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Procedures were developed for the purification of calmodulin from the eggs and from the sperm of the sea urchin Strongylocentrotus purpuratus and Arbacia punctulata. Two forms of calmodulin were isolated from A. punctulata eggs, one from each of the other three sources. All five calmodulins are similar to vertebrate calmodulin as judged by their activation of bovine brain cyclic nucleotide phosphodiesterase, calcium-dependent interaction with troponin I and with chloropromazine, increased anode mobility on sodium dodecyl sulfate gels in the presence of calcium, cross-reactivity with anti-vertebrate calmodulin antibodies and amino acid compositions including the presence of a single residue of trymethyllysine. The two forms of calmodulin from A. punctulata eggs, calmodulin A and calmodulin B, can be distinguished from one another on the basis of DEAE-cellulose chromatography, amino acid analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence or absence of calcium and competitive radioimmunoassays. There is no evidence that either form is a proteolytic product of the other.

The normal elevation of the fertilization membrane and the establishment of the block to polyspermy are retarded in Arbacia punctulata eggs by specific protease inhibitors, soybean trypsin inhibitor (SBTI), leupeptin, and antipain. Ultrastructural observations show that the vitelline layer remains attached to the plasma membrane of fertilized SBTI treated eggs at numerous sites (cortical projections). Quantitive morphometric analysis indicates that the vitelline layer elevates from about 65% of the surface of SBTI treated eggs during the first 3 min post insemination. However, the vulnerability of SBTI treated eggs to refertilization (polyspermy) only declined during the subsequent gradual detachment of the vitelline layer from the cortical projections over the next 15 min. Antipain and leupeptin (10−5 to 10−3 M) also promoted polyspermy in Arbacia eggs by a process of refertilization extending for a 10- to 15-min period after the initial monospermic insemination. Normal cleavage and development was obtained when eggs were placed in leupeptin and antipain (10−3 M) after the fertilization membrane had elevated. The data indicate that the normal secretory function (or functions) of the cortical granule protease in establishing the block to polyspermy is retarded by these protease inhibitors, and that the vitelline layer is transformed into a mechanical barrier to prevent penetration by supernumerary sperm during its detachment from the plasma membrane of the egg. Furthermore, the vitelline layer in unfertilized eggs appears to be a mosaic structure, with sperm receptor sites localized in regions of the egg's surface, which give rise to cortical projections in the presence of SBTI.

Fertilized and unfertilized Arbacia eggs were incubated with extracellular tracers in an effort to: (1) correlate the onset of pinocytosis with egg activation and its possible role in restructuring the zygote's surface and (2) determine the fate of internalized vesicles and their contents. Zygotes incubated with horseradish peroxidase (HRP), native ferritin, or cationized ferritin incorporated tracers within coated vesicles by 4–7 minutes postinsemination. In contrast, endocytosis by unfertilized eggs was not observed. Zygotes taken at intervals up to cleavage (60 minutes postinsemination) exhibited all three tracers within coated and uncoated vesicles and lysosomes. HRP reaction product was identified within Golgi cisternae in only a few zygotes; native and cationized ferritin were never observed within Golgi elements. The sequential appearance of tracers in pinocytotic vesicles and lysosomes of fertilized eggs suggests that surface-related components are interiorized and then modified or degraded. Because the zygote surface undergoes a reduction in area, temporally correlated with the onset of pinocytosis, it is possible that endocytosis may be instrumental in the accommodation of the plasmalemma. The interiorization of surface membrane in fertilized eggs may represent a mechanism for the alteration of developmentally related cell surface components.

1. 1. Evidence for the occurrence of the orotate pathway for the de novo biosynthesis of pyrimidines in the developing sea urchin embryo was provided by the incorporation of (14C) NaHCO3, (14C) carbamoylaspartate, and (6-14C) orotic acid into the pyrimidines of RNA, in vivo. 2. 2. This interpretation was supported by the observation that the radio-label from (14C) NaHCO3 appeared in the C-2 position of the uracil ring and by the demonstration of the activity of all of the enzymes of the orotate pathway in cell-free extracts of sea urchin eggs. 3. 3. Evidence was obtained for the regulation of the de novo pathway by end-product inhibition.

We have purified and partly characterized a calcium-binding protein from the unfertilized egg of the sea urchin Arbacia punctulata. This protein closely resembles the calcium-binding modulator protein of bovine brain in its molecular weight, electrophoretic mobility, amino acid analysis, and peptide map. It activates bovine brain phosphodiesterase in the presence of calcium but has no effect on the phosphodiesterase of the Arbacia egg. Densitometric scanning of acrylamide gels of arbacia egg homogenates shows the modulator protein to represent 0.1% of the total protein of the egg. At 10(-4) M free calcium, the protein binds four calcium ions per 17,000-dalton molecule. We have used a column of rabbit skeletal muscle troponin-I covalently coupled to Sepharose 4B as an affinity column to selectively purify the Arbacia egg calcium-binding protein. This column has also been used to purify bovine brain modulator protein and may prove of general use in isolating similar proteins from other sources. The technique may be particularly helpful when only small quantities of starting material are available.

The effect of altering normal cell associations and interactions on the synthesis of 5S RNA and transfer RNA (tRNA) was studied in cleaving embryos of the sea urchin, Arbacia punctulata. Cell interactions were altered: (1) by culturing cleaving embryos in the animalizing agent, Evans Blue, and in the vegetalizing agent, Li+ as LiCl and (2) by culturing dissociated cells. Control and experimental embryos each were labeled from 3 h to 6 h post fertilization with [8-3H]-guanosine. Sixteen-cell embryos, whose GTP precursor pools had been preloaded, were dissociated, labeled and cultured under conditions which prevent reaggregation. Quantitative measurements of rates of accumulation of newly synthesized 5S RNA and tRNA showed that these rates are similar in cleaving sea urchin embryos and in corresponding embryos cultured in the presence of Evans Blue and of Li+. In addition, cells dissociated from cleavage embryos and maintained under conditions which prevent reaggregation retained the ability to synthesize 5S RNA and tRNA. These results suggest that normal cell associations and interactions are not necessary for the synthesis of 5S RNA and tRNA to occur in cleaving sea urchin embryos.

