bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytological Characterization of Self Incompatibility in Gametes of the Ascidian, Ciona intestinalis
Authors: KAWAMURA KAZUO , FUJITA HIROKO , NAKAUCHI MITSUAKI
Published: January 2005

We have determined how many elements are involved in the regulation of self‐fertilization in the solitary ascidian, Ciona intestinalis that is an incompletely self‐sterile species. Animals collected in the field were repeatedly induced to spawn in order to examine their selfing ratios. About 20% of them were self‐fertile, although the ratios fluctuated considerably among respective spawnings. Naturally or acid‐induced self‐fertile gametes required much longer time for selfing than that for crossing. Egg‐suspending seawater (egg water) as such activated sperm motility, but it lowered conspicuously the self‐fertilization ratio. Self‐sterile spermatozoa could scarcely bind to the vitelline coat (VC) of glycerinated autologous eggs. or in case they bound well to it the sperm flagella ceased to beat within five min of the ‘insemination’. The staining of sectioned gametes with DAPI, a fluorescent dye of DNA, showed that in selfing the spermatozoa could hardly penetrate the VC even though they bound well to it. The results of this study show that the block of self‐fertilization can be classified into four elements from a phenomenal viewpoint, such as egg water, low affinity of sperm‐VC binding, inactivation of bound sperm and difficulty in sperm penetration through the VC.
Activation of Sea Urchin Eggs by Halothane and Its Inhibition by Dantrolene
Authors: FUJIWARA AKIKO , SUDOH KIMIHIKO , YASUMASU IKUO
Published: January 2005

Eggs of the sea urchin, Hemicentrotus pulcherrimus, were stimulated by halothane, known to induce Ca2+ release from sarcosome, to cause fertilization membrane formation in normal and Ca2+ free artificial sea water. In the absence of external Ca2+, halothane‐induced formation of fertilization membrane was inhibited by dantrolene, an inhibitor of Ca2+ release from sarcosome, but was not blocked by nifedipine, a Ca2+ antagonist specific to Ca2+ channels in plasma membrane. Ca2+ release from sedimentable fraction isolated from eggs was induced by halothane and was inhibited by dantrolene, but was not blocked by nifedipine. In normal artificial sea water, halothane‐caused egg activation was not inhibited either by dantrolene or by nifedipine, but was blocked in the presence of both compounds. 45Ca2+ influx was substantially stimulated by halothane in eggs exposed to 45CaCl2. Halothane‐induced 45Ca2+ influx into eggs was inhibited by nifedipine but was not blocked by dantrolene. When Ca2+ release from intracellular organellae is blocked, Ca2+ transport through Ca2+ channels in plasma membrane probably acts as a “fail‐safe” system to induce an increase in cytosolic Ca2+ level, resulting in egg activation.
Base Composition of Poly (A+) Nuclear RNA of Frog Embryo and Friend Erythroleukemia Cells
Authors: PINE SCOTT , FLICKINGER REED
Published: January 2005

Base composition determinations indicate that the poly (A+) nuclear RNA of frog embryo and Friend erythroleukemia cells that are transcribing a greater quantity of such RNA is more AU‐rich than that of cells transcribing a lower quantity. Filter hybridizations show that repetitive sequences of poly (A+) nuclear RNA of frog embryos are more AU‐rich than single copy sequences.
Changes of Endocytotic Activities during the Cell Cycle of Dictyostelium Cells
Authors: MAEDA YASUO
Published: January 2005

Changes of endocytotic activities during the cell cycle of the cellular slime mould Dictyostelium discoideum Ax‐2 were examined using the temperature‐shift method for inducing synchronous growth. The activity of fluid‐phase pinocytosis (FPP) was altered Ca2+‐dependently and stimulated by EGTA. On the other hand, pinocytosis was greatly enhanced by addition of Bacteriological‐peptone(BP) to the growth medium for Ax‐2 cells, irrespective of the extracellular Ca2+‐concentration. The maximal pinocytotic activity was attained in the presence of EGTA plus BP, the effects of the two substances being additive. The FPP activity was found to be high in cells in and just after the S phase, when the BP‐induced fraction of pinocytosis was rather low. Thus the total activity for pinocytosis in the growth medium remained almost constant throughout the cell cycle, indicating that the rate of nutrient uptake through pinocytosis was not a limiting factor for cell cycle regulation. The change of phagocytotic activity during the cell cycle was somewhat similar to that of the FPP activity. Possible mechanisms of such cell‐cycle related changes are discussed in relation to cytoskeletal proteins in the cell cortex. Some properties of BP‐induced pinocytosis are also described.
Micromere Differentiation in the Sea Urchin Embryo: Expression of Primary Mesenchyme Cell Specific Antigen during Development
Authors: SHIMIZU KEIKO , NORO NOBUHIRO , MATSUDA RYOICHI
Published: January 2005

The temporal and spatial expression of antigen specific for primary mesenchyme cell (PMC) lineage cells during early development of the sea urchins Hemicentrotus pulcherrimus and Stronglyocentrotus nudus was studied with a monoclonal antibody (P4). P4 was produced by a hybridoma cell line prepared by fusion of myeloma cells and spleen cells from a mouse immunized with cultured spicule‐forming cells. Immunofluorescence studies demonstrated that P4 antibody reacted strongly with the surfaces of PMC's and spicule‐forming cells of both species. Immunoblot analysis showed that P4 antibody reacted with several proteins including those of 140–kDa, 120–kDa, 53‐kDa, 43–kDa, and 41–kDa in H. pulcherrimus and with those of 130–kDa, 110–kDa, 51–kDa, and 43–kDa in S. nudus. These proteins appeared sequentially after the hatching blastula stage. Tunicamycin inhibited the expressions of these P4 antigens as well as spicule formation. Two of the P4‐reactive antigens, the 140–kDa and 43–kDa proteins, in H. pulcherrimus were synthesized de novo and shown to be identical to micromere differentiation specific proteins. These results suggest that P4 binds to specific molecules that are important in spicule formation in developing sea urchin embryos.
Phorbol Myristate Acetate Induces the Phosphorylation of Plasma Membrane‐Associated Proteins in Sea Urchin Eggs
Authors: CHANDLER DOUGLAS E. , VACQUIER VICTOR D.
Published: January 2005

