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Jurrien Dean - One of the best experts on this subject based on the ideXlab platform.
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Focus on Fertilization Insights into the molecular basis of sperm-egg recognition in mammals
2020Co-Authors: Tanya Hoodbhoy, Jurrien DeanAbstract:The zona pellucida surrounding the egg and pre-implantation embryo is required for in vivo fertility and early development. Explanatory models of sperm–egg recognition need to take into account the ability of sperm to bind to ovulated eggs, but not to two-cell embryos. For the last two decades, investigators have sought to identify an individual protein or carbohydrate side chain as the ‘sperm receptor’. However, recent genetic data in mice are more consistent with the three-dimensional structure of the zona pellucida, rather than a single protein (or carbohydrate), determining sperm binding. The mouse and human zonae pellucidae contain three glycoproteins (ZP1, ZP2, ZP3) and, following fertilization, ZP2 is proteolytically cleaved. The replacement of endogenous mouse proteins with human ZP2, ZP3 or both does not alter taxon specificity of sperm binding or prevent fertility. Surprisingly, human ZP2 is not cleaved following fertilization and intact ZP2 correlates with persistent sperm binding to two-cell embryos. Taken together, these data support a model in which the cleavage status of ZP2 modulates the three-dimensional structure of the zona pellucida and determines whether sperm bind (uncleaved) or do not (cleaved). Reproduction (2004) 127 417–422
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Reassessing the molecular biology of sperm–egg recognition with mouse genetics
BioEssays, 2020Co-Authors: Jurrien DeanAbstract:The zona pellucida is an extracellular coat that surrounds mammalian eggs and early embryos. This insoluble matrix separates germ from somatic cells during folliculogenesis and plays critical roles during fertilization and early development. The mouse and human zona pellucida contain three glycoproteins (ZP1 or ZPB, ZP2, ZP3), the primary structures of which have been deduced by molecular cloning. Targeted mutagenesis of endogenous mouse genes and transgenesis with human homologues provide models to investigate the roles of individual zona components. Collectively, the genetic data indicate that no single mouse zona pellucida protein is obligatory for taxon-specific sperm binding and that two human proteins are not sufficient to support human sperm binding. An observed post-fertilization persistence of mouse sperm binding to “humanized” zona pellucida correlates with uncleaved ZP2. These observations are consistent with a model for sperm binding in which the supramolecular structure of the zona pellucida necessary for sperm binding is modulated by the cleavage status of ZP2. BioEssays 26:29–38, 2004. Published 2003 Wiley Periodicals, Inc.
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glycan independent gamete recognition triggers egg zinc sparks and ZP2 cleavage to prevent polyspermy
Developmental Cell, 2018Co-Authors: Keizo Tokuhiro, Jurrien DeanAbstract:Summary The zona pellucida surrounding ovulated eggs regulates monospermic fertilization necessary for successful development. Using mouse transgenesis, we document that the N terminus of ZP2 is sufficient for sperm binding to the zona matrix and for in vivo fertility. Sperm binding is independent of ZP2 glycans and does not occur after complete cleavage of ZP2 by ovastacin, a zinc metalloendopeptidase stored in egg cortical granules. Immediately following fertilization, a rapid block to sperm penetration of the zona pellucida is established that precedes ZP2 cleavage but requires ovastacin enzymatic activity. This block to penetration is associated with release of zinc from cortical granules coincident with exocytosis. High levels of zinc affect forward motility of sperm to prevent their passage through the zona matrix. This transient, post-fertilization block to sperm penetration provides a temporal window to complete the cleavage of ZP2, which prevents sperm binding to ensure monospermy.
