The Regulation Of Ezrin By Sperm-Borne MiR-183 On The Structure Of Microvilli And Development Of Quality In Early Bovine Embryos

Mature sperm contain both coding and non-coding RNAs,which are carried into the oocyte during fertilization.More and more evidences show that sperm miRNAs,as small endogenous non-coding RNAs with regulatory functions,can specifically bind to the 3’ untranslated region of the target genes in oocytes,degrade or inhibit the translation of the target gene.Thus,it has an important influence on the epigenetic reprogramming,epigenetics,embryonic development and the phenotype of offspring after fertilization.Our previous studies found that the specific high expression of miRNA-183 in bovine sperm plays an important role in the development of early embryos,and through bioinformatics analysis and double fluorescein experiments,Ezrin was identified as a target gene for its function in early embryos.On this basis,this study mainly carried out the following three aspects:(1)to determine the regulatory relationship between miRNA-183 and the target gene Ezrin in early embryo;(2)to clarify the specific phenotype of early embryos by knockdown and over-expression experiments;(3)to preliminarily explore the regulatory mechanism of miRNA-183 on early embryonic development.The main research results are as follows:1.Nuclear transplanted embryos were injected with miRNA-183 mimic and NC miRNA-183 mimic by microinjection.At the same time,EZR siRNA and EZR siRNA NC were microinjected into nuclear transfer embryos as controls to verify the targeting regulation relationship between miRNA-183 and its target gene Ezrin at the RNA and protein levels.It was found that the effect of EZR siRNA injection was similar to that of miRNA-183 mimic.Meanwhile,qPCR and Western blot experiments showed that miRNA-183 could regulate the expression of Ezrin in both mRNA and protein.2.Functional gain experiments showed that miRNA-183 significantly increased the cleavage rate(76.63% vs.64.32%,P<0.05)and blastocyst rate(43.75% vs.28.26%,P<0.05)of boine cell nuclear transfer embryos,reduced the apoptosis rate of blastocyst on the seventh day(5.21% vs.12.64%,P<0.05),and increased the proportion of cell group trophoblast in blastocyst on the seventh day(32.65% vs.25.58%,P<0.05).3.Scanning electron microscopy revealed that the surface microvilli density of SCNT embryos was significantly higher than that of IVF embryos,and the surface microvilli density of embryos microinjected with miRNA-183 mimic was significantly reduced.Injection of EZR-siRNA also had the same effect,and the results showed that miRNA-183 may regulate the formation of early embryonic microvilli through the target gene Ezrin,thus ultimately affecting the development potential of the embryo.4.Bioinformatics was used to predict and screen the proteins associated with the function of microvilli and the interaction with Ezrin.Co-IP coupled mass spectrometry was used to screen the proteins.This laid a foundation for further research on the mechanism of miRNA-183 regulating the formation of early embryonic microvilli through its target gene Ezrin.Above all,on the basis of previous studies,this study further defined the miRNA-183 and its target genes regulating the relations of Ezrin,studied its bovine SCNT embryos development and the influence of microvilli formed,and screened with Ezrin protein,for further study on the subsequent miRNA-183 through its target genes Ezrin regulating the embryo of the early embryonic development mechanism.