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Background: As similar to glial cell line-derived neurotrophic factor (GDNF),the Yangjing Capsule (YC) extract could also lead to proliferation of spermatogonial stem cells (SSCs) by motivating phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway; however,the regulatory effect of YC extract on the expression of POU3F1 still remains unknown.Objective: To determine whether the transcription factor POU3F1 is up-regulated by YC extract through PI3K/AKT signaling pathway to regulate SSCs survival and proliferation.Methods: The cultured GC-1 spermatogonial (spg) cells were treated with 0.01,0.1,and 1 mg/mL YC extract for 48 h.The cell viability was analyzed using MTT assay,while POU3F1 expression was quantitatively detected using real-time polyrnerase chain reaction and Western blot analysis.POU3F 1,GDNF family receptor alpha 1 (GFRα1) short interfering ribonucleic acid (siRNA),and LY294002 (PI3K inhibitor) were applied as blockers to explore the underlying pathway.Results: After 48 h treatment with YC extract,the GC-1 spg cells were proliferated and the POU3F1 expression was significantly increased in a dose-dependent manner.The POU3F1 siRNA partially blocked those effects of YC extract.Both GFRα1 siRNA and LY294002,as upstream blockers,reduced the POU3F 1 expression induced by YC extract.Conclusion: The YC extract promotes proliferation ofGC-1 spg cells via up-regulation of POU3F1.The potential mechanism is that YC extract triggers the activation of PI3K/AKT pathway and then up-regulates POU3F1 expression.