Biochemical characterization of the medaka (Oryzias latipes) orthologue for mammalian tissue-type transglutaminase (TG2)

Transglutaminase is an enzyme family responsible for post-translational modification such as protein cross-linking and the attachment of primary amine and/or deamidation of glutamine-residue in proteins. Medaka (Oryzias latipes), a recently established model fish, has similar functional proteins to those characterized in mammals. Previously, we found the apparent orthologues that correspond to human transglutaminases in medaka. In this study, regarding the medaka orthologue of human tissue-type transglutaminase (OlTGT), recombinant protein was expressed in an active form in bacteria cultured at low temperature. Using the recombinant protein, we biochemically characterized the enzymatic activity and also obtained a monoclonal antibody that specifically recognized OlTGT. Immunochemical analysis revealed that OlTGT was not expressed ubiquitously, unlike its mammalian orthologue, but in primarily limited tissues such as the eye, brain, spinal cord, and gas gland.     

河流汞污染对青鳉鱼早期发育的毒性影响

清镇市化工厂是贵州省东门桥河汞污染的重要来源。为了评估这条河流的汞污染程度,本研究调查了东门桥河环境样品中的汞含量和分布情况。河流水体总汞(THg)和甲基汞(MeHg)的平均含量分别为(135.79±47.40)和(0.64±0.58) ng·L^-1;生物膜中THg和MeHg的平均含量分别为(3.38±1.91) mg·kg^-1和(10.47±0.40)μg·kg^-1;沉积物中THg和MeHg的平均含量分别为(23.21±20.22) mg·kg^-1和(32.74±10.96)μg·kg^-1。为了进一步了解这条河流中汞对水生生物的风险,将青鳉鱼胚胎和仔鱼分别暴露于从河中收集的被汞污染的河水、生物膜和沉积物中。青鳉鱼胚胎经15 d暴露后,孵化时间延长、孵化率显著降低,死亡率显著升高,实验暴露还导致胚胎无法破膜、卵黄囊水肿等畸形效应,以及初孵仔鱼不同程度的畸形。青鳉鱼仔鱼经24、48、96 h和21 d暴露后,各暴露组的死亡率和畸形率随着暴露时间的延长而显著升高,且表现出一系列以脊...     

Development and fine structure of the yolk nucleus of previtellogenic oocytes in the medaka Oryzias latipes

The development and fine structure of yolk nuclei in the cytoplasm of previtellogenic oocytes were examined by electron microscopy during several stages of oogenesis in the medaka, Oryzias latipes. Shortly after oogenesis starts, oocytes 20-30 microm in diameter have much electron-dense (basophilic) cytoplasm, within which a continuous or discontinuous, irregular ring-shaped lower electron-dense area of flocculent appearance (LF) begins to emerge around the nucleus. The yolk nucleus is first recognized within an LF area as a few fragments of dense granular thread measuring 20-25 nm in width. The threads consist of two rows of very dense granules resembling ribosomes or ribonucleoprotein (RNP)-like particles in size and electron density. These thread-like fragments gradually increase in number and length until they assemble into a compact, spherical mass of complicated networks. Analysis of serial sections suggests that the yolk nucleus is a complicated mass of numerous, small deformed vacuoles composed of a single lamella with double layers of ribosomes or RNP-like granules, rather than a mass of granular threads. When oocytes develop to greater than 100 microm in diameter, the yolk nucleus begins to fragment before dispersing throughout the surrounding cytoplasm, concomitantly with the disappearance of LF areas. At this stage of oogenesis, a restricted region of the granulosa cell layer adjacent to the yolk nucleus becomes somewhat columnar in morphology, fixing the vegetal pole region of the oocyte.     

