Wild-Derived XY Sex-Reversal Mutants in the Medaka, Oryzias Latipes

Wild-Derived XY Sex-Reversal Mutants in the Medaka, Oryzias latipes

Hiroyuki Otake,*,1 Ai Shinomiya,† Masaru Matsuda,‡ Satoshi Hamaguchi† and Mitsuru Sakaizumi† *Graduate School of Science and Technology and †Department of Environmental Science, Faculty of Science, Niigata University, Ikarashi, Niigata 950-2181, Japan and ‡Laboratory of Reproductive Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan Manuscript received April 5, 2006 Accepted for publication May 14, 2006

ABSTRACT The medaka, Oryzias latipes, has an XX/XY sex-determination mechanism. A Y-linked DM domain gene, DMY, has been isolated by positional cloning as a sex-determining gene in this species. Previously, we found 23 XY sex-reversed females from 11 localities by examining the genotypic sex of wild-caught medaka. Genetic analyses revealed that all these females had Y-linked gene mutations. Here, we aimed to clarify the cause of this sex reversal. To achieve this, we screened for mutations in the amino acid coding sequence of DMY and examined DMY expression at 0 days after hatching (dah) using densitometric semiquantitative RT–PCR. We found that the mutants could be classiﬁed into two groups. One contained mutations in the amino acid coding sequence of DMY, while the other had reduced DMY expression at 0 dah although the DMY coding sequence was normal. For the latter, histological analyses indicated that YwOurYwOur (YwOur, Y chromosome derived from an Oura XY female) individuals with the lowest DMY expression among the tested mutants were expected to develop into females at 0 dah. These results suggest that early testis development requires DMY expression above a threshold level. Mutants with reduced DMY expression may prove valuable for identifying DMY regulatory elements.

N vertebrates, the sex of an individual is established and DMY appears to have originated through duplica- I by the sex of the gonad and, in most cases, whether tion of an autosomal segment containing the DMRT1 a gonad becomes a testis or an ovary is determined by region (Nanda et al. 2002; Kondo et al. 2004). DMY is the genome of that individual. In most mammals, the Y specifically expressed in pre-Sertoli cells, somatic cells chromosome-specific gene SRY/Sry is the master male- that surround primordial germ cells (PGCs), in the early determining gene (Gubbay et al. 1990; Sinclair et al. gonadal primordium before any morphological sex 1990; Koopman et al. 1991). The SRY gene encodes a differences are seen (Matsuda et al. 2002; Kobayashi testis-specific transcription factor that plays a key role et al. 2004). In the medaka, the first indication of in sexual differentiation and development in males morphological sex differences is a difference in the (Lovell-Badge et al. 2002). Non-mammalian verte- number of germ cells between the sexes at hatching brates also have a male heterogametic (XX–XY) sex- (Satoh and Egami 1972; Quirk and Hamilton 1973; determination system, but no homolog of Sry could be Hamaguchi 1982; Kobayashi et al. 2004). It is consid- found. ered that one of the functions of DMY is to act as a factor In the teleost medaka fish, Oryzias latipes, which has an that regulates the proliferation of PGCs via Sertoli cells XX–XY sex-determining system (Aida 1921), DMY (DM in a sex-specific manner and controls testicular differ- domain gene on the Y chromosome) has been found entiation (Kobayashi et al. 2004). in the sex-determining region on the Y chromosome In humans, mutations in the SRY gene result in XY (Matsuda et al. 2002; Nanda et al. 2002). This gene is sex reversal and pure gonadal dysgenesis (Jager et al. the first sex-determining gene to be found among non- 1990). However, mutations in the SRY gene itself are mammalian vertebrates. DMY encodes a protein con- considered to account for only 10–15% of 46,XY gonadal taining a DM domain, which is a DNA-binding motif dysgenesis cases, and the majority of the remaining found in Drosophila melanogaster and Caenorhabditis elegans cases may have mutation(s) in SRY regulatory elements (DSX and MAB-3, respectively) that is involved in sexual or other genes involved in the sex differentiation path- development (Raymond et al. 1998). The cDNA se- way (Cameron and Sinclair 1997). A number of genes quences of medaka DMY and DMRT1 are highly similar have been identified as having roles in the sexual development pathway through analyses of human sexual anomaly cases and/or functional studies in mice (Koopman 2001). 1Corresponding author: Graduate School of Science and Technology, Niigata University, Ikarashi, Niigata 950-2181, Japan. Sex-reversal mutants in medaka are also useful for E-mail: [email protected] revealing the molecular function of DMY and identifying

