bluuebunny/crossref_metadata_2025 huggingface dataset:
Ultrastructure of Follicular Epithelia in the Ovary of Lepidochitona cinerea (L.) (Mollusca: Polyplacophora)
Authors: RICHTER H.‐PETER
Published: January 2005

In the sac‐like ovary of the polyplacophoran mollusc, Lepidochitona cinerea, nutritive tissue arises from the ventral gonadal wall of the organ as prominent folds which support the oocytes during the various stages of their development. Each oocyte is enveloped by the follicular epithelium. Approximately twenty follicle cells surround one full‐grown oocyte and by this late stage are connected to it and to each other by desmosomes. The follicle cells contain glycogen, Golgi dictyosomes, mitochondria, lipid droplets, numerous cisternae and vesicles of the rough endoplasmic reticulum, and various kinds of lysosomes. The nutritional function of these cells and their possible role forming the oocytic hulls is discussed.
Behavior of Primary Mesenchyme Cells In situ Associated with Ultrastructural Alteration of the Blastocoelic Material in the Sea Urchin, Anthocidaris crassispina
Authors: KATOW HIDEKI , AMEMIYA SHONAN
Published: January 2005

In the blastula of the sea urchin, Anthocidaris crassispina, a small number of primary mesenchyme cells (PMCs) ingressed from the blastocoel wall taking a bottle shape. The majority of the PMCs followed the first group of PMCs. These ingressed without taking the bottle shape, and became round within the blastocoel wall. After ingression, the PMCs migrated as single cells retaining their round cell contour. The average velocity of their migration was 13.3 μm/hr.

The blastocoel contained Alcian blue (pH 1.0)‐positive material which changed its light microscopic configuration from being amorphous in the hatched and mesenchyme blastulae to being fibrous in the early gastrulae. Ultrastructurally, the blastocoelic material in the hatched blastulae was composed of 27 nm diameter granules. In the mesenchyme blastulae and the early gastrulae relatively long 15 nm diameter fibers were seen in addition to the 27 nm diameter granules. The 27 nm diameter granules bound the ruthenium red while the 15 nm diameter fibers did not. The 27 nm diameter granules formed aggregates in the hatched blastulae, and were bound to the 15 nm diameter fibers in the mesenchyme blastulae and early gastrulae to form a fibrous network which was observed by a light microscope.
Pinocytosis of Ferritin from the Gut Lumen in Larvae of a Sea Star (Patiria miniata) and a Sea Urchin (Lytechinus pictus)
Authors: HUVARD ANDREA L. , HOLLAND NICHOLAS D.
Published: January 2005

Pinocytosis of macromolecules from the gut lumen is demonstrated for the first time in larval stages of invertebrates. Developing sea stars (Patiria miniata) and sea urchins (Lytechinus pictus) were incubated in seawater containing ferritin, which was detected in cell organelles by transmission electron microscopy. Pinocytotic uptake of ferritin by gut cells of Patiria could be detected as soon as the larval mouth opened before the esophagus, stomach and intestine could be distinguished from one another; in contrast, no pinocytosis was detected at the comparable developmental stage (the prism larva) of Lytechinus. Pinocytosis was first detected in developing Lytechinus in pluteus larvae, especially in the stomach and intestine. In gut cells of both kinds of echinoderm larvae, the ferritin progressed rapidly from coated pits at the luminal cell membrane to secondary lysosomes (e.g. this progression took only about 10 min in stomach cells of Patiria larvae). Phagocytosis from the gut lumen was never observed after latex beads or starch granules were fed to larvae of Patiria and Lytechinus. Moreover, there was no evidence of pinocytosis of ferritin or phagocytosis of large particles by epidermal cells of larvae of either species.
Effect of Disulfide‐Reducing Agents on Activity of 1‐Methyladenosine Ribohydrolase in Starfish Follicle Cells
Authors: MITA MASATOSHI
Published: January 2005

Production of 1‐methyladenine (1‐MeAde) in isolated starfish follicle cells incubated with 1‐methyladenosine was elevated by disulfide‐reducing agents, such as dithiothreitol, cysteine, and homocysteine. More than 0.05 mM cysteine and homocysteine brought about a six‐fold increase of 1‐MeAde production in comparison with controls without disulfide‐reducing agents. On the other hand, iodoacetamide, which is a membrane‐permeable SH‐blocking agent, decreased the 1‐methyladenosine‐induced 1‐MeAde production. A forty percent decrease of 1‐MeAde production was obtained with 0.5 mM iodoacetamide. Iodoacetamide inhibited also 1‐MeAde production induced by gonad‐stimulating substance (GSS). But a membrane‐impermeable SH‐blocking agent, N‐carboxyphenylmaleimide, failed to inhibit both 1‐methyladenosine‐induced and GSS‐induced 1‐MeAde production. Furthermore, partially purified 1‐methyladenosine ribohydrolase was activated by dithiothreitol, 2‐mercaptoethanol, and cysteine derivatives. An approximately ten‐fold activation was obtained with these disulfide‐reducing agents at concentrations of more than 5 mM. On the contrary, though the SH‐blocking agents inhibited 1‐methyladenosine ribohydrolase, the inhibition was relieved by the disulfide‐reducing agents. These results suggest that the reduced form of SH‐group in 1‐methyladenosine ribohydrolase plays an important role for enzymatic activation.
Purification of Acetylcholinesterase from Sea Urchin (Hemicentrotus pulcherrimus) Embryos by Affinity Chromatography
Authors: AKASAKA KOJI , SASAKI HIROSHI , SHIMADA HIRAKU , SHIROYA TSUGIO
Published: January 2005

Only one form of acetylcholinesterase (AchE) was detected in Hemicentrotus pulcherrimus embryos. In H. pulcherrimus embryos as well as in the other sea urchin embryos, AchE activity begins to increase rapidly after gastrula stage.

