bluuebunny/crossref_metadata_2025 huggingface dataset:
Nuclear Basic Proteins of Xenopus laevis Sperm: Their Characterization and Synthesis during Spermatogenesis
Authors: Yokota Toshio , Takamune Kazufumi , Katagiri Chiaki
Published: January 2005

Nuclear basic proteins from morphologically and functionally mature sperm of Xenopus laevis were analyzed by acid/urea/Triton X‐100 polyacrylamide gel electrophoresis (AUT‐PAGE). Six sperm‐specific proteins (SP1‐6) were identified in addition to somatic histones H3, H4 and smaller amount of H2A and H2B, but not H1. Of these, SP3–6 were unique in containing 33–41% arginine and having very low lysine/arginine ratios, while SP2 was more similar to H3 and H4 in having a lower arginine and higher lysine content. Fractionations of testicular cells at different spermatogenic stages by unit gravity sedimentation showed that primary spermatocytes and acrosomal vesicle spermatids possess typical somatic type histones but no SPs. Injection of [14C]‐arginine into the testis and its tracing by fluorography on AUT‐PAGE gels indicated that all somatic histones are synthesized during the stages between spermatogonia and primary spermatocytes, whereas SPs are synthesized at differentially regulated rates during the stages after acrosomal vesicle formation. In indirect immunofluorescence studies with anti‐SP3‐5 rabbit antiserum, a positive reaction was observed in the last step of spermiogenesis after the commencement of nuclear coiling.
Immunoelectron Microscopic Localization of Sperm‐specific Nuclear Basic Proteins during Spermatogenesis in Anuran Amphibians
Authors: Moriya Megumi , Katagiri Chiaki
Published: January 2005

Antisera were raised in rabbits against sperm‐specific nuclear basic proteins (SPs) of Bufo japonicus and Xenopus laevis. The localizations of these proteins in spermatogenic cells were then studied by electron microscopy with colloidal gold labeled antibodies as probes. The numbers of gold particles counted on ultra‐thin sections of cells at various spermatogenic stages were corrected for the density per unit area, on the basis of areas determined with a digitizer. No grains were deposited during early nuclear elongation stages. Grains appeared on nuclei at the beginning of chromatin granulation, and their density increased first gradually and then sharply at the last step of spermiogenesis. Recalculation of grain counts according to the estimated nuclear volumes of Bufo spermatogenic cells also indicated a sharp increase in the amount of SPs per nucleus in the last step of spermiogenesis. No significant localization of grains in the cytoplasm was observed at any stage of spermatogenesis.
Tissue Distribution of N‐myc Expression in the Early Organogenesis Period of the Mouse Embryo
Authors: Kato Kazuto , Kanamori Akira , Wakamatsu Yoshio , Sawai Shoji , Kondoh Hisato
Published: January 2005

N‐myc expression in the mouse embryo was examined in its organogenesis period. Northern blot analysis of total RNA of embryos from 9.5 days to 17.5 days of gestation indicated that N‐myc mRNA level was the highest at 9.5 days and decreased as development proceeded. Tissue distribution of N‐myc expression in 9.5 day embryos was histologically analyzed by in situ hybridization of the transcripts and immunofluorescent staining of N‐myc protein. In addition to the central nervous system indicated in previous studies on embryos of different stages, we found N‐myc expression in various developing tissues. Neural crest‐derived tissues generally expressed N‐myc transcripts and proteins to significant levels, e.g. facial primordia, visceral arches and dorsal root ganglia. Among mesodermal tissues, N‐myc expression was especially high in the migrating sclerotomes derived from caudal halves of the somites, primitive nephric tubules, and mesenchymes condensed around the digestive tract and in the limb buds. Expression in endodermal tissues, however, was very low. In situ hybridization and immunohistology gave consistent results, confirming the authenticity of the detection of N‐myc expression.
Possible Patterns of Differentiation in the Primitive Ectoderm of C3H/HeNBALB/cA Chimeric Blastocysts: An Inference from Quantitative Analysis of Coat‐Color Patterns
Authors: Tachi Chikashi , Yokoyama Minesuke , Yoshihara M.
Published: January 2005

Coat‐color patterns of 20 C3H/HeN++BALB/cA chimeras produced by using the same colony of mice in a series of experiments, were quantitatively analyzed, by means of a video‐image analysis system developed by Tachi (1988, 1989). In those chimeras, proportion of the two allogeneic components, i.e., C3H/HeN and BALB/cA, was relatively well balanced between the right and the left halves of the pelts, whereas between the anterior and the posterior regions it was unbalanced in favor of BALB/cA components in the anterior region. To interpret the results, a computerized geometrical model intended to simulate the hypothetical conditions of chimeric germ layers during early embryogenesis, was constructed. From the model, it was proposed that the seemingly high selection pressure for the BALB/cA components in the anterior region of this particular group of chimeras, might have been caused because the initial steps of the determination of the cranio‐caudal axis took place in the regions of the primary ectoderm where BALB/cA, rather than C3H/HeN blastomeres were predominant. Possible variations in the alleles of the genes regulating the germ layer differentiation, might conceivably be the cause for the observed tendencies.
Yolk Degradation in Tick Eggs: III. Developmentally Regulated Acidification of the Yolk Spheres
Authors: Fagotto Françis
Published: January 2005

