A role for polyglucans in a model sea urchin embryo cellular interaction.

The enzymatic activities of commercially prepared glycosidases were verified by direct chemical assays using defined substrates and fixed and live sea urchin (Lytechinus pictus) embryos to determine if a model cellular interaction of interest to developmental biologists for over a century (interaction of archenteron tip and roof of the blastocoel) was mediated by glycans. Glycosidases (active and denatured) were incubated with microdissected archenterons and blastocoel roofs in a direct assay to learn if their enzymatic activities could prevent the normal adhesive interaction. Of the five glycosidases tested only ß-amylase (an exoglycosidase) immediately inhibited the interaction at relatively low unit activity. α-Amylase (an endoglycosidase) had no measurable effect, while other glycosidases (α-glucosidase, ß-glucosidase, ß-galactosidase) only substantially inhibited adhesion after a 12-h incubation. We demonstrated that the five glycosidases were active (not inhibited) in the presence of embryo materials, and that cleaved sugars could be detected directly after incubation of some enzymes with the embryos. The biochemical purity of the enzymes was examined using gel electrophoresis under denaturing conditions, and the absence of contaminating proteases was confirmed using Azocoll™ substrate. As we cannot entirely rule out the presence of minor contaminating enzymatic activities, only inhibitions of adhesion after very short incubations with enzyme were considered significant and biologically relevant. Although glycans in indirect experiments have been implicated in mediating the interaction of the tip of the archenteron and roof of the blastocoel, to our knowledge, this is the first study that directly implicates polyglucans with terminal 1,4-linked glucose residues in this adhesive event.

Use of specific glycosidases to probe cellular interactions in the sea urchin embryo.

We present an unusual and novel model for initial investigations of a putative role for specifically conformed glycans in cellular interactions. We have used alpha- and ss-amylase and alpha- and ss-glucosidase in dose-response experiments evaluating their effects on archenteron organization using the NIH designated sea urchin embryo model. In quantitative dose-response experiments, we show that defined activity levels of alpha-glucosidase and ss-amylase inhibited archenteron organization in living Lytechinus pictus gastrula embryos, whereas all concentrations of ss-glucosidase and alpha-amylase were without substantial effects on development. Product inhibition studies suggested that the enzymes were acting by their specific glycosidase activities and polyacrylamide gel electrophoresis suggested that there was no detectable protease contamination in the active enzyme samples. The results provide evidence for a role of glycans in sea urchin embryo cellular interactions with special reference to the possible structural conformation of these glycans based on the differential activities of the alpha- and ss-glycosidases.

Exogenous hyalin and sea urchin gastrulation. Part IV: a direct adhesion assay - progress in identifying hyalin's active sites.

In Strongylocentrotus purpuratus the hyalins are a set of three to four rather large glycoproteins (hereafter referred to as 'hyalin'), which are the major constituents of the hyaline layer, the developing sea urchin embryo's extracellular matrix. Recent research from our laboratories has shown that hyalin is a cell adhesion molecule involved in sea urchin embryo-specific cellular interactions. Other laboratories have shown it to consist of 2-3% carbohydrate and a cloned, sequenced fragment demonstrated repeat domains (HYR) and non-repeat regions. Interest in this molecule has increased because HYR has been identified in organisms as diverse as bacteria, flies, worms, mice and humans, as well as sea urchins. Our laboratories have shown that hyalin appears to mediate a specific cellular interaction that has interested investigators for over a century, archenteron elongation/attachment to the blastocoel roof. We have shown this finding by localizing hyalin on the two components of the cellular interaction and by showing that hyalin and anti-hyalin antibody block the cellular interaction using a quantitative microplate assay. The microplate assay, however, has limitations because it does not directly assess hyalin's effects on the adhesion of the two components of the interaction. Here we have used an elegant direct assay that avoids the limitations, in which we microdissected the two components of the adhesive interaction and tested their re-adhesion to each other, thereby avoiding possible factors in the whole embryos that could confound or confuse results. Using both assays, we found that mild periodate treatment (6 h to 24 h in sodium acetate buffer with 0.2 M sodium periodate at 4 degrees C in the dark) of hyalin eliminates its ability to block the cellular interaction, suggesting that the carbohydrate component(s) may be involved in hyalin's specific adhesive function. This first step is important in identifying the molecular mechanisms of a well known cellular interaction in the NIH-designated sea urchin embryo model, a system that has led to the discovery of scores of physiological mechanisms, including those involved in human health and disease.

