Gonadotropin induction of a regulatory mechanism of steroidogenesis in fetal Leydig cell cultures.

Studies were conducted to define further the development of the gonadotropin induced, E2 mediated steroidogenic lesion (17-alpha-hydroxylase/17,20-desmolase) in fetal Leydig cell cultures. Analysis of dispersed fetal testes purified by centrifugal elutriation demonstrated a group of cells with sedimentation velocity 12 less than to less than 16.8 mm/h.g containing a small population of adult like "transitional" Leydig cells and homogeneous "fetal" Leydig cell population collected at greater than 19.3 mm/h.g. After cells were cultured for 3 days with addition of 1 microgram oLH at 3 day intervals, the transitional cells showed testosterone accumulation comparable to the fetal cells. In contrast, transitional cells had 10-fold higher basal and hCG-stimulated aromatase activity than fetal cells, and a lack of testosterone response to acute (3 h) hCG stimulation. At day 6, transitional cells steroidogenic ability declined markedly. The fetal population maintained in culture with LH additions every 3 days, showed typical immature Leydig cell response, with enhancement of acute testosterone response to hCG at 3 day (1-fold) and at 6 day of culture (5-fold). Higher doses of LH (5 micrograms/day) or daily treatment of 1 microgram to fetal cultures, induced a lesion of 17 alpha-hydroxylase/17,20-desmolase with reduction of enzymatic activities (P less than 0.01) and impaired testosterone production (P less than 0.01) in response to acute hCG stimulation. Also aromatase was stimulated by hCG + 140% and 50% and E2 receptors were increased by 100 and 180% at 3 days and 6 days of cultures with daily or high dose LH addition, findings consistent with the observation of the E2-mediated lesion during LH action. In conclusion, the cultured fetal Leydig cell provides a useful model to elucidate molecular mechanisms involved in the development of gonadotropin-induced estradiol-mediated desensitization. Treatment of fetal Leydig cell cultures with multiple or frequent doses of LH elevate aromatase activity to necessary levels for the induction of desensitization. We have isolated small population of transitional Leydig cells with morphological characteristics of cells found in 15 day post-natal testis but functional capabilities of adult cells. We have also demonstrated the emergence of a functional adult-like population from the fetal Leydig cell.

Acquisition of hormone-mediated mechanisms regulating testicular steroidogenesis during development.

Our studies have demonstrated that in the fetal rat Leydig cell, estradiol causes an up-regulation of its receptor and an induction of the regulatory mechanism (late steroidogenic lesion) that is similar to that observed in the adult rat Leydig cell. The absence of this regulation in fetal life is due to a very low level of aromatization capacity, with lack of up-regulation and/or induction of estrogen receptor by estradiol. Higher doses or frequent administration of LH is able to elevate aromatase activity and consequent E2-receptor-mediated action for the induction of gonadotropin-mediated desensitization in fetal cells. Our studies have revealed a small population of adult-like Leydig cells in the fetal testis and the emergence of a functional adult-like population from the fetal Leydig cell induced by gonadotropin treatment. The in vitro fetal Leydig cell culture system has permitted the analysis of cellular actions of gonadotropin with particular reference to the role of tropic hormone and estrogen in the development of late steroidogenic lesions during Leydig cell maturation. Future research with this system will help to clarify further the modulatory mechanisms responsible for emergence of the adult cell population.

Fetal Leydig cell culture--an in vitro system for the study of trophic hormone and GnRH receptors and actions.

