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• W2914909947 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. 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. The characterization of the genetic basis of male infertility represents an important challenge; more than 4,000 genes are thought to be needed for sperm production,1Jan S.Z. Vormer T.L. Jongejan A. Röling M.D. Silber S.J. de Rooij D.G. Hamer G. Repping S. van Pelt A.M.M. Unraveling transcriptome dynamics in human spermatogenesis.Development. 2017; 144: 3659-3673Crossref PubMed Scopus (96) Google Scholar and therefore defects in any of these genes can hamper spermatogenesis and induce one of many established sperm phenotypes.2Tüttelmann F. Ruckert C. Röpke A. Disorders of spermatogenesis: Perspectives for novel genetic diagnostics after 20 years of unchanged routine.Med. Genetik. 2018; 30: 12-20Google Scholar Of late, high-throughput sequencing technologies have allowed researchers to identify an increasing number of genes required for sperm production and have thus greatly facilitated efforts to explain the genetic basis of different forms of male infertility.3Krausz C. Riera-Escamilla A. Genetics of male infertility.Nat. Rev. Urol. 2018; 15: 369-384Crossref PubMed Scopus (343) Google Scholar This is especially true for teratozoospermia, qualitative spermatogenesis defects that lead to morphological sperm abnormalities.4Coutton C. Escoffier J. Martinez G. Arnoult C. Ray P.F. Teratozoospermia: spotlight on the main genetic actors in the human.Hum. Reprod. Update. 2015; 21: 455-485Crossref PubMed Scopus (188) Google Scholar, 5Ray P.F. Toure A. Metzler-Guillemain C. Mitchell M.J. Arnoult C. Coutton C. Genetic abnormalities leading to qualitative defects of sperm morphology or function.Clin. Genet. 2017; 91: 217-232Crossref PubMed Scopus (101) Google Scholar One of the most severe forms of qualitative sperm defects, the MMAF phenotype is responsible for astheno-teratozoospermia,4Coutton C. Escoffier J. Martinez G. Arnoult C. Ray P.F. Teratozoospermia: spotlight on the main genetic actors in the human.Hum. Reprod. Update. 2015; 21: 455-485Crossref PubMed Scopus (188) Google Scholar which is characterized by the presence of immotile spermatozoa presenting with a mosaic of sperm flagellum malformations, including short, coiled, and/or absent flagella and/or flagella of irregular caliber.6Ben Khelifa M. Coutton C. Zouari R. Karaouzène T. Rendu J. Bidart M. Yassine S. Pierre V. Delaroche J. Hennebicq S. et al.Mutations in DNAH1, which encodes an inner arm heavy chain dynein, lead to male infertility from multiple morphological abnormalities of the sperm flagella.Am. J. Hum. Genet. 2014; 94: 95-104Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar Whole-exome sequencing (WES) analysis revealed that mutations in DNAH1 (MIM: 603332), CFAP43 (MIM: 617558), CFAP44 (MIM: 617559), CFAP69 (MIM: 617949), FSIP2 (MIM: 615796), and WDR66 (MIM: 612573) account for the main genetic causes of MMAF.6Ben Khelifa M. Coutton C. Zouari R. Karaouzène T. Rendu J. Bidart M. Yassine S. Pierre V. Delaroche J. Hennebicq S. et al.Mutations in DNAH1, which encodes an inner arm heavy chain dynein, lead to male infertility from multiple morphological abnormalities of the sperm flagella.Am. J. Hum. Genet. 2014; 94: 95-104Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 7Amiri-Yekta A. Coutton C. Kherraf Z.-E. Karaouzène T. Le Tanno P. Sanati M.H. Sabbaghian M. Almadani N. Sadighi Gilani M.A. Hosseini S.H. et al.Whole-exome sequencing of familial cases of multiple morphological abnormalities of the sperm flagella (MMAF) reveals new DNAH1 mutations.Hum. Reprod. 2016; 31: 2872-2880Crossref PubMed Scopus (79) Google Scholar, 8Wang X. Jin H. Han F. Cui Y. Chen J. Yang C. Zhu P. Wang W. Jiao G. Wang W. et al.Homozygous DNAH1 frameshift mutation causes multiple morphological anomalies of the sperm flagella in Chinese.Clin. Genet. 