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Mammalian oocytes are enveloped by the zona pellucida (ZP), an extracellular matrix of glycoproteins. In sperm, stimulation with ZP proteins evokes a rapid Ca2+ influx via the sperm-specific, pH-sensitive Ca2+ channel CatSper. However, the physiological role and molecular mechanisms underlying ZP-dependent activation of CatSper are unknown. Here, we delineate the sequence of ZP-signaling events in mouse sperm. We show that ZP proteins evoke a rapid intracellular pH i increase that rests predominantly on Na+/H+ exchange by NHA1 and requires cAMP synthesis by the soluble adenylyl cyclase sAC as well as a sufficiently negative membrane potential set by the spem-specific K+ channel Slo3. The alkaline-activated CatSper channel translates the ZP-induced pH i increase into a Ca2+ response. Our findings reveal the molecular components underlying ZP action on mouse sperm, opening up new avenues for understanding the basic principles of sperm function and, thereby, mammalian fertilization.
The human oocyte zona pellucida (ZP) is made of four glycoproteins, ZP1-ZP4. Recently, the prostate adenocarcinoma and prostate cancer PC3 cell-line were shown to express the human oocyte ZP3 glycoprotein, which was evaluated in a single report subject to patent. To further clarify whether oocyte zona pellucida glycoproteins are expressed in prostate cancer tissue and PC3-cells, in this report we evaluated protein expression of the four ZP glycoproteins in normal prostate tissue, prostate adenocarcinoma tissue and PC3-cells, and performed quantitative mRNA expression of the four ZP glycoproteins in the PC3 cell-line. Furthermore, as PC3-cells have not yet been studied in detail regarding their ultrastructural characteristics, in the present report we bring forward the detailed ultrastructure of PC3-cells. PC3-cells were divided into pavement and aggregated cells. We observed new ultrastructural features in pavement and aggregated cells, with the later exhibiting two different cell types. In prostate carcinoma tissue and PC3-cells we found protein expression of the four oocyte glycoproteins, ZP1, ZP2, ZP3 and ZP4. In addition, mRNA expression studies revealed expression of ZP1, ZP3 and ZP4 glycoproteins, but not of ZP2. Interestingly, the ZP1 mRNA product exhibited intron retention.
Zona pellucida (ZP) is an extracellular matrix surrounding and protecting mammalian and fish oocytes, which is responsible for sperm binding. Mammalian ZP consists of three to four glycoproteins, called ZP1, ZP2, ZP3, ZP4. These proteins polymerize into long interconnected filaments, through a common structural unit, known as the ZP domain, which consists of two domains, ZP-N and ZP-C. ZP is related in function to silkmoth chorion and in an evolutionary fashion to the teleostean fish chorion, also fibrous structures protecting the oocyte and embryo, that both have been proven to be functional amyloids. Two peptides were predicted as 'aggregation-prone' by our prediction tool, AMYLPRED, from the sequence of the human ZP1-N domain. Here, we present results from transmission electron microscopy, X-ray diffraction, Congo red staining and attenuated total reflectance Fourier-transform infrared spectroscopy (ATR FT-IR), of two synthetic peptide-analogues of these predicted 'aggregation-prone' parts of the human ZP1-N domain, that we consider crucial for ZP protein polymerization, showing that they both self-assemble into amyloid-like fibrils. Based on our experimental data, we propose that human ZP (hZP) might be considered as a novel, putative, natural protective amyloid, in close analogy to silkmoth and teleostean fish chorions. Experiments are in progress to verify this proposal. We also attempt to provide insights into ZP formation, proposing a possible model for hZP1-N domain polymerization.
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.
