Title: Bi-allelic DNAH8 Variants Lead to Multiple Morphological Abnormalities of the Sperm Flagella and Primary Male Infertility
Abstract: Sperm malformation is a direct factor for male infertility. Multiple morphological abnormalities of the flagella (MMAF), a severe form of asthenoteratozoospermia, are characterized by immotile spermatozoa with malformed and/or absent flagella in the ejaculate. Previous studies indicated genetic heterogeneity in MMAF. To further define genetic factors underlying MMAF, we performed whole-exome sequencing in a cohort of 90 Chinese MMAF-affected men. Two cases (2.2%) were identified as carrying bi-allelic missense DNAH8 variants, variants which were either absent or rare in the control human population and were predicted to be deleterious by multiple bioinformatic tools. Re-analysis of exome data from a second cohort of 167 MMAF-affected men from France, Iran, and North Africa permitted the identification of an additional male carrying a DNAH8 homozygous frameshift variant. DNAH8 encodes a dynein axonemal heavy-chain component that is expressed preferentially in the testis. Hematoxylin-eosin staining and electron microscopy analyses of the spermatozoa from men harboring bi-allelic DNAH8 variants showed a highly aberrant morphology and ultrastructure of the sperm flagella. Immunofluorescence assays performed on the spermatozoa from men harboring bi-allelic DNAH8 variants revealed the absent or markedly reduced staining of DNAH8 and its associated protein DNAH17. Dnah8-knockout male mice also presented typical MMAF phenotypes and sterility. Interestingly, intracytoplasmic sperm injections using the spermatozoa from Dnah8-knockout male mice resulted in good pregnancy outcomes. Collectively, our experimental observations from humans and mice demonstrate that DNAH8 is essential for sperm flagellar formation and that bi-allelic deleterious DNAH8 variants lead to male infertility with MMAF. Sperm malformation is a direct factor for male infertility. Multiple morphological abnormalities of the flagella (MMAF), a severe form of asthenoteratozoospermia, are characterized by immotile spermatozoa with malformed and/or absent flagella in the ejaculate. Previous studies indicated genetic heterogeneity in MMAF. To further define genetic factors underlying MMAF, we performed whole-exome sequencing in a cohort of 90 Chinese MMAF-affected men. Two cases (2.2%) were identified as carrying bi-allelic missense DNAH8 variants, variants which were either absent or rare in the control human population and were predicted to be deleterious by multiple bioinformatic tools. Re-analysis of exome data from a second cohort of 167 MMAF-affected men from France, Iran, and North Africa permitted the identification of an additional male carrying a DNAH8 homozygous frameshift variant. DNAH8 encodes a dynein axonemal heavy-chain component that is expressed preferentially in the testis. Hematoxylin-eosin staining and electron microscopy analyses of the spermatozoa from men harboring bi-allelic DNAH8 variants showed a highly aberrant morphology and ultrastructure of the sperm flagella. Immunofluorescence assays performed on the spermatozoa from men harboring bi-allelic DNAH8 variants revealed the absent or markedly reduced staining of DNAH8 and its associated protein DNAH17. Dnah8-knockout male mice also presented typical MMAF phenotypes and sterility. Interestingly, intracytoplasmic sperm injections using the spermatozoa from Dnah8-knockout male mice resulted in good pregnancy outcomes. Collectively, our experimental observations from humans and mice demonstrate that DNAH8 is essential for sperm flagellar formation and that bi-allelic deleterious DNAH8 variants lead to male infertility with MMAF. Human infertility, defined as the inability to achieve a clinical pregnancy despite 12 months of regular and unprotected intercourse, has become a widespread health issue.1Hosseini B. Nourmohamadi M. Hajipour S. Taghizadeh M. Asemi Z. Keshavarz S.A. Jafarnejad S. The Effect of Omega-3 Fatty Acids, EPA, and/or DHA on Male Infertility: A Systematic Review and Meta-analysis.J. Diet. Suppl. 