Fig. 6: Identification of hemizygous X-linked ASB9 variants in men with oligoasthenozoospermia.
From: A testis-specific E3 ubiquitin ligase complex governs spermiogenesis and male fertility
a Pedigrees of four families with hemizygous ASB9 variants (M1–M3). Black squares indicate probands. b Computational structural modeling of human ASB9 wild-type and variant proteins. Predicted tertiary structures of WT ASB9 and indicated mutants were generated using AlphaFold2 and visualized by PyMOL software. c Sanger sequencing confirming hemizygous ASB9 missense variants. H&E staining (d) and TEM analysis (e) of spermatozoa from normozoospermic controls (NC) and men harboring ASB9 variants. Compared with spermatozoa in NC, malformed heads (amorphous, tapered, small) were observed in ASB9 mutant spermatozoa. For d, scale bar: 5 μm; for e, scale bar: 1 μm. Ac, acrosome; Nu, nucleus. For d, e, three independent technical replicates showed consistent results. f Western blot analysis of Flag-tagged WT or mutated ASB9 expression in HEK-293T cells. Data represent three biological replicates. g Quantification of f. h CHX assay assessing stability of Flag-tagged WT or mutated ASB9 in HEK-293T cells. Cells were treated with 100 μg/mL CHX for 0, 4, and 6 h prior to harvest. i Quantification of h. Three independent experiments showed consistent results. j Western blot analysis of Flag-tagged WT or mutated ASB9 expressions in HEK-293T cells treated with/without 20 μM proteasome inhibitor MG132 for 6 h. Data represent three biological replicates. k Quantification of j. l In vivo ubiquitination assay analyzing polyubiquitination of Flag-tagged WT or mutated ASB9 in HEK-293T cells transfected with the indicated plasmids, with/without 20 μM MG132 treatment (6 h). Three independent experiments showed consistent results. m Co-IP assay demonstrating the interactions between GFP-tagged TUBB4A and Flag-tagged WT or mutated ASB9 in HEK-293T cells transfected with the indicated plasmids. Three independent experiments showed consistent results. n The polyubiquitination of GFP-tagged TUBB4A in response to Flag-tagged WT or mutated ASB9 overexpression was examined in HEK-293T cells transfected with the indicated plasmids and treated with MG132 (20 μM) for 6 h. Three independent experiments showed consistent results. Data are shown as means ± SD; two-sided unpaired Student’s t test (i); one-way ANOVA with Dunnett’s (g) or Tukey’s (k) post hoc test.
