bluuebunny/crossref_metadata_2025 huggingface dataset:
Does the M‐Phase Promoting Factor (MPF) Activate a Major Ca2+‐and Cyclic Nucleotide‐Independent Protein Kinase in Starfish Ocytes?
Authors: LABBE JEAN CLAUDE , PICARD ANDRÉ , DOREE MARCEL
Published: January 2005

In starfish, the activity of a major Ca2+‐and cyclic nuleotide‐independent protein kinase has been shown to fluctuate in phase with that of MPF along meiotic and mitotic cell cycle (23, 25). Microinjection of α‐naphthylphosphate (α‐NP), a potent phosphatase inhibitor, increased considerably (from 15 to 546 picomoles/min/mg protein) the activity of this major cycling kinase in homogenates. Although this result supported the view that kinase phosphorylation might induce its own activation, this hypothesis was eliminated because injection of cytoplasm from hormone‐stimulated enucleated oocytes, which contained the fully activated kinase but no MPF, failed to trigger kinase activation in recipient oocytes. In contrast, kinase activation was induced in recipient oocytes injected with either cytoplasm taken from nucleated maturing oocytes, which contained high MPF and kinase activities, or cytoplasm taken later from hormone‐stimulated and ATP‐γ‐S‐injected oocytes which contained high MPF but low kinase activites. These results indicate that inhibiting dephosphorylation of some regulatory protein activates the M‐phase‐specific protein kinase. The possibility that the M‐phase or maturation‐promoting factor (MPF) might be this regulatory protein is discussed.
Timers in Early Development of Sea Urchin Embryos
Authors: MATSUMOTO YUTAKA , KOMINAMI TETSUYA , ISHIKAWA MASARU
Published: January 2005

To elucidate the timing mechanisms in the early development of sea urchin embryos, we measured the times of initiation of the first four cleavages, of ciliary movement, of primary mesenchyme cell ingression, and of gastrulation at four temperatures ranging from 11 to 20°C. The times of cleavage and of initiation of ciliary movement showed similar temperature dependency, indicating that these events may be controlled by a common timer (the first timer). Although batches of eggs often showed variation in the period between fertilization and the first cleavage, their subsequent cleavages were more regular. This indicates that the first timer may not start at fertilization. The ingression of mesenchyme cells and the onset of gastrulation showed similar temperature dependency that was higher than that of other events, suggesting the existence of a second timer. Temperature shift experiments indicate that the second timer starts at the mid‐blastula (the 8–9th cleavage) stage when divisions of blastomeres become asynchronous.
Significance of an Increase of Intracellular Adenosine Concentration for Dormancy in Starfish Blastulae
Authors: TSUCHIMORI NOBORU , MIYASHIRO SHIGEYOSHI , SHIBAI HIROSHIRO , IKEGAMI SUSUMU
Published: January 2005

Adenosine at concentrations greater than 6 μg/ml halted embryonic development of the starfish Asterina pectinifera specifically at the 256‐cell stage which corresponds to the onset of blastulation. When a fertilized egg was cultured continuously in sea water containing adenosine from fertilization, a gradual increase in intracellular concentrations of free adenosine was observed before a cessation of development took place. On the other hand, intracellular concentrations of ATP, ADP and AMP in the embryo cultured in sea water containing adenosine were nearly the same as those of an embryo cultured in sea water without adenosine. By returning the development‐arrested embryo to normal sea water the embyro developed normally to the bipinnaria stage accompanied by a gradual decrease in the intracellular cencentration of adenosine.

Treatment of fertilized eggs with 9‐β‐d‐arabinofuranosyl‐9H‐purine‐6‐amine (25 μg/ml) or 2′‐deoxyadenosine (10 μg/ml) halted development specifically before the onset of blastulation in an irreversible manner. Embryos treated with 3′‐deoxyadenosine (50 μg/ml) shortly after fertilization developed to healthy blastulae but hatching never occurred. These results exclude the possibilities that the action of adenosine is mediated by the inhibition of DNA synthesis or RNA polyadenylylation.
Studies on the Mechanism of Action of Estradiol in Regulating Follicular Progesterone Levels: Effects on cAMP Mediated Events and 3β‐Hydroxysteroid Dehydrogenase
Authors: LIN YU‐WAI PETER , KWON HYUK B. , PETRINO TERESA R. , SCHUETZ ALLEN W.
Published: January 2005

Previous studies demonstrated that estradiol interferes with pituitary‐induced progesterone production and oocyte maturation in cultured amphibian (Rana pipiens) ovarian follicles. To elucidate the mode of action of estradiol in modulating follicular progesterone accumulation we have examined its effects on cAMP‐induced progesterone production and enzymatic conversion of pregnenolone to progesterone by 3β‐hydroxysteroid dehydrogenase (3β‐HSD). Follicular cAMP levels were manipulated with forskolin (an adenylate cyclase activator), isobutyl methyl xanthine (IBMX‐phosphodiesterase inhibitor) and exogenously added cAMP. Progesterone production induced by forskolin alone or forskolin in combination with frog pituitary homogenate (FPH) was inhibited by estrogen. Addition of estradiol to culture medium markedly inhibited follicular progesterone accumulation following treatment of follicles with cAMP and IBMX. In the presence of exogenous pregnenolone, non‐FPH stimulated ovarian follicles effectively converted the 3β‐HSD substrate to progesterone. Treatment of follicles with estradiol inhibited conversion of pregnenolone to progesterone. The results indicate that estradiol acts, following FPH stimulation, at one or more steps subsequent to elevation of cAMP levels to regulate intrafollicular progesterone accumulation and oocyte maturation. Estrogen appears to directly influence the enzymatic (3β‐HSD) conversion of pregnenolone to progesterone.
Sperm Incorporation Independent of Fertilization Cone Formation in the Danio Egg
Authors: WOLENSKI JOSEPH S. , HART NATHAN H.
Published: January 2005

