Integrative Transcriptome Analysis Across Follicles Highlights Key Regulatory Pathways in Low and High-Egg-Laying Hens

Egg-laying performance in hens is regulated by complex molecular mechanisms within the hypothalamic-pituitary-gonadal (HPG) axis and ovarian follicles. This study employed integrative transcriptome profiling of primordial (PR), primary (PM), small white (SW), and small yellow (SY) follicles in hens with low and high egg-laying capacities to explain regulatory pathways influencing reproductive outcomes. Specific gene expression patterns were observed that correlated with follicular growth, steroidogenesis, and granulosa cell proliferation. Heatmap clustering and principal component analysis revealed transcriptional divergence between low- and high-laying hens, suggesting that coordinated changes in signaling pathways influence egg-laying performance. High-laying hens intricated an upregulation of the PI3K-AKT-FOXO3, TGF-β, and Wnt/β-catenin pathways, which facilitate early follicular development, granulosa cell proliferation, and folliculogenesis. Higher phosphorylation of AKT and reduced nuclear FOXO3 activity were associated with enhanced primordial follicle growth. Increased TGF-β signaling, as demonstrated by higher levels of SMAD2/3/4 and cell cycle regulators, promoted granulosa cell proliferation in primary follicles (PMF). In SWF, higher levels of β-catenin and its downstream genes, such as c-Myc and cyclin D1, promoted follicle development. High-laying hens revealed increased expression of FSHR, CYP19A1, 17β-HSD, CYP1A1, and CYP1B1 in SYF, signifying enhanced FSH level and steroidogenesis. Similarly, low-laying hens exhibited downregulation of key genes, suggesting reduced follicular development and hormone signaling. These findings identify key regulatory networks and molecular markers associated with reproductive performance, providing targets for genetic selection and interventions to enhance egg production while reducing the risk of hormonal overstimulation.