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Sea urchin (class Echinoidea) gonads are a prized delicacy in Japan and many other world cultures. The complexity of its fatty acid (FA) profile, particularly minor FA, presents a formidable analytical challenge. We applied solvent mediated (SM) covalent adduct chemical ionization (CACI) tandem mass spectrometry to comprehensive de novo structural and quantitative characterization of the FA profile of Gulf of Mexico Atlantic sea urchin (Arbacia punctulata). >100 FA were detected including many with unusual double bond structure. Gulf sea urchin gonad lipids are rich in Δ5 monounsaturated FA 20:1(5Z) at 2.7% and the polymethylene-interrupted (PMI) diene 20:2(5Z,11Z) at 4.9%, as well as common omega-3 eicosapentaenoic acid (EPA; 5Z, 8Z, 11Z, 14Z, 17Z) at 9.8%±3.1% and arachidonic acid (AA; 5Z, 8Z, 11Z, 14Z) at 6.1%±2.1%. We propose plausible desaturation/elongation-based biochemical pathways for the endogenous production of unusual unsaturates. Unusual unsaturates may modify mammalian signaling and present novel bioactivities.

The majority of aquatic toxicity data for petroleum products has been limited to a few intensively studied crude oils and Corexit chemical dispersants, and acute toxicity testing in two standard estuarine test species: mysids (Americamysis bahia) and inland silversides (Menidia beryllina). This study compared the toxicity of two chemical dispersants commonly stock piled for spill response (Corexit EC9500A®, Finasol®OSR 52), three less studied agents (Accell Clean®DWD dispersant; CytoSol® surface washing agent; Gelco200® solidifier), and three crude oils differing in hydrocarbon composition (Dorado, Endicott, Alaska North Slope). Consistent with listings on the U.S. National Contingency Plan Product Schedule, general rank order toxicity was greatest for dispersants and lowest for the solidifier. The results indicate that freshwater species can have similar sensitivity as the conventionally tested mysids and silversides, and that the sea urchin (Arbacia punctulata) appears to be a reasonable addition to increase taxa diversity in standardized oil agent testing.

Life-history models for marine invertebrate animals assume a strong correlation between the size of the egg and the time from fertilization until metamorphosis. This assumption is supported by comparative data across a wide range of phyla. However, the exact form of the relationship between egg size and development time has a strong effect on the predicted outcomes of life-history models and has been the subject of much debate. Comparative data suggest that as egg size increases the effect of egg size reductions on development time will decrease and the effects of size reductions on juvenile size will increase. I used blastomere separations to test the effects of a 50% reduction in egg volume on development time and juvenile size in three species of echinoid echinoderms (Arbacia punctulata, Strongylocentrotus purpuratus, and Dendraster excentricus) that develop from eggs of a range of sizes (80–124 μm). I also manipulated the food level given to developing embryos and larvae to investigate the potential interaction between the effects of egg size reductions and food availability. Larvae from halved zygotes took significantly longer to develop to metamorphosis than their whole size counterparts in all three species. In only one species was I able to detect a significant reduction in juvenile size for offspring developing from halved zygotes. When compared with similar manipulations in species with larger eggs (140–387 μm), egg size reductions have a stronger effect on development time in species with small eggs. As predicted, development time does not change linearly with egg size but instead increases exponentially as egg size is reduced. The relationship between egg size and juvenile size remains unclear. Further investigations into the factors, including egg size, that influence juvenile performance are warranted.

Regular echinoid skeletons, or tests, comprise plate patterns and overall shapes that have proven challenging to analyse solely on the basis of any one approach or process. Herein, we present a computational model, Holotestoid, that emulates four macrostructural ontogenic processes involved in test growth (plate growth, plate addition, plate interaction, and plate gapping). We devise a geometric representation for analysing tests and describe how we use analogies (bubble interactions and close-packing) to emulate the processes. In the computational model, the emulated processes are used to determine the plate size and plate shape and combined to simulate a growth zone. We simulated growth zones for Arbacia punctulata and for Strongylocentrotus franciscanus by changing the value for one parameter, the ambulacral column angle. We quantitatively compared morphological features for simulated forms to those for real specimens to test the computational model. Additionally, we simulated growth zones for A. punctulata, S. franciscanus, Eucidaris thouarsii, and Mellita quinquiesperforata by changing three parameters, ambulacral column angle, peristome radius to apical system radius ratio, and apical system radius to column length ratio. Holotestoid can be used to explain morphological disparity among echinoid tests.

