Molecular insights into ROS-induced aggregation in chicken primordial germ cells and its alleviation by ascorbic acid

BACKGROUND: Primordial germ cells (PGCs) are highly valuable for chicken genetic engineering; however, their cultivation is frequently hindered by spontaneous cell aggregation and slow proliferation in certain breeds. METHODS: We systematically evaluated the culture dynamics of PGCs derived from four distinct chicken breeds. To investigate the molecular mechanisms driving these phenotypes, we performed integrated transcriptomic and proteomic profiling, alongside targeted exogenous supplementation assays. RESULTS: We found that White Leghorn PGCs exhibited superior line establishment efficiency, whereas broiler breeds, particularly White Cornish, displayed severe cell aggregation and delayed growth. Transcriptomic and proteomic analyses revealed that WC PGCs exhibit an inherent deficiency in glutathione metabolism and an activation of the p53 signaling pathway. This finding was experimentally corroborated by the accumulation of reactive oxygen species (ROS) in these cells. Based on these mechanistic insights, we experimentally demonstrated that targeted exogenous supplementation with L-sulforaphane and ascorbic acid effectively mitigated excessive ROS and ameliorated the aggregation phenotype in White Cornish PGCs, with ascorbic acid notably accelerating cell proliferation. Subsequently, ascorbic acid also promoted proliferation in both Rhode Island Red and White Plymouth Rock lines, while alleviating aggregation in the White Plymouth Rock line. CONCLUSIONS: These findings demonstrate the cross-breed applicability of ascorbic acid, suggesting it could serve as a general supplement for PGC culture in other chicken breeds. Collectively, our study proposes a mechanistic model in which oxidative stress promotes PGC aggregation and offers practical antioxidant interventions to enhance the derivation efficiency of chicken PGCs, providing a valuable reference for the future optimization of in vitro culture media.