Binding of bacterial toxins to glycoproteins in the envelopes of rainbow trout eggs

Summary The ability of the vitelline and fertilization envelopes of rainbow trout eggs to trap toxins was investigated using cholera enterotoxin B and staphylococcal enterotoxin B in cytochemical or immunocytochemical experiments. Extracts from both envelopes were investigated by immunoblot analysis to identify toxin-binding proteins after SDS-PAGE. Binding studies of cholera enterotoxin B to vitelline envelopes and fertilization envelopes revealed a greater reactive intensity in the former. Treatment with neuraminidase enhanced the reactive intensity (or deposit) in the vitelline envelope and fertilization envelope outermost layers, with more conspicuous reactivity in the former. Cytochemical experiments showed that exogenous ganglioside GM₁ considerably enhanced cholera enterotoxin B binding to vitelline and fertilization envelopes. This enhancement was shown by an intense reactivity following the occurrence of new binding sites on the vitelline envelope inner surface and the inner wall of the zona radiata, a simultaneous extreme reduction in the reactivity of the vitelline envelope outermost layer, and a striking increase in reactive products in the fertilization envelope outermost layer. The surface region of the vitelline or fertilization envelope outermost layer was the binding site for staphylococcal enterotoxin B, and neuraminidase treatment caused a considerable reduction of reactive products in these areas. Immunoblot analysis of cholera enterotoxin Bor staphylococcal enterotoxin B-binding substances in extracts from the vitelline envelopes or fertilization envelopes demonstrated that the great majority of the binding substances are glycoproteins. The present results suggest that glycoproteins constituting the vitelline envelope or fertilization envelope may contribute to the protection of the egg itself or the embryo by trapping noxious toxins.     

Fertilization in a crab. II. Cytological aspects of the cortical reaction and fertilization envelope elaboration

At fertilization, the egg of Carcinus maenas undergoes cortical vesicle exocytosis, in response to the first contacts between the spermatozoon and the egg plasma membrane. This process was observed in vitro and may be connected with a cortical reaction. Carcinus maenas eggs display two populations of cortical vesicles which, during the reaction, successively release two different exudates: a fine granular material and a mass of ring-shaped granules. During the first steps of exocytosis, the two superimposed vitelline envelopes are detached from the egg surface, and the inner one gradually changes. Thus a new coating, derived from the coalescence of the secreted ring-shaped granules, is progressively elaborated under the vitelline envelopes. These events occur over a 7–8 hr period. The morphological uniqueness of the cortical vesicle exudates and the complexity of the related events are discussed in terms of the cortical reaction and of the formation of the fertilization envelope in Carcinus maenas.     

Behaviour of the vitelline envelope in Bufo arenarum oocytes matured in vitro in blockade to polyspermy

During activation of amphibian eggs, cortical granule exocytosis causes elaborate ultrastructural changes in the vitelline envelope. These changes involve modifications in the structure of the vitelline envelope and formation of a fertilization envelope (FE) that can no longer be penetrated by sperm. In Bufo arenarum, as the egg traverses the oviduct, the vitelline envelope is altered by a trypsin-like protease secreted by the oviduct, which induces an increased susceptibility of the vitelline envelope to sperm lysins. Full-grown oocytes of B. arenarum, matured in vitro by progesterone, are polyspermic, although cortical granule exocytosis seems to occur within a normal chronological sequence. These oocytes can be fertilized with or without trypsin treatment, suggesting that the vitelline envelope is totally sperm-permeable. Vitelline envelopes without trypsin treatment cannot retain either gp90 or gp96. This suggests that these glycoproteins are involved in the block to polyspermy and that trypsin treatment of matured in vitro oocytes before insemination is necessary to enable vitelline envelopes to block polyspermy. The loss of the binding capacity in vitelline envelopes isolated from B. arenarum oocytes matured in vitro with trypsin treatment and activated by electric shock suggests that previous trypsin treatment is a necessary step for sperm block to occur. When in vitro matured oocytes were incubated with the product of cortical granules obtained from in vitro matured oocytes (vCGP), vitelline envelopes with trypsin treatment were able to block sperm entry. These oocytes exhibited the characteristic signs of activation. These results support the idea that B. arenarum oocytes can be activated by external stimuli and suggest the presence of unknown oocyte surface receptors linked to the activation machinery in response to fertilization. Electrophoretic profiles obtained by SDS-PAGE of solubilized vitelline envelopes from oocytes matured in vitro revealed the conversion of gp40 (in vitro matured oocytes, without trypsin treatment) to gp38 (ascribable to trypsin activity or cortical granule product activity, CGP) and the conversion of gp70 to gp68 (ascribable to trypsin activity plus CGP activity). Taking into account that only the vitelline envelopes of in vitro matured oocytes with trypsin treatment and activated can block sperm entry, we may suggest that the conversion of gp70 to gp68 is related to the changes associated with sperm binding.     

