Synchronous production of the low- and high-molecular-weight precursors of the egg envelope subunits, in response to estrogen administration in the teleost fish Oryzias latipes.
Murata-K; Iuchi-I; Yamagami-K
Life Science Institute, Sophia University, Tokyo, Japan.
Gen-Comp-Endocrinol. 1994 Aug; 95(2): 232-9
Estrogen stimulation of production and accumulation of L-SF andH-SF, the low- and high-molecular-weight precursors of the eggenvelope subunits in Oryzias latipes, was analyzed byimmunohistochemical and immunoblotting methods. Both SFsubstances were detected in the liver of the male fish as quicklyas 16-24 hr after estrogen treatment, and their production andaccumulation in the liver and blood plasma increased as estrogentreatment continued. Immunohistochemical analysis revealed thatthe number of cells producing both SF substances was increasedin the liver as estrogen treatment continued. These resultsstrongly suggest the occurrence of synchronous production of L-SFand H-SF in response to estrogen. In liver extracts, but not inblood plasma, an additional L-SF-like immunoreactive protein wasdetected on immunoblots. Preliminary data indicate parallelappearance of vitellogenin and SF substances in the liver ofestrogenized fish.
Early life-stage effects in medaka (Oryzias latipes) following in ovo exposure to polyamine biosynthetic inhibitors.
Ortego-LS; Nimrod-AC; Brehm-WT; Parsons-GR; Benson-WH
Department of Pharmacology, School of Pharmacy, University of Mississippi, University 38677.
Ecotoxicol-Environ-Safety. 1994 Aug; 28(3): 329-39
Medaka, Oryzias latipes, were exposed in ovo to the polyamine(PA) biosynthesis inhibitors alpha-difluoromethylornithine (DFMO)and methylglyoxal bis(guanylhydrazone) (MGBG). In an additionalgroup, spermine, the end product of the PA pathway, was addedwith DFMO and MGBG for a "rescue" treatment. At 4 days posthatch,length, DNA and RNA content, and swimming endurance weremeasured. The only parameter affected by treatment was swimmingendurance which revealed decreased latent time to fatigue withincreased dose, although not statistically significant. Therescue group, however, did demonstrate a statisticallysignificant decrease in fatigue latency as compared to controls.
Cohen-C; Stiller-A; Miller-MR
Department of Biochemistry, R.C. Byrd Health Sciences Center, West Virginia University, Morgantown 26506-9142.
Arch-Environ-Contam-Toxicol. 1994 Oct; 27(3): 400-5
The objective of this study was to characterize cytochrome P4501Ainduction in medaka liver as a biomarker for detectingpolyaromatic hydrocarbon (PAH)-type compounds in samples ofprocessed coal or petroleum. Ethoxyresorufin-O-deethylase (EROD)activity in individual medaka livers was used to asses inductionof P4501A following the addition of various samples to aquariawater. Samples included a known P4501A inducer,beta-naphthoflavone, and various processed coal samples, as wellas a petroleum-pitch. The sensitivity of detecting significantEROD induction by adding samples to aquaria water wasapproximately 0.1 mg/L for most samples; however, a coal-tarpitch significantly increased EROD activity at 0.01 mg/L.Different samples induced EROD activity to different extents. Allsamples elicited a concentration-dependent increase in ERODactivity, with maximum EROD induction 2 days after a singleadministration of xenobiotics to aquaria water. Western blotstudies established that induction of EROD activity by allxenobiotics tested was associated with corresponding increasedamounts of immunoreactive P4501A. EROD induction was notinfluenced by gender, by single or multiple xenobiotic exposures,nor by feeding or fasting animals during the course of xenobioticexposure. The ability of xenobiotics to induce EROD activity inmedaka liver did not always correlate with their genotoxicpotential determined by bacterial mutagenesis assays. Inductionof P4501A in medaka liver appears to provide a convenient,economical, reliable and sensitive indicator for the presence ofPAH-type compounds in coal- or petroleum-derived samples.
The chinook salmon gonadotropin II beta subunit gene contains a strong minimal promoter with a proximal negative element.
Xiong-F; Liu-D; Elsholtz-HP; Hew-CL
Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
Mol-Endocrinol. 1994 Jun; 8(6): 771-81
The salmon pituitary expresses two distinct gonadotropins,gonadotropin I (GTHI) and gonadotropin II (GTHII). These twohormones are synthesized in distinct pituitary cells and secretedat different stages during the reproductive cycle. To study thetranscriptional regulation of the hormone- specific beta-subunitof GTHII (sGTHII beta) gene, approximately 3.5 kilobases of the5'-flanking region was characterized and sequenced. The pituitaryspecificity of sGTHII beta was examined by analyzing sGTHII betapromoter activity in homologous primary pituitary cells derivedfrom spawning rainbow trout and in a collection of heterologouscell lines. Various lengths of the 5'-flanking region of thesGTHII beta gene were ligated into a vector encoding thebacterial chloramphenicol acetyltransferase (CAT) gene, and theresulting sGTHII beta/CAT chimeric constructs were analyzed usingtransient expression systems. Several constructs (-3500CAT,-1260CAT, -563CAT, and -39CAT) displayed readily detectable CATactivity in the pituitary cells derived from spawning male andfemale rainbow trout. In contrast, three of the constructs(-3500CAT, - 1260CAT, and -563CAT) were expressed only atbackground levels in a variety of heterologous cell lines,suggesting that the 5'-flanking sequence of sGTHII beta containsinformation dictating its pituitary specificity. A silencersequence (-95 to -35, pSil) was identified, which might functionto repress sGTHII beta gene expression in the nongonadotropes orin the gonadotropes at developmental stages that precede finalmaturation and spawning.
Disruption of mesoderm and axis formation in fish by ectopic expression of activin variants: the role of maternal activin.
Wittbrodt-J; Rosa-FM
University of Basel, Department of Cell Biology, Switzerland.
Genes-Dev. 1994 Jun 15; 8(12): 1448-62
Formation of mesoderm in Xenopus embryos is the result of aninduction event in which peptides such as FGF or activins havebeen implicated. It was recently demonstrated, by the ectopicexpression of a truncated activin receptor, that activin receptorsignaling pathways are involved in the processes of mesoderm andaxis formation in vivo. However, this approach does not directlyaddress the role of activin itself nor the involvement ofactivins in the formation of mesoderm in embryos from othervertebrates. In addition, activins are expressed maternally asa protein component of the egg as well as transcribedzygotically, and it is not clear which of the two forms isinvolved in mesoderm formation. To address those three issues,we analyzed the role of activins in the development of fishembryos by generating two activin dominant-negative variants. Oneof the variants behaves as an inhibitor of activin protein. Thesecond variant was found to deplete the activin pool whencotranslated with wild-type activin. Injection of RNA encodingthese variants into the two-cell embryo of the small teleost fishOryzias latipes (Japanese medaka) demonstrates that only thematernally provided activin protein is required for mesoderm andaxis formation in fish in vivo.
Cranial nerves and brain fiber systems of the medaka fry as observed by a whole-mount staining method.
Ishikawa-Y; Hyodo-Taguchi-Y
Division of Biology, National Institute of Radiological Sciences, Chiba, Japan.
Neurosci-Res. 1994 Jun; 19(4): 379-86
The cranial nerves and the brain fiber systems of the medaka(Oryzias latipes) fry are revealed by a whole-mount stainingmethod. Newly hatched fry of an albino strain of the medaka werefixed, partially digested with trypsin, treated in 1% TritonX-100, and finally immunohistochemically stained usinganti-neurofilament protein (70K+160K+210K) antibodies. Since bothhead skin and eyes were colorless in the albino fish, thethree-dimensional distribution of of nerve fibers in the braincould be readily observed in whole specimens without interferenceof pigment cells. All cranial nerves and main fiber systems inthe adult fish were differentiated in the fry brain. Using thismethod, the distribution of nerves to the ocular muscles and theperiorbital pit organs was shown.
Ligand-dependent tumor induction in medakafish embryos by a Xmrk
receptor tyrosine kinase transgene.
Winkler-C; Wittbrodt-J; Lammers-R; Ullrich-A; Schartl-M
Department of Physiological Chemistry I, University of Wurzburg, Germany.
Oncogene. 1994 Jun; 9(6): 1517-25
Xmrk encodes a subclass I receptor tyrosine kinase (RTK) whichhas been cloned from the melanoma-inducing locus Tu of thepoeciliid fish Xiphophorus. To demonstrate a high oncogenicpotential in vivo we transferred the gene into early embryos ofthe closely related medakafish. Ectopic expression of the Xmrkoncogene under the control of a strong, constitutive promoter(CMVTk) led to the induction of embryonic tumors with highincidence, after short latency periods, and with a specificpattern of affected tissues. We demonstrate ligand-dependenttransformation in vivo using a chimeric receptor consisting ofthe extracellular and transmembrane domains of the human EGFreceptor (HER) and the cytoplasmatic domain of Xmrk. Expressionof the chimeric receptor alone does not lead to kinase activationor induction of tumors. Coexpression of the chimera with itscorresponding ligand, human transforming growth factor alpha(hTGF alpha), however, results in the activation of the chimericRTK. In injected fish embryos the induction of the neoplasticgrowth is observed with similar incidence and tissue distributionas in embryos carrying the native Xmrk oncogene suggesting thatthe ligand as well as factors downstream of the RTK are requiredfor tumor formation. In this study we show single-step inductionof tumors by ectopic expression of RTKs in vivo substantiatingthe significance of autocrine stimulation in RTK induced tumorsin vertebrates.
Structural studies of a novel type of pentaantennary largeglycan unit in the fertilization-associated carbohydrate-richglycopeptide isolated from the fertilized eggs of Oryziaslatipes.
Taguchi-T; Seko-A; Kitajima-K; Muto-Y; Inoue-S; Khoo-KH;Morris-HR; Dell-A; Inoue-Y
Department of Biophysics and Biochemistry, Faculty of Science,University of Tokyo, Japan.
J-Biol-Chem. 1994 Mar 25; 269(12): 8762-71
In a previous report (Kitajima, K., Inoue, S., and Inoue, Y.(1989) Dev. Biol. 132, 544-553), we found the presence of aheavily glycosylated polyprotein, "H-hyosophorin," isolated fromthe unfertilized eggs of Oryzias latipes. We now report ourdetailed analysis of the structure of the N-glycan chain inL- hyosophorin, the smallest repeating unit of H-hyosophorin,which was isolated from the fertilized eggs of O. latipes andformed from H-hyosophorin upon fertilization. The N-glycanstructures were defined by a combination of compositionalanalysis, methylation analysis, selective chemical degradation(i.e. mild methanolysis, periodate-Smith degradation, andhydrazinolysis-nitrous acid deamination), enzymatic(endo-beta-galactosidase, peptide:N-glycanase, and Newcastledisease virus sialidase) digestion, and instrumental analyses(one- and two-dimensional proton nuclear magnetic resonancespectroscopy and fast atom bombardment mass spectrometry) whichrevealed novel and unique features: (a) the presence of highlybranched poly-N-acetylactosamino pentaantennary structures; (b)the presence of a beta-galactosylated Lewis X antigenic epitope,Gal beta 1-->4 Gal beta 1-->4 (Fuc alpha 1-->3) GlcNAc beta 1-->;(c) the presence of a beta-galactosylated sialyl Lewis Xstructure, Gal beta 1-->4 (Neu5Ac alpha 2-->3) Gal beta 1-->4(Fucalpha 1-->3) GlcNAc beta 1-->; (d) the presence of Gal beta 1-->4Gal beta 1--> and Gal beta 1--> 4Gal beta 1-->4Gal beta 1--> asthe major and minor groupings, respectively; and (e) the presenceof the branched Gal residues, -->4GlcNAc beta 1-->3(Gal beta1-->4) Gal beta 1-->. This study represents the first detailedinvestigation regarding the nature of highly branched complexasparagine-linked pentaantennary glycans in glycoproteins. Theunique expression of such bulky multiantennary glycan units onproteins could be essential during early embryogenesis.
