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1.
Am J Hum Genet ; 107(3): 514-526, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32791035

RESUMO

Multiple morphological abnormalities of the sperm flagella (MMAF) is a severe form of asthenoteratozoospermia. Although recent studies have revealed several MMAF-associated genes and demonstrated MMAF to be a genetically heterogeneous disease, at least one-third of the cases are still not well understood for their etiology. Here, we identified bi-allelic loss-of-function variants in CFAP58 by using whole-exome sequencing in five (5.6%) unrelated individuals from a cohort of 90 MMAF-affected Chinese men. Each of the men harboring bi-allelic CFAP58 variants presented typical MMAF phenotypes. Transmission electron microscopy demonstrated striking flagellar defects with axonemal and mitochondrial sheath malformations. CFAP58 is predominantly expressed in the testis and encodes a cilia- and flagella-associated protein. Immunofluorescence assays showed that CFAP58 localized at the entire flagella of control sperm and predominantly concentrated in the mid-piece. Immunoblotting and immunofluorescence assays showed that the abundances of axoneme ultrastructure markers SPAG6 and SPEF2 and a mitochondrial sheath protein, HSP60, were significantly reduced in the spermatozoa from men harboring bi-allelic CFAP58 variants. We generated Cfap58-knockout mice via CRISPR/Cas9 technology. The male mice were infertile and presented with severe flagellar defects, consistent with the sperm phenotypes in MMAF-affected men. Overall, our findings in humans and mice strongly suggest that CFAP58 plays a vital role in sperm flagellogenesis and demonstrate that bi-allelic loss-of-function variants in CFAP58 can cause axoneme and peri-axoneme malformations leading to male infertility. This study provides crucial insights for understanding and counseling of MMAF-associated asthenoteratozoospermia.


Assuntos
Anormalidades Múltiplas/genética , Astenozoospermia/genética , Axonema/genética , Infertilidade Masculina/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Anormalidades Múltiplas/patologia , Alelos , Animais , Astenozoospermia/fisiopatologia , Axonema/patologia , Sistemas CRISPR-Cas/genética , Proteínas de Ciclo Celular/genética , Homozigoto , Humanos , Infertilidade Masculina/patologia , Mutação com Perda de Função/genética , Perda de Heterozigosidade/genética , Masculino , Camundongos , Camundongos Knockout , Proteínas dos Microtúbulos/genética , Mitocôndrias/genética , Cauda do Espermatozoide/metabolismo , Cauda do Espermatozoide/patologia , Testículo/metabolismo , Testículo/patologia , Sequenciamento Completo do Exoma
2.
PLoS Genet ; 16(1): e1008585, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31961863

RESUMO

Flagella and cilia are evolutionarily conserved cellular organelles. Abnormal formation or motility of these organelles in humans causes several syndromic diseases termed ciliopathies. The central component of flagella and cilia is the axoneme that is composed of the '9+2' microtubule arrangement, dynein arms, radial spokes, and the Nexin-Dynein Regulatory Complex (N-DRC). The N-DRC is localized between doublet microtubules and has been extensively studied in the unicellular flagellate Chlamydomonas. Recently, it has been reported that TCTE1 (DRC5), a component of the N-DRC, is essential for proper sperm motility and male fertility in mice. Further, TCTE1 has been shown to interact with FBXL13 (DRC6) and DRC7; however, functional roles of FBXL13 and DRC7 in mammals have not been elucidated. Here we show that Fbxl13 and Drc7 expression are testes-enriched in mice. Although Fbxl13 knockout (KO) mice did not show any obvious phenotypes, Drc7 KO male mice were infertile due to their short immotile spermatozoa. In Drc7 KO spermatids, the axoneme is disorganized and the '9+2' microtubule arrangement was difficult to detect. Further, other N-DRC components fail to incorporate into the flagellum without DRC7. These results indicate that Drc7, but not Fbxl13, is essential for the correct assembly of the N-DRC and flagella.


Assuntos
Dineínas/metabolismo , Flagelos/genética , Infertilidade Masculina/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Espermatozoides/metabolismo , Animais , Axonema/genética , Axonema/metabolismo , Axonema/patologia , Feminino , Flagelos/metabolismo , Flagelos/patologia , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Espermatogênese , Espermatozoides/citologia , Espermatozoides/patologia
3.
Am J Hum Genet ; 105(6): 1148-1167, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31735292