Arachidonic acid can be oxidized via the cyclooxygenase pathway to produce prostaglandins and via the 5−lipoxygenase pathway to produce leukotrienes. These substances are known to be extremely potent regulators of cellular function in somatic tissues, particularly during inflammatory reactions. Recent studies have implicated cyclooxygenase−derived products in preventing polyspermy in sea urchins [Schuel et al, Gamete Res 10:9–19, 1984]. FPL−55712, a receptor antagonist for leukotrienes in somatic tissues, causes a dose−(1–10 μM) and sperm−density−dependent induction of polyspermic fertilization in sea urchins if added before the egg completes the cortical reaction (elevation of the fertilization envelope). Eggs pretreated with FPL−55712 become polyspermic upon subsequent insemination with untreated sperm in sea water. Sperm pretreated with the drug do not cause polyspermy when used to inseminate untreated eggs. The 5−lipoxygenase inhibitor BW775C also promotes polyspermy. FPL−55712 and BW755C do not retard elevation of the fertilization envelope. These findings imply that (1) leukotrienes may be produced via the 5−lipoxygenase pathway during fertilization in sea urchins, and (2) the reaction of leukotrienes with putative receptors on the egg's surface may modulate its receptivity to sperm during the cortical reaction, and thereby help prevent polyspermy.

Phospholipase activity of egg-water treated Arbacia punctulata and Lytechinus variegatus sperm was shown to result from the sequential action of phospholipase A and lysophospholipase. A transient burst of phospholipase A activity followed induction of the acrosome reaction with egg water. The time of appearance suggested an acrosomal localization of the enzyme. The peak activity of phospholipase A correlated with initiation of sperm-egg fusion, suggesting a role for sea urchin sperm phospholipase A in membrane fusion and/or egg activation during fertilization.

Development of Arbacia punctulata embryos in the presence of bromodeoxyuridine (BUdR) was investigated under conditions which allowed about 20% substitution of BUdR for thymidine residues in DNA. Development was essentially normal from fertilization to the feeding larva. There appeared to be no restricted stretches of DNA which failed to incorporate BUdR. Even very early stages were permeable to BUdR, which entered all the nuclei. The failure of BUdR substitution to block development in Arbacia is discussed in the light of evidence that this analogue blocks the onset of differentiative product synthesis in many cellular systems.

Electron microscopic and cytochemical investigations were carried out on inseminated Arbacia oocytes comparing structural and chemical properties of their microvillous surface and fertilization cones. Early fertilization cones (up to 4 min postinsemination) were relatively small, smooth surface projections of cytoplasm that engulfed the incorporated sperm nucleus. However, in contrast to surrounding microvillous areas of the oocyte surface, enlarged fertilization cones (8 to 15 min postinsemination) had a distinctive crenated appearance that persisted until their regression. When examined by various cytochemical techniques, membrane delimiting fertilization cones had a much lower affinity for agents that stain negatively charged and carbohydrate moieties (cationized ferritin, concanavalin A, ruthenium red, and alcian blue) than did other regions of the oocyte surface. This difference in surface properties of membrane delimiting the site of sperm-egg fusion was not due solely to incorporated sperm plasma membrane and did not occur when inseminated oocytes were incubated with cytochalasin B. Little or no difference in the membrane of the fertilization cone versus microvillous areas was observed when inseminated eggs were freeze-fractured or prepared with agents (filipin and polymixin B) to demonstrate β-hydroxysterols and anionic phospholipids. These observations indicate that membrane delimiting the fertilization cone differs from the remainder of the oocyte surface and suggests that following insemination significant rearrangements of surface molecules take place within the egg plasmalemma that give rise to asymmetries in membrane topography.

Fertilization and ionophore activation of the sea urchin Arbacia punctulata were inhibited in the presence of six analogs of the dye fluorescein. The concentration of any one dye needed for blockage of sperm or Ca-ionophoremediated activation in 50% of the eggs (I50) was a function of the dye's lipid solubility. Substantially higher concentrations of each dye were required to block activation by Ca-ionophore (A23187) than were needed to inhibit sperm activation. A detailed study of the action of Erythrosin B (tetraiodofluorescein) showed that its effects were readily reversible. The I50 concentration of Erythrosin B increased as temperature increased from 10 to 25°C. The kinetics of blockage indicated that Erythrosin B blocked some early step in the program of fertilization. The results suggest that these anionic dyes may inhibit fertilization by preventing successful sperm-egg fusion.

Sea urchin eggs are known to release H2O2 during the cortical reaction at fertilization to help prevent polyspermy by inactivating excess sperm in the vicinity. This process resembles the peroxidatic killing of bacteria by phagocytic leukocytes during inflammation. Associated with these reactions in leukocytes, arachidonic acid is released from phospholipids and can be oxidized via the cyclooxygenase pathway to produce prostaglandins. Nonsteroidal anti-inflammatory drugs (NSAID) that are cyclooxygenase inhibitors in somatic cells were used to determine whether Arbacia punctulata and Strongylocentrotus purpuratus eggs use these processes to help prevent polyspermy. The potent cyclooxygenase inhibitor indomethacin causes a dose (10–100 μM) and sperm density dependent induction of polyspermy if added before the egg completes the cortical reaction. It does not retard elevation of the fertilization envelope and does not promote polyspermy by protecting sperm from peroxidatic inactivation by egg-derived H2O2. Other potent cyclooxygenase inhibitors, flufenamate and meclofenamate, also induce polyspermy at 10–60 μM. Aspirin, a weak cyclooxygenase inhibitor in somatic cells, does not cause polyspermy at 5 mM. These findings provide evidence that prostaglandins or other cyclooxygenase-derived metabolites may help assure monospermic fertilization in sea urchins.

Eggs and embryos of Arbacia punctulata were microinjected with the fluorescent dye, Lucifer yellow CH, using a simple pressure injection system. When injected into eggs that were subsequently fertilized, the dye was distributed throughout all cells of the developing embryo. If one cell of a two-cell embryo was injected, dye did not diffuse into the uninjected blastomere. During subsequent development, all progeny of the injected cell contained dye resulting in an embryo that was half-fluorescent. Blue light irradiation of a two-cell embryo, one cell of which had been injected with Lucifer yellow, caused the injected blastomere to stop further divisions while the uninjected blastomere developed normally and was free of dye. These results indicate that the first two blastomeres of Arbacia embryos are not electrically coupled, nor up to the time of hatching, is there any coupling between cells in one half of the first cleavage plane and cells in the other half.

