bluuebunny/crossref_metadata_2025 huggingface dataset:
Differences in Fine Structures between Normal and RNA‐induced Drosophila Pole Cells
Authors: Yamazaki Yoshihito , Okada Masukichi
Published: January 2005

Fine structures were compared between normal pole cells and those induced in embryos that had been uv‐irradiated and then injected with intact polar plasm or with poly(A)+RNA extracted from cleavage embryos. Nuclei in nomal pole cells were spherical. In contrast, those in the induced pole cells were deformed to variable extents depending on materials injected with. Polar granules were smaller in pole cells induced by injection of poly(A)+RNA than in normal pole cells. The size of polar granules in polar‐plasm‐induced pole cells was intermediate between those in poly(A)+RNA‐induced and normal pole cells. Small polar granules were observed in posterior cells of embryos uv‐irradiated, nevertheless those cells were columnar and with identical morphology to somatic cells. Nuclear bodies showed a similar tendency in size differences as observed in polar granules in three types of pole cells observed.
The Murine Prepuberal Oviduct Supports Early Embryo Development In Vitro
Authors: Ménézo Yves , Hamidi Jamal , Khatchadourian Ch. , Nardon C.
Published: January 2005

Swiss albino mice were used to evaluate the ability of explanted murine oviducts to support development through the block stage. One cell eggs develop as well when cultured 50 hours in oviducts explanted from pregnant mice or in oviducts from immature mice: The blastocyst formation occurs at a similar rate in both cases. The viability of the blastocysts was high (8/9) when transferred to pseudopregnant (C 57 Black) recipient mice. Only a few difference was observed in the polypeptide pattern of immature and pregnant explanted oviduct. In immature oviduct, the polypeptide secretory profile is not modified by the presence of fertilized embryos transferred into it, and so nor is it directly egg dependent. It is concluded that oviduct's ability to sustain normal early embryonic developments is not dependent upon sexual maturity.
Expression of Neural Antigens in Normal Xenopus Embryos and Induced Explants
Authors: Itoh Keiji , Kubota Hiroshi Y.
Published: January 2005

Monoclonal antibodies were raised against neural tissues of Xenopus larvae. Three monoclonal antibodies, named NEU‐1, NEU‐3, and NEU‐4, were specific for neural tissue and first bound to neural cells at stage 25 after neural tube formation (NEU‐1 and NEU‐3) or at stage 31 (NEU‐4). These antibodies bound to differentiating neural cells, but not to germinal neuroepithelial cells. NEU‐1 and NEU‐3 recognized antigens in cell bodies as well as neural fibers of neural cells, and these antigens were distributed throughout the central nervous system. NEU‐4 bound to antigens in granular materials in neural cells, and these antigens were present in head and trunk regions but not in the tail region.

These three antibodies were used as neural markers in two types of induction experiments, in which 1) the animal pole region and the dorsal blastopore lip from stage‐10 gastrulae were combined, or 2) the animal pole region and the vegetal pole region from stage‐8 blastulae were combined. In both experiments, most conjugated explants expressed the NEU‐1, NEU‐3, and NEU‐4 antigens, although the expression of NEU‐4 antigen was delayed compared with those of the NEU‐1 and NEU‐3 antigens. These results show that these antibodies are useful as markers in neural induction experiments.
Binding of [3H]Nitrendipine to Proteins in the Plasma Membrane of Sea Urchin Sperm
Authors: Mikami‐Takei Kaori , Yasumasu Ikuo
Published: January 2005

The plasma membrane fractions of the sperm of four species of sea urchin, obtained by the method by Podell et al. (24), gave similar electrophoretic profiles of proteins. Several proteins in the membrane fraction from Hemicentrotus pulcherrimus bound [3H]nitrendipine, a specific antagonist of voltage‐dependent Ca2+channels, added at concentration of about 104times those reported to be effective in muscle and nerve cells. Nifedipine, a close analogue of nitrendipine, decreased the bindings of [3H]nitrendipine to 210, 140, 130 and 110 kDa and increased its bindings to several other proteins. Diltiazem, another type of Ca2+channel blocker, enhanced the bindings of [3H]nitrendipine to proteins of 210, 140, 130 and 110 kDa, and decreased its bindings to the other proteins. This effect of diltiazem on the binding of [3H]nitrendipine to proteins in the membrane fraction was similar to its effect on the mammalian excitable membrane fraction. The proteins whose binding to [3H]nitrendipine was blocked by nifedipine and enhanced by diltiazem are Ca2+channels.
The Control of Sea Urchin Metamorphosis: Ionic Effects
Authors: Cameron R. Andrew , Tosteson Thomas R. , Hensley Valentine
Published: January 2005

Because the cascade of events which comprise sea urchin metamorphosis occur rapidly, regulatory mechanisms able to respond in minutes must function. Employing sea water solutions of altered ionic composition in the presence or absence of metamorphically active microbial films, we tested the ability of particular ions to inhibit or enhance metamorphosis in competent larvae of the sea urchin, Lytechinus variegatus. At 40 mM excess potassium maximally induces normal metamorphosis in the absence of a microbial film. In the presence of metamorphically active microbial films, 40 mM excess magnesium inhibits the process. Increasing concentrations of calcium up to an excess of 40 mM stimulates larvae to undergo metamorphosis but in smaller proportions than similar concentrations of potassium. Divalent cation‐free sea water solutions are toxic to larvae. These studies support the hypothesis that ion fluxes are involved in the regulation of metamorphosis and reveal a complexity of response that parallels the histological complexity of competent echinoid larvae.
A Monoclonal Antibody Specific to Embryonic Trunk‐Lateral Cells of the Ascidian Halocynthia roretzi Stains Coelomic Cells of Juvenile and Adult Basophilic Blood Cells
Authors: Nishide Kazuhiko , Nishikata Takahito , Satoh Noriyuki
Published: January 2005