Genetics 173: 2083–2090 (August 2006) 2084 H. Otake et al. other genes involved in sex determination and differ- GAT GCC ACC-39 and PG17ex5.2, 59-GGG AGC CAA AAA entiation. Analyses of such mutants may lead to further TGC GCC ACA TAA-39; and exon 6: PG17ex6.1, 59-GTC ATT 9 9 understanding of the molecular mechanisms of sex AAC ACA ACG CAC AAC AAC TT-3 and PG17ex6.2, 5 -AAA AAC CAG AAG ACC CGA GAG GAA G-39 (Figure 1A). The differentiation. In the present study, we identified two PCR products were sequenced directly in an ABI Prism 310 types of DMY mutants derived from wild populations of automated sequencer. medaka. The first type is composed of loss-of-function RNA extraction and densitometric semiquantitative RT– mutants that contain mutations at the 39 region of the PCR: Total RNA was extracted from embryos or fry at 9.5 days DM domain, suggesting that the 39 region of the DM after fertilization in Kesen-numa G2,ShironeG2,Aizu-Wakamatsu G2, and Oura G4 generations using an RNeasy mini kit domain is required for the normal function of DMY, (QIAGEN) and subjected to RT–PCR using a OneStep RT– male sex determination, and development. The second PCR kit (QIAGEN). Aliquots (20 ng) of the total RNA samples type is composed of reduced DMY expression mutants were used as templates in 25-ml reaction volumes. The PCR that have lower levels of DMY transcripts and contain a conditions were: 30 min at 55°; 15 min at 95°; cycles of 20 sec at number of germ cells, including oocytes, at hatching. 96°, 30 sec at 55°, and 60 sec at 72°; and 5 min at 72°. The number of cycles for each gene was adjusted to be within the Taken together, these results suggest that early testis linear range of amplification, specifically 36 cycles for DMY development requires DMY expression above a thresh- and 24 cycles for b-actin. The primers for DMY (DMYspe, 59- old level and support the hypothesis that DMY acts as TGC CGG AAC CAC AGC TTG AAG ACC-39 and 48U, 59-GGC a factor regulating PGC proliferation via Sertoli cells TGG TAG AAG TTG TAG TAG GAG GTT T-39) amplified a b 9 during early gonadal differentiation. 404-bp DNA fragment. The primers for -actin (3b, 5 -CMG TCA GGA TCT TCA TSA GG-39 and 4, 59-CAC ACC TTC TAC AAT GAG CTG A-39) amplified a 322-bp DNA fragment. Aliquots of 8 and 4 ml of the DMY and b-actin RT–PCR products, respectively, were electrophoresed in a 2% agarose MATERIALS AND METHODS gel in 13 TAE buffer and stained with ethidium bromide. The Fish and mating schemes: In our previous study, we gels were visualized by UV transillumination and photo- surveyed 2274 wild-caught fish from 40 localities throughout graphed with a Geldoc 2000 (Bio-Rad). The optical density Japan and 730 fish from 69 wild stocks from Japan, Korea, of each band was quantified by densitometry using Quantity China, and Taiwan and identified 23 XY females from 11 One Software (Bio-Rad) and represented as the ratio of DMY localities (Shinomiya et al. 2004). Genetic analyses revealed mRNA to b-actin mRNA in arbitrary units. Analysis of variance that the XY females from 8 localities produced all female XYm (ANOVA) was used to test for statistically significant differ- HNI Hd-rR (Ym, Y chromosome derived from an XY female) progeny, ences among XY ,XY , and XY sex-reversal mutants, and while those from 3 localities yielded both male and female XYm the Games/Howell post hoc test for multiple comparisons was progeny (Table 1), suggesting that all these