In vitro Oocyte Maturation in the Zebrafish Brachydanio rerio and Fertilization and Development of the Mature Egg

Maturation process of zebrafish oocyte was investigated using in vitro incubation.In medium EM-199 containing 0.5 μg/ml of 17α-hydroxyprogesterone incubated under 80％ O_2 and at 25°C,germinal vesicles(GV)of oocytes in stage Ⅳ migrated from midway between the center and theperiphery ofoocytes to the periphery in 40 minutes and the oocytes went into stage V.Half an hourlater,the oocytes underwent germinel vesicle breakdown(GVBD)with a breakdown rate of 59％.Two more hours were needed for such oocytes to complete their final maturation.The mature eggscould not come off from the follicle layer surrounding them by themselves(ovulation).By removingthe follicle and adding active sperms for insemination,we could make the mature eggs fertilized.Thechorion was elevated and blastoderm formed on the animal pole.The cleavage and development ofthese fertilized eggs followed the same course as the naturally matured and fertilized eggs.Usingblastula formation as a marker of successful fertilization of the in vitro matured egg,the fertilizationrate was 78％.This is the first report on the successful in vitro incubation of mature oocytes inzebrafish.The establishment of this in vitro oocyte maturation technology has laid the foundationfor further investigation of the transfer of foreign genes in the germinal vesicles of oocytes.     

Stages of oocyte development in the zebrafish,Brachydanio rerio

Oocyte development has been divided into five stages in the zebrafish Brachydanio rerio, based on morphological criteria and on physiological and biochemical events. In stage I (primary growth stage), oocytes reside in nests with other oocytes (Stage IA) and then within a definitive follicle (Stage IB), where they greatly increase in size. In stage II (cortical alveolus stage), oocytes are distinguished by the appearance of variably sized cortical alveoli and the vitelline envelope becomes prominent. In stage III (vitellogenesis), yolk proteins appear in oocytes and yolk bodies with crystalline yolk accrue during this major growth stage. Ooctes develop the capacity to respond in vitro to the steroid 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) by undergoing oocyte maturation. In stage IV (oocyte maturation), oocytes increase slightly in size, become translucent, and their yolk becomes non-crystalline as they undergo final meiotic maturation in vivo (and in response to DHP in vitro). In stage V (mature egg), eggs (approx. 0.75 mm) are ovulated into the ovarian lumen and are capable of fertilization. This staging series lays the foundation for future studies on the cellular processes occurring during oocyte development in zebrafish and should be useful for experimentation that requires an understanding of stage-specific events. © 1993 Wiley-Liss, Inc.     

Attraction of zebrafish, Brachydanio rerio, to alanine and its suppression by copper

Preference responses of zebrafish to 10⁻³, 10⁻⁴ and 10⁻⁵M alanine (Ala) were concentration- dependent. Behavioural responses to copper (Cu) and Cu + Ala mixtures were also assessed. Zebrafish avoided 100 and 10 μg Cu l⁻¹, but not 1 μg l⁻¹. Mixtures of 10⁻³ m Ala+ 100 μg Cu l⁻¹ and 10 ⁴ M Ala + 10 μg Cu 1⁻¹ were avoided as intensely as was Cu alone. Responses to 10⁻³ M Ala + 10 or 1 μg Cu l⁻¹ and 10 ⁴ M Ala +1 μg Cu l⁻¹ did not differ statistically from controls (no detectable preference or avoidance). These results demonstrate, firstly, that a concentration of a pollutant avoided by itself (10 μg Cu l⁻¹) may not be avoided when encountered with an attractant chemical stimulus (Ala) and may suppress the preference for an attractant stimulus, and secondly, that a concentration of a pollutant not avoided by itself and not considered deleterious (1 μg Cu l⁻¹) suppresses attraction to Ala (an important constituent of prey odours for many fishes).     

五氯酚对斑马鱼胚胎的毒性效应研究

采用斑马鱼胚胎发育技术,对环境激素类物质五氯酚的毒性进行测定,结果表明,五氯酚(PCP)对胚胎的特定作用时间段是卵产出至发育6 h之内;PCP对胚胎发育有明显的抑制作用,会造成胚胎发育的畸形或死亡,不同时间染毒产生的可观察毒理学终点各异;随着PCP对发育48 h斑马鱼胚胎作用时间的减短,其致死效应敏感性降低,其中0 hpf组的LC0值最小,为70.8μg·L^-1,24hpf组LC0值最大,为831.8μg·L^-1;斑马鱼胚胎对孵化后0时染毒的PCP最为敏感,PCP对胚胎产生急性毒性效应的敏感指标:心胞囊肿、血液循环障碍、无心律＞孵化率降低＞停滞发育作用;斑马鱼胚胎最敏感的指标为48 h血液循环障碍和48 h半致死效应.     

