Regulation of luteinizing hormone receptors and steroidogenesis in gonadotropin-desensitized leydig cells.

Abstract

The steroidogenic pathway in normal and gonadotropin-desensitized Leydig cells was analyzed by radioimmunoassay of steroid intermediates during hormonal stimulation in vitro to characterize the block in steroid biosynthesis caused by intravenous injection of human chorionic gonadotropin (hCG) (1 to 10 cg). Such cells exhibit dose-dependent loss of receptors after gonadotropin treatment, and a simultaneous defect in steroidogenesis that prevents maximum testosterone production during subsequent hormone stimulation. In normal cells pregnenolone was metabolized to androstenedione as the major precursor for testosterone. In the presence of cyanoketone, pregnenolone metabolism was diverted through the As pathway to form androstenediol. This sequence could be prevented by addition of spironolactone to inhibit 17a-hydroxylation, and quantitative assays of pregnenolone formation were performed in cells incubated with cyanoketone and spironolactone. The sensitivity of the pregnenolone response to gonadotropin in vitro was similar to that of testosterone (EDso = 0.2 PM hCG), but the kinetics of pregnenolone formation were more rapid. The inhibitory effects of cycloheximide and actinomycin D on gonadotropin-induced testosterone production were also evident when pregnenolone production was measured, locating the inhibitor-sensitive step at the earliest portion of the steroidogenic pathway. In gonadotropin-desensitized Leydig cells, the extent and nature of the block in steroidogenesis was correlated with hCG dose and the subsequent degree of receptor loss. After treatment with 1 pg of hCG, leading to loss of 70% of the receptors, testosterone production during stimulation by hCG, dibutyryl cyclic adenosine 3’:5’-monophosphate (cyclic AMP) and choleragen was reduced by 60%, and pregnenolone formation was normal or elevated. Therefore, the steroidogenic block was beyond pregnenolone synthesis, and was not dependent on impairment of cyclic AMP formation and availability. After treatment with 10 pg of hCG, leading to 90% receptor loss, testosterone production was correspondingly low, and pregnenolone formation was reduced by 50%. More extensive receptor loss (-99%) produced by higher hCG doses was accompanied by abolition of both testosterone and pregnenolone responses to hormone. In the presence of cyanoketone, desensitized cells with up to 70% receptor loss formed pregnenolone and 17a-hydroxypregnenolone during hormone stimulation, whereas progesterone and 17a-hydroxyprogesterone, pregnenolone, and 17a-hydroxypregnenolone