Thyroxine and vitamin D in the gorgonian Leptogorgia virgulata

The gorgonian coral Leptogorgia virgulata contains thyroxine, or a thyroxine-like substance, referred to here as G-T₄. The G-T₄ levels were significantly higher in colonies collected in the summer vs. winter months. Using immunocytochemical techniques, G-T₄ was localized in the axis, polyp epithelium, and within the electron dense bodies of scleroblasts (spicule-forming cells), as well as on the periphery of spicules. G-T₄ was also localized in the mesoglea between closely adjacent scleroblasts. The effects of exogenous T₄ on the uptake of Ca⁴⁵ was determined in spicule, tissue and axis fractions of L. virgulata. The uptake of Ca⁴⁵ increased in T₄ treated spicules but decreased in the tissue fraction for all time periods tested. The uptake of Ca⁴⁵ into axes was not affected by exogenous T₄ until day 10 of the study. These data suggest that G-T₄ may function in the process of spicule formation. 1,25-dihydroxyvitamin D apparently is synthesized via ultraviolet radiation. Colonies deprived of ultraviolet radiation had significantly more ‘irregular’ spicules than colonies maintained in ultraviolet radiation. Exposure to sunlight therefore may be associated with the process of normal spicule formation.     

Spicule Formation in the Invertebrates with Special Reference to the Gorgonian Leptogorgia virgulata

Many of the invertebrates possess calcium carbonate spicules.This paper is a review of the formation of these structuresin the Porifera, Coelenterata, Platyhelminthes, Mollusca, Echinodermataand Ascidiacea. Mature spicules appear to be extracellular structures.Sponge spicules initiate intercellularly then become extracellular.Alcyonarian, turbellarian, echinoid and ascidian spicule depositionbegins intracellularly and then becomes extracellular. The continuationof growth in the extracellular environment has not been documentedexcept for the echinoids. Placophoran spicules initiate andremain as extracellular structures. Early spicule growth seemsto occur from or within a single cell. However, cell aggregationand/or neighboring cells appear to be important to the processof spicule formation. The spicule forming cells, in general,are found in a collagenous medium which may be associated withspicule growth. The organic matrix from the spicules of the gorgonian Leptogorgiavirgulata is a glycoprotein. Autoradiography reveals that thismatrix is apparently synthesized in the rough endoplasmic reticulumand Golgi complexes and then transported to the spicule formingvacuole via Golgi vesicles. To gain information about the entryand transport of calcium ions, the effects of ouabain and vanadateon calcium uptake were examined. Ouabain had no effect on calciumuptake. Vanadate treatment increased the uptake of calcium inscleroblasts and epithelial tissue and decreased its uptakein spicules. This may suggest that vanadate sensitive ATPasesare involved in the pumping of calcium out of scleroblasts,out of epithelial cells into the mesoglea, and into scleroblastorganelles. Autoradiography using ⁴⁵Ca indicates that the majorityof these ions initially accumulate in the branch axis. The labelmoves through the axial epithelium to the mesoglea and reachesthe spiculeforming vacuoles in the scleroblasts via dense bodies     

Mechanism for electrosilent Ca2+ transport to cause calcification of spicules in sea urchin embryos

Embryos of the sea urchin, Hemicentrotus pulcherrimus, kept in sea water containing the calcium antagonists, diltiazem and verapamil, or an anion transport inhibitor, 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS), during a developmental period between the mesenchyme blastula and the pluteus corresponding stage, became abnormal plutei with poorly developed arms and quite small spicules. Treatment with ethacrynic acid and furosemide, inhibitors of chloride transport, during the same period of development yielded quasi-normal plutei with poor spicules and somewhat developed arms. In late gastrulae, the inhibitory effects of these calcium antagonists and DIDS on the uptake of 45Ca2+ in whole embryos were as strong as those on 45Ca deposition in spicules, whereas the effects of chloride transport inhibitors on calcium deposition in the spicules were markedly stronger than on its uptake in whole embryos. Electrosilent uptake of Ca2+ seems to be established mainly by coupled influx of chloride in the cells which mediate spicule calcification, and by concomitant influx of anions in the other cells. In swimming blastulae, 45Ca2+ uptake was inhibited by calcium antagonists and DIDS, but not by chloride transport inhibitors. Ca2+ uptake probably becomes coupled with chloride influx only in embryos in which spicule calcification occurs.     

