A new mutant that has neither male nor female secondary sex characters was found in the medaka, Oryzias latipes. Both XX and XY mature mutants had gonads with many spermatozoa, but spawning did not occur when the mutants were paired with normal males or normal females. F1 progeny were successfully obtained by artificial insemination using unfertilized eggs from wild-type females and spermatozoa of the XY mutant. The mutant phenotype did not occur in the F1 progeny from this cross. Incrossing among the F1 progeny produced 17 mutant offspring out of 68 progeny (25%), demonstrating that the mutant phenotype is caused by a single recessive mutation. This mutant was named scl (sex character-less). Because papillary processes, a male secondary sex character, were induced in the XY mutants by androgen administration, it seems that the androgen receptor is functioning normally. We found a loss-of-function type mutation in the P450c17 gene of the mutant; this gene encodes a steroidogenic enzyme required for the production of estrogen and androgen. The scl phenotype was completely linked to the mutant genotype of P450c17, strongly suggesting that mutation at the P450c17 locus is responsible for the scl mutant phenotype.
In vertebrates, gonadal sex is determined by major sex factors contained on the sex chromosomes or by environmental factors (e.g., temperature and social interaction). Following gonadal sex determination, steroid hormones secreted from the gonads determine the sex of gonadal accessory organs. Secondary sex characters are similarly induced by sex steroid hormones.
The medaka, Oryzias latipes, has been used as a model organism for examining mechanisms of sex determination and sex differentiation. This species has a firm XX/XY sex-determining system (Aida, 1921), with DMY on the Y chromosome found to be the sex-determining gene (Matsuda et al., 2002, 2007; Nanda et al., 2002). In XY fish, DMY can induce testicular development, whereas ovarian development occurs in the absence of DMY.
The medaka has clear sexual dimorphism (Fig. 1A–D). The shape of the dorsal and anal fins and the presence of papillary processes on the anal fins are conspicuous male-specific secondary sex characters. The dorsal fin of the male has a saw-toothed distal edge, and the edge between the hindmost ray and other rays has a deep notch, not observed in females. The papillary processes on the anal fin rays are present only in males. In females, on the other hand, the small anal fin has a right-triangle shape. The urinogenital papilla (UGP) is a female-specific secondary sex character (reviewed by Egami, 1975). Formation of the papillary processes can be induced by androgen (Okada and Yamashita, 1944), and the UGP by estrogen (Yamamoto and Suzuki, 1955).
Here, we found XY fish of the Hd-rR.YHNI(R1) strain (Matsuda et al., 2002) of medaka lacking both male and female secondary sex characters. Genetic analyses revealed that this trait was heritable. In this paper, we describe the phenotypic and genetic traits of this mutant and suggest a primary candidate gene for the mutation. We carried out histological observation of gonads, genetic analysis using artificial insemination, and induction of formation of papillary processes by using an artificial androgen, methylt-estosterone. We found that the phenotype was associated with a deficiency of sex steroids caused by a recessive mutation at the scl (sex character less) locus.
MATERIALS AND METHODS
All fish used in this study originated from stocks kept at the Faculty of Science, Niigata University, Japan. They were reared at 27°C±2°C, under a photoperiod of 14 h light and 10 h dark. From two inbred strains of medaka, Hd-rR and HNI (Hyodo-Taguchi and Sakaizumi, 1993), we established a Y-congenic strain, Hd-rR.YHNI. XY fish of the Hd-rR.YHNI strain have a genetic background derived from the Hd-rR strain, and a Y chromosome that includes the sex-determining gene from the HNI strain (Matsuda et al., 1998). Next, we established a recombinant strain, Hd-rR.YHNI(R1), in which recombination occurred between the X and Y chromosomes (Matsuda et al., 2002). Genotypic sex (XY or XX) of the fish used in this study was determined by establishing the presence or absence of the DMY gene by PCR on DNA extracted from a caudal fin clip (Shinomiya et al., 1999; 2004).
