High incidence of mosaic mutations induced by irradiating paternal germ cells of the medaka fish, Oryzias latipes.

Delayed-type mutations induced by radiation have recently been demonstrated in various somatic-cell systems. Such mutations are thought to result from the transmission of genetic instability through many cell divisions subsequent to a single exposure to ionizing radiation. Here, we have examined whether 'transgenerational' delayed-type mutations can arise during embryonic development of the medaka fish as a result of exposing the sperm and spermatids of live fish to 137Cs gamma-radiation. To do this, we made use of a sensitive specific-locus test (SLT) for the medaka that we have recently developed. Because the medaka has a transparent egg membrane and embryo body, both visible mosaics and whole-body mutations can be detected during development at an early-expressed pigmentation locus. When wild-type +/+ males were gamma-irradiated and then mated with wl/wl females, the frequency of F1 embryos with both wild-type orange leucophores (wl/+) and mutant-type white leucophores (wl/wl*) (mosaic mutants) was about 5.7x10(-3)/Gy. The frequency of embryos with only white leucophores (whole-body mutants) was about 1.3x10(-3)/Gy. These results suggest that delayed mutations frequently arise in medaka fish embryos that have been fertilized with irradiated sperm. Some possible mechanisms involved in the generation of these delayed mutational events (including genomic instability in the early embryos) are discussed.     

Interspecific hybridization between Oryzias latipes and Oryzias curvinotus causes XY sex reversal

The teleost fish, Oryzias curvinotus, is a closely related species to the medaka, Oryzias latipes, and both species have the DMY gene, which is required for male development in O. latipes. It suggests that the molecular function of the DMY gene and the following molecular events of sex differentiation are conserved between these two species. In the present study, we obtained interspecific hybrids between O. curvinotus and O. latipes and demonstrated sex-reversed XY females in the hybrids. The incidence of sex-reversed females in F1 XY hybrids between O. curvinotus females and O. latipes males, and hybrids between O. latipes females and O. curvinotus males were 21% and 100%, respectively. These results indicate that DMY does not always determine maleness in hybrid fish even though it is able to specify normal male development on its native genetic background and suggest that there are some differences between DMY(latipes) and DMY(curvinotus) alleles. Appearance of XY females in F1 hybrids also suggests that an autosomal or X-liked gene(s) from the maternal species interferes in the function of the paternal DMY gene in the male-determining process of the hybrid fish. These hybrid fish would supply a new experimental approach for investigating the genetic and molecular pathway of testis determination and differentiation.     

Genomic analysis of gamma-ray-induced germ-cell mutations at the b locus recovered from the medaka specific-locus test.

To study how gamma-ray-induced germ-cell mutations are fixed at the early embryonic stage of the next generation, genomic alterations in the b locus mutants (colorless melanophores) detected during development in the medaka specific-locus test (SLT) were analyzed. First, nine anonymous DNA markers linked to the b locus were cloned and mapped into the region extending about 47cM surrounding the b locus. Next, losses of paternal alleles of these DNA markers were examined in each of the 51 gamma-ray-induced b locus mutants obtained after irradiation of sperm or spermatids. In these mutants, 47 were dominant lethals, three were semi-viable and one was viable. All the mutants examined had large deletions surrounding the b locus. One viable mutant had an interstitial deletion, while all the semi-viable and dominant lethal ones appeared to have terminal deletions. Deletions extending about 20-35cM were the most frequently observed in 18 of the 51 mutants examined. The largest one extended more than 40cM. These results suggest that most of the gamma-ray induced germ cell mutations recovered as total specific-locus mutants were accompanied by large genomic deletions, which eventually led the mutant embryos to dominant lethality.     

