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

Simple Summary

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.

Abstract

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.

1. Introduction

The growth, development, and function of ovarian follicles are key determinants of egg-laying performance in poultry [1]. The follicle constitutes the fundamental structural and functional unit of the ovary, comprising the oocyte, granulosa cells (GCs), and theca cells (TCs) [2]. As the core component of the follicle, the proliferation of GCs drives follicular maturation, whereas their apoptosis leads to follicular atresia [3]. The balance between follicular maturation and atresia is tightly regulated by the hypothalamus-pituitary-ovarian (HPO) axis, which coordinates the actions of Luteinizing Hormone (LH), Follicle-Stimulating Hormone (FSH), estrogen, and progesterone [4]. Estrogen acts primarily on the ovary via estrogen receptors to regulate GC proliferation, differentiation, and steroidogenesis, thereby promoting follicle formation [5]. These effects are mediated by two distinct receptors, Estrogen Receptor 1 (ESR1) and Estrogen Receptor 2 (ESR2) [6], which differ significantly in tissue distribution and biological function yet jointly coordinate reproductive processes. Previous transcriptomic analyses by our group comparing Wuding chicken ovaries during laying and broody periods revealed that ESR2 expression is significantly upregulated during the laying phase and enriched in steroid hormone biosynthesis and GnRH signaling pathways associated with laying performance [7], suggesting ESR2 may be a crucial regulator of reproductive efficiency in this breed.

The Wuding chicken is a dual-purpose meat and egg breed native to Wuding and Luquan counties in the Chuxiong Yi Autonomous Prefecture, Yunnan Province [8]. It is valued for its adaptability to low-quality feed and excellent meat quality [9]. However, compared to commercial layer lines, the reproductive performance of Wuding chicken is low. Hens typically begin laying after 6 months of age, producing only 90–130 eggs annually. A major factor limiting productivity is intense broodiness. Hens typically enter a broody state after laying 14–16 eggs, with broodiness occurring 4–6 times per year and lasting 6–20 days per cycle. This frequent broody behavior results in the loss of approximately 30% of the laying cycle, severely restricting economic efficiency [7].

To overcome this profound reproductive limitation, understanding the molecular drivers of ovarian follicle development is crucial. Among these drivers, estrogen signaling plays a central role. The ESR2 gene (also known as ERβ) is a member of the estrogen receptor family [10]. Estrogen receptors generally mediate their effects through two distinct mechanisms: nuclear receptors (nESRs), which mediate genomic effects, and membrane-associated receptors, which initiate rapid non-genomic signaling. Classical nuclear receptors include ESR1 and ESR2, while the primary membrane receptor is the G protein-coupled estrogen receptor (GPER1) [11]. ESR2 is predominantly expressed in ovarian GCs and regulates follicular development and ovulation through two pathways [12]: feedback regulation within the HPO axis and direct action on the ovary [13]. Existing literature indicates that ESR2 maintains ovarian function by controlling key processes such as cell proliferation and differentiation [14]. For instance, Khristi et al. [15] demonstrated that both complete ESR2 knockout and DNA-binding-deficient ESR2 models in rats led to infertility due to anovulation, accompanied by ovarian atrophy, abnormal follicular development, and reduced expression of genes critical for follicle maturation. ESR2 not only regulates the transcription of target genes through classical nuclear receptor mechanisms (genomic effects) but also mediates non-genomic effects via cytoplasmic or membrane-associated estrogen receptors, thereby leading to the activation of intracellular signaling pathways such as PI3K/AKT and MAPK/ERK [16,17].

The reproductive performance of Wuding chicken is constrained by high broodiness and low laying rates, making it essential to elucidate the molecular mechanisms governing ovarian development. Given our previous transcriptomic profiling that identified ESR2 as a key differentially expressed gene, the present study primarily focuses on its regulatory role as a transcription factor. Specifically, we aim to elucidate how ESR2-mediated gene expression regulates GC proliferation and steroidogenesis, thereby influencing ovarian development and reproductive performance in Wuding chickens. To achieve this goal, we analyzed the spatiotemporal expression patterns and subcellular localization of ESR2 and validated its function using overexpression and RNA interference techniques. By investigating the potential roles of ESR2 in modulating gene expression associated with proliferation, apoptosis, and steroidogenesis in Wuding chicken GCs, this work provides a theoretical basis for understanding ovarian development and improving egg production in indigenous chicken breeds.

