bluuebunny/crossref_metadata_2025 huggingface dataset:
Pigmented Follicle Cells and the Maturation of Oocytes in the Sand Dollar, Dendraster excentricus
Authors: Burke Robert D. , Bouland Catherine
Published: January 2005

Early in the spawning season female D. excentricus can be induced to spawn oocytes in the late stages of oogenesis. Observations with light microscopy indicate that pigment cells migrate in the jelly coat away from the surface of oocytes coincident with the final growth of the oocyte and its maturation. The pigment cells undergo a series of changes in shape and the oocyte elaborates arrays of long microvilli as the cells elevate from the surface of the oocyte. Transmission electron microscopy (TEM) observations of oocytes fixed within the ovary indicate that features of the sequence observed in the spawned oocytes normally take place within the ovary, prior to spawning. Extracts of the pigment cells induce a low level of germinal vesicle breakdown in asteroid oocytes. It is proposed that these cells are homologous to the follicle cells of other echinoderms and are involved in stimulating the maturation of oocytes.
Activation of Starfish Oocytes Modifies their Hormone Dependent Period for 1‐Methyladenine in Meiosis Reinitiation
Authors: Chiba Kazuyoshi , Hoshi Motonori
Published: January 2005

Starfish oocytes reinitiate the meiosis when they are exposed to the hormone, 1‐methyladenine (1‐MA): 1‐MA must be present for a period of about 10–20 min (20°C) known as the hormone dependent period (HDP). We investigated several conditions which affect the duration of the HDP in the starfish Asterina pectinifera. Fertilization lengthened the HDP about 30–80%, when spermatozoa were added to the oocytes at the same time as 1‐MA was applied. When oocytes were inseminated before 1‐MA treatment, HDP was reduced progressively correlating with the time of the insemination coming earlier, and eventually became shorter than the control. Similarly, A23187 and caffeine lengthened the HDP if they were added to the oocytes at the same time as 1‐MA was applied, and shortened it if they are applied sufficiently in advance to 1‐MA treatment. These modifications of HDP are likely to be brought by changes in the intracellular calcium concentration. A23187 did not lengthen the HDP, if it was administered 10 min after 1‐MA application. Thus the effect of A23187 on the HDP occurred specifically during the first 5 or 10 min after 1‐MA application. These results indicate that the HDP comprises at least two distinct phases.
Involvement of Wheat Germ Agglutinin (WGA)‐Binding Protein in the Induction of the Acrosome Reaction of the Sea Urchin, Strongylocentrotus intermedius. I. WGA Affects the Ion Fluxes Associated with the Acrosome Reaction
Authors: Sendai Yutaka , Ohta Tadayuki , Aketa Kenji
Published: January 2005

The lectin wheat germ agglutinin (WGA) inhibited the egg jelly‐induced acrosome reaction (AR) of sperm of the sea urchin, Strongylocentrotus intermedius. Fluorescein‐conjugated WGA applied to sperm bound to the acrosomal region, to the midpiece, and to the tip of the flagellum. These effects were not observed in the presence of N‐acetly‐D‐glucosamine. When the egg jelly was replaced by artificial AR inducers such as A23187 or nigericin, the AR was not inhibited by WGA. Results obtained using a Ca2+ indicator fura‐2, a pH indicator 2′,7′‐bis(carboxyethyl)carboxyfluorescein (BCECF) and a membrane potential sensitive dye 3,3′‐dipentyl 2,2′‐dioxacarbocyanine [diO‐C5(3)] showed that WGA suppresses the egg jelly‐induced influx of Ca2+ and slightly suppresses the efflux of H+ caused by the egg jelly, whereas the depolarization of the plasma membrane by the egg jelly is remarkably amplified by the treatment with WGA. These results suggest that WGA affects the regulatory system of the ion fluxes associated with the AR. The target protein of WGA (WGA‐binding protein) was a membrane glycoprotein of 260 kD under non‐reducing condition.
Involvement of Wheat Germ Agglutinin (WGA)‐Binding Protein in the Induction of the Acrosome Reaction of the Sea Urchin, Strongylocentrotus intermedius. II. Antibody against WGA‐Binding Protein Induces the Acrosome Reaction
Authors: Sendai Yutaka , Aketa Kenji
Published: January 2005

We obtained a polyclonal antibody against the WGA‐binding protein (WGAbp) of Strongylocentrotus intermedius sperm, which is a membrane glycoprotein of 260 kD under non‐reducing condition. Anti‐WGAbp antibody induced increases in both intracellular Ca2+ ([Ca2+]i) and intracellular pH (pHi), resulting in the onset of the AR. The increases in [Ca2+]i and pHi required extracellular Ca2+ and Na+, respectively, and were suppressed by the pretreatment with WGA, resulting in the inhibition of the AR. Anti‐WGAbp antibody‐induced AR was inhibited also by lowered extracellular pH. elevated K+, removal of Na+ from seawater and the treatment with verapamil, a Ca2+ channel inhibitor. These inhibitory conditions are identical with those of the egg jelly‐induced AR. Monovalent Fab fragments from anti‐WGAbp antibody also induced the AR at relatively high concentration. These results suggest that the WGAbp on the sperm plasma membrane is involved in the regulation of Ca2+ influx and Na+/H+ exchange associated with the AR of S. intermedius sperm. It is a strong candidate for the receptor of the AR‐inducing substance in the egg jelly.
The Influence of Yolk Protein Proteolysis on Hydration in the Oocytes of Fundulus heteroclitus
Authors: McPherson Roger , Greeley Mark S. , Wallace Robin A.
Published: January 2005

