Effects Of Sperm-borne MiR-2285n On Development Of Early Embryo In Bovine

Sperm is a cell of highly concentrated cytoplasm.Because the genetic material carried by the sperm is far less than that in the oocyte,the research on embryonic development mostly focuses on the egg-derived material,while ignoring the crucial effects on early embryonic development of the protein,coding RNAs and non-coding RNAs(such as mi RNAs)carried by the sperm during fertilization.Our previous studies have shown that sperm-derived mi RNAs play a crucial role in regulating the development of post-fertilization embryos.Several highly expressed small RNAs including mi R-2285 n,mi R-202 and mi R-182 were found by small RNAs sequencing.Their effect on early embryonic development needs further study.The main purpose of this experiment was to explore the role of sperm-specific and highly expressed bta-mi R-2285 n in early bovine embryos.The main experiments and results of the research are as follows:1.Bta-mi R-2285 n specifically expressed in sperm: Combined with the small RNA library established by my laboratory,mi R-2285 n was determined to be a sperm-born mi RNA by the mi RNA quantitative method2.Prediction and primary screening of the target genes of mi R-2285n: By using online database and bioinformatics,the target genes of mi R-2285 n in fertilized eggs were predicted.Combined with the gene expression profiling analysis in MII stage oocytes,CTTN and PFN1 were preliminarily screened as mi R-2285n’s target genes in fertilized eggs.3.PFN1 is the target gene of bta-mi R-2285 n.First,by doing the dual-luciferase reporter gene experiment,PFN1 is verified to be the target gene at the gene binding level.Then,this experiment used Lip 3000 to co-transfect the plasmid and mi RNA mimic into 293 T cells for transfection.The co-transfection group of transfection plasmid and negative control mimic was taken as control group.We found that the fluorescence intensity of CTTN and PFN1 were significantly decreased(P < 0.01).Next,we verified the target gene on the m RNA transcription level by real-time quantitative PCR: the plasmids and negative control mimic/mi R-2285 n mimic/mi R-2285 n inhibitor were respectively co-transfected into bovine fibroblasts,the result is that the expression level of CTTN and PFN1 in mi R-2285 n mimic transfected group were decreased(P < 0.01).Finally,we used Western Blot to verify the target gene on the protein translation level: compared with the control group,the protein expression level of PFN1 in the mi R-2285 n mimic electro-transfection group was significantly reduced,proving that PFN1 is the target gene of mi R-2285 n.4.The effect of PFN1 on early bovine embryonic development: the target gene mi R-2285 n mimic was injected into embryos in different concentration,1 μM was selected as the optimal injection of mi R-2285 n mimic by q PCR.Compared with the NC injection group,PFN1 expression level in the 2-cell and 4-cell stage parthenogenetic embryos injected with1 μM mi R-2285 n mimic group was significantly decreased(P < 0.05)and was closer to the expression levels in IVF 2-cell stage embryos.After injection,the blastocyst rate was significantly increased(P < 0.05),and the number of blastocyst apoptotic cells was significantly decreased(P < 0.05).Finally,the experiment used phalloidin to stain F-actin.In2-cell and 4-cell stage PA embryos,the fluorescence intensity of F-actin in the mi R-2285 n mimic group was stronger than that in the NC group(P < 0.05)which demonstrated that PFN1 could affects cell division by affecting the actin cytoskeleton.In conclusion,the high expression of sperm-derived bta-mi R-2285 n regulates the actin skeleton by targeting PFN1,which affects cell division after fertilization then regulates the development of early bovine embryos.