Gill morphology in the zebrafish, Brachydanio rerio (Hamilton- Buchanan)

A light and electron microscopic study of the gills of the zebrafish, Brachydanio rerio , were made to serve as a morphological basis for future investigations. It was found that for fixation of B. rerio gills, a mixture of 1·5% gluturaldehyde and 1·5% paraformaldehyde gives a mucus-free surface. Morphometric measurements of structural components of the gill secondary lamellae were made. Observations at SEM were correlated with those made at TEM. The different cell types in the branchial epithelium were characterized. Chloride cells were mainly located in the interlamellar regions and on the afferent side of the primary lamellae. Two morphologically different chloride cells were seen. The first type communicates with the external environment through a reservoir-like lumen, which is normally absent in freshwater fishes. The second type of chloride cell has more direct contact with the ambient water, resembling chloride cells from other freshwater fishes. Another cell type with features similar to those of the rodlet cell was frequently observed. This cell is interposed between other types of cells in the epithelium, and sometimes junctional complexes were present between the rodlet cell and surrounding cells.     

Neurulation in the anterior trunk region of the zebrafish Brachydanio rerio

We have studied the process of neurulation within the anterior trunk region of the zebrafish by means of serial sectioning of staged embryos and labelling cells by applications of the dye Dil and intracellular injections of fluoresceine dextran amine. The first morphological manifestation of the prospective neural plate is a dorsomedial ectodermal thickening which becomes visible immediately after gastrulation. Within 1–2 h, by the time somatogenesis begins, two bilaterally symmetrical thickenings have appeared more laterally, which eventually fuse with the medial thickening to form the neural keel. The central canal forms next by separation of the cells on either side of the midline of the neural keel, beginning ventrally at the 17-somite stage and progressing towards dorsal levels. By means of fluorescent dye labelling in the late gastrula, we found that both the medial and lateral thickenings contribute to the nerve cord. The medial thickening was found to contain, exclusively, neural progenitor cells from the 90–100% epiboly stage on, whereas the adjacent regions contained a mixture of neural and epidermal progenitor cells, as well as prospective neural crest cells. Between the 90–100% epiboly and 2-somite stages, this heterogeneity of developmental capabilities is resolved into territories, with epidermogenic and neurogenic cells clearly separated from each other. To achieve this segregation into neural and epidermal anlagen, cells from the lateral thickenings have to move over a distance of roughly 400 m within 1–2 h. Epidermal overgrowth of the nerve cord occurs during the morphogenetic movements that accompany nerve cord formation. Correspondence to: J.A. Campos-Ortega     

Scanning electron microscope studies on blastodisc formation in the zebrafish,Brachydanio rerio

Upon fertilization, the zebrafish egg undergoes marked physiological and structural changes, one of which involves blastodisc formation. Before fertilization, yolk globules are rounded and the endoplasm extends throughout the oocyte. During blastodisc formation, the yolk globules become angular and the endoplasm is restricted to streamers among the yolk globules. The streamers are oriented in an anterior-posterior axis of the egg. During blastodisc formation the cytoskeleton consists of an extensive array of filamentous structures of variable width in both the cortex as well as within elongate endoplasmic streamers. Although the filamentous components in the cortex and endoplasmic streamers probably include both microfilaments and microtubules, frequently they are somewhat wider than the usual dimensions, and possible reasons for this are suggested. From their arrangement in both the cortex and endoplasm, it seems likely that the components of the cytoskeleton (e.g., microfilaments and microtubules) may provide, through contraction, the major force responsible for the streaming of the endoplasm into the forming blastodisc. It is assumed that the surface tension of the vegetal hemisphere exceeds that of the animal hemisphere, thus forcing, through differential contraction, the endoplasm to flow in the direction of the forming blastodisc. No distinct barrier between the yolk and forming blastodisc was observed. The compressed condition of the larger and many-sided yolk globules could prevent their movement into the blastodisc. Scanning electron microscopy is limited in the resolution with which it can depict the cytoskeleton, but nonetheless it provides useful information about structural interrelationships.     

