Vitelline envelope gps 63 and 75 specifically bind sperm in "in vitro" assays in Discoglossus pictus

In Xenopus, conflicting data related to sperm-vitelline envelope (VE) binding suggest that further experiments should be performed to study the role of VE glycoproteins in sperm binding. In this article, we studied the VE of Discoglossus pictus, where gp63, the product of the Dp ZP2 gene, has high molecular identity to Xenopus gp69/64 and to mouse ZP2 and only A23187-treated sperm bind to VE. Sperm bind to VE all over the egg, yet a sperm tuft was found only in the animal half of the egg, where the dimple, the site of fertilization, is located and an intense immunostain was detected in VE by an antiserum directed against gp69/64. The same antiserum inhibited sperm binding to VE. Sperm binding to beads coated with gp63, gp40, or gp75 was in the range of 62-70% for gp63-beads, 67-75% for 75 beads, and about 20% for BSA beads and gp40-coated beads. Soluble purified gp63 and gp75 competitively inhibited binding of sperm to gp63-coated beads. Similarly, the same glycoproteins inhibited sperm binding to gp75-coated beads. SDS-polyacrylamide gels (PAGE) of FE and comparison of VE and FE peptide maps showed that gp63 undergoes a minor shift to about 62 kDa in FE. In sperm binding assays with beads coated with FEs gp62, there was no binding. Following fertilization, in the region of the dimple, an F-layer is formed as well as an alteration of the VE structure. Lectin blots of the FE showed that the FE and in particular gp62 acquires a stronger affinity to Maackia amurensis agglutinin (MAA) with respect to VEs gp63. These results indicate that gps 63 and 75 are the sperm binding glycoproteins of D. pictus VE, where major post-fertilization changes occur as in other anuran species.     

Characterization of hatching-associated changes in the sea urchin fertilization envelope

The embryo of the sea urchin Strongylocentrotus purpuratus hatches from the fertilization envelope (FE) via synthesis and secretion of a hatching enzyme and by ciliary activity. Although the basic characteristics of the hatching enzyme are known, little is understood about changes in the FE during hatching. We have studied the biochemical changes in FEs during hatching. Polyacrylamide gel analysis revealed an increasingly complex polypeptide spectrum of the extractable fraction of FEs isolated during development. Immunoblotting of these polypeptides (using antiserum against the soluble polypeptides extracted from FEs isolated at 30 minutes postinsemination) revealed a decrease in the soluble FE components during hatching. Immunochemical analysis of hatching medium showed a strong correlation between the soluble FE components released and the hatching interval. Immunoblotting of hatching media indicated the presence of soluble FE polypeptides of similar and lower molecular weights than those obtained for extracts of FEs. These results imply that the hatching-associated changes in the FE of S purpuratus occur via proteolysis of FE components, which are derived from the paracrystalline protein fraction, a subset of cortical granule proteins.     

The Primary Egg Envelope of the Anuran Lepidobatrachus laevis: Physicochemical and Macromolecular Alterations During Development

The coelomic egg envelope (CE) of the frog Lepidobatrachus laevis has a network of fibrillar bundles which disperse after transit through the oviduct. Following oviposition, the egg vitelline envelope (VE) has an additional amorphous zone on the exterior surface. The fertilization envelope (FE) formed after fertilization, appears to be very similar to the VE. The CEs, VEs, FEs and hatched envelopes (FE_h) were manually isolated. The CE, VE and FE were solubilized at 100° using denaturing conditions, but were only partially solubilized in phosphate buffer, pH 7.0. All envelopes and several purified polypeptides from the VE and FE were analyzed using gel electrophoresis and one-dimensional peptide mapping. Each of the envelopes contained 9 major polypeptides ranging from 118.5 to 22 kD and 8–12 minor polypeptides. Several envelope components were added/removed in the conversions based on the results of experiments in which preparations were incubated with activated egg exudate and crude hatching enzyme; some of these transformations were mimicked by tryptic and chymotryptic digestions. Therefore, serine proteases may be involved in envelope processing in vivo. Lepidobatrachus CE polypeptides and several major components from the VE, FE and FE_h were crossreactive with antibodies against Xenopus VE^*.     

