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

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.