Dual fluorescence analysis of DNA apoptosis in sperm

OBJECTIVE: The objective was to compare fluorochrome Hoechst 33342 (Ho342) with combined Ho342/propidium iodide (PI) stains for assessment of sperm quality. STUDY DESIGN: Washed donor sperm cells were incubated in either 0, 0.15, or 15 mmol/L camptothecin (CAM) or 0.37 or 3.7 mmol/L genistein (GEN) for 4 hours at 37C. The sperm cells were analyzed for cycle-independent apoptosis and necrosis by single- compared with dual-stained fluorescence microscopy to contrast the relative effectiveness of these two approaches. RESULTS: The single-stain procedure did not detect viability differences (overall 76.1% 2.2% live). In contrast, the dual-stain procedure identified a dose-dependent decrease in viability and increased necrozoospermia for CAM and GEN treatments. Apoptosis was 2-fold higher with topoisomerase inhibitor treatment. CONCLUSION: The two topoisomerase inhibitors were associated with increased apoptosis and dose-dependent necrosis. The data suggested that the dual-stain combination Ho342/PI was more sensitive than the single Ho342 stain analysis and permitted quantifying the apoptosis and necrosis events in sperm.