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.
Rowland
Sara C
SC
Department of Gynecology and Obstetrics, Loma Linda University School of Medicine, 11370 Anderson Street, Loma Linda, CA 92354, USA.
Jacobson
John D
JD
Patton
William C
WC
King
Alan
A
Chan
Philip J
PJ
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