The alphaBbetaC integrin is expressed on the surface of the sea urchin egg and removed at fertilization

Integrins are expressed on the surface of some vertebrate eggs where they are thought to have a role in fertilization. The objective of this study is to determine if integrins are expressed on sea urchin eggs. The alphaB and betaC subunits were cloned using the homology polymerase chain reaction. Monoclonal and polyclonal antibodies were developed against bacterially expressed fragments of the extracellular domains of the betaC subunit and the alphaB subunit. As well, a monoclonal antibody was developed against a synthesized peptide corresponding to part of the cytoplasmic domain of betaC. Analysis of biotinylated egg cortex extracts immunoprecipitated with either anti-betaC or anti-alphaB yields bands of 130 and 225 kDa. Immunoblots confirm that betaC is part of the complex immunoprecipitated with anti-alphaB. Confocal immunofluorescence and immunogold electron microscopy show that betaC is present on the surface of the unfertilized egg at the tips of microvilli and in cortical granules. During the cortical reaction, immunoreactivity with antibodies to the extracellular domains of betaC and alphaB disappears from the egg surface, and microvillar casts on the fertilization envelope become immunoreactive. With antibodies to the cytoplasmic domain of betaC, immunoreactivity is lost from the surface of the egg, but the fertilization envelope does not immediately become immunoreactive. In immunoblots of egg cortex there are immunoreactive bands of the predicted sizes for alphaB and betaC. However, in fertilization envelopes, a second band that is slightly lower in molecular weight is also present. Eggs fertilized in the presence of soybean trypsin inhibitor have elongated microvilli that remain bound to the elevating fertilization envelope and immunoreactive to anti-betaC antibodies. Eggs fertilized in the presence of an ovoperoxidase inhibitor, 3-amino-1,2,4-triazole, have a patchy distribution of betaC immunoreactivity in fertilization envelopes. Together, these data suggest that alphaBbetaC integrins are expressed on the surface of unfertilized eggs and, during the cortical reaction, the extracellular domains are cleaved by proteases and cross-linked into the fertilization envelope by ovoperoxidase. The alphaBbetaC integrin receptors may have several potential functions prior to their removal at fertilization, including attachment of the vitelline envelope to the egg surface and anchoring the cortical cytoskeleton.     

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.     

Structure of the egg funiculus and deposition of embryonic envelopes in a crab

The newly laid egg of Carcinus maenas is attached to a maternal ovigerous seta by a funiculus which consists of the two superimposed vitelline envelopes 1a + 1b, highly stretched and concurrently showing important structural alterations. The funiculus is glued to the specialized seta merely owing to the strong adhesiveness of its external face comprising the outermost vitelline envelope 1a, without any added adhesive. The subjacent envelope 2, originated from the cortical reaction, is not involved in such a funiculus elaboration. In the course of the embryonic development, four new coatings are successively secreted from the ectodermal embryonic cells, underneath the (1a + 1b + 2) fertilization envelope or embryonic capsule. They will remain until hatching in this concentric order, thus giving evidence of successive embryonic moulting cycles, with apolysis but without exuviation. In addition, the successive secretory phases, regarding to the embryonic envelope elaborations, happen in presence of high concentrations of the ecdysteroid ponasterone A which might be involved consequently in such secretory processes.     