Sea urchin fertilization envelope: Uncoupling of cortical granule exocytosis from envelope assembly and isolation of an envelope intermediate from Strongylocentrotus purpuratus embryos

The sea urchin fertilization envelope (FE) is an extraembryonic coat which develops from the egg vitelline envelope (VE) and the secreted paracrystalline protein fraction of the cortical granules at fertilization. The FE undergoes further developmental changes postinsemination which are characterized by changes in envelope permeability, solubility in reducing and denaturing solvents, and morphology. We have developed a procedure to uncouple cortical granule exocytosis from assembly of the paracrystalline protein fraction onto the VE template. Egg suspensions were inseminated in normal seawater and diluted into Ca²⁺- and Mg²⁺-free seawater at 15 sec postinsemination. Phase-contrast and electron microscopic observations showed that the embryos formed a normally elevated, extremely thin envelope through which the cortical granule exudate permeated. Secretion studies showed that eggs which were diluted into divalent ion-free seawater postinsemination secreted as much protein into the surrounding seawater as eggs which had their VEs removed prior to the experiment. We have termed the envelope elevated in divalent ion-free seawater the VE1 and we believe that it is the VE structural component of the FE based on its thickness and morphology. VE1s were isolated by gentle physical means and the preparations appeared to be greater than 80% pure based on radioactive mixing experiments and on malate dehydrogenase and glucose-6-phosphate dehydrogenase marker studies. VE1s were at least 80% soluble based on extraction of radioiodinated preparations with reducing and denaturing solvents. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of VE1s showed eight major polypeptides which ranged from 30,500 to 270,000 in molecular weight.     

A comparison of fertilization envelope development in three species of Strongylocentrotus (S. franciscanus, S. droebachiensis, and S. purpuratus)

The ultrastructure of fertilization envelope (FE) development and the polypeptide spectra of Strongylocentrotus franciscanus and S. droebachiensis envelopes were compared to S. purpuratus. In S. franciscanus, the FE reached its maximum thickness of 67 nm by 3 minutes postinsemination (PI), and final structuralization was observed by 40 minutes PI. The fully formed FE did not have microvillar impressions (casts) and was symmetrical, with outer double laminar elements surrounding an amorphous central region. Isolated S. franciscanus FEs were soluble in reducing and denaturing solvents and the same set of 33 polypeptides ranging from 18.5 to 260 kD was detected in FEs isolated from 10 to 180 minutes PI. The S. droebachiensis FE retained microvillar casts, assumed its definitive form by 3 minutes PI, and was 70 nm thick between microvillar impressions. Isolated S. droebachiensis FEs were partially soluble in reducing and denaturing solvents, and the polypeptide spectra of FEs isolated between 10 and 60 minutes PI were identical and showed 14 polypeptides from 18.5 to 265 kD. Antisera against extracted FEs and the FE extract from S. purpuratus were immunologically cross-reactive (using an enzyme-linked immunosorbent assay) with S. franciscanus and S. droebachiensis FE preparations; immunoblots identified 13 and 5 cross-reactive polypeptides, respectively. Most of the cross-reactive polypeptides were of slightly different molecular weight. Based on comparative ultrastructural, solubility, and electrophoretic data, we suggest that S. droebachiensis FE development is most like that observed in S. purpuratus.     

Formation and structure of the fertilization envelope in Xenopus laevis

This paper reports the morphological events that occur when the vitelline envelope (VE) of an unfertilized egg of Xenopus laevis is transformed into the fertilization envelope (FE) surrounding the zygote. The VE is about 1 μm thick and is composed of an interlacing network of small filaments. The FE is constructed from the VE plus an electron-dense layer (fertilization layer), about 2–6 μm thick, on the outer surface of the VE, i.e., at the interface between the VE and the innermost jelly-coat layer. The fertilization layer is a stable component of the FE and is not removed by mercaptan solutions used to dejelly eggs. The events of FE formation were observed in the light and electron microscopes after dejellied eggs were activated by pricking. The FE is established when material from the cortical granules is extruded into the perivitelline space. The cortical granule material passes through the VE as the envelope lifts away from the egg surface. Some cortical granule material deposits in the interstices of the VE, but most of it forms the fertilization layer on the outer surface of the envelope. The cortical reaction is completed about 8–9 min after addition of sperm when eggs are fertilized in vitro.     