wild XY females applied. had Y-linked gene mutations. In this study, we established Histological analysis in early gonadal development: The fry mutant strains from these XY female mutants. The XY females of the Kesen-numa G2 and Oura G4 generations at 0 and 10 from northern populations were mated with XY males of an days after hatching (dah) were cut to separate the head and inbred strain Hd-rR (Hyodo-Taguchi 1996) and the XY body. The body portions were fixed in Bouin’s fixative solution females from southern populations were mated with a overnight and then embedded in paraffin. The heads were HNI atsuda used to determine the genotype. Serial cross-sections (5 mm congenic strain Hd-rR.Y (M et al. 1998). The F1 progeny from each pair were grown and their genotypes were thick) of the body portions were cut and stained with hema- determined, since the DMY gene of the northern population, toxylin and eosin. Germ cells were counted in each fry at 0 dah. including the HNI inbred strain, contains 21 nucleotide After the cell counting, the mean number and SE were calcu- deletions in intron 2 compared to the southern population, lated for the Oura G4 generation and the differences between wOur wOur HNI wOur including the Hd-rR strain (Shinomiya et al. 2004). When an Y Y and Y Y individuals were evaluated statistically m m by paired t-tests. Gonadal sex differentiation at 10 dah was XY female produced all female F1 XY progeny, an F1 XY m m m examined for the presence or absence of diplotene oocytes. female was mated with an F1 YY male to produce F2 Y Y m m m females. Next, an F2 Y Y female was mated with an F2 YY male to establish mutant strains producing YmYm females and YYm males in later generations. When an XY female produced RESULTS m m male and female F1 XY progeny, an F1 XY female was mated m m m m m Mutations in the DMY amino acid coding sequence: with an F1 YY male to produce F2 Y Y males. Next, an F2 Y Y m To identify mutations in the DMY amino acid coding male was mated with an F2 XY female to establish mutant strains producing XYm males and females and YmYm males in sequence, we sequenced exons 2–6 of DMY (Figure 1A) later generations. These mutant strains were used for RT–PCR in 23 XY sex-reversed females from 11 localities. Four and histological analyses. types of mutations were identified in 15 XY females from PCR and direct sequencing: To screen for mutations in the 7 localities. One Awara (Matsuda et al. 2002), 2 Aomori, amino acid coding sequence of DMY, exons 2–6 of DMY were PCR-amplified from caudal fin clip DNA using the following 1 Aizu-Bange, and 6 Kurobe XY females contained a C primer sets: exon 2: PG17ex2.1, 59-GGA GTC ACG TGA CCC insertion in a polyC tract specific to the northern popu- TCT TTC TTG GG-39 and PG17ex2.2, 59-TTT CGG GTG AAC lation in exon 3 (Figure 1, A and C), which caused a TCA CAT GGT TGT CG-39; exon 3: PG17ex3.1, 59-GCA ACA frameshift from residue 110 (designated N110fsinsC) GAG AGT TGG ATT TAC GTC TCA-39 and PG17ex3.2, 59-CTT and resulted in premature termination at residue 139 TTG ACT TCA GTT TGA CAC ATC AAT G-39; exon 4: PG17ex4.1, 59-CTC AGG TTT GAC TTG GAT GCT GAC (Figure 1B). An XY female from Suzu contained a C CTG A-39 and PG17ex4.2, 59-CAA ACC AGG CCA TGA CCA deletion in the same polyC tract in exon 3 (Figure 1, A TTC CGA-39; exon 5: PG17ex5.1, 59-CCG ATT CTA GCG GAT and C), which caused a frameshift from residue 109 Sex-Reversal Mutant Medaka 2085