Animal egg coats are composed of different glycoproteins collectively named zona pellucida (ZP) proteins. The characterized vertebrate genes encoding ZP proteins have been classified into six subfamilies, and exhibit low similarity to the ZP genes characterized in certain invertebrates. The origin and evolution of the vertebrate ZP genes remain obscure. A search against 97 representative metazoan species revealed various numbers (ranging from three to 33) of different putative egg-coat ZP genes in all 47 vertebrates and several ZP genes in five invertebrate species, but no putative ZP gene was found in the other 45 species. Based on phylogenetic and synteny analyses, all vertebrate egg-coat ZP genes were classified into eight ZP gene subfamilies. Lineage- and species-specific gene duplications and gene losses occurred frequently and represented the main causes of the patchy distribution of the eight ZP gene subfamilies in vertebrates. Thorough phylogenetic analyses revealed that the vertebrate ZP genes could be traced to three independent origins but were not orthologues of the characterized invertebrate ZP genes. Our results suggested that vertebrate egg-coat ZP genes should be classified into eight subfamilies, and a putative evolutionary map is proposed. These findings would aid the functional and evolutionary analyses of these reproductive genes in vertebrates.
Probing mechanical properties of cells has been identified as a means to infer information on their current state, e.g. with respect to diseases or differentiation. Oocytes have gained particular interest, since mechanical parameters are considered potential indicators of the success of in vitro fertilisation procedures. Established tests provide the structural response of the oocyte resulting from the material properties of the cell's components and their disposition. Based on dedicated experiments and numerical simulations, we here provide novel insights on the origin of this response. In particular, polarised light microscopy is used to characterise the anisotropy of the zona pellucida, the outermost layer of the oocyte composed of glycoproteins. This information is combined with data on volumetric changes and the force measured in relaxation/cyclic, compression/indentation experiments to calibrate a multi-phasic hyper-viscoelastic model through inverse finite element analysis. These simulations capture the oocyte's overall force response, the distinct volume changes observed in the zona pellucida, and the structural alterations interpreted as a realignment of the glycoproteins with applied load. The analysis reveals the presence of two distinct timescales, roughly separated by three orders of magnitude, and associated with a rapid outflow of fluid across the external boundaries and a long-term, progressive relaxation of the glycoproteins, respectively. The new results allow breaking the overall response down into the contributions from fluid transport and the mechanical properties of the zona pellucida and ooplasm. In addition to the gain in fundamental knowledge, the outcome of this study may therefore serve an improved interpretation of the data obtained with current methods for mechanical oocyte characterisation.
OVGP1 is the major non-serum glycoprotein in the oviduct fluid at the time of fertilization and early embryo development. Its activity differs among species. Here, we show that the C-terminal region of recombinant OVGP1 regulates its binding to the extracellular zona pellucida and affects its activity during fertilization. While porcine OVGP1 penetrates two-thirds of the thickness of the zona pellucida, shorter OVGP1 glycoproteins, including rabbit OVGP1, are restricted to the outer one-third of the zona matrix. Deletion of the C-terminal region reduces the ability of the glycoprotein to penetrate through the zona pellucida and prevents OVGP1 endocytosis. This affects the structure of the zona matrix and increases its resistance to protease digestion. However, only full-length porcine OVGP1 is able to increase the efficiency rate of in vitro fertilization. Thus, our findings document that the presence or absence of conserved regions in the C-terminus of OVGP1 modify its association with the zona pellucida that affects matrix structure and renders the zona matrix permissive to sperm penetration and OVGP1 endocytosis into the egg.
The mouse zona pellucida is composed of three glycoproteins (ZP1, ZP2, and ZP3), of which ZP2 is proteolytically cleaved after gamete fusion to prevent polyspermy. This cleavage is associated with exocytosis of cortical granules that are peripherally located subcellular organelles unique to ovulated eggs. Based on the cleavage site of ZP2, ovastacin was selected as a candidate protease. Encoded by the single-copy Astl gene, ovastacin is an oocyte-specific member of the astacin family of metalloendoproteases. Using specific antiserum, ovastacin was detected in cortical granules before, but not after, fertilization. Recombinant ovastacin cleaved ZP2 in native zonae pellucidae, documenting that ZP2 was a direct substrate of this metalloendoprotease. Female mice lacking ovastacin did not cleave ZP2 after fertilization, and mouse sperm bound as well to Astl-null two-cell embryos as they did to normal eggs. Ovastacin is a pioneer component of mouse cortical granules and plays a definitive role in the postfertilization block to sperm binding that ensures monospermic fertilization and successful development.