2019; 16: 245-256Crossref PubMed Scopus (21) Google Scholar Multiple morphological abnormalities of the flagella (MMAF) are defined by the combination of absent, short, bent, coiled, and/or irregular-caliber flagella.2Ben 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 (197) Google Scholar Previous genetic studies revealed a series of MMAF-associated genes in cases of primary infertility without primary ciliary dyskinesia (PCD; MIM: 244400) associated symptoms (reviewed by Touré et al.).3Baccetti B. Collodel G. Estenoz M. Manca D. Moretti E. Piomboni P. Gene deletions in an infertile man with sperm fibrous sheath dysplasia.Hum. Reprod. 2005; 20: 2790-2794Crossref PubMed Scopus (93) Google Scholar, 4Tang 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 (121) Google Scholar, 5Dong F.N. Amiri-Yekta A. Martinez G. Saut A. Tek J. Stouvenel L. Lorès P. Karaouzène T. Thierry-Mieg N. 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Commun. 2018; 9: 686Crossref PubMed Scopus (88) Google Scholar, 11Liu C. He X. Liu W. Yang S. Wang L. Li W. Wu H. Tang S. Ni X. Wang J. et al.Bi-allelic Mutations in TTC29 Cause Male Subfertility with Asthenoteratospermia in Humans and Mice.Am. J. Hum. Genet. 2019; 105: 1168-1181Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 12Lorès P. Dacheux D. Kherraf Z.E. Nsota Mbango J.F. Coutton C. Stouvenel L. Ialy-Radio C. Amiri-Yekta A. Whitfield M. Schmitt A. et al.Mutations in TTC29, Encoding an Evolutionarily Conserved Axonemal Protein, Result in Asthenozoospermia and Male Infertility.Am. J. Hum. Genet. 2019; 105: 1148-1167Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 13Whitfield M. Thomas L. Bequignon E. Schmitt A. Stouvenel L. Montantin G. Tissier S. Duquesnoy P. Copin B. Chantot S. et al.Mutations in DNAH17, Encoding a Sperm-Specific Axonemal Outer Dynein Arm Heavy Chain, Cause Isolated Male Infertility Due to Asthenozoospermia.Am. J. Hum. Genet. 2019; 105: 198-212Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 14Touré A. Martinez G. Kherraf Z.E. Cazin C. Beurois J. Arnoult C. Ray P.F. Coutton C. The genetic architecture of morphological abnormalities of the sperm tail.Hum. Genet. 2020; https://doi.org/10.1007/s00439-00020-02113-xCrossref PubMed Google Scholar However, these genetic findings account for approximately 35% to 60% of MMAF cases,11Liu C. He X. Liu W. Yang S. Wang L. Li W. Wu H. Tang S. Ni X. Wang J. et al.Bi-allelic Mutations in TTC29 Cause Male Subfertility with Asthenoteratospermia in Humans and Mice.Am. J. Hum. Genet. 2019; 105: 1168-1181Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar,12Lorès P. Dacheux D. Kherraf Z.E. Nsota Mbango J.F. Coutton C. Stouvenel L. Ialy-Radio C. Amiri-Yekta A. Whitfield M. Schmitt A. et al.Mutations in TTC29, Encoding an Evolutionarily Conserved Axonemal Protein, Result in Asthenozoospermia and Male Infertility.Am. J. Hum. Genet. 2019; 105: 1148-1167Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar demonstrating the high genetic heterogeneity of this disorder and the necessity for further genetic explorations. Cilia and flagella are hair-like organelles extending from the cell surface.15Reiter J.F. Leroux M.R. Genes and molecular pathways underpinning ciliopathies.Nat. Rev. Mol. Cell Biol. 2017; 18: 533-547Crossref PubMed Scopus (488) Google Scholar,16Zariwala M.A. Knowles M.R. Omran H. Genetic defects in ciliary structure and function.Annu. Rev. Physiol. 