bluuebunny/crossref_metadata_2025 huggingface dataset:
Dissociation of Cell Growth and DNA Synthesis and Alteration of the Nucleo‐Cytoplasmic Ratio in Growing Embryonal Carcinoma Cells
Authors: Sennerstam Roland , Strömberg Jan‐Olof
Published: January 2005

The relationship between the two subcycles ‘the cell‐growth cycle' (CGC) and the ‘DNA‐division cycle' (DDC) were examined in the pluripotent embryonal carcinoma cell line PCC3 N/I. This line shows intraclonal bimodal‐like heterogeneity in growth rate. A combined protein (mass) and DNA staining method was used to evaluate the relationship between DDC and CGC at various stages in the cell cycle. The results revealed dissociation of the two subcycles and the mass distributions at certain points in the cell cycle reflected the bimodality reported by us for intermitotic time (IDT) distribution. The results were applied to a model called ‘The Two‐subcycles Cell Cycle Model' (TSCM). This model predicts that the period of DDC (Pre‐S+S‐G2‐M) is fairly constant, while the CGC varies, being the main cause of the growth heterogeneity observed in this line. A point of growth rate regulation (PGRR) in G1 was thought to coincide with the start of CGC. These results reveal a mechanism by which the nucleo‐cytoplasmic ratio of the cells can change from one cell cycle to the next.
Does Phospholipase A2 Participate in the Acrosome Reaction of Sea Urchin Sperm?: A Pharmacological Study
Authors: Matsumura Kiyotaka , Aketa Kenji
Published: January 2005

We examined whether phospholipase A2 (PLA2) is involved in the initiation of the acrosome reaction of sperm of the sea urchin, Strongylocentrotus intermedius, using inhibitors and an activator of this enzyme. Quinacrine and p‐bromophenacyl bromide (PBPB) inhibited the egg jelly‐induced acrosome reaction at 100 μM, but not the ionomycin‐induced one. Depression of egg jelly‐induced increase of intracellular free Ca2+concentration ([Ca2+]i) by these reagents was expected and examined using fura 2. Quinacrine interfered with the flourescence of fura 2, but PBPB was found to depress at concentrations which could inhibit the acrosome reaction. Furthermore, melittin, which is known to stimulate PLA2, caused a [Ca2+]i increase and a formation of acrosomal process‐like structure on sperm head. These results suggest that PLA2 participates in the early step(s) of the acrosome reaction of sea urchin sperm.
Use of Horseradish Peroxidase Conjugated Lectins for Detection of Glycoconjugate Changes in Developing Lingual Epithelium of the Chick Embryo
Authors: Gheri Gherardo , Bryk Silvia Gheri , Sgambati Eleonora
Published: January 2005

A battery of six different horseradish peroxidase‐labelled lectins (SBA, DBA, PNA, WGA, ConA and LTA) was used to study the distribution of carbohydrate residues in glycoconjugates during the development of the chick embryo tongue anlage (7th‐21st day of incubation) and in the tongue of 3 day‐old chicken, at the dorsal and ventral epithelium. From the 7th to the 16th day β‐D‐galactose, D‐galactose‐(β1 → 3)‐N‐acetyl‐D‐galactosamine, D‐mannose and β‐N‐acetyl‐D‐glucosamine show the same epithelial distribution at two lingual aspects. From the 17th day to hatching the epithelium of the dorsal and ventral surfaces is characterized by a different distribution of sugar residues. The functional adaptation of the epithelium, as far as the saccharidic moletie distribution is concerned, seems to be completed at the 21st day of incubation.
Analysis of Epithelial Protein Profiles of Prostatic Glands Induced Heterotypically in the Bladder Epithelium of the Rat
Authors: Suematsu Naoya , Mizuno Takeo
Published: January 2005

When urinary bladder epithelia of rats were grown in association with fetal urogenital sinus mesenchyme, prostatic morphogenesis was induced. The epithelial proteins were examined by HPLC fractionation followed by sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis (PAGE). More than 500 bands of silver‐stained epithelial proteins were analyzed. The glandular epithelia induced from both adult and fetal bladder epithelia lost all of the 7 bladder‐specific bands (BE 1–7) in most recombinants and expressed a number of prostate‐specific bands. Among the 18 bands commonly found in all prostatic lobes, 13 (PE 4, 7–18) were constantly and 3 (PE 1–3) were sporadically detected, while the other 2 (PE 5 and 6) bands were not detected when the adult epithelium was used in recombination. Among the 7 prostatic lobe‐specific bands (vPE 14, dPE 1–3), most of them were detected when the fetal epithelium was used, while few of them when the adult epithelium was used. These results demonstrate that prostatic morphogenesis induced in the bladder epithelium was associated with most of biochemical features of prostate. In addition to the biochemical study, histological examination revealed that the prostatic differentiation was more complete in the fetal bladder epithelium than the adult one.
Developmental Timing in Fertilized Merogones of the Sand Dollar, Clypeaster japonicus
Authors: Yoneda Mitsuki , Nomura Akira
Published: January 2005