Ontogeny of the Solitary Chemosensory Cells in the Zebrafish, Danio rerio

Secondary epidermal solitary chemosensory cells (SCCs) are widespreadamong the primary aquatic vertebrates. They resemble taste budsensory cells in fine structure and may be innervated from facialor spinal nerves. According to previous studies, SCCs may constitutea water sampling system in the contexts of predator avoidance,habitat recognition and, in some cases, finding food. By quantitativescanning (SEM) and transmission electron microscopy (TEM) in60 specimens (57 SEM, 3 TEM) of 16 developmental stages, frompre-hatchlings to adults, we describe the ontogenetic developmentof SCC densities and shapes of sensory apices in the zebrafish,Danio rerio. This is put into perspective with the ontogenyof external taste buds. Just prior to hatching, 3 days afterfertilization (3d AF), sensory apices of SCCs penetrate betweenthe squamous epidermal cells, whereas taste bud pores only appearat the onset of exogenous feeding (5d AF). SCC densities increasesharply from hatching shortly after metamorphosis (25d AF) upto 6 x 10³ per mm² on the head and remain relatively constantin density thereafter. Conservatively estimated, there may be     

Innate and Learned Colour Preference in the Zebrafish, Danio rerio

Receiver biases towards specific sensory signals have been demonstrated in insects, birds and fish, both in the context of foraging and mate choice. In some cases, signals important in sexual selection appear to have evolved by exploiting a pre-existing bias in the sensory system. For instance, female preferences for male nuptial colouration may have arisen from selection on foraging practices. Using the zebrafish ( Danio rerio ), a species in which red is not a factor in mate choice, we tested for a foraging bias towards the colour red. We further investigated the plasticity of foraging biases by raising groups of fish on diets consisting solely of red, blue, green or white food. When we subsequently tested their colour preferences in a foraging context, each group responded most strongly to red, irrespective of the colour of food with which they had been conditioned. We also detected a significant effect of conditioning on colour preferences; fish responded more strongly to the colour that matched diet colour than to other colours. The observed receiver bias towards red may have evolved as an adaptive preference for carotenoid compounds in their diet. While the bias to red appears to be innate, our results indicate that learning is also important in shaping foraging biases.     

Zebrafish information network,the knowledgebase for Danio rerio research

The Zebrafish Information Network (zfin.org) is the central repository for Danio rerio genetic and genomic data. The Zebrafish Information Network has served the zebrafish research community since 1994, expertly curating, integrating, and displaying zebrafish data. Key data types available at the Zebrafish Information Network include, but are not limited to, genes, alleles, human disease models, gene expression, phenotype, and gene function. The Zebrafish Information Network makes zebrafish research data Findable, Accessible, Interoperable, and Reusable through nomenclature, curatorial and annotation activities, web interfaces, and data downloads. Recently, the Zebrafish Information Network and 6 other model organism knowledgebases have collaborated to form the Alliance of Genome Resources, aiming to develop sustainable genome information resources that enable the use of model organisms to understand the genetic and genomic basis of human biology and disease. Here, we provide an overview of the data available at the Zebrafish Information Network including recent updates to the gene page to provide access to single-cell RNA sequencing data, links to Alliance web pages, ribbon diagrams to summarize the biological systems and Gene Ontology terms that have annotations, and data integration with the Alliance of Genome Resources.     

The biology and use of zebrafish, Brachydanio rerio in fisheries research.

PREFACE TO THE REVIEW BY PROFESSOR H. LAALE ON THE BIOLOGY AND USE OF THE ZEBRAFISH, BRACHYDANIO RERIO IN FISHERIES RESEARCH
The growing demand for increasingly sophisticated information on the toxic hazards of potential water pollutants has focused attention on the need for a suitable 'standard' animal model which could be accepted internationally. The Zebrafish, Brachydanio rerio (Hamilton-Buchanan, 1822, 1823) is considered to be the most likely candidate. It is relatively easy to maintain and breed in laboratory aquaria and it has proved to be responsive to a wide range of mutagens, carcinogens and teratogens, as well as direct toxicants. B. rerio has been the subject of a considerable number of investigations involving a diverse spectrum of disciplines in a number of countries. Professor Hans Laale, who has himself contributed to our knowledge of the embryopathology of B. rerio , has summarized and collated the findings of 450 publications, a number of which are unlikely to have become available to fishery scientists. We hope the publication of this review will aid those who are working with B. rerio and provide comprehensive data on the advantages and limitations of B. rerio as a contender for the standard laboratory fish for the safety evaluation of aquatic pollutants.
T he E ditor     

