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1.
Nat Commun ; 15(1): 5715, 2024 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-38977659

RESUMO

Mitochondria are maternally inherited, but the mechanisms underlying paternal mitochondrial elimination after fertilization are far less clear. Using Drosophila, we show that special egg-derived multivesicular body vesicles promote paternal mitochondrial elimination by activating an LC3-associated phagocytosis-like pathway, a cellular defense pathway commonly employed against invading microbes. Upon fertilization, these egg-derived vesicles form extended vesicular sheaths around the sperm flagellum, promoting degradation of the sperm mitochondrial derivative and plasma membrane. LC3-associated phagocytosis cascade of events, including recruitment of a Rubicon-based class III PI(3)K complex to the flagellum vesicular sheaths, its activation, and consequent recruitment of Atg8/LC3, are all required for paternal mitochondrial elimination. Finally, lysosomes fuse with strings of large vesicles derived from the flagellum vesicular sheaths and contain degrading fragments of the paternal mitochondrial derivative. Given reports showing that in some mammals, the paternal mitochondria are also decorated with Atg8/LC3 and surrounded by multivesicular bodies upon fertilization, our findings suggest that a similar pathway also mediates paternal mitochondrial elimination in other flagellated sperm-producing organisms.


Assuntos
Proteínas de Drosophila , Fertilização , Mitocôndrias , Corpos Multivesiculares , Fagocitose , Espermatozoides , Animais , Mitocôndrias/metabolismo , Masculino , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Feminino , Espermatozoides/metabolismo , Corpos Multivesiculares/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Óvulo/metabolismo , Lisossomos/metabolismo , Cauda do Espermatozoide/metabolismo , Mitofagia
2.
Mol Hum Reprod ; 30(7)2024 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-38870534

RESUMO

Acephalic spermatozoa syndrome (ASS) is a severe teratospermia with decaudated, decapitated, and malformed sperm, resulting in male infertility. Nuclear envelope protein SUN5 localizes to the junction between the sperm head and tail. Mutations in the SUN5 gene have been identified most frequently (33-47%) in ASS cases, and its molecular mechanism of action is yet to be explored. In the present study, we generated Sun5 knockout mice, which presented the phenotype of ASS. Nuclear membrane protein LaminB1 and cytoskeletal GTPases Septin12 and Septin2 were identified as potential partners for interacting with SUN5 by immunoprecipitation-mass spectrometry in mouse testis. Further studies demonstrated that SUN5 connected the nucleus by interacting with LaminB1 and connected the proximal centriole by interacting with Septin12. The binding between SUN5 and Septin12 promoted their aggregation together in the sperm neck. The disruption of the LaminB1/SUN5/Septin12 complex by Sun5 deficiency caused separation of the Septin12-proximal centriole from the nucleus, leading to the breakage of the head-to-tail junction. Collectively, these data provide new insights into the pathogenesis of ASS caused by SUN5 deficiency.


Assuntos
Proteínas de Membrana , Camundongos Knockout , Membrana Nuclear , Septinas , Cabeça do Espermatozoide , Cauda do Espermatozoide , Masculino , Septinas/metabolismo , Septinas/genética , Animais , Camundongos , Cabeça do Espermatozoide/metabolismo , Cabeça do Espermatozoide/patologia , Membrana Nuclear/metabolismo , Cauda do Espermatozoide/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Lamina Tipo B/metabolismo , Lamina Tipo B/genética , Teratozoospermia/metabolismo , Teratozoospermia/genética , Infertilidade Masculina/metabolismo , Infertilidade Masculina/genética , Espermatozoides/metabolismo , Humanos
3.
Cell Mol Biol Lett ; 29(1): 74, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38750428

RESUMO

By analyzing a mouse Interspecific Recombinant Congenic Strain (IRCS), we previously identified a quantitative trait locus (QTL), called Mafq1 on mouse chromosome 1, that is associated with male hypofertility and ultrastructural sperm abnormalities. Within this locus, we identified a new candidate gene that could be implicated in a reproductive phenotype: Tex44 (Testis-expressed protein 44). We thus performed a CRISPR/Cas9-mediated complete deletion of this gene in mice in order to study its function. Tex44-KO males were severely hypofertile in vivo and in vitro due to a drastic reduction of sperm motility which itself resulted from important morphological sperm abnormalities. Namely, Tex44-KO sperm showed a disorganized junction between the midpiece and the principal piece of the flagellum, leading to a 180° flagellar bending in this region. In addition, the loss of some axonemal microtubule doublets and outer dense fibers in the flagellum's principal piece has been observed. Our results suggest that, in mice, TEX44 is implicated in the correct set-up of the sperm flagellum during spermiogenesis and its absence leads to flagellar abnormalities and consequently to severe male hypofertility.


