The Hippo Signaling Pathway Regulates Ovarian Function via the Proliferation of Ovarian Germline Stem Cells

Abstract

Objective: To improve the separation, identification and cultivation of ovarian germline stem cells (OGSCs), to clarify the relationship between the Hippo signaling pathway effector YAP1 and the proliferation and differentiation of OGSCs in vitro and to identify the major contribution of Hippo signaling to ovarian function. Methods: Two-step enzymatic separation processes and magnetic separation were used to isolate and identify OGSCs by determining the expression of Mvh, Oct4, Nanog, Fragilis and Stella markers. Then, YAP1, as the main effector molecule in the Hippo signaling pathway, was chosen as the target gene of the study. Lentivirus containing overexpressed YAP1 or a YAP1-targeted shRNA was transduced into OGSCs. The effects of modulating the Hippo signaling pathway on the proliferation, differentiation, reproduction and endocrine function of ovaries were observed by microinjecting the lentiviral vectors with overexpressed YAP1 or YAP1 shRNA into infertile mouse models or natural mice of reproductive age. Results: (1) The specific expression of Mvh, Oct4, Nanog, Fragilis and Stella markers was observed in isolated stem cells. Thus, the isolated cells were preliminarily identified as OGSCs. (2) The co-expression of LATS2, MST1, YAP1 and MVH was observed in isolated OGSCs. Mvh and Oct4 expression levels were significantly increased in OGSCs overexpressing YAP1 compared to GFP controls. Consistently, Mvh and Oct4 levels were significantly decreased in cells expressing YAP1-targeted shRNA. (3) After 14-75 days of YAP1 overexpression in infertile mouse models, we detected follicle regeneration in ovaries, the activation of primordial follicles and increased birth rate, accompanied by increasing levels of E2 and FSH. (4) However, we detected decreasing follicles in ovaries, lower birth rate, and decreasing E2 and FSH in serum from healthy mice of reproductive age following YAP1 shRNA expression. Conclusion: Methods for the isolation, identification and culture of OGSCs were successfully established. Further results indicate that isolated OGSCs can specifically recognize Hippo signaling molecules and that manipulation of YAP1 expression can be used to regulate the proliferation and differentiation of OGSCs, as well as ovarian function in mice. This study suggests that the Hippo signaling pathway may represent a new molecular target for the regulation of mouse ovarian functional remodeling.