Cellular cytotoxicity is a key component of animal innate immune responses that is one of the first lines of defense against invaders. There is increasing interest in the study of the cellular immune response, particularly non-specific cytotoxic cells and natural killer cells and their receptors. Studies of non-specific cytotoxic cell and natural killer cell recognition and killing (and the receptors involved) will reveal new and important insights into cellular mechanisms of host defense. Here we describe mAbs specific for coelomocyte sub-populations of the purple sea urchin, Arbacia punctulata, using highly purified coelomocyte populations as the antigen source. Monoclonal antibodies were selected using flow cytometric screening methods. Several of the mAbs were shown to bind to two sub-types of coelomocytes when assayed by fluorescence microscopy. Furthermore, these mAbs inhibited coelomocyte cytotoxicity against vertebrate target cells in a functional assay. The mAbs have been used in immunoprecipitation studies.

Toxicological and chemical studies were performed with a silty and a sandy marine sediment spiked with 2,6-dinitrotoluene (2,6-DNT), 2,4,6-trinitrophenylmethylnitramine (tetryl), or 2,4,6-trinitrophenol (picric acid). Whole sediment toxicity was analyzed by the 10-day survival test with the amphipod Ampelisca abdita, and porewater toxicity tests assessed macro-algae (Ulva fasciata) zoospore germination and germling growth, sea urchin (Arbacia punctulata) embryological development, and polychaete (Dinophilus gyrociliatus) survival and reproduction. Whole sediments spiked with 2,6-DNT were not toxic to amphipods. The fine-grained sediment spiked with tetryl was also not acutely toxic. The tetryl and picric acid LC50 values in the sandy sediment were 3.24 and 144 mg/kg dry weight, respectively. The fine-grained sediment spiked with picric acid generated a U-shaped concentration–response curve in the amphipod test, with increased survival both in the lowest and highest concentration. Grain-size distribution and organic carbon content strongly influenced the behavior of ordnance compounds in spiked sediments. Very low concentrations were measured in some of the treatments and irreversible binding and biodegradation are suggested as the processes responsible for the low measurements. Porewater toxicity varied with its sedimentary origin and with ordnance compound. The sea urchin embryological development test tended to be the least sensitive. Tetryl was the most toxic chemical in all porewater tests, and picric acid the least toxic. Samples spiked with 2,6-DNT contained a degradation product identified as 2-methyl-3-nitroaniline (also known as 2-amino-6-nitrotoluene), and unidentified peaks, possibly degradation products, were also seen in some of the picric acid- and tetryl-spiked samples. Degradation products may have played a role in observed toxicity.

Toxicity Identification Evaluation (TIE) methods have proven very useful in characterizing, identifying and confirming toxicants in environmental samples. This report describes the characterization of toxicants present in two effluents, industrial and municipal, discharged into the marine environment. A toxicity characterization consists of dividing an effluent sample into several relatively unique chemical fractions. Manipulations used to generate these fractions include aeration, filtration, chelation, oxidant reduction, reverse phase chromatography, and graduated pH adjustments. Marine toxicity tests used include mysids (Mysidopsis bahia), fish (Menidia beryllina and Cyprinodon variegatus), macroalga (Champia parvula) and sea urchins (Arbacia punctulata). Results of this TIE demonstrated that both effluents contained toxicity predominantly due to metals and organic contaminants. Ammonia toxicity in the municipal effluent was sample dependent. This study illustrates that the use of marine toxicity tests, TIE procedures and historical data can be combined to better understand the nature of toxicity discharged to the marine environment.

