Article pubs.acs.org/est
A Transgenic Medaka Line with Visible Markers for Genotypic and Phenotypic Sex Akira Kanamori* and Keiko Toyama Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan S Supporting Information *
ABSTRACT: Accurate genotyping of sex is required for correct interpretation in any in vivo assays with endocrine disrupting chemicals (EDCs). Visible markers for genotypic sex, if reliable, simplify assays because time-consuming PCRbased genotyping can be skipped. Here, we describe a line of Japanese medaka with a brain-expressed green fluorescent protein (GFP) transgene inserted near the sex-determining locus. When used with a white pigment cell marker, genotypic sex can be determined reliably as early as 3 days after fertilization (well before gonadal sex differentiation). No recombinants were found in more than 2000 progenies. We also introduced a strong ovarian GFP marker into the line with these genetic sex markers, so that phenotypic sex can also be determined reliably at 8 days after hatching. Well-known sex reversal protocols using exogenous steroid treatments of embryos were monitored by this transgenic line, demonstrating the line to be a useful tool for in vivo studies utilizing gonadal sex differentiation of the medaka, especially for screenings of potential estrogenic and androgenic EDCs.
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INTRODUCTION In 2011, OECD adopted the Fish Sexual Development Test (FSDT) as an assay for testing endocrine disrupting chemicals (EDCs).1 Sex reversal and vitellogenin production are its end points. Because sex reversal occurs relatively early in a fish lifecycle, FSDT is less resource consuming than full life-cycle or multigeneration tests. Japanese medaka (Oryzias latipes) has been used to test EDCs (see refs 2, 3) and is one of the species chosen by OECD in the FSDT.1 The sex of medaka is determined genetically by the XX/XY system,4 and its maledetermining gene, dmy (dmrt1bY), was identified as a duplicated copy of dmrt1,5,6 simplifying PCR-based genotyping of sex. Gonadal sex differentiation of the medaka has been extensively studied and occurs around hatching (about 10 days after fertilization; see refs 7, 8). Estrogenic and androgenic steroids are known to induce sex reversal in the medaka embryos (XY females9 and XX males10). Taken together, it is possible, in theory, to screen estrogenic and androgenic EDCs with the medaka at early stages after hatching with gonadal sex reversals as an end point. It can be even less resource consuming than FSDT. Visible markers for genotypic and phenotypic sex of the medaka would simplify this test tremendously. There are two recessive body color markers, r (no red color) and lf (leucophore-free, no white pigment cells), linked to the sex of the medaka. The r locus is strongly linked to the sex4 (0.2% recombination11), but its phenotypes only become unequivocally distinguishable more than 1 month after hatching in our hands. The lf phenotypes become discernible by 3 days after © XXXX American Chemical Society
fertilization, well before gonadal sex differentiation, but there is about 2% recombination between dmy and the lf locus.12 During a study on transcriptional regulation of f igα,13 an oocyte-expressed gene, we found a line with a green fluorescent protein (GFP) based transgene expressed in the brains of embryos. The aim of the present study was to demonstrate that this transgene is inserted in the X chromosome and is strongly linked with sex. Together with another marker also expressed in the oocytes (42Sp50:EGFP14), we propose a convenient medaka system for screening potential EDCs utilizing visible genotypic and phenotypic markers of sex.