RESUMO

In humans, structural or functional defects of the sperm flagellum induce asthenozoospermia, which accounts for the main sperm defect encountered in infertile men. Herein we focused on morphological abnormalities of the sperm flagellum (MMAF), a phenotype also termed "short tails," which constitutes one of the most severe sperm morphological defects resulting in asthenozoospermia. In previous work based on whole-exome sequencing of a cohort of 167 MMAF-affected individuals, we identified bi-allelic loss-of-function mutations in more than 30% of the tested subjects. In this study, we further analyzed this cohort and identified five individuals with homozygous truncating variants in TTC29, a gene preferentially and highly expressed in the testis, and encoding a tetratricopeptide repeat-containing protein related to the intraflagellar transport (IFT). One individual carried a frameshift variant, another one carried a homozygous stop-gain variant, and three carried the same splicing variant affecting a consensus donor site. The deleterious effect of this last variant was confirmed on the corresponding transcript and protein product. In addition, we produced and analyzed TTC29 loss-of-function models in the flagellated protist T. brucei and in M. musculus. Both models confirmed the importance of TTC29 for flagellar beating. We showed that in T. brucei the TPR structural motifs, highly conserved between the studied orthologs, are critical for TTC29 axonemal localization and flagellar beating. Overall our work demonstrates that TTC29 is a conserved axonemal protein required for flagellar structure and beating and that TTC29 mutations are a cause of male sterility due to MMAF.


Assuntos
Astenozoospermia/etiologia , Axonema/patologia , Flagelos/patologia , Infertilidade Masculina/etiologia , Proteínas Associadas aos Microtúbulos/genética , Mutação , Animais , Astenozoospermia/metabolismo , Astenozoospermia/patologia , Axonema/genética , Axonema/metabolismo , Evolução Molecular , Feminino , Fertilização In Vitro , Flagelos/genética , Flagelos/metabolismo , Humanos , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Masculino , Camundongos Endogâmicos C57BL , Trypanosoma brucei brucei/fisiologia , Tripanossomíase
4.
Hum Reprod ; 34(10): 2071-2079, 2019 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-31621862

RESUMO

The use of high-throughput sequencing techniques has allowed the identification of numerous mutations in genes responsible for severe astheno-teratozoospermia due to multiple morphological abnormalities of the sperm flagella (MMAF). However, more than half of the analysed cases remain unresolved suggesting that many yet uncharacterised gene defects account for this phenotype. Based on whole-exome sequencing data from a large cohort of 167 MMAF-affected subjects, we identified two unrelated affected individuals carrying a homozygous deleterious mutation in CFAP70, a gene not previously linked to the MMAF phenotype. One patient had a homozygous splice variant c.1723-1G>T, altering a consensus splice acceptor site of CFAP70 exon 16, and one had a likely deleterious missense variant in exon 3 (p.Phe60Ile). The CFAP70 gene encodes a regulator protein of the outer dynein arms (ODA) strongly expressed in the human testis. In the sperm cells from the patient carrying the splice variant, immunofluorescence (IF) experiments confirmed the absence of the protein in the sperm flagellum. Moreover, IF analysis showed the absence of markers for the ODAs and the central pair complex of the axoneme. Interestingly, whereas CFAP70 staining was present in sperm cells from patients with mutations in the three other MMAF-related genes ARMC2, FSIP2 and CFAP43, we observed an absence of staining in sperm cells from patients mutated in the WDR66 gene, suggesting a possible interaction between two different axonemal components. In conclusion, this work provides the first evidence that loss of CFAP70 function causes MMAF and that ODA-related proteins may be crucial for the assembly and/or stability of the flagellum axoneme in addition to its motility.


Assuntos
Astenozoospermia/genética , Proteínas Associadas aos Microtúbulos/genética , Cauda do Espermatozoide/patologia , Astenozoospermia/diagnóstico , Astenozoospermia/patologia , Axonema/patologia , Análise Mutacional de DNA , Éxons/genética , Homozigoto , Humanos , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Mutação de Sentido Incorreto , Sítios de Splice de RNA/genética , Índice de Gravidade de Doença , Sequenciamento Completo do Exoma
5.
J Assist Reprod Genet ; 36(8): 1683-1700, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31273583

RESUMO

PROPOSE: To study CCDC103 expression profiles and understand how pathogenic variants in CCDC103 affect its expression profile at mRNA and protein level. METHODS: To increase the knowledge about the CCDC103, we attempted genotype-phenotype correlations in two patients carrying novel homozygous (missense and frameshift) CCDC103 variants. Whole-exome sequencing, quantitative PCR, Western blot, electron microscopy, immunohistochemistry, immunocytochemistry, and immunogold labelling were performed to characterize CCDC103 expression profiles in reproductive and somatic cells. RESULTS: Our data demonstrate that pathogenic variants in CCDC103 gene negatively affect gene and protein expression in both patients who presented absence of DA on their axonemes. Further, we firstly report that CCDC103 is expressed at different levels in reproductive tissues and somatic cells and described that CCDC103 protein forms oligomers with tissue-specific sizes, which suggests that CCDC103 possibly undergoes post-translational modifications. Moreover, we reported that CCDC103 was restricted to the midpiece of sperm and is present at the cytoplasm of the other cells. CONCLUSIONS: Overall, our data support the CCDC103 involvement in PCD and suggest that CCDC103 may have different assemblies and roles in cilia and sperm flagella biology that are still unexplored.