2. Materials and Methods

2.1. Experimental Animals and Sample Collection

The Wuding chickens used in all experiments were sourced from the breeding farm of Yunnan Shouyu Agricultural Development Co., Ltd., Chuxiong, Yunnan, China. Birds were housed in cages under standard management conditions for breeding layers, fed a standard commercial diet, and provided water ad libitum. The temperature was maintained at 24 ± 0.5 °C with a photoperiod of 14 h light/10 h dark (14 L:10 D). Hens of similar weight and good health at 300 days of age were selected from the same batch. Based on laying records and behavioral observations, hens were divided into two groups: the laying group (laying chickens, n = 6, continuous laying for ≥7 days) and the broody group (broody chickens, n = 6, persistent nesting behavior for ≥7 days). All animals were sacrificed via cervical dislocation. Ovary, pituitary, and hypothalamus tissues were collected, flash-frozen in liquid nitrogen, and stored at −80 °C for subsequent analysis.

2.2. Isolation and Identification of the ESR2 Gene

Total RNA was extracted from Wuding chicken tissues using the RNAiso Plus kit (TaKaRa, Dalian, China). RNA integrity was assessed via 1.5% agarose gel electrophoresis, and concentration and purity were measured using a UV spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthesized using Oligo(dT)₁₈ (500 μg/mL, TaKaRa, Dalian, China) and a reverse transcription kit (TaKaRa), then stored at −20 °C.

PCR amplification was performed using ovary cDNA from a 300-day-old laying hen as the template. Primers were designed based on the reference sequence (NM_001396358.1; Table S1) and synthesized by Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China. The 10 μL PCR reaction mixture contained: 5 μL 2 × Hieff^® Ultra-Rapid II HotStart PCR Master Mix (Yeasen, Shanghai, China), 1 μL cDNA template, 0.5 μL each of forward and reverse primers, and 3 μL RNase-free ddH₂O. The amplification protocol was: 94 °C pre-denaturation for 3 min; 34 cycles of 94 °C for 10 s, 58.5 °C annealing for 20 s, and 72 °C extension for 30 s; followed by a final extension at 72 °C for 7 min and storage at 4 °C.

PCR products were verified by 1% agarose gel electrophoresis. Target bands were excised, purified, ligated into the pMD-18T vector (TaKaRa), and transformed into DH5α competent cells (TransGen Biotech, Beijing, China). Ten positive clones were randomly selected and sequenced bi-directionally by Sangon Biotech. Sequences were assembled and verified using the SeqMan and EditSeq programs in the Lasergene package (DNAStar, Madison, WI, USA). The Open Reading Frame (ORF) and Coding DNA Sequence (CDS) were identified using ORF Finder (NCBI, Bethesda, MD, USA; https://www.ncbi.nlm.nih.gov/orffinder/, accessed 30 November 2025). Homology searches were performed using NCBI BLAST (NCBI, Bethesda, MD, USA; https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&BLAST_SPEC=GeoBlast&PAGE_TYPE=BlastSearch, accessed 30 November 2025) to confirm gene identity.

2.3. Bioinformatics Analysis

To elucidate the structural characteristics of the Wuding chicken ESR2 transcript, homologous sequences from Phasianidae, Numididae, Odontophoridae, Anatidae, Hominidae, and Muridae were downloaded from the NCBI database for comparative analysis (Table S2). Various bioinformatics tools (Table S3) were employed to systematically analyze gene structure, protein physicochemical properties, functional domains, phylogenetic relationships, and structural predictions. Meanwhile, we performed protein-protein interaction (PPI) network analysis, in which the top 20 core interacting proteins were displayed accordingly.

