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Absence of Dpy19l2, a new inner nuclear membrane protein, causes globozoospermia in mice by preventing the anchoring of the acrosome to the nucleus
Abstract and Figures
Sperm-head elongation and acrosome formation, which take place during the last stages of spermatogenesis, are essential to produce competent spermatozoa that are able to cross the oocyte zona pellucida and to achieve fertilization. During acrosome biogenesis, acrosome attachment and spreading over the nucleus are still poorly understood and to date no proteins have been described to link the acrosome to the nucleus. We recently demonstrated that a deletion of DPY19L2, a gene coding for an uncharacterized protein, was responsible for a majority of cases of type I globozoospermia, a rare cause of male infertility that is characterized by the exclusive production of round-headed acrosomeless spermatozoa. Here, using Dpy19l2 knockout mice, we describe the cellular function of the Dpy19l2 protein. We demonstrate that the protein is expressed predominantly in spermatids with a very specific localization restricted to the inner nuclear membrane facing the acrosomal vesicle. We show that the absence of Dpy19l2 leads to the destabilization of both the nuclear dense lamina (NDL) and the junction between the acroplaxome and the nuclear envelope. Consequently, the acrosome and the manchette fail to be linked to the nucleus leading to the disruption of vesicular trafficking, failure of sperm nuclear shaping and eventually to the elimination of the unbound acrosomal vesicle. Finally, we show for the first time that Dpy19l3 proteins are also located in the inner nuclear envelope, therefore implying that the Dpy19 proteins constitute a new family of structural transmembrane proteins of the nuclear envelope.
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Supplementary resources (6)
... The acroplaxome-acrosome attachment 16 to the nucleus is dependent on the inner nuclear membrane protein DPY19L2 (Pierre et al. 17 2012). In mice lacking SUN3, SUN4 or DPY19L2 the manchette does not attach to the nucleus 18 and all spermatozoa have compacted round heads, showing that nuclear attachment of the 19 acroplaxome and the manchette are required for nuclear elongation and shaping but not 20 compaction (Calvi et al. 2015;Gao et al. 2020;Pasch et al. 2015;Pierre et al. 2012). 21 ...
... To investigate the form of the NE and the evolution of the nuclear lamina (NL) in round 154 spermatids in Banf2 -/-,Nup210l -/males, we used an antibody against lamin B1. In controls and 155 double-KO mice, we observed the normal pattern of labelling (Pierre et al., 2012), with lamin 156 B1 at the nuclear periphery, except under the developing acrosome in round spermatids until 157 its disappearance at the initiation of nuclear elongation, indicating that the absence of BAF-L 158 and NUP210L does not affect the dismantling of the NL in round spermatids. Nevertheless, 159 these analyses revealed a major difference in the shape of the round spermatid nucleus: in 160 controls it was round, but in Banf2 -/-,Nup210l -/males, beginning at step 1, it was deformed, 161 appearing elliptical, angular or indented (Fig. 4A). ...
During spermiogenesis, haploid round spermatids differentiate into spermatozoa. This involves nuclear elongation, chromatin compaction, cytoplasm reduction, and formation of the acrosome and the flagellum. These events are orchestrated by cytoskeletal elements - acroplaxome and manchette - that attach to the nuclear envelope (NE) except at the caudal pole where the nuclear pore complexes (NPCs) shift to form a dense array. Here, we use a genetic dissection approach to reveal function at the caudal NE, through the study of two spermatid-specific proteins, whose human orthologues persist there as spermatids elongate: NUP210L, a transmembrane nucleoporin and BAF-L, paralogue and interactor of chromatin protein BAF. In mice, inactivation of either BAF-L or NUP210L has no impact on fertility. However, we show here that in mice lacking NUP210L, two copies of BAF-L become essential for fertility; in Nup210l −/− , Banf2 +/− or Nup210l −/− , Banf2 −/− mice, most spermatids arrest during nuclear elongation (step 10-11) with mislocalized NPCs and disorganized manchette microtubules that frequently invaginate the nucleus from the caudal pole. Our results suggest that the NPC array, and BAF-L/BAF, ensure nuclear integrity at the caudal pole during spermatid remodeling.
