bluuebunny/crossref_metadata_2025 huggingface dataset:
Polysaccharide Complexes in Full‐Grown Oocytes of the Newt, Pleurodeles, and Changes in their Distribution During Progesterone‐Induced Maturation
Authors: CHARBONNEAU MICHEL , PICHERAL BERTRAND
Published: January 2005

Immature full‐grown oocytes of Pleurodeles waltlii contain large amounts of small electron‐dense polysaccharidic granules. These granules lack a limiting membrane, and have a dense but heterogeneous matrix and an apparent diameter of 24–36 nm. Their structure, organization and distribution strongly suggest that they are glycogen granules. On the other hand, mature oocytes both after oviposition or 22–24 hr after in vitro progesterone stimulation contain no polysaccharide granules or complexes. During the first 9–10 hr after hormonal stimulation, granules were abundant and present both individually and as large strands occupying most of the space between the organelles. Granules were frequently found packed together and arranged in regularly arrayed stacks within large subcortical ant cortical vacuoles. Between 4 and 6 hr after progesterone addition, oocytes released the contents of vacuoles to the outside. Between about 11 and 14 hr after progesterone addition, oocytes still contained large amounts of polysaccharide complexes, but the vacuoles were empty. From about 15 hr after progesterone treatment until the end of maturation, the complexes progressively disappeared from the cytoplasm, coincident with the detachment of the follicle cell layer from the oocytes and a reduction in the number and size of microvilli.
Differentiation of Sea Urchin Micromeres: Correlation between Specific Protein Synthesis and Spicule Formation
Authors: KITAJIMA TAKASHI
Published: January 2005

Sea urchin micromeres were isolated from the 16‐cell stage embryos and cultured until they differentiated into spicule‐forming cells. Electrophoretic analysis of proteins labeled with [35S]‐methionine showed that the differentiation accompanied the synthesis of five cell‐specific proteins. These proteins appeared prior to spicule formation and were synthesized continuously or maintained stably while the cultured micromeres formed spicules. In contrast, these proteins were hardly detectable during development of the meso‐ and macromeres. Correlation between synthesis of the specific proteins and spicule formation was further examined in culture conditions which inhibit spicule formation. In Zn2+ ‐containing or serum‐free medium, the micromere descendants failed to form spicules and exhibited markedly reduced synthesis of one of the specific proteins (32 K daltons). After removal of Zn2+, or addition of serum, however, spicules were formed with delay but concomitantly with an increase in the synthesis of this protein. This clear correlation suggests the participation of the 32 K protein in the process of spicule formation.
Effects of Low Temperature on Differentiation of Dictyostelium Cells in the Vegetative and Preaggregation Stages
Authors: MAEDA MINEKO
Published: January 2005

The effects of low temperature on differentiation of Dictyostelium discoideum cells in the vegetative and preaggregation stages were examined immunohistochemically and electronmicroscopically. Ultimately cells in both stages were found to stain with FITC‐conjugated anti‐Dictyostelium mucoroides spore antibody at low tempeature (5°C) in nutrient‐rich medium under submerged conditions. These cells were sensitive to 0.1% Triton X‐100. In contrast, cells in the late aggregation stage were converted to detergent‐resistant spores. Irrespective of their high stainability with antispore‐Ig, these cells did not contain the prespore‐specific vacuoles (PSVs) and spore‐coats characteristic of stainable cells. From these facts, it is concluded that cells in the vegetative and preaggregation stages are unable to differentiate at low temperature into prespore and spore cells, which have PSVs and spore‐coats, respectively. The composition of spore‐specific (SS) antigens accumulated by these cells was examined by immunoblotting. Results showed that under these conditions the cells mainly produced one SS‐antigen, namely SP70. The effects of cell density and components of the medium on the production of SS‐antigens were also examined.
Extracellular Matrix in the Blastocoel of Newt Gastrula: its Effects on Dissociated Embryonic Cells and some Aspects of its Biochemical Nature
Authors: KOMAZAKI SHINJI
Published: January 2005

The extracellular matrix (ECM) was removed manually from the blastocoel of freeze‐dried embryos of the newt, Cynops pyrrhogaster. In vitro examination demonstrated that the substratum of the blastocoelic ECM (B–ECM) promoted adhesion and spreading of dissociated gastrula cells, while little effect was shown on dissociated blastula cells under the same conditions. Furthermore, the B–ECM promoted locomotion of the spreading gastrula cells. SDS‐polyacrylamide gel electrophoretic analysis revealed that the B–ECM contained three major proteins (108, 97 and 30 kilodaltons), which co‐migrate with yolk proteins, and a 190‐kilodalton protein. The yolk proteins extracted from yolk platelets were found also to act effectively as an strong adhesive sub‐stratum for dissociated gastrula cells, however they did not promote cell locomotion. These results suggested that the yolk proteins in the B–ECM may act as an effective adhesion substance for dissociated gastrula cells.
Effects of Microinjected Cations on the Early Event of Fertilization in the Medaka Egg
Authors: IWAMATSU T. , ITO S.
Published: January 2005