The responses of the egg to insemination in a modified Fish Ringer's solution (FRS) were examined in eggs of the zebrafish (Brachydanio rerio) primarily by scanning electron microscopy. FRS is a physiological saline which temporarily inhibits parthenogenetic activation of the egg for 5–8 min. Spermatozoa were collected in a small volume of water and pipetted over eggs in FRS. Eggs inseminated in FRS typically incorporated the fertilizing sperm within 3–4 min. Inseminated cells showed an absence of a fertilization cone and no cortical granule exocytosis. The deep conical depression in the egg surface beneath the micropyle remained unaltered. Control eggs inseminated in tank water developed a large fertilization cone during sperm incorporation. Occasionally, eggs inseminated in water were observed to incorporate the entire sperm head prior to egg activation. Our results corroborate earlier findings showing that in the zebrafish, cortical granule exocytosis, fertilization cone formation and elevation of the sperm entry site are not triggered by the fertilizing sperm in experimental conditions (18, 19). Furthermore, sperm incorporation requires neither egg activation nor formation of a fertilization cone in this fish.
Analysis of Cellular Mosaicism in a Transgenic Mouse by Histological In Situ Hybridization
Authors: KATOH KAZUTO , YOKOYAMA MINESUKE , KIMURA SHIARI , HIRAMOTO YUKIO , KONDOH HISATO
Published: January 2005

One of the transgenic mice carrying a chicken δ‐crystallin gene was found to be mosaic with regard to the distribution of the exogenous gene. Taking advantage of the exogenous DNA sequences as a cell lineage marker detectable by histological in situ hybridization technique, we studied cellular mosaicism in mouse 7–5. This mouse carried the exogenous gene in 20–40% of its cells, probably reflecting chromosomal integration of the exogenous DNA which occurred in a blastomere of around the 4‐cell stage. The cells carrying the gene contributed to virtually any kind of tissue and their distribution varied from one tissue to another. For instance, in the neural retina, gene‐positive cells formed columns several cells wide, indicating that migration of the cells derived from the founder cells is mainly along the radial axis. However, in other tissues we examined, clusters of the marked cells were less obvious, indicating the occurrence of extensive cell mixing during histogenesis. Thus, mosaic analysis of cell lineage in mouse ontogeny appears meaningful in early developmental stages or when clonal outgrowth takes place in a tissue.
The Prosencephalon Has the Capacity to Differentiate into the Optic Tectum: Analysis by Chick‐Specific Monoclonal Antibodies in Quail‐Chick‐Chimeric Brains
Authors: NAKAMURA HARUKAZU , TAKAGI SHIN , TSUJI TOSHIAKI , MATSUI KOHJI A. , FUJISAWA HAJIME
Published: January 2005

The alar plate of the prosencephalon of the quail embryo was heterotopically transplanted into the alar plate of the mesencephalon of the chick embryo at the 7–10 somite stage. Chick and quail cells in chimeric brains were distinguished after Feulgen‐Rossenbeck staining and/or immunohistochemical staining with a species specific monoclonal antibody MAb‐37F5 which recognized cytoplasmic components of chick brain cells. Neural connections between the transplant and the host were studied by monoclonal antibodies, MAb39‐B11, which recognizes a species specific antigen on chick nerve fibers, and MAb‐29B8, which reacts to 160 kD neurofilaments of both chick and quail.

When the transplant was completely integrated into the host mesencephalon, the transplant developed a laminar morphology closely resembling that of the optic tectum. Immunohistochemical staining with MAb‐39B11 showed that the host optic nerve fibers innervated both the host tectum and the tectum‐like transplant. However, optic nerve fibers did not invade transplants that failed to develope a laminar structure characteristic of the tectum. These findings suggest that the prosencephalon has a capacity to differentiate into the optic tectum at the 7–10 somite stage.
Insights into the Molecular Mechanisms Involved in Sea Urchin Fertilization Envelope Assembly
Authors: Somers Cynthia E. , Shapiro Bennett M.
Published: January 2005

Sea urchin fertilization envelope assembly provides an ideal model system for investigating the production and modification of an extracellular matrix. The contents of secretory vesicles and the egg glycocalyx mix to initiate assembly. Limited proteolysis and covalent crosslinking by a transglutaminase act as early events to modify the nascent envelope. A subset of secreted proteins binds to this matrix through ionic interactions that require divalent cations. For example, one secreted protein, proteoliaisin, is responsible for attaching ovoperoxidase to the envelope. Ovoperoxidase hardens the envelope by using hydrogen peroxide, produced by the egg during the respiratory burst, to form dityrosine crosslinks between a subset of fertilization envelope proteins. Numerous spatial and temporal regulatory mechanisms exist to ensure that proper assembly occurs in an environment isolated from the normal cytosolic regulatory machinery.
Cloning of a Gene Encoding Bombyxin, an Insulin‐Like Brain Secretory Peptide of the Silkmoth Bombyx mori with Prothoracicotropic Activity
Authors: Iwami Masafumi , Kawakami Atsushi , Ishizaki Hironori , Takahashi Susumu Y. , Adachi Takashi , Suzuki Yoshiaki , Nagasawa Hiromichi , Suzuki Akinori
Published: January 2005

