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Cellular & Molecular Biology Letters May 2024By analyzing a mouse Interspecific Recombinant Congenic Strain (IRCS), we previously identified a quantitative trait locus (QTL), called Mafq1 on mouse chromosome 1,...
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.
Topics: Animals; Male; Infertility, Male; Sperm Motility; Sperm Tail; Mice; Mice, Knockout; Spermatozoa; Spermatogenesis; Flagella; Mice, Inbred C57BL; CRISPR-Cas Systems
PubMed: 38750428
DOI: 10.1186/s11658-024-00587-5 -
BMC Genomics May 2024The severity of COVID-19 is influenced by various factors including the presence of respiratory diseases. Studies have indicated a potential relationship between asthma...
BACKGROUND
The severity of COVID-19 is influenced by various factors including the presence of respiratory diseases. Studies have indicated a potential relationship between asthma and COVID-19 severity.
OBJECTIVE
This study aimed to conduct a genome-wide association study (GWAS) to identify genetic and clinical variants associated with the severity of COVID-19, both among patients with and without asthma.
METHODS
We analyzed data from 2131 samples sourced from the Biobanque québécoise de la COVID-19 (BQC19), with 1499 samples from patients who tested positive for COVID-19. Among these, 1110 exhibited mild-to-moderate symptoms, 389 had severe symptoms, and 58 had asthma. We conducted a comparative analysis of clinical data from individuals in these three groups and GWAS using a logistic regression model. Phenotypic data analysis resulted in the refined covariates integrated into logistic models for genetic studies.
RESULTS
Considering a significance threshold of 1 × 10, seven genetic variants were associated with severe COVID-19. These variants were located proximal to five genes: sodium voltage-gated channel alpha subunit 1 (SCN10A), desmoplakin (DSP), RP1 axonemal microtubule associated (RP1), IGF like family member 1 (IGFL1), and docking protein 5 (DOK5). The GWAS comparing individuals with severe COVID-19 with asthma to those without asthma revealed four genetic variants in transmembrane protein with EGF like and two follistatin like domains 2 (TMEFF2) and huntingtin interacting protein-1 (HIP1) genes.
CONCLUSION
This study provides significant insights into the genetic profiles of patients with severe forms of the disease, whether accompanied by asthma or not. These findings enhance our comprehension of the genetic factors that affect COVID-19 severity.
KEY MESSAGES
Seven genetic variants were associated with the severe form of COVID-19; Four genetic variants were associated with the severe form of COVID-19 in individuals with comorbid asthma; These findings help define the genetic component of the severe form of COVID-19 in relation to asthma as a comorbidity.
Topics: Humans; COVID-19; Asthma; Genome-Wide Association Study; Male; Female; Middle Aged; SARS-CoV-2; Comorbidity; Adult; Severity of Illness Index; Cohort Studies; Polymorphism, Single Nucleotide; Aged; Genomics; Genetic Predisposition to Disease
PubMed: 38750426
DOI: 10.1186/s12864-024-10342-x -
Current Biology : CB Jun 2024A primary cilium is a membrane-bound extension from the cell surface that contains receptors for perceiving and transmitting signals that modulate cell state and...
A primary cilium is a membrane-bound extension from the cell surface that contains receptors for perceiving and transmitting signals that modulate cell state and activity. Primary cilia in the brain are less accessible than cilia on cultured cells or epithelial tissues because in the brain they protrude into a deep, dense network of glial and neuronal processes. Here, we investigated cilia frequency, internal structure, shape, and position in large, high-resolution transmission electron microscopy volumes of mouse primary visual cortex. Cilia extended from the cell bodies of nearly all excitatory and inhibitory neurons, astrocytes, and oligodendrocyte precursor cells (OPCs) but were absent from oligodendrocytes and microglia. Ultrastructural comparisons revealed that the base of the cilium and the microtubule organization differed between neurons and glia. Investigating cilia-proximal features revealed that many cilia were directly adjacent to synapses, suggesting that cilia are poised to encounter locally released signaling molecules. Our analysis indicated that synapse proximity is likely due to random encounters in the neuropil, with no evidence that cilia modulate synapse activity as would be expected in tetrapartite synapses. The observed cell class differences in proximity to synapses were largely due to differences in external cilia length. Many key structural features that differed between neuronal and glial cilia influenced both cilium placement and shape and, thus, exposure to processes and synapses outside the cilium. Together, the ultrastructure both within and around neuronal and glial cilia suggest differences in cilia formation and function across cell types in the brain.