Yolk spheres in tick eggs contain a latent procathepsin L, which is activated in vivo, in parallel with yolk degradation, and in vitro by acid treatment (Fagotto, F., Arch. Insect Biochem. Physiol., 1990b in press). Mature cathepsin L hydrolyzes vitellin at acidic pH (Fagotto, F., Arch. Insect Biochem. Physiol., 1990a in press). Here, yolk spheres' pH has been estimated using acridine orange. In the early development, all yolk spheres are neutral, then an increasing number acidify, until hatching, where general acidification seems to occur. This fits well with vitellin utilized slowly during embryogenesis, more intensely at hatching (Chinzei, Y. and I. Yano, Experientia 41, 948, 1985), and can be related to sequential degradation of individual spheres observed during embryonic development, then extensive yolk liquefaction in the larva. Different yolk spheres populations have been separaded on Percoll density gradients. In freshly laid eggs, yolk spheres are dense, neutral, undegrated and contain exclusively the precursor of cathepsin L. In later stages, yolk spheres are progressively recovered in lower density fractions, displaying acidic interior and cytological signs of degradation. They cosediment with mature cathepsin L. It is concluded that acidification initiates yolk degradation through procathepsin L activation.
Receptors for Sperm‐activating Peptides, SAP‐I and SAP‐IIB, on Spermatozoa of Sea Urchins, Hemicentrotus pulcherrimus and Glyptocidaris crenularis
Authors: Harumi Tatsuo , Yamaguchi Masaaki , Suzuki Norio
Published: January 2005

Sperm‐activating peptide analogues, GYGG‐SAP‐I (Gly‐Tyr‐Gly‐Gly‐Gly‐Phe‐Asp‐Leu‐Asn‐Gly‐Gly‐Gly‐Val‐Gly) and GYGG‐SAP‐IIB (Gly‐Tyr‐Gly‐Gly‐Lys‐Leu‐Cys‐Pro‐Gly‐Gly‐Asn‐Cys‐Val) which exibit almost the same respiration‐stimulating activity as the respective original peptide were chemically synthesized, radiolabeled with Na1251, and used for experiments of binding and cross‐linking to Hemicentrotus pulcherrimus or Glyptocidaris crenularis spermatozoa. In competitive binding, SAP‐I caused 50% decrease in 125l‐GYGG‐SAP‐l binding to intact spermatozoa, sperm heads and sperm tails of H. pulcherrimus at the concentrations of 282 nM, 3 nM and 141 nM, respectively. The incubations of H. pulcherrimus sperm tails with 125l‐GYGG‐SAP‐I and the chemical cross‐linking reagent, disuccinimidyl suberate, resulted in the appearance of two radiolabeled bands with apparent molecular masses of 71 kDa and 63 kDa (determined by sodium dodecyl sulfate‐gel electrophoresis under reducing conditions). 125l‐GYGG‐SAP‐IIB bound almost equally to sperm heads and sperm tails of G. crenularis, and was cross‐linked to 172 kDa, 62 kDa and 58 kDa proteins in sperm heads, and 157 kDa and 62 kDa proteins in sperm tails with disuccinimidyl suberate.
Analysis of Hardening of the Egg Envelope (Chorion) of the Fish, Oryzias latipes
Authors: Masuda Kaori , Luchi Ichiro , Yamagami Kenjiro
Published: January 2005

Hardening of the chorion of medaka eggs was quantitated in terms of the solubility of its constituent proteins. After activation of unfertilized eggs with the Ca2+‐ionophore A23187, hardening of chorion (named ionophore‐activation hardening) proceeded and 60 min after activation the solubility of the proteins in 1 N NaOH had decreased to 20% of that of proteins of unhardened chorions. On SDS‐PAGE, the chorions of unfertilized eggs gave four major protein bands (150, 83, 78 and 51 K). After Ca2+‐ionophore activation, new two protein bands (135 and 61 K) appeared, with concurrent disappearance of all the original bands except the 51 K band. Isolated chorions of unfertilized eggs were also hardened by Ca2+and 60 min after addition of Ca2+the solubility of their proteins in 1 N NaOH had decreased to about 45% of that originally. During this type of hardening (named ‘Ca2+‐hardening), however, the SDS‐PAGE pattern of the proteins remained unchanged. Therefore, there are two mechanisms of hardening. The ‘ionophore‐activation hardening was inhibited by cadaverine. When chorions were isolated 20 min after Ca2+‐ionophore activation and kept in Ca2+‐free conditions, the ‘ionophore‐activation hardening process was arrested: further hardening was resumed on addition of Ca2+to the medium. These results suggest the presence of some hardening machinery in isolated chorions.
Change in Component Proteins of the Egg Envelope (Chorion) of Rainbow Trout during Hardening
Authors: Luchi Ichiro , Masuda Kaori , Yamagami Kenjiro
Published: January 2005

Egg envelope (chorion) of unfertilized eggs of rainbow trout, Oncorhynchus mykiss, consists of 4 major protein components whose molecular weights are approximately 110 K, 64 K, 56 K and 50 K. On hardening of the chorion after activation, 64 K, 56 K and 50 K components disappeared, some components higher than 160 K were newly formed and the solubility of the chorion in 1 N NaOH or 12 M urea‐2% SDS‐1% 2‐mercaptoethanol (Sample Buffer) decreased. This implies that probable formation of covalent crosslinks between the constituents occurs during the hardening.

Hardening occurred also in the chorion isolated from the unfertilized eggs. The conversion of the 64 K, 56 K and 50 K components into the higher molecular weight intermediates was Ca2+‐dependent, accelerated by 2‐mercaptoethanol and inhibited by a high temperature (60°C). Optimum pH of this hardening system was acidic, i.e., 5.0 to 6.0.