Scleroblast cultures from the gorgonianLeptogorgia virgulata (Lamarck) (Coelenterata: Gorgonacea)

Summary Scleroblasts were separated from fragmented tissue of growing tips ofLeptogorgia virgulata and cultured using a modification of the technique of Rannou. Replacement of fetal bovine serum with horse serum seemed to increase scleroblast viability. Cell adhesion occurred from 14 to 43 d. Cultured scleroblasts demonstrated cell aggregation, spicule formation, and extrusion of spicules into the external medium. Cells showing spicules in the process of being extruded appeared on the average after 24 d of culture. Variability among cultures was marked with respect to both division and spicule formation. Healthy cultures were maintained for more than 4 mo. This work was supported by National Science Foundation grants PCM8201389 and DCB8502698. This is contribution No. 674 of Belle W. Baruch Institute for Marine Biology and Coastal Research, University of South Carolina.     

Ultrastructural investigation of spicule formation in the gorgonianLeptogorgia virgulata (Lamarck) (Coelenterata: Gorgonacea)

Summary Ultrastructural examination of original and regenerated branch tips of the gorgonianLeptogorgia virgulata reveals that spicule formation begins with the aggregation of scleroblasts in the mesoglea. Calcite crystal deposition occurs within a Golgi vacuole containing organic matrix. Vacuole size increases while matrix incorporation and subsequent crystal growth continue, filling the vacuole. At approximately this time, the scleroblasts dissociate and wart formation begins. Further spicule growth stretches the cell into a thin envelope. Fusion of vacuole and plasma membrane followed by breach formation during spicule growth, as well as scleroblast atrophy or migration from mature spicules, result in the transition of the spicule from the intracellular to the extracellular environment. The results also reveal aborted spicules and digestive bodies, implying possible relationships among calcification, detoxification, and waste management.Contribution No 436, Belle W. Baruch Institute for Marine Biology and Coastal Research, University of South Carolina, Columbia, South Carolina, 29208, USA     

An autoradiographic study of calcium transport in spicule formation in the gorgonian Leptogorgia virgulata (Lamarck) (Coelenterata: Gorgonacea)

Summary The route of calcium transport to the sites of spicule formation in the gorgonian Leptogorgia virgulata has been examined by the use of ⁴⁵Ca as a tracer in light- and electron-microscopic autoradiography. From 1 to 15 min after the 15-min incubation the tracer accumulates in the axis. After 15 min there is a movement of label out of the axis largely to the peripheral region of the axis, the axial epithelium, and the mesoglea. By 60 min much of the label is in the spicules reaching its maximum level at 120 min. When calcium enters the scleroblast from the mesoglea, it appears to be transported to the spicule by electron-dense bodies. There does not appear to be a simultaneous release of all ionic calcium from the axis, but rather a continuously increasing efflux which levels off at 60–120 min. Not all of the calcium reaching the axis will traverse it en route to spicules; instead, a portion of it apparently precipitates as an amorphous compound.Contribution No. 550; Belle W. Baruch Institute for Marine Biology and Coastal Research, University of South Carolina, Columbia, South Carolina 29208 USA     

UV protection in the photosymbiotic ascidian Didemnum molle inhabiting different depths

Didemnum molle is a colonial ascidian that harbors the prokaryotic photosymbiont Prochloron in its cloacal cavity. Colonies occur over a relatively wide bathymetric range (approximately 0-30 m), and colony color is widely variable, partly depending on depth. Colonies in shallow sites are bright white, with densely distributed spicules, and often with brown or dark gray pigmentation, while colonies in deeper sites are less pigmented, with sparsely distributed spicules. Didemnum molle colonies contain mycosporine-like amino acids (MAAs) as UV-absorbing substances. These include mycosporine-glycine, shinorine, and porphyra-334. Among colonies from 5-, 10-, 15-, and 20-m depths, the concentration of total MAAs was significantly high at 10 m and low at 20 m. Colonies at 10 m need to maintain low spicule densities to have enough photosynthetically active radiation (PAR) to maintain the photosymbionts, and they probably concentrate MAAs to block UV radiation without attenuating PAR. Because high levels of PAR cause photoinhibition of photosynthesis, spicules and pigment cells would be more effective for photoprotection in shallow water. Colonies of D. molle may adjust the light conditions for photosymbionts by combining MAAs, spicules, and pigment cells in varying amounts.     

