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

Wu, Tiantian; Tu, Chaofeng; Feng, Yuxuan; Qu, Wenying; Chen, Jinyi; Wu, Huan; Gao, Wenxin; Xu, Bingya; Yu, Xiangling; Bao, Mingyuan; Xu, Jinfu; Zhou, Nianchao; Hu, Haoyue; Jiang, Bing; Xie, Qingsong; Meng, Lanlan; Tan, Chen; Lin, Ge; Shen, Cong; Chen, Xia; Guo, Yueshuai; Zhou, Tao; Liang, Yuting; Hua, Rong; Cao, Yunxia; Liu, Mingxi; Yu, Jun; Huang, Xiaoyan; Tan, Yue-Qiu; Zheng, Bo

doi:10.1038/s41467-026-70025-x

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Published: 24 February 2026

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

Nature Communications volume 17, Article number: 3100 (2026) Cite this article

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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.

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Introduction

Cellular proteostasis relies on dynamic regulation of protein synthesis and degradation, with temporally controlled protein abundance dictating functional outcomes across biological systems^1,2. The ubiquitin-proteasome system (UPS) serves as a central executor of protein degradation, governed by a conserved enzymatic cascade involving E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin ligases³. This cascade initiates with ATP-dependent activation of ubiquitin (Ub) by E1, forming a thioester bond between Ub’s C-terminus and the E1 catalytic cysteine. Ub is subsequently transferred to an E2 enzyme, generating a reactive E2~Ub intermediate. E3 ligases then orchestrate substrate specificity by recruiting both the E2~Ub complex and target proteins, catalyzing Ub transfer to substrate lysine residues^4,5. K48-linked polyubiquitination marks substrates for recognition and proteolysis by the 26S proteasome^6,7.

The remarkable diversity of E3 ligases (>600 in humans) reflects their evolutionary adaptation to target a broad array of substrates^8,9. E3s are classified into three structural families: RING (really interesting new gene), HECT (homologous to E6AP carboxyl terminus), and RBR (RING-between-RING)¹⁰. RING-type E3s, the largest class, utilize zinc-coordinating RING or U-box domains to bind E2~Ub and facilitate Ub transfer¹¹. Among these, cullin-RING ligases (CRLs) represent modular multi-subunit complexes assembled on cullin (CUL) scaffolds, RING-box proteins, adaptors, and substrate receptors^12,13,14. A prototypical CRL subclass is the ECS complex (Elongin B/C–CUL–SOCS-box protein), where SOCS-box proteins serve as substrate receptors by interacting with the Elongin B/C (ELOB/C) adaptor dimer^15,16. This adaptor recruits either a CUL2-RBX1 or a CUL5-RBX2 catalytic core, completing assembly of a functional E3 ligase that directs substrate polyubiquitination and proteasomal degradation¹⁷.

Notably, >40 SOCS-box proteins have been identified in mammals¹⁶, conferring tissue-specific functionality to ECS complexes. For instance, SOCS2 modulates growth hormone signaling through dual regulatory roles^18,19,20,21, while ASB3 suppresses antiviral immunity by targeting mitochondrial antiviral signaling protein (MAVS) for polyubiquitination and degradation²². Tissue-restricted ECS variants include ASB15 in skeletal muscle differentiation^23,24, and ASB6 in adipocyte insulin signaling via SH2B2 (SH2B adapter protein 2) degradation²⁵. These studies collectively demonstrate that ECS complex composition dictates substrate selectivity and functional specialization across tissues. However, the mechanisms governing ECS assembly heterogeneity and its tissue-specific regulatory logic remain unresolved, necessitating systematic exploration of ECS plasticity in vivo.

In somatic cells, ASB9 is predominantly known to assemble into an ECS complex with CUL5, where it targets mitochondrial proteins such as ubiquitous mitochondrial creatine kinase (uMtCK) for degradation, thereby regulating mitochondrial structure and bioenergetics²⁶. This establishes a paradigm where ASB9–CUL5 functions in metabolic regulation. However, whether ASB9 can form alternative, tissue-specific complexes with distinct catalytic cores and functions remains an open question. To address this gap and to systematically explore the heterogeneity of ECS complexes in vivo, we performed tissue-specific proteomic profiling of endogenous ECS complexes in mice, which led to the identification of a testis-specific ECS variant, termed ECS^ASB9, that is important for spermatogenesis and fertility in mice and humans.

Results

IP-coupled proteomic profiling identifies ECS^ASB9

To systematically assess the heterogeneity of the endogenous ECS complex across mammalian tissues, we conducted comprehensive interactome mapping in 11 adult mouse tissues using ELOB as bait (Fig. 1a and Supplementary Data 1). ELOB, together with ELOC, forms a stable adaptor dimer that is central to the assembly of ECS-type E3 ligases. This ELOB/C dimer serves a dual function: it specifically recruits SOCS-box-containing substrate receptors while simultaneously binding to the cullin (CUL2 or CUL5) scaffold, thereby bridging the substrate-recognition module to the catalytic core of the ligase. The selection of ELOB as bait was predicated on this essential adaptor role^27,28 and its relatively stable expression across tissues compared with other ECS components, as evidenced by reanalysis of published murine multi-tissue transcriptomic datasets (Fig. S1)²⁹. IP coupled to liquid chromatography-tandem mass spectrometry (IP-LC-MS/MS) profiling revealed tissue-specific variability in ECS complex composition. ELOB/C and CUL2 demonstrated constitutive presence across all examined tissues. In contrast, SOCS-box proteins displayed stringent spatial restriction, with NEURL2 and ASB10 exclusively detected in heart, while VHL showed intestinal specificity (Fig. 1a). Of note, testicular heterogeneity was observed, characterized by the co-enrichment of four SOCS-box proteins: ASB1, ASB3, ASB9, and TCEB3. Among these, TCEB3 exhibited broad distribution (detected in 5/11 tissues), whereas ASB1/3/9 showed strictly testis-specific enrichment (Fig. 1a). Notably, uterus uniquely harbored both CRL2- and CRL5-type ECS complexes, with CUL5 showing uterus-specific enrichment (Fig. 1a). Collectively, our multi-tissue interactome reveals considerable combinatorial diversity in the ECS complex.

Fig. 1: Characterization of the ECSASB9 complex in mouse testis. — **Fig. 1: Characterization of the ECS^ASB9 complex in mouse testis.**

In our previous studies, we found that Asb1 and Asb3 showed abundant expression during the postmeiotic spermatid elongation phase in mice^30,31. Notably, whereas Asb3-deficient mice maintained normal fertility and spermatogenesis³¹, ASB1 formed a functional E3 ligase complex with ELOB to orchestrate the ubiquitination of sulfide-quinone oxidoreductase (SQOR), thereby modulating hydrogen sulfide (H₂S) metabolic homeostasis during spermiogenesis³⁰. This mechanistic insight was corroborated by the phenotype of Asb1-KO mice, which exhibited compromised male fertility characterized by sperm defects and significant testicular H₂S depletion³⁰. However, little is known about the distribution and function of ASB9 during spermatogenesis. To this end, we first performed a cross-species conservation analysis of ASB9, which showed that it is highly conserved across diverse taxa (Fig. S2a). Notably, the structural integrity of both Ankyrin (ANK) and SOCS domains was maintained throughout evolution, strongly suggesting an evolutionarily conserved functional role for this protein (Fig. S2a). RT‒PCR identified Asb9 as testis-enriched in both mice and humans (Fig. S2b, c). In addition, immunostaining uncovered strict spatiotemporal distribution of ASB9 during spermatogenesis (Fig. 1b). While core ECS components ELOB/C, CUL2, and RBX1 exhibited constitutive expression across all spermatogenic steps (Fig. 1c–f), ASB9 first appeared at step 8 spermatids. Its signal intensified through steps 9-14, then sharply declined by step 15 (Fig. 1b). Intriguingly, TCEB3, a ubiquitously identified ECS substrate receptor, displayed complementary dynamics: present from pachytene spermatocytes to step 8 spermatids but undetectable in later steps (Fig. 1g). This reciprocally phased expression pattern suggests a model where timed replacement of TCEB3 by ASB9 reprograms ECS complex substrate specificity during late spermiogenesis (Fig. 1h).