2007; 69: 423-450Crossref PubMed Scopus (224) Google Scholar Both contain an important core component, termed the axoneme, which is an evolutionarily conserved structure consisting of a highly ordered “9 + 2” arrangement of nine peripheral microtubule doublets and two central microtubules.17Ishikawa T. Axoneme Structure from Motile Cilia.Cold Spring Harb. Perspect. Biol. 2017; 9: a028076Crossref PubMed Scopus (69) Google Scholar A number of multi-protein complexes (including radial spokes, nexin-dynein regulatory complex, central complex, and dynein arms) constitute the major components of the axoneme.18Loges N.T. Olbrich H. Fenske L. Mussaffi H. Horvath J. Fliegauf M. Kuhl H. Baktai G. Peterffy E. Chodhari R. et al.DNAI2 mutations cause primary ciliary dyskinesia with defects in the outer dynein arm.Am. J. Hum. Genet. 2008; 83: 547-558Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar Among the complexes, the outer and inner dynein arms (ODAs and IDAs, respectively) play an important role in the beating of cilia and flagella through ATP hydrolysis.19Ibañez-Tallon I. Heintz N. Omran H. To beat or not to beat: roles of cilia in development and disease.Hum. Mol. Genet. 2003; 12: R27-R35Crossref PubMed Google Scholar Previous studies have revealed that mutations in IDA and ODA protein complexes cause several ciliopathies and male infertility. In particular, the deficiency of DNAH1 (MIM: 603332), which encodes an important component of the IDA heavy chain, leads to isolated male infertility with MMAF.2Ben 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 (197) Google Scholar Importantly, recent studies reported that mutations in DNAH17 (MIM: 610063; encoding a sperm-specific ODA heavy-chain component) also cause isolated male infertility due to asthenoteratozoospermia.13Whitfield M. Thomas L. Bequignon E. Schmitt A. Stouvenel L. Montantin G. Tissier S. Duquesnoy P. Copin B. Chantot S. et al.Mutations in DNAH17, Encoding a Sperm-Specific Axonemal Outer Dynein Arm Heavy Chain, Cause Isolated Male Infertility Due to Asthenozoospermia.Am. J. Hum. Genet. 2019; 105: 198-212Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar,20Zhang B. Ma H. Khan T. Ma A. Li T. Zhang H. Gao J. Zhou J. Li Y. Yu C. et al.A DNAH17 missense variant causes flagella destabilization and asthenozoospermia.J. Exp. Med. 2020; 217: e20182365Crossref PubMed Scopus (31) Google Scholar These findings suggest the potential involvement of other components of dynein arms in male infertility and sperm flagellar malformations. Here, two distinct MMAF cohorts were analyzed. The first cohort comprised 90 Chinese MMAF-affected men enrolled from the First Affiliated Hospital of Anhui Medical University and the Women and Children’s Hospital of Xiamen University in China. The second cohort comprised 167 individuals with MMAF, including 83 men from North Africa (Algeria, Libya, and Tunisia; enrolled at the Clinique des Jasmins in Tunis), 52 men recruited at the Royan Institute (Reproductive Biomedicine Research Center) in Iran, and 32 men recruited in France (mainly at the Reproductive Department of the Cochin Hospital in Paris). The clinical phenotypes of the affected individuals are summarized in the Supplemental Note (see Supplemental Information). Informed consent was obtained from all subjects participating in the study. The study regarding the cohorts was approved by the institutional review boards at all of the participating institutes. To investigate the unknown genetic factors involved in human MMAF, we performed whole-exome sequencing (WES) analyses in the first cohort of 90 Chinese men with MMAF. After applying stringent bioinformatic analyses according to our previously described protocol,4Tang 