Hardening of the sea urchin fertilization envelope by peroxidase-catalyzed phenolic coupling of tyrosines

Within minutes after its elevation from the egg surface, the sea urchin fertilization envelope (FE) becomes "hardened" by a reaction that renders it resistant to agents that solubilize, denature or degrade most proteins. Peroxidase activity is released into the surrounding seawater from Stronglyocentrotus purpuratus eggs during fertilization. Evidence from several sources indicate that the catalytic action of the peroxidase is responsible for hardening the FE through the phenolic coupling of tyrosyl residues of the FE proteins. First, the peroxidase is localized within the hardened FE and within the crystalline FE precursor material released from egg cortical granules during the fertilization reaction. Second, a direct correlation is established between the effectiveness of compounds in inhibiting the cortical granule peroxidase (CGP) and their effectiveness in inhibiting hardening of the FE. Third, the CGP catalyzes the cross-linking of tyrosines in solution, a reaction known to be catalyzed by horseradish peroxidase (HRP). Fourth, acid hydrolysates of hardened FEs contain cross-linked tyrosines that are identified by comparing their chromatographic ultraviolet absorption and fluorescent characteristics to those known for cross-linked tyrosines formed by HRP. Finally, when eggs are fertilized in the presence of 125I, the CGP heavily labels proteins of the FE and of the crystalline FE precursor material released with the enzyme from the cortical granules. The iodide label reflects the localization of the CGP and may reflect the sites of peroxidase-generated tyrosyl phenyl radicals involved in the tyrosine coupling reaction. Maximal iodide labeling occurs during the first 5 min period following fertilization, corresponding to the period of FE hardening.     

Bacterial action of fertilization envelope extract from eggs of the fish Cyprinus carpio and Plecoglossus altivelis

The vitelline envelope (VE) and fertilization envelope (FE) in eggs of the fish Cyprinus carpio and Plecoglossus altivelis were purified by homogenization of eggs or embryos in 5 mM Tris-HCl buffer, pH 7.0, containing 2 mM ethylenediamine tetraacetic acid disodium salt (EDTA), except for processing of VEs in Plecoglossus eggs, and by repeated washing wih the same buffer. To extract the outermost layer material, the purified VEs and FEs were processed overnight at 4 degrees C in 5 mM Tris-HCl buffer, pH 7.0, containing 8 mM 2-mercaptoethanol, 2 mM EDTA, 0.3 M alpha-lactose, 0.3 M glucose, and 0.9% NaCl. Since extraction of the outermost layer of the VEs of Cyprinus eggs in this solution was found to be ultrastructurally incomplete, further sonication in the same buffer was necessary. The solution extracted from purified VEs or FEs was dialyzed against 5 mM Tris-HCl buffer, pH 7.0, followed by lyophilization. The extracts from the FEs from both fish species contained two kinds of lectins, one agglutinated human B-type erythrocytes and the other nonspecifically agglutinated fish spermatozoa, and both extracts had a strong bactericidal effect on Vibrio anguillarum that was isolated from diseased cultured fish, but not on Aeromonas hydrophila and Escherichia coli. The extracts of purified VEs from eggs of both fish had no bactericidal effect on the bacteria examined, nor any agglutination effect on human erythrocytes and fish spermatozoa.     

Sea urchin embryo fertilization envelope: immunological evidence that soluble envelope proteins are derived from cortical granule secretions

Although structural studies support the hypothesis that the sea urchin embryo fertilization envelope is derived from the preexisting vitelline envelope template and structural proteins secreted during the cortical reaction, biochemical evidence is minimal. We used an immunological approach to determine the subcellular origin of proteins which were extracted from the fertilization envelope. Fertilization envelopes were isolated from Stronglyocentrotus purpuratus embryos 30 min postinsemination and extracted with 6.0 M urea-0.15 M 2-mercaptoethanol, pH 10.5, for 10 min at 80°C. Extracted proteins were exhaustively dialyzed against 0.015 M 2-mercaptoethanol-0.100 M Tris-HCl at pH 8.6 and mixed with Fruend's complete adjuvant prior to injection into female New Zealand white rabbits. The antiserum which was prepared contained antibodies to six major and two minor polypeptides in the soluble fertilization envelope fraction based on two-dimensional sodium dodecyl sulfate immunoelectrophoresis. Extracts of vitelline envelopes and extracts of unfertilized egg surfaces which are known to contain viteline envelope proteins did not form immunoprecipitates with antiserum against soluble fertilization envelope polypeptides. Extracts of isolated cortical granules and the secreted paracystalline protein fraction formed four and three immunoprecipitates, respectively, which showed complete identity with the soluble fertilization envelope polypeptides based on rocket-line immunoelectrophoresis. Two-dimensional sodium dodecyl sulfate immunoelectrophoresis of cortical granule extract and the secreted paracrystalline protein fraction showed a complex pattern of immunoprecipitates, but a major finding was that cortical granules contain a 193,000-dalton polypeptide which was not found in the paracrystalline protein fraction. These results suggest that proteolytic processing of a cortical granule precursor of the paracrystalline protein fraction occurs during fertilization and that not all of the cortical granule polypeptides are incorporated into the fertilization envelope by means of di- and trityrosine crosslinks with the vitelline envelope proteins.     

