Deleterious variants in X-linked CFAP47 induce asthenoteratozoospermia and primary male infertility

Abstract

Asthenoteratozoospermia characterized by multiple morphological abnormalities of the flagella (MMAF) has been identified as a sub-type of male infertility. Recent progress has identified several MMAF-associated genes with an autosomal recessive inheritance in human affected individuals, but the etiology in approximately 40% of affected individuals remains unknown. Here, we conducted whole-exome sequencing (WES) and identified hemizygous missense variants in the X-linked CFAP47 in three unrelated Chinese individuals with MMAF. These three CFAP47 variants were absent in human control population genome databases and were predicted to be deleterious by multiple bioinformatic tools. CFAP47 encodes a cilia- and flagella-associated protein that is highly expressed in testis. Immunoblotting and immunofluorescence assays revealed obviously reduced levels of CFAP47 in spermatozoa from all three men harboring deleterious missense variants of CFAP47. Furthermore, WES data from an additional cohort of severe asthenoteratozoospermic men originating from Australia permitted the identification of a hemizygous Xp21.1 deletion removing the entire CFAP47 gene. All men harboring hemizygous CFAP47 variants displayed typical MMAF phenotypes. We also generated a Cfap47-mutated mouse model, the adult males of which were sterile and presented with reduced sperm motility and abnormal flagellar morphology and movement. However, fertility could be rescued by the use of intra-cytoplasmic sperm injections (ICSIs). Altogether, our experimental observations in humans and mice demonstrate that hemizygous mutations in CFAP47 can induce X-linked MMAF and asthenoteratozoospermia, for which good ICSI prognosis is suggested. These findings will provide important guidance for genetic counseling and assisted reproduction treatments.

Keywords: CFAP47; CFAP65; ICSI; MMAF; cilia; flagellum; male infertility; mice; sperm.

Figure 1

Identification of hemizygous variants of X-linked CFAP47 in men with asthenoteratozoospermia (A) Pedigrees of four families affected by hemizygous CFAP47 variants (M1–M4). Black filled squares indicate the male individuals with asthenoteratozoospermia. (B) Sanger sequencing confirmed hemizygous CFAP47 missense variants (M1–M3) in subjects T115 II-1, T176 II-1, and H025 II-1, respectively. The positions of variants are indicated by red arrows. WT, wild type. (C) An approximately 3.2-Mb Xp21.1 deletion affecting CFAP47 (M4) in subject MA2603 II-1. This hemizygous deletion removed the entire CFAP47 gene copy.

Figure 2

Sperm morphology and ultrastructure analyses for men harboring hemizygous CFAP47 variants (A) SEM analysis of the spermatozoa from a male control individual and men harboring hemizygous CFAP47 variants. (i) Normal morphology of the spermatozoon from a healthy control male. (ii–v) Most spermatozoa from men harboring hemizygous CFAP47 variants displayed typical MMAF phenotypes, including absent (ii), short (iii), coiled (iv), and bent flagella (v). The data of subject T115 II-1 were shown as an example. Scale bars: 5 μm. (B) TEM analysis of the spermatozoa from a male control individual and men harboring hemizygous CFAP47 variants. Cross-sections of the midpiece (i) and principal piece (iv) of the sperm flagella from a male control individual displayed typical “9+2” microtubule structure: the central pair of microtubules (CP; red arrows) and nine pairs of peripheral microtubule doublets (DMTs; blue arrows) surrounded by nine outer dense fibers (ODFs; yellow arrows). The organized mitochondrial sheath and fibrous sheath are also observed. Cross-sections of the spermatozoa from men harboring hemizygous CFAP47 variants revealed various axonemal anomalies, including misarranged ODFs (ii, iii) and missing DMTs and/or the CP (v and vi). Scale bars: 200 nm.

Figure 3

Expression analysis of CFAP47 mRNA and CFAP47 in the spermatozoa from a male control individual and men harboring hemizygous CFAP47 variants (A) Real-time quantitative PCR analysis indicated that the abundance of CFAP47 mRNA was dramatically reduced in the sperm from men harboring hemizygous CFAP47 variants when compared to that of a control male. Data represent the means ± standard error of measurement of three independent experiments. Two-tailed Student’s paired or unpaired t tests were used as appropriate (^∗∗∗p < 0.001). (B) Immunoblotting assay revealed that CFAP47 was dramatically reduced or nearly absent in the spermatozoa from men harboring CFAP47 mutations. β-actin was used as a loading control.

Figure 4

Immunofluorescence staining of CFAP47 in the spermatozoa from a male control individual and men harboring hemizygous CFAP47 variants Sperm cells were stained with anti-CFAP47 (red) and anti-α-tubulin (green) antibodies. DNA was counterstained with DAPI (4′,6-diamidino-2-phenylindole) as a marker of the cell nucleus. CFAP47 staining is concentrated at the base of sperm flagella from the control individual, but the signal was almost absent in the sperm flagella from men harboring hemizygous CFAP47 variants. Scale bars: 5 μm.

Figure 5

Abnormal CFAP65 location in the spermatozoa from men harboring hemizygous CFAP47 variants Sperm cells were stained with anti-CFAP65 (red) and anti-α-tubulin (green) antibodies. DNA was counterstained with DAPI as a marker of the cell nucleus. CFAP65 staining was observed mainly at the equatorial segment of sperm head and the base of flagella in control spermatozoa, but the signal was only diffusely clustered in the acrosome of spermatozoa from men harboring hemizygous CFAP47 variants. Scale bars: 5 μm.

Figure 6

Semen characteristics of Cfap47-mutated male mice (A‒D) Semen characteristics by computer-assisted analysis system revealed significantly lower rates of sperm motility (A) and progressive motility (B) but significantly higher rates of immotile spermatozoa (C) and non-progressive spermatozoa (D) in Cfap47-mutated (Cfap47^‒/Y) male mice when compared with those in wild-type (Cfap47⁺/Y) male mice. (E) Curvilinear velocity (VCL), straight-line velocity (VSL), and average-path velocity (VAP) were also dramatically reduced in Cfap47-mutated male mice. Error bars represent the standard error of the mean. ^∗∗p < 0.01; ^∗∗∗p < 0.001; n.s., not significant (Student’s t test). ALH, amplitude of lateral head displacement.

Figure 7

Sperm morphology analysis for Cfap47-mutated male mice H&E staining (A) and SEM analyses (B) revealed higher rates of bent flagella in the spermatozoa of Cfap47-mutated (Cfap47^‒/Y) male mice when compared with those of wild-type (Cfap47⁺/Y) male mice. Scale bars: 20 μm (A) and 5 μm (B).

Figure 8

Damaged fertilization capability by deficiency of mouse CFAP47 could be rescued by ICSI (A) Representative two-cell embryos and blastocysts from IVF in mice. The rates of both two-cell embryos and blastocysts were significantly lower in the group using the spermatozoa from Cfap47-mutated (Cfap47^‒/Y) male mice than those in the wild-type (Cfap47⁺/Y) group. Both groups consisted of four male mice. A total of 716 mouse embryos were counted. Data are represented as means ± standard error of measurement; ^∗∗∗p < 0.001. Scale bars: 200 μm. (B) Representative two-cell embryos and blastocysts from ICSI in mice. The rates of two-cell embryos and blastocysts were counted after sperm heads were injected into oocytes that were collected from superovulated wild-type female mice. Both the Cfap47⁺/Y and Cfap47^‒/Y groups consisted of three male mice. A total of 367 mouse embryos were counted. Data are represented as means ± standard error of measurement. n.s., not significant. Scale bars: 200 μm.