A genomic DNA encoding bombyxin, a 5kD brain peptide of the silkmoth Bornbyx mori with prothoracicotropic hormone activity, has been isolated. The nucleotide sequence coding for bombyxin shows high homology with insulin‐gene family members and the overall organization of the preprobombyxin gene is the same as in preproinsulin genes, indicating that bombyxin shares a common ancestral molecule with insulin‐family peptides. The bombyxin gene has no intron contrasting to other members of insulin‐gene family.
Fertilization Potential of the Medaka Egg
Authors: Ito Shizuo , Shimamoto Koji
Published: January 2005

Fertilization of the medaka egg in 10% Ringer's solution generates a depolarization of 4 mV just before the appearance of a characteristically longer hyperpolarization (25). The depolarization appears to be the result of a nonspecific leak triggered by sperm stimulation and the amplitude of the depolarization is thought to be independent of [Ca2+]o (25). We have investigated the ionic dependence of this depolarization. An initial small depolarization (3–4 mV; duration, 5–8 sec) is followed by a rising phase of a spike‐like depolarization in the range of 10–60 mV. The amplitude of this spike‐like depolarization is propodional to log [Ca2+], ranging from 0.33–18 mM. Calcium antagonists, e.g. 10 mM cobalt or 10 μg/ml verapamil in 10% Ringer do not block the depolarization in the presence of 1.1 mM CaCl2.
Vertebral Abnormalities Following Heat Shock in Xenopus Embryos
Authors: Kobayashi Hideki , Watanabe Toru , Terashita Noboru , Handa Sumio , Furuno Nobuaki
Published: January 2005

Xenopus embryos were heat‐shocked at several stages of development and vertebral abnormalities were examined by means of in situ staining. A high incidence of vertebral abnormalities was evident in larvae treated at the neurula stages (stages 15 and 20) and at the tailbud stages (stages 32 and 35). Heat shock at the neurula stages led to malformed vertebrae and their fusion following an altered arrangement of the arcualia. Heat shock at the tailbud stages induced an asymmetric arrangement of the sacrum and a change in the number of vertebrae, but the arrangement of the arcualia was not affected. Early events of the development of the vertebral column are discussed in relation to somitogenesis.
Glandular Structure Formation of LS174T Human Colon Cancer Cells Cultured with Collagen Gels
Authors: Fukamachi Hiroshi , Kim Young S.
Published: January 2005

LS174T human colon cancer cells formed glandular structures with microvilli, tight junctions, desmosomes and basal laminae arranged in order as in normal intestinal epithelial cells when combined with fetal rat mesenchymes in organ culture. When cultured with type I collagen gel, they also formed glandular structures, but less efficiently than with the mesenchyme. In collagen gel‐induced glandular structures, microvilli, tight junctions and desmosomes were arranged in order as in mesenchyme‐induced ones, but basal laminae were never observed in contrast with mesenchyme‐induced ones. These results indicate that basal lamina formation is not necessary for the glandular structure formation and that mesenchymes promote glandular structure formation of LS174T cells by supplying components necessary for basal lamina formation.
Organization of Actin Filaments and Zonula Adherens during Somitogenesis in the Chick Embryo
Authors: Murakami Ryutaro , Suzuki Miyuki , Fujii Kaoru , Yamaoka Ikuo
Published: January 2005

The organization of F‐actin during somitogenesis in the chick embryo was studied by use of rhodamine‐conjugated phalloidin and transmission electron microscopy (TEM). Separation of a somite from the segmental plate proceeded simultaneously with the organization of segmental plate cells into a hemispherical epithelial sheet whose open side was directed antero‐laterally. At the same time, intense staining of F‐actin appeared in the apical surface of the epithelial sheet. Observations by TEM showed that zonulae adherentes associated with many actin filaments increased in the apical region of cells being organized into an epithelial sheet while this junctional apparatus was only sparsely distributed in the segmental plate cells. The hemispherical sheet subsequently closed to form an epithelial vesicle, with increase in curvature of its apical surface, and narrowing of cellular apices. At the same time, the zonulae adherentes and actin filaments in the cellular apices further increased, and many cellular processes formed on the apical surface of the epithelial somites. These findings suggest that segmentation involves organization of zonulae adherentes and a contractile process caused by acin filaments anchored to the zonulae adherentes.
Dorsal Blastomeres in the Equatorial Region of the 32‐Cell Xenopus Embryo Autonomously Produce Progeny Committed to the Organizer
Authors: Takasaki Hiroko , Konishi Hisami
Published: January 2005

For testing the autonomic differentiation abilities of dorsal equatorial blastomeres of 32‐cell Xenopus embryos, their roles in head formation in normal development and the organizer‐inducing capabilities of the dorsal‐most vegetal cells, interspecific transplantations were made using Xenopus borealis and X. laevis. When transplanted into the ventral region, the dorsal blastomeres produced descendants that differentiated into prechordal mesoderm, notochord and somites in the recipient according to their fates. They induced formation of the secondary embryo with the head and tail. The prechordal mesoderm and notochord in the secondary structure consisted of progeny of the graft, whereas somites and the CNS were chimeric and the pronephros was composed of host cells. Replacement of the dorsal blastomeres by ventral equatorial cells caused complete arrest of head formation in the recipient. Without exception, the notochord was completely absent or very thin. These results confirm the assumption that the presumptive head organizer in the Xenopus embryo is localized in the dorsal equatorial region at the 32‐cell stage and comes into existence not under the inductive influence of the dorsal‐most vegetal cells, but owing to allocation of morphogenetic determinants residing in the fertilized egg to the dorsal equatorial blastomeres of the 32‐cell embryo.
Formation of the Primitive Streak and Mesoderm Cells in Mouse Embryos—Detailed Scanning Electron Microscopical Study
Authors: Hashimoto Koichiro , Nakatsuji Norio
Published: January 2005

