Zona Pellucida

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Jurrien Dean - One of the best experts on this subject based on the ideXlab platform.

  • a single domain of the zp2 Zona Pellucida protein mediates gamete recognition in mice and humans
    Journal of Cell Biology, 2014
    Co-Authors: Matteo Alessandro Avella, Boris Baibakov, Jurrien Dean
    Abstract:

    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.

  • zp2 and zp3 cytoplasmic tails prevent premature interactions and ensure incorporation into the Zona Pellucida
    Journal of Cell Science, 2011
    Co-Authors: Maria Jimenezmovilla, Jurrien Dean
    Abstract:

    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, proteinprotein 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.

  • gamete recognition in mice depends on the cleavage status of an egg s Zona Pellucida protein
    Science, 2010
    Co-Authors: Gagandeep K Gahlay, Olga Epifano, Boris Baibakov, Lyn Gauthier, Jurrien Dean
    Abstract:

    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.

  • Sperm binding to the Zona Pellucida is not sufficient to induce acrosome exocytosis.
    Development, 2007
    Co-Authors: Boris Baibakov, Lyn Gauthier, Tracy Rankin, Prue Talbot, Jurrien Dean
    Abstract:

    At fertilization, spermatozoa bind to the Zona Pellucida (ZP1, ZP2, ZP3) surrounding ovulated mouse eggs, undergo acrosome exocytosis and penetrate the Zona matrix before gamete fusion. Following fertilization, ZP2 is proteolytically cleaved and sperm no longer bind to embryos. We assessed Acr3 -EGFP sperm binding to wild-type and hu ZP2 rescue eggs in which human ZP2 replaces mouse ZP2 but remains uncleaved after fertilization. The observed de novo binding of Acr3 -EGFP sperm to embryos derived from hu ZP2 rescue mice supports a `Zona scaffold' model of sperm-egg recognition in which intact ZP2 dictates a three-dimensional structure supportive of sperm binding, independent of fertilization and cortical granule exocytosis. Surprisingly, the acrosomes of the bound sperm remain intact for at least 24 hours in the presence of uncleaved human ZP2 regardless of whether sperm are added before or after fertilization. The persistence of intact acrosomes indicates that sperm binding to the Zona Pellucida is not sufficient to induce acrosome exocytosis. A filter penetration assay suggests an alternative mechanism in which penetration into the Zona matrix initiates a mechanosensory signal transduction necessary to trigger the acrosome reaction.

  • zp2 and zp3 traffic independently within oocytes prior to assembly into the extracellular Zona Pellucida
    Molecular and Cellular Biology, 2006
    Co-Authors: Tanya Hoodbhoy, Maria Jimenezmovilla, Olga Epifano, Boris Baibakov, Lyn Gauthier, Manuel Avilés, Jurrien Dean
    Abstract:

    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.

S.k. Gupta - One of the best experts on this subject based on the ideXlab platform.

  • baculovirus expressed recombinant human Zona Pellucida glycoprotein b induces acrosomal exocytosis in capacitated spermatozoa in addition to Zona Pellucida glycoprotein c
    Molecular Human Reproduction, 2005
    Co-Authors: Sanchita Chakravarty, K Suraj, S.k. Gupta
    Abstract:

    To facilitate our understanding of the role of Zona Pellucida glycoproteins during fertilization in humans, recombinant human Zona Pellucida glycoprotein-A (hZPA), -B (hZPB) and -C (hZPC) were obtained by using Escherichia coli and baculovirus expression systems. Analysis by SDS-PAGE and Western blot of the Ni-NTA affinity purified recombinant proteins revealed that the baculovirus-expressed hZPA, hZPB and hZPC have an apparent molecular weight of ,110, ,70‐75 and ,65kDa, respectively, as compared to ,80, ,65 and ,50kDa of the respective E. coli-expressed proteins. Lectin binding studies revealed that the baculovirus-expressed recombinant Zona proteins were glycosylated. Major oligosaccharides were represented by strong reactivity with Concanavalin A (mannose a 1‐3 or mannose a 1‐6 residues) and Jacalin (a-O glycosides of Gal or GalNAc moieties). A significant increase in acrosomal exocytosis was observed when capacitated human sperm were incubated in vitro with baculovirus-expressed hZPB (P 5 0.0005) and hZPC (P 5 0.0005) The E. coli-expressed hZPB, hZPC and baculovirus-expressed hZPA failed to induce any significant increase ( P> 0.05) in acrosome reaction. In contrast to hZPC, the acrosome reaction induced by recombinant hZPB was not inhibited by pertussis toxin. These studies, for the first time, have demonstrated that in humans, ZPB also induces acrosomal exocytosis through a Gi independent pathway.

