pc1 and psc1, zebrafish homologs of Drosophila Polycomb and Posterior sex combs, encode nuclear proteins capable of complex interactions.

Drosophila Polycomb group proteins are thought to form multimeric nuclear complexes that are responsible for stable transmission of repressed states of gene expression during the proliferation of differentiating embryos. In this study, we cloned, sequenced, and characterized two Polycomb group homologs, designated pc1 and psc1, in zebrafish. Amino acid sequence analyses determined that pc1 is a structural homolog of Drosophila Polycomb and that psc1 is a homolog of Drosophila Posterior sex combs.

Does the low respiratory rate in unfertilized eggs result mainly from depression of the redox reaction catalyzed by flavoproteins? Analysis of the respiratory system by light-induced release of CO-mediated inhibition.

In unfertilized eggs of the sea urchin, the quite low respiratory rate is enhanced by tetramethyl-p-phenylenediamine (TMPD), phenazine methosulfate (PMS) and sperm and this augmentation is completely inhibited by carbon monoxide (CO). Exposure to light releases eggs from this CO-mediated inhibition. The action spectra for photoreactivation of CO-inhibited cytochrome c oxidase in isolated mitochondria and CO-blocked respiration in TMPD-treated eggs were found to be similar to the absorption spectrum of CO-bound cytochrome aa .

Change in the levels of adenine-related compounds in starfish ovarian follicle cells following treatment with gonad-stimulating substance.

The resumption of meiosis in starfish oocytes is induced by 1-methyladenine (1-MeA), which is produced by ovarian foilicle cells under the influence of a gonad-stimulating substance (GSS). It has been reported that the 1-MeA produced is newly synthesized via a process of methylation, rather than being pre-stored within follicle cells or a breakdown product of some 1-MeA-containing substance. The present study examined a possible substrate for 1-MeA biosynthesis stored in follicle cells of the starfish Asterina pectinifera.

Does phosphatidylinositol 3-kinase play a role in insulin-induced outgrowth of pseudopodial cables in cultured cells derived from micromeres of sea urchin embryos?

Cultured cells derived from micromeres isolated from sea urchin embryos at the 16-cell stage, which have insulin receptors, undergo pseudopodial cable growth and spicule rod formation in culture with horse serum and only cable growth in culture with insulin. Genistein, an inhibitor of protein tyrosine kinase, and wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3kinase), inhibited pseudopodial cable growth in micromere-derived cells cultured with insulin and also growth accompanied by spicule rod formation in horse serum-treated cells. The PI3kinase activity in the immunoprecipitates obtained by anti-phosphotyrosine antibody from the cells cultured with insulin was higher than that in cells cultured without insulin or with insulin and genistein.

Fertilization envelope formation induced by melittin in sea urchin eggs does not depend on Ca signalling.

In sea urchin eggs, 10 μg/mL melittin was found to induce fertilization envelope formation without any increase in [Ca ] (the intracellular free Ca level). On the other hand, 10 μmol/L Br-A23187 and 100 μg/mL SDS induced fertilization envelope formation associated with [Ca ] increase. If EGTA was injected into eggs to make an intracellular concentration of 2 mmol/L, [Ca ] became quite low and was not altered by melittin, or by Br-A23187 and SDS.

Several Cell Responses to Insulin of Cultured Cells Derived from Micromeres, Isolated from Sea Urchin Embryos at the 16 Cell Stage: (Sea urchin/development/morphogenesis/insulin/micromere).

In micromere-derived cells of sea urchin embryos, treatment with insulin started for up to 24 h during culture at 20°C resulted in augmentation of P incorporation into protein (protein phosphorylation) followed by activation of P incorporation into RNA (RNA synthesis) and then induced pseudopodial cable growth, accompanied by considerable decreases in the rates of protein phosphorylation and RNA synthesis. This augmentation of RNA synthesis and cable growth induced by insulin were blocked by H-7, which inhibited protein phosphorylation, and were also inhibited by actinomycin D without any inhibition of protein phosphorylation. Similar results were obtained on treatment with horse serum, found to contain insulin-like compounds.

