Study Of Exogenous Glutathione Enhance In Vitro Viability Of Ovine Oocytes And Improve The Development Abnormalities After Arsenic Exposure

The tripeptide,γ-L-glutamyl-L-cysteinyl-glycine known as glutathione（GSH）.GSH is an antioxidant capable of preventing damages caused by ROS and is an importantly cellular component,too.It has been demonstrated that the endogenous GSH can effectively protect bovine and porcine oocytes from oxidative stress,improving oocyte maturation.Thus,to investigate the effect of GSH on ovine oocyte maturation,4m M GSH was supplemented into oocyte maturation medium.Cumulus-oocyte complexes（COCs）were isolated from ovaries and the COCs were randomly divided into three groups according to layer numbers of cumulus cells surrounding each oocyte,namely,all oocytes in group A had at least 5 layers of cumulus cells,3-4 layers of cumulus cells in group B,whereas oocytes surrounded by fewer than three layers of cumulus cells were in group C.All oocytes in three groups were separately cultured in the conventional maturation medium supplemented with4m M GSH under the same culture conditions.Furthermore,the cytoplasmic and nuclear maturations in these oocytes were evaluated by immunocytochemistry.The results showed that exogenous GSH in the oocyte maturation medium could significantly improve nuclear and cytoplasmic maturations,especially for ovine oocyte maturation rate in groups B and C.Additionally,the oocytes matured in the medium supplemented with 4m M GSH showed an increase in mitochondria activity and in other anti-oxidative enzymes activities,respectively.In addition to these,addition of GSH into maturation medium also led a reduction of gene expression related to cell apoptosis in groups B and C compared to those in the control.Likewise,expressions of genes such as H3K4me3 and H3K9me3 in groups B and C elevated as well.Arsenic damaging cellular mitochondria has been well documented.,which could dysfunction damaged cells,especially for reproductive cells such as oocytes since the damaged mitochondria could lead accumulation of ROS in the cells.Moreover,to know the anti-oxidative effect of GSH on ovine oocyte maturation,ovine oocytes were exposure to Na As O₂,and then the harmful effects were determined by immunostaining to examine tublin,spindles,chromosomes,and mitochondria in the oocytes.Apparently,the results showed various abnormalities in the treated oocytes including spindle,chromosomes,and mitochondria,as well as a significant decrease in maturation rates.The evidence support arsenic is toxic to the treated ovine oocytes since the damaged mitochondria would be unable to reduce accumulated ROS in the oocytes.Therefore,when oocytes were co-treated with 4m M GSH and Na As O₂（arsenic）the oocytes were well protected with less damages by Na As O₂and the maturation rates were improved compared to the control.The results obviously demonstrated that GSH in ovine oocyte maturation medium could protect ovine oocytes from heavy metal toxicity during oocyte maturation.In conclusion,this study could provide a potential method to improve in vitro ovine embryo production and to overcome the problem with toxicity of heavy metals in ovine oocyte maturation medium.