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 ZP2(51-149). 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 ZP2(51-149) sperm-binding domain is necessary for human and mouse gamete recognition and penetration through the zona pellucida.
Mammalian eggs are surrounded by an extracellular matrix called the zona pellucida (ZP). This envelope participates in processes such as acrosome reaction induction, sperm binding, protection of the oviductal embryo, and may be involved in speciation. In eutherian mammals, this coat is formed of three or four glycoproteins (ZP1-ZP4). While Mus musculus has been used as a model to study the ZP for more than 35 years, surprisingly, it is the only eutherian species in which the ZP is formed of three glycoproteins Zp1, Zp2, and Zp3, Zp4 being a pseudogene. Zp4 was lost in the Mus lineage after it diverged from Rattus, although it is not known when precisely this loss occurred. In this work, the status of Zp4 in several murine rodents was tested by phylogenetic, molecular, and proteomic analyses. Additionally, assays of cross in vitro fertilization between three and four ZP rodents were performed to test the effect of the presence of Zp4 in murine ZP and its possible involvement in reproductive isolation. Our results showed that Zp4 pseudogenization is restricted to the subgenus Mus, which diverged around 6 MYA. Heterologous in vitro fertilization assays demonstrate that a ZP formed of four glycoproteins is not a barrier for the spermatozoa of species with a ZP formed of three glycoproteins. This study identifies the existence of several mouse species with four ZPs that can be considered suitable for use as an experimental animal model to understand the structural and functional roles of the four ZP proteins in other species, including human.
The egg is a spherical cell encapsulated by the zona pellucida (ZP) which forms a filamentous matrix composed of several glycoproteins that mediate gamete recognition at fertilization. Studies on molecular mechanisms of sperm-egg binding are limited in many mammalian species by the scarcity of eggs, by ethical concerns in harvesting eggs, and by the high cost of producing genetically modified animals. To address these limitations, we have reproduced a three-dimensional (3D) model mimicking the oocyte's shape, by means of magnetic sepharose beads coated with recombinant ZP glycoproteins (BZP) and cumulus cells. Three preparations composed of either ZP2 (C and N-termini; BZP2), ZP3 (BZP3) or ZP4 (BZP4) were obtained and characterized by protein SDS-PAGE, immunoblot and imaging with confocal and electron microscopy. The functionality of the model was validated by adhesion of cumulus cells, the ability of the glycoprotein-beads to support spermatozoa binding and induce acrosome exocytosis. Thus, our findings document that ZP-beads provide a novel 3D tool to investigate the role of specific proteins on egg-sperm interactions becoming a relevant tool as a diagnostic predictor of mammalian sperm function once transferred to the industry.
The egg coat including mammalian zona pellucida (ZP) and the avian equivalent, i.e., inner-perivitelline layer (IPVL), is a specialized extracellular matrix being composed of the ZP glycoproteins and surrounds both pre-ovulatory oocytes and ovulated egg cells in vertebrates. The egg coat is well known for its potential importance in both the reproduction and early development, although the underlying molecular mechanisms remain to be fully elucidated. Interestingly, ZP3, one of the ZP-glycoprotein family members forming scaffolds of the egg-coat matrices with other ZP glycoproteins, exhibits extreme but distinctive microheterogeneity to form a large number of isoelectric-point isoforms at least in the chicken IPVL. In the present study, we performed three-dimensional confocal imaging and two-dimensional polyacrylamide-gel electrophoresis (2D-PAGE) of chicken IPVLs that were isolated from the ovarian follicles at different growth stages before ovulation. The results suggest that the relative proportions of the ZP3 isoforms are differentially altered during the structural maturation of the egg-coat matrices. Furthermore, tandem mass spectrometry (MS/MS) analyses and ZP1 binding assays against separated ZP3 isoforms demonstrated that each ZP3 isoform contains characteristic modifications, and there are large differences among ZP3 isoforms in the ZP1 binding affinities. These results suggest that the microheterogeneity of chicken ZP3 might be regulated to be associated with the formation of egg-coat matrices during the structural maturation of chicken IPVL. Our findings may provide new insights into molecular mechanisms of egg-coat assembly processes.
