MOTILITY AND LOCOMOTION OF EMBRYONIC CELLS OF THE MEDAKA, ORYZIAS LATIPES, DURING EARLY DEVELOPMENT

The motility and locomotion of embryonic cells of the medaka, Oryzias latipes , were studied in situ with time-lapse cinematography.
In the early morula (128-cell stage), the surface of the blastomeres begins to undulate gently. By the early blastula stage, these undulations increase gradually in amplitude, and blebs appear. These blebs protrude and retract rapidly. In the mid-blastula stage they are found in most of the blastomeres. Some are found adhering to the surfaces of other cells. Blebs often expand into elongate lobopodia. Cell locomotion is first evident in the mid-blastula stage and continues throughout gastrulation and afterward. The cells move in the direction of the protrusion. In the late blastula a number of blastomeres locomote in random directions. In the thickening stage, when blastoderm epiboly begins, the cells with lamellipodia or elongate filopodia increase gradually in number, and in the early gastrula most cells change into this form. The motility, rate of movement, and mode of locomotion of embryonic cells during early development are described in detail.     

HISTOCHEMICAL AND PHYSIOLOGICAL STUDY OF CHROMAFFIN CELLS IN THE SKIN OF THE MEDAKA, ORYZIAS LATIPES.

A modified H illarp -H ökfelt 's method was applied to detect the chromaffin cells in the skin of the orange-red variety of the medaka, which has "colorless melanophores". A positive reaction was obtained in dendritic melanophore-like cells. This means that these cells contain at least dopa and/or dopa derivatives.
Effects of atropin and atrenalin on the chromaffin cells of the orange-red variety and on the melanophores of the wild type were investigated. Both kinds of these cells showed similar physiological responses to the reagents. From the above facts, the chromaffin cell was identified as the colorless melanophores.     

ACETYLCHOLINE AND ACETYLCHOLINESTERASE ACTIVITY IN EARLY EMBRYOS OF THE MEDAKA ORYZIAS LATIPES, A TELEOST

Acetylcholinesterase (AChE) activity is present in homogenates of medaka embryos during cleavage and epiboly. The levels of AChE activity change little during this period of development and are similar in embryos grown at either 15°C or 25°C. The specific activity of AChE in cells isolated from blastulae is 0.06 mmoles substrate hydrolyzed/min/g protein, a value comparable to that of chicken myoblasts and myotubes in vitro . Acetylcholinesterase activity was detected cytochemically in all cells dissociated from blastulae and gastrulae. In deep blastomeres AChE activity is present nearly throughout the cytoplasm; it is absent from peripheral regions of the cytoplasm which are involved in circus, or limnicolor, movements. Acetylcholine-like activity in extracts of the embryos was assayed using the clam heart ventricle bioassay. The active principle, which caused a decrease in both the frequency and magnitude of ventricular contractions, was inactivated by heating at pH 10 and by incubation at pH 7.0 with commercial AChE. The effect of the active principle on the clam heart was blocked by mytolon chloride, a drug which specifically blocks the effect of acetylcholine on the clam heart. The active principle migrated on Whatman #1 chromatography paper with the same R_f as authentic acetylcholine in three solvent systems. The amount of acetylcholine in blastulae is about 4 picomoles/embryo.     

CELL POPULATION KINETICS OF THE SCLEROBLAST DURING ETHISTERONE-INDUCED ANAL-FIN PROCESS FORMATION IN ADULT FEMALES OF THE MEDAKA, ORYZIAS LATIPES

Proliferation and migration of the scleroblasts and their precursor cells during ethisterone-induced anal-fin process formation in adult females of the medaka, Oryzias latipes , were investigated using a radioautographic technique with ³H-thymidine. In the posterior margin of the joint plate, rapid increases in labeling indexes and scleroblast population began simultaneously in the central portion on the third day of ethisterone treatment, and the population became approximately ten times that in the control fish on the fifth day. However, nomarked increase in scleroblast population occurred in the peripheral portion, although the scleroblasts showed nearly the same labeling index as those in the central portion throughout the experiment. These observations indicate that the scleroblasts and their precursor cells migrated from the peripheral to the central portion along the proxi-distal axis of the joint plate. Some cells in the inner and hind spaces also migrated to the posterior margins and subsequently developed into scleroblasts. It may be concluded, therefore, that the scleroblasts are derived from precursor cells situated not only along the posterior margins but also in the adjacent spaces. These precursor cells proliferate and/or migrate to the central portion of the posterior margins and aggregate to form the scleroblast mass.     

