Effects Of Endogenous SiRNA On In Vitro Maturation Of Porcine Oocytes

The role of RNA interference(RNAi)in the regulation of gene expression has been revealed in recent years,and miRNAs(microRNAs)and endo-siRNAs(endogenous small tumor RNAs)play a crucial role in this process.MiRNAs are similar to endo-siRNAs in length.However,the biogenesis of these two small RNAs are different.The formation of MiRNAs requires not only DICER,but also DROSHA and its synergistic factor DiGeorge syndrome critical region 8(DGCR8).The formation of endo-siRNAs only requires DICER.Some studies have proved that endo-siRNAs,rather than miRNAs,play a significant role in the maturation of mouse oocytes.But the results were only shown in mice,and not yet in other species.Pigs and human have many similar characteristics in physiological structure,morphology and immunology.Therefore,this study focused on the effects of miRNAs and endo-siRNAs on in vitro maturation of pig oocytes.We microinjected LNA-DICER1,LNA-DROSHA and LNA-DGCR8 to the oocytes at GV stage to knock down DICER1,DROSHA and DGCR8 expression.After in vitro maturation,we detected the extrusion of the first polar body and themorphology of oocyte chromosome and spindleby immunofluorescence.The results showed that:(1)compared with the control group,the maturation rate of oocytes in LNA-DICER1 was significantly decreased,from 75.20%to 39.23%(p<0.05).The maturation rate of oocytes in LNA-DROSHA group and LNA-DGCR8 group was not significantly changed,68.71%and 71.25%respectively.(2)Compared with the control group,the chromosome and spindle morphology of oocytes in the LNA-DICER1 group were abnormal and disordered.The chromosome and spindle morphology were normal in LNA-DROSHA group and LNA-DGCR8 group.It indicates that the deletion of DICER1,rather than DROSHA or DGCR8,results in the failure of oocyte maturation.Next,the effects of DICER1 knockdown on apoptosis of pig oocytes were detected by Annexin-V and Western Blot.We found more positive signal of exogenous phosphatidylserine.In addition,the expression of p53 increased significantly.These results show that the lack of DICER1 can lead to the pig oocyte apoptosis,suggested endo-siRNAs have important effects on the in vitro maturation of porcine oocytes.Finally,after knockdown of DICER1,we analyzed the expression of small RNAs in oocyte through bioinformatics sequencing.The results showed that the expression of 22-24 nt small RNAs was significantly reduced after microinjection of LNA-DICER1.Therefore,we classified the small RNAs from the oocytes,and found that the total sequences of miRNA and endo-siRNA were significantly reducedafter DICER1 knockdown.After further analysis,we found that lack of DICER1 could lead to the decreaseof endo-siRNAs expression.In conclusion,this study performed the knockdown of DICER1,DROSHA and DGCR8 during maturation of porcine oocytes,and proved that the deficiency of endo-siRNAs,rather than miRNAs,resulted in the abnormal maturation of pig oocytes and apoptosis.The results provide a in-depth analysis of the specific regulation mechanism of endo-siRNAs on the maturation of porcine oocytes.