RA-RAR signaling promotes mouse vaginal opening through increasing β-catenin expression and vaginal epithelial cell apoptosis

Abstract

Background: Retinoic acid (RA) plays important role in the maintenance and differentiation of the Müllerian ducts during the embryonic stage via RA receptors (RARs). However, the function and mechanism of RA-RAR signaling in the vaginal opening are unknown. Method: We used the Rarα knockout mouse model and the wild-type ovariectomized mouse models with subcutaneous injection of RA (2.5 mg/kg) or E2 (0.1 µg/kg) to study the role and mechanism of RA-RAR signaling on the vaginal opening. The effects of Rarα deletion on Ctnnb1 mRNA levels and cell apoptosis in the vaginas were analyzed by real-time PCR and immunofluorescence, respectively. The effects of RA on the expression of β-catenin and apoptosis in the vaginas were analyzed by real-time PCR and western blotting. The effects of E2 on RA signaling molecules were analyzed by real-time PCR and western blotting. Results: RA signaling molecules were expressed in vaginal epithelial cells, and the mRNA and/or protein levels of RALDH2, RALDH3, RARα and RARγ reached a peak at the time of vaginal opening. The deletion of Rarα resulted in 25.0% of females infertility due to vaginal closure, in which the mRNA (Ctnnb1, Bak and Bax) and protein (Cleaved Caspase-3) levels were significantly decreased, and Bcl2 mRNA levels were significantly increased in the vaginas. The percentage of vaginal epithelium with TUNEL- and Cleaved Caspase-3-positive signals were also significantly decreased in Rarα−/− females with vaginal closure. Furthermore, RA supplementation of ovariectomized wild-type (WT) females significantly increased the expression of β-catenin, active β-catenin, BAK and BAX, and significantly decreased BCL2 expression in the vaginas. Thus, the deletion of Rarα prevents vaginal opening by reducing the vaginal β-catenin expression and epithelial cell apoptosis. The deletion of Rarα also resulted in significant decreases in serum estradiol (E2) and vagina Raldh2/3 mRNA levels. E2 supplementation of ovariectomized WT females significantly increased the expression of RA signaling molecules in the vaginas, suggesting that the up-regulation of RA signaling molecules in the vaginas is dependent on E2 stimulation. Conclusion: Taken together, we propose that RA-RAR signaling in the vaginas promotes vaginal opening through increasing β-catenin expression and vaginal epithelial cell apoptosis.