Vitelline Envelope Research Articles

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.

Structure fine et rôle, dans la rétention hydrique, des enveloppes de l'oeuf d'un diptère subantarctique, l'Anatalanta optera Eaton (Sphaeroceridae)

Anatalanta aptera is a wingless subantarctic dipteran. Its egg is oblong with a flat upper wall separated from a convex lower wall by a ridge of respiratory horns. Just after the egg is laid, it possesses two distinct envelopes: a porous chorion and a dense vitelline membrane. The chorion is covered with a thin mucous layer. Its structure shows a complex system of cavities and a network of trabecular material. It consists of three layers: an outer, a middle, and an inner one. The chorion forms a respiratory plastron all over the egg surface. Water loss was recorded and analyzed in fertilized eggs with their two envelopes and in eggs from which the chorion had been removed. Transpiration measurements were performed in dried air (0–5% relative humidity) at temperatures rising from 5 to 50 °C at a regular rate of 0.5 °C/min. It appears that the vitelline envelope greatly reduces water loss within a fully desiccated atmosphere, at low to moderate air temperatures, but the egg is poorly adapted to dry and hot air conditions.

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.

Changes in the protein components of vitelline envelope during oogenesis of a tubiculous polychaete, Schizobranchia insignis

In sabellid polychaetes the vitelline envelopes, in which microvilli with glycocalyx structures at the tips are invested, change in structure during oogenesis. Vitelline envelopes isolated from Schizobranchia oocytes 25–100 μ m and 160–185 μ m in diameter, were analyzed in protein components by iodination, electrophoresis, Western blotting and radioactive labeling technique. The observations demonstrate that the membrane proteins of the vitelline envelopes are not consistent but variable in components during oogenesis. Most of these proteins, particularly the high molecular weight proteins, are PAS-positive glycoproteins, which may have specific carbohydrate residues binding to wheat germ agglutinin. The proteins could be labeled with [3H]valine within 36 h by incubating the whole oocytes in sea water to a high level, indicating that the proteins are actively synthesized by the growing oocytes. Synthetic rates of the proteins differ from each other at one stage and are higher in the small than in the large oocytes in general, suggesting that the membrane proteins are involved in the function of the vitelline envelopes during oogenesis.

Stages of oocyte development in the pipefish, Syngnathus scovelli.

Oocyte development occurs in a temporal and spatial pattern in the ovary of the pipefish, Syngnathus scovelli. Cytological observations combined with [3 H]thymidine uptake and cell culture were used as criteria to identify six stages of oocyte development in this species. Stage I-Oogonia, found within the germinal ridge. Stage II-Oocytes in primary growth, subdivided into (1) chromatin-nucleolus oocytes in early prophase I of meiosis, also found within the germinal ridge, and (2) perinucleolar oocytes, which possess multiple nucleoli and are within definitive follicles (the Balbiani vitelline body is the main cytoplasmic component in these latter oocytes). Stage III-Oocytes distinguished by the appearance of cortical alveoli, the vitelline envelope, and lipid. Stage IV-Vitellogenic oocytes, which accumulate yolk proteins and possess primary, transitional, and mature yolk spheres. Stage V-Maturational oocytes, which are competent to undergo final meiotic maturation in vivo and in response to the steroid 17 α-hydroxy-20β-dihydroprogesterone in vitro. Stage VI-Ovulated mature eggs, which are present in the ovary lumen and are capable of being fertilized. These studies provide a staging series for oogenesis and oocyte development in the pipefish and relate these stages to the unusual ovary in this animal.

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.

KDN-glycoprotein: A novel deaminated neuraminic acid-rich glycoprotein isolated from vitelline envelope of rainbow trout eggs

A new acidic glycoprotein containing deaminated neuraminic acid (KDN = 3-deoxy-D-glycero-D-galacto-nonulosonic acid; greater than 50%, w/w) was isolated from vitelline envelope of the unfertilized eggs of rainbow trout (Salmo gairdneri). This glycoprotein is designated as "KDN-glycoprotein" because it contains only KDN but no sialic acid as the acidic carbohydrate moieties. Other major carbohydrate components of KDN-glycoprotein were Gal and GalNAc. Thr and Ala accounted for 71% (mol/mol) of amino acid composition. A possible occurrence of KDN-KDN linkages, i.e. oligoKDN groups has been suggested in the carbohydrate chains presumably linked O-glycosidically to the core protein.