2.4. Isolation and Culture of Wuding Chicken Ovarian Granulosa Cells

Laying hens (300 days old) in the laying period were euthanized by cervical dislocation. Ovaries were removed and placed in PBS containing penicillin-streptomycin. In a sterile laminar flow hood, connective tissue and blood were carefully removed from hierarchical follicles. Follicles were punctured to drain the yolk, and the granulosa layer was separated from the theca layer. The granulosa layers were minced with ophthalmic scissors and digested in 10 mL of 0.1% Collagenase Type II (Sigma, St. Louis, MO, USA) and 0.25% Trypsin (Gibco, Grand Island, NY, USA) at 37 °C for 10 min, with gentle agitation every 2 min. Digestion was terminated by adding an equal volume of complete medium (89% DMEM high glucose, 10% Fetal Bovine Serum, 1% penicillin-streptomycin; Gibco). The suspension was filtered through a 200-mesh sieve and centrifuged at 1800 rpm for 10 min. The supernatant was discarded, and cells were washed with complete medium to remove residual enzyme and debris. After centrifugation at 1800 rpm for 5 min, the pellet was resuspended in complete medium. Cells were seeded into 6-well plates with 2 mL suspension per well and cultured at 37 °C in 5% CO₂. After 3 h of differential attachment to deplete fibroblasts, the medium was replaced; subsequent medium changes occurred every 24 h.

2.5. Construction of Overexpression Vectors, siRNA Synthesis, and Transfection

The ESR2 CDS (excluding the stop codon) was amplified from laying hen ovary cDNA (primers in Table S1) and cloned into the pMD-18T vector (TaKaRa). After double digestion with Kpn I and Hind III, the PCR product was gel-purified and ligated into the EGFP vector (Clontech Laboratories, Inc., Palo Alto, CA, USA). The recombinant plasmid (EGFP-ESR2) was verified by PCR, double digestion, and sequencing.

Two siRNAs targeting ESR2 (siESR2-1, siESR2-2) and a negative control (siNC) were synthesized by Tsingke Biotech (Beijing, China) (Table S4). GCs were transfected at ~70% confluency using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) with either EGFP-ESR2, siESR2-1 or siESR2-2, or their corresponding controls (empty EGFP vector for overexpression, and siNC for knockdown). Cells were harvested 48 h post-transfection.

2.6. Gene Expression Analysis

Total RNA was extracted from ovarian, hypothalamic, and pituitary tissues, and GCs of 300-day-old Wuding chickens using the Trizol method, followed by assessment of RNA integrity and concentration. Equal amounts of RNA were reverse-transcribed into cDNA at a final concentration of 300 ng/µL according to the manufacturer’s instructions. RT-qPCR was performed to detect the relative expression levels of ESR2 in different tissues, as well as genes associated with cell proliferation, apoptosis, and steroidogenesis in GCs. Primer sequences used for RT-qPCR are listed in Table S1. Chicken GAPDH (Accession: NM_205518.2) served as the internal control. The 20 µL PCR reaction consisted of 0.8 µL each of forward and reverse primers, 10 µL Dib^® SYBR qPCR SuperMix Plus (Aibisheng Biotechnology, Beijing, China), 6.4 µL ddH₂O and 2 µL cDNA template. Reactions followed the manufacturer’s protocol, and melting curve analysis confirmed a single specific peak. Each sample was analyzed in triplicate. Relative gene expression levels were calculated using the 2^−ΔΔCt method.

2.7. Cell Counting Kit-8 (CCK-8) and EdU Proliferation Assays

GCs were seeded in 96-well plates or 35 mm confocal dishes. Upon reaching 70% confluency, cells were transfected with EGFP-ESR2 or siESR2. Cell viability was assessed at 0, 24, 48, and 72 h post-transfection using the CCK-8 kit (Beyotime, Shanghai, China). DNA synthesis was evaluated 48 h post-transfection using the EdU kit (Beyotime). Cells were incubated with EdU for 2 h, stained with DAPI, and imaged using a confocal microscope (FV1000, Olympus, Tokyo, Japan). EdU-positive cells were quantified using ImageJ (v1.52a, NIH, Bethesda, MD, USA).

