Medium composition for effective slow freezing of embryonic cell lines derived from marine medaka (Oryzias dancena)

This study was conducted to identify optimal medium composition for freezing Oryzias dancena embryonic cell lines. Different freezing media consisting of various concentration of dimethyl sulfoxide (DMSO), fetal bovine serum (FBS), and trehalose were prepared and long-term cultured embryonic cell line was frozen in each freezing medium by conventional slow freezing program for 7 days. Through measurement of viability and growth of post-thaw cells frozen in each freezing medium, it was determined that optimal composition of three components was 10 % DMSO, 20 % FBS, and 0.1 M trehalose. The post-thaw cells frozen in optimal freezing medium showed similar morphology and growth rate with non-frozen cells. Next, this condition was applied to two different sets of experiment; (1) freezing of the same cells during expanded period (57 days) and (2) freezing of short-term cultured cells from other batches for 7 days. The viability of post-thaw cells was significantly low and comparable in set 1 and 2, respectively, when compared with the result of long term-cultured cells frozen in optimal freezing medium for 7 days and similar morphology and growth rate with non-frozen counterparts were detected in the post-thaw cells from both sets. In conclusion, this study first reports the optimal medium composition for freezing O. dancena embryonic cells, which can contribute to fish species preservation as well as improvement of cell-based biotechnology by providing stable cell storage.     

Evolution of different Y chromosomes in two medaka species, Oryzias dancena and O. latipes

Although the sex-determining gene DMY has been identified on the Y chromosome in the medaka (Oryzias latipes), this gene is absent in most Oryzias species, suggesting that closely related species have different sex-determining genes. Here, we investigated the sex-determination mechanism in O. dancena, which does not possess the DMY gene. Since heteromorphic sex chromosomes have not been reported in this species, a progeny test of sex-reversed individuals produced by hormone treatment was performed. Sex-reversed males yielded all-female progeny, indicating that O. dancena has an XX/XY sex-determination system. To uncover the cryptic sex chromosomes, sex-linked DNA markers were screened using expressed sequence tags (ESTs) established in O. latipes. Linkage analysis of isolated sex-linked ESTs showed a conserved synteny between the sex chromosomes in O. dancena and an autosome in O. latipes. Fluorescence in situ hybridization (FISH) analysis of these markers confirmed that sex chromosomes of these species are not homologous. These findings strongly suggest an independent origin of sex chromosomes in O. dancena and O. latipes. Further analysis of the sex-determining region in O. dancena should provide crucial insights into the evolution of sex-determination mechanisms in vertebrates.     

The genome of the marine medaka Oryzias melastigma

Marine medaka (Oryzias melastigma) is considered to be a useful fish model for marine and estuarine ecotoxicology studies and has good potential for field‐based population genomics because of its geographical distribution in Asian estuarine and coastal areas. In this study, we present the first whole‐genome draft of O. melastigma. The genome assembly consists of 8,602 scaffolds (N50 = 23.737 Mb) and a total genome length of 779.4 Mb. A total of 23,528 genes were predicted, and 12,670 gene families shared with three teleost species (Japanese medaka, mangrove killifish and zebrafish) were identified. Genome analyses revealed that the O. melastigma genome is highly heterozygous and contains a large number of repeat sequences. This assembly represents a useful genomic resource for fish scientists.     

Complete mitochondrial genome of the marine medaka Oryzias melastigma (Beloniformes, Adrianichthyidae)

The complete mitochondrial genome was obtained from the assembled genome data sequenced by next-generation sequencer from the marine medaka Oryzias melastigma. The mitochondrial genome sequence was 16,864 bp in size, and the gene order and contents were identical with those of previously reported fish mitochondrial genomes. Of 13 protein-coding genes (PCGs), 4 genes (CO3, ND3, ND4, and Cytb) had incomplete stop codons. The base composition of O. melastigma mitogenome showed high A+T (59.65%) and anti-G bias (8.73%) on the 3rd position of PCGs.     

Characterization of estrogen-responsive transgenic marine medaka Oryzias dancena germlines harboring red fluorescent protein gene under the control by endogenous choriogenin H promoter

Transgenic marine medaka (Oryzias dancena) germlines were generated by the microinjection of the red fluorescent protein (RFP) reporter gene (rfp) driven by the endogenous choriogenin H gene (chgH) promoter. The selected transgenic lines contained multiple copies of the transgene (3–42 copies per cell) in their genomes. Although all the founders were mosaic, the transgene was stably transmitted from the F1 generation to all subsequent generations following a Mendelian pattern. Different transgenic lines showed different responsiveness to estradiol-17β (E2) exposure at the mRNA and protein levels, and the expression efficiency was dependent upon the transgene copy number. The induction of RFP was significantly affected by the developmental stage of transgenic larvae: later-stage larvae (older than 7 days post-hatching) showed higher sensitivity to E2 exposure than earlier-stage larvae. The response of transgenic expression to E2 was fairly dependent upon the E2 dose (200–3,200 ng/L) and exposure period (1–7 days), according to both a microscopic examination of RFP intensity and a qRT–PCR assay. The transgenic marine medaka showed similar transgenic responses to E2 under freshwater, brackish, and seawater conditions. In addition to E2, the transgenic RFP signal was also successfully induced during 1-week exposure to various other natural (1 μg/L estrone and 10 μg/L estriol) and synthetic (xeno)estrogens (0.1 μg/L 17α-ethynylestradiol, 1 μg/L diethylstilbestrol, and 10 mg/L bisphenol A). The efficiency of transgene expression varied greatly among the chemicals tested. The results of this study suggest that the chgH–rfp transgenic marine medaka species will be useful in the in vivo detection of waterborne estrogens under a wide range of salinity conditions.     