Pairs of nucleate and non‐nucleate fragments prepared by manually bisecting unfertilized eggs of Clypeaster japonicus were inseminated to obtain a pair of egg fragments, one with a diploid nucleus (zygote) and the other with a male pronucleus (merogone). Haploid male pronuclei in the merogone always entered the first mitosis (NEB) later than the zygote partner. The delay in NEB ranged from 6 to 24 min in different pairs. The interval from the first NEB to formations of the karyomere and the cleavage furrow, and the durations of the second, third and fourth cell cycles were identical in the zygote‐merogone pairs. Observations of Colcemid‐treated eggs suggested that the duration of the first DNA synthetic period was prolonged in the merogone. From this observation of wide variation in the time of the first mitosis, in contrast to constant durations of subsequent mitotic intervals, we suppose that some critical event triggers the transition of development for the start of well‐ordered sequences in the mitotic cycle in sea urchin development.
Ampulla Morphogenesis in Anural and Urodele Molgulid Ascidians
Authors: Bates William R.
Published: January 2005

Vascular ampulla morphogenesis was studied in two anural‐developing molgulids and one urodeledeveloping molgulid. In all three species, each juvenile developed one longer ampulla (termed the primary ampulla) and several shorter ampullae (termed secondary ampullae). Ampulla growth was accompanied by the formation of contraction rings that moved in a proximal to distal direction. Contraction ring formation was initiated by the epidermal cells situated in the proximal region of each ampulla. The formation of an expanding bubble‐like region within each lumen preceded ring formation. These rings moved at approximately 120 μm/min. Rings were produced for several days until the ampullae retracted towards the body wall.

In all three species, hemoblast cells were evident at day 4 within the developing mantle sinus (hemocoel). Ampulla growth was studied by positioning chalk particles on the tunic surface near the tips of ampullae. During the growth phase, chalk particles moved towards the body of the zooid. This report is the first to describe contraction ring formation and the polarized transport of tunic components during ampulla morphogenesis in two anural ascidian species.
Occurrence of Lectin in the Silkgland of the Silkworm, Bombyx mori
Authors: Amanai Kazuhito , Sakai Mie , Sakurai Sho , Mori Toshio , Nikaido Osamu , Ohtaki Tetsuya
Published: January 2005

Monoclonal antibodies were prepared against the 350 kDa lectin purified from larval hemolymph of the silkworm, Bombyx mori. The antibodies inhibited the hemagglutinating activity (HA activity) and bound specifically to the hemolymph 350 kDa lectin on Western blotting analysis. Immunohistological observations revealed the occurrence of lectin in the cuticular intima of the anterior silk gland, but not the middle or posterior silk glands of fifth instar larvae of Bombyx mori. Extracts from the anterior silk glands showed HA activity and exhibited the same biochemical characteristics as those of the 350 kDa lectin in the hemolymph. These results suggested that lectin‐like molecules in epithelial tissues may be important in histolysis during molting and metamorphosis.
Sexual Development of Cellular Slime Molds
Authors: Urushihara Hideko
Published: January 2005

Macrocyst formation represents sexual development of cellular slime molds and begins with fusion between cells of compatible mating types. Homothallic and heterothallic strains as well as bisexual and asexual ones have been described. Macrocyst development requires certain environmental conditions such as darkness and excessive humidity. Sexual cell fusion has been analyzed at a molecular level in Dictyostelium discoideum, and several cell surface proteins related to it have been identified. Some of them are common to both mating types, while others are specific to one or other type. The involvement of cell‐surface carbohydrates has also been suggested, though direct evidence for this is still lacking. Macrocyst formation is regulated by diffusible, pheromone like substances. Genetic studies on sexual development are scarce, probably because no suitable mutants have been available. However, several asexual mutants, as well as antibody and nucleotide probes, have recently been obtained, so mechanisms of sexual cell fusion may be understood in the near future. Considering the unique phylogenical position of cellular slime molds, analysis of sexual development in these organisms should contribute to the understanding of the mechanism and evolution of sexual reproduction systems.
Increase of Specific Activity, Electrophoretic Type‐Transition and Gene Expression of Alkaline Phosphatase during Endodermal Differentiation of F9 Mouse Embryonal Carcinoma Cells
Authors: Yamada Kazuya , Uemura Makoto , Matsuzawa Tetsuro
Published: January 2005

In F9 mouse embryonal carcinoma cells, the specific activity of alkaline phosphatase (ALPase) increases markedly during endodermal differentiation induced by retinoic acid (RA) treatment, but the specific 5′‐nucleotidase activity of a similar ecto‐phosphatase increases only temporally. Polyacrylamide disc gel electrophoresis showed that F9 cells express only type I ALPase, whereas RA‐treated F9 cells express both type I and type II ALPases. Type II ALPase is a minor form on day 1 of RA treatment and becomes the major form on day 4. RA‐treated F9 cells also expressed mRNAs for endoderm cell‐specific molecules, such as α‐fetoprotein, type IV collagen and laminin B1 chain, but their expression of M2‐type pyruvate kinase mRNA of an essential non‐ectoenzyme remains constant throughout endodermal differentiation. Northern blot analyses showed that type I ALPase was encoded by a liver (L)/bone (B)/kidney (K)/placenta (P)‐type mRNA. The expression of L/B/K/P‐type ALPase mRNA was induced in RA‐treated F9 cells, but its increase preceded that of ALPase specific activity. These results suggest that the expression of L/B/K‐type ALPase is regulated at the translational and/or post‐translational level. The differential inhibition of ALPases by L‐phenylalanine/L‐homoarginine and the thermal inactivation (56°C for 60 min) inferred that type II ALPase was also an L/B/K‐type isozyme.
Vestigial Brain Melanocyte Development During Embryogenesis of an Anural Ascidian
Authors: Swalla Billie J. , Jeffery William R.
Published: January 2005