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a unique egg cortical granule localization motif is required for ovastacin sequestration to prevent premature ZP2 cleavage and ensure female fertility in mice
PLOS Genetics, 2017Co-Authors: Bo Xiong, Yangu Zhao, S Beall, Anna Burkart Sadusky, Jurrien DeanAbstract:Monospermic fertilization is mediated by the extracellular zona pellucida composed of ZP1, ZP2 and ZP3. Sperm bind to the N-terminus of ZP2 which is cleaved after fertilization by ovastacin (encoded by Astl) exocytosed from egg cortical granules to prevent sperm binding. AstlNull mice lack the post-fertilization block to sperm binding and the ability to rescue this phenotype with AstlmCherry transgenic mice confirms the role of ovastacin in providing a definitive block to polyspermy. During oogenesis, endogenous ovastacin traffics through the endomembrane system prior to storage in peripherally located cortical granules. Deletion mutants of ovastacinmCherry expressed in growing oocytes define a unique 7 amino acid motif near its N-terminus that is necessary and sufficient for cortical granule localization. Deletion of the 7 amino acids by CRISPR/Cas9 at the endogenous locus (AstlΔ) prevents cortical granule localization of ovastacin. The misdirected enzyme is present within the endomembrane system and ZP2 is prematurely cleaved. Sperm bind poorly to the zona pellucida of AstlΔ/Δ mice with partially cleaved ZP2 and female mice are sub-fertile.
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ZP2 peptide beads select human sperm in vitro decoy mouse sperm in vivo and provide reversible contraception
Science Translational Medicine, 2016Co-Authors: Matteo Alessandro Avella, Maria Jimenezmovilla, Boris Baibakov, Anna Burkart Sadusky, Jurrien DeanAbstract:The zona pellucida, a matrix that surrounds ovulated eggs, is the site of sperm recognition and binding, which precede sperm penetration and fertilization. Avella et al . identified a peptide from the zona pellucida called ZP2, which the authors attached to agarose beads to facilitate infertility treatment or, conversely, contraception. On the one hand, the beads with ZP2 peptide were combined with sperm in vitro to successfully select the best sperm for assisted reproductive technologies. On the other hand, the same beads placed into the uterus of fertile mice bound any incoming sperm and prevented them from reaching the eggs, thus providing a long-acting method of contraception. A related podcast discusses these findings.
Maria Jimenezmovilla - One of the best experts on this subject based on the ideXlab platform.
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ZP2 peptide beads select human sperm in vitro decoy mouse sperm in vivo and provide reversible contraception
Science Translational Medicine, 2016Co-Authors: Matteo Alessandro Avella, Maria Jimenezmovilla, Boris Baibakov, Anna Burkart Sadusky, Jurrien DeanAbstract:The zona pellucida, a matrix that surrounds ovulated eggs, is the site of sperm recognition and binding, which precede sperm penetration and fertilization. Avella et al . identified a peptide from the zona pellucida called ZP2, which the authors attached to agarose beads to facilitate infertility treatment or, conversely, contraception. On the one hand, the beads with ZP2 peptide were combined with sperm in vitro to successfully select the best sperm for assisted reproductive technologies. On the other hand, the same beads placed into the uterus of fertile mice bound any incoming sperm and prevented them from reaching the eggs, thus providing a long-acting method of contraception. A related podcast discusses these findings.
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porcine sperm bind to beads conjugated to ZP2 protein under in vitro conditions
Animal reproduction, 2016Co-Authors: Julieta G Hamze, A Canha, L Zamorano, B Algarra, M C Olivares, Raquel Romar, Maria JimenezmovillaAbstract:The oocyte is encapsulated by a filamentous structure composed of several glycoproteins termed zona pellucida (ZP),that acts as a matrix mediating interaction with sperm. It has been shown that processing of ZP2 at N terminal position (LADEN) mediates the recognition between gametes (Avella M, J Cell Biol. 205(6):801-9, 2014). The study of these molecular mechanisms is very limited due to the ethical problems, the difficulty of obtaining mature oocytes in many mammalian species, the high cost of genetically modified mice and the inability to transfer this knowledge to other species such as swine. Therefore, we propose an in vitro model that mimics the oocyte shape, allowing the research on the interaction of gametes, identification and characterization of the ZP proteins activity and the conditions for sperm recognition. In this work, the proposed model is developed by combining magnetic beads (His Mag Sepharose™ Excel) conjugated with porcine ZP2 and ZP4 recombinant proteins. This novel model besides increasing our knowledge of the oocyte-sperm interaction, could also be industry-wide implemented as an evaluator of mammalian sperm quality. For this study ZP2 and ZP4 recombinant proteins were marked with a Flag and V5 tag recognition site, respectively, and with a histidine tag for easy identification and adhesion to the beads. The proteins were expressed in mammalian cells (CHO) and once secreted, identified by electrophoresis and western blot. Then, the secreted recombinant proteins were conjugated with the magnetic beads. Groups of 40-45 ZP proteins conjugated-beads and beads raised with growth CHO-cell medium (Control group) were coincubated for 2hr with boar spermatozoa (heterospermic dose) in TALP medium at a final concentration of 200.000 spermatozoa/ml. After 2h coincubation, the beads were washed twice in PBS, fixed and stained with Hoechst. Bound sperm in each bead was scored by fluorescence microscopy and the obtained results analysed by one-way ANOVA. Three replicates in a blind analysis were done and P-value <0.05 was taken to denote statistical significance. Secreted proteins ZP2 and ZP4 were identified by electrophoresis and western blot with anti-Flag and and Anti-V5 antibodies, respectively. The ZP2 showed a molecular weight of 100 kDa and ZP4 a molecular weight of 65 kDa. Adhesion of secreted proteins to the beads was confirmed by western blot. Finally, number of sperms bound to ZP2-beads (8.56 ± 0.64, n = 230) was significantly higher (P < 0.001) than sperm bound to ZP4-beads (3.00 ± 0.27, n = 233) and control (4.00 ± 0.36, n = 207). In conclusion, a novel in vitro model combining magnetic beads with ZP proteins (ZP2 and ZP4) was developed in order to study the role of ZP2 protein on sperm-oocyte interaction. Future studies could increase our knowledge of the oocyte-sperm interaction and could be also implemented as an in vitroselection and quality evaluation technique of potentially fertile mammalian spermatozoa.
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ovastacin a cortical granule protease cleaves ZP2 in the zona pellucida to prevent polyspermy
Journal of Cell Biology, 2012Co-Authors: Anna D Burkart, Maria Jimenezmovilla, Boris Baibakov, Bo Xiong, Jurrien DeanAbstract:The mouse zona pellucida is composed of three glycoproteins (ZP1, ZP2, and ZP3), of which ZP2 is proteolytically cleaved after gamete fusion to prevent polyspermy. This cleavage is associated with exocytosis of cortical granules that are peripherally located subcellular organelles unique to ovulated eggs. Based on the cleavage site of ZP2, ovastacin was selected as a candidate protease. Encoded by the single-copy Astl gene, ovastacin is an oocyte-specific member of the astacin family of metalloendoproteases. Using specific antiserum, ovastacin was detected in cortical granules before, but not after, fertilization. Recombinant ovastacin cleaved ZP2 in native zonae pellucidae, documenting that ZP2 was a direct substrate of this metalloendoprotease. Female mice lacking ovastacin did not cleave ZP2 after fertilization, and mouse sperm bound as well to Astl-null two-cell embryos as they did to normal eggs. Ovastacin is a pioneer component of mouse cortical granules and plays a definitive role in the postfertilization block to sperm binding that ensures monospermic fertilization and successful development.
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ZP2 and zp3 cytoplasmic tails prevent premature interactions and ensure incorporation into the zona pellucida
Journal of Cell Science, 2011Co-Authors: Maria Jimenezmovilla, Jurrien DeanAbstract:The zona pellucida contains three proteins (ZP1, ZP2, ZP3), the precursors of which possess signal peptides, ‘zona’ domains and short (9–15 residue) cytoplasmic tails downstream of a transmembrane domain. The ectodomains of ZP2 and ZP3 are sufficient to form the insoluble zona matrix and yet each protein traffics through oocytes without oligomerization. ZP2 and ZP3 were fluorescently tagged and molecular interactions were assayed by fluorescent complementation in CHO cells and growing oocytes. ZP2 and ZP3 traffic independently, but colocalize at the plasma membrane. However, protein–protein interactions were observed only after release and incorporation of ZP2 and ZP3 into the extracellular matrix surrounding mouse oocytes. In the absence of their hydrophilic cytoplasmic tails, ZP2 and ZP3 interacted within the cell and did not participate in the zona pellucida. A heterologous GPI-anchored ‘zona’ domain protein fused with the cytoplasmic tails was integrated into the zona matrix. We conclude that the cytoplasmic tails are sufficient and necessary to prevent intracellular oligomerization while ensuring incorporation of processed ZP2 and ZP3 into the zona pellucida.