On the contents of the cortical granules from Xenopus laevis eggs

The extruded contents of the cortical granules in eggs of Xenopus laevis were solubilized by exposure to divalent metal ion chelators. Chelator extraction of cortical granule (CG) material from intact fertilized or artificially activated eggs was quantitated by fluorescence spectroscopy. The isolated fertilization envelope, formed upon interaction between CG material and the preexisting vitelline envelope, was also subject to extraction. An ultrastructural analysis revealed that chelator exposure resulted in the disruption of the structural integrity of the CG-derived F-component of the fertilization envelope. CG material was isolated from Xenopus ova by three procedures: (1) extrusion from artificially activated, dejellied eggs; (2) extraction of intact, fertilized eggs; and (3) extraction of isolated fertilization envelopes. Only 4–5% of the CG protein recovered by extrusion or by extraction of the intact fertilized egg could be associated with the isolated fertilization envelopes. One predominant polypeptide fraction with an identical relative mobility was demonstrated in all CG preparations upon polyacrylamide gel electrophoresis in SDS. Polymeric forms of CG protein were detected in chelator extracted preparations. The presence of an intact jelly coat during CG breakdown was a prerequisite to the transformation of the vitelline envelope to a fertilization envelope with altered physicochemical characteristics. Further, the CG-derived F-component of the fertilization envelope did not appear to play a critical role in determining the physicochemical properties of the fertilization envelope.     

Independent and hetero-oligomeric-dependent sperm binding to egg envelope glycoprotein ZPC in Xenopus laevis

Vitelline envelopes are composed of glycoproteins that participate in sperm-egg interactions during the initial stages of fertilization. In Xenopus laevis, the vitelline envelope is composed of at least 4 glycoproteins (ZPA, ZPB, ZPC, and ZPX). A sperm binding assay involving the covalent coupling of envelope glycoproteins to silanized glass slides was developed. In our assay, sperm bound to the egg envelopes derived from oviposited eggs but not activated eggs. The majority of the egg envelope ligand activity for sperm binding was derived from the complex N-linked oligosaccharides of ZPC. This sperm binding involved N-acetylglucosamine and fucose residues, as binding was abolished after treatment with cortical granule beta-N-acetylglucosaminidase and commercial beta-N-acetylglucosaminidases and was reduced by 44% after treatment with alpha-fucosidase. Although both the envelope glycoproteins ZPA and ZPC possessed independent ligand activity, ZPC was the major ligand for sperm binding (75%). Mixing of isolated ZPA, ZPB, and ZPC in a ratio of 1:4:4 (equal to that in the egg envelope) resulted in sperm binding that was greater than that of the sum of the separate components. The egg glycoproteins acted in synergy to increase sperm binding. Thus, ZPC possessed both independent and hetero-oligomeric-dependent ligand activities for sperm binding.     

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.     

Identification of the ZPC oligosaccharide ligand involved in sperm binding and the glycan structures of Xenopus laevis vitelline envelope glycoproteins

