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.     

Production and quality of bovine oocytes and embryos

Many factors influence the efficiency of the in vitro embryo production technology in cattle but the most important are the physiological conditions of the donor and the culture protocols for oocyte maturation and fertilization and for embryo culture from zygote to blastocyst. Therefore, general factors such as age, body conditions and herd management play a pivotal role together with more specific factors such as reproductive soundness and ovarian cyclicity. Given that good quality and competent oocytes are available a complex series of processes, including oocyte maturation, fertilization and culture of the derived zygotes, must be completed to generate viable embryos.     

Production and Quality of Bovine Oocytes and Embryos

Many factors influence the efficiency of the in vitro embryo production technology in cattle but the most important are the physiological conditions of the donor and the culture protocols for oocyte maturation and fertilization and for embryo culture from zygote to blastocyst. Therefore, general factors such as age, body conditions and herd management play a pivotal role together with more specific factors such as reproductive soundness and ovarian cyclicity. Given that good quality and competent oocytes are available a complex series of processes, including oocyte maturation, fertilization and culture of the derived zygotes, must be completed to generate viable embryos.     

Nitric oxide synthase expression and nitric oxide/cyclic GMP pathway in swine granulosa cells

The present investigation was undertaken to verify if the two nitric oxide synthase isoforms, eNOS and iNOS, are present in swine granulosa cells and whether the enzyme soluble guanylate cyclase is functionally active in the same cells and can account for NO effects. Using western blotting, the presence of endothelial NO synthase was demonstrated in freshly collected cells; on the contrary, iNOS expression was not observed in the same cells either before or after culture with the inflammatory cytokine hTNF-. The treatment with a strong NO donor (S-Nitroso-L-acetyl penicillamine, SNAP) determined an increase of cGMP levels in culture media, which was attenuated by the combined treatment with an inhibitor of NO-sensitive soluble guanylate cyclase, 1H-[1,2,3]oxadiaziolo [4,3a]quinoxaline −1-one (ODQ). The cGMP analog, 8 bromo-cGMP, mimicked the strong inhibitory effect exerted by SNAP on estradiol 17 β and progesterone production, while ODQ did not modify steroids concentrations in culture media. These observations demonstrate the presence of a follicular NO-generating system, which in swine granulosa cells seems to include only the endothelial NOS isoform. Furthermore, the nitric oxide/cyclic GMP system seems to be functionally active in these cells, since cGMP appears to mediate NO action, even if it cannot account completely for NO inhibitory effect on steroidogenesis.