Molecular cloning and characterization of oocyte-specific Pat1a in<i>Rana rugosa</i>frogs

Nakamura, Yoriko; Iwasaki, T; Umei, Yosuke; Saotome, Kazuhiro; Nakajima, Yukiko; Kitahara, Shoichi; Uno, Yoshinobu; Matsuda, Yoichi; Oike, Akira; Kodama, Masaya; Nakamura, Masahisa

doi:10.1002/jez.1938

Cited by 4 publications

(14 citation statements)

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“…We also determined expression of double sex and mab‐3‐related transcription factor 1 ( Dmrt1 ), involved in testis formation in R . rugosa (Shibata et al., ), and Pat1a , strongly expressed in oocytes (Nakamura et al., ), in the ZW masculinized gonads of R. rugosa . Dmrt1 expression levels were as high in ZW masculinized gonads treated with T at 2.0 ng/mL as those in the untreated Wt testis (closed squares in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Sections were stained with the Vasa antibody at a dilution of 1:1,000 or with the CYP17 antibody at a dilution of 1:2,000 followed by the secondary antibody goat anti-rabbit IgG Alexa Fluor 488 antibody (Life Technologies, Tokyo, Japan) or nonimmune serum. The specificity of the two primary antibodies had been confirmed elsewhere (Fujii et al, 2014;Nakamura et al, 2015). All frozen sections were counterstained with 4 ,6diamidino-2-phenylindole.…”

Section: Immunohistochemistrymentioning

confidence: 96%

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A Threshold Dosage of Testosterone for Female‐to‐Male Sex Reversal in Rana rugosa Frogs

et al. 2016

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Androgens play a critical role in testicular differentiation in many species of vertebrates. While female-to-male sex reversal can be induced by testosterone (T) in some species of amphibians, the mechanism still remains largely unknown even at the histological level. In this study, we determined a threshold dosage of T to induce female-to-male sex reversal in the Japanese frog Rana (R.) rugosa. Tadpoles were allowed to metamorphose into frogs with T present in the rearing water. At 0.2 ng/mL T, female frogs formed tissue comprising a mixture of ovary and testis, the so-called ovotestis, the size of which was significantly smaller than the wild-type ovary. Histological changes occurring in the oocytes of T-treated ovaries induced oocyte degeneration in the masculinizing ovaries leading to their final disappearance. In parallel, many germ cells emerged in the cortex of the ovotestis and, later, in the medulla as well. RT-PCR analysis revealed upregulated expression of CYP17 and Dmrt1 but not 17βHSD in the ovotestis, and downregulation of Pat1a expression. Furthermore, immunohistology revealed CYP17-positive signals in the cortex of the masculinizing ovary, spreading throughout the whole area as the testis developed. These results indicate that oocytes are sensitive to T in the ovary of R. rugosa and that male-type germ cells expand in the masculinizing gonad (testis) contemporaneous with oocyte disappearance.

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Section: Resultsmentioning

confidence: 99%

Section: Immunohistochemistrymentioning

confidence: 96%

A Threshold Dosage of Testosterone for Female‐to‐Male Sex Reversal in Rana rugosa Frogs

et al. 2016

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“…Most of the oocytes disappeared from the sex‐reversing ovaries by day 24 and many small germ cells had emerged. Our previous study also showed that oocytes in the female‐to‐male sex‐reversing gonads of R. rugosa disappear by 24 days after T treatment (Nakamura et al., ). In addition, basement membranes surrounded many of the oocytes in ovaries from days 0 to 16, but became disrupted by day 24 under T treatment at a concentration of 250 μg/body, suggesting that this 8‐day window is critical for transforming the ovaries into testes in R. rugosa tadpoles.…”

Section: Discussionmentioning

confidence: 74%

“…Similarly, T can change phenotypic sex from female to male in Rana rugosa (Nishioka et al., ; Nakamura, ) as well as in other frogs (Schmid et al., ; Wallace et al., ). In R. rugosa frogs, most of the oocytes disappear by 24 days after treatment of female gonads with T (Nakamura et al., ). Estradiol benzoate, on the other hand, induces feminization of developing gonads in many species of reptile (Dantchakoff, ; Pieau, , ; Crews et al.,’89).…”

