Exogenous Glutathione Promotes The Synthesis Of GSH Via ?-glutamyl Cycle In Early Bovine Embryos

A large amount of reactive oxygen species?ROS?was produced and oxidative stress occurs during in vitro culture of early embryos.Excessive ROS damage the redox balance of cells,which leads to DNA,protein damages and lipid peroxidation.Antioxidant GSH scavenges ROS,improves the embryos development rate and blastocyst quality.However,the mechanism of exogenous GSH to influence intracellular GSH level and improve the developmental rate of embryo is poorly understood.This study aims to reveal the mechanism of exogenous GSH promotes intracellular GSH synthesis and provide a foundation for improvement of embryo production efficiency in vitro.Experiment 1:Bovine IVF embryos treated with 3 mM GSH and GSX during in vitro culture for 7days were treatment groups,those cultured in media without GSH were control group.The rates of cleavage and blastocyst of embryos,the total cell number of blastocysts were calculated in three groups.The ROS and GSH contents in zygotes at different stages were detected by fluorescence staining and LC/MS.GSX was added in culture medium,and the changes of GSX content in zygotes were tracked by LC/MS.The results showed that GSH reduced significantly ROS content in embryos,and increased the blastocyst rate and the total cell number of blastocysts?P<0.05?.According to the data with fluorescence staining and LC/MS,GSH contents in zygotes of treatment group were significantly higher than that in control group?P<0.05?.Meanwhile,GSH contents in zygotes of both groups were decreased along with developmental process.After addition of GSX,GSH contents in zygotes of treatment group increased significantly?P<0.05?.However,GSX contents were low and only 1/5 of the total GSH.In addition,GSX contents in culture medium steadily decreased along with the culture time extension.Conclusively,exogenous GSH could scavenge ROS in embryos,improve embryo development rate and blastocyst quality.Moreover,LC/MS was a reliable method of quantitative analysis of GSH and isotope labeled GSH,and used to study the effects of exogenous GSH on intracellular GSH synthesis in embryo during in vitro culture.Experiment 2:Bovine IVF zygotes treated with 3 mM GSH during in vitro culture were treatment group,those cultured in media without GSH were control group.The mRNA expression levels of six genes associated with GSH synthesis?GGT,GGCT,5-OPase,GCLC,GCLM and GSS?in zygotes were determined and the activity of membrane protease GGT was detected.With GSH treatment,the expression levels of GSS,GGT and GCLM genes were significantly increased,and GGT activity was significantly increased too?P<0.05?.The expression levels of 5-OPase and GCLC genes in 24 hpi zygotes of treatment group were significantly lower than that in control group,and there was no significant difference between treatment group and control group at other time points?P>0.05?.Therefore,exogenous GSH promotes the mRNA levels and acitivity of enzymes related with GSH synthesis in?-glutamyl cycle of early bovine embryos.Experiment 3:Inhibitors of GCL and GGT enzymes,BSO and Acivicin,were added into the culture media of zygotes in control and GSX groups,respectively.The development rates of the embryo were calculated,as well as the intracellular GSX and GSH contents in zygotes at different stages of development.The activity of GCL and GGT enzymes was inhibited,and cleavage rate and blastocyst rate of embryos decreased significantly.Evenly,no blastocyst was obtained treated with Acivicin.The content of GSX and GSH decreased significantly in zygotes.Therefore,exogenous GSH improved the synthesis of intracellular GSH in embryo via the?-glutamyl cycle.In this study,isotope labeled GSH(GSH-¹³C₂¹⁵N-glycine,GSX)was added,Using LC/MS to explore GSH and GSX content in embryos,the gene expression level of GSH transportation-related enzymes was analyzed,and detected GGT activity in the zygotes,as well as the inhibition of key enzymes.The purpose of this study was to explore the synthesis pathway in embryo of exogenous GSH.