Comparison of human and mouse sperm chromatin structure by flow cytometry

Human and mouse sperm nuclei obtained by sonication or mechanical agitation of freshly isolated sperm in the presence of anionic detergent were purified through a sucrose gradient and stained with acridine orange (AO); their fluorescence intensity was measured by flow cytometry. The green fluorescence, characteristic of AO binding to DNA by intercalation, was twice lower per unit of DNA for human sperm nuclei than for human peripheral blood lymphocytes. After extraction of basic proteins with 0.08 N HCl, AO binding to DNA increased 3.2-fold for lymphocytes and only 1.3-fold for sperm indicating that, in contrast to somatic cells, the proteins restricting AO binding to DNA are essentially non-extractable from sperm at that low pH. Treatment of human and mouse nuclei with dithiothreitol (DTT), a sulfhydryl reducing agent, and trypsin, removed constraints responsible for the restriction of AO binding. Specifically, as a result of DTT treatment alone there was up to a 20–30% increase of AO binding; upon subsequent addition of trypsin there was a further rapid rise in AO binding up to a final level of approximately 5 times the original AO binding to isolated sperm nuclei. Electron microscopy of DTT-treated human sperm nuclei showed that the reducing agent caused chromatin decondensation to a level whereby 20–30 Å diameter fibers interconnecting chromatin bodies about 30–75 nm in diameter were revealed. Trypsin digestion in the presence of DTT converted the chromatin bodies into a network of fibrous structures about 150 Å in diameter. Both electron microscopy and flow cytometry demonstrated an extremely large intercellular variation among human sperm nuclei in response to DTT and trypsin treatment indicating heterogeneity of chromatin structure. In contrast, AO staining of mouse sperm nuclei increased homogeneously in response to DTT and trypsin treatment.