Non-Genomic Action of Androgens is Mediated by Rapid Phosphorylation and Regulation of Androgen Receptor Trafficking

Abstract

Background: Testosterone is critical for maintaining spermatogenesis and male fertility. The accomplishment of these processes requires the synergistic actions of the classical and non-classical signaling pathways of androgens. Methods: A murine testicular Sertoli cell line, TM4 cell was used to examine androgen actions in Sertoli cells. Western blot analysis and immunofluorescence assay were employed to study the testosterone-induced Androgen receptor (AR) translocation. Protein phosphorylation antibody array was applied to identify the phosphorylation sites under testosterone treatment, and these findings were verified by Western blot analysis. Results: We found that a physiological dose of testosterone induced fast membrane association of AR. By using a phosphorylation antibody array, several phosphorylation sites, such as MEK1/2 (Ser217/221), Akt (Ser473), and Erk1/2 (Thr202/Tyr204) were rapidly phosphorylated within 5 min of testosterone treatment. Inhibition of the MEK and Akt signaling pathways prevented AR trafficking. Blocking of AR by flutamide eliminated the stimulation effect of testosterone on kinase phosphorylation. Testosterone induced kinase Src phosphorylation, and inhibition of Src restricted AR translocation to the membrane and the nucleus. Conclusion: Findings suggested that the membrane association of AR was mediated by the MEK and Akt phosphorylation signaling pathways, which resulted in Src activation and was initiated by testosterone binding to the membrane-localized AR. This study provides new insights into the testosterone signaling pathway in Sertoli cells, which mediate spermatogenesis. In addition, the study can be used in the diagnosis and treatment of male infertility caused by disorders in spermatogenesis.