Context Hypoxia could have an impact on spermatogenesis. However, the mechanism by which hypoxia affects spermatogonial stem cells (SSC) is still unknown. Aims This study aims to elucidate the impact of hypoxia on the proliferation of mouse SSC and explore the underlying mechanism. Methods We established the hypoxia cultured C18-4 cell model using CoCl2. Cell counting kit-8 and 5-ethynyl-2′-deoxyuridine assays were used to assess C18-4 cells proliferation. RNA sequencing was performed to explore the differentially expressed genes. Western blotting and real-time quantitative polymerase chain reaction were used to detect the expression of hypoxia-inducible factor-1α and iron metabolism-related proteins. The small interfering RNA assay was used to knock down the expression of transferrin receptor complex (TFRC) in hypoxia cultured C18-4 cells. Key results We found that CoCl2 decreased the proliferation of C18-4 cells. Based on the RNA sequencing, we found that iron metabolism imbalance and ferroptosis might play crucial roles in the hypoxia-induced injury to C18-4 cells. Western blotting and real-time quantitative polymerase chain reaction analyses detected the upregulation of TFRC, ferritin heavy chain, ferritin light chain and ACSL4, as well as the downregulation of ferroportin and glutathione peroxidase 4 in hypoxia cultured C18-4 cells. Notably, knock down of Tfrc restored C18-4 cell proliferation, which had been inhibited by CoCl2. Conclusions This study demonstrates that hypoxia inhibits C18-4 cell proliferation through the effects of TFRC on iron metabolism and ferroptosis. Implications The hypoxia could induce iron metabolism imbalance and ferroptosis in SSC. Regulating the expression of TFRC could protect SSC from hypoxic injury.
Qian et al. (Wed,) studied this question.