Females of the sea urchin Strongylocentrotus purpuratus differ in the structures of their egg jelly sulfated fucans

The egg jelly coats of sea urchins contain sulfated fucans which bind to a sperm surface receptor glycoprotein to initiate the signal transduction events resulting in the sperm acrosome reaction. The acrosome reaction is an ion channel regulated exocytosis which is an obligatory event for sperm binding to, and fusion with, the egg. Approximately 90% of individual females of the sea urchin Strongylocentrotus purpuratus spawned eggs having only one of two possible sulfated fucan electrophoretic isotypes, a slow migrating (sulfated fucan I), or a fast migrating (sulfated fucan II) isotype. The remaining 10% of females spawned eggs having both sulfated fucan isotypes. The two sulfated fucan isotypes were purified from egg jelly coats and their structures determined by NMR spectroscopy and methylation analysis. Both sulfated fucans are linear polysaccharides composed of 1-->3-linked alpha-L-fucopyranosyl units. Sulfated fucan I is entirely sulfated at the O -2 position but with a heterogeneous sulfation pattern at O -4 position. Sulfated fucan II is composed of a regular repeating sequence of 3 residues, as follows: [3-alpha-L-Fuc p - 2,4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)- 1]n. Both purified sulfated fucans have approximately equal potency in inducing the sperm acrosome reaction. The significance of two structurally different sulfated fucans in the egg jelly coat of this species could relate to the finding that the sperm receptor protein which binds sulfated fucan contains two carbohydrate recognition modules of the C-type lectin variety which differ by 50% in their primary structure.      

Refertilization in eggs of the sea urchin Strongylocentrotus purpuratus

To determine the role of the sea urchin egg plasma membrane in the species-specificity of fertilization, the ability of denuded activated eggs to be heterospecifically refertilized was determined. Our initial studies included evaluating the effectiveness of three commonly used methods of vitelline envelope (VE) removal using indirect immunofluorescence microscopy with antibodies directed against the VE. Unfertilized Strongylocentrotus purpuratus eggs were extracted with 0.01 M dithiothreitol (DTT) for 3 min or digested with 1.0 mg/ml pronase for 1 hr. Eggs were also fertilized, then diluted into a divalent-free medium to produce thin, elevated envelopes (VE*s) that were mechanically removed by sieving the eggs through nylon mesh. We found that both DTT extraction and pronase digestion were not completely effective in VE removal, and mechanical removal methods gave rise to a mixed population of eggs, those that had their VEs removed and those with a collapsed envelope that was not detectable at the light microscope level. Therefore, a new method of VE removal was developed. Eggs with VE*s were prepared followed by treatment with 0.01 M DTT to solubilize the envelopes. Nearly 100% of the denuded activated eggs incorporated one or more homologous and heterologous sperm, suggesting that the egg plasma membrane does not function in determining the species-specificity of fertilization.     

Apoptosis in early development of the sea urchin, Strongylocentrotus purpuratus

Apoptosis provides metazoans remarkable developmental flexibility by (1) eliminating damaged undifferentiated cells early in development and then (2) sculpting, patterning, and restructuring tissues during successive stages thereafter. We show here that apoptotic programmed cell death is infrequent and not obligatory during early embryogenesis of the purple sea urchin, Strongylocentrotus purpuratus. During the first 30 h of urchin development, fewer than 20% of embryos exhibit any cell death. Cell death during the cleavage stages consists of necrotic or pathological cell death, while cell death during the blastula and gastrula stages is random and predominantly caspase-mediated apoptosis. Apoptosis remains infrequent during the late blastula stage followed by a gradual increase in frequency during gastrulation. Even after prolonged exposure during the cleavage period to chemical stress, apoptosis occurs in less than 50% of embryos and always around the pre-hatching stage. Embryonic suppression of apoptosis through caspase inhibition leads to functionally normal larvae that can survive to metamorphosis, but in the presence of inducers of apoptosis, caspase inhibition leads to deformed larvae and reduced survival. Remarkably, however, pharmacological induction of apoptosis, while reducing overall survival, also significantly accelerates development of the survivors such that metamorphosis occurs up to a week before controls.     

