Follicle-stimulating hormone promotes energy metabolism in bovine granulosa cells through the FOXO1-ISG15 pathway to maintain the development of multiple follicles

Abstract

As a central regulator of follicular development, follicle-stimulating hormone (FSH) is widely applied to induce synchronous multi-follicular growth in assisted reproductive technologies and livestock breeding. However, the molecular mechanism linking FSH signaling to synchronous multi-follicular development remains poorly defined. This study aimed to investigate the molecular mechanism by which porcine purified pituitary FSH (pFSH) promotes translation of multiple follicles. Here, we established a mammalian model of multi-follicular development using pFSH and integrated single-cell transcriptomics with functional analyses, which uncovered a regulatory axis governing the metabolism and proliferation of granulosa cells (GCs). Treatment with pFSH significantly increased ovarian weight, the number of antral follicles—particularly small antral follicles (3–5 mm)—and circulating estradiol levels. Single-cell RNA sequencing of GCs from small antral follicles revealed that pFSH markedly enhanced the expression of genes involved in energy metabolism, especially the oxidative phosphorylation and mitochondrial electron transport chain pathways, while concurrently downregulating FOXO1 expression. Mechanistically, pFSH activated the PI3K/AKT pathway to drive phosphorylation of FOXO1 at Ser267, a modification that is required for GC proliferation and metabolic activation. Overexpression of the dephosphorylated mutant FOXO1(S267A) abolished the pFSH-induced increases in GC proliferation, glucose uptake, oxidative phosphorylation, and ATP production, highlighting a central role of FOXO1 phosphorylation in mediating FSH responses. RNA-seq and chromatin immunoprecipitation analyses further identified ISG15 as a direct transcriptional target of FOXO1. Upon binding to the promoter region, FOXO1 activates ISG15 expression, linking FSH signaling to an interferon-related pathway and metabolic regulation. Collectively, this study revealed the pivotal role of the pFSH–FOXO1–ISG15 regulatory axis in orchestrating GC metabolic reprogramming and synchronous multi-follicular development. These finding provide a conceptual framework to clarify how endocrine signals integrate with metabolic and immune pathways to regulate reproductive processes, with potential implications to improve assisted reproduction and livestock productivity.