RESUMEN
The structural integrity of the sperm flagellum is essential for proper sperm function. Flagellar defects can result in male infertility, yet the precise mechanisms underlying this relationship are not fully understood. CCDC181, a coiled-coil domain-containing protein, is known to localize on sperm flagella and at the basal regions of motile cilia. Despite this knowledge, the specific functions of CCDC181 in flagellum biogenesis remain unclear. In this study, Ccdc181 knockout mice were generated. The absence of CCDC181 led to defective sperm head shaping and flagellum formation. Furthermore, the Ccdc181 knockout mice exhibited extremely low sperm counts, grossly aberrant sperm morphologies, markedly diminished sperm motility, and typical multiple morphological abnormalities of the flagella (MMAF). Additionally, an interaction between CCDC181 and the MMAF-related protein LRRC46 was identified, with CCDC181 regulating the localization of LRRC46 within sperm flagella. These findings suggest that CCDC181 plays a crucial role in both manchette formation and sperm flagellum biogenesis.
Asunto(s)
Ratones Noqueados , Proteínas de Microtúbulos , Cola del Espermatozoide , Animales , Masculino , Ratones , Fertilidad/fisiología , Flagelos/metabolismo , Flagelos/fisiología , Motilidad Espermática , Cola del Espermatozoide/metabolismo , Cola del Espermatozoide/fisiología , Espermatozoides/fisiología , Proteínas de Microtúbulos/genética , Proteínas de Microtúbulos/metabolismoRESUMEN
Male infertility due to asthenozoospermia is quite frequent, but its etiology is poorly understood. We recruited two infertile brothers, born to first-cousin parents from Pakistan, displaying idiopathic asthenozoospermia with mild stuttering disorder but no ciliary-related symptoms. Whole-exome sequencing identified a splicing variant (c.916+1G>A) in ARMC3, recessively co-segregating with asthenozoospermia in the family. The ARMC3 protein is evolutionarily highly conserved and is mostly expressed in the brain and testicular tissue of human. The ARMC3 splicing mutation leads to the exclusion of exon 8, resulting in a predicted truncated protein (p.Glu245_Asp305delfs*16). Quantitative real-time PCR revealed a significant decrease at mRNA level for ARMC3 and Western blot analysis did not detect ARMC3 protein in the patient's sperm. Individuals homozygous for the ARMC3 splicing variant displayed reduced sperm motility with frequent morphological abnormalities of sperm flagella. Transmission electron microscopy of the affected individual IV: 2 revealed vacuolation in sperm mitochondria at the midpiece and disrupted flagellar ultrastructure in the principal and end piece. Altogether, our results indicate that this novel homozygous ARMC3 splicing mutation destabilizes sperm flagella and leads to asthenozoospermia in our patients, providing a novel marker for genetic counseling and diagnosis of male infertility.
Asunto(s)
Astenozoospermia , Consanguinidad , Homocigoto , Linaje , Empalme del ARN , Cola del Espermatozoide , Adulto , Humanos , Masculino , Astenozoospermia/genética , Astenozoospermia/patología , Secuenciación del Exoma , Infertilidad Masculina/genética , Infertilidad Masculina/patología , Mutación , Empalme del ARN/genética , Motilidad Espermática/genética , Cola del Espermatozoide/patología , Cola del Espermatozoide/ultraestructura , Cola del Espermatozoide/metabolismo , Espermatozoides/ultraestructura , Espermatozoides/patologíaRESUMEN
BACKGROUND: Bi-allelic variants in DNAH11 have been identified as causative factors in Primary Ciliary Dyskinesia, leading to abnormal respiratory cilia. Nonetheless, the specific impact of these variants on human sperm flagellar and their involvement in male infertility remain largely unknown. METHODS: A collaborative effort involving two Chinese reproductive centers conducted a study with 975 unrelated infertile men. Whole-exome sequencing was employed for variant screening, and Sanger sequencing confirmed the identified variants. Morphological and ultrastructural analyses of sperm were conducted using Scanning Electron Microscopy and Transmission Electron Microscopy. Western Blot Analysis and Immunofluorescence Analysis were utilized to assess protein levels and localization. ICSI was performed to evaluate its efficacy in achieving favorable pregnancy outcomes for individuals with DNAH11 variants. RESULTS: In this study, we identified seven novel variants in the DNAH11 gene in four asthenoteratozoospermia subjects. These variants led the absence of DNAH11 proteins and ultrastructure defects in sperm flagella, particularly affecting the outer dynein arms (ODAs) and adjacent structures. The levels of ODA protein DNAI2 and axoneme related proteins were down regulated, instead of inner dynein arms (IDA) proteins DNAH1 and DNAH6. Two out of four individuals with DNAH11 variants achieved clinical pregnancies through ICSI. The findings confirm the association between male infertility and bi-allelic deleterious variants in DNAH11, resulting in the aberrant assembly of sperm flagella and contributing to asthenoteratozoospermia. Importantly, ICSI emerges as an effective intervention for overcoming reproductive challenges caused by DNAH11 gene variants.