We describe morphological events of the mammalian gastrulation in pre‐ to middle‐primitive‐streakstage mouse embryos by using scanning electron microscopy. The first sign of the ingression of the mesodermal cells was disruption of the epithelial structure of ectoderm and the underlying basal lamina, thus forming a semicircular area of the presumptive primitive streak. Then, cells at periphery of the semicircular region spread on the basal lamina by extending many filopodia to it. The majority of the migrating cells formed a loosely arranged cell sheet. We found solitary cells and isolated small groups of cells migrating away from the periphery of the cell sheet. These cells were well spread on the basal lamina, and had large cell processes and many filopodia in the direction of cell migration. Filopodia of these cells were attached to the basal lamina or a meshwork of the extracellular fibrils. These observations suggest that the extracellular matrix serves as the substratum for cell adhesion and migration, and plays an important role in the mammalian gastrulation.
Differentiation of Striated Muscle Fibers in Monolayer Cultures of Rat Anterior Pituitary
Authors: Watanabe Yuichi G.
Published: January 2005

Striated muscle fibers appeared in monolayer cultures of rat anterior pituitary cells maintained in αMinimum Essential Medium (αMEM). As muscle differentiation in cultures of pituitary cells under ordinary conditions has not hitherto been reported, an in vitro study was undertaken to determine what factor(s) is responsible for this myogenesis. When dispersed anterior pituitary cells were culrured in three different media, αMEM, Medium 199 and Dulbecco's Modified Eagle Medium (DMEM), only αMEM induced a high incidence of striated muscles. The nature of the serum (fetal calf, calf and horse) and its concentration (1–10%) did not affect myogenesis.

In monolayers in αMEM, the sequence of differentiation of striated muscle was as follows: 1) Elongated cells, resembling myoblasts appeared; 2) these cells fused; and finally 3) cross striations appeared. Rhythmic contraction was most intense in striated muscle fibers, but it was also obsrved in myotubes without cross striations and even in myogenic cells before fusion. The possible origin of muscles in these pituitary cultures is discussed.
Induction of the Acrosome Reaction of Hemicentrotus pulcherrimus Spermatozoa by the Egg Jelly Molecules, Fucose‐Rich Glycoconjugate and Spem‐Activating Peptide I
Authors: Yamaguchi Masaaki , Kurita Masanori , Suzuki Norio
Published: January 2005

A fucose‐rich glycoconjugate (FRG) was isolated from egg jelly of the sea urchin Hemicentrotus pulcherrimus by gel filtration. FRG induced the acrosome reaction in H. pulcherrimus spermatozoa in a concentration‐dependent manner, although it showed about half the activity of the original unfractionated jelly. Synthetic sperm‐activating peptide I (SAP‐I: Gly‐Phe‐Asp‐Leu‐Asn‐Gly‐Gly‐Gly‐Val‐Gly) increased the rate of the acrosome reaction induced by FRG; the maximal rate of the acrosome reaction with FRG and SAP‐I being that of the unfractionated jelly. The half‐maximal increase in induction of the acrosome reaction by SAP‐I with FRG occurred at 4 × 10−10 M SAP‐I, which was almost the same concentration inducing half‐maximal stimulation of sperm respiration. Pronase digestion of FRG resulted in an 50% decrease in induction of the acrosome reaction and also in the elevation of cAMP in sperm. Some reagents (monensin and 3‐isobutyl‐1‐methylxanthine) which increase intracellular pH, Ca2+ and cyclic nucleotides also increased the rates of the acrosome reaction induced by FRG or pronase‐digested FRG. However, the rates did not reach those with FRG or pronase‐digested FRG with SAP‐I. These results indicate that SAP‐I promotes induction of the acrosome reaction by acting as a specific co‐factor of FRG.
Regional Response to Cytochalasin B of the Equatorial Cell Cortex in Sea‐Urchin Eggs during the First Mitosis
Authors: Usui Noriko , Yoneda Mitsuki
Published: January 2005

Sea‐urchin eggs treated with cytochalasin B (CB) during the first mitosis were examined for topography of the cell surface and the cortical microfilaments. Scanning electron microscopy revealed microvillar elongation and clumping on the whole cell surface of interphase and prophase eggs, but such changes were restricted to the equatorial surface in eggs treated with CB after metaphase up to the end of telophase. The clumping occurred also all over the cell surface of binuclear eggs after prolonged treatment up to the following interphase. These indicate localization of a target structure(s) of CB during mid‐ and late mitotic stages. Extraction with Triton X‐100 allowed detection of CB‐induced alterations in cortical microfilaments: The microvillar microfilaments were transformed into short filaments with random orientation. The submembranous meshwork and the contractile ring microfilaments were transformed into a network of shorter filaments. The prolonged CB treatment led to incorporation of the network microfilaments into the basal portions of the microvillar clumps. Thus, CB application during mid‐ and late mitotic stages induced (a) local microvillar changes and (b) alterations in the meshwork and the contractile ring. The present results suggest that the microvillar changes are caused by alterations of underlying cortical microfilamentous structures.
Ultrastructural Observations on the Germ Plasm in the Lizard Podarcis sicula
Authors: Andreuccetti Piero
Published: January 2005

