Fertilized Sea Urchin Research Articles

Sediment quality assessment survey and toxicity identification evaluation studies in Lavaca Bay, Texas, a marine superfund site

A sediment quality assessment survey was conducted in Lavaca Bay, Texas, in proximity to a marine Superfund National Priority List (NPL) site. Previous studies at this site had focused primarily on the degree and extent of mercury contamination. The purpose of this survey was to determine the potential ecotoxicological impacts of contaminants, using sensitive sediment toxicity tests in conjunction with a comprehensive chemical analysis of the sediments. In addition, phase I toxicity identification evaluation (TIE) studies were conducted at several of the more toxic sites. Sea urchin (Arbacia punctulata) fertilization and embryological development tests with sediment pore water were employed to assess the toxicity of the sediments. Elevated levels of mercury were found in a number of samples as much as 10-20 times that of background. Six stations had total polycyclic aromatic hydrocarbon (PAH) concentration above the probable effect level (PEL) guideline value, and some stations had concentrations as much as 1000 times above background levels. Eighteen of the 24 stations exhibited toxicity in one of the toxicity tests, while 14 stations were toxic in both tests. A number of stations within the fish closure area exceeded 8-16 of the PEl guidelines. Both aeration and the C18 column treatments were effective in reducing toxicity from the pore water of the two stations included in the TIE. This information, in conjunction with the fact that two of the four stations with the highest mercury concentrations were not toxic, suggests that the toxicity observed in this study is primarily due to organics, probably PAHs, and not mercury. We recommend that these other contaminants of concern be considered in any remedial actions that are planned for this NPL site and that impacts on the ecosystem, as well as human health issues, also be considered.

SEA URCHIN FERTILIZATION ASSAY: AN EVALUATION OF ASSUMPTIONS RELATED TO SAMPLE SALINITY ADJUSTMENT AND USE OF NATURAL AND SYNTHETIC MARINE WATERS FOR TESTING

SEA URCHIN FERTILIZATION ASSAY: AN EVALUATION OF ASSUMPTIONS RELATED TO SAMPLE SALINITY ADJUSTMENT AND USE OF NATURAL AND SYNTHETIC MARINE WATERS FOR TESTING

Sediment toxicity identification evaluation (TIE) studies at marine sites suspected of ordnance contamination.

A sediment quality assessment survey and subsequent toxicity identification evaluation (TIE) study was conducted at several sites in Puget Sound, Washington. The sites were previously suspected of contamination with ordnance compounds. The initial survey employed sea urchin porewater toxicity tests to locate the most toxic stations. Sediments from the most toxic stations were selected for comprehensive chemical analyses. Based on the combined information from the toxicity and chemical data, three adjacent stations in Ostrich Bay were selected for the TIE study. The results of the phase I TIE suggested that organics and metals were primarily responsible for the observed toxicity in the sea urchin fertilization test. In addition to these contaminants, ammonia was also contributing to the toxicity for the sea urchin embryological development test. The phase II TIE study isolated the majority of the toxicity in the fraction containing nonpolar organics with high log K(ow), but chemical analyses failed to identify a compound present at a concentration high enough to be responsible for the observed toxicity. The data suggest that some organic or organometallic contaminant(s) that were not included in the comprehensive suite of chemical analyses caused the observed toxicological responses.

Use of power analysis to develop detectable significance criteria for sea urchin toxicity tests

When sufficient data are available, the statistical power of a test can be determined using power analysis procedures. The term “detectable significance” has been coined to refer to this criterion based on power analysis and past performance of a test. This power analysis procedure has been performed with sea urchin ( Arbacia punctulata) fertilization and embryological development data from sediment porewater toxicity tests. Data from 3100 and 2295 tests for the fertilization and embryological development tests, respectively, were used to calculate the criteria and regression equations describing the power curves. Using Dunnett’s test, a minimum significant difference (MSD) ( β=0.05) of 15.5% and 19% for the fertilization test, and 16.4% and 20.6% for the embryological development test, for α≤0.05 and α≤0.01, respectively, were determined. The use of this second criterion reduces type I (false positive) errors and helps to establish a critical level of difference based on the past performance of the test.