The Xenopus laevis egg vitelline envelope is composed of five glycoproteins (ZPA, ZPB, ZPC, ZPD, and ZPX). As shown previously, ZPC is the primary ligand for sperm binding to the egg envelope, and this binding involves the oligosaccharide moieties of the glycoprotein (Biol. Reprod., 62:766-774, 2000). To understand the molecular mechanism of sperm-egg envelope binding, we characterized the N-linked glycans of the vitelline envelope (VE) glycoproteins. The N-linked glycans of the VE were composed predominantly of a heterogeneous mixture of high-mannose (5-9) and neutral, complex oligosaccharides primarily derived from ZPC (the dominant glycoprotein). However, the ZPA N-linked glycans were composed of acidic-complex and high-mannose oligosaccharides, ZPX had only high-mannose oligosaccharides, and ZPB lacked N-linked oligosaccharides. The consensus sequence for N-linked glycosylation at the evolutionarily conserved residue N113 of the ZPC protein sequence was glycosylated solely with high-mannose oligosaccharides. This conserved glycosylation site may be of importance to the three-dimensional structure of the ZPC glycoproteins. One of the complex oligosaccharides of ZPC possessed terminal beta-N-acetyl-glucosamine residues. The same ZPC oligosaccharide species isolated from the activated egg envelopes lacked terminal beta-N-acetyl-glucosamine residues. We previously showed that the cortical granules contain beta-N-acetyl-glucosaminidase (J. Exp. Zool., 235:335-340, 1985). We propose that an alteration in the oligosaccharide structure of ZPC by glucosaminidase released from the cortical granule reaction is responsible for the loss of sperm binding ligand activity at fertilization.     

High titers of ecdysteroids are associated with the secretory process of embryonic envelopes in the european lobster

The newly laid egg of the lobster Homarus gammarus is surrounded by a vitelline coat. Just after fertilization, a new subjacent envelope (2), originating from the cortical reaction, is deposited beneath the vitelline coat. In the course of embryonic development, five new coatings (envelopes 3 to 7) are secreted successively from the ectodermal embryonic cells. These will remain until hatching, freeing the mysis larva in concentric order without exuviation. The concentration of both the two major ecdysteroids (ponasterone A and 20-hydroxyecdysone) and their respective precursors (25-deoxyecdysone and ecdysone) were determined as a function of the secretory phase for three embryonic envelopes (2, 3 and 6). We determined that the secretory processes proceed in the presence of high titers of 20-hydroxyecdysone during the onset of envelope secretion and of ponasterone A in the last phase of secretion.     

The Chaetopterus vitelline envelope is not necessary for the gamete interactions that lead to fertilization

It has been recently shown that, in several genera of annelids, including Chaetopterus, fertilizing sperm attach to and fuse with egg microvilli which penetrate the vitelline envelope. This suggests that the annelid vitelline envelope may have no direct or obligatory role in normal fertilization. The present study was undertaken to investigate the involvement of the vitelline envelope in fertilization in Chaetopterus experimentally, by examining the fertilization of vitelline envelope-free eggs quantitatively and qualitatively. Brief exposure of the eggs to isotonic sucrose-EDTA removed the vitelline envelope as determined by both phase-contrast and electron microscopy, rendered the eggs more sensitive to polyspermy and substantially reduced the binding of supernumerary sperm to eggs but did not decrease fertilizability as determined by sperm dilution assay and did not make the eggs more sensitive to cross-fertilization. The events of fertilization were examined by electron microscopy and found to be very similar in vitelline envelope-free eggs to those in intact eggs. We conclude that the vitelline envelope in Chaetopterus has binding sites for sperm but that it has no obligatory role in fertilization and is primarily involved in the prevention of polyspermy.     

The vitelline envelope-to-fertilization envelope transformation in the toad Bufo arenarum.

An investigation of some changes associated with the transformation of the vitelline envelope into the fertilization envelope in the egg of the toad Bufo arenarum is reported here. In most of the experiments described, the parameter used to demonstrate these changes was the stability of structural integrity of isolated envelopes when submitted to different agents and conditions. The envelopes used for this purpose exhibited a high degree of purity and remained apparently unaltered by the isolation procedure. As a quantitative method to ascertain their solubility rate, the release of uv-absorbing materials into solution was determined. Compared to the vitelline envelope, the fertilization envelope has proven to be less soluble in water, more stable in the presence of the chaotropic ion thiocyanate, and less susceptible to digestion in the presence of sperm lysin, trypsin, and pronase. In Bufo arenarum, as in other species, the vitelline envelope appears to be composed of glycoproteins. In contrast to previous results, however, disulfide bonds do not seem to be involved in their structural integrity. Thus, experiments carried out with isolated envelopes as well as with envelopes in situ have demonstrated a lack of effect of disulfide bond breaking agents on envelope stability. Evidence is presented suggesting that the solubility of envelopes in mercaptan solutions, as reported by other laboratories, is likely to be the expression of artifactual results.     