Assuntos
Axonema/patologia , Transtornos da Motilidade Ciliar/genética , Infertilidade Masculina/patologia , Síndrome de Kartagener/genética , Proteínas Associadas aos Microtúbulos/genética , Mutação , Cauda do Espermatozoide/patologia , Axonema/genética , Transtornos da Motilidade Ciliar/patologia , Dineínas/metabolismo , Feminino , Humanos , Infertilidade Masculina/etiologia , Síndrome de Kartagener/patologia , Masculino , Pessoa de Meia-Idade , Reprodução , Situs Inversus/genética , Situs Inversus/patologia , Cauda do Espermatozoide/metabolismo
6.
J Med Genet ; 56(10): 678-684, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31151990

RESUMO

BACKGROUND: Multiple morphological abnormalities of the sperm flagella (MMAF) is a kind of severe teratozoospermia. Patients with the MMAF phenotype are infertile and present aberrant spermatozoa with absent, short, coiled, bent and/or irregular flagella. Mutations in several genes can explain approximately 30%-50% of MMAF cases and more genetic pathogenies need to be explored. SPEF2 was previously demonstrated to play an essential role in sperm tail development in mice and pig. Dysfunctional mutations in SPEF2 impair sperm motility and cause a short-tail phenotype in both animal models. OBJECTIVE: Based on 42 patients with severe infertility and MMAF phenotype, we explored the new genetic cause of human MMAF phenotype. METHODS AND RESULTS: By screening gene variants in 42 patients with MMAF using whole exome sequencing, we identified the c. 12delC, c. 1745-2A > G, c. 4102 G > T and c. 4323dupA mutations in the SPEF2 gene from two patients. Both of these mutations are rare and potentially deleterious. Transmission electron microscope (TEM) analysis showed a disrupted axonemal structure with mitochondrial sheath defects in the patients' spermatozoa. The SPEF2 protein level was significantly decreased in the spermatozoa of the patients revealed by Western blot (WB) and immunofluorescence (IF) analyses. CONCLUSION: Our experimental findings indicate that loss-of-function mutations in the SPEF2 gene can cause the MMAF phenotype in human.


Assuntos
Anormalidades Múltiplas/genética , Proteínas de Ciclo Celular/genética , Infertilidade Masculina/genética , Mutação com Perda de Função , Anormalidades Múltiplas/diagnóstico por imagem , Axonema/patologia , Humanos , Infertilidade Masculina/diagnóstico por imagem , Masculino , Fenótipo , Motilidade Espermática/genética , Cauda do Espermatozoide/patologia , Espermatozoides/patologia , Sequenciamento Completo do Exoma
7.
Andrologia ; 50(10): e13128, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30132935

RESUMO

This study characterized three cases of systematic sperm tail defects using electron microscopy and immunolocalisation of centrin 1 and tubulin and explored their impact on ICSI outcome. Structural sperm tail defects of possible genetic origin were suspected as the eosin test revealed a sperm viability of >70% despite severe asthenozoospermia or the absence of motility. In Patient 1, 80%-85% of axoneme cross sections was incomplete. The fluorescent signal of tubulin was weak along the entire tail; the signal of centrin 1 was normal. After ICSI, a female healthy baby was born. Patient 2 showed spermatozoa with tails reduced in length at different levels, axonemal and periaxonemal alterations and fragility of head-tail junction. Centrin 1 was altered in 80% of sperm. After ICSI, no embryos were obtained. Patient 3 showed tails reduced in length at light and fluorescence microscopy; ultrastructural study revealed a condition of dysplasia of fibrous sheath with heterogeneity of tails' length. The signal for centrin 1 was altered in 50% of spermatozoa; two embryos were transferred without pregnancy. The correct diagnosis of sperm pathology is important in case of systematic sperm defects as it enables the clinician to improve patient's management and to provide an adequate genetic counselling.


Assuntos
Axonema/patologia , Infertilidade Masculina/terapia , Injeções de Esperma Intracitoplásmicas , Cauda do Espermatozoide/patologia , Adulto , Axonema/ultraestrutura , Proteínas de Ligação ao Cálcio/análise , Proteínas de Ciclo Celular/análise , Feminino , Humanos , Imuno-Histoquímica , Recém-Nascido , Infertilidade Masculina/fisiopatologia , Masculino , Microscopia Eletrônica de Transmissão , Gravidez , Análise do Sêmen , Motilidade Espermática , Cauda do Espermatozoide/ultraestrutura , Resultado do Tratamento , Tubulina (Proteína)/análise
8.
Clin Genet ; 93(2): 345-349, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28548327

RESUMO

Asthenozoospermia (AZS) is a common cause of male infertility, characterized by abnormal reduction in the motility of ejaculated spermatozoa. Here, in a patient from a consanguineous family, we identified a homozygous mutation (c.G4343A, p.R1448Q) in SPAG17 by whole-exome sequencing. The encoded protein, SPAG17, localizes to the axonemal central apparatus and is considered essential for flagellar waveform. In silico analysis revealed that R1448Q is a potential pathogenic mutation. Immunostaining and western blot assays showed that the R1448Q mutation may exert a negative effect on the steady-state of the SPAG17 protein. Therefore, SPAG17 may be a new pathogenic gene causing AZS.


