Methylation profiling of human Spermatogonial Stem Cells (HSSCs) at single allele resolution

Introduction: The characterization of hSSCs is crucial for clinical applications
like fertility treatment of the male adult or re-initiation of spermatogenesis
after cancer treatments in prepubertal boys. Thus, the determination
of spermatogonial subpopulations based on mRNA and protein
expression allows the establishment of cell culture conditions to enable
reproductive interventions, like transplantation in the testis for the development
into mature spermatozoa or in vitro-spermatogenesis. In our previous
work, we identified the membrane protein fibroblast growth factor
receptor 3 (FGFR3) as a specific marker for a subpopulation of undifferentiated
spermatogonia/potential hSSCs that possess self-renewal capacity
and maintain the supply of stem cells for spermatogenesis. Furthermore,
we established an approach to selectively isolate FGFR3-positive
(FGFR3+) single cells. In this study, we focus on the dynamics of DNA
methylation patterns at single DNA molecule resolution after isolation of
distinct subpopulations of FGFR3+ and FGFR3- spermatogonia from a
cell suspension of human testicular cells, using the limiting dilution (LD)
bisulfite pyrosequencing method.
Patients and Methods: Testicular biopsies were obtained from patients
with normal spermatogenesis and patients with meiotic arrest who presented
to the Department of Andrology, University Hospital Hamburg-Eppendorf,
Germany, or the Fertility Center Hamburg, Germany. We isolated
single cells and pools of 10 cells that were FGFR3+ or FGFR3-. After
DNA extraction and bisulfite conversion, DNA of single cells and 10-cell
pools was diluted to a distinct degree to obtain one DNA molecule per well
for subsequent PCR reactions (LD method). Here, the distribution of the
target molecules across the partitions can be seen as a Poisson process. In
the next step, multiplex-PCR followed by gene-specific single nested-PCR
was performed with primer pairs binding in regulatory regions of potential
hSSC marker genes (FGFR3, L1TD1, PLZF, GFRA1), pluripotency genes
(OCT4, NANOG), paternally imprinted genes (GTL2, H19) and maternally
imprinted genes (LIT1, SNRPN, PEG3). For each assay, we also analyzed
sperm DNA as well as somatic DNA for comparison of methylation
patterns. Pyrosequencing of amplicons was performed and methylation
levels were analyzed.
Results and Conclusions: Our approach allowed us to analyze methylation
profiles of multiple genes in single and pools of 10 FGFR3+ and FGFR3-
spermatogonia at single allele resolution. In both FGFR3+ and FGFR3-
cells, we found hypomethylation at the promoters of hSSC marker genes
and control regions of maternally imprinted genes as well as hypermethylation
at the promoters of pluripotency genes and control regions of paternally
imprinted genes. These methylation profiles were very similar to
those of mature sperm DNA. Therefore, we speculate that the transcriptional
program underlying human spermatogenesis is early primed already at the level of SSCs.