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.


METHODS
Washed donor sperm cells were incubated in either 0, 0.15, or 15 micromol/L camptothecin (CAM) or 0.37 or 3.7 mmol/L genistein (GEN) for 4 hours at 37 degrees C. 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.


CONCLUSIONS
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.
Department of Gynecology and Obstetrics, Loma Linda University School of Medicine, 11370 Anderson Street, Loma Linda, CA 92354, USA.
Sara C
SC
Rowland
John D
JD
Jacobson
William C
WC
Patton
Alan
A
King
Philip J
PJ
Chan
D017209:Apoptosis / Q000187:drug effects; D001562:Benzimidazoles / Q000008:administration & dosage; D002166:Camptothecin / Q000494:pharmacology; D002470:Cell Survival; D004396:Coloring Agents / Q000008:administration & dosage; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000008:administration & dosage; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D008297:Male; D008856:Microscopy, Fluorescence; D009336:Necrosis; D011419:Propidium / Q000008:administration & dosage; D013094:Spermatozoa / Q000187:drug effects / Q000473:pathology / Q000502:physiology*; D013194:Staining and Labeling
D001562:Benzimidazoles; D004396:Coloring Agents; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D011419:Propidium; D019833:Genistein; C017807:bisbenzimide ethoxide trihydrochloride; D002166:Camptothecin