日本青鳉2种WNT4基因的克隆及鉴定(英文)

Wingless-type MMTV integration site family,member 4(WNT4) is a critical signaling molecule,regulating cell-cell interactions,proliferation,differentiation,migration,and gene activation [1,2].It is a highly conserved gene with mammals and has multiple roles in organogenesis and homeeostasis,and it has special importance in the sexual differentiation of the ovary [3 - 6].     

EFFECTS OF ANDROSTENEDIONE, 19-NORETHYNYLTESTOSTERONE, PROGESTERONE, AND 17α-HYDROXYPROGESTERONE UPON THE MANIFESTATION OF SECONDARY SEXUAL CHARACTERS IN THE MEDAKA, ORYZIAS LATIPES.

Effects of androstenedione, 19-norethynyltestosterone, progesterone, and 17α-hydroxyprogesterone upon the manifestation of secondary sexual characters were studied in the orange-red variety of the medaka. The steroids were consecutively administered per os for 15 days. Androstenedione and 19-norethynyltestosterone induced formation of new segments in the fin rays and produced papillar processes on the anal fin of the female. 19-norethynyltestosterone proved to have a strong potency, estimated to be more than 300 times that of androstenedione. Progesterone and 17α-hydroxyprogesterone had neither androgenic nor estrogenic effect on the secondary sexual characters. This is quite in harmony with the fact that they have neither androtermonic nor gynotermonic potency. The administration of these steroids did not impair the fertility of females. The administered females produced offspring by mating with administered males.     

青鳉prdm14的原核表达、多克隆抗体制备及其应用

青鳉(Oryzias latipes)是研究遗传发育和细胞多能性的重要模式鱼类, 为探究prdm14同源基因的潜在作用, 实验将青鳉prmd14经原核表达后制备了兔抗Prdm14多克隆抗体。首先, 将prdm14基因的部分编码区连接到pET32a质粒中, 构建重组表达载体pET32a-prdm14?600。随后将重组载体转化至大肠杆菌(Escherichia coli)Rosetta(DE3), 经异丙基-β-d-硫代半乳糖苷(Isopropyl-β-d-thiogalactoside, IPTG)诱导表达, 获得分子量为60 kD的Prdm14重组蛋白。接着大量诱导蛋白表达并切胶纯化, 免疫家兔(Oryctolagus cuniculus), 6周后获得阳性抗体, 最后通过ELISA和Western blot检测抗体效价及其特异性。结果显示, 在37℃、0.6 mmol/L IPTG、诱导3h的条件下, 可获得Prdm14重组蛋白的高效表达; 制备的兔抗青鳉Prdm14多克隆抗体能够特异性识别青鳉组织中表达的Prdm14蛋白以及在HepG2细胞中过表达的青鳉Prdm14: EGFP融合蛋白。综上所述, 研究首次制备了一种能有效识别青鳉Prdm14的多克隆抗体, 该抗体的获得为后续研究prdm14基因在鱼类多能性干细胞中的作用提供了有力工具。     

EVOLUTION OF SOCIAL BEHAVIOR IN A RESOURCE-RICH,STRUCTURED ENVIRONMENT: SELECTION EXPERIMENTS WITH MEDAKA (ORYZIAS LATIPES)

