bluuebunny/crossref_metadata_2025 huggingface dataset:
Signal Sequences Associated with Fibroin Gene Expression are Identical in Fibroin‐Producer and ‐Nonproducer Tissues
Authors: SUZUKI YOSHIAKI , ADACHI SATOKO
Published: January 2005

DNA sequencing has been carried out for the region of nucleotide position −649 to +1261 of the fibroin DNA originally cloned from Bombyx mori pupae in which the fibroin gene is not expressed (nonproducer tissues). This sequence was compared with that from the posterior silk gland cells, where the gene is expressed (producer tissue), of a different strain. Both sequences are indentical in the regions of −297 to +333 and +746 to +1261, leaving some base changes in the remaining regions. The regions of the identical sequences contain important signals for the fibroin gene expression; −29 to +6 for promoter function, −238 to −116 and −73 to −53 for signals for transcription enhancement, and around +66 and +1037 for the exon/intron boundaries. Template activities of the fibroin DNAs from the producer and the nonproducer were indistinguishable when assayed in a cell‐free transcription system prepared from the silk glands. The result suggests that the base changes found in the regions of −649 to −298 and +334 to +745 of the fibroin DNAs from two strains are neutral mutations that do not affect the transcription activities of the genes. Thus, DNA rearrangements or somatic mutations are not likely to contribute to the tissue‐specific expression of the fibroin gene.
Distribution of the Germinal Vesicle Material during Progesterone‐Induced Oocyte Maturation in Xenopus and in Cynops
Authors: IMOH HIROSHI , MIYAZAKI YASUYO
Published: January 2005

The distribution of the germinal vesicle material in the oocyte during progesterone‐induced maturation was studied in Xenopus and in Cynops. In both species, two distinctive masses of yolkfree cytoplasm appear in specific areas of the oocyte and at definite stages of maturation. One, the primary cytoplasmic mass, is formed at the basal side of the germinal vesicle during early maturation and is very RNA‐rich. In Xenopus, a large part of the primary cytoplasmic mass persists as a mass during maturation and ends up as a thin disk at the boundary between the animal and the vegetal hemisphere in the mature oocyte. In Cynops, a rod‐like primary cytoplasmic mass extends near to the equatorial zone and becomes indistinct in the mature oocyte. The other, the secondary cytoplasmic mass, is formed at or prior to germinal vesicle breakdown in areas around the germinal vesicle and is also RNA‐rich. The secondary cytoplasmic mass is dispersed and constitutes the RNA‐rich animal hemisphere cytoplasm in the mature oocyte. Observed results suggest that the primary and the secondary cytoplasmic mass contain different germinal vesicle materials.
Immunoelectron Microscopic Demonstration of the Pre‐fertilization Layer in Xenopus eggs
Authors: YOSHIZAKI NORIO
Published: January 2005

The formation of the fertilization (F) layer in Xenopus laevis was studied by immunoelectron microscopic protein A‐gold technique, employing an antiserum specific to the secretory granules in the bottom cells at the posterior portion of the pars recta of the oviduct (pars recta 2). In unfertilized eggs, the gold particles revealing the antigenic sites were specifically present over the pre‐fertilization (PF) layer located between the vitelline coat and the jelly layer. Upon transformation of the PF layer into the F layer, the labeling became confined to the region on the outer surface of the F layer, suggesting the loss of antigenic site in the inner part of newly‐formed F layer. These results provide an ultrastructural basis for the view that the PF layer is supplied by specific cells in the oviducal pars recta 2.
Appearance of Extrachromosomal Circular DNA in Lymphocytes from Developing Chick Bursa
Authors: TODA MASAAKI , YAMAGISHI HIDEO
Published: January 2005

Lymphocytes were purified from the developing bursa of Fabricius of chick embryos and chickens and pressed with a mica sheet. Then the extruded DNA complexes were adsorbed to the mica and processed for electron microscopy. Circular DNA complexes were found at 40 to 60 copies in all bursal lymphocyte except 12‐day‐old embryonic bursas, which contained 150 copies. Circular DNA complexes of more than 1 μm (larger circular DNA) appeared in 12‐day‐old embryonic bursas at more than 20 copies per cell, and subsequently decreased in number to less than 10 copies per cell. These changes in the size distribution and copy number of circular DNA coincided with lymphocyte differentiation of the hemopoietic precursor cells in the bursa after seeding.
Neuroendocrine Regulation of the Development of Seasonal Forms of the Asian Comma Butterfly, Polygonia c‐aureum L
Authors: ENDO KATSUHIKO
Published: January 2005

In the butterfly, Polygonia c‐aureum, development of seasonal forms controlled by the photoperiod and temperature was shown to involve a neuroendocrine system of the brain‐corpus cardiacum‐corpus allatum complex.

For analysis of the neuroendocrine system concerned, the innervation of the complex was investigated first by cobalt chloride perfusion staining and then by severance of axons, ablation of the candidate cells, injection of a homogenate of these cells and transplantation of corpora cardiaca using pupae programmed to be either summer‐form or autumn‐form adults.

The results suggested that medial nerve cells produce what is called material producing the summer form.

The seasonal forms of the Asian comma butterfly, Polygonia c‐aureum L., summer and autumn forms (Fig. la, b), are determined by the photoperiod and the temperature during the larval period (1–3). Previous studies have given the following results on the physiological mechanism involved in the effect of environmental factors in inducing these seasonal forms. First, the mechanism involves neurosecretory cells located somewhere in the brain (2). Second, the nervous connections between the brain and the corpus cardiacum (NCC I+II (4)) and between the right and left brain lobes are indispensable for the effect (2, 5–7).

