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Andrology Jun 2024Dysfunction of motile cilia, including respiratory cilia and sperm flagella, typically leads to primary ciliary dyskinesia and male infertility or low fertility in...
BACKGROUND
Dysfunction of motile cilia, including respiratory cilia and sperm flagella, typically leads to primary ciliary dyskinesia and male infertility or low fertility in humans. Genetic defects of LRRC6 have been associated with primary ciliary dyskinesia and asthenozoospermia due to abnormal ultrastructure of ciliated axonemes.
OBJECTIVES
To identify novel mutations of the LRRC6 gene related to multiple morphological abnormalities of the sperm flagella and male infertility and investigate the underlying molecular mechanisms involved.
MATERIALS AND METHODS
The LRRC6 mutations were identified by whole exome sequencing and confirmed with Sanger sequencing. Papanicolaou staining, scanning, and transmission electron microscopy were performed to investigate the morphological and ultrastructural characteristics of spermatozoa. Further tandem mass tagging proteomics analyses were performed to explore the effect of mutations and confirmed by immunostaining and western blotting. Intracytoplasmic sperm injection was applied for the assisted reproductive therapy of males harboring biallelic LRRC6 mutations.
RESULTS
In this study, we identified a novel homozygous LRRC6 mutation in a consanguineous family, characterized by asthenozoospermia and primary ciliary dyskinesia. Further Semen parameter and morphology analysis demonstrate that the novel LRRC6 mutation leads to a significant reduction in sperm flagella length, a decrease in sperm progressive motility parameters, and abnormalities of sperm ultrastructure. Specifically, the absence of outer dynein arms and inner dynein arms, and incomplete mitochondrial sheath in the flagellar mid-piece were observed by transmission electron microscopy. In addition, tandem mass tagging proteomics analysis revealed that spermatozoa obtained from patients harboring the LRRC6 mutation exhibited a significant decrease in the expression levels of proteins related to the assembly and function of dynein axonemal arms. Functional analysis revealed that this novel LRRC6 mutation disrupted the function of the leucine-rich repeat containing 6 protein, which in turn affects the expression of the dynein arm proteins and leucine-rich repeat containing 6-interacting proteins CCDC40, SPAG1, and ZMYND10. Finally, we reported a successful pregnancy through assisted reproductive technology with intracytoplasmic sperm injection in the female partner of the proband.
DISCUSSION AND CONCLUSION
This study highlights the identification of a novel homozygous LRRC6 mutation in a consanguineous family and its impact on sperm progressive motility, morphology, and sperm kinetics parameters, which could facilitate the genetic diagnosis of asthenozoospermia and offer valuable perspectives for future genetic counseling endeavors.
PubMed: 38934611
DOI: 10.1111/andr.13685 -
Cytoskeleton (Hoboken, N.J.) Jun 2024Wolbachia are obligate intracellular alphaproteobacteria that enhance their spreading by altering the reproductive mechanisms of several invertebrates. Among the...
Axonemal tubules in the distal sperm tail of Wolbachia-infected Drosophila simulans males contain ring-like intraluminal structures that persist after axoneme fragmentation.
Wolbachia are obligate intracellular alphaproteobacteria that enhance their spreading by altering the reproductive mechanisms of several invertebrates. Among the reproductive alterations, Wolbachia also causes cytoplasmic incompatibility that leads to embryo death when infected males are crossed with uninfected females, thus selecting infected females. However, the presence of Wolbachia has important fitness costs and infected Drosophila simulans males produce less sperm than their uninfected counterparts. Such sperm suffer, indeed, of some structural alterations that hinder their proper function. We took advantage of the fact that several sperm have abnormal distal regions of the tail, in which the plasma membrane is broken and the axonemal components splayed, making the ultrastructural aspects clearly observable. We found that axoneme reduction in the distal region of the sperm does not follow a unique pattern as observed in other insects, but occurs by losing accessory tubules or peripheral doublets. The axonemal tubules contain distinct coaxial ring-like structures that are still observed after axoneme fragmentation and form large clusters of several units.