Assuntos
Astenozoospermia/genética , Infertilidade Masculina/genética , Proteínas dos Microtúbulos/genética , Sequenciamento Completo do Exoma , Adulto , Astenozoospermia/patologia , Axonema/genética , Axonema/patologia , Homozigoto , Humanos , Infertilidade Masculina/patologia , Masculino , Mutação , Motilidade Espermática/genética , Espermatozoides/patologia , Estudos em Gêmeos como Assunto
9.
Part Fibre Toxicol ; 14(1): 44, 2017 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-29132433

RESUMO

BACKGROUND: Multi-walled carbon nanotubes (MWCNTs) are engineered nanomaterials used for a variety of industrial and consumer products. Their high tensile strength, hydrophobicity, and semi-conductive properties have enabled many novel applications, increasing the possibility of accidental nanotube inhalation by either consumers or factory workers. While MWCNT inhalation has been previously shown to cause inflammation and pulmonary fibrosis at high doses, the susceptibility of differentiating bronchial epithelia to MWCNT exposure remains unexplored. In this study, we investigate the effect of MWCNT exposure on cilia development in a differentiating air-liquid interface (ALI) model. Primary bronchial epithelial cells (BECs) were isolated from human donors via bronchoscopy and treated with non-cytotoxic doses of MWCNTs in submerged culture for 24 h. Cultures were then allowed to differentiate in ALI for 28 days in the absence of further MWCNT exposure. At 28 days, mucociliary differentiation endpoints were assessed, including whole-mount immunofluorescent staining, histological, immunohistochemical and ultrastructural analysis, gene expression, and cilia beating analysis. RESULTS: We found a reduction in the prevalence and beating of ciliated cells in MWCNT-treated cultures, which appeared to be caused by a disruption of cellular microtubules and cytoskeleton during ciliogenesis and basal body docking. Expression of gene markers of mucociliary differentiation, such as FOXJ1 and MUC5AC/B, were not affected by treatment. Colocalization of basal body marker CEP164 with γ-tubulin during days 1-3 of ciliogenesis, as well as abundance of basal bodies up to day 14, were attenuated by treatment with MWCNTs. CONCLUSIONS: Our results suggest that a single exposure of bronchial cells to MWCNT during a vulnerable period before differentiation may impair their ability to develop into fully functional ciliated cells.


Assuntos
Brônquios/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Nanotubos de Carbono/toxicidade , Axonema/efeitos dos fármacos , Axonema/patologia , Brônquios/metabolismo , Brônquios/patologia , Células Cultivadas , Cílios/efeitos dos fármacos , Cílios/patologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Proteínas dos Microtúbulos/metabolismo , Movimento/efeitos dos fármacos , Cultura Primária de Células , Medição de Risco , Fatores de Tempo , Tubulina (Proteína)/metabolismo
10.
Nat Commun ; 8: 14279, 2017 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-28176794

RESUMO

By moving essential body fluids and molecules, motile cilia and flagella govern respiratory mucociliary clearance, laterality determination and the transport of gametes and cerebrospinal fluid. Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder frequently caused by non-assembly of dynein arm motors into cilia and flagella axonemes. Before their import into cilia and flagella, multi-subunit axonemal dynein arms are thought to be stabilized and pre-assembled in the cytoplasm through a DNAAF2-DNAAF4-HSP90 complex akin to the HSP90 co-chaperone R2TP complex. Here, we demonstrate that large genomic deletions as well as point mutations involving PIH1D3 are responsible for an X-linked form of PCD causing disruption of early axonemal dynein assembly. We propose that PIH1D3, a protein that emerges as a new player of the cytoplasmic pre-assembly pathway, is part of a complementary conserved R2TP-like HSP90 co-chaperone complex, the loss of which affects assembly of a subset of inner arm dyneins.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Dineínas do Axonema/metabolismo , Genes Ligados ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Síndrome de Kartagener/genética , Proteínas dos Microtúbulos/genética , Chaperonas Moleculares/genética , Adolescente , Adulto , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Axonema/patologia , Criança , Pré-Escolar , Cílios/patologia , Cílios/ultraestrutura , Citoplasma/patologia , Modelos Animais de Doenças , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Células HEK293 , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Recém-Nascido , Peptídeos e Proteínas de Sinalização Intracelular , Síndrome de Kartagener/patologia , Masculino , Microscopia Eletrônica de Transmissão , Linhagem , Filogenia , Mutação Puntual , Dobramento de Proteína , Alinhamento de Sequência , Deleção de Sequência , Motilidade Espermática/genética , Sequenciamento Completo do Exoma , Peixe-Zebra
11.
Tissue Cell ; 48(6): 588-595, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27823762