Exogenously supplied, C-l4 labeled organic matrix from oyster shell inhibited spicule formation by embryos of the sea urchin, Arbacia punctulata, as measured by simultaneous incorporation of inorganic C-14. Analysis of isolated spicules showed that the matrix reached the site of crystal growth and became incorporated into crystals. Comparison of the amount of matrix in spicules to inhibitory levels for matrix in an in vitro, pH-stat crystallization assay showed that the matrix became incorporated into spicules in an amount sufficient to account for the observed in vivo inhibition. The matrix did not act as a general metabolic inhibitor, as measured by respirometry, but rather seemed to have only the specific effect on spicule formation. Calculated and measured values of natural levels of matrix in spicules and urchin tests matched reasonably well with experimentally determined levels that regulated the rate of crystallization in vivo. Overall, the results support the idea that matrix fulfills a direct, re...

The ultrastructural localization of B-1,3-glucanase in three species of sea urchin eggs was determined using a monospecific antibody in an electronmicroscopic immunogold procedure. In all three species, Lytechinus variegatus, Strongylocentrotus purpuratus, and Arbacia punctulata, B-1,3-glucanase was localized specifically to the cortical granules. No other organelle within the egg contained significant label. During the fertilization reaction, B-1,3-glucanase was released from cortical granules into the perivitelline space and became associated with the hyaline layer. No significant label was found in association with the fertilization envelope.

At an extracellular pH of 6.6, a peptide (resact) isolated from the egg jelly of Arbacia punctulata increased the respiration rates of A. punctulata spermatozoa but did not activate sperm cells from Lytechinus pictus. In contrast, speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly), elevated the respiration rates of L. pictus but not A. punctulata spermatozoa. At normal seawater pH (7.6-8.0) egg jelly from A. punctulata, or egg jelly from L. pictus purified free of speract, inhibited L. pictus sperm respiration rates. Similarly, the egg jelly from L. pictus inhibited the respiration rates of A. punctulata spermatozoa. The jelly component responsible for the inhibition of respiration was nondialyzable. The inhibition of respiration induced by jelly could be reversed by the addition of speract to L. pictus spermatozoa and by the addition of resact to A. punctulata spermatozoa. Speract stimulated L. pictus sperm respiration half-maximally at about 1 nM in the presence of either heterologous or homologous (speract-free) jelly. Monensin A, an ionophore which elevates sperm intracellular pH, reversed the jelly inhibition of respiration. These results demonstrate that two peptides associated with eggs (speract and resact) can stimulate sperm motility and metabolism in the face of inhibitory components present in the egg jelly. Additionally, the peptides demonstrate species specificity.

Retinoids are being used increasingly in dermatologic practice. Fetal malformation is a major form of toxicity associated with certain retinoids. In this study, the developmental toxicity of isotretinoin, its metabolites, and a structurally related analog, tretinoin, were evaluated using the sea urchin model. The American sea urchin, Arbacia punctulata, completes its major developmental stages within 24 hr and has been previously utilized for screening human teratogens. The parent compound, isotretinoin, induced dose-dependent delayed rather than dysmorphic development of the sea urchin embryo. In contrast, its metabolites, 4-oxo-isotretinoin and 4-oxo-tretinoin, and the analog tretinoin induced strikingly dysmorphic development. This may indicate that the metabolites of isotretinoin, rather than the parent compound, may be responsible for the fetal abnormalities observed in the "isotretinoin teratogen syndrome." Therefore, the sea urchin model might serve as a discriminating and rapid screening test for identifying other potential developmentally toxic retinoids.

The microtubule-containing structures that appear in eggs during fertilization and cell division in the sea urchins Lytechinus variegatus and Arbacia punctulata were detected by antitubulin immunofluorescence microscopy of detergent extracted cytoskeletal preparations. The extraction buffer, which is composed of 0.55 mM MgCl2, 10 mM EGTA, 25 mM MES, 25% glycerol, 1% Nonidet P-40, and 25 μM PMSF, pH 6.7, allows for dramatically improved fluorescent images compared to those obtained using conventional staining procedures, with residual background staining being reduced to near zero. The immunofluorescent images obtained using this technique provide information on several motile events that occur during the first cell cycle. This technique demonstrates that all of the cytoplasmic microtubules are associated with the incorporated sperm's centrioles during female pronuclear migration. This changes during the centration of the male and female pronuclei at which time a monastral array of microtubules forms in the egg's cytoplasm. A large proportion of the monastral microtubules do not appear to be associated with the centrioles. At prophase and early metaphase, the centrioles are the dominant microtubule organizing centers (MTOCs) consistent with mitotic theories that the kinetochore catches, but does not initiate, microtubules. Observations of intercentriolar distances show that there are three stages of pole separation during the first cell cycle. The initial separation occurs during pronuclear centration, the second during the streak stage, and the final one during the late stages of mitosis. At telophase, polar microtubules appear to extend into the cortex supporting the cell surface at all regions except the presumptive cleavage site.

Studies carried out on reaggregating sea urchin embryonic cells reveal that reaggregating cells can give rise to normal pluteus larvae by three developmental pathways: (i) from clusters, (ii) from chains of beads, and (iii) from a tissue culture phase. Experiments carried out on a mixture of cells from two different species, Arbacia punctulata and Lytechinus pictus , demonstrate that reaggregation is species specific and that the sorting out of cells according to species occurs. Movement of cells within an aggregate is non-random and unidirectional. Electron microscope analysis of mixed aggregates of cells of the two species indicates that cells of the same species make initial contact and adhere to one another by means of numerous microvilli and with the formation of an intercellular hyaline-like material. Cells of the two different species adhere, but never by means of microvilli and no hyaline-like material can be detected on the cell surfaces. The results indicate a role in reaggregation of an intercellular material, possibly an intercellular cement.

1. The presence and intracellular location of serotonin binding sites in embryos of the sea urchin, Arbacia punctulata , were investigated by the binding of radiolabelled serotonin to late blastulae, gastrulae, prism larvae, early pluteus larvae, and advanced pluteus larvae subcellular fractions. 2. Although [ 3 H]serotonin binding activity was demonstrated in some cell fractions from each developmental stage examined, the subcellular distribution of binding sites was stage-specific. 3. In the older plutei, serotonin binding sites were tentatively determined to be located on cell surface membranes. 4. In younger embryos the localization of serotonin binding sites to other cell organelles is suggested. 5. [ 3 H]serotonin bound to nuclei of late blastula, gastrula, and prism embryos, while no binding to nuclear fractions of early and more advanced plutei was detected. 6. Results of binding studies support developmental stage-specific regulatory roles of serotonin during sea urchin embryogenesis.

