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Objective: To investigate the role of Pou3f1 in proliferation of GC-1 spermatogonia (spg) cells cultured by Yangjing Capsule (YC) extract.Methods: GC-1 spg cells were treated with 0.01,0.1,and 1 mg/mL YC extract.CCK-8 assay was performed to detect the cell viability.Flow cytometry was used to measure the cell cycle and apoptosis of GC-1 spg cells.Real-time PCR and western blot were applied to determine the mRNA and protein expression of Pou3f1.Gfrα1 and Pou3f1 knockdown and LY294002 (PI3K inhibitor) were applied to explore the underlying mechanism.Results:After 48 h treatment of YC,the viability of GC-1 spg cells increased significantly and the ratio of apoptotic cells reduced significantly.The increased mRNA and protein expression of Pou3f1 and down-regulation of it blocked by Pou3f1 siRNAs suggested YC promoted self-renewal of GC-1 spg cells via Up-regulation of Pou3f1.Both Gfrα1 siRNAs and LY294002 treatments held back YC extracts stimulation effects on mRNA and protein expression of Pou3f1 and consequently inhibited the proliferation of GC-1 spg cells induced by YC extract.Conclusion:YC extract could stimulate the proliferation of GC-1 spg cells.YC extract is able to trigger the activation of PI3K pathway and up-regulates the expression of Pou3f1 downstreamand finally leads to self-renewal ofGC-1 spg cells.