A Homozygous Ancestral SVA-Insertion-Mediated Deletion in WDR66 Induces Multiple Morphological Abnormalities of the Sperm Flagellum and Male Infertility.

Multiple morphological abnormalities of the sperm flagellum (MMAF) is a severe form of male infertility defined by the presence of a mosaic of anomalies, including short, bent, curled, thick, or absent flagella, resulting from a severe disorganization of the axoneme and of the peri-axonemal structures. Mutations in DNAH1, CFAP43, and CFAP44, three genes encoding axoneme-related proteins, have been described to account for approximately 30% of the MMAF cases reported so far. Here, we searched for pathological copy-number variants in whole-exome sequencing data from a cohort of 78 MMAF-affected subjects to identify additional genes associated with MMAF. In 7 of 78 affected individuals, we identified a homozygous deletion that removes the two penultimate exons of WDR66 (also named CFAP251), a gene coding for an axonemal protein preferentially localized in the testis and described to localize to the calmodulin- and spoke-associated complex at the base of radial spoke 3. Sequence analysis of the breakpoint region revealed in all deleted subjects the presence of a single chimeric SVA (SINE-VNTR-Alu) at the breakpoint site, suggesting that the initial deletion event was potentially mediated by an SVA insertion-recombination mechanism. Study of Trypanosoma WDR66's ortholog (TbWDR66) highlighted high sequence and structural analogy with the human protein and confirmed axonemal localization of the protein. Reproduction of the human deletion in TbWDR66 impaired flagellar movement, thus confirming WDR66 as a gene associated with the MMAF phenotype and highlighting the importance of the WDR66 C-terminal region.

Whole-exome sequencing identifies mutations in FSIP2 as a recurrent cause of multiple morphological abnormalities of the sperm flagella.

STUDY QUESTION: Can whole-exome sequencing (WES) of infertile patients identify new genes responsible for multiple morphological abnormalities of the sperm flagella (MMAF)? SUMMARY ANSWER: WES analysis of 78 infertile men with a MMAF phenotype permitted the identification of four homozygous mutations in the fibrous sheath (FS) interacting protein 2 (FSIP2) gene in four unrelated individuals. WHAT IS KNOWN ALREADY: The use of high-throughput sequencing techniques revealed that mutations in the dynein axonemal heavy chain 1 (DNAH1) gene, and in the cilia and flagella associated protein 43 (CFAP43) and 44 (CFAP44) genes account for approximately one-third of MMAF cases thus indicating that other relevant genes await identification. STUDY DESIGN, SIZE, DURATION: This was a retrospective genetics study of 78 patients presenting a MMAF phenotype who were recruited in three fertility clinics between 2008 and 2015. Control sperm samples were obtained from normospermic donors. Allelic frequency for control subjects was derived from large public databases. PARTICIPANTS/MATERIALS, SETTING, METHODS: WES was performed for all 78 subjects. All identified variants were confirmed by Sanger sequencing. Relative mRNA expression levels for the selected candidate gene (FSIP2) was assessed by quantitative RT-PCR in a panel of normal human and mouse tissues. To characterize the structural and ultrastructural anomalies present in patients' sperm, immunofluorescence (IF) was performed on sperm samples from two subjects with a mutation and one control and transmission electron microscopy (TEM) analyses was performed on sperm samples from one subject with a mutation and one control. MAIN RESULTS AND THE ROLE OF CHANCE: We identified four unrelated patients (4/78, 5.1%) with homozygous loss of function mutations in the FSIP2 gene, which encodes a protein of the sperm FS and is specifically expressed in human and mouse testis. None of these mutations were reported in control sequence databases. TEM analyses showed a complete disorganization of the FS associated with axonemal defects. IF analyses confirmed that the central-pair microtubules and the inner and outer dynein arms of the axoneme were abnormal in all four patients carrying FSIP2 mutations. Importantly, and in contrast to what was observed in patients with MMAF and mutations in other MMAF-related genes (DNAH1, CFAP43 and CFAP44), mutations in FSIP2 led to the absence of A-kinase anchoring protein 4 (AKAP4). LIMITATIONS, REASONS FOR CAUTION: The low number of biological samples and the absence of a reliable anti-FSIP2 antibody prevented the formal demonstration that the FSIP2 protein was absent in sperm from subjects with a FSIP2 mutation. WIDER IMPLICATIONS OF THE FINDINGS: Our findings indicate that FSIP2 is one of the main genes involved in MMAF syndrome. In humans, genes previously associated with a MMAF phenotype encoded axonemal-associated proteins (DNAH1, CFAP43 and CFAP44). We show here that FSIP2, a protein of the sperm FS, is also logically associated with MMAF syndrome as we showed that it is necessary for FS assembly and for the overall axonemal and flagellar biogenesis. As was suggested before in mouse and man, our results also suggest that defects in AKAP4, one of the main proteins interacting with FSIP2, would induce a MMAF phenotype. Finally, this work reinforces the demonstration that WES sequencing is a good strategy to reach a genetic diagnosis for patients with severe male infertility phenotypes. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the following grants: the 'MAS-Flagella' project financed by the French ANR and the DGOS for the program PRTS 2014 (14-CE15) and the 'Whole genome sequencing of patients with Flagellar Growth Defects (FGD)' project financed by the Fondation Maladies Rares for the program Séquençage à haut débit 2012. The authors have no conflict of interest.