2017; 91 (Published online November 24, 2016): 313-321Crossref PubMed Scopus (62) Google Scholar, 9Coutton C. Vargas A.S. Amiri-Yekta A. Kherraf Z.-E. Ben Mustapha S.F. Le Tanno P. Wambergue-Legrand C. Karaouzène T. Martinez G. Crouzy S. et al.Mutations in CFAP43 and CFAP44 cause male infertility and flagellum defects in Trypanosoma and human.Nat. Commun. 2018; 9: 686Crossref PubMed Scopus (122) Google Scholar, 10Tang S. Wang X. Li W. Yang X. Li Z. Liu W. Li C. Zhu Z. Wang L. Wang J. et al.Biallelic mutations in CFAP43 and CFAP44 cause male infertility with multiple morphological abnormalities of the sperm flagella.Am. J. Hum. Genet. 2017; 100: 854-864Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 11Dong F.N. Amiri-Yekta A. Martinez G. Saut A. Tek J. Stouvenel L. Lorès P. Karaouzène T. Thierry-Mieg N. Satre V. et al.Absence of CFAP69 causes male infertility due to multiple morphological abnormalities of the flagella in human and mouse.Am. J. Hum. Genet. 2018; 102: 636-648Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 12Martinez G. Kherraf Z.-E. Zouari R. Fourati Ben Mustapha S. Saut A. Pernet-Gallay K. Bertrand A. Bidart M. Hograindleur J.P. Amiri-Yekta A. et al.Whole-exome sequencing identifies mutations in FSIP2 as a recurrent cause of multiple morphological abnormalities of the sperm flagella.Hum. Reprod. 2018; 33: 1973-1984Crossref PubMed Scopus (69) Google Scholar, 13Kherraf Z.-E. Amiri-Yekta A. Dacheux D. Karaouzène T. Coutton C. Christou-Kent M. Martinez G. Landrein N. Le Tanno P. Fourati Ben Mustapha S. et al.A homozygous ancestral SVA-insertion-mediated deletion in WDR66 induces multiple morphological abnormalities of the sperm flagellum and male infertility.Am. J. Hum. Genet. 2018; 103: 400-412Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar Rarer recessive mutations in AK7 (MIM: 615364), CEP135 (MIM: 611423), and CFAP65 (MIM: 614270) were also recently identified in different familial cases of MMAF.10Tang S. Wang X. Li W. Yang X. Li Z. Liu W. Li C. Zhu Z. Wang L. Wang J. et al.Biallelic mutations in CFAP43 and CFAP44 cause male infertility with multiple morphological abnormalities of the sperm flagella.Am. J. Hum. Genet. 2017; 100: 854-864Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 14Lorès P. Coutton C. El Khouri E. Stouvenel L. Givelet M. Thomas L. Rode B. Schmitt A. Louis B. Sakheli Z. et al.Homozygous missense mutation L673P in adenylate kinase 7 (AK7) leads to primary male infertility and multiple morphological anomalies of the flagella but not to primary ciliary dyskinesia.Hum. Mol. Genet. 2018; 27: 1196-1211Crossref PubMed Scopus (68) Google Scholar, 15Sha Y.-W. Xu X. Mei L.-B. Li P. Su Z.-Y. He X.-Q. Li L. A homozygous CEP135 mutation is associated with multiple morphological abnormalities of the sperm flagella (MMAF).Gene. 2017; 633: 48-53Crossref PubMed Scopus (82) Google Scholar Despite these recent findings, more than half of the studied MMAF cases remain without a diagnosis, demonstrating the high genetic heterogeneity of this phenotype and the need for further genetic explorations.12Martinez G. Kherraf Z.-E. Zouari R. Fourati Ben Mustapha S. Saut A. Pernet-Gallay K. Bertrand A. Bidart M. Hograindleur J.P. Amiri-Yekta A. et al.Whole-exome sequencing identifies mutations in FSIP2 as a recurrent cause of multiple morphological abnormalities of the sperm flagella.Hum. Reprod. 2018; 33: 1973-1984Crossref PubMed Scopus (69) Google Scholar Here, we analyzed by WES a total of 168 MMAF-affected individuals, including 78 who were previously reported9Coutton C. Vargas A.S. Amiri-Yekta A. Kherraf Z.-E. Ben Mustapha S.F. Le Tanno P. Wambergue-Legrand C. Karaouzène T. Martinez G. Crouzy S. et al.Mutations in CFAP43 and CFAP44 cause male infertility and flagellum defects in Trypanosoma and human.Nat. Commun. 