The present study consisted of two series of experiments. One was designed to demonstrate morphologically the axonal connection of the corpus cardiacum with the corpus allatum in this butterfly, like that shown in several other insects (8–13). The other series was designed to locate the neurosecretory cells producing material related to the seasonal form and to see if this material is also present in the corpus cardiacum.
Mesoblastic Cells in the Early Phase of Primitive Streak Formation in Chick Embryos. Observations by Scanning Electron Microscopy in ovo and time‐Lapse Cinematography in vitro.
Authors: TAKEUCHI SHIGEO
Published: January 2005

During primitive streak formation in the chick embryo, mesoblastic cells were observed by SEM after removal of the hypoblast layer. Before the primitive streak began to develop, numbers of bleb cells and bleb‐like protrusions were seen on the ventral surface of the epiblast. From optical observation on the process of change of epiblastic cells into bleb cells in vitro, it was concluded that cells that had elongated became bleb cells when they emerged from the epiblast. Cell behavior during primitive streak formation is discussed on the basis of these findings.
Effects of Succinyl Concanavalin A and Tunicamycin on F9 Embryonal Carcinoma Cells: Inhibition of Cell Spreading
Authors: MURAMATSU HISAKO , MURAMATSU TAKASHI
Published: January 2005

Succinyl concanavalin A at a concentration of 50 μg/ml inhibited spreading of F9 embryonal carcinoma cells, while at this concentration it inhibited cell growth only partially. Thus, in the presence of succinyl concanavalin A, F9 cells grew as rounded cell aggregates that sometimes became detached from the substratum. Tunicamycin at a concentration of 1 μg/ml had a similar effect on F9 cells. These results suggest that a mannosyl glycoprotein(s) is involved in cell‐substratum interaction of the cells. Furthermore, tunicamycin partially inhibited the biosynthesis of embryoglycan, the large glycoprotein‐bound carbohydrates of early embryonic cells.
Stress Protein and Crystallin Synthesis During Heat Shock and Transdifferentiation of Embryonic Chick Neural Retina Cells
Authors: CARR A. , de POMERAI D.I.
Published: January 2005

Embryonic chick neural retina responds to heat shock by the synthesis of “stress” polypeptides with molecular weights of 85 and 70 kd. Both stress proteins are synthesised from newly‐transcribed messenger RNA. Sodium arsenite induces an additional stress protein of MW 25 kd. The heat shock response does not change during culture and subsequent transdifferentiation, and crystallin synthesis is not coinducible with the heat‐shock proteins. We have also examined the pattern of protein synthesis at various stages of culture in both monolayer and aggregate systems; although changes in the protein synthetic profine are evident, there is no stress protein induction above basal levels at any time. Whilst mammalian α crystallin (B2 chain) exhibits considerable homology to four small Drosophila heat‐shock proteins, no significant antigenic similarity is apparent between δ crystallin and the major avian heat shock proteins. Thus during transdifferentiation, (a) the crystallin proteins do not behave in a manner analogous to stress proteins; moreover (b) crystallin production is not mediated by stress proteins resulting from a culture‐induced stress response.
Communications of Oocyte‐Granulosa Cells in the Chum Salmon Ovary Detected by Transmission Electron Microscopy
Authors: KOBAYASHI WATARU
Published: January 2005

The communications between oocytes and granulosa cells in the ovary of the chum salmon, Oncorhynchus keta, were examined in follicles in which the oocytes were at the stage of germinal vesicle migration. Microvilli were seen extending through the radial canals of the egg envelope and terminating in either the subfollicular space or at the surface of granulosa cells. Cytoplasmic processes extending from granulosa cells toward oocytes were also observed; most of the processes appeared to end in the radial canals, but some passed through the canals and terminated as bulbs in slight depressions or indentations of the oolemma. Various types of junctional structures that participated in intimate association between these cells were seen. The granulosa cells were found to be firmly fixed on the surface of the intrafollicular oocyte by means of desmosomes or desmosome‐like junctions. It is speculated that intrafollicular oocytes communicate with surrounding granulosa cells directly through gap junctions and indirectly by endocytosis of material released from the granulosa cells.
Effect of Cysteine and Its Derivatives on 1‐Methyladenine Production by Starfish Follicle Cells
Authors: MITA MASATOSHI
Published: January 2005

Spawning of the gametes in the starfish, Asterina pectinifera, induced by a gonad‐stimulating substance (GSS) was inhibited by cysteine. This inhibitory effect of cysteine was due to the reduction of GSS activity and the inhibition of 1‐methyadenine (1‐MeAde) production in follicle cells. Homocysteine and cysteamine also reduced GSS activity, but cystamine had no effect on the activity. This suggests that the loss of GSS activity is related to a SH group. Furthermore, the effect of cysteine on 1‐MeAde production was investigated using isolated follicle cells. The half maximum decrease of the GSS‐dependent 1‐MeAde production in follicle cells was obtained with the concentration of 2.0 mM L‐cysteine. A similar effect was observed with D‐cysteine. Homo cysteine and cysteamine also reduced the GSS‐dependent 1‐MeAde production. But methionine, serine, glutamic acid and aspartic acid did not affect the 1‐MeAde production of follicle cells. In addition, cystamine also inhibited the GSS‐dependent 1‐MeAde production. These suggest that both a SH group and a SS bond of these compounds are effective on the inhibition of the GSS‐dependent 1‐MeAde biosynthesis. Cysteine and homocysteine also inhibited concanavalin A (Con A).induced 1‐MeAde production of follicle cells, but they had no effect on binding of Con A to the surface of follicle cells.
Mode of Action of some Stimulants of the Hatching Enzyme Secretion in Fish Embryos*
Authors: IUCHI ICHIRO , HAMAZAKI TATSUO , YAMAGAMI KENJIRO
Published: January 2005

