Hedgehog pathway inhibition causes primary follicle atresia and decreases female germline stem cell proliferation capacity or stemness

Background: Follicle depletion is one of the causes of premature ovarian failure (POF) and primary ovarian insufficiency (POI). Hence, maintenance of a certain number of female germline stem cells (FGSCs) is optimal to produce oocytes and replenish the primordial follicle pool. The mechanism that regulates proliferation or stemness of FGSCs could contribute to restoring ovarian function, but it remains uncharacterized in postnatal mammalian ovaries. This study aims to investigate the mechanism by which inhibiting the activity of the Hedgehog (Hh) signaling pathway regulates follicle development and FGSC proliferation.

Methods and results: To understand the role of the Hh pathway in ovarian aging, we measured Hh signaling activity at different reproductive ages and the correlation between them in physiological and pathological mice. Furthermore, we evaluated the follicle number and development and the changes in FGSC proliferation or stemness after blocking the Hh pathway in vitro and in vivo. In addition, we aimed to explain one of the mechanisms for the FGSC phenotype changes induced by treatment with the Hh pathway-specific inhibitor GANT61 via oxidative stress and Apoptosis. The results show that the activity of Hh signaling is decreased in the ovaries in physiological aging and POF models, which is consistent with the trend of expression levels of the germline stem cell markers Mvh and Oct4. In vitro, blocking the Hh pathway causes follicular developmental disorders and depletes ovarian germ cells and FGSCs after treating ovaries with GANT61. The proliferation or stemness of cultured primary FGSCs is reduced when Hh activity is blocked. Our results show that the antioxidative Enzyme level and the ratio of Bcl-2/Bax decrease, the expression level of Caspase 3 increases, the mitochondrial membrane potential is abnormal, and ROS accumulate in this system.

Conclusions: We observed that the inhibition of the Hh signaling pathway with GANT61 could reduce primordial follicle number and decrease FGSC reproductive capacity or stemness through oxidative damage and Apoptosis.