Asunto(s)
Astenozoospermia , Dineínas Axonemales , Secuenciación del Exoma , Infertilidad Masculina , Humanos , Masculino , Astenozoospermia/genética , Astenozoospermia/patología , Dineínas Axonemales/genética , Femenino , Infertilidad Masculina/genética , Infertilidad Masculina/patología , Adulto , Cola del Espermatozoide/patología , Cola del Espermatozoide/ultraestructura , Cola del Espermatozoide/metabolismo , Inyecciones de Esperma Intracitoplasmáticas , Embarazo , Espermatozoides/ultraestructura , Espermatozoides/patología , Dineínas/genéticaRESUMEN
Proper connection between the sperm head and tail is critical for sperm motility and fertilization. Head-tail linkage is mediated by the head-tail coupling apparatus (HTCA), which secures the axoneme (tail) to the nucleus (head). However, the molecular architecture of the HTCA is poorly understood. Here, we use Drosophila to investigate formation and remodeling of the HTCA throughout spermiogenesis by visualizing key components of this complex. Using structured illumination microscopy, we demonstrate that key HTCA proteins Spag4 and Yuri form a 'centriole cap' that surrounds the centriole (or basal body) as it invaginates into the surface of the nucleus. As development progresses, the centriole is laterally displaced to the side of the nucleus while the HTCA expands under the nucleus, forming what we term the 'nuclear shelf'. We next show that the proximal centriole-like (PCL) structure is positioned under the nuclear shelf, functioning as a crucial stabilizer of centriole-nucleus attachment. Together, our data indicate that the HTCA is a complex, multi-point attachment site that simultaneously engages the PCL, the centriole and the nucleus to ensure proper head-tail connection during late-stage spermiogenesis.
Asunto(s)
Núcleo Celular , Centriolos , Proteínas de Drosophila , Espermatogénesis , Espermatozoides , Centriolos/metabolismo , Centriolos/ultraestructura , Masculino , Animales , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Espermatogénesis/fisiología , Espermatozoides/metabolismo , Espermatozoides/ultraestructura , Drosophila melanogaster/metabolismo , Cola del Espermatozoide/metabolismo , Cola del Espermatozoide/ultraestructura , Cabeza del Espermatozoide/ultraestructura , Cabeza del Espermatozoide/metabolismo , Axonema/metabolismo , Axonema/ultraestructuraRESUMEN
The structure of the sperm flagellar axoneme is highly conserved across species and serves the essential function of generating motility to facilitate the meeting of spermatozoa with the egg. During spermiogenesis, the axoneme elongates from the centrosome, and subsequently the centrosome docks onto the nuclear envelope to continue tail biogenesis. Mycbpap is expressed predominantly in mouse and human testes and conserved in Chlamydomonas as FAP147. A previous cryo-electron microscopy analysis has revealed the localization of FAP147 to the central apparatus of the axoneme. Here, we generated Mycbpap-knockout mice and demonstrated the essential role of Mycbpap in male fertility. Deletion of Mycbpap led to disrupted centrosome-nuclear envelope docking and abnormal flagellar biogenesis. Furthermore, we generated transgenic mice with tagged MYCBPAP, which restored the fertility of Mycbpap-knockout males. Interactome analyses of MYCBPAP using Mycbpap transgenic mice unveiled binding partners of MYCBPAP including central apparatus proteins, such as CFAP65 and CFAP70, which constitute the C2a projection, and centrosome-associated proteins, such as CCP110. These findings provide insights into a MYCBPAP-dependent regulation of the centrosome-nuclear envelope docking and sperm tail biogenesis.
