Ultrastructure and Ultracytochemistry of Fertilization Envelope Formation in the Carp Egg

This paper describes ultrastructural and ultracytochemical events occurring during the process of transformation of the vitelline envelope (VE) into the fertilization envelope (FE) in the egg of Cyprinus carpio. The VE is composed of four layers, except the micropylar region. The outermost (first) layer can be subdivided into a double layer (upper and lower halves) by cytochemical differences. The upper half is more protein-rich and positive for acid phosphatase (AcPase) activity, while the lower one is more carbohydrate-rich and negative for AcPase.
The most striking differences between the VE and the FE appear in the first layer component and the thickness. The FE first layer, ultrastructurally and cytochemically consisting of a single layer, gradually grows to be about five-fold as thick as the VE first layer by 40 min after fertilization. This may be due to displacement of the former VE first layer by deposits of the cortical alveolar exudate.     

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.     

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.     

Ultrastructural changes during fertilization envelope assembly in Lytechinus pictus eggs revealed by quick-freeze,deep-etch electron microscopy

Morphological features of fertilization envelope assembly in egges from the sea urchin Lytechinus pictus were examind in platinum replicas of samples quick-frozen, deep-etched, and rotary-shadowed at various times after insemination. Unfertilized eggs are surrounded by the vitelline layer, a glycocalyx, which faith-fully follows the contours of the microvillus-studded egg surface. The vitelline layer is secured to the plasma membrane below via a series of short projections called vitelline posts. The vitelline matrix itself is an elaborate meshwork of uniformly sized filaments, which are decorated in places with globular particles. At fertilization, the vitelline layer elevates off the egg surface and by 1 min after insemination appears as a thin, airy network of fibers. In contrast to Strongylocentrotus purpuratus, impressions of the underlying microvilli are not retained in this species. The vitelline template appears to become filled in by the deposition of amorphous secretory material between 1 and 5 min after fertilization. This smooth, amorphous layer is then coated with a thin sheet of paracrystalline material. Paracrystalline coating is incomplete at 5 min, but by 20 min after insemination the coat is complete, consisting of ordered parallel rows of roset-telike particles.     

In vitro formation of the "S" layer, a unique component of the fertilization envelope in Xenopus laevis eggs

The extracellular matrix (ECM) of unfertilized Xenopus laevis eggs consists of an elaborate filamentous network in the perivitelline space (PS) and a thick fibrillar vitelline envelope (VE), with a thin layer of horizontal filaments (HF) separating the two. At fertilization this ECM is converted into the fertilization envelope (comprised of the fertilization (F) layer and altered VE), and a third layer, the smooth (S) layer, is formed at the upper boundary of the PS (Larabell and Chandler, 1988). In this report, we use quick-freeze, deep-etch, rotary-shadow electron microscopy to show that an intact S layer can be formed in vitro by incubation of unfertilized eggs in an exudate obtained from cortical granules. Within 5 min numerous 36-nm-diameter particles assemble in a highly ordered array at the microvillar tips. These particles appear to "melt" and to form patches of smooth material and within 10 min one continuous sheet has formed. The presence of the VE is required for formation of the S layer, and we suggest that the HF layer is the site of assembly.     

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.     

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^*.     

Regulation of extracellular matrix assembly: in vitro reconstitution of a partial fertilization envelope from isolated components