Mode of action of two stimulants of the hatching enzyme secretion, electric current (AC) and potassium cyanide, was analyzed by applying them to Medaka embryos in the presence or absence of suppressants of nervous system‐mediated secretion, tetrodotoxin or MS–222. Electric current (AC) stimulated the secretion of the hatching gland of the embryos that had been treated with these suppressants, while potassium cyanide did not. These results strongly suggest that electric current acts as a stimulant of hatching enzyme secretion directly on the gland cell itself, while potassium cyanide stimulates the secretion indirectly, probably through nervous system of the embryo.

In the present experiments, it was also shown that Ca2+ and ionophore, X‐537A, when applied directly to the hatching gland extracellularly, induced a marked secretion‐associated morphological change of the gland cells instantaneously. However, it was found that chum salmon prolactin did not induce the secretion‐associated morphological changes in the hatching gland cells when it was applied directly to the gland cells in situ or indirectly through embryonic circulation.
Histological Detection of Chicken δ‐Crystallin DNA Sequences Introduced into Mouse Teratocarcinoma Cell Lines
Authors: TAKAHASHI YOSHIKO , OKADA T. S. , KONDOH HISATO
Published: January 2005

In situ hybridization techniques to detect specific DNA sequences in histological sections were developed for the purpose of analyzing experimental chimeras produced by combination of mouse teratocarcinoma (TCC) cells stably carrying chicken δ‐crystallin DNA sequences and normal mouse embryos. Various hybridization conditions for detection of exogenous DNA sequences were compared in samples of solid tumors of TCC lines. Of the conditions examined, denaturation of DNA in alkali and hybridization at 68°C in 6x SSC in the presence of dextran sulphate was the best for detecting δ‐crystallin DNA sequences. With 3H‐labelled probe under these conditions, virtually all nuclei containing more than 100 copies of chicken δ‐crystallin sequences were labelled sufficiently to be distinguishable from nuclei without chicken sequences. This technique could be applied to other experimental chimeras in which specific DNA sequences can be used as markers of certain cell lineages.
Involvement of Glutathione in the Differentiation of the Slime Mold Physarum polycephalum
Authors: ALLEN R. G. , FARMER K. J. , TOY P. L. , NEWTON R. K. , SOHAL R. S. , NATIONS C.
Published: January 2005

The effects of experimentally‐altered glutathione concentration on differentiation of the slime mold, Physarum polycephalum were examined. Spherulation was induced by transfer of Physarum from growth medium to a salts‐only starvation medium. As differentiation proceeded, superoxide dismutase (SOD) activity in control cultures increased by as much as 21‐fold. This increase in SOD activity paralleled the rate of differentiation. Glutathione (GSH) concentration decreased during differentiation by more than 80% in all cultures, regardless of the initial concentration. The rate of differentiation was inversely related to the initial GSH concentration and directly proportional to the SOD activity. These observations suggest that a free radical mechanism may be involved in the differentiation of Physarum microplasmodia into spherules.
Laminin Fibrils in Newt Gastrulae Visualized by the Immunofluorescent Staining
Authors: NAKATSUJI NORIO , HASHIMOTO KOICHIRO , HAYASHI MASAO
Published: January 2005

An anastomosing network of extracellular fibrils on the inner surface of the ectoderm layer of amphibian gastrulae has been shown to provide an adequate substratum for attachment and migration by the mesodermal cells. These fibrils contain fibronectin as shown by immunostaining at the light and electron microscope levels. Now we report the presence of laminin, another cell adhesion glycoprotein, as a fibrillar network on the inner surface of the ectoderm layer in gastrulae of the Japanese newt (Cynops pyrrhogaster), but its absence on the blastula ectoderm layer, by the immunofluorescent staining using an antiserum specific for mouse laminin. The same antiserum was shown to stain basement membranes of adult newt organs as expected.
Properties of Intracellular Hatching enzyme in the Embryos of the Sea Urchin, Hemicentrotus pulcherrimus
Authors: NAKATSUKA MITSUNOBU
Published: January 2005

The intracellular hatching enzyme was confirmed to be particulate‐bound in the sea urchin, Hemicentrotus pulcherrimus. The enzyme was solubilized most effectively by sonication in buffer containing 12.5 mM CaCl2, and 0.5 M KCl. The intracellular hatching enzyme is suggested to be activated by an antipain‐ or elastatinal‐susceptible protease(s) on its solubilization. Since the intracellular hatching enzyme solubilized in the absence of protease inhibitors was inhibited by phenylmethylsulfonyl fluoride (PMSF) and chymostatin, the active hatching enzyme is concluded to be a chymostatin‐sensitive serine protease. The enzyme required CaCl2, and KCl or NaCl for both stability and activity. The preference of the enzyme of anions as sodium salts was as follows: Cl− > NO3− > I− > SCN−. The apparent molecular weights of the intracellular hatching enzyme (IHE) and the hatching enzyme secreted from the blastula with or without the fertilization envelope (SHE or dSHE) were estimated as 89,000, 135,000, 80,000, respectively. On incubations with isolated fertilization envelopes as an enzyme substrate, the apparent molecular weights of dSHE and IHE increased to 128,000 and 105,000, respectively.
Partial Purification from Chick Embryos of a Factor which Promotes Myoblast Proliferation and Delays Fusion
Authors: ICHIO  , OZAWA EIJIRO
Published: January 2005

