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.     

Starfish sperm-oocyte jelly binding triggers functional changes in cortical granules. A study using acid phosphatase and ruthenium red ultrastructural histochemistry

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.     

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.     

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.     

Properties of the Cortical Granule Lectin Isolated from Xenopus Eggs

The cortical granule lectin that participates in forming the fertilization layer in Xenopus laevis was isolated and partially characterized. About 400 μg of lectin was purified from 5 mg of crude exudate by chromatography on Sepharose 6B and Concanavalin A-conjugated Sepharose 4B columns and electrophoretic separation on polyacrylamide gel. The lectin has a molecular weight of 550 Kd and is composed of two species of polypeptides (46 Kd and 42 Kd). The lectin gave a single precipitin line against material in the prefertilization layer in an agglutination reaction on an agarose plate. The agglutination reaction involved D-galactoside residues and metal ions. The lectin formed an electron-dense layer on the outer surface of the vitelline coat of oviducal eggs covered with the prefertilization layer, but on the outer surface of jelly layer, not on that of the vitelline coat of jellied eggs. Although the jelly could be agglutinated by the lectin, the possibility that the jelly layer is the site of fertilization layer formation was excluded by the fact that the prefertilization layer is the first to meet the cortical granule lectin during normal fertilization.     

Relationship between p62 and p56, two proteins of the mammalian cortical granule envelope, and hyalin, the major component of the echinoderm hyaline layer, in hamsters

Mammalian cortical granules contain two polypeptides (p62 and p56) that are incorporated into the cortical granule envelope after fertilization and function in cleavage of the zygote and the preimplantation blastomeres. Since the echinoderm hyaline layer and mammalian cortical granule envelope are analogous, and since the hyaline layer protein, hyalin, functions in early echinoderm embryogenesis, this study was done to determine whether p62 and p56 and/or other components of the mammalian cortical granule envelope are related to hyalin. A polyclonal antibody (IL2) against purified S. purpuratus hyalin was shown by confocal scanning laser microscopy to bind to hamster cortical granules and to the cortical granule envelope of fertilized hamster oocytes and preimplantation embryos up to the blastocyst stage. In immunoblots, IL2 bound only to 62- and 56-kDa cortical granule proteins that were incorporated into the cortical granule envelope after fertilization. IL2 binding antigens appeared to be resynthesized by preimplantation embryos starting at the 2-cell stage of development. In vivo treatment of 2-cell-stage hamster embryos with IL2 inhibited blastomere cleavage, but treatment of morulae did not inhibit blastocyst implantation. These results support the idea that the mammalian cortical granule envelope proteins, p62/p56, share a common antigenic epitope(s) with echinoderm hyalin, and that p62/p56, like hyalin, play a role in early embryogenesis.     

p62/p56 are cortical granule proteins that contribute to formation of the cortical granule envelope and play a role in mammalian preimplantation development

The purpose of this study was to identify specific cortical granule protein(s) that form the cortical granule envelope and examine their role(s) in fertilization and preimplantation development. The polyclonal antibody A-BL2 was used to show that the cortical granules of mice, rats, hamsters, cows, and pigs contain a pair of proteins designated p62/p56. These proteins are released from hamster cortical granules at fertilization and contribute to formation of the cortical granule envelope, an extracellular matrix present in the perivitelline space of fertilized mammalian oocytes. P62/p56 were present in the cortical granule envelope throughout preimplantation development and were found in blastomere cortices of 4-cell to blastocyst stage embryos. Hamster oocytes fertilized in vivo in the presence of A-BL2 were all monospermic, suggesting that p62/p56 do not function in blocking polyspermy. Likewise treatment of morula to blastocyst stage hamster embryos with A-BL2 had no effect on the implantation of blastocysts. However, cleavage divisions were inhibited in vivo in a dose-dependent manner when fertilized oocytes or 2-cell embryos were treated with A-BL2. Inhibition of cell division was more pronounced in 2-cell embryos than in fertilized oocytes. This study identifies p62/p56 as cortical granule proteins that contribute to the formation of the cortical granule envelope and further supports the idea that after their release at fertilization, p62/p56 function in regulating preimplantation development at the level of oocyte and blastomere cleavage.     

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 .     

An immunocytochemical localization of the cortical granule lectin in fertilized and unfertilized eggs of xenopus laevis

At fertilization, the vitelline envelope surrounding the egg of Xenopus laevis is modified by the addition of an electron-dense component termed the “F layer.” The F layer functions as a block to polyspermy and as a block to the escape of macromolecules from the perivitelline space, thereby causing an osmotically driven envelope elevation. F-layer formation has been hypothesized to result from interaction between a cortical-granule lectin, released in the cortical reaction, and a jelly-coat ligand. Evidence for this hypothesis was sought by determining the location of the cortical-granule lectin both before and after fertilization, using a specific antibody conjugated to horseradish peroxidase. The cortical-granule lectin was localized only in the cortical granules of the unfertilized egg and was located predominantly in the perivitelline space and the F layer of a fertilized egg. These observations support the hypothesis that the F layer is formed by a cortical-granule-Iectin–jelly layer-ligand interaction.     

