bluuebunny/crossref_metadata_2025 huggingface dataset:
Nuclear Differentiation in Exconjugants of Paramecium caudatum: Role of Nuclear Division in Differetiation
Authors: MIKAMI KAZUYUKI
Published: January 2005

It has been known that, immediately after the third division of fertilization nucleus (synkaryon), nuclei localized near the posterior region of exconjugant are to be macronuclear anlagen and those near the anterior region are to be presumptive micronuclei in Paramecium caudatum. One of such posterior nuclei was transplanted into amicronucleate cell at vegetative phase in this work. The implanted nuclei were able to divide at every fission. Their DNA content was nearly equal to or less than ordinary micronuclei during vegetative phase. When conjugation was induced between clones obtained and amicronucleates, macronuclear anlagen developed from the division products of implanted nuclei and thereafter derivative caryonides were true to the marker gene of implanted nuclei. The results indicate that there was no intrinsic difference between nuclei localized anteriorly and those situated posteriorly in exconjugant. Differentiation of nuclei into macronucleus may be irreversible at the stage of anteroposterior localization of the nuclei. The role of nuclear division in differentiation may be only to transport the daughter nuclei into the cytoplasm/cortex differentiated anteroposteriorly.
Effect on Inhibition of Micromere Segregation on the Mitotic Pattern in the Sea Urchin Embryo
Authors: FILOSA SILVANA , ANDREUCCETTI PIERO , PARISI ELIO , MONROY ALBERTO
Published: January 2005

Detergent treatment of sea urchin eggs at the mid 4‐cell stage results in prevention of micromere segregation at the fourth cleavage. In these embryos not only the formation of the primary mesenchyme is suppressed, but synchrony of cell division, which is the rule during the first four cleavage cycles, continues for several cycles after the 16‐cell stage while the typical mitotic phase wave that sets in after micromere segregation is abolished.

These results support the hypothesis that micromeres act as coordinators of the mitotic activity of the embryo.
Tissue Differentiation in Mouse Teratocarcinomas in Lung Colonies from Ascitic Embryoid Bodies
Authors: ISHIKAWA TOMOICHI
Published: January 2005

Tissue differentiation in ascitic teratocarcinoma embryoid bodies (EBs) was investigated in the lung colony system of syngeneic 129/Sv and allogeneic Balb/c mice previously reported by us. In this report, tissues are classified as neural, epithelial and mesodermal tissues. Although multiple differentiation in these three groups of tissues became evident about a month following an injection of EBs, colonies containing only one type of tissue but not mesodermal were commonly found. There were few colonies containing only mesodermal or both neural and mesodermal tissues. This suggests that mesodermal tissue differentiation takes place, as in normal embryogenesis, through the formation of a mesodermal layer, which has not been found in vivo.
Cell Adhesion and Fusion Induced by Insemination in Zona‐Free Hamster Eggs: Electrophysiological Monitoring of the Fusion Process
Authors: URABE MASASHI , KAWAKAMI TADATAKA , IGUSA YUKIO , MIYAZAKI SHUN‐ICHI
Published: January 2005

Cell adhesion and fusion were found to occur in zona‐free hamster eggs placed in contact and then inseminated. A cytoplasmic fusion occurred in 7% of paired eggs mainly between 10 and 50 min after insemination. All other eggs that failed to fuse adhered tightly to one another within about 1 hr. Electrophysiological monitoring of the fusion process revealed that an electrical coupling (EC) suddenly appears between apposed eggs and completed within 2–15 min as the first step of cell fusion. Periodic hyperpolarizing responses (HRs), which have been found previously in fertilized hamster eggs to reflect a periodic increase in cytoplasmic Ca2+ ions, become gradually synchronized and completely coincide in paired eggs 10–30 min after the establishment of EC. Thus the paired eggs become an electrically single cell as the second step. Then the boundary between the eggs breaks down, resulting in formation of a single, spherical egg in several minutes. On the other hand, most of adhered eggs showed neither EC nor synchronization of HRs. Some adhered eggs showed only a low efficiency EC.
Inhibition of Differentiation in Dictyostelium discoideum by Anti‐Inflammatory Drugs
Authors: TOWN CHRISTOPHER D. , KRILL DIAN. , HASSID AVIV I.
Published: January 2005

Evidence is presented that a panel of non‐steroidal anti‐inflammatory drugs inhibit both developmental gene expression and terminal cell differentiation in the slime mold Dictyostelium discoideum. Incubation of developing cells with a number of these drugs also prevents the accumulation in the cells of two lipid species which have chromatographic properties similar to authentic eicosanoids. The results raise the possibility that Dicytostelium cells synthesize eicosanoid‐like lipids which are required for normal development.
A Membrane Receptor Mechanism for Steroid Hormones Reinitiating Meiosis in Xenopus Laevis Oocytes*
Authors: BAULIEU ETIENNE‐EMILE , SCHÓRDERET‐SLATKINE SABINE , LE GOASCOGNE CLAUDE , BLONDEAU JEAN‐PAUL
Published: January 2005

There is a membrane progesterone receptor in Xenopus laevis oocytes that undergo meiosis under steroid exposure. Early responses include a decrease of leucine uptake, a decrease of adenylate cyclase and alkaline phosphatase activities, and a decrease of the phosphorylation of a specific p48 protein in the membrane. These results are compatible with a decrease of membrane fluidity brought about by the hormonal message. However the above‐cited effects as well as the Ca2+‐changes are not yet enough well understood to be able to precisely delineate their role in meiosis reinitiation.
Mass Isolation of Germinal Vesicles from Starfish Oocytes*
Authors: CHIBA KAZUYOSHI , HOSHI MOTONORI
Published: January 2005

