Bi-allelic Mutations in ARMC2 Lead to Severe Astheno-Teratozoospermia Due to Sperm Flagellum Malformations in Humans and Mice

Abstract

Male infertility is a major health concern. Among its different causes, multiple morphological abnormalities of the flagella (MMAF) induces asthenozoospermia and is one of the most severe forms of qualitative sperm defects. Sperm of affected men display short, coiled, absent, and/or irregular flagella. To date, six genes (DNAH1, CFAP43, CFAP44, CFAP69, FSIP2, and WDR66) have been found to be recurrently associated with MMAF, but more than half of the cases analyzed remain unresolved, suggesting that many yet-uncharacterized gene defects account for this phenotype. Here, whole-exome sequencing (WES) was performed on 168 infertile men who had a typical MMAF phenotype. Five unrelated affected individuals carried a homozygous deleterious mutation in ARMC2, a gene not previously linked to the MMAF phenotype. Using the CRISPR-Cas9 technique, we generated homozygous Armc2 mutant mice, which also presented an MMAF phenotype, thus confirming the involvement of ARMC2 in human MMAF. Immunostaining experiments in AMRC2-mutated individuals and mutant mice evidenced the absence of the axonemal central pair complex (CPC) proteins SPAG6 and SPEF2, whereas the other tested axonemal and peri-axonemal components were present, suggesting that ARMC2 is involved in CPC assembly and/or stability. Overall, we showed that bi-allelic mutations in ARMC2 cause male infertility in humans and mice by inducing a typical MMAF phenotype, indicating that this gene is necessary for sperm flagellum structure and assembly.

Keywords: Multiple morphological anomalies of the flagella (MMAF); cilia; flagella; infertility; spermatogenesis; spermatozoa.

Figure 1

Morphology of Normal and ARMC2-Mutant Spermatozoa, and the Mutations Identified in Individuals with ARMC2 Mutations Light-microscopy analysis of spermatozoa from a fertile control individual (A) and individual ARMC_{_1} (B–D). Most spermatozoa from individuals with ARMC2 mutations have flagella that are coiled (B), short (C), and/or of irregular caliber (D). Transmission-electron-microscopy analyses of sperm cells from a control individual (E) and individual ARMC2_{_1} (F–G). (E) Cross-sections of the principal piece from a fertile control individual. The axoneme is composed of nine doublets of microtubules (DMTs) circularly arranged around a central-pair complex (CPC) of microtubules (9 + 2 organization). The axoneme is surrounded by seven outer dense fibers (ODFs) and by the fibrous sheath (FS) composed of two longitudinal columns (LCs) connected by circumferential ribs (CRs). (F) A cross-section of a sperm flagellum from individual ARMC2_{_1} shows a 9 + 0 axoneme lacking the CPC. (G) A cross-section of a sperm flagellum from individual ARMC2_{_1} shows a severe axonemal disorganization with unassembled ODFs and DMTs. Scale bars: 10 μm (A–D) and 200 nm (E–G). (H) Electrophoregrams of Sanger sequencing for the five ARMC2-mutated individuals are compared to the reference sequence. (I) Location and nature of ARMC2 mutations in ARMC2 and of alterations in the protein. Colored squares stand for armadillo repeats as predicted by the Uniprot server. Mutations are annotated in accordance to the HGVS’s recommendations.

Figure 2

Reproductive Phenotype of Heterozygous and Homozygous Armc2 Male Mice (A) A spermatozoon with normal morphology from a wild-type male mouse. (B–D) Sperm from Armc2-deficient mice show severe morphological defects. All spermatozoa had flagellar abnormalities: short, coiled, absent, and/or of irregular caliber consistent with the MMAF phenotype reported in the ARMC2-mutated individuals. Scale bars: 10 μm. (E) Fertility of wild-type (WT), Armc2 heterozygous-mutant (Armc2^+/−), and Armc homozygous-mutant (Armc2^−/−) males. (F) Testis weight from WT, Armc2^+/−, and Armc2^−/− males. (G) Total motility of sperm extracted from the cauda epididymis of WT, Armc2^+/−, and Armc2^−/− males. (H) Concentrations of sperm from the cauda epididymis from WT, heterozygous, and homozygous Armc2 males. (I) Concentration of morphologically abnormal sperm in WT, heterozygous, and homozygous Armc2 males, evaluated after Harris-Schorr coloration (expressed in million/mL [M/mL]). Data represent means ± SE; statistical differences were assessed with a t test; a probability value of less than 0.05 was considered to be statistically significant.

Figure 3

Immunostaining of SPAG6 and SPEF2 Revealed that the Central-Pair Complex Is Affected by Mutations in ARMC2 in Humans and Mice (A) Sperm cells from a fertile control individual and from individual ARMC2_{_1} were stained with anti-SPAG6 (rabbit polyclonal, HPA38440, Sigma-Aldrich, 1:500, green), which detects a protein located in the C1 microtubule, and anti-acetylated tubulin (monoclonal mouse T7451, Sigma-Aldrich, 1:2000, red) antibodies. SPAG6 staining uniformly decorates the full-length flagellum in the fertile control individual, whereas it is absent from the flagellum of sperm from individual ARMC2_{_1}. (B) Mouse sperm cells from a WT male and Armc2^−/− males stained with anti-SPEF2 (rabbit polyclonal, HPA040343, Sigma-Aldrich, 1:1000, green), a marker of the projection 1b of singlet C1, and anti-acetylated tubulin (monoclonal mouse, T7451, Sigma-Aldrich, 1:500, red) antibodies. DNA was counterstained with DAPI. Contrary to the WT, the SPEF2 immunostaining is not detectable in the sperm flagellum from the Armc2^−/− male. Scale bars: 10 μm.