Assuntos
Infertilidade Masculina , Camundongos Knockout , Motilidade dos Espermatozoides , Cauda do Espermatozoide , Animais , Masculino , Infertilidade Masculina/genética , Infertilidade Masculina/patologia , Motilidade dos Espermatozoides/genética , Cauda do Espermatozoide/patologia , Cauda do Espermatozoide/metabolismo , Camundongos , Espermatozoides/metabolismo , Espermatogênese/genética , Flagelos/genética , Flagelos/metabolismo , Camundongos Endogâmicos C57BL , Sistemas CRISPR-Cas/genética
4.
Front Endocrinol (Lausanne) ; 15: 1377780, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38745955

RESUMO

Objective: Multiple morphological abnormalities of the sperm flagella (MMAF) is characterized by abnormal flagellar phenotypes, which is a particular kind of asthenoteratozoospermia. Previous studies have reported a comparable intracytoplasmic sperm injection (ICSI) outcome in terms of fertilization rate and clinical pregnancy rate in patients with MMAF compared with those with no MMAF; however, others have conflicting opinions. Assisted reproductive technology (ART) outcomes in individuals with MMAF are still controversial and open to debate. Methods: A total of 38 patients with MMAF treated at an academic reproductive center between January 2014 and July 2022 were evaluated in the current retrospective cohort study and followed up until January 2023. Propensity score matching was used to adjust for the baseline clinical characteristics of the patients and to create a comparable control group. The genetic pathogenesis of MMAF was confirmed by whole exome sequencing. The main outcomes were the embryo developmental potential, the cumulative pregnancy rate (CLPR), and the cumulative live birth rate (CLBR). Results: Pathogenic variants in known genes of DNAH1, DNAH11, CFAP43, FSIP2, and SPEF2 were identified in patients with MMAF. Laboratory outcomes, including the fertilization rate, 2PN cleavage rate, blastocyst formation rate, and available blastocyst rate, followed a trend of decline in the MMAF group (p < 0.05). Moreover, according to the embryo transfer times and complete cycles, the CLPR in the cohort of MMAF was lower compared with the oligoasthenospermia pool (p = 0.033 and p = 0.020, respectively), while no statistical differences were observed in the neonatal outcomes. Conclusion: The current study presented decreased embryo developmental potential and compromised clinical outcomes in the MMAF cohort. These findings may provide clinicians with evidence to support genetic counseling and clinical guidance in specific patients with MMAF.


Assuntos
Desenvolvimento Embrionário , Taxa de Gravidez , Injeções de Esperma Intracitoplásmicas , Cauda do Espermatozoide , Humanos , Masculino , Feminino , Gravidez , Adulto , Estudos Retrospectivos , Cauda do Espermatozoide/patologia , Desenvolvimento Embrionário/fisiologia , Astenozoospermia/genética , Astenozoospermia/patologia , Infertilidade Masculina/genética , Infertilidade Masculina/patologia , Espermatozoides/patologia
5.
J Assist Reprod Genet ; 41(6): 1499-1505, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38775994

RESUMO

In this study, we investigated the role of a newly identified homozygous variant (c.1245 + 6T > C) in the CFAP61 gene in the development of multiple morphologically abnormal flagella (MMAF) in an infertile patient. Using exome sequencing, we identified this variant, which led to exon 12 skipping and the production of a truncated CFAP61 protein. Transmission electron microscopy analysis of the patient's spermatozoa revealed various flagellar abnormalities, including defective nuclear chromatin condensation, axoneme disorganization, and mitochondria embedded in residual cytoplasmic droplets. Despite a fertilization rate of 83.3% through ICSI, there was no successful pregnancy due to poor embryo quality.Our findings suggest a link between the identified CFAP61 variant and MMAF, indicating potential disruption in radial spokes' assembly or function crucial for normal ciliary motility. Furthermore, nearly half of the observed sperm heads displayed chromatin condensation defects, possibly contributing to the low blastulation rate. This case underscores the significance of genetic counseling and testing, particularly for couples dealing with infertility and MMAF. Early identification of such genetic variants can guide appropriate interventions and improve reproductive outcomes.


