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

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 ß-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 ß-catenin, active ß-catenin, BAK and BAX, and significantly decreased BCL2 expression in the vaginas. Thus, the deletion of Rarα prevents vaginal opening by reducing the vaginal ß-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 ß-catenin expression and vaginal epithelial cell apoptosis.

The Mature COC Promotes the Ampullary NPPC Required for Sperm Release from Porcine Oviduct Cells.

Porcine spermatozoa are stored in the oviductal isthmus after natural mating, and the number of spermatozoa is increased in the oviductal ampulla when the mature cumulus-oocyte complexes (COCs) are transferred into the ampulla. However, the mechanism is unclear. Herein, natriuretic peptide type C (NPPC) was mainly expressed in porcine ampullary epithelial cells, whereas its cognate receptor natriuretic peptide receptor 2 (NPR2) was located on the neck and the midpiece of porcine spermatozoa. NPPC increased sperm motility and intracellular Ca2+ levels, and induced sperm release from oviduct isthmic cell aggregates. These actions of NPPC were blocked by the cyclic guanosine monophosphate (cGMP)-sensitive cyclic nucleotide-gated (CNG) channel inhibitor l-cis-Diltiazem. Moreover, porcine COCs acquired the ability to promote NPPC expression in the ampullary epithelial cells when the immature COCs were induced to maturation by epidermal growth factor (EGF). Simultaneously, transforming growth factor-ß ligand 1 (TGFB1) levels were dramatically increased in the cumulus cells of the mature COCs. The addition of TGFB1 promoted NPPC expression in the ampullary epithelial cells, and the mature COC-induced NPPC was blocked by the transforming growth factor-ß type 1 receptor (TGFBR1) inhibitor SD208. Taken together, the mature COCs promote NPPC expression in the ampullae via TGF-ß signaling, and NPPC is required for the release of porcine spermatozoa from the oviduct isthmic cells.

The oocyte cumulus complex regulates mouse sperm migration in the oviduct.

As the time of ovulation draws near, mouse spermatozoa move out of the isthmic reservoir, which is a prerequisite for fertilization. However, the molecular mechanism remains unclear. The present study revealed that mouse cumulus cells of oocytes-cumulus complexes (OCCs) expressed transforming growth factor-ß ligand 1 (TGFB1), whereas ampullary epithelial cells expressed the TGF-ß receptors, TGFBR1 and TGFBR2, and all were upregulated by luteinizing hormone (LH)/human chorionic gonadotropin (hCG). OCCs and TGFB1 increased natriuretic peptide type C (NPPC) expression in cultured ampullae via TGF-ß signaling, and NPPC treatment promoted spermatozoa moving out of the isthmic reservoir of the preovulatory oviducts. Deletion of Tgfb1 in cumulus cells and Tgfbr2 in ampullary epithelial cells blocked OCC-induced NPPC expression and spermatozoa moving out of the isthmic reservoir, resulting in compromised fertilization and fertility. Oocyte-derived paracrine factors were required for promoting cumulus cell expression of TGFB1. Therefore, oocyte-dependent and cumulus cell-derived TGFB1 promotes the expression of NPPC in oviductal ampulla, which is critical for sperm migration in the oviduct and subsequent fertilization.

Lithium treatment promotes the activation of primordial follicles through PI3K/Akt signaling†.

In mammals, dormant primordial follicles represent the ovarian reserve throughout reproductive life. In vitro activation of dormant primordial follicles has been used to treat patients with premature ovarian insufficiency (POI). However, there remains a lack of effective strategies to stimulate follicle activation in vivo. In this study, we used an in vitro ovarian culture system and intraperitoneal injection to study the effect of lithium treatment on primordial follicle activation. Lithium increased the number of growing follicles in cultured mouse ovaries and promoted pre-granulosa cell proliferation. Furthermore, lithium significantly increased the levels of phosphorylated protein kinase B (Akt) and the number of oocytes with forkhead Box O3a (FOXO3a) nuclear export. Inhibition of the phosphatidylinositol 3 kinase (PI3K)/Akt pathway by LY294002 reversed lithium-promoted mouse primordial follicle activation. These results suggest that lithium promotes mouse primordial follicle activation by the PI3K/Akt signaling. Lithium also promoted primordial follicle activation and increased the levels of p-Akt in mouse ovaries in vivo and in human ovarian tissue cultured in vitro. Taken together, lithium promotes primordial follicle activation in mice and humans by the PI3K/Akt signaling. Lithium might be a potential oral drug for treating infertility in POI patients with residual dormant primordial follicles.

Sgpl1 deletion elevates S1P levels, contributing to NPR2 inactivity and p21 expression that block germ cell development.

Sphingosine phosphate lyase 1 (SGPL1) is a highly conserved enzyme that irreversibly degrades sphingosine-1-phosphate (S1P). Sgpl1-knockout mice fail to develop germ cells, resulting in infertility. However, the molecular mechanism remains unclear. The results of the present study showed that SGPL1 was expressed mainly in granulosa cells, Leydig cells, spermatocytes, and round spermatids. Sgpl1 deletion led to S1P accumulation in the gonads. In the ovary, S1P decreased natriuretic peptide receptor 2 (NPR2) activity in granulosa cells and inhibited early follicle growth. In the testis, S1P increased the levels of cyclin-dependent kinase inhibitor 1A (p21) and apoptosis in Leydig cells, thus resulting in spermatogenesis arrest. These results indicate that Sgpl1 deletion increases intracellular S1P levels, resulting in the arrest of female and male germ cell development via different signaling pathways.