Sea urchin egg hyaline layer: evidence for the localization of hyalin on the unfertilized egg surface

The sea urchin embryo hyaline layer is an extracellular investment which develops within 20 min postinsemination of Strongylocentrotus purpuratus eggs and contains a single calcium-precipitable subunit termed hyalin. Other ultrastructural and biochemical studies have suggested that hyalin is localized in the cortical granules. We have examined the hypothesis that hyalin is a cell surface protein of the unfertilized egg using vectorial lactoperoxidase-catalyzed radioiodination. Extracts of labeled unfertilized eggs contained several labeled proteins, one of which was electrophoretically indistinguishable from authentic hyalin isolated by each of three different procedures. Pronase digestion of labeled unfertilized eggs removed 75% of the label, but the labeled hyalin-like molecule was still present in whole cell extracts. Upon insemination, pronase-digested, labeled eggs formed an apparently normal hyaline layer and whole cell extracts contained the labeled hyalin-like molecule. Denuded, labeled eggs were inseminated and the hyaline layer was selectively solubilized in calcium- and magnesium-free artificial seawater. Labeled hyalin was purified from this crude hyalin preparation to constant specific radioactivity and apparent homogeneity as shown by gel electrophoresis. These data strongly suggest that hyalin or a precursor is a cell surface protein of the unfertilized sea urchin egg.     

Mechanism for Increase in Intracellular Concentration of Free Calcium in Fertilized Sea Urchin Egg : A Method for Estimating Intracellular Concentration of Free Calcium

Intracellular free calcium concentration in the sea urchin egg was calculated to increase from 0.1 mM in an unfertilized egg to 1 mM in a fertilized egg 10 min after fertilization, based on measurement of the dissociation constant between free calcium and sea urchin egg homogenate. The dissociation constant between free calcium (dialyzable calcium) and homogenate of sea urchin eggs was measured by means of dialysis equilibrium. The dissociation constant of the unfertilized egg was about 10^–4 M and that of the fertilized egg was about 10–3 M in three species of sea urchin, Hemicentrotus pulcherrimus, Anthocidaris crassispina, and Pseudocentrotus depressus. An increase in the dissociation constant of the unfertilized egg homogenate was observed after the addition of calcium ion at a concentration above 0.3 mM, the dissociation constant becoming the same as that observed in the fertilized egg homogenate after the administration of CaCl₂ at a concentration above 1 mM. Sodium ion also caused a decrease in the calcium-binding ability of the unfertilized egg homogenate. Therefore, penetration of calcium ion or sodium ion upon fertilization might induce an increase in the dissociation constant and then intracellular concentration of free calcium would increase at fertilization. Almost all calcium-binding ability of the egg homogenate was found in the microsomal fraction, and the substance which bound calcium was thought to be protein in nature, since trypsin could decrease the level of calcium-binding substance in the homogenate of the eggs.     

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.     

Storage, ultrastructural targeting and function of toposomes and hyalin in sea urchin embryogenesis.

This study compares by immunogold labeling the ultrastructural localization of a hexameric 22S glycoprotein, called toposome, with that of hyalin in unfertilized eggs and cells of hatched sea urchin blastulae. Nearly all hyalin is present in the electron translucent compartment of the cortical granules and in the translucent non-cortical pigment granules. In the blastula both of these intracellular stores have vanished and hyalin now forms a broad band below the apical lamina. By contrast, in the egg toposomes are present on the surface, as well as stored in yolk granules and in the electron dense lamellar compartment of the cortical granules. In the hatched blastula, toposomes that have been modified by limited proteolysis in the yolk granules, are associated with the plasma membranes of all newly formed cells, while the toposomes originating from the cortical granules have been incorporated as unmodified 160 kDa polypeptides into an extracellular double layer enveloping the embryo on the outside of the hyaline layer. From evidence discussed in detail, we conclude that the extracellular toposomes rivet the apical lamina to the surface and underlying cytoskeleton of the microvilli, while the modified toposomes from the yolk granules are responsible for position specific intercellular adhesion as they are released to the surface of newly formed cells. We propose that all the material stored in yolk granules is utilized for the assembly of new membranes.     