RESUMO

In the present study, we examined the morphology of cilia and expression of the dynein intermediate chain 2 (DNAI2) in the oviduct of non-obese diabetic (NOD) mice. Results obtained with immunohistochemistry showed that DNAI2 expression was reduced in oviducts of diabetic NOD (dNOD) mice, as compared to that observed in the normoglycemic NOD (cNOD) group, especially in the acyclic dNOD mice. Oviductal cilia of dNOD mice appeared to be reduced in number. Results obtained with Western blot analysis revealed that the expression of DNAI2 protein was significantly less in oviducts of dNOD mice as compared to that of cNOD mice corroborating the results obtained with immunohistochemistry. Electron microscopic examination and quantitative imaging of thin sections of Epon-embedded oviducts of both dNOD and cNOD mice confirmed the reduction of the number of cilia in the oviduct of the dNOD group which also displayed aberrant axonemal ultrastructure, including disorganization of the axoneme and alteration of microtubule doublets into singlets as well as disruption of the plasma membrane in many cilia. Taken together, the present findings suggest that structural alterations of oviductal cilia in female diabetic NOD mice might be detrimental to the normal function of these particular cell structures in gamete transport.


Assuntos
Dineínas do Axonema/biossíntese , Cílios/metabolismo , Diabetes Mellitus/genética , Tubas Uterinas/metabolismo , Animais , Axonema/metabolismo , Axonema/patologia , Axonema/ultraestrutura , Cílios/patologia , Cílios/ultraestrutura , Diabetes Mellitus/patologia , Modelos Animais de Doenças , Tubas Uterinas/patologia , Tubas Uterinas/ultraestrutura , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD/genética , Microscopia Eletrônica
12.
PLoS One ; 11(11): e0166397, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27846257

RESUMO

PURPOSE: Mutations in the EYS gene are a common cause of autosomal recessive retinitis pigmentosa (arRP), yet the role of the EYS protein in humans is presently unclear. The aim of this study was to investigate the isoform structure, expression and potential function of EYS in the mammalian retina in order to better understand its involvement in the pathogenesis of arRP. METHODS: To achieve the objective, we examined the expression of mRNA transcripts of EYS isoforms in human tissues and cell lines by RT-PCR. We also investigated the localisation of EYS in cultured cells and retinal cryo-sections by confocal fluorescence microscopy and Western blot analysis. RESULTS: RT-PCR analysis confirmed that EYS has at least four isoforms. In addition to the previously reported EYS isoforms 1 and 4, we present the experimental validation of two smaller variants referred to as EYS isoforms 2 and 3. All four isoforms are expressed in the human retina and Y79 cells and the short variants were additionally detected in the testis. Immunofluorescent confocal microscopy and Western blot analysis revealed that all EYS isoforms preferentially localise to the cytoplasm of Y79 and HeLa cells. Moreover, an enrichment of the endogenous protein was observed near the centrosomes in Y79 cells. Interestingly, EYS was observed at the ciliary axoneme in Y79 ciliated cells. In macaque retinal cryosections, EYS was found to localise in the region of the photoreceptor ciliary axoneme in both rods and cones as well as in the cytoplasm of the ganglion cells. CONCLUSION: The results obtained in this study lead us to speculate that, in photoreceptor cells, EYS could be a protein involved in maintaining the stability of the ciliary axoneme in both rods and cones. The variability of its isoform structure suggests that other roles are also possible and yet to be established.


Assuntos
Proteínas do Olho/genética , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Retinite Pigmentosa/genética , Animais , Axonema/genética , Axonema/metabolismo , Axonema/patologia , Cílios/genética , Cílios/metabolismo , Cílios/patologia , Citoplasma/genética , Citoplasma/metabolismo , Análise Mutacional de DNA , Eletrorretinografia , Proteínas do Olho/metabolismo , Células HeLa , Humanos , Indóis , Macaca/genética , Macaca/metabolismo , Mutação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Retina/metabolismo , Retina/patologia , Células Fotorreceptoras Retinianas Cones/patologia , Células Fotorreceptoras Retinianas Bastonetes/patologia , Retinite Pigmentosa/metabolismo , Retinite Pigmentosa/patologia
13.
Am J Hum Genet ; 99(2): 460-9, 2016 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-27486780

