Showing papers on "Strongylocentrotus purpuratus published in 1986"
TL;DR: Spatial patterns of expression of individual actin genes in embryos of the sea urchin Strongylocentrotus purpuratus are determined and expression of CyI and CyIIb appears to be co-ordinate, and quite distinct from that of CyIIa.
167 citations
TL;DR: These experiments suggest that either long days inhibit gametogenesis or short days enhance it in the sea urchin Strongylocentrotus purpuratus, suggesting that photoperiod determines whether energy is allocated to body and gonad growth or togametogenesis and gamete maintenance.
Abstract: Our experiments demonstrate that both gametogenesis and growth in the sea urchin Strongylocentrotus purpuratus are under photoperiodic control. Juvenile sea urchins (4–8 mm diameter) were collected in September 1979 and reared for 18 months under two photoperiodic regimes with changing daylengths as in the natural annual cycle. One regime followed inphase (ambient) photoperiods; the other was kept 6 months out of phase with ambient photoperiods. In March of both 1980 and 1981 most of the in-phase animals (like field animals) contained numerous gametes while the out-of-phase animals contained very few gametes. DNA synthesis (estimated by autoradiography with tritiated thymidine) was high in the in-phase males and low in the out-of-phase males. In September of 1980, the situation was reversed. In animals exposed to two successive “winter” photoperiod regimes, gamete production continued for 6 months; in those exposed to two successive “summers,” there was little gamete production. These experiments suggest that either long days inhibit gametogenesis or short days enhance it. The normal peak of body growth in the summer and fall was also shifted 6 months out of phase in animals kept on the out-of-phase photoperiod regime; this shift was reflected in size, in weight, and in the growth zones of the skeletal plates. Feeding rates did not differ between animals on the two photoperiod regimes, suggesting that photoperiod does not influence energy intake but rather determines whether energy is allocated to body and gonad growth or to gametogenesis and gamete maintenance.
152 citations
TL;DR: The present measurements permit calculation of the levels of each actin RNA species in the particular cell types in which each gene functions in the fully differentiated embryo, and confirm prior observations that the prevalence of actin mRNA in the unfertilized egg is low.
121 citations
TL;DR: It is proposed that this component of the larval nervous system of larvae of S. purpuratus is derived from a small number of ectodermal cells associated with the apical tuft.
Abstract: The development of the serotonergic component of the nervous system of larvae of S. purpuratus is traced using indirect immunofluorescence with a polyclonal antibody against the neurotransmitter serotonin. Initially one or two neuroblasts can be detected in the thickened epithelium of the animal plate of late gastrulae (56 hr). The number of immunoreactive cells increases to about eight during formation of the pluteus (85–90 hr). Immunoreactive axons appear simultaneously from all neuroblasts present in the 79 hr prism stage larva and form the apical ganglion. It is proposed that this component of the larval nervous system is derived from a small number of ectodermal cells associated with the apical tuft.
95 citations
TL;DR: The chapter presents current techniques used in raising these animals through their entire life cycle, including shedding gametes from gravid animals, culturing embryos, rearing larvae, and rearing juveniles, and the procedures for raising algae as a food source for the larvae are included.
Abstract: Publisher Summary This chapter describes methods for collecting, maintaining, and culturing the purple sea urchin— Strongyfocentrotus purpuratus . The chapter presents current techniques used in raising these animals through their entire life cycle, including shedding gametes from gravid animals, culturing embryos, rearing larvae, and rearing juveniles. As the larvae must feed to complete development, the procedures for raising algae as a food source for the larvae are included. The practical procedures for induction of the fragile postmetamorphic stages and raising juveniles to adults are considered. The general protocol for rearing purple sea urchins in most cases applies to other species also. The chapter discusses various methods for management of adult sea urchins. A critical variable in maintaining adult sea urchins in the laboratory is the number of animals per unit volume of seawater and the amount of oxygen required for adequate maintenance. Care must also be exercised when packing and transporting the animals. Plastic ice chests or Styrofoam containers can be used with a layer of newspaper on the bottom to further protect tubefeet and to soak up any excess water. Shipping purple sea urchins and many other species over long distances to inland laboratories can be done successfully using the packing techniques.
82 citations
TL;DR: The isolation and sequence of bindin cDNA clones prepared from Strongylocentrotus purpuratus testis RNA are reported, showing that the bindin gene appears to be productively expressed only in males and only in testes.
Abstract: Bindin, a major protein of the sea urchin acrosome granule, mediates the species-specific adhesion and binding of sperm to egg required to effect fertilization. We report the isolation and sequence of bindin cDNA clones prepared from Strongylocentrotus purpuratus testis RNA. The bindin gene appears to be productively expressed only in males and only in testes. The protein is produced from a 51-kDa precursor, which is subsequently processed to yield the mature 24-kDa bindin protein.