Effects of microinjected cations on the early events of fertilization were examined using eggs of Oryzias latipes. Microinjection of either Ca2+, Ba2+ or Sr2+ into the thin cortical cytoplasm induced breakdown of cortical alveoli (vesicles) (CABD) under Ca‐Mg‐free conditions, but microinjection of Mg2+, Mn2+ or Co2+ prevented CABD at the injected region when the eggs were inseminated in regular saline. Under Ca‐Mg‐free conditions, CABD could also be induced by microinjection of various solutions (NaCl, choline chloride, sucrose, pH buffer) without any divalent cations or ionophore A23187. Ca2+ microinjected into the cortical cytoplasm did not play a role in sperm penetration. Upon microinjection with either Ca2+, Mg2+ or K+, the resting membrane potential leakage was transiently observed. However, depolarization of the membrane followed by slow hyperpolarization was observed only upon microinjection of Ca2+. From these experiments, it was inferred that microinjected divalent cations such as Ca2+, Ba2+ or Sr2+ do not act directly upon the cortical alveolus membrane, but trigger the induction of CABD via depolarization of the membrne and increase in intracellular Ca2+.
The Wave Pattern of Free Calcium Release Upon Fertilization in Medaka and Sand Dollar Eggs
Authors: YOSHIMOTO YASUAKI , IWAMATSU TAKASHI , HIRANO KEN‐ICHI , HIRAMOTO YUKIO
Published: January 2005

A transient rise in the concentration of Ca2+ in the cortex upon fertilization was demonstrated in medaka eggs injected with aequorin. Detection of the aequorin luminescence with an ultra‐high sensitivity photonic microscope system revealed a wave of increased Ca2+ concentration starting at the site of sperm entry (animal pole) and being propagated along the cortex of the egg toward the antipode. The wave traversed the entire egg surface within 2–3 min. The peak value of the aequorin luminescence, and therefore the peak value of the Ca2+ transient, was generally higher at the site of sperm entry than in other regions. The peak values of the luminescence (and therefore of the Ca2+ concentration in the cortex) remained fairly constant during propagation of the wave. Microinjection of Ca2+ into the cortex also induced a Ca2+ wave. When the egg was stimulated by microinjection of Ca2+ at the equatorial region, the Ca2+ wave was propagated at a fairly constant speed over the egg surface, except at the region near the vegetal pole where the wave was retarded. Simultaneous recording of the Ca2+ wave and the wave of cortical change (breakdown of cortical alveoli) in eggs during fertilization revealed that the Ca2+ wave preceded the wave of cortical change.

A Ca2+ wave was also demonstrated in sand dollar eggs, although due to their smaller size the phenomenon was not as clear as in medaka eggs.
Microdissection Studies on the Polarity of Unequal Division in Grasshopper Neuroblasts: II. Cell Division in Binucleate Neuroblasts
Authors: YAMASHIKI NAOKO , KAWAMURA KEN‐YA
Published: January 2005

The grasshopper neuroblast divides unequally to produce two types of cells: a large daughter neuroblast that contains a doughnut‐shaped nucleus and repeats unequal division with definite polarity, and a small daughter ganglion cell that has a spherical nucleus with low mitotic activity. Binucleate neuroblasts were induced by preventing cytokinesis in the course of microdissection experiments, and subsequent divisions were traced to analyze the factors that determine the polarity of unequal division.

In binucleate neuroblasts, both daughter chromosome groups developed into neuroblast‐type nuclei. Mitosis of the two nuclei proceeded synchronously. Although the axes of the two mitotic apparatuses formed at late prophase were random in direction, they became parallel with the original division axis at metaphase. The two mitotic apparatuses shifted simultaneously toward the ganglion cell side during anaphase, just as in normal neuroblasts, and the binucleate cell divided unequally. These findings showed that the poearity of unequal division is strictly maintained in grasshpper neuroblasts, even when they contain two nuclei.
Cytological Effects of Heat‐Shocks on Xenopus Oocytes and Eggs
Authors: HANOCQ‐QUERTIER J. , BALTUS E. , BRACHET J.
Published: January 2005

Heat‐shocks (80 min at 34°c) induce the appearance of aster‐like fibrous structures (cytasters) in maturing Xenopus oocytes. Cytaster formation is suppressed by treatments with colchicine or nocodazole of heat‐shocked maturing oocytes. Heat‐shocks destroy the meiotic spindle, but have no effect on cytasters induced by D2O treatment.

Heat‐shocks (20 min at 36°c) also induce the formation of cytasters in unfertilized and freshly fertilized eggs of some females, but not in cleaving eggs. They suppress amphimixy, induce the regression of existing cleavage furrows and destroy the mitotic apparatuses in cleaving eggs. The arrested blastomeres often contain micronuclei (probably unfused telophase swollen chromosomes) where small basophilic nucleoli are frequently seen.

The significance of these results is discussed.
Changes in Intracellular Concentrations of Cyclic Nucleotides during the Initiation Process of Starfish Sperm Motility
Authors: NIITSU‐HOSOYA NATSUMI , ISHIDA KATSUMI , MOHRI HIDEO
Published: January 2005

Changes in the intracellular concentrations of cyclic nucleotides during the initiation of starfish sperm motility were examined. The intracellular concentration of cGMP decreased just after dilution of sperm with sea water, whereas that of cAMP increased concomitant with initiation of sperm motility. In acidic sea water, the intracellular concentration of cGMP decreased but no increase in that of cAMP was observed and sperm remained immotile. The presence of a phosphodiesterase inhibitor enhanced the rate of increase in the intracellular concentration of cAMP and the sperm motility.

