Abstract

In poultry, primordial germ cells (PGCs) play a crucial role in preserving and manipulating genetic resources for animal production. PGCs face challenges from oxidative stress during in vitro culture and manipulation processes. While the NRF2 pathway is known to centralize cellular homeostasis and stress responses in various cell types, its role in in vitro cultured PGCs remains poorly understood. This study investigates the impact of NRF2 inhibition on PGC proliferation, cellular basal characteristics, and potential underlying mechanisms. All experiments were performed with at least three biological replicates. The results showed that treatment with 24 μM ML385 significantly reduced PGC numbers after 3 d of culture compared to controls (2.83 ± 0.04 vs. 1.77 ± 0.06, × 10 5 cells), accompanied by a decrease in EdU-positive cells (28.85 ± 1.92% vs. 23.41 ± 1.49%) and a significant increase in apoptotic cells (3.50 ± 0.06% vs. 6.34 ± 0.13%). Transcriptomic sequencing analysis revealed 377 differentially expressed genes (234 up-regulated, 143 down-regulated) in ML385-treated PGCs, with significant enrichment of apoptosis-related pathways and downregulation of cell cycle pathways. NRF2 inhibition induced oxidative stress, with a 2.2-fold increase in reactive oxygen species levels and a 3.3-fold decrease in the GSH/GSSG ratio. Mitochondrial damage, including decreased mitochondrial number (27.86 ± 2.74 vs. 17.57 ± 1.55), vacuolization (9.54 ± 1.64% vs. 41.27 ± 11.18%) and hyperpolarization of mitochondrial membrane potential, was observed, along with increased markers of ferroptosis, including a 2.7-fold increase in MDA and a 2.4-fold increase in Fe²⁺ levels, and enhanced autophagy, as evidenced by increased LC3-II expression and upregulation of MAP1LC3B, ATG5 , and BECN1 . Notably, while in vivo migration efficiency of PGCs to recipient gonads was unaffected, the number