Anural development was examined in the ascidian Bostrichobranchus digonas using specific markers for differentiated urodele ascidian larval cells and tissues. In this ovoviviparous anural ascidian, eggs, embryos and developing juveniles were present in the gonads, brood sacs, and atrial cavity, respectively. Morphological studies indicated that B. digonas embryos do not develop into tailed larvae with an extended notochord and differentiated muscle cells. In addition, these embryos lack detectable expression of the muscle‐specific markers acetylcholinesterase, alpha actin, and myosin heavy chain. In striking contrast to other anural ascidian embryos, however, B. digonas embryos can develop tyrosinase in several melanocyte precursor cells and eventually form a brain pigment cell. The melanocyte does not become part of a definitive brain sensory organ (otolith) and subsequently disappears during metamorphosis. A period of tyrosinase expression was also observed following metamorphosis in which many tyrosinase‐positive cells appear in the body of the developing juvenile. The results demonstrate that different urodele features can be uncoupled during the evolution of anural development. The development of a vestigial brain melanocyte also suggests that B. digonas evolved from a urodele ancestor rather than from another anural ascidian lacking a brain pigment cell.
Organ‐Dependent Expression of Differentiated States in Fibroblasts Cultured in Vitro
Authors: Shimizu Katsuhiko , Yoshizato Katsutoshi
Published: January 2005

Mouse fibroblasts were obtained from three different organs (skin, lung and heart), cultured and investigated to know whether the fibroblasts express differentiated characters in an organ‐dependent manner. Fibroblasts showed organ‐dependent morphology at the confluent state. Fibroblasts were labeled with [35S]‐methionine, and the pattern of protein synthesized was electrophoretically analyzed for both cellular proteins and extracellular proteins. Though most proteins were common to three types of fibroblasts, some proteins were produced in an organ‐dependent manner. Experiments on DNA synthesis and colony forming ability under a low density culture revealed that skin fibroblasts were the most proliferative among the three, while heart fibroblasts were the least. When fibroblasts were three‐dimensionally cultured in collagen gels, heart fibroblasts induced the gel contraction most intensely and skin fibroblasts did the least. In accordance with the ability of contraction heart fibroblasts secreted more collagen and fibronectin than skin and lung fibroblasts. Results in the present study indicate that the fibroblasts of three organs are in the organ‐dependent states of differentiation; heart fibroblasts are well differentiated while skin and lung fibroblasts are less differentiated, i.e., more proliferative and less active in the synthesis of extracellular matrices.
Cell Migration and Induction in the Development of the Surface Ectodermal Pattern of the Xenopus laevis Tadpole
Authors: Drysdale Thomas A. , Elinson Richard P.
Published: January 2005

The surface of the Xenopus tadpole contains three specialized, transient cell types; the ciliated, hatching gland, and cement gland cells. To distinguish whether the appearance of these cell types on the surface is due to induction of surface cells or due to migration of deep ectodermal cells into the surface, we transplanted labelled surface or deep cells to unlabelled hosts at early to mid‐gastrulae. After raising the host to a tadpole (Stage 28), we examined the embryo's surface for ciliated, hatching gland, and cement gland cells, and observed which cells were labelled. We find that all ciliated cells move into the surface from the deep ectodermal layer along with other cells of unknown function. Hatching gland cells arise by induction of surface cells as do the majority of cement gland cells. A few deep cells give rise to cement gland cells. Therefore, migration of deep cells to the surface and localized induction of surface cells contribute to the final surface patterning of the Xenopus tadpole.
Mesoderm Induction in Pleurodeles waltl: Distribution of Fibronectin and Chondroitin Sulfate in Induced Explants
Authors: Løvtrup‐Rein H. , Umbhauer M. , Riou J.‐F. , Boucaut J.‐C.
Published: January 2005

In Pleurodeles, cell‐matrix interactions play a major role in promoting active mesodermal cell migration during gastrulation. It was therefore important to determine whether the expression of define matrix molecules may be dependent on mesoderm induction. Results from induction experiments done with XTC cell line‐conditioned medium show that mesoderm tissues induced in animal cap explants of Pleurodeles are identical to those from Xenopus. However, we also show that dorsally‐induced explants in Pleurodeles elongate to a lesser degree than in Xenopus. This observation agrees well with the differences observed in the role of ECM in Pleurodeles and Xenopus gastrulation, respectively. Additional immunostaining studies demonstrate that the induction of mesodermal tissues is associated with the expression of chondroitin sulfate whereas fibronectin fibrils are already assembled in uninduced animal caps. These results suggest that mesoderm cell‐matrix interactions in early amphibian embryo may be under the control of mesoderm induction.
Treatment of Starfish Sperm with Egg Jelly Induces the Degradation of Histones
Authors: Amano Tosikazu , Okita Yoshihito , Hoshi Motonori
Published: January 2005

When spermatozoa of Asterina pectinifera are treated with a solution of homologous egg jelly, besides undergoing the acrosome reaction, they begin to degrade their histones gradually. The degradation is most prominent with histone H1, almost 75% of which is degraded within one hour at 20°C. The jelly‐induced histone degradation, like the acrosome reaction, requires external Ca2+, prefers high pHs and is susceptible to Ca2+‐channel antagonists such as verapamil and diltiazem. Histone degradation is also induced by nigericin as well as monensin in normal seawater, but not in Ca2+‐free seawater. Calcium ionophore A23187, that greatly facilitates the monensin‐induced histone degradation, also induces histone degradation by itself, slightly in normal seawater and markedly in Ca2+‐enriched seawater. Concanavalin A inhibits the jelly‐induced histone degradation but not the jelly‐induced acrosome reaction. These results suggest that egg jelly induces the histone degradation by enhancing Ca2+‐influx via a Ca2+‐channel(s) and by increasing cytoplasmic pH, through a pathway which is closely related to, but not entirely the same as, the one leading to the acrosome reaction.
Change in the Activity of Na1, K+‐ATPase in Embryos of the Sea Urchin, Hemicentrotus pulcherrimus, during Early Development
Authors: Mitsunaga‐Nakatsubo Keiko , Fujiwara Akiko , Yasumasu Ikuo
Published: January 2005