Immunofluorescence imaging of testicular single-cell suspensions revealed a skirt-like structure of ASB9 signals surrounding the elongating spermatid head (Fig. 1i, j), a topography reminiscent of the transient microtubule-based manchette that is crucial for sperm head shaping³². Co-immunostaining with the manchette marker α-tubulin showed precise colocalization in elongating spermatids, confirming ASB9’s location in manchette (Figs. 1k, l and S3). Subsequently, we performed Co-IP assays using testicular lysates to validate the IP-LC-MS/MS results. Anti-ELOB antibody precipitated the immunoprecipitates comprising ELOB/C, CUL2, RBX1, and ASB9 (Fig. 1m), demonstrating constitutive assembly of an ECS complex we term ECS^ASB9. Together, these data suggest testis-specific ECS^ASB9 functions as a manchette-resident ubiquitination machinery which may be required for sperm head morphogenesis.

Impaired spermiogenesis and fertility in Asb9-KO mice

To investigate the functional significance of ECS^ASB9 in male fertility, we established an X-linked ubiquitous Asb9 knockout (Asb9-KO) mouse model via CRISPR/Cas9-mediated genome editing (Fig. S4a). Sanger sequencing verified a 13-bp deletion causing frameshift mutation in hemizygous Asb9-KO males (Fig. S4b), with subsequent Western blot and immunostaining confirming complete ASB9 protein ablation in Asb9-KO testes (Fig. S4c, d). Asb9-KO males exhibited marked subfertility accompanied by smaller testis sizes and lower testis weights compared to wild-type (WT) mice (Fig. 2a–c). Epididymal sperm analysis revealed that the sperm count, motility and progressive motility were significantly reduced in Asb9-KO mice relative to WT mice (Fig. 2d–f). Notably, head morphology defects, including amorphous, microcephalic, or tapered configurations (Fig. S5a, b), as well as acrosomal abnormalities, such as irregular shaping, fragmentation or loss of membrane integrity (Fig. 2g, h), were increased in Asb9-KO mice, while tail abnormalities remained statistically comparable between the two groups (Figs. 2g, h and S5a, b). Ultrastructurally, WT sperm displayed intact acrosomal caps overlying densely condensed nuclei, whereas Asb9-KO sperm exhibited malformed heads with acrosomal defects ranging from irregular and fragmented structures to near-complete absence (Fig. 2i and S5c, d). In contrast, the flagellar ultrastructure of Asb9-KO sperm appeared normal, exhibiting the typical “9+2” microtubule doublet arrangement characteristic of functional sperm tails (Fig. 2i). To investigate the basis of the motility defect despite normal flagellar structure, we measured intracellular ATP levels. Sperm from Asb9-KO mice exhibited a significant reduction in ATP content compared to WT controls (Fig. S5e), indicating a state of bioenergetic deficit. These collective anomalies define a classical oligoasthenoteratozoospermia (OAT) phenotype, explaining the observed subfertility in Asb9-KO males.

**Fig. 2: ASB9 ablation impairs spermatogenesis.**

Histomorphometric assessment of Asb9-KO and WT testes revealed comparable populations of spermatogonia, spermatocytes, Sertoli cells, and Leydig cells between the two groups (Fig. S6). However, morphologically compromised spermatids were evident in Asb9-KO tubules (Fig. S6a). The increased apoptotic signals in the cauda epididymis of Asb9-KO mice (Fig. S7) suggest that these deformed spermatids may undergo apoptosis. This interpretation is supported by step-resolved morphometric analysis of Periodic acid-Schiff (PAS)- and peanut agglutinin (PNA)-stained spermatids, which revealed normal spermatid numbers and head shaping through step 9 in Asb9-KO mice, but a significant reduction in spermatid number and an increase in head and acrosome defects at steps 10–16 of spermiogenesis (Figs. 2j–m and S8).

The manchette, a transient microtubular scaffold composed of α/β-tubulin heterodimers, assembles transiently during mouse spermiogenesis (steps 8–14), serving as the principal architect of postmeiotic nuclear shaping^33,34,35. Given ASB9’s manchette-specific localization (Figs. 1k, l and S3), we explored whether the deficiency of ASB9 affects manchette structure across spermiogenesis. Immunofluorescence staining and transmission electron microscopy (TEM) revealed intact manchette assembly in Asb9-KO spermatids at steps 8–9, with normally condensing nuclei and properly overlying acrosomes (Figs. 2n, o and S9a). However, at step 10, the microtubule bundles in Asb9-KO spermatids were thinner than in WT spermatids, which coincided with the onset of nuclear shaping defects and aberrant acrosomal development (Figs. 2n, o and S9b). In later steps, aberrant elongation and disorganization of the manchette in Asb9-KO spermatids were accompanied by severe malformation of the sperm head and irregular acrosomal structure (Figs. 2n, o and S9c, d). Consequently, by late spermiogenesis when the manchette disassembles, Asb9-KO spermatids failed to complete remodeling, resulting in the production of spermatozoa with misshapen heads and malformed acrosomes (Fig. S9e). All these results indicate ASB9 as an important modulator of manchette dynamics during murine spermiogenesis, with its deficiency causing OAT through microtubule-dependent nuclear shaping defects.

Given that Asb9 mRNA is detectable in several tissues (Fig. S2b), with notable expression in the kidney, we assessed whether ASB9 deficiency led to broader pathological consequences. Histopathological examination of key organs, including the brain and lung (tissues rich in motile cilia) and the kidney, revealed no overt morphological abnormalities in Asb9-KO adults compared to WT littermates (Fig. S10).

ASB9 facilitates the K48-linked ubiquitination of TUBB4A on K379

To identify substrates of the ECS^ASB9 complex critical for manchette dynamics, we performed IP-LC-MS/MS on endogenous ASB9 complexes isolated from mouse testes. Comparative analysis of two biological replicates identified 97 overlapping proteins, including ASB9, ELOB/C, and tubulin beta 4A (TUBB4A)—a β-tubulin family member (Fig. 3a and Supplementary Data 2). Reciprocal co-IP validated endogenous interactions between ASB9 and both ELOB/C and TUBB4A in testicular lysates (Fig. 3b). Immunofluorescence confirmed colocalization of ASB9 and TUBB4A in elongating spermatids (Fig. 3c, d). Notably, deficiency of ASB9 increased TUBB4A protein abundance without altering its mRNA levels (Figs. 3e–g, and S11a), implicating post-translational regulation. Consistently, TUBB4A immunoprecipitated from Asb9-KO testes exhibited reduced poly-pan and poly-K48 ubiquitination compared to WT testes, as assessed by pan-ubiquitin and K48-specific antibodies (Fig. 3h). Given the canonical role of K48 chains in proteasomal degradation, these findings suggest TUBB4A as an ECS^ASB9 substrate undergoing ubiquitin-dependent degradation.

