Common Spontaneous Sex-Reversed XX males of the Medaka Oryzias latipes
a Institute for Human Genetics, Biocenter, University of Würzburg, D-97074 Würzburg, Germany2q, http://www.100md.com
b Department of Physiological Chemistry I, Biocenter, University of Würzburg, D-97074 Würzburg, Germany2q, http://www.100md.com
ABSTRACT2q, http://www.100md.com
In the medaka, a duplicated version of the dmrt1 gene, dmrt1bY, has been identified as a candidate for the master male sex-determining gene on the Y chromosome. By screening several strains of Northern and Southern medaka we identified a considerable number of males with normal phenotype and uncompromised fertility, but lacking dmrt1bY. The frequency of such males was >10% in some strains and zero in others. Analysis for the presence of other Y-linked markers by FISH analysis, PCR, and phenotype indicated that their genotype is XX. Crossing such males with XX females led to a strong female bias in the offspring and also to a reappearance of XX males in the following generations. This indicated that the candidate male sex-determining gene dmrt1bY may not be necessary for male development in every case, but that its function can be taken over by so far unidentified autosomal modifiers.
GENETIC mechanisms that determine the development of the male and female sex have developed independently many times during evolution. This is reflected by such diverse sex-determining mechanisms as the autosome-to-one-sex-chromosome ratio and the female or male heterogamety. In some studied cases, even in established sex-determining systems, new sex chromosomes can appear due to mutation and invade the population (CHARLESWORTH 1991 ; CHARLESWORTH and CHARLESWORTH 2000 ).wdc, 百拇医药
Fish display a wide spectrum of genetic sex-determining mechanisms (for reviews see BAROILLER et al. 1999 ; DEVLIN and NAGAHAMA 2002 ). One extreme, yet very thoroughly analyzed situation, is found in the swordtails and platyfish of the genus Xiphophorus. Here, in closely related species diverse modes of sex determination like polygenic sex determination, XX/XY and WZ/ZZ systems are operating. In most populations of the Southern platyfish Xiphophorus maculatus, even three sex chromosomes, X, Y, and W, coexist. Exceptionally so-called atypical sex determination was observed. This was explained by the action of rare alleles of autosomal modifiers that can influence or override the sex chromosome genes (KALLMAN 1984 ). The sex-determining region has been mapped in these fish (MORIZOT et al. 1991 ; GUTBROD and SCHARTL 1999 ) but the sex-determining gene(s) is unknown so far.
Another classic fish model for studying genetic sex determination is the medaka Oryzias latipes. In this organism crossing over between X and Y chromosomes was shown for the first time in vertebrates (AIDA 1921 ). In line with the early theories of genetic sex determination (BRIDGES 1922 , BRIDGES 1925 ; WINGE 1934 ) the medaka was described as having a polygenic sex-determining system of multiple male- and female-determining factors spread over different chromosomes and as being polymorphic in a given population, however, with a strong epistasis of those factors that concentrate on the X and Y chromosome (AIDA 1936 ). This view was later given up (see YAMAMOTO 1975 ). Today it is generally accepted that the medaka has a firm genetic female XX/male XY sex-determination system, with a master sex-determining gene on the Y chromosome that initiates the determination of the bipotential embryonic gonad similar to the action of Sry in mammals. However, unlike in mammals and many other groups of animals, treatment with sex steroids at the sensitive period of gonadal development can result in all types of functional sex reversals (YAMAMOTO 1975 ). A single report was made on spontaneous atypical sex determination (AIDA 1936 ). Of 5000 males analyzed, 7 were detected in one laboratory strain, which, due to a linked marker, were diagnosed to be XX. In the absence of a detailed genetic map and of knowledge about the sex-determining gene, this finding remained enigmatic.
Recently, a first candidate for the male sex-determining gene from a fish species was cloned in the medaka (MATSUDA et al. 2002 ; NANDA et al. 2002 ). The male sex-determining candidate gene of medaka is a duplicated version of the autosomally located dmrt1a gene. It is designated dmrt1bY. The high similarity even in introns between dmrt1a and 1bY, in conjunction with molecular phylogenetic analysis, indicates a recent origin of dmrt1bY from its autosomal counterpart (KONDO et al. 2003 ). dmrt1bY is expressed during male embryonic development, preceding the differentiation of the gonad. In adults it is expressed exclusively in the Sertoli cells of the testis. The expression pattern is not altered by sex-reverting steroid treatment (NANDA et al. 2002 ). The strongest evidence for dmrt1bY as in fact being the master regulatory gene for male development comes from the finding that naturally occurring mutations in this gene lead to XY male-to-female sex reversals (MATSUDA et al. 2002 ). This leads to the conclusion that dmrt1bY is necessary for male development.
Conceptual translation of dmrt1bY reveals a predicted protein with all conserved residues of a DM domain containing transcription factor. Not much is known about the biochemical and biological function of vertebrate dmrt genes in general and dmrt1 in particular. There is genetic evidence—in line with the expression pattern—that dmrt1 plays an important role as a downstream sex-determination/sex-differentiation gene in male development of some reptiles, birds, and mammals (RAYMOND et al. 1999 , RAYMOND et al. 2000; SMITH et al. 1999 ; DE GRANDI et al. 2000 ; KETTLEWELL et al. 2000; MONIOT et al. 2000 ). How dmrt1bY, as a duplicate of a downstream sex-determination/sex-differentiation gene, could assume a function at the top of the sex-determination cascade and how it could act molecularly in medaka as a master male sex-determining gene is totally unclear at the moment.92, 百拇医药
Two lines of evidence indicate that sex determination in medaka is at an early stage of evolution and has not reached a similar stability as in other organisms like birds and mammals. First, both X and Y chromosomes are homomorphic, indicating that the molecular differentiation process of the sex chromosomes leading to recombinational isolation over large parts has not progressed to a stage where it becomes visible. Sex chromosomal crossovers occur over almost the entire length of the corresponding linkage groups. In fact, the Y-chromosome-specific region appears to be very small, estimated to be only a few hundred kilobases in length. Second, the genotypic sex can be easily reverted by hormone treatment. As another facet of the instability of the genetic sex-determination system we report here that in several medaka strains, fish with an XX chromosome constitution spontaneously become fully fertile males. The frequency of such XX males in some strains can be as high as >10%. The results indicate the presence of autosomal modifiers for sex determination and that under certain conditions dmrt1bY is dispensable for genetic sex determination in medaka.
MATERIALS AND METHODSd%+*{0, http://www.100md.com
Fish:d%+*{0, http://www.100md.com
All fish used in this study were taken from closed colony breeding stocks and are derived either from the highly inbred medaka lines HNI, HB32C, i-3, HdrR (HYODO-TAGUCHI and SAKAIZUMI 1993 ) Kaga, SOK (A. SHIMADA and A. SHIMA, unpublished results), and Quart (WADA et al. 1998 ) or from the noninbred Carbio and Wü strain. SOK is derived from Korea; HNI and Kaga are derived from the northern population of Japan; and HB32C, i-3, HdrR, and Quart are derived from the southern population of Japan. The Carbio strain was established during 1986–1988 from fish obtained from Carolina Biological Supplies. Carbio fish are derived from the southern population of O. latipes and are homozygous for the b' (variegated) allele at the B pigmentation locus. Wü is a wild-type-pigmented hybrid strain of HNI and HB32C, the Y chromosome being derived from the Southern population of Japanese medaka. Both strains have been bred as closed colonies since their establishment.
