Spoke Head Research Articles

Ovine fertility by artificial insemination in the breeding season could be affected by intraseasonal variations in ram sperm proteomic profile

It is important to note that seasonality could affect ram reproductive parameters, and therefore, fertility results after artificial insemination. In this work, 1) we assessed fertility rates after cervical artificial insemination of 11,805 ewes at the beginning (June 21st to July 20th) and at the end (November 20th to December 21st) of the reproductive season in the Assaf breed for the last four years, and 2) we aimed to identify male factors influencing the different reproductive success obtained depending on the time at the mating season in which ovine artificial insemination was performed. For this purpose, we evaluated certain ram reproductive and ultrasonographical parameters as well as we performed a multiparametric and proteomic sperm analysis of 6–19 rams at two very distant points in the mating season (July as Early Breeding Season –EBS– and November as Late Breeding Season –LBS–). Rutinary assessments carried out in the ovine reproduction centers (testicular volume, libido, sperm production and mass motility) showed non-significant differences (P ≥ 0.05) between both studied times, as well as the ram ultrasonographic evaluation (Resistive and Pulsatility Index as Doppler parameters; and pixels mean gray level, and hypoechoic areas percentage and density as echotexture parameters). However, at level of sperm functionality, although sperm quality appeared non-significantly lower (P ≥ 0.05) in the EBS, we identified a significantly different (P < 0.05) sperm proteomic profile between the seasonality points. The following proteins were identified with the lowest abundance in the EBS with a fold change > 4, a P = 2.40e-07, and a q = 2.23e-06: Fibrous Sheath-Interacting Protein 2, Disintegrin and Metalloproteinase Domain-Containing Protein 20-like, Phosphoinositide-Specific Phospholipase C, Tektin 5, Armadillo Repeat-Containing Protein 12 Isoform X3, Solute Carrier Family 9B1, Radial Spoke Head Protein 3 Homolog, Pro-Interleukin-16, NADH Dehydrogenase [Ubiquinone] 1 Alpha Subcomplex Subunit 8, Testis, Prostate and Placenta-Expressed Protein, and Acyl Carrier Protein Mitochondrial. In conclusion, while our basic analyses on male and sperm quality showed similar results between the beginning and the end of the breeding season, on a proteomic level we detected a lower expression of sperm proteins linked to the energy metabolism, sperm-oocyte interactions, and flagellum structure in the EBS. Probably, this different protein expression could be related to the lower fertility rate of Assaf ewes after cervical artificial insemination at this time. More importantly, sperm proteins can be used as highly effective molecular markers in predicting sperm fertilization ability related to intraseasonal variations.

Pathogenic gene variants in CCDC39, CCDC40, RSPH1, RSPH9, HYDIN, and SPEF2 cause defects of sperm flagella composition and male infertility.

Primary Ciliary Dyskinesia (PCD) is a rare genetic disorder affecting the function of motile cilia in several organ systems. In PCD, male infertility is caused by defective sperm flagella composition or deficient motile cilia function in the efferent ducts of the male reproductive system. Different PCD-associated genes encoding axonemal components involved in the regulation of ciliary and flagellar beating are also reported to cause infertility due to multiple morphological abnormalities of the sperm flagella (MMAF). Here, we performed genetic testing by next generation sequencing techniques, PCD diagnostics including immunofluorescence-, transmission electron-, and high-speed video microscopy on sperm flagella and andrological work up including semen analyses. We identified ten infertile male individuals with pathogenic variants in CCDC39 (one) and CCDC40 (two) encoding ruler proteins, RSPH1 (two) and RSPH9 (one) encoding radial spoke head proteins, and HYDIN (two) and SPEF2 (two) encoding CP-associated proteins, respectively. We demonstrate for the first time that pathogenic variants in RSPH1 and RSPH9 cause male infertility due to sperm cell dysmotility and abnormal flagellar RSPH1 and RSPH9 composition. We also provide novel evidence for MMAF in HYDIN- and RSPH1-mutant individuals. We show absence or severe reduction of CCDC39 and SPEF2 in sperm flagella of CCDC39- and CCDC40-mutant individuals and HYDIN- and SPEF2-mutant individuals, respectively. Thereby, we reveal interactions between CCDC39 and CCDC40 as well as HYDIN and SPEF2 in sperm flagella. Our findings demonstrate that immunofluorescence microscopy in sperm cells is a valuable tool to identify flagellar defects related to the axonemal ruler, radial spoke head and the central pair apparatus, thus aiding the diagnosis of male infertility. This is of particular importance to classify the pathogenicity of genetic defects, especially in cases of missense variants of unknown significance, or to interpret HYDIN variants that are confounded by the presence of the almost identical pseudogene HYDIN2.