In sea urchin sperm of Arbacia punctulata, a guanylyl cyclase (GC) serves as chemotaxis receptor that enables sperm to respond to a single molecule of the chemoattractant, resact. The efficiency of resact capture is high, because GC covers about 50% of the flagellar surface and binds resact with picomolar affinity. Furthermore, the binding affinity is controlled by the level of occupancy of the receptor. At high occupancy the resact affinity is lowered through negative cooperativity among subunits of the trimeric GC complex. The lifetime of active GC is controlled by its phosphorylation state. At rest GC is phosphorylated at six serine residues. After activation by resact, the GC becomes dephosphorylated with a biphasic time course, whereas dephosphorylation strongly coincides with the decrease of cGMP synthesis. During the initial fast phase the amplitude of GC dephosphorylation increases with the occupancy level. However, the time constant of this phase is independent of receptor occupancy. Moreover, dephosphorylation is superstoichiometric: even if only 5% of the GCs are occupied by resact, approximately 70% become dephosphorylated. We conclude from these results, that the occupied GC inactivates by auto-dephosporylation (fast phase) and additionally can dephosphorylate adjacent non-occupied GCs (slow phase). We could show for the first time that a receptor GC is regulated by auto-dephosphorylation.

Transglutaminase inhibitor (1,3,4,5-tetramethyl-2-[(2-oxopropyl)thio]imidazolium chloride or L-682777) affected the appearance of the fertilization envelope (FE) and subsequent development in Sphaerechinus granularis and Arbacia punctulata in a concentration-dependent manner. Abnormalities were first visible in forming FE′s at the lowest concentration (0.001 mM) of the inhibitor used. The wrinkled appearance of FE′s suggested that the treatment prevented the I-T transition, whereas the finding of numerous denuded cells and empty FE′s indicated that the inhibitor acted by rendering the FE′s very fragile. Cell division by the 2-cell stage was also affected by this active-site-directed transglutaminase inhibitor. With 0.05 mM of the inhibitor, about 50% of all cells were polynucleated. In view of earlier findings that other transglutaminase inhibitors can produce abnormalities with regard to FE wrinkling and deranged cell division, it can be suggested that different forms of transglutaminases might be involved at various stages in embryonic development.

Inhibition of cAMP-dependent protein kinase activity by microinjection of a specific physiologic protein inhibitor into sea urchin eggs inhibits the first cleavage after fertilization. Inhibition apparently occurs at some time prior to or during formation of the mitotic spindle. Measurement of the total protein kinase activity of sea urchin egg homogenates after fertilization showed that cAMP-dependent phosphorylation increases after fertilization and then declines prior to or at the time of the first cleavage. It is concluded that a cAMP-dependent phosphorylation plays a significant role in events leading to regulation of mitotic spindle assembly.

The structures of three new diterpenoids, acutilol A, acutilol A acetate and acutilol B have been determined by a combination of spectral methods. The new compounds are based upon the common pachydictyane carbon skeleton but possess unusual Δ 1,10 double bonds. The acutilols are potent feeding deterrents against both temperate and tropical herbivorous fishes and sea urchins, suggesting that these molecules provide an effective chemical defence.

The ability of exogenous proteins to enhance the reaggregation of aggregation-deficient butanol extracted cells has been studied using blastula cells of the sea urchin Arbacia punctulata. Butanol extracts, calcium- and magnesium-free sea-water dissociation supernatant, human plasma fibronectin and bovine serum albumin enhance reaagregation in a concentration dependent manner. Electrophoretic analysis and autoradiography of 125I surface labeled dissociated cells indicate that while butanol extracts a large number of proteins, few iodinated proteins are present in the butanol extract. The non-specificity of proteins to promote reaggregation and the probable cellular location of the butanol extracted proteins are discussed.

Individuals of Arbacia punctulata (Lamarck) from Woods Hole, Massachusetts, and the northeastern Gulf of Mexico were reared from fertilized eggs through metamorphosis under comparable laboratory conditions. Interpopulation differences in spine length development were significant between pure-bred offsprings of these two widely separated geographic areas. Spine lengths of hybrid urchins were intermediate to pure-bred animals. Interpopulation differentiation of specific portions of the genome is proposed to account for the observed phenotypic variation in spine length.