Purification of AchE from plutei has been carried out by the procedure including affinity chromatography. Purified AchE had the activity 14,600 times higher than that of homogenate, and the final yield of AchE was 8%. The enzyme seems to be electrophoretically homogeneous, and has a molecular weight of 3 × 105 as determined by Sepharose CL–6B column chromatography.
Spermiogenesis in the Marine Shrimp, Sicyonia ingentis
Authors: SHIGEKAWA KATHERINE , CLARK WALLIS H.
Published: January 2005

Spermiogenesis in the marine prawn Sicyonia ingentis was examined using transmission electron microscopy. The acrosomal vesicle, derived from the fusion of pro‐acrosomal vesicles blebbed from the nuclear envelope, contains the membrane pouches, anterior granule and a spike. The anterior granule is formed from the coalescence of granular aggregates within the proacrosomal vesicles. Primordia underlying the apical acrosomal vesicle membrane polymerize to form a spike approximately 6 μm long. The convoluted pouch membranes arise from the posterior acrosomal vesicle membrane. Lateral and apical portions of the acrosomal vesicle are surrounded by a pentalaminar membrane comprised of the spermatid plasma membrane and the acrosomal vesicle membrane. Subacrosomal structures include the dense saucer plate, granular core and crystalline lattice. These components condense just posterior to the acrosomal vesicle and are separated from the chromatin by a nuclear plate.

The spermatid nucleus becomes surrounded by rough endoplasmic reticulum (RER) and membranous lamellar bodies. RER gives rise to smooth endoplasmic reticulum. These membrane systems degenerate, forming a band of reticular elements around the lateral and posterior portions of the nucleus. The nucleus undergoes condensation followed by decondensation with concomitant breakdown of the nuclear envelope. The resultant chromatin is fibrillar in appearance.
Developmental Fates of Blastomeres of Eight‐Cell‐Stage Xenopus laevis Embryos
Authors: MASHO RIE , KUBOTA HIROSHI Y.
Published: January 2005

The developmental fates of animal, vegetal, dorsal, and ventral egg regions of Xenopus laevis embryos were examined. For this purpose, a tracer enzyme (horseradish peroxidase) was injected bilaterally into pairs of eight‐cell‐stage blastomeres and the clonal organization of marked cells in the early tail‐bud embryos was examined. The epidermis over most of the body originated from animal‐ventral micromeres, but that in the head originated from animal‐dorsal blastomeres and that in the area surrounding the anus originated from vegetal‐ventral blastomeres. The neural tube originated from animal‐dorsal, vegetal‐dorsal, and animal‐ventral blastomeres. These results were consistent with those of previous studies. But in contrast to previous findings, results showed that the entire notochord is derived from animal‐dorsal micromeres and that the somites originate from all four bilateral pairs of blastomeres in the eight‐cell stage. These results are discussed in relation to the current maps of prospective fates based on results of vital‐dye staining. Morphogenetic movements are also discussed on the basis of the clonal organization demonstrated in the present study.
Distribution of Membrane‐Associated Calcium in Fertilized Sea Urchin Eggs during Mitosis*
Authors: ŌHARA TAKANE , SATO HIDEMI
Published: January 2005

The distribution of membrane‐associated calcium in dividing sea urchin eggs was examined with chlortetracycline as a fluorescent chelate probe. The fluorescence of bound chlortetracycline in fertilized eggs was initially evenly distributed, but began to gather around the nucleus in prophase, and formed a dumb‐bell shaped condensation enclosing the mitotic apparatus by metaphase. During anaphase and telophase, the fluorescence was observed in kinetochore‐to‐pole regions of the spindle, with little fluorescence in the interkinetochore region. The astral regions showed intense fluorescence. The distribution of the chlortetracycline‐fluorescence coincided with that of ER‐like membranes seen in electron micrographs. The distribution of the fluorescence was obscure and the birefringence of spindles disappeared on perfusion on perfusion of the cells in metaphase with 1 mM tetracaine, which is known to displace membrane‐bound calcium.

These results suggest that intracellular free calcium ions are sequestered in the membrane system associated with the mitotic apparatus during mitosis.
Analysis of the Role of Astral Rays in Pronuclear Migration in Sand Dollar Eggs by the Colcemid‐UV Method
Authors: HAMAGUCHI MIYAKO S. , HIRAMOTO YUKIO
Published: January 2005

The formation and migration of the sperm aster, and the migration of male and female pronuclei during fertilization were investigated in the eggs of the sand dollar, Clypeaster japonicus using the Colcemid‐UV method. When an egg in Colcemid sea water was irradiated locally with UV light (about 365 nm wavelength) at a limited region containing sperm head, a sperm aster formed in this region, and migrated to the center of the UV‐irradiated region during its formation. When the UV‐irradiated region was displaced or its shape was changed after the formation of the sperm aster, the aster migrated to the center of the new UV‐irradiated region. The direction of the migration of the sperm aster coincided with the direction of the longest astral rays. Direct contact between astral rays and the egg surface was not essential for sperm aster migration. When a region containing both the sperm centrosome and the female pronucleus was irradiated with UV light, the female pronucleus migrated toward the center of the sperm aster after they were connected by astral rays. The migration was suppressed when UV light was shaded over the region between the aster and the female pronucleus. These results suggest that the female pronucleus migrates to the sperm aster by attractive force between them.
Studies on the Water‐Soluble Lens Proteins of the Lizard, Calotes versicolor III. Ontogenic Changes
Authors: PAL JAYANTA K. , GOEL SURESH C.
Published: January 2005