The RSPH4A Gene in Primary Ciliary Dyskinesia.

The radial spoke head protein 4 homolog A (RSPH4A) gene is one of more than 50 genes that cause Primary ciliary dyskinesia (PCD), a rare genetic ciliopathy. Genetic mutations in the RSPH4A gene alter an important protein structure involved in ciliary pathogenesis. Radial spoke proteins, such as RSPH4A, have been conserved across multiple species. In humans, ciliary function deficiency caused by RSPH4A pathogenic variants results in a clinical phenotype characterized by recurrent oto-sino-pulmonary infections. More than 30 pathogenic RSPH4A genetic variants have been associated with PCD. In Puerto Rican Hispanics, a founder mutation (RSPH4A (c.921+3_921+6delAAGT (intronic)) has been described. The spectrum of the RSPH4A PCD phenotype does not include laterality defects, which results in a challenging diagnosis. PCD diagnostic tools can combine transmission electron microscopy (TEM), nasal nitric oxide (nNO), High-Speed Video microscopy Analysis (HSVA), and immunofluorescence. The purpose of this review article is to provide a comprehensive overview of current knowledge about the RSPH4A gene in PCD, ranging from basic science to human clinical phenotype.

Expression of RSPH6A in the first wave of rat spermatogenesis and oxidative stress conditions: Attenuation by melatonin.

Here, we report, for the first time, the temporal expression and localization of axonemal radial spoke head homolog A (RSPH6A) protein during the first wave of rat spermatogenesis and in oxidative stress conditions. For the developmental study, testes were collected from rats at different developmental stages (7, 14, 21, 28, 35, 42, and 60 postnatal days); for in vivo treatment, 24 rats were treated with cadmium and/or melatonin. From each sample, western blot (WB) and immunofluorescence (IF) analyses for RSPH6A were performed. RSPH6A expression starts at 21 PND alongside the appearance of I spermatocytes (SPC) with a significant increase up to 60 PND. Data were confirmed by IF analysis, showing that RPSH6A expression is restricted to I and II SPC, spermatids, and mature sperm. In vivo experiments showed that the expression and localization of RSPH6A in the testis and epididymal spermatozoa of adult rats treated with cadmium were impaired. Interestingly, melatonin (an antioxidant), given together with Cd, can counteract its damaging effects. All combined data confirm that RSPH6A contributes to the onset of fertility by acting on sperm motility, raising the possibility of using RSPH6A as a marker for normal fertility in the general population.

