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

Abstract

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 ECS^ASB9. Genetic ablation of ECS^ASB9 in mice via ubiquitous Asb9 knockout (KO) or spermatid-specific elongin B/C conditional KO disrupts spermiogenesis and compromises fertility. Mechanistic studies reveal that ECS^ASB9 engages tubulin beta 4 A (TUBB4A) through substrate recognition, catalyzing K48-linked polyubiquitination at lysine 379 (K379) to promote proteasomal degradation. Notably, Tubb4a^K379R knock-in (KI) mice phenocopy the spermiogenesis defects observed upon ECS^ASB9 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 ECS^ASB9 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.