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Involvement of androgen receptor in sex determination in an amphibian species

PLoS ONE (2014) 9(5)
DOI: 10.1371/journal.pone.0093655
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Abstract

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. © 2014 Fujii et al.

Figures

  • Figure 1. Six geographic populations and phylogenetic tree. (A) Six geographic populations differing in their morphology of the sexdetermining chromosome. This figure, modified from Ref. [4–6], shows five local populations in Japan and one in Korea. Three local populations in Japan and one population in Korea have the XX/XY sex-determining system, whereas two populations have the ZZ/ZW. The four geographic populations in Japan are shown in different colors. Sado Island is located in northern Japan as indicated by a black arrow. The Z of northern, and X and Y of western Japan are subtelocentric. The W of northern and X of central Japan are metacentric, and the X and Y of Korea and eastern Japan are more subtelocentric. Pericentric inversions are indicated by a circle with an arrowhead. (B) Phylogenetic tree of the AR gene from different populations of R. rugosa. The tree was constructed by the UPGMA method, using Xenopus tropicalis AR (NM001090884) as an outgroup. The Korean R. rugosa AR (AB910584) is basal. All other ARs segregate into two main clusters: the X of western (AB910586), Y of central (AB910589) and Z of northern and Sado island R. rugosa (AB910592); and the X of eastern (AB910585) and central (AB910588), and the W of northern Japan (AB910591). doi:10.1371/journal.pone.0093655.g001
  • Table 1. Primers used for PCR analysis.
  • Figure 2. Histology of gonads. (A) Schematic diagram of the Tg vector, pARPAR, 1 of 2 Tg vectors used in this study. (B) Histology of Tg and Wt gonads. Tg and Wt gonads with (+) or without (2) T-treatment were taken after laparotomy (upper figures, a–g; bar = 2 mm). Sections from Wt and Tg right and left gonads (RG and LG) were stained with Hematoxylin & Eosin (middle figures, h–n; bar = 100 mm) and a laminin antibody (lower figures, o–u; bar = 100 mm). Dashed lines indicate the borders of the gonads. In the middle figures, germ and somatic cells are indicated by black and blue arrows, respectively. Magnified images of the area within the square are shown in (l), (m) and (n). doi:10.1371/journal.pone.0093655.g002
  • Figure 3. Z-AR/V5, CYP17 and Dmrt1 mRNA expression and Z-AR/ V5 and AAT genomic analysis. Genomic DNA PCR amplification was performed for the transgene (genomic Z-AR/V5). The sex of each frog was determined by genomic amplification of the ATP/ADP translocase (AAT) gene as previously described [16]. RT-PCR analysis was used to detect Z-AR/V5, CYP17 and Dmrt1 mRNA in Wt and Tg gonads treated with (+) or without (2) T. Top panel, Z-AR/V5 integration into genomic DNA; 2nd panel, Z-AR/V5 expression; 3rd panel, CYP17 expression; 4th panel, Dmrt1 expression; bottom panel, AAT genetic sex determination of each frog. doi:10.1371/journal.pone.0093655.g003
  • Figure 4. Western blot and immunohistochemical analyses. (A) Immunoblot analysis of adult testis homogenates with CYP17 and AR antibodies Black arrowheads indicate a single dominant band corresponding to CYP17 and AR with molecular weights of 56 (b) and 86 kDa (d), respectively. Panels (a and c) show the distribution pattern of testicular proteins that were electrophoresed and stained with Coomassie Blue. (B) Localization of AR and CYP17 in the testes from adult (a, b) and juvenile (c, d) frogs. Frozen sections from adult and juvenile testes were stained for AR (a, c) and CYP17 (b, d), and counter-stained with DAPI, respectively. White arrows indicate AR-positive signals in the nuclei of many interstitial cells and some germ cells in (a, c), while CYP17-positive signals in the cytoplasm of many interstitial cells are also indicated by white arrows (b, d). Magnified images of the area within the solid square are shown in (a–d). doi:10.1371/journal.pone.0093655.g004
  • Figure 5. Immunohistology of ZW ovotestes. (A) Localization of AR, CYP17 and Vasa in the ovotestis frozen sections of Wt ZW ovary, and Type 1 to 3 ovotestes were stained for AR (1–5), CYP17 (6–10) and Vasa (11–15). A single oocyte and a small Vasa-positive cell are indicated by a yellow and white arrow, respectively. The orange arrows in (3) indicate AR-positive signals. (B) Localization of AR and CYP17 in the Type 2 ovotestis. Figures 2 and 7 in (A) are enlarged to (a) and (b) in (B), respectively. Frozen sections were stained immunohistologically for AR (a) and CYP17 (b), and counterstained with DAPI. Dashed lines indicate the borders of the gonads. AR- and CYP17-positive signals are indicated by white arrows in (a) and (b), respectively. Bar = 50 mm. doi:10.1371/journal.pone.0093655.g005
  • Figure 6. Co-expression of AR and GFP in the ovotestes. (A) Schematic diagram of Tg vector, prrT2ARG, 1 of 2 Tg vectors used in this study. (B) Frozen sections from the gonads of Z-AR/GFP Tg frogs just after metamorphosis were stained for AR and GFP and counterstained with DAPI. The letters G, K and O indicate gonad, kidney and oocyte, respectively. Dashed lines in (a) indicate the border of the gonads. The gonad in the boxed area with a yellow line in (a) was enlarged and examined under DIC (b, d) and fluorescence (c, d) microscopy. Some of the GFP-immuno-positive cells are indicated by white arrows (d, f, g). AR co-expression is indicated by white arrows in the same cells (e, g). Bar = 50 mm. doi:10.1371/journal.pone.0093655.g006

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Fujii, J., Kodama, M., Oike, A., Matsuo, Y., Min, M. S., Hasebe, T., … Nakamura, M. (2014). Involvement of androgen receptor in sex determination in an amphibian species. PLoS ONE, 9(5). https://doi.org/10.1371/journal.pone.0093655

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