HCE, a constituent of the hatching enzymes of Oryzias latipes embryos, releases unique proline-rich polypeptides from its natural substrate, the hardened chorion.
Lee-KS; Yasumasu-S; Nomura-K; Iuchi-I
Life Science Institute, Sophia University, Tokyo, Japan.
FEBS-Lett. 1994 Feb 21; 339(3): 281-4
HCE, a constituent protease of the hatching enzymes of Oryzias latipes embryos [1,2], releases unique proline-rich polypeptides from its natural substrate, the hardened chorion. The polypeptides consist of repeats of Pro-X-Y, mainly Pro-Glx-X. In addition, the polypeptides contain abundant gamma-glutamyl epsilon-lysine isopeptides which are regarded to be responsible for chorion hardening. These findings suggest that HCE recognizes specific site(s) of the chorion, releases the proline-rich polypeptides from it, and makes the substrate accessible to LCE, another protease of the hatching enzymes.
A new member to the astacin family of metalloendopeptidases: anovel 1,25- dihydroxyvitamin D-3-stimulated mRNA fromchorioallantoic membrane of quail.
Elaroussi-MA; DeLuca-HF
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison 53706. Biochim-Biophys-Acta. 1994 Jan 18; 1217(1): 1-8
1,25-Dihydroxyvitamin D-3 is essential for the utilization ofeggshell calcium by avian embryo through the chorioallantoicmembrane (CAM). A cDNA library was constructed from poly(A)+ RNAextracted from vitamin D- deficient CAMs given1,25-dihydroxyvitamin D-3. Screening this library by differentialhybridization yielded a full-length (approximately 1.8 kb) cDNA,whose corresponding mRNA is increased 3-fold 2.5 h after a singleinjection of 1,25-(OH)2D3. The complete nucleotide sequence forthe full-length cDNA has been determined. An open-reading frame,corresponding to a 310 amino acid, 41 kDa protein was found.Searching protein sequence data bases revealed a strongsimilarity to the following proteases: astacin, a crayfishdigestive protease, Oryzias latipes hatching enzyme constituentprotease (Orz), Xenopus laevis developmentally regulated UVS.2protein secreted by the hatching gland of embryos, theNH2-terminal domain of human bone morphogenetic protein (BMP-1)and Drosophila dorsal- ventral patterning tolloid. The cDNA hasapproximately 36% overall identity with astacin and BMP-1, andis more than 60% identical to either Orz or UVS.2. Moreover,multiple alignment analysis indicates that 37 residues, including3 cysteine residues, are strictly conserved in the complete200- amino acid astacin sequence. All 6 proteins contain azinc-binding motif (HEXXH), found at the active site of mostmetalloendopeptidases. This motif is found within an extendedsequence of HEXXHXXGFXHE that is unique to this subgroup ofmetalloendopeptidases. In addition, the 6 proteins have 50%identity (including the present cDNA) and 79% are conserved in4 of these proteins in a 24-amino acid sequence that includes theputative active site. The level of mRNA for the new proteinreaches a maximum at day 12 of embryonic life and declinesthereafter. It is suggested that this clone corresponds to anmRNA encoding for a protease that may play a role in thedegradation of eggshell matrix.
Isolation of H-SF substances, the high-molecular-weight precursors of egg envelope proteins, from the ascites accumulated in the oestrogen-treated fish, Oryzias latipes.
Murata-K; Iuchi-I; Yamagami-K
Life Science Institute, Sophia University, Tokyo, Japan.
Zygote. 1993 Nov; 1(4): 315-24
High- and low-molecular weight spawning-female-specific (H-SF and L-SF) substances are liver-derived putative precursors of the inner layer of egg envelope of the medaka Oryzias latipes, and are also found in the ascites which are accumulated in the male fish on prolonged treatment with oestrogen. In the present study, the H-SF substances purified from the ascites of the oestrogen-treated male medaka were found to consist of three proteins. Their molecular weights ranged from 74,000 to 76,000, which corresponds to those of ZI-1 and ZI-2, the major components of medaka egg envelope. The amino acid composition of the H-SF substances was characterised by high contents of proline (17.58 mol%) and glutamic acid/glutamine (13.34 mol%). The characteristics were almost identical to those of ZI-1 and ZI-2 but differed from those of L-SF substance, a precursor of ZI-3, another major component of the egg envelope. These results confirm our view that H-SF substances are the precursors of ZI-1 and ZI-2. The average amino acid compositions of H-SF substances and L-SF substance coincided with the amino acid composition of whole egg envelope. This result strongly suggests that an equal amount of H-SF substances and L-SF substance would construct the inner layer of egg envelope after a little modification.
Cytoskeletal architecture of dermal chromatophores of the freshwater teleost Oryzias latipes.
Obika-M; Fukuzawa-T
Department of Biology, Keio University, Yokohama, Japan.
Pigment-Cell-Res. 1993 Dec; 6(6): 417-22
Cytoskeletal construction of dermal chromatophores of Oryzias latipes was studied by immunofluorescence microscopy. A microtubule system was most prominent in melanophores where a large number of microtubules emanated from the center of the cell. Xanthophores had an arrangement basically similar to that of melanophores, though the radial pattern became more irregular in the peripheral region where intersecting wavy microtubules were quite frequent. Oval-shaped leucophores exhibited the least-developed microtubule system, where the limited number of microtubules formed a loose basket-like architecture. Intermediate filaments were ubiquitously present in all types of chromatophores and were found to be vimentin-immunoreactive. Examination of doubly-labeled cells indicated that vimentin filaments had similar distribution patterns with microtubules. Orderly arranged bundles of actin filaments were found only in xanthophores, while in melanophores and xanthophores, actin expression was diffuse without displaying a conspicuous filamentous organization. Colchicine treatment induced depolymerization of microtubules and retraction of dendrites in varying degrees in cells in culture and in situ. Melanophores in culture are very sensitive to the treatment while xanthophores appeared to be more resistant in respect to the maintenance of cell morphology.
Bunton-TE
Department of Pathology, Johns Hopkins University, School of Medicine, Baltimore, MD.
Vet-Pathol. 1993 Sep; 30(5): 418-25
An increasing interest in fish species as sentinels ofenvironmental pollution and in carcinogenesis research has ledto the identification of diagnostically challenging neoplasms ofuncertain cellular origin and the need for additional diagnosticmethods. To determine the potential of using commerciallyavailable antibodies to intermediate filament proteins onparaffin-embedded fish tissues for immunocytochemistry in tumordiagnosis, the application of three antikeratin antibodies tonormal adult tissues from two fish species was assessed. Multipletissues from 12- 14-in. striped bass (Morone saxatilis) and6-month-old medaka (Oryzias latipes) of both sexes were fixed inBouin's or formalin fixatives. Formalin- fixed neoplasms fromseveral mammalian species, including cat, dog, hedgehog (Atelerixalbiventris, Erinaceus europaeus), rhesus macaque (Macacamulatta), and sloth bear (Melursus ursinus), were also used aspositive controls. Using a strepavidin horseradish peroxidasemethod on paraffin-embedded tissues, the broad spectrumantibodies AE1/AE3 (Boehringer Mannheim, Indianapolis, IN) andMAK-6 (Triton Biosciences, Alameda, CA), which recognize most ofthe 19 human cytokeratins, and CAM 5.2 (Becton Dickinson,Mountain View, CA), which recognizes cytokeratins present inhuman liver, were used as primary antibodies. Epithelia fromskin, gills, cornea, bile ducts, renal tubules, gastrointestinaltract, and thymus were strongly positive with AE1/AE3 and MAK-6in striped bass, but nonepithelial tissues such as bone andmuscle were negative. Skin, gills, cornea, and portions of thegastrointestinal tract were strongly positive in medaka with thesame antibodies, whereas bile duct, renal, and intestinalepithelia were less so. Tissue digestion improved the intensityof staining, and fixation with Bouin's fixative improved resultssomewhat compared with formalin fixation.(ABSTRACT TRUNCATED AT250 WORDS)
Sublethal effects of three pesticides on Japanese medaka.
Heath-AG; Cech-JJ Jr; Zinkl-JG; Steele-MD
Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg 24061.
Arch-Environ-Contam-Toxicol. 1993 Nov; 25(4): 485-91
One- to 2-day-old medaka (Oryzias latipes) larvae were exposedfor 4 days to the rice field pesticides methyl parathion,molinate, carbofuran and a mixture of all three. Pesticideconcentrations were one-half the 96 h LC50 ("high concentration")and levels approximating those measured in receiving waters fromrice field runoff ("low concentration"). Maximum swimming speed,spontaneous muscular activity, acetylcholinesterase activity, dryweight, RNA:DNA ratio, and five morphometric variables weredetermined at the end of the exposures. Larvae were retained foran additional 10 days in non-contaminated water, and the samemeasurements taken to investigate residual effects. Results arecompared to a parallel study on striped bass larvae to evaluatethe suitability of this species as a surrogate for the bass intoxicological studies involving sublethal exposures. There wasno relationship between mortality rate and pesticide exposureeither during the exposures or during the ten day subsequentperiod. Only the high concentration of carbofuran caused animpairment of swimming performance. Spontaneous activity wasstimulated in the high concentration of molinate and the combinedpesticides groups. Acetylcholinesterase was severely inhibitedin parathion and molinate, and this persisted in some cases after10 days in non-contaminated water. The pesticides had littleeffect on growth rate except for molinate which acted as astimulant. Combining the three pesticides caused a less thanadditive effect. Except for decreases in acetylcholinesterase,the sublethal effects of the pesticides tested at the very lowconcentrations used were subtle. Apparently, larvae of thisspecies are less sensitive to these pesticides than are stripedbass larvae.
Development of a motor nerve in the caudal fin of the medaka (Oryzias latipes).
Ishikawa-Y; Iwamatsu-T
Division of Biology, National Institute of Radiological Sciences, Chiba, Japan.
Neurosci-Res. 1993 Jul; 17(2): 101-16
The entire process of the development of the motor nerve to themiddle interradial muscle (MIR muscle) in the tail region of themedaka (Oryzias latipes) was examined by light and electronmicroscopy. The motor nerve to the MIR muscle was observed todevelop in a series of four steps. (1) Trunk- formation step: fromstage 30 to stage 31, when the MIR muscle had not yetdifferentiated to the myotube stage, the motor axons startingfrom the caudal spinal cord, including spinal segment 27, formeda nerve trunk, the ventral caudalmost nerve (VCN). The VCNbypassed the place where the MIR muscle would differentiate andextended further caudad. (2) Wandering step: at stage 32, nerveprocesses of the motor axons (short branches) wandered from thecourse of the VCN. (3) Connection step: from stage 33/34 to stage35, the mesenchyme cells differentiated into the MIR myotubes inthe vicinity of the short branches, and the nerve and the musclebecame connected. (4) Modification step: after stage 35, themorphological pattern of the motor nerve was modified by thegrowth and the shift of the short branches, and by thedisappearance of the caudalmost part of the VCN. These resultssuggest that the complex pattern of the motor nerve in the adultfish is gradually constructed by multiple steps, each of whichmay have its own mechanism.
Host specificity of Calyptospora funduli (Apicomplexa: Calyptosporidae) in atheriniform fishes.
Fournie-JW; Overstreet-RM
U.S. Environmental Protection Agency, Center for Marine and Estuarine Disease Research, Gulf Breeze, Florida 32561.
J-Parasitol. 1993 Oct; 79(5): 720-7
Calyptospora funduli has a broad host specificity, infecting at least 7 natural and 10 additional experimental definitive hosts, all atheriniform fishes within 5 families, but most in the genus Fundulus. Barriers, apparently innate ones, prevent any development of C. funduli in perciform fishes but allow incomplete or abnormal development of the parasite in a few unnatural atheriniform hosts. In the freshwater species Fundulus olivaceus and Fundulus notti, these abnormalities consisted of asynchronous development, degeneration of the parasite in early stages of development, and the formation of numerous macrophage aggregates. Rivulus marmoratus has the ability to eliminate infections with a granulomatous inflammatory response. Additional barriers that limit natural infections of C. funduli in other hosts include feeding behavior, environmental conditions, and geographic isolation.