2018; 9: 686Crossref PubMed Scopus (122) Google Scholar and an additional 90 unrelated and unpublished individuals with MMAF. All but one individual were analyzed together with the same analytical pipeline, constituting a 167-strong cohort. In this main cohort, 83 individuals were of North African origin and sought consultation for primary infertility at the Clinique des Jasmins in Tunis, 52 individuals originated from the Middle East (Iran) and were treated in Tehran at the Royan Institute (Reproductive Biomedicine Research Center) for primary infertility, and 32 subjects were recruited in France: 25 at the Cochin Institute in Paris, three in Rouen, two in Grenoble, one in Lille, and one in Caen. All individuals presented with a typical MMAF phenotype, which is characterized by severe asthenozoospermia (total sperm motility below 10%) with at least three of the following flagellar abnormalities present in >5% of the spermatozoa: short, absent, coiled, bent, or irregular flagella (Table 1, Figures 1A–1D). All individuals had a normal somatic karyotype (46, XY) with normal bilateral testicular size, hormone levels, and secondary sexual characteristics. Informed consent was obtained from all the individuals participating in the study according to local protocols and the principles of the Declaration of Helsinki. The study was approved by local ethics committees, and samples were then stored in the CRB Germethèque (certification under ISO-9001 and NF-S 96-900) according to a standardized procedure or were part of the Fertithèque collection declared to the French Ministry of health (DC-2015-2580) and the French Data Protection Authority (DR-2016-392).Table 1Detailed Semen Parameters for the Five MMAF Individuals Harboring a ARMC2 MutationARMC2-Mmutated IndividualsSemen ParametersIndividualsARMC2 MutationSperm Volume (ml)Sperm Conc.(106/mL)Total Motility 1hVitalityNormal SpermatozoaAbsent FlagellaShort FlagellaCoiled FlagellaBent FlagellaFlagella of Irregular CaliberTapered HeadThin HeadMicro-CephalicMacro-CephalicMultiple HeadsAbnormal BaseAbnormal Acrosomal RegionARMC2_1c.1023+1G>A4356NA0105016NA70182832022898ARMC2_2c.2279T>A4.54.65640143061678224204688ARMC2_3c.2353_2354delTT158.50163313723NA396090003152ARMC2_4c.1284_1288delACAAA4.22035208300011020000ARMC2_5c.421C>T9.210.11.1NA020383600NANANANANANANAReference limitsaReference limits (5th centiles and their 95% confidence intervals) according to the World Health Organization.1.5 (1.4–1.7)15 (12–16)40 (38–42)58 (55–63)23 (20–26)5 (4–6)1 (0–2)17 (15–19)13 (11-15)2 (1–3)3 (2–4)14 (12–16)7 (5–9)1 (0–2)2 (1–3)42 (39–45)60 (57–63)Values are percentages unless specified otherwise.a Reference limits (5th centiles and their 95% confidence intervals) according to the World Health Organization. Open table in a new tab Values are percentages unless specified otherwise. WES and bioinformatics analysis were performed according to an improved version of our previously described protocol9Coutton C. Vargas A.S. Amiri-Yekta A. Kherraf Z.-E. Ben Mustapha S.F. Le Tanno P. Wambergue-Legrand C. Karaouzène T. Martinez G. Crouzy S. et al.Mutations in CFAP43 and CFAP44 cause male infertility and flagellum defects in Trypanosoma and human.Nat. Commun. 2018; 9: 686Crossref PubMed Scopus (122) Google Scholar as described in the Supplemental Material and Methods. Data analysis of the whole cohort of 167 MMAF individuals identified 54 individuals (32.3%) with harmful mutations in known MMAF-related genes (Table S1). In 15 individuals, previously unreported variants were identified in CFAP43 (n = 2), CFAP44 (n = 1), DNAH1 (n = 6), WDR66 (n = 4), and FSIP2 (n = 2), thus confirming the importance of these genes in the etiology of the MMAF syndrome (Table S1). In addition, we identified four individuals (ARMC2_1–4) with a homozygous