Abstract
Follicle selection, a crucial step in maintaining continuous egg production in chickens, is a process that relies on granulosa cells (GCs). In this study, we aimed to identify the key genes that are involved in follicle selection from our previous single-cell transcriptomic data. We used a combination of techniques and assays, including quantitative real-time PCR, immunofluorescence, Oil Red O staining, transmission electron microscopy (TEM), enzyme-linked immunosorbent assay (ELISA), monodansylcadaverine (MDC) assay, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, cell counting Kit-8 (CCK-8) assay, and 5-ethynyl-2-deoxyuridine (EdU) assay. Multiple indices, such as cell proliferation, cell differentiation, progesterone synthesis, lipid droplet production, total and free cholesterol content, apoptosis, and autophagy, were measured to determine the states of GCs in vitro. The results demonstrated that overexpression of genes related to insulin-like growth factor 2 (IGF2) or IGF-binding protein 4 (IGFBP4) increases intracellular free cholesterol (progesterone precursors) and lipid droplet production, inhibits apoptosis through increased autophagy, and inhibits cell proliferation. This indicates that IGF2 or IGFBP4 can maintain the survival state and improve differentiation tendency of chicken granulosa cells in vitro. Therefore, this study provides new evidence on the functions of IGFs and IGFBPs in chickens, establishing a crucial experimental foundation for understanding the regulatory mechanisms of follicle selection. In addition, our study contributes to understanding follicular development and improves the egg-laying performance of chickens.