TY - JOUR
T1 - 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
AU - Fend-Guella, Desiree Lucia
AU - Von Kopylow, Kathrein
AU - Spiess, Andrej Nikolai
AU - Schulze, Wolfgang
AU - Salzbrunn, Andrea
AU - Diederich, Stefan
AU - Hajj, Nady El
AU - Haaf, Thomas
AU - Zechner, Ulrich
AU - Linke, Matthias
N1 - Publisher Copyright:
© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
PY - 2019/6/6
Y1 - 2019/6/6
N2 - Human spermatogonial stem cells (hSSCs) have potential in fertility preservation of prepubertal 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 labelled with magnetic beads and isolated in addition to unlabelled SPG (FGFR3−). DNA methylation was assessed by limiting dilution bisulfite pyrosequencing for paternally imprinted (H19 and MEG3), maternally imprinted (KCNQ1OT1, PEG3, and SNRPN), pluripotency (POU5F1/OCT4 and NANOG), and spermatogonial/hSSC marker (FGFR3, GFRA1, PLZF, and L1TD1) genes on either single cells or pools of 10 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, 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 SPG and sperm implies that hSSC regulation and germ cell differentiation do not occur at the DNA methylation level.
AB - Human spermatogonial stem cells (hSSCs) have potential in fertility preservation of prepubertal 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 labelled with magnetic beads and isolated in addition to unlabelled SPG (FGFR3−). DNA methylation was assessed by limiting dilution bisulfite pyrosequencing for paternally imprinted (H19 and MEG3), maternally imprinted (KCNQ1OT1, PEG3, and SNRPN), pluripotency (POU5F1/OCT4 and NANOG), and spermatogonial/hSSC marker (FGFR3, GFRA1, PLZF, and L1TD1) genes on either single cells or pools of 10 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, 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 SPG and sperm implies that hSSC regulation and germ cell differentiation do not occur at the DNA methylation level.
KW - DNA methylation
KW - Epigenetics
KW - FGFR3
KW - Limiting dilution bisulfite pyrosequencing
KW - Single allele
KW - Single cell
KW - Spermatogonia
KW - Spermatogonial stem cells
KW - Testis
UR - http://www.scopus.com/inward/record.url?scp=85067376356&partnerID=8YFLogxK
U2 - 10.1093/molehr/gaz017
DO - 10.1093/molehr/gaz017
M3 - Article
C2 - 30892608
AN - SCOPUS:85067376356
SN - 1360-9947
VL - 25
SP - 283
EP - 294
JO - Molecular Human Reproduction
JF - Molecular Human Reproduction
IS - 6
ER -