| After the birth of the cloned sheep "Dolly",somatic cell nuclear transfer(SCNT)has made great progress,and the offspring have been successfully obtained in many species.However,compared with in vitro fertilization(IVF)embryos,the in vivo development of SCNT embryos and the health status of individuals after birth have not been significantly improved,which has become a bottleneck restricting the widespread application of this technology in actual production.The inability of somatic cells to be completely reprogrammed in oocytes may be the main reason for the subsequent development of cloned embryos.Therefore,for a long time,most studies have sought to improve the efficiency of SCNTs and focus on the reprogramming factors in oocytes.In recent years,with the in-depth study of sperm function,it has been found that a small amount of proteins,m RNAs,miRNAs and other non-coding RNAs introduced by sperm during fertilization have an important influence on embryonic developmental ability.By screening the bovine sperm miRNAs expression library established in the previous period,our team found that miR-202 is a specific and highly expressed miRNA in bovine sperm,which has a significant effect on the development of early embryos.At the same time,the online prediction of miRNA target genes and the double luciferase reporter experiment indicated that SEPT7 is a target gene of sperm-borne miR-202 in fertilized embryos,but it is still unclear whether miR-202 plays a role in the development of bovine early embryos through regulation of SEPT7..Based on the different mechanisms and development of SCNT and IVF,this study first explored the relationship between miR-202 and the first cleavage time and embryonic debris production using SCNT embryos.The results showed that as a sperm-specific expression of miRNA,miR-202 plays a key role in the first cleavage time of SCNT embryos and embryogenesis during cleavage.In order to further clarify the downstream target genes directly regulated by miR-202 in this process,we injected SEPT7 si RNA into SCNT reconstructed embryos,and the results showed that the changes of the first cleavage time and fragment rate were consistent with those injected with miR-202 mimic when SEPT7 was deleted in SCNT embryo,suggesting that miR-202 plays a role in the regulation of SEPT7 in the first cleavage of bovine SCNT embryos.In vitro study of neurogenesis in mice,SEPT7 can negatively regulate the stability of microtubules through HDAC6-mediated deacetylation and promote the development of dendrites and axons,but whether there is a physical relationship between SEPT7 and HDAC6 in bovine early embryos.No reports have been reported yet.Therefore,we used proximity ligation assay(PLA)technology to explore the relationship between SEPT7 and HDAC6 during the first cleavage of SCNT and IVF embryos.The results confirmed that there is an interaction between HDAC6 and SEPT7 from the metaphase of first cleavage until the end of cytoplasmic division.Since miR-202 is an sperm-borne miRNA,it is expressed in IVF embryos but not in SCNT embryos.In order to further explore the role of miR-202 in bovine embryo development,we microinjected miR-202 inhibitor into the zygotes,and the results showed that miR-202 inhibitor can up-regulate the expression of SEPT7 by inhibiting miR-202 in bovine IVF embryos,and significantly reduce the cleavage rate and blastocyst formation of embryos while accelerating the first cleavage of embryos,the cleavage rate,blastocyst rate and blastocyst quality.This effect can be improved with simultaneous injection of SEPT7 si RNA.Deletion of miR-202 in IVF embryos would result in chromosome misalignment and reduced cytoskeletal stability in the first metaphase of cleavage.In addition,changes of SEPT7 expression did not affect the expression of HDAC6,suggesting that it might regulate the acetylation level of α-tubulin by affecting the activity of HDAC6.Although the mechanisms of IVF and SCNT embryo formation and development is different,considering that microinjection of miR-202 inhibitor can significantly accelerate the time of first cleavage of IVF embryos,change the cytoskeleton during IVF embryo cleavage,and influence the development rate and the final blastocyst quality,we injected miR-202 mimic into the SCNT reconstructed embryo,and the results confirmed that miR-202 can improve the phenomena of chromosome misarrangement and cytoskeleton instability,delay the first cleavage of embryo and significantly increase the developmental potential of embryo.Similar to IVF embryos,the existence of miR-202 inhibited the expression of SEPT7,and allowed the acetylation α-tubulin accumulation in SCNT embryos without affecting the expression of HDAC6.In summary,during the development of bovine early embryos,the sperm-borne miR-202 could regulate the activity of HDAC6 through the target gene SEPT7,and affect the acetylation level of the skeleton protein α-tubulin and the initiation of cytokinesis during the first division,so as to ensure the normal division of cells and the final blastocyst formation. |
