Fig 2: Affected individuals with variants in IQCN exhibited TFF and defects in the acrosomal ultrastructure The unfertilized oocytes were injected with spermatozoa with a variant in IQCN at 15–18 h postinjection. The white arrowheads represent the polar body (PB). PN represents pronucleus.The expression and localization of PLC? in sperm from the affected individuals. Scale bars, 3 µm.H&E staining of spermatozoa from affected individuals. The black arrow represents normal spermatozoa. The black arrowheads represent tapered-head spermatozoa with increased major axis length and normal minor axis length. The white arrowheads represent thin spermatozoa with decreased major axis length and minor axis length. Scale bars, 20 µm.The ultrastructure of spermatozoa from normal and affected individual. The white squares represent the perinuclear ring. Ac represents acrosome. N represents nucleus. Scale bars, 500 nm.The range of acrosomes in sperm heads from normal and affected individuals (n = 7). Data information: Data represent mean ± SEM. ****P < 0.0001 by unpaired Student's t-test (E). Source data are available online for this figure.

Fig 3: Potential downstream molecular changes in calmodulin regulated by IQCN Workflow of immunoprecipitation (IP) followed by LC–MS analysis of the testes in WT and Iqcn -/- male mice.Mass spectrometry results from Iqcn -/- IP and WT IP (n = 2). Red arrowheads represent the IQCN and manchette-related proteins exclusively detected in the WT group. Upregulated DEPs are shown in red; downregulated DEPs are shown in blue.Distribution and counts of up- and downregulated DEPs. Upregulated DEPs are shown in red. Downregulated DEPs are shown in blue.GO term enrichment analyses of downregulated DEPs.Protein–protein interaction network of downregulated DEPs enriched in microtubule-related processes. Yellow dots represent microtubule-based movement process; red dots represent negative regulation of the cytoskeleton organization process; green dots represent the microtubule bundle formation process; and purple dots represent the actin cytoskeleton organization process.

Fig 4: Identification of pathogenic variants of IQCN in affected individuals with total fertilization failure (TFF) Pedigree analysis of two families with TFF. The individuals (black squares) exhibited homozygous variants in IQCN; the confirmation by Sanger sequencing is shown below the pedigrees.Localization and conservation of mutant amino acids in IQCN. The yellow squares represent the localization of mutant amino acids in seven species.

Fig 5: Iqcn -/- male mice cause defects in manchette assembly during spermiogenesis Expression and localization of IQCN during spermiogenesis in the testes of WT male mice. IQCN (red) was co-localized with a-tubulin (green) from steps 8–14. Sb represents late spermatids. Scale bars, 20 µm.Ultrastructure of the testes from WT and Iqcn -/- male mice from steps 8–16. MA represents manchette. PR represents perinuclear ring. CD represents cytoplasmic droplets. Scale bars, 2 µm (steps 8–12; 15–16) and 5 µm (steps 13–14).Immunostaining of a-tubulin (green) in spermatids at steps 13–14. MA represents manchette. PR represents perinuclear ring. Scale bars, 5 µm.Length of the manchette structure in spermatids at steps 13–14 from WT and Iqcn -/- male mice (n = 10). Data information: Data represent mean ± SEM. ****P < 0.0001 by unpaired Student's t-test (D). Source data are available online for this figure.