Chick embryo extract (EE) contained an activity which promoted myoblast proliferation and delayed fusion. Various tissue extracts prepared from 12‐day embryos and adult chicken also showed the activity. We partially purified this active substance from 12‐day embryos, following procedures which included extraction at pH 3.5, CM‐Sephadex C–50 ion exchange and Sephadex G‐75 gel filtration. Judging from the dose‐response analyses, the factor was purified by some hundred‐fold when EE was used as the starting material. The activity was associated with a macro‐molecular substance (MW > 300K daltons) at first, but the apparent molecular weight of the active substance was estimated to be between 16 and 20K daltons at the final step of the preparation. It promoted myoblast proliferation and delayed myotube formation, and was active for both avian and rat myoblasts.

Since bovine pituitary gland fibroblast growth factor (FGF) showed the same activity, the factor may be FGF‐related.
Mosaic Expression of the Reeler and Normal Phenotypes in the Cerebral Cortex in Reeler‐Normal Chimeras at a Late Embryonic Stage
Authors: MIKOSHIBA KATSUHIKO , YOKOYAMA MINESUKE , NISHIMURA YOZO , KATSUKI MOTOYA , NOMURA TATSUJI , TSUKADA YASUZO
Published: January 2005

The cerebral cortex of reeler‐normal chimera embryos was studied by hematoxylin‐eosin staining and fractographic scanning electron microscopy in comparison with the cortices of normal and reeler mutant mice. The cerebral cortex of normal mice had a plexiform layer, which was composed of a fine meshwork of matrix cell processes. Spindle shaped neuroblasts formed a radial lining columnar structure, which was formed by attachment of migrating neuroblasts to the radial bundles. The cerebral cortex of reeler mutants did not show a plexiform layer and the cells were round with no radially columnar structures, and no radial bundles. In reeler‐normal chimera embryos, the thickness of the plexiform layer varied in different parts of the cerebral cortex. In parts where the plexiform layer was present, neuroblasts were spindle‐shaped and had a radially oriented columnar structure (normal type). But where the plexiform layer was absent, the neuroblasts were round with a radial architecture (reeler type). Intermediates between the reeler and control types were also observed. Since mosaic expression of the two phenotypes, was observed in chimeras, the reeler abnormality is apparently not caused by humoral factors. The possible mechanism of cell migration is discussed.
Requirement of Extracellular Calcium Ions for the Early Fertilization Events in the Medaka Egg
Authors: IWAMATSU T. , OHTA T. , OSHIMA E. , SUGIURA T.
Published: January 2005

The requirement for calcium and the change in calcium content in eggs of Oryzias iatipes during the cortical reaction and sperm penetration were examined. Naked eggs failed to exhibit the cortical reaction upon insemination under Ca Mg‐free conditions. These eggs exhibited the cortical reaction by reinsemination in the presence of extracellular Ca2+. The effect of extracellular Ca2+ on sperm penetration could be replaced by one of several divalent cations in the external medium. Unlike the cortical reaction, sperm penetration failed to be induced by microinjection to increase intracellular Ca2+. Verapamil significantly reduced the action of extracellular Ca2+ or Ba2+ of divalent cations examined in fertilization, while TEA and TTX had no effect on fertilization in the presence of these cations. No 45Ca uptake into the egg proper was recognized before completion of the cortical reaction. These observations suggest that extracellular divalent cations are indispensable for sperm stimulation of the egg and its penetration into the egg, for which an influx of Ca2+ from the external medium is not required.
Comparative Study on the Ability of Various Teratocarcinomas to Form Chimeric Mouse Embryos
Authors: HANAOKA KAZUNORI , KATO YOSHIHIRO , NOGUCHI TAKEHIKO
Published: January 2005

Various embryonal carcinoma cells of different origins were compared as to the ability to form chimeric blastocysts by means of aggregating with normal 8‐cell stage mouse embryos. The teratocarcinoma lines examined were OTT6050 and five newly established ones including a spontaneous testicular teratocarcinoma STT‐2. The present results have revealed that distinct differences existed in the ability of colonizing blastocysts among teratocarcinomas and also among embryonal carcinoma cell lines.

Since STT‐2 stem cells were found to be incorporated into blastocysts most efficiently, further development of the blastocysts were examined in utero. It was found that STT‐2 stem cells could be incorporated into the fetuses up to the 7‐to 28‐somite stages. This is the first case to demonstrate that testicular teratocarcinoma cells with the male germ cell origin have the developmental potency to participate into mouse embryogenesis.
Close Correlation of the Periodicities of the Cleavage Cycle and Cytoplasmic Cycle in Early Newt Embryos
Authors: SHINAGAWA ATSUNORI
Published: January 2005

