Egg white lysozyme is the major protein of the hen's egg vitelline membrane

Lysozyme accounts for 37% of the proteins of the hen's egg vitelline membrane. It can be extracted by salt solutions and purified by gel filtration on Sephadex G-50. There are no differences between the chemical and enzymic properties of egg white and vitelline membrane lysozymes. Vitelline membranes of ovarian eggs do not contain lysozyme. It is thus concluded that lysozyme is localized in the outer layer. Vitelline membranes from fertilized and unfertilized eggs contain the same amount of lysozyme; its percentage decreases after two days of incubation.     

Analysis of vitelline envelope synthesis and composition during early oocyte development in gilthead seabream (Sparus aurata)

The oocyte vitelline envelope (VE) of gilthead seabream is composed of four known zona pellucida (ZP) proteins, ZPBa, ZPBb, ZPC, and ZPX. We have previously shown that the gilthead seabream ZP proteins are differentially transcribed in liver and ovary, with the expression in liver being under estrogenic control. However, although mRNA was found in both liver and ovary, only low ZPBa protein levels were detected in liver and plasma. Using isoform-specific ZP antibodies we show that ZPBa and ZPX translation products are present in the cytosol of stage I and II oocytes. In addition, the zpBa and zpX mRNAs were detected in early developing oocytes. During oocyte growth (vitellogenesis), the VE increased in thickness (>10 microm), and we show that the four ZP isoforms are present in different regions of the VE. ZPX was detected closest to the oocyte plasma membrane while the intermediate region was composed of ZPBa, ZPBb, and ZPC. At the outer layer, only ZPC was detected. When oocytes reach the fully grown stage they resume meiosis and hydration. As the oocyte expands, thinning to 4 microm, the VE acquire a striped and compact appearance at the electron microscopy level. This study provides further evidence for the oocyte origin of some ZP proteins in the gilthead seabream and suggests that the ZP proteins are differentially distributed within the VE.     

Ultrastructural studies on the nematode Xiphinema diversicaudatum: Egg shell formation

The ultrastructure of the formation of the egg shell in the longidorid nematode Xiphinema diversicaudatum is described. Upon fertilization a vitelline membrane, which constitutes the vitelline layer of the egg shell, is formed. The chitinous layer is secreted in the perivitelline space, between the vitelline layer and the egg cell membrane. On completion of the chitinous layer, the material of the lipid layer is extruded from the egg cytoplasm to the outer surface, through finger-like projections. Both chitinous and lipid layers are secreted by granules in the egg cytoplasm that disappear as the layers are completed. Chitinous and lipid layers are formed during the passage of the egg through the oviduct. The vitelline layer is enriched with secretions produced by the oviduct cells and then by phospholipids secreted by the cells of the pars dilatata oviductus. The inner uterine layer is also formed by deposition of secretory products apposed on the egg shell in the distal uterine region and Z-differentiation. In the proximal part of the uterus, the egg has a discontinuous electron-dense layer, the external uterine layer. Tangential sections between chitinous and uterine layers revealed the presence of holes, possibly egg pores, delimited by the two uterine layers.     

Rapid evolution of outer egg membrane proteins in the Drosophila melanogaster subgroup: a case of ecologically driven evolution of female reproductive traits

Although sexual selection has been predominantly used to explain the rapid evolution of sexual traits, eggs of oviparous organisms directly face both the challenges of sexual selection as well as natural selection (environmental challenges, survival in niches, etc.). Being the outermost membrane in most insect eggs, the chorion layer is the interface between the embryo and the environment, thereby serving to protect the egg. Adaptive ecological radiations such as divergence in ovipositional substrate usage and host-plant specializations can therefore influence the evolution of eggshell proteins. We can hypothesize that proteins localized on the outer eggshell may be affected to a greater degree by ecological challenges compared with inner eggshell proteins, and therefore, proteins localized in the outer eggshell (chorion membrane) may evolve differently (faster) than proteins localized in the inner egg membrane (vitelline membrane). We compared the evolutionary divergence of vitelline with chorion membrane proteins in species of the melanogaster subgroup and found that chorion proteins as a group are indeed evolving faster than vitelline membrane proteins. At least one vitelline membrane protein (Vm32E), specifically localized on the outer eggshell, is also evolving faster than other vitelline membrane proteins suggesting that all proteins localized on the outer eggshell may be evolving rapidly. We also found evidence that specific codons in chorion proteins cp15 and cp16 are evolving under positive selection. Polymorphism surveys of cp16 revealed inflated levels of divergence relative to polymorphism in specific regions of the gene, indicating that these regions are under strong selection. At the morphological level, we found notable difference in eggshell surface morphologies between specialist (Drosophila sechellia and Drosophila erecta) and generalist species of Drosophila. We do not know if any of the chorion proteins actually interact with spermatozoids, therefore leaving the possibility of rapid evolution through gametic interaction wide open. At this point, however, our results support previous suggestions that divergences in ecology, particularly, ovipositional substrate divergences may be a strong force driving the evolution of eggshell proteins.     

