The Xenopus laevis cortical granule lectin: cDNA cloning, developmental expression, and identification of the eglectin family of lectins

A Xenopus laevis egg cortical granule, calcium-dependent, galactosyl-specific lectin participates in forming the fertilization layer of the egg envelope and functions in establishing a block to polyspermy. We report the cDNA cloning of the lectin, expression of the cortical granule lectin gene during oogenesis and early development, and identification of a new family of lectins. The translated cDNA for the cortical granule lectin had a signal peptide, a structural sequence of 298 amino acids, a molecular weight of 32.7 K, contained consensus sequence sites for N-glycosylation and a fibrinogen domain. The lectin cDNA was expressed during early stages of oogenesis. Lectin glycoprotein levels were constant during development with 2/3 of the lectin associated with the extracellular perivitelline space and the egg/embryo fertilization envelope. Lectin mRNA levels were from 100- to 1000-fold greater in ovary than in other adult tissues. The lectin had no sequence homology to the previously identified lectin families. The lectin had 41-88% amino acid identity with nine translated cDNA sequences from an ascidian, lamprey, frog, mouse, and human. Based on the conserved carbohydrate binding and structural properties of these glycoproteins, we propose a new family of lectins, the eglectin family.     

FORMATION OF THE CORTICAL CONCAVITY AT FERTILIZATION IN THE SEA URCHIN EGG

During the initial stages of fertilization envelope elevation in eggs of Strongylocentrotus pur puratus and S. droebachiensis a large concavity of the egg cortex was observed in the light microscope. This concavity corresponded in shape and size with the elevating fertilization envelope. However, after the vitelline layers of eggs were disrupted and the eggs inseminated, the concavity failed to develop although the eggs were fertilized and developed normally. We propose that the concavity is formed owing to increased hydrostatic pressure within the perivitelline space. To further support this hypothesis we measured total egg protein secreted during fertilization, and found that 98% was retained within the perivitelline space. Furthermore, 80% of the total protein was contributed by the hyaline layer. Presumably, colloidal osmotic pressure and/or hydration of fertilization product, trapped beneath the fertilization envelope, is responsible for increased hydrostatic pressure within the perivitelline space, and therefore promotes not only fertilization envelope elevation, but the cortical concavity as well.     

Immunoelectophoretic Identification of Jelly Coat Ligands Bound by the Cortical Granule Lectin from Xenopus laevis Eggs

The formation of the fertilization layer in the Xenopus laevis egg fertilization envelope involves a lectin-ligand interaction and establishes a block to polyspermy in the extracellular matrix of the egg. The cortical granule lectin participating in the formation of the fertilization layer has been isolated but its ligand has not. We identified three jelly coat ligands bound by the cortical granule lectin using immunoelectrophoretic analyses. Two antigens were detected with anti-jelly serum and a third was identified using anti-envelope serum. All three antigenic ligands were associated with the innermost jelly coat layer, J₁, and two of the three antigenic ligands contained sulfate. One or more of these jelly coat ligands may function in establishing a block to polyspermy at fertilization in Xenopus laevis .     

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.     

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.     

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.     

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.     

Localization of cortical granule lectin ligand in Xenopus laevis egg jelly

The block to polyspermy in Xenopus laevis involves an interaction between a cortical granule lectin, released at fertilization, and a ligand located in the egg extracellular matrix. The egg extracellular matrix in X. laevis consists of a vitelline envelope and three distinct jelly layers, designated J1, J2 and J3. To localize cortical granule lectin ligand in the egg extracellular matrix, we used enzyme-linked lectin assays that showed that cortical granule lectin ligands were absent in J2, J3 and the vitelline envelope. Cortical granule lectin bound to a ligand(s) in J1 in a galactose-dependent fashion. In addition, we separated egg jelly macromolecules electrophoretically and, in conjunction with western blotting, have shown that J1 contains two major, high molecular weight ligands for cortical granule ligand. Finally, using confocal microscopy, we demonstrated that the ligand(s) for cortical granule lectin occupies a 20–30 μm thick band in a region of J1 just proximal to the vitelline envelope.     