Measurements and observations of five early events of fertilization, singly and in pairs, from single sea urchin eggs have revealed the precise temporal sequence and spatial distribution of these events. In the Arbacia punctulata egg, a wave of surface contraction occurs coincident with membrane depolarization (t = 0). These two earliest events are followed by the onset of a rapid, propagated increase in cytoplasmic-free calcium at approximately 23 s as measured by calcium-aequorin luminescence. The luminescence reaches its peak value by 40 s after the membrane depolarization. The luminescence remains uniformly elevated for some time before its decay over several minutes. The onset of an increase in the pyridine nucleotide (NAD(P)H) fluorescence follows the membrane depolarization at approximately 51 s. The fertilization membrane begins its elevation in a wave-like fashion coincidentally with the increase in NAD(P)H fluorescence. Similar results are observed in the Lytechinus variegatus egg. The results suggest that while the increase in cytoplasmic-free calcium may be important for many changes occurring in the egg, the elevated-free calcium is not directly responsible for the propagated wave of cortical granule exocytosis.

Resact, a peptide of known sequence isolated from the jelly layer of Arbacia punctulata eggs, is a potent chemoattractant for A. punctulata spermatozoa. The chemotactic response is concentration dependent, is abolished by pretreatment of the spermatozoa with resact, and shows an absolute requirement for millimolar external calcium. A. punctulata spermatozoa do not respond to speract, a peptide isolated from the jelly layer of Strongylocentrotus purpuratus eggs. This is the first report of animal sperm chemotaxis in response to a defined egg-derived molecule.

Embryos of Arbacia punctulata, treated to remove the fertilization membrane and hyaline layer, were mixed with sperm (reinseminated) at 20–30 min (streak stage) and at 70 min (2-cell stage) postinsemination. Sperm, incorporated into embryos reinseminated at the streak stage, metamorphosed into male pronuclei which subsequently migrated to and fused with the zygote nucleus. Although blastomeres were capable of incorporating sperm and forming fertilization cones, less than 10% of the 2-cell stage embryos reinseminated. Sperm, were found entrapped within an amorphous material along the surface of 2-cell stage embryos; many had failed to undergo an acrosome reaction. These results indicate that conditions necessary for incorporation and metamorphosis of sperm nuclei into male pronuclei are present in the embryo after the normal period of fertilization.

A cytoplasmic dynein ATPase has been identified in three species of unfertilized sea urchin eggs, Strongylocentrotus droebachiensis, S. purpuratus , and Arbacia punctulata . The enzyme was partially purified by sucrose gradient density centrifugation, and its polypeptide chain weight and composition were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein has enzymatic characteristics in common with flagellar dynein. It is activated nearly equally by Mg 2+ and Ca 2+ , has no activity in the presence of K + and EDTA, shows a specificity for ATP over other nucleoside triphosphates, and is inhibited by vanadate anion. On SDS-PAGE, the enzyme shows two major bands at 320,000 and 385,000 daltons, comigrating with certain ciliary and flagellar dynein polypeptides. The enzyme, given the name “egg dynein,” constitutes 2 to 4% of the total cell protein in the unfertilized egg and maintains this high value from fertilization through the late blastula stage. It appears to be equally distributed throughout the embryo at the 16-cell stage. Possible functions of egg dynein are discussed and models for dynein-microtubule mediated movements within the cytoplasm are presented.

The structurally similar diterpenoid alcohols pachydictyol-A and dictyol-E are produced by the brown seaweed Dictyota dichotoma. This seaweed and several related species that also produce these compounds are known to be relatively low preference foods for tropical fishes and urchins. We evaluated the effect of various concentrations of these compounds on feeding by the three common types of herbivores that co-occur with Dictyota in coastal North Carolina. Fish (Diplodus holbrooki), sea urchins (Arbacia punctulata), and a mixed species group of gammarid amphipods were offered pieces of the palatable seaweed Gracilaria tikvahiae coated with either (1) dictyol-E or pachydictyol-A dissolved in diethyl ether or (2) diethyl ether alone. Dictyol-E significantly reduced consumption by fish and urchins at concentrations of 0.5 and 1.0% of algal dry mass, but had no effect on amphipod grazing. Pachydictyol-A significantly reduced fish grazing at the relatively high concentrations of 1.0 and 1.3% of plant dry mass; at 0.5% it tended to decrease grazing, but the effect was not significant (P = .07). Pachydictyol-A had no effect on urchin grazing and significantly increased amphipod grazing. When Pachydictyol-A was fed to fish as 1.0% of food dry mass, their growth rate was reduced by a significant 48%. In feeding preference experiments with several seaweeds, Dictyota ranks low for fish and urchins but high for amphipods. This is consistent with the hypothesis that the sec- ondary metabolites produced by Dictyota play a major role in determining its susceptibility to herbivores. The ability of amphipods to circumvent the chemical defenses of Dictyota, and the fact that the two species of algae most readily consumed by amphipods (Codium and Dictyota) were the two species least readily consumed by fish, suggest that predation and herbivory by fishes may be major factors selecting for amphipods that can live on, and eat, seaweeds that are unpalatable to fishes. Amphipods that fed on Dictyota did not appear to sequester the Dictyota metabolites; when exposed to fish predation, Dictyota-fed amphipods were eaten as readily as amphipods that had fed on an alga with no defensive chemistry. Tubicolous amphipods and other small marine herbivores that may spend significant portions of their lives on only a few plants may be under very different evolutionary constraints than the larger, more mobile herbivores that commonly move between many plants. Several characteristics of these smaller, less mobile, and much less studied, marine herbivores suggest that they may be ecologically similar to terrestrial insects and may play a large, but presently unappreciated, role in structuring marine plant communities.