PATL2 is a key actor of oocyte maturation whose invalidation causes infertility in women and mice.

The genetic causes of oocyte meiotic deficiency (OMD), a form of primary infertility characterised by the production of immature oocytes, remain largely unexplored. Using whole exome sequencing, we found that 26% of a cohort of 23 subjects with OMD harboured the same homozygous nonsense pathogenic mutation in PATL2, a gene encoding a putative RNA-binding protein. Using Patl2 knockout mice, we confirmed that PATL2 deficiency disturbs oocyte maturation, since oocytes and zygotes exhibit morphological and developmental defects, respectively. PATL2's amphibian orthologue is involved in the regulation of oocyte mRNA as a partner of CPEB However, Patl2's expression profile throughout oocyte development in mice, alongside colocalisation experiments with Cpeb1, Msy2 and Ddx6 (three oocyte RNA regulators) suggest an original role for Patl2 in mammals. Accordingly, transcriptomic analysis of oocytes from WT and Patl2-/- animals demonstrated that in the absence of Patl2, expression levels of a select number of highly relevant genes involved in oocyte maturation and early embryonic development are deregulated. In conclusion, PATL2 is a novel actor of mammalian oocyte maturation whose invalidation causes OMD in humans.

Absence of CFAP69 Causes Male Infertility due to Multiple Morphological Abnormalities of the Flagella in Human and Mouse.

The multiple morphological abnormalities of the flagella (MMAF) phenotype is among the most severe forms of sperm defects responsible for male infertility. The phenotype is characterized by the presence in the ejaculate of immotile spermatozoa with severe flagellar abnormalities including flagella being short, coiled, absent, and of irregular caliber. Recent studies have demonstrated that MMAF is genetically heterogeneous, and genes thus far associated with MMAF account for only one-third of cases. Here we report the identification of homozygous truncating mutations (one stop-gain and one splicing variant) in CFAP69 of two unrelated individuals by whole-exome sequencing of a cohort of 78 infertile men with MMAF. CFAP69 encodes an evolutionarily conserved protein found at high levels in the testis. Immunostaining experiments in sperm from fertile control individuals showed that CFAP69 localized to the midpiece of the flagellum, and the absence of CFAP69 was confirmed in both individuals carrying CFPA69 mutations. Additionally, we found that sperm from a Cfap69 knockout mouse model recapitulated the MMAF phenotype. Ultrastructural analysis of testicular sperm from the knockout mice showed severe disruption of flagellum structure, but histological analysis of testes from these mice revealed the presence of all stages of the seminiferous epithelium, indicating that the overall progression of spermatogenesis is preserved and that the sperm defects likely arise during spermiogenesis. Together, our data indicate that CFAP69 is necessary for flagellum assembly/stability and that in both humans and mice, biallelic truncating mutations in CFAP69 cause autosomal-recessive MMAF and primary male infertility.

Mutations in CFAP43 and CFAP44 cause male infertility and flagellum defects in Trypanosoma and human.