Fertilization of sea urchin eggs results in the rapid recruitment of stored messages into polyribosomes. Whether translational control in sea urchin eggs is mediated by macromolecules associated with the stored messages remains unknown, since preparations of messenger ribonucleoprotein complexes (mRNPs) were active in protein synthesis in a rabbit reticulocyte lysate. To facilitate the study of mRNPs, chromatography on oligo(dT)-cellulose was used to purify poly(A)-containing mRNPs from eggs and embryos of the sea urchin Strongylocentrotus purpuratus. Nonpolyribosomal mRNPs purified from eggs had a similar sedimentation in sucrose to unpurified mRNPs, a peak buoyant density in metrizamide of 1.22 g/cm3, and peak buoyant densities in Cs2SO4 in 1.42 g/cm3 after fixation with glutaraldehyde and 1.46 g/cm3 without fixation. Nonpolyribosomal mRNPs from eggs and zygotes contained 5–10 major proteins on sodium dodecylsulfate (SDS) polyacrylamide gels, and numerous minor bands. UV-irradiation of living eggs of the sea urchin Arbacia punctulata produced cross-linked mRNPs which contained a similar pattern of polypeptides to noncross-linked mRNPs. The polypeptides associated with embyronic polyribosomal mRNPs were also qualitatively similar to those present in nonpolyribosomal mRNPs, although stoichiometric differences may exist.

Basic proteins in nuclei and nucleosomes at different stages of development in Arbacia punctulata sea urchins were analyzed directly by in situ protamine release of chromosomal proteins into Triton/acid/urea-polyacrylamide gels. The predominant protein band in the H4 region of 2-cell through 64-cell stage embryos migrates with the mobility expected for diacetylated histone H4 (i.e. H4aa), whereas after blastulation (approximately 300 cells) the predominant H4 species is the unmodified form, H4O. In early embryos this H4aa band is highly labeled in vivo with [3H]acetic acid. The ratio of H4aa:H4O is more than 20-fold greater at the rapidly dividing 2-cell stage than at pluteus stage. This is true for both newly synthesized H4 labeled with [3H]lysine and total H4 (stained). Enhanced acetylation is also found in nucleosomes. The relative amount of this acetylated H4 species correlates roughly with the rate of cell doubling during early embryogenesis, and decreases as the average nucleosomal repeat increases. The results are indicative of a dynamically changing chromatin structure through development, as well as an intimate role of diacetylated histone H4 in the maturation of newly replicated chromatin.

Following studies on calcium transport by isolated smooth endoplasmic reticulum from unfertilized sea urchin eggs (Oberdorf, J. A., Head, J. F., and Kaminer, B. (1986) J. Cell Biol. 102, 2205-2210) we have purified and partially characterized a calsequestrin-like protein from this organelle isolated from eggs from Strongylocentrotus droebachiensis and Arbacia punctulata. Muscle calsequestrin from sarcoplasmic reticulum is well characterized as a calcium storage protein. The egg protein resembles calsequestrin in its behavior in purification steps, electrophoretic mobility, blue staining with Stains-all on polyacrylamide gels, and its calcium binding and amino acid composition. Purification was attained with DEAE-cellulose and hydroxyapatite chromatography. The egg protein Mr of 58,000 in the Laemmli gel system is reduced to 54,000 under Weber-Osborn (neutral) conditions, thus showing a pH dependence in its mobility, although less than occurs with muscle calsequestrins. 25% of its amino acids are acidic and 10% basic. It binds 309 nmol of Ca2+/mg of protein, within the range reported for cardiac calsequestrin. Antigenically, the sea urchin egg protein is related to cardiac calsequestrin capable of binding anti-cardiac calsequestrin antibody.

Calmodulin is highly conserved, and only in the sea urchin Arbacia punctulata have two distinct isotypes been reported. We have isolated and sequenced two cDNAs from a λgt11 library constructed from RNA from ovary tissue of A. punctulata. One clone, designated α, encodes a calmodulin isotype previously designated A. It encodes an amino acid sequence that is identical with calmodulin of most vertebrates in positions 1 through 141; however, it does not encode the last seven amino acids. The other clone, designated β, starts with an open reading frame and encodes the B form of calmodulin from position 11 through the C-terminal position 148. It has only four differences from vertebrate calmodulin, occurring at positions 78 (Asp,β Glu), 99 (Tyr, β Phe), 143 (Gln, β Ala) and 147 (Ala,β Ser). The nucleic acid sequences of the α and β cDNAs differ at 46 nucleotide positions that are distributed throughout their coding sequences. We conclude that the corresponding mRNAs are not derived from post-transcriptional processing of a single gene, and we infer that they are transcribed from two non-allelic genes. The gene duplication is inferred to have occurred prior to the divergence of the vertebrates and the echinoderms. The expression of these calmodulin mRNAs in ovary tissue and eggs of a single animal differs as judged by hybridization of probes to RNA immobilized to filters.

