Factors modulating apoptosis: an in-vitro study in swine granulosa cells

The aim of this study was to investigate the: effects of some endocrine and intra-ovarian factors on the activation/inhibition of apoptosis in swine g ranulosa cells. Upon incubation in a 10% FCS-supplemented M199, granulosa c ells from small(< 3 mm) follicles programmed their death after 24-48 h Of c ulture; in the absence of FCS, apoptosis was reduced after 24 h of culture. Cells cultured in the presence of FCS were treated with dbcAMP, LH, FSH. I nsulin-like Growth Factor-I (IGF-I) or PMSG to verify the role of these sub stances in apoptotic death: all these molecules inhibited apoptosis after 4 8 h of incubation. A further aim of the study was to investigate the possib le involvement of nitric oxide (NO), an intra-ovarian modulator. in the reg ulation of granulosa cell apoptosis and its possible role in the modulation of steroidogenesis. After a 48 h incubation with a substrate of NO synthes is (L-arginine, 0.1 and 1 mM). a NO donor [S-nitroso-N-acetyl-penicillamine (SNAP), 0.2 a nd 1 mM] or a NO synthase inhibitor [N-<omega>-nitro-L-argin ine-methyl-ester (NAME, 1 and 5mM)], the onset of apoptotic death was evalu ated: L. arginine and NAME did not induce any significant variation of apop tosis, whereas 1 mM SNAP exerted a protective action. A significant stimula tory effect of L-arginine on NO production, associated with a suppressive a ction on estradiol 17 beta concentrations was observed. NAME exerted an inh ibitory effect on NO production, associated with an increase in estradiol s ecretion; estradiol 17 beta production was markedly inhibited by SNAP. In s ummary, the depletion of FCS could induce a cell cycle arrest in G(0) where as apoptosis could be the consequence of cell cycle progression mediated by FCS; gonadotropins and IGF-I could also act as survival Factors. NO appear ed to represent a 'trophic' signal for the Follicle. whose involvement in t he regulation of ovarian function is substantiated by its modulatory action on steroidogenesis.