Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (20): 4456-4465.doi: 10.3864/j.issn.0578-1752.2021.20.018

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

Transcription Factor TEAD4 Regulates Early Embryonic Development in Pigs

ZHANG DanDan(),XU TengTeng,GAO Di,QI Xin,NING Wei,RU ZhenYuan,ZHANG XiangDong,GUO TengLong,SHENTU LuYan,YU Tong,MA YangYang,LI YunSheng,ZHANG YunHai,CAO ZuBing()   

  1. Key Laboratory of Conservation and Biological Breeding of Local Livestock and Poultry Genetic Resources/College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036
  • Received:2020-09-09 Accepted:2021-07-08 Online:2021-10-16 Published:2021-10-25
  • Contact: ZuBing CAO E-mail:1730504758@qq.com;zubingcao@ahau.edu.cn

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Abstract:

【Background】 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. 【Objective】This study aimed 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, the bioinformatics analysis of the porcine TEAD4 gene was performed by using web-based tools, including analysis of the porcine TEAD4 gene sequence, comparison of homology between pigs, human 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 the early embryos was firstly detected by fluorescence quantitative PCR. and then, siRNA targeting TEAD4 was designed and injected 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. 【Result】Sequence analysis showed that the porcine TEAD4 gene contained 11 exons and localized on chromosome 5, with spanning 37.188 kb, 1 473 bp in full mRNA length, and 1305 bp in full coding region, which encoded 434 amino acids. Homology analysis with human and mouse revealed that TEAD4 was highly conserved in different species and had 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; compared with GV-stage oocytes, the expression of TEAD4 mRNA 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, the 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 indicated that the TEAD4 gene was highly conserved across species, with the closest affinity on pigs and bovine, and that TEDA4 might be involved in regulating the development of early porcine embryos..

Key words: pig, early embryos, TEAD4, parthenogenetic activation, in vitro fertilization

Table 1

TEAD4 siRNA sequences"

干扰RNA名称 siRNA name 正向序列 Sense (5′-3′) 反向序列 Antisense (5′-3′)
siRNA-1 GGCUUUGGACAAGCCCAUUTT AAUGGGCUUGUCCAAAGCCTT
siRNA-2 GCCAGAUCUACGACAAGUUTT AACUUGUCGUAGAUCUGGCTT
siRNA-3 GCCAUUACUCCUACCGCAUTT AUGCGGUAGGAGUAAUGGCTT

Table 2

Primers used in the study"

基因名称
Gene name		 序列 (5′-3′)
Sequence (5′-3′)
TEAD1 Forward Primer: CCGAAGTTTGCCTCGGACT
Reverse primer: ATTTTGGCGGCCGGATTTTC
TEAD3 Forward Primer: CGAGTACCAGGTTGGCATCA
Reverse primer: TTGTTCTGTAGGACGCTGGC
TEAD4 Forward Primer: CATTACTCCTACCGCATCC
Reverse primer: CCTGTGTGTCTCTGTTGG
EF1a1 Forward Primer: ATTGTTGCTGCTGGTGTTG
Reverse primer: TCATATCTCTTCTGGCTGTAGG

Fig. 1

Gene structure of pig TEAD4 The numbers 1-11 represent the exon position of inTEAD4"

Table 3

Homology comparison of pig, human and mice TEAD4 gene"

比较方式
Comparison method		 猪TEAD4与人TEAD4比较
pTEAD4 vs hTEAD4		 猪TEAD4与小鼠TEAD4比较
pTEAD4 vs mTEAD4		 小鼠TEAD4与人TEAD4比较
hTEAD4 vs mTEAD4
CDS 90.96 % 77.93 % 78.54 %
AA 95.85 % 82.95 % 83.41 %

Fig. 2

The evolutionary relationship of TEAD4 among different species (AA) AA represents amino acid sequence"

Fig. 3

Expressions of TEAD4 mRNA in early porcine embryos a,b,c,d indicate significant differences, P<0.05"

Fig. 4

TEAD4 siRNA specifically targets the TEAD4 Figure Aand B: Effects of TEAD4 siRNA on TEAD4 mRNA expression in 8-cell and morula embryo. Figure Cand D: Effects of TEAD4 siRNA on mRNA expression of TEAD1 and TEAD3. a and b indicate significant differences (P<0.05). The same as below"

Fig. 5

Effect of TEAD4 knockdown on early development of porcine PA embryos (Bar = 50 μm)"

Fig. 6

Effect of TEAD4 knockdown on early development of porcine IVF embryos (Bar = 50 μm)"

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