Summary: Nucleoporin NUP210L and BAF-paralogue BAF-L function redundantly during mouse spermatid nuclear remodeling to concentrate nuclear pores to the flagellar pole, organize the manchette cytoskeleton and prevent nuclear invagination by microtubules.
... DPY19L2 is a transmembrane protein expressed in the testis [150], which is involved in the anchorage of the cytoskeleton to the nuclear membrane. Therefore, its absence/mutation leads to instability and the dissociation of the layered structure of acroplaxome, further resulting in the formation of round head spermatozoa [151]. A wide spectrum of plausible mutations of DPY19L2 has been detected in globozoospermic individuals: deletion of the whole locus, nonsense, missense, splicing mutations, and partial deletion encompassing exons 8,9,11,15,21, and intron 11 [150]. ...
Several genes are implicated in spermatogenesis and fertility regulation, and these genes are presently being analysed in clinical practice due to their involvement in male factor infertility (MFI). However, there are still few genetic analyses that are currently recommended for use in clinical practice. In this manuscript, we reviewed the genetic causes of qualitative sperm defects. We distinguished between alterations causing reduced sperm motility (asthenozoospermia) and alterations causing changes in the typical morphology of sperm (teratozoospermia). In detail, the genetic causes of reduced sperm motility may be found in the alteration of genes associated with sperm mitochondrial DNA, mitochondrial proteins, ion transport and channels, and flagellar proteins. On the other hand, the genetic causes of changes in typical sperm morphology are related to conditions with a strong genetic basis, such as macrozoospermia, globozoospermia, and acephalic spermatozoa syndrome. We tried to distinguish alterations approved for routine clinical application from those still unsupported by adequate clinical studies. The most important aspect of the study was related to the correct identification of subjects to be tested and the correct application of genetic tests based on clear clinical data. The correct application of available genetic tests in a scenario where reduced sperm motility and changes in sperm morphology have been observed enables the delivery of a defined diagnosis and plays an important role in clinical decision-making. Finally, clarifying the genetic causes of MFI might, in future, contribute to reducing the proportion of so-called idiopathic MFI, which might indeed be defined as a subtype of MFI whose cause has not yet been revealed.
... The DPY19L2 protein is found within the inner nuclear membrane of spermatozoa. Its function is to serve as an anchor, connecting the developing acrosome to the nuclear envelope through a specialized cytoskeletal plate called the acroplaxome [46]. It was revealed that spermatozoa spermatozoa from patients with mutations in the DPY19L2 gene exhibited a separated inner nuclear membrane from the outer membrane and the entire detachment of the acrosome [47]. ...
Background
Male infertility is a global health issue. The more causative genes related to human male infertility should be further explored. The essential role of Zcwpw1 in male mouse fertility has been established and the role of ZCWPW1 in human reproduction needs further investigation to verify.
Methods
An infertile man with oligoasthenoteratozoospermia phenotype and his parents were recruited from West China Second University Hospital, Sichuan University. A total of 200 healthy Han Chinese volunteers without any evidence of infertility were recruited as normal controls, while an additional 150 infertile individuals were included to assess the prevalence of ZCWPW1 variants in a sporadic male sterile population. The causative gene variant was identified by Whole-exome sequencing and Sanger sequencing. The phenotype of the oligoasthenoteratozoospermia was determined by Papanicolaou staining, immunofluorescence staining and electron microscope. In-vitro experiments, western blot and in-silicon analysis were applied to assess the pathogenicity of the identified variant. Additionally, we examined the influence of the variant on the DNA fragmentation and DNA repair capability by Sperm Chromatin Dispersion and Neutral Comet Assay.
