In vivo Nupr1 regulates oxidative stress–endoplasmic reticulum stress crosstalk and mitochondria-dependent apoptosis in varicocele-related testicular injury

Varicocele (VCL) is a major cause of male infertility and is closely associated with oxidative stress (OS)-induced testicular injury. Increasing evidence suggests that endoplasmic reticulum stress (ERS) interacts with OS and mitochondrial dysfunction in the pathogenesis of spermatogenic impairment, but the molecular regulators linking these processes remain incompletely understood. In this study, we investigated the role of Nupr1 in VCL-related testicular stress responses. Re-analysis of transcriptomic data from VCL rat testes identified Nupr1 as a candidate gene associated with OS, ERS, and apoptosis. Nupr1 expression was significantly increased in VCL testes and was similarly upregulated in GC-2 and TM4 cells under H ₂ O ₂ -induced OS or tunicamycin-induced ERS. Stable knockdown of Nupr1 in GC-2 cells reduced viability under oxidative challenge, increased intracellular ROS accumulation, decreased mitochondrial membrane potential, and enhanced apoptosis, accompanied by increased cleaved caspase-3 expression and an elevated Bax/Bcl-2 ratio. Notably, Nupr1 deficiency attenuated the adaptive ERS response, as shown by reduced expression of GRP78, IRE1/p-IRE1, PERK/p-PERK, and ATF6. Pharmacological induction of ERS with tunicamycin further worsened cell injury in Nupr1 -deficient cells, whereas 4-PBA failed to restore cell viability or GRP78 expression. Collectively, these findings indicate that Nupr1 is involved in the cellular stress response relevant to VCL and may help maintain adaptive UPR signaling and mitochondrial homeostasis in spermatogenic cells under oxidative stress.