Gonads of mature fish at about six months after hatching were dissected and fixed in Bouin's solution. Specimens were embedded in paraffin, sectioned serially at 5-μm thickness, and stained with hematoxylin and eosin for observation under a light microscope.
Treatment with methyltestosterone
Fish were reared in aged tap water containing methyltestoster-one (MT; Sigma Chemical Co., St. Louis, MO) at a concentration of 10 μg/L. The MT solution was changed every three days. At water changing, the fish were anesthetized with ethylene glycol monophenyl ether (0.025%), and the number of papillary processes on the anal fin rays was counted under a dissection microscope.
Determination of P450c17 genotypes
Genotypes of the P450c17 gene, which is responsible for both androgen and estrogen synthesis, were determined by PCR analysis. The intron and exon structure of P450c17 was estimated on the basis of the cDNA sequence from ovarian follicles of the orange-red variety of medaka (DDBJ Accession No. D87121) and the genomic sequence of the Hd-rR inbred strain (Medaka Genome Project: http://dolphin.lab.nig.ac.jp/medaka/index.php, version 1.0, scaffold 557) (Fig. 2). P450c17 PCR primers were C17-14F (5′-ACA GAA GTA CGG ACA GAC CT-3′), C17-14R (5′-AAC AGA GCG AGC AGA TGA CG-3′), and C17-24F (5′-TGT TTG GAG AGG GTT CTG-3′). The thermal cycling conditions were 5 min at 94°C for 1 cycle; 30 sec at 94°C, 30 sec at 55°C, and 60 sec at 72°C for 30 cycles; and 5 min at 72°C for 1 cycle. After the PCR reaction, PCR products were electrophoresed on 1% agarose gel.
Phenotype of XY fish with no secondary sex characters
We found XY fish with female-like fins and no UGP in the Hd-rR.YHNI(R1) strain (Fig. 1E, F). When XY fish with no secondary sex characters were paired with either normal females or normal males, spawning did not occur. We performed a histological examination of the gonads from 11 mature XY fish with no secondary sex characters at six months after hatching. All 11 XY fish had testes, and spermatogenesis appeared to have been proceeding normally (Fig. 3A, B). That is, these XY fish were neither intersex, nor neuter, nor sex-reversed female, but males without secondary sex characters.
Induction of papillary processes by exogenous MT
Since male secondary sex characters of the medaka have been reported to be induced by the administration of androgens (Okada and Yamashita, 1944), the lack of male secondary sex characters in the fish in this study was thought to be caused by either deficiency of androgen or low sensitivity to androgen. To examine the sensitivity of the fish to androgen, we treated XY fish with no secondary sex characters and normal Hd-rR females with MT. In addition, because the presence of the Y chromosome might affect the development of papillary processes, we also treated sex-reversed XY females of the Hd-rR.YHNI(R1) strain, which were induced by the treatment of embryos with estradiol-17β(Iwamatsu, 1999). We used three individuals in each group.
In all groups, the papillary processes began to appear between three and six days after the beginning of treatment, and then increased in number (Figs. 4, 5). The time-course of change in the number of papillary processes did not differ among the three groups.
Genetic features of fish with no secondary sex characters
Because the XY fish with no secondary sex characters had mature spermatozoa but did not reproduce naturally, we performed artificial insemination (Iwamatsu, 1973), using a testis from an XY fish to obtain offspring from these fish. The testis was dissected, and mature ova from the Hd-rR strain were artificially inseminated in a saline solution (111.2 mM NaCl, 5.4 mM KCl, 1.1 mM CaCl2, 0.6 mM MgSO4, pH adjusted to 7.3 with NaHCO3; Iwamatsu, 1973). We obtained 16 adult F1 fish, all of which showed secondary sex characters. We then crossed the F1 females with the F1 males to obtain 68 F2 fish, and obtained XY fish without male secondary sex characters and XX fish without a UGP (Table 1). The ratio of fish with no sex characters to normal fish in the F2 was just one-third (17:51), and there was no significant difference in this ratio between XX (8:27) and XY (9:24) fish (χ2 test, P<0.05), indicating that the phenotype of no secondary sex characters is caused by a single recessive mutation on an autosome. Therefore, we named the mutant scl (sex character less).
