1-Methyl-4-thiohistidine and Glutathione in the Developing Embryo of the Sea Urchin, Paracentrotus lividus.

4-thiohistidines occur as free amino acids in the eggs of several marine organisms, in particulary relevant concentrations in the eggs of echinoderms. Since nothing is known about their biological role or biosynthesis and metabolic fate, we measured the concentration of 1-methyl-4-thiohistidine together with the two most common small molecular weight thiols, glutathione and cysteine, during embryonic development of the sea urchin Paracentrotus lividus until the pluteus stage. The thiols were determined by high performance liquid chromatography as fluorescent derivatives of monobromobimanes. 1-methyl-4-thiohistidine and glutathione are present until the pluteus stage with an approximate ratio of 2: 1. Cysteine was detected at the blastula stage and found to increase thereafter. 1-methyl-4-thiohistidine was absent, or present in traces, in sperm and in somatic tissues of adults. It is concluded that 1-methyl-4-thiohistidine is typical of eggs and embryos and may be metabolized during metamorphosis into an as yet unknown compound.     

Phosphorylation of Deoxyribonucleosides and DNA Synthesis during Embryogenesis of the Sea Urchin

The phosphorylation of thymidine, deoxycytidine, deoxyadenosine and deoxyguanosine was studied during the embryogenesis of the sea urchin Hemicentrotus pulcherrimus. [³H]Thymidine was taken up, phosphorylated and accumulated mostly as [³H]thymidine triphosphate in the early cleavage stage embryos. As the embryos developed, the formation of [³H]thymidine triphosphate decreased and most of the [³H]thymidine taken up by the blastulae remained be phosphorylated. When [³H]deoxycytidine was added to the cleaving embryos, the resultant labeled pool consisted of almost equal amounts of [³H]deoxycytidine monophosphate and [³H]deoxycytidine triphosphate. The formation of [³H]deoxycytidine monophosphate increased up to 10 hr following fertilization and then decreased, while the formation of [³H]deoxycytidine triphosphate decreased for 10 hr following fertilization and then gradually increased. [³H]Deoxyadenosine was rapidly phosphorylated to monophosphate derivative in the cleavage stage embryos. The formation of [³H]deoxyadenosine triphosphate increased rapidly after cleavage stage with a concomitant decrease of [³H]deoxyadenosine monophosphate. The activity of phosphorylation in [³H]deoxyguanosine to triphosphate derivative increased rapidly reaching a plateau 10 hr after fertilization. At this point, 80 % of the [³H]deoxyguanosine was recovered as [³H]deoxyguanosine triphosphate. Based on the above results, it was concluded that the profile of production of each deoxyribonucleoside triphosphate changed during the embryogenesis of the sea urchin, and the in vivo rate-limiting step of phosphorylation of the individual deoxyribonucleoside was assumed to be different.     

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.     

Nucleic Acid and Histone Synthesis by Ethanol-Treated Cleavage-Arrested Sea Urchin Embryos

Abstract. It has been found that fertilized sea urchin eggs prevented from normal cleavage by solutions of isosmotic ethanol in sea water are able to complete some cellular and molecular aspects of the normal developmental program that are observed in control cultures. In both treated and control cultures, the type of RNA transcribed changes at 24 h (early gastrula) in favor of higher molecular weight rRNA. Ultrastructural studies reveal the presence of nucleoli in ethanol-treated as well as control embryos. The type of H1 histone synthesized also shifts at 24 h in favor of a higher molecular weight H1 in both ethanol-treated and control embryos. Replication of DNA proceeds at a slower rate in ethanol-treated embryos than in controls, resulting in DNA/embryo values in ethanol which are 20–30% of control values after 24 h. The results relate to the problem of differentiation without cleavage, and the role of normal partitioning, cell-cell interaction, and DNA synthesis in triggering the sequence of events in the developmental program.     

