Expression And Role Of Transcription Factor SNAIL In The Mouse Preimplantation Embryos

Objective:The mRNA expression and protein changes of Snail in the mouse preimplantation embryos at different developmental stages were observed,and the expression level of Snail gene was specifically lowered by RNA interference(RNAi)technology to study the role of Snail in the preimplantation embryo development of mice,and to further explore the relationship between Snail and zygotic genome activation.Methods:1.Mouse preimplantation embryos of different developmental stages obtained from mating of KM female mice and male mice(?KM�?KM)were collected.By using IF detection technology,the localization of SNAIL protein was detected in the preimplantation embryos.2.Preimplantation embryos of different developmental stages were collected from mating of KM female mice,and the expression of Snail mRNA was detected by Real-Time PCR.3.The 1-cell embryos of KM mice(27h after hCG)were collected,and the 1-cell embryos with obvious double prokaryotic were selected.The Snail-siRNA was introduced into the 1-cell embryos by microinjection technique,and then cultured in KSOM medium.(1)The developmental potential of 1-cell embryos after Snail-siRNA injection was observed.(2)At 48 h after hCG injection,2-cell embryos developed in vitro in the KSOM control group,the negative control group and the Snail-siRNA injection group were collected,and the mRNA expression levels of Snail,Trps1 and ZGA related gene e IF1 A were detected by Real-Time PCR.4.The localization of SNAIL protein in human ovarian epithelial cell line(IOSE-80)was observed by IF detection.5.The Snail-siRNA was introduced into human ovarian epithelial cell line(IOSE-80)by using transfection technology,and the expressions of Snail and Trps1 mRNA were detected by Real-Time PCR after 24 h.Results:1.IF detection results showed that SNAIL protein was distributed from the oocyte to the blastocyst,and was located in the nucleus from the late 2-cell embryo stage to the early morula stage,and mainly located in the cytoplasm from the late morula stage to the blastocyst stage.2.The result of Real-Time PCR showed that the expression of Snail mRNA was the highest at the stage of 1-cell embryo,and then decreased gradually.3.The results of cell culture showed that the blastocyst formation rate of the Snail-siRNA injection group was significantly lower than that of the KSOM control group and the negative control group(P < 0.001),and about 1/3 of the preimplantation embryos were suppressed before the 4-cell embryos(P < 0.001).Real-Time PCR results showed that after injection of Snail-siRNA,the Snail mRNA level of the 2-cell embryo stage was significantly lower than that of the KSOM control group and the negative control group(P < 0.01),and the mRNA level of e IF1 A was significantly increased(P< 0.01),while the mRNA level of Trps1 was significantly increased(P < 0.001).4.SNAIL protein is expressed in the nucleoplasm of the IOSE-80 cell line,mainly in the nucleus,which is similar to the localization of preimplantation embryos.5.In the IOSE-80 cell line,Trps1 mRNA level significantly increased after Snail was knockdown(P < 0.001).Conclusion:IF staining and quantitative PCR were used to analyze mouse preimplantation embryos at different developmental stages,suggesting that Snail may be involved in preimplantation zygote genomic activation.After microinjection of Snail-siRNA into1-cell embryos,it was found that Snail mainly affected the activation of zygotic genome and the transition from maternal to Zygotic.The up-regulation of e IF1 A and Trps1 mRNA expression in 2-cell embryos after knocking down low levels of Snail mRNA suggested that Snail might participate in the process of zygote genome activation by affecting the transcription of e IF1 A and Trps1.using Snail-siRNA transfection technique,the mRNA expression of Trps1 was significantly up-regulated after knocking down the level of Snail mRNA in human normal ovarian epithelial cells IOSE-80,further confirming that the transcription of Trps1 may be similarly regulated by Snail.