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During early embryogenesis, the undifferentiated gonad is bipotential and subsequently commits to an ovarian or testicular fate. In birds, double dose of the Z-linked gene DMRT1 is required for testicular differentiation in male embryos (genetically ZZ). In female birds, estrogen plays a key role in ovarian differentiation. 17β-estradiol (E2) induces gonadal feminization when applied to male embryos (ZZ). Conversely, inhibition of estrogen synthesis with the drug fadrozole (FAD) results in testicular development in genetically female embryos (ZW). However, activation of male markers in sex-reversed ZW embryos is typically delayed, raising the possibility that FAD-treated embryos may transition through an undifferentiated state before masculinization. Recently, PAX2 was identified as a marker of undifferentiated supporting cells in the chicken embryo, being downregulated in both sexes at the onset of gonadal sex determination. To investigate the supporting cell differentiation process in estrogen-mediated sex reversal, we injected 1 mg of fadrozole in 100µl of PBS or vehicle into embryonic day 3.5 (E3.5) chicken eggs. Eggs were incubated until E9.5, genotypically sexed (ZZ or ZW) and processed for qRT-PCR and immunofluorescence. Quantitative RT-PCR confirmed that sex reversal had occurred in FAD-treated females, showing a reduction of pre-granulosa cell markers aromatase (P&amp;lt;0.005) and FOXL2 (P&amp;lt;0.05), compared to the control. Interestingly, PAX2 mRNA expression was up-regulated (P&amp;lt;0.05) in sex-reversed females, suggesting an increase in undifferentiated supporting cells (n=6). To confirm this observation, immunofluorescence was used to detect aromatase, SOX9 (male marker) and PAX2. In FAD-treated females, both SOX9+ (male) and aromatase+ (female) cells co-existed in the same gonad, but in separated defined regions. Aromatase positive cells were located in the most apical region of the gonad whereas SOX9 positive cells were detected in the basal region. We detected an increase in PAX2 positive cells in the gonadal medulla between the SOX9 and aromatase positive supporting cells. No SOX9 or PAX2 positive cells were detected in control female gonads (n=3). For feminization experiments 100µl of a 1mg/ml solution of E2 or vehicle (Oil) was injected into E3.5 chicken eggs. No significant increase in PAX2 was detected by qRT-PCR (p&amp;gt;0.05, n=6) and no PAX2 positive cells were detected in E2 treated gonads at E9.5. These results suggest that in fadrozole-mediated masculinization (but not in estrogen-induced feminization) there is an increase in undifferentiated supporting cells. The absence of both estrogens (feminizing) and elevated DMRT1 (masculinizing) could explain why the supporting cells remain in an undifferentiated state in ZW (genetically female) embryos. Further research is required to evaluate the fate of these undifferentiated cells in gonadal sex differentiation.

Fadrozole-Mediated Sex Reversal Induces PAX2+Undifferentiated Supporting Cells in Female Chicken Gonads