Incorporation of ZP1 into perivitelline membrane after in vivo treatment with exogenous ZP1 in Japanese quail (Coturnix japonica)

In birds, the egg envelope surrounding the oocyte prior to ovulation is called the perivitelline membrane and it plays important roles in fertilization. In a previous study we demonstrated that one of the components of the perivitelline membrane, ZP3, which is secreted from the ovarian granulosa cells, specifically interacts with ZP1, another constituent that is synthesized in the liver of Japanese quail. In the present study, we investigated whether ZP1 injected exogenously into the blood possesses the ability to reconstruct the perivitelline membrane of Japanese quail. When ZP1 purified from the serum of laying quail was injected into other female birds, the signal of this exogenous ZP1 was detected in the perivitelline membrane. In addition, we revealed, by means of ligand blot analysis, that serum ZP1 interacts with both ZP1 and ZP3 of the perivitelline membrane. By contrast, when ZP1 derived from the perivitelline membrane was administered, it failed to become incorporated into the perivitelline membrane. Interestingly, serum ZP1 recovered from other Galliformes, including chicken and guinea fowl, could be incorporated into the quail perivitelline membrane, but the degree of interaction between quail ZP3 and ZP1 of the vitelline membrane of laid eggs from chicken and guinea fowl appeared to be weak. These results demonstrate that exogenous ZP1 purified from the serum, but not ZP1 from the perivitelline membrane, can become incorporated into the perivitelline membrane upon injection into other types of female birds. To our knowledge, this is the first demonstration that the egg envelope component, when exogenously administered to animals, can reconstruct the egg envelope in vivo.     

Isolation and biological activity of the proteases released by sea urchin eggs following fertilization.

The protease activity released from sea urchin egg cortical granules into the surrounding seawater at fertilization is involved in vitelline layer elevation and the block to polyspermy. The cortical granule protease components were isolated by isoelectric precipitation and affinity chromatography on p-aminobenzamidine-Sepharose columns. Elution profiles from affinity columns suggested heterogeneity of the proteases, and polyacrylamide-gel electrofocusing of affinity-purified preparations established the presence of two proteins. Dramatically different biological activities were resolved by affinity chromatography. Early-eluting fractions of low specific activity delaminated the vitelline layer from the egg plasma membrane; this activity is termed vitelline delaminase. Late-eluting fractions of high specific activity modified the egg vitelline layer surface such that sperm could not bind or fertilize them; this activity is referred to as sperm receptor hydrolase. The biological activities of the sea urchin proteases are apparently the result of limited action on the vitelline layer, unlike bovine trypsin which simply digests the vitelline layer. The cortical granule proteases lost biological specificity when stored at 0°C at pH 8.0. Esterase activity increased, and the preparation acquired the ability to digest the vitelline layer. Increase of the esterase activity in protease preparations was prevented by storage at low pH.The molecular weight of both enzymes was estimated by sucrose gradient centrifugation to be 47,000, whereas multiple components with molecular weights between 10⁵ and 10⁶ were demonstrated by gel filtration.     

