Spatiotemporal Expression Inversion of CYP11A1 in the HPO Axis and Its Regulation of Granulosa Cell Proliferation via the PI3K/AKT/mTOR Pathway in Wuding Chickens

TL;DR

This study shows how the CYP11A1 gene controls egg-laying and broodiness in chickens by switching activity between the ovary and brain, offering a way to improve egg production in local breeds.

Contribution

The study reveals a novel spatiotemporal expression inversion of CYP11A1 in the HPO axis and its role in regulating granulosa cell proliferation via the PI3K/AKT/mTOR pathway.

Findings

CYP11A1 expression shifts from ovary-dominant during egg-laying to hypothalamus-dominant during broodiness.

CYP11A1 promotes granulosa cell proliferation by activating the PI3K/AKT/mTOR pathway.

CYP11A1 coordinates steroid metabolism and cell growth by regulating genes like STAR and HSD3B1.

Abstract

Broodiness—a behavior where hens stop laying eggs to focus on incubating nests—greatly reduces egg production in valuable local chicken breeds like Wuding chickens, limiting their economic value for farmers. This study aimed to understand how a key gene called CYP11A1 influences broodiness and the development of egg-forming follicles in these chickens. We found that this gene’s activity shifts between the hypothalamus and ovary during different reproductive stages: it is highly active in the ovary when hens are laying eggs to support egg development, but switches to high activity in the hypothalamus when hens become broody to maintain broodiness behavior. Additionally, the gene helps follicle cells grow by triggering a natural molecular process that supports cell division. These findings show CYP11A1 acts as a “switch” linking egg production and broodiness, offering a way to breed local chickens that retain good meat quality while laying more eggs—benefiting farmers economically and ensuring a sustainable supply of high-quality poultry for consumers.

Broodiness in poultry represents a major bottleneck for reproductive performance, governed by complex remodeling of the hypothalamic-pituitary-ovarian (HPO) axis. CYP11A1, the rate-limiting enzyme in steroidogenesis, is essential for hormone synthesis; however, its spatiotemporal dynamics within the HPO axis during reproductive transitions remain unclear. Using the Wuding chicken as a model, this study characterized the expression profile and cellular function of CYP11A1. We identified a distinct “ovary-hypothalamus expression inversion” pattern: CYP11A1 expression exhibited an ovary-dominant pattern during the egg-laying period to support folliculogenesis; however, this shifted to a hypothalamus-dominant pattern during the broodiness period. This inversion indicates its active role in central neurosteroid modulation rather than a passive response to HPO axis regression. In vitro assays in granulosa cells (GCs) demonstrated that CYP11A1 overexpression significantly upregulated AKT1 and mTOR transcription, promoted the G0/G1 to S/G2/M cell cycle transition, and enhanced cell proliferation. Conversely, CYP11A1 knockdown arrested the cell cycle and suppressed the PI3K/AKT/mTOR pathway. Additionally, CYP11A1 coordinated the expression of steroidogenic genes (STAR, HSD3B1), reflecting a coupling between steroid metabolism and cell growth. These findings reveal CYP11A1 as a critical molecular node linking HPO axis remodeling, granulosa cell proliferation, and steroidogenesis, providing a potential target for molecular breeding to mitigate broodiness in indigenous chickens.

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1. Introduction

In the poultry industry, an evolutionary trade-off often exists between reproductive persistence and superior germplasm characteristics. Through decades of intensive selection, modern commercial layer breeds such as White Leghorn chickens have been effectively selected to eliminate inherent broody behavior, enabling highly efficient, year-round egg production with annual yields exceeding 300 eggs [1]. In contrast, many indigenous Chinese breeds like the Yunnan Wuding chicken—endowed with superior meat quality, strong stress resistance, and tolerance to low-quality diets—retain robust broody instincts, as they have not undergone intensive artificial selection [2]. This genetically conserved maternal behavior, characterized by cessation of egg-laying, prolonged nesting and incubation, reduced feed intake, elevated body temperature, and increased aggression [3], serves as a critical survival strategy for species propagation in the wild. However, under artificial rearing conditions, the frequent disruption of the laying cycle poses a biological bottleneck that severely restricts the large-scale propagation and economic viability of these premium local breeds. Consequently, elucidating the neuroendocrine mechanisms governing this behavioral transition is of paramount practical significance for resolving the industrial paradox of “high quality yet low fertility”.