Immediately after fertilization sea urchin eggs undergo an increase in cytoplasmic pH from 6.8 to 7.2. This pH change occurs by activation of a Na+/H+ antiporter, and is a necessary signal for later steps in metabolic activation of development. Activators of protein kinase C such as phorbol myristate acetate (PMA) and diacylglycerol produce a similar pH increase in eggs. Phosphorylation of the antiporter or a regulatory protein may be a step in activating Na+/H+ exchange. Here we show that treatment of sea urchin eggs (S. purpuratus) with PMA results in increased phosphorylation of over a dozen proteins. Of these, three proteins of Mr=240, 92 and 80 kD are located in the egg cortex; under‐representation of these bands in isolated cortical granules suggests that they are plasma membrane‐associated. Phosphorylation of the 92 kD band is concentration‐dependent over a range of 10 to 1000 nM PMA and occurs over a time‐course of 1 to 3 min. Phosphoamino acid analysis indicates that phosphorylation is on serine residues. Phosphorylation appeares to be mediated by protein kinase C since the inactive PMA analogue, 4α‐phorbol 12, 13‐didecanoate, does not induce phosphorylation nor does experimental alkalinization of the egg cytoplasm.
Introduction and Expression of Recombinant β‐Galactosidase Genes in Cleavage Stage Mouse Embryos
Authors: UENO KENJU , HIRAMOTO YUKIO , HAYASHI SHIGEO , KONDOH HISATO
Published: January 2005

In an attempt to study gene regulation in very early stages of mouse embryogenesis, we injected genes constructed by joining the coding sequence of the bacterial β‐galactosidase gene to four different animal gene enhancers/promoters and to poly (A) signals, and examined the gene expression in cleavage stage embryos.

With appropriate injection volumes for each embryonic stage, ranging from 0.2 to 1.3 pl, the majority of the injected embryos underwent at least one further cleavage. Expression of injected genes, which occurred transiently after injection, required the promoter sequences but without much distinction of the source of enhancer/promoter complexes. This result was in a sharp contrast to transfection of mouse cell lines where the recombinant genes were variably expressed reflecting differential enhancer effects.

By injection at the 1‐cell stage, expression of injected genes was low while the expression by injection at the 2‐cell or later stages was several fold higher, which may correlate with the fact that most zygotic gene expression begins after the 2‐cell stage. The low expression at the 1‐cell stage was augmented by the conditions causing clea***age arrest such as inhibition of DNA synthesis with aphidicolin.
Inhibition of Lens Regeneration by Nickel Subsulfide in the Japanese Common Newt, Cynops pyrrhogaster
Authors: OKAMOTO MITSUMASA
Published: January 2005

To investigate the effects of nickel subsulfide (Ni3S2) on lens regeneration, Ni3S2 was administered to the lentectomized newt eye by a single injection into the eye chamber. Lens regeneration was inhibited in the early stage of regeneration with about 60% of the treated eyes after 25 days, while all control eyes had a regenerating lens. The size of the regenerating lens in the treated eyes was the same as in controls. Lens regeneration was inhibited in all treated eyes containing Ni3S2 particles within the eye chamber. Inhibition of lens regeneration in the treated eyes was about 80% at 3 months, when all control eyes had already completed lens regeneration. Although the tendency toward pupil disappearance was prominent in the eyes having no regenerating lens, there were also a few eyes which had no regenerating lens and no signs of pupil disappearance. The significance of these results was discussed in relation to the regeneration and carcinogenesis.
A Fine Structural Study of the Fertilization Process of the Jellyfish Cladonema uchidai
Authors: YAMASHITA MASAKANE
Published: January 2005

This study described the fertilization process of the jellyfish Cladonema uchidai by means of transmission electron microscopy. Female pronucleus was situated in close vicinity to the animal pole of the spawned egg, where the surface of the egg was flat or slightly depressed. Microvilli were observed except on the surface at the animal pole. The egg was entirely covered with a coat composed of fibrous materials. The spermatozoon was of the primitive type, and the proacrosomal vesicles were found immediately beneath the plasma membrane of the antero‐lateral region of the sperm head. Within 15 sec after insemination, spermatozoa were incorporated in the egg cytoplasm only at the microvilli‐free surface at the animal pole. Neither opening of the proacosomal vesicles nor formation of the acrosomal process was observed. No appreciable changes of cortical cytoplasm could be detected, although the egg became sticky after fertilization. Decondensation of the incorporated sperm nucleus occurred without breakdown of the original nuclear envelope. Within 10 min after insemination, the sperm nucleus still under the process of its decondensation fused with the female pronucleus. These findings were discussed in comparison with the fertilization process of higher metazoans as well as of other cnidarians.
Stimulation of Protein Synthesis in the Mitochondria of Sea Urchin Embryos before Gastrulation
Authors: KAWASHIMA TSUYOSHI , NAKAZAWA TOHRU
Published: January 2005