Structural studies of a novel type of tetraantennary sialoglycan unit in a carbohydrate-rich glycopeptide isolated from the fertilized eggs of Indian Medaka fish, Oryzias melastigma.
Taguchi-T; Seko-A; Kitajima-K; Inoue-S; Iwamatsu-T; Khoo-KH; Morris-HR; Dell-A; Inoue-Y
Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Japan.
J-Biol-Chem. 1993 Feb 5; 268(4): 2353-62
A novel carbohydrate-rich sialoglycopolyprotein of apparentmolecular mass approximately 7000 Da was isolated from thefertilized eggs of the Medaka fish species, Oryzias melastigma.The glycoprotein was identified as a member of the L-hyosophorinfamily because it exhibited the following several distinctivefeatures of L-hyosophorin molecules: (a) it contains a highproportion of carbohydrate (90% by weight), and (b) the aminoacid sequence of the apopeptide was identical with that of theOryzias latipes L- hyosophorin which has previously beendemonstrated to be derived from a high molecular weight form ofhyosophorin, i.e. H-hyosophorin, present in the cortical vesiclesof unfertilized eggs. The apoprotein of H-hyosophorin is composedof tandem repeats of the L-hyosophorin apopeptide, i.e. it is apolyprotein. The structure of the carbohydrate portion ofpurified L-hyosophorin of O. melastigma was studied bycomposition and methylation analysis, selective chemical(periodate-Smith degradation; hydrazinolysis- nitrous aciddeamination), and enzymatic (endo-beta-galactosidase;peptide:N-glycanase) degradation, together with instrumentalmethods (fast atom bombardment-mass spectrometry and 1H NMR). O.melastigma L-hyosophorin was found to contain two types of large,branched tetraantennary glycan units capped with sialic acids.The two glycans differ with respect to the branching pattern ofthe trimannosyl core (x = 4 or 6 in Eq. A). [formula: see text]The possible physiological significance of the hyosophorin familyis discussed in the light of their unique structural features.
Vertebral malformations in medaka (teleost fish) after exposure to tritiated water in the embryonic stage.
Hyodo-Taguchi-Y; Etoh-H
Division of Biology, National Institute of Radiological Sciences, Chiba, Japan.
Radiat-Res. 1993 Sep; 135(3): 400-4
This study was designed to investigate radiation-inducedvertebral malformations in medaka embryos irradiated with betaparticles from tritium. Embryos of two inbred strains (HO4 andHO5) of medaka, Oryzias latipes, were exposed either to differentconcentrations of tritiated water (9.25-37 MBq/ml) or to 137Csgamma rays (dose rates of 0.44-1.89 Gy/day) continuously frommorula to hatching. The newly hatched fry were removed from theradiation field and kept under usual conditions for 1 month.Young fish were fixed in 10% buffered formalin, cleared in 1%KOH, stained with alizarin red S, and kept in glycerine. Therewas almost no difference in the response to radiation betweenmedaka strains. No marked reduction of hatching rate was observedafter chronic irradiation with beta particles and gamma rays, buta considerable reduction in survival of fry was detected inirradiated groups within 1 month after hatching. From observationof whole-mounted skeleton specimens, the following vertebralmalformations were found in irradiated groups: fusion of two ormore vertebrae, incomplete formation of vertebrae, and lack ofvertebral process. The incidence of vertebral malformationsincreased significantly in both groups irradiated with tritiumbeta particles and 137Cs gamma rays. A similarity in theincidences was also observed between beta-particle- andgamma-irradiated groups. The RBE of beta particles relative togamma rays was estimated to be 1 based on the dose-responserelationships observed.
Alterations in the morphology of nuages in spermatogonia of the fish, Oryzias latipes, treated with puromycin or actinomycin D.
Hamaguchi-S
Niigata University, College of General Education, Department of Biology, Japan.
Reprod-Nutr-Dev. 1993; 33(2): 137-41
A germ-cell specific organelle, nuage, in Oryzias latipes is polymorphic. Nuages with a strand-like structure and amorphous fibrous bodies can be discriminated from each other; furthermore 2 types of nuages of intermediate morphology are also present. After the administration of puromycin or actinomycin D, nuage morphology in spermatogonia was examined by electron microscopy. Puromycin as well as actinomycin D caused a significant increase in the incidence of nuages with a strand-like structure. These observations indicate that the loss of supply of some materials can induce morphological changes in nuages, suggesting that the polymorphism in nuages of O latipes possibly results from the turnover of nuage materials.
Analytical procedures and quality assurance criteria for the determination of major and minor deoxynucleosides in fish tissue DNA by liquid chromatography-ultraviolet spectroscopy and liquid chromatography-thermospray mass spectrometry.
Serrano-J; Kuehl-DW; Naumann-S
U.S. Environmental Protection Agency, Duluth, MN 55804.
J-Chromatogr. 1993 Jun 2; 615(2): 203-13
Analytical procedures and quality assurance criteria have beenestablished for enzymatic hydrolysis of fish tissue DNA to freenucleosides and their subsequent characterization by liquidchromatography-photodiode-array ultraviolet spectroscopy andliquid chromatography-thermospray mass spectrometry. Optimizationof enzymatic efficiency to assure minimal loss of modifiednucleosides is described. Variability in analyte capacity factorsand multiwavelength response have been compared for analytestandards and hydrolysates, and results have been used to derivequalitative and quantitative quality assurance criteria. Acomparison of DNA mole percent calculated using single-wavelengthquantification and multiwavelength averaging quantificationindicates that less variability in data may be expected using themultiwavelength technique. Finally, the composition of DNA fromliver of three species of fish widely used in mutagen/carcinogenlaboratory and field studies, rainbow trout (Onchorynchusmykiss), medaka (Oryzias latipes), and brown bullhead (Ictalurusnebulosus), has been determined. Identification of deoxyuridinein the DNA hydrolysates of each fish indicates that this analyteshould be measured to accurately report DNA deoxynucleoside molepercent, especially when reporting data for the methylation ofdeoxycytidine.
Deficiency of the gene B impairs differentiation of melanophores in the medaka fish, Oryzias latipes: fine structure studies.
Hirose-E; Matsumoto-J
Department of Biology, Keio University, Yokohama, Japan.
Pigment-Cell-Res. 1993 Feb; 6(1): 45-51
In an orange-colored variant of the medaka fish, Oryzias latipes,which is homozygous for b allele, the melanophores represent atissue-specific differentiation, manifesting an amelanoticappearance in the skin, an incomplete melanogenesis in thechoroid and the peritoneum, and mosaic phenotype-likemelano-iridophores in the peritoneum. In a wild-type strain ofthis species carrying the B gene, all melanophores are terminallydifferentiated irrespective of the tissues in which they arelocated. This indicates that the deficiency of B gene impairs thedifferentiation of melanophores in the medaka. Electronmicroscopy disclosed that the deficiency of B gene causesdeterioration of melanogenesis to occur inside the melanosomesand that the manner of deterioration in the melanophores in theskin, the choroid and the peritoneum is different. The ubiquitousoccurrence of reflecting platelet-laden melanophores in theperitoneum of this variant and the total absence of a mosaicismin pigment cells of the wild-type strain indicate that thedeficiency of B gene predestines melanoblasts distributed in thistissue to an ambiguous state with regard to theirdifferentiation. Little difference is observed betweenmelanosomes maturation in pigment epithelial cells of theorange-colored variant and the wild-type strain, indicating aninnocent role of the B gene in their differentiation.
Ornithine decarboxylase (ODC) activity in the liver of individual medaka (Oryzias latipes) of both sexes.
Calabrese-EJ; Leonard-DA; Baldwin-LA; Kostecki-PT
School of Public Health, University of Massachusetts, Amherst 01003.
Ecotoxicol-Environ-Safety. 1993 Feb; 25(1): 19-24
Ornithine decarboxylase (ODC) activity has been determined in the liver of individual medaka (N = 48). The results indicate a mean ODC activity of 1.69 +/- 1.77 nmol 14CO2/hr/mg protein. The fish displayed a large interindividual variation of normal ODC activity with a range of 140fold. However, no sex differences were observed. The findings also indicate that the mean ODC activity in this fish model is approximately 100fold greater than observations in the published literature as well as in our laboratory for commonly employed rodent models.
The effect of peroxisome proliferators on S-phase synthesis in primary cultures of fish hepatocytes.
Baldwin-LA; Kostecki-PT; Calabrese-EJ
Environmental Public Health Center, University of Massachusetts, Amherst 01003.
Ecotoxicol-Environ-Safety. 1993 Apr; 25(2): 193-201
The ability of seven structurally diverse peroxisome proliferators to induce S-phase synthesis was studied in primary cultures of rainbow trout and medaka hepatocytes. Cells were maintained in serum-free conditions designed to facilitate attachment, viability, and function. Lead nitrate, a well-known and potent rodent liver mitogen, was used in vitro in trout and medaka and in vivo in trout to evaluate the capacity of the species and our system to respond to mitogenic stimuli. S-phase synthesis induced by peroxosome proliferators did not result in any statistically significant increase over control cultures. These in vitro results are supported by in vivo data previously obtained and the present work with lead nitrate.
Melano-macrophage centre-like structure in the heart of the medaka, Oryzias latipes.
Nakamura-H; Shimozawa-A; Kikuchi-S
Department of Anatomy, Dokkyo University School of Medicine, Tochigi, Japan.
Anat-Anz. 1993 Feb; 175(1): 59-63
Endocardial lining cells in the heart of certain teleosts are phagocytic against foreign materials. In the medaka Oryzias latipes, in addition to the endocardial lining cells, cells in the subendocardial spaces are also phagocytic. Most subendocardial cells contain yellowish-brown pigments and are often aggregated to form nodules resembling splenic and kidney melano-macrophage centres. Subendocardial cells are positive to PAS and Schmorl's reaction. Macrophages containing large heterogeneous inclusions together with lymphocytes, were observed with the TEM. The definitive characteristics of reticular cells, including the cell sheaths, were not observed. Because of these two characteristics they were regarded as more primitive aggregates of macrophages than the ordinary melano-macrophage centres. The possible analogy between these structures and MMCs is discussed.
Toxicokinetics and metabolism of aniline and 4-chloroaniline in medaka (Oryzias latipes).
Bradbury-SP; Dady-JM; Fitzsimmons-PN; Voit-MM; Hammermeister-DE; Erickson-RJ
United States Environmental Protection Agency, Environmental Research Laboratory-Duluth, Minnesota 55804.
Toxicol-Appl-Pharmacol. 1993 Feb; 118(2): 205-14
The in vivo toxicokinetics and in vitro hepatic microsomalmetabolism of [14C]aniline and [14C]4-chloroaniline in medaka(Oryzias latipes) were investigated to provide a basis upon whichto interpret the toxicological responses of small aquarium fishto aniline derivatives. During static aqueous exposures of up to320 min, parent equivalents failed to reach steady state andresults from depuration studies clearly demonstrated biphasicelimination. Due to low elimination rates, 40 to 20% of absorbedaniline and 4-chloroaniline doses, respectively, remained withinthe fish through 330 min postexposure. Based on an analysis ofexcreted metabolites, N-acetylation was the dominant route of invivo metabolism for 4-chloroaniline, with no indication of ringhydroxylation, while the evidence suggested that polar conjugateswere the dominant in vivo aniline metabolites. The toxicokineticsand in vivo metabolism of both aniline and 4- chloroaniline werebest described by a two-compartment model that was consistentwith the assumption that metabolites of the parent amines wereaccumulating in the fish. In partial support of the hypothesisthat these amines are being metabolically activated in medaka,N-hydroxylation of aniline and 4-chloraniline tophenylhydroxylamine and 4-chlorophenylhydroxylamine werequantified in hepatic microsomal preparations.