Changes in Insulin-Binding Capacity of the Plasma Membrane Fraction during Culture in vitro of Cells Derived from Micromeres of 16-Cell-Stage Sea Urchin Embryos: (sea urchin/development/micromere/insulin/insulin receptor).

In cultured cells derived from isolated micromeres of 16-cell stage sea urchin embryos, which undergo insulin-induced pseudopodial cable growth, specific and reversible insulin binding by a 52-kDa protein, probably an insulin receptor in the plasma membrane, is augmented during 5 h of culture without any change in the dissociation constant (Kuno et al : 1994). The increase in insulin-binding capacity in micromere-derived cells was only minimally blocked by actinomycin D and cycloheximide, which inhibited [U- H]uridine incorporation into RNA and [ S]methionine incorporation into protein, respectively. Insulin binding capacity was found in the plasma membrane fraction and the microsome fraction of isolated micromeres.

Insulin-Induced Outgrowth of Pseudopodial Cables from Cultured Micromere-Derived Cells Isolated from Sea Urchin Embryos at the 16 Cell Stage, with Special Reference to the Insulin-Receptor.: (sea urchin/micromere/insulin/psedopodial cable/receptor).

Cultured cells derived from micromeres isolated from sea urchin embryos at the 16 cell stage are known to show outgrowth of pseudopodial cables followed by spicule rod formation when cultured in the presence of horse serum. Micromere-derived cells cultured with bovine insulin showed pseudopodial cable growth but did not produce spicule rods. Micromere-derived cells reversibly bound to insulin through out the period between 3 and 20 hr of culture.

ADP-Ribosylation of Histones in Nuclei Isolated from Embryos of the Sea Urchin, Hemicentrotus pulcherrimus: (histone/ADP-ribosylation/sea urchin/development/nucleus).

Two-dimensional electrophoresis (2D-PAGE) of a histone fraction isolated from nuclei of embryos of the sea urchin Hemicentrotus pulcherrimus exhibited almost all histone species at all stages examined. At the gastrula stage, a spot of H1A became evident and three spots closely associated with one another were found in place of a single spot of H2A.1.

Proteins ADP-ribosylated in Nuclei and Plasma Membrane Vesicles Isolated from Sea Urchin Embryos at Various Stages of Early Development: (ADP-ribosylation/sea urchin/development/nucleus/plasma membrane).

The ADP-ribosylations of proteins in nuclei, plasma membrane vesicles, mitochondria, microsome vesicles and the soluble fraction of sea urchin embryos isolated at various stages of development were examined by measuring the radioactivities of proteins after exposure of these subcellular fractions to [adenosine- C]NAD or [adenylate- P]NAD. ADP-ribosylation of proteins was detected only in the nuclear and plasma membrane fractions. In the nuclear fraction, the rate of ADP-ribosylation of the histone fraction did not change appreciably during early development.

Does ADP-Ribosylation of Proteins in Nuclei Contribute to Ectoderm Cell Differentiation in Sea Urchin Embryos?: (ADP-ribosylation/sea urchin/development/animalization/differentiation).

In nuclei of sea urchin embryos, marked increase in ADP-ribosyltransferase activity followed by its decrease occurrs in the pre-hatching and post-hatching periods with peaks of activity at the morula and gastrula stages. Increase in its activity was blocked by cycloheximide in the pre- and post-hatching periods and by actinomycin D only in the post-hatching period. Embryo wall cells (ectoderm cells) isolated from gastrulae exhibited markedly higher activity of this enzyme than archenteron cells and mesenchyme cells.

Expression of Na , K -ATPase α-Subunit in Animalized and Vegetalized Embryos of the Sea Urchin, Hemicentrotus pulcherrimus.

A marked increase in the Na , K -ATPase activity of sea urchin embryos occurred following an elevation of its mRNA level, revealed by Northern blotting analysis, in developmental period between the swimming blastula and the late gastrula stage. cDNA clone of Na , K -ATPase α-subunit, obtained from γgt10 cDNA library of sea urchin gastrulae, was digested with EcoRl ad Hindlll. The obtained 268 bp cDNA fragment, hybridized to a 4.