Seasonal localization of a collagenous protein in the organic matrix of spicules from the gorgonian Leptogorgia virgulata (Cnidaria: Gorgonacea)

Spicules of the gorgonian Leptogorgia virgulata possess an insoluble matrix fraction that is predominantly collagenous in summer months. This collagenous component is largely absent in winter months. Using an antibody directed against the 140 kD collagenous protein (CP) of the insoluble matrix, immuno-gold labelling was employed to localized this protein at the transmission electron-microscopy level throughout the year, and in different areas of the gorgonian colonies. Within the tip regions, the 140 kD CP varied throughout the year in the spicules, electron-dense bodies (EDBs) of scleroblasts, polyp vesicles, desmocytes and axes. In the mid and base regions, the 140 kD CP varied throughout the year in the spicules, EDBs and lysosomes of scleroblasts, desmocytes and axes. This variation in the location and density of the label suggests a dynamic annual cycling of the collagenous component of the insoluble matrix. EDBs may transport a collagenous component of the matrix to the spicule-forming vacuole. A component of the 140 kD CP may be transported and/or degraded by polyp vesicles and lysosomes, respectively. The pattern of labelling of the axial region suggests that translocation and storage of a component of the collagenous protein may occur. Environmental factors may be responsible for the triggering of matrix cycling.     

Magnetic resonance imaging of the siliceous skeleton of the demosponge Lubomirskia baicalensis

The skeletal elements (spicules) of the demosponge Lubomirskia baicalensis were analyzed; they are composed of amorphous, non-crystalline silica, and contain in a central axial canal the axial filament which consists of the enzyme silicatein. The axial filament, that orients the spicule in its longitudinal axis exists also in the center of the spines which decorate the spicule. During growth of the sponge, new serially arranged modules which are formed from longitudinally arranged spicule bundles are added at the tip of the branches. X-ray analysis revealed that these serial modules are separated from each other by septate zones (annuli). We describe that the longitudinal bundles of spicules of a new module originate from the apex of the earlier module from where they protrude. A cross section through the oscular/apical-basal axis shows that the bundle rays are organized in a concentric and radiate pattern. High resolution magnetic resonance microimaging studies showed that the silica spheres of the spicules in the cone region contain high amounts of 'mobile' water. We conclude that the radiate accretive growth pattern of sponges is initiated in the apical region (cones) by newly growing spicules which are characterized by high amounts of 'mobile' water; subsequently spicule bundles are formed laterally around the cones.     

Enhancement of spicule formation and calcium uptake by monoclonal antibodies to fibronectin-binding acid polysaccharide in cultured sea urchin embryonic cells

The effect of monoclonal antibodies to fibronectin-binding acid polysaccharide (anti-FAPS) on differentiation of primary mesenchyme cells and spicule formation was examined in cultured embryonic cells isolated from the sea urchin, Hemicentrotus pulcherrimus. Spicule formation of micromere-derived cells was enhanced by anti-FAPS. The increase of spicule formation correlated with the increase of calcium uptake into micromere-derived cells and spicules. Furthermore, both spicule formation and calcium uptake were inhibited by calcium-channel blockers (verapamil, diltiazem and nicardipine) and divalent ions (manganese and cobalt). These results suggest that FAPS, a component of the blastocoelic extracellular matrix surrounding the primary mesenchyme cells, may regulate the level of calcium uptake and spicule formation.     

