Effects of GnRH on protein kinase C activity, Ca2+ mobilization and steroidogenesis of human granulosa cells.

Abstract

Gonadotropin-releasing hormone (GnRH) and its agonists have been known to directly affect steroid hormone production in human granulosa cells. In this study, we examined effects of GnRH on Ca2+ mobilization, protein kinase C activity and steroidogenesis of human granulosa cells. Human granulosa cells were harvested by aspiration of follicles during oocyte retrieval for IVF. Test substances, that is, GnRH agonist ([D-Ser(TBu)6]-LHRH-EA), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), A23187, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and nordihydroguaiaretic acid (NDGA) were added to HTF medium with human granulosa cells. At the end of a 5-h incubation period, progesterone and estradiol in the medium were extracted and measured. GnRH agonist (10(-9)M and 10(-8)M) significantly stimulated (P < 0.05) progesterone and estradiol accumulation in the medium. TPA (10(-7)M, 10(-8)M and 10(-9)M) also significantly stimulated (P < 0.05) progesterone accumulation. A23187 (10(-6)M) significantly stimulated progesterone accumulation (P < 0.05) and significantly enhanced (P < 0.05) the stimulatory effect of TPA on progesterone production. H-7 (10(-3)M, 10(-4)M and 10(-5)M) and NDGA (10(-3)M, 10(-4)M and 10(-5)M) significantly inhibited (P < 0.05) the effect of GnRH agonist on progesterone and estradiol production by the granulosa cells. The granulosa cells were loaded with 4 microM Fura 2-AM, and the ratio of the intensities of fluorescent emission at 510 nm with excitation at 340 and 380 nm was calculated at 100-ms intervals. GnRH agonist (10(-7)M and 10(-6)M) increased (P < 0.05) [Ca2+]i significantly as compared with the control. These results suggested that the effects of GnRH on progesterone and estradiol production in human granulosa cells were mediated partially by calcium mobilization and partially by C-kinase activity, and protein kinase C, [Ca2+]i and lipoxygenase were suspected of being important intracellular messages for GnRH in human granulosa cells.