A rapid, simple and efficient isolation procedure for germinal vesicles was developed using fully grown oocytes from the starfish, Asterina pectinifera. It depends on removal of the vitelline coat by trypsin digestion, gentle cell lysis by hypotonic treatment and centrifugation on a discontinuous sucrose gradient. The germinal vesicles isolated by this method are not clumped, fairly uniform in size and morphology, and rimmed with a very thin layer of cytoplasmic embroidery. They appear morphologically very similar to those in the oocytes. Potential applications of this method and possible functions of the cytoplasmic embroidery are discussed.
Energy Metabolism and Pyridine Nucleotide Levels during Xenopus Oocyte Maturation*, 1
Authors: CICIRELLI MICHAEL F. , SMITH L. DENNIS
Published: January 2005

The rate of oxygen consumption increased in maturing Xenopus oocytes within 2 hr after progesterone addition, well before GVBD. This suggested an early requirement for energy metabolism during maturation, similar to the situation in sea urchin eggs during fertilization. Yet, the absence of similar increases in glucose‐6‐phosphate levels, glucose‐6‐phosphate dehydrogenase activity, glucose conversion to CO2, and the conversion of NAD(H) to NADP(H), indicated that carbohydrate metabolism was not being stimulated in Xenopus oocytes during maturation. The oxidation of other energy yielding substrates is discussed which might account for the finding that, within 5 min of progesterone addition, both reduced forms of the pyridine nucleotides increased 20% over control levels. This was later followed by a drop in NADH levels and a rise in NAD relative to controls. The significance of these changes in pyridine nucleotide levels and their relationship to a number of maturation events are discussed.
Cortical Expressions of Polarity in the Starfish Oocyte*
Authors: SCHROEDER THOMAS E.
Published: January 2005

The animal‐vegetal (A‐V) axis in oocytes of the starfish Pisaster ochraceus and Asterina pectinifera is described in terms of new cytological observations. Ordinarily, this axis of polarity is revealed by the eccentric germinal vesicle that is displaced toward the animal pole where polar bodies eventually form. In addition, however, the cortex at the animal pole is characterized by a pre‐meiotic aster (centrosomes and bundles of microtubules lying within folds of the nuclear envelope), an absence of large, so‐called “acidic vacuoles” that occur abundantly everywhere else, a diminished ability to form actin‐filled spikes, and local mechanical weakness. These expressions of the A‐V axis within the cortex are documented by micrographs. The problem of distinguishing if any feature is an “expression” or a “determinant” of axial differentiation is briefly discussed.
Protein Patterns and Synthetic Profiles during Distal Regeneration in Hydra oligactis
Authors: LESH‐LAURIE GEORGIA E. , BROOKS DENNIS C. , HOOD ROBERT L.
Published: January 2005

Protein patterns and synthetic profiles were examined during distal regeneration in Hydraoligactis. Electrophoretic and radioactive tracer analyses revealed qualitative changes in the general protein profile during regeneration, with a heightened period of protein synthesis between 27–30 hr of regeneration, immediately preceding emergence of the first pair of tentacles. Following this, an increase in collagen‐rich mesogleal protein secretion was observed coincident with tentacle initiation. Inhibition of collagen secretion with the proline analog L‐azetidine‐2‐carboxylic acid (LACA) inhibited tentacle formation, and resulted in the development of unique “hypostome buds” at the distal regeneration surface. At the cellular level LACA did not inhibit the nerve cell differentiation that normally precedes tentacle growth, although some predicted decline in cnidocyte production was noted. It is proposed that mesogleal collagen secretion and structural organization may play a major role in the mechanical aspects of Hydra tentacle morphogenesis.
Use of Hybrids between Xenopus laevis and Xenopus borealis in Chimera Formation: Dorsalization of Ventral Cells
Authors: KOGA M. , KAGEURA H. , YAMANA K.
Published: January 2005

The combination of Xenopus borealis and X. laevis provides an excellent cell marking system. The potential availability of this system for chimera formation has also been suggested. However, eggs and early embryos of these species differ in size and the fusion of blastomeres of different sizez results in some disturbance in arrangement of blastomeres of a chimera. This disturbance was avoided by use of embryos from X. laevis eggs fertilized with X. borealis sperm, instead of X. borealis embryos. The cells of these hybrids could also be distinguished from the cells of X. laevis.

The fate of animal ventral cells placed in the dorsal region was followed by making a chimera by fusing a right lateral half of an 8‐cell X. laevis embryo with that of an 8‐cell hybrid embryo. The animal ventral cells in the “dorsal” region were found to become “dorsalized”, giving rise to a lateral half of dorsal axial structures. This observation explains a previous finding that the replacement of dorsal cells by ventral ones had no effect on embryogenesis in a composite embryo.
Respiration in Eggs of the Echiuroid, Urechis unicinctus, Before and After Fertilization
Authors: FUJIWARA AKIKO , TAZAWA EIGORO , HINO AKIYA , ASAMI KOUICHI , YASUMASU IKUO
Published: January 2005