Loss of phospholipid asymmetry in dilauroylphosphatidylcholine induced plasma membrane vesicles from human platelets

Incubation of human platelets with unilamellar vesicles composed of dilauroylphosphatidylcholine (DLPC) induces shedding of small vesicular structures from the platelet plasma membrane. No significant cell lysis is observed during the process of shedding. Isolated spicules contain the major membrane glycoproteins, Ib, IIb, and IIIa, which are used to define the sidedness of the spicule membrane. These glycoproteins are completely susceptible to chymotrypsin treatment, whereas cytoskeletal proteins are inaccessible towards this enzyme. This demonstrates that the spicule membranes have a right-side-out orientation in as far as membrane proteins are concerned. Isolated spicules were 30-fold more active than platelets in stimulating prothrombin conversion to thrombin by the prothrombinase complex (factors Xa, Va and Ca2+). The increased prothrombinase activity reflects an increased amount of phosphatidylserine in the outer leaflet of the spicule membrane. Protein analysis of platelet spicules and native platelets reveals a number of differences, the most conspicuous of which is the virtual absence of myosin in the spicule preparations. It is proposed that a lack of myosin produces a different cytoskeletal organization in the spicules. This enables phosphatidylserine to become exposed at the outer surface of the spicule membrane.     

Distributions of H+,K+-ATPase and Cl–,HCO3–-ATPase in micromere-derived cells of sea urchin embryos

In cultured cells derived from isolated micromeres of sea urchin eggs, H+,K+-ATPase activity, which became detectable simultaneously with the initiation of spicule formation, was localized in the plasma membrane and the microsome fractions. Activities of marker enzymes for plasma membrane, 5'-nucleotidase, Na+,K+-ATPase, and adenylate cyclase, were found to be high in the plasma membrane fraction. Considerable activity of rotenone-insensitive NADPH-cytochrome c reductase, a marker enzyme for microsome, was detectable in the microsome fraction. These fractions exhibited barely any appreciable activity of markers for the other organellae. H+,K+-ATPase in plasma membrane probably mediates H+ release from the cells, in which H+ is produced in overall reaction to form CaCO3, the main component of spicules, from Ca2+, CO2 and H2O. Cl-,HCO3(-)-ATPase activity was also found in these two fractions before and after the initiation of spicule formation. After initiation, the skeletal vacuole fraction was obtained from subcellular structures containing spicules. Considerable activity of Cl-,HCO3(-)-ATPase was observed in this fraction, which exhibited a weak activity of UDP-galactose: N-acetylglucosamine galactosyltransferase, a marker enzyme for Golgi body. Cl-,HCO3(-)-ATPase in the skeletal vacuole membrane probably mediates HCO3- transport into the vacuoles to supply HCO3- for spicule formation.     

CRYSTAL PROPERTY OF THE LARVAL SEA URCHIN SPICULE*

A pair of pluteus skeletal spicules arises from a pair of calcareous granules via the triradiate form. In polarized light, each spicule behaves as though carved out of a single crystal of magnesian calcite. The optic axis lies perpendicular to the plane of the triradiate and parallel to the body rod of the pluteus. However, in the scanning electron microscope, the spicule surface appeared smooth or somewhat spongy and manifested no crystal faces. Neither etching nor fracturing revealed underlying crystalline texture. Nevertheless, rhombohedral calcite crystals could be grown epitaxially onto isolated spicules immersed in a medium containing CaCl₂ and NaHCO₃. The optic axes of all crystals coincided with the optic axis of the spicule on which they were grown. Corresponding faces of the crystals were all aligned parallel to each other despite the complex shape of each spicule. Where the left and right spicules joined, two mutually tilted sets of crystals were observed but not crystals of intermediate orientation. Thus, the sea urchin larval spicule is built from a stack of molecularly contiguous microcrystals but its overall shape is generated by the mesenchyme cells independent of the magnesian calcite crystal habit.     

An ultrastructural study on spicule formation in the pennatulid colony Renilla reniformis

Examination of the coenenchyme tissues of Renilla reniformis revealed two regions of crystal formation: the endoderm containing small oval deposits of unknown composition and the mesoglea containing larger elongated spicules composed of calcite. Spicule formation takes place intracellularly in scleroblasts and may be explained by the following sequential processes: an organic matrix consisting of a homogeneous ground substance and fibers is formed in a large vacuole. Calcite needles 0.4 μ in diameter develop in close association with matrix fibers and vesicles, and grow to form the central core of the spicule. Large electron dense bodies dominate the scleroblast cytoplasm during these early stages of growth. In a later stage, smaller needles 0.2 μ in diameter develop surrounding the core to form the distal lobes of the spicule. ‘Lollipop’-shaped vesicles containing fibers appear in the scleroblast cytoplasm at the onset of this stage. This material is released at the calcification front and presumably is incorporated into the spicule as an organic matrix of crystals in the distal lobes.     

