Effect of Heparin-Reduced Glutathione on Hamster Sperm DNA Unpacking and Nuclear Swelling

Abstract

This study examined the kinetics of sperm nuclear decondensation induced by the action of physiological concentrations of heparin and glutathione in hamster sperm nuclei as a chromatin model that contains protamine PI and P2. Sperm suspension was incubated at different temperatures (37, 40, 43, and 46°C) in media, keeping constant the concentration of either heparin or GSH and increasing concentrations of the other reagent. Spermatozoa nuclei without any treatment, incubated for 72 h, appear densely condensed. Swelling of hamster spermatozoa nuclei was observed after 30 min of incubation in the presence of efficient concentrations of heparin-GSH. The extent of this time lag was significantly reduced at higher temperatures. DNA presence was verified by the use of ethidium bromide, acridine orange, and Feulgen stain. Phase-contrast microscopy shows that nuclear decondensation begins at the equatorial levels, with DNA highly condensed at the acrosome pole, and the basal pole as the DNA attachment point. Electron microscopy observations showed that hamster sperm nuclei initiates its decompaction at the peripheral regions and this behavior remains until late stages of decondensation; nevertheless, the chromatin is organized into “hub-like” nuclear bodies that measured 10–100 nm in diameter, joined by a network of chromatin fibers with apparent reduction in number. At the decondensation full stage, the network seems to be wide open with a reduced number of hub-like nuclear bodies present in the interlace. DNA is not organized into topological constrained loop domains and is attached to the basal plate instead of to the nuclear matrix or any other structure.

Key Words:

• acridine orange
• chromatin
• heparin
• histone
• nuclear matrix
• protamine
• reduced glutathione