Cadaverine inhibited the change in solubility of chorion in NaOH or Sample Buffer, while it could not inhibit the disappearance of the 64 K, 56 K and 50 K components and the new formation of the higher molecular weight intermediates. This observation indicates existence of two processes in chorion hardening, which are tentatively named cadaverine‐insensitive and cadaverine‐sensitive processes.
Activation of Ca2+ Transport System of Sea Urchin Sperm by High External pH: 220 kD Membrane Glycoprotein is Involved in the Regulation of the Ca2+ Entry
Authors: Sendai Yutaka , Aketa Kenji
Published: January 2005

When sperm of the sea urchin, Hemicentrotus pulcherrimus, were exposed to high pH (9.0) sea water, they showed large increases in intracellular Ca2+ ([Ca2+]i) and pH (pHi) and underwent the acrosome reaction (AR) without the aid of the egg jelly. Not only [Ca2+]i increase but also pHi rise did not occur under Ca2+‐free conditions. Both the increases in [Ca2+]i and pHi and the AR by high pH were inhibited by a Ca2+ channel blockers, verapamil and nisoldipine, and by a lectin, wheat germ agglutinin (WGA) which interacts with a 220 kD membrane glycoprotein of sperm. These reagents inhibited also the AR by the egg jelly. The inhibitory effects of WGA were immediately canceled by the addition of N‐acetyl‐D‐glucosamine, a sugar which is known to remove WGA from its binding site. These results suggest that 1) the same Ca2+ transport system is activated by high external pH and the egg jelly, 2) increase in [Ca2+]i is prerequisite for the stimulation of the H+‐efflux system(s) and 3) the 220 kD WGA‐binding membrane protein functions as a regulator protein of Ca2+ transport system.
Change of Rat Embryos from a Ventrally Concave U‐Shape to a Ventrally Convex C‐Shape
Authors: Matsuda Motoko
Published: January 2005

In an early stage of development in murine embryos, axial rotation occurs and the body axis changes from a ventrally concave U‐shape to a ventrally convex C‐shape. In this study, axial rotation in Sprague‐Dawley rat embryos occurred in about 5 h in vitro (from 27 h to 32 h in cultures of head‐fold stage embryos). In sagittal sections, the somites in the mid‐region of the body changed from a trapezoidal shape with a short dorsal side and long ventral side to the reverse trapezoidal shape with a long dorsal side and a short ventral side. The dorsal part of these somites acquired the ability to react with actin‐specific antibody and developed into dermatome. On treatment with 0.1 μg/ml cytochalasin D during this 5 h period, embryos became ventrally concave with two lordosis bends. The somites in the bends had a short dorsal side, which did not show any evidence of dermatome or intense immunocytochemical staining. These results suggest that the increase in length of the dorsal side of the somites is a cause of the axial rotation and that the organization of actin filaments plays an important role in the conformational change of the somites.
Involvement of Cell Surface Carbohydrates in the Sexual Cell Fusion of Dictyostelium discoideum
Authors: Ishikawa Tomo‐o , Urushihara Hideko , Yanagisawa Kaichiro
Published: January 2005

In order to analyze the molecular mechanism of sexual cell fusion between cells of HM1 and NC4 (opposite mating type strains in Dictyostelium discoideum), monoclonal antibodies were raised against partially‐purified gp 70, a fusion‐related protein of HM1 cells. The antibodies were screened for activity to inhibit cell fusion and 9 hybridoma clones were obtained. One of the fusion‐blocking monoclonal antibodies, mAb1G7, was used for further analysis. It recognized nearly ten bands in an immunoblot of fusion competent HM1 cells, but no bands when HM1 membrane proteins had been deglycosylated. These results suggest the importance of carbohydrates in the cell fusion process. To confirm this possibility, effects of sugars or lectins on cell fusion were examined. Although inhibition by the sugars was incomplete, Con A, WGA, LCA, strongly inhibited cell fusion. Furthermore, tunicamycin inhibited the acquisition of fusion competence in HM1 cells, indicating the importance of N‐linked glycosylation of proteins in cell fusion. All above results suggest that N‐linked carbohydrates on HM1 cell surface are involved in the sexual cell fusion of D. discoideum.
The Successive Steps in the Pattern Formation of the Amphibian Central Nervous System
Authors: Nieuwkoop Pieter D.
Published: January 2005

Removal of the presumptive midbrain region of the central nervous system (C.N.S.) from stage 12/13 or 14 axolotl embryos, with or without removal of the underlying section of the archenteron roof, leads to a reformation of the midbrain when the presumptive fore‐ and hindbrain regions of the neural plate are brought into direct contact with each other. The younger the stage when the operation is performed, the more complete the reformed midbrain. These experiments suggest that the midbrain is formed by a spatial interaction between two preexisting formed domains, namely, the presumptive forebrain (generated by activation) and the hindbrain/spinal cord (generated by subsequent transformation), so that the main pattern of the C.N.S. is brought about by successive interactions between its segregating domains.
The Cytoskeleton and Nuclear Disassembly during Germinal Vesicle Breakdown in Starfish Oocytes
Authors: Stricker Stephen A. , Schatten Gerald
Published: January 2005

In response to maturation‐inducing hormone, prophase‐arrested oocytes of the starfish Pisaster ochraceus resume meiosis and undergo nuclear disassembly during a process referred to as germinal vesicle breakdown (GVBD). Time‐lapse video recordings of maturing oocytes reveal that the nucleus lengthens along the animal‐vegetal axis of the oocyte directly prior to GVBD. Neither taxol (10 μM) nor microtubule‐depolymerizing agents [colcemid (50 μM), colchicine (250 μM), or nocodazole (1 μM)] prevent the pre‐GVBD changes in nuclear shape from occurring, although correlative microscopical studies demonstrate that microtubules are nucleated (taxol) or depolymerized (colcemid, colchicine, nocodazole) at the concentrations listed above. The microtubule‐altering drugs also do not affect the time at which GVBD begins or ends. A 10 μM solution of the microfilament‐disrupting drug cytochalasin B (CB), on the other hand, essentially eliminates the pre‐GVBD elongation of the nucleus. CB also slightly delays the onset of GVBD and significantly lengthens the time required to complete GVBD. Such studies suggest that: (i) drug‐sensitive microtubules are not required for GVBD to proceed in a normal fasion; (ii) the pre‐GVBD changes in nuclear shape involve microfilament‐mediated events; and (iii) cytochalasin‐induced depolymerization of microfilaments retards the normal timing of GVBD.
Control of the Expression of AV‐1 Protein, a Position‐Specific Antigen, by ZPA (Zone of Polarizing Activity) Factor in the Chick Limb Bud
Authors: Yokouchi Yuji , Ohsugi Kojune , Uchiyama Koji , Ide Hiroyuki
Published: January 2005