SPICULE FORMATION IN VITRO BY THE DESCENDANTS OF PRECOCIOUS MICROMERE FORMED AT THE 8-CELL STAGE OF SEA URCHIN EMBRYO*

The micromeres at the 16-cell stage of sea urchin embryo have already been endowed with a faculty to self-differentiate into spicule-forming cells (11). The present experiment was designed to test whether the factor(s) necessary for such self-differentiation had already been localized at the 8-cell stage in an area corresponding to the presumptive micromere region in Hemicentrotus pulcherrimus. Since the blastomeres at the 8-cell stage are all equal in size in normal embryo, unequal 3rd cleavage, by which small blastomeres are pinched off toward the vegetal pole (precocious micromeres), was experimentally induced either by treatment with 4NQO (4-nitroquinoline-1-oxide) at the 2-cell stage or by continuous culture in Ca-free sea water. The precocious micromeres were cultured in vitro in natural sea water containing horse serum. Descendants of the precocious micromeres formed spicules. In comparison their spicule formation with that by the descendants of the micromere of normal embryo, no differences were found regarding 1) time of initiation of spicule formation, 2) rate of growth of spicule, 3) size and shape of resultant spicule and 4) percentage of clones which formed spicule. The fact indicates that factor(s) indispensable for self-differentiation into spicule-forming cells have already been localized near the vegetal pole as early as the 8-cell stage.     

Effect of thioloxidant diazene dicarboxylic acid bis-(N'-methylpiperazide) (DIP) on 45Ca2+ net uptake into rat pancreatic islets

The effect of DIP (an oxidant of glutathione) on 45Ca2+ net uptake induced by a variety of stimulators of insulin secretion was studied in rat pancreatic islets. In addition the effect of exogenous glutathione (GSH) on 45Ca2+ net uptake in response to glucose was tested. DIP (0.1 mM) inhibited the increase of 45Ca2+ net uptake in the presence of glucose (16.7 mM) and glyceraldehyde (10 mM). A similar inhibitory effect could be demonstrated, when 45Ca2+ net uptake was enhanced by tolbutamide (100 micrograms/ml), glibenclamide (0.5 micrograms/ml), b-BCH (20 mM), 2-ketoisocaproate (20 mM), arginine (20 mM) in the presence of 3 mM glucose or by high extracellular potassium (20 mM). The increase of 45Ca2+ net uptake stimulated by leucine (20 mM) plus glucose (3 mM) was further augmented by DIP. Exogenous GSH did not affect 45Ca2+ net uptake in the presence of (5.6-16.7 mM) glucose. It is suggested that 45Ca2+ net uptake of pancreatic islets depends on the redox state of islet thiols regardless of whether uptake is promoted via inhibition of potassium efflux (nutrients, sulfonylureas) or by high potassium and arginine. The voltage sensitive calcium-channel is the site of action of critical thiols. It is possible that these thiols are localized at the inner side of the plasma membrane.     

Structural investigations of octocoral sclerites

A common structural pattern (as observed under the SEM) in the main body of sectioned sclerites of the family Alcyoniidae, and octocorals in general, is the arrangement of acicular crystals in concentric layers. The crystals roughly follow the direction of the spicule axis, however, the sectioned tubercles of large Sinularia spicules have the acicular crystals oriented in the direction of the tubercles (i.e. perpendicular to the spicule axis), contrary to the sectioned tubercles of Sarcophyton and Lobophytum spicules. They reveal some rod-like structures, furcating the acute processes at the top of the tubercles. Sectioned small, club-like Sinularia sclerites show numerous tiny acicular crystals, oriented with their long axes at a fairly constant degree of inclination around a central axis. SEM studies of sectioned Cladiella sclerites show a granulate structure organized in concentric layers, but lacking acicular crystals. The Silurian Atractosella cataractaca show important characters in common with Recent alcyoniid species.     