We have generated and characterized a monoclonal antibody (McA Tg-HYL) that recognizes sea urchin hyalin as evidenced by immunofluorescence staining of the hyaline layer (HL) and immunoblot staining of the hyalin protein band. On immunoblots of HL extracts only the hyalin protein reacted with McA Tg-HYL. Immunoprecipitates of radioactive proteins from embryos incubated with [3SS]methionine yielded radioactive hyalin and 190, 140 and 105×103Mr proteins associated with hyalin. McA Tg-HYL was generated against Tripneustes gratilla embryos but reacts with hyalin from the distantly related sea urchin species, Colobocentrotus atratus, Strongylocentrotus purpuradas, Arbacia punctulate, Lytechinus variegatus and Lytechinus pictus. Developing embryos of the abovementioned six species were treated with McA Tg-HYL and did not gastrulate or form arms. Observations of treated embryos revealed areas of separation of the hyaline layer from the underlying embryonic cells, suggesting that McA Tg-HYL was interfering with binding of the cells to the HL. Using the centrifugation-based adhesion assay of McClay et al. (Proc, natn. Acad. Sci. U.S.A. 78, 4975-4979, 1981), Fab’ fragments of McA Tg-HYL were found to inhibit cell-hyalin binding. McA Tg-HYL did not inhibit hyalin gelation in vitro or the reaggregation of dissociated blastula cells. We postulate that McA Tg-HYL recognizes an evolutionarily conserved hyalin domain involved in cell-hyalin binding and required for normal epithelial folding.

A membrane protein possessing sperm-aggregating activity was partially purified from Spisula oocyest. Spisula oocytes were incubated with three different media: A) 1 M urea, 5 mM EDTA, 10 mM Tris-HCI, pH 7.4, B) 1 M urea, 10 mM Tris-HCI, pH 7.4, and C) 5 mM EDTA in artificial sea water. Oocytes incubated in media A or B at 22°C were viable up to 15 min of treatment based on the trypan blue exclusion test. After this treatment period, oocyte viability gradually decreased as demonstrated by a progressive increase in the uptake of the dye. However, oocytes excluded the dye when incubated in medium C for 2 hr or longer. Oocytes incubated in medium A or B did not undergo germinal vesicle breakdown (GVBD) on exposure to sperm, while GVBD was induced on treatment with 70 mM KCI, suggesting removal or alteration of sperm receptors by the treatment. When sperm were incubated with oocyte extract prepared by treatment with medium A or B, they aggregated and formed clusters. The clusters remained unchanged for at least 1 hr at 22–24°C and sperm within the aggreates were motile. Extracts of Spisula oocytes showed species specificity by not agglutinating sperm of Arbacia punctulata, Asterias forbesi, ovalipes ocellatus, or Chaetopterus peramentaceus. The factor was puridied by ammonium sulfate fractionation (30% saturation) and by gel filtration on a Sephadex G 100 column. Four major protein peaks were eluted. Fraction comprising the second and third peaks possessed sperm-aggregating activity at an affective does od 2.5 μg of protein per ml. The factor is a heat-stable protein with an estimated molecular weight (mol wt) of 15 to 25 kdaltons.

Several factors involved in induction of the acrosomal reaction in sperm of the sea urchin, Arbacia punctulata, have been investigated quantitatively using a simple substrate film technique to monitor extension of the acrosomal process by electron microscopy. Verification of typical acrosomal process formation has been accomplished using thin sections. Sperm were found to undergo the acrosomal reaction in artificial sea water in the absence of egg jelly coat at pH values above 9.6. In the presence of egg jelly a high percentage of sperm react at pH 8.6. At this pH, the fraction of sperm that undergo the acrosomal reaction is directly proportional to the concentration of egg jelly. The Ca2+ ionophore A23187 induces the acrosomal reaction in the absence of egg jelly at pH 8.6. The proportion of sperm that react is dependent on the concentration of ionophore and on the concentration of Ca2+ in the medium. Pretreatment of sperm with low levels of La3+ ion, which is known to be a Ca2+ ion antagonist, results in inhibition of egg jelly induction of the acrosomal reaction. These findings suggest that there are marked similarities between the acrosomal reaction in sea urchin sperm and membrane fusion dependent secretory processes in other cell types.

Evidence is presented of cell-type specificity of surface proteins from the 16-cell stage sea urchin embryo. The protein composition of the micromere cell surface has been examined by 125 I labelling of intact cells followed by SDS-PAGE. In Arbacia punctulata , four high molecular weight (HMW) proteins are detected on the surface of isolated micromeres—but not on mesomere-macromere fractions. In Strongylocentrotus droebachiensis , a micromere-specific protein of 133 K molecular weight (MW) was identified. This 133 K protein binds to wheat germ agglutinin (WGA) but not to concanavalin A (conA). Lectin binding was studied using a new technique. The procedure involves the separation, by SDS-PAGE, of iodinated cell-surface proteins followed by their electrophoretic transfer to lectin-coated nitrocellulose membranes. Using this procedure, celltype-specific surface proteins which are also lectin-binding-specific, were detected.

Newly synthesized polysomal messenger RNAs from cleavage stage embryos of the sea urchin Arbacia punctulata and Lytechinus pictus that contain putative histone mRNAs have been fractionated on 6% polyacrylamide slab gels. At least 8 RNA species with unique electrophoretic mobilities have been recognized. The complex of RNAs has been eluted from the gels in three groups, A, B, and C, in increasing order of mobility. The template activity of the three fractions and the unfractionated starting material was examined in the mouse Krebs II ascites tumor cell-free protein synthesizing system. The unfractionated messenger complex programs the synthesis of proteins that coelectrophorese exclusively with sea urchin histones in both sodium dodecyl sulfate and acid urea gel systems. The products of in vitro protein synthesis stimulated by the individual polyacrylamide gel RNA fractions were similarly examined. Each stimulated protein synthesis and was enriched for specific histone templates. We conclude that RNA fraction A is template for histone f1, C is template for histone f2a1, and B serves as template for f2b, f2a2, and f3 histones. A minor degree of contamination of the A and B RNA fractions was obvious from the production of other histones by each template. The co-electrophoresis of specific template activity with specific radiolabeled RNAs supports the concept that most or all of the labeled RNAs are indeed themselves the histone mRNAs.

The fluxes of 22Na+ and 86Rb+ in Arbacia sperm and oocytes were studied in order to determine how these cells carry out cation exchange with the sea environment. The uptake of these ions by serum followed a pattern of early rapid influx (initial 0.5 min) and subsequent efflux (1–3 min) followed by a gradual uptake (after 3 min). Neither the uptake nor the efflux of these cations by Arbacia sperm were affected by ouabain, suggesting that influx and efflux of 22Na+ and 86Rb+ in Arbacia sperm occur predominantly by passive transport. The 22Na+ uptake by Arbacia oocytes showed a steady increase after an initial rapid uptake. A slight but significant inhibition of 22Na+ uptake was observed with ouabain. However, 86Rb+ uptake by the oocytes reached an early equilibrium and was not affected by ouabain. The uptake of Rb+ by Arbacia oocyte is by passive transport while that of Na+ is both by passive and active transport.

