scholarly journals Sperm-binding regions on bovine egg zona pellucida glycoprotein ZP4 studied in a solid supported form on plastic plate

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0254234
Author(s):  
Kamila Dilimulati ◽  
Misaki Orita ◽  
Ganbat Undram ◽  
Naoto Yonezawa
Keyword(s):  
Zona Pellucida ◽  
Binding Sites ◽  
Insect Cells ◽  
Competitive Inhibition ◽  
Expression System ◽  
Binding Activity ◽  
Mammalian Oocyte ◽  
Sperm Binding ◽  
Bovine Sperm ◽  
Binding Mechanisms

The zona pellucida (ZP) is a transparent envelope that surrounds the mammalian oocyte and mediates species-selective sperm–oocyte interactions. The bovine ZP consists of the glycoproteins ZP2, ZP3, and ZP4. Sperm-binding mechanisms of the bovine ZP are not yet fully elucidated. In a previous report, we established the expression system of bovine ZP glycoproteins using Sf9 insect cells and found that the ZP3/ZP4 heterocomplex inhibits the binding of sperm to the ZP in a competitive inhibition assay, while ZP2, ZP3, ZP4, the ZP2/ZP3 complex, and the ZP2/ZP4 complex do not exhibit this activity. Here, we show that bovine sperm binds to plastic plates coated with ZP4 in the absence of ZP3. We made a series of ZP4 deletion mutants to study the sperm-binding sites. The N-terminal region, Lys-25 to Asp-136, and the middle region, Ser-290 to Lys-340, of ZP4 exhibit sperm-binding activity. These results suggest that among the three components of bovine ZP glycoproteins, ZP4 contains the major potential sperm-binding sites, and the formation of a multivalent complex is necessary for the sperm-binding activity of ZP4.

Porcine zona pellucida glycoprotein ZP4 is responsible for the sperm-binding activity of the ZP3/ZP4 complex

Zygote ◽  
2011 ◽  
Vol 20 (4) ◽  
pp. 389-397 ◽  
Author(s):  
Naoto Yonezawa ◽  
Saeko Kanai-Kitayama ◽  
Tetsushi Kitayama ◽  
Ayumi Hamano ◽  
Minoru Nakano
Keyword(s):  
Recombinant Proteins ◽  
Zona Pellucida ◽  
Expression System ◽  
Binding Activity ◽  
Carbohydrate Structure ◽  
Mammalian Oocyte ◽  
Sperm Binding ◽  
Bovine Sperm ◽  
Cell Expression ◽  
Cell Expression System

SummaryThe zona pellucida (ZP) is a transparent envelope that surrounds the mammalian oocyte and mediates species-selective sperm–egg interactions. Porcine and bovine ZPs consist of glycoproteins ZP2, ZP3, and ZP4. In both pig and bovine a heterocomplex consisting of ZP3 and ZP4 binds to sperm, however it is not clarified whether ZP3 or ZP4 in the complex is responsible for the sperm binding. Previously, we have established a baculovirus-Sf9 cell expression system for porcine ZP glycoproteins. A mixture of recombinant ZP3 (rZP3) and rZP4 displayed sperm-binding activity toward bovine sperm but not porcine sperm, probably due to differences in carbohydrate structure between the native and recombinant ZP glycoproteins. In this study, a mixture of porcine rZP3 and native ZP4 (nZP4) inhibited the binding of porcine sperm to the ZP. In contrast, a mixture of porcine nZP3 and rZP4 did not inhibit the binding of porcine sperm, although the mixture inhibited the binding of bovine sperm. The porcine rZP3/nZP4 mixture bound to the acrosomal region of porcine sperm, in a manner similar to that of the nZP3/nZP4 mixture. nZP3 was precipitated with rZP4, and nZP4 was precipitated with rZP3 by utilising the N-terminal tags on the recombinant proteins. These results indicated that nZP4, but not rZP4, is necessary for binding activity of porcine ZP3/ZP4 complex towards porcine sperm and further suggested that the carbohydrate structures of ZP4 in the porcine ZP3/ZP4 complex are responsible for porcine sperm-binding activity of the complex.

