Fig. 7
Effects of MCP-1 on stem Leydig cell proliferation and differentiation in vitro. a: regimen for stem Leydig cell (SLC) proliferation. Seminiferous tubules (STs) were cultured in MCP-1 (1–100 ng/ml) in M199 for 7 d and EdU incorporation for direct proliferation assay was conducted (① step) and the rest STs switched in the Leydig cell (LC) differentiation-inducing medium (DIM) for additional 7 d to induce SLCs to differentiate into LCs to secrete testosterone (T) into medium for indirect proliferation assay (② step) was conducted. b: regimen for SLC differentiation. STs were cultured in MCP-1 (1–100 ng/ml) in DIM for 14 d to induce SLCs to differentiate into LCs to secrete T into medium for differentiation assay was conducted. c and d: immunofluorescent staining of ST cross-section after MCP-1 (0 and 100 ng/ml) treatment from d7 to 14 of culture in DIM for 7 d (in the indirect proliferation assay), respectively. ST cross-sections with CYP11A1 staining (green color, thick arrow) showed the formation of LCs. α-smooth muscle actin (SMA) staining (red color, thin arrow) showed the peritubular myoid cells, which circled the STs. CYP11A1-positive cells were outside the SMA-positive cells, indicating that they were differentiated from SLCs on the surface of the tubules. e and f: immunofluorescent staining for EdU-incorporated SLCs (white arrowhead) on the surface of STs after MCP-1 (0 and 100 ng/ml) treatment from d1 to 7 of culture for 7 d (in the direct proliferation assay), respectively. g: quantitative data of LC number per cross section (for c and d), mean ± SEM, n = 6; h: quantitative data of EdU-positive SLC number per cm2 (for e and f), mean ± SEM, n = 6; i: quantitative data of T levels in indirect proliferation assay in the ② step in a, mean ± SEM, n = 6. j: quantitative data of T levels after 7-d MCP-1(0–100 ng/ml) and/or RS102895 treatment, R = 0.1 μM RS102895 from d7 to 14 of culture. Mean ± SEM, n = 6. The identical letters showed no significant difference between two groups at P < 0.05