Fig. 7: Asb9-KI mice mirror human phenotypes.
From: A testis-specific E3 ubiquitin ligase complex governs spermiogenesis and male fertility
a In vivo ubiquitination assay for determining the pan- and K48-linked polyubiquitination of ASB9 in testes of WT and Asb9-KI (Asb9G88E) mice. b Western blot analysis of ASB9 and TUBB4A expressions in the testes of WT and Asb9-KI mice. n = 3 mice per group. c Quantification of b. d In vivo ubiquitination assay for determining the pan- and K48-linked polyubiquitination of TUBB4A in testes of WT and Asb9-KI mice. e Fertility assessment for WT and Asb9-KI male mice. n = 4 mice per group. f Sperm count from the cauda epididymis of WT and Asb9-KI mice. g Total motile sperm percentage measured by CASA for WT and Asb9-KI sperm. h Progressive motility rate measured by CASA for WT and Tubb4a-KI sperm. For f–h, n = 8 WT mice and n = 4 Asb9-KI mice. i Co-immunostaining of PNA, ac-tubulin, and TOMM20 in caudal epididymal sperm of WT and Asb9-KI mice. Scale bar: 20 μm. j Quantification of i. n = 3 mice per group. k TEM analysis showing malformed sperm head and acrosome in Asb9-KI mice. Scale bar: 0.5 μm. l PAS staining of testicular sections in WT and Asb9-KI mice. Red arrows: deformed spermatids. Scale bar: 20 μm. m TEM analysis showing aberrant elongation of manchette microtubules (arrows) and misshapen sperm head in Asb9-KI mice. Scale bar: 2 μm. n Co-immunostaining of PNA and α-tubulin in testicular single-cell suspensions of WT and Asb9-KI mice. Scale bar: 10 μm. o Quantification of n. n = 3 mice per group. p Functional rescue of fertilization potential in Asb9-KI males via ICSI. Representative images of two-cell embryos derived from ICSI using sperm from WT or Asb9-KI male mice. Scale bar: 40 µm. q Quantification of p. n = 3 mice per group. Ac, acrosome; Nu, nucleus; Mt: manchette; Pr: perinuclear ring. For (a, d, k–m), three independent biological replicates showed consistent results. Data are shown as means ± SD; two-sided unpaired Student’s t test.
