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.     

Sexually dimorphic expression of the sex chromosome-linked genes cntfa and pdlim3a in the medaka brain

In vertebrates, sex differences in the brain have been attributed to differences in gonadal hormone secretion; however, recent evidence in mammals and birds shows that sex chromosome-linked genes, independent of gonadal hormones, also mediate sex differences in the brain. In this study, we searched for genes that were differentially expressed between the sexes in the brain of a teleost fish, medaka (Oryzias latipes), and identified two sex chromosome genes with male-biased expression, cntfa (encoding ciliary neurotrophic factor a) and pdlim3a (encoding PDZ and LIM domain 3 a). These genes were found to be located 3–4 Mb from and on opposite sides of the Y chromosome-specific region containing the sex-determining gene (the medaka X and Y chromosomes are genetically identical, differing only in this region). The male-biased expression of both genes was evident prior to the onset of sexual maturity. Sex-reversed XY females, as well as wild-type XY males, had more pronounced expression of these genes than XX males and XX females, indicating that the Y allele confers higher expression than the X allele for both genes. In addition, their expression was affected to some extent by sex steroid hormones, thereby possibly serving as focal points of the crosstalk between the genetic and hormonal pathways underlying brain sex differences. Given that sex chromosomes of lower vertebrates, including teleost fish, have evolved independently in different genera or species, sex chromosome genes with sexually dimorphic expression in the brain may contribute to genus- or species-specific sex differences in a variety of traits.     

Differences in recombination frequencies during female and male meioses of the sex chromosomes of the medaka, Oryzias latipes

In the medaka, Oryzias latipes, sex is determined chromosomally. The sex chromosomes differ from those of mammals in that the X and Y chromosomes are highly homologous. Using backcross panels for linkage analysis, we mapped 21 sequence tagged site (STS) markers on the sex chromosomes (linkage group 1). The genetic map of the sex chromosome was established using male and female meioses. The genetic length of the sex chromosome was shorter in male than in female meioses. The region where male recombination is suppressed is the region close to the sex-determining gene y, while female recombination was suppressed in both the telomeric regions. The restriction in recombination does not occur uniformly on the sex chromosome, as the genetic map distances of the markers are not proportional in male and female recombination. Thus, this observation seems to support the hypothesis that the heterogeneous sex chromosomes were derived from suppression of recombination between autosomal chromosomes. In two of the markers, Yc-2 and Casp6, which were expressed sequence-tagged (EST) sites, polymorphisms of both X and Y chromosomes were detected. The alleles of the X and Y chromosomes were also detected in O. curvinotus, a species related to the medaka. These markers could be used for genotyping the sex chromosomes in the medaka and other species, and could be used in other studies on sex chromosomes.     

Induction of mutations in males of the fish Oryzias latipes at a specific locus after gamma-irradiation

We have studied frequencies of mutations induced at the b locus of the fish, Medaka Oryzias latipes, after gamma-irradiation. Homozygotes for the b locus have colorless melanophores whose phenotypic expression can be distinguished from that of the wild type. An advantage of the use of oviparous fish for detection of skin color mutations is that the mutant phenotype can be confirmed as early as 1.5 days after fertilization because of the transparent egg membrane of the embryo. Wild-type (B/B) male fish were exposed to 4.75 or 9.5 Gy of 137Cs gamma-rays at a dose rate of 0.95 Gy/min and then mated with the female testers (b/b). A total of 77,761 F1 offspring were examined for mutation and other abnormalities. In the control, we had 1 mutant among 22,068 offspring, resulting in a mutation rate of 4.53 X 10(-5)/locus/gamete. However, this mutant embryo died before hatching. Therefore, in an attempt to present specific-locus mutation frequencies in the fish, the frequencies of color mutants that survived more than 4 days after hatching were used as frequencies of viable mutants; (number of viable color mutants)/(number of hatched fry that survived more than 4 days after hatching). In the 4.75 Gy-irradiated group the viable mutant frequencies were 45.0 X 10(-5), 69.7 X 10(-5) and 0/locus/gamete, while exposure to 9.5 Gy resulted in mutation rates of 217 X 10(-5), 130 X 10(-5) and 8.06 X 10(-5), respectively, for sperm, spermatids and spermatogonia. In comparison with viable color mutant frequencies those of the total color mutants, which include such mutants as ones that died before hatching (defined as number of total color mutants/number of fertilized eggs minus number of early deaths), were considerably higher. For sperm, spermatids, and spermatogonia after exposure to 4.75 Gy, the frequencies were 1180 X 10(-5), 629 X 10(-5) and 9.90 X 10(-5)/locus/gamete, respectively, and in 9.5-Gy-irradiated fish, the frequencies were 1940 X 10(-5), 953 X 10(-5) and 55.5 X 10(-5). Although our data are incomplete, the present results were compared with mutation induction in mice. We concluded that the frequencies of viable color mutants in the fish can be compared with those in mice.     