Asunto(s)
Centrosoma , Ratones Noqueados , Membrana Nuclear , Cola del Espermatozoide , Animales , Masculino , Membrana Nuclear/metabolismo , Centrosoma/metabolismo , Cola del Espermatozoide/metabolismo , Cola del Espermatozoide/ultraestructura , Ratones , Espermatogénesis/genética , Ratones Transgénicos , Fertilidad , Axonema/metabolismo , Axonema/ultraestructura , Espermatozoides/metabolismo , Espermatozoides/ultraestructuraRESUMEN
Ribonucleoprotein (RNP) granules are membraneless electron-dense structures rich in RNAs and proteins, and involved in various cellular processes. Two RNP granules in male germ cells, intermitochondrial cement and the chromatoid body (CB), are associated with PIWI-interacting RNAs (piRNAs) and are required for transposon silencing and spermatogenesis. Other RNP granules in male germ cells, the reticulated body and CB remnants, are also essential for spermiogenesis. In this study, we disrupted FBXO24, a testis-enriched F-box protein, in mice and found numerous membraneless electron-dense granules accumulated in sperm flagella. Fbxo24 knockout (KO) mice exhibited malformed flagellar structures, impaired sperm motility, and male infertility, likely due to the accumulation of abnormal granules. The amount and localization of known RNP granule-related proteins were not disrupted in Fbxo24 KO mice, suggesting that the accumulated granules were distinct from known RNP granules. Further studies revealed that RNAs and two importins, IPO5 and KPNB1, abnormally accumulated in Fbxo24 KO spermatozoa and that FBXO24 could ubiquitinate IPO5. In addition, IPO5 and KPNB1 were recruited to stress granules, RNP complexes, when cells were treated with oxidative stress or a proteasome inhibitor. These results suggest that FBXO24 is involved in the degradation of IPO5, disruption of which may lead to the accumulation of abnormal RNP granules in sperm flagella.
Asunto(s)
Proteínas F-Box , Infertilidad Masculina , Ratones Noqueados , Cola del Espermatozoide , Masculino , Animales , Infertilidad Masculina/genética , Infertilidad Masculina/metabolismo , Ratones , Cola del Espermatozoide/metabolismo , Proteínas F-Box/metabolismo , Proteínas F-Box/genética , Gránulos Citoplasmáticos/metabolismo , Espermatozoides/metabolismoRESUMEN
PURPOSE: To identify novel variants in ACTL9 and new phenotypes responsible for male infertility. METHODS: Genomic DNA was extracted from peripheral blood samples for whole-exome sequencing (WES). Computer-assisted sperm analysis (CASA) was used to test the motility of spermatozoa. The ultrastructure of flagella and the mitochondrial sheath were assessed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Immunostaining was used to validate the localization and expression of ACTL9 and ACTL7A. An Actl9-mutated mouse model was used to validate the phenotypes by CASA and TEM. RESULTS: We identified novel homozygous variants in ACTL9 in two independent Chinese families. Spermatozoa with ACTL9 mutations showed decreased CASA parameters and a higher proportion of spermatozoa with abnormal morphology, exhibiting coiled flagella and a thickened midpiece. The spermatozoa were characterized by chaotic or irregular '9+2' structures and irregular mitochondrial sheath arrangements in the flagellum. Actl9 knock-in mice also showed abnormal CASA parameters and irregular '9+2' structures in flagella. CONCLUSIONS: Our study expands the mutation spectrum and phenotypic spectrum of ACTL9.
