title: Single-cell gene expression analysis reveals diversity among human spermatogonia.
abstract: Is the molecular profile of human spermatogonia homogeneous or heterogeneous when analysed at the single-cell level?
  
  
  
  Heterogeneous expression profiles may be a key characteristic of human spermatogonia, supporting the existence of a heterogeneous stem cell population.
  
  
  
  Despite the fact that many studies have sought to identify specific markers for human spermatogonia, the molecular fingerprint of these cells remains hitherto unknown.
  
  
  
  Testicular tissues from patients with spermatogonial arrest (arrest, n = 1) and with qualitatively normal spermatogenesis (normal, n = 7) were selected from a pool of 179 consecutively obtained biopsies. Gene expression analyses of cell populations and single-cells (n = 105) were performed. Two OCT4-positive individual cells were selected for global transcriptional capture using shallow RNA-seq. Finally, expression of four candidate markers was assessed by immunohistochemistry.
  
  
  
  Histological analysis and blood hormone measurements for LH, FSH and testosterone were performed prior to testicular sample selection. Following enzymatic digestion of testicular tissues, differential plating and subsequent micromanipulation of individual cells was employed to enrich and isolate human spermatogonia, respectively. Endpoint analyses were qPCR analysis of cell populations and individual cells, shallow RNA-seq and immunohistochemical analyses.
  
  
  
  Unexpectedly, single-cell expression data from the arrest patient (20 cells) showed heterogeneous expression profiles. Also, from patients with normal spermatogenesis, heterogeneous expression patterns of undifferentiated (OCT4, UTF1 and MAGE A4) and differentiated marker genes (BOLL and PRM2) were obtained within each spermatogonia cluster (13 clusters with 85 cells). Shallow RNA-seq analysis of individual human spermatogonia was validated, and a spermatogonia-specific heterogeneous protein expression of selected candidate markers (DDX5, TSPY1, EEF1A1 and NGN3) was demonstrated.
  
  
  
  The heterogeneity of human spermatogonia at the RNA and protein levels is a snapshot. To further assess the functional meaning of this heterogeneity and the dynamics of stem cell populations, approaches need to be developed to facilitate the repeated analysis of individual cells.
  
  
  
  Our data suggest that heterogeneous expression profiles may be a key characteristic of human spermatogonia, supporting the model of a heterogeneous stem cell population. Future studies will assess the dynamics of spermatogonial populations in fertile and infertile patients.
  
  
  
  RNA-seq data is published in the GEO database: GSE91063.
  
  
  
  This work was supported by the Max Planck Society and the Deutsche Forschungsgemeinschaft DFG-Research Unit FOR 1041 Germ Cell Potential (grant numbers SCHO 340/7-1, SCHL394/11-2). The authors declare that there is no conflict of interest.
authors.affiliation: Centre of Reproductive Medicine and Andrology, University Hospital of Münster, Domagkstrasse 11, 48149 Münster , Germany.
authors.forename: N
authors.initials: N
authors.lastname: Neuhaus
authors.affiliation: Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Röntgenstrasse 20, 48149 Münster , Germany.
authors.forename: J
authors.initials: J
authors.lastname: Yoon
authors.affiliation: Centre of Reproductive Medicine and Andrology, University Hospital of Münster, Domagkstrasse 11, 48149 Münster , Germany.
authors.forename: N
authors.initials: N
authors.lastname: Terwort
authors.affiliation: Department of Clinical Andrology, Centre of Reproductive Medicine and Andrology, University Hospital Münster, Domagkstrasse 11, 48149 Münster , Germany.
authors.forename: S
authors.initials: S
authors.lastname: Kliesch
authors.affiliation: Institute of Human Genetics, University Hospital Münster, Vesaliusweg 12-14, 48149 Münster , Germany.
authors.forename: J
authors.initials: J
authors.lastname: Seggewiss
authors.affiliation: Core Facility Genomik, Medical Faculty of Münster, Domagkstrasse 3, 48149 Münster , Germany.
authors.forename: A
authors.initials: A
authors.lastname: Huge
authors.affiliation: Interdisciplinary Centre for Clinical Research in the Faculty of Medicine, Domagkstrasse 3, 48149 Münster , Germany.
authors.forename: R
authors.initials: R
authors.lastname: Voss
authors.affiliation: Centre of Reproductive Medicine and Andrology, University Hospital of Münster, Domagkstrasse 11, 48149 Münster , Germany.
authors.forename: S
authors.initials: S
authors.lastname: Schlatt
authors.affiliation: University Hospital Zurich, Department of Infectious Diseases and Hospital Epidemiology, 8091 Zurich , Switzerland.
authors.forename: R V
authors.initials: RV
authors.lastname: Grindberg
authors.affiliation: Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Röntgenstrasse 20, 48149 Münster , Germany.
authors.forename: H R
authors.initials: HR
authors.lastname: Schöler
mesh_terms: D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics* / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D002454:Cell Differentiation; D002469:Cell Separation / Q000379:methods; D053487:DEAD-box RNA Helicases / Q000235:genetics* / Q000378:metabolism; D005640:Follicle Stimulating Hormone / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D020869:Gene Expression Profiling; D018740:Genetic Heterogeneity; D006801:Humans; D007150:Immunohistochemistry; D007986:Luteinizing Hormone / Q000235:genetics / Q000378:metabolism; D008297:Male; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D020648:Peptide Elongation Factor 1 / Q000235:genetics* / Q000378:metabolism; D017423:Sequence Analysis, RNA; D059010:Single-Cell Analysis / Q000379:methods*; D013091:Spermatogenesis / Q000235:genetics; D013093:Spermatogonia / Q000166:cytology / Q000378:metabolism*; D013737:Testis / Q000166:cytology / Q000378:metabolism; D013739:Testosterone / Q000235:genetics / Q000378:metabolism; D059467:Transcriptome
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D015415:Biomarkers; D018797:Cell Cycle Proteins; D053487:DEAD-box RNA Helicases; D005640:Follicle Stimulating Hormone; D007986:Luteinizing Hormone; D009419:Nerve Tissue Proteins; D020648:Peptide Elongation Factor 1; D013739:Testosterone; C094814:EEF1A1 protein, human; C498695:NEUROG3 protein, human; C497749:TSPY1 protein, human; C506749:Ddx5 protein, human
keywords: human testis; micromanipulation; single-cell expression; spermatogonia; stem cell heterogeneity