Effect of exogenous glutathione supplementation on the in vitro developmental competence of ovine oocytes

Abstract

The beneficial effect of glutathione (GSH) on the in vitro maturation (IVM) of bovine/porcine oocytes has been confirmed; however, the antioxidant effect of exogenous GSH supplementation on the IVM of ovine oocytes has not been determined. In this study, ovine cumulus oocyte complexes (COCs) were classified into three groups according to the layer number of cumulus cells (the Grade A group has more than five layers, the Grade B group has three to four layers and the Grade C group has less than three layers). After in vitro culture of COCs in the presence of exogenous GSH, the meiotic competence of ovine oocytes was assessed by analyzing nuclear maturation to metaphase II (MII) stage, cortical granules (CGs) dynamics, astacin like metalloendopeptidase (ASTL) distribution, histone methylation pattern, reactive oxygen species (ROS) production, mitochondrial activities and genes expression. After in vitro fertilization (IVF), assessments of embryonic development were conducted to confirm the effects of exogenous GSH supplementation. The results showed that exogenous GSH not only enhanced the maturation rates of the Grade B and Grade C groups but also promoted CGs dynamics and ASTL distribution of the Grade A, B and C groups (p < 0.05). Exogenous GSH increased the mitochondrial activities of the Grade A, B and C groups and decreased the ROS production levels of oocytes (p < 0.05), regardless of the layer number of cumulus cells. Moreover, exogenous GSH promoted the expression levels of genes related with oocyte maturation, antioxidant activity and antiapoptotic effects in the Grade B and Grade C groups (p < 0.05). The expression levels of H3K4me3 and H3K9me3 in the Grade B and Grade C groups were promoted after exogenous GSH supplementation (p < 0.05), consistent with the expression levels of genes related with histone methylation (p < 0.05). In addition, exogenous GSH strongly promoted the embryonic developmental competence of Grade B and Grade C groups (p < 0.05). Taken together, our findings provide foundational evidence for the free radical scavenging potential of exogenous GSH in the in vitro developmental competence of ovine oocytes, especially oocytes from COCs lacking cumulus cells. These findings, which demonstrated the potential for improving the quality of ovine oocytes during IVM, will contribute to researches on GSH applications and the efficiency of assisted reproductive technology for ovine breeding.

Keywords: Embryonic development; Exogenous GSH; IVM; Ovine oocytes; Reactive oxygen species.