Results
The proband exhibits a phenotype of oligoasthenoteratozoospermia, his spermatozoa show head defects by semen examination, Papanicolaou staining and electron microscope assays. Whole-exome sequencing and Sanger sequencing found the proband carries a homozygous ZCWPW1 variant (c.1064C > T, p. P355L). Immunofluorescence analysis shows a significant decrease in ZCWPW1 expression in the proband’s sperm. By exogenous expression with ZCWPW1 mutant plasmid in vitro, the obvious declined expression of ZCWPW1 with the mutation is validated in HEK293T. After being treated by hydroxyurea, MUT-ZCWPW1 transfected cells and empty vector transfected cells have a higher level of γ-H2AX, increased tail DNA and reduced H3K9ac level than WT-ZCWPW1 transfected cells. Furthermore, the Sperm Chromatin Dispersion assay revealed the proband’s spermatozoa have high DNA fragmentation.
Conclusions
It is the first report that a novel homozygous missense mutation in ZCWPW1 caused human male infertility with sperm head defects and high DNA fragmentation. This finding enriches the gene variant spectrum and etiology of oligoasthenoteratozoospermia.
... The DPY19L2 protein is found within the inner nuclear membrane of spermatozoa. Its function is to serve as an anchor, connecting the developing acrosome to the nuclear envelope through a specialized cytoskeletal plate called the acroplaxome (33). It was revealed that spermatozoa from DPY19L2 mutated patients exhibited separated inner nuclear membrane from the outer membrane and the entire detachment of the acrosome (34). ...
Background
In about one-third of cases, the genetic causes of asthenozoospermia are unknown. The more causative genes related to human male infertility should be further explored. The essential role of ZCWPW1 in mouse male fertility has been established and the role of ZCWPW1 in human reproduction need further investigation to verify.
Methods
Whole-exome sequencing was conducted to identify causative genes in the infertile man. The phenotype of the asthenozoospermia was determined by Papanicolaou staining, immunofluorescence staining and electron microscope. In-vitro experiments, western blot and in-silicon analysis were applied to assess the pathogenicity of the identified variant. Additionally, we examined the influence of the variant on the DNA fragmentation and DNA repair capability by Sperm Chromatin Dispersion and Neutral Comet Assay.
Results
The spermatozoa of the proband exhibited low quantity, head defects in semen examination, papanicolaou staining and electron microscope. Whole-exome sequencing and sanger sequencing found the proband carried a homozygous ZCWPW1 variant (c.1064C > T, p. P355L). Immunofluorescence analysis showed that a significant decrease of ZCWPW1 expression in proband’s sperm. The obvious declined expression of ZCWPW1 with the mutation was validated in HEK293T. Furthermore, Sperm Chromatin Dispersion assay revealed the proband’s spermatozoa had high DNA fragmentation. After treated by hydroxyurea, MUT-ZCWPW1 transfected cells and empty vector transfected cells had higher level of γ-H2AX and tail DNA than WT-ZCWPW1 transfected cells.
Conclusions
It is the first report that a novel homozygous missense mutation in ZCWPW1 caused human male infertility with sperm head defect and high DNA fragmentation. This finding enriches the gene variant spectrum and etiology of asthenozoospermia.
... The acroplaxome is a F-actin-rich cytoskeletal plate of the perinuclear theca (PT) that functions in attaching the developing acrosome to the nucleus; the manchette develops during the acrosome phase, exerts mechanical force for the acrosome, and serves as a scaffold for the transport of molecules to the growing flagellum (Wei and Yang, 2018;Xiong et al., 2021). Studies of sperm from infertile men have shown that the absence of an acrosome, often accompanied by loosened or absent acroplaxome, can sometimes lead to failure of in vitro fertilization (IVF) or failure of oocyte activation post intracytoplasmic sperm injection (ICSI) owing to the deficiencies in PT-related proteins, suggesting the importance of acrosomes in normal fertilization and embryonic developmental potential (Pierre et al., 2012;Chen et al., 2021b;Huang et al., 2022). ...