2.8. Flow Cytometry Analysis

GCs cultured in 100 mm dishes were transfected at ~70% confluency. After 48 h, cells were harvested, washed three times with pre-chilled PBS, and fixed in 70% ice-cold ethanol. Cell cycle distribution was analyzed using a Cell Cycle and Apoptosis Analysis Kit (Beyotime, Shanghai, China). Staining was performed using propidium iodide (PI) and RNase A for 30 min at 37 °C in the dark. Data were acquired on a BD FACSCelesta flow cytometer (BD Biosciences, San Diego, CA, USA) and analyzed using FlowJo (v10.0.7, Tree Star, Ashland, OR, USA).

2.9. Subcellular Localization

GCs were seeded in 35 mm confocal dishes and transfected with the EGFP-ESR2 recombinant plasmid at approximately 70% confluency. After 48 h of transfection, mitochondria were stained with MitoTracker^® Red CMXRos (Beyotime, Shanghai, China). The culture medium was then removed, and cells were washed with PBS and air-dried. Cells were fixed with 4% paraformaldehyde for 30 min, washed with PBS, and permeabilized with 0.5% Triton X-100 for 20 min. After washing, cells were blocked with 5% bovine serum albumin (BSA) for 30 min. Nuclei were counterstained with DAPI (Beyotime) for 15 min. After final washing, cells were maintained in PBS and imaged using a confocal laser scanning microscope (LSCM, FV1000, Olympus, Tokyo, Japan).

2.10. Statistical Analysis

Statistical analysis and graphing were performed using GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA, USA). Statistical comparisons between experimental and control groups were carried out using a two-tailed unpaired Student’s t-test. For comparisons involving more than two groups (e.g., RNA interference efficiency experiments and tissue expression comparisons across ovary, pituitary, and hypothalamus), data were analyzed using one-way ANOVA followed by Dunnett’s post-hoc test to compare treatment groups against the control. For multi-time point assays (CCK-8), comparisons were made between the treatment and control groups at each specific time point. As the primary objective was to evaluate the efficacy of the treatment at specific duration markers rather than to model the longitudinal interaction, independent comparisons were performed at each time point. Significance levels were defined as p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***). Data are presented as mean ± SEM. Sample size was n = 6 for tissue expression analysis and n = 3 for cell culture experiments. For cell culture experiments, three independent biological replicates were performed, each assayed in technical triplicate. Data are representative of three independent biological replicates, and the SEM was calculated based on these biological replicates.

3. Results

3.1. Isolation and Identification of the Wuding Chicken ESR2 Gene

The ESR2 gene was successfully cloned from ovarian tissue of 300-day-old laying Wuding chickens (Figure S1). The amplified PCR product was 1789 bp in length and contained a 1626 bp coding sequence (CDS), encoding a polypeptide of 541 amino acids (Figure S2). Sequence alignment showed 100% identity with chicken ESR2 transcript variants N1, N2, X1, X3, and X4, but variations existed compared to variant X2. Only a single transcript was identified in this study, indicating that the cloned Wuding chicken ESR2 corresponds to the predominant ESR2 transcript detected in ovarian tissue. BLAST analysis indicated high homology (>96.41%) with other Phasianidae species. The nucleotide composition of the CDS was 27.92% A, 24.05% T, 24.23% C, and 23.80% G, with a G + C content of 48.03%. As shown in the ESR2 gene structure schematic (Figure 1), the ESR2 gene varies among species in terms of exon number, untranslated region (UTR) length, and CDS length. With the exception of the Montezuma Quail and Tufted Duck, the two alternatively spliced chicken ESR2 transcript variants (N1 and N2), together with most other Phasianidae species, consist of nine exons and eight introns (Table S5). These results indicate that the transcriptional region structure of ESR2 gene is highly conserved within the Phasianidae family.