The correlation between autonomous cyclic activity of the cytoplasm (cytoplasmic cycle) and the cleavage cycle was studied by using animal and vegetal half fragments of newt eggs formed by bisecting the uncleaved eggs after those eggs had been rotated through 90° off the vertical axis so as to alter the allocations of the cytoplasmic content in the two halves. When the bisection was made shortly after the rotation (Early Bisection), the resulting vegetal fragment showed 1.5 times longer intervals in the cytoplasmic cycle than its animal partner when cleavage was prevented by injection of colchicine, while when the bisection was made 30 min after the rotation (Late Bisection), the resulting pair of halves displayed equal intervals in the cytoplasmic cycle. The intervals of cell division of vegetal halves formed by the two kinds of bisection in the absence of colchicine were then examined. In these conditions, the vegetal half formed by Early Bisection still showed 1.5 times longer intervals in the cleavage cycle than its counterpart, and the half formed by Late Bisection displayed the same intervals in the cleavage cycle as its animal partner.
Microtubules During Blastoderm Formation in the Egg of the Silkworm, Bombyx mori L.
Authors: TAKESUE SACHIKO , KEINO HIROOMI
Published: January 2005

Microtubules in the silkworm egg, Bombyx mori, were observed by electron microscopy, in order to investigate the relationship between cytoskeletal organelles and the migration of energids, the cleavage nuclei accompanied by the associated cytoplasm, near the egg surface or during blastoderm formation. Numerous microtubules were observed in the associated cytoplasm of an energid even in the interphase of mitosis.

At about 8.5 hr after oviposition, when many energids had already cleft and distributed near the peripheral yolk granule region, long microtubules distributed radially from the perinuclear region to the periphery in the associated cytoplasm. When an energid was protruding, the microtubules above the nucleus distributed at a more acute angle than those under the nucleus. When a blastoderm cell had just been formed, the microtubules were observed only under the nucleus.

Colchicine, an inhibitor of microtubules, stopped the migration of energids and inhibited the formation of blastoderm cells even after many energids had already distributed at the peripheral yolk granule region. The relationship between the microtubules and the migration of energids near the egg surface or during blastoderm formation was discussed.
Properties of the Cortical Granule Lectin Isolated from Xenopus Eggs
Authors: YOSHIZAKI NORIO
Published: January 2005

The cortical granule lectin that participates in forming the fertilization layer in Xenopus laevis was isolated and partially characterized. About 400 μg of lectin was purified from 5 mg of crude exudate by chromatography on Sepharose 6B and Concanavalin A‐conjugated Sepharose 4B columns and electrophoretic separation on polyacrylamide gel. The lectin has a molecular weight of 550 Kd and is composed of two species of polypeptides (46 Kd and 42 Kd). The lectin gave a single precipitin line against material in the prefertilization layer in an agglutination reaction on an agarose plate. The agglutination reaction involved D‐galactoside residues and metal ions. The lectin formed an electron‐dense layer on the outer surface of the vitelline coat of oviducal eggs covered with the prefertilization layer, but on the outer surface of jelly layer, not on that of the vitelline coat of jellied eggs. Although the jelly could be agglutinated by the lectin, the possibility that the jelly layer is the site of fertilization layer formation was excluded by the fact that the prefertilization layer is the first to meet the cortical granule lectin during normal fertilization.
Accumulation and Distribution of Extracellular Matrix as Revealed by Scanning Electron Microscopy in Freeze‐Dried Newt Embryos Before and During Gastrulation
Authors: KOMAZAKI SHINJI
Published: January 2005

A scanning electron‐microscopic study was carried out on the extracellular matrices (ECMs) in freeze‐dried newt embryos from the cleavage to the gastrula stage. The results revealed the appearance, accumulation and distribution of two types of ECMs, a fibrillar ECM in the blastocoel and an amorphous ECM on the inner surface of the blastocoelic wall (BW). The fibrillar ECM first appeared in the blastocoel at the cleavage stage and increased notably in quantity at the blastula and gastrula stages. On the other hand, the amorphous ECM was initially detected on the inner surface of the BW at the beginning of gastrulation and it increased in quantity during gastrulation. With the progress of archenteron invagination, the amorphous ECM was found to be deposited in the space between the BW and migrating cells.
Development of the Nervous System of Sea Urchin Embryos: Formation of Ciliary Bands and the Appearance of Two Types of Ectoneural Cells in the Pluteus
Authors: NAKAJIMA YOKO
Published: January 2005

An ultrastructural study of the larval integument of the sea urchin, Hemicentrotus pulcherrimus, was conducted with special emphasis on the development of the nervous system in relation to the formation of ciliary bands. In the integument of 4‐armed pluteus larvae, cells associated with the ciliary band, which have 200 nm‐thick projections at their apices, and cells in the squamous epithelium, which have a cilium and long, fine radiating processes in the apical region, were observed. Both cell types have axons at their basal ends that form nerve bundles beneath the ciliary bands, where the axons make contact with ectodermal effector cells with motile cilia. The cilia and other apical projections of these ectoneural cells run parallel to the surface of the cells, and are under the hyaline layer. The axoneme of the cilium has a typical “9 + 2” microtubular arrangement, but generally has no dynein arms. These ectoneural cells are more frequent on the oral surface than on the antioral surface.
Development of Serotonergic Neurons in Embryos of the Sea Urchin, Strongylocentrotus purpuratus
Authors: BISGROVE BRENT W. , BURKE ROBERT D.
Published: January 2005

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.
Cell Fusion and Its Application to Genetic Analysis of Development in Dictyostelium discoideum
Authors: SUGIURA KATSUMI , ISHIKAWA AKIRA
Published: January 2005