Growth in oocytes of many marine teleosts can be attributed to a combination of yolk accumulation during the vitellogenic phase of development and water uptake during meiotic maturation. In the salt marsh fish, Fundulus heteroclitus, hydration associated with maturation gives rise to a greater than two‐fold increase in oocyte volume. It has been proposed that a concurrent proteolysis of specific yolk proteins may be the mechanism driving this water uptake. To test this hypothesis, we used various in vitro culture techniques to block or significantly reduce oocyte hydration while allowing meiotic maturation to continue, then examined yolk proteins by SDS‐polyacrylamide gel electrophoresis. We were able to dissociate yolk proteolysis from both hydration and nuclear maturation stimulated by a maturation‐inducing steroid, 17α‐hydroxy‐ 20β‐dihydroprogesterone. It therefore appears that the proteolysis of specific yolk proteins observed in maturing oocytes of marine teleosts is an independent developmental event, and is not directly involved in the hydration mechanism.
Plasma Membrane Domains and the Site of Sperm Entrance in Discoglossus pictus (Anura) Eggs
Authors: Gualtieri Roberto , Cafiero Gennaro , Andreuccetti Piero
Published: January 2005

Freeze‐fracture quantitative analysis reveals three different plasma membrane (PM) domains in the unfertilized egg of the anuran Discoglossus pictus. One of these is specific to the sperm entrance site (D1). where the plasma membrane shows a larger number of intramembranous particles (IMP) than the rest of the egg surface. Such an increment is due to a markedly higher number of the IMPs anchored to the P‐face. The two other domains (D2 and D3) are characterized by a lower IMP density at the P‐face with respect to D1. The IMP density decreased within 10 min after fertilization by about 33% in all domains observed, probably due to the insertion of new membrane through exocytosis. The possibility that the IMPs located in D1 may represent putative plasma membrane proteins playing a role in sperm‐egg interaction and/or in egg activation is discussed.
Concanavalin A Modifies Allo‐specific Sperm‐Egg Interactions in the Ascidian, Ciona intestinalis
Authors: Kawamura Kazuo , Fujita Hiroko , Nakauchi Mitsuaki
Published: January 2005

In the self sterile ascidian, Ciona intestinalis, the spermatozoa rarely bind to the vitelline coat of autologous eggs and never penetrate it. We report here that concanavalin A (ConA), a lectin recognizing mannose or glucose residues of carbohydrates, can modify these self‐ and nonself‐specific sperm‐egg interactions. When eggs were pretreated with 0.1–0.5 mg/ml of ConA, about two thousand spermatozoa became attached to the autologous vitelline coat within five minutes of insemination. The effect of ConA was not modified by the addition of D‐mannose or pretreatment of spermatozoa with ConA, showing that ConA does not function merely as a ligand bridging the sperm and vitelline coat. In contrast to the marked enhancement of sperm‐egg binding, ConA did not facilitate the penetration of spermatozoa through the autologous vitelline coat. Even in non‐autologous insemination, it blocked the sperm penetration and, consequently, fertilization did not occur, as shown by Rosati et al. (1978). D‐Mannose, when mixed with ConA in advance, completely abolished this inhibitory effect of ConA. Lotus agglutinin, a fucose‐binding lectin, was less effective and wheat germ agglutinin and soy bean agglutinin had no effect on sperm entry in the perivitelline space. The results of this study are discussed in relation to the possible involvement of mannosyl and/or glucosyl glycoconjugates in allo‐specific sperm‐egg interactions.
Sorting Out of Presumptive Stomach Cells of the Starfish Embryo
Authors: Dan‐Sohkawa Marina , Kaneko Hiroyuki
Published: January 2005

Presumptive stomach cells of the starfish embryo are shown to sort out and to occupy a position in the endoderm of the reconstructed embryo. A new method is introduced to specifically mark these cells by staining the embryo with a fluorescent dye, 8‐anilino‐1‐naphthalenesulfonic acid (1,8‐ANS).
Cell Fusion Promoting Factor Common to Homothallic and Heterothallic Mating Systems in Dictyostelium discoideum1
Authors: Urushihara Hideko , Saigo Tomoko , Yanagisawa Kaichiro
Published: January 2005

Cellular slime mould Dictyostelium discoideum propagates as single haploid cells and under certain environmental conditions enters into a sexual cycle called macrocyst formation. There are homothallic and heterothallic strains reported, the former being able to form macrocysts in clonal cell populations while the latter to do so only in the presence of opposite mating‐type strains. Molecular basis for differential mating systems is an intersting subject totally unknown yet. In the present study, sexual cell interactions in AC4, a homothallic strain of D. discoideum, was studied in comparison with the heterothallic mating system. The conditoned medium of AC4 cells was found to promote the sexual cell fusion among themselves. In addition, it also enhanced the cell fusion between heterothallic strains. Furthermore, the conditioned medium obtained from the mated culture of heterothallic strains reported to induce the sexual cell fusion in the heterothallic strains (Saga and Yanagisawa, 1983) was found also to promote the cell fusion in AC4. These results suggest that common regulatory mechanisms operate for sexual cell fusion among different mating systems in D. discoideum.
Concanavalin A Acts as a Factor in Establishing the Dorso‐Ventral Gradient in the Ventral Mesoderm of Newt Gastrula Embryos1
Authors: Diaz Maria R. M. , Takahashi Tadashi C. , Takeshima Kazuhito , Takata Kenzo
Published: January 2005

Con A induced dorsal differentiation in the ventral mesoderm of Cynops gastrula embryo. This process apparently requires a certain amount of Con A to be internalized as supported by the following evidence: 1) Oligomannose‐type oligosaccharide, a potent inhibitor of Con A, considerably inhibited dorsalization of ventral mesoderm by Con A. The incorporation of 125I‐Con A into the ventral mesoderm was greatly inhibited by this sugar. 2) Sepharose‐immobilized Con A did not dorsalize the ventral mesoderm. Con A‐induced dorsalization was found to be concentration‐dependent.