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hamster zona pellucida is formed by four glycoproteins zp1 ZP2 zp3 and zp4
Journal of Proteome Research, 2009Co-Authors: M J Izquierdorico, Maria Jimenezmovilla, E Llop, A B Perezoliva, J Ballesta, Ricardo Gutierrezgallego, C Jimenezcervantes, Manuel AvilesAbstract:The zona pellucida (ZP) is an extracellular glycoprotein matrix that surrounds all mammalian oocytes. Recent data have shown the presence of four glycoproteins (ZP1, ZP2, ZP3, and ZP4) in the ZP of human and rat rather than the three glycoproteins proposed in the mouse model. In the hamster (Mesocricetus auratus), it was previously described that ZP was composed of three different glycoproteins, called ZP1, ZP2, and ZP3, even though only ZP2 and ZP3 have been cloned thus far. The aim of the study was to determine whether hamster might also express four, rather than three, ZP proteins. The full-length cDNAs encoding hamster ZP glycoproteins 1 and 4 were isolated using rapid amplification cDNA ends (RACE). The cDNA of ZP1 contains an open reading frame of 1851 nucleotides encoding a polypeptide of 616 amino acid residues. The amino acid sequence of ZP1 revealed a high homology with other mammalian species like human (66%), rat (80%), and mouse (80%). The cDNA of ZP4 contains an open reading frame of 1632 nu...
K D Hinsch - One of the best experts on this subject based on the ideXlab platform.
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quantification of zp1 ZP2 and zp3 mrna of marmoset monkey callithrix jacchus oocytes from periantral and antral follicles
Andrologia, 2012Co-Authors: Lutz Konrad, K D Hinsch, S Kuhnert, P Nayudu, R Einspanier, E HinschAbstract:Summary In mammals, the oocyte and preimplantation embryo are protected by the zona pellucida (ZP) consisting mainly of ZP glycoproteins, which are responsible for sperm binding, induction of the acrosome reaction and zona pellucida hardening to prevent polyspermia. The ZP proteins become increasingly important as possible predictors for in vitro cultured oocytes competence. As little is known about the stage-dependent expression of ZP1, ZP2 and ZP3 in marmoset monkey (Callithrix jacchus) oocytes, mRNA expression was investigated with realtime RT-PCR. Total-RNA was isolated from three different classes of marmoset oocytes; Class 1 oocytes from periantral follicles ( 1000 lm, n = 9). Compared with Class 1 oocytes mRNA expression of ZP1, ZP2 and ZP3 in Class 2 oocytes was significantly decreased. In Class 3 oocytes, the transcription of ZP1, ZP2 and ZP3 genes showed also a significant decrease compared with Class 1 oocytes. In this study a differently regulated expression of the ZP genes during late folliculogenesis with an obvious downregulation of ZP1, ZP2 and ZP3 could be demonstrated for the first time in the marmoset monkey.
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immunological identification of zona pellucida 2 ZP2 protein in human oocytes
Andrologia, 2009Co-Authors: E Hinsch, W Hagele, W B Schill, Sergio Oehninger, K D HinschAbstract:The ZP2 protein is a zona pellucida glycoprotein that plays a major role in fertilization. It mediates secondary binding of spermatozoa and is one of the proteins that are involved in zona 'hardening'. ZP2 proteins were identified in various mammalian zonae pellucidae. Their primary structures are highly conserved as revealed by cDNA cloning. Antisera were used against synthetic peptides generated either against a ZP2 amino acid that is homologous in human and mouse ZP2 amino acid sequences (AS ZP2-20) or antibodies against a synthetic human ZP2 peptide (AS ZP2-26). Immunoblots showed that antiserum AS ZP2 20 and AS ZP2-26 strongly recognized human ZP2 protein with an apparent molecular mass of about 72 kDa; both antisera reacted with a minor immunoreactive polypeptide at 96 kDa. In human ovary sections, both antiscra revealed immunoreactivity to human zonae pellucidae. Immuno-electron microscopy demonstrated an equal distribution of ZP2 throughout the human zona pcllucida. Considerable amounts of immunoreactive material were observed in the ooplasm; some ramification-like extensions of zona pellucida antigen were found close to cells surrounding the oocyte. Our results indicate that antisera against synthetic ZP2 peptides can be used as specific markers for the identification of ZP2 protein in human oocytes.