scholarly journals Identification of Sperm-Binding Sites in the N-Terminal Domain of Bovine Egg Coat Glycoprotein ZP4

2022 ◽  
Vol 23 (2) ◽  
pp. 762
Author(s):  
Kamila Dilimulati ◽  
Misaki Orita ◽  
Yoshiki Yonahara ◽  
Fabiana Lica Imai ◽  
Naoto Yonezawa
Keyword(s):  
Binding Sites ◽  
Binding Activity ◽  
Binding Region ◽  
Terminal Domain ◽  
Sperm Binding ◽  
Bovine Sperm ◽  
Selective Interaction ◽  
Egg Coat

The species-selective interaction between sperm and egg at the beginning of mammalian fertilisation is partly mediated by a transparent envelope called the zona pellucida (ZP). The ZP is composed of three or four glycoproteins (ZP1–ZP4). The functions of the three proteins present in mice (ZP1–ZP3) have been extensively studied. However, the biological role of ZP4, which was found in all other mammals studied so far, has remained largely unknown. Previously, by developing a solid support assay system, we showed that ZP4 exhibits sperm-binding activity in bovines and the N-terminal domain of bovine ZP4 (bZP4 ZP-N1 domain) is a sperm-binding region. Here, we show that bovine sperm bind to the bZP4 ZP-N1 domain in a species-selective manner and that N-glycosylation is not required for sperm-binding activity. Moreover, we identified three sites involved in sperm binding (site I: from Gln-41 to Pro-46, site II: from Leu-65 to Ser-68 and site III: from Thr-108 to Ile-123) in the bZP4 ZP-N1 domain using chimeric bovine/porcine and bovine/human ZP4 recombinant proteins. These results provide in vitro experimental evidence for the role of the bZP4 ZP-N1 domain in mediating sperm binding to the ZP.

scholarly journals Egg cortical granule N-acetylglucosaminidase is required for the mouse zona block to polyspermy.

1993 ◽  
Vol 123 (6) ◽  
pp. 1431-1440 ◽  
Author(s):  
D J Miller ◽  
X Gong ◽  
G Decker ◽  
B D Shur
Keyword(s):  
Zona Pellucida ◽  
Binding Sites ◽  
Binding Activity ◽  
Cortical Granule ◽  
Cortical Granules ◽  
Sperm Binding ◽  
Competitive Inhibitors ◽  
Glycosidase Inhibitor ◽  
Egg Coat

The mammalian egg must be fertilized by only one sperm to prevent polyploidy. In most mammals studied to date, the primary block to polyspermy occurs at the zona pellucida, the mammalian egg coat, after exocytosis of the contents of the cortical granules into the perivitelline space. The exudate acts on the zona, causing it to lose its ability to bind sperm and to be penetrated by sperm previously bound to the zona. However, the cortical granule components responsible for the zona block have not been identified. Studies described herein demonstrate that N-acetylglucosaminidase is localized in cortical granules and is responsible for the loss in sperm-binding activity leading to the zona block to polyspermy. Before fertilization, sperm initially bind to the zona by an interaction between sperm surface GalTase and terminal N-acetylglucosamine residues on specific oligosaccharides of the zona glycoprotein ZP3 (Miller, D. J., M. B. Macek, and B. D. Shur. 1992. Nature (Lond.). 357:589-593). These GalTase-binding sites are lost from ZP3 after fertilization, an effect that can be duplicated by N-acetylglucosaminidase treatment. Therefore, N-acetylglucosaminidase, or a related glycosidase, may be present in cortical granules and be responsible for ZP3's loss of sperm-binding activity at fertilization. Of eight glycosidases assayed in exudates of ionophore-activated eggs, N-acetylglucosaminidase was 10-fold higher than any other activity. The enzyme was localized to cortical granules using immunoelectron microscopy. Approximately 70 or 90% of the enzyme was released from cortical granules after ionophore activation or in vivo fertilization, respectively. The isoform of N-acetylglucosaminidase found in cortical granules was identified as beta-hexosaminidase B, the beta, beta homodimer. Inhibition of N-acetylglucosaminidase released from activated eggs, with either competitive inhibitors or with specific antibodies, resulted in polyspermic binding to the zona pellucida. Another glycosidase inhibitor or nonimmune antibodies had no effect on sperm binding to activated eggs. Therefore, egg cortical granule N-acetylglucosaminidase is released at fertilization, where it inactivates the sperm GalTase-binding site, accounting for the block in sperm binding to the zona pellucida.