Poly(ADP-ribosylation) protects maternally derived histones from proteolysis after fertilization

Fertilization in sea urchins is followed by the replacement of sperm-specific histones by cleavage-stage histone variants recruited from maternal stores. Such remodelling of zygote chromatin involves a cysteine proteinase that degrades the sperm-specific histones in a selective manner, leaving the maternal cleavage-stage histone variants intact. The mechanism that determines the selectivity of the sperm-histone-selective proteinase (SpH-proteinase) was analysed by focusing on the post-translational modification status of both sets of histones. It has previously been reported that only native cleavage-stage histones are poly(ADP-ribosylated), whereas the sperm-specific histones are not modified. To determine whether the poly(ADP-ribose) moiety afforded protection from degradation, the ADP-ribose polymers were removed from the cleavage-stage histones in vitro; these proteins were then assayed as potential substrates of the SpH-proteinase. Strikingly, the cleavage-stage histone variants were extensively degraded after the enzymic removal of their ADP-ribose moieties. In addition, the SpH cysteine proteinase was not inhibited by isolated poly(ADP-ribose) polymers. Consequently, only poly(ADP-ribosylated) cleavage-stage histone variants are protected from proteolysis. These results demonstrate a novel role for this type of post-translational modification, namely the protection of nuclear proteins against nuclear proteinases after fertilization.

Poly(ADP-ribosylation) protects maternally derived histones from proteolysis after fertilization

Fertilization in sea urchins is followed by the replacement of sperm-specific histones by cleavage-stage histone variants recruited from maternal stores. Such remodelling of zygote chromatin involves a cysteine proteinase that degrades the sperm-specific histones in a selective manner, leaving the maternal cleavage-stage histone variants intact. The mechanism that determines the selectivity of the sperm-histone-selective proteinase (SpH-proteinase) was analysed by focusing on the post-translational modification status of both sets of histones. It has previously been reported that only native cleavage-stage histones are poly(ADP-ribosylated), whereas the sperm-specific histones are not modified. To determine whether the poly(ADP-ribose) moiety afforded protection from degradation, the ADP-ribose polymers were removed from the cleavage-stage histones in vitro; these proteins were then assayed as potential substrates of the SpH-proteinase. Strikingly, the cleavage-stage histone variants were extensively degraded after the enzymic removal of their ADP-ribose moieties. In addition, the SpH cysteine proteinase was not inhibited by isolated poly(ADP-ribose) polymers. Consequently, only poly(ADP-ribosylated) cleavage-stage histone variants are protected from proteolysis. These results demonstrate a novel role for this type of post-translational modification, namely the protection of nuclear proteins against nuclear proteinases after fertilization.

Combined Imaging and Chemical Sensing of Fertilization-Induced Acid Release from Single Sea Urchin Eggs

We demonstrate a microarray sensor capable of obtaining both chemical and visual information on multiple cells simultaneously with single-cell resolution. The array was fabricated by covalently immobilizing a thin, pH-sensitive polymer layer on the distal end of an optical imaging fiber. The sensor's ability to measure localized chemical dynamics in real-time was evaluated using sea urchin fertilization biochemistry as a model system. Following sea urchin fertilization, the Na+/H+ transporter is activated to exchange extracellular sodium ions for intracellular hydrogen ions, causing a release of hydrogen ions at the egg's surface. By placing the pH sensor proximal to the egg and switching between a fluorescence image and a white light image, we were able to observe both localized pH changes following fertilization as well as morphological transformations during cell division.

Inhibition of sea urchin fertilization by fatty acid ethanolamides and cannabinoids

The influence of saturated and unsaturated fatty acid ethanolamides as well as Δ 9-tetrahydrocannabinol (Δ 9-THC), WIN 55,212-2 and cannabinoid CB 1 receptor antagonist SR 141716 on sea urchin fertilization was studied. The ethanolamides of arachidonic, oleic and linoleic acids but not saturated fatty acid (C 14–C 20) derivatives inhibited fertilization when pre-incubated with sperm cells. Δ 9-THC and WIN 55,212-2 also inhibited fertilization, Δ 9-THC being ten times as potent as WIN 55,212-2. Selective cannabinoid CB 1 receptor antagonist SR 141716 also blocked fertilization and did not antagonize the action of Δ 9-THC. The obtained results indicate that different unsaturated fatty acid ethanolamides may control sea urchin fertilization, and that sea urchin sperm cell cannabinoid receptor may differ from the known cannabinoid receptor subtypes.

