Fibronectin from the ovary of the sea urchin,Pseudocentrotus depressus

Summary Fibronectin, with a subunit molecular weight of 220,000 daltons, was isolated from the ovary of the sea urchin,Pseudocentrotus depressus, using affinity chromatography on heat-denatured mammalian collagen coupled to Sepharose 4B. The distribution of fibronectin in the sea urchin ovary was examined by indirect immunofluorescence using antifibronectin serum. The basement membrane and the connective tissues exhibited strong fluorescence. The fibronectin was localized closely together with collagen bundles in the sea urchin ovary. Biochemical and immunological examinations indicate that sea urchin fibronectin has similar properties as those of mammalian fibronectin.     

Cell-to-substratum adhesion of dissociated embryonic cells of the sea urchin,Pseudocentrotus depressus

Summary Some plant lectins, Concanavalin agglutinin (Con A), succinyl Con A and wheat germ agglutinin (WGA) increased the adhesion of dissociated embryonic cells of the sea urchin,Pseudocentrotus depressus, to the substratum (plastic and glass surface) in vitro. Other plant lectins,Ulex europeus agglutinin (UEA) andDolichos biflorus agglutinin (DBA) had no effect on the cell-to-substratum interaction. A specific monocarbohydrate inhibitor of lectins, -methyl-d-mannoside, inhibited the Con A-induced cell-to-substratum adhesion of dissociated embryonic cells. This observation suggests that the Con A-induced cell-to-substratum adhesion may be attributed to the Con A-carbohydrate interaction. In Millipore-filtered sea water (MPFSW) containing Con A (0.1 mg/ml), dissociated embryonic cells adhered to the substratum for more than 6 h at 18°C, while in MPFSW as control, almost all the dissociated cells were released from the substratum after 1 h. A scanning electron microscopic study showed that dissociated embryonic cells adhered to the substratum were surrounded by an extracellular fibrous material, when the cells were cultured in MPFSW containing Con A. The induction of the extracellular fibrous material by Con A was inhibited by -methyl-d-mannoside. The appearance of this material may be related to the cell-to-substratum adhesion of dissociated cells. Sequential extractions of Con A-treated dissociated cells with Triton X 100 and urea solubilized most of the cellular components, leaving the fibrous material on the surface. Biochemical conponents of the isolated fibrous material included sea urchin fibronectin, Con A and minor components (88 and 140 kilodalton proteins). Fibronectin preformed in the cells was excreted after the dissociation, while the 88 and 140 kilodalton proteins were synthesized and released to the extracellular space.     

Feeding Characteristics of Three Species of Intertidal Sea Anemones of the South Kuril Islands

We examined the content of the coelenteron of Oulactis orientalis, Cnidopus japonicus, and Aulactinia sp. (family Actiniidae) inhabiting the intertidal area of Shikotan and Kunashir Islands (South Kuril Islands). The feeding characteristics (food spectrum, the index of filling of the coelenteron, the index of food similarity of the species, occurrence frequency, the index of trophic importance, and the proportions of certain components in the food pellet) allowed us to determine that the examined species are euryphagous.     

Matrix and Mineral in the Sea Urchin Larval Skeleton

The endoskeletal spicules of sea urchin larvae are composed of calcite, a surrounding extracellular matrix, and small amounts of occluded matrix proteins. The spicules are formed by primary mesenchyme cells (PMCs) in the blastocoel of the embryo, where they adopt stereotypical locations, thereby specifying where spicules will form. PMCs also fuse to form cytoplasmic cords connecting the cell bodies, and it is within the cords that spicules arise. The mineral phase contains 5% Mg as well as Ca, and about 0.1% of the mass is protein. The matrix and mineral form concentric plies, and the composite has different physical properties than those of pure calcite. The calcite diffracts as a single crystal and is composed of well-ordered, but not perfectly ordered, microdomains. There is evidence for adsorption of matrix proteins to specific crystal faces at domain boundaries, which may help regulate crystal growth and texture. Immature spicules contain considerable precipitated amorphous CaCO3, and PMCs also have vesicles that contain amorphous CaCO3. This suggests the hypothesis that the cellular precursor to the spicules is actually amorphous CaCO3 stabilized in the cell by protein. The spicule s enveloped by the PMC cord, but is topologically exterior to the cell. The PMC plasmalemma is tightly applied to the developing spicules, except perhaps at the elongating tip. The characteristics, localization, and possible function of the four identified matrix proteins are discussed. SM50, SM37, and PM27 all primarily enclose the mineral, though small amounts are occluded. SM30 is found in cellular vesicles and is probably the principal occluded protein of the spicule.     