The transition between reproductive states is fundamentally governed by the precise hierarchical regulation of the hypothalamic–pituitary–ovarian (HPO) axis. During the egg-laying period, the hypothalamus primarily perceives environmental cues such as light and temperature to secrete gonadotropin-releasing hormone (GnRH), which stimulates the anterior pituitary to release follicle-stimulating hormone (FSH) and luteinizing hormone (LH) [4]. These gonadotropins act on the ovary to promote follicle recruitment, selection, hierarchy establishment, and ovulation, while inducing the synthesis of steroid hormones including progesterone (P4) and estradiol (E2) [4]. When hens are induced into broodiness by environmental or endogenous signals, the HPO axis undergoes drastic functional remodeling: environmental triggers prompt a functional switch in the hypothalamus, leading to suppressed GnRH secretion and a surge in pituitary prolactin (PRL) release [5]. High concentrations of PRL antagonize gonadotropins, resulting in ovarian regression, a sharp decline in steroid synthesis, stagnation and atresia of follicle development, and ultimately the cessation of egg-laying [5]. While the central role of PRL is well established, the molecular switches that drive the HPO axis from a “reproductive mode” to a “parental care mode” remain incompletely elucidated. Specifically, cytochrome P450 family 11 subfamily a member 1 (CYP11A1)—the rate-limiting enzyme initiating all steroid hormone biosynthesis—may represent a critical yet overlooked link in understanding the spatiotemporal dynamics and non-classical cellular regulation underlying this complex physiological transition.

Canonical perspectives have largely focused on the role of CYP11A1 in ovarian granulosa cells (GCs), where it catalyzes the conversion of cholesterol to pregnenolone for sex steroid production. As the rate-limiting enzyme in steroidogenesis, CYP11A1 localizes to the inner mitochondrial membrane and exerts its catalytic activity only after cholesterol is transported to mitochondria by the steroidogenic acute regulatory protein (STAR) [6]. Recent studies confirm that STAR and CYP11A1 form a functional unit in GCs, with their coordinated expression essential for follicle selection and progesterone secretion [7]. However, accumulating neurobiological evidence indicates that the avian brain—particularly the hypothalamus—possesses the capacity for autonomous synthesis of “neurosteroids” [8]. In birds, the hypothalamus expresses key enzymes such as CYP11A1 and 3β-hydroxysteroid dehydrogenase 1 (HSD3B1), and the resulting pregnenolone, along with metabolites like 7α-hydroxypregnenolone and tetrahydroprogesterone, can regulate neural excitability by modulating GABA_A_ receptors, thereby influencing aggressive behavior, locomotor activity, and maternal behavior [9]. The calmness, increased aggression, and nest attachment exhibited by hens during the broodiness period are likely driven by these locally synthesized neurosteroids. This raises a compelling scientific question: during the specific physiological phase of broodiness, does CYP11A1, as the biosynthetic source, exhibit a spatiotemporal trade-off, or even inversion, in its expression pattern across different levels of the HPO axis? If the hypothalamus initiates an independent steroidogenic program during broodiness, it would offer a novel neuroendocrine perspective on the maintenance of broody behavior.