In eggs of the echiuroid Urechis unicinctus the respiration rate, which is not altered by fertilization, is inhibited by rotenone, antimycin A and cyanide. The respiration in echiuroid eggs is probably mediated by the mitochondrial respiratory chain. In fertilized eggs, the respiration was inhibited by oligomycin and stimulated by the uncouplers of oxidative phosphorylation 2,4‐dinitrophenol and carbonylcyanide p‐trifluoromethoxyphenylhydrazone, whereas respiration in unfertilized eggs was insensitive to these compounds. Insemination increased the respiratory rate in eggs in the presence of uncouplers and reduced it in the presence of oligomycin. These findings suggest that the capacity of electron transport in mitochondira is elevated by fertilization but becomes latent on fertilization‐induced coupling of respiration with oxidative phosphorylation. Strong stimulation of the respiration in unfertilized eggs was induced by dichlorophenol indophenol, phenazine methosulfate and tetramethyl p‐phenylenediamine, suggesting possible sites at which electron transport is regulated in unfertilized eggs. The resulting stimulation of respiration in unfertilized eggs was insensitive to uncouplers and oligomycin, but became sensitive to them after fertilization simultaneously with considerable decrease in its rate. Fertilization‐induced coupling of the respiration seemed to reduce the respiratory rate enhanced artificially by these redox compounds.
Direct Evidence for in vitro Binding of a Lectin from Arachis hypogea to Endoblastic Primordial Germ Cells of Amphibian Embryos
Authors: GIPOULOUX JEAN‐DANIEL , DELBOS MARTINE , GIPOULOUX SIMONE
Published: January 2005

Peanut agglutinin was previously shown to have a specific affinity for primordial germ cells (PGCs) from anuran amphibian embryos. For separation of these cells from endoblastic ones, suspensions of dissociated cells from the endoblastic masses of Xenopus laevis and Rana dalmatina embryos were treated with peanut agglutinin. This treatment resulted in agglutination of a small number of cells, and these aggregates were separated from unaggregated single cells by gravity in 50% calf serum medium. Histological and ultrastructural analysis of numerous sections of the aggregated cells showed that they contained the germinal plasm characteristic of PGCs. The specificity of the PGCs agglutination was confirmed by disocciation of the aggregates with 0, 2 M D‐galactose solution.

This embryonic cellular population of PGCs should be useful in further in vitro experiments.
The Cell Cycle Governs the Onset of Spherulation of Xenopus Eggs Fused by an Electric Field
Authors: STEWART‐SAVAGE J. , GREY ROBERT D.
Published: January 2005

The mechanism of electric field‐induced fusion has been studied in detail (Zimmermann, Biochim. Biophys. Acta. 694 (1982) 227), but little is known about the process by which the two fused cells become a single entity, a process we term spherulation. We observe a clear difference between activated and unactivated Xenopus eggs in the time after electic field (EF) application when spherulation starts, and in the time required for spherulation to be completed. In unactivated eggs, spherulation started 7 min after EF application and was completed within 5 1/2 min. In activated eggs, the lag between EF application and the start of spherulation increased with the cell cycle. At the end of the first cell cycle spherulation started 78 min after EF application and was competed 30 min later. The lag period is not due to delayed fusion, for electric coupling between activated eggs can be recorded before the start of spherulation. The morphology of the contact zone between paired eggs, as observed by light and electron microscopy, is also described. We suggest that the difference in the timing of spherulation reflects a difference in the lability of the cytoskeleton through the cell cycle.
Viable Chimeras between Embryonal Carcinoma Cells and Mouse Embryos: Comparison of Aggregation and Injection Methods
Authors: HANAOKA KAZUNORI , HAYASAKA MICHIKO , NOGUCHI TAKEHIKO , KATO YOSHIHIRO
Published: January 2005

An embryonal carcinoma (EC) cell line having the ability to form chimeric mice was isolated from embryo‐derived teratocarcinomas experimentally induced in BALB/cCrSlc mice. This EC cell line, B242 g, was one of the 5 EC cell lines pre‐selected based on the ability to incorporate into blastocysts by means of aggregating with 8‐cell mouse embryos.

Using the B242g EC cells, the effectiveness of producing chimeras was compared between two currently available techniques, aggregation and injection, by examining chimerism of the midgestationally recovered conceptuses and live‐born mice. The present result revealed that EC cells studied here were able to form chimeras more efficiently by injection as compared to aggregation method.
Founder Cell Differentiation and Acrasin Production in an Aggregateless Mutant of Polyspondylium violaceum
Authors: NEWTH CYNTHIA K. , ARNAL CYNTHIA , GOULART ROBERT A. , HANNA MICHAEL H.
Published: January 2005

A specific class of aggregation‐deficient mutants, aggA, of Polyshondylium violaceum are unable to aggregate unless supplied exogenously with a stimulating factor called D factor. The present study examines the effect of D factor on the induction of founder cells and on the production of the chemoattractant of aggregation, N‐propionyl‐γ‐L‐glutamyl‐L‐ornithine‐δ‐lactam ethyl ester (or glorin). Founder cells initiate aggregate formation and are morphologically distinct from the majority of the amoebae. Founder cell differentiation and oriented movement of attracted amoebae have been studied by time‐lapse videotape analysis. In wild‐type strains, on the average 90 min after the onset of starvation, a single, motile, irregularly shaped amoeba stops wandering and becomes round in shape. This founder cell has differentiated randomly from the pool of starved amoebae and within 2.5 min after the cessation of movement begins to attract and establish cellular contacts nighboring amoebae. The aggA mutants neither aggregate nor differentiate founder cells in the absence of D factor; whereas, aggregate formation and founder cell differentiation occur in the presence of physiological concentrations of purified, externally added D factor. However, in either the presence or absence of D factor, aggA amoebae produce and excrete glorin (measured using a bioassay) at levels comparable to their parental strain. These studies suggest that D factor is required for founder cell differentiation and organization of the aggregate, and that the ability to synthesize and excrete glorin is not sufficient to trigger aggregation.
Growth of Notochord and Formation of Cranial and Mesencephalic Flexures in Chicken Embryo
Authors: TAKAMATSU TETSUROU , FUJITA SETSUYA
Published: January 2005

The proliferative activity of notochordal cells, neuroepithelial cells and endodermal cells in the chicken embryo is examined during flexure formation along its long axis by using bromodeoxyuridine (BrdU)‐immunohistochemistry.

