Epithelial Estrogen Receptor Alpha is Required for Uterine Epithelial Development

Jacob M. Pru¹, Jason A. Rizo², Sarayut Winuthayanon¹, Thomas E. Spencer^1,2, and Andrew M. Kelleher¹. ¹Department of Obstetrics, Gynecology, and Women’s Health, Columbia, MO, 65211, USA; ²Division of Animal Sciences, University of Missouri, Columbia, MO, 65211, USA

Abstract Text:

The uterus and cervix originate from the Müllerian duct and, at birth, contain an undifferentiated single-layered epithelium that either develops into a multilayered epithelium with basal cells (p63, KRT5 positive) in the cervix or remains single-layered in the uterus. Tissue recombination experiments conducted during the first week of postnatal life, wherein cervicovaginal mesenchyme was recombined with neonatal uterine epithelium and cervicovaginal epithelium with uterine mesenchyme, revealed the essential role of mesenchymal-epithelial crosstalk in uterine epithelial fate specification. Clinical data and mouse models indicate that neonatal exposure to the synthetic estrogen diethylstilbestrol causes uterine epithelial stratification, suggesting a potential role for estrogen receptor alpha (ESR1) in mediating mesenchymal-derived epithelial fate specification signals. However, the function of epithelial ESR1 in uterine differentiation and development remains unclear. To address this gap, we utilized a combination of in vivo genetic mouse models and in vitro endometrial epithelial organoid (EEO) cultures to determine the epithelial-intrinsic role for ESR1 in neonatal uterine epithelial differentiation. Organoids derived from postnatal day (PND) 5 wild-type epithelium, when the epithelium is plastic, produced either multilayered EEOs demarcated by KRT5 and p63 or single-layered EEOs, with Esr1 expression restricted to the single-layered population. In contrast, EEOs derived from PND 15 wild-type uterine epithelium formed only single-layered EEOs that were universally positive for ESR1. To determine the impact of epithelial ESR1 in uterine development, transcriptomic analysis was conducted on the uterine epithelium of PND 3 and 15 control and epithelial-specific Esr1 conditional knockout (Esr1 cKO) mice. The uteri of these mice displayed no histological differences when compared to wild-type controls prior to PND 15; however, the Esr1 cKO uterus exhibited a decrease in glandular epithelium and epithelial stratification by PND 40. No significant gene expression differences were observed in the PND 3 epithelium, but PND 15 Esr1 cKO epithelium had major perturbations in gene expression (314 genes upregulated, 863 downregulated; log₂FC ± 0.9, p< 0.05), including dysregulation of Crabp1/2 and Cyp26a/c1, which suggest impaired retinoic acid (RA) signaling. To determine the impact of RA on epithelial differentiation, exogenous all-trans retinoic acid (atRA, 5uM) was added to the culture media of neonatal and adult EEOs derived from either wild-type or Esr1 cKO mice. Multilayered organoid formation was not impacted in EEOs derived from adult Esr1 cKO; however, addition of atRA to PND 5 EEO cultures resulted in the formation of only single-layered EEOs derived from both wild-type and Esr1 cKO mice. The addition of atRA did not affect the formation or survival of EEOs compared with vehicle control. Of particular interest, removal of atRA after three weeks of culture resulted in multilayered organoid development from previously formed single layered EEO derived from PND 5, from both wild-type and Esr1 cKO. Taken together, these results suggest that RA functions to maintain the unspecified epithelium as a monolayer, whereas ESR1 is required for downstream terminal differentiation into adult uterine epithelium.

Funding: NIH-R01HD112315