USP2 Attenuates Spermatogenic Cell Apoptosis Via PINK1/Parkin‐Dependent Mitophagy in Varicocoele: A Functional Study

BACKGROUND: Varicocoele is a major cause of male infertility, yet its underlying molecular mechanisms and determinants of surgical efficacy remain unclear. OBJECTIVE: This study aimed to identify key proteins involved in varicocoele-related infertility and to investigate the expression and functional role of ubiquitin-specific peptidase 2 in spermatozoa and spermatogenic cells. METHODS: Ubiquitin-specific peptidase 2 expression in spermatozoa from varicocoele patients was analyzed before and after varicocelectomy by Western blotting. In vitro, ubiquitin-specific peptidase 2 was overexpressed or silenced in mouse GC-2 spermatocyte cells to assess mitochondrial autophagy (PINK1/Parkin pathway), autophagic flux, apoptosis, and reactive oxygen species. In vivo, a left-sided varicocoele rat model was established, and ubiquitin-specific peptidase 2 activity was inhibited by intratesticular injection of ML364. Testicular histopathology and sperm motility were evaluated. RESULTS: Ubiquitin-specific peptidase 2 protein expression was significantly reduced in spermatozoa after varicocelectomy. Ubiquitin-specific peptidase 2 overexpression enhanced PINK1/Parkin-mediated mitochondrial autophagy and protected spermatogenic cells from apoptosis, whereas ubiquitin-specific peptidase 2 knockdown impaired mitochondrial autophagy and increased cell apoptosis. In varicocoele rats, ubiquitin-specific peptidase 2 inhibition aggravated seminiferous tubule damage, reduced spermatogenic cell density, and impaired sperm motility. In human spermatozoa, ML364 treatment significantly decreased progressive motility without affecting sperm concentration. CONCLUSION: Ubiquitin-specific peptidase 2 is essential for maintaining mitochondrial quality control and spermatogenic cell survival in varicocoele by regulating PINK1/Parkin-mediated autophagy and oxidative stress, highlighting its potential as a biomarker and therapeutic target for varicocoele-related male infertility.