Fertilization envelope assembly in sea urchin eggs inseminated in Cl− deficient sea water: I. Morphological effects

Elevation and hardening of the fertilization envelope (FE) occur within 15 min following insemination of the sea urchin egg. When chloride ions were replaced in the media with various anion substitutes, including methyl sulfonate, nitrates, bromide, and isethionate, the fertilization envelope failed to harden and collapsed back to the surface of the egg of Lytechinus variegatus, L. pictus, and Strongylocentrotus purpuratus. At the light microscopy level, the collapse of the envelope was accompanied by a decrease in birefringence, compared with controls. When examined with electron microscopy, the FEs of eggs inseminated in reduced Cl^? solutions failed to transform from an amorphous layer into the more robust laminar structure observed around eggs incubated in normal sea water. Furthermore, in the case of S. purpuratus, the I-T transformation of the FE did not occur. When transfer of the inseminated eggs from the Cl^?-deficient sea water to normal sea water was carried out before 10 min elapsed, the envelope did not collapse, and the birefringence of the envelope was similar to that of controls. Partial envelope collapse was also observed in a dose-dependent manner, varying with the concentration of the Cl^? in the sea water solution. The results suggest that lack of Cl^? in the media may interfere with proper fertilization envelope assembly. Possible mechanisms, including proper incorporation of the cortical granule exudate into the nascent envelope structure, are discussed.     

Immunological evidence that a 305-kilodalton vitelline envelope polypeptide isolated from sea urchin eggs is a sperm receptor

Direct isolation of the sea urchin egg vitelline envelope with intact sperm receptors is difficult because the envelope is firmly attached to the egg plasma membrane. We now report a method for producing an inseminated egg preparation in Strongylocentrotus purpuratus (using soybean trypsin inhibitor [STI] and Ca²⁺, Mg²⁺-free seawater) that contains an elevated vitelline envelope (VE*-STI). The VE*-STI is devoid of cortical granule material, and supernumerary sperm do not detach postinsemination, suggesting that the VE*-STI contains active sperm receptors. VE*-STIs contain a 305-kD polypeptide and additional components that range from 225 to 31 kD, whereas the 305-kD polypeptide was considerably reduced in VE*s. Electrophoresis of sperm receptor hydrolase digests of VE*-STIs showed that the 305-kD polypeptide and several other envelope polypeptides are protease substrates. Univalent Fab fragments against VE*s, VE*-STIs, and 305 and 225-kD polypeptides blocked sperm binding and fertilization in an Fab concentration-dependent manner. The 305 and 225-kD polypeptides were localized in the VE*-STI using indirect immunofluorescence. Enzyme-linked immunosorbent assays showed that the 305 and 225-kD polypeptides share determinants, suggesting that the 225-kD polypeptide may be derived from the 305-kD polypeptide by the proteolysis that occurs at the cell surface during fertilization. Fab fragments against S purpuratus VE*-STI antigens neither bound to nor blocked homologous sperm binding and fertilization of Lytechinus variegatus eggs. Cross fertilizability occurred to the extent of 5% or less between L variegatus and S purpuratus, therefore, we conclude that the 305 kD-polypeptide isolated from S purpuratus is a species-specific vitelline envelope sperm receptor.     

