Bovine oocytes in vitro matured in the presence of antioxidants: implications for intracellular levels of glutathione and reactive oxygen species and blastocyst development

Abstract Text:

Production of reactive oxygen species (ROS) is a physiological process that occurs mainly on mitochondrial metabolism. Some, in vitro culture conditions can lead an increase in ROS production, making the oocytes more susceptible to oxidative stress damage. This study was conducted with the main objective to assess the effects of supplementation of in vitro maturation (IVM) medium with intracellular (cysteine and cysteamine) and extracellular (catalase) antioxidants on the intracellular levels of glutathione (GSH) and ROS in bovine oocytes and its implications on the subsequently embryonic development. Cumulus-oocyte complexes were matured in TCM-199 with bicarbonate, hormones and 10% FCS without supplementation (Control group) or supplemented with 0.6 mM cysteine associated with 100 µM cysteamine (C+C group), 100 UI catalase (CAT group) or 0.6 mM cysteine associated with 100 µM cysteamine and 100 UI catalase (C+C+CAT group) for 22 h at 38.5º C in 5% CO₂ in air. A sample of matured and immature oocytes (0 h) were stained (n=192) with 5 µM of the fluorescent probe 6-carboxy-2’7’-dichlorodihydrofluorescein diacetate (H₂DCFDA; Molecular Probes, Invitrogen, Oakville, Canadá) or stained (n=246) with ThiolTracker^TM Violet (Glutathione Detection Reagent; Molecular probes, Invitrogen, Oregon, USA) in order to evaluate ROS and GSH, respectively. Stained oocytes were evaluated immediately under an epifluorescence inverted microscope (excitation 495/510-550nm and emission 404/526 nm, respectively for H₂DCFDA and ThiolTracker^TM) and the images were analyzed by Q-Capture Pro image software for determining the fluorescent intensity. Other oocytes were submitted to IVF and the presumptive zygotes were IVC in SOF medium, at 38.5°C in 5% CO₂ in air, for 7 days. The cleavage rates and embryonic development were evaluated, respectively, at 72 and 168 hpi. The differences of fluorescent intensity among groups was compared by ANOVA followed by Tukey’s test and embryonic development was analyzed by Chi-square test (P<0.05). The fluorescent intensity for ROS quantification was 1.00±0.12^a (0 h), 1.91±0.10^c (Control), 1.11±0.04^a (C+C), 1.45±0.08^b (CAT) and 1.07±0.04^a (C+C+CAT). The fluorescent intensity for GSH quantification was 1.00±0.4^a (0 h), 0.21±0.01^bd (Control), 0.47±0.02^c (C+C), 0.32±0.01^b (C+C+CAT) and 0.15±0.01^d (CAT). The cleavage rates were 73.5^a (Control), 75.7^a (C+C), 75.4^a (CAT) and 73.1^a (C+C+CAT). The blastocyst rates were 28.2%^a (Control), 31.1%^a (C+C), 33.3%^a (CAT) and 46.2%^b (C+C+CAT). In conclusion, supplementation with cysteine, cysteamine and catalase during IVM reduced intracellular ROS levels and improved the embryonic development; however; such improvement was not due to an increase on intracellular amounts of GSH.

Keywords: antioxidants; in vitro maturation; ROS; GSH; blastocyst.