76 citations
TL;DR: The results indicate that IP3, but not DAG or its analogues, may be involved in the generation of the fertilization potential triggered by the interaction of sperm with sea urchin eggs.
Abstract: The effects of inositol-1,4,5-trisphosphate (IP3) and of diacylglycerol (DAG) and its analogues on the membrane potential of eggs from the sea urchin Strongylocentrotus purpuratus were examined. Injection of IP3 into eggs resulted in a change in membrane potential that was similar in magnitude and time course to the fertilization potential elicited by sperm attachment. In low-calcium seawater, IP3 injection elicited a partial response. DAG and its analogues phorbol myristyl acetate and 1-oleoyl-2-acetylglycerol did not affect membrane potential either when applied by perfusion or when injected. The results indicate that IP3, but not DAG or its analogues, may be involved in the generation of the fertilization potential triggered by the interaction of sperm with sea urchin eggs.
62 citations
TL;DR: Binding of G6PDH to the particulate fraction lowers its catalytic activity at all substrate concentrations, and release of the enzyme into the cytoplasm may be an important part of the suite of events causing metabolic activation of the egg at fertilization.
Abstract: In unfertilized eggs of the sea urchin, Strongylocentrotus purpuratus, glucose-6-phosphate dehydrogenase (G6PDH) associates with the particulate elements remaining either after homogenization or extraction of eggs with non-ionic detergent in low ionic-strength media. At physiological ionic strength, the extent of G6PDH binding to these particulate elements is proportional to the total protein concentration in the extracts. In fertilized eggs this association is prevented by one or more low molecular weight solutes. The dissociation is reversible, and there are no permanent modifications of either G6PDH or its particulate binding site that affect binding. After fertilization, the time course of dissociation of G6PDH from particulate elements is too fast to be caused by a change in intracellular pH, but it could be triggered, but not maintained, by an increase in the intracellular calcium concentration. Binding of G6PDH to the particulate fraction lowers its catalytic activity at all substrate concentrations. Therefore, release of the enzyme into the cytoplasm may be an important part of the suite of events causing metabolic activation of the egg at fertilization.
61 citations
TL;DR: It is concluded that different members of the late H2a gene family are differentially expressed in embryos and adult tissues.
Abstract: We analyzed the histone mRNA population found in several adult tissues of the sea urchin Strongylocentrotus purpuratus and in testis of Lytechinus pictus. Unique species of H1 and H2b mRNAs encoding the sperm-specific histone subtypes can be found exclusively in testis RNA. S. purpuratus contains two distinct testis-specific H1 transcripts, while L. pictus contains one such transcript. Each of these mRNAs is larger than either early or late embryonic H1 mRNAs. Other somatic adult tissues contain transcripts derived from members of the late embryonic H1 histone gene family. S. purpuratus contains one H2b transcript found exclusively in testis, while L. pictus contains two such H2b mRNAs. Similarly, in tissues other than testis, late H2b transcripts were found. While there is no sperm-specific H2a protein, a limited set of late histone H2a genes encoding primarily the H2a-beta subtype is expressed in testis. The majority of the H2a protein found in diploid adult tissues is also the H2a-beta subtype; however, the size of the H2a transcripts differs between testis and other tissues. We conclude that different members of the late H2a gene family are differentially expressed in embryos and adult tissues. We prepared and characterized cDNA clones encoding the sperm-specific H2b protein as well as the H2a-beta protein found in testis.
44 citations
TL;DR: Results of experiments support the hypothesis that the species specificity of inhibition of fertilization observed in a competitive bioassay is conferred by the polypeptide portion of the receptor molecule.
42 citations
TL;DR: The likely age of the nuclear sequence element from the divergence between nuclear and mitochondrial sequences and from cross-hybridization with the genomes of other sea urchin species is suggested, with an age of more than 30 million years is suggested.
TL;DR: Cell-free translation analyses indicate that most of the mRNA encoding hsp90 resides in the pool of free ribonucleoprotein particles in eggs and early embryos, but shifts to polysomes by the 64-cell stage while remaining constant in mass, and the increase in synthesis of hSp90 appears to be via the selective activation of translation of a stored maternal mRNA.
TL;DR: Electron microscopy of R loops between the phage lambda recombinant clone and poly(A)+ RNA reveals multiple short exons, a feature also seen in vertebrate collagen genes.