Monoclonal antibodies have been generated to determine the fate of sperm-specific surface components of the sea urchin, Arbacia punctulata, subsequent to gamete fusion. Monoclonal antibody-7 (MAB-7) reacted with two polypeptide bands having apparent molecular masses of 33 and 35 kDa derived from the sperm plasma membrane; similar reactivity was not detected in egg preparations. Eggs and oocytes were prepared for electron microscopy at periodic intervals following insemination and reacted with MAB-7 followed by antimouse antibodies conjugated to colloidal gold. In samples prepared 30 to 60 sec postinsemination (PI) colloidal gold was confined to the surface of fused sperm with only a few gold particles associated with the egg plasma membrane. In later samples (2-10 min PI) label was present on increasingly greater areas of the egg/oocyte surface away from the site of gamete fusion so that by 20 min PI particles were located along one hemisphere of inseminated eggs and oocytes. These results demonstrate the incorporation of sperm plasma membrane polypeptides and their intermixing with egg plasmalemmal components subsequent to gamete membrane fusion. From measurements of labeled oocytes at different times PI, the diffusion coefficient for sperm surface polypeptides detected by MAB-7 was estimated to be 0.7 to 2.4 x 10(-9) cm2/sec.

Predation by various invertebrates on the burrowing sponge, Cliona celata Grant, was investigated both in the laboratory and field, in order to determine the importance of predation to a sponge population and the adaptive advantage of burrowing to escape predators. Thirty-one species of molluscs, crustaceans, polychaetes, and echinoderms commonly found on oyster beds in Beaufort, North Carolina were tested for their ability to prey on C. celata . Gastropods Diodora cayenensis Lamarck, Seila adamsi , H. C. Lea and Doriopsilla pharpa Marcus, isopod Cilicaea caudata (Say), decapods Alpheus heterochaelis Say, Panopeus herbsti Milne Edwards, Neopancpe sayi (Smith) Eurypanopeus depressus (Smith), Menippe mercenaria (Say) and Pilutnnus sayi Rathbun, and echinoids Arbacia punctulata (Lamarck) and Lytechinus variegatus (Lamarck) were all found to ingest sponge. Diodora, Seila, Doriopsilla, Cilicaea, Alpheus and Arbacia frequently eat sponges in nature. All sponge predators were able to obtain sponge ventilation papillae, which extend beyond the surface of the shell housing the sponge. Papillae lost to predation were regenerated within 12 days in Cliona celata ; little regeneration was noted in predator-damaged Haliclona permollis (Bowerbank), a nonburrowing sponge. Only Arbacia was able to breach sponge galleries in the shell and destroy Cliona celata 2 to 3 times faster than growth could replace lost sponge tissue. The paucity of subtidal shelly bottom sponge fauna and cryptic or high intertidal habits of oyster bed sponges in Beaufort Harbor suggest at least partial control of populations of several common sponges by predators.

Hatched sea urchin blastulae, which have primarily short 25-μm cilia except for some long 40-to 70-μm cilia at the apical tuft, were induced to form long (40- to 70-μm) cilia around most of their circumference when treated with trypsin (0.008–0.1%) or concanavalin A. Other animalizing agents did not induce the formation of long cilia when applied to the normal blastulae. The formation of long cilia by trypsin was both time and concentration dependent. The long cilia first appeared around the apical tuft after 6–8 hr in trypsin (21°C), and by 18–22 hr most of the blastula was covered with the long cilia. Length distribution studies on cilia isolated at various times showed that the percentage of long cilia increased from approximately 10% in the normal blastula to over 66% in the 22-hr trypsin-treated embryo, and indicated that the long cilia formed by the elongation of the original short cilia. Only the blastulae and gastrulae could be induced to form long cilia; the prisms and plutei could not. Once development was inhibited by the trypsin and the first long cilia appeared, the trypsin effect could not be reversed. When blastulae with long cilia were removed from the trypsin for 10 hr, the cilia remained long; when the long cilia were detached, the blastulae regenerated long cilia in the absence of trypsin. The induced long cilia moved poorly, similar to the long, apical tuft cilia of normal embryos. The formation of long cilia by trypsin treatment of sea urchin blastulae provides a model system for studying the mechanisms of ciliary length control.