Structure analysis of a fusogenic peptide sequence from the sea urchin fertilization protein bindin.

The structure of "B18", an 18-residue fusogenic peptide from the sea urchin fertilization protein bindin, was investigated in several membrane-mimicking environments with circular dichroism and nuclear magnetic resonance spectroscopy. The fully conserved peptide sequence represents the minimal functional part of the 24 kDa protein, which can bind to membranes and induce fusion of lipid vesicles. The B18 peptide undergoes a coil-helix transition in the presence of TFE, showing a transient tendency to self-associate. Its NMR structure in 30% TFE exhibits two helical regions at either side, connected by a flexible loop. In DPC and SDS detergent micelles, this loop becomes distinctly bent, presumably due to the high degree of curvature of the micelles. The loop contains a histidine-rich motif for binding zinc, which is required for the fusogenic function of the peptide. Therefore, we monitored the structural response of B18 and of recombinant bindin toward this ion. Like TFE, and in a mutually cooperative manner, zinc induces a partially helical structure in both the peptide and the protein. Complex formation via the histidine residues rigidifies the flexible loop and is accompanied by self-association of the molecules. The data suggest that the zinc-bound functional state is a continuous amphipathic alpha-helix, bearing some resemblance to a leucine zipper. Two hydrophobic patches on one face could favorably penetrate into a membrane, while two arginines on the other face could interact with lipid phosphate groups. The three-dimensional model of the B18 sequence thus contributes to a better understanding of peptide-induced vesicle fusion in general, and of the lipid-protein interactions of sperm bindin in particular.

Assessment of the Comparative Toxicity of Sewage Effluent from 10 Sewage Treatment Plants in the Area of Sydney, Australia using an Amphipod and Two Sea Urchin Bioassays

In 1995, Sydney Water Corporation undertook an ecological and human health risk assessment for 10 sewage treatment plants (STPs) that discharge primary (6 STPs) and secondary (4 STPs) treated effluents into coastal waters in the Sydney and Illawarra regions, NSW, Australia. A program of toxicity testing of effluent from the 10 STPs was undertaken to determine the toxic effects of the effluents and as a weight-of-evidence study for the risk assessment. Three types of bioassays were used to test multiple samples of effluent from each STP. Tests used were the sea urchin ( Heliocidaris tuberculata) fertilization and larval development bioassays and the amphipod ( Allorchestes compressa) survival bioassay. Comparing between STPs, primary treated effluent was slightly more toxic than secondary treated effluent for all three tests. The highest toxicity was recorded for those STPs that use primary treatment and discharge to the shoreline. Comparing between tests, the sea urchin fertilization bioassay was slightly more sensitive than the sea urchin larval development bioassay, with the amphipod survival bioassay being the least sensitive. This paper will describe the results of this toxicity testing program and discuss the application of the study results as weight-of-evidence for the risk assessment study.

Use of power analysis to develop detectable significance criteria for sea urchin toxicity tests

Abstract When sufficient data are available, the statistical power of a test can be determined using power analysis procedures. The term “detectable significance” has been coined to refer to this criterion based on power analysis and past performance of a test. This power analysis procedure has been performed with sea urchin (Arbacia punctulata) fertilization and embryological development data from sediment porewater toxicity tests. Data from 3100 and 2295 tests for the fertilization and embryological development tests, respectively, were used to calculate the criteria and regression equations describing the power curves. Using Dunnett's test, a minimum significant difference (MSD) (β = 0.05) of 15.5% and 19% for the fertilization test, and 16.4% and 20.6% for the embryological development test, for α ≤ 0.05 and α ≤ 0.01, respectively, were determined. The use of this second criterion reduces type I (false positive) errors and helps to establish a critical level of difference based on the past performance of the test.