Synthesis and deposition of oocyte envelopes in the Colorado beetle,Leptinotarsa decemlineata say

Summary Electron microscopical and autoradiographic methods demonstrate that the secretion vesicles (SV), which are condensed by the Golgi-complexes of the follicle cells of the Colorado beetle, contain proteins which can be labelled with ³H-leucine. The labelled proteins are transported to the oocyte during vitellogenesis. At the end of yolk deposition, a few SV, situated just above the microvilli, disintegrate and give rise to the two layers of the vitelline membrane (VM). During the laying down of the VM or perhaps at a slightly earlier stage a layer is deposited beneath the basement membrane of the follicle cells. This layer may be important in inducing the formation of the egg membranes. Once the VM has formed, the follicle cells degenerate completely. The chorionic inner layer arises from the breakdown of SV, while the chorionic outer layer is formed from the degenerated follicle cells.Dr. A. de Loof gratefully acknowledges a mandate as Aangesteld Navorser of the National Foundation of Scientific Research in Belgium. He also thanks Prof. Dr. A. Gillard for his very helpfull criticism, Dr. W. Mordue (Cambridge) for his help in correcting the language, Prof. Dr. A. Lagasse, for supplying facilities in his laboratory of EM, Mr. W. Bohyn for operating the EM and Mr. G. Maes for photography. Special thanks to Drs. G. Vrensen (Nijmegen) for the introduction in autoradiographic techniques.     

Electron Microscopic Observations on the Cortical Reaction of the Brittle-Star Amphipholis kochii Lütken, with Special Reference to its Vitelline Coat Modification as Revealed by the Surface Replica Method

This paper describes the fine structural changes of the egg of the brittle-star Amphipholis kochii Lütken during the cortical reaction. The vitelline coat is 20 nm thick, when Ruthenium Red stain is used, and consists of a dense network of fibers. The cortical granules are large, 1.5–2.0 μm in diameter, and exist in several layers in the egg cortex, unlike the monolayer arrangement found in many other animals. The contents of the cortical granules are clearly distinguished into two components: peripheral fibrous (PF) material and central fibrous (CF) material that consists of two components differing in electron density. The PF material is densely stained by periodic acid-chromic acid-silver methenamine stain, while the CF material is stained little if at all by this technique. The vitelline coat and some PF materials form the fertilization membrane, which is about 40 nm thick and consists of three layers; the outer and the inner layer of the fertilization membrane each have a trilaminated structure. The vitelline coat substances are probably located in the upper part of the fertilization membrane. The hyaline layer, 7–8 μm thick, consists mainly of CF materials. These observations on the morphology of the ophiuroid egg are discussed in comparison with those on other echinoderms, especially echinoids and asteroids.     

Blocks to polyspermy in Chaetopterus

Blocks to polyspermy may act either at the level of the egg plasma membrane to prevent gamete fusion or at the level of egg surface coats to prevent gamete attachment. The present study was undertaken to determine what type(s) of block(s) to polyspermy exist in Chaetopterus. The results showed the existence of both types. A rapid block acts at the plasma membrane level based on independence from detectable changes in the vitelline layer and is dependent on external sodium ions. A vitelline layer block had been predicted on morphological evidence and is supported here by demonstrating an increase in polyspermy following chemical disruption of the vitelline layer. However, the vitelline layer of the fertilized egg retained its ability to initiate the acrosome reaction in sperm and attach sperm which had undergone the acrosome reaction. The vitelline layer block resulted from the retraction of egg microvilli from the vitelline layer, and not from elevation of the vitelline layer per se. Thus the vitelline layer of the fertilized egg could be involved in preventing sperm penetration into the egg without being altered structurally or functionally.     

Species specificity in avian sperm:perivitelline interaction

The interaction of chicken spermatozoa with the inner perivitelline layer from different avian species in vitro during a 5 min co-incubation was measured as the number of points of hydrolysis produced per unit area of inner perivitelline layer. The average degree of interaction, as a proportion of that between chicken spermatozoa and their homologous inner perivitelline layer, was: equal to or greater than 100% within Galliformes (chicken, turkey, quail, pheasant, peafowl and guineafowl); 44% within Anseriformes (goose, duck); and less than 30% in Passeriformes (Zebra Finch) and Columbiformes (collared-dove). The homologue of the putative chicken sperm-binding proteins, chicken ZP1 and ZP3, were identified by Western blotting with anti-chicken ZP1/ZP3 antibody in the perivitelline layers of all species. The functional cross-reactivity between chicken spermatozoa and heterologous inner perivitelline layer appeared to be linked to known phylogenetic distance between the species, although it was not related to the relative affinity of the different ZP3 homologues for anti-chicken ZP3. This work demonstrates that sperm interaction with the egg investment does not represent such a stringent species-specific barrier in birds as it does in mammals and marine invertebrates. This may be a factor in the frequency of hybrid production in birds.     