Microautoradiograms of 125I‐Con A‐treated ventral mesoderm suggest that the target site (some receptor molecules) of Con A exists inside the cell.

Con A is the first pure substance reported to mimic the two properties of the organizer—neural induction of the competent ectoderm and dorsalization of the ventral mesoderm. In neural induction, Con A acts on the cell surface, while Con A apparently needs to be internalized to trigger dorsal differentiation. Interestingly, Con A‐dorsalized ventral mesoderm acquired the neural inducing function of the organizer within the early phase of dorsalization.
The Mechanism of σ‐Crystallin Gene Regulation: Cooperation of Lens‐Specific and Non‐Specific Elements1
Authors: Kondoh Hisato
Published: January 2005

Employing δ‐crystallin gene as a model, we have investigated tissue‐specific gene regulation. Our approach was to analyze regulatory elements associated with the gene utilizing gene transfer techniques. Introduction of the chicken δ1‐crystallin gene into the genome of developing mouse embryos resulted in lens‐specific expression, indicating that the elements governing the tissue specificity are located in the DNA sequence introduced. Through analysis of various regions of the δ1‐crystallin gene and the associated DNA sequences, we identified a lens‐specific enhancer in the third intron of the gene. It was demonstrated that this enhancer alone is sufficient to account for lens specificity of the δ1‐crystallin gene. Dissection of the δ1‐crystallin enhancer and functional assessment by multiplication of enhancer fragments demonstrated the cooperative interaction of lens‐specific and nonspecific elements in the enhancer. The mechanism by which heterologous elements cooperate in generating enhancer activity unquestionably provides great flexibility to the regulatory system, and may account for developmental modulation of gene activity superimposed on tissue specificity.
Developmental Potential of Haploid‐derived Parthenogenetic Cells in Mouse Chimeric Embryos1
Authors: Ishino Fumitoshi , Kaneko‐Ishino Tomoko , Ito Michihiko , Matsuhashi Michio , Yokoyama Minesuke , Katsuki Motoya
Published: January 2005

Studies were made on the contribution of haploid‐derived parthenogenetic cells to haploid parthenogenetic ↔ fertilized chimeric embryos on day 9 and 10 of pregnancy. In most cases, the contribution of haploid‐derived parthenogenetic cells to embryonic tissues was higher than that to extraembryonic tissues. The contribution of haploid‐derived cells to embryonic tissues of some chimeras was more than 90%. Chromosomal analysis showed that actively dividing cells in most chimeric embryos contained about 40 chromosomes, indicating that they were diploidized, as haploid parthenogenetic blastocysts have about 20 chromosomes. Results suggested that haploid‐derived parthehogenetic cells in chimeric embryos diploidized spontaneously after the blastocyst stage. These cells were capable of differentiating into most cell types of embryonic tissues, but scarcely differentiated into extraembryonic tissues of day 9 embryos. The fate of haploid‐derived parthenogenetic cells during postimplantational development was similar to that of diploid parthenogenetic cells that had been diploidized experimentally in the one‐cell stage.
Analysis of Embryonic Chick Periderm by Monoclonal Antibody Specific against Periderm1
Authors: Kitamura Kunio , Sezaki Mariko , Yanazawa Masako
Published: January 2005

The monoclonal antibody, 6H11, specific against embryonic chick periderm, was produced and found capable of recognizing polypeptides with 45 Kd in cytoskeletal fractions of skin and limb buds. These polypeptides were separated into two and one isoelectric variant, respectively, by two dimensional gel electrophoresis, and assigned to the acidic subfamily of cytokeratin. The ectoderm of the limb buds at stage 24 stained with 6H11. At stages 25 to 26, the experession of 6H11 in the ectoderm could no longer be detected except in a limited number of 6H11‐positive cells present in the ectoderm. These positive cells were subsequently noted on the periphery of the ectoderm. They increased in number to finally form the periderm. Periderm in the dorsolateral trunk was also formed similarly by 6H11‐positive cells but the timing of formation differed in the dorsal and lateral trunk. In the formation of skin integuments, such as feathers and scales, no regionality of 6H11‐expression was found. The expression of 6H11 eventually decreased and ceased to be evident at about the time of hatching. Its expression in the basal epidermis during embryogenesis could not be detected. In the following, a possible mechanism of the periderm formation is discussed.
Activities of Mesoderm‐Inducing Factors Secreted by Mammalian Cells in Culture1
Authors: Nakano Hiroshi , Kinoshita Kei , Ishii Koichi , Shibai Hiroshiro , Asashima Makoto
Published: January 2005