Ultrastructural aspects of the surface coatings of eggs and larvae of the starfish,Pisaster ochraceus,revealed by alcian blue

Eggs of the asteroid Pisaster ochraceus demonstrate cortical granules, a thick vitelline membrane, and a poorly stained jelly coat similar to that seen on the eggs of other echinoderms. When fixed in the presence of alcian blue the jelly coat is seen to be made up of three regions, an inner layer consisting of a meshwork of fibres, a middle layer of thicker fibres, and a dense outer layer. At fertilization the cortical granules release their contents into the potential space between the vitelline layers and a low fertilization membrane consisting of the vitelline layer and a dense component of the corticle granule is formed. Initially the remaining contents of the corticle granules form an amorphous hyaline layer that fills the space between the plasma membrane and the fertilization membrane. At hatching a distinct hyaline layer is present. It persists at least to the bipinnaria stage and consists of four distinct layers. A similar layer is also located over much of the early embryonic endoderm but is lost from the regions involved in the formation of the mesenchyme cells, coelom, and mouth just before these events take place. Numerous large clear vesicles are located in the apex of all cells associated with a hyaline layer. Where the hyaline layer is lacking, only scattered vesicles are present suggesting that the vesicles may be involved in maintenance of the layer. Attempts to identify elements of the hyaline layer by immunofluorescence demonstrated that it appears to bind both antisera and control sera in a nonspecific manner.     

Quantitative measurement of active polysomes of developing chick muscle

The hatching process in embryos of the toad Xenopus laevis consists of two temporally distinct phases. In phase 1, the embryo escapes sequentially from the two outermost jelly layers, J₃ and J₂, and during phase 2 the embryo hatches from the last remaining jelly coat layer J₁ and the fertilization envelope. Phase 1 hatching appears to be a physical process caused by water inbibition of jelly coat layer J₁ and dynamic changes in the volume enclosed by the fertilization envelope. The combined turgor pressure ruptures jelly coat layers J₃ and J₂. The subsequent phase 2 hatching is a result of both physical and chemical processes. Phase 1 hatching exposes layer J₁ to the medium which, in contrast to jelly layers J₂ and J₃ is partially soluble, and permits its gradual dissolution during Phase 2. The embryo secretes a proteolytic enzyme from the frontal region which partially digests the fertilization envelope; subsequent embryo movement ruptures the weakened envelope and completes the hatching process.     

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.     

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.     

Structure of the extraembryonic matrices around the benthic embryos of Patiriella exigua (Asteroidea) and their roles in benthic development: Comparison with the planktonic embryos of Patiriella regularis

The extracellular matrices (ECMs) surrounding the benthic embryos and larvae of the seastar Patiriella exigua and the planktonic embryos of Patiriella regularis were examined by transmission and scanning electron microscopy. Three ECMs surround unhatched embryos: An outer jelly coat, a fertilization envelope, and an inner hyaline layer. The ECMs of P. exigua are modified for supporting benthic development. The dense jelly coat attaches the embryo to the substratum, and the fertilization envelope forms a though protective case. In comparison, P. regularis has a less dense jelly coat and a thinner fertilization envelope. The hyaline layer of both species is comprised of three main regions: An intervillous layer overlying the epithelium, a supporting layer, and a coarse meshwork layer. Unhatched P. exigua have an additional outer amorphous layer that adheres to the fertilization envelope. As a result, the hyaline layer forms a continuous ECM that unites the embryonic surface with the fertilization envelope. Embryos of P. exigua removed from their fertilization envelopes lack the outer amorphous region, have a poorly developed hyaline layer, and do not develop beyond gastrulation. It appears that the substantial hyaline layer of P. exigua and its attachment to the fertilization envelope are essential for early development and that this ECM may function as a gelatinous cushioning layer around the benthic embryos. At hatching, the amorphous layer is discarded with the envelope. In contrast, an amorphous layer is absent from the hyaline layer of P. regularis. The demembranated embryos of this species have an ECM similar to that of controls and develop normally to the larval stage. © 1995 Wiley-Liss, Inc.     

Fusion of Spermatozoa with Embryonic Cells and Somatic Cells in the Sea Urchin

Spermatozoa can enter the separated blastomeres of 8- and 16-cell stage embryos, the cells of blastulae and even somatic cells of the oesophagus wall of an adult sea urchin, under certain conditions. In the presence of egg jelly solution, the rate of entrance of spermatozoa is remarkably increased. In the case of the blastomere of 8-cell stage embryos, characteristic cytoplasmic protrusions are formed at the sites of sperm entry, in succession to the formation of the cytoplasmic bulge. These protrusions elongate until 4 min after insemination, and then they retract gradually. The nucleus of penetrated sperm swells and decondenses to form a pronucleus. In most cases, the pronucleus seems to fuse with the preexisting diploid nucleus of the blastomere. When the dissociated oesophagus cells were inseminated, a certain type of the cells was found to fuse with spermatozoa, although the percentage of fused cells was very low.     

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.     

Concanavalin A inhibits the dispersion of the cortical granule contents of sand dollar eggs

Dendraster excentricus eggs fertilized in ConA (10 μg/ml) elevate vitelline layers and expel cortical granule contents into the perivitelline space. The granule material does not disperse but remains composed as discrete spheres. The elevated vitelline layer remains thin and weak. It is not a true fertilization membrane because it lacks the structural material supplied by the granules.     

Evidence for the existence of two assembly domains within the sea urchin fertilization envelope.

The sea urchin fertilization envelope (FE) is a complex, macromolecular aggregate assembled by the addition of cortical granule secretions to the vitelline layer. The completed, trilaminar structure has a dense layer sandwiched between surface coats of paracrystalline material. Two cortical granule enzymes, ovoperoxidase and protease, and a cell surface transglutaminase are required for the assembly process. We have examined, by quick-freeze, deep-etch, rotary-shadow electron microscopy, the effects of inhibiting each of these enzymes upon FE assembly. These experiments reveal two domains within the FE, distinguishable by their enzymatic requirements for proper maturation. The first domain consists of the microvillar casts which require both protease and transglutaminase activities to obtain a normal paracrystalline coat. The second domain comprises the regions between casts and appears to mature by ovoperoxidase-mediated cross-linking of paracrystalline material to the envelope.     

On the contents of the cortical granules from Xenopus laevis eggs

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

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.