Preliminary Study Of Sperm-borne Bta-mir-301a On Early Embryonic Development In Bovine

Spermatozoa have a highly concentrated genetic material.Since its RNA content is significantly lower than that of oocytes,most researchers neglected the function of sperm RNA and focused on oocyte RNA.Analysis of RNA constitution shows that sperm RNA is mainly composed of mRNAs,miRNAs,tRNAs,and siRNAs.These RNAs carried by sperm could enter the oocyte during fertilization.It is suggested that sperm-borne miRNAs and endo-siRNAs may play an important role in embryonic development.Therefore,it is very necessary to deeply study the function of sperm-borne miRNAs to improve our understanding of early embryo development events and their mechanisms.According to the miRNA sequencing data in the published article and the small RNA library constructed by our group,bta-miR-301 a was screened as subsequent research.And then,it has been confirmed that bta-miR-301 a is sperm-borne miRNA by quantitative real-time PCR assays.Multiple databases were combined to predict target genes of miR-301 a,crossed target genes were used for pathways and functions analysis,result showed that they are more likely to participate TGF-? pathway.Then predicted genes in TGF-? pathway were selected for subsequent studies.After aligning the sequences of target genes among species,querying the integrity of mRNA sequence,detecting the expression levels of target genes in MII oocyte,and conducting Dual-Luciferase Reporter Assay,ACVR1 is confirmed as a target gene of miR-301 a eventually.The results of RT-qPCR showed the expression of bta-mi R-301 a was significantly decreased during the development of pronuclear to 2-cell stage in in vitro fertilization group.The expression trends of ACVR1 in parthenogenetic group;nuclear transplantation group and in vitro fertilization group embryos were different.During the stage of MII oocyte to 2 cell,the expression of ACVR1 was significantly decreased in in vitro fertilization group,but the expression of ACVR1 was significantly increased in the somatic nuclear transfer group and parthenogenetic group.Therefore,it was speculated that the miR-301 a downregulated the expression of ACVR1 in in vitro fertilization.To determine the effect of miR-301 a on bovine early embryonic development,miR-301 a mimic was microinjected in parthenogenetically activated oocytes.ACVR1 protein in 2-cell stage of the injected group was significantly reduced compared with the control group.The first cleavage time of the injected embryos was prolonged and the blastocyst rate was increased.Therefore,it is speculated that miR-301 a could target ACVR1 at the 2-cell stage and prolong the first cleavage time,which may affect blastocyst development.In conclusion,this study preliminarily confirmed that sperm-borne miR-301 a can affect early embryonic development process in bovine.