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

TY - JOUR

T1 - Methylation profiling of human Spermatogonial Stem Cells (HSSCs) at single allele resolution

AU - Fend, Desiree

AU - von Kopylow, Kathrein

AU - Schulze, Wolfgang

AU - Salzbrunn, Andrea

AU - Spiess, Andrej-Nikolai

AU - El Hajj, Nady

AU - Haaf, Thomas

AU - Zechner, Ulrich

AU - Linke, Matthias

N1 - Document Type: Abstract

PY - 2018/2/19

Y1 - 2018/2/19

N2 - Introduction: The characterization of hSSCs is crucial for clinical applicationslike fertility treatment of the male adult or re-initiation of spermatogenesisafter cancer treatments in prepubertal boys. Thus, the determinationof spermatogonial subpopulations based on mRNA and proteinexpression allows the establishment of cell culture conditions to enablereproductive interventions, like transplantation in the testis for the developmentinto mature spermatozoa or in vitro-spermatogenesis. In our previouswork, we identified the membrane protein fibroblast growth factorreceptor 3 (FGFR3) as a specific marker for a subpopulation of undifferentiatedspermatogonia/potential hSSCs that possess self-renewal capacityand 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 DNAmethylation patterns at single DNA molecule resolution after isolation ofdistinct subpopulations of FGFR3+ and FGFR3- spermatogonia from acell suspension of human testicular cells, using the limiting dilution (LD)bisulfite pyrosequencing method.Patients and Methods: Testicular biopsies were obtained from patientswith normal spermatogenesis and patients with meiotic arrest who presentedto the Department of Andrology, University Hospital Hamburg-Eppendorf,Germany, or the Fertility Center Hamburg, Germany. We isolatedsingle cells and pools of 10 cells that were FGFR3+ or FGFR3-. AfterDNA extraction and bisulfite conversion, DNA of single cells and 10-cellpools was diluted to a distinct degree to obtain one DNA molecule per wellfor subsequent PCR reactions (LD method). Here, the distribution of thetarget molecules across the partitions can be seen as a Poisson process. Inthe next step, multiplex-PCR followed by gene-specific single nested-PCRwas performed with primer pairs binding in regulatory regions of potentialhSSC marker genes (FGFR3, L1TD1, PLZF, GFRA1), pluripotency genes(OCT4, NANOG), paternally imprinted genes (GTL2, H19) and maternallyimprinted genes (LIT1, SNRPN, PEG3). For each assay, we also analyzedsperm DNA as well as somatic DNA for comparison of methylationpatterns. Pyrosequencing of amplicons was performed and methylationlevels were analyzed.Results and Conclusions: Our approach allowed us to analyze methylationprofiles 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 genesand control regions of maternally imprinted genes as well as hypermethylationat the promoters of pluripotency genes and control regions of paternallyimprinted genes. These methylation profiles were very similar tothose of mature sperm DNA. Therefore, we speculate that the transcriptionalprogram underlying human spermatogenesis is early primed already at the level of SSCs.

AB - Introduction: The characterization of hSSCs is crucial for clinical applicationslike fertility treatment of the male adult or re-initiation of spermatogenesisafter cancer treatments in prepubertal boys. Thus, the determinationof spermatogonial subpopulations based on mRNA and proteinexpression allows the establishment of cell culture conditions to enablereproductive interventions, like transplantation in the testis for the developmentinto mature spermatozoa or in vitro-spermatogenesis. In our previouswork, we identified the membrane protein fibroblast growth factorreceptor 3 (FGFR3) as a specific marker for a subpopulation of undifferentiatedspermatogonia/potential hSSCs that possess self-renewal capacityand 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 DNAmethylation patterns at single DNA molecule resolution after isolation ofdistinct subpopulations of FGFR3+ and FGFR3- spermatogonia from acell suspension of human testicular cells, using the limiting dilution (LD)bisulfite pyrosequencing method.Patients and Methods: Testicular biopsies were obtained from patientswith normal spermatogenesis and patients with meiotic arrest who presentedto the Department of Andrology, University Hospital Hamburg-Eppendorf,Germany, or the Fertility Center Hamburg, Germany. We isolatedsingle cells and pools of 10 cells that were FGFR3+ or FGFR3-. AfterDNA extraction and bisulfite conversion, DNA of single cells and 10-cellpools was diluted to a distinct degree to obtain one DNA molecule per wellfor subsequent PCR reactions (LD method). Here, the distribution of thetarget molecules across the partitions can be seen as a Poisson process. Inthe next step, multiplex-PCR followed by gene-specific single nested-PCRwas performed with primer pairs binding in regulatory regions of potentialhSSC marker genes (FGFR3, L1TD1, PLZF, GFRA1), pluripotency genes(OCT4, NANOG), paternally imprinted genes (GTL2, H19) and maternallyimprinted genes (LIT1, SNRPN, PEG3). For each assay, we also analyzedsperm DNA as well as somatic DNA for comparison of methylationpatterns. Pyrosequencing of amplicons was performed and methylationlevels were analyzed.Results and Conclusions: Our approach allowed us to analyze methylationprofiles 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 genesand control regions of maternally imprinted genes as well as hypermethylationat the promoters of pluripotency genes and control regions of paternallyimprinted genes. These methylation profiles were very similar tothose of mature sperm DNA. Therefore, we speculate that the transcriptionalprogram underlying human spermatogenesis is early primed already at the level of SSCs.

UR - https://link.springer.com/content/pdf/10.1007%2Fs11825-018-0176-4.pdf

M3 - Conference abstract in journal

VL - 30

SP - 135

JO - MED GENET-BERLIN

JF - MED GENET-BERLIN

SN - 0936-5931

IS - 1

ER -