These results indicate that cAMP is involved in the initiation of sperm motility in starfish.
Roles for Potassium and Calcium Channels in the Initiation of Sperm Motility in Rainbow Trout.
Authors: TANIMOTO SATOMI , MORISAWA MASAAKI
Published: January 2005

It is well known that the motility of spermatozoa in rainbow trout is suppressed by K+. We showed here that although trout sperm are completely immotile in medium containing 5 mM K+, motility was initiated by the subsequent addition of several mM Ca2+, suggesting that both K+and Ca2+are related to the process of the initiation of sperm motility. It was further found that K+channel blockers tetraethylammonium, nonyltriethylammonium, Ba2+and Cs+, as well as the Ca2+channel blocker verapamil, inhibited the initiation of sperm motility at doses at which these reagents inhibit chnnel‐related functions in other cells. However, Na+channel blocker, tetrodotoxin and anion channel blocker 4, 4‐diisothiocyatatostilbene‐2, 2′‐disulfonic acid inhibited the motility only at extremely high doses. These results suggest that transport of K+and Ca2+through ion channels at the plasma membrane of spermatozoa is the first event that triggers the initiation of sperm motility in rainbow trout.
Plasma Membrane Glycoproteins of Ciona intestinalis Spermatozoa that Interact with the Egg1
Authors: CASAZZA GIANNA , DE SANTIS ROSARIA , PINTO MARIA ROSARIA
Published: January 2005

In the ascidian Ciona intestinalis the species‐specific interaction between the spermatozoon and the egg occurs between the vitelline coat (VC) of the egg and the plasma membrane of the apical part of the head of the spermatozoa. Concanavalin A (Con A)‐binding sites are present on this area of the sperm surface. We used Con A to identify and isolate the spermatozoon plasma membrane components that may be involved in the interaction with the VC. These glycoproteins have been identified on SDS‐PAGE of a sperm membrane fraction (SMF) enriched with the extermal proteins, after incubation of the gel with 3H‐Con A. Affinity chromatography on Con A‐agarose has been used for the purification of sperm plasma membrane proteins with and affinity for the lectin. The biological activity of the Con A‐retained fraction was determined with binding and fertilization assays.
Protein Synthesis Initiated by Cell Fusion in Dictyostelium discoideum
Authors: MORIYAMA RYOICHI , YANAGISAWA KAICHIRO
Published: January 2005

D. discoideum has two alternative developmental pathways. If cells of two complement mating‐type strains, NC4 and HM1, fuse sexually, a giant cell is produced which subsequently develops into a macrocyst, the sexual structure of this organism. However, if fusion fails to occur and cells are starved, a fruiting‐body is produced instead of a macrocyst. In this paper, a two‐dimensional polypeptide gel electrophoresis study showed that giant cells produce specific polypeptides which may possibly be involved in macrocyst development. Out of total 497 polypeptides which appeared in a giant cell during an incubation period of 13 hr, 92 were the specific for giant cells. Four of these polypeptides were appeared within only 1 hr after the cell fusion. The other 405 were non‐specific polypeptides which appeared in both giant cells and NC4 or/and HM1 cells. However, the patterns and the rates of production of each polypeptide during the incubation period were different between these cells.
Dual Effects of cAMP on the Stability of Prespore Vesicles and 8‐bromo cAMP‐enhanced Maturation of Spore and Stalk Cells of Dictyostelium discoideum
Authors: MAEDA MINEKO
Published: January 2005

It is well known that interconversion between prestalk and prespore cells occurs in 3‐dimensional (3–D) isolates of Dictyostelium. The present work was undertaken to examine whether or not the interconversion occurs even in monolayer sheets. The results suggested that in monolayer sheets of either prespore or prestalk cells, the interconversion does not occur. Furthermore, effects of cAMP were examined in relation to the formation or loss of prespore vesicles (PSVs). In monolayer sheets, prespore cells retain their PSVs in the presence of cAMP, though they lose them in its absence. In 3–D masses, however, cAMP induces the conversion into stalk cells, stimulating PSV loss. In the case of prestalk cells, cAMP induces the maturation of prestalk cells to stalk cells in 3–D masses, but it does not induce stalk differentiation in monolayer sheets.

8‐Bromo cAMP stimulates the maturation of prespore and prestalk cells into spore and stalk cells, respectively. However, the vegetative and the aggregative cells remain amoeboid even in its presence. These observations suggest that 8‐bromo cAMP stimulates the maturation rather than inducing prespore and prestalk differentiation.
Cellular src Gene Expression Associated with Lentoidogenesis in Transdifferentiating Cultures.
Authors: TOBAL KHALID A. , ELLIS DAVID K. , DEPOMERAI DAVID I.
Published: January 2005

We have shown (9) that elevated pp60c–src kinase activity accompanies the transdifferentiation of chick embryo neuroretinal (NR) cells into lens in vitro; moreover, most immunologically‐detectable pp60c–src protein is confined to lentoid bodies in permissive cultures (FH; 6). By contrast, pp60c–src expression is low in non‐permissive cultures where lentoid formation is blocked by high glucose (FHG; 6) or medium 199 (11). We now extend these findings in several respects. Firstly, glial‐enriched cultures in both FH and FHG media form small but sparse lentoid bodies at around 20 days, accompanied by increases in both δ crystallin and pp60c–src expression. In later FHG cultures, these lentoids increase neither in number/size nor in δ/pp60c–src expression, in contrast to permissive (FH) cultures. Thus the high glucose block on transdifferentiation is only partly mediated by neuronal influences (19). Secondly, transdifferentiating cultures of tapetal cells show higher levels of pp60c–src relative to redifferentiated or dedifferentiated (16, 17) cultures of these cells. Thirdly, we find no evidence that c‐src oncogene expression directly signals transdifferentiation. Thus v‐src expression in RSV‐transformed NR cells inhibits δ crystallin accumulation (29; this study), while a c‐src‐substituted RSV variant has little effect on NR transdifferentiation. Late cultures of NR cells in medium 199 fail to accumulate pp60c–src protein or c‐src transcripts, even though previous studies (2) showed that δ crystallin transcripts are localised within the nuclei of many cells in such cultures.
Heat‐Induced Changes in Thermosensitivity and Gene Expression during Development
Authors: BOON‐NIERMEYER ELIDA K. , DE WAAL ADRIENNE M. , SOUREN JAN E. M , van WIJK ROELAN
Published: January 2005

The thermosensitivity of developing embryos of the fresh water snail Lymnaea stagnalis was investigated from the 4‐cell stage to the 3‐day‐old trochophore larva by means of survival curves for 43.6°C. Cleavage stage embryos were extremely thermoresistant as compared with older stages, and thermosensitivity increases during the development.