The acute and regulatory phases of time-course changes in gill mitochondrion-rich cells of seawater-acclimated medaka (Oryzias dancena) when exposed to hypoosmotic environments

The recent model showed that seawater (SW) mitochondrion-rich (MR) cells with hole-type apical openings secrete Cl^? through the transporters including the Na⁺, K⁺-ATPase (NKA), Na⁺, K⁺, 2Cl^? cotransporter (NKCC), and cystic fibrosis transmembrane conductance regulator (CFTR). The present study focused on the dynamic elimination of the Cl^? secretory capacity and illustrated different phases (i.e., acute and regulatory phases) of branchial MR cells in response to hypoosmotic challenge. Time-course remodeling of the cell surfaces and the altered expressions of typical ion transporters were observed in the branchial MR cells of SW-acclimated brackish medaka (Oryzias dancena) when exposed to fresh water (FW). On the 1st day post-transfer, rapid changes were shown in the acute phase: the flat-type MR cells with large apical surfaces replaced the hole-type cells, the gene expression of both Odnkcc1a and Odcftr decreased, and the apical immunostaining signals of CFTR protein disappeared. The basolateral immunostaining signals of NKCC1a protein decreased throughout the regulatory phase (> 1 day post-transfer). During this period, the size and number of NKA-immunoreactive MR cells were significantly reduced and elevated, respectively. Branchial NKA expression and activity were maintained at constant levels in both phases. The results revealed that when SW-acclimated brackish medaka were transferred to hypoosmotic FW for 24 h, the Cl^? secretory capacity of MR cells was eliminated, whereas NKCC1a protein was retained to maintain the hypoosmoregulatory endurance of the gills. The time-course acute and regulatory phases of gill MR cells showed different strategies of the euryhaline medaka when subjected to hypoosmotic environments.     

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.     

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.     

Recombinant medaka (Oryzias melastigmus) pro-hepcidin: Multifunctional characterization

Recently, two hepcidin variant genes (Om-hep1 and Om-hep2) were identified in a model fish marine medaka and both were highly induced in vivo with bacterial challenge, suggesting that the medaka hepcidin may have a similar function to other reported teleostean hepcidins. In the present study, the antibacterial, antiviral and antitumor activities of Om-hep1 were determined using its synthetic and recombinant pro-peptides. The recombinant pro-hepcidin1 was expressed in Escherichia coli and an effective method to produce recombinant Pro-Omhep1 was developed in order to obtain a right folded structure. The results showed that both the synthetic mature peptide and recombinant pro-peptide had similar antibacterial activity against Gram-positive and negative bacteria. In particular, both the synthetic mature Om-hep1 and recombinant Pro-Omhep1 inhibited the viral replication of white spot syndrome virus in the hematopoietic tissue cells of the crayfish Cherax quadricarinatus. Om-hep1 also presented antitumor activity on the cultured human hepatocellular carcinoma cells. In addition, the antimicrobial mechanism of Om-hep1 was measured and it was found that Om-hep1 was likely to be non-membranolytic. The recombinant Pro-Omhep1 performed better biological activity compared to the synthetic mature Om-hep1. This study suggested that Om-hep1 was likely to be an important multifunction protein involved in various resistance actions in the marine medaka immune system.     

Atypical piscine mycobacteriosis in Japanese medaka (Oryzias latipes)

Japanese medaka, (Oryzias latipes), small, freshwater, tropical cyprinodonts, are principally used for toxicologic and carcinogenicity assays, but are finding more applications in developmental genetic and biological research. An increase in mortality began in brood stock of adult medaka that had been shipped and housed separately by sex. Initially, mortality averaged one fish daily and began in females two weeks after they were received. Cohabitation began eight weeks after arrival. After four to six weeks of cohabitation in different spawning aquaria, mortality was observed in males. Clinical signs of disease included loss of scale luster and color, with subsequent blanching of dorsal flank musculature, small raised nodules on various external surfaces, emaciation, fraying of fin tips, and equilibrium disturbances. Histologic examination of affected adults revealed multi-organ granulomatous inflammation with intracellular acid-fast bacilli. Specimens from 46 juvenile medaka that were spawned from affected adults, were submitted for culture and histologic evaluation. Of 18 fish, two had lesions similar to those of adults. The organism isolated from the remaining fish was identified as Mycobacterium fortuitum. Due to atypical rapid progression of disease, spread of M. fortuitum to progeny, and poor prognosis, the entire colony was euthanized.     

Specification of primordial germ cells in medaka (Oryzias latipes)

Background  

Primordial germ cells (PGCs) give rise to gametes that are responsible for the development of a new organism in the next generation. Two modes of germ line specification have been described: the inheritance of asymmetrically-localized maternally provided cytoplasmic determinants and the induction of the PGC fate by other cell types.     

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

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