PubMed: 38923204
DOI: 10.1002/cm.21891 -
Cells Jun 2024Primary ciliary dyskinesia (PCD) is a rare, genetically heterogeneous, motile ciliopathy, characterized by neonatal respiratory distress, recurrent upper and lower... (Review)
Review
Primary ciliary dyskinesia (PCD) is a rare, genetically heterogeneous, motile ciliopathy, characterized by neonatal respiratory distress, recurrent upper and lower respiratory tract infections, subfertility, and laterality defects. Diagnosis relies on a combination of tests for confirmation, including nasal nitric oxide (nNO) measurements, high-speed videomicroscopy analysis (HSVMA), immunofluorescent staining, axonemal ultrastructure analysis via transmission electron microscopy (TEM), and genetic testing. Notably, there is no single gold standard confirmatory or exclusionary test. Currently, 54 causative genes involved in cilia assembly, structure, and function have been linked to PCD; this rare disease has a spectrum of clinical manifestations and emerging genotype-phenotype relationships. In this review, we provide an overview of the structure and function of motile cilia, the emerging genetics and pathophysiology of this rare disease, as well as clinical features associated with motile ciliopathies, novel diagnostic tools, and updates on genotype-phenotype relationships in PCD.
Topics: Humans; Cilia; Ciliary Motility Disorders; Phenotype
PubMed: 38891105
DOI: 10.3390/cells13110974 -
Frontiers in Cell and Developmental... 2024The unicellular green alga, i, has played a central role in discovering much of what is currently known about the composition, assembly, and function of cilia and... (Review)
Review
The unicellular green alga, i, has played a central role in discovering much of what is currently known about the composition, assembly, and function of cilia and flagella. combines excellent genetics, such as the ability to grow cells as haploids or diploids and to perform tetrad analysis, with an unparalleled ability to detach and isolate flagella in a single step without cell lysis. The combination of genetics and biochemistry that is possible in has allowed many of the key components of the cilium to be identified by looking for proteins that are missing in a defined mutant. Few if any other model organisms allow such a seamless combination of genetic and biochemical approaches. Other major advantages of compared to other systems include the ability to induce flagella to regenerate in a highly synchronous manner, allowing the kinetics of flagellar growth to be measured, and the ability of flagella to adhere to glass coverslips allowing Intraflagellar Transport to be easily imaged inside the flagella of living cells, with quantitative precision and single-molecule resolution. These advantages continue to work in favor of as a model system going forward, and are now augmented by extensive genomic resources, a knockout strain collection, and efficient CRISPR gene editing. While has obvious limitations for studying ciliary functions related to animal development or organ physiology, when it comes to studying the fundamental biology of cilia and flagella, is simply unmatched in terms of speed, efficiency, cost, and the variety of approaches that can be brought to bear on a question.
PubMed: 38872931
DOI: 10.3389/fcell.2024.1412641 -
The Journal of Allergy and Clinical... Jun 2024Eosinophils are elusive cells involved in allergic inflammation. Single-cell RNA Sequencing (scRNA-seq) is an emerging approach to deeply characterize cellular...
BACKGROUND
Eosinophils are elusive cells involved in allergic inflammation. Single-cell RNA Sequencing (scRNA-seq) is an emerging approach to deeply characterize cellular properties, heterogeneity, and functionality.
OBJECTIVE
To comprehensively characterize the transcriptome and biological functions of human eosinophils at a site of severe allergic inflammation in the esophagus (i.e., eosinophilic esophagitis (EoE)).
METHODS
We employed a gravity-based scRNA-seq methodology to sequence blood eosinophils from patients with EoE and control individuals compared to a reanalyzed public scRNA-seq dataset of human esophageal eosinophils of EoE patients. We used flow cytometry, immunostaining, and a stimulation assay to verify mRNA findings.
RESULTS
In total, scRNA-seq was obtained from 586 eosinophils (188 from blood [n=6 individuals] and 398 from esophagus [n=6 individuals]). The esophageal eosinophils were composed of a population of activated eosinophils (enriched in 659 genes compared with peripheral blood-associated eosinophils) and a small population of eosinophils resembling peripheral blood eosinophils (enriched in 62 genes compared with esophageal eosinophils). Esophageal eosinophils expressed genes involved in sensing and responding to diverse stimuli, most notably interferon-, interleukin 10, histamine and leukotrienes, and succinate. Esophageal eosinophils were most distinguished from other esophageal populations by gene expression of the receptors CCR3, HRH4, SUCNR1, and VSTM1; transcription factors CEBPE, OLIG1, and OLIG2; protease PRSS33; and hallmark eosinophil gene CLC. A web of bidirectional eosinophil interactions with other esophageal populations was derived. Comparing esophageal eosinophils and mast cells revealed that esophageal eosinophils expressed genes involved in DAP12 interactions, IgG receptor-triggered events, immunoregulation, and IL-10 signaling.
CONCLUSIONS
In EoE, esophageal eosinophils exist as two populations, a minority population resembling blood eosinophils and the other population characterized by high de novo transcription of diverse sensing receptors and inflammatory mediators readying them to potentially intersect with diverse cell types.
