Construction and mechanism analysis of the sex reversal model of chicken gonadal somatic cells

Abstract

In chickens, sex determination is governed by genetic-hormonal interactions, but the dynamic interplay between sex hormones and receptor expression in gonadal somatic cells remains unclear. Here, we established an in vitro sex reversal model using embryonic chicken gonadal somatic cells (E18.5 days) to dissect temporal and dose-dependent regulatory mechanisms. Male (SOX9⁺) and female (FOXL2⁺) cells were isolated via two-step enzymatic digestion and maintained sex-specific markers in culture. Hormonal treatments revealed distinct phenotypic plasticity: Short-term (3 days) 50 nM fadrozole (FAD) in female cells upregulated male markers (SOX9, AMH; P < 0.001), while prolonged (13 days) exposure induced an intersex state with co-expression of male/female genes (P < 0.01). In male cells, 50 nM estradiol (E2) induced bisexual characteristics within 3 days (P < 0.0001), and 100 nM E2 triggered sex reversal by day 4 (CYP19A1, FOXL2, WNT4; P < 0.01), though extended treatment reverted to an intersex phenotype. Flow cytometry validated hormone-induced protein-level changes in sex-related genes. Receptor dynamics showed oscillatory patterns: In female cells, 50 nM FAD transiently activated ESR1 (0-7 days), inhibited ESR2/AR, and induced AR-dependent male gene expression at 8-13 days. In male cells, E2 (50/100 nM) repressed AR expression for 14 days while phase-activating ESR1/ESR2; early AR inhibition correlated with male gene peaks, whereas ESR1/ESR2 fluctuations drove female gene activation. These findings define temporal thresholds in avian sex determination, highlighting receptor-driven sexual plasticity. Elucidating this genetic-hormonal crosstalk provides a framework for optimizing avian sex control strategies.

Keywords: Chicken; Gonadal somatic cell; Sex differentiation; Sex reversal.

Fig. 1

Sex identification, morphological observation and culture of male and female gonadal cells. A. The morphology of the gonads of male and female chicken embryos at E18.5 d. Up: Female gonad; Down: Male gonad. Scale bars: 50 μm. B. Morphological observation of male and female gonadal somatic cells. Up: Female gonadal somatic cell; Down: Male gonadal somatic cell. C. Sex identification of male and female gonadal somatic cells. M: Marker; Lane 1: Male gonadal somatic cell; Lane 2: Female gonadal somatic cell.

Fig. 2

Molecular and cellular characterization of male and female gonadal somatic cells. A. Identification of the mRNA expression level of sex-related genes in male and female gonadal somatic cells by qRT-PCR. *P < 0.05, significant difference; **P < 0.01, extremely significant difference. B. Detection of the expression changes of sex-related genes in male and female gonadal somatic cells by Western blot. C. Observation of the expression of FOXL2 and SOX9 in male and female gonadal somatic cells by immunofluorescence. Anti-FOXL2/SOX9 conjugated to FITC was shown as a green fluorescence signal. Nucleus staining with DAPI was shown as a blue signal. Scale bars: 500 μm (upper), 100 μm (lower).

Fig. 3

Effects of DMSO concentration on the expression of sex - related genes in male and female gonadal somatic cells. A. Effects of DMSO concentration on the expression of sex - related genes in female gonadal somatic cells. B. Effects of DMSO concentration on the expression of sex - related genes in male gonadal somatic cells. All groups were compared to the Blank group (unstimulated cells). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; n = 3. Student’s t-test was used to perform all statistical comparisons against the respective Blank group.

Fig. 4

The mRNA expression level of male sex-related genes in female gonadal somatic cells after treatment with FAD and the expression of female sex-related genes in male gonadal somatic cells after treatment with E2 for 0-14 days by qRT-PCR. A. Expression of male sex-related genes in female gonadal somatic cells after treatment with 50 nM FAD. B. Expression of female sex-related genes in male gonadal somatic cells after treatment with 50 nM E2. C. Expression of female sex-related genes in male gonadal somatic cells after treatment with 100 nM E2. All time points were compared to the Blank group. **P < 0.01, ***P < 0.001, ****P < 0.0001; n = 3. Statistical comparisons used Student’s t-test vs. respective Blank group.

Fig. 5

The mRNA expression level of male sex-related genes in male gonadal somatic cells after treatment with FAD and the expression of female sex-related genes in female gonadal somatic cells after treatment with E2 for 0-14 days by qRT-PCR. A. Expression of female sex-related genes in female gonadal somatic cells after treatment with 50 nM FAD. B. Expression of male sex-related genes in male gonadal somatic cells after treatment with 50 nM E2. C. Expression of male sex-related genes in male gonadal somatic cells after treatment with 100 nM E2. All time points were compared to the Blank group. **P < 0.01, ***P < 0.001, ****P < 0.0001; n = 3. Against the respective Blank group, Student’s t-test was used for all statistical comparisons.

Fig. 6

Flow cytometry analysis of the expression of FOXL2 and SOX9 in male and female gonadal somatic cells after treatment with FAD (50 nM) or E2 (50 nM, 100 nM). A. The expression level of FOXL2 and SOX9 in male and female gonadal somatic cells after treatment with FAD (50 nM) or E2 (50 nM, 100 nM) for 3d, 4d, or 13d. The horizontal axis represents the green fluorescence signal (FITC) of Anti-FOXL2 conjugated to FITC. The vertical axis represents the red fluorescence signal (PE) of Anti-SOX9 conjugated to ABflo® 594. N = 3. B. Statistical analysis of the expression of SOX9 in female gonadal cells treated with 50 nM FAD for 3 days. C. Statistical analysis of the expression of SOX9 in female gonadal cells treated with 50 nM FAD for 13 days. D. Statistical analysis of the expression of FOXL2 in female gonadal cells treated with 50 nM E2 for 3 days or 100 nM E2 for 4 days. E. Statistical analysis of the expression of FOXL2 in female gonadal cells treated with 50 nM FAD or 100 nM E2 for 13 days. B-E. Statistical analysis of protein expression relative to the corresponding Blank group. **P < 0.01, ***P < 0.001, ****P < 0.0001; n = 3. All statistical comparisons were performed using Student’s t-test against the respective Blank group.

Fig. 7

The mRNA expression level of AR, ESR1 or ESR2 in female gonadal somatic cells treated continuously with 50 nM FAD and in male gonadal somatic cells treated with 50 nM or 100 nM E2 for 0-14 days by qRT-PCR. A. Expressions of AR, ESR1 and ESR2 in female gonadal somatic cells treated continuously with 50 nM FAD for 0-14 days. B. Expressions of AR, ESR1 and ESR2 in male gonadal somatic cells treated continuously with 50 nM E2 for 0-14 days. C. Expressions of AR, ESR1 and ESR2 in male gonadal somatic cells treated continuously with 100 nM E2 for 0-14 days. All time points were compared to the Blank group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; n = 3. Statistical significance was determined by Student’s t-test.