Effects Of BIX-01294 On Early Preimplantation Embryonic Development And Its Related Mechanisms In Mice

BackgroundEpigenetics is a bridge between genotype and phenotype,which allows genetic changes of gene expression without changing the underlying DNA sequence.Epigenetic changes play a very important role in the development and reproduction of mammals,and participate in normal development and specific gene expression process of mammals.As an important marking histone methylation site,histone H3 lysine 9(H3K9)takes part in gene transcriptional inhibition,and affects on the formation of heterochromation.Di-methylation of H3K9(H3K9me2),which plays a major role in transcriptional regulation,is a repressive histone mark related to the decrease of promoter activity.And there is a dynamic change of H3K9me2 during mouse zygote development.Previous studies have found asymmetric modification of H3K9me2 between paternal and maternal genomes.H3K9me2 is mainly regulated by two histone methyltransferases(HMT),G9a and G9a-like protein(GLP).G9a is an important HMT and it is the main methyltransferase of H3K9 and H3K27 methylation.With a classical SET domain,it can recruit a variety of transcription factors to inactivate downstream gene expression,resulting in inhibition of gene expression in euchromatin region.G9a-deficient mice displays growth retardation and early embryonic lethality,suggesting that H3K9 methylation mediated by G9a in euchromatin region is important for early embryogenesis.BIX-01294,a diazepin-quinazolin-amine derivative,is a small molecule that can selectively inhibit G9a/GLP.BIX-01294 is noncompetitive with the cofactor SAM and it was used in generating induced pluripotent stem(iPS)cells from mouse fetal neural precursor cells to improve the induction efficiency of induced pluripotent stem cells.BIX-01294 reduced H3K9me2 levels at several G9a target genes in mouse somatic cells and this change will be restored after the removal of inhibitors.However,the effect of BIX01294 in early preimplantation mouse embryos development and its mechanism are still unclear.In this experiment,mouse zygotes were selected as the study subjects.Therefore,Immunofluorescence staining,qRT-PCR were used to explore the effect of BIX01294 on the development of the preimplementation mouse embryos and its influence on the level of H3K9me2,the regulatory mechanism of H3K9me2 during early preimplantation embryonic development was also further explored.Objective:Explore the effect of BIX-01294 on the development of the preimplementation mouse embryos and its influence on the level of H3K9me2;explore the effect of G9a and H3K9me2 on the development of the preimplementation mouse embryos;study the effects and mechanisms of BIX-01294 on cells proliferation and apoptosis;study the role and mechanism of H3K9 methylation in the early preimplementation embryonic development in mice.Methods:Mouse zygotes were cultured with different concentration of BIX-01294(0,0.1,1,5,10?M)medium in vitro.2-cell,4-cell,morula and blastocyst stage embryo were collected from different groups to do detection.Immunofluorescence staining was used to explore the influence of BIX-01294 on the level of H3K9me2.qRT-PCR were used to explore the effects and mechanisms of BIX-01294 on cells proliferation and apoptosis.Results:1.Alteration of H3K9me2 in mouse zygotes.There is an asymmetric H3K9me2 pattern between paternal and maternal genomes.Compared with male genomes,female genomes showed high H3K9me2 levels in almost all stages.The H3K9me2 modification of male genomes of 2-cell embryos mitosis metaphase and previous zygotes is very low or none.At the 4-cell embryos,the parent genomes H3K9me2 level were similar.2.BIX-01294 stunts the development of the preimplementation mouse embryos.10?M BIX-01294 significantly stunt almost all embryos and few embryos survived.When the concentration was 5?M,almost all embryos are blocked at 2-cell stage.1?M BIX-01294 significantly reduced the morula formation and almost no embryos develop into blastula embryos,whereas 0.1?M BIX-01294 reduced blastocyst formation and had little effect on the embryo formation rate in other stages.3.The results of immunofluorescence staining showed that BrdU could be labeled by adding BIX-01294 at the concentration of 10 ?m and 5?m,which indicated that adding bix-01294 had no effect on DNA replication in early embryonic development.4.BIX-01294 reduces H3K9me2 levels in mouse embryos.Immunofluorescence staining was used to explore the influence of BIX-01294 on the level of H3K9me2.At the blastocyst stage,H3K9me2 levels were lower in embryos treated with 0.1?MBIX-01294 than in the untreated group(control group).5.The mRNA levels of apoptosis-related genes(capase-3,capase-8,capase-9)were significantly increased in the 1 ?M BIX-01294 group,suggesting that BIX01294 may affect mouse embryo development by affecting apoptosis.Conclusion:During early embryonic development of mice,asymmetric H3K9me2 modification existed in male and female pronucleus,which lasted until the metaphase of 2-cell mitosis,and increased in male pronucleus at 4-cell stage,which was similar to that in female pronucleus.BIX-01294 stunts the development of the preimplementation mouse embryos,high concentrations of BIX-01294 significantly blocked early embryonic development in mice,while low concentration of BIX01294 affected the expression of differentiation-and apoptosis-related genes.BIX01294 reduced H3K9me2 levels in mouse embryos,which suggested that BIX-01294 may affect mouse embryo development by affecting the levels of H3K9me2 and apoptosis.