cPKCγ facilitates the apoptosis of mouse GC-1 spg cells by regulating the mitophagy via inhibiting the UCHL1/HIF‑1α axis

Cryptorchidism, among the most frequent congenital urogenital anomalies in male infants, has a global prevalence ranging from 1% to 9%. Characterized by failure of testicular descent into the scrotum, such a condition exposes the testes to elevated temperature, oxidative stress, and impaired spermatogenesis, significantly increasing risks of infertility and testicular cancer. Although orchiopexy is the standard treatment, long-term spermatogenic defects often persist, highlighting the need to elucidate underlying molecular mechanisms. Although the main mechanistic experiments were performed in GC-1 spg cells through genetic manipulation of cPKCγ, an additional heat-stress experiment was included to simulate the high-temperature condition associated with cryptorchidism. In the present study, the role of conventional protein kinase C gamma (cPKCγ) in regulating mitophagy and apoptosis was investigated using mouse spermatogonial GC-1 cells. Through siRNA-mediated knockdown and adenovirus-induced overexpression, cPKCγ upregulation was demonstrated to significantly induce cellular apoptosis and disrupt mitochondrial function, indicated by elevated malondialdehyde (MDA) levels, reduced superoxide dismutase (SOD) activity, decreased ATP production, increased 8-hydroxy-2'-deoxyguanosine (8-OHdG) accumulation, increased mitochondrial reactive oxygen species (ROS), and loss of mitochondrial membrane potential. Mechanistically, cPKCγ was found to inhibit the UCHL1/HIF-1α signaling pathway, suppressing mitophagy, as evidenced by a reduced LC3-II/LC3-I ratio and decreased PINK1 and Parkin expression. Importantly, adverse effects induced by cPKCγ overexpression were substantially rescued through UCHL1 or HIF-1α overexpression, or rapamycin treatment. Conversely, knockdown of UCHL1 or HIF-1α partially counteracted the protective effects of cPKCγ silencing on cell viability, oxidative stress, and mitophagy activation. Furthermore, heat stress upregulated endogenous cPKCγ and suppressed UCHL1/HIF-1α signaling and mitophagy-related proteins in GC-1 spg cells. Collectively, cPKCγ exacerbates spermatogonial apoptosis by repressing UCHL1/HIF-1α-dependent mitophagy, suggesting a potential molecular mechanism for spermatogonial vulnerability under cryptorchidism-related heat stress.