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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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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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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, Beena Bhandari, Antonin Bukovsky, Raj K. Sharma, Pankaj Bansal, 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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'ZP domain' of human zona pellucida glycoprotein-1 binds to human spermatozoa and induces acrosomal exocytosis.
Reproductive Biology and Endocrinology, 2010Co-Authors: Anasua Ganguly, Pankaj Bansal, Tripti Gupta, Satish K GuptaAbstract:The human egg coat, zona pellucida (ZP), is composed of four glycoproteins designated as zona pellucida glycoprotein-1 (ZP1), -2 (ZP2), -3 (ZP3) and -4 (ZP4) respectively. The zona proteins possess the archetypal 'ZP domain', a signature domain comprised of approximately 260 amino acid (aa) residues. In the present manuscript, attempts have been made to delineate the functional significance of the 'ZP domain' module of human ZP1, corresponding to 273-551 aa fragment of human ZP1. Baculovirus-expressed, nickel-nitrilotriacetic acid affinity chromatography purified 'ZP domain' of human ZP1 was employed to assess its capability to bind and subsequently induce acrosomal exocytosis in capacitated human spermatozoa using tetramethyl rhodamine isothiocyanate conjugated Pisum sativum Agglutinin in absence or presence of various pharmacological inhibitors. Binding characteristics of ZP1 'ZP domain' were assessed employing fluorescein isothiocyanate (FITC) labelled recombinant protein. SDS-PAGE and immunoblot characterization of the purified recombinant protein (both from cell lysate as well as culture supernatant) revealed a doublet ranging from ~35-40 kDa. FITC- labelled 'ZP domain' of ZP1 binds primarily to the acrosomal cap of the capacitated human spermatozoa. A dose dependent increase in acrosomal exocytosis was observed when capacitated sperm were incubated with recombinant 'ZP domain' of human ZP1. The acrosome reaction mediated by recombinant protein was independent of Gi protein-coupled receptor pathway, required extra cellular calcium and involved both T- and L-type voltage operated calcium channels. Results described in the present study suggest that the 'ZP domain' module of human ZP1 has functional activity and may have a role during fertilization in humans.
Manuel Avilés - One of the best experts on this subject based on the ideXlab platform.
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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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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.
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Rabbit zona pellucida composition: a molecular, proteomic and phylogenetic approach.
Journal of Proteomics, 2012Co-Authors: I. Stetson, M. J. Izquierdo-rico, José Ballesta, Pascale Chevret, Ricardo Gutiérrez-gallego, C. Moros, Pedro Lorenzo, Pilar García Rebollar, Manuel AvilésAbstract:Abstract The zona pellucida (ZP) participates in sperm–egg interactions during the first steps of fertilization. Recent studies have shown that the ZP matrix of oocytes in several species is composed of four glycoproteins, designated as ZP1, ZP2, ZP3 and ZP4, rather than the three described in mouse, pig and cow. In this study, investigations were carried out to unveil a fourth glycoprotein in the rabbit ( Oryctolagus cuniculus ) ZP. Using total RNA isolated from rabbit ovaries, the complementary deoxyribonucleic acid (cDNA) encoding rabbit ZP1 was amplified by reverse transcribed polymerase chain reaction (RT-PCR). The ZP1 cDNA contains an open reading frame of 1825 nucleotides encoding a polypeptide of 608 amino acid residues. The deduced amino acid sequence of rabbit ZP1 showed high identity with other species: 70% identity with human and horse ZP1, and 67% identity with mouse and rat ZP1. At the proteomic level, peptides corresponding to the four proteins were detected by mass spectrometry. In addition, a molecular phylogenetic analysis of ZP1 showed that pseudogenization of this gene has occurred at least four times during the evolution of mammals. The data presented in this manuscript provide evidence, for the first time, that the rabbit ZP is composed of four glycoproteins.
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Hamster zona pellucida is formed by four glycoproteins: ZP1, ZP2, ZP3, and ZP4.
