A testis-specific E3 ubiquitin ligase complex governs spermiogenesis and male fertility

The ubiquitin-proteasome system (UPS) represents an evolutionarily conserved machinery governing proteostasis through spatiotemporal regulation of protein degradation. While spermatogenesis involves multilayered regulatory mechanisms spanning translation to dynamic post-translational modifications (PTMs), the identity of UPS-associated E3 ligases orchestrating germ cell-specific protein turnover remains elusive. Here, we identify a testis-specific E3 ubiquitin ligase complex comprising elongin B/C, Cullin-2 (CUL2), RING-box protein-1 (RBX1), and SOCS box protein ASB9, designated ECSASB9. Genetic ablation of ECSASB9 in mice via ubiquitous Asb9 knockout (KO) or spermatid-specific elongin B/C conditional KO disrupts spermiogenesis and compromises fertility. Mechanistic studies reveal that ECSASB9 engages tubulin beta 4 A (TUBB4A) through substrate recognition, catalyzing K48-linked polyubiquitination at lysine 379 (K379) to promote proteasomal degradation. Notably, Tubb4aK379R knock-in (KI) mice phenocopy the spermiogenesis defects observed upon ECSASB9 deficiency. Clinically, we identify three hemizygous missense variants in X-linked ASB9 among Chinese males with idiopathic infertility. Male mice bearing orthologous ASB9 variant exhibit oligoasthenoteratozoospermia (OAT) and subfertility, mirroring human phenotypes. Taken together, our findings establish ECSASB9 as an important regulator of spermatogenic proteostasis and provide mechanistic insights into UPS-mediated tissue-specific degradation, while implicating ASB9 variants in male infertility pathogenesis. A testis-specific E3 ubiquitin ligase complex, ECSASB9, governs spermiogenesis by promoting TUBB4A degradation. Disruption of this pathway impairs male fertility, and pathogenic ASB9 variants are found in infertile men.