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.

Hyalin is a cell adhesion molecule involved in mediating archenteron-blastocoel roof attachment.

The US National Institutes of Health has designated the sea urchin embryo as a model organism because around 25 discoveries in this system have led to insights into the physiology of higher organisms, including humans. Hyalin is a large glycoprotein in the hyaline layer of sea urchin embryos that functions to maintain general adhesive relationships in the developing embryo. It consists of the hyalin repeat domain that has been identified in organisms as diverse as bacteria, worms, flies, mice, sea urchins and humans. Here we show, using a polyclonal antibody raised against the 11.6 S species of hyalin, that it localizes at the tip of the archenteron and on the roof of the blastocoel exactly where these two structures bond in an adhesive interaction that has been of interest for over a century. In addition, the antibody blocks the interaction between the archenteron tip and blastocoel roof. These results, in addition to other recent findings from this laboratory that will be discussed, suggest that hyalin is involved in mediating this cellular interaction. This is the first demonstration that suggests that hyalin functions as a cell adhesion molecule in many organisms, including humans.

Jeltraxin, a frog egg jelly glycoprotein, has calcium-dependent lectin properties and is related to human serum pentraxins CRP and SAP.

The egg jelly that encapsulates amphibian eggs is essential for fertilization, but its molecular composition and roles remain largely unknown. We identified a calcium-dependent lectin from the pentraxin superfamily in the egg jelly coat from the South American burrowing frog, Lepidobatrachus laevis. This lectin, jeltraxin, was related to the host-response acute phase serum proteins C-reactive P component (CRP) and serum amyloid P component (SAP). The amino acid sequence of jeltraxin is 44% identical to that of Xenopus laevis CRP, 31-35% identical to those of mammalian CRP and SAP, and 21-27% identical to those of the large fusion pentraxins. Expression of jeltraxin mRNA was restricted to the oviduct, which distinguishes it as the first serum-related pentraxin not expressed in the liver. Purified jeltraxin was previously shown to exist in an oligomeric complex of approximately 250 kDa comprised of self-associating subunits. We have demonstrated by MALDI-TOF that this configuration is due to a decameric complex of 27.7 kDa subunits. Biotinylated jeltraxin bound to the high-molecular mass components of the egg jelly in a calcium-dependent manner with specificity for beta-galactose residues. On the basis of homology modeling, we predict that jeltraxin will coordinate two calcium ions. The function of jeltraxin will likely be related to its calcium-dependent lectin properties.

Thermal tolerance and heat shock protein synthesis during development in the anuran Lepidobatrachus laevis.

Development of the Paraguayan anuran Lepidobatrachus laevis is unusual in that the larvae are obligate carnivores, facultative cannibals and apparently exist at high environmental temperatures in their natural habitat. In the present study, the effect of environmental temperature on the rate of anuran development was investigated. The larvae have a thermotolerance range of 18°C for normal development between 19 and 37°C. The effect of temperature on the rate of development was dramatic; larvae that were incubated at 36.8°C develop to stage 24 (Gosner) in approximately 9 h compared with 24 h for larvae incubated at 19°C. The ability of larvae to survive heat shock was also examined; larvae did not survive a shock of 45°C for 15 min when it was administered at stages 3, 5, 9, 10 or 20. However, using the same heat shock conditions, 50% survival was observed when larvae were shocked at stage 16. To study protein synthesis during heat shock, larvae were pulsed with [35 S]-methionine during heat shock and labeled proteins were analyzed by electrophoresis under reducing and denaturing conditions. Larvae synthesized two sets of heat-shock proteins at doublet molecular weights of 83/78 and 62/59 kDa. These proteins were synthesized independently of the stage of development at which the shock was administered or the magnitude of the heat shock.