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 (121) Google Scholar we identified two men (2.2%) harboring bi-allelic missense variants in DNAH8 (MIM: 603337; NCBI: NM_001206927.2). The DNAH8-mutated alleles were c.11771C>T (p.Thr3924Met) plus c.6689A>G (p.Lys2230Arg) in subject A051 (II-1 in Figure 1A) and c.9427C>T (p.Arg3143Cys) plus c.12721G>A (p.Ala4241Thr) in subject X003 (II-1 in Figure 1B). Subsequent Sanger sequencing confirmed that these bi-allelic DNAH8 variants were inherited from heterozygous parental carriers (Figures 1A and 1B; Table S1). All of the DNAH8 variants were either absent or rare in the human genome datasets archived in the 1000 Genomes Project and gnomAD databases. These DNAH8 variants were also predicted to be damaging through the use of the PolyPhen-2, SIFT, and MutationTaster tools (Table 1).Table 1Bi-allelic DNAH8 Variants Identified in MMAF-affected Men-Subject A051Subject X003Subject F0300cDNA alterationc.11771C>Tc.6689A>Gc.9427C>Tc.12721G>Ac.6962_6968delVariant alleleheterozygousheterozygousheterozygousheterozygoushomozygousProtein alterationp.Thr3924Metp.Lys2230Argp.Arg3143Cysp.Ala4241Thrp.His2321Profs∗4Variant typemissensemissensemissensemissenseframeshiftAllele Frequency in Human Population1000 Genomes00000gnomAD (v3)0.00672900.00046770.00011870Function PredictionSIFTdamagingdamagingdamagingdamagingNAPolyPhen-2damagingdamagingdamagingdamagingNAMutationTasterdamagingdamagingdamagingdamagingdamagingNCBI reference sequence number of DNAH8 is NM_001206927.2.NA, not applicable. Open table in a new tab NCBI reference sequence number of DNAH8 is NM_001206927.2. NA, not applicable. WES analysis of the 167 MMAF-affected men from the second cohort identified an additional case in an individual of Moroccan ancestry (Figure 1C). This MMAF-affected subject F0300 (II-1 in Figure 1C) harbored a homozygous frameshift variant (c.6962_6968del [p.His2321Profs∗4]) that produced a frameshift and premature stop codon in DNAH8. No DNA was available from family members of subject F0300. His brother was reported to be infertile, although no further investigation could be performed. We note that his parents are consanguineous, thus strongly supporting the likelihood that this DNAH8 frameshift variant was also transmitted under a recessive mode of inheritance. Importantly, the residues in DNAH8 affected by these aforementioned variants are all highly conserved across species (Figure 1D). Furthermore, no bi-allelic deleterious variants in previously described MMAF- or PCD-associated genes were observed in the three men with bi-allelic DNAH8 variants. These findings further suggest that the infertility phenotypes were likely caused by the identified bi-allelic DNAH8 variants. DNAH8 contains 92 exons and encodes a predicted 4,707-amino-acid protein (NCBI: NP_001193856.1; UniProt: A0A075B6F3). The DNAH8 protein is preferentially expressed in the human testis, according to the Human Protein Atlas. Our reverse transcription polymerase chain reaction (RT-PCR) assays also indicated that mouse Dnah8 is predominantly expressed in the testis (Figure S1A). Furthermore, the expression of mouse Dnah8 mRNA in the testis began at 14 days after birth, corresponding to the pachytene stage (Figure S1B). Semen parameters of men harboring bi-allelic DNAH8 variants were analyzed in the source laboratories according to World Health Organization 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 (24) Google Scholar Sperm motility and progressive motility in the men harboring bi-allelic DNAH8 variants were dramatically lower than the normal reference values (Table 2). Hematoxylin-eosin (H&E) staining and scanning electron microscopy examination were performed to assess sperm morphology. Approximately 70% of the immotile spermatozoa displayed abnormal flagella, including absent, short, and coiled flagella, angulation, and irregular caliber (Figure 2 and Table 2).Table 2Semen Characteristics and Sperm Flagellar Morphology of Men Carrying Bi-allelic DNAH8 Variants-Subject A051Subject X003Subject F0300Reference ValuesSemen ParametersSemen volume (mL)2.23.27.1>1.5Sperm concentration (106/mL)39.142.713.6∗Abnormal values.