In spite of extensive knowledge on the structure and function of ascidian blood cells, little is known about their embryological origin. In the present investigation, the developmental fate of trunk lateral cells (TLCs) was explored using a specific monoclonal antibody. TLCs comprise a group of undefined embryonic cells of the ascidian Halocynthia roretzi, which arise from the A7.6 blastomeres of a 64‐cell embryo. The antigenicity first appeared at the middle tailbud stage in a pair of TLC‐clusters situated lateral to the brain stem of the bilaterally symmetrical embryo. The position and number of stained cells did not change during later embryogenesis until hatching. After hatching, the stained cells were found in the entire trunk region of the swimming larva. After metamorphosis, cells that expressed the antigen were present within the coelom and within the tunic layer of the juvenile. In addition, the antibody stained adult basophilic blood cells. These observations suggest a relationship of this group of embryonic cells with the prospective blood forming mesenchymal cells.
Scale Arrangement Pattern in a Lepidopteran Wing. 1. Periodic Cellular Pattern in the Pupal Wing of Pieris rapae
Authors: Yoshida Akihiro , Aoki Kiyoshi
Published: January 2005

In most species of lepidopteran insects, anteroposterior rows formed by scales are arranged at regular intervals in the adult wing; within each row two kinds of scales are alternately arranged. To investigate the cellular basis for the scale arrangement pattern, we examined cell arrangement in the epidermal monolayer of the pupal wing of a small white cabbage butterfly, Pieris rapae, by scanning electron microscopy and light microscopy.

The arrangement of scale precursor cells, closely resembling that of scales in the adult wing, was observed in the wing epidermis of the early pupa. Scale precursor cells are proximodistally elongated and form anteroposterior rows. Within a row two kinds of scale precursor cells are nearly alternately arranged, which is not so precise as the alternation of scales in the adult wing. Individual rows of scale precursor cells are separated by rows of single or double undifferentiated general epidermal cells. Occasionally, arrangement abnormalities occur both in the adult and the pupal wing. The cellular basis for the regular spacing of scale rows is discussed.
Spatial Patterns of Arylsulfatase mRNA Expression in Sea Urchin Embryo
Authors: Akasaka Koji , Ueda Takayuki , Higashinakagawa Toru , Yamada Kazumi , Shimada Hiraku
Published: January 2005

Previously we showed that the arylsulfatase (Ars) activity increases markedly after the mesenchyme blastula stage, and that the increase in its activity is due to initiation of the Ars gene transcription. In order to detect cells responsible for Ars mRNA production in the sea urchin embryo, we carried out in situ hybridization with the use of 35S‐labeled Ars RNA probe transcribed from Ars cDNA in a vector pT7T3‐19U. The Ars gene is uniformly expressed in the cells of blastula, and the expression becomes restricted to aboral ectoderm during development by gastrula stage.
A New Technique for Introducing Anti‐Fibronectin Antibodies and Fibronectin‐Related Synthetic Peptides into the Blastulae of the Sea Urchin, Clypeaster japonicus.
Authors: Katow Hideki
Published: January 2005

Migration of primary mesenchyme cells (PMCs) of the sea urchin, Clypeaster japonicus, was examined in vivo by introducing anti‐fibronectin (FN) IgG, and FN‐related synthetic peptides, Gly‐Arg‐Gly‐Asp‐Ser‐Pro‐Cys, Gly‐Arg‐Gly‐Asp‐Ser, and Arg‐Gly‐Asp‐Ser (RGDS) into the blastocoel by a new technique. In this technique the embryos are treated with cytochalasin B (CB) and part of the presumptive PMCs (PPMCs) is removed, leaving a hole in the vegetal plate. Then macromolecules are introduced into the blastocoel through this hole. Their introduction was confirmed by introducing them with polystyrene beads as a marker. The hole closes soon after introduction of the materials, and so these materials remain in the blastocoel. After this treatment, blastulae had fewer PMCs, because of partial loss of PPMCs, but their morphogenesis proceeded normally. Introduction of anti‐FN IgG or the synthetic peptides inhibited PMC migration in vivo, and this inhibition was associated with failure of the PMCs to form cell processes. These results indicate that sea urchin PMCs use the RGDS amino acid sequence in the FN molecule for migration in vivo.
Ultrastructural Studies on the Behavior of Centrioles during Meiosis of Starfish Oocytes
Authors: Kato Koichi H. , Washitani‐Nemoto Setsuko , Hino Akiya , Nemoto Shin‐ichi
Published: January 2005

The behavior of centrioles and ultrastructural changes of the nucleus were observed in maturing oocytes of the starfishes, Asterina pectinifera and Asterias amurensis. Observations were focused on the number and behavior of centrioles during two successive meiotic divisions. Examination of serial sections revealed that in meiosis I each division pole has a pair of centrioles, whereas in meiosis II each has only one centriole, confirming the observations by Sluder et al. (1989) on oocytes of Pisaster ocraceus and Asterias forbesi. The first polar body had two centrioles and the second polar body had only one. These results indicate that no duplication of centrioles occurs during the two successive meiotic divisions, and that the egg inherits one centriole from a primary oocyte.
The Acrosome Reaction in Ciona intestinalis (Ascidia, Tunicata)
Authors: Fukumoto Makoto
Published: January 2005