Journal of Proteome Research, 2009Co-Authors: M. J. Izquierdo-rico, Esteve Llop, José Ballesta, María Jiménez-movilla, A B Perez-oliva, Ricardo Gutiérrez-gallego, Celia Jiménez-cervantes, Manuel AvilésAbstract:The zona pellucida (ZP) is an extracellular glycoprotein matrix that surrounds all mammalian oocytes. Recent data have shown the presence of four glycoproteins (ZP1, ZP2, ZP3, and ZP4) in the ZP of human and rat rather than the three glycoproteins proposed in the mouse model. In the hamster (Mesocricetus auratus), it was previously described that ZP was composed of three different glycoproteins, called ZP1, ZP2, and ZP3, even though only ZP2 and ZP3 have been cloned thus far. The aim of the study was to determine whether hamster might also express four, rather than three, ZP proteins. The full-length cDNAs encoding hamster ZP glycoproteins 1 and 4 were isolated using rapid amplification cDNA ends (RACE). The cDNA of ZP1 contains an open reading frame of 1851 nucleotides encoding a polypeptide of 616 amino acid residues. The amino acid sequence of ZP1 revealed a high homology with other mammalian species like human (66%), rat (80%), and mouse (80%). The cDNA of ZP4 contains an open reading frame of 1632 nucleotides encoding a polypeptide of 543 amino acid residues. The deduced amino acid sequence of ZP4 revealed high overall homology with rat (82%) and human (78%). Subsequent mass spectrometric analysis of the hamster ZP allowed identification of peptides from all four glycoproteins. The data presented in this study provide evidence, for the first time, that the hamster ZP matrix is composed of four glycoproteins.
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hamster zona pellucida is formed by four glycoproteins ZP1 zp2 zp3 and zp4
Journal of Proteome Research, 2009Co-Authors: M J Izquierdorico, Maria Jimenezmovilla, Esteve Llop, A B Perezoliva, Ricardo Gutierrezgallego, Celia Jimenezcervantes, José Ballesta, Manuel AvilésAbstract:The zona pellucida (ZP) is an extracellular glycoprotein matrix that surrounds all mammalian oocytes. Recent data have shown the presence of four glycoproteins (ZP1, ZP2, ZP3, and ZP4) in the ZP of human and rat rather than the three glycoproteins proposed in the mouse model. In the hamster (Mesocricetus auratus), it was previously described that ZP was composed of three different glycoproteins, called ZP1, ZP2, and ZP3, even though only ZP2 and ZP3 have been cloned thus far. The aim of the study was to determine whether hamster might also express four, rather than three, ZP proteins. The full-length cDNAs encoding hamster ZP glycoproteins 1 and 4 were isolated using rapid amplification cDNA ends (RACE). The cDNA of ZP1 contains an open reading frame of 1851 nucleotides encoding a polypeptide of 616 amino acid residues. The amino acid sequence of ZP1 revealed a high homology with other mammalian species like human (66%), rat (80%), and mouse (80%). The cDNA of ZP4 contains an open reading frame of 1632 nu...
Girum Borossa - One of the best experts on this subject based on the ideXlab platform.
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subcellular distribution of ZP1 zp2 and zp3 glycoproteins during folliculogenesis and demonstration of their topographical disposition within the zona matrix of mouse ovarian oocytes
Biology of Reproduction, 2002Co-Authors: Majid Elmestrah, Philip E Castle, Girum BorossaAbstract:Abstract The zona pellucida (ZP) is an extracellular coat synthesized and secreted by the oocyte during follicular development and surrounding the plasma membrane of mammalian eggs. To date, the mechanism of synthesis and secretion, mode of assembly, and intracellular trafficking of the ZP glycoproteins have not been fully elucidated. Using antibodies against mouse ZP1, ZP2, and ZP3 in conjunction with the protein A-gold technique, we have shown an association of immunolabeling with the Golgi apparatus, secretory granules, and a complex structure called vesicular aggregate, respectively, in mouse ovarian follicles. In contrast, the neighboring granulosa cells were not reactive to any of the three antibodies used. Immunolabeling of ZP1, ZP2, and ZP3 was detected throughout the entire thickness of the ZP, irrespective of the developmental stage of ovarian follicles. Double and triple immunolocalization studies, using antibodies tagged directly to different sizes of gold particles, revealed an asymmetric spa...
