bluuebunny/crossref_metadata_2025 huggingface dataset:
Behavior of Na+/K+‐ATPase α‐subunit alleles in sea urchin eggs upon fertilization
Authors: Yamazaki Ken , Maruyama Toshio , Ihara Toshiaki , Yasumasu Ikuo
Published: October 2003

The allelic division of the Na+/K+‐ATPase α‐subunit gene was found in eggs of the sea urchin Hemicentrotus pulcherrimus by polymerase chain reaction (PCR). Two PCR products of different lengths were detected from a genome in one embryo derived from a fertilized egg, although only one product in one embryo derived from an artificially activated egg by parthenogenesis was detected, indicating one copy of the gene in a haploid genome. One of the two PCR products from each fertilized egg was identical in size to the product from an artificially activated egg in the same batch. The other PCR product was the same in length as one of the products from the sperm with which the eggs were fertilized. These results indicate that recombination of the heteromeric alleles of the Na+/K+‐ATPase α‐subunit gene occurs in the sea urchin egg due to meiosis and fertilization. The sequencing of these products demonstrated that their exon sequences were identical and that the intron, inserted in the PCR products, generated polymorphism in length due to the frequency of the repeating 53 bp sequence and insertion/deletion of other two segments.
Inhibition of gastrulation in Xenopus embryos by an antibody against a cathepsin L‐like protease
Authors: Miyata Shohei , Kubo Toshiyuki
Published: October 2003

An antibody against cathepsin L‐like protease (AACLP) was injected into one cell of 2‐celled Xenopus embryos. The blastopores of AACLP‐injected embryos either did not invaginate or failed to complete invagination. As a result of this failure to complete gastrulation, the body axes could not form normally and tail bud stage embryos were bent dorsally. Embryos injected with a control antibody (CA) developed normally through the tadpole stage. Mesodermal induction was not inhibited in embryos exhibiting this AACLP‐induced gastrulation defect, but the mesodermal structure of these embryos was organized incorrectly due to the defective gastrulation during the early stages.
Ultrastructural localization of intracellular calcium stores in Xenopus ovarian follicles as revealed by cytochemistry and X‐ray microanalysis
Authors: Bertout Marc , Flament Stephane , Browaeys‐Poly Edith , Vilain Jean‐Pierre
Published: October 2003

The ultrastructural localization of calcium in full‐grown ovarian follicles of Xenopus laevis was demonstrated after fixation in the presence of fluoride ions and by means of energy dispersive X‐ray microanalysis. In hormonally untreated follicles (prophase I‐arrested oocytes), two calcium sites were detected: follicle cells and oocyte pigment granules. In follicle cells, calcium containing deposits were preferentially associated with macrovilli, which ended by gap junctions. In human chorionic gonadotropin treated follicles (meiotically reinitiated oocytes), deposits were only seen in follicle cells. This is the first report of the cytochemical detection of intracellular Ca2+ in follicle cells of amphibians. The possible involvements of these Ca2+ stores in mediating the hormonal control of meiotic maturation are discussed.
Mdes, a mouse homolog of the Drosophila degenerative spermatocyte gene is expressed during mouse spermatogenesis
Authors: Endo Keita , Matsuda Yoichi , Kobayashi Satoru
Published: October 2003

A new mouse gene Mdes has been identified, which has a significant sequence homology with a Drosophila gene degenerative spermatocyte (des) that is required for the initiation of meiosis in spermatogenesis. The expression pattern of the Mdes transcript during mouse spermatogenesis is similar to that of the des transcript during Drosophila spermatogenesis. Based on these results, it is proposed that the products of Mdes and des have a phylogenetically conserved role in vertebrate and invertebrate spermatogenesis.
Direct evidence of an essential role for extended involution in the specification of a dorsal marginal mesoderm during Cynops gastrulation
Authors: Suzuki Akio S. , Yamamoto Yoshiyuki , Imoh Hiroshi
Published: October 2003

It has been indicated that specification of the dorsal marginal mesoderm of the Cynops gastrula is established by vertical interactions with other layers, which occur during its extended involution. In the present study, when the prospective notochordal area of the early gastrula was almost completely removed together with the dorsal mesoderm‐inducing endoderm and most of the bottle cells, the D‐less gastrulas still formed the dorsal axis with a well‐differentiated notochord; in half of them, where the involution occurred bi‐laterally, twin axes were observed. On the other hand, when the wound of a D‐less gastrula was repaired by transplanting the ventral marginal zone and ectoderm, the formation of the dorsal axis was inhibited if the involution of the lateral marginal zone was prevented by the transplanted piece. The present study suggests that: (i) cells having dorsal mesoderm‐forming potency distribute farther laterally than the fate map; and (ii) the extended involution plays an essential role in the specification of the dorsal marginal mesoderm, especially in notochordal differentiation in normal Cynops embryogenesis.
Epidermal growth factor inhibits morphogenesis of the embryonic quail uropygial gland cultured in vitro
Authors: Fukui Yuriko
Published: October 2003