Pretreatment with a mild heat exposure (10 min at 39°C) did not induce thermotolerance at the 4‐cell stage, but it did so in the early gastrula and trochophora. Development of thermotolerance in 1‐, 2‐, and 3‐day‐old stages showed an identical kinetic pattern.

After incubation in 35S‐methionine one‐dimensional gel electrophoresis was carried out with or without preheating. At the 4‐cell stage no enhanced synthesis of heat shock proteins was induced by exposure to heat. At stages of 1 day and older heat induced the enhanced synthesis of the heat shock proteins with apparent molecular weights of 38, 65 and 70 kilodaltons. The synthesis of heat shock protein 70 changes during the early development of Lymnaea both in its constitutive level and in its ability to be enhanced by heat treatment.
Deprenyl, an Inhibitor of Monoamine Oxidase B, Delays the First Two Mitotic Cycles of Sea Urchin eggs
Authors: Nicotra Antonietta , Serafino Annalucia , Arizzi Mario
Published: January 2005

Sea urchin eggs are known to display a significant level of monoamine oxidase B activity. Deprenyl, a highly selective inhibitor of monoamine oxidase B, was found to prolong the length of the first two mitotic cycles of the sea urchin egg. The prophase seems to be specifically prolonged by the drug, while the length of the other phases of the cycle is unaffected. These results suggest that the monoamine oxidase B may be a component of the monoaminergic system acting during the divisions of the sea urchin eggs.
Effects of Forskolin on Fine Structures of Medaka Follicles
Authors: Iwamatsu Takashi , Ohta Tadayuki
Published: January 2005

Effects of forskolin (FK), which stimulates production of 17α, 20β‐dihydroxy‐4‐pregnen‐3‐one and estradiol‐17β, on the fine structure of preovulatory follicles of Oryzias latipes were examined. Granulosa cells incubated in culture medium containing FK exhibited dislocation of the nucleus from the chorion side to the basement membrane side, vesiculation of conspicuous dilated endoplasmic reticulum (ER) with electron‐dense material and Golgi lamellae, and development of large oval mitochondria with an electron‐dense matrix. Moreover, a thin vesicular layer adherent to the outermost layer of the chorion was found in all immature oocytes at the end of incubation in the presence of FK. Intercellular junctions between granulosa cells and the oocyte gradually decreased during incubation in the presence of FK, and were finally lost with closure of the radial canals in the chorion at the end of the incubation. On the other hand, intrafollicular oocytes that were first incubated with FK for 10 hr, matured normally when they were incubated an additional 8 hr in plain medium. In granulosa cells of these follicles, the dilated ER and vacuolated Golgi lamellae were no longer detectable. These observations suggest that the development of dilated ER and vacuolated Golgi lamellae is characteristic of granulosa cells induced by FK.
Temporal Expression of Myosin Heavy Chain Gene during Ascidian Embryogenesis
Authors: Makabe Kazuhiro W. , Satoh Noriyuki
Published: January 2005

A muscle‐specic monoclonal antibody, termed Mu‐2, recognizes a single 220‐kd polypeptide which first appears in early tailbud‐stage embryos of the ascidian Halocynthia roretzi (17). In the present investigation, a cDNA library was prepared from poly(A)+ RNA isolated from tailbud embryos. The resulting cDNAs were inserted into the expression vector γgt11, and the library was screened with the Mu‐2. One of positive clones, containing an insert about 1.6‐kb in length, was selected and subcloned. Partial sequence analyses at the 5′ terminus and at the 3′ terminus of the 1.6‐kb cDNA allowed the amino acid sequence. The deduced sequence showed extensive homology to the rodent myosin heavy chain protein. Using an antisense RNA probe generated from the cDNA, Northern blot hybridization was carried out to measure the level of the myosin heavy chain gene transcripts during normal embryogenesis. These transcripts were not detected at pregastruia stages, after which they accumulated rapidly.
Kidney Proximal Tubule Cells Originate from Approximately Four Progenitor Cells and Make Distinct Patches in Mouse Aggregation Chimeras
Authors: Nakayama Hiroki , Kuroda Hideya , Fujita Jun , Ru Xiao‐Mei , Kitamura Yukihiko
Published: January 2005

Giant lysosomal granules of bgJ/bgJ mutant mice were used as a marker to investigate the histological composition of kidney proximal tubule cells. Embryos of the bgJ/bgJ genotype and those of the +/+ genotype were aggregated, and lysosomes of their proximal tubule cells were histochemically stained with the β‐glucuronidase activity. Tubules composed of bgJ/bgJ‐type epithelial cells alone, tubules composed of +/+‐type epithelial cells alone and tubules containing both types of epithelial cells were observed in cross sections. When the long straight portion of proximal tubules was reconstructed from serial sections, most of the tubules were of the mixed type, and distinct patches of bgJ/bgJ‐type or +/+‐type epithelial cells were detectable. These patches appeared to extend to the longitudinal rather than the circumferential direction. This suggests the clonal proliferation followed the mixing of embryonic progenitor cells for proximal tubule cells. Estimation from proportions of bgJ/bgJ‐type proximal tubules in total examined proximal tubules gave a pool size of approximately four primordial precursor cells for proximal tubules in both right and left kindeys. The fact that the proportion of bgJ/bgJ‐type components was comparable between the right and left kidneys of each chimera suggests that all proximal tubule cells in both right and left kidneys may originate from common primordial precursor cells.
The Effect of Corticosterone on the Growth of Perinatal Rat Lung Fibroblasts in Culture: Modulation by the Extracellular Matrix
Authors: Morishige Walter K. , Montes Ana M.
Published: January 2005