Dispersion of produced water in a coastal environment and its biological implications

Produced water, a pollutant associated with offshore oil production, has been shown to have adverse effects on marine organisms. We conducted a study of the dispersion of a produced water plume from an outfall in the Santa Barbara Channel near Carpinteria, CA. Biological effects were studied previously in a series of experiments which examined the toxicity of ocean waters near the outfall. To define the changing ocean conditions around the outfall, we obtained time series observations of currents and water properties from July, 1992 to January, 1994. Near-field dispersion of the produced water is simulated with a buoyant plume model. Measured currents and density profiles are used as model inputs. Far-field dispersion is simulated with the current statistics combined with an elementary solution to the diffusion equation. The modeled depth of the plume varies strongly with season due to changing stratification. In spring and summer, the modeled plume is trapped below the surface. In fall and winter it extends over most of the water column and occasionally surfaces. Minimum initial dilution is ∼100 in summer and ∼500 in winter. Far-field modeling indicates along-isobath symmetry in produced water dispersion in the mid water column. This pattern agrees with the distribution of toxic effects from the biological studies. At 1000 m from the outfall, the farthest test sites in the biological studies, minimum dilutions range from 4000 to 4×10 5 when the plume is present. These estimates exceed the threshold for sub-lethal effects found by Krause (1993) in a sea urchin fertilization bioassay. Time averaged dilutions in the far-field are larger by factors of 10 2 to 10 3.

Imaging of echinoderm fertilization.

Eggs from echinoderms (sea urchin, starfish, and sand dollars) have been an excellent subject for cell biology for more than 100 years. Among the many discoveries that have been made with echinoderm eggs are the first studies of the centrosome (Boveri, 1902 ; also see Baltzer, 1967 ), the Ca requirement for healing plasma membrane wounds (Chambers, 1917 ) with the subsequent hypothesis of a central role of Ca in cell physiology (summarized by Heilbrunn, 1958 ), relationships of the mitotic apparatus and cytokinetic furrow (Rappaport, 1986 ), and the first identification of cyclins (Evans et al., 1983 ). Why have echinoderm eggs been so productive experimentally? There are probably several reasons. Marine eggs are autonomous and develop normally in sea water, an easily reproduced environment. Echinoderm fertilization is reliable and sets into motion many fundamental cell biological processes. The eggs are relatively clear, so that changes in organization deep in the cytoplasm can be viewed by light microscopy. For biochemical studies, the eggs can be obtained as a homogeneous population in large quantities. With the resurgence of light microscopy in the last 20 years or so, new tools, techniques, and reagents have become available to make new discoveries and to see more clearly what was known or previously only deduced. These movies show several views of fertilization of sea urchin (Lytechinus variegatus and Lytechinus pictus) and starfish (Asterina miniata) eggs.

Membrane Fusion Is Induced by a Distinct Peptide Sequence of the Sea Urchin Fertilization Protein Bindin

Fertilization in the sea urchin is mediated by the membrane-associated acrosomal protein bindin, which plays a key role in the adhesion and fusion between sperm and egg. We have investigated the structure/function relationship of an 18-amino acid peptide fragment "B18," which represents the minimal membrane binding motif of the protein and resembles a putative fusion peptide. The peptide was found to mimic the behavior of its parent protein bindin with respect to (a) its high affinity for lipid bilayers, (b) the ability to aggregate and fuse vesicles, (c) the binding of Zn2+ by a histidine-rich motif, (d) the tendency to self-assemble, and (e), as indicated earlier, the adhesion to cell surface polysaccharides. Fluorescence and light scattering assays were used here to monitor peptide-induced lipid mixing, leakage, and aggregation of large unilamellar sphingomyelin/cholesterol vesicles. For these activities, B18 requires the presence of Zn2+ ions, with which it forms oligomeric complexes and assumes a partially alpha-helical conformation, as observed by circular dichroism. We conclude that aggregation and fusion involves a "trans-complex" between peptides on apposing vesicles that are connected by Zn2+ bridges.

