242 GLUTATHIONE CONTENT AND EXPRESSION OF ANTIOXIDANT ENZYME MRNAS IN SHEEP OOCYTES

Abstract

Reactive oxygen species (ROS) are normal products of embryo metabolism;; however, oxidative injury may negatively impact development if ROS production exeeds antioxidant defense mechanisms. Protection of the embryo against ROS is dependent, in part, upon the pool of antioxidant enzyme mRNA and products stored in the oocyte. Previous work has demonstrated that retinol administration to superovulated ewes, followed by natural service, resulted in embryos with improved competence to develop in vitro. In other cell systems, retinoids have been shown to participate in the antioxidant network and redox system. The objectives of this study were to analyze glutathione content and characterize antioxidant enzyme mRNA expression in mature ovine oocytes and test the hypothesis that retinol administration affects levels of these products. Ewes were superovulated and administered retinol on the first and last day of FSH injection. Oocytes were collected from oviducts 60 h later and either prepared for glutathione (GSH) analysis or subjected to RNA isolation. Glutathione was quantified by the recycling assay of the enzyme 5,5-dithiobis-(2-nitrobenzoic acid)-glutathione reductase using a total of more than 260 oocytes. Transcripts encoding for manganese superoxide dismutase (Mn-SOD), copper zinc superoxide dismutase (Cu-Zn SOD), glutathione synthetase (GS) and glutathione transferase pi (GSTp) were analyzed in a total of 86 single oocytes by semi-quantitative RT-PCR using a histone H2a transcript as the internal control. Chi-square analysis was performed to detect differences due to treatments. Glutathione content did not differ significantly between oocytes collected from retinol-treated (6.78 ± 3.81 pmol/oocyte) and control (6.38 ± 1.58 pmol/oocyte) ewes. Transcripts encoding for Mn-SOD, Cu-Zn SOD, GS and GSTp were detected in greater than 95% of the oocytes analyzed. Relative abundance of these transcripts did not differ between treatments. For the first time, glutathione content and antioxidant enzyme mRNA expression has been analyzed in in vivo-matured sheep oocytes. Retinol treatment did not appear to affect the expression of these products. Results demonstrate the existence of endogenous antioxidant defense products, stored as mRNA and protein, in sheep oocytes.