An acrosome reaction occurs by fusion between the acrosomal outer membrane and the plasmalemma enclosing the acrosome in Ciona intestinalis spermatozoa. The fusion seems to proceed along the peripheral margin of the acrosome, which causes vesiculation. The membrane bound vesicle formed by this process is probably shed by the sperm. The acrosomal inner membrane is exposed and becomes a part of the plasmalemma enclosing the anterior region of the sperm head. During this process, any acrosomal substance might be released through the opening formed by membrane fusion. The acrosome reaction most likely occurs in C. intestinalis spermatozoa, via vesiculation, in fundamentally the same way as observed in mammalian spermatozoa.
Close Correlation between the First Cleavage Plane and the Body Axis in Early Xenopus Embryos
Authors: Masho Rie
Published: January 2005

To examine whether the first cleavage plane demarcates the dorsal‐ventral axis (Klein, 1987) or it is random in relation to the plane of bilateral symmetry (Danilchik and Black, 1988), one blastomere of two‐cell‐stage Xenopus laevis embryos was labeled with injected fluorescein dextran amine. In resulting embryos, the first cleavage plane was represented by the boundary between the labeled and non‐labeled cells. In more than 90% of the embryos whose first cleavage plane had coincided with or inclined by 45 degrees to the plane of bilateral symmetry of surface pigmentation, the boundary fell within 30 degrees of the midline at the early gastrula stage. By contrast, in most embryos (10 out of 11) whose first cleavage plane was perpendicular to the plane of bilateral symmetry (up to 0.4% of a batch), the boundary fell between the dorsal and ventral regions. When randomly taken out from batches, more than 90% of the embryos had the first cleavage plane lying within 30 degrees of the dorsal midline at the initial gastrula stage. Thus the present results support neither Klein's nor Danilchik and Black's results, but confirm the currently accepted view that the first cleavage plane marks the dorsal midline with some range of deviation.
Selective Degradation of Specific Components of Fertilization Coat and Differentiation of Hatching Gland Cells during the Two Phase Hatching of Bufo japonicus Embryos
Authors: Yamasaki Hisashi , Katagiri Chiaki , Yoshizaki Norio
Published: January 2005

The embryonic hatching process in the toad, Bufo japonicus, consists of two phases: rupture of the outer jelly strings at stage 20 (neural tube) and an escape from the inner jelly layers and fertilization coat (FC) of individual embryos at stage 23 (tailbud). SDS‐PAGE analyses of FCs revealed that, of the eight major protein bands, two components with 58 K and 62 K in molecular weight gradually decreased from stage 18–19 on and totally disappeared at stage 22. When the FCs were treated with a hatching medium prepared by culturing denuded prehatching embryos, both 58 K and 62 K components of the FCs were solubilized, and in the solubilized materials 18 K and 31 K components appeared. Electron microscopy showed that a meshwork of filament bundles present in the FCs before stage 17 became dissociated at stage 19–20, and completely disappeared at stage 23, just before the hatching of embryos. Hatching gland cells (HGCs), an epidermal cell with numerous secretory granules, were first identified at stage 19, and underwent active secretion of the granules during stage 19–23. These results indicate that the hydrolytic degradation of 58K and 62 K components in FCs effected by the hatching enzyme constitutes the basic mechanism of embryonic hatching during both the first and second phases.
Statocyte and Ocellar Pigment Cell in Embryos and Larvae of the Ascidian, Styela plicata (Lesueur)
Authors: Ohtsuki Hisashi
Published: January 2005

The processes of formation of two pigmented cells, the statocyte and the ocellar pigment cell, in the cerebral vesicle of larvae of the ascidian Styela plicata were investigated in whole mount specimens and serial paraffin sections by light microscopy. The pigmentations of the two cells became visible simultaneously in embryos at the stage of tail elongation, 5–6 hr after fertilization. The pigmented cells were at first located side by side in the dorsal wall of the neurocoel. Growth of the pigment mass in the ocellus ceased at about 6.5 hr, while that in the statocyte continued through the hatching period (9–10 hr) up to the swimming stage. The pigment mass in the statocyte consisted of two blocks which joined together during their growth. The statocyte migrated from the dorsal to the ventral wall of the cerebral vesicle by the swimming stage. In swimming larvae, the more ventral of the two pigment blocks of the statocyte formed an inverted pigment cup and a cluster of protuberances projected into it from the ventral wall of the cerebral vesicle. Phylogenetically, the sensory organs in the cerebral vesicle of Styela plicata seem intermediate between those in Pyuridae and Botryllinae with respect of their structure and process of differentiation.
Do Peanut Agglutinin Receptors on Somites Control the Behavior of Neural Cells?
Authors: Asamoto Ken , Nojyo Yoshiaki , Aoyama Hirohiko
Published: January 2005

Peanut agglutinin (PNA) receptors are expressed in the caudal halves of sclerotomes in chick embryos after 3 days of incubation (stages 19–20 of Hamburger & Hamilton). The neural crest cells forming dorsal root ganglia (DRG) and motor nerves appear to avoid PNA positive regions and concentrate into rostral halves of sclerotomes. To investigate the role of PNA receptors in gangliogenesis and nerve growth, we examined PNA binding ability in quail sclerotomes and in chick‐quail chimeric embryos made by transplanting quail somites to chick embryos, comparing the development of DRG, motor nerves and sclerotomes. PNA did not bind to any part of the somites of 4.5‐day quail embryos, although dorsal root ganglia and motor nerves appeared only in the rostral halves of sclerotomes as in chick embryos. Moreover, in spite of no PNA binding ability of the transplanted quail somite in 4.5‐day chick‐quail chimeric embryos, DRG and motor nerves derived from chick tissues appeared only in the rostral halves of the sclerotomes derived from these somites. Thus, both quail and chick neural crest cells and motor nerves recognized the difference between the rostral and caudal halves of sclerotomes of quail embryos in the absence of PNA binding ability, indicating that PNA binding site on somite cells does not support the selective neural crest migration and nerve growth.
Absence of Species Specificity of Germinal Vesicle Factor Required for the Cytoplamic Cycle During Meiotic Division
Authors: Yamamoto Kenya , Nemoto Shin‐ichi
Published: January 2005