>15.0Total sperm count (106)86.0136.796.6>39.0Motility (%)4.0∗Abnormal values.2.7∗Abnormal values.8.0∗Abnormal values.>40.0Progressive motility (%)1.3∗Abnormal values.1.5∗Abnormal values.4.0∗Abnormal values.>32.0Sperm Flagellar MorphologyAbsent flagella (%)2.02.54.0<5.0Short flagella (%)7.1∗Abnormal values.9.0∗Abnormal values.8.0∗Abnormal values.<1.0Coiled flagella (%)57.1∗Abnormal values.32.0∗Abnormal values.33.0∗Abnormal values.<17.0Angulation (%)0.56.019.0∗Abnormal values.<13.0Irregular caliber (%)1.026.0∗Abnormal values.8.0∗Abnormal values.<2.0Normal flagella (%)32.324.528.0>23.0Lower and upper reference limits are shown according to the World Health Organization standards46Cooper T.G. Noonan E. von Eckardstein S. Auger J. Baker H.W. Behre H.M. Haugen T.B. Kruger T. Wang C. Mbizvo M.T. Vogelsong K.M. World Health Organization reference values for human semen characteristics.Hum. Reprod. Update. 2010; 16: 231-245Crossref PubMed Scopus (1535) Google Scholar and the distribution ranges of morphologically abnormal spermatozoa observed in fertile individuals.47Auger J. Jouannet P. Eustache F. Another look at human sperm morphology.Hum. Reprod. 2016; 31: 10-23Crossref PubMed Scopus (76) Google Scholar∗ Abnormal values. Open table in a new tab Lower and upper reference limits are shown according to the World Health Organization standards46Cooper T.G. Noonan E. von Eckardstein S. Auger J. Baker H.W. Behre H.M. Haugen T.B. Kruger T. Wang C. Mbizvo M.T. Vogelsong K.M. World Health Organization reference values for human semen characteristics.Hum. Reprod. Update. 2010; 16: 231-245Crossref PubMed Scopus (1535) Google Scholar and the distribution ranges of morphologically abnormal spermatozoa observed in fertile individuals.47Auger J. Jouannet P. Eustache F. Another look at human sperm morphology.Hum. Reprod. 2016; 31: 10-23Crossref PubMed Scopus (76) Google Scholar Various ultrastructural defects were revealed by transmission electron microscopy (TEM) in the sperm flagella from men harboring bi-allelic DNAH8 variants. The typical “9 + 2” microtubule structure was observed in the spermatozoa from control men (Figure 3). However, a dramatic disorganization in axonemal or peri-axonemal structures (including disorganized peripheral microtubule doublets and outer dense fibers, missing or disassembled ODAs, and absent central pairs) was detected in the spermatozoa from men harboring bi-allelic DNAH8 variants (Figure 3). Quantification conducted on transverse sections of the sperm flagella indicated higher rates of abnormal flagellar ultrastructure in men harboring bi-allelic DNAH8 variants than those in the normal control (Table S2). To further investigate the pathogenicity of bi-allelic DNAH8 variants, we analyzed the levels of DNAH8 mRNA and DNAH8 protein using RT-PCR (Table S3) and immunofluorescence assays, respectively. The abundance of DNAH8 mRNA in the sperm from subject A051, who harbored bi-allelic DNAH8 variants, was significantly reduced when compared to the normal control (Figure S2). As for the protein level, in the normal control man, DNAH8 immunostaining was concentrated along the mid-piece and principal piece of the sperm flagella (Figure S3). This observation in humans is consistent with previous evidence in wild-type male mice.22Samant S.A. Ogunkua O.O. Hui L. Lu J. Han Y. Orth J.M. Pilder