Histone Analysis During the First Cell Cycle of Development of the Sea Urchin Tetrapygus niger

To obtain information on the contribution of paternal and maternal histone complement to the assembly of sea urchin chromatin after fertilization, we have compared the complete set of histones of zygotes obtained at the beginning of the first S phase those of sperm and unfertilized eggs of Tetrapygus niger . The electrophoretic pattern on acetic acid-urea polyacrylamide gels and the amino acid composition of the histones isolated from zygotes are virtually identical with those isolated from unfertilized eggs. These results strongly suggest that sperm histones must be lost before the initiation of the first replication event. The histones of zygotes obtained after the completion of the first S phase have the same electrophoretic pattern as the proteins isolated from zygotes prior to the first S phase; however, differences were found in their amino acid composition. These results suggest that some new proteins may associate with chromatin during the first replication round in Tetrapygus niger development.     

Change in the Triglyceride Level in Sea Urchin Eggs and Embryos During Early Development

Triglycerides in the embryos of the sea urchin, Anthocidaris crassispina , analyzed by gas-liquid chromatography, distributed in a range of carbon numbers between 42 and 58 in the sum of three fatty acid residues. During the development until gastrulation, the levels of triglycerides with 48, 56 and 58 carbon numbers decreased at constant rates and the levels of the others decreased at specific stages different with one another, respectively. Thereafter, the amounts of all triglycerides decreased simultaneously. The amount of oxygen consumed in the embryos is enough for the oxidation of mobilized triglycerides during post-hatching period but is not during pre-hatching period. The levels of neutral glycerides increased gradually during pre-hatching period and thereafter decreased. The fatty acid level also increased during pre-hatching and post-hatching period. These suggest that the cleavage of triglycerides and the oxidation of their cleavage-products occur during whole span of early development. During pre-hatching period, the break down of triglycerides is probably higher in its rate than the rate of their oxidation, resulting in the increase in the levels of neutral glycerides, as well as fatty acids.     

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.     

ADP-Ribosylation of Histones in Nuclei Isolated from Embryos of the Sea Urchin, Hemicentrotus pulcherrimus

Two-dimensional electrophoresis (2D-PAGE) of a histone fraction isolated from nuclei of embryos of the sea urchin Hemicentrotus pulcherrimus exhibited almost all histone species at all stages examined. At the gastrula stage, a spot of H1A became evident and three spots closely associated with one another were found in place of a single spot of H2A.1. In the histone fraction isolated from [adenylate-³²P] NAD⁺-treated nuclei of all stages examined, autoradiograms of 2D-PAGE exhibited spots of mono [ADP-ribosyl] ated H1 and polymodified H2B.2, H3.1, H3.3 and H4 but did not show ADP-ribosylated H2A.1, H2A.2 or H2B.1. Poly [ADP-ribosyl] ated H3.2, found in morulae, was not detectable in blastulae and gastrulae. Treatment with dimethylsulfate, known to activate ADP-ribosylation in other cell types, induced poly [ADP-ribosyl] ation of H2A.2 and H2B.1 in embryos at all stages examined, and also polymodification of H3.2 in gastrulae. ADP-ribosylation of H1, H2B.2, H3.1 and H3.3 was hardly affected by dimethylsulfate treatment, though modification of H4 was blocked by this treatment. Probably, strong regulation of ADP-ribosyltransferase reactions causes failures of modification of H2A.2 and H2B.1 throughout early development and also of H3.2 at the gastrula stage. Regulation of histone ADP-ribosylation is thought to alter chromatin structures and the rate of gene expression, contributing to cell differentiation.     