variant in ARMC2, a gene not previously associated with any pathology; these individuals accounted for 2.4% of our cohort. ARMC2 (GenBank: NM_032131.5) is located on chromosome 6 and contains 18 exons encoding a predicted 867-amino-acid protein (NCBI: NP8115507.4; UniProtKB: Q8NEN0). Three individuals (ARMC2_1,3,4) had a loss-of-function variant, and one (ARMC22) had a likely deleterious missense variant (Table 1, Table S1). In addition, we identified a fifth individual (ARMC2_5) with an ARMC2 homozygous loss-of-function variant (Table S2). This individual was of Chinese origin and sought consultation for primary infertility at the First Affiliated Hospital of Anhui Medical University (Hefei City, Anhui Province) in 2012. He was born to first-cousin parents and presented a typical MMAF phenotype, and WES analysis for this individual was performed as described in the previous report.10Tang S. Wang X. Li W. Yang X. Li Z. Liu W. Li C. Zhu Z. Wang L. Wang J. et al.Biallelic mutations in CFAP43 and CFAP44 cause male infertility with multiple morphological abnormalities of the sperm flagella.Am. J. Hum. Genet. 2017; 100: 854-864Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar Informed consent was obtained from all tested individuals, and the study was approved by the local institutional board. The five ARMC2 variants were found in five unrelated individuals, and all were absent from control sequence databases (dbSNP, 1000 Genomes Project, NHLBI Exome Variant Server, gnomAD, and our in-house control database). The variant identified in individual ARMC2_1 is a splice variant, c.1023+1G>A, altering a consensus splice donor site of ARMC2 exon 8 (Figure 1I). To evaluate the deleterious effect of this splicing variant, we performed RT-PCR with RNA extracted from sperm cells from individual ARMC2_1. Amplification of a sequence ranging from cDNA exon 7–10 from a control individual yielded a normal band of 608 bp, whereas a single smaller band was obtained from cDNA of individual ARMC2_1 (Figure S1A). Sequence analysis of the amplified products demonstrated that the c.1023+1G>A variant results in exon 8 skipping (Figure S1B, Figure S1C), causing a shift in the translational reading frame and introducing a premature termination codon (p.Glu283AlafsTer2). Primer sequences and RT-PCR conditions are indicated in Table S3. The variant identified in individual ARMC2_2 is a missense variation, c.2279T>A (p.Ile760Asn), located in exon 16 (Figure 1I). No mRNA analysis or immunostaining could be performed on sperm cells from this individual because of the lack of biological samples. However, using prediction software for non-synonymous SNPs, we found that this missense change is predicted to be deleterious by SIFT (score of 0) and probably damaging by PolyPhen (score of 0.987). Moreover, the concerned amino acid (Ile760) was found to be conserved in ARMC2 orthologs (Figure S2). The two other variants identified in ARMC2_3 and ARMC2_4 were small frameshift indels, c.2353_2354delTT (p.Leu785MetfsTer5) and c.1284_1288delACAAA (p.Lys428AsnfsTer3), located in exon 17 and exon 10, respectively (Figure 1I), inducing a premature translation termination. The last identified variant (ARMC2_5) is a stop-gain variant, c.421C>T (p.Gln141Ter), located in exon 4 (Figure 1I). Familial study confirmed the presence of the homozygous loss-of-function variant in ARMC2_5 and indicated that his parents were both heterozygous and that his non-affected brother was homozygous wild-type (Figure S3A, B). The last three variants (in individuals ARMC2_3–5) introduce a premature stop codon and are therefore expected to induce nonsense-mediated mRNA decay that is likely to prevent protein synthesis. All ARMC2 variants are deposited in ClinVar under reference SUB4929442. Overall, a total of 168 exomes from MMAF-affected individuals were