Motile Ciliary Disorders of the Nasal Epithelium in Adults With Bronchiectasis

Motile ciliary disorder (MCD) has been implicated in chronic inflammatory airway diseases such as asthma and COPD. What are the characteristics of MCD of the nasal epithelium and its association with disease severity and inflammatory endotypes in adults with bronchiectasis? In this observational study, we recruited 167 patients with bronchiectasis and 39 healthy control participants who underwent brushing of the nasal epithelium. A subgroup of patients underwent bronchoscopy for bronchial epithelium sampling (n= 13), elective surgery for bronchial epithelium biopsy (n= 18), and blood sampling for next-generation sequencing (n= 37). We characterized systemic and airway inflammatory endotypes in bronchiectasis. We conducted immunofluorescence assays to profile ultrastructural (dynein axonemal heavy chain 5 [DNAH5], dynein intermediate chain 1 [DNAI1], radial spoke head protein 9 [RSPH9]) and ciliogenesis marker expression (Ezrin). MCD was present in 89.8%of patients with bronchiectasis, 67.6%showed secondary MCD, and 16.2%showed primary plus secondary MCD. Compared with healthy control participants, patients with bronchiectasis yielded abnormal staining patterns of DNAH5, DNAI1, and RSPH9 (but not Ezrin) that were more prominent in moderate to severe bronchiectasis. MCD pattern scores largely were consistent between upper and lower airways and between large-to-medium and small airways in bronchiectasis. Coexisting nasal diseases and asthma did not confound nasal ciliary ultrastructural marker expression significantly. The propensity of MCD was unaffected by the airway or systemic inflammatory endotypes. MCD, particularly an ultrastructural abnormality, was notable in patients with mild bronchiectasis who showed blood or sputum eosinophilia. Nasal ciliary markers profiling provides complimentary information to clinical endotyping of bronchiectasis.

IQUB deficiency causes male infertility by affecting the activity of p-ERK1/2/RSPH3.

Can new genetic factors responsible for male infertility be identified, especially for those characterized by asthenospermia despite normal sperm morphology? We identified the novel pathogenetic gene IQ motif and ubiquitin-like domain-containing (IQUB) as responsible for male infertility characterized by asthenospermia, involving sperm radial spoke defects. To date, only a few genes have been found to be responsible for asthenospermia with normal sperm morphology. Iqub, encoding the IQUB protein, is highly and specifically expressed in murine testes and interacts with the proteins radial spoke head 3 (RSPH3), CEP295 N-terminal like (CEP295NL or DDC8), glutathione S-transferase mu 1 (GSTM1) and outer dense fiber of sperm tails 1 (ODF1) in the yeast two-hybrid system. The IQUB variant was identified by whole-exome sequencing in a cohort of 126 male infertility patients with typical asthenospermia recruited between 2015 and 2020. Knockout (KO) and knockin (KI) mouse models, scanning and transmission electron microscopy (TEM), and other functional assays were performed, between 2019 and 2021. The IQUB variant was identified by whole-exome sequencing and confirmed by Sanger sequencing. Iqub KO and KI mice were constructed to mimic the phenotype of the affected individual. After recapitulating the phenotype of human male infertility, scanning and TEM were performed to check the ultrastructure of the sperm. Western blot and co-immunoprecipitation were performed to clarify the pathological mechanism of the IQUB variant. We identified a homozygous nonsense IQUB variant (NM_001282855.2:c.942T> G(p.Tyr314*)) from an infertile male. Iqub KO and KI mice mimicked the infertility phenotype and confirmed IQUB to be the pathogenetic gene. Scanning and TEM showed that sperm of both the mouse models and the affected individual had radial spoke defects. The functional assay suggested that IQUB may recruit calmodulin in lower Ca2+ environments to facilitate the normal assembly of radial spokes by inhibiting the activity of RSPH3/p-ERK1/2 (a nontypical AKAP (A-Kinase Anchoring Protein) forming by RSPH3 and phosphorylation of extracellular signal-regulated kinase 1 and 2 (p-ERK1/2)). Additional cases are needed to confirm the genetic contribution of IQUB variants to male infertility. In addition, because no IQUB antibody is available for immunofluorescence and the polyclonal antibody we generated was only effective in western blotting, immunostaining for IQUB was not performed in this study. Therefore, this study lacks direct in vivo proof to confirm the effect of the variant on IQUB protein level. Our results suggest a causal relation between IQUB variants and male infertility owing to asthenospermia, and partly clarify the pathological mechanism of IQUB variants. This expands our knowledge of the genes involved in human sperm asthenospermia and potentially provides a new genetic marker for male infertility. This work was supported by the National Key Research and Development Program of China (2021YFC2700100), the National Natural Science Foundation of China (32130029, 82171643, 81971450, 82001538, and 81971382) and the Guangdong Science and Technology Department Guangdong-Hong Kong-Macao Joint Innovation Project (2020A0505140003). There are no competing interests to declare. N/A.