The extracellular matrix of Xenopus laevis eggs: a quick-freeze, deep- etch analysis of its modification at fertilization

Eggs of the amphibian, Xenopus laevis, were quick-frozen, deep-etched, and rotary-shadowed. The structure of the extracellular matrix surrounding these eggs, including the perivitelline space and the vitelline envelope (VE), was visualized in platinum replicas by electron microscopy. The perivitelline space contains an elaborate filamentous glycocalyx which connects microvillar tips to the plasma membrane, to adjacent microvilli, and to the overlying VE. The VE is comprised of two layers, the innermost of which is a thin network of horizontal fibrils lying on the tips of the microvilli. The outermost is a thicker layer of large, cable-like fibers which twist and turn throughout the envelope. Upon fertilization, three dramatic modifications of the matrix occur. A thin sheet of smooth material, termed the smooth layer, is deposited on the tips of the microvilli and separates the egg from the overlying envelopes. The VE above is transformed from a thick band of cable-like fibers to concentric fibrous sheets, the altered VE. Finally, an ornate band of particles, corresponding to the fertilization layer in previous studies, is deposited at the altered VE/jelly interface. The altered VE and the fertilization layer comprise the fertilization envelope, which effects the structural block to polyspermy.     

The coelomic envelope to vitelline envelope conversion in eggs of Xenopus laevis

An amphibian egg recovered from the body cavity is enclosed by a coelomic egg envelope. Upon transport down the oviduct, the envelope is converted to the vitelline envelope. The coelomic and vitelline envelopes are distinct in terms of sperm penetrability, ultrastructural morphology, and radioiodination profiles. In this study, the macromolecular compositions of these two envelopes were determined. Isolated envelopes were compared by one- and two-dimensional gel electrophoresis, peptide mapping, and radiolabeling. A protein with a molecular weight of 57,000 (57K) was present in the vitelline envelope but was absent in the coelomic envelope. A glycoprotein with a molecular weight of 43K in the coelomic envelope was converted to a component with a molecular weight of 41K in the vitelline envelope. The 43K-molecular weight component of the coelomic envelopes could be radioiodinated by lactoperoxidase but no labeling of the 41K-molecular weight component occurred in the vitelline envelope. Peptide mapping using limited proteolysis established that the 43K-molecular weight component of the coelomic envelope was a precursor to the 41K-molecular weight component of the vitelline envelope. These molecular alterations may underlie the ultrastructural and physiological changes occurring in these envelopes.     

The cytochemistry of Limulus eggs.

Cytochemical studies on uninseminated mature eggs of Limulus demonstrate the presence of carbohydrates, lipids and proteins in the egg envelopes and yolk. The vitelline envelope, cortical region and yolk are rich in 1,2-glycols, with the vitelline envelope, containing fewer reactive 1,2-glycol groups than other components of the egg. Neutral mucopolysaccharides are found in the cortical region and yolk, but only the cortical region of the eggs demonstrate the presence of sulfated mucosubstances (which are in part glycoprotein in nature) and glucose-6-phosphatase. Protein is evident in all egg components. Biochemical analysis demonstrate the protein in the egg envelopes of uninseminated eggs is composed of sixteen amino acids while that of developing eggs contain seventeen amino acid residues. Electrovalent linkages and non-S-S- covalent linkages between protein chains are shown to be instrumental in maintaining the stuctural integrity of Limulus egg envelopes. Neutral lipids, unsaturated lipids, phospholipids and fatty acids are demonstrated in yolk bodies and lipoproteins, unsaturated lipids and fatty acids constitute part of the egg envelopes. DNA is concentrated in the cortical region and the yolk bodies     

Fine structure and secretion of the capsule enclosing the embryo in a crab (Carcinus maenas (L.))