All fish were raised and maintained under the same standard conditions (at 27°, with a light cycle of 14 hr light and 10 hr dark). Determination of sex ratios in the Carbio and i-3 strain was done from fish kept in large population tanks. The entire egg clutches from all females that had spawned were collected over several days and pooled and the hatched larvae were raised together until sexual maturation..5]y;e, 百拇医药
Sexing of fish:.5]y;e, 百拇医药
The phenotypic sex was first determined from secondary sex characters (shape of dorsal and anal fins, spines on male anal fin rays) and confirmed by functional egg or sperm production or gonad histology. No sterile fish with immature or mature gonads were detected in this study. The genotypic sex was diagnosed from the presence or absence of the dmrt1bY gene by PCR from fin clip DNA (ALTSCHMIED et al. 1997 ) using allele-specific primers such as DMT1k (5' CAA CTT TGT CCA AAC TCT GA 3') and DMT1l (5' AAC TAA TTC ATC CCC ATT CC 3') at an annealing temperature of 56°. Eventually a series of other dmrt1bY-specific primers were used. This information is available upon request. Furthermore, casp6, which is a polymorphic marker that differentiates the X and Y chromosome in fish of the AA2 strain derived from the Southern population of medaka (KONDO et al. 2001 ), was employed for genotyping. The primers used in this previous study amplified also casp6 from the HB32C strain. Primers intf2 (5' TAG CAC TTT CAC ATT TCC AAG C 3') and c6R (5' CGT CTC TCG ATG AGA ATA GAA ACC 3') at an annealing temperature of 60° were used.
Southern blot analysis:%hrsqwf, http://www.100md.com
DNA from individual fish was obtained from pooled organs as described (SCHARTL et al. 1995 ). Five micrograms of genomic DNA was digested with restriction enzymes, separated on 0.8% agarose gels, and blotted onto nylon membranes (Hybond N+, Amersham Buchler). Membranes were hybridized under conditions of moderate stringency (hybridization in 35% formamide, 0.1% Na-pyrophosphate, 50 mM Tris-HCl, pH 7.5, 5x SSC, 1% sodium dodecyl sulfate (SDS), 5x Denhardt's, 100 µg/ml calf thymus DNA at 42°, washing in 1x SSC/1% SDS at 60°) with the OlaDmrt1a probe (4-kb EcoRI fragment from cosmid 73K2481).%hrsqwf, http://www.100md.com
FISH analysis:%hrsqwf, http://www.100md.com
For fluorescence in situ hybridization (FISH), mitotic chromosome preparations were made directly from pooled spleen, gills, and cephalic kidney cells after exposing the fish several hours to a 0.02% solution of colchicine. Prior to hybridization, slides were subjected to pepsin (0.01%) and formaldehyde (1%) treatment and denaturation in 70% formamide in 2x SSC (pH 7.0) at 70° following the standard procedure.
Two different bacterial artificial chromosome (BAC) clones, one spanning the Y-chromosome-specific region (BAC 15H17) and one containing a flanking maker (BAC 98J17, SL1), were separately labeled by nick-translation with biotin-16-dUTP or digoxigenin-11-UTP (Roche Molecular Biochemicals, Mannheim, Germany). Labeled DNA at a concentration of 10 ng/µl was coprecipitated with 150 ng/µl calf thymus DNA and 100 ng/µl sonicated medaka genomic DNA and redissolved in 50% formamide, 10% dextran sulfate, and 2x SSC. After 10 min denaturation at 75° and reannealing at 37° for 30 min, 20 µl of probe mixture was applied to a denatured slide and sealed under a coverslip. Following overnight incubation at 37°, the slides were washed at 45° in 50% formamide, 2x SSC for 15 min and for an additional two times of 5 min each, with 1x SSC at 60°.43mruk, 百拇医药
The locations of the hybridization sites were detected with rhodamine-conjugated avidin (Vector, Burlingame, CA) and antidigoxigenin (monoclonal)-conjugated fluorescein (Sigma, St. Louis) followed by further signal enhancement of biotinylated probe using biotinylated antiavidin and rhodamine-conjugated avidin. Likewise, the sheep anti-mouse FITC conjugate was used to enhance the signal of the digoxygenated probe. Chromosomes and cell nuclei were counterstained with 4'-6-diamidino-2-phenylindole (DAPI). Slides were mounted with antifade medium and the hybridization signal was visualized on a Zeiss epifluorescence microscope equipped with a computer-controlled thermoelectronically cooled charged-coupled device camera. Digitized images of the FITC, rhodamine, and DAPI signals of metaphase spread were captured separately and merged using the Easy FISH 1.0 software (Applied Spectral Imaging). At least 20 metaphase plates for both probes were simultaneously examined to evaluate the hybridization pattern.
RESULTS$jm)in$, 百拇医药
With a candidate for the male sex-determining gene and linked molecular markers now available we wanted to reinvestigate the classical finding of AIDA 1936 of the rare, exceptional XX males. The sex ratio in two representative medaka strains, Carbio and i-3, was determined by the unambiguous phenotypic secondary sex characters and male and female gamete production. No significant deviation from the expected 1:1 ratio was found 1).$jm)in$, 百拇医药
fig.ommitteed$jm)in$, 百拇医药
Table 1. Sex ratios in medaka i-3 and Carbio strains$jm)in$, 百拇医药
When males of various strains were PCR genotyped for the medaka male sex-determining gene, we found a strikingly large number of animals that did not show the expected amplification product (1A). The lack of dmrt1bY was confirmed by Southern blot analysis. All the aberrant males showed a restriction fragment pattern like females 2). Such males were then genotyped for the linked sex chromosomal marker casp6, which is located outside the Y-chromosome-specific fragment in the pseudoautosomal region. The genetic distance between casp6 and dmrt1bY is ~
1 cM (NANDA et al. 2002 ). Primers that give a 392-bp product from the Y chromosomal allele and a 387-bp product from the X chromosomal allele of Southern medaka and a 387-bp product from the Y chromosomal allele and a 392-bp product from the X chromosomal allele of Northern medaka were used (KONDO et al. 2001 ). All aberrant males had only the X chromosomal alleles ( 1B). In the HdrR strain a dominant color marker (orange body coloration, R) on the Y chromosome was also absent from te dmrt1bY-negative males.g0w}, 百拇医药
fig.ommitteedg0w}, 百拇医药
Figure 1. PCR genotyping of medaka males and females. (a) Presence of the dmrt1bY PCR product in normal males of the i-3, Carbio, and HB32C strains. No PCR product was amplified from females and the aberrant males (*). For control, an aliquot of the same DNA was used for actin PCR. (b) Hemizygosity of normal males of the HB32C and HNI strains for caspase 6. Females and the aberrant males (*) show only the X chromosomal PCR product.g0w}, 百拇医药
fig.ommitteed
Figure 2. Southern blot analysis of EcoRI-digested DNA from female, normal male, and aberrant (dmrt1bY negative) males (*) from the Carbio strain, hybridized with the OlaDmrt1a probe. This probe detects the autosomal dmrt1a (two alleles present in Carbio) as well as the Y chromosomal dmrt1bY under conditions of moderate stringency (see NANDA et al. 2002 ).r, http://www.100md.com
FISH analysis was done on metaphase chromosomes of such males using BAC 15H17 as probe. This BAC contains only sequences from the Y-specific region, including dmrt1bY, and hybridizes only to the Y chromosome in normal males (XY,3A), but not to the X. In addition, it shows a weak cross-hybridization with the telomeric region of linkage group 9, which is the location of the autosomal dmrt gene cluster and some other sequences that were coduplicated during the event that created the Y-specific fragment. To identify both sex chromosomes visually, a marker that gives hybridization signals on the long arm of both the X and the Y chromosome was used (3A). Contrary to XY males, in all the analyzed metaphases of XX males no specific FISH signal was detectable with the Y-specific BAC on either one of the sex chromosomes 3B), indicating the absence of most of the Y-specific region in these males. However, the Y-specific probe stiloss-hybridized with the autosomal dmrt1a locus on linkage group 9 (3B). Repeated FISH experiments on XX males were carried out, which consistently corroborated the finding of the Southern hybridization experiment.