White Leghorn chicken embryos, Hamburger and Hamilton stages from 9 to 15 are cumulatively labled with BrdU for 120 min and fixed with Carnoy's fluid. Serial transverse and sagittal sections are immunostained with a monoclonal anti‐BrdU antibody. The distribution of labeled and unlabeled cells is determined on various regions of the embryos.

Before and during the external flexure development from state 10 to 14, the labeled cells are virtually absent at two regions; The cranial region including the floor of the prosencephalon and pharynx close to the pre‐chordal plate and the mesencephalic region comprising the mesencephalic notochord and mid‐ventral side of the mesencephalon. At stages 10 and 11, the cranial and mesencephalic flexure become apparent at the respective unlabeled regions. It is concluded that the regional differences in proliferative activity between the notochord, neural tube and endoderm play a causal role in the flexure formation of early morphogenesis in the embryo.
Accumulation and Spatial Distribution of Poly(A)+RNA in Oocytes and Early Embryos of Drosophila melanogaster
Authors: KOBAYASHI SATORU , MIZUNO HIDEKO , OKADA MASUKICHI
Published: January 2005

We describe the accumulation and distribution of poly (A)+RNA during oogenesis and early embryogenesis as revealed by in situ hybridization with a radio‐labeled poly (U) probe. The amount of poly (A)+RNA in nurse cell cytoplasm continuously increased untill mid‐vitellogenic stage (st. 10), then decreased with the rapid increase of poly (A)+RNA in the oocyte (st. 11). The localization of poly (A)+RNA at stage 10 was in the anterior region of the oocyte, where it is connected by cytoplasmic bridge to the nurse cells. These observations indicate that most of the poly (A)+RNA synthesized in the nurse cells is transferred to the oocyte through the cytoplasmic bridges at stage 10–11. During the remainder of oogenesis (st. 11–14) and during preblastodermal embryogenesis, poly (A)+RNA was evenly distributed over the cytoplasm of oocytes and embryos. At blastoderm stage, poly(A)+RNA became concentrated in the peripheral region of embryos. Though the somatic nuclei of the blastoderm contained a detectable amount of poly (A)+ RNA, the pole cell nuclei did not. The cytoplasmic RNA visualised by acridine orange staining and the poly (A)+RNA detected by hybridization with [3H]poly (U) exhibited identical distributions during oogenesis and early embryogenesis. These observations provide a basis to assess the unique distributions of specific RNA sequences involved in early development.
Characterization of Developmentally Regulated cAMP/Ca2+‐Independent Protein Kinases from Dictyostelium discoideum
Authors: Pelech Steven , Paddon Harry , Kwong Linda , Weeks Gerald
Published: January 2005

Cell‐free extracts of the slime mold Dictyostelium discoideum were assayed for phosphorylating activity towards endogenous proteins and towards histone H1, casein and myelin basic protein (MBP). During development, protein kinase activity towards all of these substrates steadily increased and peaked between the aggregation and the pseudoplasmodial stages. Particulate‐associated kinase activity was solubilized with 1% CHAPS, and separated into 300–400 kDa and ∼ 100 kDa components on Sephacryl S‐300. The 300–400 kDa peak exhibited the most pronounced developmental increase in MBP phosphorylating activity. It was further fractionated on DEAE‐Sephacel and heparin‐Sepharose, and in each case, it coeluted with the histone H1 phosphorylating activity. The activity of this kinase was unaffected by cAMP and calmodulin, but it was reduced to 50% by ∼ 350 mM NaCl, 5 mM NaF and 40 μg polylysine/ml. The ∼ 100 kDa peak exhibited the most pronounced increase in casein kinase activity during development. Most of the casein phosphorylating activity did not bind to DEAE‐Sephacel; it was distinct from casein kinase 2, which was not developmentally regulated. In parallel with these elevated kinase activities during development, there was increased in vitro phosphorylation of a number of Dictyostelium proteins, including two major phosphoproteins of 140 and 94 kDa.
Archenteron‐Forming Capacity in Blastomeres Isolated from Eight‐Cell Stage Embryos of the Starfish, Asterina pectinifera
Authors: Maruyama Yoshihiko K. , Shinoda Masaru
Published: January 2005

Starfish blastomeres are reported to be totipotent up to the 8‐cell stage. We reinvestigated the development of blastomeres of 8‐cell stage embryos with a regular cubic shape consisting of two tiers of 4 blastomeres. On dissociation of the embryo by disrupting the fertilization membrane at the 8‐cell stage, each of the 4 blastomeres of the vegetal hemisphere gave rise to an embryo that gastrulated, whereas blastomeres from the animal hemisphere did not. By injection of a cell lineage tracer into blastomeres of 8‐cell stage embryos, we found that only those of the vegetal hemisphere formed cells constituting the archenteron. Next, we compressed 4‐cell stage embryos along the animal‐vegetal axis so that all the blastomeres in the 8‐cell stage were in a single layer. When these 8 blastomeres were then dissociated, an average of 7 of them developed into gastrulae. By cell lineage analysis, all the blastomeres in single‐layered embryos at the 8‐cell stage were shown to have the capacity to form cells constituting an archenteron. Taken together, these findings indicate that the fate to form the archenteron is specified by a cytoplasmic factor(s) localized at the vegetal hemisphere, and that isolated blastomeres that have inherited this factor develop into gastrulae.
A New Monoclonal Antibody that Recognizes 180 kDa Polypeptide Expressed on Early Mouse Embryos and Mouse Embryonal Carcinoma Cells
Authors: Abe Kenji , Takagi Nobuo , Naiki Masaharu , Sasaki Motomichi
Published: January 2005