3.2. Structural Characteristics and Phylogenetic Analysis of ESR2 Protein in Wuding Chicken

The Wuding chicken ESR2 amino acid sequence exhibits high conservation across the Phasianidae family (97.90–100% homology), with the notable exception of the chicken X2 variant (Figure S3). The protein is hydrophilic and unstable, lacks transmembrane domains or signal peptides, and contains 60 potential phosphorylation sites and 5 types of functional modification sites (Figure S4, Tables S6 and S7). Structural analysis revealed high consistency in physicochemical properties, secondary/tertiary structures, and conserved motifs between Wuding chicken ESR2 and those of other Phasianidae species (Figure S5, Table S8). The DNA-binding domain (DBD), ligand-binding domain (LBD), N-terminal domain (NTD), and conserved motifs of ESR2 were highly conserved in Wuding chicken, chicken (except for variant X2), and species from the Phasianidae, Anatidae, and Odontophoridae families. Phylogenetic analysis indicated that the ESR2 of Wuding chickens clustered within the same major evolutionary branch as those of chickens, turkeys, ring-necked pheasants, and other Phasianidae species (Figure 2). GO annotation suggests involvement in estrogen signaling and transcriptional regulation, interacting with proteins such as ESR1, GPER1, and CYP19A1 (Figure 3).

3.3. Differential Tissue Expression of ESR2

We profiled ESR2 expression in the hypothalamic-pituitary-ovarian (HPO) axis of laying and broody hens. Expression levels of ESR2 were significantly higher across the hypothalamus, pituitary, and ovary during the laying period compared to the broody period (Figure 4A). Notably, the tissue-specific expression profile shifted between reproductive states: in laying hens, ESR2 abundance peaked in the ovary (Figure 4B), whereas in broody hens, expression was highest in the hypothalamus with minimal ovarian expression (Figure 4C).

3.4. Subcellular Localization of Wuding Chicken ESR2 in GCs

Immunofluorescence assays demonstrated that the EGFP-ESR2 fusion protein was distributed in both the cytoplasm and the nucleus of GCs (Figure 5).

3.5. ESR2 Upregulates Genes Associated with Steroidogenesis in GCs

To investigate the role of ESR2 in steroidogenesis of chicken GCs, primary cultured GCs were transfected with EGFP-ESR2 to induce gene overexpression or with siESR2 to achieve gene knockdown. RT-qPCR analysis showed that ESR2 expression was markedly increased in the EGFP-ESR2 group compared with the EGFP control (p < 0.001, Figure 6A). In contrast, RNA interference using siESR2-1 and siESR2-2 reduced mRNA levels by approximately 70% and 74%, respectively (p < 0.001, Figure 6B); therefore, siESR2-2 was selected for subsequent experiments. Functional analyses revealed that ESR2 overexpression significantly upregulated the expression of key steroidogenesis-related genes, including FSHR (p < 0.01), PTGS2 (p < 0.01), CYP19A1 (p < 0.05), and STAR (p < 0.05) (Figure 6C). Conversely, ESR2 knockdown significantly downregulated these genes (Figure 6D).

3.6. ESR2 Enhances GC Viability and Proliferation via Regulation of the PI3K/AKT/mTOR-Related Genes

CCK-8 assays indicated that ESR2 overexpression significantly increased GC viability at 48 h (p < 0.01) and 72 h (p < 0.05), whereas knockdown reduced viability at 48 h and 72 h (p < 0.05) (Figure 7A,B). EdU assays confirmed that ESR2 overexpression significantly enhanced GC proliferation rate (p < 0.01) (Figure 7C,E), while knockdown suppressed it (p < 0.01) (Figure 7D,F). Consistently, ESR2 overexpression upregulated the expression of proliferation-associated genes PI3K (p < 0.05), AKT1 (p < 0.01), and mTOR (p < 0.05) (Figure 7G), whereas ESR2 knockdown resulted in their downregulation (Figure 7H). To further elucidate the cellular mechanisms driving proliferation, we assessed DNA content distribution across cell cycle phases using flow cytometry (Figure 7I,J). Quantitative analysis demonstrated that ESR2 overexpression actively facilitated cell cycle progression, evidenced by a significantly diminished G0/G1 population (p < 0.05) and a concomitant increase in the S and G2/M phase populations (p < 0.05). This indicates a marked acceleration of the G0/G1-to-S transition. Conversely, ESR2 knockdown induced a pronounced accumulation of cells in the G0/G1 phase (p < 0.01), triggering a cell cycle arrest that mechanistically underpins the observed suppression of GC proliferation (Figure 7I–L).