At fertilization, the glycocalyx (vitelline layer) of the sea urchin egg is transformed into an elevated fertilization envelope by the association of secreted peptides and the formation of intermolecular dityrosine bonds. Dityrosine cross-links are formed by a secreted ovoperoxidase that exists in a Ca2+-stabilized complex with proteoliaisin in the fertilization envelope. By using purified proteins, we now show that proteoliaisin is necessary and sufficient to link ovoperoxidase to the egg glycocalyx. Specifically, we have found that ovoperoxidase can associate with the vitelline layer only when complexed with proteoliaisin; proteoliaisin binds to the vitelline layer independently of its association with ovoperoxidase; proteolytic modification of the vitelline layer is not required for this interaction to occur; the binding of proteoliaisin to the vitelline layer is mediated by the synergistic action of the two major seawater divalent cations, Ca2+ and Mg2+; the number of proteoliaisin-binding sites on the vitelline layer of unfertilized eggs is equivalent to the amount of proteoliaisin secreted at fertilization; and the binding of ovoperoxidase to the vitelline layer, via proteoliaisin, permits the in vitro cross-linking of these two in vivo substrates. The association of purified ovoperoxidase and proteoliaisin with the vitelline layer of unfertilized eggs reconstitutes part of the morphogenesis of the fertilization envelope.     

鲍配子识别蛋白的研究

配子相互作用的生化机制对于进一步阐明生殖过程具有重要作用,它是深入了解细胞内识别的理想体系。精卵细胞相互作用包括一系列的步骤,开始于精子与卵细胞外被的接触,终止于两性细胞的融合及精子核进入卵细胞质中,而精卵细胞的识别具有建立于各自性细胞表面成分基础上的种的特异性,鲍则是研究精卵识别的好材料。鲍精子在发生顶体反应时释放出两种蛋白质——细胞溶素(1ysin)和18ku糖蛋白(spl8),其中的细胞溶素与其卵黄膜上的受体紧密结合,并利用非酶反应在卵黄膜上穿一个小孔,整个精子则从此孔穿过卵黄膜与卵细胞融合;spl8释放后则覆盖到精子细胞膜表面,起到溶解卵细胞脂质体的作用,即spl8介导精、卵细胞膜的融合。鲍卵细胞膜上存在细胞溶素受体,它是大的不分支的糖蛋白分子,占据了卵黄膜30％的组分,可以专一性地与细胞溶素相结合。这些配子识别蛋白共同进化且速度很快,其中细胞溶素和18ku糖蛋白通过正向选择进化,而细胞溶素受体进行协同进化。     

Refertilization in eggs of the sea urchin Strongylocentrotus purpuratus

To determine the role of the sea urchin egg plasma membrane in the species-specificity of fertilization, the ability of denuded activated eggs to be heterospecifically refertilized was determined. Our initial studies included evaluating the effectiveness of three commonly used methods of vitelline envelope (VE) removal using indirect immunofluorescence microscopy with antibodies directed against the VE. Unfertilized Strongylocentrotus purpuratus eggs were extracted with 0.01 M dithiothreitol (DTT) for 3 min or digested with 1.0 mg/ml pronase for 1 hr. Eggs were also fertilized, then diluted into a divalent-free medium to produce thin, elevated envelopes (VE*s) that were mechanically removed by sieving the eggs through nylon mesh. We found that both DTT extraction and pronase digestion were not completely effective in VE removal, and mechanical removal methods gave rise to a mixed population of eggs, those that had their VEs removed and those with a collapsed envelope that was not detectable at the light microscope level. Therefore, a new method of VE removal was developed. Eggs with VE*s were prepared followed by treatment with 0.01 M DTT to solubilize the envelopes. Nearly 100% of the denuded activated eggs incorporated one or more homologous and heterologous sperm, suggesting that the egg plasma membrane does not function in determining the species-specificity of fertilization.     

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.     

Binding of antibiotics to glycoproteins of the vitelline and fertilization envelopes of cherry salmon eggs