Section: Introductionmentioning

confidence: 99%

Structural changes in female‐to‐male sex‐reversing gonads of Rana RUGOSA

et al. 2016

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The phenotypic sex of many species of amphibians is subject to reversal by steroid hormones. The mechanism of this process, however, still remains largely unknown. As a step toward understanding the histological changes during sex reversal in amphibians, we analyzed two- and three-dimensional (2D and 3D) structures of sex-reversing gonads in Rana rugosa frogs. 2D views revealed that many oocytes in the wild-type ovary disappeared during female-to-male sex-reversal concomitant with the emergence of Vasa-positive small germ cells. Some of the germ cells were labeled with BrdU. BrdU-positive germ cells were few in the testosterone (T) treated ovaries at days 8 and 16, which resembled wild-type ovaries. Basement membranes became disrupted by day 24 in T-treated ovaries. However, the membranes were later reconfigured into testis-like gonadal structures 40 days after T treatment. 3D imaging of the sex-reversing gonad using serial immunostained sections showed that germ cells were organized in linear fashion extending out from where the sex-reversing gonad attached to the mesorchium 24 days after T treatment. Germ cells were increased in number by 40 days and were localized to the cortex of the gonads. In a T-untreated testis at day 24, many germ cells were distributed throughout the cortex except in the central space, while the efferent duct ran between two sheets of the mesorchium. These results, taken together, suggest that the mesorchium plays an important role in the organization of testicular structure. This is the first report showing germ cell ontogeny and organization in the female-to-male sex-reversing gonad in a vertebrate species.

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“…Laminin is a component of the basement membrane in the male and female gonads [18]. The Wt ZW ovary was much bigger than the Wt ZZ testis (Fig 6a and 6b).…”

Section: Resultsmentioning

confidence: 99%

Participation of androgen and its receptor in sex determination of an amphibian species

et al. 2017

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IntroductionIn the Japanese frog Rana (R.) rugosa the androgen receptor (AR) gene on the W chromosome (W-AR) is barely expressed. Previously we showed that incomplete female-to-male sex-reversal occurred in Z-AR transgenic female frogs. To date, however, there is no report showing that AR with androgens can determine genetically programed male sex fate in any vertebrate species. Here, we examined whether AR together with androgens functions as a sex determinant in an amphibian species.MethodsTo examine whether complete female-to-male sex-reversal occurs in R. rugosa frogs, we produced AR-transgenic (Tg) and -knockdown (KD) female R. rugosa frogs by the I-SceI meganuclease-mediated gene trap and CRISPR/Cas9 system, respectively. AR-Tg and -KD tadpoles were reared in water containing testosterone (T) at 0 to 7.1 ng/ml. Frozen sections were prepared from the gonads of metamorphosed frogs and immunostained for laminin, Vasa, Pat1a, CYP17 and AR. We also employed PCR analysis to examine Dmrt1, Pat1a and CYP17 expression in the gonads of KD and placebo-KD female frogs.ResultsComplete female-to-male sex-reversal occurred in the AR-Tg ZW female frogs when a low dosage of T was supplied in the rearing water of tadpoles. However, no sex-reversal was observed in AR-KD ZW female frogs when the gonads were treated with dosages of T high enough to induce complete female-to-male sex-reversal even in wild type frogs.DiscussionThese results suggest that AR with its androgen ligand functions as a male sex-determinant in the ZW type R. rugosa frogs.

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Molecular cloning and characterization of oocyte-specific Pat1a inRana rugosafrogs

Cited by 4 publications

References 25 publications

A Threshold Dosage of Testosterone for Female‐to‐Male Sex Reversal in Rana rugosa Frogs

A Threshold Dosage of Testosterone for Female‐to‐Male Sex Reversal in Rana rugosa Frogs

Structural changes in female‐to‐male sex‐reversing gonads of Rana RUGOSA

Participation of androgen and its receptor in sex determination of an amphibian species

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