Formation of the uropygial papilla and glandular lumena was inhibited when the uropygial rudiment of a day 8 1/3 quail embryo was cultured for 2 days in a chemically defined medium in the presence of 50 ng/mL of epidermal growth factor (EGF). The epithelium of EGF‐treated explants remained at the placode stage, or underwent minor invagination into the mesenchyme and became stratified like that of a 12‐ or 13‐day‐old embryo. EGF promoted cellular proliferation in the uropygial epithelium and the epidermis adjacent to the gland and it shortened the lag phase of proliferation and markedly stimulated epithelial DNA synthesis, detected immunocytochemically by labeling explants with 5‐bromodeoxyuridine (BrdU). The maximal labeling index in EGF‐treated uropygial epithelium was 55% higher than in the control. Electron microscopic observation revealed that the basal lamina had become irregular in the EGF‐treated explants and that epithelial cytoplasmic processes penetrated through the basal lamina toward the mesenchyme. These same phenomena are observed in vivo when the glandular buds are formed during day 12–13. Some precocious changes occurred in the uropygial epithelium when the rudiment was cultured in the presence of EGF.
Tubulin tyrosine ligase: Protein and mRNA expression in developing rat skeletal muscle
Authors: Arregui Carlos O. , Mas Carlos R. , Argaraña Carlos E. , Barra Héctor S.
Published: October 2003

Alpha tubulin can be post‐translationally tyrosinated at the carboxy‐terminus by a specific enzyme: tubulin tyrosine ligase. The expression of tubulin tyrosine ligase mRNA and protein during the development of rat skeletal muscle was examined in the present study. A portion of the coding region of the rat ligase cDNA was isolated and sequenced. The nucleotide and amino acid sequences showed about 90% homology with previously reported porcine and bovine ligase sequences. In newborn rats, ligase mRNA and protein were highly expressed in skeletal muscle. During early postnatal development, however, both ligase mRNA and protein dropped down dramatically. Quantitative measurements revealed that ligase protein at postnatal day 20 represented only 10% or less of the level at postnatal day 1. Ligase mRNA expression was also examined during the myogenesis in vitro. A strong ligase mRNA signal was detected in both undifferentiated myoblasts and cross‐striated, contractile myotubes. The present results suggest that, during muscle differentiation, ligase function may be regulated by the amount of available mRNA. The discrepancy in the ligase expression between the in vivo and in vitro myogenesis suggests that factors controlling the levels of mRNA in vivo are lost in vitro.
Does the respiratory rate in sea urchin embryos increase during early development without proliferation of mitochondria?
Authors: Fujiwara Akiko , Yasumasu Ikuo
Published: October 2003

During early development of the sea urchin, the respiratory rate, enhanced upon fertilization, is maintained up to hatching (pre‐hatching period) and then gradually increases to a maximum at the gastrula stage (post‐gastrula period). Except for a short duration after fertilization, respiration in embryos is strongly inhibited by CN− and antimycin A. During the whole span of early development, the amounts of proteins, cytochromes and the specific activities of cytochrome c oxidase and reduced nicotinamide adenine dinucleotide (NADH) cytochrome c reductase in mitochondria are practically the same as in unfertilized eggs. A marked augmentation of mitochondrial respiration after hatching probably occurs without net increase in whole mitochondrial intrinsic capacities. Carbonylcyanide p‐trifluoromethoxyphenylhydrazone (FCCP) or tetramethyl p‐phenylenediamine (TMPD) enhances the respiratory rate in the pre‐hatching period but hardly augments the respiration in the post‐gastrula period. In the presence of both FCCP and TMPD, the respiratory rate in the pre‐hatching period was as high as in the post‐gastrula period. Probably, electron transport in the mitochondrial respiratory chain is regulated by acceptor control and limitation of cytochrome c reduction in the pre‐hatching period and released from those regulations in the post‐gastrula period. Acceptor control of respiration is experimentally reproduced in isolated mitochondria by making adenine nucleotide levels as those levels in the pre‐hatching period.
Species‐dependent migration of fish hatching gland cells that commonly express astacin‐like proteases in common
Authors: Inohaya Keiji , Yasumasu Shigeki , Araki Kazuo , Naruse Kiyoshi , Yamazaki Ken , Yasumasu Ikuo , Iuchi Ichiro , Yamagami Kenjiro
Published: October 2003

Two constituent proteases of the hatching enzyme of the medaka (Oryzias latipes), choriolysin H (HCE) and choriolysin L (LCE), belong to the astacin protease family. Astacin family proteases have a consensus amino acid sequence of HExxHxxGFxHExxRxDR motif in their active site region. In addition, HCE and LCE have a consensus sequence, SIMHYGR, in the downstream of the active site. Oligonucleotide primers were constructed that corresponded to the above‐mentioned amino acid sequences and polymerase chain reactions were performed in zebrafish (Brachydanio rerio) and masu salmon (Oncorynchus masou) embryos. Using the amplified fragments as probes, two full‐length cDNA were isolated from each cDNA library of the zebrafish and the masu salmon. The predicted amino acid sequences of the cDNA were similar to that of the medaka enzymes, more similar to HCE than to LCE, and it was conjectured that hatching enzymes of zebrafish and masu salmon also belonged to the astacin protease family. The final location of hatching gland cells in the three fish species: medaka, zebrafish and masu salmon, is different. The hatching gland cells of medaka are finally located in the epithelium of the pharyngeal cavity, those of zebrafish are in the epidermis of the yolk sac, and those of masu salmon are both in the epithelium of the pharyngeal cavity and the lateral epidermis of the head. However, in the present study, it was found that the hatching gland cells of zebrafish and masu salmon originated from the anterior end of the hypoblast, the Polster, as did those of medaka by in situ hybridization. It was clarified, therefore, that such difference in the final location of hatching gland cells among these species resulted from the difference in the migratory route of the hatching gland cells after the Polster region.
Analysis of the temporal expression of endoderm‐specific alkaline phosphatase during development of the ascidian Halocynthia roretzi
Authors: Nishida Hiroki , Kumano Gaku
Published: October 2003

