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

CAMPORINO, A.; SENGIALI, F.; CETICA, P.; MORADO, S.

CABA

Workshop; RedoxBA; 2023

Facultad de Farmacia y Bioquímica

During in vitro maturation (IVM) a series of modificationstake place in the oocyte, which lead to its developmentalcompetence and imply a variation in reactive oxygenspecies (ROS) production. The aim of this study was toevaluate meiotic progression and ROS production inporcine oocytes during IVM in the presence or absenceof carbonyl cyanide 3-chlorophenyl hydrazone (CCCP)as mitochondrial electron transport chain uncoupler.Cumulus-oocyte complexes (COCs) obtained byaspiration of antral follicles from ovaries of slaughteredgilts were incubated in 199 medium supplemented with50 μg/ml gentamicin sulfate, 10% (v/v) porcine follicularfluid, 0.57 mM cysteine, 0.5 μg/ml FSH and 0.5 μg/ml ofLH at 39oC, 5% CO2 in a humidified atmosphere for 44h.At 12h of IVM, COCs were treated with 0 (control), 0.5 or1 μM CCCP. At 0, 12, 24, 36 and 44h of IVM, cohorts ofCOCs were recovered and denuded with a glass Pasteurpipette. Using epifluorescence microscopy, meioticprogression was assessed by Hoechst 33342 and ROSproduction by 2’,7’-dichlorodihydrodiacetate (DCH2FDA).Digital microphotographs were obtained and processedusing IMAGE J software to calculate oocyte fluorescenceintensity. Results were analysed by an ANOVA followedby a Bonferroni test. Between 0 and 12h a significantdecrease in ROS production was observed (p