Novel RSPH4A Variants Associated With Primary Ciliary Dyskinesia-Related Infertility in Three Chinese Families.

Background: The radial spoke head component 4A (RSPH4A) is involved in the assembly of radial spokes, which is essential for motile cilia function. Asthenoteratozoospermia in primary ciliary dyskinesia (PCD) related to RSPH4A variants has not been reported. Materials and Methods: RSPH4A variants were identified and validated using whole-exome and Sanger sequencing in three unrelated Chinese families. High-speed video microscopy analysis (HSVA) was performed to measure the beating frequency and pattern of nasal cilia of the patients and healthy control. Papanicolaou staining and computer-aided sperm analysis were performed to analyze the morphology and motility of the sperm in patient 1. Immunofluorescence was adopted to confirm the structure deficiency of sperm and nasal cilia. Results: Patient 1 from family 1 is a 22-year-old unmarried male presented with bronchiectasis. Semen analysis and sperm Papanicolaou staining confirmed asthenoteratozoospermia. Novel compound heterozygous RSPH4A variants c.2T>C, p.(Met1Thr) and c.1774_1775del, p.(Leu592Aspfs*5) were detected in this patient. Patients 2 and 3 are from two unrelated consanguineous families; they are both females and exhibited bronchiectasis and infertility. Two homozygous RSPH4A variants c.2T>C, p.(Met1Thr) and c.351dupT, p.(Pro118Serfs*2) were detected, respectively. HSVA showed that most of the cilia in patients 1 and 3 were with abnormal rotational movement. The absence of RSPH4A and RSPH1 in patient 1’s sperm and patient 3’s respiratory cilia was indicated by immunofluorescence. Patient 2 died of pulmonary infection and respiratory failure at the age of 35 during follow-up. Conclusion: Dysfunctional sperm flagellum and motile cilia in the respiratory tract and the fallopian tube were found in patients with RSPH4A variants. Our study enriches the genetic spectrum and clinical phenotypes of RSPH4A variants in PCD, and c.2T>C, p.(Met1Thr) detected in our patients may be a hotspot RSPH4A variant in Chinese.

Motile Cilia on Kidney Proximal Tubular Epithelial Cells Are Associated With Tubular Injury and Interstitial Fibrosis.

It is well established that mammalian kidney epithelial cells contain a single non-motile primary cilium (9 + 0 pattern). However, we noted the presence of multiple motile cilia with a central microtubular pair (9 + 2 pattern) in kidney biopsies of 11 patients with various kidney diseases, using transmission electron microscopy. Immunofluorescence staining revealed the expression of the motile cilia-specific markers Radial Spoke Head Protein 4 homolog A, Forkhead-box-protein J1 and Regulatory factor X3. Multiciliated cells were exclusively observed in proximal tubuli and a relative frequent observation in human kidney tissue: in 16.7% of biopsies with tubular injury and atrophy (3 of 18 tissues), in 17.6% of biopsies from patients with membranous nephropathy (3 of 17 tissues) and in 10% of the human kidney tissues derived from the unaffected pole after tumour nephrectomy (3 of 30 tissues). However, these particular tissues showed marked tubular injury and fibrosis. Further analysis showed a significant relation between the presence of multiciliated cells and an increased expression of alpha-smooth-muscle-actin (p-value < 0.01) and presence of Kidney-injury-molecule-1 (p-value < 0.01). Interestingly, multiciliated cells co-showed staining for the scattered tubular cell markers annexin A2, annexin A3, vimentin and phosphofructokinase platelet but not with cell senescence associated markers, like (p16) and degradation of lamin B. In conclusion, multiciliated proximal tubular cells with motile cilia were frequently observed in kidney biopsies and associated with tubular injury and interstitial fibrosis. These data suggest that proximal tubular cells are able to transdifferentiate into multiciliated cells.