The activity of ouabain‐sensitive Na+, K+‐ATPase in sea urchin embryos at the morula and the swimming blastula stage was practically the same to that in unfertilized eggs. The activity increased during the period between the mesenchyme blastula and the late gastrula stages. In embryo‐wall cell fraction, which contained presumptive ectodermal cells as well as those of other cell lineages at the pre‐gastrula stage and ectodermal cells at the late gastrula stage, the Na+, K+‐ATPase activity increased in this developmental period more largely than in another cell fraction, containing mesenchyme cells and archenteron cells. Cycloheximide did not only block the activity increase in this period but also caused evident decrease in the activity in embryos at all examined stages. The activity increase in this period was strongly blocked by the treatment with actinomycin D, starting before the mesenchyme blastula stage, and was not seriously inhibited by the treatment starting at the mesenchyme blastula stage. The treatment starting at the initiation of gastrulation only slightly blocked further increase in the activity. Probably, an accumulation of mRNA encoding Na+, K+‐ATPase occurs mainly in ectodermal cells and is completed up to the early gastrula stage.
Expression of en in the Prosencephalon Heterotopically Transplanted into the Mesencephalon
Authors: Nakamura Harukazu , Itasaki Nobue
Published: January 2005

The alar plate of the prosencephalon differentiates into a tectum‐like structure when transplanted and integrated into the mesencephalon around 10‐somite stage. Homeobox containing gene, en, is expressed in the mesencephalon with caudorostral gradient from 7‐somite stage. Here expression of en in the prosencephalic transplant in the mesencephalon was studied immunohistochemically with anti‐en antibody. Transplantation was carried out between quail and chick. It was shown that the transplant was stained with anti‐en antibody at the caudal part of the mesencephalon. At the rostral part, the transplant did not express en. When the transplant was not contiguous to the caudal part of the host mesencephalon, it did not express en. The present study suggests that the prosencephalic transplant differentiates into the tectum‐like structure when it expresses en at the caudal part of the mesencephalon.
Differential Protein Distribution Related to Dorsoventral Polarity in Pleurodeles waltl Cleaving Egg
Authors: Dupont Henri , Deparis Pierre , Tujague Michel , Saboulard Didier , Ardourel Claude , Daguzan Christiane , Beetschen Jean‐Claude
Published: January 2005

The molecular basis for the initial specification of dorsoventral polarity in the Amphibian egg prior to the mid‐blastula transition still remains an open question. Regional differences in the protein pattern of Pleurodeles egg were investigated during early cleavage (8‐ and 512‐cell stages, prior to the mid‐blastula transition). Animal‐dorsal, animal‐ventral, vegetal‐dorsal and vegetal‐ventral quarters were separated and proteins were analyzed by 2D‐electrophoresis. The comparison of acidic protein patterns from dorsal and ventral quarters revealed differences between vegetal cells but no difference was detected between animal cells. One protein (p11, 30 kDa) was characterized in the dorsal side as early as the 8‐cell st. and two dorsal spots were detected at the 512‐cell st. (p11 and p5, 65 kDa). Similarly one protein (p7b, 46 kDa) appears to be ventral‐specific from the 8‐cell st. The p11 spot was shown to appear in ventral cells as a consequence of a dorsalizing LiCl‐treatment at the 32‐cell stage. Conversely, p11 disappeared from dorsal cells and p5 did not appear at 512‐cell stage after UV‐irradiation of the uncleaved egg, which results in the expression of the ventral‐specific protein p7b in the dorsal part of the egg. Therefore differential protein expression is already present at very early cleavage stages. Its significance needs further investigation.
Differential Effects of the Egg Jelly Molecules FSG and SAP‐I on Elevation of Intracellular Ca2+ and pH in Sea Urchin Spermatozoa
Authors: Hoshino Katsuaki , Shimizu Takeshi , Sendai Yutaka , Harumi Tatsuo , Suzuki Norio
Published: January 2005

We examined the effects of two egg jelly components, a fucose sulfate glycoconjugate (FSG) and sperm‐activating peptide I (SAP‐I: Gly‐Phe‐Asp‐Leu‐Asn‐Gly‐Gly‐Gly‐Val‐Gly), on the intracellular pH (pHi) and Ca2+ ([Ca2+]i) of spermatozoa of the sea urchin Hemicentrotus pulcherrimus. FSG and/or SAP‐I induced elevations of [Ca2+]; and pHi in the spermatozoa at pH 8.0. At pH 8.0, a second addition of FSG did not induced further elevation of the [Ca2+]i or pHi of spermatozoa treated with FSG, but addition or FSG after SAP‐I or of SAP‐I after FSG induced further increases of [Ca2+]i and pHi, At pH 6.6, FSG and/or SAP‐I did not induce significant elevation of the [Ca2+]i, although SAP‐I elevated the pHi, its half‐maximal effective concentration being 10 to 100 pM. At pH 8.0, tetraethyl‐ammonium, a voltage‐sensitive K+‐channel blocker, inhibited induction of the acrosome reaction and elevations of [Ca2+]i and pHi by FSG, but did not affect those by SAP‐I. These results suggest that FSG and SAP‐I activate different Ca2+ and H+ transport systems.
Germ Cell Differentiation in Starfish: The Posterior Enterocoel as the Origin of Germ Cells in Asterina pectinifera
Authors: Inoue Chiemi , Kiyomoto Masato , Shirai Hiroko
Published: January 2005