scholarly journals Conserved Surface-Exposed K/R-X-K/R Motifs and Net Positive Charge on Poxvirus Complement Control Proteins Serve as Putative Heparin Binding Sites and Contribute to Inhibition of Molecular Interactions with Human Endothelial Cells: a Novel Mechanism for Evasion of Host Defense

2000 ◽  
Vol 74 (12) ◽  
pp. 5659-5666 ◽  
Author(s):  
Scott A. Smith ◽  
Nick P. Mullin ◽  
John Parkinson ◽  
Sergei N. Shchelkunov ◽  
Alexei V. Totmenin ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Binding Sites ◽  
Positive Charge ◽  
Expression System ◽  
Binding Activity ◽  
Heparin Binding ◽  
Dna Encoding ◽  
Human Endothelial Cells ◽  
Charged Amino Acids ◽  
Pathway Activation

ABSTRACT Vaccinia virus complement control protein (VCP) has been shown to possess the ability to inhibit both classical and alternative complement pathway activation. The newly found ability of this protein to bind to heparin has been shown in previous studies to result in uptake by mast cells, possibly promoting tissue persistence. It has also been shown to reduce chemotactic migration of leukocytes by blocking chemokine binding. In addition, this study shows that VCP—through its ability to bind to glycosaminoglycans (heparin-like molecules) on the surface of human endothelial cells—is able to block antibody binding to surface major histocompatibility complex class I molecules. Since heparin binding is critical for many functions of this protein, we have attempted to characterize the molecular basis for this interaction. Segments of this protein, generated by genetic engineering of the DNA encoding VCP into the Pichia pastoris expression system, were used to localize the regions with heparin binding activity. These regions were then analyzed to more specifically define their properties for binding. It was found that the number of putative binding sites (K/R-X-K/R), the overall positive charge, and the percentage of positively charged amino acids within the protein were responsible for this interaction.

Bovine sperm raft membrane associated Glioma Pathogenesis-Related 1-like protein 1 (GliPr1L1) is modified during the epididymal transit and is potentially involved in sperm binding to the zona pellucida

2012 ◽  
Vol 227 (12) ◽  
pp. 3876-3886 ◽  
Author(s):  
Julieta Caballero ◽  
Gilles Frenette ◽  
Olivier D'Amours ◽  
Clémence Belleannée ◽  
Nicolas Lacroix-Pepin ◽  
...  
Keyword(s):  
Zona Pellucida ◽  
Sperm Binding ◽  
Bovine Sperm ◽  
Pathogenesis Related

scholarly journals Fertilization of Pronase-Treated Mouse Ova in Vitro

1981 ◽  
Vol 34 (2) ◽  
pp. 245 ◽  
Author(s):  
P Quinn ◽  
JD Stanger
Keyword(s):  
Zona Pellucida ◽  
Binding Sites ◽  
Relative Effectiveness ◽  
Fertilization Rate ◽  
Enzyme Treatment ◽  
Perivitelline Space ◽  
Sperm Binding ◽  
Mechanical Removal ◽  
Better Than