3.7. ESR2 Modulates the Expression of Apoptosis-Related Genes in GCs

ESR2 overexpression significantly upregulated the anti-apoptotic genes BCL2 (p < 0.01) and TGFB1 (p < 0.001) while downregulating the pro-apoptotic genes Caspase3 (p < 0.01) and BAX (p < 0.01) (Figure 8A). Conversely, ESR2 knockdown produced the opposite effects (Figure 8B), confirming an anti-apoptotic role for ESR2.

3.8. ESR2 Modulates Estrogen Signaling Pathways

Overexpression of ESR2 markedly increased ESR1 (p < 0.001) and MAPK3 (p < 0.001) expression but downregulated GPER1 (p < 0.001) (Figure 9A). Knockdown suppressed ESR1 and MAPK3 while upregulating GPER1 (Figure 9B). This suggests that ESR2 may be involved in both genomic and non-genomic estrogen signaling.

4. Discussion

Precise regulation of the hypothalamic-pituitary-ovarian (HPO) axis is critical for optimizing laying performance in poultry [18]. The hypothalamus secretes gonadotropin-releasing hormone (GnRH), stimulating the pituitary to release luteinizing hormone (LH) and follicle-stimulating hormone (FSH), which, in turn, drive ovarian steroidogenesis and follicle maturation [19]. In this study, we provide the first evidence that ESR2 expression in Wuding chickens is spatiotemporally regulated across the HPO axis, showing a significant ovarian upregulation during the laying period compared to broodiness. While ESR2 was detected in the hypothalamus, pituitary, and ovary during both phases, its expression levels were significantly higher in all three tissues during the laying period with the highest expression levels in the ovary. In contrast, during the broody period, ESR2 expression in the ovary dropped to its lowest level, while hypothalamic expression remained relatively high. This shift in tissue-specific expression pattern suggests that high levels of ESR2 during the laying period primarily function locally in the ovary to promote follicular development and ovulation, whereas the pronounced downregulation of ovarian ESR2 expression during broodiness may be related to changes in the endocrine environment characteristic of this reproductive stage, as well as to potential epigenetic regulation. Increased prolactin (PRL) secretion is a typical characteristic of the broody phase, and previous studies have suggested that it may affect estrogen-dependent follicular development through PRL receptor-mediated signaling pathways [20]. In addition, increased DNA methylation in the ESR2 promoter region participates in the regulation of ESR2 transcription [21], which is a likely contributing factor underlying the sustained reduction in ovarian ESR2 expression observed in this study. These findings corroborate previous studies in Leizhou black ducks [22], reinforcing ESR2 as a conserved molecular marker for active reproductive function.

Beyond tissue-level expression differences, the subcellular localization of ESR2 is a key determinant of its mechanisms of action. Although classically characterized as a nuclear transcription factor, our immunofluorescence analysis revealed that ESR2 is distributed in both the cytoplasm and the nucleus of Wuding chicken GCs. This distinct spatial distribution indicates that ESR2 regulates GC function through an integrated and coordinated mechanism, rather than through a single pathway. The nuclear localization of ESR2 provides the physical basis for genomic regulation. Simultaneously, we found that ESR2 overexpression significantly upregulates the expression of genes associated with cell proliferation and steroidogenesis, indicating a transcriptional regulatory role. By upregulating these essential components, ESR2 acts as a transcriptional priming factor, establishing an enhanced capacity and responsiveness of intracellular signaling pathways. Furthermore, the presence of ESR2 in the cytoplasm points toward a potential non-genomic regulatory role. While unliganded receptors often reside in the cytoplasm, this localization may support non-genomic signaling. Follicular development, particularly during the hierarchical follicle stage, requires rapid cellular responses to hormonal cues that cannot be fully explained by transcription-dependent genomic mechanisms alone [23]. In this regard, cytoplasmic ESR2 can interact with signaling molecules such as MAPK to trigger rapid kinase cascades [24]. Therefore, we propose that ESR2 contributes to GC homeostasis through both genomic and non-genomic modes of action.