We have examined the activities of several mesoderm‐inducing factors contained in the culture fluids of phorbol ester (4beta‐phorbol 12‐myristate 13‐acetate;PMA)‐stimulated human cell lines. Mesoderm induction was assayed by examining the differentiation of mesoderm tissues reacted with presumptive ectoderm of the Cynops blastula. The assay system also examined erythroid differentiation activity (EDF activity) in order to test the relationship between mesoderm induction and activin A (EDF). Of 22 human cell lines examined, six strains were positive for both mesoderm‐inducing activity and EDF activity. Four strains showed only mesoderm inducing activity, and one showed only EDF activity. The remaining 11 strains showed neither activity. Therefore, most cell lines secreting mesoderm‐inducing activity also possessed EDF activity. Furthermore, culture fluid of a strain (K‐562) that exhibited both types of activities, was partially fractionated by DEAE‐Toyopearl column chromatography and examined in the same way. The fractions that showed the highest amount of EDF activity were coincident with those displaying mesoderm‐inducing activity. These results suggest that a number of PMA‐stimulated mammalian cell lines have the ability to secrete mesoderm‐inducing factors which are similar to activin A (EDF).
Epithelial Metaplasia Induced Amnionic Ectoderm by the Dermis of Chicken Embryos1
Authors: Mizuno Takeo , Kitamura Kunio , Saito Miwako , Tanemura Shigeji
Published: January 2005

Amnionic ectoderm of 6.8‐day chicken embryos was associated with 6.8‐day dorsal dermis or 13–15‐day scale dermis and cultured on host chorio‐allantoic membrane for 8 days. The amnionic ectoderm, recombined and cultured with the dorsal dermis, developed feather filaments consisted of a feather root, a horny sheath, and barb ridges. With several feather keratin‐specific monoclonal antibodies (4E12 and 1F3), these structures in the induced feather filaments were shown to express feather‐specific keratin antigens.

The amnionic ectoderm, recombined and cultured with the shank dermis, became stratified squamous and developed scales. The scales were keratinized and their surface reacted only weakly with the monoclonal antibodies specific for the feather keratins. However, 1F3 reacted with two polypeptides in the cytoskeletal fraction of the scales, but not of the feather filaments.

The results confirm our previous findings from in vitro experiments with the proamnionic ectoderm (Mizuno, 1970, 1972).
Differential Transcription of the Fibroin and Sericin‐1 Genes in Cell‐Free Extracts1
Authors: Suzuki Yoshiaki , Obara Takashi , Takiya Shigeharu , Hui Chi‐chung , Matsuno Kenji , Suzuki Toshiharu , Suzuki Etsuko , Ohkubo Miyuki , Tamura Toshiki
Published: January 2005

Devices of the extraction procedues and a recovery of the final supernatant as tiers accompanied with a transcription ability assessment of the individual tiers have enabled us to develop a variety of cell‐free transcription systems. The latter step revealed that the factors necessary for faithful transcription as well as enhanced transcription form aggregates or complexes indicating an intrinsic nature of these factors to interact each other. Altogether 14 cell‐free transcription systems have been developed from Bombyx mori embryos and tissues of various developmental stages and a cultured cell line, and screened for activities enhancing the basal promoter levels of the silk genes. Using these extracts a differential transcription of plural tissue‐specific genes was tried. The fibroin gene was preferentially transcribed than the sericin‐1 gene in the extracts from the posterior silk glands where the fibroin gene is specifically transcribed in vivo, while the sericin‐1 gene was dominant to the fibroin gene in the extracts from the middle silk glands where the sericin‐1 gene is specifically transcribed. Thus, these nuclear extracts offer us a biochemical clue assaying factors responsible for the differential transcription.
Effects of Different Conditions of Retinoic Acid Treatment on Expression of MK Gene, which is Transiently Activated during Differentiation of Embryonal Carcinoma Cells1
Authors: Huang Ruo‐Pan , Muramatsu Hisako , Muramatsu Takashi
Published: January 2005

MK gene was intensely expressed, when aggregates of HM‐1 embryonal carcinoma (EC) cells were treated with retinoic acid for 2 days to induce the differntiation to nerve cells, myoblasts and extraembryonic endoderm cells. The conditions inhibiting nerve cell diffrentiation or extraembryonic endoderm cell differentiation affected MK gene expression only slightly. The maximum level of MK RNA was detected 2 days after initiation of retionic acid treatment, when cells were morphologically indistinguishable from undifferentiated EC cells. Thus, MK gene appears to be expressed in differentiating EC cells irrespective of the direction of differentiation. The degree of MK gene expression in sparsely cultured HM‐1 cells correlated with the concentration of retinoic acid, especially between 10‐8 and 10‐7 M. When retinoic acid treatment was terminated after 1 day, the amount of MK RNA started to decrease. These two results are consistent with the view that retionic acid complexed with the receptor is directly involved in expression of MK gene.
Avian Scale Development XIV: A Study of Cell Proliferation in the Epidermis of the Scaleless, sc/sc, Mutant
Authors: Sawyer Roger. H.
Published: January 2005

Embryonic induction has been demonstrated in numerous studies, yet the molecular basis for induction still eludes investigators. Components of the extracellular matrix (ECM), cell adhesion molecules (CAM), diffusable factors, as well as direct cell‐cell contact, have been implicated in the early induction of avian feathers and scales. Although feathers and scales differ in many aspects, they are similar in that they appear initially as discrete and orderly arranged epidermal placodes. In the case of scutate scales, the cells of the epidermal placode are nonproliferative, while the cells of the interplacode regions are highly proliferative. In this study, I compare the proliferative activity of normal scale cells with that of the epidermal cells from embryos of the scaleless (sc/sc) mutant chicken which does not undergo epidermal placode formation and therefore lacks scutate scales. These results show that prior to the time that placodes would normally form, the proliferative activity of the scaleless epidermal cells is similar to that seen in normal epidermal cells. Likewise, the cessation of cell proliferation seen in normal placodes occurs in the epidermal basal cells of the sc/sc shank. It is the high rate of proliferation seen for the epidermal basal cells of the normal interplacode region and the outer surface of the scale ridge that never develops in the sc/sc epidermal cells.
Two‐Dimensional Gel Electrophoretic Analysis of the Cerebella from Neuro‐Pathologically‐Mutant Weaver, Nervous and Staggerer Mice1
Authors: Asaga Hiroaki , Inoue Tsutomu , Mikoshiba Katsuhiko
Published: January 2005