Blood phagocytes of the dogfish Mustelus canis attack oocytes of the sea urchin Arbacia punctulata , first provoking a surrogate fertilization response and then killing the eggs. To test the hypothesis that secretion of lysosomal contents is critical in this model of phagocyte-mediated cell injury, we studied effects of agents that modify lysosomal enzyme secretion. Inhibitors of membrane transport (>0.1 mM) inhibited postphagocytic secretion of lysosomal β-glucuronidase from dogfish phagocytes: phloretin > ethacrynate > furosemide > 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid >> pyridoxal phosphate > ouabain. The same order of activity was found for inhibition by these agents of killing of Arbacia eggs by phagocytes. Cell activation (fertilization response) and cytotoxicity were quantitated both morphologically and by measurements of enzyme (β-glucuronidase, catalase) release. The agents neither inhibited fertilization responses of eggs to calcium ionophore A23187 nor impaired their viability. Vital staining demonstrated that ethacrynate prevented phagocytes from degranulating upon contact with zymosan particles. The data not only suggest that agents primarily known for their capacity to inhibit membrane transport systems can inhibit lysosomal enzyme secretion from phagocytes but also support the hypothesis that secretion of lysosomal contents mediates activation and killing of target cells in phagocyte-mediated tissue injury.

To test whether lysosomal degranulation of phagocytes is associated with antibody-dependent cytotoxicity, eggs of Arbacia punctulata were used as targets for blood phagocytes of Mustelus canis . Eggs were coated with heat-aggregated dogfish IgM and exposed to phagocytes, and cytolysis of eggs was observed by Nomarski optics. Phagocytes adhered, degranulated, and raised fertilization membranes resembling those induced by sperm or ionophore A23187. Lysis was then observed as damage radiating from the point of phagocyte-egg contact. By 4 hr, coated eggs exposed to phagocytes released 8.9, 12.3, and 7.4% of total catalase (EC 1.11.1.6), β-glucuronidase (EC 3.2.1.31), and superoxide dismutase (EC 1.15.1.1) into the medium. Cytotoxic enzyme release significantly exceeded that from uncoated eggs incubated with phagocytes or eggs alone (uncoated or coated). Because activated eggs release a neutral protease, it was considered possible that this enzyme might be responsible for autolysis of eggs. This possibility was excluded because ( i ) lysis of eggs was not inhibited by soybean trypsin inhibitor (SBTI) whereas the egg protease was sensitive to SBTI, and ( ii ) the major trypsin-like activity of phagocytes was not inhibited by SBTI. These experiments demonstrate that Ig-coated cells are first activated, and then killed, when exposed to degranulating phagocytes and suggest that enzymes from attacking phagocytes, and not target cells, are responsible for cell death.

The early sea urchin embryo is supported in a concentric position within the perivitelline space by elongated microvilli which are attached to the fertilization envelope by extracellular matrix fibers. This "attachment complex," of microvillus tip: extracellular matrix fibers: fertilization envelope, was revealed by two methods: the use of pronase or calcium-free sea water to dissolve the extracellular matrix fibers, thus causing the eggs to lose their concentric location, and the visualization of the "attachment complex" using video-enhanced differential interference contrast microscopy and transmission electron microscope images. The presence of the "attachment complex" helps in understanding two types of early developmental events: (1) the apparently continual change in microvillus length during cleavage stages which retains the embryos in their concentric position and (2) the hatching process.

Actin is the major extractable protein component from the tube feet of four different species of sea urchin: Arbacia punctulata, Strongylocentrotus purpuratus, Strongylocentrotus droebachiensis, and Diadema setosum. Actin made up as much as 60% of the total Coomassie Blue-staining material after SDS polyacrylamide gel electrophoresis and densitometer analysis. Two-dimensional gel electrophoresis resolved two, and possible three, species of actin for each sea urchin of which the dominant component was analogous to the beta form in vertebrates. In a cell-free system from rabbit reticulocytes, total RNA from tube feet stimulated the synthesis of one protein that represented 80% of the total methionine incorporation, migrated with the properties characteristic of actin in a two-dimensional gel system, and on proteolysis yielded fragments identical to purified rabbit actin. The mRNAs from the tube feet of two divergent species of sea urchin, Arbacia punctulata and Strongylocentrotus purpuratus, synthesized actins differing by less than 0.02 pH unit for each isospecies 90% of the DNA copied from tube foot RNA by reverse transcriptase represented a highly abundant sequence class judged by copy DNA(cDNA)-RNA excess hybridization. At least two-thirds of this class represented a low-complexity component, with a Rot1/2 about three times that expected for actin messenger RNA. The remarkable degree of conservation of the actin protein is reflected in concomitant conservation of the protein-coding nucleotide sequences of the messenger RNA, which has allowed the use of a cDNA probe to isolate actin sequences from a human phage library.

Zygotes, treated with sodium-free artificial seawater (Na-free ASW) in order to inhibit the elevation of intracellular pH at fertilization, were examined by light and electron microscopy. Although such specimens elevated fertilization membranes, male pronuclear development and sperm aster formation were suppressed. Inhibition of these events was reversed when zygotes were resuspended in seawater or in Na-free ASW containing ammonia. These results indicate that alkalinization following insemination or processes accompanying this alteration induce a pervasive change within the zygote which supports events of fertilization.

Microtubules were purified from unfertilized eggs of the sea urchins Arbacia punctulata, Lytechinus pictus, Lytechinus variegatus, and Strongylocentrotus purpuratus. Numerous densely stained particles (24 x 26 nm) are associated with microtubules isolated from each of these sea urchins. The most striking aspect of this structure is an extended, slightly curved arm that appears to attach the particles to the microtubule. Morphologically similar particles are associated with microtubules of the isolated first cleavage mitotic apparatus. The particles are attached to the microtubules by ionic interactions and contain large amounts of extractable RNA. Based upon their size and density, RNA and protein composition, and sedimentation in sucrose gradients, the microtubule-associated particles are identified as ribosomes.