fig.ommitteedgtm*%5, 百拇医药
Figure 3. FISH pattern of Y-specific (BAC 15H17) and sex-chromosome-specific (BAC 98J17) probes on XY (a) and XX (b) male metaphase chromosomes. Note the presence of three hybridization signals (red) for the BAC 15H17 in XY males as compared to two spots in XX males. The two relatively weak signals (arrows) represent the autosomal dmrt1a locus (linkage group 9), which can be seen in both XY and XX males. The additional prominent signal (*) is specific to the Y chromosome (a) that is absent in the XX males (b). BAC 98J17 (green signal) containing the marker locus SL1 detects both sex chromosomes (arrowheads).gtm*%5, 百拇医药
To investigate the frequency of the occurrence of XX males, we tested a total of eight strains for the presence of XX males by diagnostic PCRs. The frequency of XX males was highly variable ( 2). In the i-3 strain, not a single XX male was among 81 tested males. Also, in the Kaga strain, no XX male was found. A low frequency of XX males was found in Quart and HNI (3 and 4%, respectively). In other strains, XX males were more frequent, for instance, 12% in Carbio. In the HdrR strain, we found 8 XX males, which were initially identified in the population tanks by the lack of the R phenotype and were confirmed by PCR for the absence of dmrt1bY.
fig.ommitteed-]j!0-, 百拇医药
Table 2. Frequency of males lacking dmrt1bY in different strains of medaka-]j!0-, 百拇医药
To further test the XX chromosome constitution of the aberrant males, several of them were mated to single females ( 3). In every case there was a strong bias toward female offspring, ranging from 100 to 89%. When some of the rare F1 males were crossed again to single females, the strong female bias was seen again except for one case. HB32C male 4-6 had 28 female and 36 male offspring. Three of his sons were tested by crossing to i-3 females and produced all female offspring. One F1 male (Carbio 1-1) was tested in an outcross with 20 of his sisters. The female-to-male ratio here was 4:1. From all crosses several offspring males were PCR tested for the absence of dmrt1bY.-]j!0-, 百拇医药
fig.ommitteed-]j!0-, 百拇医药
Table 3. Progeny test of dmrt1bY negative males-]j!0-, 百拇医药
DISCUSSION-]j!0-, 百拇医药
Using the candidate male sex-determining gene dmrt1bY as a marker, we unexpectedly detected a high number of functional males with uncompromised fertility that did not have this gene.
A possible explanation for the absence of dmrt1bY could be that this gene is not located at the male sex-determination locus, but that it is only a linked marker. The males lacking dmrt1bY (and the closely linked Y chromosomal allele of casp6) would then be recombinants due to XY crossovers in the pseudoautosomal region. In such a case, a similar proportion of females with dmrt1bY should be present. However, in 304 females not a single individual was found with dmrt1bY.;'w, 百拇医药
The crossings of the dmrt1bY-lacking males confirmed their sex chromosomal constitution to be indeed XX. In most cases a strong female-biased offspring was obtained. The reoccurrence of XX males in the following generations can be taken as evidence that the sex reversal in the parental male may not be due to some unidentified environmental effect. The seven XX males earlier reported in medaka were explained by a lowering of the female determining potency of the X chromosome and thus polygenic autosomal male determinants becoming epistatic (AIDA 1936 ). Both XX males described in the guppy Poecilia reticulata (WINGE 1930 ) and the two XX males of the platyfish X. maculatus (OKTAY 1959 ; KALLMAN 1984 ) were all explained by the action of autosomal modifier genes. These autosomal modifiers were proposed to override the sex chromosomal genes; however, it was impossible to decide whether they suppress a female-determining locus on the X chromosome or act as male inducers.
In the medaka, it has been shown that dmrt1bY is the only functional gene in a Y-chromosomal-specific segment. This segment is absent from the X chromosome, and outside this region both sex chromosomes are homologous (NANDA et al. 2002 ). It appears reasonable to assume that the autosomal modifiers have a male-determining activity analogous to dmrt1bY. The crossing data do not support a simple monogenic dominant or recessive trait for such autosomal modifiers. The hypothesis that they are polygenic receives some support from the fact that in most crosses (e.g., Carbio males 1 and 2) the number of male offspring was lower than expected and in one (HB32C male 4-6) was much higher than expected, reflecting differences in the genotype of the females used for the cross. The autosomal modifiers may be polymorphic and strain specific and even absent or present at low frequency in certain strains, such as in i-3. This situation would explain why the number of XX males dropped to zero when males from the HB32C male 4-6 offspring were outcrossed to i-3 females.
Contrary to all earlier reports, which described XX males as an extremely rare phenomenon, our data indicate that in medaka they are very common. This now offers the possibility to identify the linkage groups carrying the autosomal modifiers after repeated backcrossing and to map and identify the genes. The high frequency of XX males may also justify reevaluating Aida's theory of a polygenic sex-determination system with epistasis of sex chromosomal genes in medaka.\ipx8o, 百拇医药
So far, three mutant Y chromosomes have been found in the medaka, all of which were found in XY sex-reversed females. One mutant Y (designated Y-) lacks most or the entire male-specific region, including dmrt1bY; one (YwAwr) has a frameshift that leads to a premature termination of the dmrt1bY protein; and the third (YwSrn) has an intact dmrt1bY coding region, although a so far unknown mutation suppresses dmrt1bY expression in the embryo. This led to the conclusion that dmrt1bY is required for normal testicular differentiation (MATSUDA et al. 2002 ).
The frequent appearance of XX medaka males makes a more differentiated view necessary. The fact that through hormonal treatment even fully fertile XX males can be obtained (YAMAMOTO 1955 ) already indicated that the Y chromosome does not contain genes required for correct differentiation of the testes and for male fertility. The appearance of sexually uncompromised spontaneous XX males clearly shows that dmrt1bY is not obligatory for male sex determination under physiological conditions. This does not question the role of the dmrt1bY gene as a sex-determining gene and as the master regulator of male sexual development in most cases, but it indicates that dmrt1bY function can become dispensable. Obviously one or several autosomal genes can induce male sexual development as well. Such autosomal modifiers have been identified through crossing analyses also in the platyfish X. maculatus (KALLMAN 1984 ). However, in this fish dmrt1bY is not present (VEITH et al. 2003 ). Thus, even if the autosomal modifiers were homologous, they probably substitute for a different molecular event.
The Y chromosome of medaka is still at an early stage of evolution and in fact may be the youngest naturally occurring sex chromosome known so far. It is, however, unclear whether the fact that autosomal modifiers can override the XY system (whose molecular correlate is the presence and expression of dmrt1bY) is a reflection of a situation in which the molecular processes bringing about the male sex-determination system are not firmly established and robust. Several cases in which new sex chromosomes that arose by mutation from the normal chromosome complement invaded a population and took over the function of the previous ones have been described. In the vole Ellobius lutescens, for instance, such a mechanism has led to the elimination of Sry (JUST et al. 1995 ), which is the male sex-determining gene in the overwhelming majority of mammals. The autosomal modifiers of dmrt1bY function in medaka may represent such newly emerging sex-determining genes.@, 百拇医药
ACKNOWLEDGMENTS
We thank G. Schneider, H. Schwind, and P. Weber for breeding of the fish. Founder fish for our medaka stock were generously supplied by Y. Hyodo-Taguchi (Chiba) and A. Shima (Tokyo). Kaga fish were kindly given to us by J. Wittbrodt (Heidelberg). This work was supported by grants supplied by the Commission of the European Community (FAIR CT 97-3796) and Fonds der Chemischen Industrie to M. Schartl and by a grant from the Deutsche Forschungsgemeinschaft (SCHM 484/18-1) to M. Schmid.y\jk:0f, 百拇医药
Manuscript received August 30, 2002; Accepted for publication October 28, 2002.y\jk:0f, 百拇医药
LITERATURE CITEDy\jk:0f, 百拇医药
AIDA, T., 1921 On the inheritance of color in a fresh-water fish Aplocheilus latipes Temmick and Schlegel, with special reference to sex-linked inheritance. Genetics 6:554-573.y\jk:0f, 百拇医药
AIDA, T., 1936 Sex reversal in Aplocheilus latipes and a new explanation of sex differentiation. Genetics 21:136-153.y\jk:0f, 百拇医药
ALTSCHMIED, J., U. HORNUNG, I. SCHLUPP, J. GADAU, and R. KOLB et al., 1997 Isolation of DNA suitable for PCR for field and laboratory work. Biotechniques 23:228-229.