Generation of transgenic medaka using modified bacterial artificial chromosome

The availability of bacterial artificial chromosome (BAC) offers a good genomic platform for a targeted integration of an exogenous gene by a homologous recombination system in Escherichia coli . In combination with microinjection technology, this system allows for the analysis of various aspects of biological phenomena occurring in vivo using Japanese medaka fish ( Oryzias latipes ). Here we describe a streamlined procedure for selecting BAC clones based on the medaka University of Tokyo genome browser (UTGB), followed by rapid modification with enhanced green fluorescent protein (EGFP) or DsRed fragments for transgenic analysis in medaka. Experimental procedures for BAC DNA preparation, microinjection of medaka embryos and screening of resulting transgenic medaka carrying EGFP/DsRed modified BAC clones are also described.     

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.     

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

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.     

Determination by GISH and FISH of hybrid status in Lilium

In the genus Lilium, plants obtained from crosses, especially between distant relatives, are not always hybrids because embryos can develop as a result of apomixis. These plants constitute genetic material of the maternal parent only. In this study, verification of hybrid status of plants which have been obtained from the crosses 'Marco Polo'xLilium henryi and 'Expression'xL. henryi was performed through the use of cytological and molecular cytogenetic methods. According to cytological analyses, all genotypes tested had 2n = 2x = 24 chromosomes. Genomic in situ hybridisation (GISH) was used for hybrid verification. In hybrid plants, this method distinguished all paternal and maternal chromosomes at the stage of somatic metaphase and prophase. For GISH, paternal genomic DNA was used as a probe and maternal DNAs were used as blocks. Fluorescence in situ hybridisation (FISH) with 5S rDNA and 25S rDNA probes was used as the second method of hybrid verification. Selected chromosome markers based on genome-specific localisation of rDNA loci were used for analysis of the F1 hybrids obtained from the crosses 'Marco Polo'xL. henryi and 'Expression'xL. henryi. The presence of marker chromosomes characteristic for each of the paternal genotypes was a confirmation that the plants obtained were hybrids.     

Erasing genomic imprinting memory in mouse clone embryos produced from day 11.5 primordial germ cells

Genomic imprinting is an epigenetic mechanism that causes functional differences between paternal and maternal genomes, and plays an essential role in mammalian development. Stage-specific changes in the DNA methylation patterns of imprinted genes suggest that their imprints are erased some time during the primordial germ cell (PGC) stage, before their gametic patterns are re-established during gametogenesis according to the sex of individuals. To define the exact timing and pattern of the erasure process, we have analyzed parental-origin-specific expression of imprinted genes and DNA methylation patterns of differentially methylated regions (DMRs) in embryos, each derived from a single day 11.5 to day 13.5 PGC by nuclear transfer. Cloned embryos produced from day 12.5 to day 13.5 PGCs showed growth retardation and early embryonic lethality around day 9.5. Imprinted genes lost their parental-origin-specific expression patterns completely and became biallelic or silenced. We confirmed that clones derived from both male and female PGCs gave the same result, demonstrating the existence of a common default state of genomic imprinting to male and female germlines. When we produced clone embryos from day 11.5 PGCs, their development was significantly improved, allowing them to survive until at least the day 11.5 embryonic stage. Interestingly, several intermediate states of genomic imprinting between somatic cell states and the default states were seen in these embryos. Loss of the monoallelic expression of imprinted genes proceeded in a step-wise manner coordinated specifically for each imprinted gene. DNA demethylation of the DMRs of the imprinted genes in exact accordance with the loss of their imprinted monoallelic expression was also observed. Analysis of DNA methylation in day 10.5 to day 12.5 PGCs demonstrated that PGC clones represented the DNA methylation status of donor PGCs well. These findings provide strong evidence that the erasure process of genomic imprinting memory proceeds in the day 10.5 to day 11.5 PGCs, with the timing precisely controlled for each imprinted gene. The nuclear transfer technique enabled us to analyze the imprinting status of each PGC and clearly demonstrated a close relationship between expression and DNA methylation patterns and the ability of imprinted genes to support development.     

