Growth differentiation factor-9 stimulates progesterone synthesis in granulosa cells via a prostaglandin E-2/EP2 receptor pathway

Growth differentiation factor-9 (GDF-9), an oocyte-secreted member of the t ransforming growth factor beta superfamily, progesterone receptor, cyclooxy genase 2 (Cox2; Ptgs2), and the EP2 prostaglandin E-2 (PGE(2)) receptor (EP 2; Ptgerep2) are required for fertility in female but not male mice. To def ine the interrelationship of these factors, we used a preovulatory granulos a cell culture system in which we added recombinant GDF-9, prostaglandins, prostaglandin receptor agonists, or cyclooxygenase inhibitors. GDF-9 stimul ated Cox2 mRNA within 2 h, and PGE(2) within 6 h; however, progesterone was not increased until 12 h after addition of GDF-9. This suggested that Cox2 is a direct downstream target of CDF-9 but that progesterone synthesis req uired an intermediate. To determine whether prostaglandin synthesis was req uired for progesterone production, we analyzed the effects of PGE(2) and cy clooxygenase inhibitors on this process. PGE(2) can stimulate progesterone synthesis by itself, although less effectively than GDF-9 (3-fold vs. 6-fol d increase over 24 h, respectively). Furthermore, indomethacin or NS-398, i nhibitors of Cox2, block basal and GDF-9-stimulated progesterone synthesis. However, addition of PGE(2) to cultures containing both GDF-9 and NS-398 o verrides the NS-398 block in progesterone synthesis. To further define the PGE(2)-dependent pathway, we show that butaprost, a specific EP2 agonist, s timulates progesterone synthesis and overrides the NS-398 block. In additio n, CDF-9 stimulates EP2 mRNA synthesis by a prostaglandin- and progesterone -independent pathway. Thus, CDF-9 induces an EP2 signal transduction pathwa y which appears to be required for progesterone synthesis in cumulus granul osa cells. These studies further demonstrate the importance of oocyte-somat ic cell interactions in female reproduction.