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Satish K Gupta - One of the best experts on this subject based on the ideXlab platform.
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The Human Egg's Zona Pellucida.
Current Topics in Developmental Biology, 2018Co-Authors: Satish K GuptaAbstract:Human zona pellucida (ZP) matrix, a delicate network of thin interconnected filaments, is primarily composed of four glycoproteins, namely, ZP1, ZP2, ZP3, and ZP4. All four zona proteins share common structural elements such as signal peptide, "ZP domain," consensus furin cleavage site, transmembrane-like domain, and short cytoplasmic tail. In addition, ZP1 and ZP4 also have "Trefoil domain." Recombinant/native human zona proteins have been used to investigate their binding characteristics to the capacitated and/or acrosome-reacted spermatozoa. These investigations revealed that ZP1, ZP3, and ZP4 primarily bind to the head region of the capacitated human spermatozoa, whereas ZP2 binds to the acrosome-reacted sperm. However, using transgenic mice, N-terminal region of human ZP2 has also been shown to play an important role in binding of sperm to the egg. ZP1, ZP3, and ZP4 lead to dose-dependent increase in acrosome reaction, suggesting that in humans more than one ZP glycoprotein is responsible for induction of acrosome reaction. Glycosylation of these proteins, in particular, N-linked glycosylation as well as sialyl-Lewisx, is essential for inducing acrosome reaction. Studies delineating downstream signaling events associated with induction of acrosome reaction reveal subtle differences between ZP3 and ZP1/ZP4 with respect to activation of Gi protein-coupled receptor and protein kinase A. The role of mutations in the zona proteins and ZP autoantibodies leading to infertility in women is suggestive and needs more rigorous experimentations for confirming their role in female infertility. The above-mentioned aspects of the human ZP glycoproteins have been discussed in this review.
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Immunogenicity and contraceptive efficacy of Escherichia coli-expressed recombinant porcine zona pellucida proteins.
American journal of reproductive immunology (New York N.Y. : 1989), 2013Co-Authors: Neha Gupta, Kausiki Chakrabarti, Krishna Prakash, Neerja Wadhwa, Tripti Gupta, Satish K GuptaAbstract:Problem To overcome availability of the purified native zona pellucida (ZP) glycoproteins for immunocontraception, porcine ZP3, and ZP4 were expressed in E. coli. Method of study Purified recombinant proteins were characterized by SDS-PAGE and Western blot, and immunogenicity and contraceptive efficacy determined in FvB/J female mice. Results Purified ZP3, ZP3 with promiscuous T-cell epitope of tetanus toxoid, ZP4 and ZP4 incorporating promiscuous T-cell epitope of bovine RNase revealed ~44-, ~49-, ~53-, and ~55-kDa bands by SDS-PAGE and Western blot, respectively. Immunization of female mice with recombinant proteins elicited high antibody titers as well as T-cell responses. Immune sera recognized mouse oocyte ZP and also inhibited in vitro fertilization. Immunized mice showed significant decrease in fertility. Recombinant proteins were able to recall memory antibody response in female mice primed with porcine native ZP. Conclusion Availability of recombinant porcine proteins will be useful in the development of contraceptive vaccine.
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Mammalian zona pellucida glycoproteins: structure and function during fertilization
Cell and Tissue Research, 2012Co-Authors: Satish K Gupta, Beena Bhandari, Abhinav Shrestha, Bichitra K. Biswal, Chetna Palaniappan, Sudha Saryu Malhotra, Neha GuptaAbstract:Zona pellucida (ZP) is a glycoproteinaceous translucent matrix that surrounds the mammalian oocyte and plays a critical role in the accomplishment of fertilization. In humans, it is composed of 4 glycoproteins designated as ZP1, ZP2, ZP3 and ZP4, whereas mouse ZP is composed of ZP1, ZP2 and ZP3 ( ZP4 being a pseudogene). In addition to a variable sequence identity of a given zona protein among various species, human ZP1 and ZP4 are paralogs and mature polypeptide chains share an identity of 47%. Employing either affinity purified native or recombinant human zona proteins, it has been demonstrated that ZP1, ZP3 and ZP4 bind to the capacitated human spermatozoa and induce an acrosome reaction, whereas in mice, ZP3 acts as the putative primary sperm receptor. Human ZP2 only binds to acrosome-reacted spermatozoa and thus may be acting as a secondary sperm receptor. In contrast to O -linked glycans of ZP3 in mice, N -linked glycans of human ZP3 and ZP4 are more relevant for induction of the acrosome reaction. Recent studies suggest that Sialyl-Lewis^x sequence present on both N - and O -glycans of human ZP play an important role in human sperm–egg binding. There are subtle differences in the downstream signaling events associated with ZP3 versus ZP1/ZP4-mediated induction of the acrosome reaction. For example, ZP3 but not ZP1/ZP4-mediated induction of the acrosome reaction is dependent on the activation of the G_i protein-coupled receptor. Thus, various studies suggest that, in contrast to mice, in humans more than one zona protein binds to spermatozoa and induces an acrosome reaction.