Furthermore, at the cellular level, follicular development relies on the rapid proliferation and functional differentiation of GCs—processes in which the PI3K/AKT/mTOR signaling pathway serves as a central hub integrating nutrient signals, energy status, and growth factors [10]. As a central hub of signal transduction, phosphorylated AKT (p-AKT) relieves the inhibition of Rheb by phosphorylating tuberous sclerosis complex 2 (TSC2), thereby activating mTOR complex 1 (mTORC1) [11]; it can also phosphorylate FOXO3a, leading to its cytoplasmic retention and alleviating the transcriptional activation of cell cycle inhibitory genes [12]. Activated mTORC1 promotes protein translation and ribosome biosynthesis by phosphorylating downstream effectors such as S6 kinase (S6K1) and eukaryotic initiation factor 4E-binding protein (4E-BP1), driving cells from the G1 to S phase [13]. While gonadotropins like FSH and growth factors such as IGF-1 are known activators of this pathway in mammalian ovaries [10], it remains unclear whether the mitochondrial enzyme CYP11A1 and its catalytic product (pregnenolone or P4) are associated with the regulation of this pathway via non-genomic mechanisms in avian species. Growing evidence suggests that steroid hormones may feedback-regulate signaling pathways—for instance, P4 can rapidly activate AKT signaling via membrane progesterone receptors (mPRs) [14]. Such a mechanism would establish a “metabolism-proliferation” positive feedback loop, crucial for maintaining the growth advantage of dominant follicles. To investigate these neuroendocrine mechanisms in a natural model, we utilized the Wuding chicken, an indigenous breed mainly distributed in Wuding County, Chuxiong Prefecture, Yunnan Province, China, has typical egg-laying habits. It exhibits behaviors such as prolonged nest-building, cessation of egg-laying, and strong egg-laying desire. It is an ideal model for studying the neuroendocrine mechanisms related to egg-laying. Accordingly, using the Wuding chicken as a model for typical broody behavior, this study aims to systematically map the spatiotemporal expression profile of CYP11A1 and explore its role in linking reproductive behavioral transitions with granulosa cell proliferation. This work seeks to reveal the functional role of CYP11A1 in reproductive regulation, providing a new perspective for analyzing the neuroendocrine mechanism of broodiness in indigenous chicken breeds and offering a theoretical basis for the molecular improvement of poultry reproductive performance.

2. Materials and Methods

2.1. Selection of Experimental Animals

Twelve Wuding chicken hens aged over 40 weeks from the same batch with similar appearance and weight were selected and raised in individual cages at the Wuding chicken Breeding Farm (Yunnan Shouyu Agricultural Development Co., Ltd., Chuxiong, Yunnan, China), under identical rearing conditions. Based on behavioral characteristics and egg-laying records, they were divided into an egg-laying group and a broodiness group (n = 6, biological replicates per group). Hens in the egg-laying group were required to lay eggs continuously for over 10 days, while the broodiness group comprised hens exhibiting persistent broody behavior (hyperthermia, nesting, and feather fluffing) for over 10 days.

2.2. Tissue Collection and RNA Quality Control

Hens from both groups were euthanized by cervical dislocation. Tissues, including the hypothalamus, pituitary, ovary, cerebrum, and cerebellum (as controls), were collected, flash-frozen in liquid nitrogen, and stored at −80 °C. For ovarian samples, the stroma and hierarchical follicles were precisely separated to ensure tissue homogeneity. Total RNA was extracted using the Trizol method. RNA integrity was assessed via gel electrophoresis RNA purity was evaluated using a NanoDrop spectrophotometer (A260/280 and A260/230 ratios ≥ 1.8). Subsequently, total RNA was reverse-transcribed into cDNA using Oligo (dT) 18 primers (500 μg/mL) and a reverse transcription kit (TaKaRa Bio Inc., Dalian, China).

2.3. Molecular Cloning and Bioinformatics Analysis

Specific primers were designed based on the Gallus gallus reference sequence (NCBI) to clone the full-length coding DNA sequence (CDS) region of Wuding chicken CYP11A1 (Table S1). The 10 μL PCR reaction included 5 μL 2× Es Taq Master Mix (C WBIO Co., Ltd., Beijing, China), 0.4 μL each of upstream and downstream primers (10 μmol/mL), 3.2 μL of ddH_2_O, and 1 μL of cDNA template (300 ng/μL). Amplification conditions were: 94 °C for 5 min; 34 cycles of 94 °C (30 s), 58.5 °C (30 s), and 72 °C (2 min); followed by 72 °C for 7 min. Bioinformatics tools including MegAlign (v7, DNASTAR Inc., Madison, WI, USA) for molecular/sequence characteristics, ProtParam (no version, https://web.expasy.org/protparam/, accessed on 24 November 2025) for protein physicochemical properties, Conserved Domains (v3.2.0, National Center for Biotechnology Information, Bethesda, MD, USA) for protein conserved domain prediction, and MEGA (v12, Molecular Evolutionary Genetics Analysis, State College, PA, USA), were used to analyze molecular characteristics, protein properties, domains, and phylogeny, with full details provided in Table S2. Meanwhile, we performed protein-protein interaction (PPI) network analysis, in which the top 20 core interacting proteins were displayed accordingly.