AV‐1 protein is a molecule which shows position‐specific expression during chick limb development, and is expected to have some important roles in limb pattern formation. In this study, to examine whether the ZPA (Zone of polarizing activity) effects the expression of the AV‐1 protein, we have removed or grafted the ZPA in chick limb buds and observed AV‐1 expression. Anterior halves of the limb buds which lack a ZPA were used as hosts. In such anterior halves, AV‐1 expression was initially observed in distal mesodermal cells including the cut surface. These anterior halves were combined with ZPA fragments, anterior fragments, posterior half limb buds, or left to develop alone, and the distribution of AV‐1 expression was examined. The results of these experiments show that AV‐1 expression requires the ZPA, and that expression occurs in the distal mesodermal cells certain distance from the ZPA.
Abnormal X‐Chromosome Dosage Compensation as a Possible Cause of Early Developmental Failure in Mice
Authors: Takagi Nobuo
Published: January 2005

An extra maternally derived X chromosome (XM) but not a paternally derived one (XP) is detrimental in early mouse embryogenesis resulting in failure to form the ectoplacental cone and extra‐embryonic ectoderm. Cytogenetic studies suggested that two XMchromosomes remain active in the trophectoderm and possibly also the primitive endoderm, in which XPis preferentially inactivated in normal female embyos. Two copies of an active X chromosome due to maternal imprinting seem to prevent further differentiation of the trophectoderm.
Embryogenesis in the Presence of Blockers of Mechanosensitive Ion Channels
Authors: Steffensen Isabella , Bates William R. , Morris Catherine E.
Published: January 2005

Certain developmental events are thought to be controlled by mechanical tension, but the nature of the transduction mechanism for sensing and responding to tension changes is unknown. A good candidate for such a sensing system would be stretch‐activated (SA) ion channels, a type of mechanosensitive (MS) ion channel found in many preparations including the oocytes or embryos of ascidians, fish, and amphibians. To test the hypothesis that SA channel activation is important for early embryogenesis, we treated amphibian and ascidian eggs and embryos with inhibitors of MS ion channels. Xenopus laevis eggs and embryos were treated with gadolinium (Gd3+) concentrations up to 100 times the Kd for SA channel inhibition. Boltenia villosa eggs and embryos were exposed to three agents (Gd3+, tubocurarine, and gallamine) which are known to block SA channels in other organisms. None of these drugs interfered with morphogenesis in a manner that would suggest SA channel activity is critical to early embryogenesis.
Time Sequence of Early Events in Fertilization in the Medaka Egg
Authors: Iwamatsu Takashi , Onitake Kazuo , Yoshimoto Yasuaki , Hiramoto Yukio
Published: January 2005

The time sequence of early events in fertilization was examined in eggs of the medaka Oryzias latipes. The mean time after insemination required for sperm attachment to the egg surface through the micropyle depended on sperm concentrations. It was 3 ± 1 sec with a range from 1 to 6 sec after insemination when concentration of spermatozoa was high (about 2 × 108/ml at 23°–25°C). The mean time from sperm attachment until cessation of its movement on the egg surface was 4 ± 1 sec with a range from 1 to 9 sec. Small cortical alveoli at the animal pole region within 15 μm of the sperm attachment point began to undergo exocytosis 9 ± 0.3 sec (range 5–16 sec) after sperm attachment. The velocity at which the exocytosis wave propagated increased from the earliest initiation point of exocytosis up to the 100 μm area, and became constant at about 12 μm/sec from 100 μm to 500 μm from the sperm attachment point. The present results suggest that at the time of fertilization in the fish egg, exocytosis of small cortical alveoli in the area about 15 μm away from the sperm attachment point occurs simultaneously.
Genes for Sperm‐specific Basic Nuclear Proteins in Bufo and Xenopus Are Expressed at Different Stages in Spermatogenesis
Authors: Mita Koichi , Takamune Kazufumi , Katagiri Chiaki
Published: January 2005

The expression of the genes for sperm‐specific basic nuclear proteins was examined, using the cDNA clones encoding protamine (P2) of Bufo japonicus and SP4 of Xenopus laevis as probes. Northern analyses showed that the mRNAs for these proteins were present only in the testes. Analyses with total RNA extracted from testicular cells at various spermatogenic stages revealed that in Bufo the transcripts of protamine genes are present in the spermatids, while in Xenopus the mRNAs for SP4 are present in both primary spermatocytes and spermatids. In situ hybridization studies with radiolabeled antisense RNA probes generated from cDNAs indicated that the Bufo protamine mRNAs accumulated first in round spermatids, while the Xenopus SP4 mRNAs did so in the pachytene stage of primary spermatocytes and thereafter.
Absence of 1‐Methyladenine Production in Follicle Cells Obtained from Starfish Ovaries in the Post‐Spawning Season
Authors: Mita Masatoshi
Published: January 2005

