Fig 1: CEP76 is required for accessory structure assembly and normal axoneme ultrastructure.(A, B, C, D, E, F, G, H, I, J, K, L) Wild-type sperm are shown in panels (A, D, G, J); sperm from Cep76 mutants are shown in panels (B, C, E, F, H, I, K, L). Top row (A, B, C)—sperm midpiece cross-sections highlighted a broadly normal axoneme in both genotypes, with evidence of mitochondrial and membrane aggregation in mutant sperm. Second row (D, E, F)—sperm principal piece cross-sections highlighted the absence of some microtubule doublets and outer dense fibres (arrow, (E)), and the displacement of some microtubule doublets (black and white arrows, (F)) in mutant cells. Third row (G, H, I)—midpiece longitudinal sections highlighted abnormal mitochondrial morphology (arrows, (H)) and aggregation (arrow, (I)) in mutant cells. Fourth row (J, K, L)—longitudinal sections of the midpiece–principal piece boundary revealed abnormal annulus formation (arrows point to predicted annulus structures, (K, L)) and the consequential mixing of mitochondria and fibrous sheath structures in mutant cells (K). Bottom row (M, N, O)—no major differences were apparent in the transition zone and early axoneme development in early elongating spermatids between genotypes. MP, midpiece; PP, principal piece; An, annulus; FS, fibrous sheath; Mito, mitochondria. Scale bars = 200 nm, except for (M, N) where scale bars = 2 μm, and (O) where the scale bar = 500 nm.
Fig 2: CEP76 human genetic variant and Cep76 mutant mouse model.(A) CEP76 mutation identified in an infertile man, including genetic coordinates and protein position. (B) CEP76 protein species alignment, including conserved amino acid (G, bolded + underlined), and total protein identity across species. Red letters denote amino acids not conserved in zebrafish (Danio rerio) and the affected amino acid in the infertile man. (C) MetaDome assessment of the affected amino acid and its tolerance to change, which was assessed as intolerant. (D) Genetic variant affected exon 5 of the human CEP76 protein, within the C2 (ciliary targeting, exons 4–6) domain. CEP76 in addition holds a TGL (transglutaminase, exons 7–9) domain. (E) Mouse full-length (and a second, shorter) Cep76 transcript and Cep76 mutant transcripts shown below (* denotes a premature stop codon in exon 4). Red arrows denote—where guide RNAs for exon 3 removal were targeted—intronic regions surrounding exon 3; purple and orange arrows denote approximate target sites of qRT–PCR primers. (F) Cep76 expression as measured by qRT–PCR in wild-type and mutant testes, relative to housekeeper Ppia expression (n ≥ 3) and normalised to wild-type levels. ****P < 0.0001.
Fig 3: Cep76 is a spermatid-enriched gene.(A) CEP76 expression across human tissues as assessed by RNA-seq. (B) Cep76 expression across major organs in mice as assessed by qRT–PCR relative to housekeeper Ppia. (C) Cep76 expression in mouse testis cell types as defined by single-cell sequencing. Spg., spermatogonia; P spc., pachytene spermatocyte; sptd., spermatid.
Fig 4: CEP76 is required for the loading of essential motility and fibrous sheath proteins into the sperm tail.(A) Wild-type versus Cep76 mutant data for (A) DNAH2 localisation in cauda epididymal sperm and (B) AKAP4 localisation in cauda epididymal sperm. Scale bars = 20 μm. Arrows point to the accumulation of DNAH2 or AKAP4 in the neck region of sperm. (C, D) Number of sperm with this neck localisation was quantified, as shown in (C, D), for DNAH2 and AKAP4, respectively. (E, F) Average tail pixel intensity (per area) of DNAH2 and AKAP4 was quantified and is shown in (E, F). ***P < 0.001, **P < 0.01, *P < 0.05. All n = 3–5.
Fig 5: CEP76 is required for normal sperm morphology, motility, and tail length.(A) Wild-type versus Cep76 mutant data for (A) sperm morphology, (B) total sperm tail length (n = 5), (C) sperm principal piece (left) and midpiece (right) length (n = 5), (D, E) total and (E) progressive sperm motility (both n ≥ 5), (F) incidence of SEPT4 staining in sperm, (G) sperm annulus staining (SEPT4; n ≥ 3) (scale bar = 2 μm), and (H) sperm annulus migration distance (n ≥ 3). **P < 0.01, ****P < 0.0001.
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