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MATERIALS AND METHODS Fish. Individuals at all developmental stages were kept in tap water under 14 h light:10 h dark light cycle at 24−27 °C except during steroid and high temperature treatments (see below). The tap water has approximately 6 × 10−3 S/m conductivity, 2 mmol/L hardness, and 7.6−8.3 pH. Methylene blue was added to tap water for embryos at 3.3 ppm. We fed powdered food consisting of Otohime (Nisshin Marubeni Shiryo, Tokyo, Japan) and Tetramin flakes (Tetra Japan kabushiki Gaisha, Japan) to adult and young fish. Hatchlings were fed paramecia. The line of medaka used in this study was the MC strain described earlier.13 The X chromosomes have mutant alleles for Received: January 23, 2013 Revised: April 21, 2013 Accepted: May 2, 2013
A
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r and lf and the Y has wild alleles for both loci. Other genotypes are gu/gu and b/b, having no guanophores and little black pigmentation. Except in recombinants, the males are redcolored with leucophores and the females are white-colored and leucophoreless. In addition, Kaga inbred line was used for mapping an insertion site of the transgene. Transgenic Fish. Transgenic fish with Trf igα:EGFP13 and those with 42Sp50:EGFP14 were described earlier. The line with both transgenes was generated by two successive crossing between the two lines. The homozygosity of the transgenes was verified by test crossing with nontransgenic fish followed by PCR-based genotyping of embryos (see below). Trfigα:EGFP primers are T2f, 5′-GCAGGGACATCTATACCAACTCCT3′, and T2r, 5′-GTTCACACCTGGACTGCTGCG-3′. 42Sp50:EGFP primer are 42f, 5′-GTCTCATCAAGCAGCACACCT-3′, and 42r, 5′-AGGGTCAGCTTGCCGTAGGTG3′. Genotype Determination by PCR. Genomic DNA was extracted from embryos or hatchlings with an Extract-N-Amp tissue kit (Sigma-Aldtrich, St. Louis, MO) according to the manufacturer’s instructions and was used in amplification with the following primer pairs. Dmy-specific primers are 17.19, 5′GAACCACAGCTTGAAGACCCCGCTGA-3′, and 17.20, 5′GCATCTGCTGGTACTGCTGGTAGTTG-3′ described earlier.5 Primer pairs, 17.z1, 5′-CCGCTGAAAGGCCACAAGCGC-3′, and 17.z2, 5′-GCCTGCTGCCTCCTCAAGGCG-3′, amplify both dmy (0.8 kb) and dmrt1 (1.1 kb). Fluorescence Observation. Embryos and hatchlings were immobilized in 2.5% methyl cellulose in DW (final viscosity ca. 2000 cP, Sigma-Aldrich, St. Louis, MO), observed under a fluorescence stereomicroscope (MZ16FA, Leica microsystems, Wetzlar, Germany) and photographed with a digital camera (DFC480, Leica Microsystems, Wetzlar, Germany). Steroid and High-Temperature Treatments on Embryos. Estradiol-17β (E2, Sigma-Aldtrich, St. Louis, MO) or 17α-methyltestosterone (MT, TCI, Tokyo, Japan) were dissolved in ethanol at 2 mg/mL or 240 μg/mL, respectively, and stored at −25 °C. About 50 0-day embryos were treated in a 90 mm dish containing 30 mL of tap water at 25 °C in darkness until hatching (usually 9−10 days at 25 °C). Ethanol (for control), E2, or MT (3.75 μL each) were added just before incubations were started. Final nominal concentrations of E2 and MT were 250 and 30 ng/mL, respectively. No water change was done until hatching. Dead embryos were removed daily. For high-temperature treatment, the control experiment above was done at 32 °C (hatching took about 8−9 days).
Figure 1. A genetic linkage map of the sex chromosome of medaka derived from male meiosis, showing inserted transgene and two body color markers, r (no red color) and lf (no leucophores). We found two recombinants out of 619 progenies between the transgene and the r locus and seven recombinants between the r and the lf loci. Map distances, cM, were calculated from recombination rates using the Kosambi function.
and in the oocytes, suggesting that the transgene was inserted in the X chromosome. To map an insertion site, recombination between the transgene and the two body color markers in male meioses were analyzed at 40 days after hatching [see Supporting Information (SI) for a crossing scheme and Table S1 for the data]. The deduced genetic linkage map is shown in Figure 1. The transgene is present near the r locus and most likely opposite of the lf locus. From G2, we established a line named faX, with females homozygous for the transgene (X* r lf X* r lf, where * denotes the trangene) and males hemizygous for the transgene (X* r lf Y++). We confirmed the location of the transgene with progeny between G7 faX males (X* r lf Y++) and nontransgenic MC females (Xr lf Xr lf). The presence or absence of the transgene can be determined by GFP expression in the brains, from 3 days after fertilization, about the same time leucophores first appear (Figure 2), onward to the adults (data not shown). At 1−1.5 months after hatching, r and lf phenotypes and the presence or absence of the transgene were determined (SI, Table S2). The expression of the transgene in the gonads (GFP fluorescence in the