The present study investigates species-specific recognition and adhesion between dissociated embryonic cells of hatched blastulae of Arbacia punctulata, Lytechinus variegatus, and Strongylocentrotus purpuratus. The assay used is a modification of one previously developed by McClay and Hausman (1975) and involves collection of labeled single probe cells to unlabeled collecting aggregates. The results indicate that probe cells fixed by glutaraldehyde or formaldehyde or disrupted by sonication do not adhere significantly to their homospecific aggregates. Moreover, fixation of aggregates by glutaraldehyde greatly diminishes binding of labeled probe cells. Thus adhesion, as measured by the present assay, requires both living probe cells and aggregates. In addition, adhesion of probe cells to their homospecific aggregates is found to be significantly greater than adhesion to heterospecific aggregates. The results demonstrate reciprocal species-specific adhesion between Arbacia punctulata versus Lytechinus varie...

Spine catch ligaments of a sea urchin Arbacia punctulata were extended under constant load. Ligaments from an undisturbed animal may show any extension rate from zero (catch state) to rapid extension to failure. Replacing the preparation bath with CA(2)- and Mg(2+)-free sea water reversibly abolishes the catch state. The fine structure of the outermuscle layer and inner ligament cone associated with the spine base is described. The unstriated paramyosin muscles bear thin flanges and form compact interlocking rows. Subsurface cisternae are associated with the plasma membrane. The muscles are innervated by glia-free axons ending in bulbous terminals containing lucent synaptic vesicles. The ligament comprises cylindrical bundles of collagen fibrils: one or more minute muscle fibers (paramyosin) lie parallel with and closely adjoining each bundle. The mean diameter of these muscles is 0.3 micrometers and they occupy 2-3% of the ligament's cross-sectional area. Axons containing electron-opaque secretory droplets accompany the muscles between the collagen bundles: the cell bodies of these neurones generally lie on the outer surface of the ligament. When an urchin points a spine, the ligament on the side of the contracting spine muscle shortens but does not buckle. A function of the intraligamental muscles is to effect this non-buckled shortening. The catch mechanism (which reside entirely within the ligament) may be due either to the intraligamental muscles and/or to a locked polymer mechanism in which matrix molecules between collagen fibrils are reversibly cross-linked by divalent cations.

Utilization of marine plants and their constituents by bacteria isolated from the guts of echinoids was investigated to determine the potential role bacteria may have in carbohydrate digestion in echinoids. Bacteria from the guts of the regular echinoids Lytechinus variegatus (Lamarck) and Arbacia punctulata (Lamarck) could physically degrade the marine plants Ulva lactuca and Eucheuma nudum, but not Caulerpa prolifera. Diplanthera wrightii and Thalassia testudinum were only slightly degraded by the gut bacteria. Bacteria from the guts of the irregular echinoids Mellita quinquiesperforata (Leske) and Encope aberrans (Martens) could not physically degrade any of these marine plants. Mixed and some isolated bacteria from the gut of L. variegatus could utilize xylose, rhamnose, glucose, galactose, laminarin, carageenan, starch and agar, but not cellulose or chitin. The results with isolates suggest that the bacteria of the echinoid gut are fairly non-selective. The bacteria of the guts of the two regular echinoids could utilize certain marine plants that the echinoids eat and certain of the plant constituents. The bacteria could not utilize plant fibers.

To study the fate of the yolk glycoproteins found in eggs and embryos of the sea urchin, Strongylocentrotus purpuratus , a polyclonal antibody to a 90-kDa polymannose glycoprotein found in the embryo was prepared. Immunoblot analysis of total proteins over the course of development showed that this antibody recognized a family of glycoproteins. Concomitant with the disappearance of the major 160-kDa yolk glycoprotein of the egg during embryogenesis, glycoproteins with a lower molecular mass appeared. These glycoproteins (115, 108, 90, 83, and 68 kDa) were purified from S. purpuratus and analyzed by limited proteolysis and peptide mapping. This analysis revealed that these glycoproteins were cleavage products derived from the major yolk glycoprotein. The antibody to the 90-kDa glycoprotein in S. purpuratus embryos was used to identify a homologous set of yolk glycoproteins with similar molecular masses in the embryos of three other species in the class Echinoidea: Arbacia punctulata, Lytechinus pictus , and Dendraster excentricus . However, eggs from other echinoderm classes and from Xenopus laevis, Drosophila melanogaster , and the chicken did not contain any cross-reactive molecules. Cross-reactivity within the class Echinoidea was not due to a common carbohydrate epitope, because the antibody recognized the glycoproteins even after the N-linked carbohydrate side chains were enzymatically removed. The major yolk glycoprotein (160–170 kDa) from each of the three sea urchin species was purified and analyzed. Comparison of the physical and chemical properties of these glycoproteins revealed striking similarities in p I and in amino acid and monosaccharide composition. The results of peptide mapping also supported the conclusion that the 160- to 170-kDa glycoproteins from the four echinoids are structurally homologous glycoproteins containing N-linked polymannose chains. Immunolocalization by electron microscopy in S. purpuratus showed that the yolk glycoproteins remained within the yolk platelet throughout development, and that externalization of the 160-kDa glycoprotein or its cleavage products was not detectable.

Sea urchin blastulae were treated with two concentrations (0.54 and 0.72 mM) of diamide, a sulfhydryl oxidant, after hatching. These treatments increased the relative synthesis of one set of embryonic proteins while decreasing that of another. This was demonstrated by quantitating the incorporation of [35S]methionine into polypeptides separated by 2-dimensional polyacrylamide gel electrophoresis (2D PAGE). These shifts were dose dependent and apparently reversible after the embryos had regenerated reduced sulfhydryls. Those proteins showing increased incorporation migrated at the same position by 2D PAGE as heat shock proteins, suggesting that diamide was inducing a stress response. Diamide also caused some developmental aberrations at low frequency, and reversibly inhibited ciliary beating.

Three rapid marine toxicity tests were evaluated to determine their potential usefulness in a toxicity testing program: early embryo growth test and sperm cell toxicity test, both using the sea urchin Arbacia punctulata, and Microtox. Toxicity values (EC50s) were derived for eight organic chemicals and five metals using each system. Results were compared with LC50 values for the same chemicals from standard aquatic tests with Pimephales romelas and Daphnia magna and for the metals with Menidia menidia and Mysidopsis bahia. The EC50 values for the sea urchin early embryo growth test for organics and the LC50 values for fish and crustaceans for organics and metals with which the rapid tests were compared were from the literature. For organic chemicals, EC50s from rapid tests closely approximated LC50s and toxicity rankings, except that the sperm cell test was particularly sensitive to 2,4-pentanedione and 2-methyl-2,4-pentanediol. EC50s for metals were generally comparable with each other and with other tests using embryo and sperm of other sea urchins and oysters but not with LC50s for Mysidopsis and Menidia. These results indicate the potential value of rapid tests to screen and rank substances for toxicity. Additional information on the usefulness of these tests will be gained through continuing comparisons using a broad spectrum of single substances and complex mixtures with varying modes of action.