The origin of germ cells of Asterina pectinifera was traced back to the posterior enterocoel (PE) of 2‐day bipinnaria by two steps. First, the cellular cluster, composed of presumptive germ cells in the coelomic epithelium at brachiolaria stage, was confirmed to be the origin of the aboral haemal sinus located near the hydroporic canal (HC‐AHS) by continuous observation of the formation process of HC‐AHS. Second, the origin of the cluster was traced back to the PE of 2‐day bipinnaria by comparison of the number of the presumptive germ cells in microsurgically PE‐removed bipinnariae with that of non‐operated control larvae. A summary of the differentiation of germ cells in Asterina pectinifera is given/presented.
Relative Timing of Stiffening with Various Combinations of Nucleate and Enucleated Egg Fragments of Xenopus laevis
Authors: Shinagawa Atsunori
Published: January 2005

Previously, I found that in Xenopus eggs, the surface contraction wave (SCW) can arise at a point close to a male, a female or a zygote nucleus or a top portion of the egg (9). This finding suggested that all types of nucleus and cytoplasm of a Xenopus egg have the potential to determine the point of initiation of the SCW. Since stiffening is closely associated with the SCW, to determine the factors governing this phenomenon, I compared the times of egg stiffening of pairs of fragments containing different types of nuclei or cytoplasm. I found that the stiffening occurs earlier in fragments containing any type of nucleus than in those containing no nucleus, and earlier in fragments containing a male nucleus than in those containing a female nucleus. These results are consistent with the notion that either a nucleus or cytoplasm that induces the SCW earliest determines the point of initiation of the SCW close to itself. I also found that DNA replication is essential for the earlier occurrence of stiffening in a fragment containing a nucleus.
The Determination of Spindle Polarity in Early Mitotic Stages of the Dividing Grasshopper Neuroblasts
Authors: Kawamura Ken‐ya , Yamashiki Naoko
Published: January 2005

Although the spindle body of the grasshopper neuroblasts at the early mitotic stages does not have any mechanical linkage to the surrounding cell cortex, a spindle axis is inevitably oriented in parallel with the original division axis. The present study analyzes how the definite orientation of spindle axis along the cap cell (CC)‐ganglion cell (GC) axis of the neuroblast is maintained during these stages by use of the microdissection technique and electron microscopy. After removing a microneedle from the cell, metaphase spindles approximately 90°. rotated were able to return autonomously to the original axis. After the middle anaphase, however, the rotated spindle could not return at all. The electron microscopic observations revealed a characteristic behavior of an electron dense layer (EDL) in the CC‐side cortex during neuroblast mitosis. The EDL first appeared at very late prophase and became most conspicuous at metaphase. It became discontinuous by the beginning of middle anaphase and then completely disappeared at middle anaphase. So long as an EDL existed in the CC‐side polar cortex, 90° rotated spindle bodies were able to return autonomously to the original axis. After the disappearance of the EDL, the autonomous return of the rotated spindle no longer occurred. From these circumstantial evidence, it is conceivable that the orientation of the spindle body along the CC‐GC axis is maintained by the interaction between the EDL and the spindle pole.
Development of Prolactin and Growth Hormone Production in the Fetal Rat Pituitary: An Immunochemical Study
Authors: Nagata Saburo , Rosenfeld Michael G. , Inoue Kinji
Published: January 2005

Western immunoblot analysis was performed to monitor contents of prolactin (PRL), growth hormone (GH) and Pit‐1, a transcription factor required for both GH and PRL gene expression, in developing fetal rat pituitaries. Both PRL and GH were first detected in pituitary extracts at 18 days of fetal age and their contents increased during the rest of the fetal period. Pit‐1 was detectable from 16 days on. Quantitative densitometry scanning analysis of Western immunoblots showed that the PRL and GH contents were less than 10 ng per pituitary at 18 days. The PRL content increased to about 19 ng at 21 days (the last day of gestation), whereas the GH content showed much larger increase in the same period to reach about 1.2 μg per pituitary. In primary culture, dispersed pituitary cells from 19 day‐old fetuses incorporated [35S]‐amino acids into immunoprecipitable GH. The incorporation into PRL was detectable after stimulation with 1 nM 17β‐estradiol. These results suggests that the rat pituitary starts to produce both PRL and GH at around 18 days of fetal age.
Structure and Macromolecular Composition of the Jelly Coats of the Urodele Ambystoma mexicanum
Authors: Carroll Edward J. , Palmer Richard , Ruibal Rodolfo
Published: January 2005

