Altered actin cytoskeleton in ageing eggs of starfish affects fertilization process.

Integrity of oocytes is of pivotal interest in the medical and zootechnical practice of in vitro fertilization. With time, oocytes undergo deterioration in quality, and ageing oocytes often exhibit compromised competence in fertilization and the subsequent embryonic development. With ageing oocytes and eggs of starfish (Astropecten aranciacus), we addressed the issue by examining changes of the subcellular structure and their performance at fertilization. Ageing eggs were simulated in two different experimental paradigms: i) oocytes were overmatured by 6 hours stimulation with 1-methyladenine (1-MA); ii) oocytes were removed from the gonad and maintained in seawater for 24 or 48 h before applying the hormonal stimulation (1-MA, 70 min). These eggs were compared with normally matured eggs (stimulated after isolation from the gonad with 1-MA for 70 min) with respect to the sperm-induced intracellular Ca2+ signaling and the structural changes of the egg surface. The cytoskeletal and ultrastructural differences in these eggs were assessed by confocal and transmission electron microscopy, respectively. In the two categories of ageing eggs, we have found remarkable structural modifications of the actin cytoskeleton and the cortical vesicles beneath the plasma membrane. At fertilization, these ageing eggs manifested an altered pattern of intracellular Ca2+ release, aberrant actin dynamics, and increased rate of polyspermy often despite full elevation of the fertilization envelope. Taken together, our results highlight the importance of spatio-temporal regulation of the actin cytoskeleton in the cortex of the eggs, and we postulate that the status of the actin cytoskeleton is one of the major determinants of the oocyte quality that ensures successful monospermic fertilization.

F-Actin nucleated on chromosomes coordinates their capture by microtubules in oocyte meiosis.

Capture of each and every chromosome by spindle microtubules is essential to prevent chromosome loss and aneuploidy. In somatic cells, astral microtubules search and capture chromosomes forming lateral attachments to kinetochores. However, this mechanism alone is insufficient in large oocytes. We have previously shown that a contractile F-actin network is additionally required to collect chromosomes scattered in the 70-µm starfish oocyte nucleus. How this F-actin-driven mechanism is coordinated with microtubule capture remained unknown. Here, we show that after nuclear envelope breakdown Arp2/3-nucleated F-actin "patches" form around chromosomes in a Ran-GTP-dependent manner, and we propose that these structures sterically block kinetochore-microtubule attachments. Once F-actin-driven chromosome transport is complete, coordinated disassembly of F-actin patches allows synchronous capture by microtubules. Our observations indicate that this coordination is necessary because early capture of chromosomes by microtubules would interfere with F-actin-driven transport leading to chromosome loss and formation of aneuploid eggs.

An integrated view of asteroid regeneration: tissues, cells and molecules.

The potential for repairing and replacing cells, tissues, organs and body parts is considered a primitive attribute of life shared by all the organisms, even though it may be expressed to a different extent and which is essential for the survival of both individual and whole species. The ability to regenerate is particularly evident and widespread within invertebrates. In spite of the wide availability of experimental models, regeneration has been comprehensively explored in only a few animal systems (i.e., hydrozoans, planarians, urodeles) leaving many other animal groups unexplored. The regenerative potential finds its maximum expression in echinoderms. Among echinoderm classes, asteroids offer an impressive range of experimental models in which to study arm regeneration at different levels. Many studies have been recently carried out in order to understand the regenerative mechanisms in asteroids and the overall morphological processes have been well documented in different starfish species, such as Asterias rubens, Leptasterias hexactis and Echinaster sepositus. In contrast, very little is known about the molecular mechanisms that control regeneration development and patterning in these models. The origin and the fate of cells involved in the regenerative process remain a matter of debate and clear insights will require the use of complementary molecular and proteomic approaches to study this problem. Here, we review the current knowledge regarding the cellular, proteomic and molecular aspects of asteroid regeneration.

Sea star tenacity mediated by a protein that fragments, then aggregates.