Germinal vesicles (GV) of starfish oocytes are known to contain some factor(s) indispensable for inducing cyclic cytoplasmic activity, which may be involved in driving meiotic cell cycle. We have examined species specificity of the factor by injecting enucleated oocytes of the starfish, Asterina pectinifera, with GV contents of either different starfish (Asterias amurensis, Astropecten scoparius) or sea cucumber (Holothuria moebi). The injected oocytes showed cyclic changes in cortical tension after treatment with 1‐methyladenine. Thus the nature of the factor appears similar among echinoderms. Growing oocytes of A. pectinifera much smaller (75% in diameter) than fully‐grown oocytes did contain the factor to rescue the enucleated oocytes, even though such small oocytes themselves did not respond to 1‐methyladenine. This result suggests that the factor begins to accumulate at the earlier stage of oogenesis.
Growth Competition between W Mutant and Wild‐type Cells in Mouse Aggregation Chimeras
Authors: Nakayama Hiroki , Ru Xiao‐Mei , Fujita Jun , Kasugai Tsutomu , Onoue Hitoshi , Hirota Seiichi , Kuroda Hideya , Kitamura Yukihiko
Published: January 2005

The dominant spotting (W) locus of the mouse has been demonstrated to be identical with the c‐kit proto‐oncogene. The c‐kit is strongly expressed in hematopoietic organs and the brain of mice. In homozygotes and double heterozygotes of the W mutant alleles (hereafter W mutant), development of erythrocytes, mast cells, melanocytes and germ cells is deficient. The deficiency of erythrocytes, mast cells and melanocytes is attributed to a defect of precursor cells, but the cause of the germ cell deficiency is not clear. We investigated the effect of the W mutation on proliferative potential of cells composing various organs by examining aggregation chimeras between W mutant and wild‐type (+/+) embryos. Proportions of +/+ components were significantly greater in the male germ cells and hematopoietic cells. In contrast, the average proportions of +/+ components were comparable to those of W mutant components in other organs including the brain. The present result suggests that the W (c‐kit) gene plays an important role in development of the male germ cells and hematopoietic cells and that it does not promote the proliferation of major cell population in the brain, in spite of the strong expression of the W (c‐kit) gene in the brain.
Homeodomain Binding Sites in the Promoter Region of Silk Protein Genes1
Authors: Hui Chi‐chung , Suzuki Yoshiaki
Published: January 2005

Several 10 bp AT‐rich repeats containing a core sequence TTAATT or its complement, AATTAA, in the distal part of the Bombyx mori fibroin gene promoter are known to bind a group of silk gland factors and to be required for a maximal level of transcription in a posterior silk gland extract. These repeats are similar to a consensus sequence (TCAATTAAAT) of the binding sites deduced for a group of Drosophila homeodomain proteins. By DNasel footprinting assay, two of these proteins, ZEN (zerknüllt) and EVE (even‐skipped), were shown to bind these sites with high affinity. The “TTAATT” core sequence was found to be important for ZEN and EVE binding by an electrophoretic mobility shift assay. Though these homeodomain proteins apparently recognize the same AT‐rich consensus sequence, we have identified a mutant sequence that enhances ZEN but not EVE binding. Alteration of sequence flanking the repeats was also found to affect the binding of these proteins. The binding of EVE and ZEN was shown to be facilitated by multiple repeats in the binding sites. Similar AT‐rich repeats can be found in the promoter region of other silk protein genes and, as shown here, two regions in the sericin‐1 gene promoter also bind these homeodomain proteins. The interaction of some homeodomain proteins and these homeodomain binding sites might be important for the developmental regulation of a group of silk protein genes.
Pigmentation Patterns of Mouse Chimaeras following Injection of Embryonic Cells into Postimplantation Embryos In Utero
Authors: Kajiwara Yuji , Kuwana Takashi , Inouye Minoru
Published: January 2005

Chimaeric mice were produced by introducing dissociated embryonic cells of C57BL/6N mice into the embryos of Jcl: ICR albino mice at mid‐gestation in utero. The patterns and the existence of pigmented areas were investigated over the long term. The pigmentation of the chimaeras was observed in several locational patterns; mainly in the head and the breast, rarely in the tail, the abdomen, the anterior and posterior trunk. During long‐term observation, the pigmentation became faint in 6 of 7 chimaeras and completely disappeared in 2 of 7 chimaeras 6 months after birth, as was true in our previous observation in rat/mouse chimaeras. The reason for this discoloration, however, is unknown at present; melanocytes derived from donor cells may have failed to function or have been eliminated. To examine the entry routes of injected cells into the embryos, pollen particles, similar to embronic cells in size, were injected as a donor material. The particles were localized mainly on the mid‐dorsal line in the head, and breast near fore‐limb buds 48 hr after injection. These patterns were similar to those of areas where the pigmentation were observed in the chimaeras. The results suggested that the cells were passively incorporated into embryos on the dorsal midline and the abdomen through the neural tube and somatopleure closure, respectively.
Histochemical Detection of Arylsulfatase Activity in Sea Urchin Embryos
Authors: Akasaka Koji , Akimoto Yoshihiro , Sato Mizuki , Hirano Hiroshi , Shimada Hiraku
Published: January 2005