Asunto(s)
Flagelos , Homocigoto , Infertilidad Masculina , Mitocondrias , Mutación , Motilidad Espermática , Cola del Espermatozoide , Espermatozoides , Masculino , Infertilidad Masculina/genética , Infertilidad Masculina/patología , Humanos , Ratones , Espermatozoides/patología , Espermatozoides/ultraestructura , Espermatozoides/metabolismo , Animales , Mitocondrias/genética , Mitocondrias/ultraestructura , Mitocondrias/patología , Mitocondrias/metabolismo , Mutación/genética , Cola del Espermatozoide/patología , Cola del Espermatozoide/metabolismo , Cola del Espermatozoide/ultraestructura , Flagelos/genética , Flagelos/ultraestructura , Flagelos/metabolismo , Motilidad Espermática/genética , Secuenciación del Exoma , Linaje , Adulto , Análisis de SemenRESUMEN
ABSTRACT: The cause of asthenozoospermia (AZS) is not well understood because of its complexity and heterogeneity. Although some gene mutations have been identified as contributing factors, they are only responsible for a small number of cases. Radial spokes (RSs) are critical for adenosine triphosphate-driven flagellar beating and axoneme stability, which is essential for flagellum motility. In this study, we found novel compound heterozygous mutations in leucine-rich repeat-containing protein 23 ( LRRC23 ; c.1018C>T: p.Q340X and c.881_897 Del: p.R295Gfs*32) in a proband from a nonconsanguineous family with AZS and male infertility. Diff-Quik staining and scanning electron microscopy revealed no abnormal sperm morphology. Western blotting and immunofluorescence staining showed that these mutations suppressed LRRC23 expression in sperm flagella. Additionally, transmission electron microscopy showed the absence of RS3 in sperm flagella, which disrupts stability of the radial spoke complex and impairs motility. Following in vitro fertilization and embryo transfer, the proband's spouse achieved successful pregnancy and delivered a healthy baby. In conclusion, our study indicates that two novel mutations in LRRC23 are associated with AZS, but successful fertility outcomes can be achieved by in vitro fertilization-embryo transfer techniques.
Asunto(s)
Astenozoospermia , Mutación , Humanos , Masculino , Astenozoospermia/genética , Adulto , Linaje , Cola del Espermatozoide/patología , Cola del Espermatozoide/ultraestructura , Cola del Espermatozoide/metabolismo , Femenino , Motilidad Espermática/genética , EmbarazoRESUMEN
TTC12 is a cytoplasmic and centromere-localized protein that plays a role in the proper assembly of dynein arm complexes in motile cilia in both respiratory cells and sperm flagella. This finding underscores its significance in cellular motility and function. However, the wide role of TTC12 in human spermatogenesis-associated primary ciliary dyskinesia (PCD) still needs to be elucidated. Whole-exome sequencing (WES) and Sanger sequencing were performed to identify potentially pathogenic variants causing PCD and multiple morphological abnormalities of sperm flagella (MMAF) in an infertile Pakistani man. Diagnostic imaging techniques were used for PCD screening in the patient. Real-time polymerase chain reaction (RTâPCR) was performed to detect the effect of mutations on the mRNA abundance of the affected genes. Papanicolaou staining and scanning electron microscopy (SEM) were carried out to examine sperm morphology. Transmission electron microscopy (TEM) was performed to examine the ultrastructure of the sperm flagella, and the results were confirmed by immunofluorescence staining. Using WES and Sanger sequencing, a novel homozygous missense variant (c.C1069T; p.Arg357Trp) in TTC12 was identified in a patient from a consanguineous family. A computed tomography scan of the paranasal sinuses confirmed the symptoms of the PCD. RT-PCR showed a decrease in TTC12 mRNA in the patient's sperm sample. Papanicolaou staining, SEM, and TEM analysis revealed a significant change in shape and a disorganized axonemal structure in the sperm flagella of the patient. Immunostaining assays revealed that TTC12 is distributed throughout the flagella and is predominantly concentrated in the midpiece in normal spermatozoa. In contrast, spermatozoa from patient deficient in TTC12 showed minimal staining intensity for TTC12 or DNAH17 (outer dynein arms components). This could lead to MMAF and result in male infertility. This novel TTC12 variant not only illuminates the underlying genetic causes of male infertility but also paves the way for potential treatments targeting these genetic factors. This study represents a significant advancement in understanding the genetic basis of PCD-related infertility.