Developmental Expression of Echinonectin, an Endogenous Lectin of the Sea Urchin Embryo

Echinonectin (EN) is a galactose-binding lectin present in eggs and embryos of the sea urchin Lytechinus variegatus . Recent studies have suggested that EN is a hyaline layer protein that may function as a substrate adhesion molecule (SAM) during development. We have used monoclonal and affinity-purified polyclonal antibodies that specifically recognize this protein to determine its spatial and temporal expression during embryogenesis. EN is stored in granules or vesicles in the unfertilized egg. After fertilization, these granules are rapidly redistributed to the apical cytoplasm of the zygote. Our results show that at subsequent stages of development the lectin is expressed by cells of all three germ layers, including cells of the developing gut, coelomic pouches, and ectoderm, and by both primary and secondary mesenchyme cells. In contrast to previous observations based solely upon light level immunofluorescent staining, immunoelectron microscopy demonstrates that EN is localized in intracellular, membrane-bounded vesicles. In epithelial cell types these vesicles have a highly polarized distribution and are found in the apical cortical cytoplasm. In mesenchyme cells the distribution of EN-containing vesicles is not obviously polarized. Steady-state levels of EN protein in the embryo remain almost constant from fertilization to the pluteus larva stage, Metabolic labeling studies show that synthesis of EN in L. variegatus begins immediately after fertilization and continues throughout embryogenesis. Monospecific antibodies raised against L. variegatus EN have also been used to determine whether this lectin is expressed in other echinoid species.     

The Vertical Distribution of Larvae of the Sea Urchin Strongylocentrotus intermedius in Superficial Desalination Conditions

The vertical distribution of larvae of the sea urchin Strongylocentrotus intermedius in conditions of superficial desalination was studied under laboratory conditions. In the earlier stages of development, the larvae accumulated in the water column zone with a salinity below the optimal level. The long-term observations of the larval accumulations revealed the deceleration of, and abnormalities in, the development and death of the larvae. The larvae at the pre-settling stages changed their behavior: when they reached layers of a salinity unfavorable for their survival or development, the larvae did not enter them and instead moved down instead. Obviously, S. intermedius larvae are only capable of actively choosing their position within the water column with the salinity gradient at the later stages of development.     

Sea Urchin Spines as a Model-System for Permeable, Light-Weight Ceramics with Graceful Failure Behavior. Part I. Mechanical Behavior of Sea Urchin Spines under Compression

The spines of pencil and lance urchins Heterocentrotus mammillatus and Phyllacanthus imperialis were studied as a modelof light-weight material with high impact resistance.The complex and variable skeleton construction (stereom) of body andspines of sea urchins consists of highly porous Mg-bearing calcium carbonate.This basically brittle material with pronouncedsingle-crystal cleavage does not fracture by spontaneous catastrophic device failure but by graceful failure over the range of tensof millimeter of bulk compression instead.This was observed in bulk compression tests and blunt indentation experiments onregular,infiltrated and latex coated sea urchin spine segments.Microstructural characterization was carried out using X-raycomputer tomography,optical and scanning electron microscopy.The behavior is interpreted to result from the hierarchicstructure of sea urchin spines from the rnacroscale down to the nanoscale.Guidelines derived from this study see ceramics withlayered porosity as a possible biomimetic construction for appropriate applications.     

Purification of a Sperm Lectin Extracted from Spermatozoa of the Sea Urchin Hemicentrotus pulcherrimus

Hemagglutinating activity for human type A erythrocytes was detected in a sperm extract obtained by treatment with Triton X-100 of spermatozoa from the sea urchin Hemicentrotus pulcherrimus. Among tested sugars only N-acetyl-D-galactosamine had any inhibitory effect on the hemagglutinating activity of the sperm extract. The lectin was purified by a combination of affinity chromatography and ion-exchange chromatography. A single band was obtained after SDS-polyacrylamide gel electrophoresis of the purified lectin, corresponding to an apparent molecular weight of 15,000 daltons. Trypsin-generated fragments of the surface of eggs significantly inhibited hemagglutination of erythrocytes by the purified lectin. The biological role of the sperm lectin is discussed.     

