Correlation Between Sperm Membrane Destabilization by Heparin and Aniline Blue Staining as Membrane Integrity Index

Abstract

Acidic aniline blue stain (AAB) was studied in relation to sperm membrane destabilization and nuclei decondensation by heparin. Untreated spermatozoa smears stained with AAB or vital stain shows 28.4% of stained and 71.6% of unstained nuclei. This behavior was also observed when incubation was done in the presence of 5 mM glutathione (GSH) used alone. In the presence of 21.6 μM heparin, staining of sperm cells commenced 10 min after heparin addition and was dependent on the incubation time. During the experiment 12.3% of the total cholesterol content and 20 μg protein/10⁸sperm cells were released. In the presence of 21.6 μM heparin-5 mM/GSH, swelling of sperm nuclei reach 95% after 150 min incubation. When this experiment was run along with AAB, the same average (45%) was seen in the first 30 min, which gives plenty of time to trigger the nuclei's decondensation mechanism. The percentage of stained cells was of 71%, indicating that the histone is not completely replaced, and insuring a positive reaction with AAB stain. It would appear that AAB stain can be used as a membrane integrity index to confirm the destabilization effect of heparin on the sperm membrane.

Key Words:

• aniline blue
• heparin
• sperm
• sperm membrane