Fusion of Spermatozoa with Embryonic Cells and Somatic Cells in the Sea Urchin

Spermatozoa can enter the separated blastomeres of 8- and 16-cell stage embryos, the cells of blastulae and even somatic cells of the oesophagus wall of an adult sea urchin, under certain conditions. In the presence of egg jelly solution, the rate of entrance of spermatozoa is remarkably increased. In the case of the blastomere of 8-cell stage embryos, characteristic cytoplasmic protrusions are formed at the sites of sperm entry, in succession to the formation of the cytoplasmic bulge. These protrusions elongate until 4 min after insemination, and then they retract gradually. The nucleus of penetrated sperm swells and decondenses to form a pronucleus. In most cases, the pronucleus seems to fuse with the preexisting diploid nucleus of the blastomere. When the dissociated oesophagus cells were inseminated, a certain type of the cells was found to fuse with spermatozoa, although the percentage of fused cells was very low.     

Expression of Cyclophilin during the Embryonic Development of the Sea Urchin

The spatial and temporal expression pattern of cyclophilin (Cyp) was examined during the embryonic development of the sea urchins Anthocidaris crassispina and Hemicentrotus pulcherrimus using Western blot analysis and indirect immunofluorescence microscopy. In this study, affinity-purified anti-human Cyp A antibody was used as the primary antibody. Western blot analysis revealed that a single 17.5 kDa immunoreactive band of Cyp was present in unfertilized eggs, in embryos during several stages of development, and in ovaries and testes of adult sea urchins. Cyp was also recognized in unfertilized eggs and embryonic sea urchin cells by indirect immunofluorescence microscopy, but its concentrations within the embryonic tissues varied significantly during embryogenesis. Expression of Cyp during the cleavage stage was thought to be attributable to maternal Cyp products, with zygotic expression appearing after gastrulation. Cyp expression appears to increase depending on the Cyp concentration in the vegetal and apical plates and primary mesenchyme cells in mesenchyme blastulae, and in the oral ectodermal ridge, gut and skeletogenetic mesenchyme cells in pluteus larvae. These results suggest that widespread embryonic distribution and an increased Cyp content occur during the gastrulation in sea urchin development.     

DNA Replication in Permeabilized Cells of Sea Urchin Embryos

For study of the regulation of DNA replication in sea urchin embryos during the early stages of development, an embryonic cell system that was permeable to exogenously supplied nucleotides was established. Embryos were permeabilized by incubating them in hypotonic buffer containing 0.3 M glucose. The permeabilized embryonic cells maintained their morphological integrity, and synthesized DNA when supplied with exogenous dNTPs.
DNA synthesis in these permeabilized embryonic cells required the presence of ATP and three other deoxyribonucleoside triphosphates in addition to labeled dTTP. DNA synthesis was almost completely inhibited by N-ethylmaleimide, and proceeded in a discontinuous fashion. Only cells permeabilized during the S phase could incorporate nucleoside triphosphates into DNA: cells permeabilized during other phases did not synthesize DNA. During a 60 min-incubation period, over 10% of the genomic DNA was replicated under the experimental conditions used.     

Elongated Microvilli on Vegetal Pole Cells in Sea Urchin Embryos

The ultrastructure of cells in the vegetal pole region of sea urchin embryos during early development to the mesenchyme blastula stage was examined by scanning electron microscopy. Vegetal pole cells in the ectoderm with longer microvilli than those of neighboring cells were first detectable at the early blastula stage just before hatching. These cells with elongated microvilli remained in the central region of the vegetal plate when most vegetal plate cells ingressed into the blastocoel to form primary mesenchyme. When first detectable in the sea urchin, Anthocidaris crassispina , four vegetal pole cells had elongated microvilli, but at the time of primary mesenchyme cell ingression, the number of cells with elongated microvilli had increased to eight, apparently by cell division. These vegetal pole cells were wedge-shaped with a broad surface adhering to the hyaline layer at the time of primary mesenchyme cell ingression. SEM observation of the outer surface of embryos showed that the microvilli extended into the hyaline layer. The reinforced attachment of vegetal pole cells to the hyaline layer through their elongated microvilli may explain why these cells could remain at the vegetal pole when the surrounding cells ingressed into the blastocoel as primary mesenchyme cells.     

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.     

