MTOR Is Involved In The Regulation Of17β-estradiol-induced Immature Boar Sertoli Cell Proliferation In Vitro

It is well known that the Sertoli cell not only plays a vital role in the sperm production by providing nutritious support with a good micro-environment, but also mediates germ cells injury by serving as a natural barrier with gap junction. In addition Sertoli cells ensure the quality and the quantity of sperm production in response to binding to the androgen.Background:Estrogen plays an important role in the proliferation of Sertoli cells, and mammalian target of rapamycin (mTOR) is also known to be involved in mammalian cell proliferation. However, it is not clear whether mTOR participates in the regulation of Sertoli cell proliferation. The objective of this study was to identify whether mTOR is involved in Sertoli cell proliferation and investigate the mechanism by which17p-estradiol regulates Sertoli cell proliferation via mTOR.Methods:Immature boar Sertoli cells were cultured and treated with17P-estradiol after pre-treatment with or without different concentrations of rapamycin. The effects of rapamycin on17β-estradiol-induced cell proliferation and the cell cycle were determined using the CCK8cell proliferation assay kit and flow cytometry. Western blotting was used to measure mTOR activation. The effects of17p-estradiol and rapamycin on the protein and mRNA expression levels of the cell cycle proteins S-phase kinase associated protein2(Skp2), proliferating cell nuclear antigen (PCNA), Cyclin D1and Cyclin E1were determined by Western blotting or RT-PCR, respectively.Results:Treatment with17p-estradiol for15min to90min significantly activated mTOR; phosphorylation of mTOR peaked after30min treatment with17β-estradiol. Rapamycin significantly reduced17β-estradiol-induced Sertoli cell proliferation by reducing the expression of Skp2、PCNA、Cyclin D1mRNA and Cyclin E1mRNA. Moreover,2μM U0126, an inhibitor of extracellular Signal-Regulated Kinase1/2(ERK1/2), or2μM10-DEBC, an inhibitor of phosphatidylinositol3-kinase/protein kinase B (PI3K/Akt), both significantly reduced17β-estradiol-induced phosphorylation of mTOR. Rapamycin also inhibited17β-estradiol-induced phosphorylation of retinoblastoma protein (Rb) and early mitotic inhibitor1(Emil) protein expression.Conclusions:This study indicates that17β-estradiol exerts an influence on the expression of Skp2、PCNA、Cyclin D1and Cyclin E1by activating mTOR via ERK1/2and PI3K/Akt. Activation of mTOR in turn activates phosphorylation of Rb and the expression of Emil, which alters the transcriptional and post-translational regulation of Skp2and promotes the proliferation of Sertoli cells.