In order to obtain consistently a large number of zona-free mouse ova for studies of sperm-egg interactions, a study was made of the relative effectiveness of removing the zona pellucida from ova by mechanical or enzymatic treatments. Ova exposed to pronase before mechanical removal of the zona pellucida in medium devoid of pronase had similar fertilization rates in vitro compared with ova mechanically denuded in the absence of pronase. Ova with pronase-weakened zonae were easier to denude and survived the mechanical manipulations better than the ova denuded by vigorous aspiration in narrow-bore pipettes. However, exposure to pronase did significantly lower the incidence of polyspermy in the naked ova, indicating that some of the enzyme may have diffused across the perivitelline space and damaged sperm-binding sites on the vitelline plasma membrane. The enzyme treatment also reduced the fertilization rate of zona-intact ova.

scholarly journals Cryo-EM Structure of Native Human Uromodulin, a Zona Pellucida Module Polymer

2020 ◽  
Author(s):  
Alena Stsiapanava ◽  
Chenrui Xu ◽  
Martina Brunati ◽  
Sara Zamora-Caballero ◽  
Céline Schaeffer ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Zona Pellucida ◽  
Binding Sites ◽  
Lateral Interaction ◽  
Biological Processes ◽  
Binding Region ◽  
Sperm Binding ◽  
Cryo Electron Microscopy ◽  
Uropathogenic Bacteria ◽  
Egg Coat

SUMMARYAssembly of extracellular filaments and matrices mediating fundamental biological processes such as morphogenesis, hearing, fertilization and antibacterial defense is driven by a ubiquitous polymerization module known as zona pellucida (ZP) “domain”. Despite the conservation of this element from hydra to human, no information is available on the filamentous conformation of any ZP module protein. Here we report the cryo-electron microscopy structure of uromodulin (UMOD)/Tamm-Horsfall protein, the most abundant protein in human urine and an archetypal ZP module-containing molecule, in its mature homopolymeric state. UMOD forms a one-start helix with an unprecedented 180-degree twist between subunits enfolded by interdomain linkers that have completely reorganized as a result of propeptide dissociation. Lateral interaction between filaments in the urine generates sheets exposing a checkerboard of binding sites to capture uropathogenic bacteria, and UMOD-based models of mammalian and avian heteromeric egg coat filaments identify a common sperm-binding region at the interface between subunits.

Differences between antigenic determinants of pig and cat zona pellucida proteins

Reproduction ◽  
2000 ◽  
pp. 15-23 ◽  
Author(s):  
K Jewgenow ◽  
M Rohleder ◽  
I Wegner
Keyword(s):  
In Vitro Fertilization ◽  
Zona Pellucida ◽  
Antigenic Determinants ◽  
Vitro Fertilization ◽  
Contraceptive Vaccine ◽  
Sperm Binding ◽  
Dependent Inhibition ◽  
Dose Dependent Inhibition ◽  
Dose Dependent

Despite many efforts, the control of reproduction in feral cat populations is still a problem in urban regions around the world. Immunocontraception is a promising approach; thus the present study examined the suitability of the widely used pig zona pellucida proteins (pZP) for contraception in feral domestic cats. Purified zona pellucida proteins obtained from pig and cat ovaries were used to produce highly specific antisera in rabbits. Antibodies against pZP raised in rabbits or lions were not effective inhibitors of either in vitro sperm binding (cat spermatozoa to cat oocytes) or in vitro fertilization in cats, whereas antibodies against feline zona pellucida proteins (fZP) raised in rabbits showed a dose-dependent inhibition of in vitro fertilization. Immunoelectrophoresis, ELISA and immunohistology of ovaries confirmed these results, showing crossreactivity of anti-fZP sera to fZP and to a lesser extent to pZP, but no interaction of anti-pZP sera with fZP. It is concluded that cat and pig zonae pellucidae express a very small number of shared antigenic determinants, making the use of pZP vaccine in cats questionable. A contraceptive vaccine based on feline zona pellucida determinants will be a better choice for the control of reproduction in feral cats if immunogenity can be achieved.

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