Topics: Animals; Cilia; Mice; Microscopy, Electron, Transmission; Mice, Inbred C57BL; Neurons; Visual Cortex; Neuroglia; Female; Synapses; Male
PubMed: 38749425
DOI: 10.1016/j.cub.2024.04.043 -
BioRxiv : the Preprint Server For... Apr 2024Proper connection between the sperm head and tail is critical for sperm motility and fertilization. The link between the head and tail is mediated by the Head-Tail...
Proper connection between the sperm head and tail is critical for sperm motility and fertilization. The link between the head and tail 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 not well understood. Here, we use to create a high-resolution map of proteins and structures at the HTCA throughout spermiogenesis. 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 is inserted, or embedded into the surface of the nucleus. As development progresses, the centriole is laterally displaces to the side of the nucleus, during which time 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 and functions as a critical stabilizer of the centriole-nuclear attachment. Together, our data indicate that the HTCA is 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.
PubMed: 38712096
DOI: 10.1101/2024.04.15.589606 -
Journal of Cell Science May 2024DPF3, along with other subunits, is a well-known component of the BAF chromatin remodeling complex, which plays a key role in regulating chromatin remodeling activity...
DPF3, along with other subunits, is a well-known component of the BAF chromatin remodeling complex, which plays a key role in regulating chromatin remodeling activity and gene expression. Here, we elucidated a non-canonical localization and role for DPF3. We showed that DPF3 dynamically localizes to the centriolar satellites in interphase and to the centrosome, spindle midzone and bridging fiber area, and midbodies during mitosis. Loss of DPF3 causes kinetochore fiber instability, unstable kinetochore-microtubule attachment and defects in chromosome alignment, resulting in altered mitotic progression, cell death and genomic instability. In addition, we also demonstrated that DPF3 localizes to centriolar satellites at the base of primary cilia and is required for ciliogenesis by regulating axoneme extension. Taken together, these findings uncover a moonlighting dual function for DPF3 during mitosis and ciliogenesis.
Topics: Animals; Humans; Mice; Axoneme; Centrioles; Centrosome; Cilia; DNA-Binding Proteins; Genomic Instability; HeLa Cells; Kinetochores; Mitosis; Spindle Apparatus; Transcription Factors
PubMed: 38661008
DOI: 10.1242/jcs.261744 -
Nature Communications Apr 2024Intraflagellar transport (IFT) orchestrates entry of proteins into primary cilia. At the ciliary base, assembled IFT trains, driven by kinesin-2 motors, can transport...
Intraflagellar transport (IFT) orchestrates entry of proteins into primary cilia. At the ciliary base, assembled IFT trains, driven by kinesin-2 motors, can transport cargo proteins into the cilium, across the crowded transition zone. How trains assemble at the base and how proteins associate with them is far from understood. Here, we use single-molecule imaging in the cilia of C. elegans chemosensory neurons to directly visualize the entry of kinesin-2 motors, kinesin-II and OSM-3, as well as anterograde cargo proteins, IFT dynein and tubulin. Single-particle tracking shows that IFT components associate with trains sequentially, both in time and space. Super-resolution maps of IFT components in wild-type and mutant worms reveal ciliary ultrastructure and show that kinesin-II is essential for axonemal organization. Finally, imaging cilia lacking kinesin-II and/or transition zone function uncovers the interplay of kinesin-II and OSM-3 in driving efficient transport of IFT trains across the transition zone.