Sea stars adhere firmly but temporarily to various substrata as a result of underwater efficient adhesive secretions released by their tube feet. Previous studies showed that this material is mainly made up of proteins, which play a key role in its adhesiveness and cohesiveness. Recently, we solubilized the majority of these proteins and obtained 43 de novo-generated peptide sequences by tandem MS. Here, one of these sequences served to recover the full-length sequence of Sea star footprint protein 1 (Sfp1), by RT-PCR and tube foot transcriptome analysis. Sfp1, a large protein of 3,853 aa, is the second most abundant constituent of the secreted adhesive. By using MS and Western blot analyses, we showed that Sfp1 is translated from a single mRNA and then cleaved into four subunits linked together by disulphide bridges in tube foot adhesive cells. The four subunits display specific protein-, carbohydrate-, and metal-binding domains. Immunohistochemistry and immunocytochemistry located Sfp1 in granules stockpiled by one of the two types of adhesive cells responsible for the secretion of the adhesive material. We also demonstrated that Sfp1 makes up the structural scaffold of the adhesive footprint that remains on the substratum after tube foot detachment. Taken together, the results suggest that Sfp1 is a major structural protein involved in footprint cohesion and possibly in adhesive interactions with the tube foot surface. In recombinant form, it could be used for the design of novel sea star-inspired biomaterials.

Mechanical mutability in connective tissue of starfish body wall.

Stiffness changes in response to mechanical and chemical stimulation were studied in muscle-free dermal samples from the body wall of the starfish Linckia laevigata. The ultrastructural study showed that the dermis was packed with collagen fibrils between which only a small number of cells were observed. Muscles were found only in the walls of coelomic extensions leading to papulae. Stress-strain tests were performed on isolated dermis containing no muscles. The tangent modulus was 27.5 MPa at 0.04% strain rate in the stress-strain tests. It was increased to 40.7 MPa by mechanical stimulation, which also increased the tensile strength and breaking-strain energy density. Dynamic mechanical tests showed that the increase in stiffness in response to mechanical stimulation was transient. Acetylcholine (10(-6)-10(-3) mol l(-1)) and artificial seawater with an elevated potassium concentration (KASW) stiffened the dermis. Mechanical stimulation caused a 12% mass loss. KASW also caused mass loss, which was inhibited by anesthesia. These results clearly showed that the stiffness changes in the starfish dermis were based on a non-muscular mechanism that was similar to that of other echinoderm connective tissues with mechanical mutability.

Injection of Acanthaster planci with thiosulfate-citrate-bile-sucrose agar (TCBS). II. Histopathological changes.

We assessed histological changes in the tissues of the crown-of-thorns starfish Acanthaster planci (COTS) after injection of thiosulfate-citrate-bile-sucrose agar (TCBS) which was used as a disease inducer (potential outbreak control method), by conventional and scanning electron microscopy. Digestive glands were processed and stained with hematoxylin and eosin to describe the histological architecture of the intestinal epithelium. Subsequently comparison of healthy versus infected tissues and Gram stains were carried out to confirm bacterial occurrence on infected tissues, characterize the structural changes induced by bacterial communities in COTS tissues, and to determine if the histopathological changes of intestinal tissues were consistent with vibrio infection. TCBS injections induced marked epithelial desquamation, hypertrophy and hypersecretion of glandular cells, epithelial cell destruction, pyknosis, reduction of thickness and disorganization of connective tissue and associated nerve plexus, presence of bacterial colonies, irregular eosinophilic foci in glandular cells, brush border disruption, atrophy and detachment of intestinal microvilli and cell debris in the lumen. All these changes were attributed to a fulminating systemic dysbiosis and were consistent with vibrio infections.

An ultrastructural study of oocyte atresia in the starfish Pisaster ochraceus.

The alterations involved in oocyte atresia of the starfish Pisaster ochraceus were investigated using both light and electron microscopy. It was shown that atresia may be defined by three patterns of cell destruction. Initially, the small electron-lucent vesicles produced by the Golgi complex underwent amalgamation into groups. This was followed by loss of vesicle membranes and consequent formation of transparent necrotic zones in the cytoplasm. The second pattern, ultrastructurally comparable with autophagic cell death, was marked by apparent amalgamation of the morphologically similar electron-lucent vesicles into growing vacuoles, giving rise to a multibranched autophagic vacuole. This vacuole engulfed the cytosol granules and ultimately came to occupy the entire space within the oocyte. In addition, the cytosol insulation inside of the 'apoptotic body-like spheres' was regularly observed. Thus, it is supposed that oocyte destruction may occur by a complex mechanism that includes elements of necrosis, autophagic cell death and apoptosis.