During embryogenesis of ascidians, endoderm cells initiate certain processes associated with differentiation and produce a tissue‐specific enzyme, alkaline phosphatase (ALP). ALP has been used as a histochemical marker of endoderm differentiation. In the present study, the temporal profile of ALP expression during embryogenesis was investigated. In Halocynthia roretzi, endoderm‐specific ALP is a membrane bound protein and is distinguishable from maternal cytoplasmic ALP by molecular mass. The activity of endodermal ALP first appeared at the early tail‐bud stage. Treatment of developing embryos with inhibitors of translation and transcription was started at various stages. The results suggested that the synthesis of endodermal ALP protein started at the early tail‐bud stage, and that the transcription of mRNA was initiated in the gastrula. In other ascidians, Ciona and Styela, it has been suggested that a significant amount of maternal ALP mRNA exists in eggs. The present study revealed that there are significant species differences in ALP expression during ascidian embryogenesis.
Cleavage furrow establishment by the moving mitotic apparatus
Authors: Rappaport Raymond
Published: October 2003

The midpoint of the mitotic apparatus is fixed in the future division plane long before the division mechanism develops, and this static relationship has been considered essential in speculations concerning division mechanism establishment. The purpose of the present investigation was to determine whether prevention of the static relationship affects the establishment process. Sand dollar eggs were reshaped into cylinders by confinement in an elastic capillary tube and, beginning about 20 min before cleavage, the mitotic apparatus was kept in reciprocal motion by alternately compressing the poles. When the movement was continuous and the excursions were 25, 50 or 75 μm, furrow activity developed near the midpoint of the region underlain by the mitotic apparatus. The acuteness of the furrow decreased as the distance the mitotic apparatus was moved increased. When the movement was made discontinuous by allowing the mitotic apparatus to pause at the end of each excursion, the results depended upon the duration of the pause. Pauses 30 s long resulted in a single furrow formed in the midpoint of the entire region underlain by the mitotic apparatus. When the pauses were 45s long, furrowing activity developed in both regions where the mitotic apparatus was allowed to pause. The results indicated that the normal static relation between the mitotic apparatus midpoint and the division plane is unnecessary for division mechanism establishment. They also demonstrate that a restricted region of contractile activity can be established in the cortex despite experimentally induced spreading and dilution of mitotic apparatus effect.
Effect of protein phosphatase inhibitors on cleavage furrow formation in newt eggs: Inhibition of normal furrow formation and concomitant induction of furrow‐like dents
Authors: Sawai Tsuyoshi
Published: October 2003

The effects of three protein phosphatase inhibitors, okadaic acid, calyculin A and tautomycin, on the formation of cleavage furrows and the induction of furrow‐like dents in the egg of the newt, Cynops pyrrhogaster, were examined. Solutions of the individual compound were injected into the animal hemisphere of one of the two presumptive blastomere regions of the embryo during the first cleavage. Injection of a solution containing any of the chemicals often disturbed the formation of a normal furrow in the injected blastomere at second cleavage. Injection with okadaic acid or calyculin A often induced furrow‐like dents on the surface of the injected blastomere at the same time as second cleavage in control embryos, while that with tautomycin usually did not induce them. In an injected blastomere, formation of dents started in the animal half and moved towards the vegetal half as the furrow in its counterpart blastomere extended from the animal half towards the vegetal. Dents gradually became slightly deeper and formed cytoplasmic projections that later degenerated, leaving a surface scar. Cytological observations on blastomeres injected with calyculin A revealed that nuclear division occurred normally.
Isolation and characterization of an endodermally derived, proteoglycan‐like extracellular matrix molecule that may be involved in larval starfish digestive tract morphogenesis
Authors: Reimer Corinne L. , Crawford Bruce J.
Published: October 2003

A monoclonal antibody, anti‐Pisaster matrix‐1 (anti‐PM1) has been developed against an extracellular matrix antigen, Pisaster matrix‐1 (PM1) found in embryos and larvae of the starfish Pisaster ochraceus. Pisaster matrix‐1 was first observed in endodermal cells of the early gastrula, and shortly thereafter it was secreted into the blastocoel where it accumulated steadily during gastrulation. During the late gastrula stage it also appeared in the extracellular matrix (ECM) of the gut lumen. Immunogold electron microscopy with anti‐PM1 revealed that PM1 was found in condensations of ECM associated with blastocoel matrix fibers, in the trans Golgi network, in Golgi‐associated vesicles in endoderm and mesenchyme cells and throughout the ECM lining the digestive tract of late gastrula and bipinnaria larvae. When blastula or early gastrula stage embryos were grown in the presence of the PM1 antibody, archenteron elongation, bending and mouth formation failed to occur. Pisaster matrix‐1 stained with alcian blue and its assembly could be disrupted with the common inhibitor of O‐linked glycosaminoglycan assembly, β‐xyloside but not by tunicamycin. It was not sensitive to enzymes that degrade vertebrate proteoglycans. Pisaster matrix‐1 is a large (600 kDa) proteoglycan‐like glycosaminoglycan, secreted exclusively by endodermal and/or endodermally derived cells that may be necessary for morphogenesis of the mouth and digestive tract of Pisaster ochraceus embryos/larvae.
An embryological study of ventralization of dorsal structures in the tail of medaka (Oryzias latipes) Da mutants
Authors: Tamiya Gen , Wakamatsu Yuko , Ozato Kenjiro
Published: October 2003