Formula omitted] pump activity in the new membrane formed at first cleavage in Cynops pyrrhogaster eggs

Resting membrane potentials (Em) increased in the negative direction during first cleavage in Cynops pyrrhogaster eggs whose new membranes formed at first cleavage were exposed to bathing solutions by removing the vitelline envelopes. Em was −11.4 and −87.2 mV at the one- and two-cell stages, respectively. Na K pump activity contributed to Em at the two-cell stage by about −30 mV. The distribution of Na K ATPase activity was cytochemically studied by Ernst's method (S. A. Ernst, 1972, J. Histochem. Cytochem. 20, 23–38) . The new membrane of the eggs at the two-cell stage showed the pump activity. But the activity was detected neither in the preexisting outer membrane of the eggs at the two-cell stage nor in the membrane at the one-cell stage.

Egg‐shells in mites. Vitelline envelope and chorion in a water mite, Limnochares aquatica L. (Acari, Limnocharidae)

Results of ultrastructural investigations (SEM and TEM) of the formation and structure of eggshells in the water mite, Limnochares aquatica, are presented. The material of the vitelline envelope is secreted in two stages by the oocyte commencing vitellogenesis. The vitelline envelope is later transformed into endochorion and during the passage through the reproductive tract it becomes covered with exochorion, produced by the female reproductive accessory gland. The exochorion swells in water to acquire a foamy structure.

Chorionic peroxidase activity in the eggs of the fish Tribolodon hakonensis

AbstractPeroxidase activity was ultracytochemically demonstrated in the vitelline and fertilization envelopes of eggs of the fish Tribolodon hakonensis. The vitelline envelope (VE) was morphologically divided into five layers, from the first to the fifth, numbered in order from the outside of the VE toward the interior, but the micropylar and surrounding region lacked the first layer. Peroxidase activity was localized in the first and second layers, but was much stronger in the latter than in the former. Phosphotungstic acid (PTA) staining was positive in the first layer alone. During the elevation of the fertilization envelope (FE) the peroxidase activity was translocated into the first layer of the FE from the second layer. This translocation resulted in much weaker activity or negativity in the FE second layer. In the micropylar and surrounding region, however, peroxidase activity remained unchanged, and there was no translocation. Transformation of the VE into the FE led to an alteration in the thickness of each layer and in the cytochemical nature of the first layer. There was almost no PTA staining in the FE first layer, except for a few scattered positive spots. A purified and lyophilized VEs‐ or FEs‐NaI‐H2O2 system exerted a cell‐killing effect on three bacterial species tested.

Immunocytochemical localization of the 35-kDa sea urchin egg trypsin-like protease and its effects upon the egg surface

Trypsin-like protease in sea urchin eggs is thought to reside in cortical granules since it is secreted at fertilization and has been isolated with cortical granule fractions from unfertilized eggs. A 35-kDa serine protease has been purified from Strongylocentrotus purpuratus eggs by soybean trypsin inhibitor-affinity chromatography. For this report the protease was localized by immunocytochemistry before and after fertilization, and its potential biological activity was examined by application of the isolated enzyme to the unfertilized egg surface. The protease was localized on sections by immunofluorescence and immunoelectron microscopy, and was found to reside in the spiral lamellae of S. purpuratus cortical granules and in the electron-dense stellate core of Arbacia punctulata granules. At fertilization the enzyme is secreted into the perivitelline space and accumulates only very briefly between the hyaline layer and the nascent fertilization envelope. Shortly thereafter the enzyme is lost from the perivitelline space and immunological reactivity is no longer associated with the egg surface. The 35-kDa cortical granule protease has vitelline delaminase activity but does not appear to destroy vitelline envelope sperm receptors as judged by the fertility of protease-treated eggs.

New evidence of anuran oviducal pars recta involvement on gamete interaction

AbstractCoelomic oocytes of the toad Bufo arenarum are not fertilizable when inseminated in egg water that assures fertilization of dejellied uterine eggs. However, when coelomic oocytes were pretreated with an extract from oviducal pars recta (PRE) and inseminated in egg water, a high frequency of fertilization was obtained.In this study the effect of PRE on acrosome breakdown (AB) was studied by means of vitelline envelope (VE) lysis and fertility tests with B. arenarum sperm, and by direct observations with the light microscope of the AB with Leptodactylus chaquensis sperm. The coelomic envelope becomes sensitive to lysin solution only after PRE treatment. The addition of sperm to PR‐treated oocytes, in a medium containing egg water, resulted in a marked swelling and softening of the VE. But this lytic effect by sperm can be observed only when egg water was present in the medium. These results indicate that jelly‐molecules are necessary for the acrosome process to begin and that PR components engulfed in the VE are not capable of inducing AB by themselves. Likewise, incubation of B. arenarum sperm with PRE did not impair their fertilizing capacity and the supernatant of this incubation medium did not have typical VE lytic activity.Moreover, when the effect of PRE on AB was studied by direct observation of L. chaquensis sperm, it was found that PRE neither induces AB nor modifies its time‐course.

