REACTIVE OXYGEN SPECIES AND ACTIVE MITOCHONDRIA DYNAMICS DURING THE IN VITRO MATURATION OF PORCINE OOCYTES

AGUSTINA CAMPORINO; PABLO CETICA; SERGIO MORADO

Buenos Aires

Jornada; XXIV Jornadas de la Sociedad Argentina de Biología; 2022

The role of reactive oxygen species (ROS) in the processes related to the culture and in vitro manipulation of gametes and embryos is still controversial. It has been proposed that ROS may act as mediating molecules that influence processes such as cell proliferation, gene expression and the activation of various signaling pathways, being the mitochondria and, particularly, the electron transport chain, the main source of ROS. Therefore, the aim of this work was to determine active mitochondria and ROS production fluctuations during the in vitro maturation (IVM) of porcine oocytes at 0, 24, and 44 h. Immature oocyte-cumulus complexes (COCs) were obtained by the aspiration of antral follicles from slaughtered gilts ovaries and only oocytes completely surrounded by an intact and dense cumulus were used. COCs were matured in 199 medium supplemented with 50 μg/ml gentamicin sulfate, 10% (v/v) porcine follicular fluid, 0.57 mM cysteine, 0.5μg/ml FSH and 0.5μg/ml of LH, under mineral oil at 39°C, 5% CO2 in air and 100% humidity for 44 h. To determine ROS production and active mitochondria dynamics, cohorts of COCs were extracted from the maturation medium at 0, 24 and 44 h, time points in which some of the most important events for IVM of porcine oocytes occur. The groups of extracted COCs were incubated with hyaluronidase at 37°C and denuded with a fine Pasteur pipette. ROS production dynamics were evaluated by fluorescein diacetate 2´,7´-dichlorodihydrodiacetate (DCH2FDA) staining, while active mitochondria were evaluated with Mitotracker Green staining. Digital microphotographs were obtained by epifluorescence microscopy and were analyzed using IMAGE J software, assessing the individual luminosity of each oocyte. Results were analyzed by means of an ANOVA followed by a Bonferroni test. A p