A cortical granule-specific enzyme,B-1,3-glucanase,in sea urchin eggs

The ultrastructural localization of B-1,3-glucanase in three species of sea urchin eggs was determined using a monospecific antibody in an electronmicroscopic immunogold procedure. In all three species, Lytechinus variegatus, Strongylocentrotus purpuratus, and Arbacia punctulata, B-1,3-glucanase was localized specifically to the cortical granules. No other organelle within the egg contained significant label. During the fertilization reaction, B-1,3-glucanase was released from cortical granules into the perivitelline space and became associated with the hyaline layer. No significant label was found in association with the fertilization envelope.     

Breakdown of the cortical alveoli of medaka eggs at the time of fertilization,with a particular reference to the possible role of spherical bodies in the alveoli

Summary The process of cortical change upon fertilization of eggs of the teleostean fish,Oryzias latipes was investigated. A cortical alveolus (CA) contains colloidal material, a spherical body, and often a membranous structure. Upon insemination, breakdown of the cortical alveoli and elevation of the chorion began around the animal pole and ended at the vegetal pole. It was found that the spherical body was extruded with the colloidal material from the CA: the spherical body swelled after the opening of an aperture and was extruded into the perivitelline space through a large aperture. The empty CA shrank and disappeared completely as a result of the transformation of its envelope to numerous microvilli. The spherical body isolated or in the perivitelline space could be digested quickly by proteolytic enzymes. When spherical bodies in the perivitelline space of a fertilized egg were digested enzymatically, the vitellus came into direct contact with the chorion. The present study seems to show that swollen spherical bodies derived from CA play a role in maintaining a certain distance between the chorion and the vitellus after fertilization.     

Fibroin-like substance is a major component of the outer layer of fertilization envelope via which carp egg adheres to the substratum

Seven cDNA encoding silkworm fibroin homologues were cloned from a carp ovarian cDNA library. The encoded proteins are denoted as carp ovarian fibroin-like substances (FLS). FLS contain a repetitive domain consisting of tandem repeats of dipeptide of Gly-X, where X may be any amino acid. Each FLS has its own unique repeating sequence, such as GQGAGQGS, GQGMGQGM, GRGQGEGHGS, and GFGFGQGS, indicating a family of FLS genes exists in carp. FLS is exclusively expressed in oocytes and is stored in cortical granules. During cortical reaction, FLS is exocytosed to perivitelline space and then gradually added to the outer layer of the fertilization envelope (FEo). The FLS of fertilization envelope is conjugated with cystatin and cathepsin-like substance (CLS) and appears in multiple bands of molecular weights ranging from 40 to 205 kDa. After fertilization or artificial activation, carp eggs adhere firmly to the substratum via FEo. FLS is a major component of FEo. The presence of transglutaminase inhibitor, cadaverine or ethylene diaminetetraacetic acid, in the cortical reaction medium can impair or block the recruitment of FLS and other substances to FEo. As a consequence, FEo is not formed or is greatly reduced, resulting in a great reduction of egg adhesion.     

Immunoelectron Microscopic Demonstration of Cortical Granule Lectins in Coelomic, Unfertilized and Fertilized Eggs of Xenopus laevis

Immunoelectron microscopic studies demonstrated cortical granule lectins (CGLs) in coelomic, unfertilized and fertilized eggs of Xenopus laevis . An antiserum raised against purified cortical granule lectin 1 specifically reacted with the CGLs in immunoblotting and agar diffusion tests. When ultrathin sections were treated with the antiserum and protein A-gold solution, gold particles, indicating antigenic sites, were seen over cortical granules of coelomic and unfertilized eggs, and over the perivitelline space, the vitelline coat and the condensed region of the fertilization layer of fertilized eggs. The pre-fertilization layer immediately adjacent to the outer margin of the vitelline coat in unfertilized eggs was free from gold particles. These observations suggest that released CGLs permeate through the vitelline coat of fertilized eggs and interact with the pre-fertilization layer mainly at the outer margin of the vitelline coat, resulting in formation of the fertilization layer which acts as a block to polyspermy.     

