CYP19A1 (aromatase) dominates female gonadal differentiation in chicken (Gallus gallus) embryos sexual differentiation

Abstract

Cytochrome P450 Family 19 SubFamily A member 1 (CYP19A1) gene encodes an aromatase which regulates the sexual differentiation in vertebrates by initiating and maintaining 17β-Estradiol (E2) synthesis. Here, we described the spatiotemporal expression pattern of CYP19A1 and its functional role in the embryonic gonad development in amphoteric chickens (Gallus gallus). Results showed that CYP19A1 exhibited a sexually dimorphic expression pattern in female gonads early at embryonic day 5.5 (HH 28) and robustly expressed within the cytoplasm in ovarian medullas. Most importantly, we induced the gonadal sex reversal by ectopically delivering the aromatase inhibitor (AI) or estradiol (E2) into chicken embryos. To further explore the role of CYP19A1 in chicken embryonic sexual differentiation, we successfully developed an effective method to deliver lentiviral particles with CYP19A1 manipulation into chicken embryos via embryonic intravascular injection. The analysis of interference and overexpression of CYP19A1 provided solid evidences that CYP19A1 is both necessary and sufficient to initiate sex differentiation toward female in chicken embryos. Collectively, this work demonstrates that CYP19A1 is a crucial sex differentiation gene in the embryonic development, which provides a foundation for understanding the mechanism of sex determination and differentiation in chickens.

Keywords: Cyp19a1; chicken embryonic; gonadal differentiation.

Figure 1. The sexually dimorphic expression pattern of CYP19A1 gene in chicken (Gallus gallus)

(A) The mRNA expression of CYP19A1 in embryos (days 0–2.5, HH 0–17) and gonads of different stages (days 2.5–21.5, HH 17–48), determined by qRT-PCR analysis; β-actin was used as a reference gene. (B) Immunofluorescence detection of CYP19A1 protein in the gonad at day 18.5 in female and male. CYP19A1 protein was localized in the cytoplasm of the medulla of the female gonad (ovary, day 18.5, HH 44). Co, cortex; Me, medulla; St, seminiferous tubule. Scale bar: 10 μm (data were shown as mean ± SEM and Student’s t test was utilized for statistical analysis.**P<0.01).

Figure 2. The effect of AI and Estradiol (E₂) on embryonic gonad development

(A) The concentration of Estradiol in different treatments. (B) The ratio of PCR and visual results in different treatment groups. (C) The histology of E18.5 gonads in different treatments (the red dot line separates the cortex and medulla; the green arrow points the spermatic cord). Scale bar: 50 μm. (D) The Sox9 and Foxl2 expression in different treatments (data were shown as mean ± SEM and Student’s t test was utilized for statistical analysis. *P<0.05, **P<0.01).

Figure 3. Establishment of an efficient lentivirus-mediated gene modulating method in chicken (Gallus gallus)

(A) Schematic diagram of CYP19A1 target sites of RNAi and CDS. (B) The efficiency of virus infection in DF-1 cells of each experimental group and control group. (C) CYP19A1 mRNA relative expression in DF-1 cells of each experimental group and control group. (D) The expression of EGFP by fluorescent microscopy at day 4.5 (HH 25). (E) The expression of CYP19A1 in gonad different groups (data were shown as mean ± SEM and Student’s t test were utilized for statistical analysis. *P<0.05, **P<0.01).

Figure 4. Feminization of male embryo gonads following CYP19A1 knockdown and masculinization of female overexpressing CYP19A1 in ovo

(A) The concentration of estradiol in different groups. (B) The ratio of PCR and visual in different treatment groups. (C) The histology of E18.5 gonads in different treatments (the red dot line separates the cortex and medulla; the green arrow point the spermatic cord). Scale bar: 50 μm. (D) Immunofluorescence detection of SOX9 (green) in the gonads at different groups. Scale bar: 50 μm. (E) The expression of Sox9 and Foxl2 at different groups (data were shown as mean ± SEM and Student’s t test were utilized for statistical analysis. *P<0.05, **P<0.01).

Figure 5. The schematic diagram of CYP19A1 function in chicken sexual differentiation