Age dependence of O6-methylguanine-DNA methyltransferase activity and its depletion after carcinogen treatment in the teleost medaka (Oryzias latipes).
Aoki-K; Nakatsuru-Y; Sakurai-J; Sato-A; Masahito-P; Ishikawa-T
Division of Biology, National Institute of Radiological Sciences, Chiba, Japan.
Mutat-Res. 1993 Mar; 293(3): 225-31
O6-Methylguanine-DNA methyltransferase (O6-MT) is considered to play an important role in the repair of DNA lesions induced by alkylating carcinogens in a wide range of animals. The activity of O6-MT was compared in liver extracts from the teleost medaka (Oryzias latipes) at various ages (3-5 years old) reared under natural conditions. O6-MT activity decreased significantly with advancing age. When medaka were exposed continuously to the alkylating agent methylazoxymethanol (MAM) acetate at levels of 0.1, 0.15 and 0.3 ppm in water, O6-MT activity was markedly reduced from days 1 to 7, with a slight increase thereafter. Furthermore, when fish were exposed to MAM acetate at levels of 1-2 ppm for 1 h and then maintained in normal tap water, O6-MT activity remained suppressed for 2 weeks, followed by a partial recovery.
Steroidal alkaloid toxicity to fish embryos.
Crawford-L; Kocan-RM
US Department of Agriculture, Western Regional Research Center, Albany, CA 94710.
Toxicol-Lett. 1993 Feb; 66(2): 175-81
Embryos of two species of fish were evaluated for their suitability as model systems for steroidal alkaloid toxicity, the Japanese rice fish, medaka (Oryzius latipes) and the rainbow trout (Oncorhynchus mykiss). Additionally, the equine neurotoxic sesquiterpene lactone repin, was also tested. A PROBIT program was used to evaluate the EC1, EC50 and EC99 as well as the associated confidence limits. The steroidal alkaloids tested were the Solanum potato glycoalkaloids alpha-chaconine, alpha-solanine, the aglyclones solanidine and solasodine and the Veratrum alkaloid, jervine. Embryo mortality, likely due to structural or functional abnormalities in the early development stages of the embryo, were the only response observed in both species. The rainbow trout exhibited a toxic response to chaconine, solasidine, repin and solanine but the medaka embryos were only affected by the compounds, chaconine and solanine. Rainbow trout may indeed serve as a good lower vertebrate model for studying the toxicity of steroidal alkaloids.
Fishes of District Sundargarh, Orissa, with special reference to their potential in mosquito control.
Yadav-RS; Padhan-K; Sharma-VP
Malaria Research Centre (Field Station), Rourkela, India.
Indian-J-Malariol. 1992 Dec; 29(4): 225-33
An extensive fish fauna survey was carried out in Sundargarh, a malaria-endemic district in Orissa, during 1988 to 1990 to identify and evaluate the indigenous larvivorous fishes for mosquito control. In all, 57 species belonging to 19 families under 6 orders were found in the local water bodies. On laboratory evaluation against anopheline and culicine larvae, six potential larvivorous fishes, viz. Aplocheilus panchax, Oryzias melastigma, Oreochromis mossambicus, Gambusia affinis, Danio (B.) rerio and Esomus danricus were selected. Feasibility of mass multiplication of these fishes in village ponds for operational use is being studied.
Analysis of heterologous and homologous promoters and enhancers in vitro and in vivo by gene transfer into Japanese medaka (Oryzias latipes) Xiphophorus.
Winkler-C; Hong-Y; Wittbrodt-J; Schartl-M
Max-Planck-Institut fur Biochemie, Martinsried, Germany.
Mol-Mar-Biol-Biotechnol. 1992 Aug-Oct; 1(4-5): 326-37
Efficient expression systems are required for analysis of gene regulation and function in teleost fish. To develop such systems, a number of inducible or constitutive promoter and enhancer sequences of fish or higher vertebrate origin were tested for activity in a variety of fish cell lines and in embryos of the Japanese medaka fish (Oryzias latipes) and Xiphophorus. The activity of the different promoter-enhancer combinations were quantitated. Considerable differences were found for some constructs if tested in vitro or in vivo. Fro the data obtained, a set of expression vectors for basic research as well as for aquaculture purposes were established.
Effects of polyoxyethylene (20) sorbitan monooleate on the acute toxicity of linear alkylbenzenesulfonate (C12LAS) to fish.
Toshima-Y; Moriya-T; Yoshimura-K
Biological Science Laboratories, Kao Corporation, Tochigi, Japan.
Ecotoxicol-Environ-Safety. 1992 Aug; 24(1): 26-36
Effects of polyoxyethylene (20) sorbitan monooleate (SMOE20) on the acute toxicity of linear alkylbenzenesulfonate (C12LAS) to fish were investigated in red killifish (Orizias latipes) and carp (Cyprinus carpio). By adding polyoxyethylene sorbitan ester to C12LAS solution, the acute toxicity of C12LAS decreased. As a decreasing toxic effect of these nonionic surfactants, the depression of gill damage was histopathologically observed. No significant hematological and blood biochemical differences were observed between two tests using a single C12LAS solution and a mixed solution of C12LAS/SMOE20. The addition of SMOE20 to C12LAS decreased the incorporation of C12LAS into blood and each organ except spleen and gall bladder as well as the adsorption of C12LAS to the gill. The farther inward the phenyl position in the alkyl side chain, the lower the adsorption of C12LAS to gill. Since the acute toxicity of LAS phenyl isomers to fish was known to decrease according to the order of adsorption, these results suggest that the decreasing toxic effect of SMOE20 on the acute toxicity of C12LAS is due to depression of more toxic C12LAS adsorption on the gill.
A purified diet for medaka (Oryzias latipes): refining a fish model for toxicological research.
DeKoven-DL; Nunez-JM; Lester-SM; Conklin-DE; Marty-GD; Parker-LM; Hinton-DE
Department of Animal Science, University of California, Davis 95616.
Lab-Anim-Sci. 1992 Apr; 42(2): 180-9
The overall nutritional adequacy of a purified casein-based diet(PC-diet) for the medaka (Oryzias latipes) was evaluated andcompared with three diets: commercially available flaked fishfood (FL-diet), live newly hatched Artemia (A-diet), and acombination of FL-diet plus A-diet (F/A-diet). Survival, growth,reproductive success, general and liver histopathology, andselected hepatic enzyme activities were compared in medaka fromfirst feeding through reproductive maturity. The PC-diet provedadequate in all of the above criteria. When compared with fishfed F/A-diet, an initial lag in early growth rates (i.e., 0 to30 days) occurred with the fish fed PC-diet. The FL-diet alonewas not nutritionally adequate for medaka, resulting in poorgrowth, reduced reproductive success, lower survival, andemaciation. A significant number of spinal deformities (5.4%)were noted in medaka fed the F/A diet. Ethoxycoumarin0-deethylase and glutathione S-transferase activities weremonitored and a trend toward increasing activity with age wasnoted. This suggests that PC- and F/A-diets provide adequatenutrition for development of the xenobiotic metabolizing enzymesnecessary for detoxification and activation of endogenous andforeign compounds. The PC- diet supported good survival, growth,reproduction, and normal histology. This diet provides astandardized, nutritionally adequate, and consistent alternativeto undefined conventional diets and is less likely to contain therange of xenobiotics possible in whole, live food.
Fish gonadotropin-releasing hormone gene and molecular approaches for control of sexual maturation: development of a transgenic fish model.
Alestrom-P; Kisen-G; Klungland-H; Andersen-O
Department of Food and Dairy Industries, Agricultural University of Norway.
Mol-Mar-Biol-Biotechnol. 1992 Aug-Oct; 1(4-5): 376-9
The prepro-GnRH gene and mRNA primary structure were fully established from Atlantic salmon (Salmo salar) and partially from rainbow trout (Oncorhynchus mykiss). Results show that the GnRH coding region of 30 base pairs is well conserved during evolution. In contrast, the GnRH-associated peptide (GAP) sequence shows very limited homology when the GnRH genes from mammalian and teleost species are compared. A simple method for selecting transgenic fish after transfer of the firefly luciferase gene was developed. The method involves bioluminescent measurement of live animals in a scintillation counter.
Integration, expression and germ-line transmission of foreign growth hormone genes in medaka (Oryzias latipes).
Lu-JK; Chen-TT; Chrisman-CL; Andrisani-OM; Dixon-JE
Center of Marine Biotechnology, University of Maryland, Baltimore 21202.
Mol-Mar-Biol-Biotechnol. 1992 Aug-Oct; 1(4-5): 366-75
Gene constructs consisting of human growth hormone (hGH) genedriven by promoter/regulatory sequence of mouse metallothionein(mMT), viral thymidine kinase (vTK), rat cholecystokinin (rCCK),or chicken beta-actin (cBA) gene were injected into the cytoplasmof fertilized medaka eggs via the micropyle. More than 49% of theinjected embryos survived at hatching. Up to 26% of the survivorsshowed integration of the introduced gene construct, asdetermined by polymerase chain reaction analysis and subsequentconfirmation by Southern blot hybridization of the genomic DNA.A significant fraction of F1 progeny, derived from crossesbetween transgenic founders and the nontransgenic individuals,inherited the transgene. Expression of hGH gene was also observedin some of the P1 founders and F1 transgenic progeny carryingmMT-hCG or cBA-hGH gene. Furthermore, the growth performance ofthe P1 mMT-hGH and cBA-hGH transgenic founders and F1 cBA-hGH F1transgenic progeny was significantly greater than their fullsibling, nontransgenic individuals. In addition to themicroinjection experiment, a gene construct containing the long-terminal repeat (LTR) sequence of avian Rous sarcoma virus(RSV) and rainbow trout (rt) GH2 cDNA was introduced into embryosof medaka by electroporation using an exponential decayelectroporator. Approximately 70% of the electroporated embryossurvived at hatching, and 20% of the survived individualsintegrated RSVLTR-rtGH2 cDNA into their genomes. These twotechniques will greatly enhance the ability to study regulationof gene expression in transgenic animals during differentiationand development.
Medaka as a model of transgenic fish.
Ozato-K; Wakamatsu-Y; Inoue-K
Department of Biology, Faculty of Liberal Arts and Sciences, Kyoto University, Japan.
Mol-Mar-Biol-Biotechnol. 1992 Aug-Oct; 1(4-5): 346-54
The medaka (Oryzias latipes) is an egg-laying fresh-water fish. We describe the medaka as a model system of transgenic fish in germs of biological characteristics, manipulation of embryos, gene expression in development, and basic research in aquaculture. The fish are small (approximately 3 cm in length) and have a short generation time (approximately 3 months). The eggs are easy to manipulate. A foreign gene (e.g., the chicken delta crystallin gene) is transferred and expressed stage-dependently in development of medaka embryos. Growth hormone genes of vertebrates are transferred and expressed and, in some cases, accelerate growth of the fish. Thus, the medaka is one of the most promising models of transgenic fish for basic research of gene expression and aquaculture.
Firefly luciferase gene transmission and expression in transgenic medaka (Oryzias latipes).
Sato-A; Komura-J; Masahito-P; Matsukuma-S; Aoki-K; Ishikawa-T
Department of Experimental Pathology and Cell Biology, Cancer Institute, Tokyo, Japan.