Abstract: We have isolated and partially characterized an ca. 20-kilobase-pair Strongylocentrotus purpuratus genomic clone, using a mouse alpha 1 (type IV) collagen cDNA probe. A 1-kilobase-pair HindIII fragment of the clone hybridizes strongly to the probe; this has been subcloned and sequenced. It contains 212 base pairs of sequence coding for (Gly-Xaa-Yaa)n (where Xaa and Yaa are different unspecified amino acids), characteristic of all known collagen genes. There is a single point of discontinuity within the repeating pattern in this exon, similar to the genomic structure of mouse type IV collagen. The (Gly-Xaa-Yaa)n-encoding element is flanked by consensus splicing sequences, and the intervening sequences on either side of it have multiple in-phase termination codons. Electron microscopy of R loops between the phage lambda recombinant clone and poly(A)+ RNA reveals multiple short exons, a feature also seen in vertebrate collagen genes. The (Gly-Xaa-Yaa)n protein-encoding sequence hybridizes to a developmentally regulated 9-kilobase mRNA; the message appears during the morula stage, rises sharply in abundance at the blastula stage, and decreases in proportion to total RNA later in development.
TL;DR: Using a light scattering assay developed to measure the initial rate of hyalin gelation, it is shown that calcium alone is capable of initiating this reaction but that calcium and magnesium are synergistic in their effect.
TL;DR: Results suggest that the antiporter which is responsible for cytoplasmic alkalinization in sea urchin eggs is activated directly or indirectly by protein kinase C in a Ca2+-independent manner.
TL;DR: Genome blots suggested that there is a single calmodulin gene in the S. purpuratus genome, and pCAL-8, a single open reading frame encoding 79 amino acids, a termination codon, and 0.9 kb of 3'-untranslated message was used to studyCalmodulin mRNA accumulation in S. Purpuratus embryos.
TL;DR: A glycoconjugate isolated from uncrosslinked fertilization envelopes prepared from eggs activated by treatment with ionophore has the properties of the previously described sperm receptor.
TL;DR: Sea urchin (Strongylocentrotus purpuratus) eggs were fixed, quick-frozen, deep-etched, and rotary-replicated, and the three-dimensional structure of the external surface of the egg visualized using stereo electron microscopy was visualized.
Abstract: Sea urchin (Strongylocentrotus purpuratus) eggs were fixed, quick-frozen, deep-etched, and rotary-replicated, and the three-dimensional structure of the external surface of the egg visualized using stereo electron microscopy The cell surface is coated with three layers of filaments: the sheetlike vitelline layer adhering closely to the plasma membrane, a second layer of oblique fibrils extending from microvillar tips to the vitelline layer below, and a third, outermost layer of horizontal filaments coursing in bundles over the microvillar tips After fertilization, the newly elevated vitelline envelope is transformed into a three-layered structure, the central layer being a tightly knit network of fine filaments decorated on each side with a loose network of thicker fibrils Subsequently, the envelope becomes coated with ‘paracrystalline’ protein released from the cortical granules, and microvillar casts are reshaped into angular, jagged peaks having two to five sides The final structure of the fertilization envelope consists of a thick central layer of compact fibrillar material that is coated on each side with thin plates of paracrystalline protein
TL;DR: Phosphorylation of histone H1 occurs when spermatozoa of the sea urchin Strongylocentrotus purpuratus are treated with the macromolecular fraction of solubilized egg jelly, and can be blocked by digesting the sperm surface with Pronase, or preincubation of sperm in wheat germ agglutinin.
TL;DR: Southern analysis indicates that most late H3 and H4 genes are organized as pairs in both sea urchin genomes, and some differences in timing of appearance of different H3 RNA species are observed.
Abstract: A gene pair coding for histones H3 and H4 expressed during late embryonic development has been cloned from the S. purpuratus genome. The organization of these genes is similar to the divergently transcribed H3-H4 gene pairs of Lytechinus pictus. Whole genome Southern analysis indicates that most late H3 and H4 genes are organized as pairs in both sea urchin genomes. The nucleotide sequences of late and early S. purpuratus H3 and H4 genes differ in the coding regions by 17.0% and 15.7%, respectively. Although there is little match between early and late genes outside the transcription unit, there are short stretches of homology in the spacers 5' to the S. purpuratus early and late H3 genes and the early and late H4 genes. Sequence comparison of the late H3-H4 S. purpuratus pair with two late H3-H4 pairs from L. pictus reveals additional striking homologies in the intergenic spacer. At least four late H3 and two H4 RNAs are distinguished by hybridization to Northern electroblots with probes derived from the cloned gene pair. Although most of these RNAs appeared to accumulate coordinately, with the most dramatic increase occurring between 13 and 17 hours of development, some differences in timing of appearance of different H3 RNA species are observed.