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RESULTS A Genotypic Marker by Transgene Insertion. In order to identify genomic sequences regulating transcription of an oocyte-expressed gene, f igα, we injected a construct with Fugu genomic sequences flanking figα fused to GFP coding sequence (Trf igα:EGFP) into medaka embryos.13 The line used was MC (female, Xr lf Xr lf; male, Xr lf Y++).13 Several G0 (injected females) with GFP fluorescence in the oocytes grew up to maturity. One of the G0 females was mated to a nontransgenic MC male. About half of G1 had GFP fluorescence in the brain in addition to the oocytes, probably due to the enhancertrapping of a neural gene, β-adducin, present in the construct (this gene is located next to f igα).13 To obtain fish homozygous for the transgene, male and female G1 with the transgene were mated. Ninety-six percent of G2 with lf phenotype (26 out of 27 hatchlings) expressed GFP fluorescence both in the brain
Figure 2. Trf igα:EGFP expression in progeny between faX males (with the transgene on the X chromosome) and nontransgenic MC females at 3 days after fertilization (stage 30), showing (A) bright-field and (B) GFP fluorescence images of the same sample. An embryo without leucophores expressed GFP fluorescence in the brain (left) and one with leucophores (autofluorescence of yellowish cells around the brain in B) did not express GFP (right). B
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oocytes) was also examined for the individuals with the transgene. As a result, there was no recombination between r and the transgene in the 523 progenies. The recombination between the transgene and lf was 7/523 (1.34 cM). All 263 F1s with the transgene were females, as determined by fluorescence in the oocytes (no recombination between dmy and r/ transgene was found in the 263 progenies). The phenotyped hatchlings were kept up to 3 months after hatching, and their sex was determined by observation of the secondary sex characteristics (shapes of the anal fins, dorsal fins, and the urogenital papilla). Ninety-eight percent (205 out of 210) of those with the transgene had female characteristics, and 98% (205 out of 210) of those without had male characteristics (SI, Table S2). The results again showed that the transgene is inserted in the X chromosome near the r locus and in the region corresponding to the genome flanking dmy on the Y chromosome. Combination with a Phenotypic Marker. In the abovementioned cross between faX males (X* r lf Y++) and nontransgenic MC females (Xr lf Xr lf), the gonadal phenotypes of XX progeny were determined by the green fluorescence from Trfigα:EGFP transgene in the oocytes. This transgene starts to be expressed in the developing oocytes as early as 2 days after hatching. However, we cannot determine gonadal phenotypes of the XY progenies at early stages after hatching because they inherit X chromosomes only from nontransgenic MC females. Therefore, we introduced another transgene, 42Sp50:EGFP, into the faX line by successive crossing. 42Sp50 is an oocyteexpressed gene like f igα, but its expression level is much higher. The ovarian GFP intensity of 42Sp50:EGFP was about 50−100 times higher than that of Trfigα:EGFP (examined at 9−14 days after hatching, data not shown). The 42Sp50:EGFP fluorescence in the ovary started to be detected at 3 days after hatching and reached 100% by 5 days after hatching (SI, Figure S1). This line, 42S-faX, now has homozygous 42Sp50:EGFP in addition to the X chromosome with Trf igα:EGFP. Next we examined progeny of the cross between 42S-faX males and nontransgenic MC females for GFP fluorescence in the brains and the gonads at 8 days after hatching (Figure 3 and SI, Table S3). We found one recombinant between Trf igα:EGFP transgene and dmy from 2236 progenies [transgene (+), leucophore (+) and XY; see SI, Table S3]. There were 53 recombinants between Trfigα:EGFP transgene and lf (2.4 cM). The leucophore (−) progeny with Trf igα:EGFP transgene were 100% female and the leucophore (+) progeny lacking the Trfigα:EGFP transgene were 100% male, as judged from the gonadal fluorescence. We randomly picked four recombinants between the transgene and lf (two with GFP in the gonad and two without) and eight nonrecombinants (four with GFP in the gonad and four without) and genotyped them by PCR. All fish with GFP in the gonad were XX and all without GFP were XY. Steroid or High-Temperature Treatments. To test whether we can monitor sex reversal in this system, we treated F1 embryos between 42S-faX males and nontransgenic MC females with steroids and high temperature, which were reported to induce sex reversal. Phenotypes were examined at 8 days after hatching. All XX embryos [leucophore (−) with the Trfigα:EGFP transgene] treated with MT at 30 ng/mL developed into hatchlings without fluorescence in the gonads, reflecting 100% sex reversal (Figure 4A,C and SI, Table S5). E2 at 250 ng/mL reversed the sex of the XY embryos [leucophore (+) lacking the Trfigα:EGFP transgene], having fluorescence in the gonads (Figure 4B and SI, Table S5). The efficacy of sex
Figure 3. Trf igα:EGFP and 42Sp50:EGFP expression in progeny between 42S-faX males (with Trf igα:EGFP on the X chromosome and homozygous 42Sp50:EGFP) and nontransgenic MC females at 8 days after fertilization, showing (A) bright-field and (B) GFP fluorescence images of the same sample. An upper hatchling without leucophores and with GFP fluorescence in the brain (arrowhead) expressed GFP fluorescence in the gonad (arrow). The gonadal fluorescence was from each developing oocyte (inset, bar = 100 μm). The lower hatchling with leucophores (autofluorescence of yellowish cells in B) and without GFP in the brain did not express GFP in the gonad.
