Evolution of Different Y Chromosomes in Two Medaka Species, Oryzias Dancena and O

Evolution of Different Y Chromosomes in Two Medaka Species, Oryzias dancena and O. latipes

Yusuke Takehana,*,1,2 Diana Demiyah,*,1 Kiyoshi Naruse,† Satoshi Hamaguchi* and Mitsuru Sakaizumi* *Graduate School of Science and Technology, Niigata University, Ikarashi, Niigata 950-2181, Japan and †Department of Biological Science, School of Science, University of Tokyo, Bunkyo-ku, Tokyo, 113-0033, Japan Manuscript received November 14, 2006 Accepted for publication December 19, 2006

ABSTRACT 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.

LMOST all vertebrates have different sexes, males brates have several DM domain-containing genes and A and females. Despite such universal occurrence, one of these, DMRT1 (DM-related transcription factor sex can be determined by a variety of different mech- 1) has been implicated in male sexual development in anisms. Among the major vertebrate groups, highly mammals, birds, reptiles, and fishes (Raymond et al. divergent genetic and environmental factors control 1999; Smith et al. 1999; De Grandi et al. 2000; Guan sex determination. These include male heterogamety et al. 2000; Kettlewell et al. 2000; Marchand et al. (XX/XY system typified by mammals) and female het- 2000). The cDNA sequences of the medaka DMY and erogamety (ZZ/ZW system in birds and snakes), as well DMRT1 showed a high similarity (80%) and DMY as environmental sex determination systems (such as appears to have arisen through a gene duplication event a temperature-dependent sex determination in alli- of an autosomal DMRT1 gene (Matsuda et al. 2002; gators). In mammals, the sex-determining gene, SRY/ Nanda et al. 2002). Sry has been identified on the Y chromosome (Gubbay In contrast to the widespread distribution of Sry in et al. 1990; Sinclair et al. 1990). However, no equivalent mammals, the DMY gene has not been detected, even in genes have been found in nonmammalian vertebrates, closely related species. Although one sister species of until recently. O. latipes has the DMY gene on a homologous Y chro- In the medaka, Oryzias latipes, which has an XX/XY mosome (Matsuda et al. 2003), the gene has not been sex determination system, DMY was identified as the found in other Oryzias species (Kondo et al. 2003). Y-specific sex-determining gene (Matsuda et al. 2002; Fishes in the genus Oryzias have been divided into three Matsuda 2005). This gene encodes a putative tran- monophyletic species groups, the latipes, javanicus, and scription factor containing a DM domain, which was celebensis groups (Takehana et al. 2005) and a recent originally described as a DNA-binding motif found in phylogenetic analysis of DMY and DMRT1 genes from two proteins, DSX in Drosophila melanogaster and MAB-3 Oryzias species suggested that duplication of the DMRT1 in Caenorhabditis elegans (Raymond et al. 1998). Verte- gene (generating the DMY gene) appears to have occurred within the latipes group lineage (Kondo et al. 2004). These findings suggested that Oryzias fishes in 1 These authors contributed equally to this work. other species groups (javanicus and celebensis groups) 2Corresponding author: Department of Environmental Science, Faculty of Science, Niigata University, 8050 Ikarashi-2, Niigata, 950-2181, Japan. must have different sex-determining genes. Accordingly, E-mail: [email protected] comparisons between closely related medaka fishes with