A transient increase in protein synthesis was observed in mitochondria at the mesenchyme blastula stage of sea urchin (Hemicentrotus pulcherrimus) embryos. This stimulated activity was inhibited by chloramphenicol but not by cycloheximide. Reconstituting experiments in which poly U‐dependent protein synthesis was carried out showed the mitochondrial peptide elongation factor to be essential for increasing the protein synthetic activity in mesenchyme blastula, but aminoacyl tRNA synthetase and ribosome fraction containing initiation factor not to be involved in this increase. These findings are discussed in relation to the differentiation of embryos at the gastrulation stage.
Lung‐Derived Growth Factors: Ontogenic Shift in Parahormone Secretion in the Perinatal Rat Lung
Authors: MONTES ANA M. , MORISHIGE WALTER K.
Published: January 2005

Primary cultures of perinatal rat fibroblasts were found to produce at least three mitogenic activities which exhibited specificity for distinct cell types. One activity, lung growth factor(LGF), was a potent mitogen for chick embryonal fibroblasts, which also stimulated fetal rat lung fibroblasts to undergo DNA synthesis, provided that these cells were first exposed to a “competence” factor such as fibroblast growth factor or platelet‐derived growth factor. Although LGF was active in the somatomedin‐C (SmC) radioimmunoassay and resembled buffalo rat liver multiplication‐stimulating activity (brlMSA) in molecular size, it appears to consist of a component that is neither SmC nor brlMSA. The second activity produced by perinatal rat lung cultures, pneumocyte‐stimulating activity (PSA), stimulated mitosis in type II pneumocytes of postnatal rats, and was found to have physical attributes that are distinct from those of the other known pneumocyte‐influencing factors. The third activity is a non‐dialyzable substance which complements the mitogenic action of LGF on fetal lung fibroblasts, and appears to be a “competence” activity. An examination of the production of LGF and PSA by rat lung fibroblasts taken at various intervals of development revealed that fetal lung fibroblasts produce maximal levels of LGF but low levels of PSA, whereas, in neonatal lung fibroblasts, the situation is reversed. This ontogenic shift in the type of parahormone produced by the developing perinatal rat lung may be an important regulatory event in postnatal lung morphogenesis in this species.
The Morphology and Physiology of the Acrosome Reaction in the Sperm of the Decapod, Sicyonia ingentis.
Authors: CLARK W. H. , GRIFFIN F. J.
Published: January 2005

Sperm of the shrimp, Sicyonia ingentis, undergo a biphasic acrosome reaction consisting of acrosomal exocytosis and acrosomal filament formation. These events are temporally separated by 10–20 min, in vivo. Using egg water preparations the complete reaction can be induced, in vitro, albeit the temporal separation of the two phases is lengthened. External Ca++ is required for the exocytotic phase, while a cytoplasmic acidification and K+ efflux are associated with polymerization of the acrosomal filament.
Larval Development of Starfishes as Revealed by Long‐term Culture of the Embryos
Authors: DAVYDOV P. V. , SHUBRAVYI O. I. , VASSETZKY S. G.
Published: January 2005

Gametes were obtained from adult starfishes kept in closed cycle aquaria with artificial sea water by stimulation with 1‐methyl‐adenine (Asterina pectinifera, Aphelasterias japonica) or during spontaneous spawning (Lysastrosoma anthosticta). The eggs were fertilized and their development until the midbipinnaria stage (Aph. japonica, L. anthosticta) and settlement and metamorphosis (A. pectinifera) was described using scanning electron microscopy. Special attention was payed to the ciliary band and ciliary cover formation.
Differential Expressions of Two Types of Alkaline Phosphatase during Developmental Growth of Embryoid Bodies of Mouse Teratocarcinoma OTT‐6050 In Vitro
Authors: MATSUZAWA TETSURO , OJIMA‐KASAHARA SAKIKO , KITAMURA MASANOBU
Published: January 2005

Ascites teratocarcinoma OTT‐6050 is a totipotent tumor line producing indefinitely the simple type of embry‐oid bodies (EBs). In culture with fetal calf serum (FCS) in Eagle's minimal essential medium (MEM), these EBs show developmental growth, only in which some differentiative events result. EBs also show this developmental growth in MEM supplemented with two fractions of FCS separated with a Amicon PM 10 membrane, i.e. a low molecular weight Fraction L (mol. wt. less than 10,000) and a high molecular weight Fraction H (mol. wt. more than 10,000). Fraction H is necessary for the survival of EBs in vitro. Fraction L enhances the uptake of 3H‐thymidine into EB cells with increase in the Vmax, but no change in the Km. On culture of EBs with both Fractions, a marked bimodal increase in alkaline phosphatase (ALPase) activity is seen on day 1–2 and 4, resulting from the differential expressions of two electrophoretically distinct ALPases (Bands I and II). The differential expressions of ALPase are also observed cytochemically, one activity being on the inner cells and the other on the surrounding cells of EBs. From the cytochemical similarity of ALPase activity to that of normal mouse embryos, Band I ALPase is inferred to be the epiblast (developmentally totipotent stem cell)‐type and Band II ALPase to be the distal (parietal) endoderm‐type.
Regionalized Cell Division during Sea Urchin Gastrulation Contributes to Archenteron Formation and Is Correlated with the Establishment of Larval Symmetry
Authors: NISLOW COREY , MORRILL JOHN B.
Published: January 2005

