The Role Of Bcl2l10 Gene In Mouse Preimplantation Embryo Development

Maternal factors are accumulated mRNA,proteins and small RNAs during oocyte production,which are involved in the regulation of sperm-oocyte fusion,chromatin remodeling and Maternal-to-Zygotic Transition during early embryo development.This experiment used single-cell transcriptome data to screen potential maternal genes and select one gene for knockdown validation.Bcl2l10 is an anti-apoptotic gene that is involved in oocyte maturation and may be involved in early embryonic development in mice.This experiment uses RNAi technology to knock down and study its role in early embryos.1.Simple analysis of single-cell transcriptome dataUsing the single-cell transcriptome data which from our laboratory to analyze during oocyte-blastocyst seven stage's embryos.First,we analyzed the expression patterns of 42 known maternal genes,according to its expression pattern,135 potential maternal genes were screened from 27238 differential genes.Finally,through the analysis of GO,Bcl2l10 was selected from these potential maternal genes for further research.2.Expression analysis of Bcl2l10 in oocytes and early embryosThe expression of Bcl2l10 in oocytes and early embryos was analyzed by qPCR.The results showed that the expression level was the highest in oocytes and pronuclei embryos,the 2-cell phase was decreased,and the expression level in 4-cell phase was sharply decreased.8-cell almost no expression was detected at the stage.The location of Bcl2l10 protein was analyzed by immunofluorescence technique,and the results showed: Bcl2l10 is localized to the cell membrane in the oocyte,1-cell and 2-cell stages,and later transferred to the cytoplasm for expression.3.The effect on the development of mouse preimplantation embryos after knockdown Bcl2l10Microinjection at the pronuclear embryo stage,and Bcl2l10 mRNA expression was inhibited at the 2-cell stage,while protein was inhibited at the 8-cell stage.Knockdown of Bcl2l10 resulted in a decrease in blastocyst development rate and a significant difference(P<0.01).At the same time,we used Morpholino to perform knockdown verification and get consistent results.The above results show that knocking down Bcl2l10 affects blastocyst development.Therefore,we examined the blastocyst marker genes Oct4,Cdx2,Nanog and Sox17.The results showed that the expression of Oct4,Nanog and Sox17 in the inner cell mass development genes were significantly decreased,indicating that Bcl2l10 is involved in the development of inner cell mass.Since the Bcl-2 family is involved in apoptosis,we examined the expression of the apoptosis-related genes Bax and Trp53.The results showed that the expression of Bax was not affected;while the expression of Trp53 in the blastocyst stage was significantly decreased.After knocking down Bcl2l10,the activity of mitochondria has also changed.This suggests that Bcl2l10 is involved in the mitochondrial apoptotic pathway and affects the expression of Trp53.In conclusion,Bcl2l10 has maternal genetic characteristics that affect the expression of Oct4,Nanog and Sox17,and participate in the development of inner cell mass.These results indicate that the maternal gene we screened has certain credibility and will provide new candidate genes for mammalian early embryo development research.