Effect Of TET3 On Bovine Early Embryo Development

DNA methylation is a major epigenetic modification in the eukaryotic genome that regulates gene expression during many biological processes,including development.Dynamic changes in the DNA methylation landscape of the genome are essential for proper gene regulation and orchestration of various developmental processes.Both replication-dependent “passive” and replication-independent “active” models of DNA demethylation have been proposed.Whereas passive DNA demethylation can also occur through DNA replication-mediated dilution,active demethylation requires TET protein to remove the methyl groups involving components of the DNA repair machinery.In mouse,TET1 is highly expressed in PGCs and is required for demethylation of imprints and germline-specific genes.TET3 is abundantly present in zygotes,where it mediates active demethylation.However,whether by TET protein catalytic DNA demethylation,which is a member of TET family in bovine early embryonic development to play a leading role in the process of demethylation,as well as the correlation of TET and bovine embryo early development content is still unknow.Not all members of the Tet family participate in demethylation in the embryo,as shown by our mRNA abundance study.Only Tet2 and Tet3 were detectable in the early embryo with a predominance of Tet3,suggesting that Tet3 is one of the major actors of the reprogramming process,and that Tet2 is not.In this study,we utilized dimethyloxallyl glycine(DMOG),a small-molecule inhibitor of the TET protein,to impede the enzymatic activity of TET and explore subsequent effects on bovine early embryo development.Treatment with 1 mM DMOG increased 5mC levels at every bovine early embryo developmental stage.At the same time,DNA methylation level of satellite DNA and pluripotency gene promoters increased significantly.Real-time PCR analysis results indicated that the transcription levels of NANOG decreased in the DMOG-treated group.Furthermore,TET inhibition negatively affected blastocyst formation,resulting in a decline in the blastocyst rate(17.1 ± 1.3% vs 24.1 ± 0.6%);however,the percentage of apoptotic cells was significantly increased according to the results of a TUNEL assay.Additionally,expression levels of the apoptosis-related gene Bax were up-regulated,while the expression of Bcl-2 was down-regulated.In conclusion,these results support that TET plays important roles in bovine parthenogenetic embryo development by influencing DNA methylation reprogramming,gene expression and apoptosis.Since the birth of the first cloned animal,Dolly,somatic cell nuclear transfer(SCNT)has been successful in a variety of mammalian species.Cattle as a domestic animal has an important economic value in our country.SCNT technology has been established for many years,but the efficiency is still very low.One of the major causes of SCNT inefficiency is incomplete DNA methylation reprogramming of the donor cell nuclei,which subsequently leads to aberrant gene expression during embryo development.We adopt the strategy of overexpressing Tet3 gene in bovine fetal fibroblasts and then using this stably expressing cell line as a donor cell to perform somatic cell nuclear transfer to increase the DNA methylation reprogramming of donor cells.In this experiment,pHBLV-CMV-Tet3-3flag-EF1-ZsGreen-T2A-Puro overexpression vector was finally constructed.Fluorescence microscopy was used to detect the fibroblasts after screening.Compared with the blank group,both the overexpression group and the negative control group expressed green fluorescent protein and emitted strong green fluorescence.Quantitative real-time PCR analysis showed that Tet3 gene mRNA expression in transfected cells was 74.5 times that of control cells compared with non-overexpressed cells.To further examine the expression of the Tet3 gene in over expressing cells,we performed immunofluorescence and Western blotting to targrt the Flag-tagged proteins that recombine with the TET3 fusion protein.The results showed that they were detected in a bovine fibroblast cell line overexpressing Tet3.Positive signals for the Flag-tagged proteins were not detected in the blank group and the negative control group.This shows that we not only successfully transfected Tet3 gene into bovine fibroblasts using lentiviral vectors,but also efficiently expressed the target protein,which laid a certain foundation for the next step to improve the efficiency of bovine SCNT.