Fig. 3: TUBB4A is a substrate of ECSASB9. — **Fig. 3: TUBB4A is a substrate of ECS^ASB9.**

To mechanistically investigate this regulation, we co-expressed Flag-ASB9 and GFP-TUBB4A in HEK-293T cells. The reciprocal interaction was confirmed by co-IP (Fig. S11b), while ASB9 overexpression dose-dependently reduced endogenous TUBB4A levels (Fig. 3i, j). Cycloheximide (CHX) chase assays revealed decreased TUBB4A stability upon ASB9 overexpression (Fig. 3k, l). Moreover, we found that MG132, a proteasome inhibitor, could efficiently restore TUBB4A protein levels even under ASB9 overexpression (Fig. 3m, n). In vitro ubiquitination assays revealed that testis-derived ASB9 complexes catalyzed TUBB4A polyubiquitination (Fig. S11c), a process that was also observed in HEK-293T cells (Fig. 3o). Ubiquitin mutagenesis narrowed chain specificity to K48, as neither K6/K11/K27/K29/K33/K63 variants nor K48R (Lys48 substituted with arginine) supported conjugation (Fig. 3p, q).

Mapping ubiquitination sites using our published ubiquitome data of mouse testis³⁶, we identified five candidate lysines (K122, K216, K252, K297, K379) on TUBB4A (Fig. 3r). Site-directed mutagenesis revealed that only K379 mutation abolished ASB9-mediated polyubiquitination (Fig. 3s). Correspondingly, TUBB4A-K379R exhibited resistance to ASB9-driven destabilization in CHX assays (Fig. 3t, u), pinpointing K379 as the key residue for ECS^ASB9-dependent proteasomal targeting.

TUBB4A mutation phenocopies the spermiogenesis defects of ASB9 deficiency

To investigate the in vivo role of TUBB4A K379 in manchette integrity, we generated Tubb4a^K379R knock-in (KI) mice via CRISPR-Cas9-mediated genome editing (Fig. S12a). Genotypic validation by Polymerase Chain Reaction (PCR) sequencing confirmed successful introduction of the K379R mutation (Fig. S12b). As anticipated, Tubb4a-KI mice displayed reduced K48-linked and pan-polyubiquitination of TUBB4A, concomitant with increased TUBB4A protein abundance in testes, while mRNA levels remained unaltered (Figs. 4a–d and S12c).

**Fig. 4: TUBB4A K379 is required for manchette assembly.**

Although Tubb4a-KI mice developed normally, they exhibited subfertility phenotypes (Fig. 4e). Testicular appearance and weight showed no significant differences between Tubb4a-KI and WT littermates (Fig. S12d, e). However, epididymal sperm analyzes revealed decreased sperm count, sperm motility, and progressive motility in Tubb4a-KI mice compared to WT mice (Fig. 4f–h). Furthermore, sperm head and acrosome defects were markedly increased in Tubb4a-KI mice, while tail abnormalities remained comparable to those in WT controls (Figs. 4i–k and S12f, g).

Histopathological examination revealed shared spermiogenesis defects between Asb9-KO and Tubb4a-KI mice. As shown in Fig. 4l–o, the elongating spermatids in Tubb4a-KI mice exhibited apparent nuclear and acrosomal deformation, coinciding with aberrant elongation of the manchette cytoskeleton. These structural anomalies correlated temporally with disrupted manchette-mediated nuclear shaping, phenocopying the defects observed in Asb9-KO spermatids.

Collectively, these findings uncover TUBB4A K379 as a critical residue governing ubiquitination-dependent regulation of microtubule dynamics during murine spermiogenesis.

ELOB/C maintain ECS^ASB9 complex integrity to ensure spermiogenesis

While ELOB/C are established binding partners of ASB-family proteins, their functional necessity in ECS^ASB9-mediated substrate recognition remained undefined. To specifically dissect the role of the late-acting ECS^ASB9 complex without perturbing earlier ECS functions, we designed a spermatid-targeted knockout strategy. Although ELOB/C are expressed in multiple germ cell types, another ECS substrate receptor, TCEB3, operates from pachytene spermatocytes through step 8 spermatids (Fig. 1c, d, g, h). Ubiquitous or earlier knockout of Elob/c would disrupt the ECS^TCEB3 complex and confound interpretation of phenotypes. To isolate the function of ECS^ASB9 during late spermiogenesis, we generated Tnp2-Cre transgenic mice (Fig. S13) and Elob/c-floxed mice (Fig. S14a, b). The Tnp2-Cre driver restricts Cre recombinase activity to spermatids beginning at step 8 (Fig. 5a, b), thereby preserving ECS^TCEB3 function while enabling selective ablation of ELOB/C in post-step 8 spermatids. Crossing these strains produced spermatid-specific Elob/c conditional knockout mice (Elob/c-cKO) (Fig. S14a, b). Immunofluorescence staining confirmed efficient depletion of ELOB and ELOC specifically in elongating spermatids of Elob-cKO and Eloc-cKO testes, respectively (Fig. S14c, d).

Fig. 5: ELOB/C ablation disrupt ECSASB9 complex integrity and impair spermiogenesis. — **Fig. 5: ELOB/C ablation disrupt ECS^ASB9 complex integrity and impair spermiogenesis.**

Breeding assays revealed that Elob/c-cKO males were subfertility despite normal mating behaviors (Fig. 5c). Testis size and weight remained comparable to WT littermates (Fig. S14e). However, histopathological analysis of Elob/c-cKO epididymal sperm and testes uncovered an OAT phenotype accompanied by sperm head shaping defects and manchette disorganization (Figs. 5d–i and S14f–k), phenocopying Asb9-KO and Tubb4a-KI models.

To dissect the molecular basis of these defects, we interrogated ECS^ASB9 complex integrity in Elob/c-cKO testes. Immunoblot analyzes revealed coordinated reductions in core components: ELOB/C, ASB9, and RBX1 (Fig. 5j–m). Additionally, TUBB4A protein levels in Elob/c-cKO testes were significantly increased (Fig. 5j–m), consistent with loss of ECS^ASB9-mediated ubiquitination.

Together, these data reveal ELOB/C as key adaptor for ECS^ASB9 complex stability, with their ablation triggering aberrant TUBB4A accumulation and consequent failure of manchette-mediated nuclear remodeling during spermiogenesis.

Identification of hemizygous ASB9 variants in four infertile men

To explore a possible role for ASB9 in human male fertility, we screened whole-exome sequencing data from a cohort of 1483 individuals with oligoasthenozoospermia. We identified four infertile men with oligoasthenozoospermia harbored hemizygous X-linked ASB9 missense variants. These included c.542C>T (p.A181V) in families F1 and F3, c.275G>A (p.G92E) in F2, and c.479T>C (p.I160T) in F4 (Fig. 6a, b). All variants were absent or exceedingly rare in public population databases (gnomAD, 1000 Genomes Project). Furthermore, we specifically examined the data from the large-scale studies^37,38 involving 2875 infertile men with primary spermatogenic failure and found that none of our reported ASB9 variants were present in that cohort. These ASB9 variants were classified as deleterious by computational predictors (SIFT, PolyPhen-2, CADD) (Table S1). Subsequent Sanger sequencing validated these variants in probands (Fig. 6c). Notably, all mutated residues reside within the evolutionarily conserved ANK domain of ASB9, as demonstrated by cross-species alignment (Fig. S15).

Fig. 6: Identification of hemizygous X-linked ASB9 variants in men with oligoasthenozoospermia. — **Fig. 6: Identification of hemizygous X-linked *ASB9* variants in men with oligoasthenozoospermia.**

Semen analysis of affected individuals showed severe reductions in sperm count, total motility, and progressive motility (Table S2). Morphological abnormalities were observed via hematoxylin and eosin (H&E) staining and TEM, including amorphous/tapered/small heads and acrosomal defects (Fig. 6d, e). These results suggest that ASB9 also plays an important role in sperm head shaping in humans, and that ASB9 variants are associated with human OAT.

Intracytoplasmic sperm injection (ICSI) using sperm from variant carriers yielded fertilized embryos and blastocysts in all cases (Table S3). Three of four couples achieved clinical pregnancies (Table S3), supporting ICSI as an efficient reproductive strategy for men harboring pathogenic ASB9 variants.