Ontogeny of lens crystallins of the garden lizard, Calotes versicolor has been studied by gel filtration, polyacrylamide gel electrophoresis, 7M urea PAGE, SDS and 6M urea isoelectric focusing. Wet weight, diameter and the soluble protein content per lens were also determined as a function of age. The amount of α‐crystallins increased slightly during development. The relative proportion of the putative δ‐crystallins was significantly higher in the embryos than in the adult. The overall polypeptide pattern of crystallins changed markedly around the time of hatching.
Implications of Differential Chemotaxis and Cohesiveness for Cell Sorting in the Development of Dictyostelium discoideum
Authors: OHMORI TAKASHI , MAEDA YASUO
Published: January 2005

Cell sorting behavior was observed during the development of Dictyostelium discoideum Ax‐2, between cells grown with [G(+) cells] and without [G(−) cells] glucose. Development of the G(−) cells was about 2‐3 hr faster, as reflected by differences in chemotactic sensitivity of the cells and cell cohesiveness. Among various mixing combinations of G(−) and G(+) cells, the most clear sorting occured when vegetative G(+) cells were mixed with G(−) cells which had been allowed to develop for 3 hr, the G(−) cells being located in the anterior prestalk region of a migrating slug. In contrast, vegetative G(−) cells moved to the posterior prespore region of a slug when mixed with G(+) cells which had developed for 6 hr. These findings indicate a close relationship of the cellular developmental stage to the sorting behavior. Possible implications of the differential chemotactic ability and cohesiveness for the sorting mechanism are disscussed.
The Ontogeny of Thymic Myoid Cells in the Chicken
Authors: NAKAMURA HARUKAZU , NAKANO KENSUKE E. , YASUDA MINEO
Published: January 2005

The ontogeny of thymic myoid cells in the chick was studied electron microscopically and immunohistochemically. An anticreatine kinase antibody which reacts specifically to skeletal muscle cells was used. This antibody reacts only to myoid cells in the thymus. Myoid cells were found in the medulla or in the interlobular region, though the number of the myoid cells was small. Immunohistochemically, myoid cells were detected on the 18th day of incubation. Mature myoid cells showed clear cross striations after immunohistochemical staining around the time of hatching. Electron microscopically, myoid cells were detectable on the 19th day of incubation. The discrepancy between immunohistochemical and electron microscopical detection may be due to the low number of myoid cells.
Cell Division Pattern during Gastrulation of the Ascidian, Halocynthia roretzi
Authors: NISHIDA HIROKI
Published: January 2005

With the aid of scanning electron microscopy, the spatial and temporal pattern of cell divisions during gastrulation of ascidian embryos was analysed and described up to the 306‐cell stage. After the 64‐cell stage asynchrony of cleavages became apparent, but the spatio‐temporal cell‐division pattern was highly invariant. The division pattern of cells in the animal hemisphere was regular, while that of cells in the vegetal hemisphere was complicated. The results obtained in the present study coincided with Conklin's description (1905) with only a few discrepancies. Gastrulation started around the 110‐cell stage and a late gastrula consisted of about 300 cells. In addition, the cell lineage of the neural tube was described.
Changes in the Cell Surface Level of GP126 during Development of Dictyostelium discoideum
Authors: CHADWICK C. M.
Published: January 2005

The developmental regulation of the vegetative cohesion molecule, gp 126, has been monitored in the cellular slime mould Dictyostelium discoideum. As judged by immunoprecipitation using an anti‐vegetative cell, cohesion blocking antibody, gp126 persisted until at least the grex stage of development although a decline in the level of the molecule was observed thereafter. Further, after the grex stage, cells showed an increasing loss of ability to absorb the cohesion‐blocking effect of an anti‐vegetative cell Fab. Therefore, the decline of gp 126 could be ascribed to a loss from the cell surface. By radio‐iodination of vegetative cells followed by liquid‐scintillation counting of gp 126, developmental‐regulation could be determined quantitatively.

At the grex stage of development, whole aggregates were embedded in wax. Longitudinal sections were then stained with a monospecific anti‐gp 126 Fab, followed by a fluorescent sheep anti‐rabbit IgG. Fluorescence was observed only at the tip (the prestalk) region, thereby showing that gp 126 is a prestalk marker.

To confirm the above result, grexes were dissociated and cells were separated into prespore and prestalk populations on a gradient of Percoll. Prestalk but not prespore cells were able to absorb the cohesion‐blocking effect of an anti‐vegetative cell Fab.

To examine the biosynthesis of gp 126, cells were pulsed with radioactive glucosamine, mannose or acetate. The pattern of incorporation of radioactivity suggested that the de novo synthesis of gp 126 ceases upon commencement of development.
Effects of Various Antibiotics on the Excitability of Newt Ectodermal Cells in vitro
Authors: ITO SHIZUO , FUKUYAMA KAZUHIRO , KATOKU KOSHIRO
Published: January 2005

With the addition of actinomycin D (AMD), cycloheximide (CH) or tunicamycin (TM) to a culture medium at 23°c, the epidermal action potentials (EAPs) of the cultured epidermal explants or monolayered cell colonies taken from newt embryos were examined for confirmation of their relationship to transcriptional and translational events. EAPs of ectodermal cells from the pregastrula were first recorded after 84 hr of monolayered cell culture in FCS‐Steinberg's medium. The addition of 10 μg/ml CH or 1 μg/ml TM for 36 hr prevented the appearance of these potentials in cells precultured in FCS‐Steinberg's medium for 60 hr. Ectodermal cells precultured in FCS‐Steinberg's medium for 68 hr generated the EAPs after they were cultured in the same concentration of CH or TM for 28 hr. Thus, it appears that certain proteins and glycoproteins may be responsible for evoking the EAPs and that these molecules are synthesized at about 68 hr in culture following the pregastrula stage. Ectodermal cells precultured in FCS‐Steinberg's medium for 48 hr generated EAPs after they were cultured in 0.5 μg/ml AMD for 72 hr. Cells precultured for 36 hr failed to produce the EAPs even when they were cultured in the same concentration of AMD for 84 hr.
Induction of the Acrosome Reaction in Starfish
Authors: MATSUI TAEI , NISHIYAMA ICHIRO , HINO AKIYA , HOSHI MOTONORI
Published: January 2005

