Expression of aquaporin-3 improves the permeability to water and cryoprotectants of immature oocytes in the medaka (Oryzias latipes)

The permeability of the plasma membrane plays a crucial role in the successful cryopreservation of oocytes and embryos. Several efforts have been made to facilitate the movement of water and cryoprotectants across the plasma membrane of fish oocytes/embryos because of their large size. Aquaporin-3 is a water/solute channel that can also transport various cryoprotectants. In this study, we tried to improve the permeability of immature medaka (Oryzias latipes) oocytes to water and cryoprotectants by artificially expressing aquaporin-3. The oocytes were injected with aquaporin-3 cRNA and cultured for 6-7 h. Then, hydraulic conductivity (L(P)) and cryoprotectant permeability (P(S)) were determined from volume changes in a hypertonic sucrose solution and various cryoprotectant solutions, respectively, at 25 degrees C. The L(P) value of the cRNA-injected oocytes was 0.22+/-0.04 microm/min/atm, nearly twice larger than that of intact or water-injected oocytes (0.14+/-0.02 and 0.14+/-0.03 microm/min/atm, respectively). P(S) values of intact oocytes for ethylene glycol, propylene glycol, and DMSO were 1.36+/-0.34, 1.97+/-0.20, and 1.17+/-0.52 x 10(-3) cm/min, respectively. The permeability to glycerol could not be calculated because oocytes remained shrunken in the glycerol solution. On the other hand, cRNA-injected oocytes had significantly higher P(S) values (glycerol, 2.20+/-1.29; ethylene glycol, 2.98+/-0.36; propylene glycol, 3.93+/-1.70; DMSO, 3.11+/-0.74 x 10(-3) cm/min) than intact oocytes. When cRNA-injected oocytes were cultured for 12-14 h, 51% matured to the metaphase II stage, and 43% of the matured oocytes were fertilized and hatched following in vitro fertilization and 14 days of culture. Thus, the permeability of medaka oocytes to water and cryoprotectants was improved by the artificial expression of aquaporin-3, and the oocytes retained the ability to develop to term.     

The permeability to water and cryoprotectants of immature and mature oocytes in the zebrafish (Danio rerio)

To identify a stage feasible for the cryopreservation of zebrafish oocytes, we investigated the permeability to water and cryoprotectants of immature (stage III) and mature (stage V) oocytes. The permeability to water (microm/min/atm) of immature oocytes at 25 degrees C (0.37) was significantly higher than that of mature oocytes (0.10). The permeability (x10(-3)cm/min) of immature oocytes to ethylene glycol, propylene glycol, and Me(2)SO (1.49-3.03) at 25 degrees C was substantially higher than that of mature oocytes approximately 0. The permeability of immature oocytes to glycerol was also high (1.75), although the permeability could not be measured in mature oocytes. Immature oocytes would be more suitable than mature oocytes for conservation of the zebrafish.     

Effects of cadmium on the reproductive axis of Japanese medaka (Oryzias latipes)

Cadmium (Cd) is a ubquitous element and a significant inorganic pollutant that has previously been found to bioaccumulate in reproductive organs of fish and disrupt important endocrine processes, especially those involved in synthesis, release and metabolism of hormones. Clearly, there is potential for reproductive effects in fish populations exposed to Cd, however, few studies have investigated the non-lethal consequences of Cd in fish. To this extent, adult male and female Japanese medaka were exposed to 0-10 ppb Cd for 7 weeks. Reproductive endpoints were monitored during weeks 6 and 7 of exposure and compared to physiological responses along the hypothalamus-pituitary-gonadal (HPG) axis, including plasma vitellogenin (VTG), hepatic estrogen receptor (ER), plasma steroids, gonadal-somatic indices (GSI), and gonadal steroid release. There were no observed effects on VTG and ER by long-term Cd exposure. However, gonadal steroid release was significantly decreased in males and females at all exposure concentrations and female plasma estradiol levels were significantly altered at concentrations higher than 5 ppb Cd. Overall, responses along the HPG axis were more sensitive to Cd exposure than the reproductive and developmental endpoints, which were not affected in this study, indicating that higher level impairment in fish might be relatively protected.     

Identification of robust hypoxia biomarker candidates from fin of medaka (Oryzias latipes)

Aquatic hypoxia caused by organic pollution and eutrophication is a pressing worldwide water pollution problem. Better methods for monitoring oxygen levels are needed to assist efforts to maintain and protect the health of natural aquatic environments. In this project, we used a Japanese ricefish (medaka, Oryzias latipes) 8K oligonucleotide array as a platform to identify potential hypoxic biomarkers in different organs (fin, gill, liver and brain) upon exposure to hypoxia. The microarray results were validated by qRT-PCR employing a subset of candidate biomarkers. Interestingly, the largest number and most significant of hypoxia responding array features were detected in hypoxia exposed fin tissues. We identified 173 array features that exhibited a significant response (over 2 fold change in expression) upon exposure to hypoxic conditions and validated a subset of these by quantitative RT-PCR. These gene targets were subjected to annotation and gene ontology mining. Positively identifiable gene targets that may be useful for development of a rapid and accurate biomarker test using fin clips are discussed in relation to previous reports on hypoxia responsive genes.     