To explore the effect of these missense mutations on ASB9 expression, HEK-293T cells were transfected with Flag-tagged ASB9-WT or mutant constructs (p.A181V, p.G92E, and p.I160T). Western blotting revealed markedly reduced ASB9-G92E protein levels compared to WT, concomitant with TUBB4A accumulation (Fig. 6f, g). CHX chase assays revealed rapid degradation of ASB9-G92E (Fig. 6h, i), which was rescued by MG132-mediated proteasome inhibition (Fig. 6j, k). Ubiquitination assays confirmed enhanced polyubiquitination of ASB9-G92E (Fig. 6l), indicating that p.G92E destabilizes ASB9 via UPS.

Notably, despite ASB9-I160T and ASB9-A181V protein levels being comparable to WT (Fig. 6f), co-IP assays revealed a marked reduction in TUBB4A binding efficiency (Fig. 6m). This diminished interaction correlated with reduced TUBB4A polyubiquitination (Fig. 6n) and consequent protein accumulation (Fig. 6f), identifying I160 and A181 as key residues for ASB9-TUBB4A interactions.

Mice bearing orthologous ASB9 variant mirror human phenotypes

Given the important role of Asb9 in murine spermatogenesis was unveiled by KO studies, we further generated CRISPR-Cas9-mediated Asb9^G88E (Asb9-KI) mice (orthologous to human ASB9^G92E) to validate its functional significance (Fig. S16a). PCR sequencing confirmed precise targeting of the variant (Fig. S16b). Similar to our in vitro findings, Asb9-KI testes exhibited increased K48-linked and pan-polyubiquitination of ASB9 alongside markedly reduced ASB9 protein levels, despite unchanged mRNA abundance (Figs. 7a–c and S16c). Conversely, TUBB4A ubiquitination was diminished in Asb9-KI mice, resulting in elevated TUBB4A protein accumulation without transcriptional alteration (Figs. 7b–d and S16d).

Fig. 7: Asb9-KI mice mirror human phenotypes. — **Fig. 7: *Asb9*-KI mice mirror human phenotypes.**

Notably, while Asb9-KI males exhibited normal mating behavior and testis size (Fig. S16e, f), they displayed significant subfertility (Fig. 7e). Histopathological analysis of Asb9-KI mice revealed OAT phenotypes (Figs. 7f–k and S16g, h), characterized by nuclear shaping defects and disorganized manchette structures (Fig. 7l–o). Additionally, ICSI using sperm from Asb9-KI males resulted in normal fertilization and development to the 2-cell stage at a rate comparable to the WT control (Fig. 7p, q), recapitulating the successful fertilization outcome observed in human carriers. These data conclusively establish the pathogenic cascade initiated by the ASB9^G92E variant across species.

Discussion

While the UPS orchestrates fundamental proteostasis across biology, its tissue-specific adaptations remain incompletely understood^3,39. Our discovery of the testis-enriched ECS^ASB9 complex bridges a critical gap by linking conserved E3 ligase function to human infertility genetics through pathogenic ASB9 variants. By integrating multi-tissue proteomics, mammalian genetics, and human genetics, we establish a clear causal chain: the testis-specific ECS^ASB9 complex catalyzes K48-linked ubiquitination of TUBB4A at K379, thereby ensuring its timely proteasomal degradation. Ablation of any core component of this complex (ASB9, ELOB/C) or genetically blocking the ubiquitination site on its substrate (TUBB4A-K379R) leads to the accumulation of TUBB4A, which in turn disrupts manchette dynamics, culminating in failed sperm head shaping and OAT. This defines an important role for ubiquitin-dependent cytoskeletal remodeling during spermiogenesis (Fig. 8). The translational relevance of this mechanism is underscored by our identification of infertile men harboring hemizygous ASB9 variants. Functional studies confirmed that these mutations operate within the same causal framework, either by destabilizing the ligase itself (G92E) or by specifically ablating its ability to engage TUBB4A (I160T, A181V). In all cases, the final common path is the failure to regulate TUBB4A abundance, thereby bridging our molecular discoveries in mice to the pathogenesis of human infertility.

Fig. 8: ECSASB9 E3 ubiquitin ligase governs male fertility via microtubule destabilization. — **Fig. 8: ECS^ASB9 E3 ubiquitin ligase governs male fertility via microtubule destabilization.**

Beyond reproductive biology, our findings suggest that the modular assembly of ECS complexes contributes to precise, context-dependent degradation programs tailored to cellular niches. This model of context-dependent E3 ligase plasticity offers a transformative framework for understanding how ubiquitination networks achieve functional specificity across tissues.

Spatiotemporal plasticity of ECS complexes dictates functional specificity

Our study reveals that ECS complexes achieve functional specificity through spatiotemporal regulation. This is exemplified by two key findings: the tissue-specific “rewiring” of catalytic cores and the developmentally timed exchange of substrate receptors.

First, the canonical ECS^VHL complex associates with CUL2-RBX1^17,40,41, while SOCS-box proteins such as ASB1/2/6/7/9/12, SOCS3, SPSB1/2, RAB40C, WSB1, and LRRC41 assemble with CUL5-RBX2^{17,40,42,43,44}. Our discovery of a testis-specific ECS^ASB9-CUL2 complex reveals a marked departure from the canonical ASB9-CUL5 architecture characterized in somatic cells²⁶. This represents a clear example of ‘catalytic core rewiring,’ where the same substrate receptor (ASB9) partners with different cullin catalytic cores (CUL2 vs. CUL5) to execute distinct biological functions in a tissue-specific manner. While the somatic ASB9-CUL5 complex regulates mitochondrial metabolism by targeting uMtCK²⁶, the testicular ECS^ASB9-CUL2 complex is uniquely positioned at the manchette to control sperm head shaping by mediating the degradation of the cytoskeletal protein TUBB4A. Such modular plasticity highlights the adaptability of E3 ligases in coordinating spatially distinct proteostatic pathways. This “catalytic core rewiring” may represent a mechanistic basis for E3 ligase adaptation, enabling conserved components like ASB9 to regulate divergent substrates by recruiting tissue-specific cullin-RING modules. This adaptive strategy could potentially be exploited in other specialized cell types like cancer or immune cells^45,46,47.

Second, we uncovered a potential layer of temporal regulation during spermatogenesis. The ECS^TCEB3 complex, a dual-function module mediating RNA polymerase II degradation and transcriptional elongation^48,49,50,51, exhibits dynamic spatiotemporal regulation. While predominantly assembling as CUL2-RBX1 across tissues, its transient expression in pachytene spermatocytes to early spermatids coincides with active transcription. Subsequent replacement by ECS^ASB9 during nuclear condensation aligns with transcriptional shutdown, suggesting a developmental handover from transcriptional to post-translational regulation^52,53,54. This temporal switch ensures step-specific proteostasis, preventing aberrant protein accumulation during spermatid remodeling, highlighting UPS-mediated proteostasis as a universal timer for cellular differentiation programs.

Ubiquitin-proteasome system orchestrates manchette dynamics

Spermiogenesis entails exquisite spatiotemporal control of cytoskeletal reorganization, yet mechanistic insights into UPS-mediated manchette regulation remain sparse. UBQLN1 (Ubiquilin 1), localized to the manchette, has been proposed to shuttle ubiquitinated substrates to proteasomes⁵⁵. Furthermore, UBL7, a testis-enriched UBL-UBA protein, interacts with the valosin-containing protein complex and proteasomes, thereby protecting essential proteins from excessive degradation during manchette development⁵⁶. Nevertheless, definitive evidence identifying specific UPS substrates and elucidating their regulatory mechanisms within this context is lacking. Our study addresses this gap by identifying ECS^ASB9-dependent TUBB4A degradation as an important regulator of manchette structure and function. The nuclear shaping defects and disorganized manchette observed in Asb9-KO/KI, Elob/c-cKO, and Tubb4a^K379R KI mice demonstrate that K48-linked ubiquitination at a single residue governs tubulin stability, thereby ensuring cytoskeletal precision.

