The sections were stained by IF staining with antibodies to MVH, a germ cell marker. Statistical analysis All data are presented as the meanS.E.M. the tumor necrosis factor-(TNFreduced germ cell apoptosis in the co-cultures of germ cells and Sertoli cells in response to MuV contamination. Local injection of MuV into the testes of mice confirmed the involvement of CXCL10 in germ cell apoptosis (TNF-inhibit steroidogenesis in Leydig cells.8, 9, 10 Moreover, TNF-upregulation induced male germ cells apoptosis in an experimental autoimmune orchitis model.11 We recently demonstrated that this C-X-C motif chemokine ligand 10 (CXCL10) expression is remarkably upregulated in Leydig and Sertoli cells in response to MuV infection,12 but the effect of the increased CXCL10 on testicular function remains unknown. CXCL10 was initially identified as an IFN-and IFN-in the absence of CXCL10 (upper panels) or presence of 5?ng/ml CXCL10 (lower panels) for 24?h. AO/EB answer was added to cultures at a dilution of 1 1:1000. After 1?min, apoptotic cells were stained as orange (arrows) and living cells were stained as green. (b) Dose- and time-dependent effects of CXCL10 on germ cell apoptosis. Germ cells were cultured in the presence of the indicated doses of CXCL10 for 24?h (left panel) or in the presence of 5?ng/ml CXCL10 for specific durations (right panel). Percentages of apoptotic cells were calculated based on AO/EB staining results. At least 500 cells were spontaneously counted. (c) Flow cytometry. Germ cells were cultured in the presence of 5?ng/ml CXCL10 for 24?h. Cells were labeled with Annexin V (AnxV)-FITC for 15?min and analyzed using BD Accuri C6 flow cytometer. Images are the representatives of at least three impartial experiments, scale bar=20?(Physique 4c). However, apoptotic germ cells were significantly increased in the conditional medium from Sertoli cells 24?h after MuV contamination (Physique 4d). Open in a separate window Physique 4 MuV-induced male germ cell apoptosis. (a) MuV-induced apoptosis of male germ cells co-cultured with Sertoli cells. Sertoli and germ cells were isolated from 4-week-old mice and co-cultured at a ratio of 1 1:5 for 24?h. The co-cultures were infected with 1 107?PFU/ml MuV (middle Acrizanib panel). Cells without MuV contamination served as controls (left panel). Apoptotic cells (arrows) were decided using AO/EB staining at 24?h after MuV contamination. Percentages of apoptotic germ cells were calculated based on AO/EB staining (right panel). At least 500 cells were spontaneously counted. (b) Caspase activation. The co-cultures of Sertoli and germ cells were infected as described in a. Germ cells were collected by treatment with hypotonic answer (20?mM Tris-HCl, pH 7.4) for 1?min. Cell lysates were subject to western blot analysis to probe caspases 3 and 8. (c) Apoptosis of male germ cells cultured alone. Male germ cells of 4-week-old mice were cultured in the absence (left panel) and presence (right panel) of MuV for 24?h. Apoptotic germ cells were determined using flow cytometry after labeling cells with AnxV-FITC. (d) Apoptosis of male germ cells in the conditional medium (CM). CM was collected by a centrifugation of the supernatant of Sertoli cells 24?h post MuV infection. Germ cells were cultured in CM for 24?h and apoptotic germ cells were analyzed by flow cytometry. The supernatant of Sertoli cells without MuV contamination served as the control (ctrl). Images are the representatives of at least three experiments. Scale bar=20?production in mouse Sertoli cells and TNF-induces CXCL10 expression,12, 21 we examined the role of autocrine TNF-in inducing CXCL10 expression in Sertoli cells after MuV contamination. Enzyme-linked immunosorbent Acrizanib assay (ELISA) results confirmed that MuV contamination significantly increased the TNF-(Physique 6a, left panel) and CXCL10 (right panel) levels in the co-cultures of WT cells. An inhibitor of TNF-secretion, pomalidomide,24 significantly reduced the TNF-level. TNF-was abolished in TNF-induced CXCL10 production at comparable levels in Sertoli and germ cell co-cultures of WT and TNF-induced germ cell apoptosis and caspase activation in WT and TNF-upregulates CXCL10 production in Sertoli cells in an autocrine manner and CXCL10 induces germ cell apoptosis. Open in a separate window Physique 6 Role of TNF-in inducing CXCL10 expression. (a) MuV-induced TNF-and CXCL10 production. Sertoli and germ cells of 4-week-old WT or TNF-secretion for 24?h. Co-cultures without MuV contamination served as controls (Ctrl). TNF-(left panel) and CXCL10 (right panel) levels in the culture Acrizanib media were measured using ELISA. (b) MuV-induced germ cell apoptosis. Co-cultures were treated as described in a. Apoptotic germ cells were determined based on AO/EB staining and confirmed by determination of caspase cleavages by Western blot analysis. (c) Induction CXCL10 production by recombinant TNF-for 24?h. CXCL10 levels in media were decided using ELISA. (d) TNF-or with TNF-in the presence of Ab-CXCR3 for 24?h. Apoptotic germ cells were decided based on AO/EB staining and caspase activation IL12RB2 was assessed by western blot analysis. Data are the meansS.E.M. of three impartial experiments. and CXCL10 in the MuV-mediated impairment of spermatogenesis. We showed that either mutation of TNF-or CXCL10 significantly increased the seminiferous tubules with elongated spermatids at 2 weeks.