Change in the Activity of Na , K -ATPase in Embryos of the Sea Urchin, Hemicentrotus pulcherrimus, during Early Development: (sea urchin embryo/Na , K -ATPase/RNA synthesis/ectodermal cell/actinomycin D).

The activity of ouabain-sensitive Na , K -ATPase in sea urchin embryos at the morula and the swimming blastula stage was practically the same to that in unfertilized eggs. The activity increased during the period between the mesenchyme blastula and the late gastrula stages. In embryo-wall cell fraction, which contained presumptive ectodermal cells as well as those of other cell lineages at the pre-gastrula stage and ectodermal cells at the late gastrula stage, the Na , K -ATPase activity increased in this developmental period more largely than in another cell fraction, containing mesenchyme cells and archenteron cells.

SCN Blocks Hardening of the Fertilization Envelope of Sea Urchin Eggs and Adhesion of Blastomeres.

Sea urchin eggs kept in artificial sea water (ASW) containing 0.01-0.3 M NaSCN in place of NaCI from within 2 min after insemination formed thin, enlarged fertilization envelopes, which were broken on mild agitation of egg suspensions more easily than those formed in Ca -free ASW.

Identification of GTP-Binding Proteins by ADP-Ribosylation in the Presence of Cholera Toxin, Pertussis Toxin and Botulinum Toxin D in Plasma Membrane Isolated from Eggs and Embryos of Sea Urchin.

In plasma membrane fraction isolated from eggs and embryos of sea urchin, P-labeled proteins were found on the fluorographs of SDS-polyacrylamide gel electrophoresis, performed after an exposure of the fraction to [adenylate- P] nicotinamide adenine dinucleotide in the presence of cholera toxin, pertussis toxin or botulinum toxin D. The molecular weights of proteins, thus ADP-ribosylated in the presence of cholera toxin and pertussis toxin are 45 and 39 K, which correspond to Gs and Gi or Go, respectively. Protein with the molecular weight of 24 K, labeled in the presence of botulinum toxin D, corresponds to small molecular weight G-protein.

Animalizing Effect of A23187 on Sea Urchin Embryos: (sea urchin/animalization/vegetalization/A23187/tetracaine).

Pulse treatment of sea urchin embryos with 3 μM A23187 for 2 hr starting at a stage in initial 10 hr period of development at 20°C, followed by a culture in normal sea water up to the pluteus corresponding stage (45 hr after fertilization), yielded many large exogastrulae with thin embryo walls. The pulse treatment starting at a time between 10 and 13 hr after fertilization yielded considerable number of large prisms and gastrulae having thin embryo walls. Probably, the pulse treatment exerts stimulating effects on ectodermal cell determination in whole span of pre-hatching period to produce animalized embryos.

Does Protein Phosphorylation by Protein Kinase C Support Pseudopodial Cable Growth in Cultured Micromere-Derived Cells of the Sea Urchin, Hemicentrotus pulcherrimus?: (sea urchin/protein kinase C/spicule formation/phorbol ester/H-7).

In cultured cells derived from micromeres, H-7 strongly inhibited the outgrowth of pseudopodial cables and the formation of spicule rods at concentrations around the Ki values for protein kinases. HA1004 did not inhibit the cable growth and spicule rod formation in these cells at higher concentrations than the Ki values for cyclic nucleotide-dependent protein kinases. Pseudopodial cable growth was also inhibited by H-7 in furosemide-treated cells which were able to undergo normal growth of the cables without the formation of spicule rods.

Vegetalization Induced by Procaine and Tetracaine in Sea Urchin Embryos: (sea urchin/development/vegetalization/procaine/tetracaine).

Vegetalization of sea urchin embryos was induced by the treatment with procaine and tetracaine, inhibitors of Ca mobilization, for 3 hr starting 3-5 hr after insemination at 20°C. The treatment starting 7 hr after insemination sometimes produced similar type of vegetalized embryos. The pulse treatment starting at the other stages hardly yielded vegetalized embryos.

Respiration and Motility in Starfish Spermatozoa at Various Times in the Breeding Season: (starfish/sperm, motility/respiration).