Third passage lung fibroblasts from rat pups between the ages of fetal day 17 and postnatal day 15 were allowed to attach onto either tissue culture plastic or an endothelial cell‐derived matrix, and were then exposed to different concentrations of corticosterone in culture medium containing 1% charcoal‐stripped serum. The effect of the hormone on growth of these cells was assessed after 48 hours of exposure by radiothymidine incorporation into DNA. Lung fibroblasts on plastic responded to the hormone in an age‐dependent manner; thus, cells which were obtained during late gestation (days 19–21) and after the third day of extrauterine life were consistently growth‐inhibited by corticosterone, whereas those which were obtained from fetal donors prior to day 19 and from neonatal donors (days 0–3 after birth) were stimulated to grow in response to the hormone. On the other hand, cells that were plated onto an endothelial cell‐derived matrix showed a different age‐related response to the hormone. Thus, lung fibroblasts from all fetal donors and from postnatal donors up until 4–5 days of age were stimulated to grow in response to corticosterone. This modulatory effect of the matrix on the fibroblast response to corticosterone was also seen in early passage fibroblasts. In an attempt to identify the modulatory agent(s) in the extracellular matrix, fibroblasts from day 19 fetal rat lung were challenged with corticosterone in the presence of laminin, fibronectin, type IV collagen, type I collagen, heparin, or chondroitin sulfate. None of these agents exerted a modulatory effect resembling that seen with the extracellular matrix. These results suggest the existence during lung development of a fibroblast‐endothelial interaction, the nature of which remains to be elucidated, which may serve to modify the effects of circulating hormones.
Development of the Basal Lamina and Its Role in Migration and Pattern Formation of Primary Mesenchyme Cells in Sea Urchin Embryos
Authors: Amemiya Shonan
Published: January 2005

The development and substructure of the basal lamina and its role in migration and pattern formation of primary mesenchyme cells (PMCs) in normal as well as Li+‐ and Zn++‐treated embryos of sea urchins were investigated by electron microscopy. Major findings were as follows. 1) Network fibrils appear along the basal surface of the blastular wall by the hatching blastula stage. The area covered with fibrils is restricted to the vegetal hemisphere at earlier stages, but extends to the animal hemisphere as development proceeds. 2) Nonfibrous fuzzy material embeds the fibrils to form a basal lamina, but in places the fibrils project from the basal lamina into the blastocoel. The major components of the fuzzy material were digested by glycosidase, which failed to digest the fibrous components. 3) The fibrils can be classified into two types, one Ca++‐independent and the other Ca++‐dependent. PMCs apparently utilize the Ca++‐indepndent fibrils as a substratum for locomotion. 4) After migration, PMCs accumulate in a specific region to form the PMC pattern. This is formed in the area of greatest concentration of Ca++‐independent fibrils. 5) PMCs in embryos treated with LiCl, in contrast to normal embryos, accumulate in the animal pole region where the Ca++‐independent fibrils are markedly concentrated.
Partially Fertilized Sea Urchin Eggs: An Electrophysiological and Morphological Study.
Authors: Dale Brian , Hagström Berndt , Santella Luigia
Published: January 2005

Propagation of the cortical reaction in sea urchin eggs may be interrupted by a mild heat shock. In such partially fertilized eggs three distinct cortical zones may be distinguished. First, an activated area where cortical granule exocytosis is complete, the fertilization membrane is elevated, and there is a cortical meshwork of polymerized actin. Second, at the antipode an area where the cortical granules are intact, actin is not polymerized, and the surface structure in general resembles that of the virgin egg. Between the two there is a transitional zone, some 10 to 20 μm wide, where a fraction of cortical granules have exocytosed, giving rise to isolated “blebs” of elevated fertilization membrane. Partially fertilized eggs have resting potentials ranging from −20 to −80 mV, and upon re‐insemination give rise to step depolarizations indicating that spermatozoa may interact and possibly fuse with the “unactivated surface”.
Ultrastructural Localization of Calcium in the Acrosome and Jelly Coat of Starfish Gametes
Authors: Sousa Mário , Azevedo Carlos
Published: January 2005

The potassium pyroantimonate technique was employed to localize calcium ultrastructurally on both male and female starfish gamete regions that first interact at fertilization. In the spermatozoon of Marthasterias glacialis, antimonate precipitates in the peripheral dense component of the acrosomal vesicle, while in the oocyte it precipitates in the jelly coat and beneath the oolemma. Calcium was identified in the precipitates by testing the chelator‐sensitivity and by X‐ray microanalysis of the precipitates.
Importance of Desmosome Formation for Glandular Organization of LS174T Human Colon Cancer Cells in Organ Culture
Authors: Fukamachi Hiroshi , Kim Young S.
Published: January 2005

