Analysis of the global methylation status of human spermatozoa and its association with the tendency of these cells to enter apoptosis.

The methylation status of human spermatozoa has been examined in relation to the isopycnic density of these cells and their tendency to spontaneously default to an apoptotic state. DNA methylation was evaluated using three independent procedures: high-pressure liquid chromatography, flow cytometry and immunocytochemistry. All three techniques revealed that poor-quality spermatozoa recovered from the low-density region of Percoll gradients were characterised by a global hypermethylation of their DNA. Hypermethylation was visualised with an anti-5-methylcytosine antibody as punctate areas of cross-reactivity randomly distributed throughout the chromatin. Immunocytochemical evidence was also obtained suggesting that the sperm mitochondrial genome exists in a heavily methylated state, as a possible buffer against unscheduled transcription. Defective human spermatozoa were also shown to exhibit a tendency to default to an apoptotic state characterised by an increase in annexin V binding. The measurement of annexin V binding levels in individual sperm populations was found to be highly correlated with sperm vitality (P < 0.001) and the methylation status of their DNA (P < 0.001). We conclude that the generation of defective, apoptotic human spermatozoa is associated with disorders of spermatogenesis that lead to a global hypermethylation of their nuclear DNA.