RESUMO

Multiprotein complexes referred to as outer dynein arms (ODAs) develop the main mechanical force to generate the ciliary and flagellar beat. ODA defects are the most common cause of primary ciliary dyskinesia (PCD), a congenital disorder of ciliary beating, characterized by recurrent infections of the upper and lower airways, as well as by progressive lung failure and randomization of left-right body asymmetry. Using a whole-exome sequencing approach, we identified recessive loss-of-function mutations within TTC25 in three individuals from two unrelated families affected by PCD. Mice generated by CRISPR/Cas9 technology and carrying a deletion of exons 2 and 3 in Ttc25 presented with laterality defects. Consistently, we observed immotile nodal cilia and missing leftward flow via particle image velocimetry. Furthermore, transmission electron microscopy (TEM) analysis in TTC25-deficient mice revealed an absence of ODAs. Consistent with our findings in mice, we were able to show loss of the ciliary ODAs in humans via TEM and immunofluorescence (IF) analyses. Additionally, IF analyses revealed an absence of the ODA docking complex (ODA-DC), along with its known components CCDC114, CCDC151, and ARMC4. Co-immunoprecipitation revealed interaction between the ODA-DC component CCDC114 and TTC25. Thus, here we report TTC25 as a new member of the ODA-DC machinery in humans and mice.


Assuntos
Axonema/genética , Axonema/metabolismo , Proteínas de Transporte/genética , Cílios/patologia , Dineínas/química , Dineínas/metabolismo , Síndrome de Kartagener/genética , Síndrome de Kartagener/patologia , Mutação , Animais , Axonema/patologia , Axonema/ultraestrutura , Cílios/metabolismo , Cílios/ultraestrutura , Dineínas/genética , Dineínas/ultraestrutura , Exoma/genética , Éxons/genética , Imunofluorescência , Genes Recessivos , Humanos , Camundongos , Microscopia Eletrônica de Transmissão , Ligação Proteica , Xenopus , Proteínas de Xenopus/deficiência , Proteínas de Xenopus/genética
14.
Genes Cells ; 21(7): 728-39, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27353389

RESUMO

Lrrc6 encodes a cytoplasmic protein that is expressed specifically in cells with motile cilia including the node, trachea and testes of the mice. A mutation of Lrrc6 has been identified in human patients with primary ciliary dyskinesia (PCD). Mutant mice lacking Lrrc6 show typical PCD defects such as hydrocephalus and laterality defects. We found that in the absence of Lrrc6, the morphology of motile cilia remained normal, but their motility was completely lost. The 9 + 2 arrangement of microtubules remained normal in Lrrc6(-/-) mice, but the outer dynein arms (ODAs), the structures essential for the ciliary beating, were absent from the cilia. In the absence of Lrrc6, ODA proteins such as DNAH5, DNAH9 and IC2, which are assembled in the cytoplasm and transported to the ciliary axoneme, remained in the cytoplasm and were not transported to the ciliary axoneme. The IC2-IC1 interaction, which is the first step of ODA assembly, was normal in Lrrc6(-/-) mice testes. Our results suggest that ODA proteins may be transported from the cytoplasm to the cilia by an Lrrc6-dependent mechanism.


Assuntos
Cílios/genética , Síndrome de Kartagener/genética , Proteínas/genética , Animais , Dineínas do Axonema/genética , Axonema/genética , Axonema/patologia , Cílios/patologia , Citoplasma/genética , Citoplasma/metabolismo , Proteínas do Citoesqueleto , Modelos Animais de Doenças , Dineínas/genética , Humanos , Síndrome de Kartagener/patologia , Camundongos , Camundongos Transgênicos , Mutação
15.
Hum Mutat ; 37(8): 776-85, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27120127

RESUMO

Primary ciliary dyskinesia (PCD) is an autosomal recessive disease characterized by chronic respiratory infections of the upper and lower airways, hypofertility, and, in approximately half of the cases, situs inversus. This complex phenotype results from defects in motile cilia and sperm flagella. Among the numerous genes involved in PCD, very few-including CCDC39 and CCDC40-carry mutations that lead to a disorganization of ciliary axonemes with microtubule misalignment. Focusing on this particular phenotype, we identified bi-allelic loss-of-function mutations in GAS8, a gene that encodes a subunit of the nexin-dynein regulatory complex (N-DRC) orthologous to DRC4 of the flagellated alga Chlamydomonas reinhardtii. Unlike the majority of PCD patients, individuals with GAS8 mutations have motile cilia, which, as documented by high-speed videomicroscopy, display a subtle beating pattern defect characterized by slightly reduced bending amplitude. Immunofluorescence studies performed on patients' respiratory cilia revealed that GAS8 is not required for the proper expression of CCDC39 and CCDC40. Rather, mutations in GAS8 affect the subcellular localization of another N-DRC subunit called DRC3. Overall, this study, which identifies GAS8 as a PCD gene, unveils the key importance of the corresponding protein in N-DRC integrity and in the proper alignment of axonemal microtubules in humans.


