Figure 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 1:5 for 24 h. The co-cultures were infected with 1 × 10⁷ PFU/ml MuV (middle panel). Cells without MuV infection served as controls (left panel). Apoptotic cells (arrows) were determined using AO/EB staining at 24 h after MuV infection. 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 solution (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 infection served as the control (ctrl). Images are the representatives of at least three experiments. Scale bar=20 μm. Data are the means±S.E.M. of three experiments. **P<0.01