A Preliminary Study Of Bovine Sperm-borne MiR-202 On Early Embryonic Development

Sperm is a kind of very specialized cells.The traditional viewindicated that the main function of sperm isa carrier of paternal genetic material andto activateembryo development following fertilization. The study has found that in the process of fertilization substances,which includemRNAs, microRNAs, tRNAs and siRNAs,carried intothe oocyteplay a vital role on the formation of the male and female pronuclei, oocyte activation, blastomere division order of time and space, embryonic development and the phenotype.MicroRNA,a single strand non-coding RNA,wasfound as a kind of endogenesis in plants, animals and some viruses and function in degrading or inhibiting genes expressionvia the base pairing with 3?-UTRbinding region of target gene(s).However, the function of sperm-bornemicroRNA in fertilization and embryo developmentremains unknown.Therefore, studying the composition taken with sperm during fertilizationshould be helpful to understand the development of early embryos.In this researchgroup,next generation sequencingwas used to construct a small RNA Library of bovine mature sperm, oocytes and bovine fibroblast cells, whichwas validated by real-time quantitative PCR approach. With the help of bioinformatics analysis, sperm-borne and high expressivemicroRNA(Bta-miR-202)wasselected.Furthermore,adopting to predict and intersect with certain online databases, a seed sequence which combined with Bta-miR-202 was identified.13 candidate target genes which were searched from NCBI containing the seed sequence were selected.A number of experiments were conducted to discover the association between SEPT7 and miR-202. Dual luciferase reporter assay showed3?-UTR region of wild type SEPT7 gene was inhibited by miR-202 mimic. When point mutation introduced in the 3?-UTR, the inhibition was weakened. In addition, quantitative PCRfound thatmiR-202 was able to lowerSEPT7 mRNA. Western Blot determined that miR-202 suppressed SEPT7 in translational regulation.To sum up, SEPT7 gene is target genes of miR-202.Embryo cleavage rate increased significantly alone with SEPT7 negatively expressedwhen microinjecting miR-202 into oocytes. Quantitative PCRresults ofparthenogenetic and in vitro fertilizationsuggestedthe reduction of SEPT7 expression wasmighty notableafter fertilizationcompared with MⅡstage.During each period of in vitro fertilization, the SEPT7 and miR-202 had a relationship of negatively correlation,which suggestedthe negative feedback mechanism.In summary, this researchcombed the microRNAs of sperm-borne and the high expression.Also, a series of bioinformatics tools and molecular biotechnologypredicted and verifiedSEPT7 was a target gene of the miR-202. Microinjectionand quantitative results found mi R-202 hada major impact on the oocyte cleavage, andSEPT7 changedsignificantlyin the embryos after fertilization, which indicatedthenegative feedback regulation mechanism.All in all,miR-202 as sperm-borne and high expressivemicroRNAhas played an important influence on early embryo development.