A key question arising from our findings is how TUBB4A stabilization perturbs manchette structure. Our data support a model wherein the primary defect lies in disrupting microtubule organization and dynamics, rather than initial assembly. The manchette in Asb9-KO spermatids forms normally at steps 8-9, indicating that the foundational machinery remains intact. However, from step 10 onwards, the structure fails to maintain its proper organization, manifesting as thinner, aberrantly elongated microtubule bundles. We propose that the accumulation of stabilized TUBB4A alters the intrinsic dynamics and mechanical properties of the microtubules, which may compromise the sculpting forces required for proper nuclear shaping. Thus, ECS^ASB9-mediated degradation of TUBB4A facilitates the dynamic remodeling of microtubule subunits necessary for the manchette’s transient yet key morphogenetic function.

These findings establish UPS activity not merely as a quality control mechanism but as an important regulator of spermatid morphogenesis. Moreover, the structural metamorphosis of spermatids exemplifies a universal challenge in cell biology: how dynamic organelles are dismantled or remodeled during differentiation. Our identification of TUBB4A K379 ubiquitination as a key node in manchette integrity reveals that targeted degradation of specific tubulin isoforms can direct cytoskeletal reorganization. This mechanism resonates beyond spermiogenesis, finding parallels in neuronal microtubule dynamics during axon guidance and mitotic/meiotic spindle disassembly in dividing cells^57,58,59. The precise recognition of a single lysine residue for ubiquitination demonstrates how site-specific post-translational modification of cytoskeletal components can orchestrate major structural transitions across diverse cell types.

While the sperm head shaping defects are attributable to disorganized manchette microtubules due to TUBB4A accumulation in Asb9-KO mice, the mechanistic link to impaired tail motility warrants further investigation. Notably, ultrastructural analysis revealed that the axonemal ‘9+2’ architecture remained largely intact in Asb9-KO sperm, ruling out gross structural defects in the flagellum as the primary cause of asthenozoospermia. However, we observed a significant reduction in intracellular ATP levels in Asb9-KO sperm, pointing to a bioenergetic deficit as a likely driver of impaired motility. Emerging evidence indicates that the manchette is not only a cytoskeletal scaffold; it functions as a transport machinery for intra-manchette transport (IMT), facilitating the targeted delivery of proteins, vesicles, and organelles critical for both head remodeling and tail assembly and function^60,61. We propose that the disruption of manchette microtubule dynamics due to aberrant TUBB4A stabilization compromises this IMT system. This impairment could lead to inefficient delivery of bioenergetic components, thereby explaining the observed ATP reduction and providing a causal link to the asthenozoospermia phenotype.

Implications for ICSI and genetic counseling in X-linked inheritance

In this study, we identified three hemizygous missense variants in ASB9 across four unrelated cases of idiopathic male infertility. Among these, the pathogenic significance of the p.G92E variant was verified through functional assays in HEK-293T cells and in Asb9^G88E mice (orthologous to human ASB9^G92E). Although the remaining two missense variants, p.A181V and p.I160T, have been confirmed to partially impair ASB9 function by diminished interaction between ASB9 and TUBB4A. The functional role of p.A181V and p.I160T in male infertility should be further determined in vivo by developing a murine model harboring the corresponding Asb9 missense mutations.

It is worth noting that the inheritance of X-linked genes follows a distinct and clinically significant pattern, making it essential to determine whether a mutation was inherited or arose de novo—a distinction critical for accurate genetic counseling and recurrence risk assessment. Although the origin of the ASB9 variants (maternal or de novo) was not determined in this study, their implications in assisted reproduction remain highly relevant. While infertile men with deleterious X-linked variants (e.g., in ASB9) and those with biallelic autosomal mutations (e.g., in DNAH10)⁶² can both achieve paternity via ICSI, the outcomes for their offspring differ profoundly. In the autosomal recessive scenario, offspring are typically fertile carriers, leading to a low recurrence risk of infertility in subsequent generations. In contrast, all daughters of a man with an X-linked ASB9 variant will be carriers, who then face a 50% risk of transmitting the variant to their own children; their sons who inherit the variant will be hemizygous and infertile. In this study, we identified hemizygous X-linked ASB9 missense variants in 0.27% (4/1483) of men with oligoasthenozoospermia, establishing ASB9 as a discrete monogenic contributor to male infertility. Despite its low prevalence, ASB9 mutation screening in men with oligoasthenozoospermia and genetic counseling are imperative to prevent trans-generational recurrence.

Limitations

While our study establishes the ECS^ASB9-TUBB4A axis as an important regulator of spermiogenesis, it is also necessary to acknowledge several limitations. First, although Asb9-KO, Tubb4a-KI, and Elob/c-cKO mice all presented with OAT, the subfertility and the severity of sperm head deformities were less pronounced in Tubb4a-KI males compared to Asb9-KO males. This observation suggests that while TUBB4A is a major physiological substrate, it is likely not the exclusive target of the ECS^ASB9 complex. Other, yet-to-be-identified substrates may contribute to the full spectrum of defects upon complete ASB9 ablation, implying a broader regulatory role for ASB9 in spermiogenesis that merits further investigation.

Second, the residual fertility observed across all mutant models, including the presence of morphologically normal germ cells, indicates that ECS^ASB9 function, while crucial, is not absolutely required. This phenomenon may be attributed to compensatory mechanisms. Functional redundancy within the Asb family is a plausible candidate, as suggested by the broad upregulation of other Asb genes observed in Asb12-KO testes⁶³. From an evolutionary perspective, this partial dependency could be advantageous. It may allow for the accumulation of genetic variation in key regulators like ASB9 without catastrophic fitness loss, while still strongly selecting for the high-fidelity spermiogenesis ensured by the intact ECS^ASB9 pathway, explaining its conservation.

Finally, the comparatively milder phenotype in Elob/c-cKO mice, characterized by a less severe reduction in fertility and a lower incidence of sperm head deformities than in Asb9-KO males, hints at potential ELOB/C-independent functions of ASB9. This possibility, while speculative, underscores the complexity of E3 ligase regulation in vivo and warrants future exploration.

In conclusion, our study addresses the question of how evolutionarily conserved E3 ligase cores achieve tissue-specific degradation programs, thereby positioning ECS ubiquitin ligase complex plasticity as a useful framework for understanding context-dependent proteostasis in development and disease. The generated tissue-resolved atlas of ECS complexes provides a roadmap for deciphering context-specific ubiquitination networks, while future exploration of analogous E3 rewiring mechanisms, particularly in specialized cell types with dynamic cytoskeletal demands, may help reveal conserved principles of spatial proteome regulation essential for cellular specialization.

Methods

Study participants

In this study, a cohort of 1483 men with idiopathic infertility and oligoasthenozoospermia was recruited from the Reproductive Genetic Hospital of CITIC-Xiangya and the First Affiliated Hospital of Anhui Medical University in China. The recruitment of participants was conducted in accordance with the guidelines of the World Health Organization (WHO, 2010)⁶⁴. A clinical investigation revealed that all affected individuals exhibited normal development of male external genitalia. The study also excluded other potential causes of infertility, including reproductive malformations, exposure to drugs, and gonadotoxic factors. Meanwhile, all individuals had a normal 46, XY karyotype and no Y - chromosome AZF microdeletion. This study was conducted in strict accordance with the Declaration of Helsinki and approved by the Ethical Committee of Reproductive Genetic Hospital of CITIC-Xiangya (Approval: LL-CC-2019-034) and the First Affiliated Hospital of Anhui Medical University (Approval: PJ2020-13-10). Written informed consent was obtained from all participants.