Molecular Cloning and Bacterial Expression of the Catalytic Domain of the SENP1 Gene of Oryzias latipes

In this study, we cloned the catalytic domain of the Oryzias latipes sentrin/SUMO-specific protease 1 (OlSENP1-CD) gene and produced the recombinant OlSENP1-CD protein in Escherichia coli. Experimental procedures designed to reveal the ability of the recombinant protein to show deSUMOylating activity in vitro should be helpful in future studies of other SENPs and the SUMO pathway.     

The Genomic and Genetic Toolbox of the Teleost Medaka (Oryzias latipes)

The Japanese medaka, Oryzias latipes, is a vertebrate teleost model with a long history of genetic research. A number of unique features and established resources distinguish medaka from other vertebrate model systems. A large number of laboratory strains from different locations are available. Due to a high tolerance to inbreeding, many highly inbred strains have been established, thus providing a rich resource for genetic studies. Furthermore, closely related species native to different habitats in Southeast Asia permit comparative evolutionary studies. The transparency of embryos, larvae, and juveniles allows a detailed in vivo analysis of development. New tools to study diverse aspects of medaka biology are constantly being generated. Thus, medaka has become an important vertebrate model organism to study development, behavior, and physiology. In this review, we provide a comprehensive overview of established genetic and molecular-genetic tools that render medaka fish a full-fledged vertebrate system.     

Participation of a metalloprotease in the fertilization-associated conversion of the egg envelope (chorion) of the fish, Oryzias latipes

The unfertilized egg envelope of medaka ( Oryzias latipes ) consists of two major groups of subunits, ZI-1,2 (74–76 kDa) and ZI-3 (49kDa). During egg envelope hardening after egg activation, both subunit groups decreased in amount, new protein bands of 57–65, 110 and 125 kDa appeared and, finally, no bands were detectable on sodium dodecylsulfate-polyacrylamide gel electrophoresis. The 110 and 125 kDa bands are intermediates formed by polymerization of such subunit groups. In contrast, treatment with iodoacetamide, an inhibitor of polymerization, revealed that the 57–65 kDa intermediates originated from ZI-1,2 by limited hydrolysis. ZI-1,2 comprises at least three distinct proteins of quite similar structure with their N -termini undetectable by Edman degradation, while the 57–65 kDa intermediates also consist of at least three proteins with the same N -terminal amino acid sequence: DGKPSNPQQPQVPQYPSK-. This fact strongly suggests a participation of a protease in the conversion of ZI-1,2 into 57–65 kDa proteins. EDTA and 1,10-phenanthrolinium inhibited the conversion and both Ca²⁺ and Zn²⁺ recovered the inhibition. These results suggest that the assumed protease is a metalloprotease.     

两种农药对青鳉抗氧化酶活性和bcl6b基因表达的影响

为了解阿特拉津和毒死蜱对青鳉(Oryzias latipes)肌肉组织中抗氧化酶活性和B细胞淋巴瘤6B基因(B cell CLL/lymphoma 6B,bcl6b)表达的影响,实验通过不同染毒浓度的阿特拉津、毒死蜱,二者单独或共同染毒,在不同的染毒时间检测其对青鳉肌肉组织中超氧化物歧化酶(SOD)活性、谷胱甘肽过氧化物酶(GSH-PX)活性及bcl6b mRNA表达的影响。结果表明:青鳉肌肉组织中的抗氧化酶(SOD,GSH-PX)活性随染毒农药浓度的升高而显著下降,bcl6b mRNA相对表达则显著升高; 30 d恢复处理后,与染毒末期相比,恢复末期中浓度组和高浓度组的这两种酶活性显著升高,bcl6b mRNA表达则显著降低。这一结果证实该两种农药单独或共同染毒,会对青鳉产生毒性作用,使青鳉抗氧化酶(SOD,GSHPX)活性和bcl6b mRNA表达发生显著变化,bcl6b mRNA表达与抗氧化酶水平的变化趋势相反;环境条件改善后,上述2种酶的活性和bcl6b mRNA表达会得到有效恢复。     

