Methionine deficiency causes spermatogonial apoptosis via oxidative stress and DNA damage response pathway

Methionine serves as an essential amino acid regulating de novo protein synthesis and redox homeostasis. Previous studies have established adverse impacts of methionine restriction and deprivation on semen quality, but effects on early spermatogenesis remain poorly characterized. In this study, methionine dietary model (0.86%, 0.17%, 0%) was used to investigate the role of methionine in early spermatogenesis. The results indicated that methionine deprivation caused spermatogenesis defects by inhibiting spermatogonial proliferation and increasing apoptosis. Further studies showed that methionine deprivation downregulated mitochondrial function-related genes (Gpx4, Fis1 and Gstm1), but upregulated ISR- (Atf4, Chac1 and Ddit3) and DNA damage response-related genes (Cdkn1a, Chek2 and Atm). Meanwhile, methionine deprivation caused mitochondrial dysfunction characterized by mitochondrial membrane potential depolarization, ROS accumulation, and MitoSOX accumulation. Methionine deprivation also caused an obvious increase in DNA damage response proteins (?H2AX, p-CHK2 and p-p53) and pro-apoptotic proteins (PUMA, BAX and c-PARP1), but suppressed anti-apoptotic protein BCL2. Furthermore, NAC effectively reversed the proliferation deficiency of GC-1 cells caused by methionine deprivation. Collectively, these findings suggest that methionine deprivation triggers ISR activation, which subsequently induces spermatogonial apoptosis via oxidative stress and the CHK2-p53/p21 signaling cascade. This study highlights the critical role of methionine in early spermatogenesis, provides mechanistic insights for optimizing dietary interventions and addresses related reproductive disorders. Overall design: Methionine restriction or deprivation was performed in mice and GC-1 cells. Proliferation, apoptosis, and blood-testis barrier integrity in the testes were examined. Finally, mechanisms were investigated through transcriptome analysis, immunofluorescence staining, and Western blotting.