Spermatogenesis defects concern millions of men worldwide, yet the vast majority remains undiagnosed. Here we report men with primary infertility due to multiple morphological abnormalities of the sperm flagella with severe disorganization of the sperm axoneme, a microtubule-based structure highly conserved throughout evolution. Whole-exome sequencing was performed on 78 patients allowing the identification of 22 men with bi-allelic mutations in DNAH1 (n = 6), CFAP43 (n = 10), and CFAP44 (n = 6). CRISPR/Cas9 created homozygous CFAP43/44 male mice that were infertile and presented severe flagellar defects confirming the human genetic results. Immunoelectron and stimulated-emission-depletion microscopy performed on CFAP43 and CFAP44 orthologs in Trypanosoma brucei evidenced that both proteins are located between the doublet microtubules 5 and 6 and the paraflagellar rod. Overall, we demonstrate that CFAP43 and CFAP44 have a similar structure with a unique axonemal localization and are necessary to produce functional flagella in species ranging from Trypanosoma to human.

SPINK2 deficiency causes infertility by inducing sperm defects in heterozygotes and azoospermia in homozygotes.

Azoospermia, characterized by the absence of spermatozoa in the ejaculate, is a common cause of male infertility with a poorly characterized etiology. Exome sequencing analysis of two azoospermic brothers allowed the identification of a homozygous splice mutation in SPINK2, encoding a serine protease inhibitor believed to target acrosin, the main sperm acrosomal protease. In accord with these findings, we observed that homozygous Spink2 KO male mice had azoospermia. Moreover, despite normal fertility, heterozygous male mice had a high rate of morphologically abnormal spermatozoa and a reduced sperm motility. Further analysis demonstrated that in the absence of Spink2, protease-induced stress initiates Golgi fragmentation and prevents acrosome biogenesis leading to spermatid differentiation arrest. We also observed a deleterious effect of acrosin overexpression in HEK cells, effect that was alleviated by SPINK2 coexpression confirming its role as acrosin inhibitor. These results demonstrate that SPINK2 is necessary to neutralize proteases during their cellular transit toward the acrosome and that its deficiency induces a pathological continuum ranging from oligoasthenoteratozoospermia in heterozygotes to azoospermia in homozygotes.

Whole-exome sequencing of familial cases of multiple morphological abnormalities of the sperm flagella (MMAF) reveals new DNAH1 mutations.

STUDY QUESTION: Can whole-exome sequencing (WES) of patients with multiple morphological abnormalities of the sperm flagella (MMAF) identify causal mutations in new genes or mutations in the previously identified dynein axonemal heavy chain 1 (DNAH1) gene? SUMMARY ANSWER: WES for six families with men affected by MMAF syndrome allowed the identification of DNAH1 mutations in four affected men distributed in two out of the six families but no new candidate genes were identified. WHAT IS KNOWN ALREADY: Mutations in DNAH1, an axonemal inner dynein arm heavy chain gene, have been shown to be responsible for male infertility due to a characteristic form of asthenozoospermia called MMAF, defined by the presence in the ejaculate of spermatozoa with a mosaic of flagellar abnormalities including absent, coiled, bent, angulated, irregular and short flagella. STUDY DESIGN, SIZE, DURATION: This was a retrospective genetics study of patients presenting a MMAF phenotype. Patients were recruited in Iran and Italy between 2008 and 2015. PARTICIPANTS/MATERIALS, SETTING, METHODS: WES was performed for a total of 10 subjects. All identified variants were confirmed by Sanger sequencing. Two additional affected family members were analyzed by direct Sanger sequencing. To establish the prevalence of the DNAH1 mutation identified in an Iranian family, we carried out targeted sequencing on 38 additional MMAF patients of the same geographical origin. RT-PCR and immunochemistry were performed on sperm samples to assess the effect of the identified mutation on RNA and protein. MAIN RESULTS AND THE ROLE OF CHANCE: WES in six families identified a causal mutations in two families. Two additional affected family members were confirmed to hold the same homozygous mutation as their sibling. In total, DNAH1 mutations were identified in 5 out of 12 analyzed subjects (41.7%). If we only include index cases, we detected two mutated subjects out of six (33%) tested MMAF individuals. Furthermore we sequenced one DNAH1 exon found to be mutated (c.8626-1G > A) in an Iranian family in an additional 38 MMAF patients from Iran. One of these patients carried the variant confirming that this variant is relatively frequent in the Iranian population. The effect of the c.8626-1G > A variant was confirmed by RT-PCR and immunochemistry as no RNA or protein could be observed in sperm from the affected men. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: WES allows the amplification of 80-90% of all coding exons. It is possible that some DNAH1 exons may not have been sequenced and that we may have missed some additional mutations. Also, WES cannot identify deep intronic mutations and it is not efficient for detection of large genomic events (deletions, insertions, inversions). We did not identify any causal mutations in DNAH1 or in other candidate genes in four out of the six tested families. This indicates that the technique and/or the analysis of our data can be improved to increase the diagnosis efficiency. WIDER IMPLICATIONS OF THE FINDINGS: Our findings confirm that DNAH1 is one of the main genes involved in MMAF syndrome. It is a large gene with 78 exons making it challenging and expensive to sequence using the traditional Sanger sequencing methods. We show that WES sequencing is good alternative to Sanger sequencing to reach a genetic diagnosis in patients with severe male infertility phenotypes. STUDY FUNDING/COMPETING INTERESTS: This work was supported by following grants: the 'MAS-Flagella' project financed by the French ANR and the DGOS for the program PRTS 2014 and the 'Whole genome sequencing of patients with Flagellar Growth Defects (FGD)' project financed by the Fondation Maladies Rares for the program Séquençage à haut débit 2012. The authors have no conflict of interest.