Characterization of sea urchin primary mesenchyme cells and spicules during biomineralization in vitro

An in vitro culture system for primary mesenchyme cells of the sea urchin embryo has been used to study the cellular characteristics of skeletal spicule formation. As judged initially by light microscopy, these cells attached to plastic substrata, migrated and fused to form syncytia in which mineral deposits accumulated in the cell bodies and in specialized filopodial templates. Subsequent examination by scanning electron microscopy revealed that the cell bodies and the filopodia and lamellipodia formed spatial associations similar to those seen in the embryo and indicated that the spicule was surrounded by a membrane-limited sheath derived by fusion of the filopodia. The spicules were dissolved from living or fixed cells by a chelator of divalent cations or by lowering the pH of the medium. However, granular deposits found in the cell bodies appeared relatively refractory to such treatments, indicating that they were inaccessible to agents that dissolved the spicules. Use of rapid freezing and an anhydrous fixative to preserve the syncytia for transmission electron microscopy and X-ray microprobe analysis, indicated that electron-dense deposits in the cell bodies contain elements (Ca, Mg and S) common to the spicule. Examination of the spicule cavity after dissolution of the spicule mineral revealed openings in the filopodia-derived sheath, coated pits within the limiting membrane and a residual matrix that stained with ruthenium red. Concanavalin A--gold applied exogenously entered the spicule cavity and bound to matrix glycoproteins. Based on these observations, we conclude that components of the spicule initially are sequestered intracellularly and that spicule elongation occurs in an extracellular cavity. Ca2+ and associated glycoconjugates may be routed in this cavity via a secretory pathway.     

Proteins of calcified endoskeleton: II partial amino acid sequences of endoskeletal proteins and the characterization of proteinaceous organic matrix of spicules from the alcyonarian, Synularia polydactyla

Calcified organic substances in the skeleton contain a protein-polysaccharide complex taking a key role in the regulation of bio-calcification. However, information concerning the matrix proteins in alcyonarian and their effect on calcification process is still unknown. For this reason, we have studied the organic matrix of endoskeletal spicules from the alcyonarian coral, Synularia polydactyla, to analyze the proteins with their sequences and investigate the functional properties by a molecular approach. The separated spicules from the colony were identified by scanning electron microscope (SEM). The soluble organic matrix comprised 0.04% of spicule weight. By recording decline of pH in the experimental design, the inhibitory effect of the matrix on CaCO3 precipitation was revealed. Prior to electrophoresis, our analysis of proteins extracted from the soluble organic matrix of the spicules revealed an abundance of proteins in molecular weight. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the preparations showed seven bands of proteins with an apparent molecular mass of 109, 83, 70, 63, 41, 30 and 22 kDa. The proteins were electrophoresed on Tricine-SDS-PAGE after electro-elution treatment, and then transferred to polyvinylidene difluoride (PVDF) membranes and their N-termini were sequenced. Two bands of proteins of about 70 and 63 kDa successfully underwent N-terminal amino acid sequencing. For the detection of calcium binding proteins, a Ca2+ overlay analysis was conducted on the extract by 45Ca autoradiography. The 109 and 63 kDa calcium binding proteins were found to be radioactive. Periodic acid schiff staining indicated that 83 and 63 kDa proteins were glycosylated. An assay for carbonic anhydrase, which is thought to play an important role in the process of calcification revealed low level of the activity. These findings suggest that the endoskeletal spicules of alcyonarian corals have protein-rich organic matrices, which might be related to the calcification process.     

Characterization of proteins from the matrix of spicules from the alcyonarian, Lobophytum crassum

The organic matrix of spicules of the alcyonarian coral, Lobophytum crassum, was studied to investigate its molecular characteristics and functional properties. The shape of the spicules was identified using scanning electron microscopy. The soluble organic matrix comprised 0.03% of the spicule weight. The SDS-PAGE analysis of the preparation showed four protein bands with apparent molecular weights of 37, 48, 67 and 102 kDa. The 67- and 102-kDa proteins appeared to be calcium binding proteins, detected as radioactive bands by ⁴⁵Ca autoradiography. The 67-kDa protein appears to be glycosylated. The N-terminal amino acid sequence of the 67 kDa was determined; 7 of 20 residues were acidic. A database search for homologous proteins did not give a clear indication of the function of the 67-kDa protein. The isolated organic matrix possesses carbonic anhydrase activity which functions in calcium carbonate crystal formation, indicating that organic matrix is not only structural protein but also a catalyst. An interpretation of these results is that the spicule of alcyonarian corals has a proteinaceous organic matrix related to the calcification process.