Egg extracellular coat proteins: from fish to mammals

The extracellular coat surrounding fish (vitelline envelope; VE) and mammalian (zona pellucida; ZP) eggs is composed of long, interconnected filaments. Fish VE and mammalian ZP proteins that make up the filaments are highly conserved groups of proteins that are related to each other, as well as to their amphibian and avian egg counterparts. The rainbow trout (O. mykiss) egg VE is composed of 3 proteins, called VEalpha (approximately 58 kDa), VEbeta (approximately 54 kDa), and VEgamma (approximately 47 kDa). The mouse (M. musculus) egg ZP also is composed of 3 proteins, called ZP1 (approximately 200 kDa), ZP2 (approximately 120 kDa), and ZP3 (approximately 83 kDa). Overall, trout VE and mouse ZP proteins share approximately 25% sequence identity and have features in common; these include an N-terminal signal sequence, a ZP domain, a consensus furin cleavage-site, and a C-terminal tail. VEalpha, VEbeta, and ZP1 also have a trefoil or P-type domain upstream of the ZP domain. VEalpha and VEbeta are very similar in sequence (approximately 65% sequence identity) and are related to ZP1 and ZP2, whereas VEgamma is related to ZP3 (approximately 25% sequence identity). Mouse ZP proteins are synthesized and secreted exclusively by growing oocytes in the ovary. Trout VE proteins are synthesized by the liver under hormonal control and transported in the bloodstream to growing oocytes in the ovary. The trout VE is assembled from VEalpha/gamma and VEbeta/gamma heterodimers. The mouse ZP is assembled from ZP2/3 heterodimers and crosslinked by ZP1. Despite approximately 400 million years separating the appearance of trout and mice, and the change from external to internal fertilization and development, trout VE and mouse ZP proteins have many common structural features; as do avian and amphibian egg VE proteins. However, the site of synthesis of trout and mouse egg extracellular coat proteins has changed over time from the liver to the ovary, necessitating some changes in the C-terminal region of the polypeptides that regulates processing, secretion, and assembly of the proteins.     

Isolation and partial characterization of the plasma membrane of the sea urchin egg

The cell surface complex (Detering et al., 1977, J. Cell Biol. 75, 899-914) of the sea urchin egg consists of two subcellular organelles: the plasma membrane, containing associated peripheral proteins and the vitelline layer, and the cortical vesicles. We have now developed a method of isolating the plasma membrane from this complex and have undertaken its biochemical characterization. Enzymatic assays of the cell surface complex revealed the presence of a plasma membrane marker enzyme, ouabain-sensitive Na+/K+ ATPase, as well as two cortical granule markers, proteoesterase and ovoperoxidase. After separation from the cortical vesicles and purification on a sucrose gradient, the purified plasma membranes are recovered as large sheets devoid of cortical vesicles. The purified plasma membranes are highly enriched in the Na+/K+ ATPase but contain only very low levels of the proteoesterase and ovoperoxidase. Ultrastructurally, the purified plasma membrane is characterized as large sheets containing a "fluffy" proteinaceous layer on the external surface, which probably represent peripheral proteins, including remnants of the vitelline layer. Extraction of these membranes with Kl removes these peripheral proteins and causes the membrane sheets to vesiculate. Polyacrylamide gel electrophoresis of the cell surface complex, plasma membranes, and Kl-extracted membranes indicates that the plasma membrane contains five to six major proteins species, as well as a large number of minor species, that are not extractable with Kl. The vitelline layer and other peripheral membrane components account for a large proportion of the membrane-associated protein and are represented by at least six to seven polypeptide components. The phospholipid composition of the Kl-extracted membranes is unique, being very rich in phosphatidylethanolamine and phosphatidylinositol. Cholesterol was found to be a major component of the plasma membrane. Before Kl extraction, the purified plasma membranes retain the same species-specific sperm binding property that is found in the intact egg. This observation indicates that the sperm receptor mechanisms remain functional in the isolated, cortical vesicle-free membrane preparation.     