S.H. The mouse t complex distorter/sterility candidate, Dnahc8, expresses a gamma-type axonemal dynein heavy chain isoform confined to the principal piece of the sperm tail.Dev. Biol. 2005; 285: 57-69Crossref PubMed Scopus (12) Google Scholar In contrast, DNAH8 immunostaining was almost absent in the sperm flagella from all three subjects harboring bi-allelic DNAH8 variants, including both missense and frameshift variants (Figure 4A and Figure S3A). We also examined the presence of DNAH17, which is required for accurate localization of DNAH8.13Whitfield M. Thomas L. Bequignon E. Schmitt A. Stouvenel L. Montantin G. Tissier S. Duquesnoy P. Copin B. Chantot S. et al.Mutations in DNAH17, Encoding a Sperm-Specific Axonemal Outer Dynein Arm Heavy Chain, Cause Isolated Male Infertility Due to Asthenozoospermia.Am. J. Hum. Genet. 2019; 105: 198-212Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar Notably, the staining of DNAH17 was dramatically reduced in the spermatozoa from men harboring bi-allelic DNAH8 variants (Figure 4B and Figure S3B). DNAH8 is highly conserved among different species during evolution. Consistent with the human data available from the Human Protein Atlas, the murine ortholog Dnah8 is also preferentially expressed in the testis, as per our RT-PCR assays conducted in a set of various mouse tissues (Figure S1, Table S4). To further investigate the role of mouse Dnah8 in sperm flagellar formation, we generated Dnah8-knockout (KO; Dnah8em1/em1) mice through the use of CRISPR-Cas9 technology. Two guide RNAs targeting the regions near the start and stop codons were used to delete the entire coding region of Dnah8 (Figure S4A). Polymerase chain reaction (PCR) and Sanger sequencing were performed to confirm the mutated allele in Dnah8-KO mice (Figures S4B and S4C). We also used an immunoblot assay to investigate the level of DNAH8 protein in the testes of wild-type and Dnah8-KO male mice. As shown in Figure S5, the signal of DNAH8 was absent in the testis from Dnah8-KO male mice. No significant differences were observed in testis weight between Dnah8-KO and heterozygous mutated male mice (Figure S6). Sperm parameters and morphology of Dnah8-KO male mice were also investigated. As shown in Table 3, Video S1, and Video S2, diminished sperm movement was observed in Dnah8-KO male mice when compared to heterozygous mutated (Dnah8wt/em1) male mice. H&E staining revealed significantly higher rates of abnormal flagella in Dnah8-KO male mice than those in heterozygous mutated male mice (Table 3 and Figure 5A). The sperm flagella of Dnah8-KO male mice also presented with absent, short, coiled, bent, and/or irregular shapes, which recapitulated the clinical phenotypes of MMAF-affected men with bi-allelic DNAH8 variants. Furthermore, TEM analysis of sperm flagella showed disorganized microtubules and outer dense fibers in the spermatozoa from Dnah8-KO male mice (Figure 5B). These experimental observations on Dnah8-KO male mice conclusively demonstrated the crucial role of DNAH8 in sperm flagellar formation.Table 3Sperm Characteristics and Flagellar Morphology of Dnah8-KO Male Mice-Heterozygous Control (Dnah8wt/em1)KO (Dnah8em1/em1)Semen ParameterMotility (%)91.7 ± 1.50 ± 0∗∗∗Sperm Flagellar MorphologyaData represent the mean ± SD of three independent experiments.Absent flagella (%)4.0 ± 2.618.5 ± 17.8∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.Short flagella (%)0.0 ± 0.025.7 ± 1.2∗∗∗Coiled flagella (%)0.0 ± 0.025.5 ± 15.6∗∗Irregular caliber (%)0.2 ± 0.320.5 ± 12.6∗∗∗Bent flagella (%)0.7 ± 0.89.8 ± 1.6∗∗∗a Data represent the mean ± SD of three independent experiments.