The impact of human follicular fluid extracellular vesicle subtypes on KGN cells

Follicular fluid extracellular vesicles (FF EVs) facilitate communication between oocytes and somatic cells within the ovarian follicle, playing a pivotal role in follicular development. This study highlights the molecular and functional distinctions between small (SEV) and large (LEV) FF EV subpopulations, revealing their specialized regulatory roles in granulosa cell (GC) biology and their consequential impact on ovarian function. Single-EV profiling uncovered distinct tetraspanin distributions, with LEVs containing a lower proportion of CD9/CD63/CD81-positive particles compared to SEVs. Fluorescent labeling confirmed uptake of both SEVs and LEVs by GCs, supporting their capacity to impact cellular behavior. Functionally, LEVs increased testosterone production by GCs, while SEVs had no effect on steroid hormone secretion, suggesting a specific role for LEVs in androgen biosynthesis. Transcriptomic analysis revealed extensive SEV-induced changes in GC gene expression, affecting pathways involved in transcription, TGF-ß signaling, extracellular matrix (ECM) remodeling, and cell cycle regulation. In contrast, LEVs elicited minimal transcriptional changes, primarily modulating genes associated with immune regulation and oxidative stress defense. Small RNA sequencing further revealed distinct non-coding RNA (ncRNA) profiles, with SEVs enriched in miRNAs targeting pathways critical for GC differentiation, while LEVs carried higher levels of piRNAs implicated in maintaining genomic stability. These findings advance our understanding of FF EV-mediated intercellular communication and underscore the importance of investigating EV subpopulations independently.