| Sperm is a highly concentrated cell of genetic material,which can form a complete individual with oocytes.In addition to genetic material,sperm can also carry proteins,coding RNA,non-coding RNA etc such as miRNAs.into the oocyte,affecting early embryo development.In the early results of the laboratory,bovine sperm-derived miR-301 a was screened by high-throughput sequencing of sperm and oocytes.In vitro acquired experiments confirmed that it was involved in the first cleavage and blastocyst formation of parthenogenetic activation(PA)embryos.At the same time,ACVR1 was identified as the target gene of miR-301 a.The purpose of this study is to investigate the role of miR-301 a in the regulation of early embryonic development and its molecular mechanism by targeting ACVR1.This study carried out the following experiments:(1)To determine the targeted regulatory relationship between miR-301 a and ACVR1 in embryos;(2)To explore the effect of knockdown ACVR1 on early embryonic development of cattle.(3)Initially explore the mechanism of miR-301 a targeting ACVR1 to regulate early embryo development.The results are as follows:1.Somatic Cell Nuclear Transfer(SCNT)embryo microinjection miR-301 a mimic or in vitro fertilization(In Vitro Fertilization,IVF)embryo microinjection miR-301 a inhibitor to detect ACVR1 m RNA and protein expression,and find that miR-301 a could down-regulate the expression of ACVR1 regardless of m RNA or protein levels,verifying the targeted regulatory relationship between miR-301 a and ACVR1.2.PA embryo microinjection si RNA to knockdown ACVR1,and detect embryo development.The results showed that after knocking down ACVR1,the first cleavage time of PA embryos was delayed,but the total cleavage rate was not affected,and the blastocyst rate and blastocyst quality of PA embryos were significantly improved.This result is the same as the mimic phenotype of miR-301 a injected into PA embryos,indicating that miR-301 a regulates the first cleavage and blastocyst development of the early bovine embryos by targeting ACVR1.3.Detect the expression of TMP-β pathway ligand BMP4 and receptors other than ACVR1 in embryos,and speculated that BMPR2 is the most likely type II receptor to interact with ACVR14.After knocking down ACVR1 in PA embryos,the phosphorylation level of Smad1 /5 and Smad2 protein in the downstream signal transduction of TGF-β pathway is reduced,and the m RNA expression of target genes ID1 and ID2 is down-regulated.SCNT embryos injected with miR-301 a mimic can also cause the phosphorylation level of Smad1/5 and Smad2 protein to decrease,however,IVF embryos injected miR-301 a inhibitor caused the phosphorylation level of these two proteins to increase.The miR-301 a targeting ACVR1 is involved in the activation of TGF-β signaling pathway5.After the SCNT embryo injection of miR-301 a mimic,the nuclear H3K27me3 fluorescence intensity increases at 2-cell stage,and there is no significant difference after the IVF embryo injection of miR-301 a inhibitor,indicating that miR-301 a can affect histone H3K27me3 modification.6.PA embryo knockdown of ACVR1 reduces the fluorescence intensity of F-actin,but the reverse is the case after IVF embryos lack miR-301 a,suggesting that miR-301 a can affect the dynamic changes of microfilament skeleton through ACVR1.Further research find that the change of F-actin in IVF embryos is mediated by p-Cofilin1 protein.In summary,sperm-derived miR-301 a can target ACVR1 to regulate the activation of TGF-β pathway,and affect the dynamic changes of microfilament skeleton and histone H3K27me3 modification,and ultimately prolong the first cleavage time and increases the blastocyst rate. |