Dictyostelium cells were fused by a modification of the polyethylene glycol method of Kuhn and Parish. In the modified method Tricine buffer and Concanavalin A were used in place of Ca++. The efficiency of genetic complementation through cell fusion was about 10 times higer by the modified method than by the original method with glycine buffer and Ca++. Complementation between developmental mutants without any selectable growth character was clearly detected by the modified system, at efficiencies of about 1 in 10–20 surviving cells.
Segregation of Primordial Germ Cells from the Endoderm of the Early Xenopus Embryo by Applying Fibronectin, an Extracellular Matrix, in vitro
Authors: CATHALOT BRIGITTE , BRUSTIS JEAN‐JACQUES
Published: January 2005

Two in vitro culture systems were used in order to identify Xenopus primordial germ cells in the early stages of their migration through the endodermal mass. For this study, whole endodermal mass and dissociated endodermal cells were cultured on fibronectin substrates. In the early stages of the explantation, each system used permits the spotting of particular cells among somatic endodermal cells. These cells exhibit an elongated shape, they present random locomotion and they move on the substrate by elongation‐contraction. Ultrastructural studies of these cells confirm their germinal quality.
Induction of Fertilization Membrane Formation and Cyanide‐insensitive Respiration in Sea Urchin Eggs by the Treatment with Dimethylsulfoxide Followed by an Incubation in an Ice Bath (dimethylsulfoxide/sea urchin egg/fertilization membrane/respiration/verapamil)
Authors: FUJIWARA AKIKO , ASAMI KOUICHI , YASUMASU IKUO
Published: January 2005

In unfertilized eggs of the sea urchin Hemicentrotus pulcherrimus, fertilization membrane formation was induced by an incubation with dimethylsulfoxide (DMSO) for several min at 20°c followed by another incubation in an ice bath. The number of eggs with fertilization membrane, thus obtained, increased in relation to the concentration of DMSO between 1 and 3% (v/v) and was higher than 75% at concentrations above 3%. Fertilization membrane formation by this treatment occurred in Ca2+ free‐ or Ca2+, Mg2+ free‐ artificial sea water containing EGTA (50 mM) and was inhibited by verapamil. In the presence of DMSO, the membrane formation was also induced by 2, 4‐dinitrophenol or cyanide in considerable number of eggs at 20°c. Eggs remained fertilizable, even when they were kept with DMSO for 1 hr at 20°c. DMSO slightly enhanced respiratory rate in unfertilized eggs and substantially reduced it in fertilized eggs. DMSO‐treated eggs exhibited cyanide‐insensitive respiratory burst following chilling in an ice bath or by adding DNP or cyanide, in a similar manner to the burst induced by sperm.
Initiation of Sperm Motility Induced by Cyclic AMP in Hamster and Boar
Authors: ISHIDA KATSUMI , OKUNO MAKOTO , MORISAWA SACHIKO , MOHRI TOSHIKO , MOHRI HIDEO , WAKU MASAYOSHI , MORISAWA MASAAKI
Published: January 2005

When the plasma membrane of hamster and boar spermatozoa was extraced by treatment with Triton X‐100 and the demembranated spermatozoa were transferred to a reactivating medium containing only ATP, axonemes were initially immotile, and then gradually became motile. Under these experimental conditions, the cAMP content in the reactivating medium increased soon. This suggests that cAMP is synthesized from ATP by adenylate cyclase involved in incompletely removed or solubilized residual sperm membrane and that the autosynthesized cAMP causes the delay in motility initiation. This delayed initiation of motility did not occur when phosphodiesterase was added to the reactivating medium and the phosphodiesterase‐dependent quiescent sperm became motile instantaneously at any time when excess cAMP was supplemented. Furthermore, demembranated sperm which were diluted in the reactivating medium containing ATP and cAMP, immediately became motile. cAMP levels in the cell increased during the initiation of sperm motility in both species. These results suggest that cAMP is the real factor indispensable for the initiation of sperm motility at ejaculation in mammals.
Pattern Regulation of the Leg Disc of the Fleshfly, Sarcophaga peregeina
Authors: KAMAI HIROYUKI , OHTAKI TETSUYA
Published: January 2005

A fate map of the hind leg disc of Sarcophaga peregrina was constructed by examining the adult structures of implanted disc fragments. The locations of presumptive adult structures in the disc were similar to those of fore leg disc of Drosophila and Sarcophaga ruficornis. However, the concentric borderlines of the segments could not be ascertained in the present case.

Pattern regulation of disc fragments was studied by culturing them either in adult females for several days or for 3 days in mature larvae placed on wet condition. Cultured disc fragments regenerated or duplicated as in Drosophila, with some exceptions. For instance, the region with a high density of positional values, the upper medial quarter, of the fore leg disc of Drosophila was not found. A characteristic difference in the rate of regeneration or duplication was observed in the implanted fragments, when cultured in larvae or adult hosts. This variable pattern regulation in larval and adult hosts could be due to different compositions of the hemolymph in which would healing of the implanted disc fragments takes place.
Immunological Relationships among Embryonic and Adult Chicken Pepsinogens: A Study with Monoclonal and Polyclonal Antibodies
Authors: YASUGI SADAO , HAYASHI KENSUKE , TAKIGUCHI KEIKO , MIZUNO TAKEO , MOCHII MAKOTO , KODAMA RYUJI , AGATA KIYOKAZU , EGUCHI GORO
Published: January 2005