Mol-Mar-Biol-Biotechnol. 1992 Aug-Oct; 1(4-5): 318-25
Plasmids containing the luciferase gene from the firefly (Photinus pyralis) fused to the Chinese hamster metallothioneine I promoter (ChMTI) were microinjected into the pronuclei of medaka (Oryzias latipes) eggs, which were then artificially inseminated. Evidence of integration into the genome was gained from observation of germ-line transmission in a mendelian fashion from the F1 to the F2 generation. However, gene expression (light emission) could not be demonstrated in the established transgenic line. In a separate program, transient expression of gene constructs containing the luciferase gene fused to various promoters was compared in medaka embryos. Plasmids were microinjected into pronuclei, and homogenates from 3-day-old embryos were measured for light emission using a luminometer. Among the various promoters tested (SV40, RSV-LTR, ChMTI, HSP70, and mouse albumin), the highest levels of luciferase gene expression were observed in gene constructs containing ChMTI and HSP70 gene promoters. Expression in these two constructs was significantly increased following administration of ZnSO4 or heat treatment, respectively. Plasmids were also introduced into goldfish fibroblast-like cells in vitro, in which enzymatically active luciferase was transiently expressed. Assaying for expression of luciferase provided a rapid and sensitive method for monitoring promoter activity. The potential usefulness of this fish species for cancer research is discussed based on accumulated information from carcinogenesis studies.
Development of an all-fish gene cassette for gene transfer in aquaculture.
Du-SJ; Gong-Z; Hew-CL; Tan-CH; Fletcher-GL
Research Institute, Hospital for Sick Children, Toronto, Canada.
Mol-Mar-Biol-Biotechnol. 1992 Aug-Oct; 1(4-5): 290-300
To develop an all-fish gene cassette suitable for gene transfer in aquaculture, the antifreeze protein (AFP) gene promoter from the ocean pout (Macrozoarces americanus) was analyzed for its ability to direct exogenous gene expression both in vitro and in vivo. The ocean pout AFP (opAFP) gene promoter fused to the bacterial chloramphenicol acetyltransferase (CAT) was functionally analyzed in two fish cell lines and in Japanese medaka embryos. The opAFP gene promoter was active in these systems, as demonstrated by the transient expression of CAT activity. These results suggest that the opAFP gene promoter is useful for many other gene transfer experiments. To facilitate use of the opAFP gene promoter as a common and versatile vehicle for fish gene transfers, an expression vector, opAFP-V, was constructed by linking the 2.1-kb opAFP gene promoter, the 63-bp opAFP gene 5' untranslated sequence, and the 1.2-kb opAFP gene 3' sequence by two unique restriction sites, Bg/II and HpaI, respectively. Thus, genes of interest can be inserted into either the Bg/II site or the HpaI site depending on the length of their 5' untranslated sequence. The complete DNA sequence of opAFP-V was determined to facilitate future detailed analysis of integration and expression of the transgene.
Expression and transmission of wild-type pigmentation in the skin of transgenic orange-colored variants of medaka (Oryzias latipes) bearing the gene for mouse tyrosinase.
Matsumoto-J; Akiyama-T; Hirose-E; Nakamura-M; Yamamoto-H; Takeuchi-T
Department of Biology, Keio University, Yokohama, Japan.
Pigment-Cell-Res. 1992 Nov; 5(5 Pt 2): 322-7
Transgenic fish carrying a reconstructed mouse tyrosinase gene, mg-Tyrs- J, were produced by microinjecting the gene into the oocyte nucleus of an orange-colored variant of medaka (Oryzias latipes). Of 64 oocytes microinjected and subsequently inseminated, 13 embryos developed normally beyond hatching and three of them exhibited brown skin pigmentation in the adult as was commonly observed in the wild type of this species. Light and electron microscopic examination disclosed a ubiquitous distribution of typical melanophores in the skin of these transgenic fish. Judging from their population density and distribution pattern, it was presumed that melanogenesis in these fish was elicited in amelanotic melanophores that resided in the skin of the orange-colored fish of this variant. Immunofluorescence with use of the anti-mouse tyrosinase antiserum lacking reactivity to medaka tyrosinase clearly disclosed that the gene introduced was expressed in the melanophores of transgenic fish. Crosses of female transgenic fish and males from an orange-colored variant yielded offspring exhibiting wild-type or orange- colored pigmentation in a ratio of 1:1, thus implying that mg-Tyrs-J integrated into the medaka genome behaves like a dominant gene. Little melanogenesis was observed in xanthophores, leucophores and iridophores in transgenic fish, suggesting possible specificity in recognition of teleostean cell types (i.e., melanophores) by the regulatory region of the mouse tyrosinase gene.
Ultrastructural alterations in liver of medaka (Oryzias latipes) exposed to diethylnitrosamine.
Braunbeck-TA; Teh-SJ; Lester-SM; Hinton-DE
Department of Medicine, School of Veterinary Medicine, University of California-Davis 95616.
Toxicol-Pathol. 1992; 20(2): 179-96
Liver cytotoxic alterations of adult medaka (Oryzias latipes) following short-term bath exposure (48 hr) to 500 mg/L diethylnitrosamine (DEN) were studied (days 3-21) by electron microscopy and cytochemistry. Control medaka displayed hepatic sexual dimorphism as described for other sexually active fish. Following DEN exposure, decreased glycogen stores with loss of cellular compartmentation obscured sexual dimorphism. A spectrum of organelle alterations, previously not reported in livers of fish, was seen. Early changes in hepatocytes included: nuclear lipid inclusions, nucleolar changes, decreased amounts of granular endoplasmic reticulum (GER), increased fractionation and steatosis of GER, proliferation of smooth ER and lysosomes, reduction in number and content of particulate lipoproteins and vitellogenin in Golgi vesicles, and reduction in number and staining intensity of peroxisomes. At day 14 and/or 21, partial to complete reversal of the above alterations indicated hepatic recovery, and fewer necrotic cells were seen at day 21 versus day 14. Lesions that did not resolve during this study were altered mitochondria and areas of spongiosis hepatis that developed at day 8 and continued to increase throughout the study. Infiltration of lymphocytes, granulocytes, and large numbers of macrophages were late changes. The description, timing, and duration of lesions are of value for consideration as biomarkers of exposure and effect in aquatic toxicology.
Identification of four FGF receptor genes in Medaka fish (Oryzias latipes).
Emori-Y; Yasuoka-A; Saigo-K
Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Japan.
FEBS-Lett. 1992 Dec 14; 314(2): 176-8
Four types of cDNA clones encoding tyrosine kinases highly homologous to mammalian fibroblast growth factor receptors (FGF-R) were isolated from Medaka fish (Oryzias latipes) by the reverse transcription-polymerase chain reaction. Comparison of the four deduced amino acid sequences with four known mammalian FGF-Rs indicated that four FGF-R species corresponding to mammalian FGF-Rs exist universally in vertebrates including fishes, although FGF-R4 might have diverged sequences between fishes and mammals. Each of four FGF-R genes is transcribed to various extents as multiple mRNAs possibly by alternative splicing in adult fishes.
Sexually differentiated mechanisms of sterility in interspecific hybrids between Oryzias latipes and O. curvinotus.
Hamaguchi-S; Sakaizumi-M
Department of Biology, College of General Education, Niigata University, Japan.
J-Exp-Zool. 1992 Sep 1; 263(3): 323-9
Fertility of interspecific hybrids between Oryzias latipes and O. curvinotus was examined. F1 females were able to lay eggs but males were sterile. Histological examination of the ovaries of hybrids revealed that oogenesis does not proceed normally in spite of the apparent fertility. Most oocytes degenerated at the pachytene stage of the meiotic prophase, and only a few entered the diplotene stage to develop into ova. Hybrid males could induce females to spawn eggs, an indication that they had differentiated completely into true males. However, they did not produce fertile sperm. Most germ cells in testes of hybrids passes through almost the entire process of spermatogenesis, but deviations from the normal course of events were observed during spermiogenesis. The condensation of chromatin in spermatids occurred, but the diameters of sperm heads were about 1.5-fold larger than those of normal ones. Prominent abnormalities were apparent in the quantity and arrangement of microtubules in the cytoplasm. Abnormal spermatozoa were phagocytized by Sertoli cells. These observations indicate that the mechanisms of impaired gametogenesis in these interspecific hybrids are sexually differentiated.
Fish egg glycophosphoproteins have species-specific N-linked glycan units previously found in a storage pool of free glycan chains.
Iwasaki-M; Seko-A; Kitajima-K; Inoue-Y; Inoue-S
School of Pharmaceutical Sciences, Showa University, Tokyo, Japan.
J-Biol-Chem. 1992 Dec 5; 267(34): 24287-96
Recent findings (Ishii, K., Iwasaki, M., Inoue, S., Kenny, P. T. M., Komura, H., and Inoue, Y. (1989) J. Biol. Chem. 264, 1623-1630; Inoue, S., Iwasaki, M., Ishii, K., Kitajima, K., and Inoue, Y. (1989) J. Biol. Chem. 264, 18520-18526) of a relatively large quantity of complex-type free sialo-oligosaccharides in the unfertilized eggs of freshwater fish, Plecoglossus altivelis and Tribolodon hakonensis, prompted us to search for their progenitor glycoproteins. First we demonstrated a third occurrence of free sialoglycans in the unfertilized eggs of Medaka fish (Oryzias latipes). Next, in all three species studied, a uniformly high level of glycophosphoproteins (GPP) was identified and found to possess N-linked glycan units. The carbohydrate structures of the GPP were determined to be identical with those of the free glycans isolated from the unfertilized eggs of the respective fish species. Thus, the most likely candidate for the progenitor of free sialoglycans appeared to be the oocyte GPPs. This implies that the liberation of the free glycans by a putative peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase may represent a necessary biochemical event during vitellogenesis or oogenesis. The present results may provide insight into a new concept of a "protein N-glycosylation/de-N-glycosylation system" recently proposed by us (Seko, A., Kitajima, K., Inoue, Y., and Inoue, S. (1991) J. Biol. Chem. 266, 22110-22114).
Detection of gamma-ray-induced DNA damages in malformed dominant lethal embryos of the Japanese medaka (Oryzias latipes) using AP-PCR fingerprinting.
Kubota-Y; Shimada-A; Shima-A
Laboratory of Radiation Biology, Faculty of Science, University of Tokyo, Japan.
Mutat-Res. 1992 Dec; 283(4): 263-70
Adult male fish of the medaka HNI strain exposed to 9.5 Gy or 19 Gy (0.95 Gy/min) of gamma-rays were mated with non-irradiated female fish of the Hd-rR strain. Genomic DNA was prepared from malformed individual embryos which were expected to be dominant lethal and used for AP-PCR fingerprinting. By the use of a part of the T3 promoter sequence (20 mer), which, to our knowledge, is not found in the medaka genome as an arbitrary primer, we found polymorphisms in genomic fingerprints which could distinguish the parental strains. On the other hand, we found that the fingerprints of F1 hybrids were the sum of those of their parents. Based on these findings, we analyzed the fingerprints of genomic DNA of each severely malformed embryo, because we expect that radiation-induced genomic damages resulting in severe malformation and eventually in dominant lethals should be detected as changes in paternal fingerprints of F1 hybrids. Indeed, we succeeded in detecting changes in genomic DNA as loss of some paternal bands in fingerprints of malformed embryos. One of 10 malformed embryos obtained from 9.5 Gy gamma-irradiated males had lost one band of the paternal origin and 4 of 12 malformed embryos obtained from 19 Gy gamma-irradiated males had lost 5 bands. These results indicated a possibility that quantitative as well as qualitative estimation of gamma-ray-induced DNA damages can be made by this method which does not require the functional selection based on a specific target gene.
Four synonymous genes encode calmodulin in the teleost fish, medaka (Oryzias latipes): conservation of the multigene one-protein principle.
Matsuo-K; Sato-K; Ikeshima-H; Shimoda-K; Takano-T
Department of Microbiology, Keio University School of Medicine, Tokyo, Japan.