BAROILLER, J. F., Y. GUIGEN, and A. FOSTIER, 1999 Endocrine and environmental aspects of sex differentiation in fish. Cell. Mol. Life Sci. 55:910-931.ez4z|^, http://www.100md.com
BRIDGES, C. B., 1922 The origin and variation in sexual and sex-linked characters. Am. Nat. 56:93-107.ez4z|^, http://www.100md.com
BRIDGES, C. B., 1925 Sex in relation to chromosomes and genes. Am. Nat. 59:127-137.ez4z|^, http://www.100md.com
CHARLESWORTH, B., 1991 The evolution of sex chromosomes. Science 251:1030-1033.ez4z|^, http://www.100md.com
CHARLESWORTH, B. and D. CHARLESWORTH, 2000 The degeneration of Y chromosomes. Philos. Trans. R. Soc. Lond. B Biol. Sci. 355:1563-1572.ez4z|^, http://www.100md.com
DE GRANDI, A., V. CALVARI, V. BERTINI, A. BULFONE, and G. PEVERALI et al., 2000 The expression pattern of a mouse doublesex-related gene is consistent with a role in gonadal differentiation. Mech. Dev. 90:323-326.ez4z|^, http://www.100md.com
DEVLIN, R. H. and Y. NAGAHAMA, 2002 Sex determination and sex differentiation in fish: an overview of genetic, physiological, and environmental influences. Aquaculture 208:191-364.
GUTBROD, H. and M. SCHARTL, 1999 Intragenic sex-chromosomal crossovers of Xmrk oncogene alleles affect pigment pattern formation and the severity of melanoma in Xiphophorus. Genetics 151:773-783.\2, http://www.100md.com
HYODO-TAGUCHI, Y. and M. SAKAIZUMI, 1993 List of inbred strains of the medaka Oryzias latipes, maintained in the Division of Biology, National Institute of Radiological Sciences. Fish Biol. J. Medaka 5:5-10.\2, http://www.100md.com
JUST, W., W. RAU, W. VOGEL, M. AKHVERDIAN, and K. FREDGA et al., 1995 Absence of Sry in species of the vole Ellobius. Nat. Genet. 11:117-118.\2, http://www.100md.com
KALLMAN, K. D., 1984 A new look at sex determination in poeciliid fishes, pp. 95–171 in Evolutionary Genetics of Fishes, edited by B. J. TURNER. Plenum Publishing, New York.\2, http://www.100md.com
KETTLEWELL, J. R., C. S. RAYMOND, and D. ZARKOWER, 2000 Temperature-dependent expression of turtle Dmrt1 prior to sexual differentiation. Genesis 26:174-178.\2, http://www.100md.com
KONDO, M., E. NAGAO, H. MITANI, and A. SHIMA, 2001 Differences in recombination frequencies during female and male meioses of the sex chromosomes of the medaka, Oryzias latipes. Genet. Res. 78:23-30.
KONDO, M., I. NANDA, U. HORNUNG, T. SASAKI, and A. SHIMIZU et al., 2003 Absence of the candidate male sex determining gene dmrt1bY of medaka from other fish species. Curr. Biol. in press.#'3, http://www.100md.com
MATSUDA, M., Y. NAGAHAMA, A. SHINOMIYA, T. SATO, and C. MATSUDA et al., 2002 DMY is a Y-specific DM-domain gene required for male development in the medaka fish. Nature 417:559-563.#'3, http://www.100md.com
MONIOT, B., P. BERTA, G. SCHERER, P. SUDBECK, and F. POULAT, 2000 Male specific expression suggests role of DMRT1 in human sex determination. Mech. Dev. 91:323-325.#'3, http://www.100md.com
MORIZOT, D. C., S. A. SLAUGENHAUPT, K. D. KALLMAN, and A. CHAKRAVARTI, 1991 Genetic linkage map of fishes of the genus Xiphophorus (Teleostei: Poeciliidae). Genetics 127:399-410.#'3, http://www.100md.com
NANDA, I., M. KONDO, U. HORNUNG, S. ASAKAWA, and C. WINKLER et al., 2002 A duplicated copy of DMRT1 in the sex determining region of the Y chromosome of the medaka, Oryzias latipes. Proc. Natl. Acad. Sci. USA 99:11778-11783.#'3, http://www.100md.com
ÖKTAY, M., 1959 Über Ausnahmemaennchen bei Platypoecilus maculatus und eine neue Sippe mit XX-Maennchen und XX-Weibchen. Rev. Fac. Sci. Univ. Istanbul Ser. B Sci. Nat. 24:75-92.
RAYMOND, C. S., J. R. KETTLEWELL, B. HIRSCH, V. J. BARDWELL, and D. ZARKOWER, 1999 Expression of Dmrt1 in the genital ridge of mouse and chicken embryos suggests a role in vertebrate sexual development. Dev. Biol. 215:208-220.#ce:[l, 百拇医药
RAYMOND, C. S., M. W. MURPHY, M. G. O'SULLIVAN, V. J. BARDWELL, and D. ZARKOWER, 2000 Dmrt1, a gene related to worm and fly sexual regulators, is required for mammalian testis differentiation. Genes Dev. 14:2587-2595.#ce:[l, 百拇医药
SCHARTL, M., B. WILDE, I. SCHLUPP, and J. PARZEFALL, 1995 Evolutionary origin of a parthenoform, the Amazon molly Poecilia formosa, on the basis of a molecular genealogy. Evolution 49:827-835.#ce:[l, 百拇医药
SMITH, C. A., P. J. MCCLIVE, P. S. WESTERN, K. J. REED, and A. H. SINCLAIR, 1999 Conservation of a sex-determining gene. Nature 402:601-602.#ce:[l, 百拇医药
VEITH, A-M., A. FROSCHAUER, C. KÖRTING, I. NANDA, and R. HANEL et al., 2003 Cloning of the dmrt1 gene of Xiphophorus maculatus: dmY/dmrt1Y is not the master sex-determining gene in the platyfish. Gene in press.#ce:[l, 百拇医药
WADA, H., A. SHIMADA, S. FUKAMACHI, K. NARUSE, and A. SHIMA, 1998 Sex-linked inheritance of the lf locus in the medaka fish (Oryzias latipes). Zool. Sci. 15:123-126.#ce:[l, 百拇医药
WINGE, O., 1930 On the occurrence of XX males in Lebistes, with remarks on Aida's so-called "non-disjunctional" males in Aplocheilus. J. Genet. 23:69-76.#ce:[l, 百拇医药
WINGE, O., 1934 The experimental alteration of sex chromosomes into autosomes and vice versa, as illustrated by Lebistes.. C. R. Trav. Lab. Carlsberg Ser. Physiol. 21:1-49.#ce:[l, 百拇医药
YAMAMOTO, T., 1955 Progeny of artificially induced sex-reversals of male genotype (XY) in the medaka (Oryzias latipes) with special reference to YY-male. Genetics 40:406-419.#ce:[l, 百拇医药
YAMAMOTO, T., 1975 Medaka (Killifish) Biology and Strains. Keigaku, Tokyo.(Indrajit Nanda Ute Hornung Mariko Kondo Michael Schmid and Manfred Schartl)
b Department of Physiological Chemistry I, Biocenter, University of Würzburg, D-97074 Würzburg, Germany2q, http://www.100md.com
ABSTRACT2q, http://www.100md.com
In the medaka, a duplicated version of the dmrt1 gene, dmrt1bY, has been identified as a candidate for the master male sex-determining gene on the Y chromosome. By screening several strains of Northern and Southern medaka we identified a considerable number of males with normal phenotype and uncompromised fertility, but lacking dmrt1bY. The frequency of such males was >10% in some strains and zero in others. Analysis for the presence of other Y-linked markers by FISH analysis, PCR, and phenotype indicated that their genotype is XX. Crossing such males with XX females led to a strong female bias in the offspring and also to a reappearance of XX males in the following generations. This indicated that the candidate male sex-determining gene dmrt1bY may not be necessary for male development in every case, but that its function can be taken over by so far unidentified autosomal modifiers.