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the zona pellucida receptors zp1 ZP2 and zp3
Andrologia, 1999Co-Authors: K D Hinsch, E HinschAbstract:The male component that is necessary for successful reproduction depends on a large variety of biological processes working in concert. The sperm-egg interaction occurs through complementary molecules and is an obligatory process for successful fertilization. However, this complex phenomenon and its molecular mechanisms remain to be fully understood. The oocyte is protected by the zona pellucida, a network of various proteins which encloses the oocyte. Depending on the species, the zona pellucida consists of different glycoproteins that are proposed to function as 'receptors' for spermatozoa. In the mouse, ZP1 is the homodimeric filament crosslinker, held together by intermolecular disulphides. ZP2 is the 'secondary receptor', which is cleaved by egg proteases after egg activation. The mouse ZP3 protein appears to be the 'primary receptor', which is responsible for species-specific binding of spermatozoa to the oocyte and the induction of the acrosome reaction. To localize zona pellucida protein and to evaluate the function of ZP2 and ZP3, polyclonal antisera were raised against synthetic ZP2 or ZP3 peptides which are specific for human or for mouse zona pellucida proteins. It could be demonstrated that anti-synthetic peptide antisera detected their respective zona pellucida proteins in immunoblots, ovary sections and native hemizonae pellucidae. Functional assays with anti-ZP3 synthetic peptide antibodies revealed that the antisera did not inhibit sperm-zona pellucida binding, whereas one of the antisera against synthetic ZP2 peptides significantly inhibited binding of spermatozoa to the zona pellucida.
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evaluation of ZP2 domains of functional importance with antisera against synthetic ZP2 peptides
Reproduction, 1998Co-Authors: E Hinsch, W Hagele, R M Bohle, W B Schill, K D HinschAbstract:The mouse zona pellucida protein ZP2 plays an important role in the process of fertilization by mediating secondary sperm binding to mammalian oocytes. ZP2 primary structures are highly conserved as revealed by cDNA cloning. The aim of the study was to identify ZP2 domains of functional relevance. Antisera were raised against synthetic peptides that are either conserved in the structure of ZP2 from different mammalian species (AS ZP2-20) or present in the human ZP2 but not in the mouse ZP2 amino acid sequence (AS ZP2-26). Antibody binding to zona pellucida proteins was assessed by assaying the antisera with human hemizonae. Using human zonae pellucidae, we demonstrated that anti-ZP2 common antibodies and anti-ZP2 human peptide antibodies react with human zona pellucida antigens. For the first time, ZP2 domains of functional relevance for human sperm-oocyte interaction could be identified applying the competitive hemizona assay. Antiserum AS ZP2-20 significantly inhibited binding of spermatozoa to test hemizonae, whereas treatment of hemizonae with AS ZP2-26 did not influence sperm-oocyte interaction. These results show that antibodies against synthetic ZP2 peptides react with ZP2 protein and that AS ZP2-20 identifies a linear ZP2 epitope that is of possible functional importance for sperm-oocyte interaction.
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The zona pellucida "receptors".
Advances in Experimental Medicine and Biology, 1997Co-Authors: E Hinsch, W Hagele, W B Schill, K D HinschAbstract:Binding of mammalian sperm to the zona pellucida and the induction of the acrosome reaction are prerequisites for successful oocyte fertilization. In the mouse model, the zona pellucida consists of three sulfated glycoproteins, ZP1, ZP2, and ZP3. Zona pellucida proteins are secreted to form a filamentous zona matrix in which ZP2 and ZP3 complex into co-polymers cross-linked by ZP1. ZP3 is the ligand for primary sperm binding and important for the induction of the acrosome reaction. The zona pellucida glycoprotein ZP2 is also crucially involved in the process of fertilization. Previous reports suggest that ZP2 mediates secondary binding of spermatozoa and that cleavage of ZP2 by proteases released through cortical granule reaction causes zona “hardening” and thus prevents polyspermy. Human and mouse ZP2 proteins differ in the primary structure as derived from cDNA clones. We designed an immunological approach to search for ZP2 domains with functional relevance. Antisera were generated against synthetic peptides derived (a) from ZP2 amino acid sequences that are homologous in human and mouse ZP2 amino acid sequences (AS ZP2—20) or (b) from human ZP2 amino acid sequences that differ from the mouse ZP2 sequence (AS ZP2—26). Immunochemical studies with microbisected bovine zonae pellucidae demonstrated that both antisera, AS ZP2—20 and AS ZP2—26, specifically detected ZP2 protein. Using the competition-hemizona-assay, sperm binding to antibody treated bovine hemizonae pellucidae were compared with control hemizonae (given as hemizona index). Antiserum AS ZP2—20 significantly inhibited binding of spermatozoa to test hemizonae (p
Boris Baibakov - One of the best experts on this subject based on the ideXlab platform.