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.     

Oogenesis of Cardiochiles nigriceps viereck (Hymenoptera : Braconidae): Histochemistry and development of the chorion with special reference to the fibrous layer

A fibrous layer on the surface of eggs of the parasitoid, Cardiochiles nigriceps (Hymenoptera : Braconidae), has been implicated by earlier studies in the evasion from encapsulation by host hemocytes. The present histochemical and ultrastructural study was undertaken to characterize fibrous layer material and to determine the source of fibrous layer and other components of the eggshell. The fibrous layer contains neutral glyco- or mucoprotein; acidic mucoproteins or glycosaminoglycans are absent. The mature eggshell is resolved into 5 morphologically distinct layers by electron microscopy: (from inner to outer) vitelline envelope, endochorion, an electron-dense “irregular layer”, papilliary layer and fibrous layer. During oogenesis each eggshell layer is laid down sequentially in the order mentioned above. Eggshell material appears to be produced by the follicle cells because these develop extensive rough endoplasmic reticulum and golgi apparatus and exhibit apparent exocytotic activity at the plasma membrane adjacent to the egg.     

The chicken egg white proteome

Using 1-D PAGE and LC-MS/MS and MS(3) we identified 78 chicken egg white proteins, 54 of which were identified in egg white for the first time. All proteins were quantitated by calculating their exponentially modified protein abundance index (emPAI). Some previously known egg white components not characterized by amino acid sequences before, such as alpha-2-macroglobulin, were associated to a sequence for the first time. The predicted sequence was confirmed by MS-sequenced peptides covering 42% of the entire sequence. alpha-2-Macroglobulin occurred in egg white at the same concentration as ovostatin with which it showed 35% identity. For other proteins, which were previously only characterized by partial sequences, such as beta-ovomucin or ovalbumin X, we identified and confirmed predicted complete sequences with a high coverage by MS-sequenced peptides. New proteins included a 7 kDa protein consisting of a single secretoglobin sequence (ovosecretoglobin), a 7 kDa protein with similarity to black swan cygnin and turkey meleagrin (gallin) and proteins involved in binding, modification, and possibly detoxification, of bacterial lipopolysaccaride. The list of egg white proteins provided is by far the most comprehensive at present and is intended to serve as a starting point for the isolation and functional characterization of interesting new proteins.     

Identification and cytogenetic localization of vitelline membrane messenger RNAs in Drosophila

During stages 9 and 10 of oogenesis in Drosophila the major proteins involved in vitelline membrane (VM) formation are synthesized and secreted by the somatic follicle cells surrounding the oocyte. To identify potential mRNAs involved in VM protein synthesis, newly synthesized poly(A)-containing RNA from egg chambers of different developmental stages was studied. Urea-agarose gel electrophoresis revealed two RNA bands in stage 10 egg chambers in the size range expected for those which encode the smaller VM proteins. These RNA bands, T₁ and T₂, are specifically enriched in stage 10 follicle cell preparations. In vitro translations in reticulocyte lysates in the absence and presence of microsomal membranes showed both RNA bands code for products that are synthesized in precursor forms which are processed to species that comigrate with VM proteins. T₂ directed the synthesis of processed species that comigrated with the 23- to 24-kDa and 17.5-kDa VM proteins (J. Fargnoli and G. L. Waring, 1982, Dev. Biol. 92, 306–314) while the T₁ translation product comigrated with the 14-kDa protein. To determine the cytogenetic location of the genes encoding T₁ and T₂ RNAs, radiolabeled T₁ and T₂ RNAs were hybridized in situ to salivary gland chromosomes. The results suggest that the structural genes coding for the small vitelline membrane proteins are localized at two sites on the second chromosome: 39DE and 42A.     