Genetics 175: 1335–1340 (March 2007) 1336 Y. Takehana et al. different sex-determination mechanisms should be im- pattern between the parents, F1 and backcross progeny were portant in understanding how these diverse develop- genotyped and assessed as to whether such polymorphisms mental mechanisms evolve. segregated with phenotypic sex. Using these isolated sex- linked ESTs, a sex-linkage map of O. dancena was constructed. In this study, as a first step to identify a sex-deter- Fluorescence in situ hybridization analysis with fosmid and mining gene in other fish species, we investigated the BAC clones: A fosmid genome library of O. dancena was sex-determination system and sex chromosomes in O. constructed from an F1 male between CB and PK using the dancena, a member of the javanicus group (Takehana Copy Control Fosmid Library Production kit (Epicentre, et al. 2005). Because a previous cytogenetic study has Madison, WI). Fosmid clones containing sex-linked ESTs were screened by colony hybridization. A fosmid clone Od38_01 not reported heteromorphic sex chromosomes in this (containing BJ014360) was used as a probe. A bacterial ar- species (as O. melastigma)(Uwa et al. 1983), a genetic tificial chromosome (BAC) genomic library, constructed from analysis of sex-reversed individuals was performed to the Hd-rR strain of O. latipes (Matsuda et al. 2001), was also demonstrate whether this species has an XX/XY system screened and three clones, Md0173J11 (containing SL1), of sex determination. Moreover, we took advantage of Md0172B19 (containing DMRT1), and Md0172I07 (containing OLd17.11a), were isolated. These BAC clones were located, genomic tools established in O. latipes to isolate sex- respectively, on the sex chromosomes (LG 1) and on auto- linked DNA markers, map the sex-determining locus, somes (LG 9 and LG 10) in O. latipes. and identify the sex chromosomes. Metaphase cells from cultured caudal fins were prepared by standard cytogenetic methods (Uwa and Ojima 1981; Matsuda et al. 1998). Fluorescence in situ hybridization (FISH) was performed as described (Matsuda and Chapman 1995). Probe MATERIALS AND METHODS DNAs of the genomic clones were labeled separately by nick Fish: All fish used in this study were supplied from a translation using biotin-16dUTP (Roche, Indianapolis) or subcenter (Niigata University) of the National BioResource digoxigenin-11-dUTP (Roche). For two-color hybridization, Project (medaka) in Japan. Wild stocks of O. dancena were equal amounts of labeled probes and a 500-fold amount of originally collected at Chidambaram (CB), India, in 1981 and autoclaved genomic DNA of O. latipes were mixed with hybridi- at Phuket (PK), Thailand, in 1988 (for details, see Takehana zation solution and preannealed for 30 min at 37°. After over- et al. 2005). Fish were maintained in aquaria under an artificial night hybridization, probes were detected with avidin–FITC photoperiod of 14 hr light:10 hr dark at 27° 6 2°. (Roche) and rhodamine-labeled anti-digoxigenin antibodies Sex steroid treatments for sex reversal and progeny testing: (Roche). Slides were counterstained with 49,6-diamidino-2- The sex-reversal experiment was performed as previously de- phenylindole (DAPI) and examined under a Nikon Eclipse 80i scribed by Hamaguchi et al. (2004). Briefly, fertilized eggs of microscope using three filters (UV-1A, B-2A, and G-2A). O. dancena (CB) were incubated in water containing estradiol- Images were captured with a DXM1200C digital camera (Nikon, 17b (E2; Sigma, St. Louis) at 0.01, 0.04, and 0.2 mg/ml or Tokyo). methyltestosterone (MT; Sigma) at 0.001, 0.005, and 0.025 m g/ml. Hatched fry were transferred to normal tap water and RESULTS fed on a commercial pet-food diet until sexual maturation. Sex of the treated fish was judged from secondary sex character- Progeny testing of sex-reversed fish: Sex ratios of the istics and treated fish were subsequently mated with normal hormone-treated fish are shown in Table 1. The sex ra- fish. Sexing of F1 progeny from the mating was carried out by histological cross-sections of fry gonads, sampled at 20 days tio of the MT-treated group was biased toward male, after hatching. suggesting that MT induced sex reversal from female to Genetic crosses: By crossing a CB female and (CB female 3 male. In the groups reared under normal conditions, PK male) an F1 male, 45 backcross progeny were obtained for the sex ratio was almost 1:1 (data not shown). There- genotyping. Phenotypic sex was determined by secondary sex fore, the MT-treated group was expected to include sex- characteristics of adult fish and reconfirmed by visual exam- ination of the gonads. We fixed adult fish in 100% ethanol and reversed males whose genetic sex is female (XX or ZW). isolated their genomic DNA from some muscle tissue using the On the other hand, the sex ratio of the E2-treated PI-50 isolation system (Kurabo). groups did not deviate toward females, suggesting that Isolation of sex-linked DNA markers and linkage analysis: E2 could not reverse the sex of the fish from male to We searched for sex-linked markers using expressed sequence female at concentrations of 0.01–0.04 mg/ml. In addi- tag (EST) markers mapped to each of the O. latipes chromosomes [linkage group (LG)] (Naruse et al. 2004). ESTs were tion, all fish died at a concentration of 0.2 mg/ml E2 amplified using previously published primers designed for before or just after hatching (data not shown). O. latipes (supplemental Table 1 at http://www.genetics.org/ Of 20 mature males obtained by the 0.025 mg/ml MT supplemental/). PCR genotyping for two loci, BJ014360 and treatment, 11 were randomly selected and subjected to BJ732639, was performed using primers that we designed on a progeny test by matings with normal females. The sex the basis of the EST sequences (http://mbase.bioweb.ne.jp/ dclust/medaka_top.html) and the draft genome sequence ratio of the offspring from each mating is shown in (http://dolphin.lab.nig.ac.jp/medaka/) of O. latipes. PCR am- Table 2. As a result, 5 of 11 males from the MT-treated plification of each EST marker was performed in a total group yielded all-female progeny, demonstrating that volume of 10 ml for 3 min at 95° followed by 35 cycles of 10 sec these males were sex-reversed XX males. This result at 95°, 30 sec at 55°–60°, 60 sec at 72°, with a final elongation reveals that O. dancena has a male-heterogametic (XX/ step of 3 min at 72°. Restriction fragment length polymorphisms (RFLPs) in PCR-amplified fragments of the parents XY) sex-determination system. (CB female and PK male) were analyzed by polyacrylamide gel Sex-linkage map of O. dancena: To isolate sex-linked electrophoresis. For those markers showing a polymorphic DNA markers in O. dancena, we searched for RFLPs Sex Chromosome Evolution in Oryzias 1337