Chromosomal distribution of repetitive DNA sequences highlights the independent differentiation of multiple sex chromosomes in two closely related fish species

The arrangement of 6 repetitive DNA sequences in the mitotic and meiotic sex chromosomes of 2 Erythrinidae fish, namely Hoplias malabaricus and Erythrinus erythrinus, both with a multiple X(1)X(1)X(2)X(2)/X(1)X(2)Y sex chromosome system, was analyzed using fluorescence in situ hybridization. The distribution patterns of the repetitive sequences were distinct for each species. While some DNA repeats were species-specific, others were present in the sex chromosomes of both species at different locations. These data, together with the different morphological types of sex chromosomes and the distinct chromosomal rearrangements associated with the formation of the neo-Y chromosomes, support the plasticity of sex chromosome differentiation in the Erythrinidae family. Our present data highlight that the sex chromosomes in fish species may follow diverse differentiation patterns, even in the same type of sex chromosome system present in cofamiliar species.     

Linkage Analysis Reveals the Independent Origin of Poeciliid Sex Chromosomes and a Case of Atypical Sex Inheritance in the Guppy (Poecilia reticulata)

Among different teleost fish species, diverse sex-determining mechanisms exist, including environmental and genetic sex determination, yet chromosomal sex determination with male heterogamety (XY) prevails. Different pairs of autosomes have evolved as sex chromosomes among species in the same genus without evidence for a master sex-determining locus being identical. Models for evolution of Y chromosomes predict that male-advantageous genes become linked to a sex-determining locus and suppressed recombination ensures their co-inheritance. In the guppy, Poecilia reticulata, a set of genes responsible for adult male ornaments are linked to the sex-determining locus on the incipient Y chromosome. We have identified >60 sex-linked molecular markers to generate a detailed map for the sex linkage group of the guppy and compared it with the syntenic autosome 12 of medaka. We mapped the sex-determining locus to the distal end of the sex chromosome. We report a sex-biased distribution of recombination events in female and male meiosis on sex chromosomes. In one mapping cross, we observed sex ratio and male phenotype deviations and propose an atypical mode of genetic sex inheritance as its basis.     

No effects of estrogen receptor overexpression on gonadal sex differentiation and reversal in medaka fish

In order to elucidate a possible role of estrogen receptor in the gonadal sex differentiation and the sex reversal with sex steroids, we examined for the formation of testis or ovary in transgenic medaka fish overexpressing the medaka estrogen receptor under the constitutive medaka beta-actin promoter. The transgenic fish underwent the genetically determined gonadal differentiation and showed the same sex-reversal rates as those of wild-type non-transgenic fish after treatments with estrogen and androgen. These results present invaluable data to reconsider the role of estrogen receptor in the gonadal sex determination.     