To investigate the immunological relationships of pepsinogen isozymes present in embryonic and adult chicken proventriculi, we obtained monoclonal and polyclonal antibodies to these pepsinogens. Zymograms and immunoblots demonstrated that monoclonal antibody Y37 reacted with both embryonic and slow‐migrating adult pepsinogens, while polyclonal antibodies against embryonic pepsinogen and fast‐migrating adult pepsinogen were specific for these respective antigens. Shift from embryonic to adult‐type pepsinogen occurred at about the time of hatching and the localizations of embryonic and adult‐type pepsinogens within proventricular gland cells were found to differ by the indirect immunofluorescence method. Results with these antibodies revealed the immunological relations of these pepsinogens and the unique properties of embryonic chicken pepsinogen.
Correlation between the Shortened Period of Cell Pairing during Genomic Exclusion and the Block in Posttransfer Nuclear Development in Tetrahymena thermophia.
Authors: GAERTIG JACEK , KACZANOWSKI ANDRZEJ
Published: January 2005

The duration of pairing in crosses of the micronuclearly defective strain A*V and the amicronuclear strain BI3840 of Tetrahymena thermophila is shorter than that in normal conjugation. In controls, pairing takes about 620 min at 30°C and 60 min more at 28°C. In contrast pairing of crosses of the A*strain took 470 min at 30°C and 490 min at 28°C, and that of crosses of BI3840 strain took 440 min at 30°C. The course of nuclear development in the tester strain crossed with the A*strain was similar to that in the control until the 3rd prezygotic division. Unilateral transfer of the pronuclei occurred later than on reciprocal transfer in control crosses; posttransfer divisions in hemikarya was completely blocked in over 90% of the cases examined. Defective cell contact in crosses of the A*V strain and an amicronuclear BI3840 strain may be correlated with block in nuclear division and in macronuclear development, since unfertilized cells from triplet conjugation remain haploid and develop normally.
Inhibition of Cell Surface Binding of Fibronectin and Fibronectin‐Promoted Cell Migration by Synthetic Peptides in Sea Urchin Primary Mesenchyme Cells In Vitro
Authors: KATOW HIDEKI
Published: January 2005

The role of a site of fibronectin molecule in the cell binding and cell migration was examined in vitro using sea urchin primary mesenchyme cells and synthetic peptides that contain a particular amino acid sequence, Arg‐Gly‐Asp‐Ser.

The binding of fibronectin to the cell surface was inhibited by addition of larger synthetic peptides, Gly‐Arg‐Gly‐Asp‐Ser‐Pro‐Cys (HP) or Arg‐Gly‐Asp‐Ser‐Pro‐Ala‐Ser‐Ser‐Lys‐Pro (DP), but not by a smaller synthetic peptide Gly‐Arg‐Gly‐Asp‐Ser (PP). The inhibition was recovered by addition of excess amount of fibronectin to the medium.

The fibronectin‐promoted cell migration, by contraries, was conspicuously inhibited by addition of the PP to the cell culture medium which has alrady contained sufficient amount of fibronectin for the migration, but not so obviously by addition of the larger peptides, the HP or the DP. The inhibition was also recovered by addition of excess amount of fibronectin.

These results indicated that fibronectin utilizes Arg‐Gly‐Asp‐Ser in the molecule as an active cell binding and a cell migration promotion site. Slightly different effects seen between the smaller peptide and the larger ones were discussed.
Isolation and Partial Characterization of a Glycoprotein Complex with Sperm‐Receptor Activity from Ciona intestinalis Ovary1
Authors: DE SANTIS ROSARIA , PINTO MARIA ROSARIA
Published: January 2005

Sperm‐egg interaction in the ascidian Ciana intestinalis is mediated by a fucosyl‐glycoprotein (FP) component of the egg vitelline coat. FP are responsible for sperm binding, sperm activation and the acrosome reaction. In this paper we report a detailed biochemical and functional characterization of FP purified from the ovaries by affinity chromatography. thic component with sperm receptor activity is a high molecular weight glycoprotein complex (>107) with a protein‐carbohydrate ratio of 2:1, which inhibits the binding of the spermatozoa to the vitelline coat and induces sperm activation and the acrosome reaction. Exhaustive proteolytic digestion of FP yields high molecular weight glycopeptides (> 4×105), which contain N‐acetylgalactosamine, fucose, galactose and rhamnose. These glycopeptides retain some receptor activity, thus raising the question of the involvement of the polypeptide backbone in the sperm‐egg binding process. However, the glycopeptide fraction fails to induce the acrosome reaction: we suggest that the polypeptide fraction plays a role in the induction of sperm activation and the acrosome reaction.
The Time‐Course of Hatching Enzyme Secretion in the Sea Urchin Strongylocentrotus purpuratus
Authors: YAGER THOMAS D. , BARRETT DENNIS
Published: January 2005

By two independent methods, we have determined approximately the time‐course of hatching enzyme secretion in the sea urchin Strongylocentrotus purpuratus. A quick‐kill method indicates that a significant fraction of the enzyme is secreted between 90% and 97% of the fertilization‐hatching interval. A direct assay method indicates that the remainder of the enzyme is secreted on either side of the 90–97%“window”. The entire period of secretion spans from 75% to 100% or more of the fertilization‐hatching interval. For embryos raised at 15°C this translates to an interval of 4.8 or more hr.
Micromere Differentiation in the Sea Urchin Embryo: Two‐Dimensional Gel Electrophoretic Analysis of Newly Synthesized Proteins
Authors: MATSUDA RYOICHI , KITAJIMA TAKASHI , OHINATA HIROSHI , KATOH YUKO , HIGASHINAKAGAWA TORU
Published: January 2005