2.4. Analysis of Differential Tissue Expression of CYP11A1

Expression of CYP11A1 in the HPO axis of Wuding chickens during the egg-laying and broodiness periods was quantified using real-time fluorescence quantitative PCR (qPCR) with a SYBR Green qPCR kit (Vazyme Biotech Co., Ltd., Nanjing, China). Data were normalized to GAPDH (primers in Table S1). The reaction system comprised 10 μL SYBR qPCR SuperMix Plus, 0.8 μL each of upstream and downstream primers (10 μmol/mL), 6.4 μL ddH_2_O, and 2 μL cDNA (200 ng/μL). Cycling conditions were: 95 °C for 2 min; 40 cycles of 95 °C (20 s), gene-specific annealing (30 s), and 72 °C (40 s); followed by 72 °C for 30 s. Specificity was confirmed via melting curve analysis and sequencing. Data are expressed as the mean ± standard error of three independent experiments and analyzed using the 2^−ΔΔCt^ method relative to the control group.

2.5. Construction of Overexpression Vectors and siRNA Screening

The CYP11A1 CDS was amplified from Wuding chicken ovarian cDNA (primers in Table S1) and ligated into the pEGFP vector using T4 ligase to generate the recombinant vector CYP11A1-EGFP (OE-CYP11A1). Sequence accuracy and insertion orientation were verified by Sanger sequencing. Specific siRNA sequences (si-243, si-885) and a negative control (si-NC) were synthesized by Shanghai Sangon Biotech (Table S3). Optimal interference efficiency was determined by qPCR.

2.6. Primary Granulosa Cell Culture and Transfection

Ovaries from healthy peak-laying Wuding chickens were removed under sterile conditions, washed in pre-cooled PBS with 3% antibiotics (penicillin-streptomycin) to remove connective tissue. Granulosa layers were isolated from hierarchical follicles after puncturing to release the yolk, minced, and digested with Type II collagenase (1 mg/mL) and 0.25% trypsin at 37 °C for 10–15 min with gentle shaking. Digestion was terminated with complete medium (DMEM + 10% FBS), filtered through a 200-mesh sieve, and centrifuged at 300× g for 5 min. Cells were resuspended in complete medium and cultured at 37 °C with 5% CO_2_. Primary granulosa cells were recovered and their identity was confirmed by positive FSHR expression, as previously described [15]. For transfection, cells were divided into four groups: overexpression (OE-CYP11A1), interference (si-885), and their respective controls (OE-NC: EGFP empty vector; si-NC: scrambled siRNA).

2.7. Subcellular Localization of CYP11A1 in Granulosa Cells

GCs cultured in 35 mm dishes were transfected with OE-CYP11A1. After 48 h, cells were fixed with 4% paraformaldehyde (30 min), washed and air-dried; permeabilized with 0.5% Triton X-100 (20 min), washed and air-dried; blocked with 5% BSA (30 min), discarded blocking solution, washed, and air-dried; then stained with DAPI (15 min), washed, and added with an appropriate amount of PBS. Imaging was performed using a confocal microscope (FV1000, Olympus Corporation, Tokyo, Japan).

2.8. Gene Expression and Signaling Pathway Analysis

GCs were transfected with OE-CYP11A1 and OE-NC, or si-885 and si-NC (three replicates). After 48 h, total RNA was extracted using the Trizol method and reverse-transcribed. qPCR was used to quantify expression of STAR, HSD3B1, cytochrome P450 family 17 subfamily a member 1 (CYP17A1), cytochrome P450 family 19 subfamily a member 1 (CYP19A1), bone morphogenetic protein 15 (BMP15), FSHR, proliferating cell nuclear antigen (PCNA), protein kinase B 1 (AKT1), and mammalian target of rapamycin (mTOR) (Table S1). All primers were validated with standard curves (amplification efficiencies > 90%), and data were analyzed as described in Section 2.4.