During gastrulation of the sea urchin, Lytechinus variegutus there is localized proliferation of cells in the vegetal plate region prior to its invagination. Cell counts show that during gastrulation the number of cells per embryo increases 60% from 1025 to 1640. Measurements of cell volumes suggest that some growth may follow these divisions. Feulgen staining shows that the greatest mitotic activity throughout gastrulation occurs in the vegetal plate region. Labelling embryos with 3H‐thymidine reveals that incorporation in the vegetal plate is confined to cells that encircle the base of the archenteron. Pulse‐chase experiments indicate that these labelled cells contribute descendants to the vegetal half of the archenteron. Additionally, 3‐dimensional reconstructions of vegetal regions at different stages reveal that by the end of gastrulation two bilateral clusters of labelled cells lie at the future sites of the post‐oral arms of the pluteus larva, thus marking the axes of bilateral and dorso‐ventral symmetry. Our findings suggest that two of the principal events of sea urchin gastrulation — the formation of the archenteron and the establishment of symmetry in the larva — are accompanied by distinct patterns of cell division.
Cell Proliferation during Bud Formation of the Quail Uropygial Gland
Authors: FUKUI YURIKO
Published: January 2005

A localized increase of labeling index (L.I.) was observed in bud‐forming epithelium of the uropygial gland of the Japanese quail embryo through computer‐aided analysis. At day 10 glandular buds are not yet formed anywhere in the epithelium and the distribution of proliferative activity in the epithelium is not differential. On the following day the peripheral buds are being formed at the externo‐lateral end of the basal luminal epithelium, L.I. increases locally in these buds. At day 12 a localized increase of L.I. takes place also at the median end; extremely high labeling indices are seen at the tip of the peripheral buds and developing central buds.
Developmental Stages of the Society Finch, Lonchura striata var. dornestica.
Authors: YAMASAKI MASAHIRO , TONOSAKI AKIRA
Published: January 2005

The society finch, a little passerin, was purposed to be utilized in embryological studies. Under control of the breeding cycle in 20 pairs, 4 to 6 eggs were used to be laid daily for several repeating week in a year. Average incubation time was 17 days in contrast to 21 in the domestic fowl. The eggs weighed 1.1 g in average and expected smallness of the embryo was regarded as favorable for morphological studies including the scanning electron microscopy. We present the first report of the complete development of the society finch. A number of embryological characteristics are described with special reference to the peculiarity of the altricial finch as compared with the precocial domestic fowl.
Arginine Vasopressin Contents of the Hypothalamus and Pituitary during Fetal and Postnatal Development in the Mouse
Authors: YAMASHITA TOMOO , KAWAMOTO KEIICHI , KAWASHIMA SEIICHIRO
Published: January 2005

The contents of arginine vasopressin (AVP) in the hypothalamus and the pituitary of the mouse during fetal and postnatal development were measured by radioimmunoassay. AVP was first detected in the hypothalamo‐pituitary system at the fetal age of 14 days (FA 14). After FA 15, the pituitary levels were higher than the hypothalamic levels except on FA 16. The greater AVP content of the hypothalamus than the pituitary only on FA 16 suggests that AVP synthesis in the perikarya of AVP‐producing neurons may significantly increase between FA 15 and FA 16, and that the hormonal transport to the pituitary may be characteristically activated between FA 16 and FA 17. The levels of AVP in both the hypothalamus and the pituitary increased exponentially until the postnatal age of 21 days (PA 21). After PA 30, the content in the pituitary continued to increase, while that in the hypothalamus was kept almost constant. The decrease in AVP content in the pituitary was found between PA 0 and PA 1, and PA 21 and PA 30. These results suggest that AVP might be released from the pituitary in response to significant changes in water metabolism elicited at birth and weaning.
Activation of Heat‐Shock Genes by Digitonin is Selectively Repressed in Preheated Drosophila Salivary Glands
Authors: MYOHARA MAROKO , OKADA MASUKICHI
Published: January 2005

Treatment of Drosophila salivary glands with a mild detergent, digitonin, activates puffing at 35 chromosome loci. These digitonin‐activated puffs include all of the nine heat‐shock puffs known in D. melanogaster. Here we show that the activation of heat‐shock genes, but not of other digitoninstimulated puffs, is repressed in salivary glands which have been subjected to and have recovered from heat shock before being treated with digitonin. The findings indicate that, (a) the activation of heat‐shock genes by digitonin, as that by temperature elevation, is self‐regulated by the heat‐shock proteins (HSPs). (b) the gene repressive activity of HSPs is heat‐shock‐gene specific, and (c) the repression mechanism of heat‐shock genes by HSPs is resistant to digitonin, in contrast to that the suppression of heat‐shock genes is prevented by the detergent in non‐heat‐shocked salivary glands. The selective repression of heat‐shock genes in preheated salivary glands suggests that the heat‐shock genes and other digitonin‐activated genes may be controlled by a different mechanism(s).
Effects of Cations and Other Medium Components on the Zona‐induced Acrosome Reaction of Hamster Spermatozoa
Authors: YOSHIMATSU NOBUHIRO , YANAGIMACHI R.
Published: January 2005

Although the acrosome reaction in lively motile hamster spermatozoa can occur independently of the egg or its investments (“spontaneous” acrosome reaction), it appears to be the egg investments, particularly the zona pellucida, that induces the acrosome reaction in fertilizing spermatozoa of many mammalian species. The latter is referred to as “zona‐induced” acrosome reaction. Experiments were conducted to determine if the zona‐induced acrosome reaction has different ion requirements from the spontaneous reaction. Like the spontaneous acrosome reaction, the zona‐induced acrosome reaction required extracellular Na+, K+ and Ca2+. The absence of Cl− and albumin in the medium inhibited the reaction. The zona‐induced acrosome reaction could occur in a HCO3−‐free medium, but far less efficiently than in medium containing this ion. Proteinase inhibitors, benzamidine and TLCK, inhibited the zona‐induced acrosome reaction. These results suggest that the chemical reactions involved in the spontaneous and zona‐induced acrosome reactions are similar although the reaction‐triggering mechanism is probably different.
Regional Ultrastructural Differences in Basal Laminae Isolated from the Starfish Pisaster ochraceus
Authors: CRAWFORD BRUCE J.
Published: January 2005

