Isolation of male germ-line stem cells; influence of GDNF.
The identification and physical isolation of testis stem cells, a subset of type A spermatogonia, is critical to our understanding of their growth regulation during the first steps of spermatogenesis. These stem cells remain poorly characterized because of the paucity of specific molecular markers that permit us to distinguish them from other germ cells. Thus, the molecular mechanisms driving the first steps of spermatogenesis are still unknown. We show in the present study that GFR alpha-1, the receptor for GDNF (glial cell line-derived neurotrophic factor), is strongly expressed by a subset of type A spermatogonia in the basal part of the seminiferous epithelium. Using this characteristic, we devised a method to specifically isolate these GFR alpha-1-positive cells from immature mouse testes. The isolated cells express Ret, a tyrosine kinase transmembrane receptor that mediates the intracellular response to GDNF via GFR alpha-1. After stimulation with rGDNF, the isolated cells proliferated in culture and underwent the first steps of germ cell differentiation. Microarray analysis revealed that GDNF induces the differential expression of a total of 1124 genes. Among the genes upregulated by GDNF were many genes involved in early mammalian development, differentiation, and the cell cycle. This report describes the first isolation of a pure population of GFR alpha-1-positive cells in the testis and identifies signaling pathways that may play a crucial role in maintaining germ-line stem cell proliferation and/or renewal.
Hofmann
Marie-Claude
MC
Department of Biology, The University of Dayton, 300 College Park, Dayton, OH 45469-2320, USA. Marie-Claude.Hofmann@notes.udayton.edu
Braydich-Stolle
Laura
L
Dym
Martin
M
D000818:Animals; D002453:Cell Cycle; D002454:Cell Differentiation; D002455:Cell Division; D002469:Cell Separation; D051100:Glial Cell Line-Derived Neurotrophic Factor; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors; D008297:Male; D051379:Mice; D009414:Nerve Growth Factors / Q000235:genetics* / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000502:physiology*; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013093:Spermatogonia / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D013737:Testis / Q000502:physiology
C495040:Gdnf protein, mouse; C494988:Gfra1 protein, mouse; D051100:Glial Cell Line-Derived Neurotrophic Factor; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors; D009414:Nerve Growth Factors; D011518:Proto-Oncogene Proteins; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases; C494999:Ret protein, mouse