TL;DR: Estrogen could be involved in the regulation of reproduction in echinoids, as it is in asteroids and vertebrates, and may ultimately lead to increased transcription of the yolk glycoprotein precursor gene.
Abstract: 1. 1. A similar high molecular weight glycoprotein is found in an asteroid egg, Pisaster giganteus , and in echinoid eggs, Dendraster excentricus and Strongylocentrotus purpuratus . 2. 2. Estrogen induced the synthesis of a novel protein in echinoid coelomocytes, 82 K daltons in D. excentricus and 78 K daltons in S. purpuratus ; but it did not increase yolk glycoprotein precursor synthesis nor protein synthesis as a whole in these cells during treatment up to 24 hr. However, the induced novel protein may ultimately lead to increased transcription of the yolk glycoprotein precursor gene. Estrogen therefore could be involved in the regulation of reproduction in echinoids, as it is in asteroids and vertebrates.
TL;DR: A gene encoding a late H1 histone subtype from the sea urchin species L. pictus is isolated and sequenced, and large blocks of sequences that are identical between the two genes are revealed.
Abstract: We have isolated and sequenced a gene encoding a late H1 histone subtype from the sea urchin species L. pictus. The primary structure of the late H1 subtype encoded by this gene is 209 amino acids in length, and has a net positive charge of 67. This gene is present in a single copy per haploid genome and encodes an mRNA of 752 nucleotides. Late H1 transcripts are detected in the unfertilized egg and are most prevalent in gastrulating embryos. Comparison of 375 bp of 5' flanking sequences of the L. pictus late H1 gene and the H1-gamma gene of a distantly related sea urchin species, S. purpuratus, reveals large blocks of sequences that are identical between the two genes. To determine if these conserved 5' sequences are present in other members of the sea urchin H1 gene family, the analogous region of S. purpuratus H1-alpha, an early H1 gene, was sequenced. The homology between the flanking sequences of the early and late families was limited to consensus sequences which are found upstream of all H1 genes. The possible regulatory implications of these findings are discussed.
TL;DR: The 305 kD-polypeptide isolated from S purpuratus is a species-specific vitelline envelope sperm receptor, and it is concluded that the 225-kD polypeptides shared determinants may be derived from the 305- kD polyPEptide by the proteolysis that occurs at the cell surface during fertilization.
Abstract: Direct isolation of the sea urchin egg vitelline envelope with intact sperm receptors is difficult because the envelope is firmly attached to the egg plasma membrane. We now report a method for producing an inseminated egg preparation in Strongylocentrotus purpuratus (using soybean trypsin inhibitor [STI] and Ca2+, Mg2+-free seawater) that contains an elevated vitelline envelope (VE*-STI). The VE*-STI is devoid of cortical granule material, and supernumerary sperm do not detach postinsemination, suggesting that the VE*-STI contains active sperm receptors. VE*-STIs contain a 305-kD polypeptide and additional components that range from 225 to 31 kD, whereas the 305-kD polypeptide was considerably reduced in VE*s. Electrophoresis of sperm receptor hydrolase digests of VE*-STIs showed that the 305-kD polypeptide and several other envelope polypeptides are protease substrates. Univalent Fab fragments against VE*s, VE*-STIs, and 305 and 225-kD polypeptides blocked sperm binding and fertilization in an Fab concentration-dependent manner. The 305 and 225-kD polypeptides were localized in the VE*-STI using indirect immunofluorescence. Enzyme-linked immunosorbent assays showed that the 305 and 225-kD polypeptides share determinants, suggesting that the 225-kD polypeptide may be derived from the 305-kD polypeptide by the proteolysis that occurs at the cell surface during fertilization. Fab fragments against S purpuratus VE*-STI antigens neither bound to nor blocked homologous sperm binding and fertilization of Lytechinus variegatus eggs. Cross fertilizability occurred to the extent of 5% or less between L variegatus and S purpuratus, therefore, we conclude that the 305 kD-polypeptide isolated from S purpuratus is a species-specific vitelline envelope sperm receptor.
TL;DR: Analysis by translation and hybridization of cloned cDNA probes to RNA blots indicates that a few mRNA species are always relatively enriched in the free RNP pool of posthatching embryos and do not serve as a store of mRNA for later translation.
TL;DR: The model of FE development is that a benzamidine-sensitive, cortical granule protease cleaves a 200 kD VE polypeptide during initial envelope elevation to set up the morphological change in FE papillae which occurs later, based on protease inhibitor experiments.