GENETIC mechanisms that determine the development of the male and female sex have developed independently many times during evolution. This is reflected by such diverse sex-determining mechanisms as the autosome-to-one-sex-chromosome ratio and the female or male heterogamety. In some studied cases, even in established sex-determining systems, new sex chromosomes can appear due to mutation and invade the population (CHARLESWORTH 1991 ; CHARLESWORTH and CHARLESWORTH 2000 ).wdc, 百拇医药
Fish display a wide spectrum of genetic sex-determining mechanisms (for reviews see BAROILLER et al. 1999 ; DEVLIN and NAGAHAMA 2002 ). One extreme, yet very thoroughly analyzed situation, is found in the swordtails and platyfish of the genus Xiphophorus. Here, in closely related species diverse modes of sex determination like polygenic sex determination, XX/XY and WZ/ZZ systems are operating. In most populations of the Southern platyfish Xiphophorus maculatus, even three sex chromosomes, X, Y, and W, coexist. Exceptionally so-called atypical sex determination was observed. This was explained by the action of rare alleles of autosomal modifiers that can influence or override the sex chromosome genes (KALLMAN 1984 ). The sex-determining region has been mapped in these fish (MORIZOT et al. 1991 ; GUTBROD and SCHARTL 1999 ) but the sex-determining gene(s) is unknown so far.
Another classic fish model for studying genetic sex determination is the medaka Oryzias latipes. In this organism crossing over between X and Y chromosomes was shown for the first time in vertebrates (AIDA 1921 ). In line with the early theories of genetic sex determination (BRIDGES 1922 , BRIDGES 1925 ; WINGE 1934 ) the medaka was described as having a polygenic sex-determining system of multiple male- and female-determining factors spread over different chromosomes and as being polymorphic in a given population, however, with a strong epistasis of those factors that concentrate on the X and Y chromosome (AIDA 1936 ). This view was later given up (see YAMAMOTO 1975 ). Today it is generally accepted that the medaka has a firm genetic female XX/male XY sex-determination system, with a master sex-determining gene on the Y chromosome that initiates the determination of the bipotential embryonic gonad similar to the action of Sry in mammals. However, unlike in mammals and many other groups of animals, treatment with sex steroids at the sensitive period of gonadal development can result in all types of functional sex reversals (YAMAMOTO 1975 ). A single report was made on spontaneous atypical sex determination (AIDA 1936 ). Of 5000 males analyzed, 7 were detected in one laboratory strain, which, due to a linked marker, were diagnosed to be XX. In the absence of a detailed genetic map and of knowledge about the sex-determining gene, this finding remained enigmatic.
Recently, a first candidate for the male sex-determining gene from a fish species was cloned in the medaka (MATSUDA et al. 2002 ; NANDA et al. 2002 ). The male sex-determining candidate gene of medaka is a duplicated version of the autosomally located dmrt1a gene. It is designated dmrt1bY. The high similarity even in introns between dmrt1a and 1bY, in conjunction with molecular phylogenetic analysis, indicates a recent origin of dmrt1bY from its autosomal counterpart (KONDO et al. 2003 ). dmrt1bY is expressed during male embryonic development, preceding the differentiation of the gonad. In adults it is expressed exclusively in the Sertoli cells of the testis. The expression pattern is not altered by sex-reverting steroid treatment (NANDA et al. 2002 ). The strongest evidence for dmrt1bY as in fact being the master regulatory gene for male development comes from the finding that naturally occurring mutations in this gene lead to XY male-to-female sex reversals (MATSUDA et al. 2002 ). This leads to the conclusion that dmrt1bY is necessary for male development.
Conceptual translation of dmrt1bY reveals a predicted protein with all conserved residues of a DM domain containing transcription factor. Not much is known about the biochemical and biological function of vertebrate dmrt genes in general and dmrt1 in particular. There is genetic evidence—in line with the expression pattern—that dmrt1 plays an important role as a downstream sex-determination/sex-differentiation gene in male development of some reptiles, birds, and mammals (RAYMOND et al. 1999 , RAYMOND et al. 2000; SMITH et al. 1999 ; DE GRANDI et al. 2000 ; KETTLEWELL et al. 2000; MONIOT et al. 2000 ). How dmrt1bY, as a duplicate of a downstream sex-determination/sex-differentiation gene, could assume a function at the top of the sex-determination cascade and how it could act molecularly in medaka as a master male sex-determining gene is totally unclear at the moment.92, 百拇医药
Two lines of evidence indicate that sex determination in medaka is at an early stage of evolution and has not reached a similar stability as in other organisms like birds and mammals. First, both X and Y chromosomes are homomorphic, indicating that the molecular differentiation process of the sex chromosomes leading to recombinational isolation over large parts has not progressed to a stage where it becomes visible. Sex chromosomal crossovers occur over almost the entire length of the corresponding linkage groups. In fact, the Y-chromosome-specific region appears to be very small, estimated to be only a few hundred kilobases in length. Second, the genotypic sex can be easily reverted by hormone treatment. As another facet of the instability of the genetic sex-determination system we report here that in several medaka strains, fish with an XX chromosome constitution spontaneously become fully fertile males. The frequency of such XX males in some strains can be as high as >10%. The results indicate the presence of autosomal modifiers for sex determination and that under certain conditions dmrt1bY is dispensable for genetic sex determination in medaka.
MATERIALS AND METHODSd%+*{0, http://www.100md.com
Fish:d%+*{0, http://www.100md.com
All fish used in this study were taken from closed colony breeding stocks and are derived either from the highly inbred medaka lines HNI, HB32C, i-3, HdrR (HYODO-TAGUCHI and SAKAIZUMI 1993 ) Kaga, SOK (A. SHIMADA and A. SHIMA, unpublished results), and Quart (WADA et al. 1998 ) or from the noninbred Carbio and Wü strain. SOK is derived from Korea; HNI and Kaga are derived from the northern population of Japan; and HB32C, i-3, HdrR, and Quart are derived from the southern population of Japan. The Carbio strain was established during 1986–1988 from fish obtained from Carolina Biological Supplies. Carbio fish are derived from the southern population of O. latipes and are homozygous for the b' (variegated) allele at the B pigmentation locus. Wü is a wild-type-pigmented hybrid strain of HNI and HB32C, the Y chromosome being derived from the Southern population of Japanese medaka. Both strains have been bred as closed colonies since their establishment.