Carcinus maenas oocyte is encased, at the end of viteilogenesis phase, by two superposed vitelline envelopes. If the outermost (envelope la) is relatively thin, the innermost (envelope 1b) is however five times much thicker. The envelope 1b, secreted mostly by the oocyte, contains proteinaceous material and a substance which strongly binds WGA. The 1b material deposition is completed when the oocyte nuclear apparatus still has the characteristics of a germinal vesicle. After the secretion phase and always at the ovarian level, envelope 1b material undergoes remarkable structural modifications. These last events occur while the oocyte nuclear apparatus is in the first meiotic metaphase stage. Later, after egg-laying, a new material appears below the two superimposed vitelline envelopes 1a and 1b. This new innermost material (envelope 2), observed as a very thick and homogeneous secretion, is secreted by the newly laid egg probably after fecondation. Envelope 2 material is wholly deposited in few hours and arises from vesicles of an oocytic origin. These three superimposed envelopes make up the embryo capsule of which the main part is constituted by envelope 2. This tripartite capsule will stay, surrounding the developmental stages, superimposed to newly secreted successive embryonic envelopes, up to the end of embryogenesis.     

The vitelline envelope to fertilization envelope conversion in eggs of Xenopus laevis

Fertilization of the Xenopus laevis egg causes the conversion of the vitelline envelope to the fertilization envelope, a change reflected in the loss of sperm penetrability of the egg and the appearance of an electron-dense layer on the outer aspect of the fertilization envelope. As seen by one-dimensional gel electrophoresis, two components with molecular weights of 69,000 and 64,000 in the vitelline envelope were converted to 66,000 and 61,000 in the fertilization envelope. By two-dimensional gel electrophoresis, the components in the 69,000 and 64,000 molecular weight regions of the vitelline envelope were seen to shift to more basic isoelectric points upon conversion to the fertilization envelope. Peptide mapping by limited proteolysis suggested that the 69,000 and 64,000 molecular weight components shared the same polypeptide chains but the smaller glycoprotein lacked a carbohydrate side chain found on the larger species. Similar sites on each glycoprotein were affected when the vitelline envelope was converted to the fertilization envelope. No N-terminal amino acids could be identified on the envelope components, indicating that these glycoproteins have blocked N-termini. Ionophore A23187-activation of jellied eggs (but not dejellied eggs) caused the molecular weight changes in the absence of sperm. Thus, factors from the jelly and the cortical granules but not from sperm apparently are involved in the processing of the 69,000 and 64,000 molecular weight components.     

Extracellular coats on the surface of Strongylocentrotus purpuratus eggs: stereo electron microscopy of quick-frozen and deep-etched specimens

Summary Sea urchin (Strongylocentrotus purpuratus) eggs were fixed, quick-frozen, deep-etched, and rotary-replicated, and the three-dimensional structure of the external surface of the egg visualized using stereo electron microscopy. The cell surface is coated with three layers of filaments: the sheetlike vitelline layer adhering closely to the plasma membrane, a second layer of oblique fibrils extending from microvillar tips to the vitelline layer below, and a third, outermost layer of horizontal filaments coursing in bundles over the microvillar tips. After fertilization, the newly elevated vitelline envelope is transformed into a three-layered structure, the central layer being a tightly knit network of fine filaments decorated on each side with a loose network of thicker fibrils. Subsequently, the envelope becomes coated with paracrystalline protein released from the cortical granules, and microvillar casts are reshaped into angular, jagged peaks having two to five sides. The final structure of the fertilization envelope consists of a thick central layer of compact fibrillar material that is coated on each side with thin plates of paracrystalline protein.     

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.