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 ultrastructure of the sea urchin egg cortex isolated before and after fertilization

The three-dimensional organization of cortices isolated from unfertilized and fertilized Strongylocentrotus purpuratus eggs has been examined by several techniques of light and electron microscopy. It has been found that when moderate shear forces are used, the isolated unfertilized egg cortex, in addition to cortical granules, contains acidic vesicles and an elaborate network of rough endoplasmic reticulum. This network provides a physical link between the cell surface and several kinds of cytoplasmic organelles (mitochondria, yolk granules, acidic vesicles) which are retained as part of the isolated cortex when gentle shear forces are applied. Furthermore a good visualization of actin in the cortex is provided: it is present as short filaments and mostly within the stubby microvilli of the egg. Finally, it has been noted that plaques exist on the inside face of the plasma membrane ready to assemble into typical clathrin coats that prefigure the burst of coated vesicle endocytosis that takes place after fertilization. The cortex isolated soon after fertilization is shown to contain coated pits and a scaffolding of filaments (mostly actin) in which many acidic vesicles are embedded.     

Exogenous hyalin and sea urchin gastrulation. Part III: biological activity of hyalin isolated from Lytechinus pictus embryos

Hyalin is a large glycoprotein, consisting of the hyalin repeat domain and non-repeated regions, and is the major component of the hyaline layer in the early sea urchin embryo of Strongylocentrotus purpuratus. The hyalin repeat domain has been identified in proteins from organisms as diverse as bacteria, sea urchins, worms, flies, mice and humans. While the specific function of hyalin and the hyalin repeat domain is incompletely understood, many studies suggest that it has a functional role in adhesive interactions. In part I of this series, we showed that hyalin isolated from the sea urchin S. purpuratus blocked archenteron elongation and attachment to the blastocoel roof occurring during gastrulation in S. purpuratus embryos, (Razinia et al., 2007). The cellular interactions that occur in the sea urchin, recognized by the U.S. National Institutes of Health as a model system, may provide insights into adhesive interactions that occur in human health and disease. In part II of this series, we showed that S. purpuratus hyalin heterospecifically blocked archenteron-ectoderm interaction in Lytechinus pictus embryos (Alvarez et al., 2007). In the current study, we have isolated hyalin from the sea urchin L. pictus and demonstrated that L. pictus hyalin homospecifically blocks archenteron-ectoderm interaction, suggesting a general role for this glycoprotein in mediating a specific set of adhesive interactions. We also found one major difference in hyalin activity in the two sea urchin species involving hyalin influence on gastrulation invagination.     

THE ORIGIN OF ECHINOID HATCHING ENZYME MESSENGER RNA*

To determine if echinoid hatching enzyme messenger RNA is newly synthesized from embryonic chromatin or is a maternal mRNA stored in the unfertilized egg, hybrid andromerogones have been constructed containing a sea urchin (Strongylocentrotus purpuratus) genome in sand dollar (Dendraster excentricus) cytoplasm. Such hybrid andromerogones developed at a normal rate to the blastula stage but failed to hatch. Diploid hybrids or merogones containing at least one complement of sand dollar genome hatched on the normal maternal schedule. Since the sea urchin hatching enzyme is not able to digest the sand dollar fertilization membrane, this failure to hatch is evidence that new mRNA synthesis from embryonic chromatin is required before hatching enzyme can be synthesized.     

Thymidine kinase activation in unfertilized sea urchin eggs by homogenization and fertilization

Kinetics of in vivo phosphorylation of ³H-thymidine taken up by sea urchin eggs was compared between unfertilized and fertilized eggs. The percentage of phosphorylated ³H-thymidine in the total acid-soluble radioactivity in the cell increased with increasing incubation time within the first several minutes of incubation in the unfertilized eggs, while nearly 100% of phosphorylation of thymidine was observed without regards to the incubation time and in spite of a tremendous increase in the net uptake of thymidine in the fertilized eggs, suggesting possible activation of thymidine kinase occurring soon after fertilization.In contrast to the in vivo finding, the thymidine kinase activity in unfertilized egg homogenates was found in general to be almost as large as that in fertilized egg homogenates. However, when the enzyme activity was assayed within a short period (30 min) after homogenization of unfertilized eggs, the activity was found to increase more or less with time after homogenization, reaching a level equal to that in fertilized egg homogenates. This enzyme activation after homogenization was especially marked in case of Pseudocentrotus eggs and sometimes amounted to a several fold increase.Preliminary investigations revealed possible involvement of some redox reaction(s) in the thymidine kinase activation during and/or after homogenization of unfertilized sea urchin eggs.     