Studies on the Cellular Pathway Involved in Assembly of the Embryonic Sea Urchin Spicule

Micromeres from the 16-cell stage sea urchin embryo were isolated and cultured in vitro in seawater containing 3% horse serum. Under these conditions these cells differentiate into spicule-forming, primary mesenchyme cells. To obtain insight into the route traveled by Ca²⁺ to form the pseudocrystalline spicule composed of CaCO₃ and matrix proteins, studies with various inhibitors were undertaken. Experiments with members of several different classes of Ca²⁺ channel blockers established that the Ca²⁺ utilized for spiculogenesis must be taken up by the cells. Moreover, studies using two agents that disrupt the endomembrane system, monensin and brefeldin A, showed that both blocked spicule formation. Based on these experiments, we conclude that extracellular Ca²⁺ must enter the primary mesenchyme cells prior to being deposited extracellularly as CaCO₃ and that this ion and/or the matrix proteins found in the spicule are routed through the secretory pathway that has been established to exist in a wide variety of other cell types.     

Micromechanics of Sea Urchin Spines

The endoskeletal structure of the Sea Urchin, Centrostephanus rodgersii, has numerous long spines whose known functions include locomotion, sensing, and protection against predators. These spines have a remarkable internal microstructure and are made of single-crystal calcite. A finite-element model of the spine’s unique porous structure, based on micro-computed tomography (microCT) and incorporating anisotropic material properties, was developed to study its response to mechanical loading. Simulations show that high stress concentrations occur at certain points in the spine’s architecture; brittle cracking would likely initiate in these regions. These analyses demonstrate that the organization of single-crystal calcite in the unique, intricate morphology of the sea urchin spine results in a strong, stiff and lightweight structure that enhances its strength despite the brittleness of its constituent material.     

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.     

Ryanodine Activates Sea Urchin Eggs

We have studied the effect on sea urchin eggs of ryanodine, a plant alkaloid that causes muscle contraction by opening calcium channels in the sarcoplasmic reticulum terminal cisternae. Ryanodine, although it is less effective that IP₃, produces full or partial activation in 62% of injected sea urchin eggs. In addition ryanodine inhibits in a dose dependant manner ⁴⁵Ca pumping in the isolated egg cortex or in eggs permeabilized with digitonin. Efflux experiments show that in fact ryanodine as IP₃ stimulates the release of calcium sequestered intracellularly. We further show that these effects of ryanodine are inhibited by Mg⁺⁺, ruthenium red and heparin. Our results suggest that ryanodine-sensitive intracellular calcium channels exist in the sea urchin egg.     

Eutacticity in Sea Urchin Evolution

An eutactic star, in a n-dimensional space, is a set of N vectors which can be viewed as the projection of N orthogonal vectors in a N-dimensional space. By adequately associating a star of vectors to a particular sea urchin, we propose that a measure of the eutacticity of the star constitutes a measure of the regularity of the sea urchin. Then, we study changes of regularity (eutacticity) in a macroevolutive and taxonomic level of sea urchins belonging to the Echinoidea class. An analysis considering changes through geological time suggests a high degree of regularity in the shape of these organisms through their evolution. Rare deviations from regularity measured in Holasteroida order are discussed.     

Synthesis and Turnover of Embryonic Sea Urchin Ciliary Proteins during Selective Inhibition of Tubulin Synthesis and Assembly

When ciliogenesis first occurs in sea urchin embryos, the major building block proteins, tubulin and dynein, exist in substantial pools, but most 9+2 architectural proteins must be synthesized de novo. Pulse-chase labeling with [³H]leucine demonstrates that these proteins are coordinately up-regulated in response to deciliation so that regeneration ensues and the tubulin and dynein pools are replenished. Protein labeling and incorporation into already-assembled cilia is high, indicating constitutive ciliary gene expression and steady-state turnover. To determine whether either the synthesis of tubulin or the size of its available pool is coupled to the synthesis or turnover of the other 9+2 proteins in some feedback manner, fully-ciliated mid- or late-gastrula stage Strongylocentrotus droebachiensis embryos were pulse labeled in the presence of colchicine or taxol at concentrations that block ciliary growth. As a consequence of tubulin autoregulation mediated by increased free tubulin, no labeling of ciliary tubulin occurred in colchicine-treated embryos. However, most other proteins were labeled and incorporated into steady-state cilia at near-control levels in the presence of colchicine or taxol. With taxol, tubulin was labeled as well. An axoneme-associated 78 kDa cognate of the molecular chaperone HSP70 correlated with length during regeneration; neither colchicine nor taxol influenced the association of this protein in steady-state cilia. These data indicate that 1) ciliary protein synthesis and turnover is independent of tubulin synthesis or tubulin pool size; 2) steady-state incorporation of labeled proteins cannot be due to formation or elongation of cilia; 3) substantial tubulin exchange takes place in fully-motile cilia; and 4) chaperone presence and association in steady-state cilia is independent of background ciliogenesis, tubulin synthesis, and tubulin assembly state.     