Asunto(s)
Homocigoto , Infertilidad Masculina , Mutación Missense , Cola del Espermatozoide , Humanos , Masculino , Mutación Missense/genética , Pakistán , Infertilidad Masculina/genética , Infertilidad Masculina/patología , Cola del Espermatozoide/patología , Cola del Espermatozoide/ultraestructura , Cola del Espermatozoide/metabolismo , Adulto , Linaje , Astenozoospermia/genética , Astenozoospermia/patología , Trastornos de la Motilidad Ciliar/genética , Trastornos de la Motilidad Ciliar/patología , Secuenciación del Exoma , Oligospermia/genética , Oligospermia/patología , Síndrome de Kartagener/genética , Síndrome de Kartagener/patologíaRESUMEN
Dynein complexes are large, multi-unit assemblies involved in many biological processes via their critical roles in protein transport and axoneme motility. Using next-generation sequencing of infertile men presenting with low or no sperm in their ejaculates, we identified damaging variants in the dynein-related gene AXDND1. We thus hypothesised that AXDND1 is a critical regulator of male fertility. To test this hypothesis, we produced a knockout mouse model. Axdnd1-/- males were sterile at all ages but presented with an evolving testis phenotype wherein they could undergo one round of histologically replete spermatogenesis followed by a rapid depletion of the seminiferous epithelium. Marker experiments identified a role for AXDND1 in maintaining the balance between differentiation-committed and self-renewing spermatogonial populations, resulting in disproportionate production of differentiating cells in the absence of AXDND1 and increased sperm production during initial spermatogenic waves. Moreover, long-term spermatogonial maintenance in the Axdnd1 knockout was compromised, ultimately leading to catastrophic germ cell loss, destruction of blood-testis barrier integrity and immune cell infiltration. In addition, sperm produced during the first wave of spermatogenesis were immotile due to abnormal axoneme structure, including the presence of ectopic vesicles and abnormalities in outer dense fibres and microtubule doublet structures. Sperm output was additionally compromised by a severe spermiation defect and abnormal sperm individualisation. Collectively these data identify AXDND1 as an atypical dynein complex-related protein with a role in protein/vesicle transport of relevance to spermatogonial function and sperm tail formation in mice and humans. This study underscores the importance of studying the consequences of gene loss-of-function on both the establishment and maintenance of male fertility.
Asunto(s)
Ratones Noqueados , Cola del Espermatozoide , Espermatogénesis , Espermatogonias , Animales , Humanos , Masculino , Ratones , Diferenciación Celular , Dineínas/metabolismo , Infertilidad Masculina/genética , Infertilidad Masculina/metabolismo , Infertilidad Masculina/patología , Ratones Endogámicos C57BL , Cola del Espermatozoide/metabolismo , Espermatogénesis/genética , Espermatogonias/metabolismo , Testículo/metabolismo , Dineínas Axonemales/genética , Dineínas Axonemales/metabolismoRESUMEN
The proper functioning and assembly of the sperm flagella structures contribute significantly to spermatozoa motility and overall male fertility. However, the fine mechanisms of assembly steps are poorly studied due to the high diversity of cell types, low solubility of the corresponding protein structures, and high tissue and cell specificity. One of the open questions for investigation is the attachment of longitudinal columns to the doublets 3 and 8 of axonemal microtubules through the outer dense fibers. A number of mutations affecting the assembly of flagella in model organisms are known. Additionally, evolutionary genomics data and comparative analysis of flagella morphology are available for a set of non-model species. This review is devoted to the analysis of diverse ultrastructures of sperm flagellum of Metazoa combined with an overview of the evolutionary distribution and function of the mammalian fibrous sheath proteins.