A hybridoma clone 1F7 producing a monoclonal antibody against OTF9‐63 mouse embryonal carcinoma cell line was isolated with the aid of the intrasplenic primary immunization technique. This antibody was capable of recognizing embryonal carcinoma cell lines originated from certain mouse strains, including 129/Sv, but not those which were established from other strains as well as human and other murine cell lines except FM3A, a mouse mammary carcinoma cell line. Indirect immunofluorescence study revealed that 1F7 antigen was expressed on early mouse embryos in a stage specific manner. In order to characterize the 1F7 antigenic molecules, we analyzed both glycoshingolipids and glycoproteins recovered from OTF9‐63 by means of immunostaining after thin layer chromatography and SDS‐polyacrylamide gel electrophoresis followed by Western blotting, respectively. It was concluded that 1F7 antigenic determinants were carried by 180 kDa polypeptides. One of the most interesting characteristics of 1F7 antigen is complete failure to express on the embryonic ectoderm of 5.5d and 6.5d embryos, one of cell types most closely related to embryonal carcinoma cells. 1F7 antibody may help analyse the process of teratocarcinogenesis in 129/Sv mice.
Structure and Macromolecular Composition of the Egg and Embryo Jelly Coats of the Anuran Lepidobatrachus laevis
Authors: Carroll Edward J. , Wei Susan H. , Nagel Glenn M. , Ruibal Rodolfo
Published: January 2005

Eggs and cleavage‐stage embryos of the frog Lepidobatrachus laevis are encased by 3 μm thick vitelline/fertilization envelope and two jelly layers, termed J1 (innermost) and J2 (outermost). Based on light and transmission electron microscopy, J1 had a dense reticular appearance whereas J2 had a laminar structure. Direct dissolution of the jelly coats was accomplished by reduction of disulfide bonds with 0.08 M 2‐mercaptoethanol at pH 10. Soluble jelly preparations were uncontaminated with nucleic acid (A280/A260=1.44) and yielded an average of 150 μg protein/egg or embryo (n=5). The biochemical composition of the jelly coats in unfertilized eggs was different from that in embryos. When examined via gel permeation chromatography, soluble jelly from unfertilized eggs contained macromolecules which were markedly larger and more heterogeneous (earlier eluting and broader peaks) than jelly from embryos. Differences in the components of jelly from unfertilized eggs and embryos were also observed by electrophoresis, however, a 29,700 molecular weight glycoprotein chain was common to both jelly preparations. The electrophoretic pattern of jelly obtained from parthenogenetically activated eggs was identical to that of unfertilized eggs, therefore the fertilization‐associated changes are not due to the exclusive action of cortical granule products.
Involvement of the Nuclear Factor I Motif in the Mouse Myelin Basic Protein Promoter in Cell‐Specific Regulation: Comparison with the Mouse Glial Fibrillary Acidic Protein Promoter
Authors: Miura Masayuki , Tamura Taka‐aki , Mikoshiba Katsuhiko
Published: January 2005

We investigated the cis‐element for cell‐specific expression in the mouse myelin basic protein (MBP) promoter by transfection assay in comparison with that of the glial fibrillary acidic protein (GFAP) promoter. The MBP promoter region including the 1.3 kb upstream region from the transcription start point was found to be highly expressed in NG108–15 cells, a nervous tissue‐derived hybrid cell line, but not in fibroblasts. In contrast, the GFAP promoter region including the region 2.6 kb upstream of the transcription start point was found to function dominantly in C6 glioma cells, but not in NG108–15 cells. Deletion analysis revealed that the NFl motif in the MBP promoter (named MBTE) resulted in the loss of preferential expression in NG108–15 cells. The GFAP promoter also possessed another NFI motif (named GFII) and its distance from the transcription start point was very similar to that of the MBP promoter. Base‐substitution mutation of GFII to MBTE in the GFAP promoter caused remarkable increase in GFAP promoter activity in NG108–15 cells but only slight increase in it activity in C6 cells. These results suggest that the NFI motif in the MBP promoter (MBTE) functions as a cis‐acting promoter element that governs cell‐specific transcription.
Expression of Embryo‐Specific Epidermal XK81 Keratin Gene in Dissociated Cells of Xenopus laevis Animal Caps Co‐cultured with Mesoderm‐Inducing Vegetal Cells
Authors: Atsuchi Yasuo , Shiokawa Koichiro
Published: January 2005

Dissociated cells of whole embryos or their animal halves were cultured until the time when sibling embryos reached the neurula stage, and expression of genes for embryo‐specific epidermal XK81 keratin, muscle‐specific α‐actin and histone H4 was studied by Northern blot analysis. In the experiments with dissociated midblastula cells, the expression of keratin gene, but not two other genes, was found to be inhibited in animal cap cells when these cells were cultured with vegetal cells. However, in the experiments with midgastrula cells, such inhibition was not observed even when animal cap cells were co‐cultured with vegetal cells. Therefore, keratin gene expression in animal cap cells is sensitive at the midblastula stage, but resistant at the midgastrula stage, to the inhibitory activity by vegetal cells.