Homozygous mutation of PLCZ1 leads to defective human oocyte activation and infertility that is not rescued by the WW-binding protein PAWP.

In mammals, sperm-oocyte fusion initiates Ca(2+) oscillations leading to a series of events called oocyte activation, which is the first stage of embryo development. Ca(2+) signaling is elicited by the delivery of an oocyte-activating factor by the sperm. A sperm-specific phospholipase C (PLCZ1) has emerged as the likely candidate to induce oocyte activation. Recently, PAWP, a sperm-born tryptophan domain-binding protein coded by WBP2NL, was proposed to serve the same purpose. Here, we studied two infertile brothers exhibiting normal sperm morphology but complete fertilization failure after intracytoplasmic sperm injection. Whole exomic sequencing evidenced a missense homozygous mutation in PLCZ1, c.1465A>T; p.Ile489Phe, converting Ile 489 into Phe. We showed the mutation is deleterious, leading to the absence of the protein in sperm, mislocalization of the protein when injected in mouse GV and MII oocytes, highly abnormal Ca(2+) transients and early embryonic arrest. Altogether these alterations are consistent with our patients' sperm inability to induce oocyte activation and initiate embryo development. In contrast, no deleterious variants were identified in WBP2NL and PAWP presented normal expression and localization. Overall we demonstrate in humans, the absence of PLCZ1 alone is sufficient to prevent oocyte activation irrespective of the presence of PAWP. Additionally, it is the first mutation located in the C2 domain of PLCZ1, a domain involved in targeting proteins to cell membranes. This opens the door to structure-function studies to identify the conserved amino acids of the C2 domain that regulate the targeting of PLCZ1 and its selectivity for its lipid substrate(s).

Progesterone-induced Acrosome Exocytosis Requires Sequential Involvement of Calcium-independent Phospholipase A2ß (iPLA2ß) and Group X Secreted Phospholipase A2 (sPLA2).

Phospholipase A2 (PLA2) activity has been shown to be involved in the sperm acrosome reaction (AR), but the molecular identity of PLA2 isoforms has remained elusive. Here, we have tested the role of two intracellular (iPLA2ß and cytosolic PLA2α) and one secreted (group X) PLA2s in spontaneous and progesterone (P4)-induced AR by using a set of specific inhibitors and knock-out mice. iPLA2ß is critical for spontaneous AR, whereas both iPLA2ß and group X secreted PLA2 are involved in P4-induced AR. Cytosolic PLA2α is dispensable in both types of AR. P4-induced AR spreads over 30 min in the mouse, and kinetic analyses suggest the presence of different sperm subpopulations, using distinct PLA2 pathways to achieve AR. At low P4 concentration (2 µm), sperm undergoing early AR (0-5 min post-P4) rely on iPLA2ß, whereas sperm undergoing late AR (20-30 min post-P4) rely on group X secreted PLA2. Moreover, the role of PLA2s in AR depends on P4 concentration, with the PLA2s being key actors at low physiological P4 concentrations (≤2 µm) but not at higher P4 concentrations (~10 µm).

Dynamics of Sun5 localization during spermatogenesis in wild type and Dpy19l2 knock-out mice indicates that Sun5 is not involved in acrosome attachment to the nuclear envelope.