Ultrastructural studies on embryos and adult females of Podarcis sicula revealed fibrogranular electron‐dense aggregates in the cytoplasm of primordial germ cells, oogonia, and oocytes. The ultrastructural similarities of these aggregates to fibrogranular aggregates in germ cells of some animal species and their relationship with mitochondria, free ribosomes, as well as cisternae of the rough endoplasmic reticulum strongly suggest that they correspond to the germ plasm.
Pluripotent Embryonal Stem Cell Lines Can Be Established from Disaggregated Mouse Morulae
Authors: Eistetter Harald R.
Published: January 2005

Mouse pluripotent embryonal stem (ES) cell lines hitherto have been conventionally isolated from the ‘inner cell mass’ of mouse blastocysts. In this report, I describe a new and simplified method for establishing pluripotent cell lines from mouse morulae of the 16‐ to 20‐cell stage, which were disaggregated by the use of EDTA. From 17 cell lines established in such a way, 7 were characterized with respect to their differentiation potential:

(i) When injected into syngeneic mice, the cells gave rise to solid, fully differentiated teratomas representing derivatives of all three germ layers. (ii) When cultured in suspension in vitro, the cells were able to differentiate into complex organized ‘embryoid bodies’ analogous to mouse early postimplantation embryos. These results strongly imply that embryonal stem cell lines isolated from mouse morulae are highly homologous to conventionally isolated ES cells.

In addition, my results indicate that murine pluripotent embryonal stem (ES) cell lines can be derived with more ease and higher efficiency from disaggregated morulae than from the ‘inner cell mass’ of blastocysts.
Asymmetric Segregation and Polarized Redistribution of Pole Plasm During Early Cleavages in the Tubifex Embryo: Role of Actin Networks and Mitotic Apparatus
Authors: Shimizu Takashi
Published: January 2005

In the precleavage zygote of Tubifex, pole plasm, which is yolk‐free cytoplasm, is located at the animal and vegetal poles. The present study describes the fate and localization pattern of the pole plasms in embryonic development of Tubifex. The process of pole plasm localization during cleavage stages is comprised of three steps. The first step is asymmetric segregation which results in bipolar localization of pole plasm masses in the D‐cell of the 4‐cell embryo. The spatial organization of pole plasm at this stage depends on F‐actin but not on microtubules. The second step is the redistribution of the vegetal pole plasm toward the animal pole and its unification with the animal pole plasm. These give rise to localization of unified pole plasm at the animal side (i.e. future dorsal side of the embryo) of the D‐quadrant. The polarized redistribution is sensitive to colchicine and topographically related to the mitotic apparatus located at the animal pole of the D‐cell. Electron microscopy shows the association with astral microtubules of constituents of pole plasm, suggesting the involvement of astral microtubules in cytoplasmic movement which gives rise to redistribution. In addition, centrifuge experiments suggest that the directional information for this polarized redistribution may be provided by some cytoplasmic organizations which are resistant to centrifugal force. The last step of the localization process is partitioning of unified pole plasm into two micromeres 2d and 4d. The spatial organization of pole plasm at this stage depends on microtubules but not on F‐actin.
Injection of Antibodies into Grasshopper Eggs as a Method for Studying Embryonic Development
Authors: Petrey Dwayne , Buster Dan , Donato Katja K. , Anderson Hilary
Published: January 2005

Procedures are described for injecting antibodies into grasshopper eggs at stages prior to 50% of embryonic development. A microdispenser is used to permit the accurate delivery of small, predetermined volumes of antibody solution. Injected antibodies are taken up by the embryo and reach their appropriate binding sites within 1 hr of injection. Antibody complexes remain stable for at least 24 hr. The method provides a useful tool for investigating developmental processes during early insect embryogenesis.
Characterization of Vegetal Plate Cells Separated from Cytochalasin B‐Treated Blastulae of the Sea Urchin, Clypeaster japonicus
Authors: Katow Hideki
Published: January 2005

Vegetal plate forming cells (VPCs) of the vegetal plate blastulae of the sea urchin, Clypeaster japonicus, had a layer of microfilaments on the basal side. The VPCs specifically protruded from the embryos after a treatment with 1 μg/ml of cytochalasin B (CB). Based on scanning electron microscopy, unlike other epithelial cells the protruded VPCs possessed neither cilium nor microvilli on their surface. The protruded VPCs were easily separated from the embryos by stirring the embryo suspension with pipette. An in vitro immunohistochemistry using a primary mesenchyme cell (PMC) surface‐specific monoclonal antibody (MAb) raised against PMCs of Strongylocentrotus purpuratus showed that the MAb also specifically bound to the PMCs in mesenchyme blastulae of C. japonicus. The MAb bound in 81% of the separated VPCs in C. japonicus vegetal plate blastulae examined. However, the MAb binding occurred only after the separated VPCs were incubated in artificial sea water (ASW) for at least 1 hr. In the VPC‐deprived embryos, gastrulation occurred after they were transferred to normal ASW. However, the PMCs and the spicules were not formed in these embryos.

In conclusion, a majority of the VPCs separated from the CB‐treated blastulae were presumptive PMCs. These VPCs provide an excellent source of presumptive PMCs.
Three Phases of Cortical Maturation during Meiosis Reinitiation in Starfish Oocytes
Authors: Chiba Kazuyoshi , Hoshi Motonori
Published: January 2005

Oocytes of the starfish, Asterina pectinifera, respond differently to calcium ionophore A23187 depending upon their stage of maturation. Oocytes not‐treated with 1‐methyladenine (1‐MA) formed only a partial fertilization envelope (FE) in response to A23187. Those treated with 1‐MA formed no FE if the ionophore was introduced to them before germinal vesicle breakdown (GVBD), in contrast with which they did fully elevate the FE if it was introduced after GVBD. Similar stage‐dependent results were obtained if the intracellular concentration of calcium was increased by microinjection of calcium‐EGTA buffers. In good accordance with the FE formation, a stage‐dependent protease release from oocytes by the ionophore was observed.