In the starfish, Asterias amurensis, at least two distinct components of the egg jelly are required for inducing the acrosome reaction: a sulfated glycoprotein named acrosome reaction‐inducing substance (ARIS) and a diffusible organic substance(s) named Co‐ARIS. The following evidence suggested that ARIS and Co‐ARIS cooperatively activate CA‐channels of the sperm plasma membrane and eventually induce dramatic changes in sperm morphology, the acrosome reaction. 1) Pronase digest of ARIS (P‐ARIS) and Co‐ARIS, either as a pure or a crude preparation (Fraction M8), were fully effective in combination for induction of the acrosome reaction in normal sea water, although they were not effective individually. P‐ ARIS alone induced the acrosome reaction fully in high Ca2+ sea water and markedly at high pHs, whereas Fraction M8 alone did not induce the reaction even in these conditions. The reaction was not induced by increase in either the Ca2+ concentration or the pH of sea water, but was markedly induced in the absence of jelly components by raising both the pH and Ca2+ concentration together. 2) The ionophore A23187 induced the acrosome reaction appreciably when present alone and fully in the presence of monensin or Fraction M8. Monesin alone was ineffective. 3) The jelly or a combination of ARIS and Fraction M8 caused abrupt Ca2+ ‐uptake by the sperm. The Ca‐channel blockers verapamil and diltiazem inhibited the jelly‐induced acrosome reaction.
Acrosome Reaction‐Inducing Substance Purified from the Egg Jelly Inhibits the Jelly‐Induced Acrosome Reaction in Starfish: An Apparent Contradiction
Authors: MATSUI TAEI , NISHIYAMA ICHIRO , HINO AKIYA , HOSHI MOTONORI
Published: January 2005

Previous studies indicated that two components of the egg jelly are required for induction of the acrosome reaction in starfish: a sulfated glycoprotein called acrosome reaction‐inducing substance (ARIS) and a diffusible organic substance(s) called Co‐ARIS. In the present study the sites of action of ARIS and Co‐ARIS and their temporal relationships were examined. When sperm had been treated for a few minutes with ARIS, or a crude preparation of Co‐ARIS (Fraction M8), or inadequate amounts of jelly, or sufficient jelly in low Ca2+ sea water, they did not undergo the acrosome reaction when the deficiencies were corrected. Moreover, they became nonresponsive to the jelly. Pronase digest of ARIS (P‐ARIS) but not of Fraction M8 retained this capacity. A steroidal saponin purified as Co‐ARIS did not have this capacity. This suggests the presence of a third jelly component, probably an oligopeptide(s), participating in induction of the acrosome reaction. Activation of Ca2+ ‐uptake seems to be at least one, if not the only, action site of ARIS and Co‐ARIS, because ARIS, P‐ARIS, and Fraction M8 inhibited jelly‐induced Ca2+ ‐uptake by sperm, and because the calcium ionophore A23187 by‐passed the blockage by these components of the jelly‐induced acrosome reaction.
Intracellular pH Changes of Starfish Sperm Upon the Acrosome Reaction
Authors: MATSUI TAEI , NISHIYAMA ICHIRO , HINO AKIYA , HOSHI MOTONORI
Published: January 2005

The acrosome reaction is accompanied by ionic changes such as increases in intracellular Ca2+ and intracellular pH (pHi). Since the two jelly components essential for inducing the acrosome reaction, ARIS and Co‐ARIS, were shown to activate Ca‐channels (accompanying paper), we examined the jelly components to determine which was responsible for the pHi‐increase using 9‐aminoacridine as a probe of pHi. This paper presents evidence that an oligopeptide(s) is responsible for the pHi‐increase. The pHi of swimming sperm is 7.4‐7.5. Within 20 sec after the addition of jelly, their pHi increased rapidly by 0.06 pH unit, then decreased by 0.2–0.3 pH unit, and reached a plateau level within 3 min. Similar changes in pHi were observed on addition of a Pronase digest of ARIS (P‐ARIS) and a diffusible fraction of jelly (Fraction M8) together. Fraction M8, but not ARIS or Co‐ARIS increased the pHi, and activated sperm respiration in sea water at pH 6.5. The two activities of Fraction M8 depended upon Na+ but not Ca2+, and were susceptible to Pronase digestion. Fraction M8 is also known to enhance induction of the acrosome reaction by the Ca‐ionophore A23187. These results suggest that the egg jelly contains a peptide(s) that is not obligatory for the acrosome reaction but facilitates the reaction by increasing the pHi of the sperm. The significance of the pHi‐increase upon the acrosome reaction is discussed.
Vitellogenin‐specific Non‐histone Chromatin Protein
Authors: NAKAYAMA TATSUO
Published: January 2005

A non‐histone chromatin protein with a molecular weight of about 10,000 daltons was purified from the liver of egg‐laying hens. The protein binds tightly to hen genomic DNA fragments carrying at least a part of the vitellogenin structural gene. This vitellogenin‐specific non‐histone chromatin protein is induced by estrogen before, or in parallel with vitellogenin synthesis in the liver of male chickens, in which vitellogenin is not usually synthesized.
Maitotoxin, A Presumed Calcium Channel Activator, Induces the Acrosome Reaction in Mussel Spermatozoa
Authors: NISHIYAMA ICHIRO , MATSUI TAEI , YASUMOTO TAKESHI , OSHIO SHIGERU , HOSHI MOTONORI
Published: January 2005