The acrosome is an organelle that is central to sperm physiology and a defective acrosome biogenesis leads to globozoospermia, a severe male infertility. The identification of the actors involved in acrosome biogenesis is therefore particularly important to decipher the molecular pathogeny of globozoospermia. We recently showed that a defect in the DPY19L2 gene is present in more than 70% of globozoospermic men and demonstrated that Dpy19l2, located in the inner nuclear membrane, is the first protein involved in the attachment of the acrosome to the nuclear envelope (NE). SUN proteins serve to link the nuclear envelope to the cytoskeleton and are therefore good candidates to participate in acrosome-nucleus attachment, potentially by interacting with DPY19L2. In order to characterize new actors of acrosomal attachment, we focused on Sun5 (also called Spag4l), which is highly expressed in male germ cells, and investigated its localization during spermatogenesis. Using immunohistochemistry and Western blot experiments in mice, we showed that Sun5 transits through different cellular compartments during meiosis. In pachytene spermatocytes, it is located in a membranous compartment different to the reticulum. In round spermatids, it progresses to the Golgi and the NE before to be located to the tail/head junction in epididymal sperm. Interestingly, we demonstrate that Sun5 is not, as initially reported, facing the acrosome but is in fact excluded from this zone. Moreover, we show that in Dpy19l2 KO spermatids, upon the detachment of the acrosome, Sun5 relocalizes to the totality of the NE suggesting that the acrosome attachment excludes Sun5 from the NE facing the acrosome. Finally, Western-blot experiments demonstrate that Sun5 is glycosylated. Overall, our work, associated with other publications, strongly suggests that the attachment of the acrosome to the nucleus does not likely depend on the formation of SUN complexes.

Dpy19l2-deficient globozoospermic sperm display altered genome packaging and DNA damage that compromises the initiation of embryo development.

We recently identified the DPY19L2 gene as the main genetic cause of human globozoospermia. Non-genetically characterized cases of globozoospermia were associated with DNA alterations, suggesting that DPY19L2-dependent globozoospermia may be associated with poor DNA quality. However the origins of such defects have not yet been characterized and the consequences on the quality of embryos generated with globozoospermic sperm remain to be determined. Using the mouse model lacking Dpy19l2, we compared several key steps of nuclear compaction. We show that the kinetics of appearance and disappearance of the histone H4 acetylation waves and of transition proteins are defective. More importantly, the nuclear invasion by protamines does not occur. As a consequence, we showed that globozoospermic sperm presented with poor sperm chromatin compaction and sperm DNA integrity breakdown. We next assessed the developmental consequences of using such faulty sperm by performing ICSI. We showed in the companion article that oocyte activation (OA) with globozoospermic sperm is very poor and due to the absence of phospholipase Cζ; therefore artificial OA (AOA) was used to bypass defective OA. Herein, we evaluated the developmental potential of embryos generated by ICSI + AOA in mice. We demonstrate that although OA was fully rescued, preimplantation development was impaired when using globozoospermic sperm. In human, a small number of embryos could be generated with sperm from DPY19L2-deleted patients in the absence of AOA and these embryos also showed a poor developmental potential. In conclusion, we show that chromatin compaction during spermiogenesis in Dpy19l2 KO mouse is defective and leads to sperm DNA damage. Most of the DNA breaks were already present when the sperm reached the epididymis, indicating that they occurred inside the testis. This result thus suggests that testicular sperm extraction in Dpy19l2-dependent globozoospermia is not recommended. These defects may largely explain the poor embryonic development of most mouse and human embryos obtained with globozoospermic sperm.

Subcellular localization of phospholipase Cζ in human sperm and its absence in DPY19L2-deficient sperm are consistent with its role in oocyte activation.

