Study On The Expression Of SSAT2 In Testis And Its Biological Roles In Leydig Cells

Objective:N1-acetyltransferase Spermidine/spermine 2(SSAT2) was originally identified based on its homology to SSAT1, a protein involved in polyamine catabolism. Although SSAT2 is related to SSAT1 with 46%sequence identity and 64% sequence similarity, SSAT2 dose not appear to play roles in ployamine catabolism. SSAT2 were detectable in a variety of species from bacteria to eukaryotes in all major lineages. In this study, we aimed to investigate the expression of SSAT2 in testis, and the function of SSAT2 in Leydig cells.Methods:The localization of SSAT2 in mice testes was analyzed by immunohistochemistry and immunofluorescence assay. The effects of busulfan on the expression levels of SSAT2 in testes were determined using Real-time PCR and Western blotting. Additionally, TM3 Leydig cells were treated by busulfan in vitro to observe the effect of busulfan on the expression of SSAT2.To investigate the biological function of SSAT2 in vitro, TM3 Leydig cells were transfected with SSAT2 lentiviruses to up-regulate the expression of SSAT2. Subsequently, the cell proliferation and cell cycle of TM3 cells were assayed by MTT and FCM assay, respectively. To analyze the effect of SSAT2 overexpression on testosterone production in TM3 Leydig cells, the cell culture supernates were collected for testosterone testing by ELISA. And the mechanism that SSAT2 involved in testosterone production induced by hCG was analysised by Western blotting.Results:1. The results of immunohistochemistry and immunofluorescence showed that SSAT2 was expressed in Leydig cells and Sertoli cells of testis.2. Real-time PCR and Western blotting results showed that the expression levels of SSAT2 in mouse testis and TM3 Leydig cells were significantly increased after treated with busulfan.3. The SSAT2 overexpression cell model was successfully constructed.The MTT and FCM results showed that SSAT2 overexpression inhibited TM3 Leydig cell proliferation and induced cell cycle arrested in the G0/G1 phase.4. Western blotting results indicated that SSAT2 overexpression affected the production of testosterone through inhibiting the up-regulationof StAR stimulated by hCG.Conclusions:Our study demonstrated that SSAT2 is expressed in mouse testis and this protein was localized within the cytoplasm of TM3 Leydig cells.Busulfan can induce the increase of SSAT2 expression in testis and Leydig cells, suggested that SSAT2 may be involved in male testicular dysfunction.This study revealed that SSAT2 overexpression can inhibit the cell proliferation and affect the cell cycle of TM3 Leydig cells. In addition,SSAT2 overexpression also decreeased testosterone synthesis by inhibiting the up-regulation of StAR induced by hCG.In summary, this study indicated that SSAT2 may be involved in the testicular dysfunction by affecting the biological function of Leydig cells.