Mechanism Of Glutathione Supplementation In Culture Medium Improves The Bovine Embryo Development After In Vitro Fertilization

There is lots of reactive oxygen species(ROS) produced in embryos during in vitro culture(IVC). The balance between production and scavenge of intracellular ROS is kept normally, but the balance destroyed will result in the excessive accumulation of ROS, cell damage and affect the developmental capacity of embryos. Glutathione(GSH) is an antioxidant which can eliminate ROS, protect embryos from oxidative stress damage, promote the development of embryos during IVC and improve embryo quality. However, the mechanism that exogenous GSH is transferred into the IVC embryos and removes effectively ROS is not clear.Therefore methods in transcriptomics, embryo engineering, cell biology and molecular biology will be used in this study to reveal the transportation of GSH, its effects on ROS and transcriptome of IVC embryo.GSH(3m M) was added in the IVC medium of bovine IVF embryo for seven days. The rates of embryo at different stage and the total cell number of blastocysts were counted. Moreover intracellular ROS and GSH contents in embryos at 8 h, 18 h, 24 h, and 32 h after timed artificial insemination(AI) were evaluated. In addition, the enzyme activity and m RNA expression levels of genes associated with GSH transportation and synthesis(GGT, GGCT, 5-OPase, GCLC, GCLM and GSS) in zygotes were determinated. The results showed that the embryo development and blastocyst quality were improved, and the intracellular ROS contents were decreased with the intracellular GSH content increasing.Furthermore, the expression level of GGT gene increased and expression level of GCLC and GSS genes decreased in embryos at 32 h after AI. The expression level of GGCT declined and that of 5-OPase and GCLM genes increased in embryos at18 h after AI. And activity of four enzymes also underwent corresponding changes.The activity of γ-GCS was higher significantly and that of GGCT decreased significantly in embryos at 18 h after AI.(P<0.05). The activity of GGT was higher significantly and that of GSS decreased significantly in embryos at 32 h after AI.(P<0.05). In brief, supplementation of GSH increased significantly in vitro embryo development.Embryos at 8-16 cell stage and morula stage were collected for high-through-put deep sequencing with Illumina platform. Gene ontology and KEGG enrichment analysis were used to explore the functions and pathways of differential genes. According to analyzed 10 genes by quantitative real-time PCR performed for validation of RNA-seq data. Select three new transcripts to predict the structure of RNA and protein. The accuracy and validity of the data were confirmed with qualitative genes expression analysis, RNA-seq data quality(error rate of base, Q20, Q30, GC contents), mapping of reads to bovine genome, and distribution region analysis. Compared to known transcriptions, four new transcripts were found in different lengths of new exons. The predicted protein structure, in addition to containing the respective conserved domains(serine /threonine catalytic domain of protein kinases(S-TKc), MAD homology domain(MH1)), but also found two new protein domains(low complexity region(LCR),dwarfins B protein family domains(DWB)). Addition GSH can result in changes in transcriptional profiles of embryos in the culture medium, the ability to significantly improve the in vitro development of embryos.