The ultrastructure of different regions of the basal laminae isolated from 5‐1/2‐6 day‐old embryos of the starfish, Pisaster ochraceus, has been described after fixation in the presence of anionic dyes. Isolated basal laminae from all regions of the embryo exhibit a lamina lucida and lamina densa. No lamina fibroreticularis is present. Instead, a coarse meshwork of thick densely stained and thinner intermediately stained fibers is embedded in the lamina densa and extends into the blastocoel forming the extracellular matrix. The coarse meshwork associated with the ectodermal basal lamina consists primarily of thick densely stained fibers with a small number of intermediate ones while that associated with the endodermal one contains much less densely stained material. These structures were morphologically identical to those found in control embryos. Examination of different regions of the endodermal basal lamina shows that the amount of dense material varies from region to region. These differences in dense material may reflect biochemical differences, particularly of proteoglycans, which could provide positional information to migrating mesenchyme cells.
Ultrastructural Studies on Development of the Tail Gland of a Cuttlefish, Sepiella japonica
Authors: MATSUNO AKIRA , OUJI MASAMI
Published: January 2005

Hatching glands in embryos of teleosts and amphibians have been reported to be indispensable for hatching of the embryos. The cephalopod has capsuled eggs, so we expected to find some exocrine organ in the embryos that functioned as a hatching gland. The tail gland (Hoyle's organ) has been suspected to be a hatching gland in the cephalopod, and therefore we examined it during the course of development of cuttlefish embryos. Cells in the tail gland appeared similar to the hatching gland cells (HGCs) of teleosts and amphibians, and contained a number of secretion granules that also resembled the hatching enzyme granules (HEGs) in HGCs of teleosts and amphibians in size, electron density and distribution in the cells. However, a few of these granules were discharged one after another from an early stages, whereas most of them were retained up to the stage just before hatching, and then discharged all at once. The former process of trickling discharge was similar to that in amphibians and the latter process of abrupt discharge resembled that in teleosts.
Ultrastructural Observations on the Origin and Differentiation of Somatic Cells during Gonadal Development in the Frog Rana nigromaculata
Authors: TANIMURA AKIHIKO , IWASAWA HISAAKI
Published: January 2005

The process of gonad development in the frog Rana nigromaculata was observed using the electron microscope. The gonadal medulla was formed by the proliferation and displacement of the epithelial cells within the primordial gonad, and a distinct continuity was observed between the cortical and medullary cells. Sex differentiation of the gonad occurred directly from the sexually indifferent primordial gonads. In the rudimentary testes, the continuity between the cortical and medullary regions increased closer, and the intermingling of cortical and medullary cells was evident. The inner region of the cortex developed into a cord‐like structure and subsequently differentiated into rudimentary seminiferous tubules. The medulla differentiated into the testicular rete and efferent duct. In the rudimentary ovaries, the cortex and medulla were separated and the ovarian cavity was formed in the medullary region. In the cortex, the cortical cells surrounding oocytes which had reached the diplotene stage, differentiated into follicular cells. The intrusion of mesenchymal or blastemal cells derived from extragonadal regions into the cortex or medulla was never observed. These findings do not support Witschi's cortico‐medullary antagonistic theory of sex differentiation.
Helper Function of Follicle Cells in Sperm‐Egg Interactions of the Ascidian, Ciona intestinalis
Authors: KAWAMURA KAZUO , FUJITA HIROKO , NAKAUCHI MITSUAKI
Published: January 2005

Studies were made on the involvement in sperm‐egg interactions of follicle cells of Ciona intestinalis, which are tall, vacuolated cells attached to the outer surface of the egg vitelline coat. The basal surface of the follicle cells is polygonal. The borders between cells could easily be observed by the binding of fluorescent SBA (soy bean agglutinin), a lectin recognizing N‐acetylgalactosamine (GaINAc) residues. At fertilization many spermatozoa aggregate along these polygonal borders of cells on the vitelline coat, through which they entered the perivitelline space. The removal of follicle cells was sometimes associated with loss of SBA‐binding sites, and in such cases the sperm did not show a hexagonal pattern of aggregation, but became dispersed all over the vitelline coat. Removal of the follicle sometimes delayed fertilization. Examination of sections of gametes stained with DAPI, a fluorescent dye staining DNA, showed that removal of the follicle reduced the number of spermatozoa bound to the vitelline coat and, more especially, the number of spermatozoa penetrating through the vitelline coat. The blockage of GalNAc residues on the vitelline coat with SBA did not appreciably affect the time course of fertilization or the number of sperm associated with eggs. These findings are discussed in relation to the role of follicle cells in facilitating sperm aggregation on the vitelline coat and their penetration through it.
Influence of Donor Age on Establishing Well‐Differentiated Clonal Cultures from Embryonic Chicken Retinal Pigmented Epithelium
Authors: VIELKIND URSULA , CRAWFORD BRUCE J.
Published: January 2005