In adult Da (double anal fin) mutants of medaka (Oryzias latipes), structures such as the dorsal fin and the dorsal half of the caudal fin are ventralized in adult fish. However, there have been few embryological studies of the development of mutant phenotypes except those of the caudal fin. In this study, development of mutant phenotypes of the tail where they typically develop was examined morphologically at various stages of embryogenesis. The arrangement of melanocytes along the dorsal midline, the shape of the dorsal fin fold, and the shape of the dorsal myotome exhibited a ventral pattern in the tail at various embryonic stages in Da mutants.
Differentiation of female chicken primordial germ cells into spermatozoa in male gonads
Authors: Tagami Takahiro , Matsubara Yuko , Hanada Hirofumi , Naito Mitsuru
Published: October 2003

In avian species, the developmental fate of different‐sex germ cells in the gonads is unclear. The present study attempted to confirm whether genetically female germ cells can differentiate into spermatozoa in male gonads using male germline chimeric chickens produced by the transfer of primordial germ cells (PGC), and employing molecular biological methods. As a result of Southern hybridization, specific sequences of the W chromosome (the female specific sex chromosome in birds) were detected in the genomic DNA extracted from one out of four male germline chimeric chickens. When two‐color in situ hybridization was conducted on the spermatozoa of this germline chimera, 0.33% (average) of the nuclei of each semen sample showed the fluorescent signal indicating the presence of the W chromosome. The present study shows that female PGC can differentiate into spermatozoa in male gonads in the chicken. However, the ratio of produced W chromosome‐bearing (W‐bearing) spermatozoa fell substantially below expectations. It is therefore concluded that most of the W‐bearing PGC could not differentiate into spermatozoa because of restricted spermatogenesis.
Activation at the germinal vesicle stage of starfish oocytes produces parthenogenetic development through the failure of polar body extrusion
Authors: Washitani‐Nemoto Setsuko , Nemoto Shin‐ichi
Published: October 2003

Starfish oocytes can be fertilized after germinal vesicle breakdown (GVBD) and artificial parthenogenesis can be induced by activating the oocytes after GVBD (post‐GVBD activation). In the present study, parthenogenotes were obtained by the activation of immature oocytes with caffeine before treatment with 1‐methyladenine (1‐MeAde) to induce oocyte maturation. Most of the caffeine‐treated eggs developed as tetraploids, as parthenogenotes produced by the post‐GVBD activation. The parthenogenotes were derived only from eggs that failed to extrude polar bodies, mostly from eggs failing to extrude a second polar body. Eggs derived from immature oocytes activated by A23187, treated with 1‐MeAde and post‐treated with cytochalasin B failed to extrude polar bodies, and eventually developed into parthenogenetic embryos. These results indicate that the present parthenogenesis mechanism shares the same characteristics as that achieved by post‐GVBD activation in the suppression of polar body formation as a key means for successful starfish parthenogenesis.
Establishment of in vitro spermatogenesis from spermatocytes in the medaka, Oryzias latipes
Authors: Saiki Atsusi , Tamura Masaru , Matsumoto Masami , Katowgi Jun , Watanabe Akihiko , Onitake Kazuo
Published: October 2003

Spermatocytes of the teleost, Oryzias latipes, at meiotic prophase were cultured without contact with somatic cells. They began to divide, progressing through the meiotic divisions and differentiating into round spermatids within 48 h. The chromosome number in both the primary and secondary spermatocytes at metaphase was n= 24. In spermatids, a single flagellum was formed and the release of residual bodies was observed in vitro. The size and shape of the flagellum were the same as those seen in vivo. The expression of protamine mRNA was detected in round spermatids. This result suggests that gene expression, as well as morphological change, is regulated by the progression of spermatogenesis in cell culture. Furthermore, when the eggs of O. latipes were inseminated with germ cells cultured for 10 days, normal embryos developed and hatched out. These results suggest that the spermatocytes of O. latipes develop into fertile sperm in cell culture.
The cause of the decreased number of primordial germ cells in albino Xenopus resides not in the micro‐environment but in the presumptive PGC
Authors: Ikenishi Kohji , Tanaka Tetsuya S.
Published: October 2003

The number of primordial germ cells (PGC) in albino tadpoles of Xenopus is significantly decreased as compared with that of the wild‐type. Whether the decreased number of PGC is caused by the presumptive PGC (pPGC) themselves or the micro‐environment surrounding those cells in the albino, or both was investigated in the present study. [3H]thymidine‐labeled pPGC of wild‐type and albino were implanted into unlabeled, host neurulae of wild‐type br albino and wild‐type, respectively. Labeled PGC in the genital ridges of experimental tadpoles were examined by autoradiography. There were no significant differences in the proportion of tadpoles with labeled PGC and in the average number of those PGC between the albino and wild‐type tadpoles, into which wild‐type pPGC had been implanted. The proportion in wild‐type tadpoles with albino pPGC was much lower than that in wild‐type tadpoles with wild‐type pPGC. These results suggest that the pPGC of the albino and not the micro‐environment are responsible for the decreased number of PGC.
Role of intercellular contacts in generating an asymmetric mitotic apparatus in the Tubifex embryo
Authors: Takahashi Hirokazu , Shimizu Takashi
Published: October 2003

