inhibin B levels show specific alterations in patients with sexual ambiguity, adding a valuable diagnostic tool to the complex differential diagnosis of male pseudohermaphroditism.     

Immunoreactive beta-core-like material in normal postmenopausal urine: human chorionic gonadotrophin or LH origin? Evidence for the existence of LH core.

Material with the immunochemical properties of the beta-core of human chorionic gonadotrophin (hCG) can be found in the urine of normal postmenopausal women. However, we have been unable to detect intact hCG (using an assay which is specific for the alpha-beta heterodimer of intact hCG) in serum of such subjects. The levels of serum LH and urinary beta-core were compared in matched samples from 28 women (serum LH: median 27 U/l, range 4-70 U/l, urinary beta-core: median 0.27 microgram/l, range less than 0.05-0.645 microgram/l). Urine (4 litres) from three postmenopausal women was concentrated, dialysed and subjected to gel exclusion chromatography on Sephadex G-100. Fractions were analysed by specific assays for LH, intact hCG, total beta-hCG (free beta-subunit and intact hCG), free alpha-subunit and beta-core. Material eluting at the expected position of the beta-core fragment of hCG was detected in all three samples by the beta-core, beta-hCG and LH assays, despite the fact that the LH antibody does not recognize the authentic beta-core of pregnancy. Electrophoresis and Western blotting of the concentrated urines revealed that material of the same molecular size as beta-core was recognized by the antibody to LH but not by a monoclonal antibody raised to free beta-hCG which also recognizes the beta-core molecule of hCG. We conclude that the predominant core-like material identified in postmenopausal urine is probably derived from the beta-subunit of LH.     

Characterization of a factor(s) from partially purified human gonadotrophin preparations which inhibit(s) the binding of radiolabelled human LH and human chorionic gonadotrophin to Candida albicans

We have shown previously that partially purified human chorionic gonadotrophin (hCG) preparations inhibited the specific binding of 125I-labelled hLH or hCG to Candida albicans membranes at much lower concentrations than did highly purified hLH or hCG preparations. We now describe the characterization and partial purification of a heat-labile glycoprotein from commercially available gonadotrophin preparations. The factor strongly inhibited LH binding to Candida membranes, but not to sheep or pig luteal LH receptors. This material had a molecular weight of 16,000-21,000 daltons, bound strongly to CM-Sepharose at physiological pH, and could be resolved completely from hCG and from epidermal growth factor-like factors present in commercial gonadotrophin preparations. Its activity was not attenuated by a range of inhibitors specific for the four major classes of proteolytic enzymes, nor did it inhibit hormone binding by causing degradation of 125I-labelled hLH or hCG tracers. Factors which inhibited hLH binding to Candida membranes were also present in partially purified human urinary and equine serum gonadotrophin preparations and in placental extracts, but were not detected in highly purified CG of hLH preparations. The properties of this factor were similar to those described for beta-core protein, a cleavage product of the beta subunit of hCG which is a contaminant of commercial gonadotrophin preparations. Highly purified beta-core protein inhibited 125I-labelled hLH binding to Candida membranes, but not to sheep luteal binding sites. Preparations of hCG depleted of inhibitor activity could stimulate adenylate cyclase activity in Candida membranes almost five fold. In contrast, partially purified inhibitor preparations strongly inhibited basal adenylate cyclase activity (to 18% of control levels). These observations suggest that endogenous LH-like factors, perhaps similar to beta-core proteins of hCG, may play a role in the regulation of morphogenesis in Candida species.