During spermiogenesis, haploid spermatids undergo dramatic morphological changes to form slender sperm flagella and cap-like acrosomes, which are required for successful fertilization. Severe deformities in flagella cause a male infertility syndrome, multiple morphological abnormalities of the flagella (MMAF), while acrosomal hypoplasia in some cases leads to sub-optimal embryonic developmental potential. However, evidence regarding the occurrence of acrosomal hypoplasia in MMAF is limited. Here, we report the generation of base-edited mice knocked out for coiled-coil domain-containing 38 (Ccdc38) via inducing a nonsense mutation and find that the males are infertile. The Ccdc38-KO sperm display acrosomal hypoplasia and typical MMAF phenotypes. We find that the acrosomal membrane is loosely anchored to the nucleus and fibrous sheaths are disorganized in Ccdc38-KO sperm. Further analyses reveal that Ccdc38 knockout causes a decreased level of TEKT3, a protein associated with acrosome biogenesis, in testes and an aberrant distribution of TEKT3 in sperm. We finally show that intracytoplasmic sperm injection overcomes Ccdc38-related infertility. Our study thus reveals a previously unknown role for CCDC38 in acrosome biogenesis and provides additional evidence for the occurrence of acrosomal hypoplasia in MMAF.
In the world, about 15% of couples are infertile, and nearly half of all infertility was caused by men. A large number of genetic mutations are thought to affect spermatogenesis by regulating acrosome formation. Here, we identified three patients harbouring the protein interacting with cyclin A1 (PROCA1) mutation by whole exome sequencing (WES) and Sanger sequencing among patients with predominantly acrosome-deficient teratozoospermia. However, the expression and roles of PROCA1 in infertile men remain unclear. We found that PROCA1 is predominantly expressed in the testis, where it is specifically localized to the acrosome of normal human sperm. Proca1 knockout (KO) mice were subsequently generated using CRISPR-Cas9 technology. However, Proca1 KO adult male mice were fertile, with testis-to-body weight ratios comparable to those of wild-type (WT) mice. Testicular tissue or sperm morphology were not significantly different in Proca1 KO mice compared to WT mice. Expression of the acrosome markers PNA and SP56 in the acrosome was comparable between Proca1 KO and WT mice. In summary, these findings suggested that the PROCA1 mutation identified in humans does not affect acrosome biogenesis in mice.
Each year, infertility affects 15% of couples worldwide, with 50% of cases attributed to men. It is assumed that sperm head shape is important for sperm-zona pellucida penetration but research has yet to elucidate why. We generated testis expressed 46 (Tex46) knockout mice to investigate the essential roles of TEX46 in mammalian reproduction. We used RT-PCR to demonstrate that Tex46 was expressed exclusively in the male reproductive tract in mice and humans. We created Tex46-/- mice using the CRISPR-Cas9 system and analyzed their fertility. Tex46 null spermatozoa underwent further evaluation using computer-assisted sperm analysis (CASA), light microscopy, and ultrastructural microscopy. We used immunoblot analysis to elucidate relationships between TEX46 and other acrosome biogenesis-related proteins. Mouse and human TEX46 are testis-enriched and encode a transmembrane protein which is conserved from amphibians to mammals. Loss of the mouse TEX46 protein causes male sterility primarily due to abnormal sperm head formation and secondary effects on sperm motility. Tex46 null spermatozoa morphologically lack the typical hooked sperm head appearance and fail to penetrate through the zona pellucida. Electron microscopy of the testicular germ cells reveals malformation of the acrosomal cap, with misshapen sperm head tips and the appearance of a gap between the acrosome head and the nucleus. TEX46 is essential for sperm head formation, sperm penetration through the zona pellucida, and male fertility in mice, and is a putative contraceptive target in men.
