Evidence for different origins of sex chromosomes in closely related Oryzias fishes: substitution of the master sex-determining gene

The medaka Oryzias latipes and its two sister species, O. curvinotus and O. luzonensis, possess an XX-XY sex-determination system. The medaka sex-determining gene DMY has been identified on the orthologous Y chromosome [O. latipes linkage group 1 (LG1)] of O. curvinotus. However, DMY has not been discovered in other Oryzias species. These results and molecular phylogeny suggest that DMY was generated recently [approximately 10 million years ago (MYA)] by gene duplication of DMRT1 in a common ancestor of O. latipes and O. curvinotus. We identified seven sex-linked markers from O. luzonensis (sister species of O. curvinotus) and constructed a sex-linkage map. Surprisingly, all seven sex-linked markers were located on an autosomal linkage group (LG12) of O. latipes. As suggested by the phylogenetic tree, the sex chromosomes of O. luzonensis should be "younger" than those of O. latipes. In the lineage leading to O. luzonensis after separation from O. curvinotus approximately 5 MYA, a novel sex-determining gene may have arisen and substituted for DMY. Oryzias species should provide a useful model for evolution of the master sex-determining gene and differentiation of sex chromosomes from autosomes.     

Diverse adaptability in oryzias species to high environmental salinity

The genus Oryzias containing freshwater (FW) and seawater (SW) species is a potential model for studying mechanisms of osmotic adaptation. In this study, we compared SW adaptability of four Oryzias species, O. javanicus, O. dancena, O. latipes and O. marmoratus inhabiting different osmotic environments. SW adaptability was evaluated at several stages of the lifecycle: (i) survival rates of adult fish after transfer from FW to 50%SW or SW, (ii) spawning ability in FW and SW, (iii) fertilization rates in FW and SW, and (iv) hatching rates in FW, 50%SW and SW. Results obtained agreed with the natural habitat of each species: O. javanicus, which inhabits SW or brackish water (BW), is fully adaptable to both SW and FW at all the stages examined. The BW species O. dancena also revealed high SW adaptability except for the hatching rate. O. marmoratus, confined in FW, exhibited low SW adaptability at all stages examined while O. latipes, another FW species, was adaptable to SW at most stages examined. Based on these results, the role of SW adaptability to the distribution area of each species is discussed.     

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.

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).     

The Tol1 element of the medaka fish, a member of the hAT transposable element family, jumps in Caenorhabditis elegans

Tol1 is a DNA-based transposable element residing in the genome of the medaka fish Oryzias latipes, and has been proven to be transposed in various vertebrate species, including mammals. This element belongs to the hAT (hobo/Activator/Tam3) transposable element family, whose members are distributed in a wide range of organisms. It is thus possible that Tol1 is mobile in organisms other than vertebrates. We here show that transposition of this element occurs in the nematode Caenorhabditis elegans. A donor plasmid containing a Tol1 element and a helper plasmid carrying the transposase gene were delivered into gonad cells and, after several generations of culturing, were recovered from worms. PCR analysis of the donor plasmid, using primers that encompassed the Tol1 element, revealed excision of the Tol1 portion from the plasmid. Analysis of genomic DNA of the worms by the inverse PCR method provided evidence that Tol1 had been integrated into the C. elegans chromosomes. Vertebrates and C. elegans are phylogenetically distantly related organisms in that the former are deuterostomes and the latter a protostome animal. Our results indicate (1) the transposition reaction of the Tol1 element requires, besides the transposase, no factors from host cells, or (2) the host factors, even if required, are those that are common to protostomes and deuterostomes. The results also have significance for the development of a gene transfer vector and other biotechnology tools for C. elegans.     

Identification of the sex-determining locus in the Thai medaka, Oryzias minutillus

A sex-determining gene, DMY, which is comparable to the SRY gene in mammals, has been identified in the medaka, Oryzias latipes. Although Oryzias curvinotus, a closely related species to O. latipes also has DMY, this gene has not been found in other Oryzias fishes. It has recently been demonstrated that the sex chromosomes of Oryzias dancena and Oryzias hubbsi differ from those of O. latipes and these species have XX/XY and ZZ/ZW systems, respectively. This may suggest that Oryzias species have evolved different sex-determining genes on different sex chromosomes. In the present study, we investigated the sex determination mechanism in Oryzias minutillus, which is closely related to O. dancena and O. hubbsi. Linkage analysis using 14 isolated sex-linked DNA markers showed that this species has an XX/XY sex determination system. These sex-linked markers were located on linkage group 8 of O. latipes, suggesting that the sex chromosomes of O. minutillus are homologous to the autosomes of other Oryzias species. Furthermore, fluorescence in situ hybridization using a tightly sex-linked marker demonstrated that the XY sex chromosomes of O. minutillus and O. dancena were not homologous. These findings provide additional evidence for independent origins of sex chromosomes and sex-determining genes in these closely related species.     