The binding of antibiotics (gentamicin, oleandomycin and chloramphenicol) to vitelline and fertilization envelopes and their extracts was investigated by immunohistochemical and immunocytochemical techniques and immunoblot analysis using mature and artificially activated eggs of the fish Oncorhynchus masou. Binding of antibiotics was detected in the vitelline and fertilization envelope outermost layers, the fertilization envelope inner surface and cortical alveolus exudates, with differences in immunoreactive intensity and deposition. The fertilization envelope outermost layer had the capacity to bind much greater amounts of the antibiotics than the vitelline envelope outermost layer. The greater capacity was caused by the deposition of cortical alveolus exudates, which were known to be responsible for functional roles of protection against bacteria, fungi and noxious materials. Treatment of the vitelline and fertilization envelopes with neuraminidase markedly reduced the binding of gentamicin and chloramphenicol but slightly increased that of oleandomycin; binding of the latter to the vitelline and fertilization envelope outermost layers was considerably reduced after treatment with alpha-fucosidase. Treatment of the two envelopes with alpha-mannosidase, beta-galactosidase or beta-SdD-glucosaminidase did not cause any alteration in immunoreactive intensity or number of immunoreactive deposits. Immunoblot analysis of the vitelline or fertilization envelope extracts indicated that many of the antibiotic-binding substances were glycoproteins, and several major bands were bound by all three antibiotics. These results suggest that the vitelline or fertilization envelopes may have the ability to protect the egg itself, or the embryo, respectively, by trapping antibiotics, and the trapping may be related to the presence of carbohydrate moieties, such as sialyl or fucosyl residues. This revised version was published online in November 2006 with corrections to the Cover Date.     

Structural features of the abalone egg extracellular matrix and its role in gamete interaction during fertilization

Abalone eggs are surrounded by a complex extracellular coat that contains three distinct elements: the jelly layer, the vitelline envelope, and the egg surface coat. In this study we used light and electron microscopy to describe these three elements in the red abalone (Haliotis rufescens) and ascribe function to each based on their interactions with sperm. The jelly coat is a spongy matrix that lies at the outermost margin of the egg and consists of variably sized fibers. Sperm pass through this layer with their acrosomes intact and then go on to bind to the vitelline envelope. The vitelline envelope is a multilamellar fibrous layer that appears to trigger the acrosome reaction after sperm binding. Next, sperm release lysin from their acrosomal granules, a nonenzymatic protein that dissolves a hole in the vitelline envelope through which the sperm swims. Sperm then contact the egg surface coat, a network of uniformly sized filaments lying directly above the egg plasma membrane. This layer mediates attachment of sperm, via their acrosomal process, to the egg surface. © 1995 Wiley-Liss, Inc.     

Intermolecular cross-linking of vitelline envelope polypeptides predominates in the hardened sea urchin fertilization envelope

At fertilization, the sea urchin egg vitelline envelope (VE) elevates, and a subset of released cortical granule proteins, paracrystalline protein fraction (PCF), associates with the VE to form the fertilization envelope (FE). Cortical granule peroxidase cross-links FE polypeptides by phenolic coupling of tyrosyl residues. We have used an immunological approach to determine which polypeptides are linked together in the hardened FE of Strongylocentrotus purpuratus. Soluble polypeptides were extracted from hardened FEs, and antibodies were prepared in rabbits against the insoluble envelope matrix (FE ghost). Whole immune serum and purified IgGs each reacted with FE ghosts when using an enzyme-linked immunosorbent assay. VEs isolated by means of three published procedures cross-reacted with the immune serum and purified IgGs. Soluble FE polypeptides also cross-reacted with whole immune serum and IgGs owing to the presence of VE polypeptides. Hyalin, a protein not found in FEs, and PCF did not cross-react with antiserum against FE ghosts. To determine which VE polypeptides were cross-linked in the hardened FE, VE polypeptides were immunoblotted by using antiserum against FE ghosts. Most of the VE polypeptides that ranged from 68,000 to 283,000 molecular weight cross-reacted with the antibody.     