It is concluded from these results that, in starfish oocytes, their ability to undergo the exocytosis of cortical granules in response to an increase in intracellular calcium greatly changes along the way of maturation.
Study of the Lineage and Cell Cycle of Small Micromeres in Embryos of the Sea Urchin, Hemicentrotus pulcherrimus
Authors: Tanaka Shoji , Dan Katsuma
Published: January 2005

A study was made of 1st cell cycle of small micromeres, segregated at the 5th cleavage cycle, in the sea urchin embryos of Hemicentrotus pulcherrimus. For identification of small micromeres, the embryos were pulse labeled with 5‐bromodeoxyuridine (BrdU) at the 1st cleavage. Using multiparametric microfluorometry equipped with a scanning stage (Tanaka, 1990), DNA content, extent of BrdU incorporation, protein content and the extent of 3H‐thymidine labeling were measured on identical individual cells dissociated from an embryo. The findings of the present study are as follows. There is a short period of time between the telophase and onset of DNA replication. The period of DNA replication is 5 hr and after which, asynchronous mitosis takes place to produce 8 cells before hatching. The long S period is 83% the total 6 hr of the cell cycle. The rate of DNA accumulation is quite small during the initial one third of S but increases later in this phase. The degree of chromatin condensation remains high even during the S phase but it is low in large micromeres. The cell cycle may possibly be related causally to the development of small micromeres. The developmental significance of cell cycle duration, particularly that of DNA replication is discussed.
Thiol Protease Inhibitor, E‐64‐d, Prevents Spindle Formation during Mouse Oocyte Maturation1
Authors: Hashimoto Naohiro , Iwashita Shintaro , Shoji‐Kasai Yoko , Kishimoto Takeo , Imahori Kazutomo
Published: January 2005

E‐64‐d, a membrane permeant derivative of E‐64, the thiol protease inhibitor, was found to prevent meiotic maturation of mouse oocytes in a dose dependent manner. When immature mouse oocytes were incubated with E‐64‐d for up to 14 hr, first polar body emission was blocked to 36% at 200 μg/ml and 6% at 400 μg/ml, but germinal vesicle breakdown occurred normally. Cytological analysis revealed that meiotic spindles were not formed, while chromosome condensation occurred. Thus, E‐64‐d prevents oocytes from progressing to the first meiotic metaphase. When exposed to E‐64‐d after 8 hr of incubation without E‐64‐d, one‐fourth of oocytes completed the first meiotic division but never progressed to the second metaphase. In three‐fourth of the oocytes inhibited to emit the first polar body, spindles disappeared after incubation with E‐64‐d. The results suggest that E‐64‐d promotes disassembly of meiotic spindles resulting in inhibition of meiotic maturation. We propose that thiol protease is involved in spindle formation in mouse meiotic maturation.
Homoiogenetic Neural Induction in Xenopus Chimeric Explants
Authors: Itoh Keiji , Kubota Hiroshi Y.
Published: January 2005

We previously raised monoclonal antibodies specific for epidermis (7) and neural tissue (8) of Xenopus for use as markers of tissue differentiation in induction experiments (8). Here we have used these monoclonal antibodies to examine homoiogenetic neural induction, by which cells induced to differentiate to neural tissues can in turn induce competent ectoderm to do the same. Presumptive anterior neural plate excised from late gastrulae of Xenopus laevis was conjugated with competent ectoderm from the initial gastrula of Xenopus borealis, either side by side or with their inner surfaces together. The chimeric explants enabled us to distinguish induced neural tissues from inducing neural tissues. In both types of explant, neural tissues identified by the neural tissue‐specific antibody, NEU‐1, were induced in the competent ectoderm by the presumptive anterior neural plate. The results suggest that homoiogenetic neural induction does occur in Xenopus embryos.
Temporal and Spatial Expression of a Muscle Actin Gene during Embryogenesis of the Ascidian Halocynthia roretzi
Authors: Kusakabe Takehiro , Suzuki Junko , Saiga Hidetoshi , Jeffery William R. , Makabe Kazuhiro W. , Satoh Noriyuki
Published: January 2005

Screening a cDNA library from tailbud embryos of the ascidian Halocynthia roretzi with a Styela plicata mantle actin probe yielded several muscle‐type actin clones. These clones differed from each other in the nucleotide sequences of their 3′non‐coding regions, although the sequences of the coding region were almost identical. One of the clones, HrcMA4, was selected and characterized. HrcMA4 contains an open reading frame of 1137 bp and a 100 bp 3′non‐coding region followed by a poly(A) tail. An antisense probe consisting of a small segment of 3′coding region and a large portion of 3′non‐coding region of the clone was constructed. In situ hybridization analysis with this probe demonstrated that expression of HrMA4 mRNAs was restricted to differentiating muscle cells in tailbud embryos. Signal was not detectable in other regions of the embryo. Northern blot analysis showed that HrMA4 mRNA was undetectable in unfertilized eggs, zygotes and cleavage‐stage embryos. A single band of the HrMA4 transcripts about 1.5 kb in length was first observed in gastrulae. The amount of HrMA4 mRNAs increased rapidly as development progressed. The mRNA was evident in tadpole larvae and newly metamorphosed juveniles. The amount of transcripts, however, decreased after metamorphosis and became undetectable by a week after metamorphosis. Thus, the HrMA4 gene showed strict zygotic expression restricted to the muscle lineage cells.
Early Determination of Developmental Fate in Presumptive Intestinal Endoderm of the Chicken Embryo
Authors: Yasugi Sadao , Takeda Hiroyuki , Fukuda Kimiko
Published: January 2005