Topics: Caenorhabditis elegans; Animals; Cilia; Caenorhabditis elegans Proteins; Kinesins; Flagella; Tubulin; Axoneme; Dyneins; Biological Transport; Single Molecule Imaging; Protein Transport
PubMed: 38658528
DOI: 10.1038/s41467-024-47807-2 -
Human Reproduction Open 2024Is the mutation causative for male infertility?
STUDY QUESTION
Is the mutation causative for male infertility?
SUMMARY ANSWER
Our collected data underline the complex and devastating effect of the single-gene mutation on the testicular molecular network, leading to male reproductive failure.
WHAT IS KNOWN ALREADY
Recent data have revealed mutations in genes related to axonemal dynein arms as causative for morphology and motility abnormalities in spermatozoa of infertile males, including dysplasia of fibrous sheath (DFS) and multiple morphological abnormalities in the sperm flagella (MMAF). The nexin-dynein regulatory complex (N-DRC) coordinates the dynein arm activity and is built from the DRC1-DRC7 proteins. DRC5 (TCTE1), one of the N-DRC elements, has already been reported as a candidate for abnormal sperm flagella beating; however, only in a restricted manner with no clear explanation of respective observations.
STUDY DESIGN SIZE DURATION
Using the CRISPR/Cas9 genome editing technique, a mouse gene knockout line was created on the basis of the C57Bl/6J strain. The mouse reproductive potential, semen characteristics, testicular gene expression levels, sperm ATP, and testis apoptosis level measurements were then assessed, followed by visualization of N-DRC proteins in sperm, and protein modeling . Also, a pilot genomic sequencing study of samples from human infertile males (n = 248) was applied for screening of variants.
PARTICIPANTS/MATERIALS SETTING METHODS
To check the reproductive potential of KO mice, adult animals were crossed for delivery of three litters per caged pair, but for no longer than for 6 months, in various combinations of zygosity. All experiments were performed for wild-type (WT, control group), heterozygous and homozygous male mice. Gross anatomy was performed on testis and epididymis samples, followed by semen analysis. Sequencing of RNA (RNAseq; Illumina) was done for mice testis tissues. STRING interactions were checked for protein-protein interactions, based on changed expression levels of corresponding genes identified in the mouse testis RNAseq experiments. Immunofluorescence staining was performed to detect the N-DRC complex proteins: Tcte1 (Drc5), Drc7, Fbxl13 (Drc6), and Eps8l1 (Drc3) in mouse spermatozoa. To determine the amount of ATP in spermatozoa, the luminescence level was measured. In addition, immunofluorescence staining was performed to check the level of apoptosis via caspase 3 visualization on mouse testis samples. DNA from whole blood samples of infertile males (n = 137 with non-obstructive azoospermia or cryptozoospermia, n = 111 samples with a spectrum of oligoasthenoteratozoospermia, including n = 47 with asthenozoospermia) was extracted to perform genomic sequencing (WGS, WES, or Sanger). Protein prediction modeling of human-identified variants and the exon 3 structure deleted in the mouse knockout was also performed.
MAIN RESULTS AND THE ROLE OF CHANCE
No progeny at all was found for the homozygous males which were revealed to have oligoasthenoteratozoospermia, while heterozygous animals were fertile but manifested oligozoospermia, suggesting haploinsufficiency. RNA-sequencing of the testicular tissue showed the influence of mutations on the expression pattern of 21 genes responsible for mitochondrial ATP processing or linked with apoptosis or spermatogenesis. In males, the protein was revealed in only residual amounts in the sperm head nucleus and was not transported to the sperm flagella, as were other N-DRC components. Decreased ATP levels (2.4-fold lower) were found in the spermatozoa of homozygous mice, together with disturbed tail:midpiece ratios, leading to abnormal sperm tail beating. Casp3-positive signals (indicating apoptosis) were observed in spermatogonia only, at a similar level in all three mouse genotypes. Mutation screening of human infertile males revealed one novel and five ultra-rare heterogeneous variants (predicted as disease-causing) in 6.05% of the patients studied. Protein prediction modeling of identified variants revealed changes in the protein surface charge potential, leading to disruption in helix flexibility or its dynamics, thus suggesting disrupted interactions of TCTE1 with its binding partners located within the axoneme.