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.
Zona pellucida (ZP) containing proteins are glycoproteins in teleost chorion and are encoded by several gene subfamilies, mainly including ZPA, ZPB, ZPC and ZPX genes. In teleost species, ZP genes are expressed either in liver under regulation of estrogen or in ovary. In the present study, five ZP gene isoforms were isolated and characterized in Gobiocypris rarus. The putative amino acid sequences of these ZP gene isoforms contain the typical trefoil motif and a ZP domain. These five G. rarus ZP gene isoforms were named as grZPB.1, grZPB.2, grZPB.3, grZPB.4 and grZPB.5. Real-time quantitative reverse transcription PCR (RT-qPCR) analysis indicated that all these ZP mRNA isoforms were exclusively expressed in ovary. G. rarus juveniles at the age of 21 days postfertilization were exposed to 17α-ethinylestradiol (EE2; 0.01, 0.1 and 1 nM), 4-nonylphenol (4-NP; 10, 100 and 1000 nM) or bisphenol A (BPA; 0.1, 1 and 10nM) for 3 days. mRNA expressions of ZPB isoforms following the exposure to xenoestrogen were detected by RT-qPCR. Data were analyzed by the 2(-△△Cq) method. The results indicate that induction by 0.1-1nM EE2 on mRNA expression of the grZPB isoforms is weaker than for vitellogenin. 4-NP exposures at three concentrations had differential effects on the grZPBs. BPA at three concentrations weakly induced mRNA expression of the grZPB isoforms.
The selectin family of cell adhesion molecules mediates initial leukocyte adhesion to vascular endothelial cells at sites of inflammation. O-glycan structural similarities between oligosaccharides from human leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) and from zona pellucida glycoproteins of porcine oocytes indicate the possible existence of a P-selectin ligand in the zona pellucida. Here, using biochemical as well as morphological approaches, we demonstrate that a P-selectin ligand is expressed in the porcine zona pellucida. In addition, a search for a specific receptor for this ligand leads to the identification of P-selectin on the acrosomal membrane of porcine sperm cells. In vitro binding of porcine acrosome-reacted sperm cells to oocytes was found to be Ca2+ dependent and inhibitable with either P-selectin, P-selectin receptor-globulin, or leukocyte adhesion blocking antibodies against P-selectin and PSGL-1. Moreover, porcine sperm cells were found to be capable of binding to human promyeloid cell line HL-60. Taken together, our findings implicate a potential role for the oocyte P-selectin ligand and the sperm P-selectin in porcine sperm-egg interactions.
The extracellular matrix (ECM) of porcine mature oocytes was revealed by transmission electron microscopy (TEM) after treatment with tannic acid and ruthenium red. Present in the perivitelline space (PVS) and on the surface of the zona pellucida (ZP), it appeared to be composed of thin filaments and granules at the interconnections of the filaments, which were interpreted respectively as hyaluronic acid chains and bound proteoglycans. In order to determine whether this material is produced by the corona cells (the same ECM was found also on the surface of the zona pellucida and between cumulus cells) or by the oocyte itself, the synthesis of glycoproteins and glycosaminoglycans was checked by autoradiography on semi-thin and thin sections observed by light and electron microscopy. Immature oocytes within or without cumulus cells, were incubated with L [3H-] fucose or L [3H-] glucosamine--precursors respectively of glycoproteins and hyaluronic acid or hyaluronan (HA) bound to proteoglycans--for various times (with or without chase) and at different stages during in vitro maturation. In the first case, incorporation was found in both cumulus cells and ooplasm (notably in the Golgi area for 3H-fucose) and labeled material accumulated in the ECM of the PVS and of the ZP surface. Labeling in the PVS with both precursors was maximum between metaphase I (MI) and metaphase II (MII) and was partially extracted by hyaluronidase but not by neuraminidase. Tunicamycin, an inhibitor of glycoprotein synthesis, significantly decreased the amount of 3H-fucose labeled molecules in the PVS and increased the incidence of polyspermic penetration during subsequent in vivo fertilization. Since cumulus-free oocytes also secreted 3H-glucosamine containing compounds, both oocyte and cumulus cells probably contribute to the production of the ECM found in the PVS of mature oocytes. ECM and particularly its HA moiety present on both sides of the ZP may constitute a favourable factor for sperm penetration.