  • update on Zona Pellucida glycoproteins based contraceptive vaccine
    Journal of Reproductive Immunology, 2004
    Co-Authors: S.k. Gupta, Neelu Srivastava, Sangeeta Choudhury, Archana Rath, Neela Sivapurapu, Gagandeep K Gahlay, Deepika Batra
    Abstract:

    Zona Pellucida (ZP) glycoproteins, due to their critical role in mammalian fertilization, have been proposed as candidate immunogens for development of a contraceptive vaccine. Active immunization studies in a variety of animal species, employing either native or recombinant Zona proteins, has established their contraceptive potential. Hence, ZP glycoprotein-based contraceptive vaccines have a very good potential for controlling wild life population. To make it a realistic proposition, additional research inputs are required to develop new potent adjuvants and novel practical strategies for vaccine delivery. The observed ovarian dysfunction, often associated with immunization by ZP glycoproteins, is one of the major obstacles for their application in the control of human population. Ongoing studies to delineate epitopes of ZP glycoproteins that will generate an immune response capable of inhibiting fertility without any untoward effects on ovarian functions will help in determining their feasibility for human use.

  • human Zona Pellucida glycoproteins characterization using antibodies against recombinant non human primate zp1 zp2 and zp3
    Molecular Human Reproduction, 1998
    Co-Authors: S.k. Gupta, Renuka Kaul, P Jethanandani, Edward C. Yurewicz, Anthony G Sacco, Chhabi K Govind
    Abstract:

    : Characterization and classification of human Zona Pellucida glycoproteins is essential to understand the functions of these components during fertilization. To achieve this, antibodies were raised in rabbits against recombinant non-human primate [Bonnet Monkey (Macaca radiata)] Zona Pellucida proteins, bmZP1, bmZP2 and bmZP3 expressed in Escherichia coli. Antibodies against the three recombinant Zona proteins reacted with human Zonae as revealed by indirect immunofluorescence. Such antibodies were used as specific probes to further characterize human Zona Pellucida glycoproteins in Western blot of heat solubilized human Zonae Pellucidae (hSIZP) resolved by one dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Under non-reduced conditions human (h) hZP1, hZP2 and hZP3 resolved as 60, 100 and 53 kDa bands respectively. Under reduced conditions, dominant reactivity of hZP1, hZP2 and hZP3 was localized to 63, 65 and 58 kDa and faint reactivity to 53, 96 and 138 kDa bands respectively. In two-dimensional SDS-PAGE, hZP1 was shown to comprise two chains at 63-58 and 55-45 kDa, each consisting of multiple isomers. hZP2 was less acidic when compared with hZP1 and hZP3 and comprised a major component of 65 kDa and a minor component of approximately 96 kDa. The 65 kDa component displayed a higher degree of charged isomers in comparison with the 96 kDa component. hZP3 comprised a broad band in the range 68-58 kDa. These studies show conclusively that the hZP1 heavy train overlaps with hZP3 and that in previous studies, hZP2 was likely to have been misinterpreted as being hZP1. Our studies failed to distinguish two distinct species of hZP3, unlike previous reports. These studies will further help in our understanding of the nature of human Zona Pellucida glycoproteins.

  • dog Zona Pellucida glycoprotein 3 zp3 expression in escherichia coli and immunological characterization
    Protein Expression and Purification, 1998
    Co-Authors: Ramasamy Santhanam, Amulya K Panda, Senthil V Kumar, S.k. Gupta
    Abstract:

    Abstract An internal fragment (978 bp) corresponding to the dog Zona Pellucida glycoprotein-3 (DZP3), excluding the N-terminal signal sequence and the C-terminal transmembrane-like domain, was amplified by polymerase chain reaction from a full-length cDNA clone. The amplified Sac I and Pst I restricted fragment was cloned in-frame downstream of the T5 promoter under lac operator control for expression in the pQE-30 vector. Recombinant DZP3 (rec-DZP3) was expressed as a polyhistidine fusion protein in Escherichia coli. Optimum expression of rec-DZP3 was observed at 1.0 mM isopropyl-β- d -thiogalactopyronoside. Immunoblots with a murine monoclonal antibody, MA-451 (raised against porcine ZP3β-a homologue of DZP3 and cross-reactive with dog Zona Pellucida), revealed a major band of 42 kDa. Localization studies revealed that the recombinant protein was present only in an insoluble intracellular fraction. Further optimization studies revealed that the level of expression of rec-DZP3 was significantly higher in Luria broth medium containing glycerol rather than glucose and maximum expression was observed when cultures were induced during the mid-log phase of growth. Batch fermentation with glycerol as the carbon source yielded 30 mg/L of rec-DZP3 compared to 4 mg/L from a shake flask culture. Immunization of two male rabbits with Ni-NTA-purified rec-DZP3 and two female dogs with the rec-DZP3 conjugated to diphtheria toxoid generated high antibody titers against rec-DZP3 as determined by enzyme-linked immunosorbent assay. Rabbit immune serum reacted with porcine ZP3β but failed to react with porcine ZP3α in a Western blot. Moreover, antisera when tested by indirect immunofluorescence on dog ovarian sections showed positive fluorescence with Zona Pellucida. The availability of rec-DZP3 will help in evaluating its efficacy for fertility regulation in stray dogs.

