title: Is the protein expression window during testicular development affected in patients at risk for stem cell loss?
abstract: OBJECTIVE
  Is the protein expression window during testicular development affected in prepubertal patients at risk for stem cell loss?
  
  
  CONCLUSIONS
  Nuclear ubiquitin carboxyl-terminal esterase L1 (UCHL1) expression in Sertoli cells and interstitial expression of inhibin α (INHA), sex-determining region Y-box 9 (SOX9) and steroidogenic acute regulatory protein (STAR) was affected in patients with Klinefelter syndrome.
  
  
  BACKGROUND
  Some patients undergoing testicular tissue banking have already been treated before the testis biopsy is taken. These treatments include chemotherapy or hydroxyurea, which can have an influence on the stem cell number and function. A germinal loss occurs in Klinefelter patients, but its cause is currently unknown.
  
  
  METHODS
  Parrafin-embedded testicular tissue from 5 fetuses, 25 prepubertal patients and 5 adults was used to characterize the spatial and temporal distribution of different testicular marker proteins during testicular development. Expression of the markers was evaluated in germ cells, Sertoli cell and interstitial cells. The integrity of this time window was analyzed in patients at risk for germ cell loss: patients treated with hydroxyurea (n = 7), patients treated with chemotherapy (n = 6) and patients affected by Klinefelter syndrome (n = 5).
  
  
  METHODS
  Immunohistochemistry was performed in normal fetal, prepubertal and adult testicular tissue to set up a timeline for the expression of melanoma antigen family A4 (MAGE-A4), ubiquitin carboxyl-terminal esterase L1 (UCHL1), octamer-binding transcription factor 4 (OCT4), stage-specific embryonic antigen-4 (SSEA4), homeobox protein NANOG, INHA, anti-Müllerian hormone, androgen receptor (AR), SOX9 and STAR. The established timeline was used to evaluate whether the expression of these markers was altered in patients at risk for germ cell loss (patients treated for sickle cell disease (hydroxyurea) or cancer (chemotherapy) and patients with Klinefelter syndrome).
  
  
  RESULTS
  A protein expression timeline was created using different markers expressed in different testicular cell types. Less positive tubules and less positive cells per tubule were observed for MAGE-A4 and UCHL1 expression in the KS compared with the non-treated group (P < 0.01). Higher nuclear UCHL1 Sertoli cell expression was observed in the KS group compared with the non-treated group (P < 0.05). Higher interstitial expression of INHA (P < 0.05), SOX9 (P < 0.01) and STAR (P < 0.05) was observed in KS compared with the non-treated group.
  
  
  CONCLUSIONS
  Important age variations exist in the prepubertal groups. Therefore, data were represented in three age groups. However, owing to the limited access to prepubertal tissue, no statistical comparison was possible between these groups. For the Klinefelter group, tissue was only available from patients older than 12 years.
  
  
  CONCLUSIONS
  The expression timeline can add knowledge to the process of spermatogenesis and be used to evaluate altered protein patterns in patients undergoing potentially gonadotoxic treatments, to monitor spermatogenesis established in vitro and to unravel causes of germ cell loss in Klinefelter patients.
authors.affiliation: Department of Reproduction, Genetics and Regenerative Medicine/Research Laboratory Biology of the Testis, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, Brussels, 1090 Belgium dorien.van.saen@vub.ac.be.
authors.forename: D
authors.initials: D
authors.lastname: Van Saen
authors.affiliation: Department of Reproduction, Genetics and Regenerative Medicine/Research Laboratory Biology of the Testis, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, Brussels, 1090 Belgium.
authors.forename: J
authors.initials: J
authors.lastname: Pino Sánchez
authors.affiliation: Pediatric Hemato-Oncology, Hôpital Universitaire des Enfants Reine Fabiola, Jean-Joseph Crocqlaan 15, Brussels, 1020 Belgium.
authors.forename: A
authors.initials: A
authors.lastname: Ferster
authors.affiliation: Department of Pediatrics, Universitair Ziekenhuis Brussel (UZ Brussel), Vrije Universiteit Brussel (VUB), Laarbeeklaan 101, Brussels, 1090, Belgium.
authors.forename: J
authors.initials: J
authors.lastname: van der Werff ten Bosch
authors.affiliation: Department of Reproduction, Genetics and Regenerative Medicine/Research Laboratory Biology of the Testis, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, Brussels, 1090 Belgium Centre for Reproductive Medicine, Universitair Ziekenhuis Brussel (UZ Brussel), Vrije Universiteit Brussel (VUB), Laarbeeklaan 101, Brussels 1090, Belgium.
authors.forename: H
authors.initials: H
authors.lastname: Tournaye
authors.affiliation: Department of Reproduction, Genetics and Regenerative Medicine/Research Laboratory Biology of the Testis, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, Brussels, 1090 Belgium.
authors.forename: E
authors.initials: E
authors.lastname: Goossens
mesh_terms: D000328:Adult; D005854:Germ Cells / Q000378:metabolism*; D006801:Humans; D007265:Inhibins / Q000378:metabolism; D007713:Klinefelter Syndrome / Q000378:metabolism; D007985:Leydig Cells / Q000378:metabolism; D008297:Male; D010750:Phosphoproteins / Q000378:metabolism; D055758:SOX9 Transcription Factor / Q000378:metabolism; D012708:Sertoli Cells / Q000378:metabolism*; D013091:Spermatogenesis; D013737:Testis / Q000254:growth & development / Q000378:metabolism*; D043222:Ubiquitin Thiolesterase / Q000235:genetics / Q000378:metabolism*; D000098682:Steroidogenic Acute Regulatory Protein
chemical_list: D010750:Phosphoproteins; D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human; C482860:UCHL1 protein, human; C094377:inhibin-alpha subunit; D000098682:Steroidogenic Acute Regulatory Protein; D007265:Inhibins; D043222:Ubiquitin Thiolesterase
keywords: Klinefelter; germ cell markers; gonadotoxic treatment; human; testicular development