218 GENE AND PROTEIN EXPRESSION OF ANTIOXIDANT ENZYMES IN BOVINE EMBRYOS EXPOSED TO HEAT SHOCK

Abstract

In a previous study, we reported that 8-cell-stage embryos exposed to a temperature of 41°C for 6 h had significantly increased embryonic mortality and intracellular reactive oxygen species (ROS). There have been some reports that ROS regulates the expression of genes encoding antioxidant enzymes in culture cells. In this study, we investigated the gene and protein expression of antioxidant enzymes in bovine 8-cell-stage embryos exposed to heat shock. In vitro-produced bovine embryos were used for the experiment. Embryos were cultured with CR1aa + 5% FCS at 38.5°C in 5% CO₂ and 5% O₂. On Day 2 after fertilization, 8-cell-stage embryos were exposed to heat shock at 41°C in 5% CO₂ and 5% O₂ for 6 h (HS). Eight-cell-stage embryos cultured at 38.5°C in 5% CO₂ and 5% O₂ were sampled at the same collection time as controls. After HS, 20 embryos were immediately collected for gene expression analysis. Expression of heat shock protein 70 (HSP70), CuZn-containing superoxide dismutase (SOD), catalase (CAT), and glutathione peroxide (GPx) genes was examined by real-time polymerase chain reaction. Twenty embryos were also collected after 3 h of HS (3 h) and at 18 h after HS (18 h) to evaluate the expression of proteins. Expression of HSP70, SOD, and CAT proteins was examined by Western blotting. Both the gene and protein expression levels of HS groups were normalized to those of the controls to obtain the relative expression levels. All results were analyzed by Student’s t-test. Expression of the HSP70 gene significantly increased in HS embryos (P < 0.05). Expression of the SOD and CAT genes tended to increase in HS embryos (P < 0.07), but there were no significant differences in expression of the GPx gene. There was no significant difference in protein expression in all the antioxidant enzymes in 3-h-sampled embryos. Expression of the HSP70 protein increased significantly in heat-shocked embryos sampled at 18 h (P < 0.05). These results indicate that expression of antioxidant enzymes was not greatly affected in 8-cell-stage embryos exposed to HS. Thus, these results suggest the possibility that the early-stage embryos were stressed and damaged from heat shock because of their poor antioxidative potency.

Table 1.  Gene and protein expression of embryos

This work was supported by KAKENHI [16780209, Grant-in-Aid for Young Scientists (B)].