We have analysed the proteins of the cerebella from mutant and control mice by applying high resolution two‐dimensional polyacrylamide gel electrophoresis. The tissue of each cerebellum and also the pallium cerebri were fractionated into water‐soluble and particulate fractions, and these were used in gel electrophoresis. In order to augment the sensitivity for detection of protein spots, we applied silver staining. We used the cerebella from weaver (granule cell deficient), nervous (Purkinje cell deficient), and staggerer (poor dendritic arborization of Purkinje cells) mutant mice. The present technique revealed at least 700 to 800 protein spots. Among the spots detected we found 12 new significantly‐changed proteins in the cerebella of the mutants. The possible significance of these proteins is discussed.
Cell Interactions in Cardiac Development
Authors: DeHaan Robert L. , Fujii Shiroh , Satin Jonathan
Published: January 2005

During early heart formation, the pre‐cardiac mesoderm becomes regionally differentiated into segments destined to form ventricle, atria and sinoatrial tissue. Each region develops a characteristic beatrate and form of action potential, shaped by current through specific ion channels and membrane pumps. Fragments of pre‐sinoatrial mesoderm that would normally have a rapid intrinsic beatrate, develop into beating heart tissue with a slow beatrate, characteristic of the ventricle, when transplanted into the prospective conoventricular region at stage 6–7. These transplants also express the ventricular isoform of myosin heavy chain, suggesting that regional commintment of the precardiac mesoderm is influenced by local cues. Application of the patch‐clamp technique to single cells isolated from the ventricle of hearts at different ages during the first week of embryonic development has revealed changes in four currents that underlie the shaping of the ventricular action potential: the excitatory sodium current, the inward rectified K current, the delayed rectifier K current, and the T‐type Ca current.
Ontogeny of an Extracellular Matrix Component of Sea Urchins and its Role in Morphogenesis
Authors: Burke Robert D. , Tamboline Colin R.
Published: January 2005

A monoclonal antibody, Sp14, recognizes fibers that form a complex meshwork within the blastocoel of embryos of the sea urchin, Strongylocentrotus purpuratus. The fibers first appear as the blastocoel begins to form and increase in density throughout development. Ultrastructural localizations using the immunoperoxidase method show bundles of 20 nm fibers that are continuous with the basal lamina and have an indistinct axial periodicity. Embryos treated with tunicamycin, β‐D‐xylopyranoside, β‐aminoproprionitrile, proline analogues, or deprived of sulfate all form immunoreactive fibers although in some treatments the pattern formed is abnormal. Immunoreactivity of extracted fibers is not affected by digestion with chondroitinase ABC, hyaluronidase, collagenase or heparinase. However, proteinase K readily destroys immunoreactivity. Fibers will form in cultures of micromeres or mesenchyme 24 to 48 hr after plating with or without horse serum. In embryos in which the blastocoelar matrix has been altered by injection with Sp14, there is inhibition of the release of secondary mesenchyme from the tip of the archenteron and in some embryos supernumerary skeletal elements are formed. It is proposed that Sp14 recognizes a component of the blastocoelar extracellular matrix that is required for the migration of mesenchyme.
Purification and Characterization of the Egg Jelly Macromolecules, Sialoglycoprotein and Fucose Sulfate Glycoconjugate, of the Sea Urchin Hemicentrotus Pulcherrimus
Authors: Shimizu Takeshi , Kinoh Hiroaki , Yamaguchi Masaaki , Suzuki Norio
Published: January 2005

A sialoglycoprotein and a fucose sulfate glycoconjugate (FSG) were purified from egg jelly of the sea urchin Hemicentrotus pulcherrimus. Sialoglycoprotein which consisted of sialic acid (90%, w/w) and protein (10%, w/w) did not cause induction of the acrosome reaction and sperm isoagglutination. FSG which contained one mol sulfate/mol fucose possessed 2.0 times protein to fucose by weight. The proteins in intact FSG were separated to two major (258 kDa and 237 kDa) and one minor (120 kDa) proteins by SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE) in the presence of 2‐mercaptoethanol (2‐ME) while the proteins could not be separated by HPLC in the presence of 0.1% SDS or SDS‐PAGE without 2‐ME. However, after carboxymethylation of FSG, two major (260 kDa and 240 kDa) proteins and two minor (140 kDa and 135 kDa) proteins were separated from the fucose sulfate moiety by HPLC in the presence of 0.1% SDS or SDS‐PAGE without 2‐ME. When FSG was first carboxymethylated with non‐radioactive iodoacetic acid and then reduced with 2‐ME and finally carboxymethylated with 14C‐iodoacetic acid, the most of radioactivity was detected in 140 kDa and 135 kDa proteins. Carboxymethylted‐FSG was less potent than intact FSG in induction of the acrosome reaction. Fucoidan, a fucose sulfate polymer, did not induce the acrosome reaction.
Vegetalization Induced by Procaine and Tetracaine in Sea Urchin Embryos
Authors: Fujiwara Akiko , Kataoka Kenji , Mikami‐Takei Kaori , Tazawa Eigoro , Yasumasu Ikuo
Published: January 2005