Gene. 1992 Oct 1; 119(2): 279-81
We cloned four distinct calmodulin (CaM)-encoding cDNAs from a small teleost fish, medaka (Oryzias latipes). The deduced amino acid (aa) sequences were exactly the same in these four genes and identical to the aa sequence of mammalian CaM, because of synonymous codon usages. The four cDNAs from medaka, termed CaM-A, -B, -C and -D, corresponded to mRNAs of 1.8, 1.4, 2.5 and 1.8 kb, respectively, in Northern blot analysis. Our results demonstrated that the 'multigene one-protein' principle of CaM synthesis is applicable to medaka, as well as to mammals whose CaM is encoded by at least three different genes.
Isolation of cDNAs for LCE and HCE, two constituent proteases of the hatching enzyme of Oryzias latipes, and concurrent expression of their mRNAs during development.
Yasumasu-S; Yamada-K; Akasaka-K; Mitsunaga-K; Iuchi-I; Shimada-H; Yamagami-K
Zoological Institute, Faculty of Science, Hiroshima University, Japan.
Dev-Biol. 1992 Oct; 153(2): 250-8
The hatching enzyme of medaka consists of two types of proteases (HCE, LCE). cDNA clones for LCE and HCE were isolated from a lambda gt11 cDNA library constructed with poly(A)+ RNA of Day 3 embryos. LCE cDNA is 936 bp long and contains an 813-bp open reading frame encoding a preproenzyme with a 20-amino-acid signal sequence, a 51-amino-acid propeptide, and a 200-amino-acid mature enzyme. For HCE, two distinct cDNAs (HCE21, HCE23) having nucleotide sequences with 92.8% similarity were obtained. These cDNAs contain open reading frames encoding preproenzymes of 279 and 270 amino acids, respectively. The mature enzyme forms of both consist of 200 amino acids, the similarity between them being 95.5%. On Northern blotting analysis, the transcripts of LCE and HCE genes were first detected coincidentally in Day 2 embryos shortly before the production of LCE and HCE, accumulated thereafter in parallel, and dramatically decreased after hatching. The amino acid sequence, the HExxH motif, which is known to constitute an active site in some Zn proteases, is also found in LCE and HCE. However, the sequence analyses strongly suggest that both the enzymes belong to the astacin (protease) family, being distinct from sea urchin hatching enzyme, which is reportedly similar to collagenase.
Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on benzo(a)pyrene hydroxylase activity in embryos of the Japanese medaka (Oryzias latipes).
Wisk-JD; Cooper-KR
Joint Graduate Program in Toxicology, Rutgers University/UMDNJ, Robert Wood Johnson Medical School, Piscataway.
Arch-Toxicol. 1992; 66(4): 245-9
When Japanese medaka embryos were exposed to 12 ng/l 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) beginning on the day of fertilization (day 0), benzo(a)pyrene hydroxylase (B(a)PH) activity was induced in the whole embryo 105,000 g fraction by day 5 of development, which coincided with liver development. The induction of B(a)PH activity also coincided with the appearance of 2,3,7,8-TCDD induced hemorrhagic and edematous lesions. B(A)PH induction only occurred in embryos exposed to toxic concentrations (greater than 10 ng/l) of 2,3,7,8-TCDD. B(a)PH induction also occurred in embryos after exposure to 10 ng/l 2,3,7,8-tetrachlorodibenzofuran (TCDF) and 50 micrograms/l 1,2,7,8-TCDD. Both 2,3,7,8-TCDF and 1,2,7,8-TCDD are toxic to Japanese medaka embryos at concentrations that resulted in the induction of B(a)PH activity. B(a)PH activity was not induced by the non- toxic congener 1,3,6,8-TCDD at concentrations as high as 50 micrograms/l. The structure activity relationship for B(a)PH induction in Japanese medaka embryos was similar to that which is observed in other species and biological systems, suggesting that the biological activities of these compounds may also be mediated through the putative Ah receptor in these fish embryos. At 50 micrograms/l, beta-naphthoflavone (BNF) induced B(a)PH activity in Japanese medaka embryos to similar levels as 2,3,7,8- TCDD did at toxic concentrations. However, at 50 micrograms/l, BNF was not toxic to Japanese medaka embryos. Therefore, the induction of B(a)PH activity probably did not directly result in the toxicity observed in these fish embryos after exposure to 2,3,7,8-TCDD.
A highly repetitive interspersed sequence isolated from genomic DNA of the Medaka, Oryzias latipes, is conserved in three other related species within the genus Oryzias.
Naruse-K; Mitani-H; Shima-A
Zoological Institute, Faculty of Science, University of Tokyo, Japan.
J-Exp-Zool. 1992 Apr 15; 262(1): 81-6
A highly repeated interspersed sequence (OLR1) was isolated from a genomic DNA library of the Medaka, Oryzias latipes. The OLR1 was about 160 base pairs (bp) in length. As judged from the results of colony hybridization experiments, OLR1 is one of the major repeated DNA sequences in the Medaka genome and is present in every 136 kb on average. Results of Southern and colony-hybridization analyses indicate that OLR1 is a small interspersed repetitive element (SINE). OLR1-related sequences were conserved in other three species (O. luzonensis, O. curvinotus, and O. mekongnensis) within the genus Oryzias as a repetitive sequence. These results lend support at the DNA level to the hypothesis that these four species form one group in the genus Oryzias, as has been suggested from an analysis of their karyotypes (Magtoon and Uwa, '85, Proc. Jpn. Acad., Ser. B, 61:157-160).
Seasonal change in the locomotor activity rhythm of the medaka, Oryzias latipes.
Yokota-T; Oishi-T
Department of Biology, Nara Women's University, Japan.
Int-J-Biometeorol. 1992 Mar; 36(1): 39-44
A group of the medaka, Oryzias latipes (Cyprinodontidae, orange-red variety, 25 males and 25 females), was kept in an aquarium, which was placed outdoors under natural conditions from December 1984 to January 1986. Locomotor activity at three layers (upper, middle, and lower layers) was recorded with a phototransistor system in each season. In summer, the fish showed typical diurnal activity at all three layers and the activity was greater than in other seasons. However, in autumn and winter, the fish became less active and showed relatively high activity at night at the upper or middle layer and diurnal activity at the lower layer. Nocturnal activity seemed to appear when the water temperature was decreased and the photoperiod was shortened. A free-running activity rhythm was also recorded under continuous darkness (DD) in each season; however, the fish showed clear free-running activity rhythms under DD only in summer.
Rotational movement of a spermatozoon around its long axis.
Ishijima-S; Hamaguchi-MS; Naruse-M; Ishijima-SA; Hamaguchi-Y
Biological Laboratory, Tokyo Institute of Technology, Japan.
J-Exp-Biol. 1992 Feb; 163: 15-31
The rotational movement of a spermatozoon around its longitudinal axis was investigated by two methods: by observing a spermatozoon attached vertically to a coverslip by the tip of its head, and by observing a spermatozoon freely swimming in a medium by means of 'double-focal microscopy', which yielded simultaneous images at two different focal planes. Similar results were obtained by these two methods. Sea urchin, starfish, medaka, human, golden hamster and bull spermatozoa rolled in both clockwise and counterclockwise directions, although there was a large difference in the proportion of spermatozoa rolling in each direction in the different species. The majority of sea urchin and starfish spermatozoa rolled in a clockwise direction when an observer viewed the cell from its anterior end, whereas the majority of medaka, golden hamster, human and bull spermatozoa rolled in a counterclockwise direction relative to the same observer. Moreover, some spermatozoa occasionally changed their rotational direction. These results suggest that the mechanism regulating the direction of rotation of the spermatozoa is lax. As rotational movement of a spermatozoon around its longitudinal axis is due to the three-dimensional component of the beat of the flagellum, the direction of the three-dimensional movement presumably changes as the spermatozoa swim.
Two constituent proteases of a teleostean hatching enzyme: concurrent syntheses and packaging in the same secretory granules in discrete arrangement.
Yasumasu-S; Katow-S; Hamazaki-TS; Iuchi-I; Yamagami-K
Life Science Institute, Sophia University, Tokyo, Japan.
Dev-Biol. 1992 Feb; 149(2): 349-56
Formation, accumulation, and storage of two components of the Oryzias latipes hatching enzyme, high and low choriolytic enzymes (HCE and LCE), were examined by immunocytochemical and immunoblotting methods. Both of the enzymes were found to be formed specifically in the hatching gland cells at the stages of lens formation to eye pigmentation and their accumulation proceeded markedly and concurrently up to Day 5.5 embryos (the stage just before hatching). The amount of HCE formed was more abundant than that of LCE. In the hatching gland cells, HCE and LCE were found to be packaged in the same secretory granules but in distinct arrangement; HCE is localized to the inside of granules whereas LCE is situated at the periphery of the same granules. Their segregated arrangement is compatible with their relative quantities formed per embryo. The results provide not only the cellular and developmental basis for a view that this hatching enzyme is an enzyme system composed of HCE and LCE but also a clue to the regulatory mechanism of concurrent syntheses of two different specific proteins in the same embryonic cell.
A ceramide analogue (PDMP) inhibits glycolipid synthesis in fish embryos.
Fenderson-BA; Ostrander-GK; Hausken-Z; Radin-NS; Hakomori-S
Biomembrane Institute, Seattle, Washington 98119.
Exp-Cell-Res. 1992 Feb; 198(2): 362-6
Glycolipids were depleted from medaka embryos using 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), an inhibitor of glucosylceramide synthetase. Embryos cultured in the presence of 20 microM PDMP exhibited a dramatic decline in glycolipid synthesis and cell surface expression. Metabolic labeling of glucosylceramide declined by 87% on Days 3-6 of development and 72% on Days 7-10 (hatching occurred on Day 10). In parallel, PDMP-treated embryos exhibited a striking loss of several tissue-specific glycolipid antigens, including 9-O-acetyl GD3 from brain and retina, GT3/GQ1C from brain, neural tube, and retina, and sulfated glycolipid from skin and gut. Despite these changes in glycolipid expression, PDMP-treated embryos were fully viable with no evidence of developmental abnormality. PDMP appears to provide a useful tool for identifying glycolipid antigens in embryos and investigating their role in development.
Histopathological effects of environmental pollutants beta-HCH and methyl mercury on reproductive organs in freshwater fish. Wester-PW
Laboratory for Pathology, National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands.
Comp-Biochem-Physiol-C. 1991; 100(1-2): 237-9
From various environmental pollutants studied so far, specific effects on the reproductive system of small fish species Poecilia reticulata (guppy) and Oryzias latipes (medaka) were noted in the case of beta-hexachlorocyclohexane (induction of vitellogenesis and hermaphroditism, both indicative of estrogenic activity; 32 micrograms/l) and methyl mercury (impaired spermatogenesis; 1.8 micrograms/l). The latter effect was attributed to a disturbance of mitosis.
The usefulness of histopathology in aquatic toxicity studies.
Wester-PW; Canton-JH
National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands.
Comp-Biochem-Physiol-C. 1991; 100(1-2): 115-7
1. A survey was carried out to examine the usefulness of histopathology for the identification of toxic effects of environmental contaminants in fish. 2. Two small fish species, Poecilia reticulata (guppy) and Oryzias latipes (medaka) were used, and two exposure periods (1 and 3 months) were chosen. 3. The following compounds were studied: beta-hexachlorocyclohexane, bis(tri-n-tributyltin)oxide, di-n-butyltindichloride, sodium bromide, methyl bromide, and methylmercury chloride. 4. The following is concluded: histopathology provides useful data in characterizing toxic effects in fish; there is a slight advantage for Poecilia reticulata over Oryzias latipes; there is no advantage for 3 months exposure vs 1 month.
Functional analysis and temporal expression of promoter regions from fish antifreeze protein genes in transgenic Japanese medaka embryos.
Gong-Z; Hew-CL; Vielkind-JR
Research Institute, Hospital for Sick Children, Toronto, Canada.