TABLE 1 Sex ratio of the hormone-treated ﬁsh

Concentration (mg/ml) Male Female Total Methyltestosterone 0.001 20 2 22 0.005 30 5 35 0.025 20 0 20 Estradiol-17b 0.01 10 12 22 0.04 10 9 19 between the parents using ESTs established in O. latipes Figure 1.—Linkage analysis of EST markers in Oryzias and genotyped F and backcross progeny. We screened dancena. Electrophoretic patterns of MF01SSA032H09 PCR 1 product digested with HinfI (A) and BJ014360 PCR product 397 ESTs and identified 65 polymorphic markers. Link- digested with HhaI (B) are shown. Genomic DNA from female age analysis showed that 8 of these 65 markers segre- (F) and male (M) fish was used as templates for PCR. Abbre- gated with the phenotypic sex. The female parent was viations: P, parents (CB female and PK male); F1,F1 progeny homozygous, and the male parent and/or F1 male were from the parents; BC1, backcross progeny. Note that the heterozygous in these markers. In the backcross prog- digested bands (arrowheads) segregated perfectly with BC1 males. eny, males had the paternal genotype, while females had the maternal genotype, confirming an XX/XY sex along the sex chromosomes is not suppressed in O. determination system (Figure 1). dancena. Indeed, we obtained 4/45 (8.9%) recombi- Using eight isolated EST markers, we constructed nants between MF01SSA032H09 and BJ732639, which a sex-linkage map of O. dancena and showed that the flanked the SD locus. On the basis of the draft genome sex-determining (SD) locus was mapped to the same position with an EST, BJ014360 (Figure 2). On the basis of the draft genome sequence data of O. latipes (http:// dolphin.lab.nig.ac.jp/medaka/), all eight ESTs were located on LG 10 in O. latipes. Comparison of gene order between the O. dancena sex-linkage map and the physical map of O. latipes LG 10 indicated a conserved synteny between the two chromosomes. These results suggest that the sex chromosomes in O. dancena are homologous to an autosome (LG 10) of O. latipes, whose sex chromosomes are LG 1. In this sex-linkage map, the total map length was 23 cM in male meiosis. The corresponding region of O. latipes has a similar length (24.4 cM) (http://dolphin. lab.nig.ac.jp/medaka/), suggesting that recombination

TABLE 2 Sex ratio of the offspring of mating between a methyltestosterone- treated male and a normal female

Mating no. Male Female Total Remark 1 0 8 8 Sex-reversed XX male 2181836 3171633 4 9 14 23 Figure 2.—Comparison of gene order between a sex- 5162137 linkage map in Oryzias dancena and a physical map of LG 10 6151328 in O. latipes. Lines between the compared chromosomes con- 7 0 43 43 Sex-reversed XX male nect positions of orthologous gene pairs in these two species. 8141933 The distances between franking markers are shown in physi- 9 0 47 47 Sex-reversed XX male cal length (left) and in centimorgans (right). Map positions 10 0 47 47 Sex-reversed XX male for genes and distances in O. dancena were derived from this study; those in O. latipes were obtained from the draft genome 11 0 57 57 Sex-reversed XX male sequence data (http://dolphin.lab.nig.ac.jp/medaka/). 1338 Y. Takehana et al.

Figure 4.—Phylogenetic relationships and sex-determination mechanisms in Oryzias ﬁshes. The phylogenetic information was taken from Takehana et al. (2005). Data of sex-determination mechanisms in O. latipes, O. curvinotus, O. luzonensis, and O. mekongensis were obtained from Matsuda et al. (2002, 2003) and Hamaguchi et al. (2004).

