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Jurrien Dean - One of the best experts on this subject based on the ideXlab platform.
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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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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, Olga Epifano, Boris Baibakov, Lyn Gauthier, Manuel Avilés, 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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Human sperm do not bind to rat zonae Pellucidae despite the presence of four homologous glycoproteins.
The Journal of biological chemistry, 2005Co-Authors: Tanya Hoodbhoy, Saurabh Joshi, Emily S. Boja, Suzannah A. Williams, Pamela Stanley, Jurrien DeanAbstract:The specificity of sperm-egg recognition in mammals is mediated primarily by the zona Pellucida surrounding ovulated eggs. Mouse sperm are quite promiscuous and bind to human eggs, but human spermatozoa will not bind to mouse eggs. The mouse zona Pellucida contains three glycoproteins, ZP1, ZP2, and ZP3, which are conserved in rat and human. The recent observation that human zonae Pellucidae contain a fourth protein raises the possibility that the presence of four zona proteins will support human sperm binding. Using mass spectrometry, four proteins that are similar in size and share 62-70% amino acid identity with human ZP1, ZP2, ZP3, and ZP4/ZPB were detected in rat zonae Pellucidae. However, although mouse and rat spermatozoa bind to eggs from each rodent, human sperm bind to neither, and the presence of human follicular fluid did not alter the specificity of sperm binding. In addition, mutant mouse eggs lacking hybrid/complex N-glycans or deficient in Core 2 O-glycans were no more able to support human sperm binding than normal mouse eggs. These data suggest that the presence of four zona proteins are not sufficient to support human sperm binding to rodent eggs and that additional determinants must be responsible for taxon-specific fertilization among mammals.
H W G Baker - One of the best experts on this subject based on the ideXlab platform.
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defective sperm zona Pellucida interaction a major cause of failure of fertilization in clinical in vitro fertilization
Human Reproduction, 2000Co-Authors: De Yi Liu, H W G BakerAbstract:Sperm-zona Pellucida binding and penetration were assessed on the oocytes that failed to fertilize from couples with >/=3 oocytes treated by standard in-vitro fertilization (IVF). There were four groups: fertilization rate 0% (n = 369), 1-25% (n = 194), 26-50% (n = 81) and 51-95% (n = 100). Of the couples with zero fertilization rate 70% had spermatozoa bound per zona Pellucida and 42% had no spermatozoa penetrating the zona Pellucida of any oocyte. In contrast, in the 51-95% fertilization rate group, only 17% had spermatozoa bound per zona Pellucida and 6% had no spermatozoa penetrating the zona Pellucida. There was a significantly higher frequency of poor sperm morphology (oocytes from 68 couples with zero fertilization rate and low sperm-zonae Pellucidae binding with fertile donor spermatozoa resulted in normal sperm-zona Pellucida binding and most zonae Pellucidae being penetrated. In conclusion, defective sperm-zona Pellucida interaction was the major cause for low fertilization rates in standard IVF. This was usually because of defects of the spermatozoa rather than defects of the oocytes. Sperm defects likely to cause failure of fertilization should be diagnosed before commencing IVF and the patients directed to intracytoplasmic sperm injection.
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INHIBITION OF HUMAN SPERM-ZONA Pellucida AND SPERM-OOLEMMA BINDING BY ANTISPERM ANTIBODIES
Fertility and sterility, 1991Co-Authors: D. Y. Liu, Gary N. Clarke, H W G BakerAbstract:We have established tests for assessing sperm binding to the zona Pellucida and oolemma with human oocytes that failed to fertilize in a clinical IVF program. These tests have provided an opportunity for us to study how sperm antibodies inhibit sperm binding to the human zona Pellucida and oolemma of zona Pellucida-free oocytes in vitro
Bayard T Storey - One of the best experts on this subject based on the ideXlab platform.