Cleavage‐stage embryos of the neotenic urodele Ambystoma mexicanum are surrounded by a fertilization envelope and four macroscopic jelly coats termed J1 (innermost) through J4 (outermost). In sections prepared for light microscopy, each of the jelly layers stained with protein stains and the periodic acid‐Schiff's reagent, but only J1 stained with alcian blue at pH 2.5. These results suggest that each layer consists of proteins and glycoproteins and that J1 uniquely contains some sulfate esters. Only J4 was solubilized with alkaline mercaptan treatment in situ, however, the isolated inner jelly complex (J1, J2 and J3) was easily dissolved in this reagent suggesting that solvent access is impaired in situ. A single alcian blue‐staining component plus one protein‐staining component were detected on reducing polyacrylamide gel electrophoresis of outer jelly (J4). In the inner jelly complex (J1, J2, J3), two protein‐staining components were detected and no alcian blue‐staining components were observed. A predominant polypeptide of 110,000 molecular weight was detected and purified to homogeneity on reducing and denaturing gels of the inner jelly complex. Amino acid analysis of the polypeptide demonstrated a slightly higher fraction of acidic over basic amino acids (Glx+Asx=18.1 mole%vs. Arg + Lys = 11.7 mole%). The N‐terminal amino acid was Glu and the sequence of the first eleven amino acids was determined.
Coiled Filamentous Structure in the Sperm Acrosome of Gastropoda, Sulculus aquatilis and Turbo cornutus
Authors: Shiroya Yoko , Sakai Yoshi T.
Published: January 2005

A helically coiled filamentous structure, termed the truncated cone originally identified in Haliotis discus, was demonstrated to exist in the apex of the acrosome subjacent to the outer acrosomal membrane of the sperm in two species of Gastropoda, Sulculus aquatilis and Turbo cornutus. Thin section and whole mount preparations revealed that in intact sperm this filamentous structure consisted of about 12 helically arranged filaments with a diameter of 10–12 nm which were tightly packed in a truncated shape. This truncated cone elongated anteriorly from the acrosomal opening and transformed into a cylinder which closely surrounds the acrosomal process during the acrosome reaction. In S. aquatilis and T. cornutus, the truncated cone elongated more than 3 and 1.5 times as long as the original height, respectively. The elongated truncated cone was characterized by striations with increase in its periodic spacing and inclinations in thin sections. The truncated cone in both the species was fundamentally analogous to that of Haliotis discus, further suggesting that the truncated cone plays a role in fertilization as a common cytoskeletal structure among the species of Gastropoda.
In Vivo Study of Vitellogenin‐Gold Transport in the Ovarian Follicle and Oocyte of Xenopus laevis
Authors: Yoshizaki Norio
Published: January 2005

The transport of injected vitellogenin (VTG)‐gold in the ovarian follicle and developing oocyte in Xenopus is described. The gold particles reached the extracellular spaces of the theca and interfollicular spaces within 1 and 2 hr, respectively, after a tracer injection at 20°C. The tracers moved through channels between the constitutive cells of both the capillary endothelium and the follicle cell layer.

Compartments in the peripheral cytoplasm of vitellogenic oocytes at stage IV, which relate to yolk formation, seemed to be segregated as follows: (a) internalization compartment consisting of coated pits and vesicles of the oolemma covering the oocyte “macrovilli”, (b) transport compartment of endosomes and multivesicular endosomes in the oocyte cortex, and (c) crystallization compartment of primordial yolk platelets (PYP) in the sub‐cortical region. The gold particles appeared in the internalization and transport compartments at 3–6 hr after the tracer injection and in the cystallization compartment at 12–18 hr. The VTG, internalized by receptor‐mediated endocytosis, was transferred from coated vesicles to multivesicular endosomes by vesicle‐to‐vesicle fusion. VTG crystallization took place in globular‐shaped PYPs of about 1 μm. At 24 hr after the tracer injection, the gold particles appeared in completely crystallized yolk platelets, most of them clustered in the superficial layer and some integrated into the crystals.
Some Properties of the Hardening Process in Chorions Isolated from Unfertilized Eggs of Medaka, Oryzias latipes
Authors: Masuda Kaori , Murata Kenji , Iuchi Ichiro , Yamagami Kenjiro
Published: January 2005

Chorions isolated from unfertilized eggs of medaka, Oryzias latipes, harden during incubation with Ca2+ ions (Masuda et al., 1991). In this process, i.e. in vitro Ca2+‐hardening, the amounts of the major proteins of unfertilized egg chorions (83 K, 78 K and 51 K, corresponding to ZI‐1, 2 and 3 of oocyte chorions reported by Hamazaki et al, 1987) decreased and new proteins having molecular weights of 148 K or more appeared. Immunoblotting analysis using anti‐ZI‐1, 2 antisera and anti‐ZI‐3 antisera showed that the 148 K protein was an intermediate formed during polymerization of the original proteins.

The mechanism of in vitro Ca2+‐hardening was studied by examining the decrease in ZI‐1, 2, and 3, the formation of 148 K protein, and the change in solubility of chorions in 6% sodium dodecylsulfate‐1% 2‐mercaptoethanol‐15% glycerol‐0.2 M Tris‐HCl (pH 6.8). In vitro Ca2+‐hardening was inhibited at temperatures higher than 70°C and its optimum pH was about 5.5. It was inhibited by neither aminotriazole nor cadaverine. The results suggested that in vitro Ca2+‐hardening was generated by some factor(s) other than ovoperoxidase and transglutaminase.
Efficacious Production of Viable Germ‐Line Chimeras between Embryonic Stem (ES) Cells and 8‐Cell Stage Embryos
Authors: Tokunaga Tomoyuki , Tsunoda Yukio
Published: January 2005

