Inhibiting Necroptosis of Spermatogonial Stem Cell as a Novel Strategy for Male Fertility Preservation

Fertility preservation is a common concern for male Cancer survivors of reproductive age. However, except for testicular tissue cryopreservation, which is not very effective, there is no feasible and precise therapy capable of protecting spermatogenesis for prepubertal boys before or during gonadotoxic treatment. This study aims to investigate the effects of inhibiting Necroptosis of spermatogonial stem cell (SSC) in fertility preservation. Male mice 12 weeks of age were used to establish gonadotoxicity with two intraperitoneal injections of busulfan at a total dose of 40 mg kg^-1. The mouse model and the primary cultured mouse SSCs were used to characterize the relationship between Necroptosis of SSC and gonadotoxicity. Meanwhile, the effects of an inhibitor of Necroptosis pathway, RIPA-56, were observed on day 36 in the mouse model of busulfan-induced gonadotoxicity. We found that the number of SSCs was decreased, but the level of Necroptosis was upregulated on day 18 after busulfan treatment in testes from gonadotoxic mice. Further experiments in primary cultured cells showed that the Necroptosis caused cell death in busulfan-treated SSCs and could be inhibited by RIPA-56. After suppressing the Necroptosis of SSCs, the busulfan-induced mice had a decreased loss of spermatogenic cells as shown by histology and an increased Johnsen's score. Moreover, the quantities of SSCs and epididymal spermatozoa were restored after intervention with RIPA-56, indicating a series of beneficial effects by targeting the Necroptosis of SSCs in mice undergoing busulfan treatment. In conclusion, our findings reveal that the Necroptosis of SSCs plays a critical role in busulfan-induced gonadotoxicity and may be a potential target for male fertility preservation.