Follicle cells from the ovaries of starfish Asterina pectinifera collected in the breeding season and then kept in an aquarium for three months did not produce 1‐methyladenine (1‐MeAde) in response to genad‐stimulating substance (GSS). The cyclic AMP content of follicle cells of the ovaries was much lower in the post‐spawning season than in the spawning season. In the post‐spawning season, the cyclic AMP level was increased slightly by GSS, but nor insufficiently for production of 1‐MeAde. Addition of 3‐isobutyl‐1‐methylxanthine, a potent phosphodiesterase inhibitor, stimulated the productions of GSS‐induced 1‐MeAde and cyclic AMP. Adenylate cyclase in membrane preparations of follicle cells from ovaries in the post‐spawning season was stimulated by nonhydrolyzable GTP analogs and forskolin. An experiment on ADP‐ribosylation with [α‐32P]NAD in the presence of cholera toxin and pertussis toxin showed the presence of two types (stimulatory and inhibitory) of guanine nucleotide‐binding regulatory proteins (G‐proteins). However, GSS has no effect on the adenylate cyclase activity regardless of the presence of GTP. These findings suggest that GSS does not bind to its receptor in follicle cells of the overy in the post‐spawning season, although these cells possess G‐proteins and adenylate cyclase. Thus the absence of 1‐MeAde production by follicle cells obained from ovaries in the post‐spawning season appears to be due to lack of receptor protein for GSS.
Isolation and Characterization of Dictyostelium Mutants Defective in Sexual Cell Fusion
Authors: Urushihara Hideko , Aiba Kazuhiro , Yanagisawa Kaichiro
Published: January 2005

Sexual cell fusion in the cellular slime mold Dictyostelium discoideum occurs between cells of opposite (heterothallic system) or same (homothallic system) mating types. It also requires certain environmental conditions such as darkness and abundance of water, and thus offers an interesting model system for analyzing mechanisms of cell recognition and of cellular response to environmental factors. We have been studying the mechanism of sexual cell fusion, using two heterothallic strains, NC4 and HM1 of D. discoideum. Two cell‐surface glycoproteins, gp70 and gp138, have been identified as relevant molecules in the cell fusion of these strains. The former is specific to mat a cells (HM1) and the latter, common to both mat a and mat A (NC4). Involvement of cell‐surface carbohydrates has also been suggested. However, the fuctions of the above fusion‐related molecules are still elusive. In the present study, we isolated fusion‐deficient mutants from a mutagenized mat A strain of D. discoideum to set up combined genetic and biochemical analyses. Among the three nonconditional mutants obtained, two were normal in the fruiting‐body formation, asexual development, but one was aggregateless (agg−). Further analysis of these mutants would provide detailed information on the mechanism of sexual cell fusion.
Whorl Formation in Polysphondylium violaceum: Relevance to Organization by Cyclic AMP
Authors: Fukushima Shinji , Maeda Yasuo
Published: January 2005

Whorl formation in Polysphondylium is a simple and good system for the study of pattern formation. The first step of whorl formation is characterized by separation of the rear cell mass from an advancing primary mass during culmination. Using the iontophoresis method, it has been shown that after the establishment of multicellular organization cells respond chemotactically to 3′,5′ ‐cyclic adenosine monophosphate (cAMP), but not to glorin, the chemoattaractant at the aggregating stage. P. violaceum cell masses were also found to secrete actively cAMP. Therefore, morphogenetic movement of P. violaceum cells after aggregation could be controlled mainly by the cAMP signalling system. Vital staining of cells with neutral red (NR) revealed that there are anterior‐like cells stained well with NR in the posterior region of a migrating P. violaceum slug, and that the staining pattern changes markedly during whorl formation. Just before separation of the whorl mass, the anterior‐like cells altered their distribution, and eventually were arranged as an equatorial band on the surface of the presumptive whorl mass, which probably would turn to organizing tip cells of the secondary masses left on the primary stalk. Thus whorl formation may be caused by separation of the strongest cAMP‐source into two regions; the primary (anterior) and secondary (posterior) tips.
Mutations Affecting Embryonic F‐Actin Reorganization also Affect Separation of Nuclei from their Sisters and from the Cortex in Drosophila Cleavage Embryos
Authors: Hatanaka Ko , Okada Masukichi
Published: January 2005

We showed in Drosophila that nuclear migration was reduced all through cleavage stages in embryos with any one of the maternal‐effect mutations, gs(1)N441 and gs(1)N26, in which F‐actin reorganization in cleavage embryos is disordered. Moreover, we determined nuclear positions in embryos at cycle 1 and 2 in the wild type and two mutants, gs(1)N441 and gs(1)N26, in order to test if the nuclear migration is regulated within a nuclear cycle. At cycle 1, there was no difference in nuclear position among the strains that we observed. At cycle 2 the two sister nuclei had already migrated posteriorly in the wild type. However, migration was not detectable at cycle 2 in the mutants. Besides, the two sister nuclei were less‐separated from each other, and orientation of the two nuclei with regard to the anteroposterior axis was random, different from the wild type. These results support the hypothesis that F‐actin is involved in the regulation to separate cleavage nuclei from each other and from the egg cortex. This regulation is apparently required for posteriorward nuclear migration, and for synchronous nuclear arrival in the whole egg cortex.
The Role of “Cytostatic Factor (CSF)” in the Control of Oocyte Cell Cycles: A Summary of 20 Years of Study
Authors: Masui Yoshio
Published: January 2005