Sorption behaviour of manganese-coated calcined-starfish and manganese-coated sand for Mn(II).

The objective of the present investigation was to explore the sorption behaviour of manganese-coated samples of calcined starfish (MCCSF) (i.e. the impregnation of calcined starfish with manganese) for the removal of low levels of an important heavy metal toxic ion, Mn(II), from aqueous solutions. The suitability of this solid was further compared with two different samples of manganese-coated sands (MCS): MCS4 and MCS9 impregnated at pH 4.0 and pH 9.0, respectively. These comparative studies were performed in both batch and column experiments. Batch data indicated that a fairly good stability of the coating was obtained for these three samples in the pH region 2.5 to 10.0. The removal efficiency of MCCSF was fairly good in comparison with the MCS4 and MCS9 samples. These last two samples possessed similar Mn(II) removal capacities. Moreover, a small dose of sodium hypochlorite further enhanced the uptake of Mn(II) by these solids. The sorbate concentration dependence data fitted reasonably well to the Freundlich adsorption isotherm. The column data indicated that MCCSF possessed a relatively higher adsorption capacity compared with the MCS4 and MCS9 samples. The breakthrough curves obtained were then used to evaluate the apparent removal capacity of these solids under the dynamic conditions using the Thomas equation. The SEM images obtained for these manganese-coated solids along with the virgin base materials, i.e. sand and calcined starfish, showed that manganese oxides occupied the surfaces or pores of the base materials and formed clusters on the base surface.

Assessment of three mitochondrial loci variability for the crown-of-thorns starfish: a first insight into Acanthaster phylogeography.

Acanthaster planci (L.) is one of the major threats to coral reefs, whose genetic diversity has been mainly studied with allozymes. Allozymes revealed the low genetic differentiation between A. planci populations in the Indo-Pacific area. We obtained sequences of A. planci from Kenya, Mayotte and Madagascar at the three loci cytochrome oxydase subunit I (COI), 16S rDNA (16S) and five tRNAs, analysed together with available sequences of Acanthaster from the Pacific Ocean. The level of genetic diversity varied among the three loci, tRNAs being on average three times less divergent than COI and 16S genes. The genus Acanthaster appeared monophyletic, the two species A. brevispinus (Fisher) and A. planci forming distinct clades in agreement with data from morphology and systematics. The A. planci clade split into a West Indian Ocean group and a Pacific group, in agreement with allozyme data on population differentiation.

[A study of quantitative dynamics of F-actin during oocyte maturation in the starfish Asterias amurensis].

We studied the actin cytoskeleton state in Asterias amurensis oocytes within 30 min after the 1-methyladenine-induced maturation until the germinal vesicle breakdown. The total amount of actin remained unchanged during oocyte maturation. In immature oocytes, the major part of actin is not a part of filaments, but in the presence of 1-methyladenine massive actin polymerization began already within 20 min. Electron immunocytochemistry methods demonstrated joint localization of actin and alpha-protein in the cytoplasm. They were redistributed from the cortex to the cytoplasm in the presence of 1-methyladenine. A possible involvement of actin cytoskeleton in transmembrane transduction of the hormonal signal at the postreceptor stages is discussed.

PM-2: an ECM epitope necessary for morphogenesis in embryos of the starfish, Pisaster ochraceus.

