Upregulation of FOXA2 in uterine luminal epithelium and vaginal basal epithelium of epiERα-/- (Esr1fl/flWnt7aCre/+) mice†.

Forkhead box protein A2 (FOXA2) is a pioneer transcription factor important for epithelial budding and morphogenesis in different organs. It has been used as a specific marker for uterine glandular epithelial cells (GE). FOXA2 has close interactions with estrogen receptor α (ERα). ERα binding to Foxa2 gene in the uterus indicates its regulation of Foxa2. The intimate interactions between ERα and FOXA2 and their essential roles in early pregnancy led us to investigate the expression of FOXA2 in the female reproductive tract of pre-implantation epiERα-/- (Esr1fl/flWnt7aCre/+) mice, in which ERα is conditionally deleted in the epithelium of reproductive tract. In the oviduct, FOXA2 is detected in the ciliated epithelial cells of ampulla but absent in the isthmus of day 3.5 post-coitum (D3.5) Esr1fl/fl control and epiERα-/- mice. In the uterus, FOXA2 expression in the GE appears to be comparable between Esr1fl/fl and epiERα-/- mice. However, FOXA2 is upregulated in the D0.5 and D3.5 but not PND25-28 epiERα-/- uterine luminal epithelial cells (LE). In the vagina, FOXA2 expression is low in the basal layer and increases toward the superficial layer of the D3.5 Esr1fl/fl vaginal epithelium, but FOXA2 is detected in the basal, intermediate, and superficial layers, with the strongest FOXA2 expression in the intermediate layers of the D3.5 epiERα-/- vaginal epithelium. This study demonstrates that loss of ERα in LE and vaginal basal layer upregulates FOXA2 expression in these epithelial cells during early pregnancy. The mechanisms for epithelial cell-type specific regulation of FOXA2 by ERα remain to be elucidated.

Visualization of preimplantation uterine fluid absorption in mice using Alexa Fluor™ 488 Hydrazide†.

Uterine fluid plays important roles in supporting early pregnancy events and its timely absorption is critical for embryo implantation. In mice, its volume is maximum on day 0.5 post-coitum (D0.5) and approaches minimum upon embryo attachment ~D4.0. Its secretion and absorption in ovariectomized rodents were shown to be promoted by estrogen and progesterone (P4), respectively. The temporal mechanisms in preimplantation uterine fluid absorption remain to be elucidated. We have established an approach using intraluminally injected Alexa Fluor™ 488 Hydrazide (AH) in preimplantation control (RhoAf/f) and P4-deficient RhoAf/fPgrCre/+ mice. In control mice, bulk entry (seen as smeared cellular staining) via uterine luminal epithelium (LE) decreases from D0.5 to D3.5. In P4-deficient RhoAf/fPgrCre/+ mice, bulk entry on D0.5 and D3.5 is impaired. Exogenous P4 treatment on D1.5 and D2.5 increases bulk entry in D3.5 P4-deficient RhoAf/fPgrCre/+ LE, while progesterone receptor (PR) antagonist RU486 treatment on D1.5 and D2.5 diminishes bulk entry in D3.5 control LE. The abundance of autofluorescent apical fine dots, presumptively endocytic vesicles to reflect endocytosis, in the LE cells is generally increased from D0.5 to D3.5 but its regulation by exogenous P4 or RU486 is not obvious under our experimental setting. In the glandular epithelium (GE), bulk entry is rarely observed and green cellular dots do not show any consistent differences among all the investigated conditions. This study demonstrates the dominant role of LE but not GE, the temporal mechanisms of bulk entry and endocytosis in the LE, and the inhibitory effects of P4-deficiency and RU486 on bulk entry in the LE in preimplantation uterine fluid absorption.