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.

Busulfan, a chemotherapeutic agent for cancer, has detrimental effects on germ cells and fertility, yet the specific mechanisms remain largely uncertain. The blood-testis barrier (BTB) maintains a suitable microenvironment for germ cells self-renewal and spermatogenesis by blocking the interference and damage of deleterious substances. Therefore, we hypothesized that BTB abnormalities might be involved in busulfan-induced oligospermia. To verify the hypothesis, thirty male Balb/c mice were randomly administered with busulfan (at a total dose of 40 mg/kg body weight) by intraperitoneal injection for 4 weeks to establish the model of oligospermia. The results displayed that busulfan caused testicular histopathological lesions and spermatogenesis disorder. Meanwhile, busulfan disrupted BTB integrity and lessened the expressions of BTB junction proteins, including Occludin, Claudin-11 and Connexin-43. Furthermore, busulfan activated the endoplasmic reticulum (ER) stress and PERK-eIF2α signaling pathway, reflected by the increased protein expressions of GRP78, p-PERK, p-eIF2α, ATF4 and CHOP. Finally, to evaluate whether the ER stress is involved in busulfan-induced BTB destruction, the ER stress inhibitor 4-Phenylbutyric acid (4-PBA, 1 mM) was used to intervene in busulfan-exposed TM4 cells. The results displayed that inhibition of ER stress alleviated the reduction of BTB junction protein expressions induced by busulfan in TM4 cells. These data collectively indicated that busulfan-induced BTB impairment was mediated by triggering ER stress and activation of the PERK-eIF2α signaling pathway, thereby damaging the spermatogenesis, providing a new therapeutic target for male infertility induced by busulfan.