ABSTRACT
Although vitrification is the current method of choice for oocyte and embryo cryopreservation, it may have detrimental effects on reduction-oxidation status and mitochondrial activity. The aim of this study was to evaluate the effect of supplementing invitro culture (IVC) media and/or vitrification solutions with the antioxidant resveratrol on active mitochondria, mitochondrial superoxide production and lipid peroxidation. Abattoir-derived oocytes were matured and fertilised invitro using standard procedures. Following IVF (21h later), zygotes were cultured in IVC medium supplemented with 0 or 0.5µM resveratrol. On Day 7, blastocysts were vitrified using the Cryotech Vitrification Kit (Cryo Tech Laboratory) with or without 0.5µM resveratrol. After warming, active mitochondria, mitochondrial superoxide production and lipid peroxidation were evaluated using Mito Tracker Green FM, MitoSOX Red and BODIPY581/591 C11 staining respectively. The vitrification-warming process significantly increased active mitochondria and mitochondrial superoxide production in bovine embryos (P<0.05, ANOVA). The addition of 0.5µM resveratrol to the IVC medium or vitrification solutions significantly attenuated the increase in active mitochondria (P<0.05), but not in mitochondrial superoxide production, whereas embryos cultured and vitrified with resveratrol showed the highest values for both parameters (P<0.05). Regarding lipid peroxidation, no significant differences were detected between treatments. In conclusion, resveratrol supplementation of IVC medium or vitrification solutions contributes to recovery of an embryo's 'quieter' state (i.e. lower oxidative metabolism) after vitrification. However, supplementation of both solutions with resveratrol seemed to have a pro-oxidant effect.
Subject(s)
Antioxidants/pharmacology , Blastocyst/drug effects , Embryonic Development/drug effects , Oxidative Stress/drug effects , Resveratrol/pharmacology , Animals , Blastocyst/metabolism , Cattle , Cryopreservation , Embryo Culture Techniques/methods , Female , Lipid Peroxidation/drug effects , Mitochondria/metabolism , Reactive Oxygen Species/metabolism , Superoxides/metabolism , VitrificationABSTRACT
The role of reactive oxygen species (ROS) in the in vitro maturation (IVM) of oocytes remains controversial. The aim of the present study was to determine possible fluctuations in ROS production during bovine oocyte IVM in the presence of different modulators of ROS generation. Cumulus-oocyte complexes were cultured in medium 199 (control) in the absence or presence of 0.6 mM cysteine, 1 mM 1-choro-2,4-dinitro benzene (CDNB), 2 microM diphenyliodonium, 0.5 mM N-nitro-L-arginine methyl ester or 10 microM sodium nitroprusside (SNP) at 39 degrees C, in 5% CO2 in humidified air for 22 h. In addition, the respiratory chain effectors potassium cyanide (KCN; 1 mM) and carbonyl cyanide m-chlorophenylhydrazone (0.42 microM) were used. Meiotic maturation was determined by the presence of MII. ROS production was evaluated in denuded oocytes at different time points as the ratio of 2',7'-dichlorodihydrofluorescein diacetate (DCHF-DA) to fluorescein diacetate (FDA). ROS levels, expressed as DCHF-DA:FDA, fluctuated throughout the 22 h of maturation depending on the treatment applied. At 12 h incubation in the presence of KCN and SNP, ROS levels were increased, whereas ROS levels after 12 h in the presence of cysteine were reduced (P<0.05). Both CDNB and SNP impaired meiotic progression. The higher metabolic activity demand during bovine oocyte maturation coincides with a concomitant reduction in ROS generation. These results suggest that 12 h would be a critical point for bovine oocyte IVM because it is closely related to the production of ROS at this time.