Whole exome sequencing (WES), bioinformatic analysis, and Sanger sequencing

Blood samples were collected from participating individuals, and genomic DNA was extracted using a DNA extraction kit (Qiagen, Hilden, Germany). The exome capture was performed using the Agilent SureSelect Human All Exon V6 Kit (Agilent Technologies, Santa Clara, CA, USA), and the sequencing was carried out on the Illumina HiSeq 2000 platform (Illumina, San Diego, CA, USA). Then, the raw sequencing data were aligned to the human genome reference assembly (GRCh37/hg19) using Burrows–Wheeler Aligner software. PCR duplicates were removed, and the quality of variants was evaluated using the Picard software. Functional variants, including single-nucleotide variants and small insertions or deletions, were annotated using ANNOVAR software.

Candidate pathogenic variants were screened using the following criteria: bi-allelic or hemizygous variants, variants with a minor allele frequency below 5% in the public databases, such as the gnomAD database and the 1000 Genomes Project; variants predicted to be deleterious using multiple tools, including Polyphen-2 (genetics.bwh.harvard.edu/pph2), MutationTaster (www.mutationtaster.org/), SIFT (sift.jcvi.org/) and Combined Annotation Dependent Depletion (cadd.gs.washington.edu/). Subsequently, the candidate variants were further validated through Sanger sequencing in the affected individuals, with the primers listed in Table S4.

Mice

Asb9, Tubb4a, and Elob/c mutant lines were generated via pronuclear co-injection of Cas9 mRNA and target-specific sgRNAs (Table S5) into C57BL/6J zygotes, followed by oviduct transfer to pseudopregnant females. Founders were backcrossed for five generations with WT C57BL/6J mice to minimize off-target effects.

The Tnp2-Cre line was established using the PiggyBac transposon system⁶⁵. A donor plasmid containing the Tnp2 promoter (−549/−48 bp relative to TSS), Cre recombinase cDNA, and polyA signal was co-injected with transposase mRNA into zygotes prior to embryo transfer. Tnp2-Cre mice were crossed with ROSA26-mT/mG dual-fluorescent reporter mice (Jackson Laboratory, stock no.007676)⁶⁶. Cre-mediated recombination triggered membrane-localized GFP (mG) expression, enabling spatial tracking of recombinase activity.

All genotypes were validated by PCR (primers in Table S4). For fertility assessment, adult males (8 weeks) were individually co-housed with two WT females under standard conditions for 2 months; litter sizes were recorded for statistical analysis.

All male C57BL/6J mice used in this study were 8–12 weeks old. The exact number of animals per experimental group is clearly indicated in the corresponding figure legends. Mice were housed under specific pathogen-free conditions in individually ventilated cages (maximally 5 per cage) with a 12-h light/dark cycle, ambient temperature of 22–26 °C, and relative humidity of 50–70%. Sterile food and water were provided ad libitum. Animal health and welfare were monitored daily by trained animal facility staff and the research team; no unexpected mortality or morbidity occurred during the study. At experimental endpoints, mice were euthanized by carbon dioxide inhalation followed by cervical dislocation, and all efforts were made to minimize suffering. All experimental procedures were approved by the Animal Ethical and Welfare Committee of Nanjing Medical University (IACUC licenses: 2004020 and 2402015) and performed in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Cell culture and transfection

HEK-293T cells (American Type Culture Collection; ATCC, Manassas, VA, USA) were maintained in Dulbecco's Modifi ed Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin (Gibco) at 37 °C/5% CO₂. Expression constructs were generated by cloning the following cDNAs into specified vectors: Flag-tagged ASB9 (WT/mutant) into the pcDNA3.1 vector, HA-tagged Ub (WT/mutant) into the pRK5 vector, and GFP-tagged TUBB4A (WT/mutant) into the pEGFP-N1 vector. Transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) per manufacturer’s protocol. Cells were harvested 48 h post transfection for analysis. Plasmid concentrations were standardized at 0.5 μg/mL unless otherwise indicated in figures. All expression constructs used in this study are detailed in Table S6.

RNA extraction, RT‒PCR, and real-time q‒PCR

Total RNA was isolated from tissues using TRIzol reagent (Invitrogen), with subsequent cDNA synthesis performed via reverse transcription using HiScript III RT SuperMix (Vazyme, Nanjing, China). Commercial human multi-tissue cDNA was purchased from TaKaRa (Shiga, Japan). Gene-specific primers (Table S4) were employed for both RT-PCR (amplification products resolved on 1.5% ethidium bromide-stained agarose gels to confirm amplicon size) and quantitative real-time PCR using SYBR Green chemistry (AceQ Master Mix, Vazyme) on a StepOne System (Applied Biosystems, Waltham, MA, USA). Gene expression levels were calculated by the 2^(−ΔΔCt) method, normalized to 18S rRNA as the internal control.

Western blotting

Protein lysates were prepared in RIPA buffer (Beyotime, Shanghai, China) containing 1% protease inhibitor cocktail (Selleck, Houston, TX, USA), incubated 30 min on ice, and centrifuged (12,000 × g, 50 min, 4 °C). Protein concentration was determined by BCA assay (Beyotime). Samples were separated by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and transferred to PVDF membranes (0.45 μm, Millipore, Burlington, MA, USA). Membranes were blocked in 5% non-fat milk/tris-buffered saline Tween-20 (TBST) for 2 h followed by primary antibody incubation (overnight, 4 °C; antibodies in Table S7). After TBST washes, HRP-conjugated secondary antibodies were applied (2 h; room temperature, RT). Protein signals were detected using SuperSignal West Femto Substrate (Thermo Fisher Scientific, Waltham, MA, USA) on a Tanon 5200 imaging system.

Sperm analysis

Spermatozoa were collected from adult mouse cauda epididymides by mincing in 200 μL modified human tubal fluid (HTF) medium (FUJIFILM Irvine Scientific, Santa Ana, CA, USA) supplemented with 10% FBS (37 °C, 10 min). Concentration was quantified via hemocytometer⁶⁷, while motility parameters (total and progressive) were assessed using a Ceros II analyzer (Hamilton Thorne, Beverly, MA, USA), assessing >200 sperm per sample. For morphology analysis, sperm smears were prepared and fixed with 4% paraformaldehyde (PFA) for 15 min. After PBS washes, the smears were stained with Hematoxylin (Solarbio, Wuhan, China) for 8 min, followed by bluing in running water and counterstaining of the cytoplasm with Eosin Y (Solarbio) for 2 min. Finally, the slides were dehydrated through a graded ethanol series, cleared in xylene, and mounted with neutral balsam for morphological examination under a microscope (Axioskop2 Plus, Zeiss, Oberkochen, Germany).

ATP measurement

Caudal epididymal sperm were lysed using the ATP Assay Kit (Beyotime) according to the manufacturer’s instructions. Following a 15-min centrifugation at 12,000 × g and 4 °C, the supernatant was collected for analysis. A standard curve was generated using serial dilutions of ATP stock solution in the provided lysis buffer, yielding final concentrations of 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 μM. The working reagent was equilibrated at room temperature for 3 min prior to use. Supernatants were then mixed with the working reagent, and chemiluminescence was measured using a modular multimode microplate reader (BioTek, Winooski, VT, USA). Total protein concentration in each sample was determined using a BCA Protein Assay Kit (Beyotime). ATP levels were normalized to protein content and expressed as nmol/mg protein.

