Effects Of Sheep Sperm MiRNAs On H3k9me3 In Early Cloned Embryos

Object: An important reason affecting the success rate of somatic cloning technology is the abnormal genome reprogramming of early cloned embryos,which is caused by the lack of miRNA in sperm.In order to determine the role of miRNA and spermatogenic miR-449 b on early cloned embryo development,this study added miRNA and miR-449 b to early cloned embryos of sheep and improved the rate of clonal embryo development.Methods: Fresh sperm was collected,purified by Percoll density gradient centrifugation and PBS solution,extracted total RNA by 65℃ TRIzol,kit and TRIzol binding method,reverse transcribed c DNA,amplified PRM1 m RNA and CDH1 m RNA,15% urea denaturing polyacrylamide gel,recovered sheep sperm miRNA,and verified sperm specific miR-34 c and miR-449 b by q RT-PCR.Through the method of microinjection of sperm miRNA,control mimic into somatic cloned embryos,with normal cloned embryos and in vitro fertilized embryos as a control,developmental statistical analysis of early embryos,collect prokaryotic stage,2 cells,4 cells,8 cells and blastocyst stage early embryos,using immunofluorescence staining to compare the change of histone H3K9me3.Collect purified sheep sperm,mature oocytes and fibroblasts,compare the relative expression of miR-449 b in sheep sperm and miR-449 b by real-time PCR,conduct developmental statistical analysis of early embryos,collect prokaryotic stage,2 cells,4 cells,8cells and blastocyst embryos by immunofluorescence staining.Results: Compared with direct washing with PBS solution,sperm purified by Percoll density gradient centrifugation have higher vitality and no other cell contamination;65℃ TRIzol method and kit RNA can extract PRM1 band,CDH1,but miRNA separation cannot be achieved after purification of the kit.Sheep sperm miRNA was successfully isolated on a high-concentration polyacrylamide gel,and specific miR-449 b and miR-34 c were successfully amplified to obtain sperm miRNA that could be used for subsequent experiments.The cleavage rate of the miRNA injection group was significantly higher than that of the control injection group,significantly higher than that of the normal cloned embryo group,and the cleavage rate was statistically different from that of the in vitro fertilization group;The blastocyst rate of the miRNA injection group was significantly higher than that of the control group and the normal cloned embryo group,which was statistically different from that of the in vitro fertilization group.It promoted the early development of cloned embryos,reduced the histone H3K9me3 level of cloned embryos at prokaryotic stage,2 cell stage,4 cell stage,8 cell stage and blastocyst stage,and reduced the abnormal reprogramming of cloned embryos.The expression of miR-449 b in sperm was significantly higher than that in mature oocytes and fibroblasts;The cleavage rate of the IVF group was significantly different from that of the miR-449 b injection group,which was significantly higher than that of the normal cloned embryo group and extremely significantly higher than that of the control group;The blastocyst rate in the miR-449 b group was significantly higher than that in the control group and the normal cloned embryo group,and the cleavage rate was statistical different from the in vitro fertilization group,promoting the early development of cloned embryos,reducing the histone H3K9me3 levels of prokaryotic,2,4,8,and blastocyst stages,and promoting the reprogramming of cloned embryos.Conclusion: In this study,sperm miRNA and miR-449 b increased cleavage and blastocyst rates,and significantly reduced histone H3K9me3 methylation levels,not significantly different from in vitro fertilized embryos,which provided a reference for studying the effect of sheep sperm-derived miRNA on the development of early cloned embryos.