1. 1. Specific activities (nmol substrate hydrolyzed/min/mg total protein) of crude homogenates of the gut tissues of Arbacia punctulata, Diadema antillarum, Echinometra lucunter, Eucidaris tribuloides and Lytechinus variegatus ranged from 34 ± 19 to 462 ± 48 for α-glucosidase, 11 ± 2 to 47 ± 9 for β-glucosidase, 3 ± 1 to 6 ± 3 for α-galactosidase, and 7 ± 2 to 15 ± 3 for β-galactosidase (species means ± 1 standard deviation at pH of maximal activity). 2. 2. Maximal specific activity was at pH 7.0–7.5. 3. 3. The K m (measured at pH 7.5) ranged from 0.09 to 0.14 ± 0.03 mM for α-glucosidase, 0.03 ± 0.01 to 0.21 mM for β-glucosidase, and 0.04 ± 0.01 to 0.06 ± 0.03 for β-galactosidase. 4. 4. The calculated maximal velocity ( V max , specific activity) ranged from 37 ± 17 to 479 ± 41 for α-glucosidase, 9 ± 1 to 43 ± 6 for β-glucosid ase, and 7 to 10 ± 1 for β-galactosidase. This reflects the levels of enzymes. 5. 5. No trypsin activity was detected.

When offered single species of seaweeds in excess amounts, the sea urchin Arbacia punctulata (Lamarck) Philippi consumed significantly more Gracilaria foliifera (Forsskal) Borgesen and Codiumisthmocladium Vickers than Sargassum filipendula C. Agardh, Padina vickersiae Hoyt, or Dictyota dichotoma (Hudson) Lamouroux. When all five species were offered to urchins simultaneously, patterns of palatability remained the same but absolute consumption of favored species decreased significantly while consumption of less palatable species remained unchanged. During daytime hours, Arbacia is significantly more chemotactic toward Gracilaria (a rapidly eaten species) and Sargassum (a slowly eaten species) than toward Codium, Padina, or Dictyota. At night, Arbacia 's attraction for Gracilaria increases significantly and its attraction toward Sargassum declines significantly. We propose that the day-night changes in chemotaxis may minimize fish predation on Arbacia by tending to keep them hidden beneath Sargassum canopies during the day and allowing them to increase their foraging for favored foods at night when visual predators are less active.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

A peptide (resact) associated with the eggs of the sea urchin, Arbacia punctulata, which stimulates sperm respiration rates by 5-10-fold, was purified and its amino acid sequence was determined. The sequence was found to be Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-Leu-NH2. The peptide was subsequently synthesized by solid phase methods, amidated at the carboxyl-terminal Leu, and shown to be identical to the isolated, native material. The peptide half-maximally stimulated A. punctulata spermatozoan respiration at 0.5 nM and half-maximally elevated cyclic GMP concentrations at 25 nM at an extracellular pH of 6.6. The increase in oxygen consumption was coupled with a stimulation of motility. However, at elevated extracellular pH (pH 8.0), resact failed to appreciably stimulate respiration while the elevations of cyclic GMP continued to occur. Resact did not cross-react with sperm cells obtained from Lytechinus pictus or Strongylocentrotus purpuratus; a peptide (speract) obtained from S. purpuratus eggs (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) which activates S. purpuratus sperm respiration did not stimulate A. punctulata spermatozoa. Resact caused a shift in the apparent molecular weight (160,000-150,000) of a major sperm plasma membrane protein; as with cyclic GMP elevations, this response was evident at extracellular pH values of both 6.6 and 8.0. The protein exists in the cell as a phosphoprotein and 32P is released coincident with the molecular weight change. Approximately 115 nM resact caused one-half-maximal conversion of the 160,000-dalton protein after 1 min of incubation. Resact caused the apparent molecular weight conversion of the protein within 5 s and appeared to do so in an irreversible manner. The molecular weight change of the protein was also observed after the addition of monensin A (25 microM) and NH4Cl (40 mM), two agents known to elevate intracellular pH and to increase sperm respiration rates. The membrane protein appears to be the enzyme guanylate cyclase, but since concentrations of resact causing one-half-maximal conversion of the Mr = 160,000 form of the enzyme are about 250 times higher than those causing one-half-maximal stimulation of respiration, the relationship of the apparent molecular weight conversion to a subsequent physiological event remains unclear.