On the other hand, the BAC clone (Md0173J11), containing an O. latipes sex-chromosomal marker (SL1), was not located on the sex chromosomes in O. dancena (Fig- ure 3C), confirming that sex chromosomes are different between these two species. Furthermore, the hybridization signals of the DMRT1-positive BAC (Md0172B19) Figure 3.—FISH analysis of male metaphase chromosomes were detected only on a pair of autosomes (Figure 3D). in Oryzias dancena using O. dancena fosmid and O. latipes BAC In O. latipes, previous FISH analyses revealed the clones. (A) FISH mapping of a fosmid clone (Od38_01) con- presence of DMRT1 at the tip of a pair of autosomes, taining a sex-linked EST, BJ014360. A specific hybridization as well as at one of the sex chromosome pair (namely the signal (red) was located on a homomorphic acrocentric chro- ondo mosome pair. (B) Gene ordering of the sex-linked ESTs, DMY gene on the Y chromosomes) (K et al. 2004, BJ014360 (fosmid Od38_01) and OLd17.11a (BAC Md0172I07), 2006). Although we found a similar FISH pattern to that on the sex chromosomes. The locations of the markers are of autosomal DMRT1 in O. latipes, the additional Y- visualized as red and green signals, respectively. Arrowheads chromosomal location (corresponding to the DMY) was indicate the centromere positions. (C) Chromosomal loca- not observed in O. dancena. Hence, these results strongly tion of the O. latipes sex-chromosomal marker SL1 (BAC Md0173J11, green) and the O. dancena sex-chromosomal suggest that Y chromosomes of O. dancena and O. latipes marker BJ014360 (fosmid Od38_01, red). (D) Chromosomal are not homologous. location of the DMRT1 gene (BAC Md0172B19, green) with the BJ014360 fosmid, Od38_01 (red). DISCUSSION sequence, this region was calculated to be 3.0 Mb in O. O. dancena has an XX/XY sex-determination system: latipes. These findings suggest a high recombination fre- It has been demonstrated that, on the basis of sex ratios quency around the sex-determining region in O. dancena. in the progeny of sex-reversed fish and segregation Identification of sex chromosomes by FISH map- patterns of sex-linked DNA markers, O. dancena in the ping: The karyotype of O. dancena consists of 24 acro- javanicus species group has a male-heterogametic (XX/ centric chromosome pairs with no heteromorphic sex XY) sex-determination system. A previous study showed chromosomes (Uwa et al. 1983). To identify the sex that all four fishes in the latipes group also have an chromosomes in O. dancena, we conducted a FISH anal- XX/XY sex-determination system (Hamaguchi et al. ysis of male chromosomes using a fosmid clone con- 2004) (Figure 4). Two of these four species, O. latipes and taining BJ014360 as a probe, which is tightly linked to O. curvinotus, have the common sex-determining gene, the SD locus. Clear hybridization signals were observed DMY, on the homologous Y chromosomes (Matsuda on the middle position of an acrocentric chromosome et al. 2002, 2003). On the other hand, the gene has not pair, identifying the cryptic sex chromosomes (Figure been detected in the other two species, O. luzonensis and 3A). We also found sex chromosome localization of the O. mekongensis (Kondo et al. 2003, 2004). On the basis of O. latipes BAC clone (Md0172I07) containing another these findings with a phylogenetic analysis of DMY and sex-linked EST, OLd17.11a. Two-color FISH indicated DMRT1,Kondo et al. (2004) suggested that the DMY that the BJ014360 signal was located more proximal to gene appears to have occurred in the common ancestor the centromere than the OLd17.11a signal on the sex of O. latipes, O. curvinotus, and O. luzonensis. Therefore, chromosomes (Figure 3B). Morphological differences the DMY gene is considered to be recently lost in O. between the X and Y chromosomes were not observed luzonensis and originally absent in O. mekongensis and in in this study, indicating that O. dancena has homomor- other Oryzias species, such as O. dancena. This suggests phic sex chromosomes. that different sex-determining genes have evolved in Sex Chromosome Evolution in Oryzias 1339

