Developmental Patterns in Histones and Histone Synthesis During Early Embryogenesis of the Sea Urchin Paracentrotus lividus

Abstract. Striking developmental changes in histone and histone synthesis in sea urchin embryos were observed in three histone classes, H1, H2A and H2B. In each case there is a shift in histone synthesis from the early cleavage stage types to other types of histones at the morula stage; Two new forms appear after the blastula stage. In addition, multiple changes in histone types were found during gameto-genesis in the male and female gonads where specific histones, different from the embryonic histones, were observed.     

First Insights into the Biochemistry of Tube Foot Adhesive from the Sea Urchin Paracentrotus lividus (Echinoidea, Echinodermata)

Sea urchins are common inhabitants of wave-swept shores. To withstand the action of waves, they rely on highly specialized independent adhesive organs, the adoral tube feet. The latter are extremely well-designed for temporary adhesion being composed by two functional subunits: (1) an apical disc that produces an adhesive secretion to fasten the sea urchin to the substratum, as well as a deadhesive secretion to allow the animal to move and (2) a stem that bears the tensions placed on the animal by hydrodynamism. Despite their technological potential for the development of new biomimetic underwater adhesives, very little is known about the biochemical composition of sea urchin adhesives. A characterization of sea urchin adhesives is presented using footprints. The latter contain inorganic residues (45.5%), proteins (6.4%), neutral sugars (1.2%), and lipids (2.5%). Moreover, the amino acid composition of the soluble protein fraction revealed a bias toward six amino acids: glycine, alanine, valine, serine, threonine, and asparagine/aspartic acid, which comprise 56.8% of the total residues. In addition, it also presents higher levels of proline (6.8%) and half-cystine (2.6%) than average eukaryotic proteins. Footprint insolubility was partially overcome using strong denaturing and reducing buffers, enabling the visualization of 13 proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The conjugation of mass spectrometry with homology–database search allowed the identification of six proteins: alpha and beta tubulin, actin, and histones H2B, H3, H2A, and H4, whose location and function in the adhesive are discussed but require further investigation. For the remaining unidentified proteins, five de novo-generated peptide sequences were found that were not present in the available protein databases, suggesting that they might be novel or modified proteins.     

Spicule Formation-Inducing Substance in Sea Urchin Embryo

Isolated micromeres of sea urchin produced spicules in sea water containing blastocoelic fluid (BCF) taken from embryos, or in a medium in which embryos had previously been dissociated (dissociated solution, DS). When isolated micromeres were cultured in vitro , their descendants initiated spicule formation only when BCF was added to the culture medium by the time when, in normal development, primary mesenchyme cells form two aggregates in the vegetal region. After the initiation of spicule formation, growth of spicules occurred under the continuous influence of DS. Spicule formation-inducing (SFI) activity in DS was first detected at the mesenchme blastula stage. The activity in BCF was heat-labile and was inactivated by trypsin.     

Asymmetrical localization and segregation of Paracentrotus lividus Bep4 maternal protein.

Asymmetric divisions that produce two distinct cells play a fundamental role in generating different cell types during development. Here we investigate the role of the cortex region and mitotic apparatus in asymmetrical localization and segregation of Bep4 protein in Paracentrotus lividus egg. By centrifugation of eggs with or without drugs we established an involvement of the cortex region in localization of Bep4 protein, confirmed by immunohistochemistry of isolated cortex. Association with the mitotic apparatus during cell division permits selective partitioning of Bep4 protein into the daughter cells. Direct association with spindle was also demonstrated both by Western blot and immunohistochemistry after isolation of the mitotic apparatus.     

Embryotoxicity of fenbendazole in Paracentrotus lividus

The aim of the present work was to evaluate the embryotoxicity of Fenbendazole, a benzimidazole carbamate-derived anthelmintic drug widely employed in Veterinary Medicine, by using the embryonal development of Paracentrotus lividus (sea urchin) as a experimental model. Embryos were obtained by in vitro eggs fertilization and cultured in seawater. Five embryo suspensions were added by Fenbendazole reaching a final concentration of 5 micrograms/l, 7.5 micrograms/l, 10 micrograms/l, 12.5 micrograms/l and 25 micrograms/l; a suspension was kept drug-free as a control. Embryo development was evaluated by microscopical examination of suspensions at 3 and 40 hours. Our results show that a concentration of 5 micrograms/l of the drug determines a considerable delay of the embryonal development in the 95 percent of the elements observed, and a concentration of 25 micrograms/l produces a block of the embryogenesis at the phase of morula and blastula in all embryos. Results confirm that the effects observed are probably due to an extended inhibition of several enzyme complexes of the embryo cells.     

Spicule Formation by Isolated Micromeres of the Sea Urchin Embryo

Micromeres are isolated at the 16-cell stage from three speciesof Japanese sea urchins, Hemicentrotus pulcherrimus, Pseudocentrotusdepressus, and Anthocidaris crassispina, and are cultured insea water containing a small amount of horse serum. In all speciesused, isolated micromeres first divide unequally as they doin vivo. The pattern and number of the subsequent cleavagesare also the same as in vivo, although they are not necessarilyclear in all cases, since the border of the adjacent cells becomeinvisible at each resting stage in some batches of embryos. After cleavage, passing through the stage when the contoursof the individual cell are obscure, decendants of the isolatedmicromeres form cell aggregates similar to the group of primarymesenchyme cells in a blastula. Within such aggregates, a spicularrudiment appears which develops either into a triradiate spiculeas in normal gastrulae or into a rod. The triradiate spiculegrows into a three-dimensional skeleton which is very similarto the normal pluteus skeleton, not only in its final shapeand size including species-specific characters but in its developmentalcourse and crystallographic nature. The rod, on the other hand,develops into either a one-dimensional or two-dimensional skeleton.These skeletons probably correspond to a part of the completeskeleton.     