LS174T human colon cancer cells exhibited typical epithelial polarity and 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 or collagen gels in organ culture. Quantitative analysis showed that the number of desmosomes per unit area was always much greater in subluminal areas of lumen‐forming LS174T cells than in other areas irrespective of the culture period or culture method, and this was also the case with rat intestinal epithelial cells in normal development. In contrast, no such clear relationship could be found between the glandular organization of LS174T cells and formation of other ultrastructural components examined (microvilli, tight junctions and basal laminae). These results suggest that desmosome formation plays important role in the glandular organization of intestinal epithelial cells.
Gene Expression, DNA Synthesis and Protein Synthesis in Cells from Dissociated Sea Urchin Embryos
Authors: Stephens Laurie E. , Shiflet George W. , Wilt Fred H.
Published: January 2005

DNA synthesis, protein synthesis, and the accumulation of two tissue‐specific transcripts were examined in cultures of sea urchin embryos that were completely dissociated during early cleavage stages and raised in Ca2+ ‐free sea water for 24 hr. In Strongylocentrotus purpuratus, both DNA and protein synthesis were severely reduced by dissociation treatment. Previous studies using this species indicated that aboral ectoderm‐specific transcript accumulation is severely reduced while mesenchyme‐specific transcripts generally accumulate more normally (11, 17). We therefore examined the accumulation of tissue‐specific transcripts in another specific of sea urchin (Lytechinus pictus) whose DNA and protein synthetic rates were found to be less affected by dissociation. We find that accumulation of an aboral ectoderm‐specific transcript (Spec 1) is also extremely low in L. pictus while a primary measenchyme‐specific transcript (LSM) accumulates to normal levels. When DNA synthesis was restored to 77% of normal by keeping dissociatesd cells apposed with the fertilization membranes, Spec 1 accumulation was not improved. Adding Ca2+ to the sea water 8 hours after fertilization and allowing cells to reassociate into small, tight clusters restored Spec 1 accumulation to 70% of controls. Hence, there is no direct correlation between the reduced metabolic acitivities and lowered Spec 1 accumulation in dispersed cell cultures.
Heterogenous Macromolecular Contributions to Early Mouse Embryo Development
Authors: Hsu Yu‐Chin
Published: January 2005

Mouse embryos have been cultured for more than one half of its gestation period by providing sera from definite species of animals as the inducer at the proper stage of development. The processes of normal development at four different but discrete phases have been described.

(1) Fertilized ovum (stage 1) is able to grow up to denuded blastocyst stage and attaches to the culture dish (stage 7) in balanced salt solution with bovine serum albumin as the sole macromolecule. (2) Embryoblast or inner cell mass (ICM) of denuded blastocyst (stage 7) is able to develop in fetal calf serum (FCS), human placenta cord serum (FCS), or sera from mouse (MS), rat (RS) or rabbit (RbS) to the early cylinder stage (stage 11). (3) The early egg cylinder stage (stage 11) of mouse embryo is able to grow in HCS and RS, but not in FCS nor RbS, to the stage of early somite stage (stage 15). (4) Beyond early somite stage (stage 15), mouse embryo is able to develop neural tissues in rat serum. The macromolecular nature of these growth factors in serum has been described (Hsu, 1980).

It indicates that the differential biological activity which induces the early mouse embryogenesis among the sera from different animal species is due to the various degree of sequence homology between the growth factor family among the different species of animals.
Heat‐Induced Reversal of Dorsal‐Ventral Polarity in Xenopus Eggs
Authors: Sakai Masao
Published: January 2005

Heat‐treatment of fertilized Xenopus laevis eggs at 30°C induced; 1. conspicuous concentration of the pigment toward the sperm entry point (SEP), 2. eccentric first cleavage furrow formation, and 3. reversal of the dorsal‐ventral polarity of the embryos. The optimal treatment was for 2.5 min applied at 20 min postfertilization (p.f.). The rotation movement of the Nile‐blue stained spots in the vegetal hemisphere of the heated eggs accurately located the future dorsal midline as in untreated embryos (ref. 22). Exposure of eggs to D2O also reversed the dorsal‐ventral polarity of the embryo suggesting that stabilization of microtubules is involved in the dorsal‐ventral axis reversal.
Mitochondrial Protein Synthesis During Embryonic Development of Cricket
Authors: Asada Satoshi , Tateishi Chiyuki , Nakazawa Tohru
Published: January 2005

Protein synthetic activity in homogenate of embryos of cricket, Gryllus bimaculatus, increased transiently 25 hr after oviposition, though the activity in mitochondrial fraction increased successively thereafter. The decrease in the activity after the peak in homogenate was caused apparently by increase in the amino acid content in cytosol during development. To analyze increase in the mitochondrial protein synthetic activity 25 hr after oviposition, effect of various components was examined in the reconstituted mitochondrial system. The nucleic acids extracted from 25‐hr embryos most effectively stimulated the protein synthetic activity, but those from 0‐hr embryos did not. The results were discussed on the role of mitochondrial protein synthesis during development of cricket embryos.
Achromosomal Division of Early Starfish Embryos Cultured in the Presence of Actinomycin D
Authors: Ikegami Susumu , Ozaki Yoshihiro , Ooe Yasunori , Itoh Noriyuki
Published: January 2005

