Figure 1.
Ectopic expression of aromatase in embryonic gonads.
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.
Figure 2.
Phenotypic effects of aromatase overexpression in embryonic gonads.
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.
Figure 3.
Immunostaining of feminised male embryonic gonads following aromatase overexpression.
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.
Figure 4.
Progression of aromatase-mediated sex-reversal during gonad development.
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).
Figure 5.
Expression of testis developmental genes in sex-reversed gonads.
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.
Figure 6.
Expression of ovarian developmental genes and germ cell markers in sex-reversed gonads.
Left gonads of control and RCASBP-Aromatase infected embryos were immunostained for FOXL2, RSPO1, CVH or SCP3 (green), and DAPI (blue) (20×magnification). The medulla (m) and cortex (c) are indicated and boxed areas are shown as high power images. White dashed lines indicate medulla/cortex boundaries. (A) E7.5 left gonads: FOXL2 expression in control and RCASBP-Aromatase infected females occurs throughout the gonad and is confined to the nucleus of positive cells. Background staining can be seen in the control male, whereas high levels of nuclear FOXL2 can been seen in the RCASBP-Aromatase infected male. E7.5 left gonads: RSPO1 expression in control and RCASBP-Aromatase infected females occurs throughout the gonad and is more intense in the cortex. Staining in the cords is evident in the control male, whereas the RCASBP-Aromatase infected male shows an expression pattern that is similar to the females. (B) E18.5 left gonads: The control male has CVH positive germ cells scattered throughout the medulla, whereas control female and the RCASBP-Aromatase male have only a few CVH positive germ cells in the medulla and large numbers localised within the outer cortex. No SCP3 positive cells are present in the control male. The female control and the RCASBP-Aromatase infected male have SCP3 positive germ cells in the outer cortex.