# 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.

## Key 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.