Epigenetic modification in human male germ line

The purpose of this study was to examine the methylation status of human sperm DNA in relation to the functional competence of these cells. In order to achieve this aim discontinuous Percoll gradient density centrifugation was used to generate sperm populations that were either normal (high-density Percoll fraction) or functionally impaired (low density Percoll fraction). The methylation status of these cells was then examined using HPLC, immunocytochemistry or flow cytometry and ultimately correlated with additional markers reflecting the tendency of these cells to default to an intrinsic apoptotic pathway. The results of this study suggest that the mitochondrial genome is heavily methylated during spermatogenesis possibly as a means of suppressing expression of the paternal mitochondrial genome following fertilization. Extensive methylation of the mitochondrial genome appeared to be a ubiquitous, consistent feature of these cells and was present in both the high and low quality sperm populations. By contrast, the methylation status of the nuclear genome appeared to change dramatically in relation to the functional competence of the spermatozoa, such that defective cells exhibited a statistically significant increase in nuclear DNA methylation. Such hypermethylation of defective cells was confirmed by all 3 of the techniques used in this study (HPLC, immunocytochemistry and flow cytometry). Different patterns of 5-methylcytosine expression were observed sperm nuclei by immunocytochemistry and possible interpretations offered in terms of the packaging of chromosomes into the nucleus during sperm differentiation. Furthermore, the methylation status of these cells was negatively correlated with various aspects of sperm function including sperm motility and the tendency of these cells to become apoptotic as reflected by the expression of activated caspases and Annexin-V binding. In addition the hypermethylation of human spermatozoa was highly correlated with their capacity to bind chromomycin3A, a marker that reflects the efficiency of sperm chromatin protamination. These results clearly suggest that defective human spermatozoa are associated with hypermethylation of their nuclear genome, possibly as a consequence of the defective control of DNA methyltransferase activity during spermiogenesis. These hypermethylated cells are functionally defective and exhibit a tendency to default to an apoptotic cascade that features activation of endogenous caspases, phosphatidylserine exteriorization and DNA damage. These results have important implications for the safety of assisted conception procedures that frequently involve the forced fertilization of oocytes with defective spermatozoa