Growth and maturation of melanosomes in the melanophores of a teleost,Oryzias latipes

Summary Formation of melanosomes in melanophores of a teleost, Oryzias latipes, was studied by means of electron microscopy. Two distinct types of premelanosomes are observed in the same cell: (i) multivesicular premelanosomes, which later develop into melanosomes with electron-lucent hollows in the center, appear at early embryonic stages; (ii) premelanosomes with highly organized, fibrous internal structure are formed at later stages of development and give rise to melanosomes with a filamentous center. Melanosomes are generally ellipsoid in shape, and the difference in the dimensions of fibrillar premelanosomes, melanosomes in the cells at younger developmental stages and those developed fully in melanophores of adults indicates that these organelles grow during development. The growth is achieved by fusion of small unmelanized vesicles or fibrillar premelanosomes to preformed melanosome and by fusion of two or more premelanosomes to form a larger organelle. The addition of the matrix of fibrillar premelanosomes around preformed melanosomes, which are derived from either multivesicular or fibrillar premelanosomes, forms a concentric outer deposit, and the fusion of small vesicles produces electron-lucent pits which are scattered irregularly in mature melanosomes.     

Behavioural and growth responses to the intensity of intraspecific social interaction among medaka, Oryzias latipes (Temminck and Schlegel) (Pisces, Cyprinodontidae)

When access to food is restricted, faster growing fish may be those whose behaviour is relatively unaffected by the presence of nearby conspecifics. Behavioural experiments were carried out to determine the relation between growth and motor activity levels in crowded and uncrowded conditions, and measures of aversion/attraction to groups of conspecifics. Two experimental groups of Or)-ius laripes (Temminck and Schlegel) were grown for several weeks in two environments manipulated so as to maximize differences in social interactions. In the high interaction environment (HI), food was provided inside a floating cork ring. In the low interaction environment (LI), food was spread over the container's surface. Fish were measured at the end of the growth period and tested for their activity levels in the presence of conspecifics and for their preference for, or tolerance of crowded conditions. The correlation between motor activity and growth was significantly more positive in the HI environment than in the LI environment. The relationship between preference for crowded conditions and growth was negative for both groups of fish, although less so for HI than for LI. We conclude that artificial selection for faster growth may produce more aggressive fish only under very high levels of forced social interaction (competition), if at all. Under conditions of reduced social interaction, the social-aversive or socially indifferent fish grow faster.     

Formation of the Dermal Chromatophore Unit (DCU) in the Tree Frog Hyla Arborea

In the tadpole of the tree frog Hyla arborea, the color of the dorsal skin was dark brown. Dermal melanophores, xanthophores, and iridophores were scattered randomly under the subepidermal collagen layer (SCL). After metamorphosis, the dorsal color of the animal changed to green and the animal acquired the ability of dramatic color change, demonstrating that the dermal chromatophore unit (DCU) was formed at metamorphosis. Fibroblasts invaded the SCL and divided it into two parts: the stratum spongiosum (SS) and the stratum compactum (SC). The activity of collagenase increased at metamorphosis. The fibroblasts appeared to dissolve the collagen matrix as they invaded the SCL. Then, three types of chromatophores migrated through the SCL and the DCU was formed in the SS. The mechanism how the three types of chromatophores were organized into a DCU is uncertain, but different migration rates of the three chromatophore types may be a factor that determines the position of the chromatophores in the DCU. Almost an equal number of each chromatophore type is necessary to form the DCUs. However, the number of dermal melanophores in the tadpoles was less than the number of xanthophores and iridophores. It was suggested that epidermal melanophores migrated to the dermis at metamorphosis and developed into dermal melanophores. This change may account for smaller number of dermal melanophores available to form the DCU_s.     

