NITRIC-OXIDE - AN AUTOCRINE REGULATOR OF HUMAN GRANULOSA-LUTEAL CELL STEROIDOGENESIS

We investigated the presence of nitric oxide (NO) synthase in ovarian follicular cells obtained from women undergoing in vitro fertilization procedures. Endothelial NO synthase messenger RNA was demonstrated by polymerase chain reaction amplification of reverse transcribed RNA. N O synthase was localized to granulosa-luteal cells by immunocytochemis try, using a monoclonal antibody. Ovarian follicular cell NO synthase enzyme activity was confirmed by measuring the conversion of L-arginin e to citrulline. To investigate the effect of NO on granulosa-luteal c ell steroidogenesis, NO synthase inhibitors and NO donors were added t o cell cultures. NG-Monomethyl-L-arginine and N-nitro-arginase methyl ester, selective inhibitors of NO synthase, Nitroso-L-acetyl penicilla mine (S-NAP) and S-nitroso glutathione, NO donors, caused a dose-depen dent decrease in both estradiol and progesterone secretion. The decrea se by S-NAP was reversed by hemoglobin, which binds free NO. Although S-NAP increased the concentration of cGMP in granulosa-luteal cells, c GMP analogs had no effect on steroidogenesis in cell cultures. S-NAP a nd native NO in solution decreased cellular and microsomal aromatase a ctivities. We conclude that NO synthase is present in human granulosa- luteal cells and that NO inhibits estradiol secretion independent of c GMP by directly inhibiting aromatase.