凋亡精子DNA两种染色法的比较

目的:对荧光素Hoechst33342和Hoechst33342/碘化丙啶(PI)两种评价精子质量的染色法进行比较分析。 方法:将已分离提取的精子悬液与DNA拓扑异构酶抑制剂浓度为0、0.15、15μmol/L喜树碱(CAM)或浓度为0.37、 3.7mmol/Lgenistein(GEN)混合后,置37℃孵育4h。通过Hoechst3342单染或Hoechst3342/PI复染,荧光显微镜检 查,对具独立细胞周期的凋亡和坏死精子进行分析,以比较这两种染色方法的效果。　结果:单染法不能区分精子 活率;复染法能鉴定经不同剂量CAM和GEN处理后精子活率下降程度和坏死精子的增加量。CAM和GEN处理可 使凋亡精子数量增多两倍。　结论:精子凋亡和剂量依赖性坏死增多与两种拓扑异构酶抑制剂相关。Ho echst33342/PI复染比Hoechst33342单染法对精子的分析敏感度更高,并可对精子坏死和凋亡作出定量分析。

Comparison between Two Methods for Staining DNA of Apoptotic Spermatozoa

OBJECTIVE: To compare two fluorochrome staining methods for the assessment of sperm quality. METHODS: Washed sperm cells were incubated in 0, 0.15, or 15 micromol/L camptothecin (CAM), or 0.37 or 3.7 mmol/L genistein (GEN) at 37 degrees C for 4 hours. The sperm cells were analyzed for cycle-independent apoptosis and necrosis by single-stain compared with dual-stain fluorescence microscopy to contrast the relative effectiveness of these two approaches. RESULTS: The single-stain procedure could not detect the sperm viability differences. In contrast, the dual-stain procedure identified a dosage-dependent decrease in the viability and increased necrozoospermia after topoisomerase inhibitor CAM and GEN treatments. Apoptosis was 2-fold higher with topoisomerase inhibitor treatment. CONCLUSION: The two topoisomerase inhibitors were associated with increased apoptosis and dosage-dependent necrosis. The data suggested that the dual-stain combination Hoechst 33342/PI was more sensitive than the single Hoechst 33342 stain analysis and permitted quantitative analysis of the apoptosis and necrosis in sperm.