Expression Of MCRS1,CDX2,NANOG In Endothelial Cells And Trophoblast Cells Of Bovine Blastocyst After In Vitro Fertilization And Nuclear Transfer

Placental dysplasia is the main reason for the failure of development in vivo of cloned cattle.The first cell differentiation of mammalian early embryos is extremely complex and influenced by a variety of regulatory factors,among which MCRS1,CDX2 and NANOG play a very important role in the development of embryos.MCRS1 is mainly involved in cell proliferation and transformation.The main role of CDX2 in embryo development is to maintain embryo growth,induce cell differentiation and organ formation.NANOG gene plays a key role in the self-renewal of embryonic stem cells and the maintenance of pluripotency in early embryos,and it is considered to be an important marker of universal or pluripotent stem cells.At present,studies about the expression patterns and functions of MCRS1,CDX2,and NANOG cells during In vitro fertilization(IVF)and nuclear transplantation of intracytoblastocyst(ICM)and trophoblast(TE)cells are not perfect,mainly because the intracytoblastocyst and trophoblast cells are not easily separated for specific assays.In this study,IVF and nuclear transfer blastocysts were obtained by in vitro fertilization and nuclear transplantation,and then ICM and TE cells of blastocysts were isolated and purified by magnetic bead sorting.The mRNA expression patterns and differences of MCRS1,CDX2 and NANOG in ICM and TE cells of the two blastocysts were further studied by qrt-pcr,and the effects of MCRS1,CDX2 and NANOG on the growth and development of bovine embryos and cell differentiation in the early embryonic development were analyzed.The results are as follows:1.ICM and TE cells in blastocysts were separated by magnetic bead sorting technology.The two cells were labeled with cona-fitc and Hoescht 33342.The results showed that the method was effective in separating two kinds of cells.The results of statistical analysis showed that the purity of ICM was 89.9%±1.0%,and that of TE cells was 90.3%±1.0%,indicating that the established sorting method could be used for subsequent tests,laying a foundation for the subsequent study of gene expression in ICM and TE cells.2.Fluorescence quantitative PCR results showed that the relative expressions of MCRS1 mRNA and CDX2 mRNA in TE cells were significantly higher than those in ICM cells in IVF blastocysts.NANOG mRNA showed the opposite result,and its relative expression level in ICM cells was significantly higher than that in TE cells.3.In nuclear blastocyst transplantation,fluorescence quantitative PCR results showed that the relative expression of MCRS1 mRNA in TE cells was significantly higher than that in ICM cells,but significantly lower than that in IVF.The relative expression of CDX2 mRNA in TE cells was significantly higher than that in ICM cells,but it was not significantly different from that in IVF blastocyst.The expression of NANOG mRNA in ICM cells was significantly higher than that in TE cells,but slightly lower than that in IVF blastocyst.Conclusion: at the early stage of embryo development,blastocysts were differentiated for the first time.MCRS1,CDX2 and NANOG genes were differentially expressed in ICM and TE cells.MCRS1 and CDX2 mRNA in IVF and nuclear transfer relative expression of TE cells were higher than in the blastocyst ICM cells,the former relative expression in nuclear transplantation blastula was lower than those of IVF,CDX2 mRNA in two kinds of blastocyst has no obvious expression differences,indicates that MCRS1 and CDX2 to the cow trophoblastic cells split in early embryonic development plays an important role,MCRS1 has important effects on nuclear transfer embryonic development.The relative expression of NANOG mRNA in bovine IVF and nuclear transfer blastocyst ICM cells was significantly higher than that in TE cells,and the relative expression of NANOG mRNA in nuclear transfer blastocyst was lower than that in IVF blastocyst,suggesting that NANOG plays a key role in maintaining the pluripotency of embryonic cells and dividing into embryo bodies.