Reactive oxygen species, not Ca²⁺ , mediates methotrexate-induced autophagy and apoptosis in spermatocyte cell line

Methotrexate (MTX) is widely used to treat cancers and systemic autoimmune diseases. However, it is severely toxic to healthy cells, especially those of the reproductive system, and therefore poses a great risk to patient fertility. In addition, the underlying mechanism of MTX-induced reproductive toxicity has not yet been fully elucidated. Here, a spermatocyte cell line (GC2) was used as an in vitro model system to determine whether MTX induces autophagy and apoptosis, and to elucidate the role of reactive oxygen species (ROS) and Ca²⁺ in these two processes. Treatment with MTX resulted in a dramatic decrease in cell viability, inhibition of cell proliferation, collapse of the mitochondrial membrane potential and activation of caspase 3, suggesting that MTX induced apoptosis. Moreover, MTX activated autophagy, as indicated by conversion of LC3-I to LC3-II (microtubule-associated protein 1 light chain 3) and an increase in the number of LC3 puncta. Furthermore, MTX triggered ROS overproduction, rather than a Ca²⁺ burst. Intriguingly, eliminating excess ROS significantly alleviated MTX-induced apoptosis and autophagy. In addition, inhibiting autophagy significantly reversed apoptosis and promoted cell survival, indicating that autophagy aggravated MTX-induced apoptosis in GC2 cells. Taken together, these results suggest that ROS signalling, not Ca²⁺ , is critical in mediating MTX-induced autophagy and apoptosis and autophagy serves as a promoted partner of apoptosis to deteriorate MTX-induced cytotoxicity in GC2 cells. The findings from this study provide new perspectives for evaluating the reproductive toxicity of MTX.