Maitotoxin, a presumed activator of the voltage‐sensitive calcium channel, induced the acrosome reaction in the mussel, Mytilus edulis at physiological pH and in the starfish, Asterias amurensis at pH 9.5. The induction of acrosome reaction by maitotoxin depended upon external Ca2+ and was inhibited by two types of calcium channel blockers; verapamil and diltiazem. These results suggest that the activation of the voltage‐sensitive calcium channel takes an important part in the initiation of acrosome reaction in Mytilus and other animals.
Microinjected Polystyrene Beads Move Along Astral Rays in Sand Dollar Eggs
Authors: HAMAGUCHI MIYAKO S. , HAMAGUCHI YUKIHISA , HIRAMOTO YUKIO
Published: January 2005

Movements of polystyrene beads along astral rays of the sperm aster and the mitotic aster were investigated in eggs of the sand dollars, Clypeaster japonicus and Scaphechinus mirabilis. Polystyrene beads injected into the unfertilized egg were at a standstill in the protoplasm. After fertilization, these beads exhibited movements toward the center of the sperm aster along the rays, and finally gathered around the astral center. They were distributed in blastomeres together with the mitotic centers during successive cleavages. When injected into eggs during mitosis, beads moved to the centers of the mitotic asters along astral rays. The injected beads did not move when the aster was disorganized by treatment with Colcemid, and moved when it formed after UV‐irradiation. These results indicate that microtubules of astral rays are essential to the movement of polystyrene beads. The movement of small polystyrene beads (0.2–0.3 μm in diameter) resembled the saltatory movement of endogenous cytoplasmic granules, and the movement of large beads (ca. 1 μm in diameter) resembled the female pronuclear migration. All of these movements observed in fertilized eggs were demonstrated to be microtubule‐dependent, perhaps sharing the same basic mechanisms.
Regulation of Prestalk‐specific Acid Phosphatase in Dictyostelium discoideum
Authors: TASAKA MASAO , KAY CAROLYN A. , SOMMERVILLE SHARON M. , GRANT AROLINE E. , TSANG ADRIAN S.
Published: January 2005

Acid phosphatase–2, as characterized by gel electrophoresis under non‐denaturing conditions, is a convenient marker for prestalk cells in Dictyostelium discoideum. We have purified this prestalk‐specific enzyme and have examined its regulation during development. Under denaturing conditions, the enzyme has a molecular weight of 50,000 and an isoelectric point of 4.0. On the other hand, acid phosphatase‐I have a Mr‐55,000 polypeptide (AP1–55) and a minor Mr‐50,000 polypeptide (AP1–50) and both have diffuse isoelectric point from 3.4 to 4.1. Using monoclonal antibodies directed against acid phosphatase‐2 as probes, we showed that some acid phosphatase‐2 are newly synthesized at slug stage and some are converted from AP1–50 which was synthesized during ealy development.
Mass Isolation of Muscle Lineage Blastomeres from Ascidian Embryos
Authors: MITA‐MIYAZAWA IZUMI , SATOH NORIYUKI
Published: January 2005

The aim of this investigation was to establish an experimental system for studying the causal relationship between DNA replication and tissue‐specific enzyme development in ascidian embryos. Blastomeres were dissociated from 44∽64‐ceIl Halocynthia roretzi embryos and fractionated by centrifugation through a discontinuous Percoll density gradient. When cells harvested from the fraction at the bottom of the tube were division‐arrested with cytochalasin B (an inhibitor of cytokinesis) soon after their isolation, more than 70% of them developed histochemically‐detectable muscle‐specific acetylcholinesterase (AChE) activity, suggesting that they were almost all blastomeres of muscle lineage. When these cells were arrested with aphidicolin (an inhibitor of DNA replication) and cytochalasin B immediately after their isolation, however, none of them showed AChE activity. When they were allowed to divide once and then arrested with the inhibitors, nearly 40 % of them developed AChE activity, and when they were allowed to divide twice before arrest, about 70% of them showed AChE activity.
Metabolism of Round Spermatids: Pyruvate cannot Maintain the ATP Level
Authors: NAKAMURA MASAHISA , KAMACHI TERUYUKI , OKINAGA SHOICHI , ARAI KIYOSHI
Published: January 2005

Round spermatids (steps 1–8) were isolated from rat testes and the effect of pyruvate on their intracellular ATP level was examined. Results showed that although the spermatids consumed a considerable amounts of pyruvate, this substrate alone did not maintain their ATP level. However, their ATP level was maintained in the presence of both pyruvate and α‐ketovalerate or α‐ketobutyrate. Maintenance of the ATP level by these substrates was associated with electron trasnport and oxidative phosphorylation. α‐Ketoacid inhibited pyruvate reduction to lactate in the lactate dehydrogenase (LDH) reaction, but increased pyruvate oxidation to CO2. The NADH level in spermatids was too low to be detectable, but the NAD level remained unchanged in the presence of pyruvate and α‐ketovalerate. These results suggest that pyruvate by itself is not an adequate energy‐yielding substrate for spermatids and that a high NADH/NAD ratio may be essential for maintenance of their ATP level.
Metabolism of Round Spermatids: A Possible Regulation of Hexose Transport
Authors: NAKAMURA MASAHISA , YAMAGUCHI KEIICHIRO , SUZUKI AKIRA , OKINAGA SHOICHI , ARAI KIYOSHI
Published: January 2005

Round spermatids (steps 1–8) were isolated from rat testes and glucose transport into the cells was examined. The exposure of spermatids to glucose resulted in an extremely low level of ATP. In contrast, the level of ATP remained constant in the presence of pyruvate. Transport of a glucose analogue, 2‐deoxy‐D‐[3H]glucose ([3H]dGlc) into spermatids was correlated with intracellular levels of ATP and was much greater in cells with higher rather than lower levels of ATP. [3H]dGlc transport into spermatids with low levels of ATP was partially reversed when the cells were incubated with pyruvate. Inhibition of [3H]dGlc transport was exerted on Vmax and not on Km. Moreover, glucose acted as a competitive inhibitor of [3H]dGlc uptake (Km increased; Vmax unaltered). These results suggest that glucose transport into spermatids is active in vitro and probably regulated by the intracellular level of ATP.
How Do Retinal Axons Arrive at Their Targets?: Cellular and Molecular Approaches
Authors: FUJISAWA HAJIME
Published: January 2005

