Nitric oxide and reactive oxygen species during bovine oocyte in vitro maturation

SERGIO MORADO; PABLO CETICA; MARTHA BECONI; GABRIEL DALVIT

Rosario (Argentina)

Congreso; XXXV Reunión Anual de la Sociedad Argentina de Biofísica; 2006

Sociedad Argentina de Biofísica

Free radicals and oxidative damage Nitric Oxide and Reactive Oxygen Species during 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. [1] 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 [2-3] and the stimulation of the O2- production by inhibition of electron transfer at complex III. [4] NO and O2- metabolism in the mitochondrial matrix are linked by the very fast reaction between NO and O2- to produce peroxynitrite (ONOO-). [5] 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 22 h. Meiotic maturation was determined by the presence of metaphase II. [6] ROS production was determined in denuded oocytes at 0, 6, 12, 18 and 22 h by the ratio between 2’,7’- dichlorodihydrofluorescein diacetate (DCHFDA) and fluorescein diacetate (FDA) assays. [7] Maturation percentages were 80, 81 and 58 % for control, L-NAME and SNP, respectively. ROS levels expressed as DCHFDA/FDA ratio fluctuated throughout the 22 h of maturation, showing a similar pattern with the different treatments. A significant decrease was observed in oocytes cultured in the presence of SNP at  6 and 22 h (P<0,05). These results suggest that exogenous NO reduces the production of mitochondrial oxidative energy, required for biosynthetic processes, with the consequent increase in O2- generation. The reaction with O2- could reduce H2O2 production, necessary for intracellular signals involved in the maturation process.  References: [1]  Riley JC. and Behrman HR., Proc. Soc. Exp. Biol. Med., 1991, 198, 781-791. [2]  Brown GC. and Cooper CE., FEBS Lett., 1994, 356, 295-298. [3]  Antunes F., Boveris A. and Cadenas E., Proc. Natl. Acad. Sci. U.S.A., 2004, 101, 16774-16779. [4]  Poderoso JJ., Carreras MC., Lisdero C., Riobo N., Schöpfer F. and Boveris A., Arch. Biochem. Biophys., 1996, 328, 85-92. [5]  Kissner R., Nauser T., Bugnon P., Lye PG. and Koppenol WH., Chem. Res. Toxicol., 1997, 10, 1285-1292. [6]  Tarkowski AK., Cytogenetics, 1966, 5, 394-400. [7]  Lane L., Maybach JM. And Gardner DK., Hum. Reprod., 2002, 1, 2686-2693. Acknowledgments: This work was supported by UBACyT. The authors would like to thank “Deltacar” abbatoir for lending us slaughtered cow ovaries and JICA (Japanese International Cooperation Agency) for the equipment donated to our laboratory.