A genome-wide analysis of biomineralization-related proteins in the sea urchin Strongylocentrotus purpuratus

Biomineralization, the biologically controlled formation of mineral deposits, is of widespread importance in biology, medicine, and engineering. Mineralized structures are found in most metazoan phyla and often have supportive, protective, or feeding functions. Among deuterostomes, only echinoderms and vertebrates produce extensive biomineralized structures. Although skeletons appeared independently in these two groups, ancestors of the vertebrates and echinoderms may have utilized similar components of a shared genetic "toolkit" to carry out biomineralization. The present study had two goals. First, we sought to expand our understanding of the proteins involved in biomineralization in the sea urchin, a powerful model system for analyzing the basic cellular and molecular mechanisms that underlie this process. Second, we sought to shed light on the possible evolutionary relationships between biomineralization in echinoderms and vertebrates. We used several computational methods to survey the genome of the purple sea urchin Strongylocentrotus purpuratus for gene products involved in biomineralization. Our analysis has greatly expanded the collection of biomineralization-related proteins. We have found that these proteins are often members of small families encoded by genes that are clustered in the genome. Most of the proteins are sea urchin-specific; that is, they have no apparent homologues in other invertebrate deuterostomes or vertebrates. Similarly, many of the vertebrate proteins that mediate mineral deposition do not have counterparts in the S. purpuratus genome. Our findings therefore reveal substantial differences in the primary sequences of proteins that mediate biomineral formation in echinoderms and vertebrates, possibly reflecting loose constraints on the primary structures of the proteins involved. On the other hand, certain cellular and molecular processes associated with earlier events in skeletogenesis appear similar in echinoderms and vertebrates, leaving open the possibility of deeper evolutionary relationships.     

Development of the esophageal muscles in embryos of the sea urchin Strongylocentrotus purpuratus

Summary Development of the esophageal muscles in embryonic sea urchins is described using light- and electron microscopy. The muscles develop from processes of about 14 cells of the coelomic epithelium that become immunore-active to anti-actin at about 60 h (12–14° C). Initially, eachmyoblast extends a single process with numerous fine filopodia around the esophagus. By 72 h the processes have reached the midline and fused with those from cells of the contralateral coelomic sac. Myoblasts begin to migrate out of the coelomic epithelium between 72 and 84 h. By 72 h the processes stain with the F-actin specific probe NBD-phallacidin. The contractile apparatus is not evident in transmission electron-microscopic preparations of embryos at 70 h, but by 84 h the contractile apparatus is present and the muscle cells are capable of contraction. Because the myoblasts migrate free of the coelomic epithelium and are situated on the blastocoelar side of the basal lamina, it is suggested that that they should be considered as a class of mesenchymal cells.     

Developmental distribution of a cell surface glycoprotein in the sea urchin Strongylocentrotus purpuratus

Recent studies from this laboratory have shown that an antigen recognized by a monoclonal antibody (MAb 1223) displays a bimodal distribution of expression in development of the embryo of Strongylocentrotus purpuratus. This molecule is specifically localized to the primary mesenchyme cells of the embryo, but is also found within the egg. In the current study, immunoelectron microscopy was used to determine the subcellular distribution of the antigen and to determine its fate during early stages of development of the embryo. In eggs, the epitope recognized by MAb 1223 was localized to the cortical vesicles. Immunoblot analysis of an isolated cell surface complex (CSC) that contained the cortical vesicles revealed the presence of a 130-kDa protein, as well as immunoreactive components of higher molecular weight. Upon fertilization, the antigen was exocytosed from the cortical vesicles and became associated with the hyaline layer, the fertilization envelope, and the plasma membrane. Subsequently, the epitope could be detected within small vesicles and yolk platelets. By 60 min postfertilization, the amount of epitope detected intracellularly or in the perivitelline compartment was greatly reduced. At later stages of development, when formation of the embryonic skeleton occurred, the 1223 antigen was principally localized to the Golgi complex and to the syncytial cell surface of the primary mesenchyme cells. Thus, the results of this study suggest that in S. purpuratus the 1223 antigen is stored and secreted from the cortical vesicles of the egg, degraded after fertilization, and then later expressed on the surface of the primary mesenchyme cells.     

Calcium in sea urchin egg during fertilization

Calcium plays a strikingly important role in two of the major events in developmental biology: cell activation and differentiation. In this review we begin with the location and quantity of intracellular calcium in sea urchin oocytes, and then discuss the changes that occur during fertilization and egg activation, placing special emphasis on the mobilization and redistribution of intracellular calcium. We also discuss the propagation of the calcium wave and the role of the burst of calcium on the process of reorganizing the egg cortex at fertilization.