CFAP61 is required for sperm flagellum formation and male fertility in human and mouse

ABSTRACT Defects in the structure or motility of cilia and flagella may lead to severe diseases such as primary ciliary dyskinesia (PCD), a multisystemic disorder with heterogeneous manifestations affecting primarily respiratory and reproductive functions. We report that CFAP61 is a conserved component of the calmodulin- and radial spoke-associated complex (CSC) of cilia. We find that a CFAP61 splice variant, c.143+5G&amp;gt;A, causes exon skipping/intron retention in human, inducing a multiple morphological abnormalities of the flagella (MMAF) phenotype. We generated Cfap61 knockout mice that recapitulate the infertility phenotype of the human CFAP61 mutation, but without other symptoms usually observed in PCD. We find that CFAP61 interacts with the CSC, radial spoke stalk and head. During early stages of Cfap61−/− spermatid development, the assembly of radial spoke components is impaired. As spermiogenesis progresses, the axoneme in Cfap61−/− cells becomes unstable and scatters, and the distribution of intraflagellar transport proteins is disrupted. This study reveals an organ-specific mechanism of axoneme stabilization that is related to male infertility.

Sperm flagellar 2 (SPEF2) is essential for sperm flagellar assembly in humans.

Spermiogenesis is a complex and tightly regulated process, consisting of acrosomal biogenesis, condensation of chromatin, flagellar assembly, and disposal of extra cytoplasm. Previous studies have reported that sperm flagellar 2 (SPEF2) deficiency causes severe asthenoteratozoospermia owing to spermiogenesis failure, but the underlying molecular mechanism in humans remains unclear. Here, we performed proteomic analysis on spermatozoa from three SPEF2 mutant patients to study the functional role of SPEF2 during sperm tail development. A total of 1262 differentially expressed proteins were detected, including 486 upregulated and 776 downregulated. The constructed heat map of the differentially expressed proteins showed similar trends. Among these, the expression of proteins related to flagellar assembly, including SPEF2, sperm associated antigen 6 (SPAG6), dynein light chain tctex-type 1 (DYNLT1), radial spoke head component 1 (RSPH1), translocase of outer mitochondrial membrane 20 (TOM20), EF-hand domain containing 1 (EFHC1), meiosis-specific nuclear structural 1 (MNS1) and intraflagellar transport 20 (IFT20), was verified by western blot. Functional clustering analysis indicated that these differentially expressed proteins were specifically enriched for terms such as spermatid development and flagellar assembly. Furthermore, we showed that SPEF2 interacts with radial spoke head component 9 (RSPH9) and IFT20 in vitro, which are well-studied components of radial spokes or intra-flagellar transport and are essential for flagellar assembly. These results provide a rich resource for further investigation into the molecular mechanism underlying the role that SPEF2 plays in sperm tail development and could provide a theoretical basis for gene therapy in SPEF2 mutant patients in the future.

Distinct architecture and composition of mouse axonemal radial spoke head revealed by cryo-EM

The radial spoke (RS) heads of motile cilia and flagella contact projections of the central pair (CP) apparatus to coordinate motility, but the morphology is distinct for protozoa and metazoa. Here we show the murine RS head is compositionally distinct from that of Chlamydomonas Our reconstituted murine RS head core complex consists of Rsph1, Rsph3b, Rsph4a, and Rsph9, lacking Rsph6a and Rsph10b, whose orthologs exist in the protozoan RS head. We resolve its cryo-electron microscopy (cryo-EM) structure at 3.2-Å resolution. Our atomic model further reveals a twofold symmetric brake pad-shaped structure, in which Rsph4a and Rsph9 form a compact body extended laterally with two long arms of twisted Rsph1 β-sheets and potentially connected dorsally via Rsph3b to the RS stalk. Furthermore, our modeling suggests that the core complex contacts the periodic CP projections either rigidly through its tooth-shaped Rsph4a regions or elastically through both arms for optimized RS-CP interactions and mechanosignal transduction.

Structure of the radial spoke head and insights into its role in mechanoregulation of ciliary beating.