Figure 1.—Structure and sequences of DMY. (A) DMY structure of the Hd-rR.Y HNI congenic strain. Open boxes, shaded boxes, and the hori- zontal line indicate exons, the DM domain, and introns, respectively. Open arrowheads indicate the translation start site ATG and stop site TGA. Numbers represent the nucleotide sequence length (bp). Solid arrowheads indicate the positions of insertions or deletions. Solid ar- rows indicate the primer positions. (B) Amino acid sequences of the Hd-rR.Y HNI congenic strain, Carbio strain from the southern population, and wild-derived sex-reversal mutants. The DM domain is boxed. Italicized letters indicate missense coding. (C) Nucleotide sequences of Hd-rR.Y HNI, Carbio, and wild-derived sex-reversal mutants at the polyC tract in exon 3 depicted by the dotted box in B. The polyC tract speciﬁc to the northern population is underlined. Solid arrowheads indicate the positions of nucleotide insertions or deletions. The Suzu XY female has a cytosine deletion at amino acid 109 (designated P109fsdelC), the Awara XY female has a cytosine insertion at amino acid 110 (designated N110fsinsC), and the Aki XY female has a cytosine deletion at amino acid 102 (designated S102fsdelC), each of which results in a frameshift and premature termination.

(designated 109fsdelC) and resulted in premature ter- from a Saigo XY female) fry at 0 dah did not produce any mination at residue 203 (Figure 1B). An XY female from ampliﬁed bands (data not shown), suggesting that the Aki contained a C deletion in another site in exon 3 DMY gene of these Saigo XY females may contain a large (Figure 1, A and C), which caused a frameshift from insertion or deletion in intron 5 and/or exon 6 and residue 102 (designated S102fsdelC) and resulted in have no polyadenylation signal. The XY females from premature termination at residue 104 (Figure 1, B and Awara, Aomori, Aizu-Bange, Kurobe, Suzu, Aki, and Saigo C). Three XY females from Saigo had normal DMY exons that contained mutations in the amino acid coding m 2–5, but PCR for exon 6 (using primers PG17ex6.1 and sequence produced all female XY progeny in the G1 PG17ex6.2; Figure 1A) and 39-RACE using RNA ex- generation (Table 1), indicating their mutant DMY genes wSai wSai wSai tracted from G2 Y Y (Y , Y chromosome derived were nonfunctional.

TABLE 1 List of XY sex-reversal mutants from wild populations of medaka

m Collection site N Phenotypic sex of XY F1 Population Aomori, Aomori Prefecture 2 All female N Aizu-wakamatsu, Fukushima Prefecture 2 Males and females N Aizu-bange, Fukushima Prefecture 1 All female N Shirone, Niigata Prefecture 2 Males and females N Kurobe, Toyama Prefecture 6 All female N Suzu, Ishikawa Prefecture 1 All female N Awara, Fukui Prefecture 1 All female N Kesen-numa, Miyagi Prefecture 2 Males and females S Saigo, Shimane Prefecture 3 All female S Aki, Kochi Prefecture 1 All female S Oura, Kagoshima Prefecture 2 All female S From Shinomiya et al. (2004). N, number of XY females; Ym, Y chromosome derived from an XY female; N, northern Population; S, southern Population (Sakaizumi et al. 1983; Sakaizumi 1986). 2086 H. Otake et al.