In fetal and neonatal rat Leydig cells cultured in the presence of LH, gonadotropin and GnRH receptors and acute testosterone responses to hCG, were maintained for up to 78 days. Addition of GnRH agonists markedly inhibited steroid production in LH-treated cultures, and abolished the acute testosterone response to hCG. GnRH receptors were not detectable in fetal testes but were present post-natally and increased markedly with age. In cultured fetal testis, GnRH receptors were detected on the third day, and were increased by exposure to GnRH agonists. In LH-treated cultures, GnRH sites were reduced by about 50% and did not increase during incubation with GnRH agonists. LH supported 17 alpha-hydroxylase/17-20 desmolase activities and pregnenolone formation were observed in short (1-4 days) and long-term cultures. Also, LH stimulation of 3 beta-hydroxysteroid dehydrogenase was observed by histochemical studies at 7 days of culture. GnRH agonists inhibited LH dependent steroid production in a dose-dependent fashion and abolished the acute testosterone response to human chorionic gonadotropin. The major component of the steroid inhibitory effect of GnRH agonist occurs beyond cAMP production. A distal lesion of the microsomal enzymes of the androgen pathway is largely responsible for the GnRH-induced decreases in LH-supported androgen production. The expression of functional GnRH receptors during culture and their suppression by LH suggest that pituitary gonadotropins exert a tonic inhibitory effect upon testicular GnRH receptors. The presence of functional GnRH receptors and inhibitory actions in cultured fetal and neonatal Leydig cells indicates that GnRH-related peptides can influence the actions of gonadotropins on the fetal Leydig cell population.

Receptors and inhibitory actions of gonadotropin-releasing hormone in the fetal Leydig cell.

The receptors and actions of gonadotropin-releasing hormone (GnRH) were analyzed in cultured testicular cells from 20.5-day fetal rats, in which treatment with luteinizing hormone (LH) maintained Leydig cell steroidogenesis and gonadotropic responses for up to 2 weeks. Testicular GnRH receptors were present on the 5th postnatal day, but were not demonstrable in fetal testes or 2-day cultures thereof. However, GnRH receptors were readily detectable in 4-day cultured fetal testes and were increased by exposure to GnRH agonists. In LH-treated cultures, GnRH sites were reduced by about 50% and did not increase during incubation with GnRH agonists. In such cultures, GnRH agonists inhibited LH-dependent steroid production and abolished the acute testosterone response to human chorionic gonadotropin. The half-maximal inhibitory concentration of [D-Ala6]des-Gly10-GnRH-N-ethylamide (3 X 10(-10)M) was commensurate with its binding affinity for testis receptors (Kd = 1.4 X 10(-10)M). In contrast, GnRH agonists had no inhibitory effects in 2-day cultures prior to the detection of GnRH receptors. The expression of functional GnRH receptors during culture in the absence of gonadotropin and their suppression in LH-treated cultures suggest that pituitary gonadotropins exert a tonic inhibitory effect upon testicular GnRH receptors. The demonstrated inhibitory actions of GnRH on steroidogenesis, with the expression of GnRH receptors in cultured fetal testes and 5-day postnatal testes, indicate that GnRH agonists could influence the actions of gonadotropins upon Leydig cell function in the neonatal testis.

Serum concentrations of 24,25(OH)2D in uremic children: a reflection of renal function.

The mean serum concentration of 24,25(OH)2D determined by competitive protein-binding radioassay was significantly lower in ten uremic children maintained on hemodialysis (0.82 +/- 0.43[SD] ng/ml) than in ten patients with impaired renal function not requiring hemodialysis (1.30 +/- 0.54 ng/ml, P less than 0.05), or in 12 normal children (2.98 +/- 1.57 ng/ml, P less than 0.01). The serum levels of 250HD were similar in all groups. There were significant (P less than 0.01) positive correlations between the serum concentration of 24,25(OH)2D or the ratio 24,25(OH)2D/25OHD and the creatinine clearance. The serum concentration of 24,25(OH)2D was significantly decreased also in six anephric adults relative to normal adult values. The data indicate that production of 24,25(OH)2D is impaired in subjects with compromised renal function. Inasmuch as the major active metabolite of Vitamin D, i.e., 1,25(OH)2D, is requried for renal synthesis of 24,25(OH)2D measurement of the latter metabolite may provide a convenient method for assessment of renal vitamin D metabolism. The role of this metabolite in the pathogenesis of renal osteodystrophy remains speculative.