PCR detection of excision suggests mobility of the medaka fish Tol1 transposable element in the frog Xenopus laevis

Tol1 is a DNA-based transposable element identified in the medaka fish Oryzias latipes and a member of the hAT (hobo/Activator/Tam3) transposable element family. Its mobility has already been demonstrated in the human and mouse, in addition to its original host species. This element is thus expected to be useful in a wide range of vertebrates as a genomic manipulation tool. Herein, we show that the Tol1 element can undergo excision in the African clawed frog Xenopus laevis, a major model organism for vertebrate genetics and developmental biology. An indicator plasmid carrying a Tol1 element was injected into 2- or 4-cell-stage embryos together with either a helper plasmid coding for the full-length Tol1 transposase or a modified helper plasmid yielding a truncated protein, and recovered from tailbud-stage embryos. Deletion of the Tol1 region of the indicator plasmid was observed in the experiment with the full-length transposase, and not in the other case. The deletion was associated with various footprint sequences at breakpoints, as frequently observed with many DNA-based transposable elements. These results indicate that the Tol1 element was excised from the indicator plasmid by catalysis of the transposase, and suggest that the Tol1 element is mobile in this frog species.     

Targeted reduction of the DNA methylation level with 5-azacytidine promotes excision of the medaka fish Tol2 transposable element

The Tol2 element of the medaka fish Oryzias latipes is a member of the hAT (hobo/Activator/Tam3) transposable element family. There is evidence for rapid expansion in the genome and throughout the species in the past but a high spontaneous transposition rate is not observed with current fish materials, suggesting that the Tol2 element and its host species have already acquired an interactive mechanism to control the transposition frequency. DNA methylation is a possible contributing factor, given its involvement with many other transposable elements. We therefore soaked embryos in 5-azacytidine, a reagent that causes reduction in the DNA methylation level, and examined amounts of PCR products reflecting the somatic excision frequency, obtaining direct evidence that exposure promotes Tol2 excision. Our results thus suggest that methylation of the genome DNA is a factor included in the putative mechanisms of control of transposition of the Tol2 element.     

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.     

Tol1转座子研究进展

Tol1和Tol2是在青鳉基因组中发现的具有自主活性的DNA转座子,而Tol1转座子的自主活性是新近才发现的,因此对它的报道较少。较之Tol2,Tol1可以携带更大片段的DNA进行转座,且Tol1的转座不受转座酶"过量表达抑制"的影响。研究已证实,Tol1转座子在秀丽线虫、斑马鱼、爪蟾和人等多种生物中具有转座活性。因此,在动物转基因和基因功能研究等方面有重要的应用前景。从Tol1转座子的结构特征、转座机制和作为基因转移载体的优点,以及应用研究等方面进行了简要的综述。     

The XX-XY sex-determination system in Oryzias luzonensis and O. mekongensis revealed by the sex ratio of the progeny of sex-reversed fish

The sex-determining gene in Oryzias latipes and O. curvinotus has been proved to be DMY. Although O. curvinotus has the DMY gene on the Y chromosome which is homologous to the Y chromosome of O. latipes, the sex-determining mechanism of other Oryzias fishes has not been identified. In order to uncover the sex-determining mechanism of O. luzonensis and O. mekongensis, which are most closely related species to O. latipes and O. curvinotus, we analyzed the sex ratio of the progeny of sex-reversed fish. We were able to obtain sex-reversed males by the administration of methyltestosterone, and found that these yielded all-female offspring in both species. These results indicate that O. luzonensis and O. mekongensis have the XX-XY sex-determination system.     

Detection of de novo insertion of the medaka fish transposable element Tol2

Tol2 is a terminal-inverted-repeat transposable element of the medaka fish Oryzias latipes. It is a member of the hAT (hobo/Activator/Tam3) transposable element family that is distributed in a wide range of organisms. We here document direct evidence for de novo insertion of this element. A Tol2 clone marked with the bacterial tetracycline-resistance gene was microinjected into fertilized eggs together with a target plasmid, and the plasmid was recovered from embryos. The screening of plasmid molecules after transformation into Escherichia coli demonstrated transposition of tet into the plasmid and, by inference, precise insertion of Tol2 in medaka fish cells. De novo excision of Tol2 has previously been demonstrated. The present study provides direct evidence that the Tol2 element has the entire activity necessary for cut-and-paste transposition. Some elements of the mariner/Tc1 family, another widespread group, have already been applied to development of gene tagging systems in vertebrates. The Tol2 element of the hAT family, having different features from mariner/Tc1 family elements, also has potential as an alternative gene tagging tool in vertebrates.     

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.     