Retinal pigmented epithelia (RPE) isolated from chicken embryos of various developmental stages were dissociated into single cells, and their ability to re‐express defferentiated characteristics in clonal culture was investigated. The lighty pigmented, columnar cells isolated from stage 25 to 29 embryos dissociated more easily than the heavily pigmeted, cuboidal cells from embryos of stages 30 to 34. The yield of RPE cells per embryo increased with donor age, paralleling the growth of the epithelium in vivo. However, the potential these cells to attach, to proliferate, and to form typical, welldifferentiated RPE colonies declined with donor age. Cells from stage 25 embryos developed exclusively into large, typical epithelial colonies which expressed all stages of differentiation from flat, unpigmented cells at the margin to cuboidal, pigmented cells in the centre. At the other end of the spectrum, cells from stage 34 embryos frequently formed small, atypical colonies of unpigmented cells, in addition to typical but relatively small colonies. The plating efficiency (calculated on the basis of pigmented colonies formed within 3 weeks) dropped from more than 2% at stage 25 to 0.01% at stage 34.
Expression Pattern of E‐ and P‐Cadherin in Mouse Embryos and Uteri during the Periimplantation Period
Authors: Kadokawa Yuzo , Fuketa Izumi , Nose Akinao , Takeichi Masatoshi , Nakatsuji Norio
Published: January 2005

Periimplantation mouse embryos and uterine tissues were examined by means of immunohistochemistry for their expression of the Ca2+ dependent cell‐cell adhesion molecules, E‐ and P‐cadherin. E‐cadherin was detected in all embryonic cells during periimplantation stages, and also detected in the uterine epithelium. When blastocysts attached to the uterine epithelium, E‐cadherin was detected at implantation sites between the mural trophectoderm and the uterine epithelium on 5 day of pregnancy. P‐cadherin was first detected in the mural trophectoderm on 4.5‐day blastocysts, and then detected in the ectoplacental cone, giant cells and visceral endoderm from 5.5 day.

P‐cadherin was also detected in the maternal uterine decidual cells from 5.5 day. After degeneration of uterine epithelial cells, giant cells make direct contact with uterine decidual cells, and P‐cadherin was detected at contact sites between these cells.

Thus, the complicated process of implantation seems to be supported by temporal and spatial expression of the multiple classes of cadherins.
Exocytosis of Cortical Alveoli and Its Initiation Time in Medaka Eggs Induced by Microinjection of Various Agents
Authors: Iwamatsu Takashi
Published: January 2005

Various agents were microinjected into the cortical cytoplasm at the animal pole of unfertilized eggs of Oryzias latipes under Ca‐free conditions. The agents that triggered a wave of the cortical alveolus exocytosis were Ca2+, inositol, 1, 4, 5‐trisphosphate (IP3), Ca‐ionophore A23187, cGMP, GMP, GTP and guanosine 5′‐0‐(2‐thio‐triphosphate)(GTP‐γ‐s), while CAMP, ATP, gnanosine 5′‐0‐(2‐thio‐triphosphate)(GDP‐ β‐s), inositol monophosphate (IMP) and inositol triphosphate (ITP) were ineffective. Ca2+, IP3 and A231 87 induced the propagative exocytosis after a time lag (5–8 sec), irrespective of the presence of Co2+. The time lag was shorter than that (13–28 sec) following microinjection of cGMP or GTP, while were not effective in the presence of Co2+. The present data suggest that (1) free cytoplasmic Ca2+ participates in both an early and a late step in exocytosis, and (2) cGMP or GTP acts on an early step before initiation of Ca2+ release during exocytosis in the medaka egg.
The Synthesis and Localization of Envelope Glycoproteins in Oocytes of Xenopus laevis using Immunocytochemical Methods
Authors: Yamaguchi Seika , Hedrick Jerry L. , Katagiri Chiaki
Published: January 2005

The site of origin and the mode of differentiation of the coelomic envelope (CE) in growing oocytes of Xenopus laevis were studied using the rabbit antiserum raised against the isolated envelope from oviposited eggs. The antiserum preabsorbed with egg extracts reacted with most components of CE glycoproteins detectable by SDS‐PAGE, and stained specifically the CE of full‐grown (st. VI) oocytes using indirect immunofluorescence methods. Transmission electron microscopy employing IgG‐conjugated colloidal gold demonstrated that the CE antigens were distributed throughout the whole cytoplasm of st. I oocytes, and began to be deposited around the oocyte surface at late st. I. During st. II to VI the density of CE antigens in the oocyte cytoplasm decreased markedly, while the deposition of extracellular CE antigens increased gradually in association with the formation of a fibrillar network. The CE antigens were observed in and around the highly extended oocyte microvilli during st. II to IV, but were never found in follicle cells at any stages of oocyte growth. On western blot analyses, the extracellular CE components appeared first at st. II, and increased both in amount and number of bands during st. III ‐ V to attain a typical electrophoretic profiles of well‐developed CE. The cytosols of growing oocytes, however, possessed several antigenic components which were distinct from those of extracellular CE, suggesting the occurrence of intracellular precursor molecules for CE. The CEs of st. IV oocytes defolliculated and cultured in [3H] leucine contained certain CE components that expressed the radiolabel on fluorography. These results indicate that in Xenopus laevis the oocyte is directly involved in the synthesis and assembly, as well as secretion of CE with least contribution by the follicle cells.
Ultrastructure and Atrial Natriuretic Peptide(ANP)‐like Immunoreactivity of Cardiocytes in the Larval, Metamorphosing and Adult Specimens of the Japanese Toad, Bufo japonicus formosus
Authors: Hirohama Tohru , Uemura Haruko , Nakamura Sumio , Naruse Mitsuhide , Aoto Tomoji
Published: January 2005

