Over-expression of DMRT1 from <i>SF1p</i> in embryonic gonads.

<p>Immunostaining for key testis and ovarian developmental genes (red) in RCANBP-SF1p-EGFP infected E7.5 embryos (magnification 10×). Low-level over-expression of DMRT1 was evident in the gonads of RCANBP-SF1p-DMRT1 infected embryos compared to the control female. This over-expression did not cause any change in the expression of SOX9 in the female or aromatase in the male.</p

Wholemount fluorescent microscopy of novel gonad promoter expressed EGFP.

<p>Tissues from E7.5 embryos infected with RCANBP viruses containing <i>SV40</i> (<i>SV40p</i>), <i>WT1</i> (<i>WT1p</i>), <i>SF1</i> (<i>SF1p</i>), <i>AMH</i> (<i>AMHp</i>) and <i>aromatase</i> (<i>AROMp</i>) promoters. Dashed white lines delineate the left (Lg) and right (Rg) gonads, which sit on top of the mesonephros (Ms). A: Strong EGFP expression was evident for <i>SV40p</i> and <i>AROMp</i>, however, this was not confined to the urogenital systems. <i>WT1p</i> and <i>AMHp</i> produced low-level expression in the urogenital systems. EGFP expressed from <i>SF1p</i> was moderate in the urogenital system, and included the gonads. B: RCANBP-SF1p-EGFP infected E7.5 embryo; in addition to EGFP expression in the urogenital system, embryos also showed EGFP expression in the liver, forelimb (FL) and hind limb (HL).</p

Cellular location of <i>SF1</i> promoter expressed EGFP compared to germ cells.

<p>Immunostaining for EGFP (green) and CVH (red) in RCANBP-SF1p-EGFP infected E7.5 embryos (magnification 20×). The expression of EGFP from <i>SF1p</i> did not overlap with any cells that were positive for the germ cell marker CVH.</p

Schematic representation of putative gonad promoter sequences.

<p>All numbers shown are relative to the transcriptional start site (TSS) for each putative promoter sequence. The <i>SF1p</i> contains several promoter elements that have been described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101811#pone.0101811-Kudo1" target="_blank">[16]</a>. Both <i>aromatase</i> and <i>AMH</i> promoters contain TATA boxes and consensus SF1 binding sites. The <i>AMH</i> promoter also contains an estrogen responsive element (ERE). The <i>WT1</i> promoter is TATA-less and no other binding elements were identified. All promoter sequences were cloned into the RCANBP viral vector directly upstream of the EGFP open reading frame.</p

Overexpression of Aromatase Alone is Sufficient for Ovarian Development in Genetically Male Chicken Embryos

<div><p>Estrogens play a key role in sexual differentiation of both the gonads and external traits in birds. The production of estrogen occurs via a well-characterised steroidogenic pathway, which is a multi-step process involving several enzymes, including cytochrome P450 aromatase. In chicken embryos, the aromatase gene (<i>CYP19A1</i>) is expressed female-specifically from the time of gonadal sex differentiation. To further explore the role of aromatase in sex determination, we ectopically delivered this enzyme using the retroviral vector RCASBP <i>in ovo</i>. Aromatase overexpression in male chicken embryos induced gonadal sex-reversal characterised by an enlargement of the left gonad and development of ovarian structures such as a thickened outer cortex and medulla with lacunae. In addition, the expression of key male gonad developmental genes (DMRT1, SOX9 and Anti-Müllerian hormone (AMH)) was suppressed, and the distribution of germ cells in sex-reversed males followed the female pattern. The detection of SCP3 protein in late stage sex-reversed male embryonic gonads indicated that these genetically male germ cells had entered meiosis, a process that normally only occurs in female embryonic germ cells. This work shows for the first time that the addition of aromatase into a developing male embryo is sufficient to direct ovarian development, suggesting that male gonads have the complete capacity to develop as ovaries if provided with aromatase.</p></div

Expression of testis developmental genes in sex-reversed gonads.

<p>Left gonads of control and RCASBP-Aromatase infected embryos were immunostained for DMRT1, SOX9 and AMH (green) (20×magnification). The medulla (m) and cortex (c) are indicated and white dashed lines show the medulla/cortex boundaries. Control males show strong expression throughout testis cords for each, whereas both the control females and the sex reversed aromatase overexpressing males have little to no expression of these genes.</p

Phenotypic effects of aromatase overexpression in embryonic gonads.

<p>The urogenital systems of control and RCASBP-Aromatase virus infected E10.5 embryos. (A) Wholemount brightfield imaging (4×magnification) shows the mesonephric kidneys (Ms), and the right gonad (Rg) and left gonad (Lg), which is delineated by the black dashed lines. Gonadal asymmetry by enlargement of the left gonad of the RCASBP-Aromatase infected male is evident. (B) Haematoxylin and eosin staining of the left gonads of control and RCASBP-Aromatase injected embryos. The cortex and medulla regions for each are indicated and arrows highlight selected germ cells. Control males have a minimal cortex and large medulla, and very few germ cells, whereas the control female, and the RCASBP-Aromatase infected female and male have a thickened outer cortex and large numbers of germ cells.</p

Progression of aromatase-mediated sex-reversal during gonad development.

<p>The gonads of control and RCASBP-Aromatase infected embryos immunostained for aromatase (green), fibronectin (red) and DAPI (blue) as indicated. The medulla (m) and cortex (c) are indicated and boxed areas are shown adjacently as high power images. White dashed lines indicate medulla/cortex boundaries. (A) Time course analysis of E5.5, E7.5, E10.5 and E18.5 left gonads (Lg) of control males and females, and RCASBP-Aromatase infected males (20×magnification). The control male features characteristic seminiferous cord structures within the medulla from E7.5 onwards. The control female and the RCASBP-Aromatase infected male have a thickened outer cortex, which increases in size progressively across all time points, and both have vacuolated medullary spaces (arrows). (B) RCASBP-Aromatase infected genetic male with an ovotestis. Left panel: a left gonad that contains areas of low (i) and high (ii) aromatase expression (10×magnification). (i) Normal male development indicated by the formation of cord structures (arrows), (ii) typical female morphology indicated by the formation of a thickened outer cortex (arrows) and disruption of cord structures (20×magnification).</p

Ectopic expression of aromatase in embryonic gonads.

<p>RCASBP-Aromatase virus was injected into blastoderms and aromatase expression was assessed at embryonic day 10.5 by immunostaining (10×magnification). For each, the left (Lg) and right (Rg) gonads are shown with aromatase staining (green). No expression was detected in control males, while control females showed robust aromatase expression in the medulla. RCASBP-Aromatase infected embryos showed gonadal aromatase expression in both males and females.</p

Immunostaining of feminised male embryonic gonads following aromatase overexpression.

<p>Gonads of control and RCASBP-Aromatase virus infected E10.5 embryos immunostained for aromatase (green) and fibronectin (red) (20×magnification). The medulla (m) and cortex (c) are indicated and boxed areas are shown adjacently as high power images. White dashed lines indicate medulla:cortex boundaries. (A) The left gonads of the control male and female show normal gonadal development. The male has a medulla with characteristic cord structures (arrows) and no aromatase expression. Females show strong aromatase expression and have a vacuolated medulla with lacunae (arrows) and a thickened outer cortex. The left gonad of an RCASBP-Aromatase infected male has strong aromatase expression and like the control female, features a thickened outer cortex, lacks cords in the medulla and contains lacunae (arrow). (B) The right gonads of a control males and females show normal gonad development. The right gonad of a male overexpressing aromatase shows lower aromatase expression compared to the control female, but appears to have disrupted formation of the testis cords.</p