Mechanisms involved in the formation of specific neuronal networks are the central problem to be studied in the developmental neurobiology. This review article concerns the modes of growth of retinal axons toward their targets in lower vertebrates. Basing on the cellular mechanisms in target searching by retinal axons, the related molecular mechanisms are discussed.
Establishment of the Embryo‐derived Stem (ES) Cell Lines from Mouse Blastocysts: Effects of the Feeder Cell Layer.
Authors: SUEMORI HIROFUMI , NAKATSUJI NORIO
Published: January 2005

The ES ceii lines are embryo‐derived stem cell lines directly isolated from the inner cell mass of mouse blastocysts using feeder cell layer. We have established a number of ES cell lines from 129 or C57BL/6 strain mice by using the feeder layer of the STO cells (from ATCC) or the primary embryonic fibroblasts, which was obtained by trypsinizing the 16‐day‐old BALB/c mouse fetus. The ES cell lines established on the STO feeder layer showed differentiation into various tissues in solid tumors when injected into syngenic mice. Karyotype was, however, nearly tetraploid. The ES cell lines established on the primary fibroblasts exhibited differentiation into larger variety of tissues in solid tumors. Karyotype was almost diploid and majority of the cells kept normal set of chromosomes in G‐banding. We conclude that the primary fibroblasts are better feeder layer than the STO cells for establishment and maintenance of the ES cell lines.
The Effect of 17 β‐Estradiol and Estrone on Intermediary Metabolism of the Pyloric Caeca of the Asteroid Luidia clathrata (Say) Maintained under Different Nutritional Regimes
Authors: WATTS STEPHEN A. , LAWRENCE JOHN M.
Published: January 2005

Luidia clathrata were fed either a high or low level of food and injected with estradiol, estrone, or seawater for 16 days. In individuals maintained at a low level of food, the weight of the pyloric caeca decreased in all individuals, regardless of hormone injections. Activities of all enzymes in these individuals were lower than in individuals maintained at a high level of food. In the individuals maintained at a low level of food, the activity of GPDH was higher in the pyloric caeca of individuals injected with estradiol and estrone than in those receiving seawater injections; the activity of 6‐PGDH decreased in those injected with estrone. In individuals maintained at a high level of food, the activities of glucose‐6‐phosphate dehydrogenase (G–6–PDH) and 6‐phosphogluconate dehydrogenase (6–PGDH) were higher in individuals injected with estradiol than in those receiving seawater injections. Activities of glycerol phosphate dehydrogenase (GPDH) and pyruvate kinase (PK) were not affected. A slight decrease (P<0.1) in the activity of 6–PGDH was seen with injection of estrone. These results suggest that steroids have a role in regulating cellular metabolism in the pyloric caeca, and that nutritional condition influences the response of the pyloric caeca to steroids. In addition, these results indicate that the nutritional condition of the individual directly influences enzyme activities.
Purification of Co‐ARIS, a Cofactor for Acrosome Reaction‐Inducing Substance, from the Egg Jelly of Starfish
Authors: NISHIYAMA ICHIRO , MATSUI TAEI , HOSHI MOTONORI
Published: January 2005

In the starfish, Asterias amurensis, the acrosome reaction‐inducing capacity of the egg jelly has been attributed to two components in the egg jelly: a sulfated glycoprotein (ARIS) and an unidentified low‐molecular weight substance (Co‐ARIS). In the process of purification of Co‐ARIS, we found that Co‐ARIS is not a single chemical entity but a series of closely related molecules. Co‐ARIS was first group‐separated by using octadecylsilane and anion‐exchanger. Three major Co‐ARIS' (I, II and III) were then purified by successive chromatographies using octadecylsilane and silica gel to the homogeneity in thin layer chromatography. Each of purified Co‐ARIS', together with ARIS or the Pronase‐digest of ARIS, induced the acrosome reaction at high ratios in normal seawater.
Correlation Between the Molecular Structure and the Biological Activity of Co‐ARIS, a Cofactor for Acrosome Reaction‐Inducing Substance
Authors: NISHIYAMA ICHIRO , MATSUI TAEI , FUJIMOTO YOSHINORI , IKEKAWA NOBUO , HOSHI MOTONORI
Published: January 2005

Two components in the egg jelly are required for inducing the acrosome reaction in starfish; a sulfated glycoprotein called acrosome reaction‐inducing substance (ARIS) and its cofactor called Co‐ARIS. Three distinct molecules were isolated as the major Co‐ARIS' and designated as Co‐ARIS' I, II and III. Structural analysis of Co‐ARIS' revealed that they are steroidal saponins comprising a sulfated steroid and a pentasaccharide chain. Co‐ARIS' I and II differ only in the steroidal side chain. In the presence of ARIS, each Co‐ARIS induced the acrosome reaction with a maximal effect at 100–200 μM (Co‐ARIS I) or 25–50 μM (Co‐ARIS' II and III). Mixtures of Co‐ARIS' I, II and III were more effective than the individuals. The activity of Co‐ARIS was considerably reduced by solvolytic desulfation but was not affected at all by periodate oxidation. Reduction by NaBH4 decreased the activity of Co‐ARIS I and enhanced that of Co‐ARIS II. Treatment of Co‐ARIS III with NaBH4 did not affect the activity as anticipated from its structure. These results suggest that the sulfate moiety and the side chain of steroid are important for the activity of Co‐ARIS. The saccharide chain, however, seems not necessarily to be strictly specified for the activity.
Maternal Messenger RNA as a Determinant of Pole Cell Formation in Drosophila Embryos
Authors: OKADA MASUKICHI , KOBAYASHI SATORU
Published: January 2005