Mate copying versus preference for actively courting males by female Japanese medaka (Oryzias latipes)

We determined whether female Japanese medaka (Oryzias laiipes)copied the choice of other females or independently preferredactively courting males. We allowed focal females to observeand subsequently choose between a male that courted and potentiallyspawned with a receptive female and a male that courted andwas rejected by an unreceptive female. When the receptive femaledid not spawn, the focal female preferred the male with thehigher courtship rate; whereas when the receptive female spawned,the focal female preferred the spawning male. Our results suggestthat female medaka prefer actively courting males, unless theyhave the opportunity to copy the mate choice of another female.     

The ultrastructure of the ovarian follicle of medaka,Oryzias latipes

Summary The follicular cells in the oocytes of Oryzias latipes were studied by electron microscopy in order to clarify the fine structure, and the role of the cells during yolk formation and ovulation. The smallest follicles were observed during the early phase of peri-nucleolus stage of the oocyte. The cells have flattened nuclei, and perikarya with undeveloped organelles. But when the oocytes attain diameter of about 250 (yolk vesicle stage), both types of endoplasmic reticula are present. Moreover, the microvilli of the plasma membrane of oocyte as well as the follicles protrude into the pore canals of the zona radiata. In the oocytes of yolk stage the rough-surfaced endoplasmic-reticulum is typically developed and observed around the nuclei. Other organelles (lysosomes, mitochondria and Golgi) increase in number. The relation between the changes of cytoarchitecture in the follicles and yolk formation is discussed.At 17.00 p.m. on the day preceding ovulation the microvilli withdraw somewhat. Ribosomes are attached to the vesicular and cisternal endoplasmic reticula. When the oocytes attain complete maturation (24.00 p.m. at near ovulation), striking changes of the follicles are observed. The microvilli are almost withdrawn. In the degenerating follicles the lamellar structure is formed, and lipids are deposited at the center. At this time the contents of lysosomes have mostly disappeared.     

Deficiency of the Gene B Impairs Differentiation of Melanophores in the Medaka Fish,Oryzias latipes: Fine Structure Studies

In an orange-colored variant of the medaka fish, Oryzias latipes, which is homozygous for b allele, the melanophores represent a tissue-specific differentiation, manifesting an amelanotic appearance in the skin, an incomplete melanogenesis in the choroid and the peritoneum, and mosaic phenotype-like melano-iridophores in the peritoneum. In a wild-type strain of this species carrying the B gene, all melanophores are terminally differentiated irrespective of the tissues in which they are located. This indicates that the deficiency of B gene impairs the differentiation of melanophores in the medaka. Electron microscopy disclosed that the deficiency of B gene causes deterioration of melanogenesis to occur inside the melanosomes and that the manner of deterioration in the melanophores in the skin, the choroid and the peritoneum is different. The ubiquitous occurrence of reflecting platelet-laden melanophores in the peritoneum of this variant and the total absence of a mosaicism in pigment cells of the wild-type strain indicate that the deficiency of B gene predestines melanoblasts distributed in this tissue to an ambiguous state with regard to their differentiation. Little difference is observed between melanosomes maturation in pigment epithelial cells of the orange-colored variant and the wild-type strain, indicating an innocent role of the B gene in their differentiation.     

Effects of pentachlorophenol on the reproduction of Japanese medaka (Oryzias latipes)

Pentachlorophenol (PCP) is widely used to control termites and protect wood from fungal-rot and wood-boring insects, and is often detected in the aquatic environment. Few studies have evaluated PCP as an environmental endocrine disruptor. In the present work, Japanese medaka (Oryzias latipes) was exposed to PCP for 28 days (F0 generation) with subsequent measurements of vitellogenin (VTG), hepatic 7-ethoxyresorufin-O-deethylase (EROD), and reproductive endpoints. Plasma VTG significantly increased in male fish treated with PCP concentrations lower than 200 microg/l and decreased in male and female animals exposed to 200 microg/l. Hepatic EROD from female fish increased when PCP exposure concentrations exceeded 20 microg/l, but decreased in the 200 microg/l PCP treatment group. Fecundity and mean fertility of female medaka decreased significantly in the second and third week following exposure concentrations greater than 100 microg/l, and testis-ova of male medaka was observed at PCP concentrations greater than 50 microg/l. Histological lesions of liver and kidney occurred when exposure concentrations exceeded 50 microg/l. In F1 generations, the hatching rates and time to hatch of offspring were significantly affected in fish exposed to 200 microg/l. These results indicated that PCP exposure caused responses consistent with estrogen and aryl hydrocarbon receptor activation as well as reproductive impairment at environmentally relevant concentrations.     

