Impact of Neutral Sphingomyelinase Inhibition on Small Extracellular Vesicle Production by Mural Granulosa Cells and In Vitro Folliculogenesis in Mice.

Small extracellular vesicles (sEVs) function as critical regulators of ovarian follicular development. Although several pathways, including one involving neutral sphingomyelinase (nSMase), contribute to sEV production, the specific pathway active in ovarian follicles has not been clearly identified. In this study, we investigated GW4869, a specific inhibitor of nSMase activity, to determine its impact on sEV production by mouse mural granulosa cells (MGCs), the primary source of follicular sEVs. We also examined how nSMase inhibition affects the in vitro growth of oocyte‒granulosa cell complexes (OGCs) derived from secondary follicles. Transcripts encoding nSMases (Smpd2 and Smpd4) were detected in MGCs, and GW4869 treatment significantly reduced sEV production in MGC monolayer cultures. Control OGCs developed into antral follicle-like structures, with the antrum-like structure separating granulosa cells into cumulus-like and MGC-like cells. However, GW4869 treatment impaired OGC development. MGC-like cells from GW4869-treated OGCs exhibited significantly lower Cyp19a1 levels, whereas adding MGC-derived sEVs promoted Cyp19a1 expression. These results suggest that nSMase activity, likely involving Smpd2 and Smpd4, is required for sEV production by MGCs and that follicular sEVs may regulate Cyp19a1 expression in MGCs.