Nitric oxide in bovine oocyte in vitro maturation

SERGIO MORADO; PABLO CETICA; MARTHA BECONI; GABRIEL DALVIT

Montevideo (Uruguay)

Congreso; V Meeting of The Society for Free Radical Biology and Medicine (SFRBM), V International Conference on Peroxynitrite and Reactive Nitrogen Species; 2007

Society for Free Radical Biology and Medicine – South American Group

Nitric Oxide in Bovine Oocyte in vitro maturation   S.A. Morado, P.D. Cetica, M.T. Beconi and G.C. Dalvit   Biochemistry, School of Veterinary Science, University of Buenos Aires, Chorroarín 280, C1427CWO, Buenos Aires, Argentina. e-mail: smorado@fvet.uba.ar   In reproductive processes, cellular adaptations have been observed, not only for protection against reactive oxygen species (ROS) harmful effect, but also to use its reactive nature for beneficial purposes. However, ROS involvement in in vitro maturation (IVM) and their influence on embryonic development is still controversial. Nitric oxide (NO) at sub-micromolar concentrations exhibit two main efects on the mitochondrial respiratory chain: the competitive inhibition of cytochrome oxidase and the stimulation of the O2- production by inhibition of electron transfer at complex III. The aim of this work was to determine NO participation in meiotic maturation and ROS variation during the process. Cumulus-oocyte complexes were recovered by aspiration of antral follicles from ovaries obtained from slaughtered cows and cultured in medium 199 (control), 0,5 mM N-nitro-L-arginine methyl ester (L-NAME, endothelial NO synthase inhibitor) and 10 mM sodium nitroprusside (SNP, NO donor) at 39°C, 5% CO2 in humidified air for 22h. Meiotic maturation was determined by the presence of metaphase II. ROS production was determined in denuded oocytes at 0, 6, 12, 18 and 22h by the ratio between 2’,7’- dichlorodihydrofluorescein diacetate (DCHFDA) and fluorescein diacetate (FDA) assays. Maturation percentages were 80, 81 and 58% for control, L-NAME and SNP, respectively. ROS levels expressed as DCHFDA/FDA ratio fluctuated throughout the 22h of maturation, showing a similar pattern with the different treatments. A decrease at 6h and an increase at 12h, both significant, were observed in oocytes cultured in the presence of SNP compared to the control (P<0,05). These results suggest that exogenous NO reduces H2O2 production and mitochondrial oxidative energy, impairing the maturation process.