The GTP-binding protein RhoA localizes to the cortical granules of Strongylocentrotus purpuratas sea urchin egg and is secreted during fertilization

The sea urchin egg has thousands of secretory vesicles known as cortical granules. Upon fertilization, these vesicles undergo a Ca2+-dependent exocytosis. G-protein-linked mechanisms may take place during the egg activation. In somatic cells from mammals, GTP-binding proteins of the Rho family regulate a number of cellular processes, including organization of the actin cytoskeleton. We report here that a crude membrane fraction from homogenates of Strongylocentrotus purpuratus sea urchin eggs, incubated with C3 (which ADP-ribosylates specifically Rho proteins) and [32P]NAD, displayed an [32P]ADP-ribosylated protein of 25 kDa that had the following characteristics: i) identical electrophoretic mobility in SDS-PAGE gels as the [32P]ADP-ribosylated Rho from sea urchin sperm; ii) identical mobility in isoelectro focusing gels as human RhoA; iii) positive cross-reactivity by immunoblotting with an antibody against mammalian RhoA. Thus, unfertilized S. purpuratus eggs contain a mammalian RhoA-like protein. Immunocytochemical analyses indicated that RhoA was localized preferentially to the cortical granules; this was confirmed by experiments of [32P]ADP-ribosylation with C3 in isolated cortical granules. Rho was secreted and retained in the fertilization membrane after insemination or activation with A23187. It was observed that the Rho protein present in the sea urchin sperm acrosome was also secreted during the exocytotic acrosome reaction. Thus, Rho could participate in those processes related to the cortical granules, i.e., in the Ca2+-regulated exocytosis or actin reorganization that accompany the egg activation.     

Uptake of deuterium into proteins of fertilized and unfertilized Arbacia eggs suspended in heavy water

When fertilized and unfertilized eggs of Arbacia punctulata are suspended in heavy water, deuterium is incorporated into stable positions in the egg proteins. The rate of incorporation of the isotope is considerably greater in fertilized than in unfertilized eggs, and is accelerated at the time of formation of the blastula. The result of calculation of the maximum deuterium concentration which would be reached on complete turnover indicates that at least one out of every ten stably bound hydrogen atoms of the egg proteins is a deuterium atom. This has been interpreted as evidence that at the time of formation of the sea urchin blastula and in the period of development which follows, synthesis and breakdown are simultaneous processes leading to the redistribution of amino acids among the egg proteins.     

Adaptation of cultured human lymphoblasts to growth in citrulline

DNA synthesis is initiated in unfertilized sea urchin eggs (Strongylocentrotus purpuratus and Lytechinus pictus) by exposing them to NH₄OH-sea water (ordinary sea water titrated to pH 9–9.1 with NH₄OH). The eggs are considered to be unfertilized eggs by visual and electro-biological criteria and because they can later be fertilized and then do give visible and electrobiological fertilization reactions. The incorporation of ³H-thymidine proceeds in rounds, the magnitude increasing in successive rounds. It is also reported that the treatment with NH₄OH activates the uptake of thymidine by the eggs, although the internal thymidine builds up more slowly in unfertilized eggs treated with NH₄OH than it does in fertilized eggs. The magnitude of the incorporation of exogenously supplied labelled thymidine into DNA is lower in the NH₄OH-treated unfertilized eggs than in normal fertilized eggs. This difference is not attributed to differences in the amount of DNA synthesized and the explanation is sought in thymidine uptake and nucleotide pathways.     

Exogenous hyalin and sea urchin gastrulation, Part II: hyalin, an interspecies cell adhesion molecule.