We have examined the kinetics of nuclease digestion of chromatin from committed and uncommitted cells in experiments where the nuclei are mixed and co-digested. Cultures of the sea urchin, Arbacia punctulata, were grown to the 16-cell stage in either [3H]thymidine or [14C]thymidine and the macromere, mesomere, and micromere cell types separated. After isolation, sets of nuclei with two different blastomere types (each having different radionucleotide tagging) were mixed and co-digested with micrococcal nuclease or DNase. I. The extent of digestion was monitored by solubility in 5% perchloric acid (PCA). We find no significant differences in initial digestion rates or limit digests among the different cell types when co-digested with either nuclease. Differences in nuclease sensitivity observed when nuclei are digested separately are abolished when nuclei are probed in a mixing experiment. The results support the hypothesis that phenotypic differences in digestibility among different cell types in vitro reflect differences in chromatin-condensing factors which can diffuse between nuclei.

Fertilization of the eggs of the sea urchin Arbacia punctulata is followed by the phosphorylation of ribosomal protein S6. The increase in phosphorylation starts at the same time that protein synthesis begins to increase, and leads to the appearance of mono-, di-, and triphosphorylated S6 derivatives. Essentially all the S6 is phosphorylated by first cleavage. This phosphorylation requires the occurrence of both the normal Ca2+ transient and the consequent Na+H+ exchange. Protein synthesis can be partially activated by an increase in intracellular pH brought about by weak bases, but this neither causes S6 phosphorylation, nor the inactivation of the specific S6 phosphatase present in unfertilized Arbacia eggs.

To examine the integration and fate of the sperm plasma membrane following its incorporation into the oocyte plasma membrane, we have fertilized sea urchin (Arbacia punctulata) gametes reciprocally labeled with cationized ferritin. When unlabeled oocytes were inseminated with labeled sperm, cationized ferritin acceptors moved laterally from the sperm plasma membrane into the fertilization cone and surrounding microvilli, mixing with components of the oocyte plasmalemma. Labeled oocytes inseminated with unlabeled sperm produced extremely large fertilization cones, completely devoid of cationized ferritin, while the remainder of the oocyte surface remained heavily labeled. Surface area measurements indicated that if all the sperm plasmalemma were utilized to delimit a fertilization cone it would provide less than 10% of the total surface membrane. Evidence is presented indicating that a principal source of membrane to the expanding fertilization cone of inseminated oocytes is from microvilli, i.e., microvilli are retracted to accommodate fertilization cone formation. Membrane delimiting the fertilization cone has a much lower affinity for agents (cationized ferritin and concanavalin A) that stain negatively charged and carbohydrate moieties compared to other regions of the oocyte surface. These ultrastructural observations indicate that significant rearrangements occur in the oocyte and sperm plasma membranes following gamete fusion which give rise to asymmetries in membrane topography; components of both membranes are redistributed within the bilayer adjacent to and delimiting the fertilization cone.

Spermatozoa of the sea urchin Arbacia punctulata possess a phosphorylated guanylate cyclase as a major glycoprotein of the flagellar plasma membrane. When sperm cells contact the jelly layer surrounding the egg, the peptide “resact” binds the sperm cell surface and triggers the dephosphorylation of the cyclase. A large decrease in cyclase activity accompanies dephosphorylation. Before treatment of sperm cells with egg jelly the enzyme contains 17.95 ± 1.24 moles phosphate per mole cyclase. After treatment of sperm cells with egg jelly this number decreases to 2.57 ± 0.42. Based on a molecular weight of 137,250 for the peptide chain, approximately 15 phosphate groups are lost per molecule of guanylate cyclase at fertilization.

An elastase-like enzyme was demonstrated in the unfertilized eggs of Arbacia punctulata . The enzyme was discovered in the fertilization product of A. punctulata , but was also found to be present in the seawater surrounding unfertilized eggs. Isolation of the elastase-like enzyme was accomplished by preparation of a 100,000 g supernatant from a homogenization of unfertilized eggs. Its presence and specificity were determined by assay using a synthetic peptide substrate. The elastase-like activity was nondialyzable, heat labile, and unstable at pH 4. The enzyme was inhibited by antipain, elastatinal, and DFP, but not by leupeptin or soybean trypsin inhibitor. Eggs were fertilized and developed normally in the presence of 1.0 m M elastatinal. Trypsin-like and chymotrypsin-like enzymes were also found in the seawater surrounding fertilized eggs. The trypsin-like enzyme was isolated from this source and characterized by inhibitor profile.