The distribution of an atrial natriuretic peptide (ANP)‐like material in the cardiocytes of larval, metamorphosing, and adult specimens (both breeding and non‐breeding) of the toad, Bufo japonicus formosus, was studied immunohistochemically, ultrastructurally and immunocytochemically. Histochemically, ANP‐immunoreaction was positive in the atrium and ventricle in stage 33 larvae, while negative in the ventricle in stage 40 larvae. In adult toads, the reaction was stronger in the right than in the left atrium but quite weak in the ventricles, particularly those of non‐breeding specimens. Electron microscopy indicated a very small number of secretory granules in the atrial and ventricular cardiocytes of embryos as early as the limb‐bud stage (stage 28), and as development proceeded, the number of these granules increased rapidly in atrial but not in ventricular cardiocytes. In metamorphosing animals, a small population of larger granules (200–250 nm) was noted next to those of ordinary size (the median, 110 nm) in the same cell. In adult toads, granules of about 120 nm and 200 nm in median size were found in the same cell. Postembedding immunogold staining consistently indicated ANP‐immunoreactivity in these granules in atrial and ventricular cardiocytes. The plasma content of immunoreactive ANP was considerably higher in breeding (20.5 ± 5.9 pg/ml) than in non‐breeding toads (5.4 ± 1.7 pg/ml). These results are discussed in relation to presently available data on the physiological role of ANP.
A Freeze‐Sectioning Method for Preparation of the Detergent‐Resistant Cytoskeleton Identifies Stage‐Specific Cytoskeleal Proteins and Associated mRNA in Xenopus Oocytes and Embryos
Authors: Hauptman Ronald J. , Perry Brian A. , Capco David G.
Published: January 2005

Proteins of the detergent‐resistant cytoskeleton fraction and the detergent‐soluble fraction from Xenopus oocytes and embryos are examined using a procedure which allows rapid and uniform extraction of tissues and large, single cells. SDS‐polyacrylamide gels reveal only a few prominent cytoskeletal proteins in the early embryo, however qualitatively different proteins begin to appear after gastrulation. Incorporation of [35S]‐methionine into newly synthesized proteins indicates that there is synthesis and assembly of proteins into the cytoskeleton, but the amount remains low until after gastrulation. The use of nucleic acid probes for alpha‐tubulin and actin mRNA indicates that about 80% of these mRNAs in the oocyte and meiotically mature egg are bound to the detergent‐resistant cytoskeleton.
Changes in the Activities of H+, K+‐ATPase and Na+, K+‐ATPase in Cultured Cells Derived from Micromeres of Sea Urchin Embryos with Special Reference to Their Roles in Spicule Rod Formation
Authors: Mitsunaga Keiko , Fujiwara Akiko , Fujino Yukio , Yasumasu Ikuo
Published: January 2005

In cultured cells derived from micromeres isolated at the 16‐cell stage of sea urchin embryos, the activity of H+, K+‐ATPase became detectable after 15 hr of culture, when the cells started to form spicules, and then increased reaching a plateau from 25 hr of culture. The Na+, K+‐ATPase activity of isolated micromeres increased to a maximum at 20 hr of culture and thereafter decreased gradually. Allylisothiocyanate, an inhibitor of H+, K+‐ATPase, caused a decrease in intracellular pH (pHi) accompanied by blockage of 45Ca deposition in spicule rods in spicule‐forming cells at 30 hr of culture. Ouabain and amiloride had scarcely any effect on the pHi or 45, deposition. In cultured cells exposed to nifedipine, which blocked 45Ca deposition in spicule rods, allylisothiocyanate did not cause any decrease in pHi. These results show that H+, which is generated in the overall reaction to produce CaCO3 from Ca2+ and HCO3−, is probably released from the cells mainly in the reaction catalyzed by H+, K+‐ATPase to maintain successive production of CaCO3.
Stimulation of Tubulin Synthesis by Lactate in Isolated Spermatogenic Cells
Authors: Nakamura Masahisa , Komukai Masayuki , Matsuda Ryoichi , Okinaga Shoichi , Arai Kiyoshi
Published: January 2005

The effect of lactate on synthesis of new proteins in isolated spermatids and spermatocytes of rats was examined. Lactate stimulated[35S]methionine ([35S]met) incorporation into both spermatids and spermatocytes. The rate of protein synthesis was positively correlated with the intracellular level of ATP. The [35S]met‐labeled proteins in the two types of cells were compared by one and two dimensional polyacrylamide gel electrophoresis (1D and 2D‐PAGE) and autoradiography. The syntheses of several stagespecific and non‐specific proteins were observed. When spermatids and spermatocytes were cultured in medium without lactate, two major proteins of molecular weight (Mr) 43 kD and 55 kD were detected in the water‐soluble fraction (105,000 g supernatant), and one major protein of Mr 24 kD was observed in the membrane‐rich fraction. Addition of lactate to the incubation medium dramatically increased the synthesis of six proteins (Mr 14 kD, 16 kD, 43 kD, 55 kD, 84 kD and 135 kD) in the water‐soluble fractions of spermatids and spermatocytes, but did not stimulate the synthesis of the Mr 24 kD protein in the membrane‐rich fraction. In addition, after 1D and 2D‐PAGE and electrophoretic transfer to nitrocellulose, two proteins of Mr 43 kD and 55 kD were identified as actin and tubulin, respectively, on the basis of their reactivities with specific antisera. Tubulin was also produced by in vitro translation using a spermatid lysate. These results suggest that lactate may play an important role in changing the cell structure and shape during spermatogenesis by regulating the syntheses of actin and tubulin.
Synthesis of Collagen‐Like Proteins in Embryonic Organs of the Sea Urchin, Hemicentrotus pulcherrimus
Authors: Mizoguchi Hazime , Fujiwara Akiko , Yasumasu Ikuo
Published: January 2005