A critical stage in spermatogenesis for radiation-induced cell death in the medaka fish, Oryzias latipes

To ensure the high-fidelity transmission by reproductive cells of genetic information from generation to generation, cells have evolved surveillance systems to eliminate genomic lesions by inducing cell suicide and/or DNA repair. In this report, gamma-ray-induced cell death was investigated using the medaka fish, Oryzias latipes, because of the ease with which the differentiation stages of its spermatogenic cells can be identified. After 4.75 Gy gamma irradiation, the maximum rate of death of spermatogonial stem cells was observed at 18 h, and that of differentiating spermatogonia was at 12 h, followed by a peak in the extent of DNA fragmentation detected by the TUNEL assay. Dose-response curves for the death rate showed an obvious increase in the death rate for early-differentiating spermatogonia even after 0.11 Gy irradiation, whereas there were no such increases for spermatogonial stem cells and late-differentiating spermatogonia. In the male germ cells of this fish, the stage during spermatogenesis most sensitive to radiation-induced cell death is in early-differentiating spermatogonia, the immediate descendants of the stem cells. These spermatogonia may have a rigorous surveillance system for genomic lesions induced in spermatogonial stem cells.     

雌核发育银鲫子代中微卫星特异序列分析

雌核发育个体的基因型基本上完全与母本相同,这是源于卵子发生过程中没有经过减数分裂.父本的遗传物质是在随机水平、亚基因组水平或基因组水平参与到子代的遗传重组过程,从而对长期突变积累的雌核发育生物基因组进行补偿,一直是遗传学家关注的问题.本文对雌核发育银鲫特异个体及父母本5个微卫星位点的扩增条带进行了克隆测序,相似性比对结果显示,特异个体所表现的父咎匾霥NA条带,序列结果与父本完全相同或相似(SCM4、SCM9、YJ5),并在某些位点上保留了母本的特异条带(YJ5),而个体本身特异的DNA条带与父母本的相似性均较高(SCM13).同时,所检测到的个体经越冬后查验为雌性个体,进一步进行同源繁育,研究变异条带在繁殖中的命运.连续2代的繁育检测结果表明,融合了父本特异性条带的银鲫个体在繁殖过程中仍行雌核发育的生殖方式,变异来的条带能够传递给子代,进一步证实同源雌核发育银鲫通过小概率两性融合事件丰富银鲫种群的遗传多样性[动物学报 53(3):537-544,2007].     

Sex reversal of genetic females (XX) induced by the transplantation of XY somatic cells in the medaka,Oryzias latipes

In order to investigate the function of gonadal somatic cells in the sex differentiation of germ cells, we produced chimera fish containing both male (XY) and female (XX) cells by means of cell transplantation between blastula embryos in the medaka, Oryzias latipes. Sexually mature chimera fish were obtained from all combinations of recipient and donor genotypes. Most chimeras developed according to the genetic sex of the recipients, whose cells are thought to be dominant in the gonads of chimeras. However, among XX/XY (recipient/donor) chimeras, we obtained three males that differentiated into the donor's sex. Genotyping of their progeny and of strain-specific DNA fragments in their testes showed that, although two of them produced progeny from only XX spermatogenic cells, their testes all contained XY cells. That is, in the two XX/XY chimeras, germ cells consisted of XX cells but testicular somatic cells contained both XX and XY cells, suggesting that the XY somatic cells induced sex reversal of the XX germ cells and the XX somatic cells. The histological examination of developing gonads of XX/XY chimera fry showed that XY donor cells affect the early sex differentiation of germ cells. These results suggest that XY somatic cells start to differentiate into male cells depending on their sex chromosome composition, and that, in the environment produced by XY somatic cells in the medaka, germ cells differentiate into male cells regardless of their sex chromosome composition.     