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Mapping of epitopes relevant for induction of acrosome reaction on human zona pellucida glycoprotein-4 using monoclonal antibodies.
American journal of reproductive immunology (New York N.Y. : 1989), 2012Co-Authors: Beena Bhandari, Satish K Gupta, Haiping Tang, Sanchita Chaudhary, Pankaj Talwar, Jian WangAbstract:Problem To decipher structural and functional aspects of human zona pellucida glycoprotein-4 (ZP4), the epitopes recognized by monoclonal antibodies (MAbs) have been mapped. Method of study Recombinant human ZP4-mediated induction of acrosome reaction in human sperm was studied in the absence and presence of ZP4-specific MAbs. The epitopes of MAbs were mapped using recombinant peptides expressed in Escherichia coli. Results Monoclonal antibodies (MA-1662, MA-1671) against human ZP4 showed specific binding to ZP matrix of human eggs in an indirect immunofluorescence assay. Both the antibodies showed significant (P
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Acrosome reaction: relevance of zona pellucida glycoproteins
Asian Journal of Andrology, 2010Co-Authors: Satish K Gupta, Beena BhandariAbstract:During mammalian fertilisation, the zona pellucida (ZP) matrix surrounding the oocyte is responsible for the binding of the spermatozoa to the oocyte and induction of the acrosome reaction (AR) in the ZP-bound spermatozoon. The AR is crucial for the penetration of the ZP matrix by spermatozoa. The ZP matrix in mice is composed of three glycoproteins designated ZP1, ZP2 and ZP3, whereas in humans, it is composed of four (ZP1, ZP2, ZP3 and ZP4). ZP3 acts as the putative primary sperm receptor and is responsible for AR induction in mice, whereas in humans (in addition to ZP3), ZP1 and ZP4 also induce the AR. The ability of ZP3 to induce the AR resides in its C-terminal fragment. O-linked glycans are critical for the murine ZP3-mediated AR. However, N-linked glycans of human ZP1, ZP3 and ZP4 have important roles in the induction of the AR. Studies with pharmacological inhibitors showed that the ZP3-induced AR involves the activation of the Gi-coupled receptor pathway, whereas ZP1- and ZP4-mediated ARs are independent of this pathway. The ZP3-induced AR involves the activation of T-type voltage-operated calcium channels (VOCCs), whereas ZP1- and ZP4-induced ARs involve both T- and L-type VOCCs. To conclude, in mice, ZP3 is primarily responsible for the binding of capacitated spermatozoa to the ZP matrix and induction of the AR, whereas in humans (in addition to ZP3), ZP1 and ZP4 also participate in these stages of fertilisation.
Manuel Avilés - One of the best experts on this subject based on the ideXlab platform.
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ZP4 confers structural properties to the zona pellucida essential for embryo development.
eLife, 2019Co-Authors: Ismael Lamas-toranzo, M. J. Izquierdo-rico, Manuel Avilés, Leopoldo González-brusi, Noelia Fonseca Balvís, Ana Querejeta-fernández, Pedro Lorenzo, Pilar García-rebollar, Pablo Bermejo-alvarezAbstract:Zona pellucida (ZP), the extracellular matrix sheltering mammalian oocytes and embryos, is composed by 3 to 4 proteins. The roles of the three proteins present in mice have been elucidated by KO models, but the function of the fourth component (ZP4), present in all other eutherian mammals studied so far, has remained elusive. Herein, we report that ZP4 ablation impairs fertility in female rabbits. Ovulation, fertilization and in vitro development to blastocyst were not affected by ZP4 ablation. However, in vivo development is severely impaired in embryos covered by a ZP4-devoided zona, suggesting a defective ZP protective capacity in the absence of ZP4. ZP4-null ZP was significantly thinner, more permeable, and exhibited a more disorganized and fenestrated structure. The evolutionary conservation of ZP4 in other mammals, including humans, suggests that the structural properties conferred by this protein are required to ensure proper embryo sheltering during in vivo preimplantation development.