Development and application of a marine sediment pore‐water toxicity test using Ulva fasciata zoospores

AbstractAn acute (96 h) pore‐water toxicity test protocol using germination and growth of Ulva fasciata zoospores as endpoints was developed to test the toxicity of marine and estuarine sediment pore‐water samples. Tests with an organic toxicant (sodium dodecyl sulfate; SDS), three metals (Cd, Cu, and Zn), and ammonia (NH3) were conducted to determine zoospore sensitivity. Zoospore germination and gametophyte growth were as sensitive to SDS as sea urchin (Arbacia punctulata) fertilization and embryological development. Zoospore sensitivity to metals was greater than or comparable to that of adult macroalgae. Zoospores were less sensitive to NH3 than were other commonly used toxicity test organisms. Test results using this algal assay with sediment pore‐water samples with high NH3 concentrations were compared with results from sea urchin fertilization and embryological development tests for the same samples. Ulva fasciata zoospore germination was not affected by samples with high NH3 concentrations that were toxic in both sea urchin tests. Zoospore tolerance of NH3 and sensitivity to other contaminants indicate that their response may be useful in toxicity identification evaluation studies with pore‐water samples that contain high concentrations of unionized NH3.

Inhibition of sea urchin fertilization by jaspisin, a specific inhibitor of matrix metalloendoproteinase.

Jaspisin, originally isolated from a marine sponge as an inhibitor of the hatching of the sea urchin (Hemicentrotus pulcherrimus) embryo, causes inhibition of sea urchin fertilization. Electron microscopic examination revealed that the acrosome reaction was induced in jaspisin-treated sperm when they were incubated with an intact egg. The acrosome-reacted sperm bound to the vitelline layer by the acrosomal material surrounding the acrosomal process. However, fusion of the acrosomal process and the egg plasma membrane failed to take place. Membrane potential changes were monitored using eggs preloaded with a membrane potential-sensitive fluorochrome, di-8-ANEPPS. Depolarization of the membrane potential, normally observed in the fertilized egg was not observed in the egg inseminated in the presence of jaspisin, indicating the absence of electrical continuity between the jaspisin-treated egg and sperm. Jaspisin inhibited the activities of matrix metallo-endoproteinase members but not of other types of proteinases. These results provide strong, albeit indirect, evidence that a matrix metallo-endoproteinase(s) is involved in the process of gamete fusion during sea urchin fertilization.

The Fucose Sulfate Polymer of Egg Jelly Binds to Sperm REJ and Is the Inducer of the Sea Urchin Sperm Acrosome Reaction

The sea urchin sperm cell is an advantageous model for studying ligand-mediated exocytosis. Sperm can be obtained in vast quantities and induced to undergo exocytosis of the acrosomal vesicle with great synchrony. During sea urchin fertilization, egg jelly (EJ) triggers the sperm acrosome reaction (AR) which is required for sperm binding and fusion with the egg. Uncertainty exists as to the exact biochemical nature of the AR inducer. The following study was performed in an attempt to clarify the nature of the inducer. EJ from individual females (Strongylocentrotus purpuratus) was analyzed on SDS–PAGE gels. Each female had a unique composition of EJ macromolecules, but all females possessed the previously described fucose sulfate polymer (FSP). Two electrophoretic isotypes of FSP were discovered; 87% of females had only one isotype and 13% had both. Both FSP isotypes bound to the REJ protein (receptor for egg jelly) purified from sperm. The two FSP isotypes had almost equal potency in inducing the AR. EJ was fractionated by DEAE chromatography in 6 M urea/4% β-mercaptoethanol. All AR-inducing activity coeluted with FSP. FSP, purified by trypsin digestion followed by dialysis, was twice as active as the non-trypsin-digested control at inducing the AR. EJ was digested with proteinase K, boiled in detergent and β-mercaptoethanol, and subjected to sucrose density gradient sedimentation. The FSP and AR activity had superimposable sedimentation patterns. Purified FSP had no associated peptide component. Sperm from individual males differed in the concentration dependency of purified FSP to induce the AR. The data indicate that the 138/82 kDa EJ glycoproteins, previously thought to act as AR inducers, do not appear to be involved in triggering the AR. The data are consistent with the hypothesis that FSP is the only inducer of the AR of this sea urchin species.

