Potential Mechanism of Co-cultured Sertolli-Germ Cells Apoptosis Induced by Fluorochloridone

Objective To observe the effects and explore potential mechanism of fluorochloridone (FLC) on co-cultured sertolli-germ cells.

Methods Primary sertolli-germ cells were isolated after two-step enzyme digestion using 0.1% collagenase and 0.1% hyaluronidase from rat testes and co-cultured for 24 h. The co-cultured cells were treated with 0.01, 0.10, and 1.00 μmol/L FLC for another 24 h. Detached germ cells induced by FLC was detected by detached germ cell trial. Cell apoptosis was detected by flow cytometry. Real-time polymerase chain reaction was used to measure the mRNA expression of apoptosis related pathway key genes and p38 MAPK.

Results FLC exposure caused dose-dependent increases of the percentages of detached germ cells up to (238.17±3.78)%, (265.89±9.51)%, and (308.73±17.05)% in the sertolli-germ cell co-cultured systems treated with 0.01, 0.10, and 1.00 μmol/L FLC, respectively. The proportions of early apoptotic cells in above three groups were significantly raised to (10.80±1.86)%, (13.52±0.72)%, and (16.62±0.35)%, respectively, and there were statistical differences compared with the control group (P < 0.05 or P < 0.001). Up-regulation in the expressions of mitochondrial apoptotic pathway related genes including Bax, Cyt-c, Caspase-9, and Caspase-3 were observed at FLC concentrations of 0.10 and 1.00 μmol/L (P < 0.05 or P < 0.001), while the levels of Bcl-2 mRNA expression were decreased (P < 0.05). FLC also raised FasL and p38 MAPK mRNA expression levels at the concentrations of 1.00 μmol/L (P < 0.05).

Conclusion FLC exposure might affect the mRNA expressions of apoptosis related pathway key genes, and the activation of p38 MAPK signaling pathway is probably involved in FLC induced sertolli-germ cell apoptosis.