reversal was about 80%. High temperature, 32 °C, did not affect the phenotypic sex of the embryos (Figure 4C and SI, Table S5).
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DISCUSSION Exact genotyping still requires PCR-based analyses. However, visible markers would be useful for initial screening of various chemicals or treatments, making assays less resource intensive. Therefore, genotypic sex markers with transgene insertions have already been reported in model animals such as mice15 and fruit flies.16 Especially in mice, random GFP insertions were systematically made to mark specific chromosomes.17 In medaka, two body color markers, r (no red color) and lf (no leucophores), were known and have been used for rough genotyping of sex (see ref 18). The r marker is tightly linked to sex (0.2% recombination11) but needs at least one month after hatching for precise phenotyping in our hands (data not shown). Figure 3A depicts an XrXr female with no red color and an XrY+ male with red color at 8 days after hatching. We could not confidently differentiate their phenotypes. The lf marker is distinguishable as early as 3 days after fertilization but has about 2% recombination with sex.12 Transgenic medaka lines with dmy-drived GFP gene ligated with lens-specific red fluorescent protein gene have been reported.19 In this case, red eye phenotype was always linked to the presence of dmy, hence maleness. However, this is an artificial situation. At present, we cannot conclude that these transgenic males possess all characteristics of natural males. Therefore, in the present C
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Figure 4. Steroid-induced sex reversal as demonstrated by Trf igα:EGFP and 42Sp50:EGFP expression in progeny between 42S-faX males (with Trf igα:EGFP on the X chromosome and homozygous 42Sp50:EGFP) and nontransgenic MC females at 8 days after fertilization. (A) After MT treatment, neither XX hatchling (upper, no leucophores with GFP in the brain, arrowhead) nor XY hatchling (lower, no GFP in the brain with leucophores) expressed GFP in the gonads. (B) After E2 treatment, both XX hatchling (upper, no leucophores with GFP in the brain, arrowhead) and XY hatchling (lower, no GFP in the brain with leucophores) expressed GFP in the gonads (arrows). (C) Rate of sex reversal with steroids or high temperature (32 °C). Blue or green bar represents XY females or XX males, respectively.
laboratory, medaka do not change their sex once sex is determined around hatching. We also demonstrated that expression of 42Sp50:EGFP is a reliable marker for the phenotypic sex; all leucophore (−) hatchlings (most likely XX) expressed GFP in the gonads by 5 days after hatching (SI, Figure S1). This is significantly earlier than 2 months after hatching, when secondary sex characteristics such as fin shape become apparent (see ref 24). Finally, we checked whether the present system of visible markers can depict steroid- and high-temperature-induced sex reversal. The doses used in the present study, E2 (250 ng/mL)9 and MT (30 ng/mL),10,25 were reported to induce sex reversal in one-time embryo treatments. lose relatives of the medaka, Oryzias luzonensis and Oryzias mekongensis, showed sex reversal at similar doses of E2 and MT.26 Here, the steroid treatments changed the expression of fluorescence in the gonads of XX [leucophore (−) with the GFP in the brain] and XY [leucophore (+) with no GFP in the brain] individuals, respectively. A straightforward explanation is that the present system is capable of detecting sex reversal. The efficacy of steroid treatments in the present system were 100% for MT and 80% for E2. High-temperature (32 °C) treatment of the embryos did not change GFP expression in the gonads. There are several reports showing that 32 °C treatments do induce XX sex reversal,27−29 and this may be caused via corticosteroids.30 However, there is genetic variation in the sensitivity to the high-temperature treatments27,28 among several medaka strains. In conclusion, the system presented here, i.e., use of progeny between 42S-faX males (X* r lf Y ++ with homozygous 42Sp50:EGFP) and nontransgenic MC females (Xr lf Xr lf), reliably predict genotypes of the offspring. All leucophore (−) progeny with the transgene Trfigα:EGFP (>1000) were females and all leucophore (+) progeny without the transgene (>1000) were males. Both the lf marker and the transgene expression become apparent as early as 3 days after fertilization (well before gonadal sex differentiation). Their sexual phenotypes are also visualized by the presence or absence of gonadal fluorescence from the other transgene 42Sp50:EGFP. This system enables noninvasive genotyping without PCR and