Many embryonic stem (ES) cell lines have been isolated from various mouse strains, but production of germ‐line chimeras has been achieved with only strain 129. This report describes the isolation of a new ES cell line, F1/1, from a mouse blastocyst with the C57BL/6 X CBA male genotype and tests on its ability to produce germ‐line chimeras by two techniques, blastocyst injection and 8‐cell embryo injection. Chimera production using CD‐1 blastocysts as a host was low (20%), as reported by others. But by the 8‐cell embryo injection method, in which F1/1 cells were injected into the perivitelline space through a slit in the zona pellucida of 8‐cell embryos, chimeric mice with extremely high chimerism were obtained at a rate of 80%. Breeding tests showed that 89% of the fertile males were germ‐line chimeras and in most case, the majority of the sperms in their testes were derived from F1/1 cells. This F1/1 cell line with a different genotype from the 129 strain shows high ability to produce functional germ cells, moreover, the 8‐cell embryo injection method using F1/1 cells seems to be an efficient way to produce viable germ‐line chimeras.
Co‐Expression of Cytokeratins and Vimentin in Sheep Cumulus‐Oocyte Complexes. Alteration of Intermediate Filament Distribution by Acrylamide
Authors: Gall Laurence , De Smedt Veronique , Ruffini Sylvie
Published: January 2005

The association between germ cells and somatic granulosa cells persists throughout the growth of the oocyte by means of foot processes of the cumulus corona cells that cross the zona pellucida. During meiotic maturation important nuclear and cytoplasmic events occur in cumulus‐oocyte complex suggesting implication of cytoskeletal elements. Immunoblotting analysis of cytoskeletal proteins of the cumulus cells revealed the presence of vimentin polypeptide and of at least two cytokeratin polypeptides. Using immunofluorescence techniques on cryostat sections through frozen tissue, we provided evidence for the presence of cytokeratins of the simple epithelial type in addition to vimentin in sheep cumulus cells. These two types of intermediate filaments were localized throughout the cytoplasm and especially in the foot processes which cross the zona pellucida. The contact area between the two cell types was also labelled with the antibodies. Acrylamide treatment of cumulus‐oocyte complexes involved a drastic disorganization of the intermediate filament network and triggered the isolation of the oocyte from its cumulus cells. This isolation resulted in resumption of meiosis. From these results it appears that intermediate filaments could participate in the process of gap junction loss and indirectly in the control of meiosis resumption.
Changes in the Titer of Bombyxin‐Immunoreactive Material in Hemolymph during the Postembryonic Development of the Silkmoth Bombyx mori
Authors: Saegusa Hironao , Mizoguchi Akira , Kitahora Haruko , Nagasawa Hiromichi , Suzuki Akinori , Ishizaki Hironori
Published: January 2005

Monoclonal antibodies were raised against pure, native bombyxin‐II (bombyxin‐II antibody) and against a synthetic nonapeptide corresponding to the amino‐terminus of the C‐peptide of the bombyxin precursor protein (C‐peptide antibody). The bombyxin‐II antibody recognized both bombyxin and probombyxin. A radioimmunoassay for bombyxin using the bombyxin‐II antibody was developed, and developmental change in the titer of bombyxin immunoreactivity in the Bombyx hemolymph was investigated. The titer was low and almost constant during the fourth and early fifth instars. In the male, the titer rose abruptly 3 days after the beginning of wandering. One day after pupation it rose again steeply to reach the maximal level which lasted until the middle of the developing adult stage. The titer decreased thereafter and increased again at adult emergence. In the female, the pattern of titer fluctuation was similar to that in the male, but the female titers during pupal‐adult development were 2–3 times higher than the male titers.
Spatiotemporal Patterning of discoidin I and II during Development of Dictyostelium discoideum
Authors: Fukuzawa Masashi , Ochiai Hiroshi
Published: January 2005

We have examined the distribution of Dictyostelium lectins (discoidin I and II) during development by means of a sample preparation method of a whole mount. Monoclonal antibodies which were bound to discoidins revealed unique patterns of discoidin distribution. Discoidin I was localized mainly at the periphery of the aggregates, while the base of the aggregates was devoid of discoidin I staining. Discoidin I was not prominent in the body of the aggregates but when a migrating slug culminated, discoidin I staining appeared in the prestalk region, this suggested that prestalk cells begin to express discoidin I at the onset of culmination. During fruit formation we observed discoidin I staining at the foremost anterior prestalk region of the culminant, which implies a heterogeneity of discoidin I expression among prestalk cells; such a heterogenous pattern has also been found in other prestalk‐specific proteins. In addition, anterior‐like cells (ALC), which were sorted at the apex and basal parts of a spore mass during culmination, were also strongly stained with anti‐discoidin I mAb; interestingly, we observed the staining of ALC from the slug stage through fruit formation. No discoidin II was observed in a migrating slug that had already accumulated prespore antigen ligands for discoidin II; it appeared in prespore cells after the onset of culmination. The present results indicate that, in addition to the early expression of discoidin I, both discoidin I and II are expressed during culmination, and these lectins also seem to be involved in the late development of Dictyostelium.
Induction and Inhibition of Epithelial Differentiation by the Mixed Cell Aggregates of the Mesenchymes from the Chicken Embryonic Digestive Tract
Authors: Urase Koko , Yasugi Sadao
Published: January 2005