2.9. Cell Proliferation and Cell Cycle Detection

Transfected GCs were seeded in 96-well plates, 35 mm confocal dishes, and 100 mm plates (OE-CYP11A1, OE-NC, si-885, si-NC) and assessed for proliferation using a CCK-8 kit (Beyotime Biotechnology, Shanghai, China) at 0, 24, 48, and 72 h post-transfection. EdU assays were conducted at 48 h using an EdU kit (Beyotime Biotechnology, Shanghai, China): Cells were incubated with EdU reagent for 2 h, DAPI-stained, and analyzed via confocal microscopy (FV1000, Olympus Corporation, Tokyo, Japan) and ImageJ (v1.52a, National Institutes of Health, Bethesda, MD, USA); For cell cycle analysis, cells collected at 48 h post-transfection were washed three times with cold PBS, fixed in 70% cold ethanol, stained with propidium iodide (PI) and RNase A in the dark at 37 °C for 30 min, and analyzed on a BD FACSCelesta flow cytometer (BD Biosciences, San Diego, CA, USA) using FlowJo (v10.0.7, Tree Star, Ashland, OR, USA).

2.10. Statistical Analysis

Data analysis and visualization were performed using GraphPad Prism (v8, GraphPad Software Inc., La Jolla, CA, USA). Normality of the data distribution was assessed using the Shapiro–Wilk test, and homogeneity of variance was verified via Levene’s test; all experimental data met the normality and homogeneity assumptions required for parametric testing (p > 0.05). Differences between the two groups were assessed by a two-tailed independent samples t-test; comparisons among multiple groups used one-way ANOVA with Tukey’s post hoc test. Normality and homogeneity of variance were verified prior to analysis. p < 0.05 was considered significant. Data are presented as the mean ± standard error of the mean (SEM), with n = 6 biological replicates for all tissue-based experiments (Wuding chicken hen tissues) and n = 3 biological replicates for all in vitro cell experiments (each cell experiment was performed with three technical replicates).

3. Results

3.1. Molecular Identification and Conservation Verification of Wuding Chicken CYP11A1

To ensure functional specificity, we cloned the full-length CDS of CYP11A1 from egg-laying Wuding chickens. A 1723 bp PCR product was obtained (Figure S1), containing a 1527 bp CDS encoding 508 amino acids (Figure S2). Structural comparisons of the CYP11A1 transcriptional region revealed high conservation of core functional motifs within Phasianidae, such as Gallus gallus and Coturnix japonica, and cross-species similarity in the key coding and regulatory regions with mammalian orthologs, such as Homo sapiens and Mus musculus, while the overall intronexon arrangement and non-coding region length showed significant divergence (Figure 1, Table S4).

Protein sequence analysis confirmed 100% identity with the Gallus gallus reference and 96.5–97.2% identity with other Phasianidae species (Figure S3). The protein contains the characteristic cytochrome P450 cysteine heme-ligand motif and a conserved steroid-binding domain. Phylogenetic clustering placed Wuding chicken CYP11A1 with Gallus gallus and Coturnix japonica (Figure 2).

Physicochemical properties (Table S5), secondary structure (Table S6), and tertiary structure (Figure S4) were highly conserved, suggesting that the biochemical mechanism of cholesterol side-chain cleavage is evolutionarily preserved. Interaction network analysis identified STAR, CYP17A1, and FSHR—key regulators of steroidogenesis—as primary interactors shared by three Phasianidae species (Figure 3).

Based on the interaction network analysis, we selected STAR, CYP17A1 and FSHR—the core conserved functional regulators associated with CYP11A1-mediated steroidogenesis and avian ovarian reproductive regulation—as the key interactors for subsequent functional inference.