Characterization of the vitelline envelope of the sea urchin Strongylocentrotus purpuratus

The vitelline envelope (VE) is an extremely thin, acellular, proteinaceous coat that surrounds the extracellular surface of sea urchin eggs. Despite previous studies on VE composition, structure and function, our understanding of the envelope is still incomplete at the molecular level. We have isolated VE components from intact, unfertilized Strongylocentrotus purpuratus eggs by reduction with alkaline dithiothreitol-sea water solutions and have characterized the macromolecules by SDS-PAGE. There were eight major glycoprotein bands, including two high molecular weight components at 265 and 300 kDa, and several minor components. We have revealed, by lectin blot analysis, that most components contain mannose, while a subset of glycoproteins contain fucose and N -acetylglucosamine; galactose and sialic acid were also detected. The components in the VE preparations were compared with cell surface complex preparations by immunoblot analysis, using antisera against a VE preparation, a 305 kDa electrophoretically purified VE glycoprotein and an extracellular portion of the sea urchin egg recombinant 350 kDa sperm receptor. Serum against the recombinant sperm receptor reacted with a component of ∼350 kDa on blots, but did not react with the 300 kDa component found in VE preparations. Therefore, we suggest these two glycoproteins are not the same.     

Identification of a major polypeptide component of the sea urchin fertilization envelope

A monoclonal antibody (MAb No. 25-16), raised against purified cortical secretory vesicles (CVs) from the eggs of Strongylocentrotus purpuratus, has been used to identify a previously uncharacterized CV-derived polypeptide component of the sea urchin fertilization envelope (FE). MAb No. 25-16, an IgG1, bound to a group of proteins with Mr approximately 200,000 on immunoblots of CVs. This same group of proteins also was detected in fertilization product and in soft FEs prepared from early embryos, indicating that the antigen is released at fertilization by CV exocytosis and becomes incorporated into the FE. The multiple components recognized by MAb No. 25-16 apparently did not result from proteolysis during sample preparation or differential N-linked glycosylation. No simplification of the SDS-gel or immunoblot patterns was observed when samples of fertilization product or cell surface complex were prepared in the presence of a cocktail of protease inhibitors; nor was a change in mobility of any of the antigen forms detected following treatment with endoglycosidase F. Upon partial denaturation and reduction of the protein by incubation at room temperature in the presence of SDS and dithiothreitol, the antigen was shown to undergo a decrease in relative mobility on SDS-PAGE. Complete reduction and denaturation, by boiling in dithiothreitol-containing SDS sample buffer or by an on-blot reduction technique, resulted in loss of the epitope. The protein component recognized by MAb No. 25-16 underwent a striking increase in mobility on SDS-PAGE after chelation of calcium ions with EGTA. Immunogold labeling on thin sections of unfertilized eggs revealed that the antigen is located in the spiral lamellar cores of all CVs. In fertilized eggs, fixed 5 min after insemination, the antigen was detected in the FE. Based on these biochemical and immunological data, we suggest that the antigen recognized by MAb No. 25-16 is released exocytotically from the CVs into the perivitelline space at fertilization and becomes incorporated into the FE. The abundance of this antigen suggests that it may function as a structural component of the FE.     

Physicochemical characterization of progressive changes in the Xenopus laevis egg envelope following oviductal transport and fertilization

Previous studies have shown that the Xenopus laevis egg envelope exists in three forms with differing ultrastructural, macromolecular, and sperm penetrability properties. The coelomic envelope (CE) is derived from eggs released from the ovary into the body cavity of the female, the vitelline envelope (VE) from eggs which have passed through the oviduct, and the fertilization envelope (FE) from fertilized eggs. In the present study, the physicochemical characteristics of these three envelope types were differentiated. Investigation of envelope solubility, deformability, sulfhydryl reactivity, and hydrophobic dye and ferritin binding capacity demonstrated that profound physicochemical changes occur in envelope conversions CE----VE----FE. The physical strength of the envelopes, as evidenced by deformability studies, ranked FE greater than CE greater than VE. These differences were not accountable by differences in the number of disulfide bonds, although the CE sulfhydryl groups were significantly less accessible than those in the VE or FE. All three envelope forms were hydrophilic in nature, exhibiting little ability to bind 1-anilino-8-naphthalenesulfonic acid. The CE bound greater amounts of ferritin in comparison to the VE and FE, indicating the presence of a basic domain, presumably in the 43-kDa glycoprotein, which is lost upon proteolysis to 41 kDa during the CE----VE conversion. The envelope integrity of all three forms was maintained by both noncovalent and covalent (disulfide) bonds. Measurements of the effect of pH on envelope solubilization indicated the involvement of an ionizable group with pKa of 8.0 in maintaining envelope structure.     