analyzed, and five unrelated affected individuals were shown to carry a homozygous deleterious mutation in ARMC2. No other candidate variants reported to be associated with cilia, flagella, or male fertility were present in any of the five individuals with ARMC2 mutatations. We also note that none of the individuals analyzed here carried a homozygous deleterious variant in any two (or more) of the pathological MMAF-associated genes (DNAH1, CFAP43, CFAP44, CFAP69, FSIP2, WDR66, and ARMC2), i.e., the 54 individuals with an established causal mutation did not carry another candidate variant. All mutations identified by WES were validated by Sanger sequencing, as previously described9Coutton C. Vargas A.S. Amiri-Yekta A. Kherraf Z.-E. Ben Mustapha S.F. Le Tanno P. Wambergue-Legrand C. Karaouzène T. Martinez G. Crouzy S. et al.Mutations in CFAP43 and CFAP44 cause male infertility and flagellum defects in Trypanosoma and human.Nat. Commun. 2018; 9: 686Crossref PubMed Scopus (122) Google Scholar, 10Tang S. Wang X. Li W. Yang X. Li Z. Liu W. Li C. Zhu Z. Wang L. Wang J. et al.Biallelic mutations in CFAP43 and CFAP44 cause male infertility with multiple morphological abnormalities of the sperm flagella.Am. J. Hum. Genet. 2017; 100: 854-864Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar and as illustrated in Figure 1H. PCR primers and protocols used for each individual are listed in Table S4. ARMC2 is preferentially expressed in the testis according to data from ENCODE, FANTOM, and GTEx16Gerstein M.B. Kundaje A. Hariharan M. Landt S.G. Yan K.-K. Cheng C. Mu X.J. Khurana E. Rozowsky J. Alexander R. et al.Architecture of the human regulatory network derived from ENCODE data.Nature. 2012; 489: 91-100Crossref PubMed Scopus (1069) Google Scholar, 17Lizio M. Harshbarger J. Abugessaisa I. Noguchi S. Kondo A. Severin J. Mungall C. Arenillas D. Mathelier A. Medvedeva Y.A. et al.Update of the FANTOM web resource: high resolution transcriptome of diverse cell types in mammals.Nucleic Acids Res. 2017; 45: D737-D743Crossref PubMed Scopus (74) Google Scholar, 18GTEx ConsortiumHuman genomics. The Genotype-Tissue Expression (GTEx) pilot analysis: multitissue gene regulation in humans.Science. 2015; 348: 648-660Crossref PubMed Scopus (3074) Google Scholar and is described as being associated with cilia and flagella.19Ivliev A.E. ’t Hoen P.A.C. van Roon-Mom W.M.C. Peters D.J.M. Sergeeva M.G. Exploring the transcriptome of ciliated cells using in silico dissection of human tissues.PLoS ONE. 2012; 7: e35618Crossref PubMed Scopus (61) Google Scholar We confirmed these data by RT-qPCR experiments in a panel of human tissues; these experiments indicated that expression of ARMC2 mRNA in testis is significantly higher than in other tested tissues (Figure S4). Primer sequences and RT-qPCR conditions are indicated in Table S5. According to the Uniprot server, ARMC2 is an armadillo protein composed of 12 armadillo repeats (ARM-repeat) flanked by unique C-terminal and N-terminal domains20The UniProt ConsortiumUniProt: the universal protein knowledgebase.Nucleic Acids Res. 2017; 45: D158-D169Crossref PubMed Scopus (3122) Google Scholar (Figure 1I). Sperm analysis was carried out in the source laboratories during routine biological examination of the individuals according to World Health Organization (WHO) guidelines.21Wang Y. Yang J. Jia Y. Xiong C. Meng T. Guan H. Xia W. Ding M. Yuchi M. Variability in the morphologic assessment of human sperm: use of the strict criteria recommended by the World Health Organization in 2010.Fertil. Steril. 