ESR2 Regulates Granulosa Cell Proliferation and Steroidogenesis via the PI3K/AKT/mTOR Signaling Pathway in Wuding Chickens

The Wuding chicken, a renowned indigenous breed in Yunnan Province, is prized for its superior meat quality; however, its economic potential is limited by pronounced broodiness and suboptimal egg production. Central to alleviating these constraints is the precise regulation of ovarian granulosa cell (GC) proliferation and steroidogenic processes that dictate follicular development and laying performance. While Estrogen Receptor 2 (ESR2) is a known transcription factor implicated in follicular maturation, its spatiotemporal dynamics within the hypothalamic-pituitary-ovarian (HPO) axis and its specific regulatory mechanisms in Wuding chicken remain elusive. This study characterizes <i>ESR2</i> expression across the HPO axis during the laying and broody periods and functionally validates its role in GCs. We observed that <i>ESR2</i> expression was significantly higher throughout the HPO axis during the laying period compared to the broody period, with the most pronounced differential expression occurring in the ovary. Notably, subcellular localization analysis revealed that ESR2 is distributed in both the nucleus and the cytoplasm, indicating involvement in both nuclear transcriptional regulation and cytoplasmic signaling. Functional assays demonstrated that ESR2 modulates the expression of genes associated with GC proliferation, steroidogenesis, and apoptosis, involving the PI3K/AKT/mTOR signaling pathway. Our findings indicate that this process involves a synergistic interplay between genomic and potential non-genomic actions. Specifically, <i>ESR2</i> overexpression upregulates the expression of key signaling components and steroidogenic genes, including <i>CYP19A1</i>, <i>STAR</i>, <i>PTGS2</i>, and <i>FSHR</i>, while its cytoplasmic localization suggests a role in non‑genomic interactions. Together, these coordinated mechanisms synergistically maintain GC functional homeostasis. Collectively, these results prove that ESR2 plays an important role in regulating GC homeostasis and follicular development through genomic and non-genomic modes of action. These findings provide a molecular basis for the role of ESR2 in avian follicular development and offer potential targets for improving the reproductive efficiency of Wuding chickens.