Schooling and aggression in fish are known to be partially inherited traits. Their genetic relationship to growth rate and to each other is, however, not fully understood. In this study we present evidence that schooling, social tolerance, and agonistic behavior in medaka (Oryzias latipes) are altered as an indirect result of selection on growth in two environments that differed in the intensity of social interactions required to obtain access to food. In the high interaction environment food was provided to excess inside a floating cork ring, which limited access to the food and allowed fish to attempt to monopolize it. In the low interaction environment the same amount of food was spread over the container's surface. After two generations of selection on growth the correlation of agonistic behavior and mean growth of broods was negative in the line selected for fast growth, when selection took place in a high interaction environment, but not when it took place in a low interaction environment. School cohesion was higher in the lines selected for fast growth than in those selected for slow growth when selection and rearing environments were both the same, either high or low interaction, but not when they were different. The correlation of social tolerance with growth was significantly more positive in the line selected for fast growth than in that selected for slow growth, but only when selection took place under high social interaction. It appears from these experiments that when resources are aggregated, but unlimited in quantity, competition favors individuals that avoid wasting time and energy on unnecessary and ultimately futile attempts to monopolize food and that also exhibit higher tolerance of nearby conspecifics. The results are interpreted in terms of a hypothesized stimulus-response threshold level for agonistic responses to conspecifics. We suggest that this threshold, which is altered by selection on growth, could provide a common causal (genetic) link between growth and the observed aspects of social behavior. By combining the probabilities from the separate behavioral experiments to obtain an overall significance of our hypothesis we conclude that the probability of no change in threshold is in fact low (P < 0.01).     

青鳉p53基因克隆、结构分析及同源重组载体构建

应用“Long PCR”技术 ,用 6对 p5 3引物从青胚胎干细胞基因组DNA中扩增出 6个相互重叠的片段 ,其中最大的片段长达 4 5kb ,这 6个PCR片段覆盖了整个 p5 3基因。序列分析表明青p5 3基因长约 8 7kb ,由 11个外显子和 10个内含子组成。结构比较表明 ,青 p5 3基因在大小上与人和小鼠 p5 3基因存在较大差异。青p5 3基因的内含子 1仅为 0 85kb ,而人和小鼠p5 3基因的内含子 1则分别长达 10kb和 6kb ;青 p5 3基因的外显子 3(86bp)明显大于人和小鼠 p5 3基因的外显子 3(2 2bp) ;外显子 4 (170bp)比人 (2 80bp)和小鼠 (2 6 0bp)的外显子 4小 ;内含子 10 (3 5kb)则比人和小鼠内含子 10 (0 7kb和 0 9kb)大得多。用SVTK neo基因作正选择标记基因 ,用SVTK tk基因作负选择标记基因 ,用青 p5 3基因组片段作同源序列 ,构建了鱼类 p5 3基因同源重组载体。将此载体转染青胚胎干细胞 ,并经G4 18和Ganc药物选择后证明上述正、负选择标记基因在干细胞中能够有效表达 ,并提供对G4 18的抗性和对Ganc的敏感性。     

CELLULAR BASIS OF EPIBOLY OF THE ENVELOPING LAYER IN THE EMBRYO OF MEDAKA, ORYZIAS LATIPES. I. CELL ARCHITECTURE REVEALED BY SILVER STAINING METHOD

A modification of silver nitrate staining, when applied to embryos of Oryzias latipes , was found to make clear not only the boundary of enveloping layer during epiboly, but also the outline of individual cells of the layer. The linear speed of advance of the enveloping layer was constant at fixed temperatures (20°, 25° or 30°C), except for the start and the end of the epibloy. Observation of the shape and arrangement of cells in the layer stained at successive stages of epiboly revealed that the enveloping layer expands uniformly over the yolk until late gastrula (3/4 epiboly). No cytokinetic figure was observed during epiboly until the blastopore was going to close. Total cell number of the layer remained constant during epiboly. Thus the expansion of the enveloping layer is accomplished without an accompanying increase in the number of constituent cells. In the last phase of epiboly, the surface area occupied by individual cells reduced locally at the region above the embryonic body, which suggests the occurrence in teleost of the convergence of cell sheets commonly observed in amphibian embryos.     

