Resveratrol treatment during goat oocytes maturation enhances developmental competence of parthenogenetic and hand-made cloned blastocysts by modulating intracellular glutathione level and embryonic gene expression

Abstract

The aim of the present study was to determine whether supplementation of resveratrol, a stilbenoid antioxidant with therapeutic significance, influences goat (Capra hircus) oocyte maturation and subsequent embryonic development and expression of apoptosis and early embryonic development-related genes. Five different concentrations of resveratrol (0.1, 0.25, 0.5, 2.0 and 5.0 μM) were used in in vitro maturation (IVM) medium. Cell tracker blue and 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) fluorescent stains were used to assay intracellular glutathione and reactive oxygen species levels in mature oocytes. Parthenogenetic activation and hand-made cloning were performed to check the developmental potential following resveratrol treatment. We used quantitative real-time PCR to analyze embryonic gene expression. Compared to control, no significant improvement was observed in nuclear maturation in resveratrol-treated groups and at 5.0 μM concentration maturation rate decreased significantly (P < 0.05). But resveratrol treatment at the concentrations of 0.25, 0.5 μM significantly reduced intracellular ROS, and increased GSH concentrations. Oocytes treated with 0.25, 0.5 μM resveratrol when subsequently used for PA and HMC, higher extent of blastocyst yields were observed. Expression analysis of proapoptotic (Bax) gene in mature oocytes, cumulus cells, and HMC-derived blastocysts revealed lesser transcript abundances in various resveratrol-treated groups., however no change in the same was observed for antiapoptotic gene (Bcl2). Differential expression of genes associated with developmental competence and nuclear reprogramming was also observed in HMC-derived blastocysts. Our results show that resveratrol treatment at optimum concentrations (0.25 and 0.5 μM) during IVM produced beneficial microenvironment within oocytes by increasing the intracellular GSH, decreasing ROS level and this in turn, stimulated embryonic development and regulated gene expression.