Effects And Mechanisms Of Mfn2on The Development Of Preimplantation Embryos In Mouse

Objectives: The interference sequence (Small interfering RNA, siRNA) of Mfn2(Mitofusin2, Mfn2) in mouse cells, and the best transfection concentration of siRNAare determined.Methods: Transfect20nM、30nM、50nM and100nM siRNA labeled Cy3redfluorescent into TM4cells and then observe by fluorescence microscopy respectivelyto screen the optimal transfection concentration. We transfect three pre-selectedMfn2-siRNA into TM4cells by Lipofectamine2000, and detecte the relative quantityof Mfn2in transfected cells by qRT-PCR.Results: In Cy3-siRNA transfected cells, the transfection efficiency of20nM,30nM,50nM and100nM are20%,40%,70%and90%respectively. The fluorescence intensity is significantly enhanced and the transfection efficiency is high in50nM and100nM; in the pre-selected three Mfn2-siRNA sequences, the expression level ofMfn2are6.20%,3.08%and17.68%in Mfn2-siRNA1, Mfn2-siRNA2andMfn2-siRNA3transfencted group respectively compared with control-siRNAtransfencted group, and the Mfn2-siRNA transfencted group is significantly decreased(P<0.05). Therefore, the relative quantity of Mfn2mRNA is the lowest inMfn2-siRNA2transfected group.Conclusion: The transfected efficiency is reached a desired level at the concentrationof50nM, so we selected50nM as the subsequent transfection at next experiments. Inthe pre-selected three sequences of Mfn2, the Mfn2-siRNA2(5’CUGGACAGCUGGAUUGAUAdTdT3’，3’dTdTGACCUGUCGACCUAACUAU5’) is the best, andwe would synthetize chemically modified Mfn2-siRNA according to it. Objectives: Observe the effects of Mfn2-siRNA on the cleavage speed and blastocystformation rate of mouse zygotes in vitro.Methods: Synthetized chemically modified siRNA according to the mouse optimaltransfection sequence (Mfn2-siRNA2) and the control sequence (control-siRNA), andtranfected them to2cells zygotes which had isolated from mice oviducts, thendetected the silence efficiency using quantity real-time PCR and observed the zytotecleavage speed and blastocyst formation rate.Results: The Mfn2expression of Mfn2-siRNA transfected group per the control-siRNA transfected group was0.8%, significantly lower(P<0.05), at the sametime compared with the control-siRNA transfected group the zygotes’ growth rate isslowed down and the blastocyst formation rate is decreased in Mfn2-siRNA group.Conclusion: In the Mfn2-siRNA transfected group, the blastocyst formation rate issignificantly decreased, indicating that Mfn2can affect the early development offertilized eggs and the normal expression of Mfn2play an important role on thedevelopment of zygotes in mouse. Objective: Determine the effects of Mfn2on mitochondria function during thedevelopment of zygote, and explore the mechanisms of Mfn2-siRNA on thepreimplantation embryo development of mouse.Method: Collect30blastocysts form Mfn2-siRNA and control-siRNA transfectedgroup respectively, and detect the ATP content by firefly luciferase. Measure themichondrial membrane potential with JC-1dye. Using FITC-labeled recombinanthuman Annexin Ⅴ detect the early mouse embryos’apoptosis.Results: The ATP content of Mfn2-siRNA transfected group is significantlly lower(P<0.05) and the mitochondrial membrane potential is lower than that in thecontrol-siRNA group; the apoptosis cells in Mfn2-siRNA transfected group issignificantly more than that in control-siRNA transfected group.Conclusion: Compared with the control-siRNA transfected group, the ATP is reducedin Mfn2-siRNA transfected group, and the mitochondrial membrane potential isdecreased, suggesting that mitochondrial energy metabolic should be inhibited; at the same time, the rate of apoptosis in Mfn2-siRNA transfected group is increased,indicates the apoptosis induced through the mitochondrial is increased. These resultssuggest that Mfn2may be involved in early embryonic development by effecting themitochondrial energy metabolism and apoptosis in mouse preimplantation embryos.