study, we devised a convenient medaka system that can reliably predict genotypic sex by a combination of neural GFP expression and a leucophore marker. The transgene, Trf igα:EGFP, was unequivocally shown to be inserted near the r locus. Recombination rate between the r locus and the transgene was 2/619 in initial characterization (Figure 1 and SI, Table S1) and 0/523 in G7 experiment (SI, Table S2). The exact physical location of the transgene relative to the r and lf loci was not determined. However, if we consider crossing-over interference20,21 and restricted recombination between the X and Y chromosomes in male meioses,10,22 it is safe to assume that no double-recombination occurred and, therefore, that the r locus is present between the transgene and the lf locus (Figure 1). Next, we asked how reliable this transgene was as a genotypic marker of sex. We examined how often dmy and the transgene recombination occurred. From the G7 experiment (SI, Table S2), all the fish with the transgene were r phenotype and expressed transgene in the gonads, showing there was no recombination in 263 progeny. Ninety-eight percent (205 out of 210) of the fish without the transgene developed into males (SI, Table S2), also demonstrating low recombination rates between the transgene and dmy. Finally, in the 42S-faX experiment (SI, Table S3), we obtained only one recombinant between the transgene and sex from more than 2000 progenies. All leucophore (−) progeny with the transgene showed GFP in the gonads and all leucophore (+) progeny lacking the transgene did not express fluorescence in the gonads. The results strongly support the following interpretations: (1) The combination of the markers, leucophore (−) with the transgene or leucophore (+) without the transgene, corresponded to XX or XY genotypes, respectively. (2) All XX individuals expressed GFP in the gonads and all XY did not; there was no spontaneous sex reversal in this experiment (There is spontaneous XX sex reversal common to several medaka lines23). This low recombination rate can significantly reduce the number of fish used in various chemical assays. Additionally, the present experiments examined progeny at 8 days, 1−1.5 months, and 3 months after hatching, and there was no discrepancy among the results. These results suggest that in the D
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(8) Kondo, M.; Nanda, I.; Schmid, M.; Schartl, M. Sex determination and sex chromosome evolution: Insights from medaka. Sex. Dev. 2009, 3 (2−3), 88−98. (9) Iwamatsu, T. Convenient method for sex reversal in a freshwater teleost, the medaka. J. Exp. Zool. 1999, 283 (2), 210−214. (10) Matsuda, M.; Sotoyama, S.; Hamaguchi, S.; Sakaizumi, M. Malespecific restriction of recombination frequency in the sex chromosomes of the medaka Oryzias latipes. Genet. Res. 1999, 73 (3), 225− 231. (11) Yamamoto, T. Linkage map of sex chromosomes in the medaka, Oryzias latipes. Genetics 1964, 50 (1), 59−64. (12) Wada, H.; Shimada, A.; Fukamachi, S.; Naruse, K.; Shima, A. Sex-linked inheritance of the lf locus in the medaka fish (Oryzias latipes). Zool. Sci. 1998, 15 (1), 123−126. (13) Kanamori, A.; Toyama, K.; Kitagawa, S.; Kamehara, A.; Higuchi, T.; Kamachi, Y.; Kinoshita, M.; Hori, H. Comparative genomics approach to the expression of f igα, one of the earliest marker genes of oocyte differentiation in medaka (Oryzias latipes). Gene 2008, 423 (2), 180−187. (14) Kinoshita, M.; Okamoto, G.; Hirata, T.; Shinomiya, A.; Kobayashi, T.; Kubo, Y.; Hori, H.; Kanamori, A. Transgenic medaka enables easy oocytes detection in live fish. Mol. Reprod. Dev. 2009, 76 (2), 