The Roles Of Transcription Factor SNAI1 In The Zygotic Genome Activation Of Mouse Preimplantation Embryos

Observe:To investigate whether SNAI1 has important effects on the zygotic genome activation of mouse preimplantation embryos,we observed SNAI1 expression in mouse preimplantation embryos and the results of SNAI1-specific antibody culture in vitro.Basic research content:1.To observed the localization and distribution of SNAI1 in mouse 1-cell embryos and 2-cell embryos.2.To investigate the affection of SNAI1-specific antibody on the development of mouse preimplantation embryo,zygotic genome activation(ZGA),DNA replication of 2-cell embryo.Meanwhile,we analyzed and the expression of G1/S phase transition factors in 2-cell embryos in vitro.Methods:1.The KM mouse 1-cell embryos and 2-cell embryos were collected during 18h~55h after hCG injection.Detection of SNAI1 protein localization and expression at different periods by fluorescence after immunostaining.2.The KM mouse 1-cell embryos were collected and cultured in KSOM culture media,KSOM culture media containing different concentrations of SNAI1 specific antibodies,and KSOM culture media containing rabbit IgG,and the in vitro development of mouse 1-cell embryos was observed.3.The KM mouse 1-cell embryos were collected and cultured in KSOM media containing rabbit IgG(1 μg/ml)and KSOM medium containing SNAI1 specific antibody(1 μg/ml).Two-cell embryos were obtained at 45 h after hCG injection for the following steps:(1)To detect mRNA expression levels of MuERV-L,eIF1 A,Zscan4d,and Hsp70.1 by Realtime-PCR.(2)To observe the localization and expression of eIF1 A and Zscan4 by immunostaining.4.The KM mouse 1-cell embryos were collected and cultured in KSOM media containing rabbit IgG(1 μg/ml)and SNAI1 specific antibody(1 μg/ml).Mouse 2-cell embryos at 36h~42h post-hCG were collected for BrdU incorporation,then the DNA replication in S phase was observed by immunostaining.The KM mouse 1-cell embryos and 2-cell embryos were collected in the same way above,and then the expression of G1/S phase transition factors CyclinD1,CyclinE and Nanog were detected in 2-cell embryos by the immunostaining.Results:1.Immunofluorescence results showed that the SNAI1 protein level of nuclear expression was significantly increased during S and G2 phases in 2-cell embryos;and nuclear localization of SNAI1 evenly distributed throughout the cell during 1-cell embryos,2-cell embryos G1 and 2-cell embryos mitosis.2.Compared with the KSOM control group,1.0 μg/ml rabbit IgG and 0.1ug/ml SNAI1 specific antibody had not significantly affected the in vitro development of mouse 1-cell embryos.SNAI1 specific antibody(0.5 μg/ml and 1.0 μg/ml)had inhibited the in vitro development of mouse 1-cell embryos,The development ratios of 4-cell embryos and morula embryos was decreased in 0.5 μg/ml SNAI1 antibody group(P<0.05);1.0 μg/ml SNAI1-specific antibody could significantly inhibit 1-cell embryo development and lead to 2-cell arrest(P<0.001).3.The expression of eIF1 A mRNA in the 2-cell embryos of the SNAI1-specific antibody group(1 μg/ml)was significantly increased(P<0.05),while MuERV-L,Zscan4 d,and Hsp70.1 mRNA were not affected.Immunofluorescence results showed that the fluorescence intensity of eIF1 A in nuclei in the SNAI1-specific antibody group(1 μg/ml)was higher than that of rabbit IgG group(P<0.05);There was no significant difference in total and nuclear fluorescence intensity between the two groups of the protein of Zscan4(P>0.05).4.The SNAI1-specific antibody(1 μg/ml)can affect DNA replication of mouse 2-cell embryo(P<0.001).The incorporation rate of BrdU in the rabbit IgG(1.0 μg/ml)group was 85.7%,and the BrdU incorporation rate of the SNAI1 specific antibody group(1.0 μg/ml)was 15.0%.The G1/S phase transition factors CyclinD1,CyclinE,and Nanog was not affected significantly by the SNAI1-specific antibody(1 μg/ml)(P>0.05).Conclusion:1.The appearance of SNAI1 protein in the nucleus of 2-cell embryos is related to the time of ZGA,and SNAI1-specific antibody significantly raised eIF1 A.That suggests SNAI1 involves in mouse ZGA.2.SNAI1 may take part in the ZGA by regulating the progression of S phase in mouse 2-cell embryos.