PubMed: 38871184
DOI: 10.1016/j.jaci.2024.05.029 -
Asian Journal of Andrology Jun 2024Male infertility is a worldwide health issue, affecting 8%-12% of the global population. Oligoasthenoteratozoospermia (OAT) represents a severe type of male infertility,...
Male infertility is a worldwide health issue, affecting 8%-12% of the global population. Oligoasthenoteratozoospermia (OAT) represents a severe type of male infertility, characterized by reduced sperm count and motility and an increased frequency of sperm with aberrant morphology. Using whole-exome sequencing, this study identified a novel missense mutation (c.848C>A, p.A283E) in the coiled-coil domain-containing 34 gene (CCDC34) in a consanguineous Pakistani family. This rare mutation was predicted to be deleterious and to affect the protein stability. Hematoxylin and eosin staining of spermatozoa from the patient with OAT revealed multiple morphological abnormalities of the flagella and transmission electron microscopy indicated axonemal ultrastructural defects with a lack of outer dynein arms. These findings indicated that CCDC34 plays a role in maintaining the axonemal ultrastructure and the assembly or stability of the outer dynein arms, thus expanding the phenotypic spectrum of CCDC34 missense mutations.
PubMed: 38856307
DOI: 10.4103/aja202432 -
Brain : a Journal of Neurology Jun 2024Intracellular trafficking involves an intricate machinery of motor complexes including the dynein complex to shuttle cargo for autophagolysosomal degradation. Deficiency...
Intracellular trafficking involves an intricate machinery of motor complexes including the dynein complex to shuttle cargo for autophagolysosomal degradation. Deficiency in dynein axonemal chains as well as cytoplasmic light and intermediate chains have been linked with ciliary dyskinesia and skeletal dysplasia. The cytoplasmic dynein 1 heavy chain protein (DYNC1H1) serves as a core complex for retrograde trafficking in neuronal axons. Dominant pathogenic variants in DYNC1H1 have been previously implicated in peripheral neuromuscular disorders (NMD) and neurodevelopmental disorders (NDD). As heavy-chain dynein is ubiquitously expressed, the apparent selectivity of heavy-chain dyneinopathy for motor neuronal phenotypes remains currently unaccounted for. Here, we aimed to evaluate the full DYNC1H1-related clinical, molecular and imaging spectrum, including multisystem features and novel phenotypes presenting throughout life. We identified 47 cases from 43 families with pathogenic heterozygous variants in DYNC1H1 (aged 0-59 years) and collected phenotypic data via a comprehensive standardized survey and clinical follow-up appointments. Most patients presented with divergent and previously unrecognized neurological and multisystem features, leading to significant delays in genetic testing and establishing the correct diagnosis. Neurological phenotypes include novel autonomic features, previously rarely described behavioral disorders, movement disorders, and periventricular lesions. Sensory neuropathy was identified in nine patients (median age of onset 10.6 years), of which five were only diagnosed after the second decade of life, and three had a progressive age-dependent sensory neuropathy. Novel multisystem features included primary immunodeficiency, bilateral sensorineural hearing loss, organ anomalies, and skeletal manifestations, resembling the phenotypic spectrum of other dyneinopathies. We also identified an age-dependent biphasic disease course with developmental regression in the first decade and, following a period of stability, neurodegenerative progression after the second decade of life. Of note, we observed several cases in whom neurodegeneration appeared to be prompted by intercurrent systemic infections with double-stranded DNA viruses (Herpesviridae) or single-stranded RNA viruses (Ross-River fever, SARS-CoV-2). Moreover, the disease course appeared to be exacerbated by viral infections regardless of age and/or severity of NDD manifestations, indicating a role of dynein in anti-viral immunity and neuronal health. In summary, our findings expand the clinical, imaging, and molecular spectrum of pathogenic DYNC1H1 variants beyond motor neuropathy disorders and suggest a life-long continuum and age-related progression due to deficient intracellular trafficking. This study will facilitate early diagnosis and improve counselling and health surveillance of affected patients.
PubMed: 38848546
DOI: 10.1093/brain/awae183 -
The Journal of Cell Biology Sep 2024Two sets of motor proteins underpin motile cilia/flagella function. The axoneme-associated inner and outer dynein arms drive sliding of adjacent axoneme microtubule...