Evolutionary origin of the medaka Y chromosome

Genetic sex determination in an XX-XY chromosome system can be realized through a locus on the Y chromosome that makes the undifferentiated gonad develop into a testis. Although this mechanism is widespread, only in two cases so far have the corresponding master male sex-determining genes been identified. One is Sry, which initiates testes determination in most mammals. The other is dmrt1bY (syn. dmy), from the fish medaka, Oryzias latipes. The mammalian Y is roughly estimated to be over 200 million years old. The medaka Y may be considerably younger. A comparative analysis of the genus Oryzias revealed that one sister species of the medaka has dmrt1bY on a homologous Y chromosome, whereas in another closely related species only a non-sex-linked pseudogene is present. In all other species, dmrt1bY was not detected. The divergence time for the different species was determined with mitochondrial DNA sequences. The timing was confirmed by independent calculations based on dmrt1 sequences. We show that the medaka sex-determining gene originated approximately 10 million years ago. This makes dmrt1bY and the corresponding Y chromosome the youngest male sex-determining system, at least in vertebrates, known so far.     

Cross talk between germ cells and gonadal somatic cells is critical for sex differentiation of the gonads in the teleost fish, medaka (Oryzias latipes)

To evaluate the possible role of germ cells on sex differentiation of the gonads in vertebrates, the teleost fish, medaka ( Oryzias latipes ), was used to generate a gonad without germ cells. The germ cell-deficient medaka reveals multiple effects of germ cells on the process of sex differentiation. The previously isolated mutant medaka, hotei , with the excessive number of germ cells may support the contention that the proliferation of germ cells is related to feminization of the gonad. Futhermore, we show that two modes of proliferation for either maintenance of germ cells or commitment to gametogenesis are important components of the sex differentiation of medaka developing gonads. An intimate cross talk between germ cells and gonadal somatic cells during the sex differentiation will be discussed.     

Construction of a BAC library derived from the inbred Hd-rR strain of the teleost fish, Oryzias latipes

A large insert genomic bacterial artificial chromosome (BAC) library was constructed from the inbred Hd-rR strain of the medaka, Oryzias latipes. Approximately 92,000 clones were gridded on high-density replica filters. Insert analysis of randomly selected clones indicated a mean insert size of 210 kb and predicted a 24 times coverage of the medaka genome. The library was hybridized with a single locus DNA fragment, and the resulting positive clones were characterized and shown to be compatible with a 24-fold redundant library. This first large insert genomic library of the medaka should increase the speed of genomic analyses for this fish species.     

Estrogen-Independent Ovary Formation in the Medaka Fish, Oryzias latipes

To investigate whether a female sex steroid, estrogen, acts as a natural inducer of female gonadal sex determination (or ovary formation) in the medaka fish, Oryzias latipes, the effects of an aromatase inhibitor and anti-estrogens on sexual differentiation of gonads were examined. We found that both drugs did not show any discernible effects on the genetically determined sex differentiation in both sexes. However, the aromatase inhibitor impaired the paradoxical effects of androgen (a male sex steroid), and the anti-estrogens inhibited the male-to-female sex reversal caused by estrogen. Treatments of the fertilized eggs with androgen disturbed the gonadal sex developments in both sexes, suggesting that sex steroid synthesis is detrimental to the gonadal sex developments in the medaka embryos. These results are consistent with the previous observation that sex steroids are not synthesized before the onset of gonadal sex differentiation, and suggest that ovary formation in the genetic females of the medaka fish is not dependent on estrogen.     

A genetic screen for mutations affecting embryonic development in medaka fish (Oryzias latipes)

In a pilot screen, we assayed the efficiency of ethylnitrosourea (ENU) as a chemical mutagen to induce mutations that lead to early embryonic and larval lethal phenotypes in the Japanese medaka fish, Oryzias latipes. ENU acts as a very efficient mutagen inducing mutations at high rates in germ cells. Three repeated treatments of male fish in 3 mM ENU for 1 h results in locus specific mutation rates of 1.1-1.95 x10(-3). Mutagenized males were outcrossed to wild type females and the F1 offspring was used to establish F2 families. F2 siblings were intercrossed and the F3 progeny was scored 24, 48 and 72 h after fertilization for morphological alterations affecting eye development. The presented mutant phenotypes were identified using morphological criteria and occur during early developmental stages of medaka. They are stably inherited in a Mendelian fashion. The high efficiency of ENU to induce mutations in this pilot screen indicates that chemical mutagenesis and screening for morphologically visible phenotypes in medaka fish allows the genetic analysis of specific aspects of vertebrate development complementing the screens performed in other vertebrate model systems.     