Polymerization of a vitelline envelope (VE)-like structure produced by using fractionated VE extracts from carp eggs

Fractionation of vitelline envelope (VE) extracts from carp eggs made possible the efficient polymerization of a VE-like structure. The structure corresponded to the fourth layer of the VE or fertilization envelope (FE), and its organization was achieved by reassembly in vitro after solubilization of the sheets composed of filamentous substances or network-like aggregates which were induced by a cortical alveolus sialoglycoprotein or thrombin. The sialoglycoprotein was a serine proteinase and immunolocalized only in the structure at the periphery of cortical alveoli, not in the VE and yolk granules. Ultrastructural features of the VE-like structure suggested that reassembly in vitro occurred via several intermediates in the process of polymerization. A polyclonal antibody produced against one of the assembled VE components, a 64 kDa protein, more intensely immunostained the outer periphery of the VEs than other areas, and immunoelectron microscopy showed that immunogold particles specifically labeled reassembled VE-like structures and major skeletons of the networks or network-like sheets. The protein with a molecular weight of 64 kDa was found to be a DNase. Thus, these results suggest a new approach to investigating not only the FE assembly process in vitro but also the organizing relationship between the major skeleton of the VE or FE and other additional constituents.     

Relationship between p62 and p56, two proteins of the mammalian cortical granule envelope, and hyalin, the major component of the echinoderm hyaline layer, in hamsters

Mammalian cortical granules contain two polypeptides (p62 and p56) that are incorporated into the cortical granule envelope after fertilization and function in cleavage of the zygote and the preimplantation blastomeres. Since the echinoderm hyaline layer and mammalian cortical granule envelope are analogous, and since the hyaline layer protein, hyalin, functions in early echinoderm embryogenesis, this study was done to determine whether p62 and p56 and/or other components of the mammalian cortical granule envelope are related to hyalin. A polyclonal antibody (IL2) against purified S. purpuratus hyalin was shown by confocal scanning laser microscopy to bind to hamster cortical granules and to the cortical granule envelope of fertilized hamster oocytes and preimplantation embryos up to the blastocyst stage. In immunoblots, IL2 bound only to 62- and 56-kDa cortical granule proteins that were incorporated into the cortical granule envelope after fertilization. IL2 binding antigens appeared to be resynthesized by preimplantation embryos starting at the 2-cell stage of development. In vivo treatment of 2-cell-stage hamster embryos with IL2 inhibited blastomere cleavage, but treatment of morulae did not inhibit blastocyst implantation. These results support the idea that the mammalian cortical granule envelope proteins, p62/p56, share a common antigenic epitope(s) with echinoderm hyalin, and that p62/p56, like hyalin, play a role in early embryogenesis.     

Fertilization envelope assembly in sea urchin eggs inseminated in chloride-deficient sea water: II. Biochemical effects

Eggs of the sea urchin Strongylocentrotus purpuratus were fertilized in normal and in several chloride-deficient sea waters ([ Cl-]: normal greater than isethionate greater than methyl sulfonate greater than bromide). The fertilization envelopes (FE) were thinner and failed to harden, and the characteristic I-T transition did not occur. The permeability of the experimental FEs, as determined by release of protein from the perivitelline space, increased in the order of decreasing [Cl-]. Release of the enzymes beta-1,3-glucanase and cortical granule protease were not significantly altered. On the other hand, release of ovoperoxidase was increased three to four times in bromide sea water. Furthermore, a dose-response was observed in varying concentrations of bromide-normal sea water. With decreasing chloride (increasing bromide) concentration, more ovoperoxidase activity was observed. Cytochemical localization of ovoperoxidase activity with diaminobenzidine revealed almost a total lack of staining of FEs from bromide-substituted sea water. The results suggest that in chloride-deficient sea waters protein incorporation into the nascent FE is impaired. At least in the case of bromide, the incorporation of ovoperoxidase into the nascent FE was also inhibited.     