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ZP2 peptide beads select human sperm in vitro decoy mouse sperm in vivo and provide reversible contraception
Science Translational Medicine, 2016Co-Authors: Matteo Alessandro Avella, Maria Jimenezmovilla, Boris Baibakov, Anna Burkart Sadusky, Jurrien DeanAbstract:The zona pellucida, a matrix that surrounds ovulated eggs, is the site of sperm recognition and binding, which precede sperm penetration and fertilization. Avella et al . identified a peptide from the zona pellucida called ZP2, which the authors attached to agarose beads to facilitate infertility treatment or, conversely, contraception. On the one hand, the beads with ZP2 peptide were combined with sperm in vitro to successfully select the best sperm for assisted reproductive technologies. On the other hand, the same beads placed into the uterus of fertile mice bound any incoming sperm and prevented them from reaching the eggs, thus providing a long-acting method of contraception. A related podcast discusses these findings.
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a single domain of the ZP2 zona pellucida protein mediates gamete recognition in mice and humans
Journal of Cell Biology, 2014Co-Authors: Matteo Alessandro Avella, Boris Baibakov, Jurrien DeanAbstract:The extracellular zona pellucida surrounds ovulated eggs and mediates gamete recognition that is essential for mammalian fertilization. Zonae matrices contain three (mouse) or four (human) glycoproteins (ZP1–4), but which protein binds sperm remains controversial. A defining characteristic of an essential zona ligand is sterility after genetic ablation. We have established transgenic mice expressing human ZP4 that form zonae pellucidae in the absence of mouse or human ZP2. Neither mouse nor human sperm bound to these ovulated eggs, and these female mice were sterile after in vivo insemination or natural mating. The same phenotype was observed with truncated ZP2 that lacks a restricted domain within ZP251–149. Chimeric human/mouse ZP2 isoforms expressed in transgenic mice and recombinant peptide bead assays confirmed that this region accounts for the taxon specificity observed in human–mouse gamete recognition. These observations in transgenic mice document that the ZP251–149 sperm-binding domain is necessary for human and mouse gamete recognition and penetration through the zona pellucida.
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human sperm bind to the n terminal domain of ZP2 in humanized zonae pellucidae in transgenic mice
Journal of Cell Biology, 2012Co-Authors: Boris Baibakov, Nathan A Boggs, Belinda J Yauger, Galina Baibakov, Jurrien DeanAbstract:Fertilization requires taxon-specific gamete recognition, and human sperm do not bind to zonae pellucidae (ZP1–3) surrounding mouse eggs. Using transgenesis to replace endogenous mouse proteins with human homologues, gain-of-function sperm-binding assays were established to evaluate human gamete recognition. Human sperm bound only to zonae pellucidae containing human ZP2, either alone or coexpressed with other human zona proteins. Binding to the humanized matrix was a dominant effect that resulted in human sperm penetration of the zona pellucida and accumulation in the perivitelline space, where they were unable to fuse with mouse eggs. Using recombinant peptides, the site of gamete recognition was located to a defined domain in the N terminus of ZP2. These results provide experimental evidence for the role of ZP2 in mediating sperm binding to the zona pellucida and support a model in which human sperm–egg recognition is dependent on an N-terminal domain of ZP2, which is degraded after fertilization to provide a definitive block to polyspermy.
