The DNA methylation profile of human spermatogonia at single-cell- and single-allele-resolution refutes its role in spermatogonial stem cell function and germ cell differentiation.

Human spermatogonial stem cells (hSSCs) have potential in fertility preservation of pre-pubertal boys or in treatment of male adults suffering from meiotic arrest. Prior to therapeutic application, in-vitro propagation of rare hSSCs is mandatory. As the published data points to epigenetic alterations in long-term cell culture of spermatogonia (SPG), an initial characterisation of their DNA methylation state is important. Testicular biopsies from five adult normogonadotropic patients were converted into aggregate-free cell suspensions. FGFR3-positive (FGFR3+) SPG, resembling a very early stem cell state, were labeled with magnetic beads and isolated in addition to unlabeled SPG (FGFR3-). DNA methylation was assessed by limiting dilution bisulfite pyrosequencing for paternally imprinted (H19, MEG3), maternally imprinted (KCNQ1OT1, PEG3, SNRPN), pluripotency (POU5F1/OCT4, NANOG) and spermatogonial/hSSC marker (FGFR3, GFRA1, PLZF, L1TD1) genes on either single cells or pools of ten cells. Both spermatogonial subpopulations exhibited a methylation pattern largely equivalent to sperm, with hypomethylation of hSSC marker and maternally imprinted genes and hypermethylation of pluripotency and paternally imprinted genes. Interestingly, we detected fine differences between the two spermatogonial subpopulations, which were reflected by an inverse methylation pattern of imprinted genes, i.e. decreasing methylation in hypomethylated genes and increasing methylation in hypermethylated genes, respectively, from FGFR3+ through FGFR3- SPG to sperm. Limitations of this study are due to it not being performed on a genome-wide level and being based on previously published regulatory gene regions. However the concordance of DNA methylation between spermatogonia and sperm implies that hSSC regulation and germ cell differentiation does not occur at the DNA methylation level.