Cytostatic Factor (CSF) is a cytoplasmic factor found in unfertilized eggs of the frog that causes metaphase arrest of cell cycles in the oocyte and zygote. CSF appears in maturing oocyte cytoplasm, but disappears during egg activation. CSF‐injected zygotes are arrested at metaphase and show morphology and cellular activities strikingly similar to those of unfertilized eggs. Fresh cytosols extracted from unfertilized eggs contain unstable CSF, called “primary” CSF, which is highly sensitive to Ca ions. Cytosols incubated with Ca ions develop stable CSF, called “secondary” CSF, which is resistant to Ca ions. It has been hypothesized that primary CSF is responsible for the metaphase arrest of meiosis in the unfertilized egg, and its inactivation by a surge of Ca ions during fertilization releases the egg from metaphase arrest. Studies of molecular characteristics of partially purified primary and secondary CSFs suggest that they are both proteins. Recent studies in other laboratories indicate that primary CSF is the c‐mos proto‐oncogene product. The effect of CSF appears to be primarily stabilization of maturation‐promoting factor (MPF), another oocyte cytoplasmic factor, that causes transition of the cell from interphase to metaphase. This paper will summarize the studies on CSF in the author's laboratory over the past 20 years, describe the development of the concept of CSF as a cell cycle regulator, and speculate on the mechanism of its action based on current knowledge.
Nuclear Behavior in Artificially Induced Multi‐Nuclear Cells of Dictyostelium discoideum
Authors: Habata Yugo , Yanagisawa Kaichiro
Published: January 2005

The culture medium of the strain CK‐8 of the cellular slime mold Polysphondylium pallidum contains a cell‐fusion induction factor. Cells of the two opposite mating type strains NC‐4 and HM1 of Dictyostelium discoideum were treated to induce cell fusion with the diluted fraction of CK‐8 cultures, F2, which contains the factor and consequently numerous multinuclear cells were produced. NC‐4 and HM1 usually fuse in the sexual cycle and form large multinuclear cells, called giant cells, which develop into macrocysts. These cells are very similar in morphology to the multinuclear cells produced following F2 treatment, however, the latter cells did not develop into macrocysts. In the sexually formed multinuclear cells, only two haploid nuclei fused to form a diploid nucleus and all others degenerate as previously reported. However, in the artificially produced multinuclear cells, no nuclear‐fusion and degeneration took place. They stayed as heterokaryons and seem to lyse within 20 h incubation.
Radial Columnar Patches in the Chimeric Cerebral Cortex Visualized by Use of Mouse Embryonic Stem Cells Expressing β‐Galactosidase
Authors: Nakatsuji Norio , Kadokawa Yuzo , Suemori Hirofumi
Published: January 2005

Presumed radial migration of neuroblasts from the ventricular to pial surface during formation of the cerebral cortex predicts radial columnar patches in chimeric brains. Lack of adequate cell marker for neurons, however, has hindered such chimera analysis. We used a mouse embryonic stem cell line expressing β‐galactosidase gene to produce chimeric brains. Patches of the labeled cells were examined by whole mount staining and also by computer‐assisted three‐dimensional reconstruction from serial paraffin sections. Our study revealed presence of coherent radial columnar patches in the prenatal cerebral cortex, thus giving a direct evidence for the radial migration of neurons. These columnar patches were less clear in adult brains, suggesting cell mixing during later development and maturation.
Isolation of cDNA Clones for Epidermis‐Specific Genes of the Ascidian Embryo
Authors: Ueki Tatsuya , Makabe Kazuhiro W. , Satoh Noriyuki
Published: January 2005

The present investigation was conducted to isolate cDNA clones that correspond to epidermis‐specific genes of the ascidian embryo. When cleavage of fertilized eggs of Halocynthia roretzi is blocked by treatment with cytochalasin B and the arrested eggs are reared as one‐celled embryos for about 30 hr, they develop features of differentiation of the epidermis only. Translation in vitro of poly(A)+ RNA from cleavage‐arrested embryos and analysis of the products by two‐dimensional gel electrophoresis revealed several predominant polypeptides that were not detected in a similar analysis of fertilized eggs, suggesting the appearance of epidermis‐specific mRNAs in cleavage‐arrested embryos. A cDNA library was constructed from arrested one‐celled embryos. Differential screening of the library with a total cDNA probe from cleavage‐arrested embryos and with a similar probe from fertilized eggs yielded eight different cDNA clones specific for the cleavage‐arrested embryos. Northern blot analysis revealed that the mRNAs that corresponded to these cDNAs were present in normal tailbud embryos. In addition, in situ hybridization of whole‐mount specimens showed that the mRNAs were restricted to the epidermal cells of tailbud embryos.
Dynamics of Ubiquitin Pools in Developing Sea Urchin Embryos
Authors: Pickart Cecile M. , Summers Robert G. , Shim Hyunbo , Kasperek Eileen M.
Published: January 2005

The sea urchin embryo is a closed metabolic system in which embryogenesis is accompanied by significant protein degradation. We report results which are consistent with a function for the ubiquitinmediated proteolytic pathway in selective protein degradation during embryogenesis in this system. Quantitative solid‐ and solution‐phase immunochemical assays, employing anti‐ubiquitin antibodies, showed that unfertilized eggs of Strongylocentrotus purpuratus have a high content of unconjugated ubiquitin (ca. 8 × 108 molecules), and also contain abundant conjugates involving ubiquitin and maternal proteins. The absolute content of ubiquitin in the conjugated form increases about 13‐fold between fertilization and the pluteus larva stage; 90% or more of embryonic ubiquitin molecules are conjugated to embryonic proteins in hatched blastulae and later‐stage embryos. Qualitatively similar results were obtained with embryos of Lytechinus variegatus. The results of pulse‐labeling and immunoprecipitation experiments indicate that synthesis of ubiquitin in S. purpuratus is developmentally regulated, with an overall increase in synthetic rate of 12‐fold between fertilization and hatching. Regulation is likely to occur at the level of translation, since others have shown that levels of ubiquitin‐encoding mRNA remain virtually constant in echinoid embryos during this developmental interval. The sea urchin embryo should be a useful system for characterizing the role of ubiquitination in embryogenesis.
The Cycle of the Seminiferous Epithelium of the Turkish Hamster (Mesocricetus brandti)
Authors: Myoga Hiroshi , Sung. Weng Kong , Jagiello Georgiana
Published: January 2005