Histological analysis

Testes, brains, lungs, and kidneys were dissected and immediately fixed in modified Davidson’s fluid for a minimum of 48 h at room temperature. The fixed tissues were then processed through a graded ethanol series (70%, 80%, 95%, and 100%) for dehydration, cleared in xylene, and infiltrated with and embedded in paraffin. For histological evaluation, sections of all tissues (5 μm) were deparaffinized in xylene and rehydrated through a graded ethanol series to distilled water. Testicular sections were subjected to PAS staining, while sections of brain, lung, and kidney were stained with Hematoxylin and Eosin. After dehydration through graded ethanols, clearing in xylene, and mounting with neutral balsam (Solarbio), testicular sections were imaged using a bright-field microscope (Axioskop2 Plus, Zeiss), while brain, lung, and kidney sections were examined using a DS-Ri2 microscope (Nikon, Tokyo, Japan).

For TEM, tissues and sperm were fixed in 2% glutaraldehyde overnight at 4 °C. The samples were then post-fixed with 1% osmium tetroxide (OsO₄) for 2 h at room temperature. Following fixation, the samples were dehydrated through a graded ethanol series and infiltrated with a mixture of propylene oxide and Araldite 502 epoxy resin before being embedded in pure resin. Ultrathin sections (80 nm) were cut using an ultramicrotome (UC7, Leica Microsystems, Wetzlar, Germany), collected on copper grids, and counterstained with uranyl acetate and lead citrate. The grids were examined using a JEM-1410 transmission electron microscope (JEOL, Tokyo, Japan) operated at 80 kV.

For SEM, the sperm samples were processed following the similar fixation and dehydration procedures as described for TEM. The dehydrated samples were then dried using a Critical Point Dryer (K850, Quorum, Laughton, UK), sputter-coated with gold, and observed under a scanning electron microscope (JEOL JSM-7900F).

Immunofluorescence analysis

For tissue sections, paraffin-embedded testicular and lung sections (5 μm) were deparaffinized in xylene and rehydrated through a graded ethanol series to distilled water. Antigen retrieval was performed by incubating the sections in pre-heated 10 mM sodium citrate buffer (pH 6.0) and heating in a microwave oven for 15 min. After washing three times with PBS (pH 7.4), sections were blocked with 1% bovine serum albumin (BSA; Solarbio) for 2 h at room temperature. Subsequently, sections were incubated overnight at 4 °C with primary antibodies (Table S7). After PBS washes, Alexa Fluor-conjugated secondaries were applied for 2 h at room temperature in the dark. After three additional PBS washes, nuclei were counterstained with DAPI (5 μg/mL; Beyotime). Finally, the slides were washed with PBS and mounted with anti-fade fluorescent mounting medium (Beyotime). Images were acquired using an LSM800 confocal microscope (Zeiss). For single-cell suspensions, testes were enzymatically digested with collagenase IV (1 mg/mL, Invitrogen) for 15 min at 37 °C and trypsin (0.25 %, Gibco) for 10 min at 37 °C, respectively. Reactions were quenched with DMEM/10% FBS. The cell suspension was passed through a 40-μm cell strainer to remove aggregates, and the cells were pelleted by centrifugation at 800 × g for 5 min. Pelleted cells were resuspended, fixed on slides using 4% PFA, and washed three times with PBS. Subsequently, samples were blocked with 1% BSA (Solarbio) for 2 h at room temperature. Immunostaining was then performed following the similar procedure as described for tissue sections.

For sperm, sperm smears were prepared and fixed with 4% PFA for 15 min at room temperature. After PBS washes, samples were blocked with 1% BSA (Solarbio) for 2 h at room temperature. Subsequent immunofluorescence staining steps were performed identically to those described for tissue sections.

Terminal deoxynucleotidyl transferase-dUTP nick-end labeling (TUNEL)

Apoptosis was detected using a commercial kit (Vazyme) according to the manufacturer’s instructions. Briefly, deparaffinized cauda epididymis sections were rehydrated, permeabilized with proteinase K (10 μg/mL) for 10 min at room temperature. After washing three times with PBS, the sections were incubated with Equilibration Buffer for 30 min at room temperature and incubated with BrightRed reagent at 37 °C for 60 min. After PBS washes, nuclei were counterstained with DAPI prior to confocal imaging (LSM800, Zeiss).

IP-LC‒MS/MS analysis

Tissues from 8-week-old mice were homogenized on ice in RIPA lysis buffer (Beyotime) containing 1% protease inhibitor cocktail (Selleck). The homogenates were incubated on ice for 30 min and then centrifuged at 16,000 × g for 30 min at 4 °C to remove insoluble debris. Lysates were pre-cleared with 20 μL of Dynabeads™ Protein A (Invitrogen) for 3 h at 4 °C. The pre-cleared lysates were then incubated overnight at 4 °C with gentle rotation with the specified antibody (Table S7). The following day, 40 μL of Dynabeads™ Protein A was added to the lysate-antibody mixture and incubated for an additional 3 h at 4 °C with rotation. Precipitates were washed (3× RIPA buffer), boiled in 2× SDS buffer (5 min), and resolved by SDS-PAGE. Gel bands were excised (≈1 mm³), destained (H₂O → 50% ACN → 100% ACN), reduced (56 °C, 1 h), alkylated (45 min, RT, dark), and digested with trypsin (37 °C, 12 h). Peptides were acidified (0.1% TFA), extracted, SpeedVac-dried, desalted via StageTip, and analyzed by LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific).

Raw files were processed in MaxQuant (v1.3.0.5) against the UniProt mouse database with tryptic specificity (≤2 missed cleavages), 1% FDR thresholds, fixed carbamidomethylation (+57.0215 Da), and variable modifications (methionine oxidation, N-terminal acetylation). Label-free quantification ratios were computed, with >10-fold changes defining candidate interactors.

Co-IP assay

For exogenous co-IP, transfected HEK-293T cells were lysed on ice for 30 min in RIPA lysis buffer supplemented with 1% protease inhibitor cocktail. The lysates were centrifuged at 16,000 × g for 15 min at 4 °C, and the supernatant was collected. A small aliquot (10%) of the supernatant was saved as the “Input” control. The remaining lysates were incubated overnight at 4 °C with anti-Flag magnetic beads (Sigma, St. Louis, MO, USA) or anti-GFP magnetic beads (Proteintech, Rosemont, IL, USA). For endogenous co-IP, testicular lysates underwent primary antibody incubation (overnight, 4 °C) followed by 3-h capture with Dynabeads™ Protein A (Invitrogen) at 4 °C. In both methodologies, bead-bound complexes were collected using a magnetic rack and subsequently washed with lysis buffer (Beyotime). After the final wash, the bound proteins were eluted by boiling the beads in 2× SDS buffer at 95 °C for 10 min. Both the Input controls and the Co-IP eluates were then subjected to immunoblotting with target-specific antibodies (Table S7).

Cycloheximide chase analysis

Protein stability was assessed using a cycloheximide (CHX) chase assay. In short, HEK-293T cells in 6-well plates were transfected for 48 h. To inhibit new protein synthesis, the cell culture medium was replaced with fresh medium containing 100 μg/mL CHX (Sigma). Cells were then harvested at specific time points post-CHX treatment. Protein degradation kinetics were assessed by immunoblotting.

In vitro ubiquitination assay

ASB9 immunoprecipitates were isolated from mouse testicular lysates via immunoprecipitation and then incubated in reaction buffer (20 mM HEPES pH 7.4, 5 mM MgCl₂, 1 mM DTT, 2 mM ATP) containing 100 nM E1, 2 µM UbcH5a (E2), 2.5 µM Ub, and 10 µg His-TUBB4A (Sangon, Shanghai, China). The reaction was incubated at 37 °C for 2 h with gentle shaking. Reactions were terminated with SDS buffer (95 °C, 10 min) and immunoblotted.

