Oligogenic heterozygous inheritance of sperm abnormalities in mouse

Martinez, Guillaume; Coutton, Charles; Loeuillet, Corinne; Cazin, Caroline; Muroňová, Jana; Boguenet, Magalie; Lambert, Emeline; Dhellemmes, Magali; Chevalier, G.; Hograindleur, Jean-Pascal; Vilpreux, Charline; Neirijnck, Yasmine; Escoffier, Jessica; Nef, Serge; Ray, Pierre F.; Arnoult, Christophe

doi:10.7554/elife.75373

Cited by 19 publications

(15 citation statements)

References 61 publications

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“…Ccdc146 KO mice were generated using the CRISPR/Cas9 technology as previously described [53]. Briefly, to maximize the chances of generating deleterious mutations, two gRNAs located in two distinct coding exons located at the beginning of the targeted gene were used.…”

Section: Methodsmentioning

confidence: 99%

Lack of CCDC146, a ubiquitous centriole and microtubule-associated protein, leads to non-syndromic male infertility in human and mouse

Muroňová

Giordani

Eckert

et al. 2023

Preprint

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Genetic mutations are a recurrent cause of male infertility. Multiple morphological abnormalities of the flagellum (MMAF) syndrome is a heterogeneous genetic disease, with which more than 50 genes have been linked. Nevertheless, for 50% of patients with this condition, no genetic cause is identified. From a study of a cohort of 167 MMAF patients, pathogenic bi-allelic mutations were identified in theCCDC146gene in two patients. This gene encodes a poorly characterized centrosomal protein which we studied in detail here. First, protein localization was studied in two cell lines. We confirmed the centrosomal localization in somatic cells and showed that the protein also presents multiple microtubule-related localizations during mitotic division, suggesting that it is a microtubule-associated protein (MAP). To better understand the function of the protein at the sperm level, and the molecular pathogenesis of infertility associated withCCDC146mutations, two genetically modified mouse models were created: aCcdc146knock-out (KO) and a knock-in (KI) expressing a HA-tagged CCDC146 protein. KO male mice were completely infertile, and sperm exhibited a phenotype identical to our two MMAF patient’s phenotype withCCDC146mutations. No other pathology was observed, and the animals were viable. CCDC146 expression starts during late spermiogenesis, at the time of flagellum biogenesis. In the spermatozoon, the protein is conserved but is not localized to centrioles, unlike in somatic cells, rather it is present in the axoneme at the level of microtubule doublets. Expansion microscopy associated with the use of the detergent sarkosyl to solubilize microtubule doublets, provided evidence that the protein could be a microtubule inner protein (MIP). At the subcellular level, the absence of CCDC146 affected the formation, localization and morphology of all microtubule-based organelles such as the manchette, the head–tail coupling apparatus (HTCA), and the axoneme. Through this study, we have characterized a new genetic cause of infertility, identified a new factor in the formation and/or structure of the sperm axoneme, and demonstrated that the CCDC146 protein plays several cellular roles, depending on the cell type and the stages in the cell cycle.

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Section: Methodsmentioning

confidence: 99%

Lack of CCDC146, a ubiquitous centriole and microtubule-associated protein, leads to non-syndromic male infertility in human and mouse

Muroňová

Giordani

Eckert

et al. 2023

Preprint

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“…Since initial publication, it is now being used by other research groups (e.g. Stark-Dykema et al (2022), Martinez et al (2022)) to investigate the impact of genetic insults on sperm shape and fertility.…”

Section: Statement Of Needmentioning

confidence: 99%

Nuclear Morphology Analysis 2.0.0: Improved image analysis software for measuring nuclear shape

Skinner¹

2022

JOSS

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We generally imagine cells to have spherical or ellipsoidal nuclei, but many cell types are far more interesting, with great variation in nuclear shapes and sizes (Skinner & Johnson, 2017). Measuring and comparing shapes of cell nuclei in esoteric cell types such as sperm has traditionally been performed by manual assessment. Semi-automated approaches have been developed: geometric morphometric methods allow distinction of subtle phenotypic differences, as in the software MorphoJ (Klingenberg, 2011), but still require manual landmark annotation in each image (e.g. Varea-Sánchez et al. ( 2016)). Decomposing nuclear outlines into elliptic fourier descriptors is simpler and allows high-throughput analysis, as in the SHAPE software (Iwata & Ukai, 2002), but does not describe sharp corners well (e.g. Ostermeier et al. (2001), Mashiko et al. ( 2017)). Consequently, these methods have problems in reproducibility, scalability, or ease of use for biologists. At the other end of the spectrum are open source tools designed for high-throughput image processing such as CellProfiler (Stirling et al., 2021), with clear documentation and tutorials, but which do not have the ability to natively perform specialised morphometric analysis.Nuclear Morphology Analysis is a tool for measuring and comparing the shapes of cell nuclei. The software uses a modified Zahn-Roskies transformation (Zahn & Roskies, 1972) to convert the outlines of detected objects into linear profiles, with rules to identify landmarks of interest from these profiles. This approach allows high-throughput and reproducible analyses, detecting subtle variation in nuclear shape that is beyond the scope of manual assessment.