Unusual triploid males in a microchromosome-carrying clone of the Amazon molly, Poecilia formosa

The Amazon molly, Poecilia formosa, is an all-female fish of hybrid origin which reproduces by gynogenesis, i.e. it depends on sperm of males of closely related species to trigger parthenogenetic development of the embryo. Therefore the offspring is clonal and identical to the mother. In rare cases the exclusion mechanism fails and paternal introgression occurs. This may result either in triploid offspring - if the whole haploid chromosome set of the sperm fuses with the diploid egg nucleus - or in siblings with microchromosomes - if only subgenomic amounts of paternal DNA are included. In one of our diploid, microchromosome-carrying laboratory stocks we observed eight triploid individuals which all developed into males. We investigated the mitotic and meiotic chromosomes, the synaptonemal complex (SC), and sperm production of these males, and compared them to males of the gonochoristic parental species (P. latipinna and P. mexicana) and their hybrids. This comparison revealed that P. formosa males are functional males with reduced effective fertility. They show a deviation from the typical 23 bivalents in the synaptonemal complexes as well as in diakinesis due to the triploid state. They produced offspring but only with gynogenetic Amazon molly females. This shows that the probably aneuploid sperm from P. formosa males can trigger parthenogenetic development of unreduced eggs.     

Oryzias curvinotus has DMY,a gene that is required for male development in the medaka,O. latipes

DMY is a Y-specific DM-domain gene required for male development and appears to be the sex-determining gene in the teleost fish medaka, Oryzias latipes. Although the genomic region containing DMY appears to have originated through duplication of the DMRT1 region, it is unknown when the duplication occurred. Here we show that O. curvinotus also has the DMY gene on the Y chromosome, which is homologous to the Y chromosome of medaka, and that DMY is expressed in XY embryos. A phylogenetic tree based on the amino acid sequence including the DM-domain shows that DMY was derived from DMRT1 immediately before speciation of O. latipes and O. curvinotus.     

Genomic structure and expression of the soluble guanylyl cyclase alpha2 subunit gene in the medaka fish Oryzias latipes

A cDNA clone encoding the soluble guanylyl cyclase alpha2 subunit was isolated from medaka fish (Oryzias latipes) and designated as OlGCS-alpha2. The OlGCS-alpha2 cDNA was 3,192 bp in length and the open reading frame (ORF) encodes a protein of 805 amino acids. The deduced amino acid sequence has high similarity to that of the mammalian alpha2 subunit gene except for the N-terminal regulatory domain. The C-terminal 5 amino acids, "RETSL", which have been reported to interact with the post synaptic density protein (PSD)-95 were conserved. An RNase protection assay with adult fish organs showed that OlGCS-alpha2 was expressed mainly in the brain and testis. The complete nucleotide sequence (about 41 kbp) of the OlGCS-alpha2 genomic DNA clone isolated from a medaka fish BAC library indicated that the OlGCS-alpha2 gene consisted of 9 exons and 8 introns. The 5'-flanking region and larger introns, such as introns 1, 4, and 7, contained the several fragments conserved in the nucleotide sequences of Rex6 (non-long terminal repeat retrotransposon), MHC class I genomic region, and OlGC1, the medaka fish homolog of the mammalian guanylyl cyclase B gene. Linkage analysis on the medaka fish chromosome demonstrated that the OlGCS-alpha2 gene was mapped to LG13; this mapping position was different from those for the OlGCS-alpha1 and OlGCS-beta1 genes (LG1).     

Radiosensitivity of Drosophila lines. VII. The effect of the maternal genotype on the yield of recessive and dominant lethal mutations induced by the gamma irradiation of males

The effect of material repair on induction of paternal mutations was tested with radiosensitive rad(2)201G1 mutant. Basc males were irradiated at doses from 0 to 60 Gy of gamma-rays and mated to the radiosensitive mutant or control females. Frequencies of sex-linked recessive lethals and dominant lethals (induced in the paternal genome) were determined. With control females, the rate of recessive lethals increased linearly from 0 to 60 Gy. With rad(2)201G1 mutant, an increase in spontaneous and induced rates of paternal dominant lethals was observed; the rate of sex-linked recessive lethals increased non-linearly from 0 to 60 Gy.