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Introduction: In 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. Methods: To 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. Results: Complete 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. Discussion: These results suggest that AR with its androgen ligand functions as a male sex-determinant in the ZW type R. rugosa frogs. [ABSTRACT FROM AUTHOR]

In mice and humans, the androgen receptor (AR) gene, located on the X chromosome, is not known to be involved in sex determination. In the Japanese frog Rana rugosa the AR is located on the sex chromosomes (X, Y, Z and W). Phylogenetic analysis shows that the AR on the X chromosome (X-AR) of the Korean R. rugosa is basal and segregates into two clusters: one containing W-AR of Japanese R. rugosa, the other containing Y-AR. AR expression is twice as high in ZZ (male) compared to ZW (female) embryos in which the W-AR is barely expressed. Higher AR-expression may be associated with male sex determination in this species. To examine whether the Z-AR is involved in sex determination in R. rugosa, we produced transgenic (Tg) frogs carrying an exogenous Z-AR. Analysis of ZW Tg frogs revealed development of masculinized gonads or ‘ovotestes’. Expression of CYP17 and Dmrt1, genes known to be activated during normal male gonadal development, were up-regulated in the ZW ovotestis. Testosterone, supplied to the rearing water, completed the female-to-male sex-reversal in the AR-Tg ZW frogs. Here we report that Z-AR is involved in male sex-determination in an amphibian species. [ABSTRACT FROM AUTHOR]

The article presents a study on the isolation and characterization of Vasa in the frog Rana rugosa. They cloned complementary DNA (cDNA) encoding Vasa, a member of the DEAD Asp-Glu-Ala-Asp family of proteins, from the ovary of the frog Rana rugosa. It states that comparative alignment of amino acid sequences with Vasa from several vertebrate species indicated that the Rana rugosa orthologue shares eight conserved regions with Vasa from other vertebrates. It notes that Vasa expression indicated no sexual dimorphism in sex determination in Rana rugosa and became higher in the ovary thereafter.

Abstract: In vertebrates, gonadal production of steroid hormones is regulated by follicle-stimulating hormone (FSH) and luteinizing hormone (LH) via their receptors designated FSHR and LHR, respectively. We have shown recently that steroid hormones are synthesized in the differentiating gonad of tadpoles during sex determination in the frog Rana rugosa. To elucidate the role of gonadotropins (GTHs) and their receptors in the production of gonadal steroid hormones during sex determination, we isolated the full-length FSHβ, LHβ, FSHR and LHR cDNAs from R. rugosa and determined gonadal expression of FSHR (FSH receptor) and LHR (LH receptor) as well as brain expression of FSHβ and LHβ during sex determination in this species. The molecular structures of these four glycoproteins are conserved among different classes of vertebrates. FSHβ expression was observed at similar levels in the whole brain (including the pituitary) of tadpoles, but it showed no sexual dimorphism during gonadal sex determination. By contrast, LHβ mRNA was undetectable in the whole brain of tadpoles. FSHβ-immunopositive cells were observed in the pituitary of female tadpoles with a differentiating gonad. Furthermore, FSHR expression was significantly higher in the gonad of female tadpoles during sex determination than in that of males, whereas LHR was expressed at similar levels in males and females. The results collectively suggest that FSHR, probably in conjunction with FSH, is involved in the steroid-hormone production during female-sex determination in R. rugosa. [Copyright &y& Elsevier]

Abstract: In certain species of amphibians gonadal differentiation is influenced by steroid hormones. In the case of the frog Rana rugosa testosterone given to tadpoles reverses sex from female to male, while the opposite reversal – male to female – can be achieved using estradiol-17β. In this study, we investigated whether CYP19 (P450 aromatase), the enzyme responsible for a production of estradiol-17β, was present in the differentiating gonad of R. rugosa. Initially, we immunized rabbits against frog CYP19 peptides and performed immunostaining using specific antibodies purified from that serum. CYP19-reactive signals were observed in gonadal somatic cells of the female, but not male tadpoles at stage (St.) I (the stage prior to phenotypic sex determination in tadpoles of R. rugosa). Immunopositive signals were also produced in ovarian somatic cells froglets at St. XXV (just after the completion of metamorphosis). We also examined the enzymatic activity of CYP19 in the differentiating gonad of R. rugosa. Reverse-phase HPLC (high performance liquid chromatography) analysis revealed that [3H]testosterone was converted to [3H]estradiol-17β in the gonad of tadpoles at St. I. Interestingly, the rate of conversion was much higher in females than in males. To the best of our knowledge, this is the first report on the biosynthesis of estradiol-17β in the gonad of amphibians, and the co-incident identification of active CYP19 enzyme in the differentiating gonad of R. rugosa. Based on our results, we conclude that estradiol-17β may be involved in ovarian differentiation in this species. [ABSTRACT FROM AUTHOR]

Abstract: Cyp19 is expressed at a high level in the gonad of the female tadpole of the frog Rana rugosa during sex determination. To identify sequence elements important for expression of Cyp19, we isolated a genomic clone (∼40 kbp) carrying R. rugosa Cyp19 and analyzed the nucleotide sequence of the 5′-flanking region to search for potential transcription factor binding sites. Sox (SRY-related HMG box) protein and Sf1 binding sites were found in the ovary-specific promoter region of Cyp19. Because Sox3 is located on the sex chromosome in R. rugosa, we conducted the luciferase reporter assay in Xenopus A6 cells using the promoter region. Sox3 drove the reporter gene in the cells, but Sf1 did not. When sequential deletion of the 2.7 kbp Cyp19-promoter region was undertaken, a fragment spanning nucleotides −191 to +48 was sufficient to drive the transcription of the reporter gene. In site-directed mutagenesis, the binding site at −57 in the region was critical for Sox3 responsiveness. Sox3 lacking the HMG box had no ability to promote Cyp19 transcription. In addition, a chromatin immunoprecipitation (ChIP) assay showed that DNA fragments were enriched 8-fold, as determined by real-time PCR, when chromatin was immunoprecipitated with the anti-His antibody against His-tagged Sox3. The results, taken together, suggest that Sox3 activates Cyp19 transcription by its direct binding to the binding site of the Cyp19 promoter region. Sox3 appears to be a factor that directs indifferent gonads to develop into an ovary in R. rugosa. [Copyright &y& Elsevier]