From a cohort of 167 infertile patients suffering from multiple morphological abnormalities of the flagellum (MMAF), pathogenic bi-allelic mutations were identified in the CCDC146 gene. In somatic cells, CCDC146 is located at the centrosome and at multiple microtubule-related organelles during mitotic division, suggesting that it is a microtubule-associated protein (MAP). To decipher the molecular pathogenesis of infertility associated with CCDC146 mutations, a Ccdc146 knock-out (KO) mouse line was created. KO male mice were infertile, and sperm exhibited a phenotype identical to CCDC146 mutated patients. CCDC146 expression starts during late spermiogenesis. 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 suggests that the protein may be a microtubule inner protein (MIP). At the subcellular level, the absence of CCDC146 impacted all microtubule-based organelles such as the manchette, the head–tail coupling apparatus (HTCA), and the axoneme. Through this study, a new genetic cause of infertility and a new factor in the formation and/or structure of the sperm axoneme were characterized.
Fertility‐targeted gene drives have been proposed as an ethical genetic approach for managing wild populations of vertebrate pests for public health and conservation benefit. This manuscript introduces a framework to identify and evaluate target gene suitability based on biological gene function, gene expression and results from mouse knockout models. This framework identified 16 genes essential for male fertility and 12 genes important for female fertility that may be feasible targets for mammalian gene drives and other non‐drive genetic pest control technology. Further, a comparative genomics analysis demonstrates the conservation of the identified genes across several globally significant invasive mammals. In addition to providing important considerations for identifying candidate genes, our framework and the genes identified in this study may have utility in developing additional pest control tools such as wildlife contraceptives.
Spermatids generate diverse and unusual actin and microtubule populations during spermiogenesis to fulfill mechanical and cargo transport functions assisted by motor and non-motor proteins. Disruption of cargo transport may lead to teratozoospermia and consequent male infertility. How motor and non-motor proteins utilize the cytoskeleton to transport cargos during sperm development is not clear. Filamentous actin (F-actin) and the associated motor protein myosin Va participate in the transport of Golgi-derived proacrosomal vesicles to the acrosome and along the manchette. The acrosome is stabilized by the acroplaxome, a cytoskeletal plate anchored to the nuclear envelope. The acroplaxome plate harbors F-actin and actin-like proteins as well as several other proteins, including keratin 5/Sak57, Ran GTPase, Hook1, dynactin p150Glued, cenexin-derived ODF2, testis-expressed profilin-3 and profilin-4, testis-expressed Fer tyrosine kinase (FerT), members of the ubiquitin-proteasome system and cortactin. Spermatids express transcripts encoding the non-spliced form of cortactin, a F-actin-regulatory protein. Tyrosine phosphorylated cortactin and FerT coexist in the acrosome-acroplaxome complex. Hook1 and p150Glued, known to participate in vesicle cargo transport, are sequentially seen from the acroplaxome to the manchette to the head-tail coupling apparatus (HTCA). The golgin Golgi-microtubule associated protein GMAP210 resides in the cis-Golgi whereas the intraflagellar protein IFT88 localizes in the trans-Golgi network. Like Hook1 and p150Glued, GMAP210 and IFT88 colocalize at the cytosolic side of proacrosomal vesicles and, following vesicle fusion, become part of the outer and inner acrosomal membranes before relocating to the acroplaxome, manchette and HTCA. A hallmark of the manchette and axoneme is microtubule heterogeneity, determined by the abundance of acetylated, tysosinated and glutamylated tubulin isoforms produced by post-translational modifications. We postulate that the construction of the male gamete requires microtubule and F-actin tracks and specific molecular motors and associated non-motor proteins for the directional positioning of vesicular and non-vesicular cargos at specific intracellular sites.