The 330 kDa fibrillar glycoprotein hyalin is a well known component of the sea urchin embryo extracellular hyaline layer. Only recently, the main component of hyalin, the hyalin repeat domain, has been identified in organisms as widely divergent as bacteria and humans using the GenBank database and therefore its possible function has garnered a great deal of interest. In the sea urchin, hyalin serves as an adhesive substrate in the developing embryo and we have recently shown that exogenously added purified hyalin from Strongylocentrotus purpuratus embryos blocks a model cellular interaction in these embryos, archenteron elongation/attachment to the blastocoel roof. It is important to demonstrate the generality of this result by observing if hyalin from one species of sea urchin blocks archenteron elongation/attachment in another species. Here we show in three repeated experiments, with 30 replicate samples for each condition, that the same concentration of S. purpuratus hyalin (57 microg/ml) that blocked the interaction in living S. purpuratus embryos blocked the same interaction in living Lytechinus pictus embryos. These results correspond with the known crossreactivity of antibody against S. purpuratus hyalin with L. pictus hyalin. We propose that hyalin-hyalin receptor binding may mediate this adhesive interaction. The use of a microplate assay that allows precise quantification of developmental effects should help facilitate identification of the function of hyalin in organisms as divergent as bacteria and humans.     

A marker of animal-vegetal polarity in the egg of the sea urchin Paracentrotus lividus. The pigment band

We have examined the subequatorial accumulation of pigment granules (the so-called 'pigment band') in the egg of the sea urchin Paracentrotus lividus, which constitutes an unambiguous marker of animal-vegetal polarity. Most of the reddish pigment granules are situated at the periphery of the egg. They exhibit occasional saltatory movements and can aggregate into large patches. Pigment granules are retained as a band in the isolated cortex when the egg surface complex is isolated by shearing eggs attached to polylysine-coated surfaces with calcium-free isotonic solutions. Pigment granules remain as the main vesicular component of fertilized egg cortices or of unfertilized egg cortices perfused with calcium to provoke cortical granule exocytosis. They may be anchored to the isolated cortex through associations with the plasma membrane and with an extensive subsurface network of rough endoplasmic reticulum (rough ER). Pigment granules contain antimonate-precipitable calcium and, in this respect and many others, resemble acidic vesicles recently identified in the cortex of unpigmented sea urchin eggs. We discuss the similarities observed between granules and acidic vesicles in various urchin egg species and their possible functions.     

Two antigenically distinct forms of β-1,3-glucanase in sea urchin embryonic development

The enzyme β-1,3-glucanase is contained in the unfertilized eggs of most species of sea urchin. In some species, including Lytechinus variegatus, there is also substantial activity following gastrulation, and during remaining larval development. To determine if the same form of β-1,3-glucanase is present in both unfertilized eggs and after gut differentiation, an affinity purification procedure was utilized to isolate enzyme from unfertilized Lytechinus eggs. β-1,3-Glucanase is a 70,000-Da protein in this species, similar to the molecular weight of enzyme isolated from Strongylocentrotus purpuratus. Purified enzyme was used to generate an antibody that specifically recognized a 70,000-Da protein in unfertilized eggs by Western blot analysis, and stained the cortical granules of unfertilized eggs by immunofluorescence. The antibody also specifically immunoprecipitated β-1,3-glucanase activity from egg sonicates. The antibody was used to demonstrate that the form of β-1,3-glucanase present following gastrulation is antigenically distinct from the egg form. The 70,000-Da protein recognized by the antibody was no longer present by 24 hr, but embryos of this and later stages contained substantial amounts of activity, indicating the enzyme at these stages differs from the egg-specific form. In addition, the antibody was not capable of immunoprecipitating enzyme activity from pluteus sonicates. β-1,3-Glucanase has been partially purified from pluteus stage embryos, and appears to be a complex of approximately 200,000 Da. The enzyme is specific to endoderm and appears following differentiation of the gut, suggesting that it may function in larval digestion.     

A novel procedure for obtaining denuded sea urchin eggs and observations on the role of the vitelline layer in sperm reception and egg activation

A procedure is described for the complete removal of the vitelline layer of the eggs of the sea urchin, Strongylocentrotus purpuratus. The method involves treatment of unfertilized eggs with an S. purpuratus cortical granule protease preparation followed by incubation in an alkaline dithiothreitol seawater solution. Eggs denuded of their vitelline layers react metabolically to parthenogenetic agents and sperm like unfertilized eggs, whereas the fertilizability of denuded eggs and receptivity to sperm is much less than controls. The present method is superior to previous methods using mercaptans in that all of the vitelline layer is removed and to procedures using other proteolytic enzymes in that no ¹²⁵I-labelled plasma membrane proteins are extensively modified. Thus the cortical granule protease dithiothreitol procedure is ideal for studies of the plasma membrane of the unfertilized egg and for studies on the role of the vitelline layer in normal fertilization and development.     