Assuntos
Homozigoto , Infertilidade Masculina , Humanos , Masculino , Infertilidade Masculina/genética , Infertilidade Masculina/patologia , Feminino , Adulto , Espermatozoides/patologia , Espermatozoides/ultraestrutura , Cauda do Espermatozoide/patologia , Cauda do Espermatozoide/ultraestrutura , Gravidez , Flagelos/genética , Flagelos/ultraestrutura , Injeções de Esperma Intracitoplásmicas , Sequenciamento do Exoma , Splicing de RNA/genética
6.
J Assist Reprod Genet ; 41(6): 1481-1484, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38676841

RESUMO

In a recent journal article, Chen et al. identified a germ cell-specific cofactor, STYXL1, associated with male fertility function. Deletion of STYXL1 prevents the LEGO player CCT complex from properly folding key microtubule proteins of the sperm flagellum, which affects sperm motility and male fertility function.


Assuntos
Infertilidade Masculina , Motilidade dos Espermatozoides , Masculino , Humanos , Infertilidade Masculina/genética , Infertilidade Masculina/patologia , Motilidade dos Espermatozoides/genética , Espermatozoides , Cauda do Espermatozoide/patologia , Cauda do Espermatozoide/metabolismo , Microtúbulos/genética , Microtúbulos/metabolismo
7.
Life Sci Alliance ; 7(6)2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38570187

RESUMO

The transition zone is a specialised gate at the base of cilia/flagella, which separates the ciliary compartment from the cytoplasm and strictly regulates protein entry. We identified a potential new regulator of the male germ cell transition zone, CEP76. We demonstrated that CEP76 was involved in the selective entry and incorporation of key proteins required for sperm function and fertility into the ciliary compartment and ultimately the sperm tail. In the mutant, sperm tails were shorter and immotile as a consequence of deficits in essential sperm motility proteins including DNAH2 and AKAP4, which accumulated at the sperm neck in the mutant. Severe annulus, fibrous sheath, and outer dense fibre abnormalities were also detected in sperm lacking CEP76. Finally, we identified that CEP76 dictates annulus positioning and structure. This study suggests CEP76 as a male germ cell transition zone protein and adds further evidence to the hypothesis that the spermatid transition zone and annulus are part of the same functional structure.


Assuntos
Infertilidade Masculina , Cauda do Espermatozoide , Humanos , Masculino , Cauda do Espermatozoide/metabolismo , Motilidade dos Espermatozoides/genética , Sêmen , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Mutação/genética
8.
Reprod Biomed Online ; 48(5): 103765, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38492416

RESUMO

RESEARCH QUESTION: Is the novel homozygous nonsense variant of AK7 associated with multiple morphological abnormalities of the sperm flagella (MMAF), a specific type of oligoasthenoteratozoospermia leading to male infertility? DESIGN: Whole-exome sequencing and Sanger sequencing were performed to identify potential gene variants. Immunoblotting and immunofluorescence were applied to confirm the relationship between mutated genes and disease phenotypes. The concentration of reactive oxygen species and the rate of apoptosis were measured to evaluate the mitochondrial function of spermatozoa. Transmission electron microscopy and scanning electron microscopy were employed to observe sperm ultrastructure. RESULTS: A novel homozygous nonsense variant of AK7, c.1153A>T (p. Lys385*), was identified in two infertile siblings with asthenoteratozoospermia through whole-exome sequencing. Both immunoblotting and immunofluorescence assays showed practically complete absence of AK7 in the patient's spermatozoa. Additionally, the individual with the novel AK7 variant exhibited a phenotype characterized by severe oxidative stress and apoptosis caused by mitochondrial metabolic dysfunction of spermatozoa. Notably, remarkable flagellar defects with multiple axonemes in uniflagellate spermatozoa, accompanied by mitochondrial vacuolization, were observed; this has not been reported previously in patients with other AK7 variants. CONCLUSIONS: This study found that a novel identified homozygous nonsense variant of AK7 may be associated with MMAF-related asthenoteratozoospermia. The observed functional associations between mitochondria and sperm flagellar assembly provide evidence for potential mutual regulation between AK7 and flagella-associated proteins during spermatogenesis.