3.2. Spatiotemporal Expression Inversion of CYP11A1 in the HPO Axis

qPCR profiling revealed that CYP11A1 is expressed in the ovary, cerebrum, cerebellum, hypothalamus, and pituitary of Wuding chickens during both egg-laying and broodiness periods (Figure 4A,B). During the egg-laying period, compared with other tissues in the same period, expression was highest in the ovary, followed by the pituitary and hypothalamus, with the lowest levels in the cerebrum and cerebellum (p < 0.01). In contrast, during the broodiness period, expression peaked in the hypothalamus, followed by the cerebellum, with significantly reduced levels in the pituitary, ovary, and cerebrum (p < 0.01)—consistent with ovarian regression and cessation of progesterone synthesis. Comparative analysis highlighted a significant inversion: ovarian and pituitary expression were markedly higher during egg-laying (p < 0.01), whereas hypothalamic and cerebellar expression were significantly elevated during broodiness (p < 0.01) (Figure 4C).

3.3. Subcellular Localization of CYP11A1

Confocal microscopy of Wuding chicken GCs transfected with OE-CYP11A1 revealed that the CYP11A1 protein (green) colocalized substantially with the mitochondrial probe (red) and was distinct from the nucleus (blue) (Figure 5). Confocal imaging confirmed the mitochondrial localization of CYP11A1 in GCs.

3.4. CYP11A1 Coordinates the Expression of Steroidogenic Enzymes

Using the most effective siRNA (si-885) (Figure 6A), we modulated CYP11A1 expression in Wuding chicken GCs to assess downstream effects on steroidogenic genes (Figure 6B,C). The interference efficiency of si-885 was verified by qPCR with consistent results across three biological replicates and three technical replicates (Figure 6A). Overexpression of CYP11A1 significantly downregulated BMP15 and FSHR (p < 0.01) and E2-synthesis genes (CYP17A1, CYP19A1, p < 0.01), while upregulating P4-synthesis genes (STAR, p < 0.01, HSD3B1, p < 0.05). Conversely, CYP11A1 knockdown suppressed HSD3B1 (p < 0.05) but upregulated STAR, BMP15, FSHR, CYP17A1, and CYP19A1 (p < 0.01).

3.5. CYP11A1 Affects Granulosa Cell Proliferation and G1/S Transition

Cell viability assays (CCK-8) demonstrated that CYP11A1 overexpression significantly enhanced Wuding chicken GC proliferation at 24, 48, and 72 h (p < 0.01), whereas knockdown inhibited it (p < 0.01) (Figure 7A,B). EdU incorporation assays confirmed these findings, showing a significantly higher EdU-positive rate in overexpression groups and a lower rate in knockdown groups (p < 0.01) (Figure 7C–F). To further elucidate the cellular mechanisms driving proliferation, we assessed DNA content distribution across cell cycle phases using flow cytometry (Figure 7G,H). Quantitative analysis demonstrated that CYP11A1 overexpression actively facilitated cell cycle progression, evidenced by a significantly diminished G0/G1 population (p < 0.01) and a concomitant increase in the S (p < 0.05) and G2/M (p < 0.01) phase populations. This indicates a robust acceleration of the G0/G1-to-S transition. Conversely, siRNA-mediated knockdown of CYP11A1 induced a pronounced accumulation of cells in the G0/G1 phase, triggering a cell cycle arrest that mechanistically underpins the observed suppression of granulosa cell proliferation (Figure 7G–J). These results indicate that CYP11A1 affects the G0/G1-to-S and G2/M transition, consistent with cell cycle regulation via the PI3K/AKT/mTOR pathway. All proliferation and cell cycle data were obtained from in vitro granulosa cell cultures.

3.6. CYP11A1 Induces Transcriptional Upregulation of PI3K/AKT/mTOR Pathway Components

To elucidate the molecular mechanism underlying the observed proliferation, we quantified expression of proliferation markers and pathway components (AKT1, PCNA, mTOR) (Figure 8A,B). CYP11A1 overexpression significantly increased mRNA levels of AKT1, PCNA, and mTOR (p < 0.01), while knockdown significantly suppressed them (p < 0.01). These findings establish a correlative link between mitochondrial steroidogenic activity and nuclear proliferation signals, with increased mRNA levels of key pathway components reflecting transcriptional upregulation.