2014; 101: 945-949Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar The morphology of the sperm cells was assessed with Papanicolaou staining (Figures 1A–1D). Detailed semen parameters of the five individuals with ARMC2 mutations are presented in Table 1, and the average semen parameters of the studied MMAF cohort, separated by genotypes, are described in the Table S6. Among the different parameters studied, only viability, total motility, and “lack of flagellum” presented a statistical difference between the different groups, according to their genotype (one-way ANOVA test). For parameters with a positive ANOVA test, a pairwise statistical Fisher’s LSD test was employed so that significant differences between individuals with different genotypes could be identified (Figure S5). Concerning vitality, sperm from individuals with WDR66 mutations and non-characterized individuals (unknown) presented a significant increase in comparison with individuals harboring CFAP69, CFAP44, FSIP2, and ARMC2 mutations. With regard to motility, CFAP43 and CFAP44 showed the most pronounced alteration, as a result of a significant increase in sperm without a flagellum, and individuals harboring CFAP44 mutations displayed a significant increase in “no tail” sperm (Figure S5). Sperm samples for additional phenotypic characterization could only be obtained from individual ARMC2_1. We studied the ultrastructure of sperm cells from individual ARMC2_1 by transmission electron microscopy (TEM) (Figures 1E–1G) according to the protocol previously described.9Coutton C. Vargas A.S. Amiri-Yekta A. Kherraf Z.-E. Ben Mustapha S.F. Le Tanno P. Wambergue-Legrand C. Karaouzène T. Martinez G. Crouzy S. et al.Mutations in CFAP43 and CFAP44 cause male infertility and flagellum defects in Trypanosoma and human.Nat. Commun. 2018; 9: 686Crossref PubMed Scopus (122) Google Scholar For details, see Supplemental Material and Methods. Because of the low number of sperm cells available, only a few cross-sections (<10) were of sufficient quality for analysis. Among these sections, all were abnormal, and the main defect observed was the absence of the CPC (9 + 0 conformation) (Figure 1F). In some sections we observed a dramatic axonemal disorganization associated with peri-axonemal structural defects such as unassembled outer dense fibers (ODFs) (Figure 1G), a defect already observed in sperm from MMAF-affected individuals carrying mutations in other genes. Observation of rare longitudinal sections showed severe abnormalities such as truncated flagella or the presence of cytoplasmic structures encompassing unassembled axonemal components (not shown). To assess the impact the absence of ARMC2 has on mouse spermatogenesis, we used the CRISPR-Cas9 technology (as previously described) to generate Armc2 mutant animals.9Coutton C. Vargas A.S. Amiri-Yekta A. Kherraf Z.-E. Ben Mustapha S.F. Le Tanno P. Wambergue-Legrand C. Karaouzène T. Martinez G. Crouzy S. et al.Mutations in CFAP43 and CFAP44 cause male infertility and flagellum defects in Trypanosoma and human.Nat. Commun. 2018; 9: 686Crossref PubMed Scopus (122) Google Scholar, 22Kherraf Z.-E. Conne B. Amiri-Yekta A. Kent M.C. Coutton C. Escoffier J. Nef S. Arnoult C. Ray P.F. Creation of knock out and knock in mice by CRISPR/Cas9 to validate candidate genes for human male infertility, interest, difficulties and feasibility.Mol. Cell. Endocrinol. 2018; 468: 70-80Crossref PubMed Scopus (16) Google Scholar For all experiments involving mice, animals were handled and euthanized in accordance with methods approved by the animal ethics committees of Grenoble and Geneva. All mice were adult (6 weeks or older) mice. We generated a strain with a one-nucleotide duplication in exon 4 (DupT), inducing a translational frameshift expected to lead to the complete absence of the protein or the production of a truncated protein. mRNA was extracted from Armc2 homozygous-mutant mice (Armc2mutant) and amplified by RT-PCR. The level of Armc2mutant mRNA amplification was much lower in mutant animals than in controls (Figure S6). Sanger sequencing of mRNA from Armc2 homozygous-mutant mice