The 2‐cell stage embryo of Tubifex is composed of a smaller cell, AB, and a larger cell, CD. At the second cleavage, the CD‐cell divides unequally. The mitotic apparatus (MA) involved in this division is organized asymmetrically: the MA pole to be segregated to a smaller cell is flattened and truncated, and associated with the anterior cortex facing the AB‐cell, while the other pole is symmetric and located more centrally. The present study was undertaken to elucidate the mechanism that generates asymmetry in the MA organization in CD‐cells. When CD‐cell nuclei, which are normally located near the anterior cortex, were displaced toward the posterior end of the cell (i.e. opposite AB‐cells) by centrifugation, MA assembled ectopically there, and were bilaterally symmetric in organization. Similar symmetric MA were formed in isolated CD‐cells, which divided more equally than intact cells. This equality of cell division was dramatically reduced if the anterior surface of isolated CD‐cells formed contact with other cells, such as AB‐, C‐ and 4D‐cells. The MA that formed in these reconstituted embryos were asymmetric in organization; one MA pole was always found to be truncated and apposed to the cortical site at the cell contact. Symmetric MA were also observed in cytochalasin‐treated embryos. Together with the finding that one of the MA poles is physically attached to the anterior cortex of the intact CD‐cell, these results suggest that factors generating asymmetry in the spatial organization of MA poles reside at the anterior cortex of the CD‐cell and that this cortical mechanism is dependent upon cell contacts.
Isolation of a differentiation‐defective myoblastic cell line, INC2, expressing muscle LIM protein under differentiation‐inducing conditions
Authors: Hashimoto Naohiro , Ogashiwa Masayo
Published: October 2003

A non‐differentiating myoblastic cell line, INC2, and a differentiating cell line, COM3, were established from the mouse myoblastic cell line C2C12. Under differentiation conditions, both COM3 and INC2 cells stopped proliferation in a similar manner. The COM3 cells then differentiated into myotubes during the 4‐day differentiation culture. In contrast, almost none of the INC2 cells differentiated into myotubes even in differentiation medium. Reverse transcriptase‐polymerase chain reaction (RT‐PCR) and immunoblot analyses showed that the levels of myogenin and MyoD proteins were significantly decreased in INC2 cells. The differentiation marker sarcomeric myosin heavy chain (MHC) was expressed in COM3 but not in INC2 cells. In contrast, both INC2 and COM3 cells expressed another myogenic regulatory factor, muscle LIM protein (MLP), in a differentiation condition‐dependent manner. These results suggest that MLP gene expression is regulated in a myogenin/MyoD‐independent manner. Enforced expression of the myogenin gene induced MHC expression in INC2 cells. Thus, the signaling pathway situated downstream is assumed to be intact in INC2 cells and suppression of myogenin gene expression may be a primary defect in INC2 cells.
Activation of protein kinase C induces cortical granule exocytosis in a Ca2+‐independent manner, but not the resumption of cell cycle in porcine eggs
Authors: Sun Qing‐Yuan , Wang Wei‐Hua , Hosoe Misa , Taniguchi Toshiaki , Chen Da‐Yuan , Shioya Yasuo
Published: October 2003

The effects of protein kinase C (PKC) activation on meiotic resumption and cortical granule (CG) exocytosis as well as its dependence on Ca2+ in porcine eggs matured in vitro were studied. Cortical granule release was judged by both confocal laser microscopy after the eggs were labeled with fluorescein isothiocyanate‐peanut agglutinin (FITC‐PNA) and electron microscopy. Meiotic resumption and pronuclear formation were observed after eggs were stained with acetic orcein. When eggs were treated with PKC activators, 1‐oleyl‐2‐acetyl‐glycerol (OAG) or phorbol 12‐myristate 13‐acetate (PMA), the pronuclear formation percentage was significantly lower than that of Ca2+ ionophore A23187‐treated group, but not statistically different from that in negative control group (P > 0.05), and most of the eggs were still arrested at metaphase II stage, suggesting that PKC activation does not induce the resumption of meiosis and pronuclear formation. In contrast, PKC activation induced 89.1% to 100% of the eggs completely or partially released their CG in different groups, not statistically different from A23187‐treated group, and this effect could be overcome by PKC inhibition. When the intracellular free Ca2+ was chelated with acetoxymethal ester form of 1,2‐bis(0‐aminophenoxy)‐ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA‐AM), and then treated with PMA or OAG in Ca2+‐free medium, the proportions of eggs with CG release were 90.9% and 78.1%, respectively, not statistically different from the above‐treated groups, suggesting that CG exocytosis induced by PKC activation is independent of Ca2+ rise. The results indicate that different events of porcine egg activation may be uncoupled from one another.
Probable participation of phospholipase A2 reaction in the process of fertilization‐induced activation of sea urchin eggs
Authors: Kamata Yasuyuki , Mita Masatoshi , Fujiwara Akiko , Tojo Tadashi , Takano Jiro , Ide Akira , Yasumasu Ikuo
Published: October 2003

