Objective: Testosterone plays an essential role in maintaining spermatogenesis and male fertility,and the primary known source of testosterone is testicular Leydig cells,which are regulated by luteinizing hormone (LH).However,whether any other ways of testosterone secretion exist still remains unknown.Methods: Transmission electron microscopy was used to detect testicular extracellular vesicles (EVs),which were isolated by an ultracentrifuge process.Separately,the concentrations of follicle-stimulating hormone (FSH),LH,and testosterone were measured by enzyme-linked immunosorbent assay.Results: Some EVs were found by tail vein injection to be present in mouse testes that elevate the circulating testosterone and LH levels in the blood,but do not affect FSH.Separately,they also promote testosterone production in the TM3 Leydig cell line in vitro.To determine whether the EVs from spermatogonia were involved in the secretion of testosterone,we used spermatogonial st/progenitor cell line C18-4 cells and revealed that C 18-4 cells promote production of testosterone in the TM3 Leydig cell line using the EVs.Conclusions: EVs in mouse testes likely originate from spermatogonia and involved in the regulation of the serum testosterone.Our results provide a new mechanism for the regulation of testosterone production.