Given the localization of ESR2 in both the cytoplasm and the nucleus, this subcellular distribution suggests a potential role in transcriptional regulation. We therefore further analyzed the involvement of ESR2 in steroid hormone biosynthesis in GCs. The synthesis of ovarian steroid hormones relies on the two-cell model involving theca and GCs, as well as the synergistic action of FSH and LH [25]. In this process, STAR (Steroidogenic Acute Regulatory protein) facilitates the transport of cholesterol to the mitochondria, representing the rate-limiting step for steroid synthesis, followed by enzymatic conversion to androstenedione, which subsequently diffuses into GCs for aromatization to estradiol [26,27]. Moreover, PTGS2, a rate-limiting enzyme in prostaglandin synthesis—especially PGE₂—is essential for follicle rupture, ovulation, and luteinization [28]. Previous studies have identified ESR2 as a key regulatory factor in gonadotropin-induced follicular maturation, controlling the expression of multiple LH/FSH-responsive genes in GCs, including CYP19A1, PTGS2, and FSHR [29]. Consistent with this, our results show that overexpression of ESR2 significantly upregulated the expression of key genes including STAR, CYP19A1, FSHR, and PTGS2, whereas interfering with ESR2 produced the opposite effect. Collectively, these results suggest that ESR2 is associated with the upregulation of multiple genes involved in gonadotropin signaling and steroid hormone biosynthesis in GCs. This coordinated gene expression pattern suggests a potential involvement of ESR2 in the regulation of steroidogenesis-related genes during follicular development.

The PI3K/AKT/mTOR pathway serves as a central regulator of cell proliferation and metabolism [30]. In GCs, activation of this pathway not only drives cell growth and follicular maturation but also supports steroidogenic activity [31]. Previous studies have demonstrated that ESR2 can activate PI3K/AKT signaling to promote cell survival [32]. In our study, ESR2 overexpression significantly upregulated the mRNA levels of PI3K, AKT1, and mTOR, which was accompanied by enhanced GC viability and increased DNA synthesis. Furthermore, flow cytometric analysis revealed that ESR2 accelerates cell cycle progression by promoting the G0/G1 to S and G2/M transition. Our data indicate that ESR2 modulates the expression of key components of the PI3K/AKT/mTOR pathway, thereby promoting GC proliferation, alongside its involvement in rapid non-genomic signaling. The observed changes in the expression of these signaling genes, together with the subcellular localization of ESR2 in both the nucleus and cytoplasm, suggest a potential coordinated regulatory pattern. ESR2 likely contributes to the intracellular abundance of signaling molecules by regulating their expression, while its cytoplasmic presence supports the spatial assembly or rapid activation of these signaling complexes in response to hormonal cues. Collectively, this coordinated pattern may help provide the molecular foundation and functional responsiveness necessary for follicular development. Specifically, our data indicate that ESR2 regulates the expression of genes in the PI3K/AKT/mTOR signaling pathway, thereby potentially enhancing signaling activity and promoting GC proliferation.

The balance between cell proliferation and apoptosis is a critical determinant of follicular fate, with ESR2 serving as a pivotal anti-apoptotic mediator in this process. BCL2 and TGFB1 are widely recognized as anti-apoptotic markers, whereas Caspase3 and BAX are commonly used indicators of pro-apoptotic signaling. Similar to other reproductive regulators such as kisspeptin, which has been shown to reduce apoptosis in ovarian GCs by upregulating BCL2 and downregulating Caspase3 and BAX [33], our findings suggest that ESR2 may exert a comparable anti-apoptotic effect in GCs. In addition, TGFB1 has been reported to suppress GC apoptosis through the SMAD4/BMF signaling pathway, thereby promoting follicular-stage proliferation and cellular viability in Small Tail Han sheep [34]. Further evidence suggests that TGFB1 is positively associated with ESR2 expression during GC differentiation [35]. In the present study, ESR2 overexpression significantly increased the expression levels of BCL2 and TGFB1 while markedly reducing Caspase3 and BAX expression in Wuding chicken GCs. These findings suggest that ESR2 enhances GC survival by modulating apoptosis-related gene expression, thereby contributing to follicular development.