Number and genetic and phenotypic sex in F1 and F2 offspring from an XY scl mutant and Hd-rR female cross.
We performed a histological examination of gonads from five XX fish with no UGP at about six months after hatching. Spermatogenesis had occurred in the gonads of all five fish; however, oocytes at the diplotene stage were also present in two of them. In the testes of all 11 scl XY fish, cysts of type B spermatogonia, spermatocytes, and spermatids were aligned in seminiferous tubules toward the central region. In contrast, in gonads of the XX fish with no UGP, these cysts were in disorderly alignment in comparison with those in the testes of normal XY and scl-XY fish, and neither an ovarian cavity nor efferent duct was identified. That is, the structure of these XX gonads appeared to be different from those of both normal ovaries and testes (Fig. 3A, C, D, E).
Identification of a candidate gene for scl
The induction of papillary processes by MT in the scl XY mutant suggests that a lack of androgens might be responsible for the scl phenotype. In addition, the scl XX mutants had no UGP. Therefore, estrogen might also be lacking in the scl mutants. The fact that both male and female secondary sex characters were absent in the scl XX and XY mutants indicates that the scl phenotype might be induced by the deficiency of an enzyme related to an early steroid hormone-producing pathway.
Of the genes that encode for enzymes related to the early steroidogenic pathway in the medaka, only the P450c17 gene had been cloned when we began this study. P450c17 encodes an enzyme with multiple catalyzing functions at a branching point of the biosynthesis pathway of cortisol and sex steroids, including androgens and estrogens. Thus, we believed that P450c17 could be a candidate scl mutant gene. We examined the P450c17 genotypes of wild-type, scl, and F1 fish. Using a combination of C17-24F and C17-14R primers, a 1.2-kilobase pair (kb) band was detected in all genotypes. On the other hand, with a combination of C17-14F and C17-14R primers, a 1.5-kb band was observed only in the wild type and the F1, but not in the scl mutants (Fig. 6). In other words, the C17-24F/C17-14R primer set could amplify both the wild-type and mutant alleles, but the C17-14F/C17-14R primer set could not amplify the mutant allele. Thus, the scl mutation was thought to be located between primer sites C17-14F and C17-24F.
Next, we carried out linkage analysis between the P450c17 genotype and scl phenotype. All 30 scl mutants lacked the 1.5-kb band, demonstrating that the scl phenotype was completely linked to the mutant genotype of P450c17. Analysis of medaka genome data (Medaka Genome Project: http://dolphin.lab.nig.ac.jp/medaka/index.php, version 1.0, scaffold 557) showed that the P450c17 gene is located in autosomal linkage group 15, consistent with the results of our genetic cross, i.e., that the scl phenotype is caused by a single recessive mutation on an auto-some.
Expression mechanism of scl phenotypes
Secondary sex characters are known to be induced by sex steroid hormones secreted from gonads in vertebrates, including the medaka. Here, in the scl mutants, neither male nor female secondary sex characters were seen. Moreover, the induction of papillary processes in the scl mutant treated with MT suggests that the processes of expression of male secondary sex characters after binding of androgen to the receptor are normal. Therefore, the ability to synthesize androgen seems defective in the scl mutant. Furthermore, since a UGP, which is a female secondary sex character, does not appear in the scl mutant, the ability to synthesize estrogen also seems defective. These results strongly suggest that the scl phenotype is caused by a lack of sex steroids—both androgens and estrogens. In fact, concentrations of testosterone and 17β-estradiol did not reach detectable levels in the scl mutants (Suzuki et al., in press). Another mutant that lacks male secondary sex characters and has testes in XY individuals is the testicular feminization mutant (Tfm) in mammals. However, Tfm exhibits complete androgen insensitivity caused by a mutation of the androgen receptor (Lyon and Hawkes, 1970). Accordingly, we suggest that scl is a novel sex differentiation mutant in vertebrates.