Localization of arylsulfatase activity in the sea urchin embryo was determined histochemically by light and electron microscopy. Histochemical observations by light microscopy revealed that the arylsulfatase activity appears after the gastrula stage and that it is restricted to the cells of the aboral ectoderm. The enzyme activity is mainly located in the apical cellular cytoplasm and is associated with lysosome‐like structures that are frequently fused with yolk granules. Intense activity is also detected in the region of the endoplasmic reticulum and Golgi apparatus. No enzyme activity is found in the extracellular spaces of embryos.
Effects of Aphidicolin on Arylsulfatase Gene Expression in Sea Urchin Embryos
Authors: Akasaka Koji , Yamada Kazumi , Sekine Koji , Shimada Hiraku
Published: January 2005

Expression of the arylsulfatase (Ars) gene in sea urchin embryos begins just before hatching and ceases at the pluteus stage. Initiation of the Ars gene expression is inhibited by aphidicolin, which inhibits DNA synthesis without arresting the total RNA synthesis. Based on these finding it is supposed that DNA replication is a prerequisite for initiation of the Ars gene expression in developing sea urchin embryos.
Fertilization Membrane Formation in Sea Urchin Eggs Induced by Drugs Known to Cause Ca2+Release from Isolated Sarcoplasmic Reticulum
Authors: Fujiwara Akiko , Taguchi Katsunari , Yasumasu Ikuo
Published: January 2005

Ryanodine, miconazole, clotrimazole, doxorubicin, quercetin, halothane, caffeine and chloroform, which activate Ca2+‐induced Ca2+release from Ca2+stores, induced Ca2+release from a particulate fraction isolated from sea urchin eggs, Ca2+influx into eggs and formation of a fertilization membrane in an appreciable number of eggs. Their minimum effective concentrations for inducing a fertilization membrane increased in the order of these drugs listed above, and this order was also the same as that of their minimum effective concentrations for inducing Ca2+release from the isolated particulate fraction. Their effect in inducing a fertilization membrane was blocked by ruthenium red and procaine, which inhibit Ca2+release from Ca2+stores. Thus these drugs probably induced sufficient Ca2+release to make the cytosolic Ca2+level high enough in many eggs for formation of a fertilization membrane. In the absence of external Ca2+, fewer eggs treated with these drugs formed a fertilization membrane and more eggs did so on further treatment with either A23187 or carbonylcyanide‐p‐trifluoromethoxy‐phenylhydrazone (FCCP). Thus, a high level of Ca2+is probably derived from Ca2+release through Ca2+releasing channels (by A23187), from mitochondria (by FCCP) and its transport from the external medium.
Construction of a DNA Library Enriched with Mouse 4xChromosome of T(X;4)37H Translocation
Authors: Takahashi Ichiro , Ueda Takayuki , Kameoka Yosuke , Abe Kenji , Takagi Nobuo , Hashimoto Katsuyuki
Published: January 2005

Normal mouse chromosomes are routinely separated into only 5 peaks by the current flow cytometry. Since this limited resolution hindered isolation of the normal mouse X chromosome with an appropriate purity, we attempted to sort the mouse 4x chromosome, a larger translocation chromosome of T(X;4)37H, consisting of nearly the entire chromosome 4 and chromosome X by flow cytometry. To obtain a large number of cells having 4x chromosome for flow sorting, we isolated a somatic hybrid cell line MHH‐1 formed between S194 myelome cell line and normal splenocytes from a male mouse carrying T(X;4)37H. Flow karyotyping of propidium iodide‐stained chromosomes from MHH‐1 cell line revealed an additional peak containing 4x chromosomes at about 80%. DNA purified from sorted 4x chromosomes was cloned into phage lambda gtWES after complete digestion with EcoRl restriction endonuclease. Thus a 4x chromosome‐enriched library of about 4.4 × 104 recombinant phages was made and 13 single copy DNA clones specific to the X chromosome were isolated from the library so far.
Changes in Polyamine Contents Development of the Frog, Microhyla ornata
Authors: Joseph Kusumam , Baby Tholanikunnel G.
Published: January 2005

Putrescine, spermidine and spermine levels were measured during development, metamorphosis and adult life of the frog, Microhyla ornata. Development of Microhyla was accompanied by high fluctuating levels of putrescine and spermidine with low and steady levels of spermine. Putrescine was the major polyamine during development from egg to mature tadpole. During metamorphosis both putrescine and spermidine decreased significantly; but the decrease in putrescine content was more rapid than that of spermidine. Thus, in the freshly metamorphosed frog, the concentration of spermidine exceeded that of putrescine. In most of the adult tissues also spermidine concentration was higher than putrescine and spermine. While the free form of putrescine and spermidine increased during early development of the fertilized egg to tadpole, the levels of protein conjugated polyamines decreased. In the free form, putrescine was the major polyamine while in the protein conjugated form spermidine concentration was higher than putrescine and spermine. Thus polyamine pattern is different in early development, during metamorphosis and in differentiated adult tissues of this frog. ∞‐Difluoromethylornithine treatment at early blastula stage did not interfere with the normal development of Microhyla embryos.
Probable Contribution of Protein Phosphorylation by Protein Kinase C to Spicule Formation in Sea Urchin Embryos
Authors: Mitsunaga Keiko , Shinohara Shio , Yasumasu Ikuo
Published: January 2005

