Evidence of Putative Epithelial Stem/progenitor Cells in the Bovine Endometrium: Implications for Fertility

Postpartum fertility complications in lactating dairy cows challenge the economic value of most dairy herds, restricting the industry’s efforts to extend herd survivability. Impaired fertility is strongly associated with high prevalence of postpartum uterine disease, resulting from invasion of pathogenic bacteria into the uterus during parturition. Sustained inflammation hinders the reestablishment of the uterine glandular epithelium (GE) by suppressing cellular proliferation at the base of the endometrium (stratum basalis), resulting in incomplete epithelial repair. Previously, our group identified an epithelial population at the basalis endometrium of the adult bovine uterus that exhibits unique stem/progenitor transcriptional signatures. These findings led us to hypothesize that decreased fertility may be attributed to the effect of postpartum uterine disease on a population of long-lived epithelial stem/progenitor cells (eS/P), which become established at the basalis endometrium postnatally, persist into adulthood, and play a key role in coordinating epithelial regeneration following parturition. To test this, we combined spatial transcriptomic datasets generated from bovine uterine cross-sections collected during critical developmental and regenerative states (n = 2 per time point), including 1) onset of postnatal GE branching morphogenesis [postnatal day (PND) 28], 2) completion of postnatal adenogenesis (gland development; PND 56) and 3) completion of postpartum repair [postpartum day (PPD) 30]. Here, we characterized the signatures of stem cell activity unique to basalis GE throughout development and repair, to elucidate the identity of this putative eS/P cell population. Integrative UMAP analysis revealed two GE clusters consistently mapped to the basalis endometrium of postnatal and postpartum uteri, which expressed N-cadherin (CDH2), a marker associated with human endometrial eS/P cells. The CDH2+ clusters displayed transcriptional hallmarks of eS/P activity, including retinoic acid synthesis (CDH2+ALDH1A1+) and canonical Wnt signaling (CDH2+RSPO3+). Differential expression analysis showed dynamic shifts in cell activity between the two CDH2+ populations across time points, including enrichment for proliferation markers at PND28 and PPD30 within the CDH2+ALDH1A1+ population, consistent with a state of eS/P activation. Conversely, at PND56, signatures of cell cycle arrest and telomere maintenance suggested a predominantly quiescent state, indicative of a regenerative program that may alternate between activation and dormancy in the adult endometrium. Using flow cytometry, we quantified CDH2+ and ALDHbright (ALDH activity by Aldefluor Assay) endometrial epithelial cells (EPCAM+) at the designated time points. We observed a significant decrease in the CDH2+ and ALDHbright epithelia from branching morphogenesis (PND28, CDH2+ = 59.7%; ALDHbright = 3.6%) to completion of adenogenesis (PND56, CDH2 + = 2.86%; ALDHbright = 1.25%). Notably, during postpartum GE regeneration (PPD 30), CDH2+ and ALDH bright epithelia expanded comprising 65.5% and 5.25% of the epithelial population, respectively. In summary, these data support the existence of a long-lived, putative stem/progenitor population within the GE of the bovine endometrium that is established postnatally and maintained throughout adulthood.