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 chromo-some 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.
The genus Oryzias consists of 14 species endemic to Asia from India to Japan (Nelson 1994). From cytogenetical studies, Uwa (1986) divided these species into three groups: the mono-armed chromosome group, the bi-armed chromosome group, and the fused-chromosome group. This classification has been confirmed by molecular phylogenetic studies (Naruse et al., 1992). The bi-armed group consists of four species, O. latipes, O. curvinotus, O. luzonensis and O. mekongensis, and the phylogenetical vicinity of these species has been evidenced by analyses of the nucleotide sequences of the mitochondrial cytochrome b gene as well as the 12S and 16S rRNA (Naruse 1996, Takehana et al., in preparation).
Studies on the sex-determining mechanism of O. latipes began when Aida (1921) indicated that the r gene, which controls the presence or absence of yellow color pigment in xanthophores, was linked to a sex chromosome, and demonstrated that O. latipes had the XX-XY sex-determination system. Recently, we have identified molecular markers on the sex chromosomes (Matsuda et al., 1997), and succeeded in visualizing the sex chromosomes (Matsuda et al., 1998). In addition, through constructing a precise genetic map of the sex chromosomes (Sato et al., 2001) and a BAC-library (Matsuda et al., 2001), we cloned a candidate for the sex-determining gene of O. latipes, DMY (Matsuda et al., 2002). The introduction of DMY into XX eggs induced the development of testes and caused functional sex-reversal from female to male (Matsuda et al., in preparation), proving that DMY is the sex-determining gene of this fish.
In O. curvinotus, a Y-linked gene homologous to DMY has been identified (Matsuda et al., 2003), and a syntenic relationship between the sex-chromosomes of O. latipes and O. curvinotus has been demonstrated (Kondo et al., 2001, Sato et al., unpublished data), indicating that these fishes have a common sex-determining mechanism. However, no DMY homologues were detected in other Oryzias fishes by a PCR survey of Oryzias fishes using several primer sets for the DMY gene of O. latipes. In addition, molecular phylogenetical studies have indicated that DMY originated from a duplication of DMRT1 that occurred just prior to the speciation of O. latipes and O. curvinotus (Matsuda et al., 2003). These facts suggest the possibility that fishes other than O. latipes and O. curvinotus adopt different sex-determining mechanisms. Therefore, from an evolutionary point of view, it is important to examine the sex-determining mechanism of Oryzias fishes other than O. latipes and O. curvinotus. In the present study, we investigated the sex-determination system of O. luzonensis and O. mekongensis to demonstrate whether they have the XX-XY type of sex-determination system.
MATERIALS AND METHODS
Fish
The Oryzias luzonensis and O. mekongensis used in this study were from stocks in the Faculty of Science, Niigata University (O. luzonensis: collected by Formacion and Uwa 1982; O. mekongensis: collected by Magtoon and Uwa in 1984). They were kept indoors at 27±2°C, under a photoperiod of 14L-10D.
Sex steroid treatments for sex reversal
The sex-reversal experiment was carried out basically according to the method of Iwamatsu (1999). Briefly, hormones (methyltestosterone (MT) and estradiol-17β (E2); Sigma Chemical Co., St. Lous, MO) were dissolved in ethanol to make stock solutions and stored in a refrigerator. Just prior to use, stock solutions were diluted with aged tap water. Naturally spawned eggs were collected and incubated in the hormone-containing water at concentrations of 0.04 and 0.2 μg/ml of E2, and 0.001, 0.005 and 0.025 μg/ml of MT. Hatched fry were transferred to normal tap water and fed on a commercial pet-food until sexual maturation. We examined the secondary sex characters of the treated fish and judged the sex of the individuals. Treated fish were mated with normal males or females, and the sex of the offspring was examined histologically.
Histological method and judgment of the sex of the fry
At 20 days after hatching, fry were fixed in toto in Bouin's solution, sectioned at 6 μm in paraffin, and stained with hematoxylin and eosin for microscopic observation. We identified the sex of each fry from the histology of their developing gonads.
RESULTS
1) Sex ratios in the hormone-treated groups
The sex ratios of the E2-treated groups deviated toward females, suggesting that E2 induced sex reversal from male to female in both O. mekongensis and O. luzonensis (Table 1). In the groups reared under normal conditions, the sex ratios of O. luzonensis and O. mekongensis were almost 1:1 (data not shown). Therefore, the E2-treated groups are expected to contain sex-reversed females whose genetic sex is male (XY or ZZ).
Table 1
Sex ratios of the hormone-treated fishes of O. luzonensis and O. mekongensis.
The results for the MT-treated groups are also shown in Table 1. The sex ratios deviated from 1:1 toward males, indicating that MT could reverse the sex of the fish from female to male, that is, XX or ZW males are included in the treated fish.