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Subcellular Distribution of ZP1, ZP2, and ZP3 Glycoproteins During Folliculogenesis and Demonstration of Their Topographical Disposition Within the Zona Matrix of Mouse Ovarian Oocytes
Biology of Reproduction, 2002Co-Authors: Majid El-mestrah, Girum Borossa, Philip E Castle, Frederick W. K. KanAbstract:The zona pellucida (ZP) is an extracellular coat synthesized and secreted by the oocyte during follicular development and surrounding the plasma membrane of mammalian eggs. To date, the mechanism of synthesis and secretion, mode of assembly, and intracellular trafficking of the ZP glycoproteins have not been fully elucidated. Using antibodies against mouse ZP1, ZP2, and ZP3 in conjunction with the protein A-gold technique, we have shown an association of immunolabeling with the Golgi apparatus, secretory granules, and a complex structure called vesicular aggregate, respectively, in mouse ovarian follicles. In contrast, the neighboring granulosa cells were not reactive to any of the three antibodies used. Immunolabeling of ZP1, ZP2, and ZP3 was detected throughout the entire thickness of the ZP, irrespective of the developmental stage of ovarian follicles. Double and triple immunolocalization studies, using antibodies tagged directly to different sizes of gold particles, revealed an asymmetric spatial distribution of the three ZP glycoproteins in the zona matrix at various stages of follicular development. All three glycoproteins were specifically localized over small patches of darkly stained flocculent substance dispersed throughout the zona matrix. Very often, ZP1, ZP2, and ZP3 were found in close association. These results confirm findings from previous studies demonstrating that ovarian oocytes and not granulosa cells are the only source for mouse ZP glycoproteins. In addition, results from our morphological and immunocytochemical experiments suggest that the vesicular aggregates in the ooplasm are likely to serve as an intermediary in the synthesis and secretion of ZP glycoproteins. The stoichiometric disposition of ZP1, ZP2, and ZP3 in the zona matrix as revealed by double and triple immunolocalization studies provide further insight into some of the unanswered questions pertinent to the current model of mouse ZP structure proposed by the Wassarman group.
Jurrien Dean - One of the best experts on this subject based on the ideXlab platform.
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Structural Characterization of Native Mouse Zona Pellucida Proteins Using Mass Spectrometry
Journal of Biological Chemistry, 2003Co-Authors: Emily S Boja, Tanya Hoodbhoy, Henry M. Fales, Jurrien DeanAbstract:Abstract The zona pellucida is an extracellular matrix consisting of three glycoproteins that surrounds mammalian eggs and mediates fertilization. The primary structures of mouse ZP1, ZP2, and ZP3 have been deduced from cDNA. Each has a predicted signal peptide and a transmembrane domain from which an ectodomain must be released. All three zona proteins undergo extensive co- and post-translational modifications important for secretion and assembly of the zona matrix. In this report, native zonae pellucidae were isolated and structural features of individual zona proteins within the mixture were determined by high resolution electrospray mass spectrometry. Complete coverage of the primary structure of native ZP3, 96% of ZP2, and 56% of ZP1, the least abundant zona protein, was obtained. Partial disulfide bond assignments were made for each zona protein, and the size of the processed, native protein was determined. The N termini of ZP1 and ZP3, but not ZP2, were blocked by cyclization of glutamine to pyroglutamate. The C termini of ZP1, ZP2, and ZP3 lie upstream of a dibasic motif, which is part of, but distinct from, a proprotein convertase cleavage site. The zona proteins are highly glycosylated and 4/4 potential N-linkage sites on ZP1, 6/6 on ZP2, and 5/6 on ZP3 are occupied. Potential O-linked carbohydrate sites are more ubiquitous, but less utilized.
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Structural Characterization of Native Mouse Zona Pellucida Proteins Using Mass Spectrometry
Journal of Biological Chemistry, 2003Co-Authors: Emily S Boja, Tanya Hoodbhoy, Henry M. Fales, Jurrien DeanAbstract:Abstract The zona pellucida is an extracellular matrix consisting of three glycoproteins that surrounds mammalian eggs and mediates fertilization. The primary structures of mouse ZP1, ZP2, and ZP3 have been deduced from cDNA. Each has a predicted signal peptide and a transmembrane domain from which an ectodomain must be released. All three zona proteins undergo extensive co- and post-translational modifications important for secretion and assembly of the zona matrix. In this report, native zonae pellucidae were isolated and structural features of individual zona proteins within the mixture were determined by high resolution electrospray mass spectrometry. Complete coverage of the primary structure of native ZP3, 96% of ZP2, and 56% of ZP1, the least abundant zona protein, was obtained. Partial disulfide bond assignments were made for each zona protein, and the size of the processed, native protein was determined. The N termini of ZP1 and ZP3, but not ZP2, were blocked by cyclization of glutamine to pyroglutamate. The C termini of ZP1, ZP2, and ZP3 lie upstream of a dibasic motif, which is part of, but distinct from, a proprotein convertase cleavage site. The zona proteins are highly glycosylated and 4/4 potential N-linkage sites on ZP1, 6/6 on ZP2, and 5/6 on ZP3 are occupied. Potential O-linked carbohydrate sites are more ubiquitous, but less utilized.
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Abnormal zonae pellucidae in mice lacking ZP1 result in early embryonic loss.
