When the effects of heat stress are detrimental during maturation, cumulus cells are intimately associated with the oocyte. To determine the extent to which heat stress affects these cells, in this study, transcriptome profiles of the cumulus that surrounded control and heat-stressed oocytes (41 8C during the first 12 h only and then shifted back to 38.5 8C) during in vitro maturation (IVM) were compared using Affymetrix bovine microarrays. The comparison of cumulus-derived profiles revealed a number of transcripts whose levels were increased (nZ11) or decreased (nZ13) R twofold after heat stress exposure (P!0.01), sufficient to reduce the development of blastocysts by 46.4%. In a separate study, quantitative PCR (qPCR) was used to confirm heat-induced differences in the relative abundances of the transcripts of five different genes (caveolin 1, matrix metallopeptidase 9, FSH receptor, Indian hedgehog homolog, and inducible nitric oxide synthase). Heat stress exposure resulted in O1.7-fold decrease in the protein levels of latent matrix metallopeptidase 9 (proMMP9). Heat-induced reductions in transcript levels were noted at 6 h IVM with reductions in proMMP9 protein levels at 18 h IVM (PZ0.0002). Independent of temperature, proMMP9 levels at 24 h IVM were positively correlated with the development rate of blastocysts (R 2 Z0.36; PZ0.002). The production of progesterone increased during maturation; heat-induced increases were evident by 12 h IVM (PZ0.002). Both MMP9 and progesterone are associated with the developmental competence of the oocyte; thus, it seems plausible for some of the negative consequences of heat stress on the cumulus-oocyte complex to be mediated through heat-induced perturbations occurring in the surrounding cumulus.

Hyperthermia during estrus has direct consequences on the maturing oocyte that carries over to the resultant embryo to compromise its ability to continue in development. Because early embryonic development is reliant upon maternal transcripts and other ooplasmic components, we examined impact of heat stress on bovine oocyte transcripts using microarray. Oocytes were matured at 38.5ºC for 24 h or 41.0ºC for the first 12 h of in vitro maturation; 38.5ºC thereafter. Transcriptome profile was performed on total (adenylated + deadenylated) RNA and polyadenylated mRNA populations. Heat stress exposure altered the abundance of several transcripts important for mitochondrial function. The extent to which transcript differences are coincident with functional changes was evaluated by examining reactive oxygen species, ATP content, and glutathione levels. Mitochondrial reactive oxygen species levels were increased by 6 h exposure to 41.0ºC while cytoplasmic levels were reduced compared to controls (P < 0.0001). Exposure to 41.0ºC for 12 h increased total and reduced glutathione levels in oocytes at 12 h but reduced them by 24 h (time × temperature P < 0.001). ATP content was higher in heat-stressed oocytes at 24 h (P < 0.0001). Heat-induced increases in ATP content of matured oocytes persisted in early cleavage-stage embryos (8- to 16-cell embryos; P < 0.05) but were no longer apparent in blastocysts (P > 0.05). Collectively, results indicate that direct exposure of maturing oocytes to heat stress may alter oocyte mitochondrial processes/function, which is inherited by the early embryo after fertilization.

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.