During corticogenesis, the regulation of neuronal migration is crucial for the functional organization of the neocortex. Glutamatergic neurons are major excitatory components of the mammalian neocortex. In order to elucidate the specific molecular mechanisms underlying their development, we used single-cell microarray analysis to screen for mouse genes that are highly expressed in developing glutamatergic neurons. We identified dpy-19-like 1 (Dpy19l1), a homolog of C. elegans dpy-19, which encodes a putative multi-transmembrane protein shown to regulate directed migration of Q neuroblasts in C. elegans. At embryonic stages Dpy19l1 is highly expressed in glutamatergic neurons in the mouse cerebral cortex, whereas in the subpallium, where GABAergic neurons are generated, expression was below detectable levels. Downregulation of Dpy19l1 mediated by shRNA resulted in defective radial migration of glutamatergic neurons in vivo, which was restored by the expression of shRNA-insensitive Dpy19l1. Many Dpy19l1-knockdown cells were aberrantly arrested in the intermediate zone and the deep layer and, additionally, some extended single long processes towards the pial surface. Furthermore, we observed defective radial migration of bipolar cells in Dpy19l1-knockdown brains. Despite these migration defects, these cells correctly expressed Cux1, which is a marker for upper layer neurons, suggesting that Dpy19l1 knockdown results in migration defects but does not affect cell type specification. These results indicate that Dpy19l1 is required for the proper radial migration of glutamatergic neurons, and suggest an evolutionarily conserved role for the Dpy19 family in neuronal migration.
Spermatogenesis is a complex biological process that requires a highly specialized control of gene expression. In the past decade, small non-coding RNAs have emerged as critical regulators of gene expression both at the transcriptional and post-transcriptional level. DICER1, an RNAse III endonuclease, is essential for the biogenesis of several classes of small RNAs, including microRNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs), but is also critical for the degradation of toxic transposable elements. In this study, we investigated to which extent DICER1 is required for germ cell development and the progress of spermatogenesis in mice.
We show that the selective ablation of Dicer1 at the early onset of male germ cell development leads to infertility, due to multiple cumulative defects at the meiotic and post-meiotic stages culminating with the absence of functional spermatozoa. Alterations were observed in the first spermatogenic wave and include delayed progression of spermatocytes to prophase I and increased apoptosis, resulting in a reduced number of round spermatids. The transition from round to mature spermatozoa was also severely affected, since the few spermatozoa formed in mutant animals were immobile and misshapen, exhibiting morphological defects of the head and flagellum. We also found evidence that the expression of transposable elements of the SINE family is up-regulated in Dicer1-depleted spermatocytes.
Our findings indicate that DICER1 is dispensable for spermatogonial stem cell renewal and mitotic proliferation, but is required for germ cell differentiation through the meiotic and haploid phases of spermatogenesis.
Nearly 7% of men are afflicted by male infertility worldwide, and genetic factors are suspected to play a significant role in the majority of these patients. Although sperm morphology is an important parameter measured in the semen analysis, only a few genetic causes of teratozoospermia are currently known. The objective of this study was to define the association between alterations in the genes encoding the Golgi-associated PDZ- and coiled-coil motif containing protein (GOPC), the protein interacting with C kinase 1 (PICK1) and the acrosomal protein zona pellucida binding protein 1 (ZPBP1/sp38) with abnormal sperm head morphology in infertile men. Previous reports demonstrated that mice lacking Gopc, Pick1 and Zpbp1 are infertile due to abnormal head morphology. Herein, using our validated RNA-based method, we studied spermatozoal cDNA encoding the human GOPC, PICK1 and ZPBP1 genes in 381 teratozoospermic and 240 controls patients via direct sequencing. Among these genes, we identified missense and splicing mutations in the sperm cDNA encoding ZPBP1 in 3.9% (15/381) of men with abnormal sperm head morphology. These mutations were not observed in 240 matched controls and the dbSNP database (χ(2) = 9.3, P = 0.002). In contrast, statistically significant and functionally relevant mutations were not discovered in the GOPC and PICK1 genes. In our study ZPBP1 mutations are associated with abnormal sperm head morphology, defined according to strict criteria, resembling the mouse Zpbp1 null phenotype. We hypothesize that missense mutations exert a dominant-negative effect due to altered ZPBP1 protein folding and protein:protein interactions in the acrosome.
