title: The production of glial cell line-derived neurotrophic factor by human sertoli cells is substantially reduced in sertoli cell-only testes.
abstract: Do human Sertoli cells in testes that exhibit the Sertoli cell-only (SCO) phenotype produce substantially less glial cell line-derived neurotrophic factor (GDNF) than Sertoli cells in normal testes?
  
  
  
  In human SCO testes, both the amounts of GDNF mRNA per testis and the concentration of GDNF protein per Sertoli cell are markedly reduced as compared to normal testes.
  
  
  
  In vivo, GDNF is required to sustain the numbers and function of mouse spermatogonial stem cells (SSCs) and their immediate progeny, transit-amplifying progenitor spermatogonia. GDNF is expressed in the human testis, and the ligand-binding domain of the GDNF receptor, GFRA1, has been detected on human SSCs. The numbers and/or function of these stem cells are markedly reduced in some infertile men, resulting in the SCO histological phenotype.
  
  
  
  We determined the numbers of human spermatogonia per mm2 of seminiferous tubule surface that express GFRA1 and/or UCHL1, another marker of human SSCs. We measured GFRA1 mRNA expression in order to document the reduced numbers and/or function of SSCs in SCO testes. We quantified GDNF mRNA in testes of humans and mice, a species with GDNF-dependent SSCs. We also compared GDNF mRNA expression in human testes with normal spermatogenesis to that in testes exhibiting the SCO phenotype. As controls, we also measured transcripts encoding two other Sertoli cell products, kit ligand (KITL) and clusterin (CLU). Finally, we compared the amounts of GDNF per Sertoli cell in normal and SCO testes.
  
  
  
  Normal human testes were obtained from beating heart organ donors. Biopsies of testes from men who were infertile due to maturation arrest or the SCO phenotype were obtained as part of standard care during micro-testicular surgical sperm extraction. Cells expressing GFRA1, UCHL1 or both on whole mounts of normal human seminiferous tubules were identified by immunohistochemistry and confocal microscopy and their numbers were determined by image analysis. Human GDNF mRNA and GFRA1 mRNA were quantified by use of digital PCR and Taqman primers. Transcripts encoding mouse GDNF and human KITL, CLU and 18 S rRNA, used for normalization of data, were quantified by use of real-time PCR and Taqman primers. Finally, we used two independent methods, flow cytometric analysis of single cells and ELISA assays of homogenates of whole testis biopsies, to compare amounts of GDNF per Sertoli cell in normal and SCO testes.
  
  
  
  Normal human testes contain a large population of SSCs that express GFRA1, the ligand-binding domain of the GDNF receptor. In human SCO testes, GFRA1 mRNA was detected but at markedly reduced levels. Expression of GDNF mRNA and the amount of GDNF protein per Sertoli cell were also significantly reduced in SCO testes. These results were observed in multiple, independent samples, and the reduced amount of GDNF in Sertoli cells of SCO testes was demonstrated using two different analytical approaches.
  
  
  
  N/A.
  
  
  
  There currently are no approved protocols for the in vivo manipulation of human testis GDNF concentrations. Thus, while our data suggest that insufficient GDNF may be the proximal cause of some cases of human male infertility, our results are correlative in nature.
  
  
  
  We propose that insufficient GDNF expression may contribute to the infertility of some men with an SCO testicular phenotype. If their testes contain some SSCs, an approach that increases their testicular GDNF concentrations might expand stem cell numbers and possibly sperm production.
  
  
  
  This research was funded by the Eunice Kennedy Shriver National Institute for Child Health and Human Development, National Centers for Translational Research in Reproduction and Infertility Program (NCTRI) Grant 1R01HD074542-04, as well as grants R01 HD076412-02 and P01 HD075795-02 and the U.S.-Israel Binational Science Foundation. Support for this research was also provided by NIH P50 HD076210, the Robert Dow Foundation, the Frederick & Theresa Dow Wallace Fund of the New York Community Trust and the Brady Urological Foundation. There are no competing interests.
authors.affiliation: Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, 615N. Wolfe Street, Baltimore, MD 21205, USA.
authors.forename: D
authors.initials: D
authors.lastname: Singh
authors.affiliation: Department of Urology, James Buchanan Brady Foundation, and Cornell Reproductive Medicine Institute, Weill Cornell Medicine, 525 East 68th Street, New York, NY 10065, USA.
authors.forename: D A
authors.initials: DA
authors.lastname: Paduch
authors.affiliation: Department of Urology, James Buchanan Brady Foundation, and Cornell Reproductive Medicine Institute, Weill Cornell Medicine, 525 East 68th Street, New York, NY 10065, USA.
authors.forename: P N
authors.initials: PN
authors.lastname: Schlegel
authors.affiliation: Department of Obstetrics, Gynecology and Reproductive Sciences, Magee-Womens Research Institute, University of Pittsburgh School of Medicine, 204 Craft Avenue, Pittsburgh, PA 15213, USA.
authors.forename: K E
authors.initials: KE
authors.lastname: Orwig
authors.affiliation: Department of Urology, James Buchanan Brady Foundation, and Cornell Reproductive Medicine Institute, Weill Cornell Medicine, 525 East 68th Street, New York, NY 10065, USA.
authors.forename: A
authors.initials: A
authors.lastname: Mielnik
authors.affiliation: Department of Urology, James Buchanan Brady Foundation, and Cornell Reproductive Medicine Institute, Weill Cornell Medicine, 525 East 68th Street, New York, NY 10065, USA.
authors.forename: A
authors.initials: A
authors.lastname: Bolyakov
authors.affiliation: Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, 615N. Wolfe Street, Baltimore, MD 21205, USA.
authors.forename: W W
authors.initials: WW
authors.lastname: Wright
mesh_terms: D000818:Animals; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007248:Infertility, Male / Q000378:metabolism*; D008297:Male; D051379:Mice; D012333:RNA, Messenger; D012708:Sertoli Cells / Q000166:cytology / Q000378:metabolism*; D013093:Spermatogonia / Q000166:cytology / Q000378:metabolism*; D013737:Testis / Q000166:cytology / Q000378:metabolism*; D014746:Vimentin / Q000378:metabolism
chemical_list: D051100:Glial Cell Line-Derived Neurotrophic Factor; D012333:RNA, Messenger; D014746:Vimentin
keywords: GDNF; human; progenitor spermatogonia; spermatogonial stem cells