Cryopreservation Disrupts TRIM21‐Mediated AKAP3 Ubiquitination and Triggers Stress Granule Formation in Human Sperm

ABSTRACT Background Sperm cryopreservation causes cryodamage, but the roles of posttranslational modifications (PTMs) and stress granules (SGs) in this process remain unclear. Objectives To investigate the molecular mechanism of cryodamage via the TRIM21–AKAP3 axis, PTMs, and SGs. Materials and Methods Human sperm were divided into fresh and cryopreserved groups. Global ubiquitinome, phosphoproteome, and acetylome profiling were performed, combined with co‐immunoprecipitation (Co‐IP), Western blotting, and immunofluorescence for functional validation. Sperm cryopreservation with FAZ‐3532 intervention was further conducted to verify SG‐mediated injury. Results Cryopreservation‐induced SG formation in mature human sperm is reported here for the first time. TRIM21 acted as the E3 ubiquitin ligase for AKAP3, targeting K31/K425; cryopreservation redirected TRIM21 into SGs, reducing AKAP3 ubiquitination and causing its accumulation. AKAP3 showed differential phosphorylation (22 sites) and acetylation (K433). Inhibiting SG assembly with FAZ‐3532 remodeled AKAP3 ubiquitination patterns and partially improved post‐thaw progressive motility. Discussion and Conclusion PTM networks and SGs are critical for sperm cryodamage. TRIM21, AKAP3, and G3BP1 are potential targets to optimize sperm cryopreservation protocols, advancing male fertility preservation.