Motile cilia power cell locomotion and drive extracellular fluid flow by propagating bending waves from their base to tip. The coordinated bending of cilia requires mechanoregulation by the radial spoke (RS) protein complexes and the microtubule central pair (CP). Despite their importance for ciliary motility across eukaryotes, the molecular function of the RSs is unknown. Here, we reconstituted the Chlamydomonas reinhardtii RS head that abuts the CP and determined its structure using single-particle cryo-EM to 3.1 Å resolution, revealing a flat, negatively-charged surface supported by a rigid core of tightly intertwined proteins. Mutations in this core, corresponding to those involved in human ciliopathies, compromised stability of the recombinant complex, providing a molecular basis for disease. Partially reversing the negative charge on the RS surface impaired motility in C. reinhardtii. We propose that the RS head architecture is well-suited for mechanoregulation of ciliary beating through physical collisions with the CP.

Super-Resolution Microscopy and FIB-SEM Imaging Reveal Parental Centriole-Derived, Hybrid Cilium in Mammalian Multiciliated Cells.

Motile cilia are cellular beating machines that play a critical role in mucociliary clearance, cerebrospinal fluid movement, and fertility. In the airways, hundreds of motile cilia present on the surface of a multiciliated epithelia cell beat coordinately to protect the epithelium from bacteria, viruses, and harmful particulates. During multiciliated cell differentiation, motile cilia are templated from basal bodies, each extending a basal foot-an appendage linking motile cilia together to ensure coordinated beating. Here, we demonstrate that among the many motile cilia of a multiciliated cell, a hybrid cilium with structural features of both primary and motile cilia is harbored. The hybrid cilium is conserved in mammalian multiciliated cells, originates from parental centrioles, and its cellular position is biased and dependent on ciliary beating. Furthermore, we show that the hybrid cilium emerges independently of other motile cilia and functions in regulating basal body alignment.

The structure and symmetry of the radial spoke protein complex in Chlamydomonas flagella.

The radial spoke is a key element in a transducer apparatus controlling the motility of eukaryotic cilia. The transduction biomechanics is a long-standing question in cilia biology. The radial spoke has three regions - a spoke head, a bifurcated neck and a stalk. Although the neck and the stalk are asymmetric, twofold symmetry of the head has remained controversial. In this work we used single particle cryo-electron microscopy (cryo-EM) analysis to generate a 3D structure of the whole radial spoke at unprecedented resolution. We show the head region at 15 Å (1.5 nm) resolution and confirm twofold symmetry. Using distance constraints generated by cross-linking mass spectrometry, we locate two components, RSP2 and RSP4, at the head and neck regions. Our biophysical analysis of isolated RSP4, RSP9, and RSP10 affirmed their oligomeric state. Our results enable us to redefine the boundaries of the regions and propose a model of organization of the radial spoke component proteins.

Genes with evidence of positive selection as potentially related to coloniality and the evolution of morphological features among the lophophorates and entoprocts.

Evolutionary mechanisms that underlie the origins of coloniality among organisms are diverse. Some animal colonies may be comprised strictly of clonal individuals formed from asexual budding or comprised of a chimera of clonal and sexually produced individuals that fuse secondarily. This investigation focuses on select members of the lophophorates and entoprocts whose evolutionary relationships remain enigmatic even in the age of genomics. Using transcriptomic data sets, two coloniality-based hypotheses are tested in a phylogenetic context to find candidate genes showing evidence of positive selection and potentially convergent molecular signatures among solitary species and taxa-forming colonies from aggregate groups or clonal budding. Approximately 22% of the 387 orthogroups tested showed evidence of positive selection in at least one of the three branch-site tests (CODEML, BUSTED, and aBSREL). Only 12 genes could be reliably associated with a developmental function related to traits linked with coloniality, neuroanatomy, or ciliary fields. Genes testing for both positive selection and convergent molecular characters include orthologues of Radial spoke head, Elongation translation initiation factors, SEC13, and Immediate early response gene5. Maximum likelihood analyses included here resulted in tree topologies typical of other phylogenetic investigations based on wider genomic information. Further genomic and experimental evidence will be needed to resolve whether a solitary ancestor with multiciliated cells that formed aggregate groups gave rise to colonial forms in bryozoans (and perhaps the entoprocts) or that the morphological differences exhibited by phoronids and brachiopods represent trait modifications from a colonial ancestor.

