Effect of a NADPH generating system on the steroidogenic response in rat luteal cells

The effect of a NADPH generating system (NADPH-GS) on the function of rat luteal cells was studied. Cells were obtained from pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) primed immature rats and further incubated with a NADPH-GS. This system produced an increase in progesterone production and maximal stimulation was achieved at 1 mM NADP+ (10- to 15-fold). This effect was enhanced by addition of luteinizing hormone (LH 0.25 nM) to the incubation medium. On the contrary, insulin (2 nM) inhibited the effect observed with the NADPH-GS. The conversion of progesterone into 20 alpha-hydroxy-progesterone was not responsible for the changes observed. To analyze the site of NADPH action, pregnenolone and progesterone were measured using two inhibitors of steroid biosynthesis; aminoglutethimide and cyanoketone. The results confirm the specific site of action of NADPH at the mitochondrial conversion of cholesterol to pregnenolone. The effect of NADPH-GS was also observed in cultured purified luteal cells suggesting that the action of NADPH could be mediated by a free entry of the cofactor across the luteal cell plasma membrane. It can be concluded that the addition of NADPH improves the luteal cell incubation conditions and contributes to understanding the regulatory action of LH and insulin on the ovarian steroidogenic process.