Changes in Adrenergic Innervation to Chromatophores During Prolonged Background Adaptation in the Medaka,Oryzias latipes

The pattern of adrenergic innervation to scale chromatophores of the wild-type medaka, Oryzias latipes, was examined by autoradiography with ³H-norepinephrine and found for the first time to be changed reversibly during prolonged background adaptation. In scales of the medaka, which was adapted to a black background for 10-15 days, a great number of melanophores and dense networks of varicose fibers were observed: many fibers built up a radial plexus around each melanophore. However, the dense distribution of varicose fibers disappeared with a decrease in the number of melanophores during long-term adaptation to a white background. As to the changes in the innervation pattern to amelanotic melanophores of the medaka, orange-red variety, a similar result was obtained. Although the increase in the number of leucophores was observed in the medaka adapted to a white background, no exact plexuses of labeled fibers were confirmed around leucophores. From these results, it is concluded that the density of chromatic nerve fibers changes in parallel with the variation of the number of melanophores during prolonged background adaptation.     

Fibronectin-induced migration of melanophores in vitro in scales of medaka,Oryzias latipes

Summary The effects of fibronectin on melanophores were examined in two mutant strains of medaka, Oryzias latipes: mm (BmmR), which has condensed melanophores and normal dendritic melanophores; and cm (BcmR), which has condensed melanophores. When medaka scales were cultured in the presence of fibronectin, melanophores of the wild type and dendritic melanophores of the mm mutant changed their shape and migrated, whereas melanophore migration was rarely seen in the absence of fibronectin. Melanophores of the cm mutant and condensed melanophores of the mm mutant did not migrate even in the presence of fibronectin. When melanophores of the wild type and mm mutant were condensed by adrenalin, they did not migrate. On the other hand, when melanophores of the cm mutant were dispersed by theophylline, they were able to migrate. These results indicate that fibronectin induces the migration of melanophores and that dispersion of melanin granules may be requisite for such migration.     

Gene-centromere mapping of medaka sex chromosomes using triploid hybrids between Oryzias latipes and O. luzonensis

Four sex-linked genetic markers (SL1, SL2, B2.38 and stsOPQ05-1) on the sex chromosomes of the medaka, O. latipes, were mapped in relation to the centromere by means of triploid hybrids between O. latipes and O. luzonensis. Female F₁ hybrid O. latipes of two inbred strains, Hd-rR and HNI, were crossed with male O. luzonensis. Triploidization was induced by heat-shock treatment. Hatching rate of heat-shock treated eggs was 59%, and that of untreated hybrid eggs was 2%, indicating that most of the hatched fry were triploid. Using these triploid hatched fry, the map distances between the four loci and the centromere were examined. The order was SL2 – centromere – SL1 – B2.38 – stsOPQ05-1 and the map distances were: SL2 – centromere, 1%; centromere – SL1, 18%; SL1 – B2.38, 19%; B2.38 – stsOPQ05-1, 9%. Previous studies using FISH showed that SL2 is located on the short arm of large submetacentric chromosomes, and SL1 was closely linked to SDF (sex-determining factor). The results of gene-centromere mapping of this study show that SL1, B2.38 and stsOPQ05-1 are located on the long arm, and that, SDF is thus also on the long arm of the sex chromosomes.