Design and Simulation Analysis of Cutter in New Sewer Dredging Device

The existing sewer dredging method can not meet the needs of high-speed operation of the city. Through the research and analysis of the cutterhead of the shield tunneling machine, combined with the requirements of the sewer environment and dredging work, three-dimensional model, Ansys simulation analysis and physical experiments are adopted. In this way, a new type of spoke panel cutter head was designed. The cutter head adopts a new type of scraper. The structure is exquisite and the assembly is simple. It can effectively clear the sediment in the sewer environment and complete the initial recovery of the silt.

Rsph4a is essential for the triplet radial spoke head assembly of the mouse motile cilia.

Motile cilia/flagella are essential for swimming and generating extracellular fluid flow in eukaryotes. Motile cilia harbor a 9+2 arrangement consisting of nine doublet microtubules with dynein arms at the periphery and a pair of singlet microtubules at the center (central pair). In the central system, the radial spoke has a T-shaped architecture and regulates the motility and motion pattern of cilia. Recent cryoelectron tomography data reveal three types of radial spokes (RS1, RS2, and RS3) in the 96 nm axoneme repeat unit; however, the molecular composition of the third radial spoke, RS3 is unknown. In human pathology, it is well known mutation of the radial spoke head-related genes causes primary ciliary dyskinesia (PCD) including respiratory defect and infertility. Here, we describe the role of the primary ciliary dyskinesia protein Rsph4a in the mouse motile cilia. Cryoelectron tomography reveals that the mouse trachea cilia harbor three types of radial spoke as with the other vertebrates and that all triplet spoke heads are lacking in the trachea cilia of Rsph4a-deficient mice. Furthermore, observation of ciliary movement and immunofluorescence analysis indicates that Rsph4a contributes to the generation of the planar beating of motile cilia by building the distal architecture of radial spokes in the trachea, the ependymal tissues, and the oviduct. Although detailed mechanism of RSs assembly remains unknown, our results suggest Rsph4a is a generic component of radial spoke heads, and could explain the severe phenotype of human PCD patients with RSPH4A mutation.

An Integrated Analysis of Radial Spoke Head and Outer Dynein Arm Protein Defects and Ciliogenesis Abnormality in Nasal Polyps.

Background: Nasal polyp (NP) is a chronic upper airway inflammatory disease that is frequently triggered by defective host-defense. However, the mechanisms underlying the impaired barrier function such as cilia-mediated mucociliary clearance remain poorly understood. Objective: To assess ciliary ultrastructural and ciliogenesis marker expression and the phenotypes of ciliated cells in NP. Methods: NP biopsy samples were obtained from 97 NP patients and inferior turbinate from 32 healthy controls. Immunofluorescence staining, quantitative polymerase chain reaction, and single-cell cytospin staining were performed. We classified the patterns of radial spoke head protein (RSPH) 1, 4A (RSPH4A), 9 (RSPH9), and dynein axonemal heavy chain 5 (DNAH5) localization. A semi-quantitative scoring system was developed to assess their expression patterns and associations with ciliogenesis markers [centrosomal protein 110 (CP110) and forkhead box j1 (FOXJ1)]. Results: Median scores of RSPH1, RSPH4A, RSPH9, and DNAH5 were significantly higher in NP than in healthy controls, particularly in eosinophilic NPs. Expression pattern scores of RSPH1, RSPH4A, RSPH9, and DNAH5 correlated positively with each other in both groups. In primary-cell specimens, abnormal expression patterns were significantly more common in NP. The total fluorescence intensity of CP110 and FOXJ1 was significantly higher in NPs and correlated positively with expression pattern scores of RSPH1, RSPH4A, RSPH9, and DNAH5. A trend towards lengthened cilia was observed in NP. Conclusion: In the chronic airway inflammatory milieu, the up-regulated ciliogenesis correlates with the abnormal expression of ciliary ultrastructural markers (i.e., DNAH5) in NP (particularly eosinophilic NP).

