Effect Of Simazine On SPAG6-mediated AP-1 Transcription Activity On Apoptosis Of Mouse Spermatogenic Cells

Objective:To study the effect of simazine on mouse spermatocytes（GC-2 spd）proliferation and explore the underlying mechanism.Methods:The Spag6 coding region was amplified by PCR based on cDNA from mice testis tissue to construct recombinant plasmid of SPAG6/pEGFP-N₂,the enzyme digestion was used to determine whether the construction was successful.COS-7 cells were co-transfected with SPAG6/pEGFP-N₂、COPS5/pCS3 and pAP1-luc.Effect of SPAG6 on AP-1 transcriptional activity was examined by luciferase reporter assays.COS-7 cells were co-transfected with COPS5/pCS3,c-Jun/pcDNA3 and SPAG6/pEGFP-N₂ expression plasmids.The expression level of the phosphorylated c-Jun protein was examined by Western blot when SPAG6 was overexpressed to investigate the mechanism of SPAG6 on AP-1 transcriptional activity.Simazine was dissolved in 0.5%dimethyl sulfoxide（DMSO）.GC-2 spd cells were cultured with different concentrations of simazine（0、5、25、125μg/ml）for 24,48 and 72 hours and CCK-8 assay was used to estimate cell survival rate.The expression levels of SPAG6,COPS5,phosphorylated c-Jun and Bax in the GC-2 spd cells were examined by Western blot after 24 hours of simazine exposure.Real-time PCR was used to explore the expression levels of pro-apoptotic Bax and anti-apoptotic Bcl-2 mRNA.Results:The recombinant plasmid of SPAG6/p EGFP-N₂ was successfully constructed.Overexpression of SPAG6 repressed AP-1 transcriptional activity and significantly reduced the expression of phosphorylated c-Jun in the COS-7 cells.When exposed to 25and 125μg/ml simazine,the survival rate of GC-2 spd cells and the SPAG6 protein expression level were decreased;the phosphorylated c-Jun protein,Bax mRNA and Bax protein expression levels were increased;however,COPS5 protein and Bcl-2 mRNA remained similar,compared to the control.Conclusion:Simazine has a toxic effect on the proliferation of GC-2 spd cells,and could regulate the expression of AP-1 mediated apoptotic genes via repression of SPAG6 and induce spermatogenic cell apoptosis.