Crystal structure and subunit dynamics of the abalone sperm lysin dimer: egg envelopes dissociate dimers, the monomer is the active species

Lysin is a 16-kD acrosomal protein used by abalone spermatozoa to create a hole in the egg vitelline envelope (VE) by a nonenzymatic mechanism. The crystal structure of the lysin monomer is known at 1.9 A resolution. The surface of the molecule reveals two tracks of basic residues running the length of one surface of the molecule and a patch of solvent-exposed hydrophobic residues on the opposite surface. Here we report that lysin dimerizes via interaction of the hydrophobic patches of monomers. Triton X-100 dissociates the dimer. The crystal structure of the dimer is described at 2.75 A resolution. Fluorescence energy transfer experiments show that the dimer has an approximate KD of 1 microM and that monomers exchange rapidly between dimers. Addition of isolated egg VE dissociates dimers, implicating monomers as the active species in the dissolution reaction. This work represents the first step in the elucidation of the mechanism by which lysin enables abalone spermatozoa to create a hole in the egg envelope during fertilization.     

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.     

The Program of and Mechanisms of Fertilization in the Echinoderm Egg

Current research on the mechanisms of sperm-egg fusion, theblock to polyspermy, and metabolic activation are described.A cinemicrographic analysis of fertilization reveals that fusionof sperm and egg occurs between non-motile gametes, indicatingthat the flagellar motion of sperm is not required. The blockto polyspermy is reviewed, emphasizing recent work on the roleof cortical granule protease in altering sperm receptors ofthe vitelline layer. Metabolic activation or derepression at fertilization is highlyregulated and occurs in a definite sequence. The primary eventappears to be release of intracellular Ca²⁺. The timing of metabolicderepression is different in starfish oocytes. Here, a partof the derepression occurs during maturation and another partat fertilization.     

Electron microscopic study of the cortical reaction of an ophiuroid echinoderm.

The egg coats of an ophiuroid echinoderm (Ophiopholis aculeata) are described by electron microscopy before and after fertilization. The unfertilized egg is closely invested by a vitelline coat about 40 A thick, and the peripheral cytoplasm is crowded with cortical granules five or six deep. During the cortical reaction, which rapidly follows insemination, exocytosis of cortical granules takes place. Some of the cortical granule material is evidently added to the vitelline coat to form a composite structure, the fertilization envelope, which is made up of a 400 A thick middle layer separating inner and outer dense layers, each about 50 A thick. The elevation of the fertilization envelope from the egg surface creates a perivitelline space in which the hyaline layer soon forms. The hyaline layer is about 2 micron thick, finely granular, and apparently derived from cortical granule material. The extracellular layers of the early developmental stages of ophiuroids and echinoids are quite similar in comparison to those of asteroids; this finding helps support Hyman's argument that the ophiuroids are more closely related to the echinoids than to the asteroids.     

Gamete Interactions in Xenopus laevis: Identification of Sperm Binding Glycoproteins in the Egg Vitelline Envelope

A quantitative assay was developed to study the interaction of Xenopus laevis sperm and eggs. Using this assay it was found that sperm bound in approximately equal numbers to the surface of both hemispheres of the unfertilized egg, but not to the surface of the fertilized egg. To understand the molecular basis of sperm binding to the egg vitelline envelope (VE), a competition assay was used and it was found that solubilized total VE proteins inhibited sperm-egg binding in a concentration-dependent manner. Individual VE proteins were then isolated and tested for their ability to inhibit sperm binding. Of the seven proteins in the VE, two related glycoproteins, gp69 and gp64, inhibited sperm-egg binding. Polyclonal antibody was prepared that specifically recognized gp69 and gp64. This gp69/64 specific antibody bound to the VE surface and blocked sperm binding, as well as fertilization. Moreover, agarose beads coated with gp69/64 showed high sperm binding activity, while beads coated with other VE proteins bound few sperm. Treatment of unfertilized eggs with crude collagenase resulted in proteolytic modification of only the gp69/64 components of the VE, and this modification abolished sperm-egg binding. Small glycopeptides generated by Pronase digestion of gp69/64 also inhibited sperm-egg binding and this inhibition was abolished by treatment of the glycopeptides with periodate. Based on these observations, we conclude that the gp69/64 glycoproteins in the egg vitelline envelope mediate sperm-egg binding, an initial step in Xenopus fertilization, and that the oligosaccharide chains of these glycoproteins may play a critical role in this process.