ULTRASTRUCTURAL STUDY ON THE SCLEROBLAST OF ORYZIAS LATIPES DURING ETHISTERONE-INDUCED ANAL-FIN PROCESS FORMATION

Cytodifferentiation of the scleroblast during ethisterone-induced anal-fin process formation in adult females of Oryzias latipes was observed electron-microscopically. Scleroblasts seem to arise from immature mesenchymal cells located not only along the postero-external margin of the joint plates, but also in the space between paired joint plates. These cells contained a small nucleus with condensed chromatin and a very small amount of cytoplasm with a marked paucity of formed organelles. These cells proliferated and/or migrated to the process-forming site of the joint plates and aggregated to the mass. Young scleroblasts were spindle-shaped in profile and contained an enlarged nucleus with dispersed chromatin. Rough-surfaced endoplasmic reticulum was developed in the cytoplasm. Scleroblasts became oval in shape and commenced to secrete bone matrix. Well-developed scleroblasts were characterized by an extensive development of rough-surfaced endoplasmic reticulum. This seems to correlate with the active protein synthesis required by the scleroblast for laying down bone matrix. As the young horny process developed, scleroblasts covered its apex, and became flattened. Some scleroblasts which became enclosed in the developing matrix had much the same characteristics as when they were lying on its surface.     

ULTRASTRUCTURAL STUDY ON THE ATTACHING FILAMENTS AND VILLI OF THE OOCYTE OF ORYZIAS LATIPES DURING OOGENESIS

The formation of the attaching filaments and villi on the surface of the oocyte of Oryzias latipes were studied electron-microscopically. The oocyte at the early stage has almost smooth surface with a few tufts of microvilli. Some parts of the surface of the oocyte are in contact with the follicle cell, and these parts subsequently become protrusions. As maturation proceeds, a mass of fine granules appears in the space between the protrusion and the follicle cell. Similar granules begin to appear also in the space between the microvilli. These granules later become the outer layer of the chorion. The protrusions are reduced in height, and consequently become almost flat. At the same time, there appears some amorphous material of high electron density on the above-mentioned granules on the flat part. A bundle of parallel microtubules is formed in the material. The tubule is 180–200 A in diameter, and its wall consists of 12 or 13 subunits. The bundle increases in volume, and becomes the attaching filament or villus.     

SCANNING ELECTRON MICROSCOPIC STUDY ON THE SURFACE CHANGE OF EGGS OF THE TELEOST, ORYZIAS LATIPES, AT THE TIME OF FERTILIZATION

The surface change of the egg of the teleost, Oryzias latipes , during fertilization was observed with a scanning electron microscope. The microvilli of the outer surface of the unfertilized egg show a slight difference in density between the animal and vegetal pole areas. In the initial step of the breakdown of cortical alveoli (CA), several small holes or gapes are formed at the apical part of the CA membrane, becoming a large aperture from which the alveolar contents are discharged. The formation of microvilli is observed on the inner surface of the exposed cavity left by the CA, starting from the periphery of the aperture and propagating throughout the whole inner surface in accompaniment with the release of the alveolar contents. After the completion of CA breakdown, the CA membrane cannot be distinguished from the original egg plasma membrane.     

INHIBITORY EFFECT OF RABBIT SERUM FACTOR ON IN VITRO OOCYTE MATURATION OF THE TELEOST FISH,ORYZIAS LATIPES*

The present study indicates that a factor in rabbit serum inhibits the in vitro steroid- and gonadotropin-induced maturation of oocytes of the teleost fish, Oryzias latipes. Such inhibitory activity could not be recognized in the serum of this fish or in the fluids from mammalian follicles. Passage of the serum inhibitor through a cellulose membrane indicated that it has a molecular weight of less than 3,500. The inhibitory activity on steroid-induced oocyte maturation was not destroyed by heating, by repeated freezing and thawing or by treatment with proteolytic enzymes, lipase or amylases. However, its activity could be removed by extraction with charcoal but not with ethyl ether or toluene. The inhibitory action of the heat-stable and dialyzable serum factor was reversible. The factor appears to exert its inhibitory effect upon the oocyte itself in an early step of maturation, rather than upon the follicle cells.     

DIFFERENTIATION OF THE RETINA AND RETINAL LACTATE DEHYDROGENASE ISOENZYMES IN THE TELEOST, ORYZIAS LATIPES

The activity of lactate dehydrogenase in the teleost Oryzias latipes , shows a gradual increase during embryonic development, followed by an abrupt rise on hatching, which coincides with the appearance of the retinal isoenzymes. By the time of hatching, the retina has differentiated, and no significant change is observed in the retinal structure until 2 days after hatching. It was assumed that metabolic changes which occur on hatching may trigger the synthesis of retinal lactate dehydrogenase isoenzymes.     