wOur HNI wOur G2 XY female and a G2 Y Y male developed as female (Figure 2A). This result confirmed that YwOur has lost the male-determining function. wKsn wKsn For Kesen-numa, G1 XY (Y , Y chromosome derived from a Kesen-numa XY female) progeny produced from a mating between the XY female and an Hd- rR.Y HNI male were either male or female, similar to the case for the Shirone XY female (Matsuda et al. 2002). Interestingly, we observed only 4 females among 18 XYwKsn wKsn in the G1 generation, while 22 XY females were ob- wKsn servedamong35XY in the G2 generation (Figure 2B). wKsn wKsn Conversely, all 41 G2 Y Y produced from a mating wKsn HNI wKsn between a G1 XY female and a G1 Y Y male developed as male (Figure 2B), suggesting that two YwKsn chromosomes are sufficient for male determination and development. Y HNIYwKsn and Y HNIYwOur, which contained normal Y chromosomes from the Hd-rR.Y HNI congenic strain, developed into normal males in adulthood. Mutants with reduced DMY expression at the sex- determining period: The XY females from Shirone had reduced or faint DMY expression at 0 dah (Matsuda et al. 2002). To define the relationship between sex reversal and the DMY expression level, we detected DMY transcripts at 0 dah for Kesen-numa G2, Aizu-Wakamatsu G2, Shirone G2, and Oura G4 generations that produced sex-reversed females, despite a normal amino acid coding sequence of DMY. To measure the DMY expression levels semiquantitatively, we performed densitometric RT–PCR. The mRNA levels of DMY were Figure 2.—Schemes illustrating genetic crosses for XY fe- calibrated by those of b-actin (Figure 3A) and are shown males from Oura and Kesen-numa. (A) The Oura XY female as the ratios of DMY mRNA to b-actin mRNA in arbitrary wOur produced all female XY progeny in the G1,G2, and G3 gen- units (Figure 3B). Statistical analyses using the Games/ erations (boxes with a solid line). Since Y HNIYwOur did not appear in the G generation (underlined), a G XYwOur female Howell post hoc test revealed that the DMY mRNA level 1 1 wKsn wKsn Hd-rR was mated with the Hd-rR.Y HNI male again. All 33 YwOurYwOur was significantly lower in Y Y than in males of XY HNI wKsn wKsn individuals developed into females in the G3 generation (dot- and XY , although Y Y developed as all male in ted box). (B) The Kesen-numa XY female produced male and adulthood. The statistical analyses further revealed that wKsn female XY progeny in the G1 and G2 generations (boxes with wKsn wKsn wKsn the DMY mRNA levels were significantly lower in XY , a solid line). All 41 Y Y individuals developed into males wSrn wSrn in the G generation (dotted box). Y HNIYwOur and Y HNIYwKsn in- XY (Y , Y chromosome derived from a Shirone XY 2 wWkm wWkm dividuals developed into normal males in adulthood. female), and XY (Y , Y chromosome derived from an Aizu-Wakamatsu XY female), which developed as male or female in adulthood, as well as in YwOurYwOur, Patterns of inheritance for sex reversal in Oura and which developed as all female in adulthood, than in Kesen-numa medaka: The XY females from Shirone, males of XYHd-rR,XYHNI, and YwKsnYwKsn (Figure 3B). Aizu-Wakamatsu, Kesen-numa, and Oura produced sex- Early gonadal development of mutants with reduced m reversed XY female progeny in the G1 generation, DMY expression: We carried out histological observa- although the amino acid coding sequence of DMY was tions for the Oura G4 and Kesen-numa G2 generations normal. To investigate the patterns of inheritance for at 0 and 10 dah to reveal the early gonadal development sex reversal in more detail, we performed further ge- of the mutants with reduced DMY expression. It is well netic analyses for the Oura and Kesen-numa XY females. known that the number of germ cells in many non- wOur wOur For Oura, G1 XY (Y , Y chromosome derived mammalian females is greater than that in males around from an Oura XY female) progeny produced from a the time of morphological sex differentiation (Van mating between the XY female and an Hd-rR.Y HNI male Limborgh 1975; Zust and Dixon 1977; Nakamura developed as all female (Figure 2A), suggesting that YwOur et al. 1998). Thereafter, the germ cells in females con- had lost the male-determining function. Since Y HNIYwOur tinue to proliferate and then enter into meiosis, while wOur did not appear in the G1 generation, another G1 XY the male germ cells arrest in mitosis. We counted the HNI female was mated with the Hd-rR.Y male. All 33 G3 number of germ cells at 0 dah (Figure 4), which repre- YwOurYwOur progeny produced from a mating between a sents the time of the initial appearance of morphological Sex-Reversal Mutant Medaka 2087

Figure 4.—Germ-cell numbers of mutants with reduced DMY expression at 0 dah in the Oura G4 (A) and Kesen-numa HNI G2 (B) generations. Y indicates the Y chromosome derived from the Hd-rR.Y HNI congenic strain with intact DMY. Open circles indicate gonads containing oocytes. Solid circles indicate gonads without oocytes. The numbers of samples are shown in parentheses. There is a signiﬁcant difference between the germ- cell numbers for YwOurYwOur and Y HNI YwOur (*P , 0.01).