Vegetalization of sea urchin embryos was induced by the treatment with procaine and tetracaine, inhibitors of Ca2+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. The stages at which these compounds were effective to produce vegetalized embryos were almost the same to those for Li+to make embryos vegetalized. On the basis of known inhibitory effects of tetracaine, procaine and Li+on Ca2+mobilization, we postulate that Ca2+dependent reactions participate in the process of cell determination at these stages. Inhibitory effects of procaine, tetracaine and Li+on Ca2+dependent induction of fertilization membrane formation, found in the present study, indicate that these compounds block Ca2+mobilization in sea urchin eggs.
Hyaluronan Disappears Intercellularly and Appears at the Basement Membrane Region during Formation of Embryonic Epithelia
Authors: van Straaten Henny W. M. , Hooper Kevin C. , Bernfield Merton
Published: January 2005

The distribution of tissue hyaluronan has been assessed in the neuraxial region of 8.5 to 10.5 day mouse embryos using a fragment of bovine nasal cartilage proteoglycan that binds specifically to hyaluronan. Hyaluronan is abundant in all mesenchymal tissues, predominantly intercellularly, but markedly diminishes when mesenchymal cells organize into epithelia, as in the formation of somites. Hyaluronan reappears in abundance when epithelia (e.g. sclerotome) disperse into mesenchyme. Hyaluronan is present between cells of early epithelia (e.g. neural plate), but is lost during their subsequent development when it becomes abundant at their basement membrane regions. These results show for the first time changes in hyaluronan distribution during the development of embryonic epithelia. The hyaluronan distribution found is consistent with the functions proposed for hyaluronan in embryonic mesenchyme: intercellular hyaluronan would allow the epithelial cells to move and reduced hyaluronan would allow the cells to associate. The absence of intercellular hyaluronan in later epithelia would allow increased membrane contacts that lead to the formation of intercellular junctions. The restriction of hyaluronan to basement membrane regions in later epithelia further substantiates the suggestion that hyaluronan is a bona fide component of the basal lamina and that it is involved in maintaining epithelial morphology.
Explanted Fish Neural Tubes Give Rise to Differentiating Neural Crest Cells
Authors: Sadaghiani Bahram , Vielkind Juergen R.
Published: January 2005

Neural tubes were explanted from the trunk of various embryonic stages of three teleost fish, Xiphophorus maculatus (platyfish), X. helleri (swordtail), and Oryzias latipes (Japanese medaka) with the aim to obtain in vitro differentiating neural crest cells. Outgrowth of cells was observed immediately after attachment of the explants on dishes coated with fibronectin. The outgrowing cells stained with the HNK‐1 monoclonal antibody indicating that they were neural crest cells. Maximum cell outgrowth was obtained from explants of stage 9 of Xiphophorus and 19 of medaka, i.e., from stages characteristic of maximal neural crest cell segregation, and by the use of Leibovitz's (L‐15) medium supplemented with 20% FBS. In this medium cells survived for more than two weeks; M199 also gave satisfactory results but DMEM allowed only poor cell growth and survival. Neuronal cells could be observed in all cultures after 48 hr, in some medaka cultures these cells were mixed with pigment cells but homogeneous pigment cell cultures were also observed. This in vitro system will be invaluable for the study of the developmental potential of fish neural crest cells and the contributions of extrinsic factors in neural crest cell fate.
Dynamics of Mitotic Apparatus Formation and Tubulin Content during Oocyte Maturation in Starfish
Authors: Shirai Hiroko , Hosoya Natsumi , Sawada Tomoo , Nagahama Yoshitaka , Mohri Hideo
Published: January 2005

To follow the topo‐temporal behavior of structures containing tubulin and the change in tubulin content during oocyte maturation, starfish oocytes were extracted with a medium containing detergent so that morphological observation and biochemical analysis could be conducted on the same residual oocyte preparation simultaneously. Before 1‐methyladenine (1‐MeAde) stimulation, “pre‐meiotic asters” were observed on the germinal vesicle at the animal pole. 1‐MeAde caused the appearance of distinct asters at the position of the aster precursor. When germinal vesicle breakdown (GVBD) took place, chromosomes were condensed. Chromosome gathering was concurrent with a reduction in the size of nuclear matrix. The mitotic apparatus was first constructed parallel to the cortex and then changed its axis perpendicularly. Fluorescence of tubulin due to indirect immunofluorescence in the cytoplasm other than the mitotic apparatus decreased rapidly along the course of maturation at least up to the first metaphase. Despite these dynamic morphological change, the tubulin content in the whole oocyte and the residual structures, measured by SDS‐PAGE and immunostaining, did not show remarkable (statistically significant) changes through the course of maturation, although the content tended to decrease a little before the second polar body formation and to increase thereafter in the latter.
Pattern of Segregation of Mitochondria into Muscle Lineage Cells during Embryogenesis of the Ascidian Halocynthia roretzi
Authors: Fujiwara Shigeki , Satoh Noriyuki
Published: January 2005

Unequal partition of preexisting egg cytoplasmic components is one of possible cues to produce various types of cell in development. Segregation during ascidian embryogenesis of mitochondria into muscle lineage cells is a well‐known example of cytoplasmic localization and segregation. In this study, using a monoclonal antibody specific to mitochondria, we re‐examined changes in the distribution of mitochondria during oogenesis and embryogenesis of the ascidian, Halocynthia roretzi. The quantification method of the relative amount of the mitochondria‐specific antigen revealed differences in the amount of mitochondria contained in four blastomere‐pairs of an 8‐cell embryo; the primary muscle lineage B4.1‐pair contained about 40% of the total amount of mitochondria, while the secondary lineage b4.2‐ and A4.1‐pairs contained about 23% and 20% respectively, and non‐muscle lineage a 4.2‐pair about 17%. In addition, it was shown that the total amount of mitochondria‐specific antigen in the embryo remained constant throughout H. roretzi embryonic development. These results suggest that preferential segregation of preexisting mitochondria causes the characteristic distribution pattern of mitochondria within the embryo.
Effects of Inhibitors of Myosin Light Chain Kinase and Other Protein Kinases on the First Cell Division of Sea Urchin Eggs
Authors: Mabuchi Issei , Takano‐Ohmuro Hiromi
Published: January 2005