SPERMIOGENESIS OF THE TELEOST,ORYZIAS LATIPES,WITH SPECIAL REFERENCE TO THE FORMATION OF FLAGELLAR MEMBRANE *

In the early stage of Oryzias spermiogenesis, an axonemal bud appears at the distal end of a centriole characterized by its electron dense accessories. When the axoneme begins to grow in the cytoplasm, small vesicles come to surround it. These vesicles are similar to those produced by the Golgi apparatus which lies close to the growing axoneme. At this stage, the spermatid cell membranes disappear, causing transformation of the mononuclear spermatids into a multinucleated syncytium. As each axoneme elongates in the syncytium, it is enveloped by a cylindrical array of vesicles which are most likely derived from the Golgi apparatus. Shortly after this stage, the syncytium is again partitioned by cell membranes, restoring the existence of mononuclear spermatids. The arrayed vesicles fuse with each other to form two concentric membranes surrounding the axoneme. The inner membrane becomes the flagellar membrane and the outer one, the membrane of a flagellar sheath. These observations lead to the conclusion that the formation of the flagellar membrane is due to the fusion of vesicles surrounding the axoneme which are derived from the Golgi apparatus. In the course of spermiogenesis, no indication of an acrosomal structure is observed.     

RAPID BEHAVIORAL CHANGES IN MEDAKA (ORYZIAS LATIPES) CAUSED BY SELECTION FOR COMPETITIVE AND NONCOMPETITIVE GROWTH

Behavior is a major component of growth rate variation in fish, but the genetic relationship between behavior and growth is largely unknown. In particular, it is not known how behavior responds to natural or artificial selection on growth. It has been argued that artificial selection for fast growth in competitive environments might lead to higher levels of overall aggression, and therefore there would be no net gain in assimilation efficiency or growth. The contrary has also been argued. It is not immediately obvious, therefore, whether fish that avoid interacting with conspecifics should grow faster, or whether more aggressive, dominant individuals should grow faster. In the present study, we artificially selected fish on the basis of growth in two environments that differed in the intensity of social interactions. In the high interaction environment (HI), food was provided to excess inside a floating cork ring, which limited access to the food and allowed fish to attempt to monopolize the food supply. In the low interaction environment (LI), an equivalent amount of food was spread over the container's surface. In each social environment, fish were selected within family for fast and for slow growth rate during two generations (G₁ and G₂). The response was measured in the third generation (G₃) on the directly selected trait (growth) and on a correlated trait (agonistic behavior). The magnitude of the direct response to selection on growth in medaka was directly related to population density, i.e., selection was most effective at high densities. Agonistic behavior was inversely related to growth when individuals were raised and selected in an environment where enforced social interaction took place, so long as food was not limited in quantity.     

EFFECTS OF HYPOPHYSECTOMY ON RNA METABOLISM IN RAT BRAIN STEM

Abstract— Ribosomal aggregates were isolated from rat brain stem and characterized as polysomes by sedimentation analysis and by their sensitivity to RNase and EDTA treatment.
Three weeks following hypophysectomy there was a significant decrease in the content of large polysomes in the rat brain stem. The incorporation of radioactive uridine into RNA was studied using a double-labelling technique with [³H]- and [¹⁴C]uridine and labelling periods of 70 and 180 min. It was found that after hypophysectomy the incorporation of radioactive uridine into total, nuclear and cytoplasmic RNA and in polysomes was decreased after 70 and 180 min. Information on the nature of the rapidly-labelled RNA in the various subcellular fractions was obtained by sucrose gradient sedimentation analysis.
After 70 min of labelling the nucleus contained heterogeneous RNA with a considerable fraction of RNA sedimenting faster than 28 S. In the cytoplasmic fraction heterogeneous 4 to 30 S RNA was found, presumably associated with RNP particles, whereas after 180 min the polyribosomal aggregates were also labelled.
The present results indicate a profound effect of hypophysectomy on the metabolism of all species of brain RNA investigated.