Eggs of the sea urchins Arbacia punctulata (Ap), Lytechinus pictus (Lp), and Strongylocentrotus purpuratus (Sp) were labeled to equilibrium with 32 PO 3− 4 . Approximately 65–70% of the label in extractable adenine nucleotides comigrates chromatographically with ATP. Autoradiograms of one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) slab gels show that each species possesses a distinct complement of phosphate-exchangeable phosphoproteins. No changes in the phosphoprotein composition are detected in Lp and Sp eggs as a result of fertilization or development for 2.5 hr (with the possible exception of a 43,000 M r protein in Lp). In Ap, increases in the phosphorylation of bands at M r 's 30,000, 55,000, and 105,000 are seen during the first 10 min postinsemination. The 30,000 M r band in Ap eggs has previously been identified as ribosomal protein S6 and the hypothesis presented that its increased phosphorylation may be an important step in the activation of protein synthesis at fertilization ( D. G. Ballinger and T. Hunt, 1981 , Dev. Biol. 87, 277–285). In Lp and Sp eggs S6 (identified by two-dimensional PAGE) is heavily phosphorylated in the unfertilized state and the extent of labeling does not increase after fertilization. If the increased phosphorylation of S6 seen in Ap is indeed related to translational activation, then these results suggest that different sea urchin species may rely on different mechanisms for the activation of protein synthesis.

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.

Eggs of the sea urchins Strongylocentrotus purpuratus and Arbacia punctulata bind sperm with a high degree of species specificity. By use of an in vitro assay that utilizes bindin (the protein from sperm that mediates sperm-egg binding) egg surface-derived glycoconjugates that function as receptors in this adhesion process have been identified and purified. These glycoconjugates are of extraordinarily high molecular weight and exhibit some properties expected for a proteoglycan. The isolated receptors from both species bind to sperm and inhibit fertilization species specifically. Both receptors contain active carbohydrate-rich fragments that can be liberated by proteolytic digestion. The carbohydrate-rich receptor fragment from S. purpuratus is a very high-molecular-weight (greater than 10(6)), negatively charged glycosaminoglycan-like polymer containing fucose, galactosamine, iduronic acid, and sulfate esters. By contrast, the carbohydrate-rich fragment derived from the A. punctulata receptor is of defined molecular weight (6000) and has no net charge. Incubation of acrosome-reacted sperm with nanomolar amounts of the carbohydrate-rich fragments from either species results in inhibition of fertilization, indicating that these receptor fragments retain sperm binding activity. However, studies utilizing heterologous gametes show that the carbohydrate-rich receptor fragments are not species specific in binding. Thus, it appears that although the carbohydrate chains of the receptor are an adhesive element of the receptor, the intact glycoconjugate is required for species-specific binding.

Phosphoproteins of hatched blastulae, gastrulae, and pluteus larvae of the sea urchin, Arbacia punctulata, were labeled in vivo with [32P]O4 and analysed by 2-dimensional polyacrylamide gel electrophoresis and autoradiography. At least 60 phosphoproteins were resolved. Some of these showed different relative intensities of labeling at the embryonic periods monitored. Some embryonic phosphoproteins were characterized by cell fractionation and by comparing autoradiograms with Coomassie-blue staining patterns and [35S]methionine labeling patterns. Neither actin nor tubulin phosphorylation was detected. No differences in phosphorylation were detected in dissociated and partially reassociated blastula cells relative to each other and to intact embryonic controls.

Benzohydroxamic acid (BHA) is a competitive inhibitor of the sea urchin sperm peroxidase. We now report that addition of BHA to fertilization cultures of Arbacia punctulata promotes polyspermy. This effect is dose and sperm density dependent. The cortical reaction (elevation of the fertilization envelope) is not retarded by BHA. BHA must be added to the cultures before the eggs complete the cortical reaction at 60 sec post insemination in order to induce polyspermy. Since sea urchin eggs release H2O2 during the cortical reaction at fertilization, these findings support our hypothesis that the sperm peroxidase has a functional role in helping to prevent polyspermy.

The relative abundance patterns of several sessile epifaunal species occurring subtidally on large artificial substrata (pilings) were examined under experimental conditions involving the manipulation of densities of the echinoid Arbacia punctulata (Lamarck). The foraging activities of this predator could denude the substratum of most species with notable exceptions including the colonial hydroid Hydractinia echinata Fleming and the sponge Xestospongia halichondroides (Wilson). Moreover, these were the only two of the twenty most common species which did not significantly change in relative abundance over the experimental period. Both species had low recruitment rates and were commonly associated with substrata which had been submerged for several years. Neither species aggressively interacted with adjacent spatial competitors but instead, appeared to employ a defensive space utilization ‘strategy’. Provision of unoccupied substrata by Arbacia was apparently the major factor favoring both recruitment and growth of Hydractinia, which covered up to 30% of the area on the oldest pilings. More recently submerged substrata were covered by species such as Schizoporella errata (Waters) which had a much higher recruitment rate but was commonly overgrown by several other species. Recruitment rate, competitive ability, and vegetative growth are discussed in terms of the size of the substratum and the possibility of biased sampling in fouling studies. The widespread introduction of large artificial substrata into the natural environment has considerably altered the structure of the natural habitat and constitutes a potentially important selective force for changes in settlement preferences, especially among species such as Hydractinia which persist and become abundant on these substrata.

Investigations were carried out to examine more closely the aggregations of microfilaments associated with the elongation of microvilli and formation of fertilization cones and the effects of cytochalasin B (CB) on these processes in Arbacia eggs following insemination. At 1 to 5 min postinsemination fertilized eggs were treated with 1–10 μg/ml CB and then prepared for electron microscopy at periodic intervals. Examination of CB-treated and untreated specimens demonstrated that: (1) Reorganization of the egg's microvilli took place soon after insemination; this process, as well as formation of fertilization cones, was correlated with the appearance of fascicles of microfilaments. (2) CB inhibited the formation of fertilization cones and the elongation of microvilli. Bundles of microfilaments were not observed in CB-treated zygotes. (3) CB prevented the normal movements (rotation) of the incorporating spermatozoon into the egg cortex but did not inhibit the migration or fusion of the male and female pronuclei.