Fluorescein isothiocyanate-conjugated wheat germ agglutinin (WGA-FITC) binds exclusively to the primary mesenchyme cells when the lectin is microinjected into the blastocoels of living Lytechinus pictus and Strongylocentrotus droebachiensis embryos. WGA-FITC binding increases throughout the period of primary mesenchyme cell migration and aggregation. Similar binding is observed in embryos cultured in sulfate-free seawater (SFSW) but not in seawater (ASW) containing tunicamycin. The temporal expression of WGA-FITC binding sites in vivo is also correlated with the pattern of binding observed in vitro. Sixteen-cell stage Arbacia punctulata embryos were dissociated in Ca2+ and Mg2+-free seawater (CMFSW) and the micromeres isolated using sucrose gradients. Arbacia micromeres, cultured in ASW containing calf serum, first bind WGA-FITC during the period when primary mesenchyme cell ingression occurs in control embryos. Micromeres cultured in the presence of tunicamycin do not develop WGA binding sites. The temporal expression of WGA-FITC binding in micromere cultures is unaffected by the absence of sulfate, but the size and morphology of aggregates cultured in SFSW differ from that of the controls.

This paper describes a rapid and efficient way to isolate primary mesenchyme cells (PMCs) of the sea urchin embryo. The procedure involves three simple steps: 1. 1. Dissociation of mesenchyme blastulae in calcium-free artificial seawater. 2. 2. Incubation of the resulting cell suspension on dishes that have been coated with wheat germ agglutinin (WGA), to which the PMCs adhere more firmly than do other cell types. 3. 3. Gentle rinsing of the dishes to remove loosely attached cells, followed by more vigorous rinsing to remove PMCs. This panning procedure has been applied to embryos of three species of sea urchins, Lytechinus variegatus, L. pictus and Arbacia punctulata, and yields populations of PMCs that are 95–99% pure as determined by the proportion of cells that stain with fluorescein isothiocynate (FITC)-WGA and with a monoclonal antibody that binds specifically to PMCs. The yield of PMCs is 4–5 × 106 cells/100-mm dish, or 1–2 × 107 PMCs/ml of packed embryos. The principal advantages of this procedure are that it can be carried out rapidly and simply, and it yields pure populations of PMCs.

Mitochondrial ultrastructural transformations have been examined in intact eggs and embryos from three sea urchins, Arbacia punctulata, Strongylocentrotus purpuratus, and Lytechinus pictus. Following fertilization, naturally occurring ultrastructural transformations (designated as condensed to orthodox) were observed to occur in mitochondria of all three families. The ratios of the soluble ADP and ATP pools were examined in eggs of S. purpuratus before and after fertilization in order to test whether the ultrastructural transformations reflect a decrease in relative size of the ADP pool following fertilization. Our data indicate that there is a decrease in the ADP:ATP ratio at fertilization. These findings and their implications are discussed with respect to presently accepted theories on mitochondrial regulation.

Jelly coat, a multicomponent extracellular matrix surrounding the sea urchin egg, induces the acrosome reaction in sperm. The jelly coats of the four species studied, Arbacia punctulata, Strongylocentrotus purpuratus, Strongylocentrotus drobachiensis , and Lytechinus variegatus , were found to be very similar in chemical composition. A sialoprotein (approximately 20% of the mass of the jelly coat) and a fucose sulfate polysaccharide (approximately 80%) are the major macromolecular components of the jelly coat. The acrosome reaction inducing capacity resides solely in the fucose sulfate polysaccharide. Induction of the acrosome reaction ranges from highly species specific to nonspecific. Thus, A. punctulata and S. drobachiensis sperm are induced to undergo the acrosome reaction only with their homologous jelly coat, while S. purpuratus sperm react equally well with homologous or L. variegatus jelly coat, but not with A. punctulata jelly coat. L. variegatus sperm seem to be relatively nonspecific in response. Species-specific induction of the acrosome reaction resides solely in the fucose sulfate polysaccharide, suggesting that there must be structural differences in this polysaccharide in the various species. Therefore, in some species, fertilization appears to involve sperm-egg recognition at the level of the jelly coat as well as at the level of sperm-egg receptors.