∗ p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Open table in a new tab eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI3MWJjZDVlNzNlMTc2NjIxZWY5ZDE4YjJiOGM0MGY0ZiIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjQxNzMyNDY2fQ.Gz_RmQzsPp_XkIBzuhZzfGVmKptGPOxo0yCSxoHPluF6BX_yDDnh5zzHL8TvjStTEUJIsDEeGsGNcih3G6Ht7kRN9iQgaO8bwriXq2Izof9t6k9S2mSiqKJDbX-sCGki7NtHWrT_w9noMOhy1Gq5QaFI9U3onf0bh93JofGnAzABmEj3w4Ciat4uksV-ViEp2Aj8mPSL_sQBkHDcKLdbm9tlMRrG73N2Hr3XPv7ciubmDMSpsjpdaajY-tyz8l3VsMytu9xakJ6qtja2OVWH2CQ0x0p_AxDJfXOvIKm6m7LVA5JmROY7Ex_hmJoTu_Dmp38gyJeDImkWbbMSrHuFvQ Download .mp4 (0.23 MB) Help with .mp4 files Video S1. Sperm Motility in Dnah8-KO (Dnah8em1/em1) Male Mice eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiIwMmIyNzVkYjQzYzc4NmZhMDYxNTdkNTU4ZjI5ODQ3MiIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjQxNzMyNDY2fQ.d8ACnWHk7EQw4Wx0dayvxD82YrHECnjxfKfBfdqxV3AVF58mwwDGj8AkGqHLur13JUrVzFw_mSLg9EGYGLZBBu06LzJbk4BTfvqIUfbUM7shfgI7LedcVPV1Cuw88OC-4A_Yz30JN0Wqh9c51Hi-4mLfblI18kGxCJD4wEj-mMjZl8yNy00PD857PE42eaj122Nwd5p1-M1aQJ3tVI6ac8C9jSOYIZlXL-hOGOWkXFIe5FMOBhCflW5x-YVV04CwtFekBgfs0OhgyAwdzdK6mjPkt-NpY8X_5U4QcLUeIlbijHYfEd6GUqMGdtXZyN9gJzRr0sbhpkY3751LA4k-Ig Download .mp4 (0.98 MB) Help with .mp4 files Video S2. Sperm Motility in Heterozygous Mutated (Dnah8wt/em1) Male MiceDiminished sperm motility was evident in Dnah8-KO male mice when compared to Dnah8 heterozygous mutated male mice. To further investigate the role of DNAH8 in spermatogenesis, we performed H&E staining on the testes of Dnah8-KO and heterozygous mutated male mice (Figure 6A). In stage VII–VIII seminiferous tubules, no elongated tails were observed in the testes from Dnah8-KO male mice, but normal round spermatids were observed, indicating the involvement of DNAH8 in sperm flagellar formation. Periodic acid–Schiff (PAS) staining of the cauda epididymis from Dnah8-KO male mice displayed fewer sperm heads than did those from heterozygous mutated mice (Figure 6B). Collectively, these data suggest that DNAH8 deficiency can result in MMAF and male infertility in both humans and mice. To assess the fertility and reproductive behavior of Dnah8-KO male mice, sexually mature Dnah8-KO and heterozygous mutated male mice were individually caged with 8-week-old wild-type B6D2F1 female mice (one male with three females) for 2 months, and plugs were checked every morning. Pups were counted on the day of birth. As shown in Figure S7, normal mounting and copulatory plugs were observed for both groups of Dnah8-KO and heterozygous mutated male mice. However, Dnah8-KO male mice failed to produce any offspring over 2 months of breeding, whereas heterozygous mutated males routinely produced offspring (Figure S7). These experimental observations indicate that DNAH8 is necessary for male fertility in mice. Intracytoplasmic sperm injection (ICSI) has been reported to be efficient for most MMAF-associated asthenoteratozoospermia.23Chemes H.E. Alvarez Sedo C. Tales of the tail and sperm head aches: changing concepts on the prognostic significance of sperm pathologies affecting the head, neck and tail.Asian J. Androl. 2012; 14: 14-23Crossref PubMed Scopus (78) Google Scholar To examine whether DNAH8-associated male infertility could also be overcome via ICSI, we conducted experiments using the sperm from wild-type and Dnah8-KO male mice. As shown in Figure 7, pups were successfully obtained upon ICSI using the spermatozoa from Dnah8-KO male mice after the transfer of two-cell embryos to pseudopregnant ICR females. Genotyping assays confirmed that all these pups were heterozygous Dnah8-mutated carriers, as anticipated. Our findings indicated that Dnah8-associated KO male infertility in mice could be overcome by ICSI. Consistent with these experimental observations, the second ICSI attempt performed using the sperm from subject F0300 (who harbored a homozygous D