Über die Degeneration von Eizellen des Zebrafisches,Brachydanio rerio,in vitro

Zusammenfassung In Ovarkulturen des Zebrafisches, Brachydanio rerio, wird der Degenerationsverlauf der Eizellen verschiedener Entwicklungsstadien beschrieben. Junge dotterlose Oocyten überstehen die 4 wöchige Kultivierung ohne wesentliche morphologische oder histochemisch nachweisbare Veränderungen. Oocyten mittleren Alters, die bereits Rindenvakuolen enthalten, verlieren im Laufe der Kultivierung ihre cytoplasmatische Basophilie und ihre in vivo zahlreichen Nukleolen. Bei einem Teil dieser Eizellen sind als Zeichen beginnender Degeneration Follikelepithelzellen im randständigen Cytoplasma eingewandert. Reife Oocyten beginnen nach 1–2 Wochen mit der Degeneration. Das in der Kultur auswachsende Follikelepithel behält seine Fähigkeit bei, in degenerierende Eizellen einzuwandern und Dotter zu phagocytieren.

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Oocyte degeneration in the zebrafish, Brachydanio rerio, in vitro

Summary In cultured ovarian tissue of the zebrafish, Brachydanio rerio, the degeneration of oocytes in various developmental stages is described. Young yolk-free oocytes survive the 4 weeks cultivation time without significant morphological and histochemical alterations. In medium sized oocytes, which contain cortical granules already, the cytoplasmic basophilia and the number of nucleoli decrease in the culture. Some of these oocytes show follicular epithelial cells invading in the peripheral cytoplasm. Mature oocytes become atretic after 1 or 2 weeks cultivation time. The outgrowing follicular epithelial cells retain their ability to penetrate degenerating oocytes and to take up yolk by means of phagocytosis.

     

Relationships between annulate lamellae and filament bundles in oocytes of the zebrafish,Brachydanio rerio

Summary Bundles of filaments have been observed in the vitellogenic oocyte of the zebrafish, Brachydanio rerio; and these filaments illustrate a close spatial and structural relationship to annulate lamellae. The filaments range from 6–8 nm in diameter, and the annulate lamellae may cap both rounded ends of the bundle as well as extend parallel to the surface of the filament bundles. The ends of the filaments can be observed to exhibit an apparent termination in close relation to pore margins of the annulate lamellae, the membrane of the interpore regions of the annulate lamellae, as well as many nearby polyribosomes. The possible functional significance of this unique relationship is discussed in reference to a recent hypothesis regarding the function of annulate lamellae.     

Cloning and characterization of class I Mhc genes of the zebrafish,Brachydanio rerio

The zebrafish (Brachydanio rerio) offers many advantages for immunological and immunogenetic research and has the potential for becoming one of the most important nonmammalian vertebrate research models. With this in mind, we initiated a systematic study of the zebrafish major histocompatibility complex (Mhc) genes. In this report, we describe the cloning and characteristics of the zebrafish class I A genes coding for the chains of the heterodimer and thus complete the identification of all four classes and subclasses of the Mhc in this species. We describe the full class I cDNA sequence as well as the exon-intron organization of the class I A genes, including intron sequences. We identify three families of class I A genes which we designate Bree-UAA,-UBA, and -UCA. The three families originated about the time of the divergence of cyprinid and salmonid fishes. All three families are members of an ancient lineage that diverged from another, older lineage also represented in cyprinid fishes before the radiation of teleost orders. The fish class I A genes therefore evolve differently from mammalian class I A genes, in which the establishment of lineages and families mostly postdates the divergence of orders.The nucleotide sequence data reported in this Papershave been submitted to the EMBL/GenBank nucleotide sequence databases and have been assigned the accession numbers Z46776–Z46779     

