Coelomocytes and post-traumatic response in the common sea star Asterias rubens

Coelomocytes are recognized as the main cellular component of the echinoderm immune system. They are the first line of defense and their number and type can vary dramatically during infections or following injury. Sea stars have been used as a model system to study the regeneration process after autotomy or predation. In the present study we examined the cellular and biochemical responses of coelomocytes from the European sea star Asterias rubens to traumatic stress using immunochemical and biochemical approaches. In terms of trauma and post-traumatic stress period, here we consider the experimental arm amputation and the repair phase involved in the first 24 hours post-amputation, which mimicked a natural predation event. Four cell morphotypes were distinguishable in the coelomic fluid of both control and post-traumatic-stressed animals (phagocytes, amoebocytes, vibratile cells, hemocytes), but phagocytes were the major components, accounting for about 95% of the total population. Thus, the effects measured relate to the overall population of coelomocytes. A modest increase in the total number of freely circulating coelomocytes was observed 6 hours post-amputation. Interestingly, a monoclonal antibody (McAb) to a sea urchin embryo adhesion protein (toposome) cross-reacted with isolated sea star coelomocytes and stained the coelomic epithelium of control animals with an increase in trauma-stressed arms. In addition, coelomocytes from trauma-stressed animals showed a time-dependent increase in Hsp70 levels, as detected by both immunocytochemistry and immunoblotting within 24 hours after arm tip amputation, with a peak at 6 hours after amputation. Our findings indicate a clear role for coelomocytes and classic stress molecules in the post-traumatic stress associated with the early repair phase of regeneration.     

Identification of a macrophage-like mSubset in the axial organ sea star Asterias rubens.

The axial organ of the sea star Asterias rubens is a primitive immune organ. In addition to T and B-like cells phagocytic cells were also identified. In this report we demonstrate that these cells can be identified using immunocytochemistry together with the mouse to human monoclonal antibody CD68 KP1, normally used as a macrophage marker.     

Specific immune response in the sea star Asterias rubens: production of "antibody-like" factors

Axial organ cells from the sea star (Asterias rubens) inoculated 7 days before with TNP- or FITC-haptenated PAA beads and subsequently stimulated in vitro with the same antigen, produced and released a specific, soluble "antibody-like" substance that induced lysis of haptenated sheep erythrocytes. Fresh normal rabbit or guinea pig serum was essential for the lysis, suggesting the participation of complement components. The factor was produced by the total population of axial organ cells but not by nylon-wool adherent (B-like) or nonadherent (T-like) cells. These results provide further evidence of the existence, in the sea star, of a primitive immune system with characteristics reminiscent of the immune system of vertebrates.     

Human kappa-like expression in the axial organ of the sea star Asterias rubens (Echinoderma)

The axial organ of sea star Asterias rubens is a primitive immune organ which in certain conditions produces an antibody-like factor (ALF). This A.L.F. presents homologies with the human kappa chain. The ALF could be composed of many kappa chains devoid of any disulfide bond. The ontogeny of kappa expression was studied. Just after metamorphosis of the sea star, certain mesodermic cells which become axial organ cells express human kappa-like activity.     

Micro- and nanostructure of the adhesive material secreted by the tube feet of the sea star Asterias rubens

To attach to underwater surfaces, sea stars rely on adhesive secretions produced by specialised organs, the tube feet. Adhesion is temporary and tube feet can also voluntarily become detached. The adhesive material is produced by two types of adhesive secretory cells located in the epidermis of the tube foot disc, and is deposited between the disc surface and the substratum. After detachment, this material remains on the substratum as a footprint. Using LM, SEM, and AFM, we described the fine structure of footprints deposited on various substrata by individuals of Asterias rubens. Ultrastructure of the adhesive layer of attached tube feet was also investigated using TEM. Whatever the method used, the adhesive material appeared as made up of globular nanostructures forming a meshwork deposited on a thin homogeneous film. This appearance did not differ according to whether the footprints were fixed or not, and whether they were observed hydrated or dry. TEM observations suggest that type 2 adhesive cells would be responsible for the release of the material constituting the homogeneous film whereas type 1 adhesive cells would produce the material forming the meshwork. This reticulated pattern would originate from the arrangement of the adhesive cell secretory pores on the disc surface.     