Assuntos
Axonema/patologia , Proteínas do Citoesqueleto/genética , Síndrome de Kartagener/genética , Mutação , Proteínas de Neoplasias/genética , Adulto , Criança , Proteínas do Citoesqueleto/metabolismo , Feminino , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Síndrome de Kartagener/metabolismo , Síndrome de Kartagener/patologia , Masculino , Proteínas de Neoplasias/metabolismo , Análise de Sequência de DNA
16.
J Biol Chem ; 291(13): 6923-35, 2016 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-26846852

RESUMO

CUL4B ubiquitin ligase belongs to the cullin-RING ubiquitin ligase family. Although sharing many sequence and structural similarities, CUL4B plays distinct roles in spermatogenesis from its homologous protein CUL4A. We previously reported that genetic ablation ofCul4ain mice led to male infertility because of aberrant meiotic progression. In the present study, we generated Cul4bgerm cell-specific conditional knock-out (Cul4b(Vasa)),as well asCul4bglobal knock-out (Cul4b(Sox2)) mouse, to investigate its roles in spermatogenesis. Germ cell-specific deletion of Cul4bled to male infertility, despite normal testicular morphology and comparable numbers of spermatozoa. Notably, significantly impaired sperm mobility caused by reduced mitochondrial activity and glycolysis level were observed in the majority of the mutant spermatozoa, manifested by low, if any, sperm ATP production. Furthermore,Cul4b(Vasa)spermatozoa exhibited defective arrangement of axonemal microtubules and flagella outer dense fibers. Our mass spectrometry analysis identified INSL6 as a novel CUL4B substrate in male germ cells, evidenced by its direct polyubiquination and degradation by CUL4B E3 ligase. Nevertheless,Cul4bglobal knock-out males lost their germ cells in an age-dependent manner, implying failure of maintaining the spermatogonial stem cell niche in somatic cells. Taken together, our results show that CUL4B is indispensable to spermatogenesis, and it functions cell autonomously in male germ cells to ensure spermatozoa motility, whereas it functions non-cell-autonomously in somatic cells to maintain spermatogonial stemness. Thus, CUL4B links two distinct spermatogenetic processes to a single E3 ligase, highlighting the significance of ubiquitin modification during spermatogenesis.


Assuntos
Proteínas Culina/genética , Infertilidade Masculina/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Espermatogênese/genética , Espermatozoides/metabolismo , Ubiquitina-Proteína Ligases/genética , Trifosfato de Adenosina/biossíntese , Animais , Axonema/metabolismo , Axonema/patologia , Proteínas Culina/metabolismo , Deleção de Genes , Regulação da Expressão Gênica , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Camundongos , Camundongos Knockout , Microtúbulos/metabolismo , Microtúbulos/patologia , Proteólise , Transdução de Sinais , Contagem de Espermatozoides , Motilidade Espermática , Espermatozoides/patologia , Nicho de Células-Tronco/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
17.
J Assist Reprod Genet ; 33(2): 141-56, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26825807

RESUMO

This review article provides a critical analysis of the structure and molecular mechanisms of the microtubule axoneme of cilia and sperm flagella and their associated elements required for male fertility.A broad range of genetic and molecular defects (ciliopathies) are considered in the context of human diseases involving impaired motility in cilia and sperm flagella, providing provocative thought for future research in the area of male infertility.


Assuntos
Infertilidade Masculina/patologia , Técnicas de Reprodução Assistida , Espermatozoides/ultraestrutura , Axonema/patologia , Axonema/ultraestrutura , Cílios , Flagelos/patologia , Flagelos/ultraestrutura , Humanos , Infertilidade Masculina/genética , Masculino , Espermatozoides/patologia
18.
Andrologia ; 47(2): 214-20, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24611953

RESUMO

Although electron microscopy provides a detailed analysis of ultrastructural abnormalities, this technique is not available in all laboratories. We sought to determine whether certain characteristics of the flagellum as assessed by light microscopy were related to axonemal abnormalities. Forty-one patients with an absence of outer dynein arms (type I), a lack of a central complex (type III) and an absence of peripheral doublets (type IV) were studied. Sperm morphology was scored according to David's modified classification. Flagella with an irregular thickness were classified as being of normal length, short or broken. There were correlations between missing outer dynein arms and abnormal, short or coiled flagellum. Type III patients showed the highest flagellar defects (a short (P = 0.0027) or an absent flagellum (P = 0.011)). Just over 68% of the irregular flagella were short in Type III patients, whereas this value was only 34.5% in type I and 26.4% in type IV (P = 0.002). There was a negative correlation between misassembly and spermatozoa of irregular flagella (r = -0.79; P = 0.019). It is concluded that light microscopy analysis of flagellum abnormalities may help provide a correct diagnosis, identify sperm abnormalities with fertility potentials and outcomes in assisted reproduction technologies and assess the genetic risk.


