Epigenetic signatures in the ageing male germ cell.

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In western countries there is a strong trend for delaying parenthood. While the consequences of postponed maternity, such as an increased risk for chromosomal anomalies, are well known, our knowledge on male reproductive ageing (i.e. ageing affecting the male germ cells) is scant. There is strong evidence for a significant effect of paternal age on male germ cell function e.g. increase in the prevalence of certain genetic disorders in the offspring. However, we do not know to which extent ageing affects epigenetic signatures, especially DNA methylation, and whether infertile men are at an additive risk for these epimutations. We hypothesize that reproductive ageing affects the epigenetic signatures of male germ cells: spermatogonial stem cells (SSCs) age differently when compared to somatic cells and spermatozoa, deriving from these SSCs, reflect this different ageing process.We will address whether noticeable epigenetic, genetic and cellular changes occur in the germ line of healthy men during ageing and if these dynamics are altered in infertile men. A fully-characterized collection of blood and semen samples from a healthy study men cohort (18-75 years) will constitute a vital tool for studying the effects of ageing. Telomere length (TL) will be measured in blood and sperm DNA of age-matched healthy and infertile men to determine if sperm TL changes with age and to which extent infertility affects ageing. Biological/epigenetic age can be determined by measuring the DNA methylation of distinct CpG sites in the genome. Therefore blood, sperm and spermatogonial DNA from our healthy age groups will be investigated to determine whether the germ line ages in a similar or distinct way from somatic cells. By studying the sperm epigenetic heterogeneity we could deduce an age-dependent change in the clonal origin of sperm and in the cellular SSC composition.At the functional level we will evaluate whether the SSC population itself changes with age. A histomorphometric analysis on fixed testicular tissues delineates the extent to which their stem cell features are affected by age. The clonal expansion of SSC will be studied by immunohistochemistry with stem cell specific and DNA methylation markers. Native SSCs will be isolated from testicular biopsies and characterized by RNA profiling and immunostaining .Using the enriched SSCs we head to whether age-dependent epigenetic changes are restricted to only a few genes or affect multiple regions in the genome. For this we will perform a whole-methylome sequencing in spermatogonial DNA, which will allow us to identify putative candidate loci for age-related methylation changes in the male germ line. This project will greatly increase our knowledge of reproductive ageing in men, and help to identify the risks associated with delayed paternity.