We recently identified the DPY19L2 gene as the main genetic cause of human globozoospermia (70%) and described that Dpy19l2 knockout (KO) mice faithfully reproduce the human phenotype of globozoospermia making it an excellent model to characterize the molecular physiopathology of globozoospermia. Recent case studies on non-genetically characterized men with globozoospermia showed that phospholipase C, zeta (PLCζ), the sperm factor thought to induce the Ca(2+) oscillations at fertilization, was absent from their sperm, explaining the poor fertilization potential of these spermatozoa. Since 30% of globozoospermic men remain genetically uncharacterized, the absence of PLCζ in DPY19L2 globozoospermic men remains to be formally established. Moreover, the precise localization of PLCζ and the reasons underlying its loss during spermatogenesis in globozoospermic patients are still not understood. Herein, we show that PLCζ is absent, or its presence highly reduced, in human and mouse sperm with DPY19L2-associated globozoospermia. As a consequence, fertilization with sperm from Dpy19l2 KO mice failed to initiate Ca(2+) oscillations and injected oocytes remained arrested at the metaphase II stage, although a few human oocytes injected with DPY19L2-defective sperm showed formation of 2-pronuclei embryos. We report for the first time the subcellular localization of PLCζ in control human sperm, which is along the inner acrosomal membrane and in the perinuclear theca, in the area corresponding to the equatorial region. Because these cellular components are absent in globozoospermic sperm, the loss of PLCζ in globozoospermic sperm is thus consistent and reinforces the role of PLCζ as an oocyte activation factor necessary for oocyte activation. In our companion article, we showed that chromatin compaction during spermiogenesis in Dpy19l2 KO mouse is defective and leads to sperm DNA damage. Together, these defects explain the poor fertilization potential of DPY19L2-globozoospermic sperm and the compromised developmental potential of embryos obtained using sperm from patients with a deletion of the DPY19L2 gene.

Mutations in DNAH1, which encodes an inner arm heavy chain dynein, lead to male infertility from multiple morphological abnormalities of the sperm flagella.

Ten to fifteen percent of couples are confronted with infertility and a male factor is involved in approximately half the cases. A genetic etiology is likely in most cases yet only few genes have been formally correlated with male infertility. Homozygosity mapping was carried out on a cohort of 20 North African individuals, including 18 index cases, presenting with primary infertility resulting from impaired sperm motility caused by a mosaic of multiple morphological abnormalities of the flagella (MMAF) including absent, short, coiled, bent, and irregular flagella. Five unrelated subjects out of 18 (28%) carried a homozygous variant in DNAH1, which encodes an inner dynein heavy chain and is expressed in testis. RT-PCR, immunostaining, and electronic microscopy were carried out on samples from one of the subjects with a mutation located on a donor splice site. Neither the transcript nor the protein was observed in this individual, confirming the pathogenicity of this variant. A general axonemal disorganization including mislocalization of the microtubule doublets and loss of the inner dynein arms was observed. Although DNAH1 is also expressed in other ciliated cells, infertility was the only symptom of primary ciliary dyskinesia observed in affected subjects, suggesting that DNAH1 function in cilium is not as critical as in sperm flagellum.

Fine characterisation of a recombination hotspot at the DPY19L2 locus and resolution of the paradoxical excess of duplications over deletions in the general population.

We demonstrated previously that 75% of infertile men with round, acrosomeless spermatozoa (globozoospermia) had a homozygous 200-Kb deletion removing the totality of DPY19L2. We showed that this deletion occurred by Non-Allelic Homologous Recombination (NAHR) between two homologous 28-Kb Low Copy Repeats (LCRs) located on each side of the gene. The accepted NAHR model predicts that inter-chromatid and inter-chromosome NAHR create a deleted and a duplicated recombined allele, while intra-chromatid events only generate deletions. Therefore more deletions are expected to be produced de novo. Surprisingly, array CGH data show that, in the general population, DPY19L2 duplicated alleles are approximately three times as frequent as deleted alleles. In order to shed light on this paradox, we developed a sperm-based assay to measure the de novo rates of deletions and duplications at this locus. As predicted by the NAHR model, we identified an excess of de novo deletions over duplications. We calculated that the excess of de novo deletion was compensated by evolutionary loss, whereas duplications, not subjected to selection, increased gradually. Purifying selection against sterile, homozygous deleted men may be sufficient for this compensation, but heterozygously deleted men might also suffer a small fitness penalty. The recombined alleles were sequenced to pinpoint the localisation of the breakpoints. We analysed a total of 15 homozygous deleted patients and 17 heterozygous individuals carrying either a deletion (n = 4) or a duplication (n = 13). All but two alleles fell within a 1.2-Kb region central to the 28-Kb LCR, indicating that >90% of the NAHR took place in that region. We showed that a PRDM9 13-mer recognition sequence is located right in the centre of that region. Our results therefore strengthen the link between this consensus sequence and the occurrence of NAHR.

Comparative testicular transcriptome of wild type and globozoospermic Dpy19l2 knock out mice.