In sea urchin eggs activated by sperm, A23187 or melittin, BPB (4‐bromophenacyl bromide, a phospholipase A2 inhibitor) blocked fertilization envelope formation and transient CN−‐insensitive respiration in a concentration‐dependent manner. BPB had virtually no effect on the increase in [Ca2+]i, (cytosolic Ca2+ level), the activity of phosphorylase a and the rate of protein synthesis, as well as acid production and augmentation of CN−‐sensitive respiration. BPB also inhibited fertilization envelope formation and augmentation of CN−‐insensitive respiration induced by melittin. Melittin, known to be an activator of phospholipase A2, induced the envelope formation, acid production, augmentation of CN−‐insensitive and sensitive respiration, but did not cause any increase in [Ca2+]i, the phosphorylase a activity and the rate of protein synthesis. An activation of phospholipase A2 induced by Ca2+ or melittin seems to result in cortical vesicle discharge and production of fatty acids, which are to be utilized in CN−‐insensitive lipid peroxidase reactions. Activation of other examined cell functions in eggs activated by sperm or A23187, probably results from Ca2+‐triggered sequential reactions other than Ca2+‐caused activation of phospholipase A2.
Cell ablation by ectopic expression of cell death genes, ced‐3 and Ice, in Drosophila
Authors: Shigenaga Ayako , Kimura Ken‐ichi , Kobayakawa Yoshitaka , Tsujimoto Yoshihide , Tanimura Teiichi
Published: October 2003

We have developed a system for killing specific cells in Drosophila using ectopic expression of cell death genes. CED‐3 and ICE (caspase‐1) are proteins required for programmed cell death in the nematode Caenorhabditis elegans and in mammals, respectively. Our previous study has shown that both ced‐3 and Ice can elicit cell death in Drosophila. By expressing ced‐3 or Ice in several kinds of cells using a GAL4‐UAS system and examining the resulting morphological defects, we show that these abnormalities are thought to be caused by the action of ced‐3 or Ice genes. As cells are killed by apoptosis in our system, we could eliminate the possibility of harmful effects on the neighboring cells. Our system provides an alternative and novel cell ablation method to elucidate mechanisms of cell differentiation and cell‐cell interactions during development in Drosophila.
Specific cellular localization of tyrosinase mRNA during Ciona intestinalis larval development
Authors: Caracciolo Anna , Gesualdo Ida , Branno Margherita , Aniello Francesco , Di Lauro Roberto , Palumbo Anna
Published: October 2003

A Ciona intestinalis cDNA clone that encodes a protein highly homologous to other tyrosinases was isolated. Northern blot analysis showed that expression of Ciona tyrosinase starts at the early neurula stage and continues throughout the tail‐bud and tadpole larval stages. The earliest tyrosinase expression was detected, by in situ hybridization, at the neural plate stage, in pigment precursor cells located along the two neural folds, in the animal region of the embryo. In the course of embryonic development the strong hybridization signal was always localized, within the rostral part of the developing brain, in the pigment precursor cells and was later detected in the otolith and ocellus. These results are discussed in relation to tyrosinase as an early marker of neural induction.
Bh (black at hatch) gene appears to be expressed in melanocytes of feather germs in the Japanese quail (Coturnix coturnix japonica)
Authors: Satoh Hiroshi , Nakamura Akira , Shiojiri Nobuyoshi
Published: October 2003

The Bh (black at hatch) gene was examined to determine whether it is expressed in plumage melanocytes by analyzing pigmentation patterns of Bh melanocytes placed in the micro‐environment of the feather germs of quail embryos with pink eyes. These host quails genetically lack a large part of plumage melanin. The Bh locus in these almost white quails is wild‐type. When Bh neural crest cells were transplanted orthotopically into the host embryos, wild‐type and Bh/+ melanocytes, which differentiated from the transplanted neural crest cells, formed plumage pigmentation patterns characteristic of each genotype in the micro‐environment of the host feather germs. Brown plumage pigmentation, which was very similar to that of 10‐day Bh/Bh embryos, was also observed in the feather germs of host embryos that received Bh neural crest cells, although the genotype of the donors could not be determined. These donors died before pigmentation of their feather germs occurred. The results demonstrate that pigmentation patterns of Bh menalocytes are not altered in the micro‐environment of the host germs, suggesting that the Bh gene is autonomous in Bh melanocytes and is expressed in melanocytes of both Bh and the host feather germs, and that it causes the normal pigmentation pattern to be altered.
Conditions for a heat shock response during oogenesis and embryogenesis of the amphibian Pleurodeles waltl
Authors: Prudhomme Christelle , Moreau Nicole , Angelier Nicole
Published: October 2003

The optimal conditions capable of inducing an increase in HSP70 neosynthesis during development of the urodele amphibian Pleurodeles waltl were determined in this study. These conditions depend on temperature, heat shock duration and recovery duration. In oocytes, a heat shock response was repeatedly obtained at 37°C for 15 min followed by 1 h recovery. These results provided evidence for heat shock response at every stage considered. An increase in HSP70 synthesis was noted throughout oogenesis, but it did not lead to an increase in the amount of soluble HSP70, except for stage VI oocytes. Such results suggest that from stage II to stage IV oocytes, an equilibrium occurs between the HSP70 used and the HSP70 neosynthesized. In contrast, in stage VI oocytes, heat shock led to overproduction of HSP70. During early development, the heat shock response was repeatedly obtained only from the gastrula stage with a 37°C shock and a 15min duration of treatment. Surprisingly, during cleavage stage, the soluble HSP70 total amount increased after heat shock at a time when no HSP70 neosynthesis occurred.
Color pattern formation on the wing of a butterfly Pieris rapae. 2. Color determination and scale development
Authors: Takayama Eriko , Motoyama Mayumi , Yoshida Akihiro
Published: October 2003