Asunto(s)
Cola del Espermatozoide , Espermatozoides , Masculino , Animales , Espermatozoides/metabolismo , Espermatozoides/ultraestructura , Espermatozoides/fisiología , Cola del Espermatozoide/ultraestructura , Cola del Espermatozoide/metabolismo , Humanos , Axonema/ultraestructura , Axonema/metabolismo , Motilidad Espermática/fisiologíaRESUMEN
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.
Asunto(s)
Proteínas de Drosophila , Fertilización , Mitocondrias , Cuerpos Multivesiculares , Fagocitosis , Espermatozoides , Animales , Mitocondrias/metabolismo , Masculino , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Femenino , Espermatozoides/metabolismo , Cuerpos Multivesiculares/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Óvulo/metabolismo , Lisosomas/metabolismo , Cola del Espermatozoide/metabolismo , MitofagiaRESUMEN
Aims: Asthenozoospermia is the most common factor of male infertility, mainly caused by multiple morphological abnormalities of the sperm flagella (MMAF) and primary ciliary dyskinesia (PCD). Previous studies have shown that genetic factors may contribute to MMAF and PCD. The study aimed to identify novel potentially pathogenic gene mutations in a Chinese infertile man with MMAF and PCD-like phenotypes. Methods: A Chinese infertile man with MMAF and PCD was enrolled in this study. Whole exome sequencing and Sanger sequencing were performed to identify potential causative genes and mutations. Results: A novel homozygous missense mutation (c.1450G>A; p.E484K) of CCDC40 was finally identified and Sanger sequencing confirmed that the patient carried the homozygous mutation, which was inherited from his parents. We reported the first homozygous missense CCDC40 mutation in infertile men with MMAF but had other milder PCD symptoms. Conclusion: Our findings not only broaden the disease-causing mutation spectrum of CCDC40 but also provide new insight into the correlation between CCDC40 mutations and MMAF.
Asunto(s)
Pueblo Asiatico , Homocigoto , Infertilidad Masculina , Mutación Missense , Fenotipo , Cola del Espermatozoide , Humanos , Masculino , Infertilidad Masculina/genética , Mutación Missense/genética , Adulto , China , Pueblo Asiatico/genética , Cola del Espermatozoide/metabolismo , Cola del Espermatozoide/patología , Trastornos de la Motilidad Ciliar/genética , Secuenciación del Exoma/métodos , Linaje , Mutación , Astenozoospermia/genética , Pueblos del Este de AsiaRESUMEN
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.
Asunto(s)
Proteínas de la Membrana , Ratones Noqueados , Membrana Nuclear , Septinas , Cabeza del Espermatozoide , Cola del Espermatozoide , Animales , Humanos , Masculino , Ratones , Infertilidad Masculina/metabolismo , Infertilidad Masculina/genética , Lamina Tipo B/metabolismo , Lamina Tipo B/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Membrana Nuclear/metabolismo , Septinas/metabolismo , Septinas/genética , Cabeza del Espermatozoide/metabolismo , Cabeza del Espermatozoide/patología , Cola del Espermatozoide/metabolismo , Espermatozoides/metabolismo , Teratozoospermia/metabolismo , Teratozoospermia/genéticaRESUMEN
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.
Asunto(s)
Infertilidad Masculina , Motilidad Espermática , Cola del Espermatozoide , Animales , Masculino , Ratones , Sistemas CRISPR-Cas/genética , Flagelos/genética , Flagelos/metabolismo , Infertilidad Masculina/genética , Infertilidad Masculina/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Motilidad Espermática/genética , Cola del Espermatozoide/patología , Cola del Espermatozoide/metabolismo , Espermatogénesis/genética , Espermatozoides/metabolismoRESUMEN
Human infertility affects 10-15% of couples. Asthenozoospermia accounts for 18% of men with infertility and is a common male infertility phenotype. The nexin-dynein regulatory complex (N-DRC) is a large protein complex in the sperm flagellum that connects adjacent doublets of microtubules. Defects in the N-DRC can disrupt cilia/flagellum movement, resulting in primary ciliary dyskinesia and male infertility. Using whole-exome sequencing, we identified a pathological homozygous variant of the dynein regulatory complex subunit 3 (DRC3) gene, which expresses leucine-rich repeat-containing protein 48, a component of the N-DRC, in a patient with asthenozoospermia. The variant ENST00000313838.12: c.644dup (p. Glu216GlyfsTer36) causes premature translational arrest of DRC3, resulting in a dysfunctional DRC3 protein. The patient's semen count, color, and pH were normal according to the reference values of the World Health Organization guidelines; however, sperm motility and progressive motility were reduced. DRC3 protein was not detected in the patient's sperm and the ultrastructure of the patient's sperm flagella was destroyed. More importantly, the DRC3 variant reduced its interaction with other components of the N-DRC, including dynein regulatory complex subunits 1, 2, 4, 5, 7, and 8. Our data not only revealed the essential biological functions of DRC3 in sperm flagellum movement and structure but also provided a new basis for the clinical genetic diagnosis of male infertility.