LARGE SCALE DATA
All data generated or analyzed during this study are included in this published article and its supplementary information files. RNAseq data are available in the GEO database (https://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE207805. The results described in the publication are based on whole-genome or exome sequencing data which includes sensitive information in the form of patient-specific germline variants. Information regarding such variants must not be shared publicly following European Union legislation, therefore access to raw data that support the findings of this study are available from the corresponding author upon reasonable request.
LIMITATIONS REASONS FOR CAUTION
In the study, the fertilization performance of sperm from homozygous male mice was not checked.
WIDER IMPLICATIONS OF THE FINDINGS
This study contains novel and comprehensive data concerning the role of in male infertility. The gene is the next one that should be added to the 'male infertility list' because of its crucial role in spermatogenesis and proper sperm functioning.
STUDY FUNDING/COMPETING INTERESTS
This work was supported by National Science Centre in Poland, grants no.: 2015/17/B/NZ2/01157 and 2020/37/B/NZ5/00549 (to M.K.), 2017/26/D/NZ5/00789 (to A.M.), and HD096723, GM127569-03, NIH SAP #4100085736 PA DoH (to A.N.Y.). The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
PubMed: 38650655
DOI: 10.1093/hropen/hoae020 -
Phenomics (Cham, Switzerland) Feb 2024Primary ciliary dyskinesia (PCD) is a rare disorder characterized by extensive genetic heterogeneity. However, in the genetic pathogenesis of PCD, copy number...
UNLABELLED
Primary ciliary dyskinesia (PCD) is a rare disorder characterized by extensive genetic heterogeneity. However, in the genetic pathogenesis of PCD, copy number variation (CNV) has not received sufficient attention and has rarely been reported, especially in China. Next-generation sequencing (NGS) followed by targeted CNV analysis was used in patients highly suspected to have PCD with negative results in routine whole-exome sequencing (WES) analysis. Quantitative real-time polymerase chain reaction (qPCR) and Sanger sequencing were used to confirm these CNVs. To further characterize the ciliary phenotypes, high-speed video microscopy analysis (HSVA), transmission electron microscopy (TEM), and immunofluorescence (IF) analysis were used. Patient 1 (F1: II-1), a 0.6-year-old girl, came from a nonconsanguineous family-I. She presented with situs inversus totalis, neonatal respiratory distress, and sinusitis. The nasal nitric oxide level was markedly reduced. The respiratory cilia beat with reduced amplitude. TEM revealed shortened outer dynein arms (ODA) of cilia. chr5:13717907-13722661del spanning exons 71-72 was identified by NGS-based CNV analysis. Patient 2 (F2: IV-4), a 37-year-old man, and his eldest brother Patient 3 (F2: IV-2) came from a consanguineous family-II. Both had sinusitis, bronchiectasis and situs inversus totalis. The respiratory cilia of Patient 2 and Patient 3 were found to be uniformly immotile, with ODA defects. Two novel homozygous deletions chr5:13720087_13733030delinsGTTTTC and chr5:13649539_1 3707643del, spanning exons 69-71 and exons 77-79 were identified by NGS-based CNV analysis. Abnormalities in DNA copy number were confirmed by qPCR amplification. IF showed that the respiratory cilia of Patient 1 and Patient 2 were deficient in dynein axonemal heavy chain 5 (DNAH5) protein expression. This report identified three novel disease-associated variants by WES-based CNV analysis. Our study expands the genetic spectrum of PCD with in the Chinese population.
