The bovine endometrial epithelial cells promote the differentiation of trophoblast stem-like cells to binucleate trophoblast cells

Abstract

Endometrial epithelial cells (EECs) cultured in vitro are valuable tools for investigating embryo implantation and trophoblast differentiation. In this study, we have established the bovine EECs and trophoblast stem-like (TS) coculture system, and used it to investigate the binucleate cell formation of ungulates. The EECs was derived from the uterine horn ipsilateral to the corpus luteum by using collagenase I and deoxyribonuclease I, which exhibited typical epithelial morphology and were expressing bovine uterine epithelial marker such as IFNAR1, IFNAR2, Era, PGR, ESR1 and KRT18. The cells immunostained positively by epithelial and trophectoderm marker cytokeratin 18 (KRT18) and stromal marker vimentin antibodies, and the KRT18 positive cells reached 99 %. The EECs can be cultured for up to 20 passages in vitro with no significant morphology changes and uterine epithelial marker gene expression alteration. The bTS cells were established in a dual inhibitor system and exhibited typical trophoblast stem cell characteristics. When bTS cells were cultured with EECs, the bTS cells adhered to the EECs as adhering to feeder cells. Binucleate cells began appearing on day 4 of coculture and reached approximately 18.47 % of the differentiated cells. Quantitative real-time PCR or immunofluorescence analyses were performed on bTS cells cocultured at day 6 and day 12. The results showed that the expression level of KRT18 was down-regulated while the expression level of trophoblast differentiation marker MASH2, HAND1, GCM1 and CDX2 was upregulated in bTS cells. In conclusion, bovine EECs can be obtained from the uterine horn ipsilateral to the corpus luteum via treatment with collagenase I and deoxyribonuclease I, and the EECs-bTS cells coculture system presents an ideal tool for studying the differentiation of bTS cells to trophoblast binucleate cells.

>b<Deciphering the embryo-maternal interactome:>/b< Embryo-dependent programming of endometrial function during early pregnancy in cattle

“…We stablished an endometrial epithelial cell line in a manner that in every replicate, aliquots containing cells originated from the same pool were applied. Regarding the biological accuracy of our in vitro model, the co-expression of cytokeratin and vimentin in our BEEC line is in line with previous reports describing the expression of vimentin by cytokeratinpositive epithelial cells maintained in culture (Wang et al, 2000;Zeiler et al, 2007;Li et al, 2016) via a process described as epithelial-mesenchymal transition (Kalluri & Weinberg, 2009). However, even after three passages the BEECs displayed no changes on abundance of transcripts for epithelial-cell origin (KRT18) and functional markers (ESR1, IFNAR1 and PTGS2) rendering our BEEC line valid to address the aims of the study.…”

Conceptus-induced, interferon tau-dependent gene expression in bovine endometrial epithelial and stromal cells†

“…Using a similar bovine endometrial cell isolation and culture technique as described here, Herath et al [46] reported epithelial and SF cell culture purities greater than 95% with undetectable levels of CD45 (common leukocyte antigen) mRNA and protein. Li et al [35] also reported that primary bovine endometrial epithelial cells did not exhibit significant changes in morphology or cell marker gene expression alterations after 20 passages. Similarly, we did not notice changes in the epithelial or SF cell morphology after multiple passages or days in culture.…”

“…Vimentin was detected within both cell types. Vimentin expression in isolated bovine endometrial epithelial cells has been reported and has been attributed to loss off cell to cell contact during isolation resulting in a limited epithelial to mesenchyme transition [35].…”

Cytobrush: A tool for sequential evaluation of gene expression in bovine endometrium

“…The former contained a greater proportion of endometrial stroma (VIM) and vascular cell (FLT1) markers than cytobrush samples (Johnson, Austin, Collins, Murdoch, & Hansen, ). Because of the more superficial sampling compared to biopsies, cytobrush samples were enriched in endometrial epithelial cells (KTR18) and immune cells (CD3D) markers (Li et al., ). It is unlike that either probing technique reached the myometrial layer, because abundance of MSTN was similar between groups.…”