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ovastacin a cortical granule protease cleaves ZP2 in the zona pellucida to prevent polyspermy
Journal of Cell Biology, 2012Co-Authors: Anna D Burkart, Maria Jimenezmovilla, Boris Baibakov, Bo Xiong, Jurrien DeanAbstract:The mouse zona pellucida is composed of three glycoproteins (ZP1, ZP2, and ZP3), of which ZP2 is proteolytically cleaved after gamete fusion to prevent polyspermy. This cleavage is associated with exocytosis of cortical granules that are peripherally located subcellular organelles unique to ovulated eggs. Based on the cleavage site of ZP2, ovastacin was selected as a candidate protease. Encoded by the single-copy Astl gene, ovastacin is an oocyte-specific member of the astacin family of metalloendoproteases. Using specific antiserum, ovastacin was detected in cortical granules before, but not after, fertilization. Recombinant ovastacin cleaved ZP2 in native zonae pellucidae, documenting that ZP2 was a direct substrate of this metalloendoprotease. Female mice lacking ovastacin did not cleave ZP2 after fertilization, and mouse sperm bound as well to Astl-null two-cell embryos as they did to normal eggs. Ovastacin is a pioneer component of mouse cortical granules and plays a definitive role in the postfertilization block to sperm binding that ensures monospermic fertilization and successful development.
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Gamete Recognition in Mice Depends on the Cleavage Status of an Egg’s Zona Pellucida Protein
Science, 2010Co-Authors: Gagandeep K. Gahlay, Boris Baibakov, Olga Epifano, Lyn Gauthier, Jurrien DeanAbstract:At fertilization, mouse sperm bind to the zona pellucida (which consists of glycoproteins ZP1, ZP2, and ZP3) that surrounds eggs. A ZP2 cleavage model of gamete recognition requires intact ZP2, and a glycan release model postulates that zona glycans are ligands for sperm. These two models were tested by replacing endogenous protein with ZP2 that cannot be cleaved (ZP2Mut) or with ZP3 lacking implicated O glycans (Zp3Mut). Sperm bound to two-cell ZP2Mut embryos despite fertilization and cortical granule exocytosis. Contrary to prediction, sperm fertilized Zp3Mut eggs. Sperm at the surface of the zona pellucida remained acrosome-intact for more than 2 hours and were displaced by additional sperm. These data indicate that sperm-egg recognition depends on the cleavage status of ZP2 and that binding at the surface of the zona is not sufficient to induce sperm acrosome exocytosis.
Manuel Aviles - One of the best experts on this subject based on the ideXlab platform.
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Composition of marsupial zona pellucida: a molecular and phylogenetic approach.
Reproduction Fertility and Development, 2017Co-Authors: Carla Moros-nicolás, Pascale Chevret, M. J. Izquierdo-rico, William V. Holt, Daniela Esteban-díaz, Manel Lopez-bejar, Eva Martínez-nevado, Maria A. Nilsson, José Ballesta, Manuel AvilesAbstract:The zona pellucida (ZP) is an extracellular matrix that surrounds mammalian oocytes. In eutherians it is formed from three or four proteins (ZP1, ZP2, ZP3, ZP4). In the few marsupials that have been studied, however, only three of these have been characterised (ZP2, ZP3, ZP4). Nevertheless, the composition in marsupials may be more complex, since a duplication of the ZP3 gene was recently described in one species. The aim of this work was to elucidate the ZP composition in marsupials and relate it to the evolution of the ZP gene family. For that, an in silico and molecular analysis was undertaken, focusing on two South American species (gray short-tailed opossum and common opossum) and five Australian species (brushtail possum, koala, Bennett’s wallaby, Tammar wallaby and Tasmanian devil). This analysis identified the presence of ZP1 mRNA and mRNA from two or three paralogues of ZP3 in marsupials. Furthermore, evidence for ZP1 and ZP4 pseudogenes in the South American subfamily Didelphinae and for ZP3 pseudogenes in two marsupials is provided. In conclusion, two different composition models are proposed for marsupials: a model with four proteins (ZP1, ZP2 and ZP3 (two copies)) for the South American species and a model with six proteins (ZP1, ZP2, ZP3 (three copies) and ZP4) for the Australasian species.