The sex steroidogenic pathway and associated enzymes have been clarified (reviewed by Devlin and Nagahama, 2002), and P450c17, a sex steroid synthesis-related gene, has been cloned in the medaka (DDBJ Accession No. D87121). Our results suggest that the P450c17 gene of the scl mutants is distinguishable from that of normal fish. Using the C17-14F and C17-24F primer set, a 1.5-kb band was amplified in normal and F1 fish, but not in the scl mutants. We then confirmed that the DNA sequence of the Hd-rR inbred strain was the same as that of the scl mutant at the C17-14F primer site (data not shown). These results indicate that a very large DNA fragment is inserted in the region between the C17-14F and C17-24F primer sites. Therefore, scl may be a loss-of-function P450c17 mutant caused by the insertion.
P450c17 deficiency in humans results in impaired production of cortisol and sex steroids, leading to overproduction of mineralocorticoids, as well as hypertension, pseudohermaphroditism, and a delay in sexual maturation (Kater and Biglieri, 1994; Yanase, 1995; New, 2003). In the scl mutant, the loss-of-function P450c17 mutation appears to cause a loss of sex steroids, apparently resulting in the complete absence of both male and female secondary sex characters. In addition, XY fish with the scl mutation do not show the pseudohermaphroditism evident in humans with the P450c17 mutation.
Mutation of P450c17 appears to cause impaired production of cortisol, which is vital to the existence of vertebrates, including fish. Why, then, is the scl mutant alive in the absence of P450c17 function? Recently, Zhou et al. (2007a and 2007b) identified a novel type of P450c17 (P450c17-II) in several fishes, including the medaka. P450c17-II in the medaka and tilapia possesses only 17α-hydroxylase activity, without any 17,20 lyase activity, unlike conventional P450c17 (P450c17-I), which has both of these activities. In the head kidney, P450c17-II was strongly expressed, whereas no signal for P450c17-I expression was detected. Our data suggest that scl is a loss-of-function mutant of P450c17-I. Taken together, these results indicate that because of the presence of a normal P450c17-II, which is responsible for the production of cortisol, the scl mutant is viable despite its deficiency in the P450c17-I gene.
Role of androgens in medaka spermatogenesis
The scl XY fish had well-developed testes with many spermatozoa, similarly to normal male fish. Artificial insemination using a testis from an scl XY fish showed that the spermatozoa of the scl XY fish were normal. Androgens are believed to be necessary in mammals for expression of the normal male phenotype, including the initiation and maintenance of spermatogenesis (reviewed by Collins et al., 2003; Holdcraft and Broun, 2004). In Tfm mutants and androgen receptor knockout mice, spermatogenesis is arrested in pachytene spermatocytes (Lyon and Hawkes, 1970; reviewed by Yeh et al., 2002). A deficiency in P450c17 causes infertility in male mice, and many sperm of these mice are morphologically abnormal (Liu et al., 2005). In teleost fish, androgens are also believed to be important for spermatogenesis. When 11-ketotestosterone, an androgen of fish, was added to testicular organ culture, spermatogenesis, from the proliferation of spermatogonia to spermiogenesis, was induced in the Japanese eel, Anguilla japonica (Miura et al., 1991). The action of 11-ketotestosterone in the promotion of spermatogenesis has also been recognized in goldfish (Kobayashi et al., 1991) and Hucho perryi (Amer et al., 2001). However, our results from the scl mutant suggest that spermatogenesis in medaka can occur without androgens. Saiki et al. (1997) showed that the spermatocytes of the medaka develop into fertile sperm in cell culture without contact with somatic cells. The report is consistent with normal spermatogenesis in XY scl mutants. Therefore, the scl mutant may provide novel insight into the relationship between spermatogenesis and androgens in vertebrates.