Assuntos
Códon sem Sentido , Homozigoto , Cauda do Espermatozoide , Humanos , Masculino , Cauda do Espermatozoide/patologia , Cauda do Espermatozoide/ultraestrutura , Infertilidade Masculina/genética , Infertilidade Masculina/patologia , Astenozoospermia/genética , Astenozoospermia/patologia , Adulto , Espermatozoides/ultraestrutura , Espermatozoides/anormalidades , Sequenciamento do Exoma , Mitocôndrias/ultraestrutura , Mitocôndrias/genética , Mitocôndrias/patologia , Linhagem
9.
J Assist Reprod Genet ; 41(5): 1297-1306, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38492154

RESUMO

PURPOSE: To identify the genetic causes of multiple morphological abnormalities in sperm flagella (MMAF) and male infertility in patients from two unrelated Han Chinese families. METHODS: Whole-exome sequencing was conducted using blood samples from the two individuals with MMAF and male infertility. Hematoxylin and eosin staining and scanning electron microscopy were performed to evaluate sperm morphology. Ultrastructural and immunostaining analyses of the spermatozoa were performed. The HEK293T cells were used to confirm the pathogenicity of the variants. RESULTS: We identified two novel homozygous missense ARMC2 variants: c.314C > T: p.P105L and c.2227A > G: p.N743D. Both variants are absent or rare in the human population genome data and are predicted to be deleterious. In vitro experiments indicated that both ARMC2 variants caused a slightly increased protein expression. ARMC2-mutant spermatozoa showed multiple morphological abnormalities (bent, short, coiled, absent, and irregular) in the flagella. In addition, the spermatozoa of the patients revealed a frequent absence of the central pair complex and disrupted axonemal ultrastructure. CONCLUSION: We identified two novel ARMC2 variants that caused male infertility and MMAF in Han Chinese patients. These findings expand the mutational spectrum of ARMC2 and provide insights into the complex causes and pathogenesis of MMAF.


Assuntos
Astenozoospermia , Sequenciamento do Exoma , Homozigoto , Infertilidade Masculina , Cauda do Espermatozoide , Espermatozoides , Humanos , Masculino , Cauda do Espermatozoide/patologia , Cauda do Espermatozoide/ultraestrutura , Cauda do Espermatozoide/metabolismo , Infertilidade Masculina/genética , Infertilidade Masculina/patologia , Astenozoospermia/genética , Astenozoospermia/patologia , Adulto , Espermatozoides/patologia , Espermatozoides/ultraestrutura , Mutação/genética , Linhagem , Células HEK293 , Povo Asiático/genética
10.
Hum Reprod ; 39(4): 674-688, 2024 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-38366201