Regulation of Microtubule Synthesis and Utilization during Early Embryonic Development of the Sea Urchin

Microtubules deployed during early development of the sea urchinembryo are derived both from a preexisting pool of subunitspresent in the egg and from microtubule protein subunits synthesizedin the embryo. Several aspects of microtubule protein synthesisand utilization are reviewed. Microtubule protein synthesisin early development utilizes oogenetic messenger RNA species.Translation of this mRNA is under regulation. Microtubule proteinsynthesis rises concomitantly with overall protein synthesisat fertilization, but rises at a relatively higher rate laterin cleavage stages. Microtubule protein labeled with [³H]-leucinein early development is incorporated into cilia, indicatingthat newly synthesized protein enters the pool of subunits usedin organelle assembly. The microtubule protein pool comprisesabout 1%of the soluble protein of the egg, and remains constantin size at least until the blastula stage. Direct pool sizeestimates are consistent with results of experiments on recruitmentof microtubule protein subunits into the mitotic apparatus andinto regenerating cilia. Soluble and particulate colchicinebinding fractions, which have been reported from several systems,appear to be present in sea urchin embryos. The possible roleof such fractions are discussed, as are aspects of the regulationof ciliary assembly.     

Timers in Early Development of Sea Urchin Embryos

To elucidate the timing mechanisms in the early development of sea urchin embryos, we measured the times of initiation of the first four cleavages, of ciliary movement, of primary mesenchyme cell ingression, and of gastrulation at four temperatures ranging from 11 to 20°C. The times of cleavage and of initiation of ciliary movement showed similar temperature dependency, indicating that these events may be controlled by a common timer (the first timer). Although batches of eggs often showed variation in the period between fertilization and the first cleavage, their subsequent cleavages were more regular. This indicates that the first timer may not start at fertilization. The ingression of mesenchyme cells and the onset of gastrulation showed similar temperature dependency that was higher than that of other events, suggesting the existence of a second timer. Temperature shift experiments indicate that the second timer starts at the mid-blastula (the 8–9th cleavage) stage when divisions of blastomeres become asynchronous.     

The Interactions of Sea Urchin Gametes During Fertilization

The interactions between sea urchin spermatozoa and ova duringfertilization usually exhibit a high degree of species specificity.Under natural conditions and reasonable gamete concentrations,most interspecific inseminations fail to yield zygotes. Macromoleculeson the external surfaces of the apposing gametes must surelybe responsible for successful gamete recognition, adhesion andfusion. Species specific recognition between surface componentsof sperm and egg could occur during at least three events comprisingthe fertilization process. The first event is the interactionof the sperm plasma membrane with the egg jelly coat. This inducesthe sperm acrosome reaction resulting in the exocytosis of the"bindin" -containing acrosome granule and also the extrusionof the acrosome process from the anterior tip of the sperm.The second event is the adhesion of the bindin-coated acrosomeprocess to glycoprotein "bindin receptors" on the external surfaceof the egg vitelline layer. The third event is the penetrationof the vitelline layer and the fusion of sperm and egg plasmamembranes. With the isolations of the component of egg jellywhich induces the acrosome reaction, sperm bindin from the acrosomevesicle and the egg surface bindin receptor from the vitellinelayer, there is hope of discovering the molecular basis of thismost interesting intercellular interaction which results inthe activation of embryonic development.     

Determination During Early Embryogenesis in Drosophila melanogaster

Ligation of developing embryos of Drosophila melanogaster wasperformed at three different stages of nuclear multiplicationand at the cellular blastoderm stage. Egg fragments of variablesizes are able to continue development up to the hatching stage.Partial embryos differentiate larval structures, anterior fragmentsforming larval head and posterior fragments larval abdominalstructures. These fragments differentiate a variable numberof the twelve larval cuticular bands formed by intact embryos.We found that ligation at cellular blastoderm can lead to anteriorand posterior fragments which differentiate together all thetwelve bands, indicating that at this stage the embryo developsthese patterns in a mosaic fashion. Ligation of younger embryosprevents the differentiation of some intermediate larval cuticularbands, while the terminal ones are consistently differentiated.The number and position of the deleted bands is correlated withthe time and position of ligation. This indicates that the mosaicpattern present in the egg at blastoderm is not fully formedat earlier stages in development.