Recombination is suppressed over a large region of the rainbow trout Y chromosome

The previous genetic mapping data have suggested that most of the rainbow trout sex chromosome pair is pseudoautosomal, with very small X-specific and Y-specific regions. We have prepared an updated genetic and cytogenetic map of the male rainbow trout sex linkage group. Selected sex-linked markers spanning the X chromosome of the female genetic map have been mapped cytogenetically in normal males and genetically in crosses between the OSU female clonal line and four different male clonal lines as well as in outcrosses involving outbred OSU and hybrids between the OSU line and the male clonal lines. The cytogenetic maps of the X and Y chromosomes were very similar to the female genetic map for the X chromosome. Five markers on the male maps are genetically very close to the sex determination locus ( SEX ), but more widely spaced on the female genetic map and on the cytogenetic map, indicating a large region of suppressed recombination on the Y chromosome surrounding the SEX locus. The male map is greatly extended at the telomere. A BAC clone containing the SCAR (sequence characterized amplified region) Omy - 163 marker, which maps close to SEX , was subjected to shotgun sequencing. Two carbonyl reductase genes and a gene homologous to the vertebrate skeletal ryanodine receptor were identified. Carbonyl reductase is a key enzyme involved in production of trout ovarian maturation hormone. This brings the number of type I genes mapped to the sex chromosome to six and has allowed us to identify a region on zebrafish chromosome 10 and medaka chromosome 13 which may be homologous to the distal portion of the long arm of the rainbow trout Y chromosome.     

A trans-species missense SNP in Amhr2 is associated with sex determination in the tiger pufferfish, Takifugu rubripes (fugu)

Heterogametic sex chromosomes have evolved independently in various lineages of vertebrates. Such sex chromosome pairs often contain nonrecombining regions, with one of the chromosomes harboring a master sex-determining (SD) gene. It is hypothesized that these sex chromosomes evolved from a pair of autosomes that diverged after acquiring the SD gene. By linkage and association mapping of the SD locus in fugu (Takifugu rubripes), we show that a SNP (C/G) in the anti-Müllerian hormone receptor type II (Amhr2) gene is the only polymorphism associated with phenotypic sex. This SNP changes an amino acid (His/Asp384) in the kinase domain. While females are homozygous (His/His384), males are heterozygous. Sex in fugu is most likely determined by a combination of the two alleles of Amhr2. Consistent with this model, the medaka hotei mutant carrying a substitution in the kinase domain of Amhr2 causes a female phenotype. The association of the Amhr2 SNP with phenotypic sex is conserved in two other species of Takifugu but not in Tetraodon. The fugu SD locus shows no sign of recombination suppression between X and Y chromosomes. Thus, fugu sex chromosomes represent an unusual example of proto-sex chromosomes. Such undifferentiated X-Y chromosomes may be more common in vertebrates than previously thought.     

The chromosomal distribution of microsatellite repeats in the genome of the wolf fish Hoplias malabaricus, focusing on the sex chromosomes

Distribution of 12 mono-, di- and tri-nucleotide microsatellites on the chromosomes of 2 karyomorphs with 2 distinct sex chromosome systems (a simple XX/XY - karyomorph B and a multiple X(1)X(1)X(2)X(2)/X(1)X(2)Y - karyomorph D) in Hoplias malabaricus, commonly referred to as wolf fish, was studied using their physical mapping with fluorescence in situ hybridization (FISH). The distribution patterns of different microsatellites along the chromosomes varied considerably. Strong hybridization signals were observed at subtelomeric and heterochromatic regions of several autosomes, with a different accumulation on the sex chromosomes. A massive accumulation was found in the heterochromatic region of the X chromosome of karyomorph B, whereas microsatellites were gathered at centromeres of both X chromosomes as well as in corresponding regions of the neo-Y chromosome in karyomorph D. Our findings are likely in agreement with models that predict the accumulation of repetitive DNA sequences in regions with very low recombination. This process is however in contrast with what was observed in multiple systems, where such a reduction might be facilitated by the chromosomal rearrangements that are directly associated with the origin of these systems.