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6 crispr generated ZP4 knockout rabbits exhibit impaired in vivo pre implantation development
Reproduction Fertility and Development, 2018Co-Authors: I Lamastoranzo, Manuel Avilés, N F Balvis, P L Lorenzo, Pilar Garcia Rebollar, Pablo BermejoalvarezAbstract:Mammalian oocytes are surrounded by a glycoprotein layer termed the zona pellucida (ZP), which acts as a protective barrier during pre-implantation embryo development and plays essential roles during follicular development and fertilization. This structure is composed of 4 different proteins in several species such as rabbits and humans. However, the oocytes of the only species for which knockout (KO) embryos are readily available, the laboratory mouse, lacks one of these proteins (ZP4), so its function remains elusive. To determine the role of ZP4, we have generated ZP4 KO rabbits by CRISPR and compared their reproductive function to that of heterozygous (Hz) and wild type (wt) rabbits. Following mating with wt males, female KO rabbits showed a clear impairment in reproductive performance compared with Hz or wt females, with only 1 out of 5 KO females producing a small litter of 4 pups, whereas all mated Hz and wt females exhibited normal litter sizes (wt 9.2 ± 0.6; Hz 10.6 ± 0.5; mean ± SEM). In order to elucidate the cause for the impaired delivery rate, ovulation rates and cleavage rates were initially assessed, with no differences observed between groups (oocytes ovulated: wt 11.7 ± 1; Hz 15 ± 2.9; KO 13.3 ± 2.9; cleavage rates (%): wt 81.7 ± 0.1; Hz 95.5 ± 0.1; KO 87.3 ± 0.1), which suggests that ZP4 does not play a critical role in folliculogenesis or fertilization. However, when expanded blastocysts were recovered at Day 6 after mating, embryo development was found to be clearly impaired in embryos lacking ZP4, as none of the embryos obtained from 2 KO females were able to undergo blastocyst expansion. Morphological differences were observed in the zona pellucida from KO rabbits, being irregular, noticeably less elastic and easier to deform, and significantly thinner (thickness: wt 15.2 ± 1.5 µm; Hz 15.3 ± 1.4 µm; KO 10.9 ± 0.7 µm; ANOVA P < 0.05). These morphological differences suggest that rabbit ZP without ZP4 may fail to protect the embryo during development or may physically impede blastocyst expansion. This study is supported by the projects AGL2014-58739-R and RYC-2012-10193 (to PBA), AGL2015-70159-P (to MA) and AGL2015-65572-C2-1-R (to PGR and PL). ILT and NFB are supported by FPI grants.
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Analysis of ZP1 gene reveals differences in zona pellucida composition in carnivores.
Reproduction Fertility and Development, 2018Co-Authors: Carla Moros-nicolás, Manel Lopez-bejar, José Ballesta, Manuel Avilés, Pascale Chevret, A. Leza, A. Guillén-martínez, Leopoldo González-brusi, F. Boué, M. J. Izquierdo-ricoAbstract:The zona pellucida (ZP) is an extracellular envelope that surrounds mammalian oocytes. This coat participates in the interaction between gametes, induction of the acrosome reaction, block of polyspermy and protection of the oviductal embryo. Previous studies suggested that carnivore ZP was formed by three glycoproteins (ZP2, ZP3 and ZP4), with ZP1 being a pseudogene. However, a recent study in the cat found that all four proteins were expressed. In the present study, in silico and molecular analyses were performed in several carnivores to clarify the ZP composition in this order of mammals. The in silico analysis demonstrated the presence of the ZP1 gene in five carnivores: cheetah, panda, polar bear, tiger and walrus, whereas in the Antarctic fur seal and the Weddell seal there was evidence of pseudogenisation. Molecular analysis showed the presence of four ZP transcripts in ferret ovaries (ZP1, ZP2, ZP3 and ZP4) and three in fox ovaries (ZP2, ZP3 and ZP4). Analysis of the fox ZP1 gene showed the presence of a stop codon. The results strongly suggest that all four ZP genes are expressed in most carnivores, whereas ZP1 pseudogenisation seems to have independently affected three families (Canidae, Otariidae and Phocidae) of the carnivore tree.