Anti-PM-2 is a monoclonal antibody that has been developed against the ECM of embryo/larvae of the starfish Pisaster ochraceus. Immunofluorescent staining shows that the PM-2 epitope is present in the cortical granules of unfertilized eggs and is released into the perivitelline space on fertilization. At the blastula stage, staining is very faint and limited to the blastocoel and a few granules within the cells. Strong staining appears in the embryonic/larval body cavity shortly after gastrulation and continues to increase in both the embryonic/larval body cavity and lumen of the gut at least until the bipinnaria stage. The presence of PM-2 in the Golgi apparatus, its susceptibility to enzymes that attack carbohydrates, and inhibition of PM-2 synthesis by tunicamycin, a drug that inhibits the linkage of carbohydrate moieties to protein backbone chains, suggest that the PM-2 epitope is or contains carbohydrate. Western blots of the whole embryo homogenates show bands at molecular weights of 130, 122, 100, 70, and 50 kDa. As embryos grow, two other high molecular weight (greater than 200 kDa) bands also appear. This suggests that the epitope is present on a series of molecules and that some of the lower MW molecules are precursors of the higher MW ones. A single 24-h exposure to the antibody just posthatching appears to inhibit normal mesenchymal migration at the gastrula stage, and if development of these treated embryos/larvae is allowed to continue to the bipinnaria stage, the embryos are stunted and have a smaller oral hood and esophagus. Long-term exposure results in stunted animals with distorted shapes. Such animals develop a very small embryonic/larval body cavity or none at all and differentiation of the larval GI tract fails to occur. The results suggest that molecules exhibiting the PM-2 epitope are necessary for the proper formation of the blastocoel, for mesenchyme cell movement and for proper development of the larvae GI tract.

Characterization of hydroxyapatite by electron microscopy.

The obtention of hydroxyapatite (HAp) is reported using brushite (CaHPO4.2H2O) and the skeleton of a starfish (Mellita eduardobarrosoi sp. nov.), primarily composed of magnesian calcite ((Ca,Mg)CO3) as precursors. Stoichiometric amounts of both were reacted under hydrothermal conditions: a pressure of 5.8 MPa and a temperature of 200 degrees C for 2, 4, 6, 8, 10, and 20 h of reaction times. The samples obtained were characterized by means of scanning electron microscopy, X-ray diffraction, infrared spectroscopy, and transmission electron microscopy. Two defined populations of HAp fibers were found: A bundle of fibers 75 mum in length and 1-13 mum in diameter, and a second bundle of fibers 5 mum in length and less than 0.5 mum in diameter. Furthermore, an increase in HAp formation and a Ca/P ratio as a function of reaction time were observed. The growth mechanism of HAp is also discussed.

Role of specialized microvilli and the fertilization envelope in the spatial positioning of blastomeres in early development of embryos of the starfish Astropecten scoparius.

In the eggs of a wide range of animal species, various factors that determine the blastomeres' presumptive fate are known to locate unevenly within the egg. In the embryos of these animals, cleavage occurs not just to increase cell numbers, but also to distribute the factors to the respective blastomeres, resulting in cell specialization at the later stages. In the early cleavage stages, before the establishment of a device such as desmosomes to directly join the blastomeres, some other means is needed to keep the blastomeres together and maintain the relative positions among them. In this study, we found that the embryos of the starfish Astropecten scoparius lack the hyaline layer seen in sea urchin embryos and that blastomeres adhere to the fertilization envelope (FE) via filamentous cellular projections (fixing processes). Electron microscopy revealed the fixing processes to be specialized microvilli formed, after the elevation of the FE, by the elongation of short microvilli that pre-exist in unfertilized eggs. After the first cleavage, the two blastomeres separate from each other and finally attach to the FE. In the subsequent cleavages, the blastomeres undergo repeated cell division without separating from the FE. Between the blastomeres and the FE, only shortened fixing processes were observed. Destruction of the fixing processes caused release of the blastomeres from the FE and disturbance of the relative positions of the blastomeres, resulting in abnormal development of the embryos. These observations suggest that the fixing process is a device to keep the egg placed centrally in the FE up to the first cleavage, and after the first cleavage and beyond to anchor the blastomeres to the FE so that the FE can be used as a scaffold for morphogenesis. Electron microscopy also suggests that the inner layer of the FE, which is derived from the contents of cortical granules, reinforces the adhesion of the fixing processes to the FE. Immuno-electron microscopy, using an antibody against sea urchin hyaline layer, showed that the inner layer of the FE of starfish eggs and the hyaline layer of sea urchin eggs, which are both derived from cortical granules, contain some common elements.