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“…It is known that mutations in genes encoding factors involved in ciliogenesis process, lead to defects in structure and/or functioning of cilia [Girardet et al , 2019; Sironen et al , 2020; Smith et al , 2021; Kumar and Reiter 2021] There is a plenty of data concerning respiratory cilia, however, still little is known about the axonemal dynein arms functioning in sperm tail. Latest data revealed mutations in genes related to axonemal dynein arms as causative for morphology and motility abnormalities in spermatozoa of infertile males [Girardet et al , 2019; Sironen et al , 2020; Aprea et al , 2021; Lorès et al , 2021; Touré et al , 2021; Martinez et al , 2022]. Two definitions were established: (i) for sperm cells with disorganization in axonemal structures, so called dysplasia of fibrous sheath (DFS) [Chemes et al , 1998] and (ii) for multiple morphological abnormalities in the sperm flagella (MMAF), concerning all possible defects of sperm tail, mainly short and thick flagella, and a lack of the central pair (CP) of microtubules or dynein arms [Khelifa et al , 2014].…”

Section: Introductionmentioning

confidence: 99%

“…The copyright holder for this this version posted November 25, 2022. ; https://doi.org/10.1101/2022.11.17.22282339 doi: medRxiv preprint Sironen et al, 2020;Aprea et al, 2021;Lorès et al, 2021;Touré et al, 2021;Martinez et al, 2022]. Two definitions were established: (i) for sperm cells with disorganization in axonemal structures, so called dysplasia of fibrous sheath (DFS) [Chemes et al, 1998] and (ii) for multiple morphological abnormalities in the sperm flagella (MMAF), concerning all possible defects of sperm tail, mainly short and thick flagella, and a lack of the central pair (CP) of microtubules or dynein arms [Khelifa et al, 2014].…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Tcte1knockout influence on energy chain transportation, apoptosis and spermatogenesis – implications for male infertility

Olszewska

Malcher

Stokowy

et al. 2022

Preprint

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STUDY QUESTION: Is Tcte1 mutation causative for male infertility? SUMMARY ANSWER: Collected data underline the complex and devastating effect of the single-gene mutation on testicular molecular network, leading to male reproductive failure. WHAT IS KNOWN ALREADY: Latest data 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 DRC1-DRC7 proteins. DRC5 (TCTE1) - one of N-DRC element, has been already reported as a candidate for abnormal sperm flagella beating, however, only in restricted manner with no clear explanation of respective observations. STUDY DESIGN, SIZE, DURATION: Using CRISPR/Cas9 genome editing technique, mouse knockout line of Tcte1 gene was created on the basis of C57Bl/6J strain. Then, the mouse reproductive potential, semen characteristics, testicular gene expression level, sperm ATP and testis apoptosis level measurements have been performed, followed by visualization of N-DRC proteins in sperm, and protein modeling in silico. Also, a pilot genomic sequencing study of samples from human infertile males (n=248) was applied for screening of TCTE1 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 no longer than for 6 months, in various combinations of zygosity. All experiments were performed for wild type (WT - control group), heterozygous Tcte1+/-, and homozygous Tcte1-/- male mice. Gross anatomy was performed on testis and epididymis, followed by semen analysis. Sequencing of RNA (RNAseq; Illumina) has been done for mice testis tissues. STRING interactions have been checked for protein-protein interactions, based on changed expression level of corresponding genes identified in the mouse testis RNAseq experiments. Immunofluorescence in situ staining was performed to detect the N-DRC complex proteins: Tcte1 (Drc5), Drc7, Fbxl13 (Drc6), and Eps8l1 (Drc3) in mouse spermatozoa. To determine the ATP amount in spermatozoa, the luminescence level was measured. Also, immunofluorescent in situ 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 non-obstructive azoospermia or cryptozoospermia, n=111 samples with spectrum of oligoasthenoteratozoospermia, including n=47 with asthenozoospermia) has been extracted to perform genomic sequencing (WGS, WES or Sanger). Protein prediction modeling of human identified variants and the exon 3 structure deleted in mouse knockout has been also performed. MAIN RESULTS AND THE ROLE OF CHANCE: No progeny at all was found for homozygous males with revealed oligoasthenoteratozoospermia, while heterozygous animals (fertile) manifested oligozoospermia, suggesting haploinsufficiency. RNA-sequencing of the testicular tissue showed the influence of Tcte1 mutations on the expression pattern of 21 genes responsible for mitochondrial ATP processing, linked with apoptosis, or spermatogenesis. In Tcte1-/- males the protein revealed only residual amounts in sperm head nucleus, and was not transported to sperm flagella, as other N-DRC components. Decreased ATP level (2.4-fold lower) was found in spermatozoa of homozygous mice, together with disturbed tail:midpiece ratio, leading to abnormal sperm tail beating. Casp3-positive signals (indicating apoptosis) were observed in spermatogonia only, at similar level in all three mouse genotypes. Mutation screening of human infertile males revealed 1 novel and 5 ultrarare heterogeneous variants (predicted as disease causing) in 6.05% of 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 the disrupted TCTE1 interaction with its binding partners located within the axoneme.

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Oligogenic heterozygous inheritance of sperm abnormalities in mouse

Cited by 19 publications

References 61 publications

Lack of CCDC146, a ubiquitous centriole and microtubule-associated protein, leads to non-syndromic male infertility in human and mouse

Lack of CCDC146, a ubiquitous centriole and microtubule-associated protein, leads to non-syndromic male infertility in human and mouse

Nuclear Morphology Analysis 2.0.0: Improved image analysis software for measuring nuclear shape

Tcte1knockout influence on energy chain transportation, apoptosis and spermatogenesis – implications for male infertility

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