Figure 3.—Expression levels of the DMY transcript at 0 dah in XY HNI,XYHd-rR, and XY sex-reversal mutants. (A) Ethidium with a normal Y chromosome from the Hd-rR.Y HNI bromide-staining of the DMY transcripts following gel elec- congenic strain, had a signiﬁcantly lower germ-cell trophoresis. b-Actin was used as a loading control. (B) Semi- number at 0 dah than YwOurYwOur (Figure 4A), and no quantitative densitometric analysis of the DMY transcripts. Columns show the ratio of DMY mRNA to b-actin mRNA in oocytes were observed at either 0 or 10 dah (Table 2; arbitrary units. The ratios are given as the mean 6 standard Figure 5A). These results suggest that Y HNIYwOur develops error of six independent assays. Asterisks indicate statistically as male in the same manner as normal XY males. signiﬁcant differences between the mean values: *P , 0.05 For Kesen-numa, YwKsnYwKsn and XYwKsn, with reduced HNI Hd-rR compared with XY and XY ;**P , 0.05 compared with DMY expression, had a number of germ cells, including XY HNI,XYHd-rR, and YwKsnYwKsn. oocytes, at 0 dah (Figure 4B). However, at 10 dah all eight YwKsnYwKsn had a small number of germ cells and sex differences in medaka (Satoh and Egami 1972; no diplotene oocytes (Table 2; Figure 5F), similar to uirk amilton amaguchi normal males. These results suggest that some of the Q and H ,1973; H 1982). Around wKsn wKsn hatching, germ cells in XX individuals outnumber those Y Y germ cells proliferate like those in normal amaguchi obayashi females at hatching, but the mutants develop as male by in XY individuals (H 1982; K et al. wKsn 2004) and subsequently germ cells enter mitotic arrest 10 dah. Conversely, 14 of 16 XY had diplotene oocytes at 10 dah (Table 2; Figure 5D) and the remaining in XY fry, whereas they go into meiosis in XX fry after wKsn hatching (Satoh and Egami 1972). Further, we exam- 2XY had a small number of germ cells and no diplotene oocytes at 10 dah (Table 2; Figure 5C). These ined the early gonadal development according to the wKsn presence or absence of diplotene oocytes at 10 dah results are consistent with the observations that XY (Table 2; Figure 5). In this period, the female germ cells were either male or female in adulthood and that the number of females was greater than that of males in the are recognizable as oocytes from histological observa- wKsn tions since the fry in putative male contain no germ cells G2 generation. Some XY individuals would probably in the meiotic prophase, while those in putative female develop as females in the same manner as normal females. XY HNI and Y HNI YwKsn, with a normal Y chromo- contain germ cells both in mitosis and in the meiotic HNI prophase (Satoh and Egami 1972). some from the Hd-rR.Y congenic strain, contained For Oura, YwOurYwOur, with the lowest DMY expression no diplotene oocytes at either 0 or 10 dah (Table 2; levels among the tested mutants, contained a number Figure 5E), indicating that these individuals would of germ cells, including oocytes, at 0 dah (Figure 4A), develop as normal males. similar to normal XX females, and all eight YwOurYwOur DISCUSSION had diplotene oocytes at 10 dah (Table 2; Figure 5B). These results suggest that YwOurYwOur develops as female Characteristics of the DMY mutations: The results of in the same manner as normal XX females. Y HNIYwOur, this study indicate that all XY sex-reversal mutants 2088 H. Otake et al.