202−207. (15) Isotani, A.; Nakanishi, T.; Kobayashi, S.; Lee, J.; Chuma, S.; Nakatsuji, N.; Ishino, F.; Okabe, M. Genomic imprinting of XX spermatogonia and XX oocytes recovered from XX↔XY chimeric testes. Proc. Natl. Acad. Sci. U. S. A. 2005, 102 (11), 4039−4044. (16) Hayashi, S. Male-specific GFP marker strain of Drosophila melanogaster. Dros. Inf. Serv. 2010, 224. (17) Nakanishi, T.; Kuroiwa, A.; Yamada, S.; Isotani, A.; Yamashita, A.; Tairaka, A.; Hayashi, T.; Takagi, T.; Ikawa, M.; Matsuda, Y.; Okabe, M. FISH analysis of 142 EGFP transgene integration sites into the mouse genome. Genomics 2002, 80 (6), 564−574. (18) Wakamatsu, Y., Ozato, K. Suitability of medaka fish as a test organism and new medaka strains. In Medaka: Development of Test Methods and Suitability of Medaka as Test Oganism for Detection of Endocrine Disrupting Chemicals; Ministry of the Environment, Japan, and Chemicals Evaluation and Research Institute, Japan: Tokyo, Japan; 2004; pp 7−20; http://www.env.go.jp/chemi/end/sympo/ medaka2003/medaka_mats/jmedaka_all.pdf (19) Otake, H.; Masuyama, H.; Mashima, Y.; Shinomiya, A.; Myosho, T.; Nagahama, Y.; Matsuda, M.; Hamaguchi, S.; Sakaizumi, M. Heritable artificial sex chromosomes in the medaka, Oryzias latipes. Heredity 2010, 105 (3), 247−56. (20) Naruse, K.; Shimada, A.; Shima, A. Gene-centromere mapping for 5 visible mutant loci in multiple recessive tester stock of the medaka (Oryzias latipes). Zool. Sci. 1988, 5 (2), 489−492. (21) Naruse, K.; Shima, A. Linkage relationships of gene loci in the medaka, Oryzias latipes (Pisces: Oryziattidae), determined by backcrosses and gynogenesis. Biochem. Genet. 1989, 27 (3−4), 183−198. (22) Kondo, M.; Nagao, E.; Mitani, H.; Shima, A. Differences in recombination frequencies during female and male meioses of the sex chromosomes of the medaka, Oryzias latipes. Genet. Res. 2001, 78 (1), 23−30. (23) Nanda, I.; Hornung, U.; Kondo, M.; Schmid, M.; Schartl, M. Common spontaneous sex-reversed XX males of the medaka Oryzias latipes. Genetics 2003, 163 (1), 245−251. (24) Kinoshita, M., Murata, K., Naruse, K., Tanaka, M., Eds. A Laboratory Manual for Medaka Biology; Wiley-Blackwell: Ames, IA, 2009. (25) Iwamatsu, T.; Kobayashi, H.; Sagegami, R.; Shuo, T. Testosterone content of developing eggs and sex reversal in the medaka (Oryzias latipes). Gen. Comp. Endrocrinol. 2006, 145 (1), 67− 74. (26) Hamaguchi, S.; Toyozaki, Y.; Shinomiya, A.; Sakaizumi, M. The XX-XY Sex-determination system in Oryzias luzonensis and O. mekongensis revealed by the sex ratio of the progeny of sex-reversed fish. Zool. Sci. 2004, 21 (10), 1015−1018.
phenotyping much earlier than by observation of secondary sexual characteristics. Both GFP markers can be observed easily with conventional fluorescence stereomicroscopes. Therefore, this line can be utilized in various studies on sex differentiation, such as finding sexual differences by analyzing transcriptomes or proteomes, screening mutants on sex differentiation, phenotype characterization of the mutants, effects of temperature or other environmental parameters, and especially screening of potential estrogenic and androgenic EDCs.
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ASSOCIATED CONTENT
S Supporting Information *
Crossing schemes for mapping the insertion site of the transgene and the results (Table S1), the raw data from the G7 faX and MC cross (Table S2), the raw data from the 42SfaX and MC cross (Table S3), the raw data from the sex reversal experiment (Table S4), and the time course of 42Sp50:EGFP expression after hatching (Figure S1). This material is available free of charge via the Internet at http:// pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +81-52-789-2537; fax: +81-52-789-2511; e-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank Drs. Y. Nagahama and T. Iguchi for advice, Dr. A. Shimada for a generous gift of the Kaga line, Dr. H. Hori for various support, and Dr. E.F. Orland for critically reading the manuscript. We also appreciate helpful comments from the reviewers. This work was supported in part by a Grant-in-Aid for Scientific Research (19040010 to A.K.).
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REFERENCES
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