The formation of spicules and development of pluteus arms in sea urchin embryos were strongly blocked by H‐7 (1‐(5‐isoquinolinesulfonyl)‐2‐methylpiperazine dihydrochloride) but were not affected by HA1004 (N‐(2‐guanidinoethyl)‐5‐isoquinolinesulfonamide hydrochloride). Archenteron formation occurred normally in the presence of these compounds. Late gastrulae (28 hr after fertilization) were exposed to 32Pi for 30 min at 20°C, and then dissociated and their primary mesenchyme cells with spicules, embryo‐wall cells and archenteron cells were separated. Then, the radioactivities in the protein fractions of these separated cells were measured. Results showed that culture of embryos with H‐7 strongly inhibited 32p incorporation into proteins in primary mesenchyme cells but caused little inhibition of its incorporations in embryo‐wall cells and archenteron cells. The effective concentrations of H‐7 for inhibition of 32p incorporation were within the range that blocked spicule formation and growth of pluteus arms in embryos. HA1004 only slightly inhibited 32p incorporation into protein in mesenchyme cells, embryo‐wall cells and archenteron cells of embryos exposed to 32Pi. Protein kinase C activity was detectable only in isolated primary mesenchyme cells associated with spicule structures. These suggest that phosphorylation of proteins by protein kinase C contributes to the formation of spicule structures.
Localization and Characterization of Lectins in Yolk Platelets of Xenopus Oocytes
Authors: Yoshizaki Norio
Published: January 2005

The localization and characteristics of yolk platelet lectins (YLs) in Xenopus laevis oocytes were studied with antiserum against cortical granule lectins (CGLs) as a probe. In oocytes at stages I, II and III‐IV, specific, immunofluorescent staining for the lectins was observed on the cortical cytoplasm extending about 2, 4 and 20 μm, respectively, from the egg surface. In stage III‐IV oocytes, the superficial layer of the yolk platelets was also stained. The cortical cytoplasm included cortical granules, coated pits, coated vesicles, multivesicular bodies and primordial yolk platelets. The YLs were incorporated into the oocytes by endocytosis as demonstrated using gold‐labeled YLs. On PAGE, native YLs gave two bands of CGL‐like proteins and proteins that appeared as a single diffuse band. The YLs and the CGLs shared antigenicity and hemagglutination activity specific to D‐galactoside residues. However, the proteins of the diffuse band had little or no activity for either hemagglutination or jelly‐precipitation, suggesting that they were monomers with a single reactive site. These results indicate that the YLs are supplied to the oocytes, presumably from extracellular sources, polymerized to CGL‐like molecules in the cortical cytoplasm and accumulated in the superficial layer of the yolk platelets.
Organization of the Hamster Cumulus Extracellular Matrix: A Hyaluronate‐Glycoprotein Gel which Modulates Sperm Access to the Oocyte
Authors: Cherr Gary N. , Yudin Ashley I. , Katz David F.
Published: January 2005

The mammalian oocyte‐cumulus complex contains an extracellular matrix rich in hyaluronate. Recently, the microstructure of the hamster cumulus extracellular matrix was described (52). In the present work, we investigated the organization of this matrix. We employed freeze‐substitution methodologies to investigate ultrastructural effects of various treatments, including sperm enzymes, on the matrix. Protease treatment resulted in disruption with a loss of the fibrillar structures and some expansion; in contrast, hyaluronidase treatment completely solubilized the matrix. EDTA extraction revealed that the fibrils are composed of fine filaments. A discrete region of the matrix immediately surrounding the oocyte, the corona radiata, was resistant to EDTA disruption. We found that hyaluronate is an ubiquitous constituent of the microstructural elements of this extracellular matrix. The matrix exhibits a carbohydrate:protein ratio of approximately 2:1. SDS‐PAGE revealed that glycosylated polypeptides are bound to the matrix. The lectins LCA and WGA had differing affinities for these polypeptides, and bound ubiquitously to the intact matrix. The present data suggest that glycoprotein‐hyaluronate interaction is critical for maintenance of the cumulus extracellular matrix microstructure and for its physical properties.
Fusion of Neural Folds in the Rhombencephalic Region of Rat Embryos
Authors: Matsuda Motoko
Published: January 2005

The progression of fusion of neural folds in the rhombencephalic region of rat embryos was examined using actin‐specific antibody and scanning electron microscopy. In the region of unfused neural folds, actin was distributed over the entire luminal surface of the neural plate. Subsequently, the actin became localized on the luminal surface of outwardly opened, dorsolateral portions of the neural plate. Reduction in the area over which actin was distributed corresponded to the decrease in the area of the luminal surface of outwardly opened, dorsolateral portions of the neural plate. Decrease in the area of the luminal surface synchronized with elevation of the neural folds and their approach to the midline and, finally, actin became localized in areas of the apices of the neural plate that formed the bridge between the two opposing neural folds. These results suggest that formation of the bridge is orchestrated and supported by microfilaments. The procession of fusion in the rostral direction may cause approach of the neural folds to the middle, and this approach may be guaranteed by reduction in the area of the luminal surface over which actin is distributed.
Spermiation in the Mouse: Contribution of the Invading Sertoli Cell Process to Adluminal Displacement of the Spermatid Head
Authors: Sakai Yasuhiro , Yamashina Shohei
Published: January 2005