Epithelial‐mesenchymal interaction plays an important role in the differentiation of digestive tract. However, the factors of these mesenchymes involved in induction of the epithelial differentiation of each organs are still unknown. In the present study, we made reconstituted mesenchymal cell aggregates by mixing proventricular mesenchymal cells with other mesenchymal cells, recombined the reconstituted mesenchyme with gizzard epithelium, and observed the differentiation of the gizzard epithelium in the explants with special attention to the appearance of embryonic chicken pepsinogen, one of the molecular marker of the proventricular epithelial cells, in the gizzard epithelium. The results showed that the proventricular mesenchymal cells induce gland formation and pepsinogen in the gizzard epithelium and that the esophageal and gizzard mesenchymal cells have the inhibitory influence on the differentiation of epithelia toward proventricular epithelium. The cells from small‐intestinal, lung and dorsal dermal mesenchyme have no such effect. Based on the results obtained so far, a hypothesis was presented to explain the mechanism regulating the differentiation of the epithelium in the digestive tract in the chicken embryo.
A Stereometric Analysis of Karyokinesis, Cytokinesis and Cell Arrangements during and following Fourth Cleavage Period in the Sea Urchin, Lytechinus variegatus
Authors: Summers Robert G. , Morrill John B. , Leith Ardean , Marko Michael , Piston David W. , Stonebraker Alan T.
Published: January 2005

Fourth cleavage of the sea urchin embryo produces 16 blastomeres that are the starting point for analyses of cell lineages and bilateral symmetry. We used optical sectioning, scanning electron microscopy and analytical 3‐D reconstructions to obtain stereo images of patterns of karyokinesis and cell arrangements between 4th and 6th cleavage. At 4th cleavage, 8 mesomeres result from a variant, oblique cleavage of the animal quartet with the mesomeres arranged in a staggered, offset pattern and not a planar ring. This oblique, non‐radial cleavage pattern and polygonal packing of cells persists in the animal hemisphere throughout the cleavage period. Contrarily, at 4th cleavage, the 4 vegetal quartet nuclei migrate toward the vegetal pole during interphase; mitosis and cytokinesis are latitudinal and subequatorial. The 4 macromeres and 4 micromeres form before the animal quartet divides to produce a 12‐cell stage. Subsequently, macromeres and their derivatives divide synchronously and radially through 8th cleavage according to the Sachs‐Hertwig rule. At 5th cleavage, mesomeres and macromeres divide first; then the micromeres divide latitudinally and unequally to form the small and large micromeres. This temporal sequence produces 28‐and 32‐cell stages. At 6th cleavage, macromere and mesomere descendants divide synchronously before the 4 large micromeres divide parasynchronously to produce 56‐ and 60‐cell stages.
Does ADP‐Ribosylation of Proteins in Nuclei Contribute to Ectoderm Cell Differentiation in Sea Urchin Embryos?
Authors: Kamata Yasuyuki , Fujiwara Akiko , Furuya Shigehisa , Yasumasu Ikuo
Published: January 2005

In nuclei of sea urchin embryos, marked increase in ADP‐ribosyltransferase activity followed by its decrease occurrs in the pre‐hatching and post‐hatching periods with peaks of activity at the morula and gastrula stages. Increase in its activity was blocked by cycloheximide in the pre‐ and post‐hatching periods and by actinomycin D only in the post‐hatching period. Embryo wall cells (ectoderm cells) isolated from gastrulae exhibited markedly higher activity of this enzyme than archenteron cells and mesenchyme cells. Probably, the increase in the activity of this enzyme in the post‐hatching period results from expression of the gene for this enzyme mainly in ectoderm cells. In the post‐hatching period, the activity increased more in animalized embryos than in normal ones, and increased little in vegetalized embryos. 3‐Aminobenzamide (3‐ABA), as well as luminol and nicotinamide, inhibited formation of ectoderm structures more than that of endoderm structures, such as the archenteron, in normal and animalized embryos, but had no appreciable effect on morphogenesis in vegetalized embryos. The reaction catalyzed by ADP‐ribosyltransferase probably contributes to ectoderm cell differentiation. Treatment of embryos with 3‐ABA in the pre‐hatching period had little inhibitory effect on the morphogenesis in the post‐hatching period, though it caused death of many embryos.
The Determinant for Archenteron Formation in Starfish: Co‐Culture of an Animal Egg Fragment‐Derived Cell Cluster and a Selected Blastomere
Authors: Kiyomoto Masato , Shirai Hiroko
Published: January 2005

A method of detecting blastomeres that carrying the determinant for archenteron formation was established, based on the reported localization of the determinant in the vegetal cytoplasm (17, 24). The essence of the method was to co‐culture a selected blastomere with an animal egg fragment‐derived cell cluster, so as to generate one joined embryo. The presence of the determinant in the blastomere was assessed by the formation of the archenteron in the developed joined embryos. The vegetal blastomeres that carried the determinant sometimes induced animal egg fragment‐derived cells to form part of the archenteron.
Reconstruction of Starfish Eggs by Electric Cell Fusion: A New Method of Detect the Cytoplasmic Determinant for Archenteron Formation
Authors: Kiyomoto Masato , Shirai Hiroko
Published: January 2005

A method of detecting cytoplasm carrying the determinant for archenteron formation in starfish was established. Animal egg fragments (AEFs) which had been severed from the vegetal halves were fused electrically into pairs with fragments prepared from various regions of immature oocytes. It has been previously shown that the vegetal halves are exclusively endowed with the ability to form the archenteron; AEFs alone develop into so‐called permanent blastulae. Eggs thus reconstructed were allowed to develop in order to assess the presence of the determinant in the added fragments. Only AEFs fused with fragments from near the vegetal pole of the oocytes formed the archenteron and developed into bipinnariae and juveniles.

Comparison between the inner and outer (including cortex) cytoplasm of small vegetal fragment showed that the outer cytoplasm gave the reconstructed egg a greater ability to form an archenteron than the inner cytoplasm.