TABLE 2 Gonadal development at 10 dah in mutant lines

No. of fry Mutant line and generation Genotype No. of fry examined With DO Without DO

wOur wOur Oura G4 Y Y 880 YwOurYHNI 808

HNI Kesen-numa G2 XY 808 XYwKsn 16 14 2 YwKsnYHNI 505 YwKsnYwKsn 808 DO, diplotene oocyte; YwOur, Y chromosome derived from an Oura XY female; YHNI, Y chromosome derived from an Hd-rR.YHNI congenic strain male; YwKsn, Y chromosome from a Kesen-numa XY female. discovered among wild populations of medaka are gene, which encodes the high-mobility group (HMG) associated with defective DMY and can be classified into domain (Harley et al. 2003). In total, 43 of 53 missense two types. One type contains mutations in the amino mutations identified in the SRY gene open reading acid coding sequence of DMY (Awara, Aomori, Aizu- frame are located in the HMG domain (Shahid et al. Bange, Kurobe, Suzu, Aki, and Saigo), while the other 2004), strongly suggesting that the DNA-binding motif type has a normal coding sequence but reduced DMY is essential in vivo. Conversely, only 10 mutations that lie expression at 0 dah (Shirone, Aizu-Wakamatsu, Kesen- outside the HMG domain have been detected. Among numa, and Oura). these, 7 are located in the 59 region and 3 are located in In humans, mutations in SRY are associated with the 39 region of the HMG domain and all have different male-to-female sex reversal and a number of mutations effects on the patient phenotype (Shahid et al. 2004). It have been identified within the SRY gene open reading is hypothesized that the region outside the HMG domain frame (Shahid et al. 2004). Although nonsense muta- may be required to stabilize protein binding and/or to tions are scattered throughout the SRY gene, missense generate specificity by helping to discriminate between mutations tend to cluster in the central region of the protein partners (Wilson and Koopman 2002).

Figure 5.—Gonadal sex differentiation in the Kesen-numa and Oura mutant strains at 10 dah. The gonads of YwOurYwOur (B) and most XYwKsn (D) developed as female, while the gonads of Y HNIYwOur (A), the remaining XYwKsn (C), Y HNIYwKsn (E), and YwKsnYwKsn (F) developed as male. Solid arrowheads indicate germ cells. Open arrowheads indicate diplotene stage oocytes. n, pronephric duct; g, gut. Bars, 10 mm. Sex-Reversal Mutant Medaka 2089

