A reappraisal of mesenchymal-to-epithelial transition within the endometrium

Abstract

A controversial topic, mesenchymal-epithelial transition (MET) is thought to be a mechanism involved in regeneration of the uterine epithelial layer following pregnancy and menstruation. Little is known about this process though, requiring further exploration. Previously, MET was thought to only occur as a damage-repair mechanism following parturition and menses-like events in mouse models. However, in the current study we hypothesized that MET would also occur in other endometrial epithelialization events as a mechanism of homeostatic epithelial turnover. To identify mesenchymal-derived cells within the adult uterine epithelium, an Amhr2-Cre; Rosa26-EYFP reporter mouse line was used. Mice were staged by vaginal cytology prior to the isolation of uterine epithelial and stromal cells, which were then stained with EpCAM to identify epithelial cells and analyzed by flow cytometry. EpCAM+YFP+ cells were identified in all stages of the estrous cycle except diestrus, indicating a proportion of epithelial cells were derived from the stroma (i.e., mesenchyme). Up to 80 percent of the uterine epithelia was EpCAM+YFP+ during estrogen-dominant stages (proestrus and estrus) of the cycle, with negligible amounts found during progesterone-dominant stages (metestrus and diestrus), suggesting this population may be responsive to estrogen. Uteri were also evaluated direct fluorescence and immunofluorescence in tissue sections. Immunofluorescence for EpCAM, forkhead box protein A2 (FOXA2), Ki67, estrogen receptor alpha (ESR1), and progesterone receptor (PGR) was performed to assess epithelial characteristics and potential functionality. To further investigate the role of MET in epithelialization and assess the temporal origin of mesenchymal-derived epithelial cells, we evaluated key postnatal (P) developmental time points using Amhr2-Cre; Rosa26-tTA; H2B-GFP reporter mice. Flow cytometry data indicated that MET may initially occur immediately after birth at P 0.5, with results varying from negligible amounts (0.21 percent) to approximately 82 percent. Similar results were found at P 3, but with decreasing variation; the highest EpCAM+GFP+ population representing approximately 50 percent of the epithelium. Between P 3 and early adenogenesis at P 8, this population decreased to average less than 2 percent. By the completion of adenogenesis initiation at P 14, approximately 10 percent of epithelial cells were EpCAM+GFP+, suggesting MET may occur during adenogenesis initiation and is maintained through P 21. Together, these results suggest that MET may be a more ubiquitous mechanism of epithelialization than originally thought and is likely hormone regulated. This research will help elucidate the role of MET in uterine epithelialization with potential for insights into dysregulation of MET in diseases such as endometrial adenocarcinoma and endometriosis.