Reactive oxygen species are required during bovine oocyte in vitro maturation

SERGIO MORADO; PABLO CETICA; MARTHA BECONI; GABRIEL DALVIT

Buenos Aires (Argentina)

Jornada; VIII Jornadas Multidisciplinarias de la Sociedad Argentina de Biología; 2006

Sociedad Argentina de Biología

REACTIVE OXYGEN SPECIES ARE REQUIRED DURING BOVINE OOCYTE IN VITRO MATURATION Morado S., Cetica P., Beconi M. and Dalvit G. Química Biológica, Facultad de Ciencias Veterinarias, Universidad de Buenos Aires. smorado@fvet.uba.ar   Immature and matured bovine oocytes generate similar reactive oxygen species (ROS) levels. The addition of cysteine to the maturation medium increase reduced glutathione (GSH). Inhibition of  respiratory chain increases ROS generation. The aim of this work was to determine ROS production in the presence of different treatments during bovine oocyte in vitro maturation. Cumulus-oocyte complexes were recovered by aspiration of antral follicles from ovaries obtained from slaughtered cows and cultured in medium 199 (control), 0.6 mM cysteine, 1 mM choro-dinitro benzene (CDNB, GSH removal) and 2 mM diphenyliodonium (IDP, NADPH oxidase inhibitor), at 39ºC, 5% CO2 in humidified air for 22 h. Respiratory chain effectors, 1 mM potassium cyanide (KCN, cytochrome oxidase inhibitor) and 0,42 mM carbonyl cyanide m-chlorophenylhydrazone (CCCP, mitochondrial uncoupler) were used.  Meiotic maturation was determined by the presence of metaphase II. ROS production was determined in denuded oocytes at 0, 6, 10, 18 and 22 h by the ratio between 2´,7´-dichlorodihydrofluorescein diacetate (DCHFDA) and fluorescein diacetate (FDA) assays, analysing digital microphotographs. Maturation percentages of control, cysteine, IDP and CDNB were 78, 80, 75 and 32%, respectively. ROS levels expressed as DCHFDA/FDA ratio fluctuated throughout the 22 h of maturation, showing a similar pattern with different treatments. A significant decrease at 10 h was observed (P<0.05), recovering the initial value at the end of maturation. Potassium cyanide increased ROS levels at 10 h (P<0.05). The GSH removal, which does not increase ROS production, impaires normal maturation. ROS levels, generated mainly in mitochondrial respiratory chain, may be linked to key events associated with the maturation process.