2) Sex ratios of the offspring of the treated fish
For mating in O. luzonensis treated with sex hormones, we used 5 females obtained from the 0.2 μg/ml E2 treatment group and 7 males from the 0.025 μg/ml MT treatment group. For O. mekongensis, we used 8 females from the 0.04 μg/ml E2 treatment group and 9 males from the 0.001 μg/ml MT treatment group. The sex ratios of the offspring from each mating are shown in Table 2 (O. luzonensis) and Table 3 (O. mekongensis).
Table 2
Sex ratios of the offspring of mating between normal and hormone-treated fishes of O. luzonensis.
Table 3
Sex ratios of the offspring of mating between normal and hormone-treated fishes of O. mekongensis.
All females from E2 treatment group in both species produced both males and females, although the sex ratios were variable. On the other hand, 4 of 7 males from MT treatment group in O. luzonensis yielded all-female progeny. In O. mekongensis, all offspring from 4 of 9 males treated with MT were females. These results indicated that the MT-treatment groups contained XX sex-reversed males of O. luzonensis as well as O. mekongensis, that is, both species adopt an XX-XY sex-determination system.
DISCUSSION
The sex-determining mechanism in vertebrates is variable. Almost all mammalian species have the XX-XY sex determination system, with Sry as the common male-determining gene, whereas birds have ZZ-ZW type sex chromosomes (reviewed by Solari 1994). In lower vertebrates, amphibians and fishes, both XX-XY and ZZ-ZW systems are known. Even within a genus or species, it has been shown that there are a variety of systems. In Rana rugosa, there is a geographical diversity in the sex chromosome types, that is, in west Japan they have XX-XY sex chromosomes, while the northern part of Japan populations have been reported to have ZZ-ZW sex chromosomes (Nishioka et al., 1993, 1994). In fishes of the genus Xiphophorus, X, Y and Z sex chromosomes are known to exist (Volff and Schartl 2001).
With the exception of mammals, sex-determining genes were not identified until quite recently, and therefore it has been impossible to examine the diversity in the sex-determining mechanism at the molecular level. The recent identification of the sex-determining gene DMY in the medaka (Matsuda et al., 2002) was a breakthrough in this regard. Now, we can utilize the variety of sex-determining mechanisms that occur in fish, especially in Oryzias fishes, to approach a common sex-determining mechanism in vertebrates.
In the present study, we investigated the sex-determination system as the first step to approaching the sex-determining genes in O. mekongensis and O. luzonensis which are the most closely related species to O. latipes. As summarized in Fig. 1, our results demonstrated that both fishes have male heterogametic system (XX-XY) as O. latipes and O. curvinotus.
Figure 1
The sex-determination systems and the sex-determining gene in bi-armed group of Oryzias fishes. The phylogentical relationship of the four Oryzias fishes is drawn. The present study indicated that O. mekongensis and O. luzonensis have XX-XY system as O. latipes and O. curvinotus. The DMY has proven to be originated in the common ancestor of O. curvinotus and O. luzonensis, and we failed to identify in O. luzonensis a homologous gene to DMY, therefore the sex determining genes of O. mekongensis and O. luzonensis may possibly be other genes than DMY homologues.
In O. latipes and O. curvinotus, DMY has been proved to be the common male-determining gene on the Y chromo-some (Matsuda et al., 2003), but it does not seem to be the common sex-determining gene in a variety of Oryzias fishes. DMY originates from DMRT1, which is believed to be a common important gene for male development in vertebrates. A duplicated copy of DMRT1 became incorporated into a chromosome, and thereafter it acquired the male-determining function to become DMY. The molecular phylogeny of DMY and DMRT1 shows that the duplication of DMRT1 occurred just prior to the speciation of O. latipes and O. curvinotus (Matsuda et al., 2003). This indicates that O. mekongensis, which first branched off from other Oryzias fishes of the bi-armed chromosome group, can hardly use DMY as the sex-determining gene. In fact, DMY has not been identified in O. mekongensis (Kondo et al., 2003). Considering these facts, it can safely be said that O. mekongensis has a male-determining gene other than DMY, in other words, the Y chromo-some of this species may not be homologous to that of O. latipes and O. curvinotus.
O. luzonensis and O. curvinotus are a sister species pair to O. latipes, that is, they have a common ancestor, but we failed to demonstrate the presence of DMY in O. luzon-ensis (data not shown). It may also be probable that the Y chromosome of O. luzonensis is homologous to an auto-some of O. latipes. Currently, we are searching for the sex chromosomes of O. luzonensis and O. mekongensis, using DNA markers established in O. latipes.
Acknowledgments
This work was partly supported by a Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (11839006) to S.H., and (11236206) to M.S.