4. Discussion

The distinct “ovary–hypothalamus” spatiotemporal expression pattern of CYP11A1 in Wuding chickens links its expression to reproductive physiology and maternal behavior [3]. As a rare indigenous breed not subjected to excessive artificial selection, the Wuding chicken retains the complex physiological regulatory mechanisms underlying broodiness, making it an ideal model for dissecting the plasticity of the hypothalamic–pituitary–ovarian (HPO) axis [2]. Transcriptomic analyses of other avian species have similarly validated the critical role of HPO axis remodeling in transitions between reproductive behaviors [4], underscoring the universality of this regulatory framework. Our findings suggest that the HPO axis undergoes a spatiotemporal expression shift of CYP11A1 during the transition to broodiness, indicating a more complex regulatory role beyond simple negative feedback.

During the egg-laying phase, the HPO axis prioritizes peripheral reproductive metabolism. The ovary serves as the primary site of CYP11A1 expression, where high levels of this enzyme drive the rapid conversion of cholesterol to pregnenolone—a process dependent on cholesterol transport to mitochondria by STAR [6]. This coordinated expression of STAR and CYP11A1 forms a functional unit that sustains the high hormonal milieu required for follicle recruitment, maturation, and selection [7]. Consistent with our qPCR results, CYP11A1 expression in the ovary during the egg-laying period was significantly elevated compared to the broodiness period. In this state, hypothalamic CYP11A1 expression remains low, aligning with classical negative feedback theory, wherein elevated peripheral steroids suppress central steroid synthesis [3].

However, the onset of broodiness induces a dramatic functional reversal. The precipitous decline in ovarian CYP11A1 acts as a molecular “brake” on reproductive function, leading to impaired steroid synthesis, rapid ovarian regression, and subsequent cessation of egg-laying [5]. Concurrently, the significant upregulation of CYP11A1 in the hypothalamus indicates this is not a passive endocrine collapse but an active neural remodeling process, which aligns with the “central neurosteroid” hypothesis [9]. Unlike peripheral hormones that regulate reproductive morphology, locally synthesized pregnenolone and its metabolites—such as 7α-hydroxypregnenolone and tetrahydroprogesterone—function primarily as neuromodulators [8]. These neurosteroids are known to potentiate GABA_A_ receptors, inducing sedation, anxiolysis, and reduced locomotor activity characteristic of broody hens [9,16]—traits essential for sustaining long-term incubation, reducing feed intake, and stabilizing nest temperature. Thus, hypothalamic CYP11A1 upregulation suggests a potential role as a neurobiological regulator contributing to the persistence of broody behavior by maintaining a neuroendocrine state that favors incubation over ovulation.

Beyond this macroscopic tissue-specific shift, CYP11A1 dictates the functional fate of GCs at the cellular level. Granulosa cell development and function are governed by a complex network of signaling pathways [12], and our data demonstrate that CYP11A1 is a key regulatory node within this network. Specifically, CYP11A1 overexpression directs GCs toward a specialized “progesterone-dominant” developmental trajectory. We observed a marked upregulation of STAR and HSD3B1, accompanied by significant suppression of estradiol-synthesis genes (CYP17A1, CYP19A1) and follicle markers (BMP15, FSHR) [17]. Conversely, CYP11A1 knockdown suppressed HSD3B1 but upregulated STAR, BMP15, FSHR, CYP17A1, and CYP19A1. BMP15 itself plays a pivotal role in follicle selection, and its downregulation may reflect a functional adaptation to CYP11A1-mediated progesterone synthesis [18,19]. This gene expression profile mirrors the physiological demands of follicle selection, wherein dominant follicles must transition from FSH-dependent proliferation to robust progesterone production [20]. The paradoxical downregulation of FSHR and BMP15 likely represents a compensatory negative feedback loop—GCs sense enhanced intracellular steroidogenic capacity and consequently desensitize themselves to external signaling, prioritizing the rapid accumulation of progesterone [21]. Notably, high CYP11A1 levels direct GCs to prioritize P4 synthesis over E2, which aligns with the physiological requirement for selected follicles to mount a robust P4 response to FSH signals [21]. Thus, CYP11A1 is a rate-limiting enzyme for steroidogenesis, and its regulation of steroidogenic gene expression correlates with granulosa cell functional differentiation during follicle selection. It is worth noting that other steroidogenic enzymes (e.g., CYP21A1) also influence follicle development [22], but CYP11A1’s unique spatiotemporal expression pattern distinguishes it as a critical switch in reproductive transitions.

