Metabolism of arachidonic acid to epoxyeicosatrienoic acids by human granulosa cells may mediate steroidogenesis

Epoxyeicosatrienoic acids (EETs), cytochrome P-450 metabolites of arachidonic acid, have attracted attention because of their effects on stimulus-response coupling in endocrine, renal, and vascular cells. To investigate a possible role for EETs in ovarian physiology, we conducted a series of experiments using human luteinized granulosa cells. Granulosa cell microsomes produce EETs, which are identified by their comigration with known standards using reverse phase high pressure liquid chromatography. EET synthesis by granulosa cells is NADPH dependent and inhibited by ketoconazole, suggesting an enzymatic mechanism of production. Intact granulosa cells synthesize EETs from exogenous arachidonic acid, and EET production is increased by hCG stimulation of the cells. To investigate whether EETs have a role in ovarian steroidogenesis, they were added to cultures of granulosa cells. Varying concentrations of 14,15-EET differentially affected estradiol secretion; 0.001-0.05 microM stimulated estradiol production, whereas 14,15-EET concentrations of 10-50 microM inhibited estradiol production. hCG-stimulated estradiol secretion was also inhibited by 10-50 microM 14,15-EET. In contrast, progesterone secretion was not affected by any concentration of 14,15-EET tested. The cellular concentration of cAMP was not affected by the addition of EETs. These findings suggest that hCG stimulates granulosa cell production of EETs via an NADPH-supported, cytochrome P-450-dependent enzymatic mechanism. EETs may have an important autocrine or paracrine role in regulating ovarian granulosa cell estrogen synthesis.