Embryos of the starfish Asterina pectinifera were examined for their ability to undergo the early events of embryonic development in the presence of actinomycin D, a most widely used inhibitor of RNA synthesis. Fertilized eggs continued to divide eight or nine times in the presence of 25 μg ml−1actinomycin D, although delay of development was observed. Chromatin disintegrated in the blastomeres of actinomycin D‐treated embryos specifically at the 32‐cell stage and the nucleus was undetectable at later stages. Before the 32‐cell stage, RNA synthesis was not affected by the presence of actinomycin D whereas DNA synthesis was severely inhibited. The stage when achromosomal divisions cease and embryos begin to die corresponds to the period just before onset of blastulation, suggesting that the presence of the nucleus and chromosomes is a prerequisite for blastula formation and development beyond the 512‐cell stage in this species.
Influence of Melatonin on the Rate of Rana pipiens Tadpole Metamorphosis In Vivo and Regression of Thyroxine‐Treated Tail Tips In Vitro
Authors: Wright Mary L. , Cykowski Lori J. , Mayrand Shawn M. , Blanchard Linda S. , Kraszewska Anna A. , Margot Gonzales T. , Patnaude Martha
Published: January 2005

Metamorphosis of Rana pipiens tadpoles may be retarded when the light phase of the light/dark (LD) cycle is shortened or when thyroxine (T4) is given in the dark because melatonin peaks during the dark. Injection of premetamorphic tadpoles in spontaneous metamorphosis with melatonin (15 μg) retarded tail growth and hindlimb development on 18L:6D but had no significant effect on 6L:18D. During induced metamorphosis (30 μg/liter T4), melatonin injections retarded tail resorption on 18L:6D and accelerated it on 6L:18D, but did not affect the hindlimb. When melatonin was injected during T4 immersion at different times in the photophase on 18L:6D (L onset 0800 hr), tail regression was retarded by melatonin at 1430 or 2030 hr. At 0830 hr, shrinkage of tail length was accelerated whereas tail height was not affected. Tail tips in vitro induced to resorb by 0.2 μg/ml T4 in Niu‐Twitty solution regressed more slowly in the presence of melatonin (10 or 15 μg/ml) than with T4 alone on both 6L:18D and 18L:6D. The findings implicate melatonin in LD cycle effects on tadpole metamorphic rate in vivo, show the importance of the time of melatonin injections, and indicate that melatonin antagonizes the metamorphic action of T4 at the tissue level.
Protein Synthesis during Neural and Epidermal Differentiation in Cynops Embryo
Authors: Takabatake Takashi , Takahashi Tadashi C. , Takeshima Kazuhito , Takata Kenzo
Published: January 2005

Two‐dimensional gel electrophoresis was used to analyze protein synthesis in relation to neural and epidermal differentiation in Cynops pyrrhogaster embryo. Various regions of embryos at different developmental stages, from late morula to early neurula stages, were excised, radiolabelled with 35S‐methionine, and the pattern of protein synthesis were compared. The following four types of protein spots were observed: (1) six proteins synthesized characteristically in the epidermal region of the embryo after gastrulation, (2) two proteins synthesized in both epidermal and endodermal regions, but not in other regions, after gastrulation, (3) a protein first detected at early blastula stage, of which expression was nearly constant in presumptive epidermis region but declined in the other regions, (4) the candidate for neural plate specific protein synthesized at a very high level in ectoderm explants treated with concanavalin A, a substance which evokes neural induction.
Transcriptional Regulation of Cell‐Type‐Enriched Genes during Development in Dictyostelium discoideum Analyzed by Nuclear Run‐On Assays
Authors: Morio Takahiro , Ozaki Toshinori , Takeuchi Ikuo , Tasaka Masao
Published: January 2005

We have previously isolated several cell‐type‐enriched mRNAs of Dictyostelium discoideum. Since the temporal pattern of appearance and cell‐type‐enrichment of these RNAs were examined only by determining their accumulation, it was unclear whether their accumulation is regulated at the transcription level or the post‐transcriptional level. To distinguish between these two possibilities, we examined the temporal and cell‐type‐enriched transcription of several of these genes by nuclear run‐on assay. The results suggest that some genes are controlled in both temporal accumulation and cell‐type‐enrichment at the transcriptional level, but post‐transcriptional regulation is also important for regulating cell‐type enrichment in the case of some other genes.
Mechanism of an Alternate Type of Echinoderm Blastula Formation: The Wrinkled Blastula of the Sea Urchin Heliocidaris erythrogramma
Authors: Henry Jonathan J. , Wray Gregory A. , Raff Rudolf A.
Published: January 2005

While most indirect‐developing echinoderms (possessing a feeding larval stage) form a hollow, smooth‐walled blastula, most direct‐developing species form a wrinkled blastula. The process of wrinkled blastula formation was examined in the direct‐developing sea urchin, Heliocidaris erythrogramma. Approximately 5 hrs after fertilization the blastula epithelium contains folds along one, two or three orthogonal planes, which superficially appear like 2‐, 4‐ or 8‐cell stages, respectively. Microinjection of fluorescent dye into individual blastomeres of 2‐, 4‐ and 8‐cell embryos revealed that the wrinkles correspond with the first, second and third cleavage planes. Two factors appear to generate the wrinkled blastula epithelium. First, blastomeres undergo a partial separation along the first, second and third cleavage planes during early cleavage. Subsequent cell divisions are oriented such that the blastula epithelium is constructed with deep creases along these planes of cell separation. Second, there is no room for the expansion of the developing blastoderm within the tightly fitting fertilization envelope. Prior to hatching from the fertilization envelope, wrinkles in the blastula epithelium disappear, due to an increased packing and elongation of the cells. In addition, a substantial volume of cellular material is removed as lipids are secreted into the blastocoel in an apocrine fashion.
Development of Microtubule‐Dependence of the Chromosome Cycle at the Midblastula Transition in Xenopus laevis Embryos
Authors: Clute Paul , Masui Yoshio
Published: January 2005