To determine when such transition occurs, we prepared animal cap cells from embryos at different stages and co‐cultured them with vegetal cells from midblastulae. Results obtained showed that the transition takes place at the late blastula stage (stage 9). These results suggest that the transcriptional system for the embryo‐specific epidermal XK81 keratin gene is stabilized in animal cap cells prior to gastrulation, so that it is expressed autonomously, without being inhibited by contact with vegetal cells.
Self‐Nonself Recognition Activity Extracted from Self‐Sterile Eggs of the Ascidian, Ciona intestinalis
Authors: Kawamura Kazuo , Nomura Maki , Kameda Tomoko , Shimamoto Hiroko , Nakauchi Mitsuaki
Published: January 2005

Eggs of the hermaphrodite, self‐sterile ascidian, Ciona intestinalis, were washed with acid seawater (pH 3.2), and the washing solution was then adjusted to pH 8.2. This solution was found to inhibit only the binding of non‐autologous sperm to the vitelline coat (VC) of eggs, indicating that it contained self‐nonself recognition activity. This activity was heat‐stable and insensitive to trypsin, but was destroyed by V‐8 protease and α‐glucosidase. Both the hydrophobic and hydrophilic components of a lyophilized powder of the extract showed allo‐recognizing activity. On TLC, the hydrophobic components gave a major spot of glucose (Glc) and a peptide spot(s) containing mainly glutamic acid and/or glutamine (Glx). The glucosyl conjugate was purified by HPLC and shown to block sperm‐egg binding to various extents. Individual peptide subfractions had no inhibitory activity, but in combination they showed inhibitory activity. These findings suggest that the acid extract of Ciona eggs contains a Glc‐enriched nonspecific inhibitor of sperm‐egg binding, which could be the primary effector of self‐incompatibility, and Glx‐enriched modulators, which serve as acceptors of allo‐sperm. The cooperative interactions of these components may be responsible for the diversity of allo‐recognition in Ciona gametes.
Possible MIS Production by Follicle Cells in Spontaneous Oocyte Maturation of the Ascidian, Halocynthia roretzi
Authors: Sakairi Kazuhiro , Shirai Hiroko
Published: January 2005

Outer and inner follicle cell‐enclosed oocytes (oocyte complexes) of Halocynthia roretzi underwent germinal vesicle breakdown (GVBD) within 2 hr when transferred from ovaries to normal seawater of pH 8 (NSW). Extrusion of test cells (TC) into the perivitelline space and elevation of the chorion also occurred. This phenomenon was designated as spontaneous oocyte maturation.

Seawater of low pH, protease inhibitors such as leupeptin or soybean trypsin inhibitor (SBTI), and calcium deficiency inhibited the spontaneous maturation only when introduced to the NSW during the first 10 minutes of incubation. GVBD‐blocked complexes underwent GVBD after addition of trypsin regardless of pH or the absence of calcium ions. The oocytes from which follicle cells were removed with glycosidase did not undergo GVBD in NSW, but addition of trypsin triggered GVBD in these defolliculated oocytes (TC oocytes). Furthermore, incubation media in which spontaneous maturation had occurred, induced GVBD in the TC oocytes. This GVBD‐inducing activity was heat‐labile and was inhibited by leupeptin.

These results indicate that in the first step of the spontaneous oocyte maturation, outer and/or inner follicle cells give a signal to the oocyte itself or TC oocyte. This signal is likely to be trypsin‐like.
Spicule Formation‐Inducing Substance in Sea Urchin Embryo
Authors: Kiyomoto Masato , Tsukahara Junzo
Published: January 2005

Isolated micromeres of sea urchin produced spicules in sea water containing blastocoelic fluid (BCF) taken from embryos, or in a medium in which embryos had previously been dissociated (dissociated solution, DS). When isolated micromeres were cultured in vitro, their descendants initiated spicule formation only when BCF was added to the culture medium by the time when, in normal development, primary mesenchyme cells form two aggregates in the vegetal region. After the initiation of spicule formation, growth of spicules occurred under the continuous influence of DS. Spicule formation‐inducing (SFI) activity in DS was first detected at the mesenchme blastula stage. The activity in BCF was heat‐labile and was inactivated by trypsin.
Spawning Female‐Specific Egg Envelope Glycoprotein‐Like Substances in Oryzias latipes
Authors: Murata Kenji , Hamazaki Tatsuo S. , Iuchi Ichiro , Yamagami Kenjiro
Published: January 2005

In addition to the spawning female‐specific (SF) substance (3, 6), a group of new higher molecular weight proteins cross‐reacting with anti‐egg envelope (chorion) glycoprotein (F1) antibody (original antibody) were found in the liver, blood plasma and the ovary of spawning female fish and in the ascites of the estrogenized fish of Oryzias latipes. Exploiting the antibodies specific for the SF substance and the new proteins, which were made of the original antibody by absorbing with the new proteins or the SF substance, the new proteins were found to behave very similarly to the SF substance concerning their localization in the inner layer of the oocyte envelope, intrahepatic formation in response to estrogen etc. They include the protein bands corresponding to Zl‐1 and ‐2, two major constituent glycoproteins of the oocyte envelope, while the SF substance corresponds to ZI‐3, the third major constituent of the envelope. Thus the three major constituent proteins of the inner layer of oocyte envelope are probably formed in the liver under the influence of estrogen in this fish.
A Monoclonal Antibody Against the PSTAIR Sequence of p34cdc2, Catalytic Subunit of Maturation‐Promoting Factor and Key Regulator of the Cell Cycle
Authors: Yamashita Masakane , Yoshikuni Michiyasu , Hirai Toshiaki , Fukada Sachiko , Nagahama Yoshitaka
Published: January 2005

