Urolithin A maintains primordial follicle dormancy and prevents cyclophosphamide-induced follicle loss by inhibiting the PI3K/Akt signaling

Urolithin A, a natural gut microbial metabolite, exerts multiple beneficial effects upon supplementation, including prolonging lifespan, mitigating diseases, restoring the quality of aged oocytes and alleviating drug toxicity. The study aims to investigate the ovarian protective role of Urolithin A, focusing on its mechanisms for inhibiting primordial follicle activation and mitigating cyclophosphamide (CY)-induced follicle apoptosis. The results showed that Urolithin A significantly decreased the number of growing follicles, downregulated the expression of oocyte growth-related genes (Gdf9 and Zp3) and protein (DDX4), as well as Ki-67 and BrdU-positive signals. Further studies revealed that Urolithin A downregulated the levels of phosphorylated Akt and FOXO3a and decreased the percentage of oocytes with FOXO3a nuclear export. Molecular docking showed a strong binding ability between Urolithin A and its downregulated gene Pik3cg (PI3K?). Moreover, Urolithin A attenuated CY- and 4-HC-induced increases in Cleaved caspase-3 and cleaved PARP1 positive signals. Meanwhile, RNA-seq analysis indicated that Urolithin A downregulated CY-induced expression of DNA damage-related genes (Trp73 and Trim29). In short, Urolithin A inhibits primordial follicle activation by reducing PI3K/Akt pathway reactivity. Furthermore, Urolithin A prevents CY-induced follicle apoptosis. The study provides valuable insights into Urolithin A treatment for chemotherapy-induced infertility. Overall design: Ovaries from 3 days postpartum (dpp) mice were cultured in Urolithin A (5-20 µM) for 4 days to determine the optimal concentration. For the in vitro chemotherapy model, ovaries were treated with vehicle, 4-Hydroperoxy cyclophosphamide (4-HC) or 4-HC + Urolithin A. For the in vivo chemotherapy model, 4 dpp mice received intraperitoneal injections of vehicle, Urolithin A, CY or CY + Urolithin A. Follicles were quantified using hematoxylin staining. RT-qPCR, RNA-seq, immunofluorescence and Western blotting were performed to detect activity of PI3K/Akt and mTOR pathways, as well as apoptotic signaling. Molecular docking analysis was used to predict the potential interactions between Urolithin A and PI3K/Akt.