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.     

Characterization of bep1 and bep4 antigens involved in cell interactions during Paracentrotus lividus development

We have identified and partially characterised two antigens, extracted with 3% butanol, from Paracentrotus lividus embryos dissociated at the blastula stage, and encoded by the cDNA clones previously described as bep1 and bep4 (bep-butanol extracted proteins). The cDNA fragments containing the specific central portions of bep1 and bep4 were expressed as MS2 polymerase fusion proteins in Escherichia coli. These two fusion proteins, called 1C1 (bep1) and 4A1 (bep4), were injected subcutaneously into rabbits and the corresponding polyclonal antibodies generated. Western blot analysis of proteins, extracted with 3% butanol, from sea urchin embryos at the blastula stage (b.e.p.), established that both antibodies recognize two 33 KDa proteins. Reducing and non-reducing electrophoretic conditions show that both antibodies against bep1 and bep4 related proteins react also with a protein band of a molecular weight 66 KDa, indicating that these two antigens probably exist as dimers. Immunolocalization with anti 1C1 and 4A1 antibodies shows the presence of the related antigens also on the cell surface. Fab fragments of the polyclonal antibodies against 1C1 and 4A1 inhibited reaggregation of sea urchin embryonic cells, dissociated from blastula stage embryos. This prevention of reaggregation indicates that these proteins probably play a role in cell interaction during sea urchin embryonic development.     

Binding properties of Paracentrotus lividus (Echinoidea) hemolysin.

1. Paracentrotus lividus hemolysin binds erythrocytes, zymosan particles, lipopolysaccharide and laminarin surfaces but not auto and allogeneic cell membranes. 2. The binding could, at least for erythrocytes, involve phospholipids and cholesterol. 3. The protease activity of the coelomic fluid is not related to hemolysis. 4. The finding that very low concentrations of Zn2+ inactivate the hemolysin suggests a possible regulative function of the ion in the hemolytic reaction. 5. Ultrastructural observations on rabbit erythrocyte membranes indicate that most likely the transmembrane pores are induced by the lytic molecules.     

Dynamics of Ubiquitin Pools in Developing Sea Urchin Embryos

The sea urchin embryo is a closed metabolic system in which embryogenesis is accompanied by significant protein degradation. We report results which are consistent with a function for the ubiquitinmediated proteolytic pathway in selective protein degradation during embryogenesis in this system. Quantitative solid- and solution-phase immunochemical assays, employing anti-ubiquitin antibodies, showed that unfertilized eggs of Strongylocentrotus purpuratus have a high content of unconjugated ubiquitin ( ca . 8 × 10⁸ molecules), and also contain abundant conjugates involving ubiquitin and maternal proteins. The absolute content of ubiquitin in the conjugated form increases about 13-fold between fertilization and the pluteus larva stage; 90% or more of embryonic ubiquitin molecules are conjugated to embryonic proteins in hatched blastulae and later-stage embryos. Qualitatively similar results were obtained with embryos of Lytechinus variegatus . The results of pulse-labeling and immunoprecipitation experiments indicate that synthesis of ubiquitin in S. purpuratus is developmentally regulated, with an overall increase in synthetic rate of 12-fold between fertilization and hatching. Regulation is likely to occur at the level of translation, since others have shown that levels of ubiquitin-encoding mRNA remain virtually constant in echinoid embryos during this developmental interval. The sea urchin embryo should be a useful system for characterizing the role of ubiquitination in embryogenesis.     

Mifepristone affects fertility and development in the sea urchin Paracentrotus lividus

Drugs such as oral contraceptives and hormone replacement therapies are known to find their way into rivers, lakes and seas, and have the potential to affect reproduction and development of the wildlife. The knowledge of the reproductive mechanisms and their regulation in aquatic species is of fundamental importance for predicting and preventing the damage by the increasing release of such chemicals in the environment. Mifepristone, a synthetic steroid used as a drug for chemical abortion, works by blocking the effects of progesterone. Its presence in fresh and salt water has been reported, representing a danger for aquatic species. In this frame, we evaluated in both acute and chronic exposures, the effects of mifepristone on the reproductive performance of the sea urchin P. lividus. In both acute and chronic exposures, mifepristone did not affect the histological structure of the gonads. However, mifepristone administered to females caused the decrease of the percentage of normal developed plutei larvae compared with the control, whereas it did not alter sperm motility parameters and fertilization success in males. The immunohistological localization of progesterone receptor‐like immunoreactivity on the plasma membrane of oocytes and ova and the molecular weight of a progesterone receptor‐like immunoband identified by western blotting, are in agreement with a membrane progesterone receptor deducted from the genome sequence of the sea urchin Strongylocentrotus purpuratus and suggest that in P. lividus mifepristone actions may be mediated by a progesterone receptor.