Inhibition of ferroptosis attenuates busulfan-induced oligospermia in mice

Busulfan is commonly used for cancer chemotherapy, nevertheless it cause male infertility via damaging the germ cells. Therefore, the underlying mechanism should be explored. In the present study, we demonstrated for the first time that ferroptosis was involved in busulfan-induced oligospermia in mice. Mice were given testicular injection of busulfan on both sides at the dose of 4 mg/kg body weight to establish the model of oligospermia. Four weeks later, the results showed that busulfan-treated mice exhibited decreased sperm concentration and motility, along with features of typical ferroptosis in testis, such as increased malondialdehyde (MDA) content and prostaglandin-endoperoxide synthase (PTGS2) mRNA expression, and decreased NADPH content. Inhibition of ferroptosis by ferrostatin-1 (Fer-1) or deferoxamine (DFO) partially alleviated busulfan-induced oligospermia in mice. Additionally, we also revealed that busulfan treatment induced spermatogenic cells ferroptosis by down-regulating nuclear factor-E2-related factor 2 (Nrf2) and glutathione peroxidase 4 (GPX4) expressions, and decreasing iron efflux through reduction of ferroportin 1 (FPN1) expression. Fer-1 or DFO obviously reversed busulfan-induced ferroptosis by increasing Nrf2, GPX4 and FPN1 expressions. Furthermore, after activation of Nrf2 by sulforaphane, sperm concentration and motility in busulfan-treated mice increased, accompanied by enhanced expressions of GPX4 and FPN1. These findings imply that busulfan-induced ferroptosis might be mediated via inhibition of Nrf2-GPX4 (FPN1) signaling pathway, and highlight that targeting ferroptosis serves as a potential strategy for prevention of busulfan-induced damage and male infertility.