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 granulosa cells. Upon incubation in a 10% FCS‐supplemented M199, granulosa cells from small (< 3 mm) follicles programmed their death after 24–48 h of culture; in the absence of FCS, apoptosis was reduced after 24 h of culture. Cells cultured in the presence of FCS were treated with db‐cAMP, LH, FSH, Insulin‐like Growth Factor‐I IGF‐I or PMSG to verify the role of these substances in apoptotic death: all these molecules inhibited apoptosis after 48 h of incubation. A further aim of the study was to investigate the possible involvement of nitric oxide (NO), an intra‐ovarian modulator, in the regulation of granulosa cell apoptosis and its possible role in the modulation of steroidogenesis. After a 48 h incubation with a substrate of NO synthesis ( l ‐arginine, 0.1 and 1 m m ), a NO donor S‐nitroso‐N‐acetyl‐penicillamine (SNAP) , 0.2 and 1 m m ] or a NO synthase inhibitor N^ω‐nitro‐ l ‐arginine‐methyl‐ester (NAME, 1 and 5 m m )], the onset of apoptotic death was evaluated: l . arginine and NAME did not induce any significant variation of apoptosis, whereas 1 m m SNAP exerted a protective action. A significant stimulatory effect of l ‐arginine on NO production, associated with a suppressive action on estradiol 17β concentrations was observed; NAME exerted an inhibitory effect on NO production, associated with an increase in estradiol secretion; estradiol 17β production was markedly inhibited by SNAP. In summary, the depletion of FCS could induce a cell cycle arrest in G₀ whereas apoptosis could be the consequence of cell cycle progression mediated by FCS; gonadotropins and IGF‐I could also act as survival factors. NO appeared to represent a ‘trophic’ signal for the follicle, whose involvement in the regulation of ovarian function is substantiated by its modulatory action on steroidogenesis.