The Control of Sea Urchin Metamorphosis: Ionic Effects

Because the cascade of events which comprise sea urchin metamorphosis occur rapidly, regulatory mechanisms able to respond in minutes must function. Employing sea water solutions of altered ionic composition in the presence or absence of metamorphically active microbial films, we tested the ability of particular ions to inhibit or enhance metamorphosis in competent larvae of the sea urchin, Lytechinus variegatus . At 40 mM excess potassium maximally induces normal metamorphosis in the absence of a microbial film. In the presence of metamorphically active microbial films, 40 mM excess magnesium inhibits the process. Increasing concentrations of calcium up to an excess of 40 mM stimulates larvae to undergo metamorphosis but in smaller proportions than similar concentrations of potassium. Divalent cation-free sea water solutions are toxic to larvae. These studies support the hypothesis that ion fluxes are involved in the regulation of metamorphosis and reveal a complexity of response that parallels the histological complexity of competent echinoid larvae.     

The Sea Urchin Blastula: Extent of Cellular Determination

The extent of cellular determination at the blastula stage ofsea urchins has been investigated by using as parameters themorphological differentiation and cellular interactions of dissociatedblastula cells under conditions that either promote or discouragetheir reaggregation and reconstitution into embryos. The dissociatedblastula cells have the capacity to differentiate into at leastthree different specific cell types that occur in the normallydeveloping sea urchin embryo. Of these, ciliated epitheliumis known to originate in the animal half, and spindle-shapedcells and polyfilamentous cells are of mesenchymal origin andare derived from the vegetal half. The formation of ciliatedepithelium and blastulation requires calcium and hyalin; thecellular differentiation and interactions of the mesenchymalcells do not. This basic difference in calcium requirement isexplored in terms of differential hyalin synthesis or secretionby the cells at either equatorial half of the embryo. The animalizingeffect of zinc and the vegetalizing effect of lithium can beexplained in part by their enhancement and reduction, respectively,of hyalin gelation by calcium. Literature relating to otherbasic differences along the animal-vegetal axis of the embryois appraised. These differences include the use and the assemblyof microtubule protein and quantitative RNA synthesis duringboth the cleavage and blastula stages.     

A Century of Sea Urchin Development

SYNOPSIS. In the last quarter of the nineteenth century severalinvestigators including Richard and Oskar Hertwig, Theodor Boveri,Hans Driesch, Curt Herbst, T. H. Morgan and others turned theirattention to sea urchin eggs and early embryos. This favorablecombination of outstanding investigators and the sea urchinembryo as an experimental organism contributed to a fundamentalunderstanding of the cell, fertilization and heredity. The advantagesof the sea urchin continued to be recognized as experimentalembryologists used these embryos to develop the concepts ofgradients, regulative development and inductive interactions.Then, as developmental biology arose from chemical embryology,the sea urchin embryo once again emerged as an ideal experimentalanimal, pivotal in the understanding of the molecular and developmentalbiology of eukaryotic organisms.     

Electric Field-induced Fusion of Sea Urchin Eggs

An electrical method is described which permits the fusion of denuded eggs of the sea urchin species Paracentrotus lividus . In a nearly non-conductive medium, containing 1.2 M glucose at pH 6.0–8.4, eggs or fertilized stages are brought into close membrane contact by dielectrophoresis arising from the application of a highly inhomogeneous alternating electric field. During this process the eggs aligne parallel to the field forming egg-chains. In well pigmented eggs pigmentcapping is observed in the areas of cell contact. After completion of the alignment, the application of an additional single high field pulse of μs duration induces fusion of two or more eggs. The mechanism underlying the fusion process is the reversible electric breakdown of membranes in the zones of cell-to-cell contact. Fusion proceeds within 1–10 min at 10–20°C. Fused eggs have intact nuclei, can be fertilized, but undergo abortive cleavage.     

海胆Runx基因的研究进展

海胆Runx(Runt box)基因是Runx基因家族的成员之一,在海胆的胚胎发育、细胞的增殖和分化过程中起重要的作用.海胆Runx基因表达的缺失或下调,将会影响其它相关基因的表达,进而影响胚胎的正常发育.综述了海胆Runx基因的研究进展.