All fish were raised and maintained under the same standard conditions (at 27°, with a light cycle of 14 hr light and 10 hr dark). Determination of sex ratios in the Carbio and i-3 strain was done from fish kept in large population tanks. The entire egg clutches from all females that had spawned were collected over several days and pooled and the hatched larvae were raised together until sexual maturation..5]y;e, 百拇医药
Sexing of fish:.5]y;e, 百拇医药
The phenotypic sex was first determined from secondary sex characters (shape of dorsal and anal fins, spines on male anal fin rays) and confirmed by functional egg or sperm production or gonad histology. No sterile fish with immature or mature gonads were detected in this study. The genotypic sex was diagnosed from the presence or absence of the dmrt1bY gene by PCR from fin clip DNA (ALTSCHMIED et al. 1997 ) using allele-specific primers such as DMT1k (5' CAA CTT TGT CCA AAC TCT GA 3') and DMT1l (5' AAC TAA TTC ATC CCC ATT CC 3') at an annealing temperature of 56°. Eventually a series of other dmrt1bY-specific primers were used. This information is available upon request. Furthermore, casp6, which is a polymorphic marker that differentiates the X and Y chromosome in fish of the AA2 strain derived from the Southern population of medaka (KONDO et al. 2001 ), was employed for genotyping. The primers used in this previous study amplified also casp6 from the HB32C strain. Primers intf2 (5' TAG CAC TTT CAC ATT TCC AAG C 3') and c6R (5' CGT CTC TCG ATG AGA ATA GAA ACC 3') at an annealing temperature of 60° were used.
Southern blot analysis:%hrsqwf, http://www.100md.com
DNA from individual fish was obtained from pooled organs as described (SCHARTL et al. 1995 ). Five micrograms of genomic DNA was digested with restriction enzymes, separated on 0.8% agarose gels, and blotted onto nylon membranes (Hybond N+, Amersham Buchler). Membranes were hybridized under conditions of moderate stringency (hybridization in 35% formamide, 0.1% Na-pyrophosphate, 50 mM Tris-HCl, pH 7.5, 5x SSC, 1% sodium dodecyl sulfate (SDS), 5x Denhardt's, 100 µg/ml calf thymus DNA at 42°, washing in 1x SSC/1% SDS at 60°) with the OlaDmrt1a probe (4-kb EcoRI fragment from cosmid 73K2481).%hrsqwf, http://www.100md.com
FISH analysis:%hrsqwf, http://www.100md.com
For fluorescence in situ hybridization (FISH), mitotic chromosome preparations were made directly from pooled spleen, gills, and cephalic kidney cells after exposing the fish several hours to a 0.02% solution of colchicine. Prior to hybridization, slides were subjected to pepsin (0.01%) and formaldehyde (1%) treatment and denaturation in 70% formamide in 2x SSC (pH 7.0) at 70° following the standard procedure.
Two different bacterial artificial chromosome (BAC) clones, one spanning the Y-chromosome-specific region (BAC 15H17) and one containing a flanking maker (BAC 98J17, SL1), were separately labeled by nick-translation with biotin-16-dUTP or digoxigenin-11-UTP (Roche Molecular Biochemicals, Mannheim, Germany). Labeled DNA at a concentration of 10 ng/µl was coprecipitated with 150 ng/µl calf thymus DNA and 100 ng/µl sonicated medaka genomic DNA and redissolved in 50% formamide, 10% dextran sulfate, and 2x SSC. After 10 min denaturation at 75° and reannealing at 37° for 30 min, 20 µl of probe mixture was applied to a denatured slide and sealed under a coverslip. Following overnight incubation at 37°, the slides were washed at 45° in 50% formamide, 2x SSC for 15 min and for an additional two times of 5 min each, with 1x SSC at 60°.43mruk, 百拇医药
The locations of the hybridization sites were detected with rhodamine-conjugated avidin (Vector, Burlingame, CA) and antidigoxigenin (monoclonal)-conjugated fluorescein (Sigma, St. Louis) followed by further signal enhancement of biotinylated probe using biotinylated antiavidin and rhodamine-conjugated avidin. Likewise, the sheep anti-mouse FITC conjugate was used to enhance the signal of the digoxygenated probe. Chromosomes and cell nuclei were counterstained with 4'-6-diamidino-2-phenylindole (DAPI). Slides were mounted with antifade medium and the hybridization signal was visualized on a Zeiss epifluorescence microscope equipped with a computer-controlled thermoelectronically cooled charged-coupled device camera. Digitized images of the FITC, rhodamine, and DAPI signals of metaphase spread were captured separately and merged using the Easy FISH 1.0 software (Applied Spectral Imaging). At least 20 metaphase plates for both probes were simultaneously examined to evaluate the hybridization pattern.
RESULTS$jm)in$, 百拇医药
With a candidate for the male sex-determining gene and linked molecular markers now available we wanted to reinvestigate the classical finding of AIDA 1936 of the rare, exceptional XX males. The sex ratio in two representative medaka strains, Carbio and i-3, was determined by the unambiguous phenotypic secondary sex characters and male and female gamete production. No significant deviation from the expected 1:1 ratio was found 1).$jm)in$, 百拇医药
fig.ommitteed$jm)in$, 百拇医药
Table 1. Sex ratios in medaka i-3 and Carbio strains$jm)in$, 百拇医药
When males of various strains were PCR genotyped for the medaka male sex-determining gene, we found a strikingly large number of animals that did not show the expected amplification product (1A). The lack of dmrt1bY was confirmed by Southern blot analysis. All the aberrant males showed a restriction fragment pattern like females 2). Such males were then genotyped for the linked sex chromosomal marker casp6, which is located outside the Y-chromosome-specific fragment in the pseudoautosomal region. The genetic distance between casp6 and dmrt1bY is ~
1 cM (NANDA et al. 2002 ). Primers that give a 392-bp product from the Y chromosomal allele and a 387-bp product from the X chromosomal allele of Southern medaka and a 387-bp product from the Y chromosomal allele and a 392-bp product from the X chromosomal allele of Northern medaka were used (KONDO et al. 2001 ). All aberrant males had only the X chromosomal alleles ( 1B). In the HdrR strain a dominant color marker (orange body coloration, R) on the Y chromosome was also absent from te dmrt1bY-negative males.g0w}, 百拇医药
fig.ommitteedg0w}, 百拇医药
Figure 1. PCR genotyping of medaka males and females. (a) Presence of the dmrt1bY PCR product in normal males of the i-3, Carbio, and HB32C strains. No PCR product was amplified from females and the aberrant males (*). For control, an aliquot of the same DNA was used for actin PCR. (b) Hemizygosity of normal males of the HB32C and HNI strains for caspase 6. Females and the aberrant males (*) show only the X chromosomal PCR product.g0w}, 百拇医药
fig.ommitteed
Figure 2. Southern blot analysis of EcoRI-digested DNA from female, normal male, and aberrant (dmrt1bY negative) males (*) from the Carbio strain, hybridized with the OlaDmrt1a probe. This probe detects the autosomal dmrt1a (two alleles present in Carbio) as well as the Y chromosomal dmrt1bY under conditions of moderate stringency (see NANDA et al. 2002 ).r, http://www.100md.com
FISH analysis was done on metaphase chromosomes of such males using BAC 15H17 as probe. This BAC contains only sequences from the Y-specific region, including dmrt1bY, and hybridizes only to the Y chromosome in normal males (XY,3A), but not to the X. In addition, it shows a weak cross-hybridization with the telomeric region of linkage group 9, which is the location of the autosomal dmrt gene cluster and some other sequences that were coduplicated during the event that created the Y-specific fragment. To identify both sex chromosomes visually, a marker that gives hybridization signals on the long arm of both the X and the Y chromosome was used (3A). Contrary to XY males, in all the analyzed metaphases of XX males no specific FISH signal was detectable with the Y-specific BAC on either one of the sex chromosomes 3B), indicating the absence of most of the Y-specific region in these males. However, the Y-specific probe stiloss-hybridized with the autosomal dmrt1a locus on linkage group 9 (3B). Repeated FISH experiments on XX males were carried out, which consistently corroborated the finding of the Southern hybridization experiment.
