Insights into speckled eggs provided by single-cell transcriptomics in high-yield laying hens

Eggshell quality is a major concern in the poultry industry. The speckle phenotype in eggshell affects eggshell color, thickness, and Haugh Unit (HU) and significantly affects the hatchability rate. The rate of speckled eggs increases during the late-laying period, which can lead to notable economic losses. Eggshell speckles have medium heritability; however, the genetic architecture of speckled eggshells remains unclear. In this study, the chickens were divided into two groups based on the rate of egg speckling. Uterine tissues were collected and analyzed using 10X Genomics single-cell RNA sequencing to delineate the molecular signatures of the various cell types. A total of 10 cell types were identified: immune (B, T, monocyte macrophages, and plasma cells), endothelial, epithelial, stromal, neuroendocrine, serous, and protoporphyrin IX pigment cells. The trends of cell type distributions were consistent in two groups. Functional enrichment analysis showed that unique biological processes, such as IgA production and Toll-like C-type lectin receptor signaling pathways, were enriched in immune cells, whereas TGF-beta, Wnt, and NOD-like receptor signaling pathways were enriched in epithelial cells. Subsequently, we identified differentially expressed genes in the two groups, among which SPP1 (secreted phosphoprotein 1) was down-regulated in all cell types in the speckled group. The cellular interaction signals of speckled group were significantly enhanced compared to the control group. The cell proliferation and differentiation pathways mediated by WNT, SPP1, and BMP were inhibited and the interaction related to cell migration was activated in the speckled group. This study explored cell heterogeneity, extended prior findings by adding single-cell resolution atlas, and identified cell type specific patterns that can guide future functional studies of speckled eggs.