Electron Microscopic Study of Development in a Sea Cucumber,Stichopus tremulus (Holothuroidea), from Unfertilized Egg through Hatched Blastula

In this, the first fine structural study of sea cucumber embryology, eggs and embryos of Stichopus tremulus developing at 7.5°C are described from spawning through hatched blastulae. Spawned eggs are at about first meiotic metaphase and are surrounded by a jelly layer that remains around the embryos until hatching. No vitelline coat can be demonstrated, but whether it is truly absent or removed by electron microscopic processing is not known. Insemination initiates a rapid cortical reaction, completed within 2 min., which involves a wave of cortical granule exocytosis and fertilization envelope formation. The compactly fibrous fertilization envelope is about 50 nm thick and appears to consist entirely of ejected cortical granule material (if one assumes that there is no vitelline coat). As the fertilization envelope elevates, no hyaline layer appears in the perivitelline space. The first and second polar bodies are emitted, respectively, at about 9 and 15 min. after insemination. The first seven or so cleavages are equal, radial, and occur approximately every 4 hr. The blastocoel opens up at the four-cell stage and, during the earlier cleavages, remains connected with the perivitelline space via numerous gaps between the roughly spherical blastomeres. At the 64-cell stage, these gaps begin to close as the blastomeres start to become cuboidal; in addition, an embryonic cuticle is produced on the apical surface of each blastomere. In embryos of several hundred cells, the blastomeres become associated apicolaterally by junctional complexes, each consisting of a zonula adherens and a septate junction. Several hours before hatching, a single cilium is produced at the apical surface of most blastomeres. At hatching (about 50 hr after insemination), the ciliated blastula leaves behind the fertilization envelope and jelly layer. Swimming blastulae soon begin to elongate in the animal-vegetal axis, and a basal lamina develops on blastomere surfaces facing the blastocoel. The discussion includes a fine structural comparison of egg coats among the five classes of the phylum Echinodermata.     

Fertilization in Oikopleura dioica (Tunicata,Appendicularia): Acrosome reaction,cortical reaction and sperm-egg fusion

Summary Fine structural changes in the egg and sperm are described during gamete interaction in Oikopleura dioica, an appendicularian tunicate. The unfertilized egg has a vitelline layer 80 nm thick and a perivitelline space about 5 m wide. In the peripheral cytoplasm are a few cortical granules 0.6×0.7 m in diameter and areas rich in parallel cisternae of rough endoplasmic reticulum alternating with areas rich in long mitochondria. In the deeper cytoplasm the predominant organelles are multivesicular bodies. From 25 s to 60 s after insemination, the egg transiently elongates, although with no obvious cytoplasmic rearrangement, and the egg surface becomes bumpy. During this interval sperm enter the egg, and the cortical granules undergo exocytosis. After expulsion into the perivitelline space, the cortical granule contents do not appear to change their shape or blend with the vitelline layer, which neither elevates further nor loses its ability to bind sperm. On encountering the egg, the sperm undergoes an acrosome reaction involving exocytosis of the acrosome and production of an acrosomal tubule. The acrosomal contents bind the sperm to the vitelline layer, and the posterior portion of the acrosomal membrane and the anterior portion of the nuclear envelope evaginate together to form an acrosomal tubule, which fuses with the egg plasma membrane to form a fertilization cone. By 45 s after insemination, the sperm nucleus, centriole, mitochondrion and at least the anterior portion of the axoneme are within the fertilization cone. By 60 s sperm entry is complete. In having eggs with a cortical reaction and sperm with an acrosome reaction, O. dioica resembles echinoderms and enteropneusts and differs markedly from ascidian tunicates, which lack both these features. The relatively unmodified pattern of gamete interaction in O. dioica in comparison with the highly modified pattern in ascidians is difficult to reconcile with the neoteny theory that appendicularians have evolved via ascidian ancestors. The present results are more consistent with the idea that an appendicularian-like ancestor gave rise to ascidians.     