The acrosome is a unique organelle that plays an important role at fertilization and during sperm morphogenesis and that is absent in globozoospermia, an inherited infertility syndrome in humans. At the light of recent experimental evidence, the acrosome is considered a lysosome-related organelle to whose biogenesis both the endocytic and biosynthetic pathways contribute. Vps54 is a vesicular sorting protein involved in the retrograde traffic; the recessive Vps54(L967Q) mutation in the mouse results in the wobbler phenotype, characterized by motor-neuron degeneration and male infertility. Here we have investigated the spatio-temporal occurrence/progression of the wobbler fertility disorder starting from mice at post-natal day 35, the day of the first event of spermiation. We show that the pathogenesis of wobbler infertility originates at the first spermiogenetic wave, affecting acrosome formation and sperm head elongation. Vps54(L967Q)-labeled vesicles, on the contrary of the wild-type Vps54-labeled ones, are not able to coalesce into a larger vesicle that develops, flattens and shapes to give rise to the acrosome. Evidence that it is the malfunctioning of the endocytic traffic to hamper the development of the acrosome comes out from the study on UBPy. UBPy, a deubiquitinating enzyme, is a marker of acrosome biogenesis from the endocytic pathway. In wobbler spermatids UBPy-positive endosomes remain single, scattered vesicles that do not contribute to acrosome formation. As secondary defect of wobbler spermiogenesis, spermatid mitochondria are misorted; moreover, with the progression of the age/disease also Sertoli-germ cell adhesions are compromised suggesting a derailment in the endocytic route that underlies their restructuring.
The transmembrane inner nuclear membrane (INM) protein Samp1 is required for anchoring centrosomes near the nuclei. Using high-resolution fluorescence microscopy we show that Samp1 is distributed in a distinct and characteristic pattern in the nuclear envelope (NE), where it partially colocalizes with the LINC complex protein Sun1. By studying the localization of Samp1 deletion mutants and fusion proteins, we conclude that the cysteine-rich N-terminal half of Samp1 is nucleoplasmically exposed and is responsible for targeting to the INM. It contains four conserved CxxC motifs with the potential to form zinc fingers. The distribution of cysteine-to-alanine substitution mutants, designed to prevent zinc finger formation, showed that NE localization of Samp1 depends on intact CxxC motifs. Overexpression of Samp1 zinc finger mutants produced an abnormal dominant phenotype characterized by disrupted organization of a selective subset NE proteins, including emerin, Sun1, endogenous Samp1 and, in some cases, lamin A/C, but not lamin B, Sun2 or nucleoporins. Silencing of Samp1 expression showed that emerin depends on Samp1 for its correct localization in the NE. Our results demonstrate that Samp1 is functionally associated with the LINC complex protein Sun1 and proteins of the A-type lamina network.
In einer andrologischen Praxis wurden bei einem infertilen Mann im Ejakulat ausschließlich rundköpfige Spermatozoen beobachtet. Mit Hilfe der Semidünnschnitt-Technik und der Elektronenmikroskopie konnte an Ejakulat- und Biopsiematerial geklärt werden, daß in der Differenzierungsphase der Spermatogenese eine Mißbildung am Akrosom auftritt: Das Akrosom entwickelt sich unabhaägig vom Kern im Zytoplasma der Spermatide, der Kern macht einen normalen Kondensationsprozeß durch. Da das Akrosom somit keinen Einfluß auf die Formgebung des Spermatozoenkernes ausübt, bleibt der Spermatozoenkopf rund. Im Ejakulat erscheinen nur akrosomlose Spermatozoen, die deshalb nicht befruchtungsfähig sind. Die cytologischen Besonderheiten bei der Differenzierung der Spermatide zum Spermatozoon werden im Einzelnen beschrieben und diskutiert.