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Kinetics of onset of mouse sperm acrosome reaction induced by solubilized zona Pellucida: fluorimetric determination of loss of pH gradient between acrosomal lumen and medium monitored by Dapoxyl (2-aminoethyl) sulfonamide and of intracellular Ca2+ c
Molecular reproduction and development, 2000Co-Authors: Pamela L. Rockwell, Bayard T StoreyAbstract:The onset of the zona Pellucida-induced acrosome reaction in mouse sperm is marked by loss of the pH gradient existing in acrosome-intact sperm between the acidic acrosomal lumen and the suspending medium, due to pore formation between outer acrosomal and plasma membranes. In earlier work, it was shown that this pH gradient loss occurred in single sperm bound to structurally intact zonae Pellucidae with a half-time of 2.1 min; the extended kinetics of this loss determined in a sperm population bound to intact zonae was due to a 180-min range of variable lag times. We hypothesized that this lag time range was due to steric constraints imposed by the three-dimensional structure of the structurally intact zona Pellucida, and that this constraint should be removed in solubilized zonae. The fluorescent probe, Dapoxyl(TM) (2-aminoethyl)sulfonamide (DAES) allowed a test of this hypothesis in a population of sperm cells. It is a weak base that is non-fluorescent in aqueous solution, but which accumulates in the acidic acrosomal compartment due to the pH gradient with highly enhanced fluorescence; loss of the pH gradient leads to a decrease in fluorescence. The half-time for DAES fluorescence loss in a population of capacitated, acrosome-intact sperm in response to solubilized zona Pellucida protein was 2.13 +/- 0.10 min (SEM, n = 9). The agreement between single cell and cell population kinetics validates the hypothesis of steric constraint in the structurally intact zona Pellucida. The change in intracellular Ca(2+) concentration in response to solubilized zona Pellucida, as monitored with fluo-3, was a rapid increase, followed by a decrease, with a half-time of 0.85 +/- 0.09 min (SEM, n = 6) to a steady state level higher than the initial level, indicating this Ca(2+) transient as the precursor reaction to onset of the zona-induced acrosome reaction.
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calcium influx into mouse spermatozoa activated by solubilized mouse zona Pellucida monitored with the calcium fluorescent indicator fluo 3 inhibition of the influx by three inhibitors of the zona Pellucida induced acrosome reaction tyrphostin a48 pe
Molecular Reproduction and Development, 1994Co-Authors: Janice L Bailey, Bayard T StoreyAbstract:The fluorescent calcium indicator, fluo-3, was loaded as the membrane permeant tetraacetoxymethyl (AM) ester into cauda epididymal mouse sperm at 25 degrees C for 20 min in the absence of bovine serum albumin (BSA) and presence of the dispersant, Pluronic F-127. Excess indicator was removed by two centrifugation washes at 100g for 10 min, a procedure that did not impair sperm motility. Upon resuspension in medium containing 20 mg/ml BSA to promote capacitation, the sperm cells exhibited readily detectable fluorescence uniformly distributed in the cytoplasm. Cell fluorescence was stable over the time of the experiments and was responsive to changes in intracellular calcium concentration, [Ca2+]i. Initial [Ca2+]i was 231 +/- 58 nM (+/- SE, n = 43). Addition of heat-solubilized mouse zonae Pellucidae to capacitated sperm increased [Ca2+]i by 106 +/- 19 nM (+/- SE, n = 18), the higher steady-state concentration being reached after 30 min. Subsequent addition of the non-fluorescent calcium ionophore Br-A23187 resulted in a further increase of 114 +/- 18 nM (+/- SE, n = 18), the higher steady-state concentration being reached after 6 min. The increase in [Ca2+]i induced by solubilized zonae Pellucidae was largely blocked by 3-quinuclidinyl benzilate (QNB), an antagonist of muscarinic receptors that was earlier shown to block the zona Pellucida induced acrosome reaction in mouse sperm (Florman and Storey, 1982: Dev Biol 91:121-130). This [Ca2+]i increase was completely blocked by the tyrosine kinase inhibitor, tyrphostin A48, and by the inactivator of G1 proteins, pertussis toxin. At the concentrations at which they blocked the zona Pellucida-induced increase in [Ca2+]i, all three inhibitors also blocked the zona Pellucida-induced acrosome reaction. These results indicate that [Ca2+]i increase in is an early, if not the initial, reaction in the sequence leading to zona Pellucida induced acrosomal exocytosis in mouse sperm. The observation that the three inhibitors, each having a different mode of action, all block the zona Pellucida induced [Ca2+]i suggests that the sperm plasma membrane receptors mediating the zona Pellucida induced acrosome reaction may function as a complex, whose formation is activated by zona Pellucida ligand binding.
Klaus Dieter Hinsch - One of the best experts on this subject based on the ideXlab platform.
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immunological identification of zona Pellucida 2 zp2 protein in human oocytes
Andrologia, 2009Co-Authors: E Hinsch, W Hagele, Wolf-bernhard Schill, Sergio Oehninger, Klaus Dieter 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: Klaus Dieter 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, Wolf-bernhard Schill, Rainer M Bohle, Klaus Dieter 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, Wolf-bernhard Schill, Klaus Dieter 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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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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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, Olga Epifano, Boris Baibakov, Lyn Gauthier, Manuel Avilés, 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.