Two sets of motor proteins underpin motile cilia/flagella function. The axoneme-associated inner and outer dynein arms drive sliding of adjacent axoneme microtubule doublets to periodically bend the flagellum for beating, while intraflagellar transport (IFT) kinesins and dyneins carry IFT trains bidirectionally along the axoneme. Despite assembling motile cilia and flagella, IFT train speeds have only previously been quantified in immobilized flagella-mechanical immobilization or genetic paralysis. This has limited investigation of the interaction between IFT and flagellar beating. Here, in uniflagellate Leishmania parasites, we use high-frequency, dual-color fluorescence microscopy to visualize IFT train movement in beating flagella. We discovered that adhesion of flagella to a microscope slide is detrimental, reducing IFT train speed and increasing train stalling. In flagella free to move, IFT train speed is not strongly dependent on flagella beat type; however, permanent disruption of flagella beating by deletion of genes necessary for formation or regulation of beating showed an inverse correlation of beat frequency and IFT train speed.
Topics: Axoneme; Biological Transport; Cilia; Dyneins; Flagella; Kinesins; Leishmania; Protozoan Proteins; Microtubules
PubMed: 38829962
DOI: 10.1083/jcb.202401154 -
Journal of Reproductive Immunology May 2024Sperm-immobilizing antibodies (SI-Abs) are detected in the sera of 3 % of infertile women. SI-Abs are occasionally produced as allogeneic antibodies against sperm,...
Sperm-immobilizing antibodies (SI-Abs) are detected in the sera of 3 % of infertile women. SI-Abs are occasionally produced as allogeneic antibodies against sperm, causing immune infertility. SI-Abs inhibit the passage of sperm through the female reproductive tract. Research on anti-sperm antibodies (ASA) remains of great importance for population control. We aimed to identify the antigens recognized by SI-Abs and elucidate the pathogenesis of immune infertility. Twelve sperm-immobilization test (SIT)-positive and fourteen SIT-negative sera were analyzed by two-dimensional electrophoresis and western blotting. Antigenic materials were extracted from well-motile sperm prepared using 0.1 % sodium dodecyl sulfate. In total, 22 different spots were detected in the 12 positive sera. Among these, three positive serum samples showed two positive signals with similar migration patterns. The significant positive spots were Mr: 49 K, pI: 5.1 and Mr: 51 K, pI: 5.6. All these positive spots were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS); tubulin beta-4A (TBB4A) was identified from the spot Mr: 49 K, pI: 5.1. TBB4A is a major component of tubulin and constitutes the axoneme in the sperm tail and the centrosome in the sperm neck; it is generally located inside the cell. An authentic antibody against TBB4A showed a positive reaction in the sperm neck and tail regions in an immunofluorescence study. This antibody also inhibited sperm motility in a complement-dependent manner. Sperm membrane permeability reportedly changes during swimming and capacitation. We identified TBB4A as an antigenic molecule recognized by SI-Abs, which may be relevant to immunological contraception in the future.
PubMed: 38824771
DOI: 10.1016/j.jri.2024.104259 -
Nature Communications Jun 2024Differentiation of male gametocytes into flagellated fertile male gametes relies on the assembly of axoneme, a major component of male development for mosquito...
Differentiation of male gametocytes into flagellated fertile male gametes relies on the assembly of axoneme, a major component of male development for mosquito transmission of the malaria parasite. RNA-binding protein (RBP)-mediated post-transcriptional regulation of mRNA plays important roles in eukaryotic sexual development, including the development of female Plasmodium. However, the role of RBP in defining the Plasmodium male transcriptome and its function in male gametogenesis remains incompletely understood. Here, we performed genome-wide screening for gender-specific RBPs and identified an undescribed male-specific RBP gene Rbpm1 in the Plasmodium. RBPm1 is localized in the nucleus of male gametocytes. RBPm1-deficient parasites fail to assemble the axoneme for male gametogenesis and thus mosquito transmission. RBPm1 interacts with the spliceosome E complex and regulates the splicing initiation of certain introns in a group of 26 axonemal genes. RBPm1 deficiency results in intron retention and protein loss of these axonemal genes. Intron deletion restores axonemal protein expression and partially rectifies axonemal defects in RBPm1-null gametocytes. Further splicing assays in both reporter and endogenous genes exhibit stringent recognition of the axonemal introns by RBPm1. The splicing activator RBPm1 and its target introns constitute an axonemal intron splicing program in the post-transcriptional regulation essential for Plasmodium male development.
Topics: Introns; RNA Splicing; RNA-Binding Proteins; Animals; Protozoan Proteins; Male; Axoneme; Female; Gametogenesis; Spliceosomes; Plasmodium berghei; Malaria; Plasmodium
PubMed: 38824128
DOI: 10.1038/s41467-024-49002-9