Lors de l'examen d'un homme consultant pour stérilité les auteurs n'observent dans le sperme recueilli que des spermatozoïdes ayant une tête ronde. Le sperme et l'histologie testiculaire sont étudiés en coupes semi-fines au microscope optique, en coupes ultra-minces au microscope électronique. Il a pu être ainsi démontré qu'une malformation acrosomiale se développe pendant la phase de differentiation de la spermatogenèse: l'acrosome se développe indépendament du noyau dans le cytoplasme des spermatides, le noyau luimême subissant une condensation normale. Comme l'acrosome n'influence pas la morphogenèse du noyau du spermatozoïde il résulte que la tête du spermatozoïde restera arrondie. Le sperme recueilli ne montre que des spermatozoïdes dépourvus de leur acrosome et sont done non fertilisant. Les observations mises en évidence à propos de la différenciation des spermatides en spermatozoïdes font l'objet d'une description et d'une discussion détaillée.
An increasing number of couples require medical assistance to achieve a pregnancy, and more than 2% of the births in Western countries now result from assisted reproductive technologies. To identify genetic variants responsible for male infertility, we performed a whole-genome SNP scan on patients presenting with total globozoospermia, a primary infertility phenotype characterized by the presence of 100% round acrosomeless spermatozoa in the ejaculate. This strategy allowed us to identify in most patients (15/20) a 200 kb homozygous deletion encompassing only DPY19L2, which is highly expressed in the testis. Although there was no known function for DPY19L2 in humans, previous work indicated that its ortholog in C. elegans is involved in cell polarity. In man, the DPY19L2 region has been described as a copy-number variant (CNV) found to be duplicated and heterozygously deleted in healthy individuals. We show here that the breakpoints of the deletions are located on a highly homologous 28 kb low copy repeat (LCR) sequence present on each side of DPY19L2, indicating that the identified deletions were probably produced by nonallelic homologous recombination (NAHR) between these two regions. We demonstrate that patients with globozoospermia have a homozygous deletion of DPY19L2, thus indicating that DPY19L2 is necessary in men for sperm head elongation and acrosome formation. A molecular diagnosis can now be proposed to affected men; the presence of the deletion confirms the diagnosis of globozoospermia and assigns a poor prognosis for the success of in vitro fertilization.
Globozoospermia, characterized by round-headed spermatozoa, is a rare (< 0.1% in male infertile patients) and severe teratozoospermia consisting primarily of spermatozoa lacking an acrosome. Studying a Jordanian consanguineous family in which five brothers were diagnosed with complete globozoospermia, we showed that the four out of five analyzed infertile brothers carried a homozygous deletion of 200 kb on chromosome 12 encompassing only DPY19L2. Very similar deletions were found in three additional unrelated patients, suggesting that DPY19L2 deletion is a major cause of globozoospermia, given that 19% (4 of 21) of the analyzed patients had such deletion. The deletion is most probably due to a nonallelic homologous recombination (NAHR), because the gene is surrounded by two low copy repeats (LCRs). We found DPY19L2 deletion in patients from three different origins and two different breakpoints, strongly suggesting that the deletion results from recurrent events linked to the specific architectural feature of this locus rather than from a founder effect, without fully excluding a recent founder effect. DPY19L2 is associated with a complete form of globozoospermia, as is the case for the first two genes found to be associated with globozoospermia, SPATA16 or PICK1. However, in contrast to SPATA16, for which no pregnancy was reported, pregnancies were achieved, via intracytoplasmic sperm injection, for two patients with DPY19L2 deletion, who then fathered three children.
To study the role of Hsp90β1, an endoplasmic chaperone, we have built a conditional knockout by crossing Hsp90β1(flox/flox) with the Vasa-Cre transgenic line. Spermatozoa deficient in Hsp90β1 could not naturally fertilize oocytes and exhibited large and globular heads with abnormal intermediate pieces, a phenotype reminiscent of human globozoospermia.