Regulation of steroidogenesis in rat leydig cells in culture: Effect of human chorionic gonadotropin and dibutyryl cyclic AMP on the synthesis of cholesterol side chain cleavage cytochrome P-450 and adrenodoxin

Abstract

Rat Leydig cells in primary culture were used as a model system to investigate the effects of human chorionic gonadotropin (hCG) and dibutyryl cyclic AMP (Bt 2cAMP) on the synthesis of cholesterol side chain cleavage cytochrome P-450 (cytochrome P-450 scc) and the iron-sulfur protein, adrenodoxin. Leydig cells isolated from the testes of mature rats were placed in monolayer culture in the absence of stimulatory factors for 8 days. HCG (10 mIU/ml) or Bt 2cAMP (1 m m) were then added to some of the cultures and the incubations were continued for up to 48 h. Testosterone production was increased markedly in cells incubated with hCG or Bt 2cAMP. A significant accumulation of pregnenolone in the medium of cells treated with Bt 2cAMP was also observed. Both hCG and Bt 2cAMP increased the rates of synthesis of cytochrome P-450 scc and adrenodoxin. In hCG-treated cells the apparent rate of synthesis of cytochrome P-450 scc was increased 13-fold over that of controls after 48 h of incubation; the rate of adrenodoxin synthesis was increased 4-fold by hCG treatment. In Bt 2cAMP-treated cells the rate of synthesis of cytochrome P-450 scc was 37-fold greater than that of control cells after 48 h of incubation; adrenodoxin synthesis was increased 36-fold over controls. In hCG- and Bt 2cAMP-treated cells, the concentration of immunoreactive cytochrome P-450 scc and adrenodoxin increased with increasing time of incubation, and were correlated with the stimulatory effects of these agents on cytochrome P-450 scc activity and on total steroid production. The results of this study are indicative that the maintenance by LH/hCG of elevated levels of testosterone synthesis by the Leydig cell is mediated, in part, by induction of the synthesis of cytochrome P-450 scc and its associated protein, adrenodoxin. Since Bt 2cAMP had effects similar to those observed with hCG, it is suggested that the stimulatory effects of hCG on the synthesis of cytochrome P-450 scc and adrenodoxin are mediated by increased cyclic AMP formation.