The endodermal epithelia of esophagus, proventriculus and gizzard of 6‐day chicken embryos can form glands and express embryonic chicken pepsinogen (ECPg), when they are subjected to the influence of proventricular mesenchyme, while intestinal epithelium of the same age cannot respond to the inductive influence of proventricular mesenchyme. We attempted in this paper to know whether this regional difference of epithelia to respond to mesenchymal influence originates very early in development or it is established gradually in the course of development of digestive tract.

The young presumptive intestinal endoderm taken from embryos having 15–20 somites was associated and cultivated with 6‐day proventricular mesenchyme. The presumptive intestinal endoderm never expressed ECPg although it formed gland‐like structures. In the control explants composed of presumptive stomach endoderm and proventricular mesenchyme, glands were formed and gland cells expressed ECPg detected by immunocytochemistry and in situ hybridization.

These results indicate that the developmental fate of presumptive intestinal endoderm is determined rather strictly at very early developmental stage, and suggest that the segregation of at least two cell lineages occurs early in the development; one which can express ECPg under the influence of proventricular mesenchyme, and another one which cannot express ECPg and differentiates mainly into intestinal epithelium.
Induction of Secondary Embryos by Intra‐ and Interspecific Grafts of Center Cells under the blastopore in Horseshoe Crabs
Authors: Itow Tomio , Kenmochi Shigeki , Mochizuki Taeko
Published: January 2005

When the cell mass (center cells) of the early gastrulae in both American and Asian horseshoe crabs was grafted into the embryo of the homologous species, secondary embryos were formed as a result of these grafts. Secondary embryos were also formed in a similar way when the center cells of heterologous embryos were grafted between the American and Asian horseshoe crab embryos. The characteristics of the secondary embryos were similar to the host embryos in both cases, indicating that the center cells played the roles same as those by the amphibian organizer. The homogenate of center cells also induced the formation of secondary embryos. This is the first published study in which secondary embryos of horseshoe crabs have been induced by grafting. The fact may mean that this type of embryonic induction is widespread in the animal kingdom.
Gray and Red Fragments of the Egg of the Ascidian Ciona savignyi: Preferential Development of Muscle Cells from Gray Fragments
Authors: Marikawa Yusuke , Satoh Noriyuki
Published: January 2005

The egg of the ascidian Ciona savignyi is pinkish red with brownish myoplasm that contains the putative determinants responsible for differentiation of muscle cells. When dechorionated unfertilized eggs were centrifuged at moderate speed, eggs were divided into centripetal, small gray fragments and centrifugal, large red fragments. The former contained the female pronucleus and clear cytoplasm, while most of the latter was filled with yolk granules. An antibody raised against the myoplasm of C. intestinalis eggs extensively stained the cortical region of gray fragments, while the antibody stained only small regions of the red fragments. After insemination, both fragments cleaved and gave rise to partial embryos. When development of muscle and epidermal cells in the partial embryos was examined with specific antibodies, muscle development was conspicuous in gray partial embryos, while epidermal differentiation was extensive in red partial embryos. Furthermore, when expression of markers of differentiation was examined in cleavage‐arrested gray and red fragments, the number of arrested gray fragments exhibiting the muscle marker was about three‐fold greater than in controls. These results suggest that putative muscle determinants are concentrated into gray fragments.
Biochemical and Immunological Characterization of Collagenase in Tissues of Metamorphosing Bullfrog Tadpoles
Authors: Oofusa Ken , Yoshizato Katsutoshi
Published: January 2005

Tissue inhibitor of metalloproteinases (TIMP, a specific inhibitor of collagenase) was found to inhibit thyroid hormone‐induced tail regression, suggesting the important role of collagenase in this process. Collagenase was purified from culture media of back skin of tadpole of bullfrog, Rana catesbeiana. Anti‐tadpole collagenase polyclonal antisera were obtained against the purified enzyme. The antibody inhibited the activity of tadpole collagenase. The antisera reacted to tissues of adult bullfrogs, tadpoles of african clawed frog, Xenopus laevis, and adult newts, Cynopus pyrrhogaster, and also reacted to human fibroblast collagenase. Immunoblot analyses suggested that tadpole collagenase lacks the procollagenase which is generally found in mammalian collagenases. Intense immunological stains were observed for the tissues of thyroid hormone‐treated tadpoles as compared to those of untreated animals. Thyroid hormone increased amounts of collagenase not only in epidermal layer but also in mesenchymal tissues including fibroblastic cells.
The Effects of lithium and Zinc Ions on the Pattern of Acidic Glycans in the Sea Urchin (Hemicentrotus pulcherrimus) Embryo
Authors: Taniguchi Yasushi , Terayama Hiroshi
Published: January 2005