William S.b. Yeung - One of the best experts on this subject based on the ideXlab platform.

  • Effects of Native Human Zona Pellucida Glycoproteins 3 and 4 on Acrosome Reaction and Zona Pellucida Binding of Human Spermatozoa
    Biology of reproduction, 2008
    Co-Authors: Philip C.n. Chiu, Satish K. Gupta, Ben S.t. Wong, Mk Chung, Kevin K.w. Lam, Ronald T.k. Pang, Kai-fai Lee, Sutiman Bambang Sumitro, William S.b. Yeung
    Abstract:

    Acrosome reaction is crucial to the penetration of spermatozoa through the Zona Pellucida (ZP). Glycosylation of ZP glycoproteins is important in spermatozoa-ZP interaction. Human ZP glycoprotein-3 (ZP3) is believed to initiate acrosome reaction. Recently, human ZP4 was also implicated in inducing acrosome reaction. These studies were based on recombinant human ZP proteins with glycosylation different from their native counterparts. In the present study, the effects of native human ZP3 and ZP4 on acrosome reaction and spermatozoa-ZP binding were investigated. Native human ZP3 and ZP4 were immunoaffinity-purified. They induced acrosome reaction and inhibited spermatozoa-ZP binding time- and dose-dependently to different extents. These biological activities of human ZP3 and ZP4 depended partly on their glycosylation, with N-linked glycosylation contributing much more significantly than O-linked glycosylation. Studies with inhibitors showed that both human ZP3- and ZP4-induced acrosome reactions were protein kinaseC, protein tyrosine kinase, T-type Ca 2 þ channels, and extracellular Ca 2þ dependent. G-protein also participated in human ZP3- but not in ZP4-induced acrosome reaction. On the other hand, protein kinase-A and L-type Ca 2þ channels took part only in human ZP4-induced acrosome reaction. This manuscript describes for the first time the actions of purified native human ZP3 and ZP4 on acrosome reaction and spermatozoa-ZP binding. acrosome reaction, fertilization, sperm, Zona Pellucida

  • cumulus oophorus associated glycodelin c displaces sperm bound glycodelin a and f and stimulates spermatozoa Zona Pellucida binding
    Journal of Biological Chemistry, 2007
    Co-Authors: Philip C.n. Chiu, Mk Chung, Kai-fai Lee, Riitta Koistinen, Hannu Koistinen, Markku Seppala, William S.b. Yeung
    Abstract:

    Abstract Spermatozoa have to swim through the oviduct and the cumulus oophorus before fertilization in vivo. In the oviduct, spermatozoa are exposed to glycodelin-A and -F that inhibit spermatozoa-Zona Pellucida binding. In this study, we determined whether these glycodelins would inhibit fertilization. The data showed that the spermatozoa without previous exposure to glycodelin-A and -F acquired glycodelin immunoreactivity during their passage through the cumulus oophorus. On the other hand, when glycodelin-A or -F-pretreated spermatozoa were exposed to the cumulus oophorus, the Zona Pellucida binding inhibitory activity of glycodelin-A and -F was not only removed, but the spermatozoa acquired enhanced Zona Pellucida binding ability. These actions of the cumulus oophorus were due to the presence of a cumulus isoform of glycodelin, designated as glycodelin-C. The cumulus cells could convert exogenous glycodelin-A and -F to glycodelin-C, which was then released into the surrounding medium. The protein core of glycodelin-C was identical to that in other glycodelin isoforms, as demonstrated by mass spectrum, peptide mapping, and affinity to anti-glycodelin antibody recognizing the protein core of glycodelin. In addition to having a smaller size and a higher isoelectric point, glycodelin-C also had lectin binding properties different from other isoforms. Glycodelin-C stimulated spermatozoaZona Pellucida binding in a dose-dependent manner, and it effectively displaced sperm-bound glycodelin-A and -F. In conclusion, the cumulus cells transform glycodelin-A and -F to glycodelin-C, which in turn removes the spermatozoaZona binding inhibitory glycodelin isoforms and enhances the Zona binding capacity of spermatozoa passing through the cumulus oophorus.