Mice with a Deletion of Rsph1 Exhibit a Low Level of Mucociliary Clearance and Develop a Primary Ciliary Dyskinesia Phenotype.

Primary ciliary dyskinesia (PCD) is a genetically and phenotypically heterogeneous disease caused by mutations in over 40 different genes. Individuals with PCD caused by mutations in RSPH1 (radial spoke head 1 homolog) have been reported to have a milder phenotype than other individuals with PCD, as evidenced by a lower incidence of neonatal respiratory distress, higher nasal nitric oxide concentrations, and better lung function. To better understand genotype-phenotype relationships in PCD, we have characterized a mutant mouse model with a deletion of Rsph1. Approximately 50% of cilia from Rsph1-/- cells appeared normal by transmission EM, whereas the remaining cilia revealed a range of defects, primarily transpositions or a missing central pair. Ciliary beat frequency in Rsph1-/- cells was significantly lower than in control cells (20.2 ± 0.8 vs. 25.0 ± 0.9 Hz), and the cilia exhibited an aberrant rotational waveform. Young Rsph1-/- animals demonstrated a low rate of mucociliary clearance in the nasopharynx that was reduced to zero by about 1 month of age. Rsph1-/- animals accumulated mucus in the nasal cavity but had a lower bacterial burden than animals with a deletion of dynein axonemal intermediate chain 1 (Dnaic1-/-). Thus, Rsph1-/- mice display a PCD phenotype similar to but less severe than that observed in Dnaic1-/- mice, similar to what has been observed in humans. The results suggest that some individuals with PCD may not have a complete loss of mucociliary clearance and further suggest that early diagnosis and intervention may be important to maintain this low amount of clearance.

Wide phenotypic variability in RSPH9-associated primary ciliary dyskinesia: review of a case-series from Cyprus.

Primary ciliary dyskinesia (PCD) is an inherited ciliary motility disorder caused by mutations in at least 40 genes. RSPH9 gene mutations encoding aberrant radial spoke head proteins have been linked with PCD. The clinical spectrum extent of RSPH9 gene mutations remains to date largely unknown. We aimed to describe the diagnostic and clinical phenotype in a case-series of RSPH9-associated PCD. We performed whole exome sequencing in suspect patients from Cyprus who on repeated cilia biopsies demonstrated loss of the central pair apparatus on Transmission Electron Microscopy (TEM) and rotary beating patterns on High Speed Video Microscopy (HSVM), compatible to findings described previously in PCD patients bearing pathogenic RSPH9 mutations. In cases confirmed by genetic testing, we reviewed diagnostic, demographic and clinical data, as well as anthropometric and spirometric measurements. We diagnosed 7 individuals (5 females) homozygous for the novel RSPH9 splice site mutation c.670+2T>C in intron 4, who originated from two families. Despite bearing the same genetic variant, patients presented a highly variable age (median 47.9 years; range, 6.6 to 51.4 years) and with a diverse clinical picture, all reporting a history of chronic or recurrent wet cough (100%), and at varying frequencies neonatal respiratory distress (43%), chronic rhinosinusitis (71%), and wheezing (43%). Complications such as bronchiectasis (71%), history of pneumonia(s) (57%) and surgical interventions (43%) clustered in some patients displaying typical PCD, but not in others with milder phenotypes. BMI-z scores (median: 0.53; range, -0.69 to 1.52), FEV1-z scores (median: -0.37; range: -1.79 to 0.22) and FVC z-scores (median: -0.80; range: -2.01 to 0.36) were on average within the normal range, although slightly reduced. In conclusion, RSPH9-associated PCD disease demonstrates wide phenotypic variability. In some cases, mild clinical presentation is difficult to justify diagnostic work-up, highlighting the importance of wider adoption of genetic diagnostics. Larger studies are needed to assess variability of clinical spectrum associated to alterations of PCD genes.