Testicular tissue cryopreservation is essential for fertility preservation in prepubertal boys who cannot produce semen, but the process triggers oxidative stress, mitochondrial dysfunction, and excessive mitophagy that deplete spermatogonial stem cells (SSCs) and compromise later spermatogenesis. We evaluated whether melatonin (MLT), a mitochondria-targeted antioxidant, mitigates cryoinjury in a prepubertal C57BL/6 mouse model using a controlled slow-freezing protocol supplemented with graded MLT doses. Across histology, immunofluorescence, Western blotting, transmission electron microscopy, flow cytometry, ELISAs, and integrated transcriptomic-metabolomic profiling, cryopreservation alone induced reactive oxygen species (ROS) overproduction, disrupted mitochondrial integrity, activated PINK1/Parkin-mediated mitophagy, and increased apoptosis with loss of undifferentiated spermatogonial marker (PLZF) and SSC marker (UCHL1). MLT at an optimal concentration of 10 M preserved testicular architecture, maintained undifferentiated spermatogonia and SSC marker expression, suppressed ROS accumulation, restored redox balance, and alleviated mitochondrial damage while tempering excessive PINK1/Parkin-dependent mitophagy. Omics analyses showed that MLT reprogrammed cryo-induced disturbances in oxidative stress and metabolic pathways and was associated with suppression of MAPK-mediated apoptotic signaling. In an ectopic transplantation (xenograft) model, grafts pretreated with MLT exhibited improved spermatogenic recovery and reduced fibrotic remodeling compared with untreated controls. Pharmacologic activation of mitophagy with CCCP supported the role of mitophagy modulation in MLT's protective effects. Collectively, these data indicate that 10 M MLT effectively safeguards cryopreserved testicular tissue by maintaining SSC viability and mitochondrial/redox homeostasis while limiting maladaptive mitophagy and apoptosis, supporting its use as a practical adjunct to male fertility preservation protocols for prepubertal patients undergoing gonadotoxic therapy. -7 -7