The Turkish hamster (Mesocricetus brandti) has become a desirable species for experimentation in testicular function, photoperiod, reproductive hormones and hibernation. Basic data on the kinetics of the seminiferous epithelium have not yet been published. In the present study, the cycle of the seminiferous epithelium was divided into eight stages based on the overall cellular associations of 1540 cross sections of tubules. The mean relative frequencies for stages 1 through 8 were 5.9, 3.3, 11.7, 6.7, 7.2, 28.5, 21.6 and 15.1%, respectively. The absolute duration of the cycle of the seminiferous epithelium was determined by administration of 3H‐thymidine, removal of testes at intevals after injection and autoradiography. The mean duration of one cycle was estimated at 8.0 days and the duration of stages 1 through 8 was 0.5, 0.3, 0.9, 0.5, 0.6, 2.3, 1.7 and 1.2 days, respectively. The duration of meiotic prophase was 11.5 days and of spermiogenesis was 13.8 days. The life span of preleptotene cell was estimated at 1.21 days, leptotene, 0.73 days, zygotene, 0.94 days and pachytene, 7.37 days. The total cycle length of spermatogenesis as usually calculated was 32.0 days.
Change in Intracellular Calcium Ions upon Maturation in Starfish Oocytes
Authors: Kikuyama Munehiro , Hiramoto Yukio
Published: January 2005

A transient increase in intracellular Ca2+ upon maturation in starfish oocyte was revealed by light emission of aequorin microinjected into the cell. One minute application of 1‐methyladenine (1‐MeAde) to a limited area of the oocyte surface was sufficient to induce the Ca2+ transient over the entire cell though it did not induce the germinal vesicle breakdown (GVBD). Ten minutes application of 1‐MeAde induced a similar Ca2+ transient followed by GVBD. Even when the transient increase of Ca2+ was inhibited by injecting EGTA into the oocyte, 1‐MeAde treatment for a long period induced GVBD. These facts indicate that the Ca2+ increase is neither necessary nor sufficient for maturation of the starfish oocyte.

When the oocyte, which had been treated with 1‐MeAde for 1 min at a limited area around the animal pole, was treated again with 1‐MeAde for 10 min starting about 15 min after the first treatment, a Ca2+ transient similar to the first one was induced and was followed by GVBD. By contrast, in the oocyte treated with 1‐MeAde at an area around the vegetal pole, neither Ca2+ transient nor GVBD was induced by the second treatment with 1‐MeAde. These results indicate a difference in responsiveness to the hormone between the animal hemisphere and the vegetal hemisphere of the oocyte.
Expression of the Xhox3 Homeobox Protein in Xenopus Embryos: Blocking Its Early Function Suggests the Requirement of Xhox3 for Normal Posterior Development
Authors: Ruiz i Altaba A. , Choi T. , Melton D. A.
Published: January 2005

Antibodies directed against the product of the Xenopus homeobox gene Xhox3 were raised and used to localize the expression of Xhox3 in the embryo at different stages of development. These studies suggest that endogenous Xhox3 protein is distributed in a graded fashion in the nuclei of mesodermal cells along the anterior‐posterior (A‐P) and dorso‐ventral (D‐V) axes in the postgastrula embryo with low levels in anterior and ventral regions and higher levels in posterior and dorsal regions. Xhox3 protein is also detected at different times in the midbrain, spinal cord and hindbrain. In the hindbrain, Xhox3 displays different metameric expression patterns in dorsal and ventral regions during early embryogenesis and metamorphosis. We have tested for the early function of Xhox3 by injecting antibodies against the Xhox3 protein into the cytoplasm of developing embryos. A significant number of embryos injected with Xhox3 antibodies show posterior (trunk and tail) deficiencies. This posterior deficient phenotype constitutes the opposite of the anterior (head) deficient phenotype obtained after overexpresson of Xhox3 reported previously. These results suggest that expression of Xhox3 in the posterior mesoderm is necessary for posterior development and that the graded distribution of Xhox3 in the embryonic mesoderm is required for the development of normal embryonic axial pattern.
Migration Behavior of Granule Cell Neurons in Cerebellar Cultures. II. An Electron Microscopic Study
Authors: Ono Katsuhiko , Nakatsuji Norio , Nagata Isao
Published: January 2005

We examined the fine structure of migrating granule cell neurons in cerebellar microexplant cultures. Radially migrating bipolar cells extended microspikes or small filopodia from their soma and processes and frequently made contact with neighboring cells. These microspikes contained microfilaments but no microtubules. At the later phase of the migration, in which they had symmetrical bipolar long processes, filopodia extending from perikarial region of cells contained microtubules, suggesting that they are precursors of the future thick perpendicular processes. When cell bodies changed orientation from radial to perpendicular, microtubules that were nucleated from perinuclear centrioles frequently extended into both thick radial and perpendicular processes from the perikarial region. Bundles of 10nm intermediate filaments also appeared in these processes. During migration by the perpendicular contact guidance, many filopodia extending from both the thick leading processes and thin trailing processes made close contacts with the radial parallel neurite. These findings suggest that; 1) The direct contact of the filopodia from both the growth cones and their processes of the granule cells to the neurite bundle plays roles in both the parallel and perpendicular contact guidances. 2) The spacial and temporal changes of cytoskeletons and the association of microtubules with perinuclear centrioles are important for the formation of perpendicular processes and initiation of the perpendicular contact guidance.
Male Germ Cell‐Expressed Mouse Gene TAZ83 Encodes a Putative, Cysteine‐rich Transmembrane Protein (cyritestin) Sharing Homologies with Snake Toxins and Sperm‐Egg Fusion Proteins
Authors: Heinlein Uwe A. O. , Wallat Sabine , Senftleben Annette , Lemaire Lydia
Published: January 2005

