Activation of protein kinase-C stimulates human granulosa-luteal cell prostacyclin production

To study the production of prostacyclin (PGI2) by human granulosa-luteal cells in vitro, individual follicles from clomiphene/human menopausal gonadotropin-stimulated cycles of patients undergoing in vitro fertilization were aspirated 36 h after the administration of hCG. Granulosa cells were isolated and cultured for 48 h to regain responsiveness to hCG. The cells were then recultured for 3 days in the presence of a protein kinase-C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), hCG, cholera toxin, and indomethacin. The media were assayed for 6-keto-prostaglandin F1 alpha (PGF1 alpha), a stable hydration product of PGI2. TPA increased granulosa cell PGI2 production 20-fold. The increase in PG synthesis was detectable at 12 h, was maximal at 72 h, and was prevented by indomethacin. The increase in PG production was specific to TPA and did not occur when an inactive phorbol ester, 4 alpha-phorbol-12,13-didecanoate, was used. hCG and cholera toxin stimulated granulosa cell cAMP production, but not PGI2 synthesis. Thus, human granulosa cells produce PGI2 by protein kinase-C-mediated mechanisms.