Among several acidic glycan components found in Hemicentrotus embryos, the “F”‐ and “S”‐components were specifically affected by treatment with Li+and Zn2+, respectively. The amount of the “F”‐component in Li+‐treated embryos was about 60% that in normal embryos. This fact was in accordance with the reduced alcian blue staining of the surfaces in Li+‐treated embryos. Moreover, the “F”‐component in Li+‐treated embryos appeared to be composed of two subcomponents, while in normal and Zn2+‐treated embryos it appeared to be single. The “S”‐component in Zn2+‐treated embryos was about 8% that in normal embryos. According to histochemistry with a lectin probe, it was found that UEA‐I was much more strongly associated with a hyaline layer in Li+‐treated than in normal and Zn2+‐treated embryos. Li+‐treated embryos developed into exogastrulas, which were divided by a constriction into two parts; an animal half which stained intensely with alcian blue, and a vegetal half which stained poorly. On the other hand, Zn2+‐treated embryos remained as permanent blastulas. Considering the above, it is suggested that change in the acidic glycan pattern leads to alterations in the morphogenesis of sea urchin embryos.
Partial Characterization of Macromolecular Components in Fetal Bovine Serum Required for Development of Mouse Blastocysts Cultured in vitro
Authors: Tachi Chikashi
Published: January 2005

Elucidation of the mechanisms underlying implantation of blastocysts in eutherian mammals have been severely hampered by the lack of a suitable system for culture of blastocysts in vitro. Successful culture methods for mouse peri‐implantation embryos in vitro have been described by Hsu (1971, 1973, 1978, 1990), Chen and Hsu (1982) and Naruse et al. (1985), but these methods are too complex for routine experimental purposes. We attempted, therefore, to establish a standard culture method suitable for quantitative analysis of early embryogenesis in the mouse. Our system allows the development of peri‐implantation blastocysts from the time of shedding of the zona to formation of the proamniotic cavity under well defined conditions. Using this system, the macromolecular components in the sera required for the development of periimplantation mouse blastocysts in vitro were partially characterized. Results indicated that substances with molecular weight (MW) of 30 × 103 to 100 × 103 in the serum are capable of inducing the early phase of the trophoblast spreading. Furthermore, serum factors above MW 100 × 103 were found to be essential for the successful differentiation and/or development of ICM and ectoplacental cones.
Ammonia Determines the Alternative Pathways of Sexual or Asexual Development in the Cellular Slime Mold Dictyostelium discoideum
Authors: Fang Hui , Sakuma Takako , Yanagisawa Kaichiro
Published: January 2005

The sexual development, macrocyst formation, of Dictyostelium discoideum is initiated by sexual fusion of cells. The sexual fusion is only taken place under the culture conditions of excess water and darkness. Under these conditions, cells acquire the fusion competence, but lose it when cell density is high. The loss of the fusion competence is caused by accumulation of ammonia excreted by cells in a culture. Ammonia suppresses the fusion competence of cells at a certain concentration, and consequently inhibits formation of macrocysts and induces fruiting‐body formation. Thus, excess water induces the sexual development by diluting ammonia and lack of water induces the asexual development.
Factors Controlling Sperm Entry into the Micropyles of Salmonid and Herring Eggs
Authors: Yanagimachi Ryuzo , Cherr Gary N. , Pillai Muralidharan C. , Baldwin John D.
Published: January 2005

When the micropyle area of salmonid (trout and salmon) eggs was observed continuously from the moment of insemination, spermatozoa were seen moving along the surface of the chorion and entering the micropyle one by one in a directed fashion. The ability of spermatozoa to enter the micropyle was reduced after the treatment of chorions with pronase; this reduction in sperm entry was observed even before the outer opening of the micropyle channel was narrowed due to gradual swelling of the chorion by pronase treatment. Herring spermatozoa, unlike spermatozoa of most other marine fishes, were motionless in seawater. However, they became vigorously motile on contact with the micropyle area of the herring egg chorion and entered the micropyle rapidly and efficiently. Motility initiation of herring spermatozoa in the micropyle area was dependent on extracellular calcium and potassium. Sodium also appears to be intricately involved in this process as demonstrated by the initiation of sperm movement in sodium‐free seawater. When herring eggs were treated with acidic seawater, organic solvents, or glutaraldehyde, spermatozoa did not initiate movement in the micropyle area, and sperm entry was not observed. Herring spermatozoa did not initiate movement in the micropyle area of salmonid eggs. These and other observations suggest that the micropyle areas of salmonid and herring eggs possess some sperm guidance factors which facilitate entry of homologous spermatozoa into the micropyle.
Ascidian Budding as a Transdifferentiation‐Like System: Multipotent Epithelium is not Undifferentiated
Authors: Fujiwara Shigeki , Kawamura Kazuo
Published: January 2005

During bud development of the ascidian Polyandrocarpa misakiensis, most of the new tissues are formed from foldings of atrial epithelium. Although the atrial epithelium has been believed to be undifferentiated, we found that this epithelium of P. misakiensis strongly expressed a tissue‐restricted antigen, named Pae 1. Cross‐reactivity of the antibody was found only in a few differentiated tissues such as branchial epithelium and phagocyte‐like cells. In developing buds, the antigen disappeared selectively from the regions where the atrial epithelium forms organ rudiments. These regions corresponded with that of mitotic activity, thickening of the epithelium, swelling of nuclei, the appearance of nucleoli and accumulation of a large amount of RNA. From these observations, we assume that the change in antigen expression indicates a change in the state of differentiation of the atrial epithelium. Although Pae 1 antigen was never detected in functional gut, it was detected in the invaginating gut epithelium. This result indicates that gut cells were derived from the cells which had expressed the antigen. We therefore conclude that the conversion of the atrial epithelium into gut can be regarded as a transdifferentiation‐like process.