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.     

Egg cortical granule N-acetylglucosaminidase is required for the mouse zona block to polyspermy

The mammalian egg must be fertilized by only one sperm to prevent polyploidy. In most mammals studied to date, the primary block to polyspermy occurs at the zona pellucida, the mammalian egg coat, after exocytosis of the contents of the cortical granules into the perivitelline space. The exudate acts on the zona, causing it to lose its ability to bind sperm and to be penetrated by sperm previously bound to the zona. However, the cortical granule components responsible for the zona block have not been identified. Studies described herein demonstrate that N-acetylglucosaminidase is localized in cortical granules and is responsible for the loss in sperm-binding activity leading to the zona block to polyspermy. Before fertilization, sperm initially bind to the zona by an interaction between sperm surface GalTase and terminal N-acetylglucosamine residues on specific oligosaccharides of the zona glycoprotein ZP3 (Miller, D. J., M. B. Macek, and B. D. Shur. 1992. Nature (Lond.). 357:589-593). These GalTase-binding sites are lost from ZP3 after fertilization, an effect that can be duplicated by N-acetylglucosaminidase treatment. Therefore, N-acetylglucosaminidase, or a related glycosidase, may be present in cortical granules and be responsible for ZP3's loss of sperm-binding activity at fertilization. Of eight glycosidases assayed in exudates of ionophore-activated eggs, N-acetylglucosaminidase was 10-fold higher than any other activity. The enzyme was localized to cortical granules using immunoelectron microscopy. Approximately 70 or 90% of the enzyme was released from cortical granules after ionophore activation or in vivo fertilization, respectively. The isoform of N- acetylglucosaminidase found in cortical granules was identified as beta- hexosaminidase B, the beta, beta homodimer. Inhibition of N- acetylglucosaminidase released from activated eggs, with either competitive inhibitors or with specific antibodies, resulted in polyspermic binding to the zona pellucida. Another glycosidase inhibitor or nonimmune antibodies had no effect on sperm binding to activated eggs. Therefore, egg cortical granule N-acetylglucosaminidase is released at fertilization, where it inactivates the sperm GalTase- binding site, accounting for the block in sperm binding to the zona pellucida.     

A complex cortical reaction leads to formation of the fertilization envelope in the lobster,Homarus

We have examined the formation of the fertilization envelope in the lobsters Homarus americanus and H gammarus. Oocytes were fixed for electron microscopy either in the ovary or following extrusion from the gonopore. Mature ovarian oocytes are surrounded by a coat (envelope 1), which is comprised of small electron-dense granules and structures resembling “bottlebrushes.” At least part of this coat is synthesized by the follicle cells of the ovary. The cortex of ovarian oocytes contains four types of vesicles that we refer to as high-density vesicles (HDV), low-density vesicles (LDV), moderately dense vesicles (MDV), and ring vesicles (RV). Oocytes that were electrically extruded from the gonopore and fixed immediately had an envelope identical to that of ovarian oocytes. The cortex of gonopore oocytes contained the four types of vesicles found in ovarian oocytes. When unfertilized gonopore oocytes were allowed to incubate in sea water, the oocyte cortex appeared unaltered, but envelope 1 swelled and the bottlebrushes dispersed. When recently fertilized oocytes were fixed during natural spawning or following in-vitro fertilization, each type of vesicle was released in sequence from the cortex of the oocyte. The contents of the HDV and LDV appeared first in the perivitelline space, but their fate could not be determined at later times. The ring-shaped elements of the RV and the moderately electron-dense material of the MDV were released exocytotically somewhat later; these materials coalesced in the perivitelline space to form a new coat (envelope 2). Envelope 1 subsequently condensed to its original thickness and appeared firmly attached to envelope 2. Our results show that the fertilized lobster egg is surrounded by two discrete coats. The outer coat, which is formed in the ovary, undergoes a swelling/condensation cycle at spawning. The inner coat originates from a complex cortical reaction. Together these coats comprise the fertilization envelope of the lobster egg.     