Assuntos
Axonema/patologia , Infertilidade Masculina/patologia , Microscopia/métodos , Cauda do Espermatozoide/patologia , Adulto , Axonema/ultraestrutura , Dineínas/ultraestrutura , Humanos , Infertilidade Masculina/classificação , Infertilidade Masculina/diagnóstico , Masculino , Microscopia Eletrônica , Microtúbulos/patologia , Microtúbulos/ultraestrutura , Pessoa de Meia-Idade , Análise do Sêmen , Cauda do Espermatozoide/ultraestrutura
19.
Am J Hum Genet ; 94(1): 95-104, 2014 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-24360805

RESUMO

Ten to fifteen percent of couples are confronted with infertility and a male factor is involved in approximately half the cases. A genetic etiology is likely in most cases yet only few genes have been formally correlated with male infertility. Homozygosity mapping was carried out on a cohort of 20 North African individuals, including 18 index cases, presenting with primary infertility resulting from impaired sperm motility caused by a mosaic of multiple morphological abnormalities of the flagella (MMAF) including absent, short, coiled, bent, and irregular flagella. Five unrelated subjects out of 18 (28%) carried a homozygous variant in DNAH1, which encodes an inner dynein heavy chain and is expressed in testis. RT-PCR, immunostaining, and electronic microscopy were carried out on samples from one of the subjects with a mutation located on a donor splice site. Neither the transcript nor the protein was observed in this individual, confirming the pathogenicity of this variant. A general axonemal disorganization including mislocalization of the microtubule doublets and loss of the inner dynein arms was observed. Although DNAH1 is also expressed in other ciliated cells, infertility was the only symptom of primary ciliary dyskinesia observed in affected subjects, suggesting that DNAH1 function in cilium is not as critical as in sperm flagellum.


Assuntos
Dineínas do Axonema/genética , Infertilidade Masculina/genética , Mutação , Cauda do Espermatozoide/patologia , Axonema/genética , Axonema/patologia , Cílios/genética , Cílios/patologia , Flagelos/patologia , Variação Genética , Homozigoto , Humanos , Síndrome de Kartagener/genética , Masculino , Sítios de Splice de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Motilidade Espermática , Testículo/citologia , Testículo/patologia
20.
Am J Hum Genet ; 93(2): 346-56, 2013 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-23891471

RESUMO

Primary ciliary dyskinesia (PCD) is a ciliopathy characterized by airway disease, infertility, and laterality defects, often caused by dual loss of the inner dynein arms (IDAs) and outer dynein arms (ODAs), which power cilia and flagella beating. Using whole-exome and candidate-gene Sanger resequencing in PCD-affected families afflicted with combined IDA and ODA defects, we found that 6/38 (16%) carried biallelic mutations in the conserved zinc-finger gene BLU (ZMYND10). ZMYND10 mutations conferred dynein-arm loss seen at the ultrastructural and immunofluorescence level and complete cilia immotility, except in hypomorphic p.Val16Gly (c.47T>G) homozygote individuals, whose cilia retained a stiff and slowed beat. In mice, Zmynd10 mRNA is restricted to regions containing motile cilia. In a Drosophila model of PCD, Zmynd10 is exclusively expressed in cells with motile cilia: chordotonal sensory neurons and sperm. In these cells, P-element-mediated gene silencing caused IDA and ODA defects, proprioception deficits, and sterility due to immotile sperm. Drosophila Zmynd10 with an equivalent c.47T>G (p.Val16Gly) missense change rescued mutant male sterility less than the wild-type did. Tagged Drosophila ZMYND10 is localized primarily to the cytoplasm, and human ZMYND10 interacts with LRRC6, another cytoplasmically localized protein altered in PCD. Using a fly model of PCD, we conclude that ZMYND10 is a cytoplasmic protein required for IDA and ODA assembly and that its variants cause ciliary dysmotility and PCD with laterality defects.


Assuntos
Cílios/genética , Dineínas/genética , Infertilidade Masculina/genética , Síndrome de Kartagener/genética , Proteínas/genética , Sistema Respiratório/metabolismo , Proteínas Supressoras de Tumor/genética , Animais , Axonema/genética , Axonema/metabolismo , Axonema/patologia , Cílios/metabolismo , Cílios/patologia , Proteínas do Citoesqueleto , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Dineínas/metabolismo , Exoma , Feminino , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Síndrome de Kartagener/metabolismo , Síndrome de Kartagener/patologia , Masculino , Camundongos , Mutação , Linhagem , Estrutura Terciária de Proteína , Proteínas/metabolismo , Sistema Respiratório/patologia , Proteínas Supressoras de Tumor/metabolismo
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