Molecular phylogeny of the medaka fishes genus Oryzias (Beloniformes: Adrianichthyidae) based on nuclear and mitochondrial DNA sequences

The phylogenetic relationships among medaka fishes of 2 genera, Oryzias and Xenopoecilus, were studied using the nuclear tyrosinase and mitochondrial 12S and 16S rRNA genes. Of the 23 species currently described for these genera, 13 species of Oryzias and 2 species of Xenopoecilus were examined. The tree topologies obtained from the nuclear and mitochondrial data were consistent, indicating that Xenopoecilus is a polyphyletic genus nested within Oryzias. This result suggested the necessity for a systematic study and taxonomic revision of Xenopoecilus. The combined data analysis of all data partitions resulted in a well-resolved tree, with most internal branches supported by high statistical values. Based on our combined data phylogeny, we divided the Oryzias species into three major species groups, namely the latipes, javanicus, and celebensis groups. These three groups corresponded to the three chromosomal groups (biarmed, monoarmed, and fused chromosome groups) previously proposed from karyological analyses. The phylogeographic pattern suggests historical vicariance between Sulawesi Island and the continental shelf.     

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.     

Cytogenetic studies on the origin and species differentiation of the Philippine medaka, Oryzias luzonensis

The origin and species differentiation of the Philippine medaka, Oryzias luzonensis , was studied by means of karyotype analyses and interspecific hybridyzations. The karyotype revealed 2n = 48 consisting of 24 pairs of biarmed chromosomes (NF = 96). Characteristic C-bands were found in three sub-metacentric pairs. Nucleolar organizer regions (NORs) were detected on the short arms of the satellited sub-metacentric pair. Estimated DNA-value was 1.9 pg per nucleus. Interspecific crosses revealed that O. luzonensis was allied most closely to O. latipes (biarmed chromosome type), less closely to O. celebensis (fused chromosome type), and least closely to O. melastigma and O.javanicus (monoarmed chromosome type), which are distributed in east Asia, the Sulawesi, and south-west Asia and west side of south-east Asia, respectively. The external appearance of O. luzonensis was also more similar to that of O. latipes . From these results, we concluded that O. luzonensis is a member of the biarmed chromosome group and the nearest relative to O. latipes . Thus, the Philippine medaka seems to be of east Asiatic origin, having migrated to northern Luzon from the China continent via Formosa.     

Spatial-temporal expression of pum1 and pum2 in medaka Oryzias latipes

Two pumilios, pum1 and pum2, were identified in medaka Oryzias latipes. Oryzias latipes pum1 and pum2 are ubiquitous in the adult tissues but with specific expression in the germ cells of gonads, ovary and testis. Pum1 is expressed in the spermatogonia to spermatocytes whilst pum2 presents in spermatocytes of testis only. Oryzias latipes pum1 and pum2 are maternally supplied RNA with ubiquitous expression in the early stages, and embryonic expression of pum1 and pum2 may begin from early gastrula. Both pum1 and pum2 are expressed in the tissues including brain, eye and trunk, and both are expressed in the gonads after hatching. Taken together, Pum1 and Pum2 may play important roles in embryonic and germ cell development of O. latipes.     

Homogeneity in the structure of the medaka fish transposable element Tol2

The hAT family is a group of transposable elements of the terminal inverted repeat class, which includes Ac of maize, hobo of Drosophila and Tam3 of Antirrhinum (snapdragon). All the members of this family so far examined are known to comprise complete and defective copies, with a good correspondence to autonomous and non-autonomous elements, respectively. Internal deletion is the most common cause of defective copies. Tol2, a transposable element of the medaka fish Oryzias latipes, is a member of the hAT family. We examined, mainly by the genomic Southern blot analysis, variation in the structure of copies of this element, and revealed that there are few or no internally deleted copies. This situation is unusual in a member of the hAT family. Possible causes of this anomaly are discussed.     

金鱼hAT家族转座子Tgf2的克隆及其结构

hAT家族转座子以果蝇hobo、玉米Ac和金鱼草(Ceratophyllum demersum L.)Tam3为代表,以"剪切-粘帖"方式进行DNA转座。1996年,日本学者首次在白化青鳉(Oryzias latipes)中发现具有天然活性的脊椎动物hAT家族转座子,即青鳉Tol2转座子,该转座子已在模式生物斑马鱼转基因、基因和启动子捕获方面进行了广泛应用。文章根据玉米Ac与青鳉Tol2转座子序列保守区设计一对引物,在19种不同鱼类物种或品系中进行PCR筛选,最后发现此类hAT家族转座子在我国不同品系金鱼中存在,命名为金鱼Tgf2转座子。金鱼Tgf2转座子全长4720bp,由4个阅读框组成,与青鳉Tol2转座子的相似度为97%。金鱼Tgf2与青鳉Tol2转座子在末端倒位重复和亚末端重复上存在一定差异,此外,金鱼Tgf2转座子的中间反向重复序列(1453bp到2091bp)可形成一种"十"字结构,明显有别于青鳉Tol2转座子形成的茎环结构,这些区域与转座活性密切相关。文章预示金鱼Tgf2转座子可能具有更高的天然转座活性,构建高效金鱼Tgf2转基因元件可供鱼类转基因和基因捕获研究。