196 ELEVATED TEMPERATURE DURING BOVINE OOCYTE MATURATION ALTERS MITOCHONDRIAL ACTIVITY

Exposure of maturing cumulus-oocyte complexes to a physiologically relevant heat stress altered the transcriptome in oocytes, especially certain transcripts important for mitochondrial function. To determine if perturbations are coincident with changes in mitochondrial activity, generation of reactive oxygen species (ROS), glutathione and ATP content were examined in maturing oocytes experiencing heat stress. In the first study, ROS content was assessed after 6 h at 38.5, 41, or 42°C using 37 µM 6-carboxy-2′7′-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) or 60 µM dihydrofluorescein diacetate for staining cytoplasmic and mitochondrial ROS, respectively. In a second study, cumulus-oocyte complexes were matured for 0, 12 or 24 h at 38.5°C (control) or 41°C for 12 h (heat stress; 38.5°C thereafter). Total glutathione (GSSG + GSH) and reduced glutathione (GSH) and ATP levels were assessed in cumulus denuded-zona free oocytes as per manufacturer (GSH-Glo Glutathione assay, Promega, Madison, WI, USA; 1 to 25 pmol standards) and ATP determination kit (Life Technologies, Carlsbad, CA, USA; 0.078 to 10 pmol standards). Next, ATP content was assessed in control and heat-stressed oocytes matured for 24 h and in embryos resulting after IVF of control or heat-stressed oocytes (i.e. 2-, 4-, and 8-to-16 cell and blastocyst-stage embryos). Data were analysed as a randomised block design with fixed effects of maturation temperature (and hours of maturation where appropriate), blocking on replicate, using PROC MIXED (SAS 9.2, SAS Institute, Cary, NC, USA). Culture at 41°C for the first 6 h in vitro-matured (IVM) reduced levels of cytoplasmic ROS compared to non-stressed controls (1.02 v. 0.84 fluorescent ratio for 38.5 v. 41°C, SEM = 0.16, P < 0.0001), whereas levels after 42°C were similar to controls (0.94). Mitochondrial ROS were higher after 41°C (1.42) than after 38.5 (0.98) or 42°C (0.98, SEM = 0.14, P < 0.0001). Heat stress exposure increased total glutathione content at 12 h (4.4 v. 5.2 pmol for 38.5 v. 41°C) but levels were decreased by 24 h (6.6 v. 5.9 pmol for 38.5 v. 41°C; SEM = 1.26, P = 0.0002). A similar pattern was found for GSH (P = 0.0001); GSSG was similar across treatments (P = 0.50). Levels of ATP increased during maturation (1.10, 1.20, and 1.65 pmol for 38.5°C at 0, 12, and 24 h) and were increased by heat stress at 24 h (1.65 and 2.01 pmol for 38.5 and 41°C; SEM = 0.31, P = 0.001). In embryos, ATP content was higher in 8-to-16 cell embryos derived from heat-stressed oocytes than in those from control oocytes (P = 0.03); levels were similar in blastocyst-stage embryos (P = 0.21) regardless of origin. In conclusion, application of a physiologically relevant heat stress during maturation altered mitochondrial activity in bovine oocytes. Carryover of effects to the early embryo may explain some of the reductions in embryo development experienced by heat-stressed oocytes. This research was supported in part by USDA National Institute of Food and Agriculture, Hatch Project No. 227701, the state of Tennessee through University of Tennessee AgResearch, Department of Animal Science, and East Tennessee Research and Education Center.