Crucially, this study identifies a correlation between CYP11A1 activity and altered mRNA levels of PI3K/AKT/mTOR pathway components, linking its expression to granulosa cell cycle progression [11]. To evaluate PI3K/AKT/mTOR pathway activation, we quantified the expression of its core components (AKT1, mTOR) and the proliferation marker PCNA [23,24,25,26,27,28]. The upregulation of AKT1, mTOR, and PCNA mRNA—along with our functional data (enhanced proliferation, G0/G1-to-S/G2/M transition)—confirms a transcriptional association between CYP11A1 and the pathway. Subcellular localization shows CYP11A1 localizes to mitochondria, and its association with cytoplasmic signaling changes is likely mediated by non-genomic effects of its metabolic products (pregnenolone or P4) [14]. P4 can rapidly activate the PI3K/AKT cascade via membrane progesterone receptors (mPRs) [14,28], and our results demonstrate CYP11A1 overexpression elevates AKT1 and mTOR transcription, associated with G0/G1-to-S/G2/M transition [25], forming a “metabolism-proliferation” positive feedback loop critical for functionally proliferating GCs [29].

Consequently, CYP11A1 functions as a metabolic checkpoint for follicle survival. During the intense competition of follicle selection, only GCs with high CYP11A1 expression and robust steroidogenic capacity can produce sufficient autocrine/paracrine signals (e.g., P4) to induce transcriptional upregulation of their intrinsic PI3K/AKT/mTOR pathway [30]. Conversely, during broodiness, the downregulation of ovarian CYP11A1 severs this growth signal, leading to reduced pathway activity and cell cycle arrest, providing a molecular explanation for the cessation of egg-laying in broody hens.

To fully characterize the regulatory landscape of CYP11A1 in avian broodiness, future investigations will focus on integrating multi-omics approaches and in vivo functional validation. While our study establishes a robust transcriptional framework and cellular phenotypes, subsequent research will aim to corroborate these findings at the post-translational level, utilizing Western blotting and specific phosphorylation assays (e.g., p-AKT, p-mTOR) to dissect the precise signaling kinetics. Furthermore, integrating LC-MS/MS-based steroid profiling will allow for the direct correlation of CYP11A1 expression with tissue-specific concentrations of neurosteroids and peripheral hormones. Finally, to definitively map the causal neuroendocrine circuitry, future studies will employ hypothalamus-specific gene modulation (e.g., viral vector-mediated knockdown or overexpression) in laying hens. These prospective studies will build upon the molecular node identified here, ultimately providing precise targets for molecular breeding strategies to balance meat quality and reproductive performance in indigenous poultry.

5. Conclusions

In summary, using the Wuding chicken as a model, this study identifies the dual associative roles of CYP11A1 in reproduction and granulosa cell function, based on transcriptional correlations and in vitro phenotypic observations. Key findings include: CYP11A1 exhibits a unique “ovary–hypothalamus” spatiotemporal expression inversion in the HPO axis, characterized by ovary-dominant expression during the egg-laying period and hypothalamus-dominant expression during broodiness; hypothalamic upregulation of CYP11A1 during broodiness suggests a potential role in neurosteroid synthesis; its overexpression is associated with granulosa cell proliferation and G0/G1-to-S/G2/M transition, along with upregulated mRNA levels of PI3K/AKT/mTOR pathway components (AKT1, mTOR); additionally, CYP11A1 coordinates steroidogenic gene expression, reflecting potential feedback mechanisms. These findings identify CYP11A1 as a critical molecular switch linking HPO axis remodeling, granulosa cell function, and steroidogenesis—deepening our understanding of broodiness mechanisms in indigenous chickens. Targeted modulation of CYP11A1 or its downstream pathway could reduce broodiness while preserving meat quality, addressing the industrial trade-off between quality and fertility, and providing a candidate target for molecular breeding to support the sustainable development of local poultry industries and enrich avian reproductive physiology theory.