Some polar plasm components are UV‐sensitive. Messenger RNA extracted from oocytes or cleavage embryos can to induce pole cells in embryos that have been deprived of ability to form pole cells by UV‐irradiation. This article reviews studies on the role of this mRNA in the developmental pathway leading to germ cell formation.
The Properties of the Oviducal Pars Recta Protease which Mediates Gamete Interaction by Affecting the Vitelline Coat of a Toad Egg
Authors: TAKAMUNE KAZUFUMI , KATAGIRI CHIAKI
Published: January 2005

SDS‐PAGE analyses of the vitelline coats (VCs) of coelomic eggs (CEVC) and uterine eggs (UEVC) of Bufo japonicus revealed that the UEVC lacks the 40K–52K molecular weight components present in the CEVC; this is concomitant with the increased stainability of a 39K component and the appearance of a 36K component. These macromolecular alterations, accompanied by the acquisition of egg fertilizability, were induced when coelomic eggs were treated with the contents of secretory granules obtained from the oviducal pars recta (PRG). Gel‐filtration of PRG in combination with hydrolytic assays employing either fluorescamine‐labeled CEVC or a variety of synthetic substrates showed that the CEVC to UEVC alterations are ascribable to the action of a protease hydrolyzing specifically peptidyl‐Arg‐MCAs in a highly Ca2+‐dependent way. This enzyme, which has an optimal pH of 8.0–8.2, is inhibited by soybean trypsin inhibitor and leupeptin, as well as by such serine protease inhibitors as DFP and p‐APMSF. On the basis of a SDS‐PAGE analysis, its molecular weight is estimated to be 66K. Treatment of coelomic eggs with the partially purified PR protease did not render the eggs fertilizable, although CEVC to UEVC macromolecular alterations were effected. We conclude that the action of this oviducal protease in partially hydrolyzing the VC is a prerequisite but insufficient in itself to render the coelomic eggs fully accessible to a fertilizing sperm.
The Embryos Obtained from the Karyoplasts of Starfish Oocytes: Their Development and Sensitivity to Cytostatic Neurotransmitter Antagonists
Authors: BUZNIKOV GENNADY A. , MALCHENKO LYUDMILA A. , DELONE GALINA V. , BRODSKY VSEVOLOD Ya.
Published: January 2005

Karyoplasts obtained from full‐grown oocytes of the starfish Aphelasterias japonica have practically no cytoplams and are incapable of maturation. Karyoplasts of oocytes of starfishes Marthasterias glacialis and Acanthaster planci have the cytoplasm (10%–15% of the total karyoplast volume) and are often capable of maturation, fertilization and one or several cleavage divisions. The embryoskaryoplasts completely lose supersensitivity and retain usual sensitivity to cytostatic antagonists of neurotransmitters. The assumption is made that the incapability or limited capability of this embryos for development might be due to a deficiency of certain components of the “prenervous” neurotransmitter systems.
Selective Identification of Sperm Fused with the Surface of Echinoderm Eggs by DNA‐Specific Bisbenzimide (Hoechst) Fluorochromes
Authors: HINKLEY ROBERT E. , EDELSTEIN RICHARD N. , INVONNET PEDRO I.
Published: January 2005

When unfertilized echinoderm eggs are treated with the DNA‐specific bisbenzimide fluorochrome Hoechst 33342 and then fertilized with unlabeled sperm, a single spermatozoan bound to the egg surface becomes fluorescent. Several lines of evidence, including correlative scanning electron microscopic studies, indicate that the fluorescent sperm is, in fact, the fertilizing sperm which acquires fluorescence as a consequence of membrane fusion between the sperm and egg. Comparative studies show that several fluorochromes structurally related to H33342 can be used to selectively identify the fertilizing sperm at the egg surface and that H33258 possesses a distinct advantage when used to visualize the male and female pronuclei in eggs fixed prior to fluorochrome exposure. Finally, none of the fluorochromes tested in these studies have any discernible effect on development from the first cell division through the pluteus larva stage. These observations suggest that the fluorochrome‐transfer technique for identifying the fertilizing sperm may be useful in a wide variety of studies of gamete interaction as a simple and rapid cytological indicator for sperm‐egg fusion.
Purification of Mesodermal‐inducing Substances from Carp Swim Bladders I. Extraction and Isoelectric Focusing
Authors: ASASHIMA M. , NAKANO H. , MATSUNAGA T. , SUGIMOTO M. , TAKANO H.
Published: January 2005

Mesodermal‐inducing substances were isolated from the swim bladders of carp (Carassius auratus). These substances, extracted with 8 M urea, can induce the development of early gastrula ectoderm cells of newts into mesodermal tissues, such as muscles, notochords, mesenchyme, renal tubules, and so on, to a high degree. Isoelectric focusing was used to isolate them as a first purifying step, and five component parts with different pH ranges were obtained. Each part was tested for mesodermal‐inducing activity. On the basis of the extent and percentage of the induced tissues, two of them (parts C and D with pH ranges 6.0 to 7.0 and 7.0 to 9.0, respectively) were found to have high inducing activity, and the others had lower activity. The substances present in part C showed only low mesodermal‐inducing activity, when diluted with gamma‐globulin 100‐fold. Apparently highly active mesodermal‐inducing substances exist in the two parts C and D.
Half‐Life of Histone H4 mRNA in Xenopus laevis Embryonic Cells at Different Stages
Authors: ATSUCHI YASUO , TASHIRO KOSUKE , FU YUCHANG , SHIOKAWA KOICHIRO
Published: January 2005

By Northern blotting methods we have recently found that the relative amount of histone H4 mRNA per Xenopus embryo is constant during cleavage, doubles during the gastrula stage, then decreases at the neurula stage to the level slightly lower than that of the cleavage‐embryo. At each developmental stage, the level of mRNA will depend on three parameters: amount of maternal mRNA remaining, rate of the synthesis of the mRNA and rate of the decay of the mRNA. In the present experiment, we compared rates of the decay of H4 mRNA in Xenopus embryonic cells at different stages by pulse‐chase experiments with actinomycin D as an inhibitor of transcription.