A homolog of the serine/threonine protein kinase (p34cdc2), encoded by the cdc2+ gene of the fission yeast (Schizosaccharomyces pombe), is a catalytic subunit of maturation‐promoting factor and a key regulator of the cell cycle. We have raised a monoclonal antibody against the most conserved amino acid sequence, the PSTAIR sequence (EGVPSTAIREISLLKE) of p34cdc2 This antibody recognizes 31–34 kDa proteins by immunoblotting in all species examined so far. The proteins recognized by the anti‐PSTAIR antibody are probably either p34cdc2 itself or proteins highly homologous to p34cdc2 in the given species, since, in all species studies to date, they are all precipitated with p13suc1, the fission yeast suc1+ gene product, which binds to p34cdc2 with high specificity. The anti‐PSTAIR immunoprecipitate had no histone H1 kinase activity and did not contain cyclin B, suggesting that the PSTAIR region is masked when p34cdc2 forms a complex with cyclin B as an active kinase. Immunoblotting with the anti‐PSTAIR antibody demonstrated that the fastest‐migrating form of p34cdc2 homologues becomes abundant, when oocytes mature or the cell enters M phase. The possible significance of this observation is discussed in relation to the phosphorylation and activity state of p34cdc2 The observed broad cross‐reactivity of the anti‐PSTAIR antibody against p34cdc2 homologues in various species should permit us to examine the role of p34cdc2 homologues in the regulation of the cell cycle in a variety of organisms.
Involvement in Meiotic Prophase of H1 Histone Kinase and p34cdc2 Homologues in Lily (Lilium longiflorum) Microsporocytes
Authors: Yamaguchi Akihiko , Yamashita Masakane , Yoshikuni Michiyasu , Hotta Yasuo , Nurse Paul , Nagahama Yoshitaka
Published: January 2005

We have taken advantage of the synchrony of meiotic prophase I in Lilium microsporocytes to investigate the presence and involvement in four stages of meiotic prophase I (leptotene, zygotene, pachytene, and diplotene) of the p34cdc2 H1 histone kinase, a component of MPF and a key participant in division control in other eukaryotes. H1 kinase activity showed a peak pattern during meiotic prophase I with the highest kinase activity at pachytene. A monoclonal antibody directed against a highly conserved region of p34cdc2 (termed the ‘PSTAIR’) recognized three major protein forms by immunoblotting. The highest level of the fastest‐migrating form was observed at pachytene, coinciding with the highest activity of H1 kinase. Both the proteins recognized by the anti‐PSTAIR antibody and H1 histone kinase activity were retained on beads conjugated with p13suc1, a protein known to physically associate with p34cdc2. These observations suggest that p34cdc2 or protein(s) highly homologous to p34cdc2 is a component of Lilium H1 histone kinase and plays a role in regulating meiotic prophase I.
An Ascidian Homolog of the Mouse Brachyury (T) Gene is Expressed Exclusively in Notochord Cells at the Fate Restricted Stage
Authors: Yasuo Hitoyoshi , Satoh Noriyuki
Published: January 2005

Ascidians are primitive chordates in which a well‐organized notochord is formed in the tail of the tadpole larva. The Brachyury (T) gene in the mouse is essential for formation of mesoderm and, in particular, of notochord. We report here the expression of an ascidian homolog (As‐T) of the mouse T gene. A cDNA clone for the As‐T gene contains a single open reading frame that encodes a polypeptide of 471 amino acids. Although the overall degree of amino acid identity was not very high (39.9%, ascidian/mouse), in the N‐terminal half the extent of amino acid identity was 78.5% (ascidian/mouse). The expression of As‐T was transient. A single band corresponding to a 2.2 kb mRNA was first detected at the 64‐cell stage, and a distinct band was found at the gastrula stage, at which time accumulation of the transcripts was maximal. The transcript was barely detectable at the larval stage and was undetectable in adult tissues. The occurrence of As‐T transcripts was restricted to notochord‐lineage cells, and no other cell types expressed the As‐T gene. In addition, the timing of As‐T expression coincided with the time of restriction of developmental fate exclusively to notochord in the notochord‐lineage cells. It is likely that the Brachyury gene was present when the ancestors of chordates with a notochord emerged and that the primary function of this gene is to specify notochord cells.
Migration Behavior of Granule Cell Neurons in Cerebellar Cultures I. A PKH26 Labeling Study in Microexplant and Organotypic Cultures
Authors: Nagata Isao , Nakatsuji Norio
Published: January 2005

Granule cells were dissociated from early postnatal mouse cerebella and labeled with a fluorescent dye probe PKH26. Small number of the labeled cells were mixed with cerebellar cortical microexplant cultures or transplanted into cerebellar cortical organotypic explants, and their time‐dependent morphological changes during cultures were examined with fluorescence microscopy. Granule cell neurons first extended asymmetrical short bipolar processes in both cultures, and migrated actively in microexplant cultures. After elongation of symmetrically bipolar long and thin neurites, they sprouted short thick processes from cell bodies and migrated perpendicular to neurite bundles that were devoid of glia in microexplant cultures, or migrated vertically inward into the internal granular layer in the organotypic explant. During such migrations, they extended short thick processes in front and thin processes behind the cell body. The latter processes were connected to thin long neurites with T‐ or Y‐shaped junctions in both cultures. Finally, they extended many short thick processes from cell bodies in both cultures. Such behaviors of granule cell neurons in microexplant cultures were, thus, similar to those in organotypic explant cultures despite of the absence of Bergmann glial cells. These migration patterns may be closely related to migration of granule cells in histogenesis of the cerebellar cortex.
Expression of a Gene for Major Mitochondrial Protein, ADP/ATP Translocase, during Embryogenesis in the Ascidian Halocynthia roretzi
Authors: Miya Takahito , Makabe Kazuhiro W. , Satoh Noriyuki
Published: January 2005