Egg-Shells in mites: Cytological aspects of vitelline envelope and chorion formation inPergamasus barbarusberlese (Gamasida, Pergamasidae)

Abstract Transformations leading to the appearance of the perivitelline space and vitelline envelope, the structure of the vitelline envelope, and the structure and origin of the chorion in Pergamasus barbarus were the subjects of ultrastructural studies. The follicular cells participate in the perivitelline space formation. The material of the vitelline envelope is secreted by exocytosis from the oocyte at the beginning of vitellogenesis. Later, the vitelline envelope is transformed into endochorion protecting the embryo from mechanical damage. Exochorion is secreted onto the endochorion during egg deposition. Double role of exochorion as plastron and as a structure protecting the embryo from dessication is suggested.

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.

Lectin binding sites in the vitelline envelope of Bufo arenarum oocytes: Role in fertilization

Incubation of dejellied spawned oocytes from Bufo arenarum with different lectins results in a decrease of oocyte fertility. Concanavalin A was the most effective lectin; phytohemagglutinin P and wheat germ lectin were less effective. Agglutinin from soybean was scarcely active. These lectin effects could be ascribed to a hindering of specific sites for some proteases, since the same treatment renders the oocyte vitelline envelope insensitive to spermatolysin (an essential requisite for fertilization) and to trypsin. Also in this case concanavalin A was the most effective lectin. Univalent concanavalin A was also effective in blocking the fertility of dejellied oocytes. These results indicate that the residues of alpha-D-glucose and alpha-D-mannose present in the vitelline envelope are involved in gamete interactions in Bufo arenarum. This idea is also supported by the finding that dejellied oocytes (fertilizables) have a number of binding sites for concanavalin A that is three or four orders of magnitude higher than coelomic or fertilized oocytes (both not penetrable by spermatozoa).

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.

Cortical granules products and fertility prevention in Bufo arenarum oocytes

AbstractA description of some properties of the cortical granule products (CGP) is provided. After activation, cortical granules (CG) discharge their content to the perivitelline space where most of these products remain. Two types of CGP preparations were used: one obtained by activating oocytes deprived of their vitelline envelope (tCGP), and the other obtained by activating oocytes covered by the vitelline envelope (vCGP). Both types of CGP were similar as regarding their capacity to modify oocyte sensitivity to spermatolysin. CGP modify the vitelline envelope (VE) of unactivated dejellied oocytes, which becomes insensitive to spermatolysin. This activity depends on both CGP concentration in the incubation media and the length of the incubation period. Once the effect of CGP on the oocyte is established it can not be completely eliminated by extensive washings, suggesting a lectin‐type association with the componenets of the VE. Treatment of dejellied oocytes with CGP results in fertility loss. The biological activity of CGP is related to thermolabile and nondialyzable substances. Lytic activity of spermatolysin on isolated VE is inhibited by CGP at a concentration at which trypsin inhibitors from soybean, lima bean, and ovomucoid are ineffective. The electrophoretic pattern of CGP indicates that most of the proteins present in the vCGP are derived from the VE. Ultrastructural modifications are observed in dejellied deposed oocytes after incubation with CGP.

Eggshell fine structure of three lepidopteran pests: Cydia pomonella (L.) (Tortricidae), Heliothis virescens (Fabr.), and Spodoptera littoralis (Boisd.) (Noctuidae)

The eggshells of 3 moths, Cydia pomonella (Tortricidae), Heliothis virescens, and Spodoptera littoralis (Noctuidae) were investigated by scanning (SEM) and transmission (TEM) electron microscopy. The surface of the noctuid eggs shows structural elements (micropylar rosette, ribs, cross-ribs, and aeropyles) and regional differentiation, all typical of Lepidoptera. The egg of C. pomonella shows a different regional morphology due to its watch-glass shape and its position, lying on the flank. The micropylar structures are on the lower egg face in contact with the substrate. For S. littoralis, the surface structure (sculpturing) of the egg is not species-specific, being indistinguishable from that of S. frugiperda (Salkeld, 1984). In all 3 moths, the eggshell fine structure is basically identical, as revealed by TEM. Both the vitelline envelope and the chorion consist of several distinct layers. The vitelline envelope, bi-layered and several μm thick, undergoes a marked structural change when embryogenesis begins. At the same time, Golgi vesicles bearing dense particles, appear in the periplasm of the egg cell in fertilized eggs of H. virescens and S. littoralis. The chorion of all 3 species consists of a basal layer (C-1), a cavity layer (C-2) supported by trabecles and opening to the exterior via aeropylar canals, and a lamellar layer (C-3), which probably consists of helicoidally arranged stacks of fibrils. In H. virescens and S. littoralis, an additional epicuticle-like layer (C-4) is present. Available data from the literature are summarized and a basic scheme of the radial eggshell fine structure of ditrysian Lepidoptera is proposed.

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

AbstractDirect 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.

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

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.