Regulation of aromatase activity of rat granulosa cells: induction of synthesis of NADPH-cytochrome P-450 reductase by FSH and dibutyryl cyclic AMP

Aromatase is an enzyme complex that is composed of a specific form of cytochrome P-450 and a flavoprotein, NADPH-cytochrome P-450 reductase. Aromatase activity of granulosa cells is increased markedly by follicle-stimulating hormone (FSH) and by analogs of cyclic AMP. It was the objective of the present study to investigate the effects of FSH and dibutyryl cyclic AMP (Bt₂cAMP) on the synthesis of NADPH-cytochrome P-450 reductase in rat granulosa cells maintained in vitro. Granulosa cells were obtained from the ovaries of diethylstilbestrol (DES)-treated immature rats and were incubated in the presence of DES (10^-7 M), DES + FSH (250 ng/ml), or DES + Bt₂cAMP (1 mM) for up to 72 h. After 72 h of incubation, aromatase activity of cells incubated with DES alone was 5 pmoles estrogen formed 2 h^-1 mg^-1 protein and was increased > 60-fold in cells incubated with FSH or Bt₂cAMP. NADPH-cytochrome P-450 reductase was immunoisolated from [³⁵S]methionine-labeled lysates of granulosa cells incubated for 72 h in the absence or presence of stimulatory factors. The rate of synthesis of reductase was found to be increased about 3-fold in cells incubated with DES + FSH or DES + Bt₂cAMP as compared to cells incubated with DES alone. By immunoblot analysis we found that the cellular content of reductase was increased about 2-fold by FSH and Bt₂cAMP treatment. Reductase specific activity was 10 nmoles min^-1 mg^-1 protein in membrane fractions of DES-treated cells and was increased 1.6-fold by FSH treatment. These findings are indicative that FSH increases the rate of synthesis, cellular content and specific activity of NADPH-cytochrome P-450 reductase in rat granulosa cells in vitro. The finding that Bt₂cAMP causes a similar induction of reductase synthesis is suggestive that the stimulatory effect of FSH on this component of the aromatase enzyme complex is mediated by an increase in cyclic AMP formation.