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6 CRISPR-Generated ZP4 Knockout Rabbits Exhibit Impaired In Vivo Pre-Implantation Development
Reproduction Fertility and Development, 2018Co-Authors: I. Lamas-toranzo, Manuel Avilés, Pedro Lorenzo, N F Balvis, Pilar Garcia Rebollar, Pablo Bermejo-alvarezAbstract:Mammalian oocytes are surrounded by a glycoprotein layer termed the zona pellucida (ZP), which acts as a protective barrier during pre-implantation embryo development and plays essential roles during follicular development and fertilization. This structure is composed of 4 different proteins in several species such as rabbits and humans. However, the oocytes of the only species for which knockout (KO) embryos are readily available, the laboratory mouse, lacks one of these proteins (ZP4), so its function remains elusive. To determine the role of ZP4, we have generated ZP4 KO rabbits by CRISPR and compared their reproductive function to that of heterozygous (Hz) and wild type (wt) rabbits. Following mating with wt males, female KO rabbits showed a clear impairment in reproductive performance compared with Hz or wt females, with only 1 out of 5 KO females producing a small litter of 4 pups, whereas all mated Hz and wt females exhibited normal litter sizes (wt 9.2 ± 0.6; Hz 10.6 ± 0.5; mean ± SEM). In order to elucidate the cause for the impaired delivery rate, ovulation rates and cleavage rates were initially assessed, with no differences observed between groups (oocytes ovulated: wt 11.7 ± 1; Hz 15 ± 2.9; KO 13.3 ± 2.9; cleavage rates (%): wt 81.7 ± 0.1; Hz 95.5 ± 0.1; KO 87.3 ± 0.1), which suggests that ZP4 does not play a critical role in folliculogenesis or fertilization. However, when expanded blastocysts were recovered at Day 6 after mating, embryo development was found to be clearly impaired in embryos lacking ZP4, as none of the embryos obtained from 2 KO females were able to undergo blastocyst expansion. Morphological differences were observed in the zona pellucida from KO rabbits, being irregular, noticeably less elastic and easier to deform, and significantly thinner (thickness: wt 15.2 ± 1.5 µm; Hz 15.3 ± 1.4 µm; KO 10.9 ± 0.7 µm; ANOVA P
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Composition of marsupial zona pellucida: a molecular and phylogenetic approach.
Reproduction Fertility and Development, 2017Co-Authors: Carla Moros-nicolás, M. J. Izquierdo-rico, Daniela Esteban-díaz, Manel Lopez-bejar, Eva Martínez-nevado, Maria A Nilsson, José Ballesta, William V Holt, Pascale Chevret, Manuel AvilésAbstract: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.
Minoru Nakano - One of the best experts on this subject based on the ideXlab platform.
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porcine zona pellucida glycoprotein ZP4 is responsible for the sperm binding activity of the zp3 ZP4 complex
Zygote, 2012Co-Authors: Naoto Yonezawa, Tetsushi Kitayama, Ayumi Hamano, Saeko Kanaikitayama, Minoru NakanoAbstract:The 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.
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Porcine zona pellucida glycoprotein ZP4 is responsible for the sperm-binding activity of the ZP3/ZP4 complex.
Zygote (Cambridge England), 2011Co-Authors: Naoto Yonezawa, Saeko Kanai-kitayama, Tetsushi Kitayama, Ayumi Hamano, Minoru NakanoAbstract:The 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.
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Disulfide linkage patterns of pig zona pellucida glycoproteins ZP3 and ZP4
Molecular reproduction and development, 2008Co-Authors: Saeko Kanai, Naoto Yonezawa, Tetsushi Kitayama, Masaru Tanokura, Yoriko Sawano, Minoru NakanoAbstract:Zona pellucida, a transparent envelope surrounding the mammalian oocyte, plays major roles in fertilization and consists of three or four glycoproteins. Primary structures, and especially the positions of cysteine (Cys) residues in the zona glycoproteins, are well conserved among mammals. In this study, we analyzed the disulfide linkages of pig ZP3 and ZP4 purified from ovaries. While disulfide linkage patterns of four Cys residues in the N-terminal halves of the ZP domains of ZP3 and ZP4 were identical to those previously reported for mice, rats, humans, and fish, the disulfide linkage patterns of six Cys residues in the C-terminal half of the ZP domain in ZP4, as well as eight Cys residues in the C-terminal region of the ZP domain and a following region unique to ZP3, were different from those previously reported. Thus, higher-order structures of zona glycoproteins might not be conserved in the C-terminal regions. Mol. Reprod. Dev. 75: 847–856, 2008. © 2007 Wiley-Liss, Inc.