Gyrodactylus medaka n. sp. (Monogenea: Gyrodactylidae) parasitic on wild and laboratory-reared medaka Oryzias latipes (Beloniformes: Adrianichthyidae) in Japan

Gyrodactylus medaka n. sp. (Monogenea: Gyrodactylidae) is described from the skin, fins, and gills of medaka Oryzias latipes (Beloniformes: Adrianichthyidae) from Japan. This new species was collected from wild medaka in Hiroshima, Aichi, Saga, and Kumamoto prefectures, and laboratory-reared medaka in Chiba and Aichi prefectures. The small marginal hook sickle (≤4?μm) and the length of the marginal hook of the new species are the diagnostic morphological characters differentiated from other gyrodactylids reported from Asia. The pairwise sequence divergences for the interspecific variation in ITS regions and the phylogenetic analysis suggest that the populations of G. medaka n. sp. may have a similar genetic variation as the medaka populations in Japan. Gyrodactylus medaka n. sp. and Dactylogyrus oryziasi (Monogenea: Dactylogyridae) can maintain their populations in laboratory aquaria using medaka as their hosts, and these monogeneans and medaka have the potential as experimental model animals for clarifying various aspects of their host-parasite relationships. In addition, we report the composition of modified ammonium picrate-glycerin (APG) and show it is advantageous for monogenean taxonomy.     

Seasonal change in the locomotor activity rhythm of the medaka,Oryzias latipes

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.     

Transient expression of foreign DNA during embryonic and larval development of the medaka fish (Oryzias latipes)

Summary 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.This work contains part of the PhD thesis of C. Winkler     

Influence of parental and developmental cadmium exposure on endocrine and reproductive function in Japanese medaka (Oryzias latipes)

Cadmium (Cd) is a ubiquitous element and an important anthropogenic metal contaminant. A series of assays were modified or developed for Japanese medaka (Oryzias latipes), and used to compare the effects of Cd exposure on indicators of endocrine function in adult animals previously exposed in ovo or as hatchlings. Adults were raised either from eggs produced during a 2 week exposure to 0-10 microg/l Cd or from fry exposed for 2 weeks beginning 2 days after hatching. The reproductive capacity of the resulting adults was determined during a 2 week period during which half of the animals were re-exposed to Cd. Two week Cd exposure did not result in reproductive impairment despite producing some changes in circulating steroid concentration. In addition, 1 microg/l cadmium exposure in ovo elevated male hepatic vitellogenin (VTG) relative to controls. Hence, steroid parameters were a better biomarker of cadmium exposure than changes in VTG. However, reproductive impairment was not correlated to change in VTG or plasma steroids.     

Germ cell-less expression in medaka (Oryzias latipes)

The gene germ cell-less (gcl) plays an important role in the early differentiation of germ cells in Drosophila. We isolated the gcl homolog of the model teleost medaka (Oryzias latipes) using degenerated primers and an ovary cDNA bank. The predicted amino acid sequence of medaka gcl showed 92, 68 and 31% overall identity to mouse, human and Drosophila gcl respectively. RT-PCR revealed stronger expression in the ovary and weaker expression in testis, brain, heart, liver and muscle tissue. Expression in early embryos indicates the presence of maternal mRNA. By in situ hybridisation (ISH), gcl could not be detected in embryos. In contrast to vasa, ISH revealed expression of gcl in the ovary but not in the testis. Mol. Reprod. Dev. 67: 15-18, 2004.     

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

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

Immunoglobulin light chains in medaka (Oryzias latipes)

The gene segments encoding antibodies have been studied in many capacities and represent some of the best-characterized gene families in traditional animal disease models (mice and humans). To date, multiple immunoglobulin light chain (IgL) isotypes have been found in vertebrates and it is unclear as to which isotypes might be more primordial in nature. Sequence data emerging from an array of fish genome projects is a valuable resource for discerning complex multigene assemblages in this critical branch point of vertebrate phylogeny. Herein, we have analyzed the genomic organization of medaka (Oryzias latipes) IgL gene segments based on recently released genome data. The medaka IgL locus located on chromosome 11 contains at least three clusters of IgL gene segments comprised of multiple gene assemblages of the kappa light chain isotype. These data suggest that medaka IgL gene segments may undergo both intra- and inter-cluster rearrangements as a means to generate additional diversity. Alignments of expressed sequence tags to concordant gene segments which revealed each of the three IgL clusters are expressed. Collectively, these data provide a genomic framework for IgL genes in medaka and indicate that Ig diversity in this species is achieved from at least three distinct chromosomal regions.     