We note that a progestin, 17α,20β-dihydroxy-4-pregnen-3-one (DHP), also plays an important role not only in final maturation but also in the initiation of meiosis in Japanese eel spermatogenesis (Miura et al., 2006). However, relationship between DHP and spermatogenesis in the scl mutants is unclear.
Spermatogenesis in the scl XX mutant
Mature scl XX fish had a testis instead of ovaries, and spermatogenesis seemed to occur normally, although oocytes at the diplotene stage were observed in the gonads of two scl XX fish. Several studies in teleost fish have suggested that estrogens are involved in ovarian differentiation and development (reviewed by Fostier et al., 1983). Suzuki et al. (2004) reported that in the medaka, aromatase inhibitor (AI) treatment suppressed ovarian cavity formation, and spermatogenesis was occasionally induced in the ovaries of AI-treated adult fish. However, AI treatment did not affect early oogenesis. These results suggest that endogenous estrogen is not essential for sex determination nor early oogenesis; we thus assume that early-stage oogenesis occurs normally in scl-XX fish, but that the ovarian cavity does not develop in the absence of estrogen. Subsequently, spermatogenesis may occur in scl XX gonads after they reach maturity and ultimately only spermatogenesis may be observed, but not oogenesis. In fact, in scl XX gonads, early oogenesis is not affected, but ectopic spermatogenesis occurs in gonads at the adult stage (from 70 days after hatching) (Suzuki et al., in press). At that time, the cysts may be aligned in a disorderly fashion, unlike those in the testes of normal males.
Estrogen receptor α and β knockout (αβERKO) mice show normal ovarian development at an early stage; however, the ovaries of adult αβERKO XX individuals exhibit transdifferentiation of follicles to structures resembling seminiferous tubules of the testes after puberty (Couse et al., 1999; Dupont et al., 2000). These reports suggest that in mammals, estrogen is required for the maintenance of ovaries, but not for early ovarian development. Since the gonadal development in αβERKO XX mice is similar to that of the scl XX medaka, it appears that estrogen is required in vertebrates for maintenance of the ovary, but not for sex determination nor early oogenesis. However, the mechanism of the sex reversal in the XX scl mutant is unknown, and it is unclear whether steroids other than androgens and estrogens (for example, DHP) are involved. Thus, the mechanism of the sex reversal needs further investigation.
We found a viable P450c17 loss-of-function mutant in medaka. Although P450c17 has been isolated in many vertebrates, including several teleost fish (for example, rainbow trout [Sakai et al., 1992], Japanese eel [Kazeto et al., 2000] and zebra fish [Wang and Ge, 2004]), a P450c17 loss-of-function mutant had not yet been found in vertebrates. Thus, scl may serve as an in-vivo animal model for analyzing the role of sex steroids in sex determination and gametogenesis.
This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (17770187 to T. S.; 2126444 to S. H.).