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Recombinant bovine zona pellucida glycoproteins ZP3 and ZP4 coexpressed in Sf9 cells form a sperm-binding active hetero-complex.
The FEBS journal, 2007Co-Authors: Saeko Kanai, Naoto Yonezawa, Yuichiro Ishii, Masaru Tanokura, Minoru NakanoAbstract:The zona pellucida (ZP) is a transparent envelope that surrounds the mammalian oocyte and mediates species-selective sperm-egg interactions. Porcine and bovine ZPs are composed of the glycoproteins ZP2, ZP3, and ZP4. We previously established an expression system for porcine ZP glycoproteins (ZPGs) using baculovirus in insect Sf9 cells. Here we established a similar method for expression of bovine ZPGs. The recombinant ZPGs were secreted into the medium and purified by metal-chelating column chromatography. A mixture of bovine recombinant ZP3 (rZP3) and rZP4 coexpressed in Sf9 cells exhibited inhibitory activity for bovine sperm-ZP binding similar to that of a native bovine ZPG mixture, whereas neither bovine rZP3 nor rZP4 inhibited binding. An immunoprecipitation assay revealed that the coexpressed rZP3/rZP4 formed a hetero-complex. We examined the functional domain structure of bovine rZP4 by constructing ZP4 mutants lacking the N-terminal domain or lacking both the N-terminal and trefoil domains. When either of these mutant proteins was coexpressed with bovine rZP3, the resulting mixtures exhibited inhibitory activity comparable to that of the bovine rZP3/rZP4 complex. Hetero-complexes of bovine rZP3 and porcine rZP4, or porcine rZP3 and bovine rZP4, also inhibited bovine sperm-ZP binding. Our results demonstrate that the N-terminal and trefoil domains of bovine rZP4 are dispensable for formation of the sperm-binding active bovine rZP3/rZP4 complex and, furthermore, that the molecular interactions between rZP3 and rZP4 are conserved in the bovine and porcine systems.
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Structural characterization of the N-linked carbohydrate chains from mouse zona pellucida glycoproteins ZP2 and ZP3.
Biochimica et Biophysica Acta, 1993Co-Authors: Satoru Noguchi, Minoru NakanoAbstract:Abstract N-linked oligosaccharides of mouse zona pellucida glycoproteins ZP2 and ZP3 were prepared as pyridylaminated derivatives. Anion-exchange HPLC revealed that 95% or more of them are acidic. About 80% of these acidic chains were neutralized by digestion with Arthrobacter ureafaciens sialidase and a small amount of sulfate was found in the residual acidic chains. The neutralized fractions (SN fractiions) from ZP2 and ZP3 were similarly fractionated by size-fractionation HPLC into many peaks having different numbers of LacNAc in the non-reducing regions. The SN fractions from ZP2 were cleaved by endo-β-galactosidase into the core and non-reducing regions. The tri- and tetra-antennary complex-type chains with a Fuc residue were predominant in the core region. On the other hand, four kinds of fragment containing the LacNAc sequence were obtained from the non-reducing region. Sialic acids were shown to be linked to the core fragments, as well as the non-reducing fragments. The N-linked oligosaccharides of ZP3 were suggested to have essentially the same structures as those of ZP2.
Naoto Yonezawa - One of the best experts on this subject based on the ideXlab platform.
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The Hinge Region of Bovine Zona Pellucida Glycoprotein ZP3 Is Involved in the Formation of the Sperm-Binding Active ZP3/ZP4 Complex.