We investigated effects of protein kinase inhibitors on the first cell division in sea urchin eggs on the assumption that phosphorylation of myosin is requisite for the formation and/or the contraction of the contractile ring. ML‐7 or ML‐9, which inhibits myosin light chain kinase (MLCK), inhibited cytokinesis with a half maximal inhibition at 0.1–0.2 mM. The nuclear division was accomplished normally at 0.2–0.25 mM where the cytokinesis was completely blocked. Fluorescent staining of actin filaments with rhodamine‐labeled phalloidin revealed that the contractile ring was not formed in the cleavage‐inhibited eggs. H‐7 which inhibits cAMP‐dependent protein kinase, cGMP‐dependent protein kinase and protein kinase C arrested the process of the division at mid‐cleavage at 0.25–0.3 mM and at metaphase or anaphase at 0.5 mM. H‐8 and HA1004, which inhibit cAMP‐dependent and cGMP‐dependent protein kinases did not show significant effect at millimolar order. In the presence of micromolar concentrations of staurosporine which preferentially inhibits protein kinase C and MLCK small mitotic apparatuses were formed, in which chromosomes did not form the metaphase plate. The role of phosphorylation in the cell division is discussed.
Extreme Toxicity of Ethanol During Activation of Sea Urchin Eggs
Authors: Carroll Edward J. , Heath Robert L. , Izuishi Kohji
Published: January 2005

We have examined the effects of ethanol on early fertilization events and later development in the sea urchin Strongylocentrotus purpuratus. Eggs can still be fertilized in ethanol concentrations as high as 480 mM (2.0%); egg cytolysis was rapidly observed postinsemination in 50% of the cells at 220 mM ethanol. Yet, sperm motility was essentially normal in 250 mM ethanol; 940 mM ethanol was required to affect a 50% reduction. To determine the effect of ethanol on K+‐efflux from eggs induced by fertilization, we used parthenogenetic activation induced by the Ca2+‐ionophore A23187. Surprisingly, ethanol at only 0.2 mM caused an abnormal K+‐efflux, but only when added between 1 and 3 min after induction of activation. The K+‐efflux rates of unfertilized eggs were not influenced by up to 730 mM ethanol. Finally, normal embryonic development through the mesenchyme blastula stage was observed in egg suspensions which were treated for 30 min with ethanol concentrations as high as 240 mM, but washed with normal seawater prior to insemination. Normal plutei were obtained from cultures which were continuously cultured in 24 mM ethanol from 15 min postinsemination. We conclude that an extreme ethanol sensitivity of embryogenesis is apparent only during the cortical reaction.
Estradiol Secretion by Tadpole Ovaries Masculinized by Implanted Capsules of Cyanoketone
Authors: Chih‐Yun Hsu , Nai‐Wen Yu , Hung‐Hai Ku , Li‐Tze Chang , Hwan‐Wun Liu
Published: January 2005

Female tadpoles of Rana catesbeiana were laparotomized at metamorphic stages XI‐XIII and an empty capsule or one containing cyanoketone (CK), which is an inhibitor of Δ5‐3β‐hydroxysteroid dehydrogenase (Δ5‐3β‐HSD), was implanted intraperitoneally. Ovarian activity of Δ5‐3β‐HSD was examined histochemically 2 months later, estradiol‐17β (E2) secretion by the ovaries was measured by RIA 4 months later and histological changes of the ovaries were examined 6 months later. The Δ5‐3β‐HSD activity of the CK‐treated ovaries was much lower than that of controls. E2 secretion per froglet by CK‐treated ovaries was about one third that of controls (p<0.001). Histological examination showed various degrees of masculinization of the ovaries, about 28% of which were totally transformed into testis‐like structures.

As a result of suppressed Δ5‐3β‐HSD activity, dehydroepiandrosterone would have accumulated, resulting in deficient E2 secretion and, therefore, ovarian masculinization. In tadpoles, this effect does not depend on the pituitary, whereas interrenal hyperplasia and hyperactivity do, indicating that interrenal function is not essential for ovarian masculinization. From these findings and our previous results, we suggest that disturbance of steroidogenesis by CK in the ovaries results in their masculinization.
Origin of Germ Cells and Early Differentiation of Gonads in the Starfish, Asterina pectinifera
Authors: Inoue Chiemi , Shirai Hiroko
Published: January 2005

The origin of the germ cells and the development of the genital system in the annually spawning starfish, Asterina pectinifera, were studied by light and electron microscopy. Characteristic germ cells were first characterized in gonads after spawning: the gonia are larger than somatic cells, have large nuclei (with electron‐lucent nucleoplasm), and show mitochondrial aggregation associated with nuage (electron‐dense bodies). In young starfish without gonads similar cells were detected in the haemal sinus, where they were termed primordial germ cells (PGCs). Brachiolariae and metamorphosed juveniles had a cellular cluster in the coelomic epithelium, near the hydroporic canal. The cluster was comprised of cells endowed with the above‐mentioned characteristics of the germ cells. The germ cell counts indicated that PGCs migrate from the aboral haemal sinus near the hydroporic canal, through the haemal sinus to the gonads, where they settle, proliferate, and differentiate into gonia.
Activation of Proteasome in Sea Urchin Sperm by Lysophosphatidylinositol and by Sperm Lipids
Authors: Matsumura Kiyotaka , Aketa Kenji
Published: January 2005

