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.
Keywords: GPX4; HIF-1α; TRFC; ferroptosis; hypoxia; iron metabolism; proliferation; spermatogonial stem cell.
© 2026 The Author(s) (or their employer(s)). Published by CSIRO Publishing.