Data obtained by cloning of a mouse cDNA (TAZ83) are presented. Its corresponding gene is expressed in meiotic and haploid testicular germ cells. The gene encodes a putative, cysteine‐rich transmembrane protein with a deduced molecular weight of 90 kilodaltons and an isoelectric point of 5.4. Cysteine patterns within the predicted amino acid sequence of the TAZ83 gene product (cyritestin, cysteine‐rich, testicular) are highly conserved when compared to various snake toxins of the disintegrin metalloproteinase type. The cysteine pattern conservation between cyritestin and a guinea pig sperm‐egg fusion protein suggests that TAZ83 codes for a mouse protein with comparable properties or function.
The Loss of the Expression of α Catenin, the 102 kD Cadherin Associated Protein, in Central Nervous Tissues during Development
Authors: Nagafuchi Akira , Tsukita Shoichiro
Published: January 2005

A monoclonal antibody specific for α catenin, the 102kD cadherin‐associated protein, has been characterized and used to describe the expression and distribution pattern of α catenin in adult mice and mouse embryos. This monoclonal antibody recognized an epitope in the middle part of the α catenin molecule of various vertebrate species, and bound to neither vinculin nor αN catenin, which are cytoskeletal proteins with sequence similarity to α catenin. At the early mouse embryo stage (neurulae stage) α catenin was expressed and concentrated at cell‐to‐cell contact sites together with various types of cadherins in all tissues. In embryos at 12.5 days of gestation, the α catenin expression was gradually diminished selectively in central nervous tissues such as brain and spinal cord, and in most of the adult central nervous tissues the α catenin expression was hardly detected. In adult non‐nervous tissues most of the cells examined expressed α catenin. Especially in well‐polarized tissues such as epithelial cells, α catenin appeared to be highly concentrated at cell‐to‐cell adherens junctions where cadherins act as adhesion molecules.

This loss of α catenin expression in central nervous tissues was observed not only in mice but also in other vertebrate species such as fish and newt, suggesting that this phenomenon has important implications from the view point of nervous tissue development.
Urea is Necessary for the Culture of Embryos of the Marsupial Frog Gastrotheca riobambae, and is Tolerated by Embryos of the Aquatic Frog Xenopus laevis
Authors: del Pino Eugenia M. , Alcocer Iliana , Grunz Horst
Published: January 2005

Urea was found in the capsular fluid that bathes Gastrotheca riobambae embryos during incubation in the maternal pouch. The urea concentration in this fluid is higher than in blood from the mother, indicating that urea is accumulated by the embryo during the period of maternal incubation. Gastrotheca tadpoles tolerate up to 500 mM urea with 86% survival after 24 hours and die in solutions of 0.5 mM ammonia. These findings suggest that urea plays a role in the adaptation of G. riobambae embryos to the conditions of water stress within the maternal pouch. To improve the in vitro culture conditions of early embryos taken from the maternal pouch, a saline solution that contains urea was designed (GRS). GRS plus 30 mM urea was used for the culture of cleavage to the neurula stage embryos of G. riobambae. During organogenesis, the urea concentration was raised to 60 mM. Early embryos of Xenopus laevis tolerate urea, and in addition, no inducing effects of urea have been detected in animal cap explants of Xenopus.
Steroidogenesis in the Ovarian Follicle of Medaka (Oryzias latipes, a daily spawner) during Oocyte Maturation
Authors: Fukada Sachiko , Sakai Noriyoshi , Adachi Shinji , Nagahama Yoshitaka
Published: January 2005

The metabolism of 14C‐labeled steroid precursors by cell‐free homogenates of medaka (Oryzias latipes, a daily spawner) ovarian follicles at 12 different developmental stages was examined using thin layer chromatography (TLC). The radioactive metabolites produced were identified and tested for their ability to induce germinal vesicle breakdown (GVBD) in oocytes in an in vitro homologous bioassay. When homogenates of follicles isolated during oocyte maturation were incubated with 14C‐labeled 17α‐hydroxyprogesterone, 13 metabolites were detected in TLC. Among these metabolites, one metabolite exhibited very high maturation inducing activity by the in vitro bioassay. This metabolite was identified as 17α,20β‐dihydroxy‐4‐pregnen‐3‐one (17α,20β‐DP) by its chromatographic mobility in TLC and recrystallization to constant specific activity. 17α,20β‐DP production was high in follicles collected between 10 and 6 hr before spawning. A much less biologically active metabolite, 17α,20β‐dihydroxy‐5β‐pregnane‐3‐one appeared in follicles immediately after the formation of 17α,20β‐DP. A similar pattern of steroidogenesis was observed when the follicles were incubated with 14C‐labeled pregnenolone and progesterone. The timely synthesis of 17α,20β‐DP in medaka at the onset of oocyte maturation, together with the demonstration that this progestogen is the most potent inducer of oocyte maturation in vitro, provides further evidence that 17α,20β‐DP is the naturally occurring maturation‐inducing hormone in the medaka. The results also suggest that the conversion of 17α,20β‐DP to its 5β‐reduced metabolite may be an inactivation process.