Complementary molecules on the surface of both gametes are responsible for the interaction of sperm protein receptors with zona pellucida (ZP) saccharide structures, and many primary sperm receptors for ZP glycoproteins have been disclosed in various mammals. For our study, proteins were obtained from the surface of ejaculated and in vitro capacitated boar sperm. The isolated proteins were characterized by 1D- and 2D-electrophoretic protein profiles, and by glycoprotein staining. Our results show quantitative and qualitative differences in protein and glycoprotein patterns between ejaculated and capacitated sperm. Far-western blotting with ZP glycoproteins identified 17 interactions in the subproteome of the ejaculated sperm and 14 interactions in the subproteome of the capacitated sperm. High-molecular-mass proteins, coincident with binding to ZP, were sequence-identified. Angiotensin-converting enzyme (ACE), polycystic kidney disease receptor and egg jelly receptor (PKDREJ), and acrosin precursor were successfully identified. This is the first time PKDREJ has been identified on the surface of boar spermatozoa.
It has been established that mammalian egg zona pellucida (ZP) glycoproteins are responsible for species-restricted binding of sperm to unfertilized eggs, inducing the sperm acrosome reaction, and preventing polyspermy. In mammals, ZP apparently represents a barrier to heterospecific fertilization and thus probably contributes to reproductive isolation between species. The evolutionary relationships between some members of the tribe Bovini are complex and highly debatable, particularly, those involving Bos and Bison species for which interspecific hybridization is extensively documented. Because reproductive isolation is known to be a major precursor of species divergence, testing evolutionary patterns of ZP glycoproteins may shed some light into the speciation process of these species. To this end, we have examined intraspecific and interspecific genetic variation of two ZP genes (Zp2 and Zp3) for seven representative species (111 individuals) from the Bovini tribe, including five species from Bos and Bison, and two species each from genera Bubalus and Syncerus.
Biological tubes must develop and maintain their proper diameter to transport materials efficiently. These tubes are molded and protected in part by apical extracellular matrices (aECMs) that line their lumens. Despite their importance, aECMs are difficult to image in vivo and therefore poorly understood. The Caenorhabditis elegans vulva has been a paradigm for understanding many aspects of organogenesis. Here we describe the vulva luminal matrix, which contains chondroitin proteoglycans, Zona Pellucida (ZP) domain proteins, and other glycoproteins and lipid transporters related to those in mammals. Confocal and transmission electron microscopy revealed, with unprecedented detail, a complex and dynamic aECM. Different matrix factors assemble on the apical surfaces of each vulva cell type, with clear distinctions seen between Ras-dependent (1°) and Notch-dependent (2°) cell types. Genetic perturbations suggest that chondroitin and other aECM factors together generate a structured scaffold that both expands and constricts lumen shape.
The rapid evolution of fertilization proteins has generated remarkable diversity in molecular structure and function. Glycoproteins of vertebrate egg coats contain multiple zona pellucida (ZP)-N domains (1-6 copies) that facilitate multiple reproductive functions, including species-specific sperm recognition. In this report, we integrate phylogenetics and machine learning to investigate how ZP-N domains diversify in structure and function. The most C-terminal ZP-N domain of each paralog is associated with another domain type (ZP-C), which together form a "ZP module." All modular ZP-N domains are phylogenetically distinct from nonmodular or free ZP-N domains. Machine learning-based classification identifies eight residues that form a stabilizing network in modular ZP-N domains that is absent in free domains. Positive selection is identified in some free ZP-N domains. Our findings support that strong purifying selection has conserved an essential structural core in modular ZP-N domains, with the relaxation of this structural constraint allowing free N-terminal domains to functionally diversify.
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