In vivo ubiquitination assay

Tissues or transfected HEK-293T cells were lysed in SDS-denaturing buffer (1% SDS, 50 mM Tris-HCl pH 7.5, 150 mM NaCl). The samples were immediately boiled for 10 min to denature proteins and inactivate cellular enzymes. The lysates were then cooled on ice and diluted tenfold with NP-40 lysis buffer. The diluted lysates were centrifuged at 16,000 × g for 15 min at 4 °C, and the supernatants were subjected to immunoprecipitation overnight at 4 °C with the appropriate antibody (Table S7). Complexes were resolved by SDS-PAGE and immunoblotted.

ICSI for mice

The sperm from the cauda epididymides of adult WT and Asb9-KI mice was retrieved and added into the HTF medium. For ICSI, cumulusintact oocytes were collected from the oviduct of superovulated 4-week-old C57BL/6J female mice. Subsequently, hyaluronidase from bovine testes (Sigma, H4272) was used to remove the cumulus cells. The sperm head was injected into an oocyte. Then, the injected oocytes were cultured in KSOM medium (Millipore) in 6% CO₂ at 37 °C. The 2-cell embryo was counted 20 h later.

ICSI for ASB9 mutation-associated infertile men

Four females, whose partners harboring hemizygous ASB9 missense mutations, consented to ICSI treatment. They underwent controlled ovarian hyperstimulation, oocyte retrieval, ICSI, and had pregnancy confirmed. Pregnancy confirmation was achieved through two hCG tests conducted 14 days after embryo transfer. A clinical pregnancy was defined as the visualization of at least a gestational sac with a fetal heartbeat via ultrasound screening 28 days post embryo transfer.

Statistics and reproducibility

Data represent mean ± SD. All experiments were performed at least three independent replicates. Analyzes used GraphPad Prism 8.0: two-tailed unpaired t-tests (two groups) or one-way Analysis of Variance (ANOVA) with Dunnett’s test (multiple groups). Significance was defined as p < 0.05. Biological replicates are specified in figure legends. All schematic diagrams and cartoons presented in this study were originally created by our team using Adobe Illustrator (version 2021).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The murine multi-tissue transcriptomic datasets are obtained from the NCBI SRA database repository (https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA375882). The mass spectrometry-based proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE repository (http://www.ebi.ac.uk/pride) under accession codes PXD064742 and PXD064743. Source data are provided with this paper.

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Acknowledgements

We thank Jiahao Sha from Nanjing Medical University for helpful comments. This work was supported by the National Natural Science Foundation of China (82271633 and 82471641 to B.Z., 32370903 to X.H., and 32470899 to J.Y.), the Natural Science Foundation of Jiangsu Province (BK20221376 to J.Y. and BK20230004 to M.L.), and the Natural Science Foundation of Gusu School of Nanjing Medical University (GSKY202510201 to B.Z.).

Author information

These authors contributed equally: Tiantian Wu, Chaofeng Tu, Yuxuan Feng, Wenying Qu, Jinyi Chen, Huan Wu.

Authors and Affiliations

State Key Laboratory of Reproductive Medicine and Offspring Health, Center for Reproduction and Genetics, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou Municipal Hospital, Gusu School, Nanjing Medical University, Suzhou, China
Tiantian Wu, Wenying Qu, Bingya Xu, Xiangling Yu, Nianchao Zhou, Haoyue Hu, Bing Jiang, Cong Shen & Bo Zheng
Department of Histology and Embryology, School of Basic Medical Sciences, State Key Laboratory of Reproductive Medicine and Offspring Health, Nanjing Medical University, Nanjing, China
Tiantian Wu, Yuxuan Feng, Jinyi Chen, Wenxin Gao, Mingyuan Bao, Jinfu Xu, Yueshuai Guo, Mingxi Liu & Xiaoyan Huang
Institute of Reproductive and Stem Cell Engineering, NHC Key Laboratory of Human Stem Cell and Reproductive Engineering, Xiangya School of Basic Medical Science, Central South University, Changsha, China
Chaofeng Tu, Lanlan Meng, Chen Tan, Ge Lin & Yue-Qiu Tan
Clinical Research Center for Reproduction and Genetics in Hunan Province, Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, China
Chaofeng Tu, Lanlan Meng, Ge Lin & Yue-Qiu Tan
Department of Obstetrics and Gynecology, Reproductive Medicine Center, The First Affiliated Hospital of Anhui Medical University, Hefei, China
Huan Wu, Qingsong Xie, Rong Hua & Yunxia Cao
NHC Key Laboratory of Study on Abnormal Gametes and Reproductive Tract (Anhui Medical University), Hefei, China
Huan Wu, Qingsong Xie, Rong Hua & Yunxia Cao
Key Laboratory of Population Health Across Life Cycle (Anhui Medical University), Ministry of Education of the People’s Republic of China, Hefei, China
Huan Wu, Qingsong Xie, Rong Hua & Yunxia Cao
Department of Obstetrics and Gynecology, Center of Reproductive Medicine, Affiliated Hospital of Nantong University, Nantong, China
Xia Chen
Scientific Research Center, Gannan Medical University, Ganzhou, China
Tao Zhou
Center for Clinical Laboratory, The First Affiliated Hospital of Soochow University, Suzhou, China
Yuting Liang
Department of Orthopedic Surgery and Rehabilitation Medicine, Molecular Oncology Laboratory, The University of Chicago Medical Center, Chicago, IL, USA
Yuting Liang
Institute of Reproductive Medicine, Medical School of Nantong University, Nantong University, Nantong, China
Jun Yu

Authors

Tiantian Wu
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Chaofeng Tu
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Yuxuan Feng
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Wenying Qu
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Jinyi Chen
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Huan Wu
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Wenxin Gao
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Bingya Xu
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Xiangling Yu
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Mingyuan Bao
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Jinfu Xu
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Nianchao Zhou
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Haoyue Hu
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Bing Jiang
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Lanlan Meng
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Chen Tan
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Cong Shen
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Xia Chen
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Yueshuai Guo
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Tao Zhou
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Yuting Liang
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Mingxi Liu
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Jun Yu
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Xiaoyan Huang
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Yue-Qiu Tan
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Bo Zheng
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Contributions

T.W.: Data curation, Investigation, Methodology, Validation. C.T.: Data curation, Investigation, Methodology. Y.F.: Investigation, Visualization. W.Q.: Investigation. J.C.: Investigation, Methodology. H.W.: Data curation, Investigation. W.G.: Investigation. B.X.: Investigation. X.Y.: Investigation. M.B.: Investigation. J.X.: Investigation. N.Z.: Investigation. H.H.: Investigation. B.J.: Investigation. Q.X.: Investigation. L.M.: Investigation. C.T.: Investigation. G.L.: Supervision. C.S.: Data curation. X.C.: Data curation. Y.G.: Methodology, Software. T.Z.: Methodology, Software. Y.L.: Supervision. R.H.: Data curation, Investigation. Y.C.: Conceptualization, Supervision. M.L.: Conceptualization, Supervision. J.Y.: Data curation, Funding acquisition, Investigation. X.H.: Conceptualization, Funding acquisition, Supervision. Y.-Q.T.: Conceptualization, Supervision. B.Z.: Conceptualization, Funding acquisition, Project administration, Supervision, Writing – review & editing.

Corresponding authors

Correspondence to Mingxi Liu, Jun Yu, Xiaoyan Huang, Yue-Qiu Tan or Bo Zheng.

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Wu, T., Tu, C., Feng, Y. et al. A testis-specific E3 ubiquitin ligase complex governs spermiogenesis and male fertility. Nat Commun 17, 3100 (2026). https://doi.org/10.1038/s41467-026-70025-x

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Received: 04 October 2025
Accepted: 16 February 2026
Published: 24 February 2026
Version of record: 31 March 2026
DOI: https://doi.org/10.1038/s41467-026-70025-x