Biomolecules, 2015Co-Authors: Kaori Suzuki, Ayumi Hamano, Nanami Tatebe, Sayuri Kojima, Misaki Orita, Naoto YonezawaAbstract:The zona pellucida (ZP) surrounds the mammalian oocyte and mediates species-selective sperm-oocyte interactions. Bovine ZP consists of glycoproteins ZP2, ZP3, and ZP4. Neither ZP3 nor ZP4 alone shows inhibitory activity for the binding of sperm to the ZP; however, this activity is seen with the ZP3/ZP4 heterocomplex. Here, we constructed a series of bovine ZP3 mutants to identify the ZP4-binding site on ZP3. Each ZP3 mutant was co-expressed with ZP4 using a baculovirus-Sf9 cell expression system and examined for interaction with ZP4 as well as inhibitory activity for sperm-ZP binding. N-terminal fragment Arg-32 to Arg-160 of ZP3 interacted with ZP4 and inhibited sperm-ZP binding, whereas fragment Arg-32 to Thr-155 showed much weaker interaction with ZP4. Mutation of N-glycosylated Asn-146 to Asp in the N-terminal fragment Arg-32 to Glu-178 of ZP3 did not interrupt the interaction of this fragment with ZP4, but it did reduce the inhibitory activity of the complex for sperm-ZP binding. In contrast, mutation of N-glycosylated Asn-124 to Asp did not significantly reduce the activity. Taken together, these results suggest that one of the ZP4 binding sites exists in the flexible hinge region of ZP3 and that the N-glycosylation in this region is involved in the sperm binding.
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Involvement of Carbohydrate Residues of the Zona Pellucida in In Vitro Sperm Recognition in Pigs and Cattle
Sexual Reproduction in Animals and Plants, 2014Co-Authors: Naoto YonezawaAbstract:The zona pellucida (ZP), which surrounds the mammalian oocyte, plays roles in species-selective sperm–oocyte interactions. In pigs and cattle, the ZP consists of ZP2, ZP3, and ZP4. Nonreducing terminal β-galactosyl (Gal) residues of neutral N-linked carbohydrate chains of the ZP are necessary for porcine sperm–ZP binding, and nonreducing terminal α-mannosyl (Man) residues of high-mannose-type chains are necessary for bovine sperm–ZP binding. Acrosome-intact porcine sperm prefer β-Gal, whereas acrosome-intact bovine sperm prefer α-Man, as shown using glycolipid analogues. The major N-linked chains of recombinant porcine and bovine ZP glycoproteins expressed using the baculovirus-Sf9 cell expression system are pauci- and high-mannose-type chains that are different in structure from the major neutral N-linked chains of the native porcine ZP but similar to those of the native bovine ZP. Porcine and bovine ZP3/ZP4 complexes coexpressed in Sf9 cells bind to bovine sperm but not to porcine sperm. Hybrid complexes consisting of native porcine ZP4 and recombinant porcine ZP3 bind to porcine sperm, whereas complexes consisting of native porcine ZP3 and recombinant porcine ZP4 do not bind to porcine sperm. These data indicate that the sugar preference of sperm is consistent with the nonreducing terminal residues of N-linked chains of sperm binding-active ZP glycoproteins and suggest that, in the in vitro assay system, the nonreducing terminal sugar residues are essential for species-selective recognition of sperm in pigs and cattle.
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porcine zona pellucida glycoprotein ZP4 is responsible for the sperm binding activity of the zp3 ZP4 complex
Zygote, 2012Co-Authors: Naoto Yonezawa, Tetsushi Kitayama, Ayumi Hamano, Saeko Kanaikitayama, Minoru NakanoAbstract:The 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.
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Porcine zona pellucida glycoprotein ZP4 is responsible for the sperm-binding activity of the ZP3/ZP4 complex.
Zygote (Cambridge England), 2011Co-Authors: Naoto Yonezawa, Saeko Kanai-kitayama, Tetsushi Kitayama, Ayumi Hamano, Minoru NakanoAbstract:The 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.
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Disulfide linkage patterns of pig zona pellucida glycoproteins ZP3 and ZP4
Molecular reproduction and development, 2008Co-Authors: Saeko Kanai, Naoto Yonezawa, Tetsushi Kitayama, Masaru Tanokura, Yoriko Sawano, Minoru NakanoAbstract:Zona pellucida, a transparent envelope surrounding the mammalian oocyte, plays major roles in fertilization and consists of three or four glycoproteins. Primary structures, and especially the positions of cysteine (Cys) residues in the zona glycoproteins, are well conserved among mammals. In this study, we analyzed the disulfide linkages of pig ZP3 and ZP4 purified from ovaries. While disulfide linkage patterns of four Cys residues in the N-terminal halves of the ZP domains of ZP3 and ZP4 were identical to those previously reported for mice, rats, humans, and fish, the disulfide linkage patterns of six Cys residues in the C-terminal half of the ZP domain in ZP4, as well as eight Cys residues in the C-terminal region of the ZP domain and a following region unique to ZP3, were different from those previously reported. Thus, higher-order structures of zona glycoproteins might not be conserved in the C-terminal regions. Mol. Reprod. Dev. 75: 847–856, 2008. © 2007 Wiley-Liss, Inc.
