Impact of carbonyl cyanide 3-chlorophenyl hydrazone (CCCP) treatment in ROS production and meiotic progression during the in vitro maturation of porcine oocytes

AGUSTINA CAMPORINO; FIAMMA SENGIALI; PABLO CETICA; SERGIO MORADO

Workshop; REDOX BA 2023. Workshop on Redox Nutrition and Toxicology; 2023

During in vitro maturation (IVM) a series of modifications take place in the oocyte, which lead to itsdevelopmental competence and imply a variation in reactive oxygen species (ROS) production. The aim of this study was to evaluate meiotic progression and ROS production in porcine oocytes during IVM in the presence or absence of carbonyl cyanide 3-chlorophenyl hydrazone (CCCP) as mitochondrial electron transport chain uncoupler. Cumulus-oocyte complexes (COCs) obtained by aspiration of antral follicles from ovaries of slaughtered gilts were incubated in 199 medium supplemented with 50 μg/ml gentamicin sulfate, 10% (v/v) porcine follicular fluid, 0.57 mM cysteine, 0.5 μg/ml FSH and 0.5 μg/ml of LH at 39ºC, 5% CO 2 in a humidified atmosphere for 44h. At 12h of IVM, COCs were treated with 0 (control), 0.5 or 1 µM CCCP. At 0, 12, 24, 36 and 44h of IVM, cohorts of COCs were recovered and denuded with a glass Pasteur pipette. Using epifluorescence microscopy, meiotic progression was assessed by Hoechst 33342 and ROS production by 2’,7’-dichlorodihydrodiacetate (DCH 2 FDA). Digital microphotographs were obtained and processed using IMAGE J software to calculate oocyte fluorescence intensity. Results were analysed by an ANOVA followed by a Bonferroni test. Between 0 and 12h a significant decrease in ROS production was observed (p