Mol-Mar-Biol-Biotechnol. 1991 Sep; 1(1): 64-72
Several series of sequences that are upstream of the transcriptional start site of different types of fish AFP genes were fused to the bacterial CAT gene, and their transcriptional role was examined in a transient expression assay after microinjection into Japanese medaka (Oryzias latipes) embryos at the 1-4 cell stage. Our studies demonstrated that the AFP genes have functional promoter regions containing positive as well as negative regulatory regions, indicating that these genes could be regulated at multiple sites. We also observed a promoter-specific pattern of temporal expression. Typically, the CAT expression was low in the first 4 days of embryonic development or before the stage of body pigmentation, followed by a sharp increase. The high level was maintained until hatching (11-13 days after fertilization), by which time the activity decreased to a very low level.
Endothelin-like immunoreactivity in the nervous system of invertebrates and fish.
Kasuya-Y; Kobayashi-H; Uemura-H
Biological Laboratory, Kanagawa Dental College, Yokosuka, Japan.
J-Cardiovasc-Pharmacol. 1991; 17 Suppl 7: S463-6
There have been no reports on the distribution of immunoreactive endothelin (ir-ET) in lower vertebrates and invertebrates, except for our previous studies on the nereid Neanthes diversicolor and the earthworm Eisenia foetida. In the present study, we found ET-like immunoreactivity in five species of invertebrates and two species of fish with antiserum against synthetic endothelin-1 (ET-1). Immunoreactive perikarya and nerve fibers were observed in the central nervous system of the slug Limax marginatus, the freshwater snail Indoplanorbis exustus, and the mussel Mytilus edulis in mollusks, the field cricket Gryllus bimaculatus in insects, and the tube tunicate Ciona intestinalis in protochordates. In the medaka, Oryzias latipes, ir-ET was found in the hypothalamoneurohypophysial system, the caudal neurosecretory system, the gill, and the kidney. Immunoreactive cells were also found in the mucous gland of the slug and in the adenohypophysis of the lamprey, Lampetra japonica. The wide distribution of ET-like substances in invertebrates and fish provides evidence for the case that ET found in mammals has a long evolutionary history.
The energetics of embryonic growth and development. I. Oxygen consumption, biomass growth, and heat production.
Pearson-SD; Ackerman-RA; Seagrave-RC
Iowa State University, Ames 50011.
J-Theor-Biol. 1991 Sep 21; 152(2): 223-40
A quantitative phenomenological model to describe the relationships between biomass growth rate, oxygen consumption, and heat production in developing embryos has been developed and tested using a wide range of experimental data. The model employs generalized material and energy balances, principles of enzyme kinetics, and an overall metabolic model scheme based on known biochemical principles. The phosphorylation concentration ratio of ATP and ADP occurs naturally and becomes a significant parameter in the analysis. The model is applied to the growth of Escherichia coli, Oryzias latipes, chick spinal cord, and whole chicken eggs. Excellent agreement between the model and the experimental data is obtained. In a succeeding paper (Part II) environmental effects and growth efficiency are discussed.
Slow calcium waves accompany cytokinesis in medaka fish eggs.
Fluck-RA; Miller-AL; Jaffe-LF
Marine Biological Laboratory, Woods Hole, Massachusetts 02543.
J-Cell-Biol. 1991 Dec; 115(5): 1259-65
Animal cells are cleaved by the formation and contraction of an extremely thin actomyosin band. In most cases this contractile band seems to form synchronously around the whole equator of the cleaving cell; however in giant cells it first forms near the mitotic apparatus and then slowly grows outwards over the cell. We studied the relationship of calcium to such contractile band growth using aequorin injected medaka fish eggs: we see two successive waves of faint luminescence moving along each of the first three cleavage furrows at approximately 0.5 micron/s. The first, narrower waves accompany furrow extension, while the second, broader ones, accompany the subsequent apposition or slow zipping together of the separating cells. If the first waves travel within the assembling contractile band, they would indicate local increases of free calcium to concentrations of about five to eight micromolar. This is the first report to visualize high free calcium within cleavage furrows. Moreover, this is also the first report to visualize slow (0.3-1.0 micron/s) as opposed to fast (10-100 microns/s) calcium waves. We suggest that these first waves are needed for furrow growth; that in part they further furrow growth by speeding actomyosin filament shortening, while such shortening in turn acts to mechanically release calcium and thus propagates these waves as well as furrow growth. We also suggest that the second waves act to induce the exocytosis which provides new furrow membrane.
Identification of free glycan chain liberated by de-N-glycosylation of the cortical alveolar glycopolyprotein (hyosophorin) during early embryogenesis of the Medaka fish, Oryzias latipes.
Seko-A; Kitajima-K; Inoue-S; Inoue-Y
Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Japan.
Biochem-Biophys-Res-Commun. 1991 Nov 14; 180(3): 1165-71
In Medaka embryos (at the stages of blastulation to organogenesis), we found the presence of free glycan of which structure is identical with the multiantennary N-linked sugar chain of L-hyosophorin molecules which were originally present in the cortical alveoli of the unfertilized eggs in their precursor high molecular form. The free glycan-enriched fraction was separated from L-hyosophorin by chromatography on DEAE-Sephadex A-25 and Sephadex G-50 after removal of the sialic acid residues with exo- sialidase. Composition analysis, 400-MHz 1H NMR spectroscopy, and pyridylamination-hydrazinolysis-nitrous acid deamination of the free glycan showed the presence of di-N-acetylchitobiosyl structure at the reducing end, suggesting that the free glycan chain was derived from L-hyosophorin by the action of a specific peptide:N-glycosidase (PNGase). When we combine the previous finding of the hyosophorin-derived unique pentaantennary free glycan chain in the flounder embryos [A. Seko et al. (1989) J. Biol. Chem. 264, 15922-15929], it is anticipated that PNGase-catalyzed de-N-glycosylation of L-hyosophorin would be required at a certain stage of embryogenesis for L-hyosophorin to play a yet undefined functional role during early development.
Peptide:N-glycosidase activity found in the early embryos of Oryzias latipes (Medaka fish). The first demonstration of the occurrence of peptide:N-glycosidase in animal cells and its implication for the presence of a de-N-glycosylation system in living organisms.
Seko-A; Kitajima-K; Inoue-Y; Inoue-S
Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Japan.
J-Biol-Chem. 1991 Nov 25; 266(33): 22110-4
The recent discovery of free oligosaccharides typical for the complex type of glycan chains terminating with a free di-N-acetylchitobiosyl structure in certain fish eggs and early embryos (Ishii, K., Iwasaki, M., Inoue, S., Kenny, P. T. M., Komura, H., and Inoue, Y. (1989) J. Biol. Chem. 264, 1623-1630; Seko, A., Kitajima, K., Iwasaki, M., Inoue, S., and Inoue, Y. (1989) J. Biol. Chem. 264, 15922-15929; Inoue, S., Iwasaki, M., Ishii, K., Kitajima, K., and Inoue, Y. (1989) J. Biol. Chem. 264, 18520-18526) led us to find an enzyme responsible for detachment of N-linked glycan chains from glycoproteins by hydrolyzing the beta-aspartyl-glucosylamine linkage in Oryzias latipes embryos. The enzyme, peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase or peptide:N-glycosidase (PNGase), was partially (2090-fold) purified, and the reaction site at which this enzyme acts was specified by analysis and identification of the reaction products. This is the first demonstration showing PNGase in animal sources, although the presence of PNGases was reported in a variety of plant extracts and bacteria. Thus, the commonality of this type of enzyme is now demonstrated, and the possible physiological role of PNGase in de-N-glycosylation as a basic biologic process is proposed.
Changes in arginine vasotocin content in the pituitary of the Medaka (Oryzias latipes) during osmotic stress.
Haruta-K; Yamashita-T; Kawashima-S
Zoological Institute, Faculty of Science, Hiroshima University, Japan.
Gen-Comp-Endocrinol. 1991 Sep; 83(3): 327-36
Changes in the arginine vasotocin (AVT) neurons of the medaka, Oryzias latipes, during osmotic stress were studied by means of immunohistochemistry and radioimmunoassay. AVT neurons were identified by their immunoreactivity to anti-arginine vasopressin serum, which crossreacts with AVT. When freshwater (FW)-adapted fish were transferred to seawater (SW), the number of immunoreactive magnocellular neurons decreased, while the cell nuclear size increased. AVT content in the pituitary significantly decreased 2 hr or 1 day after transfer to SW, but returned to approximately the initial level 1 week after transfer. However, when SW-adapted fish were transferred to FW, the number of immunoreactive magnocellular neurons increased from 1 hr after readaptation to FW, but the cell nuclear size failed to show significant changes. AVT content in the pituitary significantly increased 1 or 2 hr after transfer. These results seem to indicate that AVT secretion in the medaka was temporarily accelerated by exposure to SW and inhibited immediately after transfer to FW.
Transient expression of foreign DNA during embryonic and larval development of the medaka fish (Oryzias latipes).
Winkler-C; Vielkind-JR; Schartl-M
Genzentrum, Max-Planck-Institut fur Biochemie, Martinsried, FRG.
Mol-Gen-Genet. 1991 Apr; 226(1-2): 129-40
Species of small fish are becoming useful tools for studies on vertebrate development. We have investigated the developing embryo of the Japanese medaka for its application as a transient expression system for the in vivo analysis of gene regulation and function. The temporal and spatial expression patterns of bacterial chloramphenicol acetyltransferase and galactosidase reporter genes injected in supercoiled plasmid form into the cytoplasm of one cell of the two-cell stage embryo was promoter-specific. The transient expression was found to be mosaic within the tissue and organs reflecting the unequal distribution of extrachromosomal foreign DNA and the intensive cell mixing movements that occur in fish embryogenesis. The expression data are consistent with data on DNA fate. Foreign DNA persisted during embryogenesis and was still detectable in some 3- and 9- month-old adult fish; it was found in high molecular weight form as well as in circular plasmid conformations. The DNA was replicated during early and late embryogenesis. Our data indicate that the developing medaka embryo is a powerful in vivo assay system for studies of gene regulation and function.
U.S. Fish and Wildlife Service, National Fisheries Contaminant Research Center, Columbia, Missouri 65201.
Arch-Environ-Contam-Toxicol. 1991 Jul; 21(1): 17-34
Sediments from four inshore industrial sites and a reference site in the Great Lakes were extracted with organic solvents to produce a crude extract, which was separated on alumina into two fractions: predominantly polycyclic aromatic hydrocarbons; and predominantly nitrogen-containing polycyclic aromatic compounds. Crude extracts were redissolved in acetone and analyzed by gas chromatography and gas chromatography-mass spectrometry. The acetone-redissolved crude extracts from the four industrialized sites contained 5.6-313.3 micrograms total polycyclic aromatic compounds/g sediment and 3.0-36.4 micrograms other compounds/g sediment. In addition to the typical EPA priority pollutants, a substantial amount (228.7 micrograms/g sediment) of alkyl-polycyclic- aromatic compounds was detected in sediments from one of the industrialized sites. Extracts from the reference site contained 1.55 micrograms total polycyclic aromatic compounds/g sediment. Medaka (Oryzias latipes) were exposed to multiple pulse doses of acetone-redissolved extracts and fractions. Medaka were also exposed to a known carcinogen, methylazoxymethanol acetate, to verify that chemicals produced tumors in the test fish. Acetone-redissolved extracts and fractions from contaminated sediments were toxic to medaka. Fin erosion and non-neoplastic liver abnormalities were more prevalent in medaka after exposure to acetone-redissolved extracts and fractions from contaminated sediments. Neoplasms previously associated with chemical exposure in wild fishes were induced in medaka exposed to acetone-redissolved extracts and fractions from two of the contaminated sites, but not from the reference site or controls. These findings further support the hypothesis that chemical contaminants in sediments are involved in epizootics of neoplasms in wild fishes at contaminated sites.
Differences between freshwater and seawater killifish (Oryzias latipes) in the accumulation and elimination of pentachlorophenol.
Tachikawa-M; Sawamura-R; Okada-S; Hamada-A
College of Pharmacy, Nihon University, Funbasi, Chiba, Japan.