Beena Bhandari - One of the best experts on this subject based on the ideXlab platform.
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Mammalian zona pellucida glycoproteins: structure and function during fertilization
Cell and Tissue Research, 2012Co-Authors: Satish K Gupta, Beena Bhandari, Abhinav Shrestha, Bichitra K. Biswal, Chetna Palaniappan, Sudha Saryu Malhotra, Neha GuptaAbstract:Zona pellucida (ZP) is a glycoproteinaceous translucent matrix that surrounds the mammalian oocyte and plays a critical role in the accomplishment of fertilization. In humans, it is composed of 4 glycoproteins designated as ZP1, ZP2, ZP3 and ZP4, whereas mouse ZP is composed of ZP1, ZP2 and ZP3 ( ZP4 being a pseudogene). In addition to a variable sequence identity of a given zona protein among various species, human ZP1 and ZP4 are paralogs and mature polypeptide chains share an identity of 47%. Employing either affinity purified native or recombinant human zona proteins, it has been demonstrated that ZP1, ZP3 and ZP4 bind to the capacitated human spermatozoa and induce an acrosome reaction, whereas in mice, ZP3 acts as the putative primary sperm receptor. Human ZP2 only binds to acrosome-reacted spermatozoa and thus may be acting as a secondary sperm receptor. In contrast to O -linked glycans of ZP3 in mice, N -linked glycans of human ZP3 and ZP4 are more relevant for induction of the acrosome reaction. Recent studies suggest that Sialyl-Lewis^x sequence present on both N - and O -glycans of human ZP play an important role in human sperm–egg binding. There are subtle differences in the downstream signaling events associated with ZP3 versus ZP1/ZP4-mediated induction of the acrosome reaction. For example, ZP3 but not ZP1/ZP4-mediated induction of the acrosome reaction is dependent on the activation of the G_i protein-coupled receptor. Thus, various studies suggest that, in contrast to mice, in humans more than one zona protein binds to spermatozoa and induces an acrosome reaction.
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Mapping of epitopes relevant for induction of acrosome reaction on human zona pellucida glycoprotein-4 using monoclonal antibodies.
American journal of reproductive immunology (New York N.Y. : 1989), 2012Co-Authors: Beena Bhandari, Satish K Gupta, Haiping Tang, Sanchita Chaudhary, Pankaj Talwar, Jian WangAbstract:Problem To decipher structural and functional aspects of human zona pellucida glycoprotein-4 (ZP4), the epitopes recognized by monoclonal antibodies (MAbs) have been mapped. Method of study Recombinant human ZP4-mediated induction of acrosome reaction in human sperm was studied in the absence and presence of ZP4-specific MAbs. The epitopes of MAbs were mapped using recombinant peptides expressed in Escherichia coli. Results Monoclonal antibodies (MA-1662, MA-1671) against human ZP4 showed specific binding to ZP matrix of human eggs in an indirect immunofluorescence assay. Both the antibodies showed significant (P
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Acrosome reaction: relevance of zona pellucida glycoproteins
Asian Journal of Andrology, 2010Co-Authors: Satish K Gupta, Beena BhandariAbstract:During mammalian fertilisation, the zona pellucida (ZP) matrix surrounding the oocyte is responsible for the binding of the spermatozoa to the oocyte and induction of the acrosome reaction (AR) in the ZP-bound spermatozoon. The AR is crucial for the penetration of the ZP matrix by spermatozoa. The ZP matrix in mice is composed of three glycoproteins designated ZP1, ZP2 and ZP3, whereas in humans, it is composed of four (ZP1, ZP2, ZP3 and ZP4). ZP3 acts as the putative primary sperm receptor and is responsible for AR induction in mice, whereas in humans (in addition to ZP3), ZP1 and ZP4 also induce the AR. The ability of ZP3 to induce the AR resides in its C-terminal fragment. O-linked glycans are critical for the murine ZP3-mediated AR. However, N-linked glycans of human ZP1, ZP3 and ZP4 have important roles in the induction of the AR. Studies with pharmacological inhibitors showed that the ZP3-induced AR involves the activation of the Gi-coupled receptor pathway, whereas ZP1- and ZP4-mediated ARs are independent of this pathway. The ZP3-induced AR involves the activation of T-type voltage-operated calcium channels (VOCCs), whereas ZP1- and ZP4-induced ARs involve both T- and L-type VOCCs. To conclude, in mice, ZP3 is primarily responsible for the binding of capacitated spermatozoa to the ZP matrix and induction of the AR, whereas in humans (in addition to ZP3), ZP1 and ZP4 also participate in these stages of fertilisation.