Oviduct histochemistry and site of synthesis of a 29.7 kDa jelly coat glycoprotein in the anuran Lepidobatrachus laevis.

The oviduct of the anuran Lepidobatrachus laevis contains three morphological regions, each of which contains a histochemically distinct luminal mucosa. In the pars recta, the most anterior portion of the oviduct, there are periodic acid-Schiff base (PAS)-positive simple glands and epithelia. In the pars convoluta, there are alcian blue-positive, combined alcian blue- and PAS-positive and PAS-positive gland types. The most posterior region, the pars uterina, contains alcian blue-positive and alcian blue-negative epithelial cells. Previous work has shown that solubilized egg jelly contains a major 29.7 kDa glycoprotein subunit that was detected in oviduct tissue extracts from the pars convoluta in the present study. Rabbit antisera to the 29.7 kDa egg jelly glycoprotein of L. laevis reacted with the major pars convoluta glycoprotein and there were no immunoreactive components in the pars uterina. The slight immunoreactivity detected at 29.0-37.0 kDa in pars recta extracts is not believed to be the jelly molecule, based on low immunoreactivity and subunit molecular weight measurements. We conclude that the synthesis of the 29.7 kDa egg jelly glycoprotein is restricted to the pars convoluta region of the oviduct.

The Primary Egg Envelope of the Anuran Lepidobatrachus laevis: Physicochemical and Macromolecular Alterations During Development: (egg envelopes).

The coelomic egg envelope (CE) of the frog Lepidobatrachus laevis has a network of fibrillar bundles which disperse after transit through the oviduct. Following oviposition, the egg vitelline envelope (VE) has an additional amorphous zone on the exterior surface. The fertilization envelope (FE) formed after fertilization, appears to be very similar to the VE. The CEs, VEs, FEs and hatched envelopes (FEh ) were manually isolated. The CE, VE and FE were solubilized at 100° using denaturing conditions, but were only partially solubilized in phosphate buffer, pH 7.0. All envelopes and several purified polypeptides from the VE and FE were analyzed using gel electrophoresis and one-dimensional peptide mapping. Each of the envelopes contained 9 major polypeptides ranging from 118.5 to 22 kD and 8-12 minor polypeptides. Several envelope components were added/removed in the conversions based on the results of experiments in which preparations were incubated with activated egg exudate and crude hatching enzyme; some of these transformations were mimicked by tryptic and chymotryptic digestions. Therefore, serine proteases may be involved in envelope processing in vivo. Lepidobatrachus CE polypeptides and several major components from the VE, FE and FEh were crossreactive with antibodies against Xenopus VE* .

Structure and Macromolecular Composition of the Jelly Coats of the Urodele Ambystoma mexicanum: (egg envelopes).

Cleavage-stage embryos of the neotenic urodele Ambystoma mexicanum are surrounded by a fertilization envelope and four macroscopic jelly coats termed J1 (innermost) through J4 (outermost). In sections prepared for light microscopy, each of the jelly layers stained with protein stains and the periodic acid-Schiff's reagent, but only J1 stained with alcian blue at pH 2.5. These results suggest that each layer consists of proteins and glycoproteins and that J1 uniquely contains some sulfate esters. Only J4 was solubilized with alkaline mercaptan treatment in situ, however, the isolated inner jelly complex (J1 , J2 and J3 ) was easily dissolved in this reagent suggesting that solvent access is impaired in situ. A single alcian blue-staining component plus one protein-staining component were detected on reducing polyacrylamide gel electrophoresis of outer jelly (J4 ). In the inner jelly complex (J1 , J2 , J3 ), two protein-staining components were detected and no alcian blue-staining components were observed. A predominant polypeptide of 110,000 molecular weight was detected and purified to homogeneity on reducing and denaturing gels of the inner jelly complex. Amino acid analysis of the polypeptide demonstrated a slightly higher fraction of acidic over basic amino acids (Glx+Asx=18.1 mole%vs. Arg + Lys = 11.7 mole%). The N-terminal amino acid was Glu and the sequence of the first eleven amino acids was determined.

