Role of nitric oxide in swamp buffalo oocyte maturation

Abstract

The purposes of this study were to investigate the role of nitric oxide (NO) and its signaling pathway in nuclear and cytoplasmic maturation of swamp buffalo oocytes cultured in vitro. Cumulus-oocyte complexes (COCs) and denuded oocytes (DOs) were cultured in the basal medium M199 supplemented with S-nitroso-L-acetyl penicillamine (SNAP, a NO donor) at 10[superscript-7] M, 10[superscript-6] M, 10[superscript-5] M, and 10[superscript-4] M. SNAP inhibited the spontaneous nuclear maturation, significantly delayed germinal vesicle breakdown (GVBD) during the first 8 h and further delayed metaphase II (M II) stage at the 24 h of culture in a dose-dependent manner. Cumulus-oocyte complexes and DOs were cultured in the basal medium alone or supplemented with 10[superscript-4] M SNAP or 10 micrometre 1H-[1,2,4] oxadiazolo-[4,3,-alpha] quinoxalin-1-one (ODQ), a soluble guanylyl cyclase (sGC) inhibitor, or in combination with SNAP and ODQ. The results showed that SNAP significantly decreased the percentages of germinal vesicle breakdown (GVBD) in both COCs and DOs whereas ODQ could reverse SNAP-inhibited spontaneous oocyte maturation in only COCs. The treatment with SNAP determined an increase intraoocyte cyclic guanosine monophosphate (cGMP) concentration, which was attenuated by the combined treatment with ODQ only in COCs. Intraoocyte cyclic adenosine monophosphate (cAMP) concentrations were not correlated with cGMP concentrations, cAMP declined to basal levels after 2 h of culture in both SNAP and control groups. These results suggested that NO had an inhibitory effect on nuclear maturation and NO exerted its effect partly by cGMP which was not mediated by cAMP in COCs. In the contrary, NO might inhibit nuclear maturation of DOs through another signaling pathway. The effect of NO on cytoplasmic maturation was also characterized by the activities of maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK). MPF and MAPK were activated around the time of GVBD. Nitric oxide decreased MPF activity only at the time of GVBD, while MAPK activity was suppressed throughout the period of culture. Transmission electron microscopic study showed that immature forms of mitochondria and cortical granules were more abundant in NO treated group which implied the delay of cytoplasmic maturation. It is therefore suggested that NO plays a role in delaying both nuclear and in swamp buffalo oocytes. The inhibitory effect of NO may act through NO/cGMP dependent and independent signaling pathways but is not mediated by cAMP.