The Apical Lamina and its Role in Cell Adhesion in Sea Urchin Embryos

The hyaline layer (HL) is an extracellular matrix surrounding sea urchin embryos which has been implicated in a cell adhesion and morphogenesis. The apical lamina (AL) is a fibrous meshwork that remains after removal of hyalin from the HL and the fibropellins (FP) are glycoproteins thought to be the principal components of the AL. Using anti-FP antibodies (AL-1 and AL-2) we report immunoprecipitations and affinity purifications yield a high molecular weight complex comprised of the FP glycoproteins. The three components form a complex, stabilized by disulphide cross-linking and have stochiometric ratios of 2 FPIa molecules to 1 each of FPIb and FPIII. Pulse chase experiments indicate all 3 FP's are synthesized throughout development with peaks in synthesis during cleavage and a sustained peak beginning at hatching. Using immunogold and immunoperoxidase localization, the FP localize to a fibrillar complex forming the innermost layer of the HL. In cell adhesion experiments, cells adhere to affinity purified FP in a temperature, time and concentration dependent manner. Cell adhesion to Fp is about 70% of that seen when hyalin is used as a substrate. Pretreating with AG1 and AG2 reduces in vitro cell adhesion by about 65%. We conclude FP's form a fibrillar complex, which is synthesized throughout early development and functions, with other components of the HL, as a substrate for cell adhesion.     

Energy Absorption in Functionally Graded Concrete Bioinspired by Sea Urchin Spines

Functionally Graded Concrete (FGC) is fabricated at the Institute for Lightweight Structures and Conceptual Design (ILEK) by using a layer-by-layer technique with two different technological procedures:casting and dry spraying.Functional gradations are developed from two reference mixtures with diametrically opposed characteristics in terms of density,porosity,compression strength and elasticity modulus.In this study the first mixture consists of Normal Density Concrete (NDC),with density about 2160 kg·m-3 while the second mixture helps to obtain a very lightweight concrete,with density about 830 kg·m-3.The FGC specimens have layers with different alternating porosities and provide superior deformability capacity under bulk compression compared to NDC specimens.In addition,the FGC specimens experienced a graceful failure behaviour,absorbing high amounts of energy during extended compression paths.The porosity variation inside the layout of tested specimens is inspired by the internal structure of sea urchin spines of heterocentrotus mammillatus,a promising role model for energy absorption in biomimetic engineering.     

The Effect of Estradiol Dipropionate on the Rate of Protein Synthesis in the Testicle of the Sea Urchin Strongylocentrotus nudus

The effect of estradiol dipropionate on the rate of protein synthesis in the testicle of the mature sea urchin Strongylocentrotus nuduswas studied. The injection of this estrogen considerably intensified ³H-leucine incorporation into the gonad. The change in the level of cell synthetic activity is assumed to be associated with a rise in effective incorporation of ³H-leucine into proteins following an increase in its intracellular level, and with an increase in the protein synthesis rate. The participation of sex hormones in the regulation of protein synthesis in the S. nudusgonad is discussed.     

Maternal Exogastrula-Inducing Peptides (EGIPs) and Their Changes during Development in the Sea Urchin Anthocidaris crassispina

Exogastrula-inducing activity was examined in eggs and embryos of the sea urchin Anthocidaris crassispina at various stages. During fractionation on a column of DEAE-cellulose, the exogastrula-inducing activity was found in the flow-through fraction at all developmental stages. In particular, the activity present in the flow-through fraction of unfertilized eggs represents the presence of maternal exogastrula-inducing peptides (EGIPs). The flow-through fractions from the column of DEAE-cellulose were applied to a column of Sephadex G-100 and the activities in the eluate were assayed. The active low-molecular-weight fraction was obtained in all cases with the exception of pluteus larvae, extracts of which contained another active fraction. Immunoblots of protein samples from eggs and embryos probed with antiserum against EGIP-D indicated that there is a major immunoreactive protein that migrates with an apparent molecular weight of about 6 kDa in all cases with the exception of pluteus larvae, and that there are two major immunoreactive proteins that migrate with apparent molecular weights of 6 kDa and 35 kDa, respectively, in pluteus larvae.     

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.     

Phagocytic Activity of Accessory Cells in the Gonad of the Sea Urchin <Emphasis Type="Italic">Strongylocentrotus nudus</Emphasis>

The ultrastructural mechanism of postspawning sperm resorption in the testes of the sea urchin Strongylocentrotus nudus is described. Two types of phagosomes (containing sperm and containing residual bodies) are formed in the cytoplasm of nutritive phagocytes. The phagosomes fuse with electron-dense globules, and their contents are gradually destroyed. Afterward, phagosomes are transformed into electron-transparent vacuoles, which are finally compressed by the surrounding cytoplasm.     