Zur Genese des Dottersystems in der Oocyte von Brachydanio rerio

Zusammenfassung Am Zebrafisch Brachydanio rerio wurde nach Injektion tritiierter Aminosäuren autoradiographisch untersucht, ob die Dotterproteine endogen oder exogen synthetisiert werden. Um die Verfügungszeit der markierten Aminosäuren zu bestimmen, wurde deren Einbau in Gewebe mit hohem Proteinmetabolismus, nämlich Leberparenchym und Darmepithel, erfaßt. Dort wird das Maximum der Markierung nach 3 h überschritten, d. h. nach dieser Zeit sind freie markierte Aminosäuren praktisch nicht mehr vorhanden. Die Oocyten enthalten zwei morphologisch unterscheidbare Dottersysteme, die intravesikulären und die an Anzahl und Größe überwiegenden extravesikulären Dotterkugeln. In die ersteren wird der Tracer während der Verfügungszeit eingebaut. Das spricht für eine Synthese in loco. Markierte extravesikuläre Dotterschollen erscheinen in der Peripherie der Oocyte erst am Ende der Verfügungszeit und reichern sich noch nach 24 h und später an. Diese Dotterkugeln werden demnach unter wesentlicher Beteiligung einer exogenen Proteinkomponente gebildet. Das Markierungsmaximum des Blutes folgt dem der Leber und liegt vor dem Oocytenmaximum. Dies spricht in Übereinstimmung mit elektronenmikroskopischen Untersuchungen für eine pinocytäre Aufnahme von Blutproteinen in das extravesikuläre Dottersystem. Trypanblau reichert sich in der Zona radiata an. Es stört den Einbau von Aminosäuren in das Ooplasma nicht, verhindert aber die Markierung extravesikulärer Dotterschollen, vermutlich durch Blockierung der Pinocytose an der Oocytenoberfläche.

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Summary After injection of tritiated amino acids the zebrafish oocytes were investigated by means of radioautography to see whether the yolk proteins are synthetized endogenous or exogenous. To estimate how long the labeled amino acids are available their incorporation in tissues with high protein metabolism like liver parenchym and intestine epithelium was investigated. There the maximum of labeling is exceeded after 3 h. That shows that after this time practically no free labeled amino acids are available any longer. The oocytes contain two morphological different yolk systems: intravesicular yolk spheres and extravesicular ones which dominate in size and number. In the former the tracer is incorporated within 3 h of incubation time, that means a synthesis in loco. Labeled yolk spheres appear in the periphery of the oocytes only at the end of the time the tracer is available and accumulate even after 24 h and more. According to this the extravesicular yolk spheres are formed under essential participation of an exogenous protein component. The maximum of radioactivity in the blood follows that of the liver and precedes that of the oocytes. In agreement with electron microscopic observations these results indicate the pinocytotic uptake of blood proteins into the extravesicular yolk system. Trypan blue accumulates in the zona radiata. It does not inhibit the incorporation of amino acids into the ooplasma but prevents the labeling of extravesicular yolk probably by blocking the pinocytotic activity on the surface of oocytes.

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Herrn Prof. Dr. K. Bier danke ich für die Überlassung des Themas, sein ständiges Interesse an der Arbeit und seine Unterstützung bei der Durchführung der Untersuchungen, die von der Deutschen Forschungsgemeinschaft gefördert wurden.     

Cytochalasin B binding by human platelets

Intact human platelets bind cytochalasin B (CB) with a capacity of 100– 120 p mols CB/mg protein or approximately 7 × 10⁴ molecules/cell and dissociation constants (K_D) ranging from 2 × 10^?8 to 10^?6 M. Up to 85% of this saturable binding is displaced by 10^?5 M cytochalasin E (CE). This CE-sensitive binding also appears heterogeneous with K_D similar to those of the overall binding. The CE-insensitive binding, however, appears as a single component with K_D ≌ 4 × 10^?7 M. The sedimentable constituents from frozen, thawed, and washed cells also bind CB with K_D ranging from 2.4 × 10^?8 to 1.5 × 10^?6 M and a total capacity of approximately 39 p mols/mg protein which accounts for only 4% of the ligand binding to the intact cell. The major portion (60–80%) of this CB binding is displaceable by 500 mM D-glucose and has a K_D of 1.5 × 10^?6M, while only 10–15% is CE-sensitive with a K_D of 2.4 ± 10^?8 M. It is concluded that 95% of the saturable CB binding in platelets is associated with the cytosol of which 80–85% is sensitive to CE and that only 3% of the cellular binding is glucose sensitive, membrane-associated binding. If the CE-sensitive binding associated with the cytosol is entirely to actin, the stoichiometry of this binding is approximately one CB to 30 actin monomers, which is greater by an order of magnitude than that for CB binding to muscle actin.