Absence of CFAP69 Causes Male Infertility due to Multiple Morphological Abnormalities of the Flagella in Human and Mouse

Abstract

The multiple morphological abnormalities of the flagella (MMAF) phenotype is among the most severe forms of sperm defects responsible for male infertility. The phenotype is characterized by the presence in the ejaculate of immotile spermatozoa with severe flagellar abnormalities including flagella being short, coiled, absent, and of irregular caliber. Recent studies have demonstrated that MMAF is genetically heterogeneous, and genes thus far associated with MMAF account for only one-third of cases. Here we report the identification of homozygous truncating mutations (one stop-gain and one splicing variant) in CFAP69 of two unrelated individuals by whole-exome sequencing of a cohort of 78 infertile men with MMAF. CFAP69 encodes an evolutionarily conserved protein found at high levels in the testis. Immunostaining experiments in sperm from fertile control individuals showed that CFAP69 localized to the midpiece of the flagellum, and the absence of CFAP69 was confirmed in both individuals carrying CFPA69 mutations. Additionally, we found that sperm from a Cfap69 knockout mouse model recapitulated the MMAF phenotype. Ultrastructural analysis of testicular sperm from the knockout mice showed severe disruption of flagellum structure, but histological analysis of testes from these mice revealed the presence of all stages of the seminiferous epithelium, indicating that the overall progression of spermatogenesis is preserved and that the sperm defects likely arise during spermiogenesis. Together, our data indicate that CFAP69 is necessary for flagellum assembly/stability and that in both humans and mice, biallelic truncating mutations in CFAP69 cause autosomal-recessive MMAF and primary male infertility.

Keywords: CFAP69; KO mouse model; MMAF; asthenozoospermia; infertility genetics; male infertility; manchette; sperm flagellum; teratozoospermia; whole-exome sequencing.

Figure 1

Morphology of Normal and CFAP69 Mutant Spermatozoa, and the Mutations Identified in CFAP69-Mutant Individuals (A–C) Morphology of spermatozoa from fertile control subjects (A), individual CFAP69_{_1} (B), and individual CFAP69_{_2} (C). Most spermatozoa from CFAP69 individuals have flagella that are short (#), absent (^∗), of irregular caliber (&), or coiled (@). Head malformations were also observed (Δ). (D) Location and nature of CFAP69 mutations in CFAP69 and protein. Colored squares stand for Armadillo-like helical (orange) repeats and AH/BAR domain (yellow) as predicted by InterPro server. Mutations are annotated in accordance to the HGVS’s recommendations. n = number of alleles.

Figure 2

CFAP69 Immunostaining in Human Spermatozoa from Control Subjects and CFAP69 Mutant Individuals (A) Sperm cells from a fertile control individual and the two CFAP69-mutant individuals stained with anti-CFAP69 (green) and anti-acetylated tubulin (red) antibodies. DNA was counterstained with Hoechst 33342. In the fertile control, the CFAP69 immunostaining (green) is concentrated in the midpiece of the spermatozoa (white arrows) and is not detectable in the principle piece. CFAP69 staining is absent in sperm flagellum from individual CFAP69_{_1} and individual CFAP69_{_2}. Scale bars: 10 μm. (B) Sperm cell from a fertile control individual stained with anti-HSP60 (red), which detects a mitochondrial protein located in the midpiece, and anti-CFAP69 (green) antibodies. The merged image shows that in control sperm, CFAP69 and HSP60 staining superimpose. Scale bars: 5 μm.

Figure 3

SPAG6 and SPEF2 Immunostainings Are Affected by Mutations in CFAP69 (A) Sperm cells from a fertile control subject and CFAP69_{_2} stained with anti-SPAG6 (green), which detects a protein located in the CPC, and anti-acetylated tubulin (red) antibodies. DNA was counterstained with Hoechst 33342. SPAG6 staining uniformly decorates the full-length flagellum in the fertile control subject whereas it is absent from the flagellum of sperm from individual CFAP69_{_2}. SPAG6 also shows atypical localization in the midpiece and in the acrosomal region of these spermatozoa. (B) Sperm cells from a fertile control and CFAP69_{_2} stained with anti-SPEF2 (green) and anti-acetylated tubulin (red) antibodies. DNA was counterstained with Hoechst 33342. In sperm from the fertile control, SPEF2 staining appears mainly located in the basal body and lightly decorates the sperm flagellum. In sperm cells from the individual CFAP69_{_2}, SPEF2 staining is strongly reduced or totally absent. Scale bars: 10 μm.