Asunto(s)
Astenozoospermia , Homocigoto , Infertilidad Masculina , Humanos , Masculino , Astenozoospermia/genética , Astenozoospermia/patología , Infertilidad Masculina/genética , Infertilidad Masculina/patología , Motilidad Espermática/genética , Adulto , Espermatozoides/metabolismo , Espermatozoides/patología , Secuenciación del Exoma , Cola del Espermatozoide/metabolismo , Cola del Espermatozoide/patología , Dineínas/genética , Dineínas/metabolismo , MutaciónRESUMEN
The complexities of energy transfer mechanisms in the flagella of mammalian sperm flagella have been intensively investigated and demonstrate significant diversity across species. Enzymatic shuttles, particularly adenylate kinase (AK) and creatine kinase (CK), are pivotal in the efficient transfer of intracellular ATP, showing distinct tissue- and species-specificity. Here, the expression profiles of AK and CK were investigated in mice and found to fall into four subgroups, of which Subgroup III AKs were observed to be unique to the male reproductive system and conserved across chordates. Both AK8 and AK9 were found to be indispensable to male reproduction after analysis of an infertile male cohort. Knockout mouse models showed that AK8 and AK9 were central to promoting sperm motility. Immunoprecipitation combined with mass spectrometry revealed that AK8 and AK9 interact with the radial spoke (RS) of the axoneme. Examination of various human and mouse sperm samples with substructural damage, including the presence of multiple RS subunits, showed that the head of radial spoke 3 acts as an adapter for AK9 in the flagellar axoneme. Using an ATP probe together with metabolomic analysis, it was found that AK8 and AK9 cooperatively regulated ATP transfer in the axoneme, and were concentrated at sites associated with energy consumption in the flagellum. These findings indicate a novel function for RS beyond its structural role, namely, the regulation of ATP transfer. In conclusion, the results expand the functional spectrum of AK proteins and suggest a fresh model regarding ATP transfer within mammalian flagella.
Asunto(s)
Adenosina Trifosfato , Adenilato Quinasa , Axonema , Ratones Noqueados , Motilidad Espermática , Cola del Espermatozoide , Animales , Adenilato Quinasa/metabolismo , Masculino , Ratones , Axonema/metabolismo , Motilidad Espermática/fisiología , Cola del Espermatozoide/metabolismo , Adenosina Trifosfato/metabolismo , Humanos , Metabolismo Energético , Espermatozoides/metabolismo , Flagelos/metabolismo , Creatina Quinasa/metabolismo , Infertilidad Masculina/metabolismo , Infertilidad Masculina/genéticaRESUMEN
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.
Asunto(s)
Infertilidad Masculina , Motilidad Espermática , Masculino , Humanos , Infertilidad Masculina/genética , Infertilidad Masculina/patología , Motilidad Espermática/genética , Espermatozoides , Cola del Espermatozoide/patología , Cola del Espermatozoide/metabolismo , Microtúbulos/genética , Microtúbulos/metabolismoRESUMEN
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.
Asunto(s)
Infertilidad Masculina , Cola del Espermatozoide , Humanos , Masculino , Cola del Espermatozoide/metabolismo , Motilidad Espermática/genética , Semen , Infertilidad Masculina/genética , Infertilidad Masculina/metabolismo , Mutación/genéticaRESUMEN
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.