RESUMO

STUDY QUESTION: Whether and how do Na+/H+ exchangers (NHEs) regulate the physiological functions of human sperm? SUMMARY ANSWER: NHE-mediated flagellar intracellular pH (pHi) homeostasis facilitates the activation of the pH-sensitive, sperm-specific Ca2+ channel (CatSper) and the sperm-specific K+ channel (KSper), which subsequently modulate sperm motility, hyperactivation, flagellar tyrosine phosphorylation, and the progesterone (P4)-induced acrosome reaction. WHAT IS KNOWN ALREADY: Sperm pHi alkalization is an essential prerequisite for the acquisition of sperm-fertilizing capacity. Different sperm functions are strictly controlled by particular pHi regulatory mechanisms. NHEs are suggested to modulate sperm H+ efflux. STUDY DESIGN, SIZE, DURATION: This was a laboratory study that used samples from >50 sperm donors over a period of 1 year. To evaluate NHE action on human sperm function, 5-(N,N-dimethyl)-amiloride (DMA), a highly selective inhibitor of NHEs, was utilized. All experiments were repeated at least five times using different individual sperm samples or cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: By utilizing the pH fluorescent indicator pHrodo Red-AM, we detected alterations in single-cell pHi value in human sperm. The currents of CatSper and KSper in human sperm were recorded by the whole-cell patch-clamp technique. Changes in population and single-cell Ca2+ concentrations ([Ca2+]i) of human sperm loaded with Fluo 4-AM were measured. Membrane potential (Vm) and population pHi were quantitatively examined by a multimode plate reader after sperm were loaded with 3,3'-dipropylthiadicarbocyanine iodide and 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester, respectively. Sperm motility parameters were assessed by a computer-assisted semen analysis system. Tyrosine phosphorylation was determined by immunofluorescence, and sperm acrosome reaction was evaluated by Pisum sativum agglutinin-FITC staining. MAIN RESULTS AND THE ROLE OF CHANCE: DMA-induced NHEs inhibition severely acidified the human sperm flagellar pHi from 7.20 ± 0.04 to 6.38 ± 0.12 (mean ± SEM), while the effect of DMA on acrosomal pHi was less obvious (from 5.90 ± 0.13 to 5.57 ± 0.12, mean ± SEM). The whole-cell patch-clamp recordings revealed that NHE inhibition remarkably suppressed alkalization-induced activation of CatSper and KSper. As a consequence, impairment of [Ca2+]i homeostasis and Vm maintenance were detected in the presence of DMA. During the capacitation process, pre-treatment with DMA for 2 h potently decreased sperm pHi, which in turn decreased sperm motility and kinetic parameters. Sperm capacitation-associated functions, including hyperactivation, tyrosine phosphorylation, and P4-induced acrosome reaction, were also compromised by NHE inhibition. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This was an in vitro study. Caution should be taken when extrapolating these results to in vivo applications. WIDER IMPLICATIONS OF THE FINDINGS: This study revealed that NHEs are important physiological regulators for human CatSper and KSper, which are indispensable for human sperm fertility, suggesting that malfunction of NHEs could be an underlying mechanism for the pathogenesis of male infertility. FUNDING/COMPETING INTEREST(S): This work was supported by the National Natural Science Foundation of China (32271167 and 81871202 to X.Z.), Jiangsu Innovation and Entrepreneurship Talent Plan (JSSCRC20211543 to X.Z.), the Social Development Project of Jiangsu Province (No. BE2022765 to X.Z.), the Society and livelihood Project of Nantong City (No. MS22022087 to X.Z.), and the Natural Science Foundation of Jiangsu Province (BK20220608 to H.K.). The authors have no competing interests to declare.


Assuntos
Canais de Cálcio , Sêmen , Trocadores de Sódio-Hidrogênio , Humanos , Masculino , Equilíbrio Ácido-Base , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Sêmen/metabolismo , Motilidade dos Espermatozoides , Espermatozoides/metabolismo , Tirosina/metabolismo , Tirosina/farmacologia , Cauda do Espermatozoide/metabolismo , Cauda do Espermatozoide/fisiologia , Trocadores de Sódio-Hidrogênio/metabolismo
11.
Reproduction ; 167(2)2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38271797

RESUMO

After fertilization, the human sperm tail lost its fibrous sheath at the pronuclear stage to become coiled and then attached to one of the first mitotic spindle poles. The tip of the sperm tail was branched and its degree did not change from the pronuclear stage to the second mitosis.


Assuntos
Fertilização , Cauda do Espermatozoide , Humanos , Masculino , Sêmen , Espermatozoides , Mitose
12.
Biol Reprod ; 110(5): 877-894, 2024 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-38236177

RESUMO

The centrosome is critical for maintaining the sperm head-tail connection and the formation of flagellar microtubules. In this study, we found that in mouse testes, CCDC159 (coiled-coil domain-containing protein 159) is specifically localized to the head-tail coupling apparatus (HTCA) of spermatids, a structure that ensures sperm head-tail tight conjunction. CCDC159 contains a C-terminal coiled-coil domain that functions as the centrosomal localization signal. Gene knockout (KO) of Ccdc159 in mice resulted in acephalic spermatozoa, abnormal flagella, and male infertility. To explore the mechanism behind CCDC159 regulating spermatogenesis, we identified CCDC159-binding proteins using a yeast two-hybrid screen and speculated that CCDC159 participates in HTCA assembly by regulating protein phosphatase PP1 activity. Further RNA-sequencing analyses of Ccdc159 KO testes revealed numerous genes involved in male gamete generation that were downregulated. Together, our results show that CCDC159 in spermatids is a novel centrosomal protein anchoring the sperm head to the tail. Considering the limitation of KO mouse model in clarifying the biological function of CCDC159 in spermatogenesis, a gene-rescue experiment will be performed in the future.


