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

a Validation strategy for Tnp2-Cre mice. Tnp2-Cre⁺ mice were crossed with mT/mG dual-color fluorescent reporter mice to generate mT/mG; Tnp2-Cre⁺ mice. Cre-expressing cells exhibited membrane-localized GFP (mG) fluorescence, replacing the tdTomato (mT) red fluorescence. b Immunofluorescence staining and schematic representation of step-specific GFP expression in mT/mG; Tnp2-Cre⁺ tubules (stages I-XII). GFP signal is restricted to spermatids, confirming spermatid-specific recombination. Expression initiates at step 8 of spermiogenesis and persists through elongation and maturation phases. Scale bar: 50  μm. c Fertility assessment of WT, Elob-cKO, and Eloc-cKO male mice. n = 3 mice per group. d Co-immunostaining of PNA, ac-tubulin, and TOMM20 in caudal epididymal sperm of WT, Elob-cKO, and Eloc-cKO mice. e Quantification of d. n = 3 mice per group. f TEM analysis showing malformed sperm heads in Elob-cKO and Eloc-cKO mice. Scale bar: 1 μm. g TEM analysis showing aberrant elongation of manchette microtubules (arrows) and misshapen sperm head and acrosome in Elob-cKO and Eloc-cKO mice. Scale bar: 2 μm. h Co-immunostaining of PNA and α-tubulin in testicular single-cell suspensions of WT, Elob-cKO, and Eloc-cKO mice. Scale bar: 10 μm. i Quantification of h. n = 3 mice per group. Western blot analysis showing reduced ELOB/C, RBX1 and ASB9 expression, concomitant with increased TUBB4A expression in the testes of Elob-cKO (j) and Eloc-cKO (k) mice compared with WT mice, with CUL2 expression remaining unaltered. n = 3 mice per group. l Quantification of j. m Quantification of k. Ac, acrosome; Nu, nucleus; Mt: manchette; Pr: perinuclear ring. For (b, f, g), three independent biological replicates showed consistent results. Data are shown as means ± SD; two-sided unpaired Student’s t test (l, m); one-way ANOVA with Dunnett’s post hoc test (c, e, i).