Involvement of Androgen Receptor in Sex Determination in an Amphibian Species

<div><p>In mice and humans, the androgen receptor (<i>AR</i>) gene, located on the X chromosome, is not known to be involved in sex determination. In the Japanese frog <i>Rana rugosa</i> the <i>AR</i> is located on the sex chromosomes (X, Y, Z and W). Phylogenetic analysis shows that the <i>AR</i> on the X chromosome (<i>X-AR</i>) of the Korean <i>R. rugosa</i> is basal and segregates into two clusters: one containing <i>W-AR</i> of Japanese <i>R. rugosa</i>, the other containing <i>Y</i>-<i>AR</i>. <i>AR</i> expression is twice as high in ZZ (male) compared to ZW (female) embryos in which the <i>W</i>-<i>AR</i> is barely expressed. Higher <i>AR</i>-expression may be associated with male sex determination in this species. To examine whether the <i>Z</i>-<i>AR</i> is involved in sex determination in <i>R. rugosa</i>, we produced transgenic (Tg) frogs carrying an exogenous <i>Z</i>-<i>AR</i>. Analysis of ZW Tg frogs revealed development of masculinized gonads or ‘ovotestes’. Expression of <i>CYP17</i> and <i>Dmrt1</i>, 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 <i>AR</i>-Tg ZW frogs. Here we report that <i>Z</i>-<i>AR</i> is involved in male sex-determination in an amphibian species.</p></div

Primers used for PCR analysis.

<p>F:Forward primer, R:Reverse primer.</p

<i>Z-AR/V5</i>, <i>CYP17</i> and <i>Dmrt1</i> mRNA expression and <i>Z-AR/V5</i> and <i>AAT</i> genomic analysis.

<p>Genomic DNA PCR amplification was performed for the transgene (genomic <i>Z-AR/V5</i>). The sex of each frog was determined by genomic amplification of the ATP/ADP translocase (<i>AAT</i>) gene as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093655#pone.0093655-Yokoyama1" target="_blank">[16]</a>. RT-PCR analysis was used to detect <i>Z-AR/V5</i>, <i>CYP17</i> and <i>Dmrt1</i> mRNA in Wt and Tg gonads treated with (+) or without (−) T. Top panel, <i>Z-AR</i>/<i>V5</i> integration into genomic DNA; 2nd panel, <i>Z-AR</i>/<i>V5</i> expression; 3rd panel, <i>CYP17</i> expression; 4th panel, <i>Dmrt1</i> expression; bottom panel, <i>AAT</i> genetic sex determination of each frog.</p

Western blot and immunohistochemical analyses.

<p>(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).</p

Immunohistology of ZW ovotestes.

<p>(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. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093655#pone-0093655-g002" target="_blank">Figures 2</a> 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 µm.</p

Adipose tissue is the first colonization site of <i>Leptospira interrogans</i> in subcutaneously infected hamsters

<div><p>Leptospirosis is one of the most widespread zoonoses in the world, and its most severe form in humans, “Weil’s disease,” may lead to jaundice, hemorrhage, renal failure, pulmonary hemorrhage syndrome, and sometimes,fatal multiple organ failure. Although the mechanisms underlying jaundice in leptospirosis have been gradually unraveled, the pathophysiology and distribution of leptospires during the early stage of infection are not well understood. Therefore, we investigated the hamster leptospirosis model, which is the accepted animal model of human Weil’s disease, by using an <i>in vivo</i> imaging system to observe the whole bodies of animals infected with <i>Leptospira interrogans</i> and to identify the colonization and growth sites of the leptospires during the early phase of infection. Hamsters, infected subcutaneously with 10⁴ bioluminescent leptospires, were analyzed by <i>in vivo</i> imaging, organ culture, and microscopy. The results showed that the luminescence from the leptospires spread through each hamster’s body sequentially. The luminescence was first detected at the injection site only, and finally spread to the central abdomen, in the liver area. Additionally, the luminescence observed in the adipose tissue was the earliest detectable compared with the other organs, indicating that the leptospires colonized the adipose tissue at the early stage of leptospirosis. Adipose tissue cultures of the leptospires became positive earlier than the blood cultures. Microscopic analysis revealed that the leptospires colonized the inner walls of the blood vessels in the adipose tissue. In conclusion, this is the first study to report that adipose tissue is an important colonization site for leptospires, as demonstrated by microscopy and culture analyses of adipose tissue in the hamster model of Weil’s disease.</p></div

Bioluminescence dissemination of <i>Leptospira</i> in hamsters.

<p>(A) The survival rate of Golden Syrian hamsters (n = 8) infected subcutaneously with 10⁴ <i>L</i>. <i>interrogans</i> strain M1307 into the right inguinal region, and representative ventral view photographic images tracking the hamster infections on different days post-infection. Images depict photographs overlaid with color representations of luminescence intensity, measured in photons/second/cm²/sr as indicated on the scales, where red is the most intense (3×10⁵) and purple is the least intense (3×10⁴). (B,C) Average luminescence intensities in each ROI of injection site (B) and abdominal center (C) at different days post-infection. Data are expressed as the means ± SEM of total flux in photons/second in each ROI in eight infected hamsters (●) and two uninfected controls (◦). <i>p</i> values (*<i>p</i><0.05), between groups.</p

Investigation of Encephalopathy Caused by Shiga Toxin 2c-Producing <em>Escherichia coli</em> Infection in Mice

<div><p>A large outbreak of Shiga toxin (Stx)-producing enteroaggregative <i>Escherichia coli</i> (EAEC) O104:H4 occurred in northern Germany. From this outbreak, at least 900 patients developed hemolytic uremic syndrome (HUS), resulting in more than 50 deaths. Thirty percent of the HUS patients showed encephalopathy. We previously established a mouse model with encephalopathy associated with blood brain barrier (BBB) damage after oral infection with the Shiga toxin (Stx) 2c-producing <i>Escherichia coli</i> O157: H- strain E32511 (E32511). In this model, we detected high expression of the Stx receptor synthase enzyme, glycosphingolipid globotriaosylceramide (Gb3) synthase, in endothelial cells (ECs) and neurons in the reticular formation of the medulla oblongata by <i>in situ</i> hybridization. Caspase-3 was activated in neurons in the reticular formation of the medulla oblongata and the anterior horn of the spinal cord. Astrocytes (ASTs) were activated in the medulla oblongata and spinal cord, and a decrease in aquaporin 4 around the ECs suggested that BBB integrity was compromised directly by Stx2c or through the activation of ASTs. We also report the effectiveness of azithromycin (AZM) in our model. Moreover, AZM strongly inhibited the release of Stx2c from E32511 <i>in vitro</i>.</p> </div

<i>Leptospira</i> distribution in skin and subcutaneous tissue.

<p>Representative light field (A, C) and fluorescence images (B, D) of the skin and subcutaneous tissue (A, B) or adipose tissue (C, D) around the injection sites of M1307 collected from infected hamsters at phase 4. Fluorescence images (B, D) showing cell nuclei stained with DAPI (blue), autofluorescence of the skin and subcutaneous tissue (green, not shown in panel D), and leptospires stained with rabbit polyclonal antiserum and Cy5-conjugated anti-rabbit monoclonal antibody (red). The framed area in (B) is enlarged at the upper right. Scale bars: 100 μm (A, B), 500 μm (C, D).</p

Histology of gonads.

<p>(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 (−) 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 µm) and a laminin antibody (lower figures, o–u; bar = 100 µm). 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).</p