The results of the present study demonstrate the Lower levels of DMY transcripts lead the gonads to occurrence of three types of DMY frameshift mutations. develop as phonotypic females: In mice, Sry is ex- N110fsinsC was found in four populations (Awara, pressed in pre-Sertoli cells (Albrecht and Eicher Aomori, Aizu-Bange, and Kurobe), while P109fsdelC 2001) and required for male determination by means was found in Suzu and S102fsdelC was found in Aki. The of promotion of Sertoli cell differentiation (Swain and N110fsinsC and P109fsdelC frameshift mutations are Lovell-Badge 1999). In medaka, DMY is expressed in present in the same polyC tract in exon 3, which is the pre-Sertoli cells (Matsuda et al. 2002; Kobayashi specific to the northern population. These mutations et al. 2004) and assumed to act as a factor that induces may have occurred independently in different popula- the development of pre-Sertoli cells into Sertoli cells in tions, implying that this region is a mutational hot spot XY gonads, which is involved in the regulation of PGC in the DMY gene, similar to the case for the human proliferation as well as construction of the testicular transcription factor hepatocyte nuclear factor (HNF)- tissue architecture (Matsuda 2005). 1a, which contains a polyC tract in exon 4 (Kaisaki In the present study, we demonstrated reduced DMY et al.1997). The finding that all XYm containing each expression at 0 dah in sex-reversed mutant strains. of N110fsinsC, P109fsdelC, and S102fsdelC in DMY There are two possible causes for this reduced DMY developed as female in adulthood indicates that the expression: (1) the number of DMY-expressing cells is mutated DMY genes do not have the male-determining normal but the level of DMY transcription per cell is function, although they contain the intact DM domain. severely reduced or (2) the number of DMY-expressing Taken together, these results suggest that the 39 region cells is significantly reduced but the level of DMY of the DM domain is indispensable for fulfilling the transcription per cell is normal. Either of these possi- male-determining function of DMY. bilities could severely affect the development of pre- XY sex reversal is associated with reduced DMY ex- Sertoli cells into Sertoli cells. It is interesting to note that pression: In mice, XY sex reversal is associated with two some XX recipient and XY donor chimeras of me- types of mutation that appear to exert their effects by daka develop into males that have only XX germ cells suppressing the Sry expression level. First, deletion of (Shinomiya et al. 2002). These results suggest that the repeat sequences at some distance proximal to Sry on XY somatic donor cells, the minor component in these the mouse Y short arm results in reduced expression chimeras, could induce sex reversal of the XX germ cells and sex reversal (Capel et al. 1993). Second, the sex re- and the XX somatic cells. From these results, we infer versal observed when crossing a Mus mus musculus strain that the latter possibility is unlikely to cause sex reversal, Poschiavinus Y chromosome (Y POS) onto a M. m. domes- and it is reasonable to assume that the former possibility ticus background (in particular C57BL6) (Eicher et al. is the cause of the reduced DMY expression. 1982) has been attributed to a reduced Sry expression Our finding that YwOurYwOur develops as female in the level from Y POS. Using a semiquantitative RT–PCR assay, same manner as normal XX females indicates that a low Nagamine et al. (1999) showed that the levels of Sry DMY mRNA level below the threshold level may lead to expression from different mouse Y chromosomes are failure of DMY-positive cells to further differentiate into correlated with the degree of sex reversal they cause on Sertoli cells. Since Sertoli cells are necessary for regu- an M. m. domesticus background. It appears that a critical lating PGC proliferation, lower levels of DMY transcripts threshold level must be achieved by a certain stage of lead to gonad development as phenotypic females. genital ridge development for the supporting cell pop- In contrast, the fact that YwKsnYwKsn develops as all male ulation to be pushed toward Sertoli cell differentiation. by 10 dah suggests that a DMY mRNA level above the In medaka, DMY expression first appears just before threshold level at the sex-determining period is neces- hatching and morphological sex differences are seen, sary for pre-Sertoli cells to undergo further differenti- whereas the expression of autosomal gene DMRT1 first ation. However, some YwKsnYwKsn individuals contain a occurs at 20–30 dah (Kobayashi et al. 2004). The number of germ cells, including oocytes, at 0 dah. This present results demonstrate that the DMY expression result implies that slightly reduced DMY expression may levels at 0 dah in XYwKsn,XYwSrn,XYwWk, and YwOurYwOu, lead to a reduction in pre-Sertoli cell development into which produced sex-reversed females, are significantly Sertoli cells. Moreover, some XYwKsn develop as females reduced to less than half the levels in males of XY HNI and and the remainder as males by 10 dah, although the XYHd-rR. These results suggest that an underlying cause DMY mRNA levels at 0 dah did not differ significantly of XY sex reversal is a reduced level of DMY mRNA among the six tested XYwKsn individuals (data not shown). during a critical period of sex determination. It is The increase in the sex-reversal ratio seen in Kesen- wKsn wKsn interesting to note that Y Y developed as all male numa G2 suggests the possibility that autosomal loci are in adulthood, although their DMY mRNA level was related to the sex reversal in addition to a reduced DMY significantly lower than that in XY HNI and XY Hd-rR. There- expression level, similar to the case for the tda genes in fore, we consider that the certain threshold level of mice (Eicher et al. 1996). DMY mRNA required for male determination at 0 dah The mutants with reduced DMY expression may lies between the YwKsnYwKsn and XYwKsn mRNA levels. contain regulatory mutations in the flanking region of 2090 H. Otake et al.

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