BACKGROUND: Globozoospermia is a male infertility phenotype characterized by the presence in the ejaculate of near 100% acrosomeless round-headed spermatozoa with normal chromosomal content. Following intracytoplasmic sperm injection (ICSI) these spermatozoa give a poor fertilization rate and embryonic development. We showed previously that most patients have a 200 kb homozygous deletion, which includes DPY19L2 whole coding sequence. Furthermore we showed that the DPY19L2 protein is located in the inner nuclear membrane of spermatids during spermiogenesis and that it is necessary to anchor the acrosome to the nucleus thus performing a function similar to that realized by Sun proteins within the LINC-complex (Linker of Nucleoskeleton and Cytoskeleton). SUN1 was described to be necessary for gametogenesis and was shown to interact with the telomeres. It is therefore possible that Dpy19l2 could also interact, directly or indirectly, with the DNA and modulate gene expression during spermatogenesis. In this study, we compared the transcriptome of testes from Dpy19l2 knock out and wild type mice in order to identify a potential deregulation of transcripts that could explain the poor fertilization potential of Dpy19l2 mutated spermatozoa. METHODS: RNA was extracted from testes from DPY19L2 knock out and wild type mice. The transcriptome was carried out using GeneChip® Mouse Exon 1.0 ST Arrays. The biological processes and molecular functions of the differentially regulated genes were analyzed with the PANTHER software. RESULTS: A total of 76 genes were deregulated, 70 were up-regulated and 6 (including Dpy19l2) were down-regulated. These genes were found to be involved in DNA/RNA binding, structural organization, transport and catalytic activity. CONCLUSIONS: We describe that an important number of genes are differentially expressed in Dpy19l2 mice. This work could help improving our understanding of Dpy19l2 functions and lead to a better comprehension of the molecular mechanism involved in spermatogenesis.

CONTEXTE: La globozoospermie est caractérisée par la présence dans l'éjaculat de près de 100% de spermatozoïdes ronds et dépourvus d'acrosome qui présentent un contenu chromosomique normal. L'injection intracytoplasmique (ICSI) de ces spermatozoïdes donne cependant un taux de fécondation et de développement embryonnaire particulièrement bas. Nous avons montré précédemment que la plupart des patients globozoospermes présentent une délétion homozygote de 200 Kb qui inclue la totalité de la séquence codante du gène DPY19L2. De plus nous avons montré que la protéine DPY19L2 était localisée dans la membrane interne des noyaux des spermatides pendant la spermatogénèse et qu'elle est nécessaire pour fixer l'acrosome au noyau, réalisant ainsi une fonction similaire à celle des protéines Sun au sein du complexe LINC (Linker of Nucleoskeleton and Cytoskeleton). Il a par ailleurs été montré que SUN1 était nécessaire à la spermatogénèse et que cette protéine interagit avec les télomères chromosomiques. Il est donc possible que Dpy19l2 interagisse également, directement ou indirectement avec l'ADN et module l'expression génique lors de la spermatogénèse. Dans cette étude nous avons donc comparé le transcriptome de testicules de souris invalidées (KO) pour le gène Dpy19l2 à celui de souris sauvage afin d'identifier une éventuelle dérégulation génique qui pourrait expliquer le faible potentiel reproductif des spermatozoïdes globozoocéphales. MÉTHODE: L'ARN a été extrait de testicules de souris KO pour Dpy19l2 et de souris sauvages. Le transcriptome a été réalisé en utilisant des puces d'expression ® Mouse Exon 1.0 ST Arrays. Les processus biologiques et les fonctions des gènes dérégulés ont été analysés en utilisant le logiciel PANTHER. RÉSULTATS: Un total de 76 gènes a été identifié comme étant dérégulés, 70 gènes étaient surexprimés et 6 (incluant Dpy19l2) étaient sous-exprimés. Il s'agit de gènes principalement impliqués dans des interactions avec des acides nucléiques (ADN/ARN), ou ayant un rôle structural, dans le transport, ou présentant une activité catalytique. CONCLUSIONS: Cette étude nous a permis d'identifier et de décrire un nombre important de gènes exprimés de manière différentielle chez les souris KO pour Dpy19l2. Ce travail peut permettre d'améliorer notre compréhension des fonctions de Dpy19l2 et peut contribuer à obtenir une meilleure compréhension des mécanismes moléculaires nécessaire à la spermatogénèse.