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In humans, zona pellucida glycoprotein-1 binds to spermatozoa and induces acrosomal exocytosis
Human Reproduction, 2010Co-Authors: Anasua Ganguly, Antonin Bukovsky, Beena Bhandari, Pankaj Bansal, Raj K. Sharma, Satish K GuptaAbstract:background: It has been suggested that the zona pellucida (ZP) may mediate species-specific fertilization. In human the ZP is composed of four glycoproteins: ZP1, ZP2, ZP3 and ZP4. In the present study, the expression profile of ZP1 in human oocytes and ovaries, and its role during fertilization, is presented. methods: Human ZP1 (amino acid residues 26 –551) was cloned and expressed in both non-glycosylated and glycosylated forms and its ability to bind to the capacitated human spermatozoa and to induce acrosomal exocytosis was studied. Monoclonal antibodies (MAbs), specific for human ZP1 and devoid of reactivity with ZP2, ZP3 and ZP4 were generated and used to localize native ZP1 in oocytes and ovarian tissues. results: The MAbs generated against ZP1 recognized specifically the zona matrix of secondary and antral follicles, ovulated oocytes, atretic follicles and degenerating intravascular oocytes, but failed to react with the Fallopian tube, endometrium, ectocervix and kidney. Escherichia coli and baculovirus-expressed recombinant human ZP1 revealed bands of � 75 and � 85 kDa, respectively, in western blot. Lectin binding studies revealed the presence of both N- and O-linked glycosylation in baculovirus-expressed ZP1. Fluorescein isothiocyanate-labelled E. coli- and baculovirus-expressed recombinant ZP1 bound to the anterior head of capacitated spermatozoa, however, only baculovirus-expressed ZP1 induced acrosomal exocytosis in capacitated sperm suggesting the importance of glycosylation in mediating the acrosome reaction. The human ZP1-mediated acrosome reaction involved the activation of both T- and L-type voltage-operated calcium channels, but does not activate the Gi-coupled receptor pathway. Inhibition of protein kinase A and C significantly also reduced the ZP1-mediated induction of the acrosome reaction. conclusion: These studies revealed for the first time that in humans ZP1, in addition to ZP3 and ZP4, binds to capacitated spermatozoa and induces acrosomal exocytosis.
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Human zona pellucida glycoproteins: functional relevance during fertilization.
Journal of Reproductive Immunology, 2009Co-Authors: Satish K Gupta, Beena Bhandari, Pankaj Bansal, Anasua Ganguly, Kausiki ChakrabartiAbstract:The zona pellucida (ZP), a glycoproteinaceous matrix surrounding the mammalian oocyte plays an important role in species-specific sperm-egg binding, induction of acrosome reaction in the ZP-bound spermatozoa, avoidance of polyspermy and protection of the embryo prior to implantation. In contrast to mouse, human ZP matrix is composed of 4 glycoproteins designated as ZP1, ZP2, ZP3 and ZP4 (ZP4 pseudogene in mouse). Recent studies employing recombinant and immunoaffinity purified human zona proteins revealed that in addition to ZP3, capacitated acrosome-intact spermatozoa also bind ZP4. Human ZP2 primarily binds to the acrosome-reacted spermatozoa, supporting its role as secondary sperm receptor, as delineated in the murine model. For binding of human zona proteins to spermatozoa, glycosylation is not critical. Both human ZP3 and ZP4 induce dose-dependent acrosomal exocytosis in capacitated sperm. In contrast to the murine model, N-linked glycosylation is more critical for the human ZP3/ZP4 mediated induction of acrosomal exocytosis. Subtle differences in the downstream signaling events associated with ZP3 vs. ZP4 mediated induction of acrosomal exocytosis have been observed. To conclude, in humans, ZP3 and ZP4 are involved in binding of the spermatozoa to the egg and subsequent induction of acrosome reaction. The contribution, if any, of human ZP glycoprotein-1 (ZP1) during these stages of fertilization remains to be elucidated.
