Role of epithelial–mesenchymal transition regulated by twist basic helix-loop-helix transcription factor 2 (Twist2) in embryo implantation in mice
Jinhai Gou
A B * , Tingwenyi Hu A B * , Lin Li A B , Luqi Xue A B , Xia Zhao A B C , Tao Yi B C and Zhengyu Li A B D
A Department of Gynecology and Obstetrics, West China Second University Hospital, Sichuan University, Chengdu 610041, Sichuan Province, PR China.
B Key Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University), Ministry of Education, Chengdu 610041, Sichuan Province, PR China.
C Sichuan Key Laboratory of Gynecologic Oncology, West China Second University Hospital, Sichuan University, Chengdu 610041, Sichuan Province, PR China.
D Corresponding author. Email: zhengyuli@scu.edu.cn
Reproduction, Fertility and Development 31(5) 932-940 https://doi.org/10.1071/RD18314
Submitted: 14 November 2018 Accepted: 19 December 2018 Published: 20 February 2019
Abstract
In a previous study we found the expression of epithelial–mesenchymal transition (EMT) biomarkers, including E-cadherin and N-cadherin, was significantly altered in uterine endometrium during embryo implantation via regulation by microRNA (miRNA)-429 and protocadherin-8 (Pcdh8). As a natural continuation of the previous study, the aim of the present study was to explore the role of EMT during embryo implantation and the potential activity of twist basic helix-loop-helix transcription factor 2 (Twist2) in regulating embryo implantation. A pregnancy model was established by naturally mating adult female ICR mice with fertile males. A pseudopregnancy model was established by mating fertile female ICR mice with vasectomised males. An in vitro model of embryo implantation was established by the coculture of Ishikawa and JAR spheroids. Endometrial tissue during the peri-implantation period was collected, as were Ishikawa cells, JAR cells and cocultured cells. The expression of EMT markers (E-cadherin, N-cadherin, vimentin and cytokeratin) and Twist2 was detected in vivo and in vitro using the western blot analysis during embryo implantation. The expression of N-cadherin and vimentin (mesenchymal markers) was upregulated in the in vitro implantation model, with downregulation of E-cadherin and cytokeratin (epithelial markers) expression. The expression of N-cadherin, vimentin and Twist2 increased significantly at the implantation sites at the time of implantation (Day 5), whereas the expression of E-cadherin and cytokeratin decreased. Location of Twist2 during embryo implantation was detected by immunohistochemistry (IHC), which revealed that it was extensively expressed in endometrial glandular epithelium and luminal epithelium at implantation sites on Day 5. The effect of the expression of Twist2 on embryo implantation was evaluated by suppressing Twist2 using Twist2-short interference (si) RNA in in vivo and in vitro models. The numbers of implanted embryos and the implantation rate were compared in vivo and in vitro. Western blot analysis showed that suppression of Twist2 led to upregulation of E-cadherin and cytokeratin, accompanied by downregulation of N-cadherin and vimentin (P < 0.05). The number of implanted embryos after Twist2-siRNA interference was lower than in normal pregnancy (mean (± s.d.) 2.4 ± 0.5 vs 6.8 ± 1.3 respectively; P < 0.05). These findings suggest the involvement of EMT in embryo implantation. The suppression of Twist2 could suppress embryo implantation by regulating EMT.
Additional keywords: in vitro implantation assay, short interference RNA.
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