Assuntos
Camundongos Knockout , Cabeça do Espermatozoide , Cauda do Espermatozoide , Espermátides , Espermatogênese , Animais , Masculino , Camundongos , Espermátides/metabolismo , Cauda do Espermatozoide/metabolismo , Espermatogênese/fisiologia , Cabeça do Espermatozoide/metabolismo , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Testículo/metabolismo , Centrossomo/metabolismo
13.
J Cell Biol ; 223(3)2024 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-38197861

RESUMO

Proper compartmentalization of the sperm flagellum is essential for fertility. The annulus is a septin-based ring that demarcates the midpiece (MP) and the principal piece (PP). It is assembled at the flagellar base, migrates caudally, and halts upon arriving at the PP. However, the mechanisms governing annulus positioning remain unknown. We report that a Chibby3 (Cby3)/Cby1-interacting BAR domain-containing 1 (ciBAR1) complex is required for this process. Ablation of either gene in mice results in male fertility defects, caused by kinked sperm flagella with the annulus mispositioned in the PP. Cby3 and ciBAR1 interact and colocalize to the annulus near the curved membrane invagination at the flagellar pocket. In the absence of Cby3, periannular membranes appear to be deformed, allowing the annulus to migrate over the fibrous sheath into the PP. Collectively, our results suggest that the Cby3/ciBAR1 complex regulates local membrane properties to position the annulus at the MP/PP junction.


Assuntos
Proteínas de Transporte , Proteínas Nucleares , Sêmen , Cauda do Espermatozoide , Espermatogênese , Animais , Masculino , Camundongos , Cílios , Citoesqueleto , Espermatogênese/genética , Proteínas Nucleares/genética , Proteínas de Transporte/genética
14.
Andrology ; 12(2): 349-364, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37302001

RESUMO

BACKGROUND: Multiple morphological abnormalities of sperm flagella is an idiopathic asthenoteratozoospermia characterized by absent, short, coiled, angulation, and irregular-caliber flagella. Genetic variants of DNAH1 gene have been identified as a causative factor of multiple morphological abnormalities of sperm flagella and intracytoplasmic sperm injection is an available strategy for infertile males with dynein axonemal heavy chain 1 defects to conceive. OBJECTIVES: To identify novel variants and candidate mutant hotspots of DNAH1 gene related to multiple morphological abnormalities of sperm flagella and male infertility in humans. MATERIALS AND METHODS: The DNAH1 variants were identified by whole exome sequencing and confirmed with Sanger sequencing. Papanicolaou staining, scanning and transmission electron microscopy, and immunostaining were performed to investigate the morphological and ultrastructural characteristics of spermatozoa. Intracytoplasmic sperm injection was applied for the assisted reproductive therapy of males harboring biallelic DNAH1 variants. RESULTS: We identified 18 different DNAH1 variants in 11 unrelated families, including nine missense variants (p.A2564T, p.T3657R, p.G1862R, p.L2296P, p.T4041I, p.L611P, p.A913D, p.R1932Q, p.R2356W) and nine loss-of-function variants (c.2301-1G>T, p.Q1518*, p.R1702*, p.D2845Mfs*2, p.P3909Rfs*33, p.Q4040Dfs*33, p.Q4058*, p.E4060Pfs*61, p.V4071Cfs*54). A total of 66.7% (12/18) of the identified variants were novel. Morphological analysis based on Papanicolaou staining and scanning electron microscopy demonstrated the typical multiple morphological abnormalities of sperm flagella characteristics of dynein axonemal heavy chain 1-deficient spermatozoa. Immunostaining further revealed the absence of inner dynein arms but not outer dynein arms, which induced a general ultrastructural disorganization, such as the loss of central pair and mis-localization of the microtubule doublets and outer dense fibers. To date, seven affected couples have accepted the intracytoplasmic sperm injection treatment, and three of them have given birth to five healthy babies. DISCUSSION AND CONCLUSION: These findings further expand the variant spectrum of DNAH1 gene related to multiple morphological abnormalities of sperm flagella and male infertility in humans, thus providing new information for the molecular diagnosis of asthenoteratozoospermia. The favorable fertility outcomes of intracytoplasmic sperm injection will facilitate the genetic counseling and clinical treatment of infertile males with multiple morphological abnormalities of sperm flagella in the future.