Figure 4

Expression of CFAP69 in the Mouse Testis (A) X-gal staining of whole-mount preparations of testis and epididymis from wild-type and Cfap69^tm1b/+ mice. (B–D) X-gal staining of cryosections of cauda epididymis (B) and testis (C and D) from wild-type and Cfap69^tm1b/+ mice. X-gal staining is shown in red, acrosomes are labeled by peanut agglutinin (PNA, green), and nuclei are stained with DAPI (blue). (B) X-gal staining is found in epithelial cells of both wild-type and Cfap69^tm1b/+ epididymides. However, X-gal staining is found in the sperm of Cfap69^tm1b/+ mice only and not in those of wild-type mice. (C) X-gal staining is found in all seminiferous tubules of Cfap69^tm1b/+ testes but not in those of wild-type testes. (D) In xi, white arrows indicate zygotene spermatocytes, and white solid arrowheads indicate diplotene spermatocytes within stage xi seminiferous epithelia. In Cfap69^tm1b/+ testes, strong X-gal staining is found in elongating spermatids, weak X-gal staining, often appearing as a dot, is found in diplotene spermatocytes, and no X-gal staining is found in zygotene spermatocytes. In iii, white chevrons indicate round spermatids, and white cleft arrowheads indicate pachytene spermatocytes within stage iii seminiferous epithelia. In Cfap69^tm1b/+ mice, weak X-gal staining appearing as dots is found in pachytene spermatocytes, and stronger X-gal staining in round spermatids. Dashed white lines outline the basement membrane. (E) Western blotting analysis of whole testis lysates from WT and Cfap69^tm1b/tm1b (Cfap69 KO) mice. The arrow points to the band representing CFAP69. Scale bars: (A) 0.5 cm; (B) 20 μm; (C) 50 μm; (D) 5 μm.

Figure 5

Scanning Electron Microscopy Analysis of Sperm from Epididymides of Wild-Type and Cfap69 KO Mice (A) Sperm from wild-type mice. White arrow indicates junction between midpiece and principal piece, and white arrowhead indicates junction between principal piece and end piece. (B) Sperm from Cfap69 KO mice show severe morphology defects. The thickness and length of the mutant midpiece is variable (compare II, III, VI, VII). The flagella also frequently show splaying into many thin filaments along the entire length of the principal piece (III, VI) or in certain regions (IV, V). These filaments adopt a variety of conformations, including coiled, looped, and tangled. Some sperm essentially lack a flagellum (IX), while others have unusual structures perforated with thin filaments in place of their flagellum (VIII). Head morphology defects ranging from mild (III) to severe (V) are observed, though only a subset of sperm is affected. Scale bar: 5 μm.

Figure 6

The Progression of Spermatogenesis in Cfap69 KO Mice Is Preserved, but Flagellum Components Are Disorganized (A and B) Light micrographs of semi-thin, EMbed812 sections of wild-type and Cfap69 KO testis stained with toluidine blue. (A) Red arrows indicate spermatogonia, yellow arrows indicate spermatocytes, yellow arrowheads indicate round spermatids, red arrowheads indicate spermatozoa, and red asterisks indicate Sertoli cells, all of which are present in both wild-type and Cfap69 KO seminiferous epithelia. (B) Seminiferous epithelia from both wild-type and Cfap69 KO mice of stages vii, ix, x, and xii are shown. Stages are determined by acrosome and nuclear morphology of germ cells, as well as by cell composition of the epithelium. In (A) and (B), long flagella are conspicuously absent from the lumens of seminiferous tubules in Cfap69 KO sections. (C–K) Transmission electron micrographs of testicular sperm from wild-type (C–F) and Cfap69 KO (G–K) mice. In (C)–(K), yellow arrows indicate mitochondria, yellow arrowheads indicate outer dense fibers, red arrows indicate fibrous sheath, red arrowheads indicates axoneme microtubules, and yellow “N” indicates the nucleus. In (H), yellow asterisk indicates split microtubule doublets. Scale bars: (A) 10 μm; (B) 5 μm; (C) 1 μm; (D) 250 nm; (E and F) 100 nm; (G), 1 μm; (H–K) 100 nm.