Animal‐cap cells isolated from Xenopus laevis morulae and blastulae are cultured for 2 to 6 hr in medium containing nocodazole, Colcemid or taxol, at concentrations completely inhibiting cell division. At 20°C, cells from each control embryo undergo synchronous cell cycles up to the 12th, with a period of 32 min, of which 60% represents the chromosome condensation (mitotic or M‐) phase, and the average mitotic index remains near 50%. Cells treated with nocodazole, Colcemid or taxol before 12th cleavage undergo chromosome cycles with a similar period as controls, albeit without chromosome segregation, and the average mitotic index remains near 50%. From the 12th to 15th cycles, control cycles become asynchronous, their period gradually increases 2 to 3 times, and the mitotic index declines to 10%. In cells treated after 12th cleavage with taxol, the mitotic index declines, similarly to control cells. However, in nocodazole‐treated cells, it increases steadily, and exceeds 70% at 2 hr of treatment, but gradually declines to 40% at 6 hr. Therefore, while inhibition of microtubule activities does not significantly alter the timing of chromosome condensation cycles during synchronous cleavage, inhibition of microtubule assembly can prolong M‐phase during asynchronous cleavage after the midblastula transition.
Ryanodine Activates Sea Urchin Eggs
Authors: Sardet C. , Gillot I. , Ruscher A. , Payan P. , Girard J.‐P. , de Renzis G.
Published: January 2005

We have studied the effect on sea urchin eggs of ryanodine, a plant alkaloid that causes muscle contraction by opening calcium channels in the sarcoplasmic reticulum terminal cisternae. Ryanodine, although it is less effective that IP3, produces full or partial activation in 62% of injected sea urchin eggs. In addition ryanodine inhibits in a dose dependant manner 45Ca pumping in the isolated egg cortex or in eggs permeabilized with digitonin. Efflux experiments show that in fact ryanodine as IP3 stimulates the release of calcium sequestered intracellularly. We further show that these effects of ryanodine are inhibited by Mg++, ruthenium red and heparin. Our results suggest that ryanodine‐sensitive intracellular calcium channels exist in the sea urchin egg.
Cytological Studies of Large Nucleus‐Like Structures Formed by Exogenously‐Injected Linear and Circular DNAs in Fertilized Eggs of Xenopus laevis
Authors: Shiokawa Koichiro , Yoshida Manabu , Fukamachi Hiroshi , Fu Yuchang , Tashiro Kosuke , Sameshima Munefumi
Published: January 2005

Relatively large amounts of linear or circular DNAs were injected into the animal or vegetal cytoplasm of fertilized eggs of Xenopus laevis and the nature of large nucleus‐like structures formed by the injected DNAs was studied cytologically and electron microscopically. Results of fluorescent microscopic examination combined with immunohistochemical analysis strongly suggested that the injected DNAs were assembled into the nucleus‐like structures probably after being complexed with maternal histones. The assemblage of nucleus‐like structures preferentially took place in the animal most region of the egg, and the size of the nucleus‐like structures formed depended on the amount of the injected DNA. The nucleus‐like structures were surrounded by double membranes equipped with nuclear pore complexes, but there were at least two abnormal features in their ultrastructures. First, nucleus‐like structures contained cytoplasmic particulate materials, most probably ribosomes and/or glycogen granules. Secondly, many of the nuclear pore complexes on the “nuclear envelope” appeared to be incomplete, with blebs formed from inner leaflet and protruded into the perinuclear space. Injected circular plasmid DNAs were also assembled into large nucleus‐like structures in the animal most region, and appeared to be partitioned into descendant cells during the cleavage.
Immunoelectophoretic Identification of Jelly Coat Ligands Bound by the Cortical Granule Lectin from Xenopus laevis Eggs
Authors: Birr Cynthia A. , Hedrick Jerry L.
Published: January 2005

The formation of the fertilization layer in the Xenopus laevis egg fertilization envelope involves a lectin‐ligand interaction and establishes a block to polyspermy in the extracellular matrix of the egg. The cortical granule lectin participating in the formation of the fertilization layer has been isolated but its ligand has not. We identified three jelly coat ligands bound by the cortical granule lectin using immunoelectrophoretic analyses. Two antigens were detected with anti‐jelly serum and a third was identified using anti‐envelope serum. All three antigenic ligands were associated with the innermost jelly coat layer, J1, and two of the three antigenic ligands contained sulfate. One or more of these jelly coat ligands may function in establishing a block to polyspermy at fertilization in Xenopus laevis.
Behavior and Ultrastructure of Primary Mesenchyme Cells at Sessile Site during Termination of Cell Migration in Early Gastrulae
Authors: Katow Hideki , Nakajima Yoko
Published: January 2005

The behavior and ultrastructure of primary mesenchyme cells at two ventrolateral sessile sites in early gastrulae were examined by time‐lapse videomicroscopy, scanning electron microscopy, and immunotrans‐mission electron microscopy using the sea urchin, Hemicentrotus pulcherrimus and the sand dollar. Clypeaster japonicus. At sessile sites in early gastrulae, PMCs terminated their migration after “touch‐and‐go” behavior, and even after the termination they retained a pulsatile movement. These behaviors indicate that the termination of PMC migration is not due to deprivation of cell motility nor the establishment of firm adhesion between PMCs and the site. PMCs used short cell processes during migration, and extended longer ones during the early period of migration termination. During the final period of migration at the sessile sites, PMCs extended characteristically thin and long cell processes to the basal lamina. These cell processes, as far as present results indicate, never attach to the blastocoel wall cells through the basal lamina. Thus it is indicated that the primary interaction site for PMCs to terminate their migration is the basal lamina.