Assuntos
Astenozoospermia , Infertilidade Masculina , Masculino , Humanos , Injeções de Esperma Intracitoplásmicas , Astenozoospermia/genética , Mutação , Sêmen , Cauda do Espermatozoide , Espermatozoides , Infertilidade Masculina/genética , Infertilidade Masculina/terapia , Fertilidade , Dineínas/genética , China , Flagelos/genética
16.
Clin Genet ; 105(3): 317-322, 2024 03.
Artigo em Inglês | MEDLINE | ID: mdl-37975235

RESUMO

Sperm flagella share an evolutionary conserved microtubule-based structure with motile cilia expressed at the surface of several cell types, such as the airways epithelial cells. As a result, male infertility can be observed as an isolated condition or a syndromic trait, illustrated by Primary Cilia Dyskinesia (PCD). We report two unrelated patients showing multiple morphological abnormalities of the sperm flagella (MMAF) and carrying distinct homozygous truncating variants in the PCD-associated gene CCDC65. We characterized one of the identified variants (c.1208del; p.Asn403Ilefs*9), which induces the near absence of CCDC65 protein in patient sperm. In Chlamydomonas, CCDC65 ortholog (DRC2, FAP250) is a component of the Nexin-Dynein Regulatory complex (N-DRC), which interconnects microtubule doublets and coordinates dynein arms activity. In sperm cells from the patient, we also show the loss of GAS8, another component of the N-DRC, supporting a structural/functional link between the two proteins. Our work indicates that, similarly to ciliary axoneme, CCDC65 is required for sperm flagellum structure. Importantly, our work provides first evidence that mutations in the PCD-associated gene CCDC65 also cause asthenozoospermia.


Assuntos
Infertilidade Masculina , Cauda do Espermatozoide , Humanos , Masculino , Cauda do Espermatozoide/metabolismo , Axonema/genética , Sementes/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Mutação/genética , Dineínas/genética , Infertilidade Masculina/genética , Glicoproteínas/genética
17.
Clin Genet ; 105(2): 220-225, 2024 02.
Artigo em Inglês | MEDLINE | ID: mdl-37950557

RESUMO

Motile cilia and flagella are closely related organelles structured around a highly conserved axoneme whose formation and maintenance involve proteins from hundreds of genes. Defects in many of these genes have been described to induce primary ciliary dyskinesia (PCD) mainly characterized by chronic respiratory infections, situs inversus and/or infertility. In men, cilia/flagella-related infertility is usually caused by asthenozoospermia due to multiple morphological abnormalities of the sperm flagella (MMAF). Here, we investigated a cohort of 196 infertile men displaying a typical MMAF phenotype without any other PCD symptoms. Analysis of WES data identified a single case carrying a deleterious homozygous GAS8 variant altering a splice donor consensus site. This gene, also known as DRC4, encodes a subunit of the Nexin-Dynein Regulatory Complex (N-DRC), and has been already associated to male infertility and mild PCD. Confirming the deleterious effect of the candidate variant, GAS8 staining by immunofluorescence did not evidence any signal from the patient's spermatozoa whereas a strong signal was present along the whole flagella length in control cells. Concordant with its role in the N-DRC, transmission electron microscopy evidenced peripheral microtubule doublets misalignments. We confirm here the importance of GAS8 in the N-DRC and observed that its absence induces a typical MMAF phenotype not necessarily accompanied by other PCD symptoms.


Assuntos
Axonema , Infertilidade Masculina , Masculino , Humanos , Axonema/genética , Mutação , Sêmen , Cauda do Espermatozoide , Infertilidade Masculina/genética , Espermatozoides , Flagelos , Proteínas Associadas aos Microtúbulos/genética , Dineínas/genética
18.
J Assist Reprod Genet ; 41(1): 109-120, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37831349

RESUMO

PURPOSE: Asthenozoospermia is an important cause of male infertility, and the most serious type is characterized by multiple morphological abnormalities of the sperm flagella (MMAF). However, the precise etiology of MMAF remains unknown. In the current study, we recruited a consanguineous Pakistani family with two infertile brothers suffering from primary infertility due to MMAF without obvious signs of PCD. METHODS: We performed whole-exome sequencing on DNAs of the patients, their parents, and a fertile brother and identified the homozygous missense variant (c.1490C > G (p.P497R) in NPHP4 as the candidate mutation for male infertility in this family. RESULTS: Sanger sequencing confirmed that this mutation recessively co-segregated with the MMAF in this family. In silico analysis revealed that the mutation site is conserved across different species, and the identified mutation also causes abnormalities in the structure and hydrophobic interactions of the NPHP4 protein. Different bioinformatics tools predict that NPHP4p.P497R mutation is pathogenic. Furthermore, Papanicolaou staining and scanning electron microscopy of sperm revealed that affected individuals displayed typical MMAF phenotype with a high percentage of coiled, bent, short, absent, and/or irregular flagella. Transmission electron microscopy images of the patient's spermatozoa revealed significant anomalies in the sperm flagella with the absence of a central pair of microtubules (9 + 0) in every section scored. CONCLUSIONS: Taken together, these results show that the homozygous missense mutation in NPHP4 is associated with MMAF.


