| SOX2 is a member of the SRY-related HMG-box transcription factor B family and is a core pluripotent transcription factor that plays an important regulatory role in early mammalian embryonic lineage differentiation.Pig pre-implantation embryo development is a lengthy process,and there is species-specific regulation of lineage differentiation regulatory networks.SOX2 exhibits heterogeneous expression between blastomeres in pig morula embryos,with only some blastomeres exhibiting SOX2 positivity,while in mouse morula embryos,SOX2 exhibits homogeneous expression between blastomeres,with all blastomeres exhibiting SOX2 positivity.This developmental phenomenon indicates that there is species-specific expression regulation of SOX2 in early pig embryo development.To clarify the species-specific expression regulation mechanism of SOX2 in early pig embryo development,this study systematically tracked the dynamic expression pattern of SOX2 in pre-implantation pig embryos and explored the regulatory mechanisms of the Rho-ROCK and Hippo signaling pathways on SOX2 expression in early pig embryos.This study is of great reference value for a deeper understanding of the species-specific regulatory mechanisms of early mammalian embryonic development.To further clarify the expression pattern of SOX2 in pre-implantation pig embryos,we used fluorescent quantitative PCR(RT-q PCR)and immunofluorescence technology(IF)to detect the expression pattern of SOX2 at the transcriptional and translational levels.The experimental results showed that SOX2 m RNA expression increased from the 4-cell stage and reached its highest level at the pig morula stage on the fourth day,gradually decreasing at the pig morula stage on the fourth and a half day and reaching the lowest level at the blastocyst stage.SOX2 protein was located in the cell nucleus starting from the 4-cell stage and was expressed in all blastomeres of the embryo before the 4.5-day stage,after which it began to be expressed specifically in some cells and was only expressed in the inner cell mass(ICM)during the blastocyst stage,not in the trophectoderm(TE).To explore the role of SOX2 in the early embryonic development of pigs,we used RNA interference technology to knock down the function of SOX2.The results of immunofluorescence showed that after SOX2 interference,about(15.33±3.05)% of the embryos could develop to the blastocyst stage,but these blastocysts could not form the ICM,and the total number of cells significantly decreased.RT-q PCR showed that knocking down SOX2 led to significant downregulation of ICM-related gene expression except for OCT4.These results indicate that SOX2 regulates pig pre-implantation embryonic lineage differentiation and cell proliferation in an OCT4-independent manner.To clarify the regulatory role of the Rho-ROCK and Hippo signaling pathways on SOX2 expression and early pig embryo development,we specifically blocked the Rho-ROCK pathway and the key components of the Hippo signaling pathway(LATS2,YAP1,and TEAD4)using small molecular inhibitors and RNA interference during early pig embryo development.The results showed that when the Rho-ROCK signaling pathway was inhibited,LATS2 transcription was upregulated,while YAP1 transcription was downregulated.The expression of the TE-specific gene CDX2 was downregulated,while the expression of the ICM-specific genes SOX2 and NANOG was upregulated.Knocking down LATS2 significantly reduced the development rate of morula and blastocysts,with downregulation of SOX2 and OCT4 expression and upregulation of CDX2 expression.Knocking down YAP1 significantly reduced the blastocyst rate,with upregulation of SOX2 and OCT4 expression and downregulation of CDX2 expression.Knocking down TEAD4 did not affect embryo development,and there was no significant change in the expression of transcription factors such as SOX2 and OCT4.The above results indicate that the Hippo signaling pathway in pig embryos may play a regulatory role in an TEAD4-independent form. |
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