Gastric Pepsin in an Anuran Larva: (Lepidobatrachus laevis/pepsinogen).

Larval stomach development was studied in the obligate carnivorous larva of the frog Lepidobatrachus laevis. Pepsin producing cells of the larval stomach were identified using rabbit anti-porcine pepsin and immunohistochemical techniques. Pepsin production was detected at a very early stage of development (stage 24: during opercular development) when the larvae were first competent for feeding. Peptic activity in isolated larval stomachs was demonstrated in a microassay using acid denatured hemoglobin at pH 1.7. The total activity per stomach increased 5,400 fold through the beginning of metamorphosis and the specific activity increased 345 fold through the same period. Electrophoretic analysis of the larval pepsinogens, using a caseinolytic assay revealed the presence of one major pepsinogen at stage 24; two additional isozymes were observed during later larval development. The molecular weight of the isopepsinogens was 34,800.

Structure and Macromolecular Composition of the Egg and Embryo Jelly Coats of the Anuran Lepidobatrachus laevis: (frog jelly coat/fertilization/glycoprotein).

Eggs and cleavage-stage embryos of the frog Lepidobatrachus laevis are encased by 3 µm thick vitelline/fertilization envelope and two jelly layers, termed J1 (innermost) and J2 (outermost). Based on light and transmission electron microscopy, J1 had a dense reticular appearance whereas J2 had a laminar structure. Direct dissolution of the jelly coats was accomplished by reduction of disulfide bonds with 0.08 M 2-mercaptoethanol at pH 10. Soluble jelly preparations were uncontaminated with nucleic acid (A280 /A260 =1.44) and yielded an average of 150 µg protein/egg or embryo (n=5). The biochemical composition of the jelly coats in unfertilized eggs was different from that in embryos. When examined via gel permeation chromatography, soluble jelly from unfertilized eggs contained macromolecules which were markedly larger and more heterogeneous (earlier eluting and broader peaks) than jelly from embryos. Differences in the components of jelly from unfertilized eggs and embryos were also observed by electrophoresis, however, a 29,700 molecular weight glycoprotein chain was common to both jelly preparations. The electrophoretic pattern of jelly obtained from parthenogenetically activated eggs was identical to that of unfertilized eggs, therefore the fertilization-associated changes are not due to the exclusive action of cortical granule products.

Extreme Toxicity of Ethanol During Activation of Sea Urchin Eggs: (ethanol toxicity/ionophore A23187/parthenogenesis/Strongylocentrotus purpuratus/K+ -permeability).

We have examined the effects of ethanol on early fertilization events and later development in the sea urchin Strongylocentrotus purpuratus. Eggs can still be fertilized in ethanol concentrations as high as 480 mM (2.0%); egg cytolysis was rapidly observed postinsemination in 50% of the cells at 220 mM ethanol. Yet, sperm motility was essentially normal in 250 mM ethanol; 940 mM ethanol was required to affect a 50% reduction. To determine the effect of ethanol on K+ -efflux from eggs induced by fertilization, we used parthenogenetic activation induced by the Ca2+ -ionophore A23187. Surprisingly, ethanol at only 0.2 mM caused an abnormal K+ -efflux, but only when added between 1 and 3 min after induction of activation. The K+ -efflux rates of unfertilized eggs were not influenced by up to 730 mM ethanol. Finally, normal embryonic development through the mesenchyme blastula stage was observed in egg suspensions which were treated for 30 min with ethanol concentrations as high as 240 mM, but washed with normal seawater prior to insemination. Normal plutei were obtained from cultures which were continuously cultured in 24 mM ethanol from 15 min postinsemination. We conclude that an extreme ethanol sensitivity of embryogenesis is apparent only during the cortical reaction.