Identification And Characterization Of TSEG-2, A Novel Testis-specific Gene Associated With The Apoptosis Of Spermatogenic Cells

Part 1 Construction of the eukaryotic expression vector of TSEG-2 and its expression within cultured cellsObjective To construct the eukaryotic expression vector of TSEG-2 and explore its expression and function in cultured cells.Methods TSEG-2 gene fragment with restrictive sites Hind III BamH I was cloned from mouse testis cDNA by RT-PCR, and was inserted into eukaryotic expression vector pEGFP-N1. Under the induction of polyethylenimine, the recombinant vector was transfected into cultured spermatocyte GC-2spd cells. The expression of TSEG-2 was observed under a flurosecent microscopy. The cell viabilities of GC-2spd were observed by MTT assay. Cell apoptosis was inspected by AO/EB fluorescent staining, Hoechst 33258 fluorescent staining, and Annexin V-FITC/propidium iodide staining flow cytometry. the mitochondrial membrane potential in TSEG-2-transferred GC-2spd cells was revealed by JC-1 staining flow cytometry.The mRNA levels of Fas, Bcl-2 and Bax were detected by real-time PCR.Results The fusion expression vector pEGFP-TSEG2 was constructed. Forty-eight hours post-transfetion, fusion protein expression was observed in the cytoplasm of cultured cells. Transfection of TSEG-2 into spermatocyte GC-2spd cells, resulted in decrease of cell viabilities by 39.2%(P<0.05), and obvious morphological changes of apoptosis. The apoptosis rates were 28.3%(P<0.05). Downregulation of Fas and Bcl-2, and upregulation of Bax were observed in TSEG-2-transfected cells than those in control group (P<0.05).Conclusions The eukaryotic expression vector of TSEG-2 was successfully constructed, resulting its expression and apoptosis in cultured spermatocytes, which laid the basis for subsequent research of its function at cellular and animal levels. Part 2 Expression of testis specific expressed gene 2 in mouse testicular torsion/detorsion modelObjectives:To explore the expression profiles of testis specific expressed gene 2 (TSEG-2) in mouse testicular torsion/detorsion model. Methods:Thirty six Kunming mice were randomly divided into control (n=6), sham (n=6), and experimental (n=24) groups. In experimental group, the left testes were rotated 720 degree for 2 hrs and detorsed. After 1 day and 7 days of detorsion, the testes were collected for morphological observation via HE and TUNEL staining. The activities of superoxide dismutase (SOD) and malondialdehycle (MDA) were measured by xanthine superoxidase and thiobarbiuric acid, respectively. The localization of TSEG-2 was observed by in situ hybridization. The TSEG-2 expression in testes was measured by real-time quantitative PCR. Results:Compared with the regular seminiferous tubules in control and sham groups, the seminiferous tubules of experimental group were irregular, with sparse structure and obvious apoptosis of spermatogenic cells. After 1 day and 7 days of detorsion, the Johnsen's score was reduced by 23.4% and 64.1% (P<0.01), the SOD activities were decreased by 11.6% and 22.2%(P<0.05), with those of MDA increased by 69.6% and 93.2%(P<0.01). The TSEG-2 mRNA localized at the spermatogonia and spermatocytes of seminiferous tubules. After 1 day and 7 days of detorsion, the TSEG-2 expression was enhanced by 2.2 and 6.6 times, respectively, when compared with control group. Conclusions:The mouse testicular torsion/detorsion model was successfully established. The enhanced TSEG-2 expression may be correlated with impaired anti-oxidative activities and apoptosis of spermatogenic cells. Part 3 Expression pattern of testis-specific expressed gene 2 in cryptorchidism model and its role in apoptosis of spermatogenic cellsIn our previous study, we identified a novel testis-specific expressed gene 2 (TSEG-2) from mouse testis. To further investigate its preliminary function, thirty-five male BALB/C mice (8 weeks old) were divided into cryptorchidism group (n=20), sham group (n=10), and control group (n=5). In cryptorchidism group, the right testes were anchored to the inner lateral abdominal wall. Real-time quantitative PCR was undertaken to detect the expression of TSEG-2 gene. Meanwhile, under the mediation of polyethylenimine (PEI), the recombinant vector pEGFP-TSEG-2 (n=5) or empty vector (mock, n=5) was transfected into the testis of male mice. The transfection efficiencies were measured under a fluorescence microscope. The apoptosis of spermatogenic cells was detected by terminal deoxynuleotidyl-mediated nick end labeling (TUNEL). The results showed that the TSEG-2 transcript was significantly enhanced (P<0.05) and correlated with apoptosis of spermatogenic cells in cryptorchid testes (P<0.05),when compared with sham and control groups. PEI was efficient in mediating transfection of TSEG-2 into seminiferous tubules of testis. One week post-transfection, intratesticular injection of TSEG-2 resulted in increased apoptosis of spermatogenic cells in vivo (P<0.05). These results indicate that TSEG-2 may participate in the apoptosis of spermatogenic cells and the pathogenesis of cryptorchidism.