Epithelial-mesenchymal transition and bi- and multi-nucleated trophoblast cell formation in ovine conceptuses during the peri-implantation period

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 3 that these sequential events are required if pregnancy is to be established in ruminants.

Endogenous Retroviruses and Placental Evolution, Development, and Diversity

“…Seo and coworkers provided evidence that ovine trophectodermal cells went through homotypic cell fusion, not heterotypic, and formed syncytiotrophoblast-like fused cells [ 55 ]. This observation agrees with our recent publication in which bi-nucleated and multi-nucleated cells observed in vivo are all homotypic cell fusions [ 56 ].…”

“…In the ruminants, including Bovidae, syncytiotrophoblast cells are not formed; rather, trinucleate cells, resulting from the fusion between binucleate trophoblast cells and maternal epithelial cell often appear in the uterine stroma [ 64 ]. Recently, we [ 56 ] and others [ 55 ] demonstrated that binucleate and/or multi-nucleate trophoblast cells are derived from the fusion of trophoblast cells. In animals with epitheliochorial placentation, such as horses, camels, pigs, hippopotamuses, and cetaceans, the syncytiotrophoblast layer is not formed; however, binucleate trophoblast cells are found in the horse placenta.…”

Uncovering Novel Features of the Pc Locus in Horn Development from Gene-Edited Holstein Cattle by RNA-Sequencing Analysis

“…The results of this experiment revealed that the expression of genes expressing immunoglobulin adhesion molecules, such as L1CAM , LAMC2 , and LAMA5 , was significantly lower in EH horn buds compared to WH ( Table S1 ), suggesting that EMT was inhibited in EH horn buds, which may lead to failure of neural crest cell migration initiation. Similarly, the top 20 Hub proteins from the protein interaction analysis also screened for multiple proteins associated with cell adhesion, such as cadherin 1 (CDH1), integrin subunit beta 2 (ITGB2) and integrin subunit beta 3 (ITGB3) were involved in the biological process of the regulation of cell adhesion molecule in these results [ 26 , 27 , 28 ]. It suggested that the Pc locus may inhibit EMT in neural crest cells by regulating the expression of cell adhesion molecules, particularly E-cadherin.…”

Progressive Exaptation of Endogenous Retroviruses in Placental Evolution in Cattle

“…It has long been believed that the BNCs fuse with endometrial cells on the surface of the placenta on the maternal side, which invade the endometria and transport hormones to the mother, thereby maintaining pregnancy [58]. It was recently shown by two laboratories that the fusion between trophoblast cells in sheep is not limited to the formation of BNCs, and new BNCs involve the lateral fusion between growing syncytial plaques [61,62].…”

“…It is possible that BERV-K1 became a key player in bovine placentation (Table 2). Interestingly, BERV-K1 also played a role in the formation of trophoblast hybrid cells in heterologous trophoblast cells (sheep trophoblast cells) [62]. In addition to Syncytin-Rum1 and BERV-K1, other ERV envs such as BERV-K2 [49], bERVE-A [50], and bERVE-B [50] have been identified.…”