fig.ommitteedgtm*%5, 百拇医药
Figure 3. FISH pattern of Y-specific (BAC 15H17) and sex-chromosome-specific (BAC 98J17) probes on XY (a) and XX (b) male metaphase chromosomes. Note the presence of three hybridization signals (red) for the BAC 15H17 in XY males as compared to two spots in XX males. The two relatively weak signals (arrows) represent the autosomal dmrt1a locus (linkage group 9), which can be seen in both XY and XX males. The additional prominent signal (*) is specific to the Y chromosome (a) that is absent in the XX males (b). BAC 98J17 (green signal) containing the marker locus SL1 detects both sex chromosomes (arrowheads).gtm*%5, 百拇医药
To investigate the frequency of the occurrence of XX males, we tested a total of eight strains for the presence of XX males by diagnostic PCRs. The frequency of XX males was highly variable ( 2). In the i-3 strain, not a single XX male was among 81 tested males. Also, in the Kaga strain, no XX male was found. A low frequency of XX males was found in Quart and HNI (3 and 4%, respectively). In other strains, XX males were more frequent, for instance, 12% in Carbio. In the HdrR strain, we found 8 XX males, which were initially identified in the population tanks by the lack of the R phenotype and were confirmed by PCR for the absence of dmrt1bY.
fig.ommitteed-]j!0-, 百拇医药
Table 2. Frequency of males lacking dmrt1bY in different strains of medaka-]j!0-, 百拇医药
To further test the XX chromosome constitution of the aberrant males, several of them were mated to single females ( 3). In every case there was a strong bias toward female offspring, ranging from 100 to 89%. When some of the rare F1 males were crossed again to single females, the strong female bias was seen again except for one case. HB32C male 4-6 had 28 female and 36 male offspring. Three of his sons were tested by crossing to i-3 females and produced all female offspring. One F1 male (Carbio 1-1) was tested in an outcross with 20 of his sisters. The female-to-male ratio here was 4:1. From all crosses several offspring males were PCR tested for the absence of dmrt1bY.-]j!0-, 百拇医药
fig.ommitteed-]j!0-, 百拇医药
Table 3. Progeny test of dmrt1bY negative males-]j!0-, 百拇医药
DISCUSSION-]j!0-, 百拇医药
Using the candidate male sex-determining gene dmrt1bY as a marker, we unexpectedly detected a high number of functional males with uncompromised fertility that did not have this gene.
A possible explanation for the absence of dmrt1bY could be that this gene is not located at the male sex-determination locus, but that it is only a linked marker. The males lacking dmrt1bY (and the closely linked Y chromosomal allele of casp6) would then be recombinants due to XY crossovers in the pseudoautosomal region. In such a case, a similar proportion of females with dmrt1bY should be present. However, in 304 females not a single individual was found with dmrt1bY.;'w, 百拇医药
The crossings of the dmrt1bY-lacking males confirmed their sex chromosomal constitution to be indeed XX. In most cases a strong female-biased offspring was obtained. The reoccurrence of XX males in the following generations can be taken as evidence that the sex reversal in the parental male may not be due to some unidentified environmental effect. The seven XX males earlier reported in medaka were explained by a lowering of the female determining potency of the X chromosome and thus polygenic autosomal male determinants becoming epistatic (AIDA 1936 ). Both XX males described in the guppy Poecilia reticulata (WINGE 1930 ) and the two XX males of the platyfish X. maculatus (OKTAY 1959 ; KALLMAN 1984 ) were all explained by the action of autosomal modifier genes. These autosomal modifiers were proposed to override the sex chromosomal genes; however, it was impossible to decide whether they suppress a female-determining locus on the X chromosome or act as male inducers.
In the medaka, it has been shown that dmrt1bY is the only functional gene in a Y-chromosomal-specific segment. This segment is absent from the X chromosome, and outside this region both sex chromosomes are homologous (NANDA et al. 2002 ). It appears reasonable to assume that the autosomal modifiers have a male-determining activity analogous to dmrt1bY. The crossing data do not support a simple monogenic dominant or recessive trait for such autosomal modifiers. The hypothesis that they are polygenic receives some support from the fact that in most crosses (e.g., Carbio males 1 and 2) the number of male offspring was lower than expected and in one (HB32C male 4-6) was much higher than expected, reflecting differences in the genotype of the females used for the cross. The autosomal modifiers may be polymorphic and strain specific and even absent or present at low frequency in certain strains, such as in i-3. This situation would explain why the number of XX males dropped to zero when males from the HB32C male 4-6 offspring were outcrossed to i-3 females.
Contrary to all earlier reports, which described XX males as an extremely rare phenomenon, our data indicate that in medaka they are very common. This now offers the possibility to identify the linkage groups carrying the autosomal modifiers after repeated backcrossing and to map and identify the genes. The high frequency of XX males may also justify reevaluating Aida's theory of a polygenic sex-determination system with epistasis of sex chromosomal genes in medaka.\ipx8o, 百拇医药
So far, three mutant Y chromosomes have been found in the medaka, all of which were found in XY sex-reversed females. One mutant Y (designated Y-) lacks most or the entire male-specific region, including dmrt1bY; one (YwAwr) has a frameshift that leads to a premature termination of the dmrt1bY protein; and the third (YwSrn) has an intact dmrt1bY coding region, although a so far unknown mutation suppresses dmrt1bY expression in the embryo. This led to the conclusion that dmrt1bY is required for normal testicular differentiation (MATSUDA et al. 2002 ).
The frequent appearance of XX medaka males makes a more differentiated view necessary. The fact that through hormonal treatment even fully fertile XX males can be obtained (YAMAMOTO 1955 ) already indicated that the Y chromosome does not contain genes required for correct differentiation of the testes and for male fertility. The appearance of sexually uncompromised spontaneous XX males clearly shows that dmrt1bY is not obligatory for male sex determination under physiological conditions. This does not question the role of the dmrt1bY gene as a sex-determining gene and as the master regulator of male sexual development in most cases, but it indicates that dmrt1bY function can become dispensable. Obviously one or several autosomal genes can induce male sexual development as well. Such autosomal modifiers have been identified through crossing analyses also in the platyfish X. maculatus (KALLMAN 1984 ). However, in this fish dmrt1bY is not present (VEITH et al. 2003 ). Thus, even if the autosomal modifiers were homologous, they probably substitute for a different molecular event.
The Y chromosome of medaka is still at an early stage of evolution and in fact may be the youngest naturally occurring sex chromosome known so far. It is, however, unclear whether the fact that autosomal modifiers can override the XY system (whose molecular correlate is the presence and expression of dmrt1bY) is a reflection of a situation in which the molecular processes bringing about the male sex-determination system are not firmly established and robust. Several cases in which new sex chromosomes that arose by mutation from the normal chromosome complement invaded a population and took over the function of the previous ones have been described. In the vole Ellobius lutescens, for instance, such a mechanism has led to the elimination of Sry (JUST et al. 1995 ), which is the male sex-determining gene in the overwhelming majority of mammals. The autosomal modifiers of dmrt1bY function in medaka may represent such newly emerging sex-determining genes.@, 百拇医药
ACKNOWLEDGMENTS
We thank G. Schneider, H. Schwind, and P. Weber for breeding of the fish. Founder fish for our medaka stock were generously supplied by Y. Hyodo-Taguchi (Chiba) and A. Shima (Tokyo). Kaga fish were kindly given to us by J. Wittbrodt (Heidelberg). This work was supported by grants supplied by the Commission of the European Community (FAIR CT 97-3796) and Fonds der Chemischen Industrie to M. Schartl and by a grant from the Deutsche Forschungsgemeinschaft (SCHM 484/18-1) to M. Schmid.y\jk:0f, 百拇医药
Manuscript received August 30, 2002; Accepted for publication October 28, 2002.y\jk:0f, 百拇医药
LITERATURE CITEDy\jk:0f, 百拇医药
AIDA, T., 1921 On the inheritance of color in a fresh-water fish Aplocheilus latipes Temmick and Schlegel, with special reference to sex-linked inheritance. Genetics 6:554-573.y\jk:0f, 百拇医药
AIDA, T., 1936 Sex reversal in Aplocheilus latipes and a new explanation of sex differentiation. Genetics 21:136-153.y\jk:0f, 百拇医药
ALTSCHMIED, J., U. HORNUNG, I. SCHLUPP, J. GADAU, and R. KOLB et al., 1997 Isolation of DNA suitable for PCR for field and laboratory work. Biotechniques 23:228-229.