Ultrastructural aspects of the development of the hyaline layer and extracellular matrix lining the gastrointestinal tract in embryos and larvae of the starfish Pisaster ochraceus preserved by freeze substitution.

Embryos and larvae of the starfish Pisaster ochraceus are surrounded by a complex extracellular matrix (ECM) layer called the hyaline layer (HL). A similar but less well-organized ECM layer lines some regions of the larval gut. Examination of material preserved by freeze substitution shows that the HL consists of a coarse outer meshwork, a boundary layer, a supporting layer, which is divided into three sublayers, H1, H2, and H3, and an intervillus layer. The development of the HL has been studied in material preserved by freeze substitution. Development begins at fertilization when exocytosis of the cortical granules releases ECM into the perivitelline space and elevates the fertilization membrane. Shortly after, plaques of dense material with attached fibers are present on the outer surface of the egg plasmalemma. Following this, these plaques and fibers are associated with the tips of short microvilli, suggesting that they may induce microvillus formation. Next, the tips of some of the microvilli are joined by short regions of the H1 sublayer. Some of these H1 regions have short segments of boundary layer material associated with their outer surfaces while others are naked. Just prior to hatching, the H1 and boundary layers completely surround the embryo, separating the developing coarse meshwork and intervillus layers. Short segments of the H2 and H3 sublayers are also present. Posthatching, the microvilli and all HL layers increase in thickness and density, particularly the H2, boundary, and coarse outer meshwork layers. The results suggest a sequential organization of HL components from ECM that is secreted into the perivitelline space.

Evolution of larval form in the sea star genus Patiriella: conservation and change in the larval nervous system.

The organization of the peptidergic system in the larvae of Patiriella species with divergent ontogenies was compared to determine which aspects of neurogenesis are conserved and which are altered in the evolution of development in these sea stars. P. regularis has ancestral-type feeding bipinnaria and brachiolaria larvae and the organization of the nervous system, in association with feeding structures, paralleled the bilateral larval body plan. P. calcar and P. exigua have non-feeding planktonic and benthic brachiolariae, respectively, and there was no trace of the neuronal architecture involved with feeding. The nervous system in the attachment stage brachiolaria was similar in all three species and neuronal organization reflected larval symmetry. Delayed expression of peptidergic lineages to the brachiolaria stage in the lecithotrophs indicates heterochronic change in the timing of neurogenesis or deletion of the ancestral early neurogenic program. The bipinnarial program is suggested to be a developmental module autonomous from the brachiolar one. With a divergence time of less than 10 Ma, the evolution of development in Patiriella has resulted in extensive reduction in the complexity of the larval nervous system in parallel with simplification in larval form. There is, however, strong conservation in the morphology and neuronal architecture of structures involved with settlement.

Regeneration of spines and pedicellariae in echinoderms: a review.

Morphogenesis of tissues during regeneration of echinoderm spines and pedicellariae is reviewed. Regeneration of the skeleton is rather well documented while that of associated soft tissues is poorly investigated. In particular, little information is available on the early regeneration stages which follow wound healing. From the available information, it is suggested that regeneration of broken spines proceeds through a morphallactic process of which the organizational information, as well as the involved cells, lies in the stump. In contrast, regeneration of removed spines and pedicellariae may depend on an epimorphic process whose organizational information could be located in the mutable connective tissue that joins the appendage to the main body wall.

Localization and partial characterization of a molecule found in the plasma membrane of starfish and sea urchin embryos using a novel monoclonal antibody.

A monoclonal antibody, P212, has been developed which reacts against 120 kD and 90 kD proteins, P212 antigens found in the plasma membranes of both starfish and sea urchin embryos. Immunocytochemical studies with colloidal gold tagged P212 showed that the P212 antigens are also found on the inner membranes of several different types of granules and vesicles located in the eggs and cytoplasm of embryonic cells, and in the trans region of the Golgi apparatus. One or both of the P212 antigens are also found in the cortical granules, granules which are exocytosed at fertilization. They are not found on intracellular membrane structures such as the rough endoplasmic reticulum, the nuclear membrane, or a number of deeper granular structures. The P212 antigens are probably located on the membranes of vesicles which are on the exocytosis-endocytosis pathway and may be involved in cell trafficking.