Arch-Environ-Contam-Toxicol. 1991 Jul; 21(1): 146-51
Freshwater and seawater acclimated (FWA and SWA) killifish (Oryzias latipes) were exposed to pentachlorophenol (PCP) for 3-10 days. Uptake and clearance rates of FWA and SWA killifish were determined. The estimated bioaccumulation factors (BCF) of PCP for FWA and SWA killifish were 1680 and 370, respectively. The smaller uptake rate and faster clearance rate resulted in the lower BCF for SWA killifish. Fresh- and seawater killifish excreted the PCP metabolites, the glucuronide and sulfate conjugates of PCP; the major metabolite of freshwater killifish was PCP sulfate; for seawater acclimated fish, it was PCP glucuronide. The greater excretion of PCP glucuronide by seawater killifish may be responsible for the rapid elimination of PCP. PCP accumulation in killifish decreased with higher pH levels in both freshwater and seawater environments, but these differences were less than the effect of salinity. The results indicate that salinity can affect the accumulation and elimination of environmental pollutants in killifish.
The life cycle of Echinochasmus bagulai (Trematoda: Echinostomatidae).
Dhanumkumari-C; Hanumantha-Rao-K; Shyamasundari-K
Department of Zoology, Andhra University, Waltair, India.
Int-J-Parasitol. 1991 Apr; 21(2): 259-63
The life history of Echinochasmus bagulai, an echinostomatid trematode in birds, is reported and stages in the life cycle are described. Natural infections with cercariae, which are of the gymnocephalous type, were found in the thiarid snail Thiara tuberculata. Metacercarial cysts were found in the gills of Aplocheilus panchax, Oryzias melastigma, Gambusia affinis and Channa punctata. Adults were obtained in the small intestine of Ardeola grayi. In laboratory experiements development of cercariae into infective metacercariae took 10 days in the gills of A. panchax. Mature flukes were recovered in 10 days in the small intestine of experimentally infected 1-day-old leghorn chicks. The eggs collected from the faeces of these infected chicks were incubated at 39 degrees C. The free-swimming miracidia were found on the fifth day of incubation.
Spontaneous ovarian tumour in a medaka (Oryzias latipes). Harada-T; Okazaki-N; Kubota-SS; Hatanaka-J; Enomoto-M
Biological Laboratory, Nippon Veterinary and Zootechnical College, Tokyo, Japan.
J-Comp-Pathol. 1991 Feb; 104(2): 187-93
Swelling of the abdomen was found in an adult female medaka (Oryzias latipes). Multiple tumour masses occupying most of the abdominal cavity were found at necropsy. Histologically, the tumour consisted of homogeneous round cells arranged in islands and occasional trabecular structures with thin capsules. Ova at various stages of maturation were seen in the periphery of the tumour mass. Metastasis was observed in the vicinity of the thyroid gland with accompanying infiltration by lymphocytic cells and also abdominal muscles. The tumour cells were characterized by a large ovoid nucleus with no aggregation of heterochromatin, occasional mitotic figures and prominent nucleoli. Examination by electron microscopy showed cells with scant cytoplasm but many ribosomes and a few other organelles. Annulate lamellae and dense cytoplasmic masses were also frequently observed. Based on the above features, the tumour was diagnosed as an ovarian dysgerminoma, the first reported case in a medaka.
Alteration of pectoral fin nerves following ablation of fin buds and by ectopic fin buds in the Japanese medaka fish.
Okamoto-H; Kuwada-JY
Department of Biology, University of Michigan, Ann Arbor 48109.
Dev-Biol. 1991 Jul; 146(1): 62-71
The role of the pectoral fin bud for outgrowth by fin axons was assessed by ablation of pectoral fin buds and by transplantation of fin buds to ectopic sites in the embryos of the Japanese medaka fish (Oryzias latipes). Normally nerves from segments 1-4 (S1-4) and less frequently the S5 nerve converged at the base of the fin bud by extending toward the fin bud on the ventral surface of the axial muscles (H. Okamoto and J. Y. Kuwada, 1991, Dev. Biol. 146). Following ablation of the fin bud before motor growth cones have begun to extend laterally, nerves in S1-5 followed a trajectory down the middle of each segment parallel to the borders of the metamerically arranged axial muscles rather than converging. This trajectory was similar to that of more posterior segmental nerves which do not converge toward the fin bud. When fin buds were transplanted to more posterior segments, nerves from S1-5 often changed their trajectories and extended to the base of ectopic buds. Furthermore, motor nerves from segments posterior to S5, which normally do not innervate the fin bud, also extended to the ectopic fin bud. When faced with both the host and ectopic fin bud, motor nerves extended to either fin bud or branched and extended to both fin buds. These results demonstrate that the early fin bud is necessary for correct outgrowth of fin nerves and suggest that the fin bud normally attracts fin nerves to its base. One possible mechanism for the attraction of motor growth cones by the fin bud is a long distance cue emitted by the fin bud.
Outgrowth by fin motor axons in wildtype and a finless mutant of the Japanese medaka fish.
Okamoto-H; Kuwada-JY
Department of Biology, University of Michigan, Ann Arbor 48109.
Dev-Biol. 1991 Jul; 146(1): 49-61
The outgrowth of motor axons to the developing pectoral fin of the Japanese medaka fish (Oryzias latipes) was investigated both in wildtype embryos and in the pectoral finless (pl) mutants in which adults are missing pectoral fins. Late in embryogenesis the pectoral fin is a simple limb which contains two antagonist muscles which are innervated by presumptive motor neurons from the first four spinal segments (S1-4). The pectoral fin develops from a fin bud located in S1 and S2 centered on the border between S1 and S2 and, as with other limbs, one of the earliest signs of differentiation is the apical ectodermal ridge (AER). By the time the AER is well formed the growth cones of the presumptive motor neurons have reached the base of the fin bud and formed a plexus by extending toward the fin bud upon emergence from the spinal cord. This is especially evident on the ventral surface of the metamerically arranged axial muscles. For example, growth cones from S2 extend in a diagonal direction (both anterior and lateral) towards the fin bud. One hypothesis which can account for the pattern of motor outgrowth is that growth cones are attracted to the base of the fin bud, perhaps via a long distance cue. This hypothesis was tested by examining outgrowth of segmental nerves in pl embryos in which the fin buds arrest early in development following the initial appearance of the AER. In pl, nerves from S1-4 converged to form a plexus at the base of the abnormal fin bud, but the pattern of outgrowth varied from wildtype in a way consistent with a diminished capacity of the fin bud to attract segmental nerves to it.
Aquatic toxicity testing for multicomponent compounds with special reference to preparation of test solution.
Tadokoro-H; Maeda-M; Kawashima-Y; Kitano-M; Hwang-DF; Yoshida-T
Chemical Biotesting Center, Chemicals Inspection and Testing Institute, Tokyo, Japan.
Ecotoxicol-Environ-Safety. 1991 Feb; 21(1): 57-67
An adequate method of determining the toxicity of a compound consisting of multiple components, such as creosote, coal tar, and coal tar pitch, was studied for different test solution preparation methods, i.e., direct dosing without filtration, diluting the stock solution of saturated concentration, and dispersing with acetone. Killifish, Oryzias latipes, as a freshwater fish; red sea bream, Pagrus major, as a saltwater fish; and daphnia, Daphnia magna, as a representative crustacean, were used for testing. The chemical analysis of each preparation of test solution with gas chromatography revealed an entirely different profile of the components. The highest toxicity was obtained with preparation by acetone dispersion. That was followed by the preparations with direct dosing method and with the method of dilution of saturated concentration stock solution. Considering the results obtained, the direct dosing method with a suitable settling time may provide useful information enabling extrapolation of the test results to the natural environment for complex multicomponent compounds.
DNA alterations and enzyme activities in Japanese medaka (Oryzias latipes) exposed to diethylnitrosamine.
McCarthy-JF; Gardner-H; Wolfe-MJ; Shugart-LR
Environmental Sciences Division, Oak Ridge National Laboratory, TN 37831-6036.
Neurosci-Biobehav-Rev. 1991 Spring; 15(1): 99-102
Several molecular and biochemical markers of genotoxicity were adapted for measurement in the medaka, and were used to describe the effects of treatment of the organism with diethylnitrosamine (DEN). DEN treatment inhibited the activity of a detoxication enzyme activity (ethoxyresorufin-O-deethylase) and increased the activity of glutathione-S-transferase. This pattern of response has been described in preneoplastic rodent cells. No O6-ethyl guanine adducts were detected, and a slight, but statistically significant, increase in DNA strand breaks was observed. These results are consistent with the hypothesis that prolonged exposure to high levels of DEN induced alkyltransferase activity which enzymatically removes any O6-ethyl guanine adducts but does not result in strand breaks or hypomethylation of the DNA such as might be expected from excision repair of chemically modified DNA.
Ultrastructure of hepatic hemangiopericytoma in the medaka (Oryzias latipes).
Bunton-TE
Department of Pathology, Johns Hopkins University, Baltimore, Maryland 21205.
Exp-Mol-Pathol. 1991 Apr; 54(2): 87-98
The histologic and ultrastructural features of hepatic hemangiopericytoma from a medaka (Oryzias latipes) exposed for 48 hr to 400 mg/liter of diethylnitrosamine at 14 days of age are described. The predominant histologic pattern was of spindle-shaped cells forming numerous whorls around central capillaries, vacuolated areas, or necrotic debris. The predominant cell type was a spindle-shaped cell with oval nuclei, elongated cell processes, and abundant organelles converging upon normal appearing capillaries. Occasionally, however, they converged upon cells swollen with cytoplasmic filaments and/or containing large fenestrated or debris-filled cytoplasmic vacuoles. These features were reminiscent of endothelial cells undergoing intracellular canalization seen in angiogenesis or neovascularization. Individual capillaries were also seen in the mass independent of whorls. It was not clear, as is the case in man, if capillary formation was an integral part of the neoplastic process or a reactive response. Although the liver is an unusual location for hemangiopericytoma in man, many of the cellular features in the fish tumor were similar to the human tumor. The ultrastructural characterization of tumor cells in fish carcinogenesis correlated with histologic patterns of growth will expand our understanding of how fish cells respond when transformed, and augment the development and use of aquatic bioassays for carcinogenesis research.
Development of a possible nonmammalian test system for radiation-induced germ-cell mutagenesis using a fish, the Japanese medaka (Oryzias latipes).
Shima-A; Shimada-A
Laboratory of Radiation Biology, Faculty of Science, University of Tokyo, Japan.
Proc-Natl-Acad-Sci-U-S-A. 1991 Mar 15; 88(6): 2545-9
To develop a specific-locus test (SLT) system for environmental mutagenesis using vertebrate species other than the mouse, we first established a tester stock of the fish medaka (Oryzias latipes) that is homozygous recessive at three loci. The phenotypic expression of these loci can be easily recognized early in embryonic development by observation through the transparent egg membrane. We irradiated wild-type males with 137Cs gamma-rays to determine the dose-response relationships for dominant lethal and specific-locus mutations induced in sperm, spermatids, and spermatogonia. Through observation of 322,666 loci in control offspring and 374,026 loci in offspring obtained from 0.64-, 4.75-, or 9.50-Gy- irradiated gametes, specific-locus mutations were phenotypically detected during early development. These putative mutations, designated "total mutation," can be recognized only in embryos of oviparous animals. The developmental fate of these mutant embryos was precisely followed. During subsequent embryonic development, a large fraction died and thus was unavailable for test-crossing, which was used to identify "viable mutations." Our medaka SLT system demonstrates that the vast majority of total mutations is associated with dominant lethal mutations. Thus far only one spontaneous viable mutation has been observed, so that all doubling calculations involving this endpoint carry a large error. With these reservations, however, we conclude that the quantitative data so far obtained from the medaka SLT are quite comparable to those from the mouse SLT and, hence, indicate the validity of the medaka SLT as a possible nonmammalian test system.