Ultrastructural studies of the formation of the egg capsule in the hermaphroditic species, Isohypsibius granulifer granulifer Thulin, 1928 (Eutardigrada: Hypsibiidae)

The egg capsule of Isohypsibius granulifer granulifer Thulin 1928 (Eutardigrada: Hypsibiidae) is composed of two shells: the thin vitelline envelope and the multilayered chorion. The process of the formation of the egg shell begins in middle vitellogenesis. The I. g. granulifer vitelline envelope is of the primary type (secreted by the oocyte), but the chorion should be regarded as a mixed type: primary (secreted by the oocyte), and secondary (produced by the cells of gonad wall). During early choriogenesis, the parts of the chorion are produced and then connected into a permanent layer. The completely developed chorion consists of three layers: (1) the inner, medium electron dense layer; (2) the middle labyrinthine layer; (3) the outer, medium electron dense layer. After the formation of the chorion, a vitelline envelope is secreted by the oocyte.     

Structure and morphogenesis of the eggshell and micropylar apparatus in the olive fly, Dacus oleae (Diptera: Tephritidae)

The egg of the olive fly, Dacus oleae (Diptera, Tephritidae), is laid inside olives and the larva eventually destroys the fruit. The oocyte is surrounded by several distinct layers which are produced during choriogenesis. The chorion covering the main body of the egg outside of the vitelline membrane includes a "wax" layer, an innermost chorionic layer, an endochorion consisting of inner and outer layers separated by pillars and cavities similar to their counterparts in Drosophila melanogaster, as well as inner and outer exochorionic layers. The anterior pole is shaped like an inverted cup, which is chiefly hollow around its base and has very large openings communicating with the environment. Holes through the surface of the endochorion result from deposition of endochorionic substance around follicular cell microvilli. An opening at the apex of the cup provides an entrance for sperm entering the micropylar canal, which traverses the endochorion and continues into a "pocket" in a thickened vitelline protrusion. The micropylar canal is formed by deposition of endochorion and vitelline membrane around an elongated pair of follicular cell extensions. These extensions later degenerate and leave an empty canal about 5 microns in diameter and the narrower pocket about 1 micron in diameter. Respiration is thought to be facilitated by openings at the base of the anterior pole as well as by openings through the "plastron" around the main body of the shell.     

Changes in the proteins of the vitelline membrane of hens' eggs during storage

Vitelline membranes from fresh and stored eggs were treated with either 0.5 M sodium chloride or 1% sodium dodecyl sulphate (SDS) and the resulting solutions examined by polyacrylamide gel electrophoresis. Several differences between the fresh and stored membranes were evident, the most noticeable being the loss of protein VMO I (vitelline membrane outer I) and the formation of a dimer of lysozyme during storage.     

Purified trout egg vitelline envelope proteins VEbeta and VEgamma polymerize into homomeric fibrils from dimers in vitro

The rainbow trout egg vitelline envelope (VE) is composed of three proteins, called VEalpha ( approximately 58-60kDa Mr), VEbeta ( approximately 52kDa Mr), and VEgamma ( approximately 47kDa Mr). Each of these proteins is related to mouse egg zona pellucida (ZP) glycoproteins, called ZP1, ZP2, and ZP3, and possesses a ZP domain that has been implicated in the polymerization of the proteins into long, interconnected fibrils or filaments. Here, trout egg VEbeta and VEgamma were purified to homogeneity and analyzed under various experimental conditions (SDS-PAGE, Blue Native-(BN-)PAGE, size-exclusion chromatography, and transmission electron microscopy) to determine whether individual VE proteins would polymerize into fibrils in vitro. Such analyses revealed that in the presence of 6M urea each VE protein is present primarily as monomers and as small oligomers (dimers, tetramers, etc.). However, either a reduction in urea concentration or a complete removal of urea results in the polymerization of VEbeta and VEgamma dimers into very large oligomers. Mixtures of VEbeta and VEgamma also give rise to large oligomers. Under these conditions, VE proteins are visualized by transmission electron microscopy as aggregates of long fibrils, with each fibril composed of contiguous beads located periodically along the fibril. The relationship between the behavior of fish egg VE proteins and mouse ZP glycoproteins, as well as other ZP domain-containing proteins, is discussed.