It has been shown that microcautery on the prospective apical black region of the early pupal forewing of a butterfly, Pieris rapae, causes alteration of the scale color on the adult wing and a delay in histogenesis of the pupal wing. From these results, it has been assumed that the developmental delay of scale cells in the pupal wing alters their developmental fate and the hypothesis that different color fates of scales are determined by differences in the developmental timetables between scale cells is proposed. In this study, we attempted to find the developmental timetables of individual scales expressing specific color to test this hypothesis. It was found that the holes on the upper surface of a scale become larger as they develop and the hole sizes of scales in the white region are always larger than in the black region on the same wings either during pupal period or after eclosion. This suggests that the scale hole size is a good index that reflects developmental rate of the scale and a difference in the hole size between adult scales is attributed to a difference in the developmental timetables when their ancestral scale precursor cells were in the pupal period. A comparison of the hole sizes between adult scales in different color regions suggested that normal white scales were in a more advanced state than were the black ones but white scales induced by microcautery were in a less advanced state than black ones on the same wing. This supports our hypothesis.
Nuclear envelope dynamics during male pronuclear development
Authors: Poccia Dominic , Collas Philippe
Published: October 2003

Upon fertilization, the sperm nucleus undergoes reactivation. The poreless sperm nuclear envelope is replaced by a functional male pronuclear envelope and the highly compact male chromatin decondenses. Here some recent evidence is examined: that disassembly of the sperm lamina is required for chromatin decondensation, that remnant portions of the sperm nuclear envelope target the binding of egg membrane vesicles that form the male pronuclear envelope, that functional male pronuclear envelopes containing lamin B receptor assemble prior to lamin import and lamina formation, and that lamina assembly drives male pronuclear swelling. Several unresolved issues are discussed.
Identification of sperm plasma membrane proteins exhibiting binding affinity for the ascidian egg coat
Authors: Bossi Thomas , Honegger Thomas G.
Published: October 2003

In the initial stage of ascidian fertilization sequential sperm–egg coat interactions assure successful species‐specific fertilization. Sperm recognize, bind to, and then penetrate the egg investment that consists of follicle cells (FC) and an acellular vitelline coat (VC). To identify plasma proteins that recognize the egg coat, a membrane fraction was prepared from Phallusia mammillata sperm using nitrogen cavitation followed by three centrifugation steps. The purity of the membrane fractions was assessed by transmission electron microscopy and marker enzymes. Comparison of the electrophoretic pattern of sperm extracellular membrane domains labeled by radio‐iodination or biotinylation and recorded by autoradiography or enhanced chemiluminescence, respectively, showed the non‐radioactive procedure to be a convenient and efficient method. Isolated sperm membrane components were found to inhibit fertilization in a concentration‐dependent manner and to bind mainly to the FC. Eggs were used as an affinity matrix to determine which of the solubilized sperm membrane proteins possess egg‐binding activity. Three biotinylated proteins (66kDa, 120kDa and 140kDa) were found to bind to the VC. Assays probing heterospecific binding to Ascidia mentula eggs revealed that the 120kDa protein possesses species‐specific binding activity. Thus, the current data suggest the 120 kDa sperm membrane protein as a candidate adhesion molecule with a possible role in gamete binding and species‐specific recognition in P. mammillata.
Alterations in sarcomere structure, collagen organization, mitochondrial activity, and protein metabolism in the avian low score normal muscle weakness
Authors: Velleman Sandra G. , McFarland Douglas C. , Li Zaikai , Ferrin Neal H. , Whitmoyer Robert , Dennis James E.
Published: October 2003

Skeletal muscle fibers are surrounded by an extracellular matrix. The extracellular matrix is composed of glycoproteins, collagen, and proteoglycans. Proteoglycans have been suggested to play an important functional role in tissue differentiation. However, an understanding of how the extracellular matrix affects skeletal muscle development and function is largely unknown. In the avian genetic muscle weakness, low score normal (LSN), a late embryonic increase in the expression of decorin is followed by a subsequent increase in collagen crosslinking. The sarcomere organization, collagen fibril diameter and organization were investigated using transmission electron microscopy. Measurements were made at 20 days of embryonic development and 6 weeks posthatch. These studies showed changes in sarcomere organization and deterioration of muscle fibril structure in the LSN pectoral muscle. In vitro satellite cell cultures were developed and assayed for mitochondrial activity, and protein synthesis and degradation. In these analyses, mitochondrial activity from LSN satellite cells was significantly higher than those from normal pectoral muscle satellite cells. Protein synthesis rates between the normal and LSN satellite cell‐derived myotubes were similar, but protein degradation rates were higher in the LSN cultures. Based on the reported functions of decorin as a regulator of cell proliferation and collagen fibril organization, it is possible that the late embryonic increase in decorin may be influencing the alterations in LSN sarcomere and collagen organization.
Reciprocal transplantation of wing discs between a wing deficient mutant (fl) and wild type of the silkworm, Bombyx mori
Authors: Hojyo Takashi , Fujiwara Haruhiko
Published: October 2003

