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

## TL;DR

This study explores how ESR2 regulates granulosa cell function in Wuding chickens, impacting reproduction and egg production.

## Contribution

The study reveals ESR2's dual genomic and non-genomic roles in regulating granulosa cell function via the PI3K/AKT/mTOR pathway in Wuding chickens.

## Key findings

- ESR2 upregulates PI3K/AKT/mTOR pathway genes to enhance granulosa cell proliferation and steroidogenesis.
- ESR2 is localized in both the nucleus and cytoplasm, indicating roles in transcriptional and signaling processes.
- ESR2 overexpression increases key steroidogenic gene expression, including CYP19A1, STAR, PTGS2, and FSHR.

## Abstract

Low egg production and intense broodiness limit the breeding efficiency and economic value of indigenous poultry breeds. The Wuding chicken, a local breed from Yunnan Province, is valued for its superior meat quality but exhibits suboptimal reproductive performance. Elucidating the regulatory mechanisms underlying reproductive performance in indigenous chickens is therefore essential for sustainable poultry development. Estrogen Receptor 2 (ESR2) is involved in ovarian development and ovulation, yet its specific function in granulosa cells (GCs) remains unclear. This study investigated how ESR2 influences follicular development and GC function in Wuding chickens. These findings indicate that ESR2 regulates GC function through a synergistic interplay between genomic and non-genomic actions. As a transcription factor, ESR2 transcriptionally upregulates key components of the PI3K/AKT/mTOR signaling pathway, thereby enhancing the expression of genes associated with GC proliferation and steroidogenesis. Meanwhile, its cytoplasmic localization suggests that ESR2 is involved in rapid signaling events and non-genomic regulatory processes. Together with its nuclear localization, these observations indicate an integrated regulatory mode of genomic and non-genomic actions in GCs of Wuding chicken. These results provide new insights into reproductive regulation in indigenous chicken breeds and offer theoretical support for improving breeding efficiency and advancing the development of characteristic poultry industries.

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 ESR2 expression across the HPO axis during the laying and broody periods and functionally validates its role in GCs. We observed that ESR2 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, ESR2 overexpression upregulates the expression of key signaling components and steroidogenic genes, including CYP19A1, STAR, PTGS2, and FSHR, 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.