- T. Aida 1921. On the inheritance of color in a fresh water fish, Aplocheilus latipes Temminck and Schlegel, with special reference to sex-linked inheritance. Genetics 6:554–573. Google Scholar
- M. A. Amer, T. Miura, C. Miura, and K. Yamauchi . 2001. Involvement of sex steroid hormones in the early stages of spermatogenesis in Japanese Huchen, Hucho perryi. Biol Reprod 65:1057–1066. Google Scholar
- L. L. Collins, H. J. Lee, Y. T. Chen, H. Y. Hsu, S. Yeh, and C. Chang . 2003. The androgen receptor in spermatogenesis. Cytogenet Genome Res 103:299–301. Google Scholar
- J. F. Couse, S. C. Hewitt, D. O. Bunch, M. Sar, V. R. Walker, B. J. Davis, and K. S. Korach . 1999. Postnatal sex reversal of the ovaries in mice lacking estrogen receptors α and β. Science 286:2328–2331. Google Scholar
- R. H. Devlin and Y. Nagahama . 2002. Sex determination and sex differentiation in fish: an overview of genetic, physiological, and environmental influences. Aquaculture 208:191–364. Google Scholar
- S. Dupont, A. Krust, A. Gansmuller, A. Dierich, P. Chambon, and M. Mark . 2000. Effect of single and compound knockouts of estrogen receptor alpha (ERα) and beta (ERβ) on mouse reproductive phenotypes. Development 127:4277–4291. Google Scholar
- N. Egami 1975. Secondary sexual characters. In “Medaka (Killifish) Biology and Strains”. Ed by T. Yamamoto , editor. Keigaku Publishing Company. Tokyo. pp. 109–125. Google Scholar
- A. Fostier, B. Jalabert, R. Billard, B. Berton, and Y. Zohar . 1983. The gonadal steroids. In “Fish Physiology vol 9A”. Ed by W. S. Hoar, D. J. Randall, and E. M. Donaldson , editors. Academic Press. New York. pp. 277–372. Google Scholar
- R. W. Holdcraft and R. E. Broun . 2004. Hormonal regulation of spermatogenesis. Int J Androl 27:335–342. Google Scholar
- Y. Hyodo-Taguchi and M. Sakaizumi . 1993. List of inbred strains of the medaka, Oryzias latipes, maintained in the Division of Biology, National Institute of Radiological Science. Fish Biol J Medaka 5:29–30. Google Scholar
- T. Iwamatsu 1973. Studies on oocyte maturation of the medaka, Oryzias latipes. Improvemant of culture medium for oocytes in vitro. Jap J Ichthyol 20:218–224. Google Scholar
- T. Iwamatsu 1999. Convenient method for sex reversal in a freshwater teleost, the medaka. J Exp Zool 283:210–214. Google Scholar
- C. E. Kater and F. G. Biglieri . 1994. Disorders of steroid 17-hydroxylase deficiency. Endocrinol Metab Clin North Am 23:341–357. Google Scholar
- Y. Kazeto, S. Ijiri, T. Todo, S. Adachi, and K. Yamauchi . 2000. Molecular cloning and characterization of Japanese eel ovarian P450c17 (CYP17) cDNA. Gen Comp Endocrinol 118:123–133. Google Scholar
- M. Kobayashi, K. Aida, and N. E. Stacey . 1991. Induction of testis development by implantation of 11-ketotestosterone in female goldfish. Zool Sci 8:389–393. Google Scholar
- Y. Liu, et al 2005. Haploinsufficiency of cytochrome P450 17α-hydroxylase/17,20 lyase (CYP17) causes infertility in male mice. Mol Endocrinol 19:2380–2389. Google Scholar
- M. F. Lyon and S. G. Hawkes . 1970. X-linked gene for testicular feminization in the mouse. Nature 227:1217–1219. Google Scholar
- M. Matsuda, C. Matsuda, S. Hamaguchi, and M. Sakaizumi . 1998. Identification of the sex chromosomes of the medaka, Oryzias latipes, by fluorescence in situ hybridization. Cytogenet Cell Genet 82:257–262. Google Scholar
- M. Matsuda, et al 2002. DMY is a Y-specific DM-domain gene required for male development in the medaka fish. Nature 417:559–563. Google Scholar
- M. Matsuda, A. Shinomiya, A. Suzuki, T. Kobayashi, B. Paul-Prasanth, E-I. Lau, S. Hamaguchi, M. Sakaizumi, and Y. Nagahama . 2007. DMY gene induces male development in genetically female (XX) medaka fish. Proc Natl Acad Sci USA 104:3865–3870. Google Scholar