SUPPLEMENTARY INFORMATION
The online version contains supplementary material available at 10.1007/s43657-023-00130-0.
PubMed: 38605905
DOI: 10.1007/s43657-023-00130-0 -
Cellular and Molecular Life Sciences :... Apr 2024Mature spermatozoa with normal morphology and motility are essential for male reproduction. The epididymis has an important role in the proper maturation and function of...
Mature spermatozoa with normal morphology and motility are essential for male reproduction. The epididymis has an important role in the proper maturation and function of spermatozoa for fertilization. However, factors related to the processes involved in spermatozoa modifications are still unclear. Here we demonstrated that CCDC28A, a member of the CCDC family proteins, is highly expressed in testes and the CCDC28A deletion leads to male infertility. We found CCDC28A deletion had a mild effect on spermatogenesis. And epididymal sperm collected from Ccdc28a mice showed bent sperm heads, acrosomal defects, reduced motility and decreased in vitro fertilization competence whereas their axoneme, outer dense fibers, and fibrous sheath were all normal. Furthermore, we found that CCDC28A interacted with sperm acrosome membrane-associated protein 1 (SPACA1) and glycogen synthase kinase 3a (GSK3A), and deficiencies in both proteins in mice led to bent heads and abnormal acrosomes, respectively. Altogether, our results reveal the essential role of CCDC28A in regulating sperm morphology and motility and suggesting a potential marker for male infertility.
Topics: Male; Animals; Mice; Humans; Sperm Motility; Semen; Infertility, Male; Sperm Head; Spermatozoa
PubMed: 38597936
DOI: 10.1007/s00018-024-05184-5 -
Asian Journal of Andrology Jul 2024The second-to-fourth digit (2D:4D) ratio is thought to be associated with prenatal androgen exposure. However, the relationship between the 2D:4D ratio and hypospadias...
The second-to-fourth digit (2D:4D) ratio is thought to be associated with prenatal androgen exposure. However, the relationship between the 2D:4D ratio and hypospadias is poorly understood, and its molecular mechanism is not clear. In this study, by analyzing the hand digit length of 142 boys with hypospadias (23 distal, 68 middle, and 51 proximal) and 196 controls enrolled in Shanghai Children's Hospital (Shanghai, China) from December 2020 to December 2021, we found that the 2D:4D ratio was significantly increased in boys with hypospadias ( P < 0.001) and it was positively correlated with the severity of the hypospadias. This was further verified by the comparison of control mice and prenatal low testosterone mice model obtained by knocking out the risk gene (dynein axonemal heavy chain 8 [ DNAH8 ]) associated with hypospadias. Furthermore, the discrepancy was mainly caused by a shift in 4D. Proteomic characterization of a mouse model validated that low testosterone levels during pregnancy can impair the growth and development of 4D. Comprehensive mechanistic explorations revealed that during the androgen-sensitive window, the downregulation of the androgen receptor (AR) caused by low testosterone levels, as well as the suppressed expression of chondrocyte proliferation-related genes such as Wnt family member 5a ( Wnt5a ), Wnt5b , Smad family member 2 ( Smad2 ), and Smad3 ; mitochondrial function-related genes in cartilage such as AMP-activated protein kinase ( AMPK ) and nuclear respiratory factor 1 ( Nrf-1 ); and vascular development-related genes such as myosin light chain ( MLC ), notch receptor 3 ( Notch3 ), and sphingosine kinase 1 ( Sphk1 ), are responsible for the limitation of 4D growth, which results in a higher 2D:4D ratio in boys with hypospadias via decreased endochondral ossification. This study indicates that the ratio of 2D:4D is a risk marker of hypospadias and provides a potential molecular mechanism.
Topics: Hypospadias; Male; Animals; Humans; Fingers; Mice; Female; Testosterone; Receptors, Androgen; Pregnancy; Child, Preschool; Child; Case-Control Studies
PubMed: 38563741
DOI: 10.4103/aja202377