关键词: 猪, 早期胚胎, TEAD4, 孤雌激活, 体外受精

Abstract: 【Objective】 TEA domain transcription factor 4 (TEAD4) is known to be a member of the TEAD family of transcription factors and plays a key role in determining the characteristics of the preimplantation embryo in rodents. In mouse embryos, it was found to be involved in regulating the genealogical differentiation of trophectoderm cells in preimplantation embryos by promoting Cdx2 expression. The absence of the TEAD4 gene in mouse embryos can lead to failure of mouse blastocyst formation. However, the role of TEAD4 in early porcine embryonic development is still unclear. Here we aim to preliminarily elucidate the effect of TEAD4 on early porcine embryonic development in order to lay the theoretical foundation for further exploring the molecular mechanisms of transcription factors on early porcine embryonic development.【Method】In this study, bioinformatics analysis of the porcine TEAD4 gene was performed using web-based tools, including analysis of the porcine TEAD4 gene sequence, comparison of homology between pigs and humans and mice, and comparison of the evolutionary relationship of TEAD4 between different species. The role of TEAD4 in early embryonic development in pigs was then tested. The mRNA expression level of TEAD4 gene in porcine oocytes and early embryos was firstly detected by fluorescence quantitative PCR, and then by designing siRNA targeting TEAD4 and injecting it into mature oocytes by microinjection technique to reduce the level of endogenous TEAD4 gene in the oocyte cytoplasm, and to determine that TEAD4 siRNA acts only on TEAD4 gene, with a view to determining the role of TEAD4 gene in early porcine embryonic development.【Results】.Sequence analysis showed that the porcine TEAD4 gene contains 11 exons, localized on chromosome 5, spanning 37.188 kb, 1473 bp in full mRNA length, 1305 bp in full coding region, and encoding 434 amino acids. Homology analysis with human and mouse revealed that TEAD4 is highly conserved in different species and has the closest affinity on pig and cow. The results of fluorescence quantitative PCR showed that TEAD4 mRNA was expressed in both porcine oocytes and early embryos, and when compared with GV-stage oocytes, the expression was lowest in MII-stage oocytes and remained low until the 4-cell stage, but reached the highest expression in the 8-cell stage, and then gradually decreased in the morula and blastocyst stages. Microinjection of siRNA targeting TEAD4 revealed that TEAD4 siRNA only acted on the endogenous TEAD4 gene in oocytes, but not on TEAD1 and TEAD3, and compared with the control and negative control siRNA groups, injection of TEAD4 siRNA significantly reduced TEAD4 mRNA expression at the 8-cell and morula embryo periods. When TEAD4 gene expression was knocked down, observation of the developmental efficiency of porcine orphan activation and in vitro fertilization embryos showed that the developmental efficiency of TEAD4 siRNA knockdown group from 8-cell to blastocyst stage was significantly reduced compared to the control and negative control siRNA groups.【Conclusion】 The results of this study indicate that the TEAD4 gene is highly conserved across species, with the closest affinity on pigs and bovine, and that TEDA4 may be involved in regulating the development of early porcine embryos.

Key words: Pig, Early embryos, TEAD4, Parthenogenetic activation, In vitro fertilization