BAROILLER, J. F., Y. GUIGEN, and A. FOSTIER, 1999 Endocrine and environmental aspects of sex differentiation in fish. Cell. Mol. Life Sci. 55:910-931.ez4z|^, http://www.100md.com
BRIDGES, C. B., 1922 The origin and variation in sexual and sex-linked characters. Am. Nat. 56:93-107.ez4z|^, http://www.100md.com
BRIDGES, C. B., 1925 Sex in relation to chromosomes and genes. Am. Nat. 59:127-137.ez4z|^, http://www.100md.com
CHARLESWORTH, B., 1991 The evolution of sex chromosomes. Science 251:1030-1033.ez4z|^, http://www.100md.com
CHARLESWORTH, B. and D. CHARLESWORTH, 2000 The degeneration of Y chromosomes. Philos. Trans. R. Soc. Lond. B Biol. Sci. 355:1563-1572.ez4z|^, http://www.100md.com
DE GRANDI, A., V. CALVARI, V. BERTINI, A. BULFONE, and G. PEVERALI et al., 2000 The expression pattern of a mouse doublesex-related gene is consistent with a role in gonadal differentiation. Mech. Dev. 90:323-326.ez4z|^, http://www.100md.com
DEVLIN, R. H. and Y. NAGAHAMA, 2002 Sex determination and sex differentiation in fish: an overview of genetic, physiological, and environmental influences. Aquaculture 208:191-364.
GUTBROD, H. and M. SCHARTL, 1999 Intragenic sex-chromosomal crossovers of Xmrk oncogene alleles affect pigment pattern formation and the severity of melanoma in Xiphophorus. Genetics 151:773-783.\2, http://www.100md.com
HYODO-TAGUCHI, Y. and M. SAKAIZUMI, 1993 List of inbred strains of the medaka Oryzias latipes, maintained in the Division of Biology, National Institute of Radiological Sciences. Fish Biol. J. Medaka 5:5-10.\2, http://www.100md.com
JUST, W., W. RAU, W. VOGEL, M. AKHVERDIAN, and K. FREDGA et al., 1995 Absence of Sry in species of the vole Ellobius. Nat. Genet. 11:117-118.\2, http://www.100md.com
KALLMAN, K. D., 1984 A new look at sex determination in poeciliid fishes, pp. 95–171 in Evolutionary Genetics of Fishes, edited by B. J. TURNER. Plenum Publishing, New York.\2, http://www.100md.com
KETTLEWELL, J. R., C. S. RAYMOND, and D. ZARKOWER, 2000 Temperature-dependent expression of turtle Dmrt1 prior to sexual differentiation. Genesis 26:174-178.\2, http://www.100md.com
KONDO, M., E. NAGAO, H. MITANI, and A. SHIMA, 2001 Differences in recombination frequencies during female and male meioses of the sex chromosomes of the medaka, Oryzias latipes. Genet. Res. 78:23-30.
KONDO, M., I. NANDA, U. HORNUNG, T. SASAKI, and A. SHIMIZU et al., 2003 Absence of the candidate male sex determining gene dmrt1bY of medaka from other fish species. Curr. Biol. in press.#'3, http://www.100md.com
MATSUDA, M., Y. NAGAHAMA, A. SHINOMIYA, T. SATO, and C. MATSUDA et al., 2002 DMY is a Y-specific DM-domain gene required for male development in the medaka fish. Nature 417:559-563.#'3, http://www.100md.com
MONIOT, B., P. BERTA, G. SCHERER, P. SUDBECK, and F. POULAT, 2000 Male specific expression suggests role of DMRT1 in human sex determination. Mech. Dev. 91:323-325.#'3, http://www.100md.com
MORIZOT, D. C., S. A. SLAUGENHAUPT, K. D. KALLMAN, and A. CHAKRAVARTI, 1991 Genetic linkage map of fishes of the genus Xiphophorus (Teleostei: Poeciliidae). Genetics 127:399-410.#'3, http://www.100md.com
NANDA, I., M. KONDO, U. HORNUNG, S. ASAKAWA, and C. WINKLER et al., 2002 A duplicated copy of DMRT1 in the sex determining region of the Y chromosome of the medaka, Oryzias latipes. Proc. Natl. Acad. Sci. USA 99:11778-11783.#'3, http://www.100md.com
ÖKTAY, M., 1959 Über Ausnahmemaennchen bei Platypoecilus maculatus und eine neue Sippe mit XX-Maennchen und XX-Weibchen. Rev. Fac. Sci. Univ. Istanbul Ser. B Sci. Nat. 24:75-92.
RAYMOND, C. S., J. R. KETTLEWELL, B. HIRSCH, V. J. BARDWELL, and D. ZARKOWER, 1999 Expression of Dmrt1 in the genital ridge of mouse and chicken embryos suggests a role in vertebrate sexual development. Dev. Biol. 215:208-220.#ce:[l, 百拇医药
RAYMOND, C. S., M. W. MURPHY, M. G. O'SULLIVAN, V. J. BARDWELL, and D. ZARKOWER, 2000 Dmrt1, a gene related to worm and fly sexual regulators, is required for mammalian testis differentiation. Genes Dev. 14:2587-2595.#ce:[l, 百拇医药
SCHARTL, M., B. WILDE, I. SCHLUPP, and J. PARZEFALL, 1995 Evolutionary origin of a parthenoform, the Amazon molly Poecilia formosa, on the basis of a molecular genealogy. Evolution 49:827-835.#ce:[l, 百拇医药
SMITH, C. A., P. J. MCCLIVE, P. S. WESTERN, K. J. REED, and A. H. SINCLAIR, 1999 Conservation of a sex-determining gene. Nature 402:601-602.#ce:[l, 百拇医药
VEITH, A-M., A. FROSCHAUER, C. KÖRTING, I. NANDA, and R. HANEL et al., 2003 Cloning of the dmrt1 gene of Xiphophorus maculatus: dmY/dmrt1Y is not the master sex-determining gene in the platyfish. Gene in press.#ce:[l, 百拇医药
WADA, H., A. SHIMADA, S. FUKAMACHI, K. NARUSE, and A. SHIMA, 1998 Sex-linked inheritance of the lf locus in the medaka fish (Oryzias latipes). Zool. Sci. 15:123-126.#ce:[l, 百拇医药
WINGE, O., 1930 On the occurrence of XX males in Lebistes, with remarks on Aida's so-called "non-disjunctional" males in Aplocheilus. J. Genet. 23:69-76.#ce:[l, 百拇医药
WINGE, O., 1934 The experimental alteration of sex chromosomes into autosomes and vice versa, as illustrated by Lebistes.. C. R. Trav. Lab. Carlsberg Ser. Physiol. 21:1-49.#ce:[l, 百拇医药
YAMAMOTO, T., 1955 Progeny of artificially induced sex-reversals of male genotype (XY) in the medaka (Oryzias latipes) with special reference to YY-male. Genetics 40:406-419.#ce:[l, 百拇医药
YAMAMOTO, T., 1975 Medaka (Killifish) Biology and Strains. Keigaku, Tokyo.(Indrajit Nanda Ute Hornung Mariko Kondo Michael Schmid and Manfred Schartl)