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hamster zona pellucida is formed by four glycoproteins zp1 ZP2 zp3 and zp4
Journal of Proteome Research, 2009Co-Authors: M J Izquierdorico, Maria Jimenezmovilla, E Llop, A B Perezoliva, J Ballesta, Ricardo Gutierrezgallego, C Jimenezcervantes, Manuel AvilesAbstract:The zona pellucida (ZP) is an extracellular glycoprotein matrix that surrounds all mammalian oocytes. Recent data have shown the presence of four glycoproteins (ZP1, ZP2, ZP3, and ZP4) in the ZP of human and rat rather than the three glycoproteins proposed in the mouse model. In the hamster (Mesocricetus auratus), it was previously described that ZP was composed of three different glycoproteins, called ZP1, ZP2, and ZP3, even though only ZP2 and ZP3 have been cloned thus far. The aim of the study was to determine whether hamster might also express four, rather than three, ZP proteins. The full-length cDNAs encoding hamster ZP glycoproteins 1 and 4 were isolated using rapid amplification cDNA ends (RACE). The cDNA of ZP1 contains an open reading frame of 1851 nucleotides encoding a polypeptide of 616 amino acid residues. The amino acid sequence of ZP1 revealed a high homology with other mammalian species like human (66%), rat (80%), and mouse (80%). The cDNA of ZP4 contains an open reading frame of 1632 nu...
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ZP2 and zp3 traffic independently within oocytes prior to assembly into the extracellular zona pellucida
Molecular and Cellular Biology, 2006Co-Authors: Tanya Hoodbhoy, Maria Jimenezmovilla, Manuel Aviles, Olga Epifano, Boris Baibakov, Lyn Gauthier, Jurrien DeanAbstract:The extracellular zona pellucida surrounds mammalian eggs and mediates taxon-specific sperm-egg recognition at fertilization. In mice, the zona pellucida is composed of three glycoproteins, but the presence of ZP2 and ZP3 is sufficient to form a biologically functional structure. Each zona pellucida glycoprotein is synthesized in growing oocytes and traffics through the endomembrane system to the cell surface, where it is released from a transmembrane domain and assembled into the insoluble zona pellucida matrix. ZP2 and ZP3 colocalize in the endoplasmic reticulum and in 1- to 5-μm post-Golgi structures comprising multivesicular aggregates (MVA), but a coimmunoprecipitation assay does not detect physical interactions. In addition, ZP2 traffics normally in growing oocytes in the absence of ZP3 or if ZP3 has been mutated to prevent incorporation into the zona pellucida matrix, complementing earlier studies indicating the independence of ZP3 secretion in ZP2 null mice. N glycosylation has been implicated in correct protein folding and intracellular trafficking of secreted proteins. Although ZP3 contain five N-glycans, enhanced green fluorescent protein-tagged ZP3 lacking N glycosylation sites is present in MVA and is incorporated into the zona pellucida matrix of transgenic mice. Thus, ZP2 secretion is seemingly unaffected by ZP3 lacking N-glycans. Taken together, these observations indicate that ZP2 and ZP3 traffic independently through the oocyte prior to assembly into the zona pellucida.
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modifications of carbohydrate residues and ZP2 and zp3 glycoproteins in the mouse zona pellucida after fertilization
Biology of Reproduction, 1997Co-Authors: Louay Jaber, Manuel Aviles, M T Castells, J BallestaAbstract:Oligosaccharide side chains of zona pellucida (ZP) glycoproteins play a key role in the sperm-egg interaction phenomena during fertilization. In the present study, modifications of the ZP glycoproteins during the fertilization process in the mouse were studied by the lectin-gold technique and immunocytochemistry in conjunction with quantitative analysis. Binding of PNA, RCA I, DSA, LFA, MAA, AAA, and anti-ZP2 and anti-ZP3 antibodies was observed throughout the ZP of both unfertilized and fertilized eggs. However, HPA and BSAIB4 labeling was found only in the inner region of the ZP. After neuraminidase treatment (Neu), HPA showed an affinity for the entire ZP. Labeling by LFA, WGA, MAA, PNA, BSAIB4, and AAA decreased in the ZP of fertilized eggs; however, there was an increase in the binding of RCA I. HPA and Neu-HPA increased only in the inner region of the ZP. Immunoreactivity to antibodies against ZP2 and ZP3 also decreased after fertilization. The present results demonstrate that 1) terminal carbohydrate residues contained in the ZP glycoproteins are modified after fertilization and 2) inner and outer regions of the ZP contain different oligosaccharide side chains.