In the spermatozoa of Asterias amurensis, patterns of changes in the respiratory rate and motility following dilution of dry sperm in sea water varied among batches and were classified into three types. The type I spermatozoa were immotile and exhibited quite low respiratory rate. Ethylenediamine tetraacetic acid made the type I spermatozoa motile and elevated their respiratory rate.

Probable Contribution of Protein Phosphorylation by Protein Kinase C to Spicule Formation in Sea Urchin Embryos: (sea urchin/protein kinase C/spicule formation/H-7/HA1004).

The formation of spicules and development of pluteus arms in sea urchin embryos were strongly blocked by H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride) but were not affected by HA1004 (N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride). Archenteron formation occurred normally in the presence of these compounds. Late gastrulae (28 hr after fertilization) were exposed to Pi for 30 min at 20°C, and then dissociated and their primary mesenchyme cells with spicules, embryo-wall cells and archenteron cells were separated.

Fertilization Membrane Formation in Sea Urchin Eggs Induced by Drugs Known to Cause Ca Release from Isolated Sarcoplasmic Reticulum: (sea urchin egg/fertilization membrane/ryanodine/micronazole/procaine).

Ryanodine, miconazole, clotrimazole, doxorubicin, quercetin, halothane, caffeine and chloroform, which activate Ca -induced Ca release from Ca stores, induced Ca release from a particulate fraction isolated from sea urchin eggs, Ca influx into eggs and formation of a fertilization membrane in an appreciable number of eggs. Their minimum effective concentrations for inducing a fertilization membrane increased in the order of these drugs listed above, and this order was also the same as that of their minimum effective concentrations for inducing Ca release from the isolated particulate fraction. Their effect in inducing a fertilization membrane was blocked by ruthenium red and procaine, which inhibit Ca release from Ca stores.

Pulse treatment of sea urchin embryos with A23187 blocks their hatching out.

Pulse treatment of sea urchin embryos with 3 µM A23187 for 2 h at 20° C, starting from 3 to 6 h of development, prevented the embryos from hatching. Many embryos thus treated with A23187 produced mesenchyme cells and underwent gastrulation while still enclosed within the fertilization membrane. The pulse treatment in this pre-hatching period exerts markedly stronger inhibitory effects on hatching than on other events in early development.

Binding of [ H]Nitrendipine to Proteins in the Plasma Membrane of Sea Urchin Sperm: (sea urchin/sperm/membrane fraction/[ H]nitrendipine/Ca channel).

The plasma membrane fractions of the sperm of four species of sea urchin, obtained by the method by Podell et al. (24), gave similar electrophoretic profiles of proteins. Several proteins in the membrane fraction from Hemicentrotus pulcherrimus bound [ H]nitrendipine, a specific antagonist of voltage-dependent Ca channels, added at concentration of about 10 times those reported to be effective in muscle and nerve cells.

Synthesis of Proteins Enriched in Li - Induced Vegetalized Embryos of Sea Urchin during Early Development: (protein synthesis/vegetalization / sea urchin).

Sea urchin embryos were vegetalized by a pulse treatment with 60 mM Li between 2.5 hr and 6 hr after fertilization at 20°C. Normal and Li -treated embryos were exposed to [ S]-methionine for 2 hr at various stages and [ S]-labeled proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE).

Synthesis of Collagen-Like Proteins in Embryonic Organs of the Sea Urchin, Hemicentrotus pulcherrimus: (sea urchin embryo/collagen synthesis/embryonic organ/embryogenesis).

In sea urchin embryos exposed to C-proline at 20°C for 3 hr at the gastrula, prism or pluteus stage, C-radioactivity was found in hot acid-extractable proteins, in which more than 4% of the radioactivity was detectable in hydroxyproline residues. In these embryos, C-radioactivity in collagen-like proteins was found in the archenteron, spicule and embryo-wall cells. The rate of synthesis of collagen-like proteins was highest in the archenteron in the mid-gastrula stage, in the embryo-wall cells in the prism stage and in the spicule in the pluteus stage.