Under the conditions used, the half‐life of H4 mRNA was estimated to be 90, 80, and 100 min, for the late blastula, late gastrula and neurula stages, respectively. The decay of H4 mRNA with a half‐life of 90 min at the late blastula stage predicts exhaustion of most of maternal H4 mRNA by the early gastruia stage. The slightly longer half‐life of H4 mRNA in neurula than in late gastrula cells suggests that H4 mRNA is more stable in neurula cells than in late gastrula cells, and therefore, the large decrease in the level of H4 mRNA observed at the neurula stage does not depend on the increase in the turnover‐rate of H4 mRNA. Probably, neurula cells synthesize less H4 mRNA than late gastrula cells but utilize it for longer time.
Scanning Electron Microscopic Study of the Initial Phase of Encapsulation in Samia cynthia ricini
Authors: TAKAHASHI SOHJI , ENOMOTO GINKO
Published: January 2005

When a piece of cellulose acetate membrane (CEA) was implanted into the haemocoel of a full‐grown larva of Samia cynthia ricini, a number of haemocytes became attached to it and extended cell processes. These cells all lysed within 4 min after implantation, with the appearance of a fibrous network on and near the tips of the processes. The network increased with inclusion of cell debris and finally coated the implant completely. Extensive lysis of spheroidal cells was seen on the outside of the network formed 20–60 min after implantation, together with flattened cells which were accumulating to form a capsule. The relationship of cell lysis and network formation is discussed.
pH in the Jelly Layer of Starfish Oocytes
Authors: CICCARELLI C. , RUSSO P. , DE SANTIS A. , DALE B.
Published: January 2005

Using liquid ion exchanger micro‐electrodes we have studied the pH in the vicinity of starfish oocytes and eggs and also in their jelly layers. Although highly variable, the pH in these zones was consistently lower (6.3–7.6) than the pH of sea water (7.8–8.2). Aged oocytes/eggs presented more acid environments than fresh oocytes. The pH indicator Phenol Red shows that the ovarian fluid is more acid than seawater, ranging from 6.0 to 7.5. Our data suggests that starfish oocytes, from their time in the ovary until several hr after their release, are surrounded by an acid environment. This micro‐environment may play a role in metabolic repression in the ovary and in the regulation of sperm‐egg interaction following spawning.
Early Reciprocal Interactions between Ectoderm and Chordamesoderm: Statistical Evaluation of Classical Embryological Experiments
Authors: LEYHAUSEN CLAUDIA
Published: January 2005

Embryos of the urodele Ambystoma mexicanum were used in time set experiments for the analysis of ectoderm‐chrodamesoderm interactions in “primary embryonic induction”. The influence of chordamesoderm‐age (CM‐age), ectoderm‐age (CM‐age), ectoderm‐mass (E‐mass) on differentiation processes were investigated. The results revealed that early interactions exist, which, however, seem to be dominated by the ectoderm in various respect: a) the segregations of the CNS is due to the autonomous change in ectoderm competence phases; b) based on the temporal sequence of competence phases, influences are exerted by the ecto‐neuroderm which support the chordamesoderm in its self‐differentiation capability to chorda and other mesodermal tissues. Consequently, reciprocal impulses from these newly differentiated tissues are warranted (feedback to ectoderm); c) co‐ordination of the cranialization effect and corresponding ectodermal competence phase is achieved; d) the inductive efficiency of the chordamesoderm is strongly supported.

Three statistical tests (regression, correlation, information analyses) were conducted (dependant variables: quality and quantity of the differentiated structures; independant variables: experimental factors). All three tests equally rendered statistical significance for the following order of decreasing importance the experimental factors play in induction and differentiation processes: E‐age, E‐mass, CM‐age.
Thinning of the basement Membrane and Localized Cell Proliferation during Gland Formation of the Chick Proventriculus
Authors: HAYASHI KENSUKE
Published: January 2005

The early processes of proventricular gland formation in the chick embryo were investigated. The glands appeared as intra‐epithelial invaginations of the proventricular endoderm on day 6 of incubation. By day 6.5 they began to protrude into the mesenchyme and elongated without branching until day 9. Before elongation of the glands, the immunofluorescence of laminin and the ultrastructure of the basal lamina were consistently observed in the intra‐epithelial invaginations as well as in other regions, and the mitotic activity in the gland rudiments was not different from that in other regions. However, at the tips of the elongating glands, little laminin was detected and the basal lamina were thin and discontinuous. The mitotic activity at the tip of the glands was higher than that in non‐glandular epithelium or in the stalk of the glands. These results suggest causal relationships between thinning of the basement membrane and localized epithelial cell proliferation at the tip of the elongating glands.
Internalization of Embryonal Carcinoma Cells when Aggregated with Normal Mouse Embryos
Authors: HANAOKA KAZUNORI , KONDO SHUNZO , HAYASAKA MICHIKO , KATO YOSHIHIRO
Published: January 2005

In the present study, we examined in detail the process of forming chimeric blastocysts between B242g embryonal carcinoma (EC) cells and normal mouse embryos. Electron microscopic observations of the developing aggregates revealed that the embryonic cells spread over the surface of the EC cells, resulting in the internalization of EC cells in the aggregates. When a single blastomere of an 8‐cell embryo was aggregated with EC cells, the blastomere spread over and engulfed the EC cells. These results strongly suggest that EC cells are segregated and become situated in the inside position as the development of an aggregate proceeds, and then they are incorporated into the ICM of a blastocyst.