Fate of the sea urchin egg receptor for sperm following fertilization

The sea urchin egg receptor for sperm is a 350 kDa glycoprotein containing a large extracellular domain that contains the sperm binding site, a transmembrane domain and a short COOH- terminal intracellular domain. During oogenesis, the receptor protein is first detected in Golgi-associated vesicles and cortical granules. Not until the egg is mature does the receptor appear on the cell surface; at this stage the intact receptor is found in approximately equal quantities on the egg cell surface and in cortical granules. As a potentially unique type of receptor, we were interested in its fate following fertilization. Several techniques have revealed that, following sperm binding, the amount of receptor markedly decreases. Using western blot analysis as well as direct measurement of the receptor protein, it was found that the membrane-bound form of the receptor rapidly disappeared following sperm binding to the egg, with only 3% of the receptor remaining after 30 s. Analysis by immupoelectron microscopy revealed that 30 s after sperm binding, 30% of the initial level of receptor was present. This remaining 30% was found mostly within the perivitelline space formed by the raised fertilization envelope. The disparity between these two sets of results (i.e. 3 vs 30%) is most likely accounted for by the exocytosis of receptor molecules from cortical granules; this fraction of the receptor would have been lost during isolation of the membrane-bound form of the receptor. Thus, unlike other cell surface receptors, the sea urchin egg receptor for sperm is not endocytosed and recycled following ligand binding. Rather, it disappears, presumably as a result of proteolysis. Transiently, the cortical granule form of the receptor is found released into the perivitelline space where it may bind to sperm and thereby prevent polyspermy. Despite the apparent secretion of this form of the receptor, experiments with antibodies to the extracellular and intracellular domains indicate that the receptors in cortical granules and in the plasmic membrane are similar, if not identical.     

Purification and characterization of an N-acetyl-beta-D-glucosaminidase from cortical granules of Xenopus laevis eggs

The enzyme N-acetyl-beta-D-glucosaminidase was purified from the cortical granules of Xenopus laevis eggs using affinity chromatography, gel filtration, and density gradient centrifugation. The enzyme had a molecular weight of 37,000-40,000 as determined by polyacrylamide gel electrophoresis and density gradient centrifugation, had a Km for p-nitrophenyl-beta-D-N-acetyl-glucosaminide of 0.66 mM and a Ki for glucosamine of 4.3 mM. The kinetic properties of the cortical granule enzyme were similar to the enzyme isolated from jack bean. Treatment of unfertilized eggs with the enzyme isolated from cortical granules or jack bean rendered eggs unfertilizable. Loss of fertilizability was proportional to the product of time and enzyme concentration, consistent with an enzymatic mechanism being responsible for the loss of fertilizability. The amount of enzyme present in the perivitelline space was approximately the same as that which reduced fertilizability by 50% in one hour. We suggest that the action of cortical granule N-acetyl-beta-D-glucosaminidase on egg integuments may function as a block to polyspermy at fertilization.     

Starfish sperm-oocyte jelly binding triggers functional changes in cortical granules

Summary Starfish oocytes were examined before fertilization, immediately after insemination, and during the cortical reaction by means of acid phosphatase and ruthenium red ultrastructural histochemistry. Oocyte cortical granules are composed of a lamellar body and a surrounding matrix which is subdivided into dense and light portions. In unfertilized oocytes cortical granules are not stained by ruthenium red but show a weak acid phosphatase activity in the light portion of the granule matrix. Immediately after the adhesion of the spermatozoon to the oocyte jelly coat, the light matrix portion of cortical granules appears stained by ruthenium red and shows a strong acid phosphatase activity. During the cortical reaction, cortical granules are released into the perivitelline space and the lamellar body, surrounded by the stained matrix, fuses with the fertilization envelope. Our data suggest that membrane permeability changes and enzyme activation occur in the egg when the spermatozoon binds to the oocyte jelly coat.