Phosphorylation of sperm histone H1 is induced by the egg jelly layer in the sea urchin Strongylocentrotus purpuratus

Phosphorylation of histone H1 occurs when spermatozoa of the sea urchin Strongylocentrotus purpuratus are treated with the macromolecular fraction of solubilized egg jelly. Phosphorylation is on serine residues in the N-terminal fragment of H1 bisected with N-bromosuccinimide. Phosphorylation is maximal by 4-8 min and dependent on Ca2+, but independent of Na+ or increased intracellular pH. Phosphorylation of H1 can be dissociated from the induction of the acrosome reaction. Only a fraction of the H1 molecules become phosphorylated upon treatment of sperm with egg jelly. The amount of phosphate per mole of H1 increases from 0.15 moles before jelly treatment to 0.46 moles after maximal phosphorylation. Phosphorylation of H1 occurs in a cAMP-dependent manner as indicated by the ability of the phosphodiesterase inhibitors IBMX and SQ20009 to induce H1 phosphorylation. This phosphorylation reaction can be blocked by digesting the sperm surface with Pronase, or preincubation of sperm in wheat germ agglutinin, showing that a ligand in egg jelly must interact with a sperm surface receptor to activate the kinase phosphorylating H1.     

Distribution of tubulin-containing structures in the egg of the sea urchin Strongylocentrotus purpuratus from fertilization through first cleavage

Eggs of the sea urchin Strongylocentrotus purpuratus were examined by indirect immunofluorescence microscopy for tubulin-containing structures at intervals from fertilization through first cleavage. The staining revealed that the monaster is made up not only of the sperm aster but also of tubulin-staining fibers originating elsewhere in the egg. The monaster does not divide directly but is broken down first before the amphiaster or interphase asters begin to form. The interphase asters reach a peak of development at the streak stage and are in turn broken down before the formation of the mitotic apparatus. The breakdown of the monaster, interphase asters, as well as the asters of the mitotic apparatus proceeds from the cell center or aster centers to the periphery of the cell and is followed by growth of new asters, also proceeding outward from the aster centers. The pattern suggests a transient wavelike movement of some condition, or factor, which favors microtubule depolymerization.     

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.     

The larval stages of the sea urchin, Strongylocentrotus purpuratus

The adult body plan of Strongylocentrotus purpuratus is established within the imaginal rudiment during the larval stages. To facilitate the study of these stages, we have defined a larval staging scheme, which consists of seven stages: Stage I, four-arm stage; Stage II, eight-arm stage; Stage III, vestibular invagination stage; Stage IV, rudiment initiation stage; Stage V, pentagonal disc stage; Stage VI, advanced rudiment stage; and Stage VI, tube-foot protrusion stage. Each stage is characterized by significant morphological features observed for the first time at that stage. This scheme is intended as a guide for determining the degree of larval development, and for identifying larval and adult structures. Larval anatomy was visualized using light and confocal microscopy as required on living material, whole mount fixed specimens, and serial sections. Antibody staining to localize specific gene products was also used. Detailed analysis of these data has furthered our understanding of the morphogenesis of the rudiment, and has suggested provocative questions regarding the molecular basis for these events. We intend this work to be of use to investigators studying gene expression and morphogenesis in postembryonic larvae.     

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.     

Characterization of the vitelline envelope of the sea urchin Strongylocentrotus purpuratus

The vitelline envelope (VE) is an extremely thin, acellular, proteinaceous coat that surrounds the extracellular surface of sea urchin eggs. Despite previous studies on VE composition, structure and function, our understanding of the envelope is still incomplete at the molecular level. We have isolated VE components from intact, unfertilized Strongylocentrotus purpuratus eggs by reduction with alkaline dithiothreitol-sea water solutions and have characterized the macromolecules by SDS-PAGE. There were eight major glycoprotein bands, including two high molecular weight components at 265 and 300 kDa, and several minor components. We have revealed, by lectin blot analysis, that most components contain mannose, while a subset of glycoproteins contain fucose and N -acetylglucosamine; galactose and sialic acid were also detected. The components in the VE preparations were compared with cell surface complex preparations by immunoblot analysis, using antisera against a VE preparation, a 305 kDa electrophoretically purified VE glycoprotein and an extracellular portion of the sea urchin egg recombinant 350 kDa sperm receptor. Serum against the recombinant sperm receptor reacted with a component of ∼350 kDa on blots, but did not react with the 300 kDa component found in VE preparations. Therefore, we suggest these two glycoproteins are not the same.     

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 studies of sea urchin chromatin. Chromatin isolated from spermatozoa of the sea urchin Strongylocentrotus purpuratus

Chromatin was isolated from spermatozoa of the sea urchin Strongylocentrotus purpuratus. The isolated chromatin shows less absorptivity ratio of 230 nm : 260 nm and possesses less protein than does embryonic chromatin. The ratio of histone : DNA is 1.02; nonhistone : DNA 0.13; RNA : DNA 0.04. Sperm chromatin melts in two steps with T_ms 70°C and 84°C in 2.5 × 10⁻⁴, M EDTA in contrast to embryonic chromatin with a single T_m = 72°C. Disc electrophoresis of basic proteins of sperm revealed one minor component with extremely fast mobility and three major components. The one with the slowest mobility is characteristic of sperm. The embryo has in turn its characteristic histone which also migrated slowly in disc electrophoresis. Both of these unique histone fractions are selectively extracted from chromatin by 5% perchloric acid. Amino acid analyses of these chromatographically purified unique fractions show that both contain a large amount of lysine, while that from sperm, in addition, contains also a large amount of arginine. Minimal molecular weights of 33,000 for sperm and 16,200 for embryo unique histone were estimated from these analyses. Sperm chromatin supports a level of RNA synthesis in vitro with exogeneously supplied RNA polymerase about 2% that of the corresponding free DNA.     

The origin of pigment cells in embryos of the sea urchin Strongylocentrotus purpuratus

A monoclonal antibody (SP1/20.3.1) that recognizes a cell surface epitope expressed by pigment cells in the pluteus larva of Strongylocentrotus purpuratus has been produced. Using indirect immunofluorescence, the epitope is first detected in nonpigmented cells of the vegetal plate after primary mesenchyme ingression. Between the beginning of gastrulation, and when the archenteron is one-third the distance across the blastocoel, SP1/20.3.1-positive cells are free within the blastocoel, at the tip of the archenteron, and dispersed within the blastoderm. Cells at the tip of the archenteron, and mesenchyme near the tip in later stages of gastrulation (secondary mesenchyme), do not express the SP1/20.3.1 antigen. By the completion of gastrulation all SP1/20.3.1-positive cells are dispersed throughout the epidermis. It has been concluded that in S. purpuratus pigment cell precursors are released from the vegetal plate during the initial phase of gastrulation. The cells migrate first to the vegetal ectoderm, and subsequently disperse throughout the ectoderm and develop pigment granules.