Increasing methylation of sperm rDNA and other repetitive elements in the aging male mammalian germline

In somatic cells/tissues, methylation of ribosomal DNA (rDNA) increases with age and age-related pathologies, which has a direct impact on the regulation of nucleolar activity and cellular metabolism. Here, we used bisulfite pyrosequencing and show that methylation of the rDNA transcription unit including upstream control element (UCE), core promoter, 18S and 28S rDNA in human sperm also significantly increases with donor's age. This positive correlation between sperm rDNA methylation and biological age is evolutionarily conserved among mammals with widely different life spans such as humans, marmoset, bovine, and mouse. Similar to the tandemly repeated rDNA, methylation of human alpha-satellite and interspersed LINE1 repeats, marmoset alpha-satellite, bovine alpha- and testis-satellite I, mouse minor and major satellite, and LINE1-T repeats increases in the aging male germline, probably related to their sperm histone packaging. Deep bisulfite sequencing of single rDNA molecules in human sperm revealed that methylation does not only depend on donor's age, but also on sequence context (A vs. G alleles). Both average rDNA methylation of all analyzed DNA molecules and the number of fully (> 50%) methylated alleles, which are thought to be epigenetically silenced, increases with donor's age. All analyzed CpGs in the external transcribed spacer, UCE, and core promoter show comparable age effects. Unlike other epigenetic aging markers, the rDNA clock appears to operate in similar ways in germline and soma in different mammalian species. We propose that sperm rDNA methylation, directly or indirectly, influences nucleolar formation and developmental potential in the early embryo.