Transcriptome analysis reveals KRAS as a crucial regulator of ovarian follicle development in Wuding chickens

This study investigated the molecular basis of follicular development and egg production in Wuding chickens, an indigenous Chinese breed known for exceptional meat quality and nutrient-rich eggs, but suboptimal egg-laying performance, by comparing them with high-yielding Jingfen No.1 chickens. High-throughput transcriptome sequencing of ovarian tissues identified 6,790 differentially expressed genes (DEGs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed these DEGs were significantly implicated in reproductive pathways, including steroid biosynthesis, cell cycle, MAPK signaling, calcium signaling, oocyte meiosis, and progesterone-mediated oocyte maturation. A protein–protein interaction (PPI) network highlighted Kirsten rat sarcoma viral oncogene (KRAS) as a crucial hub gene. Further functional experiments focused on the role of KRAS in chicken ovarian granulosa cells (GCs). Overexpression of KRAS significantly enhanced the production of progesterone (P4) and estradiol (E2) and upregulated the expression of steroidogenic genes (e.g., STAR, CYP19A1, HSD3B1, PTGS2). KRAS also promoted GC proliferation by increasing cell viability, stimulating DNA synthesis, and upregulating proliferation markers (AKT1, mTOR, JAK2, STAT3, PCNA). Additionally, KRAS facilitated cell cycle progression by reducing the G0/G1 phase population and increasing the S phase, alongside upregulating cell cycle regulators (CCND1, CCND2, CCNE2, CDK2). Importantly, KRAS inhibited GC apoptosis by increasing antiapoptotic genes (BCL2, TGF-β) and decreasing proapoptotic genes (Caspase 3, BAX). Finally, KRAS delayed cellular senescence by reducing SA-β-gal activity and reciprocally regulating P21 (downregulation) and SIRT1 (upregulation). Collectively, these findings demonstrate that KRAS is a pivotal regulator of follicular development in Wuding chickens, influencing GC proliferation, apoptosis, steroid hormone synthesis, and senescence. This research provides valuable insights for genetic improvement programs aimed at enhancing egg production in indigenous chicken breeds.