| Although somatic cell nuclear transfer(SCNT)shows a wide range of application prospects,low efficiency is a bottleneck that restricts the application of the technology in production practice.The basic research in the previous research group showed that there were differences in the first cleavage time between SCNT embryos and IVF embryos.There was an abnormality in acetylation levels of α-tubulin and H3K9me3 in early SCNT embryos.Therefore,finding the regulatory factors that affect the development of cloned embryos has always been the study hot spots in the field.Recently,more and more studies have found that some non-genetic materials derived from sperm are crucial for early embryonic development and the absence of these substances may be an important cause of abnormal embryonic development of SCNT.In this study,miRNAs from bovine sperm were isolated using a One-Stop mi RNA Urea-PAGE Pack kit,and the extracted sperm miRNAs were injected into the SCNT embryos.The results showed that the first cleavage of SCNT embryos in the experimental group occurred later than the control group,and the blastocyst quality was improved.In order to further study the mechanism of the first cleavage time difference between SCNT and IVF embryos caused by sperm miRNAs,this experiment used immunofluorescence staining to detect effects of sperm miRNAs supplementation on cytoskeletal skeletal protein and DNA histone remodeling in SCNT embryos from both nuclear and cytoplasmic aspects.The results showed that after sperm miRNAs injection,the H3K9me3 methylation level of SCNT embryos decreased significantly in the pronuclear and 2-cell stage,and the α-tubulin acetylation levels at anaphase and telophase of the first cleavage increased significantly.Compared with the control group,the formation rate of two pronuclear-like structures and cleavage rate at 48 h of the sperm mi RNA-supplemented group significantly increased,and the time of first cleavage was prolonged.The blastocyst rate on the 7th day was significantly increased and the apoptosis was decreased.These results suggest that sperm miRNAs may regulate embryonic first cleavage and the development of embryo by affecting the apparent modification of DNA histones and embryonic cytoskeletal proteins simultaneously.This study shows that a series of developmental events in the pronuclear phase are critical to the subsequent development of early SCNT embryos.Sperm miRNAs may play an important role in the regulation of embryonic development during this period.The development of this research provides new ideas for improving the efficiency of SCNT.. |