Development (Cambridge England), 1999Co-Authors: Tracy Rankin, Prue Talbot, Eric Lee, Jurrien DeanAbstract:All vertebrates have an egg shell that surrounds ovulated eggs and plays critical roles in gamete recognition. This extracellular matrix is known as the zona pellucida in eutherian mammals and consists of three glycoproteins, ZP1, ZP2 and ZP3 in the mouse. To investigate the role of ZP1 in fertilization and early development, we have used targeted mutagenesis in embryonic stem cells to create mouse lines (ZP1(tm/tm)) lacking ZP1. Although a zona pellucida composed of ZP2 and ZP3 was formed around growing ZP1(tm/tm) oocytes, the matrix was more loosely organized than zonae around normal oocytes. In some ZP1 null follicles, this structural abnormality resulted in ectopic clusters of granulosa cells, lodged between the zona matrix and the oolemma, that perturbed normal folliculogenesis. Comparable numbers of eggs were ovulated from ZP1 null females and normal females following hormonal stimulation. However, after mating with males, fewer two-cell embryos were recovered from ZP1 null females, and their litters were significantly smaller than those produced by normal mice. Therefore, although mouse ZP1 is not essential for sperm binding or fertilization, it is required for the structural integrity of the zona pellucida to minimize precocious hatching and reduced fecundity.
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Mouse ZP1 encodes a zona pellucida protein homologous to egg envelope proteins in mammals and fish.
Journal of Biological Chemistry, 1995Co-Authors: Olga Epifano, Li-fang Liang, Jurrien DeanAbstract:Abstract ZP1 encodes one of the three major glycoproteins of the zona pellucida, an extracellular matrix that surrounds growing oocytes, ovulated eggs, and preimplantation embryos. The mouse gene is composed of 12 exons ranging in size from 82 to 364 base pairs and spans 6.5 kilobase pairs on chromosome 19 (2.13 ± 1.5 centimorgans distal to D19Bir1). The ZP1 exon map is similar to ZPB, a human orthologue, and an E-box (CANNTG), implicated in oocyte-specific gene expression of mouse Zp2 and Zp3, is similarly located upstream of the transcription start site. The single copy ZP1 gene encodes a 623-amino acid protein, the carboxyl-terminal half of which is significantly similar to a corresponding region of mouse ZP2. The conservation of this same region in a fish egg envelope protein suggests that not only has this protein domain been duplicated in mammals but that it has been conserved and used as an egg envelope protein in species that diverged 650 million years ago.
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Coordinate expression of the three zona pellucida genes during mouse oogenesis.
Development (Cambridge England), 1995Co-Authors: Olga Epifano, Li-fang Liang, Mary Familari, Malcolm Moos, Jurrien DeanAbstract:The mammalian zona pellucida is an extracellular matrix that surrounds growing oocytes, ovulated eggs and early embryos. The mouse zona is composed of three sulfated glycoproteins: ZP1, ZP2 and ZP3. Each is critically involved in fertilization, the postfertilization block to polyspermy and protection of the preimplantation embryo. We have previously isolated cDNAs encoding mouse ZP2 and ZP3 and now report the isolation of a full-length cDNA encoding ZP1. Mouse ZP1 is composed of a 623 amino acid polypeptide chain with a signal peptide and a carboxyl terminal transmembrane domain, typical of all zona proteins. Sequence comparison demonstrate that mouse ZP1 is an orthologue of a rabbit zona protein, R55. The expression of R55 has been reported previously in both oocytes and granulosa cells. However, by northern analysis and in situ hybridization with 33P-labelled antisense probes to each of the three mouse zona mRNAs, we have determined that the expression of each mouse zona gene is restricted to the oocyte. ZP2 transcripts, but not ZP1 or ZP3, are detected in resting (15 microns diameter) oocytes, and all three zona transcripts coordinately accumulate as oocytes begin to grow. Together they represent approximately 1.5% of the total poly(A)+ RNA in 50–60 microns oocytes. In the latter stages of oogenesis, their abundance declines and each zona transcript is present in ovulated eggs at less than 5% of its maximal level. No zona transcripts were detected above background signal in granulosa cells. We conclude that, in mice, the three zona pellucida genes are expressed in a coordinate, oocyte-specific manner during the growth phase of oogenesis. Our data support the hypothesis that the transcription of the zona genes is controlled, in part, by shared regulatory element(s).
Chhabi K Govind - One of the best experts on this subject based on the ideXlab platform.
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human zona pellucida glycoproteins characterization using antibodies against recombinant non human primate ZP1 zp2 and zp3
Molecular Human Reproduction, 1998Co-Authors: S. K Gupta, Renuka Kaul, P Jethanandani, Edward C. Yurewicz, Anthony G Sacco, Chhabi K GovindAbstract:: 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.