Finally, ESR2 may participate in mediating estrogen signaling pathways in Wuding chicken GCs. In the present study, ESR2 overexpression markedly upregulated ESR1 expression, indicating a strong positive regulatory relationship between these two nuclear estrogen receptors. Based on previous studies, ESR2 participates in genomic regulation through heterodimerization with ESR1 in the nucleus, thereby modulating target gene binding and coactivator recruitment [36]. This effect likely regulates the transcription of genes essential for GC proliferation and differentiation. Consistent with this notion, ESR2 overexpression significantly increased MAPK3 expression. As a key component of the MAPK cascade, MAPK3 act as a molecular link between rapid extranuclear signaling and downstream nuclear transcriptional responses [37], potentially supporting GC proliferation and functional maintenance. In contrast, ESR2 overexpression was accompanied by marked suppression of GPER1, whereas ESR2 knockdown resulted in GPER1 mRNA upregulation, indicating that ESR2 and GPER1 exhibit opposite mRNA expression patterns. GPER1 has been reported to cross-talk with nuclear estrogen receptors (ESR1/ESR2) at shared downstream kinase and transcriptional networks. This interplay facilitates a functional competition and compensatory regulation, whereby GPER1 signaling is upregulated when nuclear ER pathways are diminished [38]. Based on the observed expression patterns in the present study, together with relevant published evidence, we speculate that ESR2-mediated intracellular signaling and GPER1-dependent membrane signaling may engage in a competitive or compensatory relationship in Wuding chicken GCs. We therefore postulate that the upregulation of GPER1 following ESR2 suppression represents a compensatory mechanism that could help maintain estrogen signaling homeostasis when nuclear receptor-mediated pathways are attenuated. Future studies using specific GPER1 modulators, such as the agonist G1 or the antagonist G15, will be necessary to directly test this hypothesis and to clarify the potential crosstalk between genomic and non-genomic estrogen signaling pathways. Collectively, these findings indicate that ESR2 is likely involved in both genomic and non-genomic estrogen signaling in Wuding chicken GCs, based on the protein subcellular localization pattern of ESR2 and its correlated mRNA expression patterns with ESR1, MAPK3 and GPER1.

While the present study characterizes the transcriptional regulatory role of ESR2 in Wuding chicken GCs, several avenues for future investigation remain. The development of avian-specific, ChIP-grade ESR2 antibodies will be instrumental in directly mapping the genomic binding landscapes and confirming the recruitment of ESR2 to specific promoter regions. Furthermore, although our transcriptomic evidence strongly points toward the activation of the PI3K/AKT/mTOR axis, future research incorporating protein phosphorylation profiling and pathway-specific inhibitors will further elucidate the kinetics of these non-genomic signaling events. Finally, building upon the robust mechanistic insights gained from our primary GC model, subsequent in vivo studies—such as targeted ovarian interventions—will be essential to translate these cellular findings into a holistic understanding of follicular development and reproductive efficiency in indigenous poultry.

5. Conclusions

This study establishes ESR2 as a critical positive regulator of ovarian function in Wuding chicken. Our results demonstrate that the high expression of ESR2 in the HPO axis, particularly in the ovary, is indispensable for maintaining the laying state. In addition, ESR2 is localized in both the cytoplasm and the nucleus, a spatial distribution that could support dual roles: ESR2 transcriptionally regulates key components of the PI3K/AKT/mTOR pathway to drive the expression of genes essential for GC proliferation and steroidogenesis; simultaneously, its cytoplasmic localization mediates rapid non-genomic signaling cascades. Therefore, rather than acting through a single dominant pathway, ESR2 exhibits an integrated regulatory mode combining genomic transcriptional regulation with non-genomic signaling, which contributes to the functional maintenance of Wuding chicken GCs. Together, these findings highlight ESR2 as a key molecular target for regulating ovarian activity and improving reproductive performance. They also provide a theoretical framework for molecular breeding strategies aimed at reducing broodiness and enhancing egg production in indigenous chicken breeds.