The ADP/ATP translocase is the most abundant integral protein of the inner mitochondrial membrane and it is encoded by the nuclear DNA. Because mitochondria in the ascidian egg appear to be segregated into blastomeres of muscle lineage, we examined the expression of a gene for ADP/ATP translocase during embryogenesis of the ascidian Halocynthia roretzi. Sequence analysis of a cDNA clone for the ascidian ADP/ATP translocase indicated that it contains a single open reading frame that encodes a polypeptide of 304 amino acids. The polypeptide showed extensive similarity to mammalian ADP/ATP translocases, with as much as 74% identity. The genome of H. roretzi contains a single gene, or two genes at most, for the protein. A large amount of maternal mRNA for ADP/ATP translocase was found in unfertilized eggs and early embryos. The amount of this mRNA decreased after the 64‐cell stage, and the mRNA became barely detectable in tailbud embryos, although zygotic transcript for the protein was evident in adult tissues. Both in situ hybridization and Northern blot analyses demonstrated that the mRNA is distributed in the entire cytoplasm of unfertilized eggs. The mRNA is segregated during embryogenesis not only into blastomeres of muscle lineage but also into those of non‐muscle lineage.
A New Method for Fibroblast‐less Primary Skeletal Muscle Cell Culture by the Use of Hydroxyurea
Authors: Hagiwara Yasuko , Ozawa Eijiro
Published: January 2005

A method was developed to suppress growth of fibroblasts in chicken and mouse primary skeletal muscle cell cultures. Addition of hydroxyurea to the culture medium at appropriate time and concentrations suppressed the proliferation of fibroblasts whereas leaving myotubes grow and differentiate. The most favorable time for the addition was soon after myotube formation. The optimal concentrations for our purpose ranged from 0.5 to 1.0 mM. In the presence of hydroxyurea at these concentrations, myotubes grew larger and well differentiated, whereas fibroblasts remained in the suppressed state. In chicken myotubes cultured with hydroxyurea, cross‐striations, spontaneous twitching and myosin heavy chain appeared as in myotubes without hydroxyurea. In mouse myotubes cultured with hydroxyurea, myosin heavy chain and dystrophin appeared, as in control myotubes.
Identification of a New Gene Expressed Specifically in Early Mouse Embryos
Authors: Wei Li‐Na , Hsu Yu‐Chih
Published: January 2005

To identify genes involved in retinoic acid signaling during early embryogenesis, specifically during implantation and early postimplantation, cDNA libraries constructed from mouse embryos at e4.5 and e5.5, respectively, have been screened. Based upon DNA sequence homology, one clone has been isolated by using mouse retinoic acid receptor α (RARα) as the probe. This clone, designated as 80.3, is expressed in the embryonic portion at early egg cylinder stage, and is highly expressed in the entire embryo at e6.5. Its expression decreases in embryos older than e9.5 and can not be detected in any adult tissues.In vitrotranscription/translation of this cDNA has produced a protein product with a molecular weight of approximately 50 kDa. The central to C‐terminal portion of this gene is highly homologous to a human orphan receptor, TR‐2. This homologous region contains a potential zinc‐finger DNA binding motif followed by a putative ligand‐binding domain. However, this gene is very different from TR2 in the N‐terminal region and appears to be a newly identified gene with a specific pattern of expression during early embryogenesis.
Induction of Mucous Metaplasia in Chick Embryonic Skin by Retinol‐Pretreated Embryonic Chick or Quail Dermal Fibroblasts through Cell‐Cell Interaction: Correlation of a Transient Increase in Retinoic Acid Receptor β mRNA in Retinol‐Treated Dermal Fibroblasts with Their Competence to Induce Epidermal Mucous Metaplasia
Authors: Obinata Akiko , Akimoto Yoshihiro , Kawamata Takashi , Hirano Hiroshi
Published: January 2005

Epidermal mucous metaplasia of 13‐day‐old chick embryonic tarsometatarsal skin can be induced by culture in medium containing 20 μM retinol for only 8 hr and then in a chemically defined medium without retinol for 2 days. Retinol primarily affects the dermal cells, which then transform the epithelial cells into mucus‐secreting cells. In this study, we developed a system using a combination of retinol‐pretreated chick or quail dermal fibroblasts and chick skin, and showed that retinol‐pretreated quail embryonic dermal fibroblasts invaded the dermis of chick embryonic skin to beneath the epidermal basal cells within 1 day of culture and induced metaplasia, suggesting that epidermal mucous metaplasia of the skin was induced by the direct interaction of retinol‐pretreated dermal fibroblasts with the epidermal cells or by low diffusible paracrine factor produced by the fibroblasts.

Increase in retinoic acid receptor β (RARβ) mRNA in dermal fibroblasts was observed after 8 hr‐treatment with retinol which preceded morphological changes induced by retinol and this increase was correlated with the competence of the dermal fibroblasts to induce epidermal mucous metaplasia. Thus some gene product(s) controlled by RARβ in dermal fibroblasts may be an essential signal for induction of epidermal mucous metaplasia.
A simplified and efficient method for in situ hybridization to whole Drosophila embryos, using electrophoresis for removing non‐hybridized probes
Authors: Kobayashi Satoru , Saito Hiromitsu , Okada Masukichi
Published: January 2005

We report a simplified and reliable method for non‐radioactive in situ hybridization to whole Drosophila embryos. In the previous method (Tautz and Pfeifle, 1989) the post‐hybridization wash, or the procedure for washing non‐hybridized probe away from embryos depends simply on diffusion. We modified the method with application of electrophoresis to the wash. After hybridized with RNA probe, embryos were transferred to a small well where an electric charge was given to drive non‐hybridized probe away from the embryos. This procedure enables us to acquire a much higher signal‐to‐noise ratio than that obtained from a conventional method. Furthermore, this is a time‐saving method. We propose a term “electro‐wash” for this procedure.