Nuclear histones of unfertilized sea urchin eggs

Histones have been isolated from the nuclei of unfertilized eggs of the sea urchin Strongylocentrotus purpuratus. The electrophoresis of these histones exhibits a pattern different from that of the sperm or embryo of the same species.     

Compartmentation of thymidine kinase in unfertilized sea urchin eggs and possible release of thymidine kinase from particulates in activated eggs

The thymidine kinase activity of homogenates of unfertilized eggs of the sea urchin, Hemicentrotus pulcherrimus, in 1 M NaCl was always lower than that of homogenates of the unfertilized eggs in hypotonic media or homogenates of the fertilized or ammonia-activated eggs in 1 M NaCl by 30–50%. Sonication of the unfertilized egg homogenates in 1 M NaCl resulted in the elevation of thymidine kinase activity up to a level in the fertilized or ammonia-activated egg homogenates which is not affected by sonication. Differential centrifugation of unfertilized egg homogenates in 1 M NaCl revealed that the latent thymidine kinase is associated with the 1500g pellet or even with the 200g pellet. Exposure of the 1500g pellet to sonication, hypotonic media, 0.3% Triton X-100 in 1 M NaCl, and 2 M propyleneglycol resulted in the elevation of thymidine kinase, which was eventually shown to be no longer bound to the pellet fraction. Latent thymidine kinase was not detected in the 1500g pellet prepared from the fertilized egg homogenate in 1 M NaCl. These findings seem to suggest that thymidine kinase in unfertilized eggs may be sequestered, at least partly, in some large intracellular structures but may be released from them upon fertilization or ammonia activation, in accordance with our earlier observation on the apparent activation of thymidine kinase afer fertilization.     

A 9.6 S protein is the third calcium-insoluble component of the sea urchin hyaline layer.

A third major, calcium-insoluble component of the sea urchin (Strongylocentrotus purpuratus) hyaline layer has been purified and physically characterized. In the absence of divalent cations, the native, soluble protein has a sedimentation coefficient of 9.6 S and a molecular weight of 4.5 +/- 0.1 x 10(5). These data indicate that this large protein assumes an elongated, nonspherical conformation in solution. Its sedimentation behavior and its mobility on nondenaturing electrophoretic gels distinguish the 9.6 S protein from the 11.6 S and 6.4 S hyalin proteins we have previously characterized. That the 6.4 S, 9.6 S, and 11.6 S proteins are the major calcium-insoluble structural components of the hyaline layer is supported by the fact that we have found them in a variety of hyalin protein fractions prepared by a number of standard approaches. All three proteins are precipitated by calcium ions, thus fitting the operational definition of hyalin. Evidence is presented that the 11.6 S protein may overlie the 9.6 S protein in the hyaline layer.     

Fibropellins, products of an EGF repeat-containing gene, form a unique extracellular matrix structure that surrounds the sea urchin embryo

The sea urchin SpEGF 1 gene belongs to a growing family of developmentally important genes which encode proteins that contain repeated epidermal growth factor-like motifs. To characterize the embryonic expression of the protein products of this gene from Strongylocentrotus purpuratus, we generated polyclonal antisera from SpEGF I fusion proteins. These antibodies recognize two glycoproteins of 145 and 185 kDa, which we have named fibropellins. These proteins are present in unfertilized oocytes and throughout early development. The fibropellins are stored in cytoplasmic vesicles in the oocyte and are released soon after fertilization in a distinct secretory event following the exocytosis of cortical granule contents. Following secretion the proteins are localized in the basal surface of the hyaline layer. At the blastula stage the fibropellins become organized into distinct fibers which form a mesh-like network over the surface of the embryo. During subsequent development to the pluteus larva stage this network increases in overall morphological complexity and becomes regionally distinct. The molecular weights of the fibropellins and their pattern of embryonic localization indicate that these proteins form a component of the hyaline layer previously described as the apical lamina.