Abstract
Epithelial-mesenchymal transition (EMT), which is common in cancer metastasis, is also observed during developmental processes such as embryo implantation into the maternal endometrium in humans and rodents. However, this process has not been well characterized in the non-invasive type of implantation that occurs in ruminants. To understand whether EMT occurs in ruminant ungulates, ovine conceptuses (embryo plus extraembryonic membranes) from days 15 (P15: pre-attachment), 17 (P17: during attachment), and 21 (P21: post-attachment, day 0 = day of estrus) were evaluated. RNA-seq analysis revealed that the expression of EMT-related transcripts increased on P21. Real-time PCR and western blotting analyses indicated that levels of transcripts and proteins indicative of mesenchyme-related molecules increased on P21, but a minor expression of epithelium-related molecules remained. Immunohistochemical analysis revealed that E-cadherin (CDH1) was localized in the elongated trophectoderm on P15 and P17. On P21, CDH1 was localized to the trophectoderm and on the conceptus cells undergoing differentiation. Vimentin (VIM) was localized in the uterine stroma on P15 and P17, and its expression was observed at the edge of elongating trophoblast on P21. Further, it was found that some bi-nucleated trophoblast cells were present on P17; however, numerous bi- and multi-nucleated trophoblast cells on the uterine epithelium or next to the uterine stroma were found on P21. A minor expression of pregnancy-associated glycoprotein (PAG) transcripts was found on P15 and P17, but a definitive expression of PAGs, transcripts, and proteins was found on P21. Although further investigation is required, these observations indicate that bi-nucleated trophoblast cell formation begins on the day conceptus implantation to the maternal endometrium is initiated, followed by EMT in trophoblast cells. These results suggest that these sequential events are required if pregnancy is to be established in ruminants.