Identification of a new sea urchin vitelline envelope sperm binding glycoprotein

The sea urchin egg vitelline envelope (VE) is composed of eight major glycopolypeptides that are heavily mannosylated and contain fucose and N-acetylglucosamine moieties based on lectin staining. In the present study, the macromolecular composition of the VE and the potential role of a purified VE glycoprotein in initial gamete binding was investigated. The VE components were solubilized from the surface of intact, dejellied eggs with dithiothreitol in divalent cation-free seawater, and analyzed using native, reduced electrophoresis and immunoblotting. Three major VE glycoproteins, VE-A, VE-B and VE-C, and one minor component, VE-D, were identified with antisera against whole VE preparations and against glutaraldehyde-fixed, unfertilized eggs. The electrophoretically purified glycoproteins resolved into a common subunit doublet and one unique subunit each of decreasing size on blots of sodium dodecylsulfate polyacrylamide gels. Lectin affinity chromatography was used for analysis and purification of reduced VE components; a glycoprotein eluted from Con A columns with methyl-mannoside comigrated with VE-B when analyzed by immunoblotting. Whole VE preparations and VE-B obtained from Con A columns were found to inhibit fertilization when preincubated with sperm, thus directly establishing a role for VE-B in gamete binding.     

Cross-linking of ZP2 and ZP3 by transglutaminase is required for the formation of the outer layer of fertilization envelope of carp egg

A transglutaminase (TGase) cDNA was cloned from carp ovary. It was highly homologous to zebrafish TGase. Immunoblot and enzymatical assay showed that TGase was present on the chorion and in the cytoplasm of carp eggs. Addition of TGase inhibitor, cadaverine or ethylene diaminetetracetic acid (EDTA) to the cortical reaction medium impaired the formation of the outer layer of fertilization envelope (FE(o)), the adhesive structure of carp egg. Fibroin-like substance (FLS), cystatin, cathepsin-like substance (CLS), and FEO-1 were the components of FE(o), wherein the majority of the former three were conjugated to form macromolecules of 90-205 kDa while the latter one was present in monomer of 22 kDa. Cadaverine interfered slightly the discharge of FLS conjugates out of the perivitelline space (PVS) but affected profoundly the recruitment of FLS conjugates to FE, whereas EDTA completely inhibited both the release and the recruitment of FLS conjugates to FE. Both EDTA and cadaverine did not inhibit the discharge of FEO-1 out of PVS but could inhibit the recruitment of FEO-1 to FE. The mechanism was studied. ZP2 and ZP3, the major constituents of inner layer of FE, were cross-linked during cortical reaction, which rendered FE hardened. In the presence of EDTA, the cross-linking of ZP2 and ZP3 were inhibited, thus FE remained soft. The PVS of an egg with a hardened FE was less expanded than an egg with a soft FE. It was assumed that a less expanded PVS would generate a higher fluid pressure than a more expanded PVS did. Therefore, the transportation of the macromolecules such as the FLS-cystatin-CLS conjugates out of PVS was facilitated in control and cadaverine-treated eggs whose FE were hardened but was blocked in EDTA-treated eggs whose FE were unhardened. On the other hand, the transportation of small molecules such as FEO-1 out of FE was not restrained, so they were discharged out of the PVS of the control and TGase inhibitor-treated eggs. In addition, TGase activity was also required for the recruitment of FLS conjugates to FE.     

The envelopes of amphibian oocytes: physiological modifications in Bufo arenarum

A characterization of the Amphibian Bufo arenarum oocyte envelope is presented. It was made in different functional conditions of the oocyte: 1) when it has been released into the coelomic cavity during ovulation (surrounded by the coelomic envelope, (CE), 2) after it has passed through the oviduct and is deposed (surrounded by the viteline envelope, (VE), and 3) after oocyte activation (surrounded by the fertilization envelope, (FE). The characterization was made by SDS-PAGE followed by staining for protein and glycoproteins. Labeled lectins were used to identify glycosidic residues both in separated components on nitrocellulose membranes or in intact oocytes and embryos. Proteolytic properties of the content of the cortical granules were also analyzed. After SDS-PAGE of CE and VE, a different protein pattern was observed. This is probably due to the activity of a protease present in the pars recta of the oviduct. Comparison of the SDS-PAGE pattern of VE and FE showed a different mobility for one of the glycoproteins, gp75. VE and FE proved to have different sugar residues in their oligosaccharide chains. Mannose residues are only present in gp120 of the three envelopes. N-acetyl-galactosamine residues are present in all of the components, except for gp69 in the FE. Galactose residues are present mainly in gp120 of FE. Lectin-binding assays indicate the presence of glucosamine, galactose and N-acetyl galactosamine residues and the absence (or non-availability) of N-acetyl-glucosamine or fucose residues on the envelopes surface. The cortical granule product (CGP) shows proteolytic activity on gp75 of the VE.