Human Chorionic Gonadotropin (hCG)-Induced Remodeling of the Granulosa Cell Exosomal Proteome: Implications for...

Human follicular development depends on coordinated communication between granulosa cells (GCs) and oocytes through endocrine cues, direct contacts, and extracellular vesicles (EVs). Exosomes are key EV mediators of intrafollicular signaling, but their cargo and functions in gonadotropin-stimulated GCs remain poorly defined. The human granulosa-like tumor cell line KGN was used to investigate exosome secretion and protein composition following human chorionic gonadotropin (hCG) stimulation. Exosomes were isolated by ultracentrifugation, characterized via nanoparticle tracking analysis (NTA), Scanning Electron Microscopy (SEM) and Western blotting, and analyzed using high-resolution mass spectrometry. Comparative proteomics integrating exosomal profiles with the whole secretome were performed, followed by bioinformatic analyses of protein networks, gene ontology, and pathway enrichment. hCG reshaped exosomal cargo, identifying 59 proteins enriched in exosomes, including Integrin α3 (ITGα3), Galectin-3-binding protein (LGALS3BP), tetraspanins (CD63, CD151), and proteasome subunits. Functional enrichment indicated roles in extracellular matrix remodeling, integrin signaling, proteostasis, and steroidogenesis. Comparison with the secretome revealed distinct protein distributions, supporting selective exosomal packaging. Western blot confirmed increased ITGα3 and LGALS3BP levels in exosomes upon hCG treatment. In conclusion, hCG modulates exosome cargo composition in granulosa cells, uncovering a novel mechanism of extracellular regulation.