Characterization of the protein fraction of the temporary adhesive secreted by the tube feet of the sea star Asterias rubens

Sea stars are able to make firm but temporary attachments to various substrata by secretions released by their tube feet. After tube foot detachment, the adhesive secretions remain on the substratum as a footprint. Proteins presumably play a key role in sea star adhesion, as evidenced by the removal of footprints from surfaces after a treatment with trypsin. However, until now, characterisation was hampered by their high insolubility. In this study, a non-hydrolytic method was used to render most of the proteins constituting the adhesive footprints soluble. After analysis by SDS-PAGE, the proteins separated into about 25 bands, which ranged from 25 to 450 kDa in apparent molecular weight. Using mass spectrometry and a homology-database search, it was shown that several of the proteins are known intracellular proteins, presumably resulting from contamination of footprint material with tube foot epidermal cells. However, 11 protein bands, comprising the most abundant proteins, were not identified and might correspond to novel adhesive proteins. They were named ‘Sea star footprint proteins’ (Sfps). Tandem mass spectrometry analysis of the protein bands yielded 43 de novo-generated peptide sequences. Most of them were shared by several, if not all, Sfps. Polyclonal antibodies were raised against one of the peptides (HEASGEYYR from Sfp-115) and were used in immunoblotting. They specifically labelled Sfp-115 and other bands with lower apparent molecular weights. The different results suggest that all Sfps might belong to a single family of related proteins sharing similar motifs or, alternatively, they are the products of polymerization and/or degradation processes.     

An ultrastructural study of relationships between the ovarian haemal system,follicle cells,and primary oocytes in the sea star,Asterias rubens

Summary The genital haemal sinus, present throughout the gonad wall of sea stars, is supposed to be the site of ultimate accumulation of nutrients for the germinal epithelium. Early vitellogenic pear-shaped oocytes are attached to this sinus by stalk-like processes. The ultrastructure of this association and of the oocyte-follicle cell complex is described with emphasis on mechanisms involved in oocyte nutrition.The genital haemal sinus, and sometimes portions of the surrounding genital coelomic sinus, contain a fine granular ground substance and amoeboid cells. Material similar to the haemal ground substance also fills vacuities in the inner basal laminae of the haemal sinus and intervenes between this layer and adjacent germinal and follicle cells in the ovarian lumen.Vitellogenesis is first detectable as numerous vacuoles accumulate within the oocyte-stalk near the haemal sinus; they contain flocculent material and often fuse with adjacent lysosome-like vacuoles. As vitellogenesis proceeds, oocytes develop complex and tenuous connections with the haemal sinus. These consist of a network of pseudopodia that interdigitate with thin sheet-like extensions of follicle cells. These cells are attached to the oolemma by microfilamentous processes and contain regularly arranged concentrations of glycogen granules and well developed rough endoplasmic reticulum.It is concluded, (1) that follicle cells provide each oocyte with a compartmentalized microenvironment within the ovarian lumen, (2) that such compartments are intimately associated with the nutrient laden haemal sinus, and (3) that nutritive and vitellogenic substances, derived extragonadally and stored temporarily in the ovarian wall, can pass through the oocyte-stalk.     

Androgen metabolism in somatic and germinal tissues of the sea star Asterias vulgaris.

1. Cell-free homogenates of male and female pyloric caeca, body wall, testis and ovary were incubated with radiolabeled 3H-androstenedione. 2. Pyloric caeca had highest rates of androstenedione conversion. The predominant metabolites in the pyloric caeca were testosterone, 5 alpha-androstane-3 beta, 17 beta-diol and 5 beta-androstane-3 beta, 17 beta-diol. 3. In body wall, testicular and ovarian homogenates, androstenedione was converted primarily to testosterone and also to 5 alpha-androstanedione and epiandrosterone. 4. Qualitative and quantitative differences in androgen metabolism in somatic and germinal tissues may be related to tissue-specific regulation of cellular metabolism.     

Histones from gonads of the star-fish Asterias rubens.

1. Histones were isolated from gonads of the star-fish Asterias rubens and characterized by their amino acid composition and their electrophoretic migration. 2. Comparative studies with calf thymus homologous histones show the highly conservative structure of the histones H3 and H4, and the variability of the other histones namely H1 and H2B.     