Effects of phospholipids, their metabolites and endogenous sperm lipids on the chymotrypsin‐like activity of proteasome purified from sperm of the sea urchin, Strongylocentrotus intermedius were examined. Some lysophospholipids remarkably enhanced the activity. The most prominent activation was found in lysophosphatidylinositol (LPI) which enhanced about 12‐fold at 2.5 μg/ml. On the other hand, higher concentrations (above 250 μg/ml) were required for the enhancement of the activity by some saturated fatty acids and phospholipids. Lipids extracted from sperm also were effective in the enhancement, and those from sperm which were treated for 15 sec in egg jelly were more effective than those from untreated sperm. These results suggest that certain metabolites belonging to lysophospholipids are produced during the acrosome reaction and activate sperm proteasome. Also, they are not inconsistent with our view that the chymotrypsin‐like activity of sperm proteasome participates in the acrosome reaction (23, 24).
Polarization of the Surface Membrane and Cortical Layer of Sea Urchin Blastomeres, and its Inhibition by Cytochalasin B
Authors: Yazaki Ikuko
Published: January 2005

Sea‐urchin blastomeres have two domains of the plasma membrane which can be distinguished immunocytochemically. An egg‐surface antibody (anti‐ES), which binds to the membrane of the entire surface region of eggs before cleavage, binds to the membrane of the outer surface region of blastomeres after cleavage, but not to that of the cleavage furrow region or interblastomeric surface region.

The anti‐ES binding sites on the egg membrane were chased after cleavage by labeling the egg plasma membrane with FITC conjugated monovalent anti‐ES (FITC‐Fab anti‐ES) before the first cleavage, and then allowing the eggs to cleave. The surface fluorescence increased in intensity in the cleavage furrow region with progress of furrowing, but after completion of the furrowing, the fluorescence became uniform and finally decreased in the interblastomeric surface region.

The distributions of pigment granules and NBD‐phallacidin stainable microfilaments in the cortex after completion of furrowing were polarized in the same way as the anti‐ES binding area. As cytochalasin B completely inhibited the polarization in both the surface and cortical layer but colchicine did not, polarization of the anti‐ES binding area was concluded to be due to the post‐cleavage polarized distribution of submembranous microfilaments in the cortical layer.
Animalizing Effect of A23187 on Sea Urchin Embryos
Authors: Fujiwara Akiko , Yasumasu Ikuo
Published: January 2005

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. On the other hand, pulse treatment with A23187 in pre‐hatching period exerts stage‐specific effects on gut formation. Embryos, thus treated for 2 hr starting at stages between 3 and 5 hr after fertilization, produced quite small exoguts but those treated at stages between 7 and 8 hr formed well developed and long exoguts. In embryos treated at the other stages than above, guts or exoguts were almost the same in their size to those in normal ones. These effects of A23187 on morphogenesis were canceled by procaine, tetracaine and ruthenium red. Probably, artificial Ca2+signal induced by A23187 alters the determination of cell fates, programmed in pre‐hatching period.
Contribution of Ventral Blood Island (VBI)‐Derived Cells to Postembryonic Liver Erythropoiesis in Xenopus laevis
Authors: Ohinata Hiroshi , Enami Tomonori
Published: January 2005

The ventral blood islands (VBI) of Xenopus laevis embryos are known as the hemopoietic site where the initial erythropoiesis takes place at st. 28. To determine the site of postembryonic erythropoiesis, larvae were induced anemic by phenylhydrazine (PHZ) at st. 31 and 40, and the tissue distribution of regenerating erythrocytes was determined with an anti‐larval hemoglobin (LHb) monoclonal antibody. Three days after total anemia induction, the LHb+cells were detected first in the liver and the digestive tract, followed by the appearance of a few LHb+cells in the blood vessels. The lavae which had been hepatectomized and cardiectomized before the PHZ treatment showed a remarkable reduction in recovery of the LHb+cells. Induction of anemia in the chimeric individuals containing cytogenetically labelled VBI tissues demonstrated that the VBI‐derived cells contribute to the regenerating LHb+cells in all experimental individuals. These results suggest that the larval liver is the major site where the VBI‐derived hemopoietic cells reside and differentiate into erythrocytes.
Subcortical Rotation and Specification of the Dorsoventral Axis in Newt Eggs
Authors: Fujisue Megumi , Sakai Masao , Yamanat Kiyotaka
Published: January 2005

The specification of the dorsoventral axis in naturally polyspermic eggs of the Japanese newt, Cynops pyrrhogaster, was first examined by studies on the spatial relationship between the dorsal midline of the future body plan and the sperm entrance points (SEPs1). On local insemination, the dorsal blastopore lip was usually found to be formed opposite the SEPs, as in anuran monospermic eggs. Next the movements of the subcortical layer and the cortex were analyzed. “Subcortical rotation” was observed, similar to that of Xenopus laevis eggs with respect to its timing and extent, and its direction was shown to predict the embryonic axis of the eggs. Thus, the dorsoventral axis was concluded to be determined by essentially the same mechanism in the newt as in Xenopus.

Owing to their large size and long first cell cycle, newt eggs appear to be suitable material for study of subcortical rotation, but their behavior is unique in that subcortical rotation occurs in only the vegetal hemisphere so that the subcortical layer stretches in the future dorsal side. Studies on the movement of Nile blue spots suggested that the cytoplasm under the cortex in newt eggs consists of two layers.