139 OXIDATIVE STRESS INDUCED CHANGES OF GLUTATHIONE CONCENTRATION IN IN VITRO-PRODUCED BOVINE BLASTOCYSTS

Abstract

Glutathione (GSH) plays a critical role in the elimination of reactive oxygen species (ROS) by acting as substrate for GSH peroxidase (GPX) which results in the oxidation of GSH. Glutathione reductase (GSR) regenerates GSH from oxidized glutathione (GSSG). In bovine oocytes, GSH concentration increases and is thought to have beneficial effects on embryo development. The goal of the present study was to analyze the GSH content in bovine blastocysts during blastocoel expansion and to determine the effects of tertiary-butyl hydroperoxide (tBH) in causing oxidative stress. It is metabolized by GPX with conversion of GSH to GSSG. Blastocysts were generated with standard protocols of in vitro production (IVP) using SOF medium supplemented with FAF-BSA and were collected at Days 7/8 of culture (Day 0 = IVF). Diameters of blastocysts were measured and embryos were allocated to 4 groups: early blastocysts (ebla, less than 135 µm), blastocysts (bla, 135–180 µm), expanded blastocysts (exbla, greater than 180 µm), and hatched blastocysts (hbla). Pools of the different categories (3 replicates, 36 embryos in total) were frozen at -80^°C until assayed for GSH by HPLC. Total cell numbers were determined using the Hoechst 33342 stain. Blastocysts from the 4 groups were cultured together for 30 min in tBH (20 µM) containing SOF medium to induce oxidative stress. This was followed by another culture period for 1 or 8 h. Samples of blastocysts were frozen at -80^°C at 0 h, 30 min, 1 h, and 8 h to assess GSH levels (Cereser et al. 2001 J. Chromatogr. B. Biomed. Sci. Appl. 752, 123–132) and transcript abundance. Semi-quantitative real-time RT-PCR was used to determine the relative abundance (RA) of GPX and GSR transcripts. Differences between groups were tested using ANOVA followed by a Tukey or Student's test (P ≤ 0.05). The grade of expansion significantly increased GSH concentration in bovine blastocysts. Expanded blastocysts (0.38 ± 0.04 ng) and hatched blastocysts (0.46 ± 0.05 ng) had a higher GSH content than early blastocysts (0.21 ± 0.01 ng) and blastocysts (0.27 ± 0.02 ng). However, GSH concentration remained constant in blastomeres during expansion and hatching as the number of cells increased from 54.9 ± 3.5 (ebla) to 152.3 ± 4.7 (hbla). After induction of oxidative stress, blastocysts showed a significant decrease in GSH content after a 1-h recovery period, followed by an increase in their GSH content after a period of 8 h (0 h: 0.26 ± 0.01 ng; 30 min: 0.25 ± 0.01 ng; 1 h: 0.20 ± 0.01 ng; 8 h: 0.32 ± 0.01 ng, respectively). This was accompanied by a significant increase of the RA of GSR transcripts in blastocysts collected after the 8-h recovery period compared to embryos at the other time points of the treatment. GPX mRNA abundance remained stable during the entire culture period. In the present study, GSH concentration was determined in bovine embryos during blastocoel expansion for the first time. The increased GSH content might to some extent be due to de novo GSH synthesis. GSSG is reduced to GSH most likely by GSR. Results show that bovine IVP embryos may serve as a new model for the study of oxidative stress.