Immunocompetent cells in the starfish Asterias rubens. An ultrastructural study

Cells from the axial organ of the starfish Asterias rubens were fractionated into two populations, adherent and non-adherent to nylon wool. In both populations the ultrastructural study revealed the presence of cells resembling the lymphocytes of the vertebrates, as well as phagocytic, peroxidase positive cells. The lymphocyte-like cells in the non-adherent population (average diameter 4 mu) have a high nucleo-cytoplasmatic ratio and are morphologically similar to Th lymphocytes while the adherent cells (average diameter 5.5 mu) are more similar to Bm lymphocytes. These observations are in line with the hypothesis that there exist, in the starfish, a primitive immune system with characteristics resembling those of the immune system of vertebrates.     

Isolation and characterization of a sialidase from the starfish Asterias rubens

The starfish Asterias rubens contains a soluble sialidase (1.4 mU/mg homogenate protein), which was purified over 500-fold to apparent homogeneity by ammonium sulfate precipitation, gel filtration and affinity chromatography on immobilized 2-deoxy-2,3-didehydroneuraminic acid. The native sialidase has a molecular mass of 230 kDa (gel filtration) and consists of 4 subunits of each 63 kDa, as determined by SDS-gel electrophoresis. Its isoelectric point is at pH 4.9, the activity is optimum at pH 4.2 and 37 degrees C, and it hydrolyses preferably 4-methylumbelliferyl-alpha-N-acetyl-neuraminic acid, followed by sialyllactose and glycoproteins. The hydrolysis rate is decreased or stopped by the presence of O-acetyl groups on the sialic-acid residue to be cleaved. N-Glycoloyl residues also retard enzyme action, as well as alpha(2-6) bonds when compared with alpha(2-3) linkages. This relatively stable enzyme is inhibited by mercury or copper ions, 2-deoxy-2,3-didehydro-N-acetylneuraminic acid and by the increase of ionic strength. The evolutionary significance of starfish sialidase is discussed.     

Anatomy of the ovaries of the starfish Asterias rubens (Echinodermata)

Summary The ovaries of the starfish Asterias rubens were studied histologically and ultrastructurally. The reproductive system in female specimens consists of ten separate ovaries, two in each ray. Each ovary is made up of a rachis with lateral primary and secondary folds: the acini maiores and acini minores. The ovarian wall is composed of an outer and an inner part, separated by the genital coelomic sinus. The ovarian lumen contains oocytes in various phases of oogenesis, follicle cells, nurse cells, phagocytosing cells and steroid-synthesizing cells.Oogenesis is divided into four phases: (i) multiplication phase of oogonia, (ii) initial growth phase of oocytes I, (iii) growth phase proper of oocytes I, and (iv) post-growth phase of oocytes I. The granular endoplasmic reticulum and the Golgi complex of the oocytes appear to be involved in yolk formation, while the haemal system, haemal fluid and nurse cells may also be important for vitellogenesis. The haemal system is discussed as most likely being involved in synchronizing the development of the ovaries during the annual reproductive cycle and in inducing, stimulating and regulating the function of the ovaries.Steroid-synthesizing cells are present during vitellogenesis; a correlation between the presence of these cells and vitellogenesis is discussed.     

Isolation and characterization of cytidine-5'-monophosphate-N-acetylneuraminate hydroxylase from the starfish Asterias rubens

The sialic acid N-glycolylneuraminic acid (Neu5Gc) is formed by cytidine-5'-monophosphate-N-acetylneuraminic acid (CMP-Neu5Ac) hydroxylase (EC 1.14.13.45). The enzyme from mammals exhibits several unusual characteristics, raising questions about its evolution. Since echinoderms are the most primitive organisms possessing glycoconjugate-bound Neu5Gc, studies on the hydroxylase from members of this phylum may yield insights into the origin and development of the hydroxylase. Investigations on crude CMP-Neu5Ac hydroxylase in gonads from the starfish Asterias rubens revealed that it shares many properties with its mammalian counterpart. However, the echinoderm hydroxylase also exhibits fundamental differences, particularly its association with a membrane and a requirement for high ionic strength for optimal activity. Here, we describe the isolation of the CMP-Neu5Ac hydroxylase from A. rubens gonads using anion exchange chromatography and chromatography on immobilized cytochrome b(5). The enzyme was enriched 137-fold with a yield of 13%. The preparation exhibited a main polypeptide of 76 kDa, consistent with a cDNA sequence published earlier, and a minor protein of 64 kDa. A kinetic characterization showed that salt activation of this enzyme results from an increase in affinity for CMP-Neu5Ac. Evidence for the formation of a ternary complex of hydroxylase, CMP-Neu5Ac and cytochrome b(5) is also presented. The mechanistic and physiological significance of these results is discussed.