Loss-of-function mutations in CFAP57 cause multiple morphological abnormalities of the flagella in humans and mice

Abstract

Multiple morphological abnormalities of the sperm flagella (MMAF) are the most severe form of asthenozoospermia due to impaired axoneme structure in sperm flagella. Dynein arms are necessary components of the sperm flagellar axoneme. In this study, we recruited 3 unrelated consanguineous Pakistani families with multiple MMAF-affected individuals, who had no overt ciliary symptoms. Whole-exome sequencing and Sanger sequencing identified 2 cilia and flagella associated protein 57 (CFAP57) loss-of-function mutations (c.2872C>T, p. R958*; and c.2737C>T, p. R913*) recessively segregating with male infertility. A mouse model mimicking the mutation (c.2872C>T) was generated and recapitulated the typical MMAF phenotype of CFAP57-mutated individuals. Both CFAP57 mutations caused loss of the long transcript-encoded CFAP57 protein in spermatozoa from MMAF-affected individuals or from the Cfap57-mutant mouse model while the short transcript was not affected. Subsequent examinations of the spermatozoa from Cfap57-mutant mice revealed that CFAP57 deficiency disrupted the inner dynein arm (IDA) assembly in sperm flagella and that single-headed IDAs were more likely to be affected. Thus, our study identified 2 pathogenic mutations in CFAP57 in MMAF-affected individuals and reported a conserved and pivotal role for the long transcript-encoded CFAP57 in IDAs' assembly and male fertility.

Keywords: Fertility; Genetic diseases; Mouse models; Reproductive Biology.

Figure 1. Affected individuals from 3 unrelated consanguineous Pakistani families.

(A) Pedigrees of family 1 with 4 infertile cases, P1 (IV:5), P2 (IV:6), and P3 (IV:7); family 2 with 2 infertile cases, P4 (II:2) and P5 (II:4); and family 3 with 2 infertile cases, P6 (IV:3) and P7 (IV:9). Arrows point to the individuals for which WES was performed. Slashes denote deceased family members, and double horizontal lines represent consanguineous marriages. (B) Representative SEM micrographs showing sperm morphological abnormalities observed in P2, including absent, short, coiled, bent, and irregular-caliber flagella. A representative spermatozoon with normal morphology from the fertile control was shown. Scale bars represent 5 μm.

Figure 2. CFAP57 loss-of-function variants identified in MMAF cases.

(A) CFAP57 variants are located in the regions of homozygosity (RoHs) of affected individuals. RoHs are marked in red. (B–D) Verification of the CFAP57 variants, p.R958* (NC_000001.10:g.43692660C>T) in family 1 (B) and p.R913* (NC_000001.10:g.43689747C>T) in family 2 (C) and family 3 (D), by Sanger sequencing using genomic DNA from all the available family members. Arrowheads, the mutation sites; F, female; M, male; WT, wild-type allele; MT, the mutant allele; M1, c.2872C>T; M2, c.2737C>T. (E) The positions of the 2 variants in CFAP57 at transcript level (NM_001195831.2) and protein level (NP_001182760.2). (F) Representative images of spermatozoa from a fertile control and P7 costained by anti–α-tubulin antibodies and anti-CFAP57 antibodies. Scale bars represent 10 μm.

Figure 3. Cfap57^M/M mice exhibit typical MMAF phenotype.

(A) Immunoblotting with lysates of testes using anti-CFAP57 antibody. GAPDH was used as the loading control. The red arrow points to the full length of CFAP57, and the green arrow points to the predicted truncated protein. (B) Representative images of spermatozoa from Cfap57^+/M and Cfap57^M/M mice costained with anti–α-tubulin and anti-CFAP57 antibodies. Scale bars represent 10 μm. (C) Representative images of testes and epididymides from Cfap57^+/M and Cfap57^M/M mice. The scale bar represents 5 mm. (D) Testes/body weight ratio of Cfap57^+/M and Cfap57^M/M mice. (E) Spermatozoa from 1 epididymis were counted for Cfap57^+/M and Cfap57^M/M mice. (F) Representative images of testis and epididymis sections of Cfap57^+/M and Cfap57^M/M mice after H&E staining. Scale bars represent 50 μm. (G) Representative images of spermatozoa after H&E staining showing normal flagella from Cfap57^+/M mice and absent, short, coiled, and bent flagella and flagella of irregular caliber from Cfap57^M/M mice. At least 200 sperm were examined. Scale bars represent 10 μm. In all the above experiments, 8-week-old mice were used. ****P < 0.0001 by unpaired Student’s 2-tailed t test.

Figure 4. Loss of IDA in spermatozoa from the infertile case and Cfap57^M/M mice.

(A) Representative TEM micrographs showing cross sections of midpiece, principal piece, and end piece of sperm flagella from a fertile control and P7. (B) Representative TEM micrographs showing cross sections of sperm flagella from Cfap57^+/M and Cfap57^M/M mice. The yellow triangle marks the IDAs, and the red triangle marks the position where IDAs are absent. Scale bars represent 100 nm.

Figure 5. CFAP57 is required for the assembly of IDAs.

(A and B) Representative images of spermatozoa from Cfap57^+/M and Cfap57^M/M mice costained by anti–α-tubulin and anti-DNAH6 antibodies (A) or anti-DNALI1 antibodies (B). Scale bars represent 10 μm. (C) Immunoblotting with lysates of testes using anti-DNAH1, anti-DNAH6, and anti-DNALI1. DNAI1 and GAPDH were used as the loading controls.