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. 2018 Sep 6;103(3):413-420.
doi: 10.1016/j.ajhg.2018.07.013. Epub 2018 Aug 16.

Loss of Calmodulin- And Radial-Spoke-Associated Complex Protein CFAP251 Leads to Immotile Spermatozoa Lacking Mitochondria and Infertility in Men

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Loss of Calmodulin- And Radial-Spoke-Associated Complex Protein CFAP251 Leads to Immotile Spermatozoa Lacking Mitochondria and Infertility in Men

Yasmina Auguste et al. Am J Hum Genet. .
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Abstract

Flagella and motile cilia share a 9 + 2 microtubule-doublet axoneme structure, and asthenozoospermia (reduced spermatozoa motility) is found in 76% of men with primary ciliary dyskinesia (PCD). Nevertheless, causal genetic variants in a conserved axonemal component have been found in cases of isolated asthenozoospermia: 30% of men with multiple morphological anomalies of sperm flagella (MMAF) carry bi-allelic mutations in DNAH1, encoding one of the seven inner-arm dynein heavy chains of the 9 + 2 axoneme. To further understand the basis for isolated asthenozoospermia, we used whole-exome and Sanger sequencing to study two brothers and two independent men with MMAF. In three men, we found bi-allelic loss-of-function mutations in WDR66, encoding cilia- and flagella-associated protein 251 (CFAP251): the two brothers were homozygous for the frameshift chr12: g.122359334delA (p.Asp42Metfs4), and the third individual was compound heterozygous for chr12: g.122359542G>T (p.Glu111) and chr12: g.122395032_122395033delCT (p.Leu530Valfs4). We show that CFAP251 is normally located along the flagellum but is absent in men carrying WDR66 mutations and reveal a spermatozoa-specific isoform probably generated during spermatozoon maturation. CFAP251 is a component of the calmodulin- and radial-spoke- associated complex, located adjacent to DNAH1, on the inner surface of the peripheral microtubule doublets of the axoneme. In Tetrahymena, the CFAP251 ortholog is necessary for efficient coordinated ciliary beating. Using immunofluorescent and transmission electron microscopy, we provide evidence that loss of CFAP251 affects the formation of the mitochondrial sheath. We propose that CFAP251 plays a structural role during biogenesis of the spermatozoon flagellum in vertebrates.

Keywords: asthenozoospermia; flagellum; genetic disorder; human; male infertility; mitochondrial sheath; spermatogenesis.

Figures

Figure 1
Figure 1
Bi-allelic LoF WDR66 Mutations in Two Independent Cases of Asthenozoospermia Associated with MMAF (A–C) Pedigree of family LIBM (A), Sanger sequencing of variant sites in asthenozoospermic brothers 18668 and 18671 and fertile brothers 18669 and 18670 (B), and representative morphologies observed on histological analysis of spermatozoa from individual 18668 (C). (D–F) Pedigree of family FNBM (D), Sanger sequencing of variant sites in asthenozoospermic man 21141 and his parents (E), and representative morphologies observed on histological analysis of spermatozoa from individual 21141 (F). In (B) and (E), the reference sequence at the variant site is underlined in homozygotes, the presence of the variant is indicated by inverted black triangles, and the position of CFAP251 codons is indicated by the thin line above the base codes. The reference sequence for genomic nomenclature is RefSeq: NC000012.11.
Figure 2
Figure 2
CFAP251 Localizes along the Spermatozoon Flagellum and Is Absent from Spermatozoa in Two Independent MMAF Individuals Carrying Distinct Bi-allelic LoF Mutations in WDR66 (A) Immunofluorescence analysis of spermatozoa from a control individual. The second antibody alone gives no labeling. With the anti-CFAP251 antibody, control spermatozoa are labeled (red) along the full length of the flagellum. (B) No CFAP251 signal was detected on spermatozoa from individual 21141. (C) Lysates of purified spermatozoa from affected individuals 18668 and 21141 were migrated with control samples from purified spermatozoa and testis and incubated with an anti-CFAP251 antibody (Sigma, HPA040005). No bands detected by the anti-CFAP251 antibody in control samples were detected in affected individual 18668 or 21141, indicating that they are specific to CFAP251. The predicted molecular weight of the full-length CFAP251 is 130 kDa, and its expected mobility is at 140 kDa (Figure S2). Of note is the strong spermatozoa-specific fragment at approximately 100 kDa. Because the antibody is against a C-terminal domain, this could correspond to an isoform from which the Glu-rich N-terminal domain has been removed during spermatozoa maturation. Lysate from 5 × 104 spermatozoa was loaded except for lanes marked with an asterisk, where lysate from 2 × 106 spermatozoa was loaded. Loading was controlled with an anti-α-tubulin antibody (Abcam, ab15246).
Figure 3
Figure 3
The Mitochondrial Sheath Is Abnormal on the Spermatozoa Flagella of Affected Individual 21141 (A and B) IF analysis of spermatozoa from a normospermic man (A) and individual 21141 (B) with an antibody against the mitochondrial outer-membrane protein VDAC1 (red). No labeling was seen on the flagella in the absence of the anti-VDAC1 antibody (secondary [2°] antibody only). DNA was labeled with DAPI (blue). The fluorescent merge is presented alone and overlayed with the transmitted light (TL) image to show the extent of the flagella. (C) Comparison of the mitochondrial labeling observed in individual 21141 and the control individual. The percentage of different types of labeling is represented (n = 100 for the control individual and individual 21141). In individual 21141, normal labeling of the proximal flagellum by anti-VDAC1 (4%) was much lower than in the control individual (69%). Additional images showing isolated anti-VDAC1-labeled structures, specific to individual 21141, that could be detached MS fragments are shown in Figure S3.
Figure 4
Figure 4
TEM Analysis of Spermatozoa from Affected Individual 21141 Reveals a Short Midpiece with Low Mitochondrial Density Representative images of spermatozoa from a normozoospermic man (A and B) and spermatozoa from individual 21141 (C and D). The arrows point to the transition between the mitochondrial sheath (MS) and the fibrous sheath (FS). In contrast to the MS of the control individual (B), the MS from individual 21141 does not extend far beyond the segmented column (SC) of the connecting piece. Normally the MS of the spermatozoon is 1–1.5 times the length of the head. The annulus (AN) is seen in individual 21141 (C) correctly positioned at the junction between the short MS and the FS, as in the control individual (B). Further images are shown in Figure S4 and together represent all observed sections with a contiguous head, midpiece, and proximal principal piece. No midpiece of normal length was observed.

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