[Localization, kinetic parameters, and functions of cholinesterase of the starfish ampulla]. / Lokalizatsiia, kineticheskie parametry i funktsional'naia rol' kholinésterazy ampul morskikh zvezd.

The starfish amplullae cholinesterase was shown to represent acetylcholinesterase and enhance its activity along with increasing motility of the starfish. Bundles of muscle fibres containing cholinesterase were found in the ampullae. Cholinesterase was shown to be localized in the muscle cells and in collagen layer in vicinity of the muscle cells. The data obtained suggest participation of the starfish ampullae cholinesterase in non-synaptic cholinergic transmission between the radial nerve axons and the muscle fibre extension. Besides, the enzyme could take part in functional relationship between the muscle cells and the outer epithelial cells of the starfish ampullae.

Ultrastructure and synthesis of the extracellular matrix of Pisaster ochraceus embryos preserved by freeze substitution.

When asteroid embryos cryoprotected with propylene glycol are rapidly frozen in liquid propane and freeze substituted with ethanol, preservation of the cells and extracellular matrix (ECM) is excellent. The basal lamina, although thicker and less well defined than in conventionally fixed embryos, demonstrates a region of decreased density just below the cells that corresponds to the lamina lucida and a lamina densa. The former region is often occupied by fibrous material. In addition, as was previously described in conventionally fixed issues, the basal lamina of the ectoderm is generally thicker and more substantial than that of the endoderm, reinforcing an earlier suggestion that the structure of the basal is different in different regions of the embryo. The ECM of the blastocoel consists of thin "twig-like" elements that form a loose meshwork evenly distributed throughout the blastocoel. Bundles of 20 nm fibers, located within the meshwork, are oriented parallel to the base of the cells of the stomodeum. In the long axis of the embryo, similar fibers are present in the dorsal aspect of the animal between the stomach and the ectoderm and radiate out from the esophagus crossing the region between it and the ectoderm. Immunocytochemical work with three different monoclonal antibodies shows that glycoprotein molecules, synthesized in the Golgi apparatus, are also secreted here and form part of the matrix structure. The results suggest that the blastocoel is filled with a gel-like material reinforced with bundles of 20-nm fibers. The manner in which the observed arrangement could contribute to the development and maintainence of the shape of the embryo is discussed.

Structural changes in the endoplasmic reticulum of starfish oocytes during meiotic maturation and fertilization.

The endoplasmic reticulum (ER) of live starfish oocytes was observed during meiotic maturation and fertilization. The ER was visualized by injection into the cytoplasm of an oil drop saturated with the fluorescent lipophilic dye DiI; DiI spread throughout the oocyte endoplasmic reticulum and the pattern was imaged by confocal microscopy. The ER in the immature (germinal vesicle stage) oocyte was composed of interconnected membrane sheets. In response to 1-methyladenine, the sheets of ER appeared to become associated with the yolk platelets, forming spherical shells. A few of these spherical shells could sometimes be seen in immature oocytes, but their number was much greater in the egg at the first meiotic spindle stage. At about the time that the first polar body formed, the spherical shells disappeared, and the ER returned to a form like that of the immature oocyte. The spherical shells did not reappear during the second meiotic cycle. During maturation, the ER also began to move; the movement was apparent by the time of germinal vesicle breakdown and continued throughout both meiotic cycles and in eggs with second polar bodies. When eggs at the first meiotic spindle stage were fertilized, the form of the ER changed. Within 1 min after sperm addition to the observation chamber, the circular cross sections of the spherical shells of the unfertilized egg ER were no longer distinct. At this point, the form of the ER could not be discerned with the resolution of the light microscope; however, the rate of spreading of DiI from an injected oil drop decreased, providing strong evidence that the ER had become fragmented. The ER remained in this form for several minutes and then gradually, the appearance of the ER and the rate of DiI spreading returned to be like those of the unfertilized egg. Injection of inositol trisphosphate caused a similar change in the ER structure. These results indicate that the ER is a dynamic structure, the form of which changes during oocyte maturation and fertilization.