- T. Miura, K. Yamauchi, H. Takahahi, and Y. Nagahama . 1991. Hormonal induction of all stages of spermatogenesis in vitro in the male Japanese eel (Anguilla japonica). Proc Natl Acad Sci USA 88:5774–5778. Google Scholar
- T. Miura, M. Higuchi, Y. Ozaki, O. Takashi, and C. Miura . 2006. Progestin is essential factor for the initiation of the meiosis in spermatogenetic cells of the eel. Proc Natl Acad Sci USA 103:7333–7338. Google Scholar
- I. Nanda, 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. Google Scholar
- M. I. New 2003. Inborn errors of adrenal steroidogenesis. Mol Cell Endocrinol 211:75–83. Google Scholar
- Y. K. Okada and H. Yamashita . 1944. Experimental investigation of the manifestation of secondary sexual characters in fish, using the medaka, Oryzias latipes (Temminck and Schlegel) as material. J Fac Sci Tokyo Imp Univ (IV-Zool) 6:383–437. Google Scholar
- A. Saiki, M. Tamura, M. Matsumoto, J. Katowgi, A. Watanabe, and K. Onitake . 1997. Establishment of in vitro spermatogenesis from spermatocytes in the medaka, Oryzias latipes. Dev Growth Differ 39:337–344. Google Scholar
- N. Sakai, M. Tanaka, S. Adachi, W. L. Miller, and Y. Nagahama . 1992. Rainbow trout cytochrome P-450c17 (17a-hydroxylase/17, 20-lyase)-cDNA cloning, enzymatic properties and temporal pattern of ovarian P-450c17 mRNA expression during oogenesis. FEBS Lett 301:60–64. Google Scholar
- A. Shinomiya, M. Matsuda, S. Hamaguchi, and M. Sakaizumi . 1999. Identification of genetic sex of the medaka, Oryzias latipes, by PCR. Fish Biol J Medaka 10:31–32. Google Scholar
- A. Shinomiya, H. Otake, K. Togashi, S. Hamaguchi, and M. Sakaizumi . 2004. Field survey of sex-reversals in the medaka, Oryzias latipes: genotypic sexing of wild populations. Zool Sci 21:613–619. Google Scholar
- A. Suzuki, M. Tanaka, and N. Shibata . 2004. Expression of aromatase mRNA and effects of aromatase inhibitor during ovarian development in the medaka, Oryzias latipes. J Exp Zool 301:266–273. Google Scholar
- A. Suzuki, M. Nakamoto, D. Kobayashi, M. Tanaka, Y. Nagahama, T. Sato, S. Hamaguchi, M. Sakaizumi, and N. Shibata . 2007. Sex reversal in germ cell and somatic cell in sex steroid deficient ovary after maturation in the medaka, Oryzias latipes. Mol Reprod Dev In press. Google Scholar
- Y. Wang and W. Ge . 2004. Cloning of zebrafish ovarian P450c17 (CYP17, 17α-hydroxylase/17,20 lyase) and characterization of its expression in gonadal and extra-gonadal tissues. Gen Comp Endocrinol 135:241–249. Google Scholar
- T. Yamamoto and H. Suzuki . 1955. The manifestation of the urinogenital papillae of the medaka (Oryzias latipes) by sex-hormones. Embryologia 2:133–144. Google Scholar
- T. Yanase 1995. 17α-hydroxylase/17,20 lyase defects. J Steroid Biochem Mol Biol 53:153–157. Google Scholar
- S. Yeh, et al 2002. Generation and characterization of androgen receptor knockout (ARKO) mice: an in vivo model for the study of androgen functions in selective tissues. Proc Natl Acad Sci USA 99:13498–13503. Google Scholar
- L-Y. Zhou, D-S. Wang, T. Kobayashi, A. Yano, B. Paul-Prasanth, A. Suzuki, F. Sakai, and Y. Nagahama . 2007a. A novel type P450c17 lacking the lyase activity is responsible for C21-steroid biosynthesis in the fish ovary and head kidney. Endocrinology 148:4282–4291. Google Scholar
- L-Y. Zhou, D-S. Wang, Y. Shibata, B. Paul-Prasanth, A. Suzuki, and Y. Nagahama . 2007b. Characterization, expression and transcriptional regulation of P450c17-I and -II in the medaka, Oryzias latipes. Biochem Biophys Res Commun 362:619–625. Google Scholar