The wingless mutant flügellos (fl) of the silkworm lacks all four wings. Although wing discs of the fl seem to develop normally until the fourth larval instar, wing morphogenesis stops after the fourth larval ecdysis, probably caused by aberrant expression of an unidentified factor, referred to as fl. To characterize factor fl, the wing discs dissected from the wild‐type (WT) and fl larvae were transplanted into other larvae and developmental changes of the discs were examined. When the wing disc from a WT larva was transplanted into another WT larva and allowed to grow until emergence, a small wing appeared that was covered with scales. Thus, the transplanted wing discs can develop autonomously, form scales and evert from adult skin. The WT wing discs transplanted into the fl larvae also developed at a high rate. However, the fl wing discs transplanted into the WT larvae did not develop during the larval to pupal developmental stages. These data suggest that the fl gene product (factor fl) works in the wing disc cells during wing morphogenesis. Its function cannot be complemented by hemolymph in the WT larva. It is also implied that the level of humoral factors and hormones required for wing morphogenesis are normally maintained in the fl larva.
Analysis of mRNA levels for developmentally regulated prespore specific glutamine synthetase in Dictyostelium discoideum
Authors: Dunbar Andrew J. , Wheldrake John F.
Published: October 2003

The enzyme glutamine synthetase (GS) of Dictyostelium discoideum is developmentally regulated, preferentially localized in prespore cells and is likely to play an important role in controlling the levels of ammonia, a known morphogen, in this organism. To further investigate the regulation of GS, a portion of the GS gene was isolated and used as a probe to examine the changes in GS mRNA throughout development and the level of GS mRNA in the two precursor cell types. The amino acid sequence of the cloned DNA fragment isolated is highly homologous to other eukaryotic GS genes. DNA blot analysis demonstrated that the GS gene exists as a single copy in D. discoideum. Analysis of RNA indicated that there is a single 1.7 kb GS transcript that increased during development to peak at the initial stages of culmination. Furthermore, GS mRNA was highly localized in prespore cells, which is consistent with a proposed source–sink model for ammonia assimilation in this organism.
Proliferation, differentiation and morphogenesis of fetal rat glandular stomach transplanted under the kidney capsule of syngeneic hosts
Authors: Ichinose Masao , Tsukada Shinko , Fujimitsu Yasunobu , Tatematsu Masae , Matsubara Yasuo , Yahagi Naohisa , Oka Masashi , Suzuki Takehisa , Shimizu Yasuhito , Yonezawa Satoshi , Kageyama Takashi , Miki Kazumasa , Fukamachi Hiroshi
Published: October 2003

Undifferentiated glandular stomach tissue fragments from 16.5‐day fetal rats were transplanted under the kidney capsule of syngeneic adult rats, and the proliferation, differentiation and morphogenesis of the transplanted tissues were investigated. Gastric epithelial cells began to invaginate 3–4 days after the transplantation and immature glands were formed after 1 week. During the period, there was a gradual increase in the expression of pepsinogen and cathepsin E, markers of cytodifferentiation of the stomach epithelia, both at protein and mRNA levels. Cathepsin E was weakly expressed in undifferentiated gastric epithelial cells at 16.5 days of gestation, and a higher level of the expression was observed in differentiated epithelia of the transplants. In contrast, the pepsinogen‐producing cells first appeared around days 3–4 after transplantation and gradually increased in number to about 30% of the epithelial cells and became localized at the bottom of the gland. During the period of the experiment up to 1 month, the pepsinogen‐producing cells were all positive for class III mucin and cathepsin E, indicating the immature character of these cells. In addition, no parietal cells were observed. When the tissue fragments were transplanted into adrenalectomized animals, the epithelial differentiation and morphogenesis was suppressed, but its proliferation was enhanced. The observed changes were reversed by hydrocortisone replacement. These results suggest that the development of the 16.5‐day fetal stomach is regulated intrinsically to a certain extent by the genetic program of the cells involved and various gastric functions develop in the absence of luminal stimulation, stage‐specific systemic hormonal change, neuronal regulation or other systemic influences, and that glucocorticoids modulate the developmental program of the fetal stomach tissues.
Early specification of intestinal epithelium in the chicken embryo: A study on the localization and regulation of CdxA expression
Authors: Ishii Yasuo , Fukuda Kimiko , Saiga Hidetoshi , Matsushita Susumu , Yasugi Sadao
Published: October 2003

CdxA, a chicken homeobox‐containing gene related to caudal in Drosophila, has been implicated in the regionalization of endoderm. It is reported here that, in the development of the chicken embryo, CdxA expression appears in the endoderm at day 1.5 of development as bilateral bands on either side of the splanchnopleure which later contribute to intestinal epithelium. The CdxA‐expressing area extends medially and caudally as formation of the gut tube progresses. It is also shown that the rostral limit of CdxA expression demarcates the boundary between stomach and duodenum after day 3 of development. CdxA is not expressed in digestive tract appendages which open into the intestine, such as pancreas, liver and allantois. Early restriction of CdxA expression in intestinal lineage suggests that the intestinal specification involving CdxA expression commences before the gut tube is formed. The expression of CdxA in epithelial‐mesenchymal tissue recombinants suggests that mesenchymal influence regulating CdxA expression plays an important role in confirming the boundary between the stomach and intestine. Chronological change in the spatial distribution of CdxA transcripts and the results of tissue recombination experiments, together with precise fate maps of early endoderm and splanchnic mesoderm, lead to a model of mechanisms by which intestinal specification is brought about.