Abstract: The sea urchin fertilization envelope (FE) is formed following initial sperm-egg interaction from the egg surface vitelline envelope (VE) and the paracrystalline protein fraction (PCF), derived from cortical granules. Although mature FEs are physicochemically hardened postinsemination, a major protein fraction consisting of seven major polypeptides was extracted from Strongylocentrotus purpuratus FEs and the major, separated components were immunologically cross-reactive with the principal polypeptides in PCF and isolated cortical granules. Antibodies prepared against extracted, core FEs were immunologically crossreactive with isolated VEs, but not with PCF, suggesting that only VE components are covalently crosslinked. Based on protease inhibitor experiments, our model of FE development is that a benzamidine-sensitive, cortical granule protease cleaves a 200 kD VE polypeptide during initial envelope elevation to set up the morphological change in FE papillae which occurs later. Divalent cations precipitate the PCF and form metal proteinate bridges between the VE and PCF. Based on peroxidase inhibitor experiments, we suggest that the cortical granule peroxidase crosslinks VE polypeptides, beginning at 2-3 min postinsemination, to restrict the permeability of the VE so that normal envelope thickening occurs. A 305 kD VE polypeptide was isolated and appears to be important in sperm-egg interaction based on inhibition of sperm binding and fertilization by antibodies against the purified polypeptide.
TL;DR: Data suggest that sperm receptor hydrolases are serine proteases, one of two classes of cortical granule proteoesterases, which was purified approximately 30-fold with 80% yield from Strongylocentrotus purpuratus corticalgranule exudate.
Abstract: Sperm receptor hydrolase, one of two classes of cortical granule proteoesterases (E.C.3.4.4.4) was purified approximately 30-fold with 80% yield from Strongylocentrotus purpuratus cortical granule exudate. Sperm receptor hydrolase preparations were free of vitelline delaminase activity (the other class of cortical granule proteoesterase) and had less than 1% of the starting levels of cortical granule peroxidase and β-1,3-glucanohydro-lase activities. Native polyacrylamide gel electrophoresis coupled with a protease activity stain showed that three proteases were present in the most highly purified preparations of sperm receptor hydrolase. Each of the three proteases has the same molecular weight of 60,000, but different isoelectric points of 2.4, 3.8, and 5.5. The Km value of the mixture of proteases for α-N-benzoyl-L-arginine ethyl ester as substrate was 263 μM at pH 8.4 and 30°C; the pH dependence of Vm showed a single prototrophic group with a pK of 6.7 and an enthalpy of ionization of 8.6 kcal-mol−1. The values of these kinetic parameters are consistent with an enzyme-active site containing histidine. Phenylmethyl sulfonyl fluoride, tosyl lysine chloromethyl ketone, several proteinaceous trypsin inhibitors, and p-aminobenzamidine inhibited the esterase activity of the proteases. These data suggest that sperm receptor hydrolases are serine proteases.
TL;DR: A simple method is described for the isolation of spicules from pluteus embryos of the sea urchin, Strongylocentrotus purpuratus, where radio-iodination of the demineralized matrix reveals six bands on SDS protein gels.
TL;DR: Ciliary Dynein and sperm flagellar dynein, although derived from very similar organelles and from the same species of sea urchin, are immunologically distinct.
TL;DR: The antibody was used to demonstrate that the form of β-1,3-glucanase present following gastrulation is antigenically distinct from the egg form, suggesting that it may function in larval digestion.
TL;DR: Since speract fails to cause responses in A. punctulata spermatozoa to the egg peptides, it can be concluded that the events are receptor-mediated.
Abstract: Speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly), a peptide obtained from the culture medium of Strongylocentrotus purpuratus eggs, stimulates the respiration and motility of S. purpuratus spermatozoa under appropriate conditions. Resact (Cys-Val-Thr-Gly- Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-LeuNH2), a peptide obtained from Arbacia punctulata eggs also stimulates the metabolism and motility of A. punctulata spermatozoa, however, it fails to stimulate S. purpuratus spermatozoa. Early biochemical responses of the spermatozoa to the egg peptides include a net H+ efflux and elevations of cyclic AMP and cyclic GMP concentrations. In addition, in A. punctulata spermatozoa, a major plasma membrane protein is modified in response to resact such that its apparent molecular weight shifts from 160,000 to 150,000. If cells are incubated with 32 P, the 160,000 molecular weight form of the protein becomes radiolabeled; subsequent addition of resact causes a rapid loss of 32P from the protein. The plasma membrane protein appears to be the enzyme, guanylate cyclase; coincident with the shift in apparent molecular weight, enzyme activity decreases by as much as 90%. Since speract fails to cause these responses in A. punctulata, it can be concluded that the events are receptor-mediated.