In sea urchin embryos exposed to 14C‐proline at 20°C for 3 hr at the gastrula, prism or pluteus stage, 14C‐radioactivity was found in hot acid‐extractable proteins, in which more than 4% of the radioactivity was detectable in hydroxyproline residues. In these embryos, 14C‐radioactivity in collagen‐like proteins was found in the archenteron, spicule and embryo‐wall cells. The rate of synthesis of collagen‐like proteins was highest in the archenteron in the mid‐gastrula stage, in the embryo‐wall cells in the prism stage and in the spicule in the pluteus stage. The rate of synthesis decreased in the archenteron and increased in embryo‐wall cells in the period between the mid‐ and late‐gastrula stages, when the rate of synthesis in the spicule was quite low. Thereafter, the rate decreased slightly in the embryo‐wall cells, was maintained in archenteron and increased markedly in the spicule. The rates of synthesis of collagen‐like proteins are high in these embryonic organs at stages at which development and growth respectively, occur in embryos. Therefore, synthesis of collagen‐like proteins probably supports morphogenesis in these embryonic organs.
Specification and Establishment of Dorsal‐Ventral Polarity in Eggs and Embryos of Xenopus laevis
Authors: Wakahara Masami
Published: January 2005

Dorsal‐ventral (D‐V) polarization in Xenopus eggs and embryos is achieved by passing through a series of complicated phenomena such as initial specification of the polarity before first cleavage, establishment of polarity during cleavage stages resulting in an acquisition of a unique developmental capacity by each blastomere, regional differentiation of mesoderm, and finally neural induction by Spemann's organizer. In order to gain an insight into basic mechanisms which govern D‐V polarization, experimental modifications or perturbations of the body axis of embryos, including physical or chemical treatments of eggs, altered orientation of eggs under the normal gravity, centrifugation, manipulation of blastomeres, cytoplasmic withdrawal, and bisection or partial ligation of fertilized eggs are reviewed: all data are consistent with the concept that a cytoplasmic activity which becomes localized in the dorsal side of the egg is responsible or indispensable for the establishment of the D‐V axis. The cytoplasmic activity is tentatively called “anterodorsal structure‐forming activity.” A model which explains the specification, establishment, and realization of D‐V polarity in Xenopus laevis is proposed.
The Maternal Origin of Acid Hydrolases in Drosophila and Their Relation with Yolk Degradation
Authors: Medina Miguel , Vallejo Carmen G.
Published: January 2005

The acid hydrolases of Drosophila are of maternal origin and appear subjected to differentiated control during embryogenesis. The enzymes are found associated with yolk granules. This association decreases during embryogenesis, in parallel with yolk degradation. As suggested before (Medina et al. Arch. Biochem. Biophys., 263, 355–363) the acid proteinase seems to be involved in the degradation of the yolk protein. The developmental profile of activity of the proteinase fits rather well with its involvement in the degradation of yolk granules. We have isolated intermediates of degradation of these subcellular structures. The intermediates have acid hydrolase activity and decrease in buoyant density during embryogenesis, in parallel with yolk degradation. The electron microscopic analysis has revealed that they are morphologically heterogenuous. A population of yolk granules appears to store mitochondria in their interior. The mitochondrial marker cytochrome oxidase is detected in density gradients associated with the intermediates of degradation, also supporting the storage of mitochondria in yolk granules in early development. The fact that the acid hydrolases are of maternal origin suggests that they have a role during embryogenesis. We propose that acid hydrolase(s) are involved in yolk degradation.
Nuclear Involvement in Localization of the Initiation Site of Surface Contraction Waves in Xenopus Eggs
Authors: Shinagawa Atsunori , Konno Seiko , Yoshimoto Yasuaki , Hiramoto Yukio
Published: January 2005

The initiation site of surface contraction waves (SCWs) was examined in fertilized, parthenogenetically activated and enucleated Xenopus eggs after either rotation through 90° off the vertical axis or injection of colchicine. In enucleated eggs, SCWs always started from a top site of the egg under all conditions examined. In fertilized or activated eggs, SCWs started, depending on the experimental conditions, from either the sperm entry point, the animal pole region located sideward or the top site of the egg. Histological examinations of fertilized and activated eggs revealed that the nucleus was in most cases positioned close to the initiation site of SCWs under various experimental conditions. It is suggested from these results that the egg cytoplasm has an intrinsic capability of causing the surface to generate SCWs, and that the nucleus is generally involved in localizing the initiation site of SCWs in fertilized or activated Xenopus eggs. A possible mechanism for localizing the initiation site of SCWs in Xenopus eggs is proposed.
The Location of Dorsal Information in Frog Early Development
Authors: Elinson Richard P. , Kao Kenneth R.
Published: January 2005

Dorsal information is necessary for the development of the dorsal axial structures which characterize the vertebrates. The nature of dorsal information in early embryos is not known, but its presence is required for the formation of dorsal mesoderm with Spermann organizer activity. In frogs, the dorsoventral axis is specified by a cortical/cytoplasmic rotation in the egg shortly after fertilization, and this dorsal information is limited to a few cells in the equatorial and vegetal region of early cleavage embryos. At the 8‐cell stage, 2–4 cells can promote dorsal development, and at the 32‐cell stage, 4–6 cells have dorsal information. Recent experiments have shown that growth factors can induce cells to form dorsal mesoderm and that lithium can act in those cells to enhance the induction. It will be important to determine the relationship between the location of dorsal information defined embryologically and factors involved in the development of dorsal mesoderm.