SCN— Blocks Hardening of the Fertilization Envelope of Sea Urchin Eggs and Adhesion of Blastomeres

Sea urchin eggs kept in artificial sea water (ASW) containing 0.01–0.3 M NaSCN in place of NaCI from within 2 min after insemination formed thin, enlarged fertilization envelopes, which were broken on mild agitation of egg suspensions more easily than those formed in Ca²⁺-free ASW. The blastomeres of almost all embryos derived from eggs treated with 0.2M SCN^— for 1 hr dissociated spontaneously, and did not reassociate with other blastomeres appreciably. Thus SCN^— probably denaturated some compound(s) participating in blastomere binding and hardening of the fertilization envelope. Abnormal arrangements of blastomeres, probably due to incomplete blastomere dissociation, were observed in embryos derived from eggs treated with 0.1 M SCN^— for 1 hr. Treatment of fertilized or unfertilized eggs with 0.05–0.1 M SCN^— for a short period caused concentration-dependent block of morphogenic processes such as formation of the archenteron and pluteus arms in the post-hatching period. The effects of SCN^— on morphogenesis were not inhibited by furosemide or 4,4'-diisothiocyano 2,2'-disulfonic stilbene. Presumably, the denaturation of several compounds in the egg surface by SCN^— causes abnormal morphogenesis of embryos. The inhibitory effects of SCN^— on hardening of the fertilization envelope, blastomere binding and morphogenesis were greater in the absence of Ca²⁺.     

Gastrulation in the Sea Urchin,Strongylocentrotus purpuratus,Is Disrupted by the Small Laminin Peptides YIGSR and IKVAV

Laminin is present on the apical and basolateral sides of epithelial cells of very early sea urchin blastulae. We investigated whether small laminin-peptides, known to have cell binding activities, alter the development of sea urchin embryos. The peptide YIGSR-NH₂ (850 μM) and the peptide PA22-2 (5 μM), which contains the peptide sequence IKVAV (Tashiro et al., J. Biol. Chem. 264, 16174, 1989), typically blocked archenteron formation when added to the sea water soon after fertilization. At lower doses, the YIGSR peptide allowed invagination of the archenteron but blocked archenteron extension and differentiation and evagination of the feeding arms. The effect of YIGSR and PA22-2 peptides declined when added to progressively older stages until no effect was seen when added at the mesenchyme blastula stage (24 hours after fertilization). Control peptides GRGDS, YIGSE, and SHA22, a dodeca-peptide with a scrambled IKVAV sequence, had no effect on development. The YIGSK peptide containing a conserved amino acid modification had only a small effect on gastrulation. The results suggest that YIGSR and IKVAV peptides specifically disrupt cell/extracellular matrix interactions required for normal development of the archenteron and feeding arms. Our recent finding that YTGIR is at the cell binding site of the B1 chain of S. purpuratus laminin supports this conclusion. Evidently, laminin or other laminin-like molecules are among the many extracellular matrix components needed for the invagination and extension of the archenteron during the gastrulation movements of these embryos.     

Hox基因及其在海胆中的研究进展

Hox(homeobox genes)基因是存在于染色体上的一段高度保守序列,其蛋白产物作为转录因子也是高度保守的。这些基因调节胚胎发育,尤其在脊椎动物前-后轴(antero-posterior axis)的发育过程中起着重要作用,并指导脊椎动物的体型形成。海胆是海洋中一类比较常见的无脊椎动物和主要的海水养殖动物,它的Hox基因对它动-植物轴(animal-vegetalaxis)的形成有调控作用,并对进化研究和养殖生产具有重要意义。综述了近年来Hox基因在海胆中的研究进展。     

Predation and the Control of the Sea Urchin Echinometra viridisand Fleshy Algae in the Patch Reefs of Glovers Reef, Belize

The massive reduction in sea urchin Diadema antillarum populations since the mid-1980s has been associated with large increases in the abundance of fleshy algae on many Caribbean reefs despite the availability of other sea urchin and finfish grazers. This study examined the ecology of a grazer living sympatrically with D. antillarum, the common and abundant sea urchin Echinometra viridis. I examined the role that finfish and invertebrate predators play in controlling the distribution of E. viridis as well as the ability of this sea urchin to control exposed fleshy algae on the patch reefs of the Glovers Reef Atoll lagoon. I found that the major predators of this sea urchin were Calamus bajonado (jolthead porgy), Balistes vetula and Canthidermis sufflamen (queen and ocean triggerfish), Lachnolaimus maximus (hogfish), and a gastropod, probably Cassis madagascariensis. The abundance of E. viridis is constrained by predation, which restricts E. viridis to cryptic locations, such as crevices. Sea urchins bit a smaller percentage of experimental algal assays than finfish. Finfish herbivory was associated positively with patch reef topographic complexity. Unexpectedly, E. viridis abundance was positively correlated with fleshy algal abundance, but negatively correlated with the frequency of finfish bites. Predators restrict E. viridis to crevices and therefore reduce their influence on exposed fleshy algae, even at moderately high population densities (up to 10 per square meter). Since net benthic primary production of coral reefs is most strongly associated with herbivory on exposed surfaces, it would appear that E. viridis is unable to maintain the same production as reefs dominated by D. antillarum. Received 5 November 1998; accepted 2 June 1999.