Assuntos
Infertilidade Masculina , Irmãos , Humanos , Masculino , Flagelos/genética , Infertilidade Masculina/genética , Infertilidade Masculina/patologia , Mutação , Mutação de Sentido Incorreto/genética , Proteínas/genética , Sêmen , Cauda do Espermatozoide/patologia , Espermatozoides/patologia
19.
Elife ; 122023 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-38091523

RESUMO

Radial spokes (RS) are T-shaped multiprotein complexes on the axonemal microtubules. Repeated RS1, RS2, and RS3 couple the central pair to modulate ciliary and flagellar motility. Despite the cell type specificity of RS3 substructures, their molecular components remain largely unknown. Here, we report that a leucine-rich repeat-containing protein, LRRC23, is an RS3 head component essential for its head assembly and flagellar motility in mammalian spermatozoa. From infertile male patients with defective sperm motility, we identified a splice site variant of LRRC23. A mutant mouse model mimicking this variant produces a truncated LRRC23 at the C-terminus that fails to localize to the sperm tail, causing male infertility due to defective sperm motility. LRRC23 was previously proposed to be an ortholog of the RS stalk protein RSP15. However, we found that purified recombinant LRRC23 interacts with an RS head protein RSPH9, which is abolished by the C-terminal truncation. Evolutionary and structural comparison also shows that LRRC34, not LRRC23, is the RSP15 ortholog. Cryo-electron tomography clearly revealed that the absence of the RS3 head and the sperm-specific RS2-RS3 bridge structure in LRRC23 mutant spermatozoa. Our study provides new insights into the structure and function of RS3 in mammalian spermatozoa and the molecular pathogenicity of LRRC23 underlying reduced sperm motility in infertile human males.


Assuntos
Infertilidade Masculina , Motilidade dos Espermatozoides , Camundongos , Animais , Masculino , Humanos , Sêmen , Axonema/metabolismo , Cauda do Espermatozoide , Proteínas/metabolismo , Espermatozoides , Infertilidade Masculina/genética , Flagelos/metabolismo , Mamíferos
20.
Cell Mol Life Sci ; 81(1): 1, 2023 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-38038747

RESUMO

Multiple morphological abnormalities of the flagella (MMAF) is a severe disease of male infertility, while the pathogenetic mechanisms of MMAF are still incompletely understood. Previously, we found that the deficiency of Ccdc38 might be associated with MMAF. To understand the underlying mechanism of this disease, we identified the potential partner of this protein and found that the coiled-coil domain containing 146 (CCDC146) can interact with CCDC38. It is predominantly expressed in the testes, and the knockout of this gene resulted in complete infertility in male mice but not in females. The knockout of Ccdc146 impaired spermiogenesis, mainly due to flagellum and manchette organization defects, finally led to MMAF-like phenotype. Furthermore, we demonstrated that CCDC146 could interact with both CCDC38 and CCDC42. It also interacts with intraflagellar transport (IFT) complexes IFT88 and IFT20. The knockout of this gene led to the decrease of ODF2, IFT88, and IFT20 protein levels, but did not affect CCDC38, CCDC42, or ODF1 expression. Additionally, we predicted and validated the detailed interactions between CCDC146 and CCDC38 or CCDC42, and built the interaction models at the atomic level. Our results suggest that the testis predominantly expressed gene Ccdc146 is essential for sperm flagellum biogenesis and male fertility, and its mutations might be associated with MMAF in some patients.


Assuntos
Infertilidade Masculina , Proteínas Associadas aos Microtúbulos , Cauda do Espermatozoide , Animais , Masculino , Camundongos , Fertilidade/genética , Proteínas de Choque Térmico/metabolismo , Infertilidade Masculina/metabolismo , Camundongos Knockout , Sêmen , Cauda do Espermatozoide/metabolismo , Cauda do Espermatozoide/patologia , Espermatozoides/metabolismo , Testículo/metabolismo , Proteínas Associadas aos Microtúbulos/genética
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