A method for large‐scale culture of isolated blastomeres of sea urchin embryos in spinner flasks was developed. Micromeres and meso‐, macromeres isolated from sea urchin embryos at the 16‐cell stage were cultured by this method and the patterns of protein synthesis by their descendants were examined by two‐dimensional gel electrophoresis of [35S] methionine‐labeled proteins. Six distinct proteins with molecular weights of 140–kDa, 105–kDa, 43–kDa, 32–kDa, and 28–kDa (two components) were specifically synthesized by differentiating micromeres. Quantitative analysis of the two‐dimensional gel patterns demonstrated that all these proteins, except the 32–kDa protein, appeared at the time of ingression of primary mesenchyme cells (PMC's) in vivo, several hours earlier than the onset of spicule formation. The synthesis of 32–kDa protein was paralleled to active spicule formation and the uptake of Ca2+. Cell‐free translation products directed by poly (A)+ RNAs isolated from descendant cells of micromeres and meso‐, macromeres were compared by two‐dimensional gel electrophoresis. Several spots specific to the micromere lineage were detected. However, none of them comigrated with the proteins synthesized specifically by the cultured micromeres. The results suggest that the expression of these proteins specific to differentiating micromeres may involve post‐translational modification.
Urea‐Induced Meiosis Reinitiation in Oocytes of the Starfish, Marthasterias glacialis
Authors: GUERRIER P. , NÉANT I. , CLÉDON P.
Published: January 2005

In the absence of hormone stimulation, prophase‐blocked oocytes of Marthasterias glacialis have been induced to undergo meiosis reinitiation up to female pronucleus formation by pulse incubation in isoosmotic urea‐sea water solutions. Even when this procedure was not effective all along the breeding season, it could trigger full maturation when applied to so‐called “incompetent oocytes” that did not complete maturation following microinjection‐induced mixing of their nucleoplasm and cytoplasm.

32P phosphate incorporation into proteins and cell fusion experiments demonstrate that this treatment produces an increased protein phosphorylation which appears tightly associated with the production of M‐phase promoting factor (MPF). Instead, when oocytes are maintained in the inducing medium, dephosphorylation soon occurs and MPF is no longer present to support meiosis. Under these conditions, the GV‐disrupted oocytes present a permanent nucleolus and do not form a meiotic spindle. The same cytological aspect was also obtained when the oocytes were treated in the presence of 90 μM emetine or 150 μM of the intracellular chelator Quin 2‐AM.

These data suggest that urea‐induced maturation may involve an intracellular Ca2+ shift which would be required to activate both MPF precursor molecules and the resting female centers which stand in the animal cortex outside the nucleus and give rise to the poles of the first maturation spindle. They also show that nuclear disruption alone, without protein phosphorylation, cannot trigger meiosis reinitiation of incompetent oocytes.
The Acrosome Reaction Induced by Dimethylsulfoxide in Sea Urchin Sperm
Authors: MIKAMI‐TAKEI KAORI , FUJIWARA AKIKO , YASUMASU IKUO
Published: January 2005

The acrosome reaction in sea urchin sperm, as judged by disappearance of the acrosomal vesicles in Nomarski optics, was induced by dimethylsulfoxide (DMSO) at concentration above 0.1% in normal artificial sea water. The number of the acrosome‐reacted spermatozoa increased in proportion to DMSO concentration. The DMSO‐induced acrosome reaction, as well as the jelly water‐ or A23187‐induced one, was inhibited by nifedipine and hardly occurred in Ca2+‐free artificial sea water. However, the DMSO‐induced acrosome reaction was found in a few number of spermatozoa in the presence of Ca2+at above 0.5 mM, though the jelly water‐ or A23187‐induced acrosome reaction did not occur at external Ca2+levels lower than 1 mM. Dependency of the acrosome reaction by DMSO on external Ca2+is somewhat lower than that of the reaction by jelly water. In Ca2+‐free artificial sea water, the acrosomal regions of DMSO‐treated spermatozoa attached to their own tails. In some cases, spermatozoa thus treated with DMSO in Ca2+free artificial sea water caused formation of fertilization membrane in a few number of eggs kept in Ca2+‐free artificial sea water. Even in the absence of extermal Ca2+, preliminary step of the acrosome reaction seems to be completed probably by DMSO‐induced weak Ca2+‐mobilization in spermatozoa.
The Participation of Speract in the Acrosome Reaction of Hemicentrotus pulcherrimus
Authors: YAMAGUCHI MASAAKI , NIWA TOSHIAKI , KURITA MASANORI , SUZUKI NORIO
Published: January 2005

A speract‐free macromolecular fraction was prepared from the egg jelly of the sea urchin Hemicentrotus pulcherrimus by gel filtration and tested for ability to induce the acrosome reaction in H. pulcherrimus spermatozoa with or without exogenously added synthetic speract. The macromolecular fraction without speract showed only about half the activity of the original unfractionated jelly for induction of the acrosome reaction. The rates of the acrosome reaction induced by the fraction with speract were comparable with those induced by the unfractionated jelly at all pHs tested. Speract itself, however, did not induce the acrosome reaction in the absence of the macromolecular fraction of jelly. The acrosome reaction was associated with incerase of the cyclic AMP concentration in sperm cells, the extent of incerase depending on the concentration of the macromolecular fraction. Addition of speract to the fraction enhanced both induction of the acrosome reaction and increase in the cyclic AMP concentration induced by the fraction. These results suggest that a major factor(s) responsible for the acrosome reaction is a macromolecular component(s) of the jelly and that speract promotes the reaction as a co‐factor.