At the maturation phase of spermiogenesis in mice, the spermatid heads that are embedded deeply in the epithelium of the seminiferous tubules dislocate toward the luminal surface. In the present study, to clarify the manner in which the spermatid head is displaced toward the lumen, morphological changes in spermatids and Sertoli cells were examined on ultrathin and thick sections stained with adenosine triphosphatase cytochemistry. During adluminal displacement, the spermatid head is surrounded by an invading process of Sertoli cell which invaginates into the spermatid cytoplasm to form complicated passages called the canal complex. At the site of the spermatid head, the wall of an invading Sertoli cell process folds to form a sheath in which the spermatid head is located. The sheath correspond to a structure known as ectoplasmic specialization. The invading Sertoli cell process also shows branching and swelling at the site where spermatid heads are present. The present results suggest that the canal complex is directly involved in the adluminal displacement of the spermatid head. Dynamic changes of invading Sertoli cell processes may produce the motive force for adluminal displacment of the spermatid head. Also, ectoplasmic specialization may contribute to the adluminal displacement of the spermatid possibly by mediating cell to cell interaction between the spermatid nucleus and the invading Sertoli cell process.
Cell‐division inhibitor produced by a killer strain of cellular slime mold Polysphondylium pallidum
Authors: Mizutani Akiko , Yanagisawa Kaichiro
Published: January 2005

The killer strain CK‐8 of cellular slime mold Polysphondylium pallidum produces a cell‐division inhibitor, in addition to a killer factor. This inhibitor represses cell division of many strains and species of cellular slime molds, except CK‐8 itself and its complementary mating‐type strain. It is sensitive to both heat and trypsin, and capable of binding to Con A. Its apparent molecular mass is more than 100 kDa. Repression of cell division by this inhibitor is reversed by trypsin treatment of the cells.
The Blood Island is a Site of Formation of the Primary Embryo Thrombocyte in the Chick Blastoderm
Authors: Tahara Yutaka , Morinaka Toshiyuki
Published: January 2005

Using the immunohistological technique we inquired at what developmental stage and in which site of chick blastoderm does the embryo thrombocyte (ET) begin to differentiate. An anti‐ET antibody was raised against rabbits by injecting ETs isolated from blood of 10 day chick embryos. By applying the indirect staining method to smear preparations of blood collected from developing embryos it was confirmed that cytoplasm of the ET showed more intense staining than that of the erythroid cell and that the ET population could be distinguished from the erythrocyte population by this antibody. Cells showing the intense staining could be detected first in blood islands of the area opaca vasculosa of stage 9+ blastoderms. These embryo thromboblasts were found singly or in groups of a small number at dorsal periphery of cell clusters in the blood island. The electron microscopy revealed that embryo thromboblasts appeared in the same position in the stage 9+ blastoderm. At stage 10+ or later embryo thromboblasts were also present adhering to the vascular endothelium or free in the vessel lumen. We conclude that ETs start differentiating from primitive mesenchymal cells localized in the blood island of the area opaca vasculosa at stage 9 or earlier, migrate thereafter to vessel lumen, and enter the blood stream.
Gavity and Microtubules in Dorsoventral Polarization of The Xenopus Egg
Authors: Zisckind Nava , Elinson Richard P.
Published: January 2005

In Xenopus laevis, the dorsoventral axis of the embryo is specified by a 30° relative rotation between the cortex and the cytoplasm of the fertilized egg, and a cortical array of parallel microtubules may be part of the rotation machinery (7). The parallel microtubules are aligned with the sperm entry point in most of the eggs as expected, since the dorsoventral axis is usually defined by the sperm entry point. We show that gravity can play two roles in the formation of the dorsoventral axis. First, a simple 90° tilt off‐axis before the start of the rotation overcomes the influence of the sperm and determines the orientation of the parallel microtubules. Second, a 90° tilt off‐axis can specify the dorsoventral axis even in the absence of the parallel microtubules. Therefore, gravity can affect dorsoventral polarity by orienting the parallel microtubules or by moving cytoplasm directly without microtubules.
Homoiogenetic Neural Inducing Activity of the Presumptive Neural Plate of Xenopus Laevis
Authors: Grunz Horst
Published: January 2005

Heteroplastic combinations were made between Xenopus laevis presumptive neural plate and competent ectoderm of Xenopus borealis. Primarily induced presumptive neural plate cells (Xenopus laevis) can easily be distinguished from Xenopus borealis cells by specific quinacrine fluorescence of the nuclei. It was clearly shown that presumptive neural plate, which has primarily been induced by the underlying chordamesoderm exerts homoiogenetic inducing activity on competent ectoderm. The inducing activity is increased in pieces of presumptive neural plates, when the superficial layer has been removed from the adjacent deep layers. The enhancement can be explained by the fact that the removal of the superficial layer acting as barrier allows the inducing stimulus to be easily propagated from the apical (distal) side of the deep layers of the presumptive neural plate.
Immunohistochemical Localization of Prothoracicotropic Hormone‐Producing Neurosecretory Cells in the Brain of Bombyx mori
Authors: Mizoguchi Akira , Oka Tadanori , Kataoka Hiroshi , Nagasawa Hiromichi , Suzuki Akinori , Ishizaki Hironori
Published: January 2005

A monoclonal antibody that recognized the Bombyx prothoracicotropic hormone (PTTH) was produced by immunizing mice with a synthetic pentadecapeptide corresponding to the amino‐terminal portion of Bombyx PTTH. The antibody recognized both intact and reduced forms of PTTH. Immunohistochemistry with this antibody has demonstrated that PTTH is produced by two pairs of dorso‐lateral neurosecretory cells of the brain and transported to the corpora allata by axons running through the contralateral hemisphere of the brain. Immunoreactive axon terminals in the corpora allata were localized between the glandular cells, suggesting that PTTH is released at the inner part of this organ.
Mechanism of Dorso‐Ventral Axis Specification in Nuclear Transplanted Eggs of Xenopus laevis
Authors: Satoh Hiroyuki , Shinagawa Atsunori
Published: January 2005