Philip C.n. Chiu - One of the best experts on this subject based on the ideXlab platform.

  • Effects of Native Human Zona Pellucida Glycoproteins 3 and 4 on Acrosome Reaction and Zona Pellucida Binding of Human Spermatozoa
    Biology of reproduction, 2008
    Co-Authors: Philip C.n. Chiu, Satish K. Gupta, Ben S.t. Wong, Mk Chung, Kevin K.w. Lam, Ronald T.k. Pang, Kai-fai Lee, Sutiman Bambang Sumitro, William S.b. Yeung
    Abstract:

    Acrosome reaction is crucial to the penetration of spermatozoa through the Zona Pellucida (ZP). Glycosylation of ZP glycoproteins is important in spermatozoa-ZP interaction. Human ZP glycoprotein-3 (ZP3) is believed to initiate acrosome reaction. Recently, human ZP4 was also implicated in inducing acrosome reaction. These studies were based on recombinant human ZP proteins with glycosylation different from their native counterparts. In the present study, the effects of native human ZP3 and ZP4 on acrosome reaction and spermatozoa-ZP binding were investigated. Native human ZP3 and ZP4 were immunoaffinity-purified. They induced acrosome reaction and inhibited spermatozoa-ZP binding time- and dose-dependently to different extents. These biological activities of human ZP3 and ZP4 depended partly on their glycosylation, with N-linked glycosylation contributing much more significantly than O-linked glycosylation. Studies with inhibitors showed that both human ZP3- and ZP4-induced acrosome reactions were protein kinaseC, protein tyrosine kinase, T-type Ca 2 þ channels, and extracellular Ca 2þ dependent. G-protein also participated in human ZP3- but not in ZP4-induced acrosome reaction. On the other hand, protein kinase-A and L-type Ca 2þ channels took part only in human ZP4-induced acrosome reaction. This manuscript describes for the first time the actions of purified native human ZP3 and ZP4 on acrosome reaction and spermatozoa-ZP binding. acrosome reaction, fertilization, sperm, Zona Pellucida

  • cumulus oophorus associated glycodelin c displaces sperm bound glycodelin a and f and stimulates spermatozoa Zona Pellucida binding
    Journal of Biological Chemistry, 2007
    Co-Authors: Philip C.n. Chiu, Mk Chung, Kai-fai Lee, Riitta Koistinen, Hannu Koistinen, Markku Seppala, William S.b. Yeung
    Abstract:

    Abstract Spermatozoa have to swim through the oviduct and the cumulus oophorus before fertilization in vivo. In the oviduct, spermatozoa are exposed to glycodelin-A and -F that inhibit spermatozoa-Zona Pellucida binding. In this study, we determined whether these glycodelins would inhibit fertilization. The data showed that the spermatozoa without previous exposure to glycodelin-A and -F acquired glycodelin immunoreactivity during their passage through the cumulus oophorus. On the other hand, when glycodelin-A or -F-pretreated spermatozoa were exposed to the cumulus oophorus, the Zona Pellucida binding inhibitory activity of glycodelin-A and -F was not only removed, but the spermatozoa acquired enhanced Zona Pellucida binding ability. These actions of the cumulus oophorus were due to the presence of a cumulus isoform of glycodelin, designated as glycodelin-C. The cumulus cells could convert exogenous glycodelin-A and -F to glycodelin-C, which was then released into the surrounding medium. The protein core of glycodelin-C was identical to that in other glycodelin isoforms, as demonstrated by mass spectrum, peptide mapping, and affinity to anti-glycodelin antibody recognizing the protein core of glycodelin. In addition to having a smaller size and a higher isoelectric point, glycodelin-C also had lectin binding properties different from other isoforms. Glycodelin-C stimulated spermatozoaZona Pellucida binding in a dose-dependent manner, and it effectively displaced sperm-bound glycodelin-A and -F. In conclusion, the cumulus cells transform glycodelin-A and -F to glycodelin-C, which in turn removes the spermatozoaZona binding inhibitory glycodelin isoforms and enhances the Zona binding capacity of spermatozoa passing through the cumulus oophorus.

Bayard T Storey - One of the best experts on this subject based on the ideXlab platform.

  • 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, 1994
    Co-Authors: Janice L Bailey, Bayard T Storey
    Abstract:

    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.