Cryoprotectant permeability of aquaporin-3 expressed in Xenopus oocytes

It has been shown that aquaporin-3, a water channel, is expressed in mouse embryos. This type of aquaporin transports not only water but also neutral solutes, including cell-permeating cryoprotectants. Therefore, the expression of this channel may have significant influence on the survival of cryopreserved embryos. However, permeability coefficients of aquaporin-3 to cryoprotectants have not been determined except for glycerol. In addition, permeability coefficients under concentration gradients are important for developing and improving cryopreservation protocols. In this study, we examined the permeability of aquaporin-3 to various cryoprotectants using Xenopus oocytes. The permeability of aquaporin-3 to cryoprotectants was measured by the volume change of aquaporin-3 cRNA-injected oocytes in modified Barth's solution containing either 10% glycerol, 8% ethylene glycol, 10% propylene glycol, 1.5 M acetamide, or 9.5% DMSO (1.51-1.83 Osm/kg) at 25 degrees C. Permeability coefficients of aquaporin-3 for ethylene glycol and propylene glycol were 33.50 and 31.45 x 10(-3) cm/min, respectively, which were as high as the value for glycerol (36.13 x 10(-3) cm/min). These values were much higher than those for water-injected control oocytes (0.04-0.11 x 10(-3) cm/min). On the other hand, the coefficients for acetamide and DMSO were not well determined because the volume data were poorly fitted by the two parameter model, possibly because of membrane damage. To avoid this, the permeability for these cryoprotectants was measured under a low concentration gradient by suspending oocytes in aqueous solutions containing low concentrations of acetamide or DMSO dissolved in water (0.20 Osm/kg). The coefficient for acetamide (24.60 x 10(-3) cm/min) was as high as the coefficients for glycerol, ethylene glycol, and propylene glycol, and was significantly higher than the value for control (6.50 x 10(-3) cm/min). The value for DMSO (6.33 x 10(-3) cm/min) was relatively low, although higher than the value for control (0.79 x 10(-3) cm/min). This is the first reported observation of DMSO transport by aquaporin-3.     

Absence of global genomic cytosine methylation pattern erasure during medaka (Oryzias latipes) early embryo development

Two techniques were used to analyze global genomic 5-methyl cytosine methylation at CCGG sites of medaka embryo DNA. DNA was labeled by incorporation of microinjected radiolabeled deoxynucleotide into one-cell embryos. After Hpa II or Msp I digestion the radiolabeled DNA was fractionated in agarose gels and the distribution of label quantified throughout each sample lane to detect differences in fragment distribution. Alternately isolated DNA was digested with Hpa II or Msp I and the resulting generated termini end-labeled. The end-labeled digestion products were then analyzed for fragment distribution after gel fractionation. These techniques proved to be extremely sensitive, allowing comparison of genomic DNA methylation values from as few as 640 fish cells. The data suggest that in medaka embryos the vast majority (>90%) of genomic DNA is methylated at CCGG sites. Furthermore, these data support the conclusion that the extent of methylation at these sites does not change or changes very little during embryogenesis (from 16 cells to the hatchling). These data argue against active demethylation, or loss of methylation patterns by dilution, during the developmental stages between the one cell zygote and gastrulation. From a comparative viewpoint, these data may indicate that mammals and fishes methylate and demethylate their genomes in very different manners during development.     

A high-throughput histoarray for quantitative molecular profiling of multiple, uniformly oriented medaka (Oryzias latipes) embryos

Embryos of aquatic animal model fish have proven to be useful organisms for developmental biology and for early life stage toxicity tests. By virtue of their transparent chorions, imaging of normal and abnormal development can be detected and related to exposure or to alterations due to environmental factors. However, the detection of changes at sub-individual levels of organization is hampered by time required to detect important events within cells and tissues of affected organisms. We describe herein development of a highly cost effective embryo chip enabling stringent inter-individual comparisons and multiplex detection in embryos and eleutheroembryos. As a proof of principle we examine cell proliferation and controlled cell death in normoxic and hypoxic conditions and relate these to tissue turnover in individual organisms. Coupled with a recently developed whole adult animal platform, we can now move beyond the common approach focusing on single target organ to the detection and characterization of systemic phenomena (syndromes) affecting the organism. Taken together, we can now determine adult consequences of early life stage exposure and assess ability of exposed individuals to respond to stresses superimposed along the axis of time.     

Enhancement by fibronectin of spreading of isolated melanophores of the medaka,Oryzias latipes

Summary Cell spreading of isolated melanophores in medium containing fibronectin was observed in the wild type and two mutants of the medaka, Oryzias latipes. Isolated and cultured melanophores of the wild type and the mm mutant were different in appearance from those within scales but dendritic in shape and with fully dispersed pigment granules. Isolated melanophores of the cm mutant were stellate with dispersed pigment granules, whereas in scales the pigment granules are condensed. In the presence of fibronectin, spreading of cultured melanophores of wild type and cm mutant was observed. Spreading of melanophores from the mm mutant was observed only among dendritic melanophores, but not among condensed melanophores. The increase of spreading was inhibited by antibody against fibronectin. To test the involvement of cytoskeletal elements, colchicine, vinblastine or cytochalasin B were added to the culture medium; spreading did not increase, even in the presence of fibronectin. These results suggest that fibronectin-induced melanophore spreading is correlated with the state of pigment granule dispersal and that microtubules and microfilaments may play a role in the mechanism of spreading.Department of Zoology NJ-15, University of Washington, Seattle, Wa 98195, USA.