Pimpled eggs have defective shells, which severely impacts hatching rates and transportation safety. In this study, we constructed single-cell resolution transcriptomic and chromatin accessibility maps from uterine tissues of chickens using single-cell RNA sequencing (scRNA-seq) and single-cell ATAC sequencing (scATAC-seq). We identified 11 major cell types and characterized their marker genes, along with specific transcription factors (TFs) that determine cell fate. CellChat analysis showed that fibroblasts had the most extensive intercellular communication network and that the chickens laying pimpled eggs had amplified immune-related signaling pathways. Differential expression and enrichment analyses indicated that inflammation in pimpled egg-laying chickens may lead to disruptions in their circadian rhythm and changes in the expression of ion transport-related genes, which negatively impacts eggshell quality. We then integrated TF analysis to construct a regulatory network involving TF–target gene–Gene Ontology associations related to pimpled eggs. We found that the transcription factors ATF3, ATF4, JUN, and FOS regulate uterine activities upstream, while the downregulation of ion pumps and genes associated with metal ion binding directly promotes the formation of pimpled eggs. Finally, by integrating the results of scRNA-seq and scATAC-seq, we identified a rare cell type—ionocytes. Our study constructed single-cell resolution transcriptomic and chromatin accessibility maps of chicken uterine tissue and explored the molecular regulatory mechanisms underlying pimpled egg formation. Our findings provide deeper insights into the structure and function of the chicken uterus, as well as the molecular mechanisms of eggshell formation.
