| Objective:To observe the localization of Eukaryotic initiation factor 1A(eIF1A)in different stages of mouse preimplantation embryo and the changes of mRNA level,and to investigate the role of eIF1 A in mouse preimplantation embryos development by using RNA interference(RNAi)technology to specifically reduce the expression of eIF1 A gene.Methods1.Different developmental stages of mouse preimplantation embryos were collected separately after mating KM female mice with KM male mice(?KM×?KM).Laser confocal microscopy was used to observe the protein localization of eIF1 A protein in each stage of preimplantation embryo.2.Preimplantation embryos of KM mice at various stages were collected and the expressions of eIF1 A mRNA were detected by Real-Time PCR.3.One-cell embryos of KM mice(18h after hCG)were collected and cultured in KSOM medium for 3h.1-cell embryos with double prokaryotes were collected and divided into three groups which were KSOM blank control group,negative-siRNA group(negative control group)and eIF1A-siRNA group,theninjected by applying micromanipulation,and placed in KSOM medium for further in vitro cultivation.1)Observe the development of preimplantation embryos after injection.2)At 50 h after hCG,mouse preimplantation embryos of KSOM control group,negative-siRNA group and eIF1A-siRNA group were collected.Real-Time PCR was used to detect the expression levels of eIF1 A,MuERV-L,Zscan4 d and Hsp70.1 and stem cell transfer factors Sox2 and Oct4.3)At 90 h afterhCG,mouse preimplantation embryos of KSOM control group,negative-siRNA group and eIF1A-siRNA group were collected.and the morula was observed by laser confocal microscope for the expression of CDX2?OCT4 and SOX2 proteins.Results:1.The results of immunofluorescence showed that eIF1 A protein began to locate in the nucleus of the 1-cell embryo,and its nuclear staining was most obvious at the 2-cell embryo stage,and then its nuclear expression beganed to weak and enhance in the cytoplasm,and was only expressed in the cytoplasm at the morula and blastocyst stages.2.Real-Time PCR was used to detect eIF1 A mRNA level shows that,the expression of eIF1 A mRNA continued to increase in 1-cell embryo,2-cell embryo,4-cell embryo and 8-cell embryo,and the expression was highest in 8-cell embryo period.3.The culture results showed that the blastocyst transition rate in the eIF1A-siRNA injection group was significantly lower than that in the KSOM control group and negative control group(P< 0.001).and about 30% of the preimplantation embryos were inhibited in the morula stage(P< 0.001).Real-Time PCR results showed that after the injection of eIF1A-siRNA,the mRNA level of eIF1 A at the 2-embryo stage was significantly lower than that of KSOM control group and negative control group(P< 0.001),while the mRNA level of Hsp70.1 was also significantly reduced(P< 0.01).CDX2 protein localization was also significantly down-regulated in morula stage.Conclusion:eIF1A was expressed in the nucleus before 8-cell embryo stage,and in the cytoplasm during morula and blastocyst,suggesting that it may play the transcriptional regulatory role in the early stage,but the translation function in the later stage.After microinjection of eIF1A-siRNA into 1-cell embryos,the cells were blocked in the morula stage,indicating that eIF1 A was involved in the regulation of blastocyst formation.After the injection of eIF1A-siRNA,it was found that the expression of Hsp70.1 in the marker gene of zygote genome activationwas down-regulated,and the protein expression of CDX2 in morula cells was significantly decreased,suggesting that eIF1 A may affect the differentiation of blastocyst trophoblastic ectoderm and thereby affect the formation of blastocyst by regulating the expression of Hsp70.1 in the marker gene of zygote genome activation. |
PDF Full Text Request