Stage-Specific lncRNA-mRNA Co-Expression Networks in Chicken Granulosa Cells Across Hierarchical Follicle Development

Long non-coding RNAs (lncRNAs) regulate granulosa cell function, but their stage-specific dynamics across the chicken follicle hierarchy remain unclear under matched endocrine conditions. We performed Ribo-Zero RNA sequencing on granulosa cells from small yellow follicles (SYF), F5, F2, and F1 follicles collected at the preovulatory luteinizing hormone (LH) surge. A multi-predictor pipeline (CPC2, CNCI, CPAT, PfamScan) identified 26,923 stringently filtered lncRNAs together with 15,838 mRNAs. Consecutive stage comparisons detected 2094, 1085, and 4318 differentially expressed genes and 671, 267, and 2762 differentially expressed lncRNAs in F5 vs. SYF, F2 vs. F5, and F1 vs. F2, respectively, with the most extensive remodeling at the F2-to-F1 transition. The F1 vs. SYF contrast captured the cumulative transcriptional difference across the hierarchy. Enrichment and temporal clustering showed that early hierarchical stages were characterized by proliferative, metabolic, and steroidogenic programs, whereas F1 granulosa cells were enriched for extracellular-matrix remodeling, MAPK signaling, and calcium ion binding. Weighted gene co-expression network analysis identified 10 stage-associated modules and highlighted candidate lncRNAs linked to lipid metabolism, angiogenesis, extracellular-matrix remodeling, and DNA repair, including G5825-MYLIP, G66587-VEGFA, and G60212-CKS1B. qPCR validation confirmed concordant expression trends for eight representative pairs. These results define a stage-resolved lncRNA-mRNA landscape across chicken follicle development and provide candidates for mechanistic studies of follicle maturation and periovulatory remodeling.