Oryzias species, although male heterogamety is likely to First, phenotypic sex was easily converted by sex hor- be common in the genus. mones, and the resultant sex-reversed fish were fully Identification of sex chromosomes in O. dancena: fertile. Furthermore, YY males have been obtained Oryzias species with no DMY gene must have different from estrogen-induced XY females (Y. Takehana and sex chromosomes from those in O. latipes; however, no D. Demiyah, unpublished results), indicating viability sex chromosomes have been identified in these species of YY individuals. Second, sex-chromosomal crossing so far. In this study, we screened sex-linked markers of O. over occurred over almost the entire length of the chro- dancena using ESTs established in O. latipes. As a result, mosome, indicating that this recombining section is most ESTs primers worked well in O. dancena, and we considered to be a pseudoautosomal region. In addition, could successfully isolate eight sex-linked EST markers. the region between MF01SSA032H09 and BJ732639, Linkage analysis showed that these ESTs are located on which flanked the SD locus in O. dancena, was calculated an autosome (LG 10) in O. latipes with the same gene to be 3.0 Mb in O. latipes, on the basis of the draft genome order, indicating a conserved synteny between sex chro- sequence. This suggests that the Y-specific region of mosomes in O. dancena and an autosome of O. latipes.In O. dancena is likely to be very small. Finally, FISH analy- addition, FISH analysis confirmed that sex chromo- sis demonstrated that the Y chromosome is not cyto- somes in O. dancena and O. latipes were not homologous. genetically distinguished from the X, although the These results indicated an independent origin of X/Y sex-determination system in the species is male het- chromosomes in these two species, suggesting that a erogametic. Taken together, these findings suggest that novel sex-determining gene is located on the sex chro- there are no functional differences between X and Y sex mosomes in O. dancena. chromosomes, other than the sex-determining gene. The lack of conservation of sex chromosomes among Only two sex-determining genes, Sry and DMY, have closely related fish species is probably common. Al- been identified in vertebrates so far. In this study, we though all salmonid fishes have a male heterogametic have shown that O. dancena should have a novel sex- system, closely related species have evolved different determining gene on the sex chromosomes homolo- sex chromosomes, as evidenced by a comparative link- gous to an autosome (LG 10) in O. latipes.Oneapproach age analysis (Woram et al. 2003) and FISH analyses to identify sex-determining genes is positional clon- (Phillips et al. 2001, 2005). In sticklebacks, Gasterosteus ing. This method has been successfully used to isolate aculeatus and G. wheatlandi also appear to have different human and medaka sex-determining genes. Two major X/Y sex chromosomes, and Apeltes quadracus has hetero- conditions are required for using this method: a firm morphic Z/W sex chromosomes (Peichel et al. 2004). genetic sex determination and a feasibility of genetic These studies suggest that fishes may use a variety of mapping for the SD locus (Matsuda 2005). O. dancena sex-determining genes. has a strict genetic sex determination (XX/XY system) Sex-determining region in O. dancena: Linkage anal- and a high recombination frequency around the SD ysis mapped the SD locus between MF01SSA032H09 locus, satisfying both requirements. In addition, O. and BJ732639 andshowedthatBJ014360 is tightly linked dancena and O. latipes share a similar character of sex to the SD locus with no recombinants (0/45). This sug- chromosomes, a very small Y-specific region, suggesting gests that a single chromosomal region controls sex that this method will also be effective in O. dancena. in O. dancena. FISH analysis indicated that the sex- Construction of a high-resolution recombination map determining region is located on the middle of one around the SD locus and chromosome walking to the SD acrocentric chromosome pair, in accordance with the locus are necessary steps to isolate the sex-determining linkage analysis. Therefore, only this region should be gene using the positional cloning method. This is cur- different between X and Y chromosomes. rently in progress. Comparative analyses of sex-deter- Standard models of sex chromosome evolution hy- mining genes and sex chromosomes among Oryzias pothesized that the first step is the occurrence of a novel species will prove useful in unraveling the evolution of single locus on an autosome, in which heterozygosity sex-determination mechanisms in vertebrates. leads to the development of one sex and homozygosity We thank Yoichi Matsuda, Takahiro Murakami (Hokkaido Univer- to the other sex, thereby establishing a protosex chro- sity), and Masaru Matsuda (National Institute for Basic Biology) for mosome system. Heteromorphic sex chromosomes are their helpful technical advice on FISH analysis and Wichian Magtoon considered to arise through the recombination isola- (Srinakharinwirot University) for his generous help in the collection tion between such homologous sex chromosomes. This of materials. This work was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, suppression of recombination maintained one chromo- Science, and Technology of Japan (16370094) to M.S. some (such as Y) in a constant heterozygous state in one sex and the subsequent degeneration process spreads LITERATURE CITED the sex-specific region over almost the entire chromosome, as in mammals (reviewed in Graves 2006). De Grandi, A., V. Calvari,V.Bertini,A.Bulfone,G.Peverali et al., 2000 The expression pattern of a mouse doublesex-related Our results indicate that sex chromosomes in O. gene is consistent with a role in gonadal differentiation. Mech. dancena are at an early stage of evolution, as in O. latipes. Dev. 90: 323–326. 1340 Y. Takehana et al.

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