GSK3β inhibits the differentiation of follicular granulosa cells by promoting lipid accumulation through autophagy in chickens.

Granulosa cells (GCs) are integral to the process of follicular development in poultry, with their differentiation and hormone synthesis being critical for follicle selection. While glycogen synthase kinase 3 beta (GSK3β) is recognized as a significant regulator of energy metabolism, differentiation, and autophagy, its specific function within GCs remains unclear. Elucidating the role of GSK3β in GCs is essential for deciphering the mechanisms governing follicle selection. Our in vitro studies in GCs from prehierarchical follicles demonstrated that overexpression of GSK3β inhibited both differentiation and proliferation, while simultaneously promoting steroid hormone synthesis. Conversely, GSK3β knockdown yielded the opposite effects. Transcriptomic analyses, supplemented by further validation, revealed that overexpression of GSK3β initiated autophagy and lipid metabolism but impeded autophagic flux, as evidenced by increased LC3-II levels and elevated p62 accumulation. Furthermore, GSK3β overexpression resulted in enhanced intracellular lipid droplet accumulation. These effects, along with the observed rise in progesterone levels and reduction in FSHR levels, were attenuated by co-treatment with rapamycin (Rapa). Mechanistically, our findings suggest that the impairment of autophagic flux induced by GSK3β triggers lipid accumulation, leading to elevated mitochondrial damage and lipid peroxidation, which in turn negatively affects GC differentiation. In conclusion, GSK3β disrupts follicular GC function by initiating autophagy while blocking its flux. This disruption induces excessive lipid accumulation, ultimately inhibiting GC differentiation. This study provides novel insights into the role of GSK3β in poultry follicular development and offers a new theoretical framework for understanding the mechanisms of follicle selection.