confirmed the production of abnormal transcripts with a premature stop codon 12 nucleotides after the first modified codon at position 135 (GenBank: NM_001034858.3 [c.403dupT]; NCBI: NP_001030030.2 [p.Tyr135LeufsTer12]) (Figure S6). The guide RNA sequence for CRISPR-Cas9 mice generation and the list of primers used for mice genotyping and RT-PCR are indicated in Table S7 and Table S8. We studied sperm morphology and observed that in contrast to what is observed in WT animals (Figure 2A), epididymal sperm from Armc2mutant males displayed a phenotype identical to the typical human MMAF phenotype: 100% of spermatozoa had short, thick, and/or coiled flagella, whereas sperm heads conserved an overall typical hooked shape (Figures 2B–2D). As could be expected, homozygous Armc2mutant males exhibited complete infertility when mated with WT females (Figure 2E), whereas homozygous Armc2mutant females were fully fertile and gave litters of normal size compared to those of WT females (8.33 ± 1.11 versus 9.67 ± 1.01 pups/litter [mean ± SE, n = 6 versus n = 3]). There was no obvious testicular anomaly; there was no difference in weight between Armc2mutant and WT testes (94 ± 20 and 92 ± 10.67 mg per testis; mean ± SE, n = 6 and n = 3, respectively) (Figure 2F). Structural defects were observed in close to 100% of spermatozoa from Armc2mutant males (Figure 2G) and were associated with a complete motility deficiency (Figure 2H). Sperm production was also affected, as shown by Armc2mutant sperm concentrations of 4.47 ± 1.29 × 106 sperm/mL versus concentrations of 20.75 ± 8.33 × 106 sperm/mL (mean ± SE, n = 6 and n = 3) in WT littermates (Figure 2I). To define the ultrastructural defects evidenced by TEM and to characterize the molecular defects induced by ARMC2 mutation in human sperm, we studied by immunofluorescence the presence and localization of several proteins belonging to different sub-structures of the axoneme. We investigated the presence of the following proteins: SPAG6 as a marker of the CPC; DNAI2 and DNALI1 as markers of outer and inner dynein arms (ODAs and IDAs), respectively; RSPH1 as a marker of the radial spokes (RS), GAS8 as a marker of the nexin-dynein regulatory complex (N-DRC), and AKAP4 as marker of the fibrous sheath (FS). We observed that in sperm from individual ARMC2_1, staining of SPAG6, an axoneme central-pair complex protein,23Sapiro R. Tarantino L.M. Velazquez F. Kiriakidou M. Hecht N.B. Bucan M. Strauss 3rd, J.F. Sperm antigen 6 is the murine homologue of the Chlamydomonas reinhardtii central apparatus protein encoded by the PF16 locus.Biol. Reprod. 2000; 62: 511-518Crossref PubMed Scopus (52) Google Scholar was totally absent from the flagellum (Figure 3A). In contrast, immunostaining for AKAP4, DNALI1, DNAH5, RSPH1, and GAS8 was similar to that of controls, suggesting that FS, IDAs, ODAs, RS, and the N-DRC, respectively, were not directly affected by mutations in ARMC2 (Figure S7). Because of limited sample availability, these analyses could not be repeated on sperm from other individuals with ARMC2 mutations. Also, we regret that we could not obtain any specific ARMC2 antibodies allowing the localization of the protein in human and mouse sperm. In addition, IF experiments performed in Armc2mutant animals showed that SPEF2 staining, another marker of the CPC,24Teves M.E. Nagarkatti-Gude D.R. Zhang Z. Strauss 3rd, J.F. Mammalian axoneme central pair complex proteins: Broader roles revealed by gene knockout phenotypes.Cytoskeleton (Hoboken). 2016; 73: 3-22Crossref PubMed Scopus (39) Google Scholar was totally absent, supporting the CPC defects observed in sperm samples from the MMAF-affected individual ARMC2_1 (Figure 3B). We showed that the presence of bi-allelic ARMC2 mutations induces a typical MMAF phenotype" @default.
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