Sulfated Polysaccharides from the Egg Jelly Layer Are Species-specific Inducers of Acrosomal Reaction in Sperms of Sea Urchins

We have characterized the fine structure of sulfated polysaccharides from the egg jelly layer of three species of sea urchins and tested the ability of these purified polysaccharides to induce the acrosome reaction in spermatozoa. The sea urchin Echinometra lucunter contains a homopolymer of 2-sulfated, 3-linked alpha-L-galactan. The species Arbacia lixula and Lytechinus variegatus contain linear sulfated alpha-L-fucans with regular tetrasaccharide repeating units. Each of these sulfated polysaccharides induces the acrosome reaction in conspecific but not in heterospecific spermatozoa. These results demonstrate that species specificity of fertilization in sea urchins depends in part on the fine structure of egg jelly sulfated polysaccharide.

Estimates of the Spatial Extent of Sediment Toxicity in Major U.S. Estuaries

Acute laboratory toxicity tests were performed on surficial sediments collected from 1176 locations throughout 22 estuarine areas in the United States. Toxicity was determined with three standardized procedures : 10-day amphipod survival tests with solid-phase (bulk) sediments ; 5-min microbial bioluminescence tests with organic solvent extracts of the sediments ; and either 1-h sea urchin fertilization tests or 48-h mollusc embryo tests with the porewater of the sediments. Test results were weighted to the sizes (km 2 ) of the geographic strata in which samples were collected. Selected bays and estuaries were sampled along the Atlantic and Gulf of Mexico coasts and in southern California along the Pacific coast of the United States. The individual survey areas ranged in size from <1.0 km 2 to over 550 km 2 . Collectively, approximately 2532 km 2 were sampled in the 22 surveys. Toxicity was observed in the amphipod survival tests of bulk sediments in approximately 10.9% of the combined area. These data suggest that acute toxicity as measured with adult crustaceans was restricted to small portions of these urbanized estuaries, principally maritime harbors and industrialized bayous and waterways. Toxicity was much more pervasive in the tests of solvent extracts and 100% porewater, indicating that approximately 61% and 43%, respectively, ofthe combined areas sampled were toxic. The spatial extent of toxicity decreased to 11% and 5% in tests of 50% and 25% porewater, respectively. In most areas each test indicated different patterns in toxicity, and consequently, the concordance in estimates oftoxicity among different tests was very small. Estimates of the spatial extent of toxicity probably would differ if tests were performed elsewhere and/or with different organisms.

Sediment quality assessment studies of Tampa bay, Florida

AbstractA survey of the toxicity of sediments throughout the Tampa Bay estuary was performed as part of the National Oceanic and Atmospheric Administration's National Status and Trends Program. The objectives of the survey were to determine the spatial extent and severity of toxicity and to identify relationships between chemical contamination and toxicity. Three independent toxicity tests were performed: a 10‐d amphipod survival test of the whole sediments with Ampelisca abdita, a sea urchin fertilization test of sediment pore water with Arbacia punctulata, and a 5‐min Microtox® bioluminescence test with solvent extracts of the sediments. Seventy‐three percent of the 165 undiluted sediment pore‐water samples were significantly toxic relative to reference samples with the sea urchin fertilization test. In contrast, only 2% of the 165 samples were significantly toxic in the amphipod tests. The causes of toxicity were not determined. However, concentrations of numerous trace metals, pesticides, polychlorinated biphenyl (PCB) congeners, polycyclic aromatic hydrocarbons (PAHs), and ammonia were highly correlated with pore‐water toxicity. Concentrations of many substances, especially total dichlorodiphenyltrichloroethanes (DDTs), endrin, total PCBs, certain PAHs, lead, and zinc, occurred at concentrations in the toxic samples that equaled or exceeded concentrations that have been previously associated with sediment toxicity.