The rotation of subcortical cytoplasm relative to egg surface (“subcortical rotation”, 28) is known to specify the dorso‐ventral axis of fertilized Xenopus eggs. However, except for brief descriptions (14, 15), no information has been provided on the process of the dorso‐ventral axis specification in nuclear transplanted Xenopus eggs. This study shows that (i), among the events that occur in association with dorso‐ventral axis specification in fertilized Xenopus eggs, only the subcortical rotation occurs in a normal manner in both nuclear transplanted and non‐nuclear transplanted (enucleated) Xenopus eggs, (ii), in these eggs, there is no factor that appears to affect the direction of subcortical rotation as the sperm entry point does in fertilized eggs: neither the site of pricking for activation nor that of injection for nuclear transplantation affects the direction of the subcortical rotation in those eggs and (iii) the subcortical rotation in nuclear transplanted eggs predicts very precisely the direction of their dorso‐ventral axis as it does in fertilized eggs. These results suggest that matured Xenopus oocytes have a cytoplasmic potential to induce subcortical rotation along a particular meridian in the absence of a sperm aster and that the subcortical rotation induced by this cytoplasmic potential specifies the dorso‐ventral axis in nuclear transplanted Xenopus eggs as in fertilized eggs.
Micromere Differentiation in the Sea Urchin Embryo: Immunochemical Characterization of Primary Mesenchyme Cell‐Specific Antigen and Its Biological Roles
Authors: Shimizu‐Nishikawa Keiko , Katow Hideki , Matsuda Ryoichi
Published: January 2005

Primary mesenchyme cell (PMC)‐specific antigens in developing sea urchin embryos of five different species have been studied by using two different monoclonal antibodies, P4 and B2C2. Like B2C2 in Strongylocentrotus purpuratus (Anstrom et al., 1987) P4 reacted with the N‐linked carbohydrate in Strongylocentrotus intermedius embryo. Although both antibodies recognize the same group of glycoproteins in S. intermedius, P4 epitopes appeared earlier than B2C2 epitopes in Clypeaster japonicus embryo. PMCs of Anthocidaris crassispina blastulae raised in sulfate‐deficient sea water were immuno‐reactive with P4 but not with B2C2, although the embryos raised in normal sea water reacted with both antibodies at similar intensity. These results suggest that the epitopes of P4 and B2C2 are formed by glycosylation and sulfation, respectively. PMCs may display differential modification in their surface glycoprotein synthesis during differentiation. Furthermore, P4 inhibited cultured micromere descendant cells of Hemicentrotus pulcherrimus from attaching to the plastic dishes and forming spicules in vitro without detectable cytotoxic effect. P4‐reactive glycoproteins may play important roles in cell‐substrate interaction and spicule formation.
Developmental Changes in the Bombyxin‐and Insulin‐like Immunoreactive Neurosecretory System in the Wax Moth, Galleria mellonella
Authors: Žitňn Dušn , Sehnal František , Mizoguchi Akira , Ishizaki Hironori , Nagasawa Hiromichi , Suzuki Akinori
Published: January 2005

Four medial neurosecretory cells (MNC) and 4 lateral neurosecretory cells (LNC) in each brain hemisphere, and one pair of cells in each thoracic ganglion (TG) of Galleria larva react with antibodies against bombyxin and insulin. Material secreted from the MNC and LNC is released mainly in the corpora allata, and that from the TG through the ventral median nerves. Intrinsic secretory cells of the corpora cardiaca (CC) also contain bombyxin‐like, but not insulin‐like material. The immunoreactivities all disappear during molts and reappear with resumption of feeding. In the MNC and TG they reappear for less than a day, but in cells of the CC immunoreactivity reappears for the whole feeding period. Before pupation, the LNC become temporarily immunopositive towards the end of feeding period, and the MNC and TG during the wandering period, i.e. at the time of prothoracic gland stimulation. Immunoreactivity disappears during the pupal molt. In pupae it is present in the 4 pairs of MNC and 1–2 pairs of LNC 12–48 hr after ecdysis, and in cells of the CC from 12 hr after ecdysis until the end of the pupal instar. In adult, immunoreactivity is restricted to 2 pairs of the LNC and to CC cells.
Does Protein Phosphorylation by Protein Kinase C Support Pseudopodial Cable Growth in Cultured Micromere‐Derived Cells of the Sea Urchin, Hemicentrotus pulcherrimus?
Authors: Mitsunaga Keiko , Shinohara Shio , Yasumasu Ikuo
Published: January 2005

In cultured cells derived from micromeres, H‐7 strongly inhibited the outgrowth of pseudopodial cables and the formation of spicule rods at concentrations around the Ki values for protein kinases. HA1004 did not inhibit the cable growth and spicule rod formation in these cells at higher concentrations than the Ki values for cyclic nucleotide‐dependent protein kinases. Pseudopodial cable growth was also inhibited by H‐7 in furosemide‐treated cells which were able to undergo normal growth of the cables without the formation of spicule rods. Protein phosphorylation, measured by 32P incorporation into proteins in the cells exposed to 32Pi, was inhibited by H‐7 at the concentrations for the blockage of the cable growth but was hardly blocked by HA1004. The cable growth and protein phosphorylation were activated by phorbol 12‐myristate 13‐acetate. The activity of Ca2+, phospholipid‐dependent protein kinase (protein kinase C), which was inhibited by H‐7, became appreciably high in micromere‐derived cells at 16 hr of culture at 20°C, at which the outgrowth of pseudopodial cables was going to be initiated and gradually increased keeping pace with the cable growth. These suggest that the outgrowth of the cables is supported by protein phosphorylation catalyzed by protein kinase C.