title: TET enzymes are successively expressed during human spermatogenesis and their expression level is pivotal for male fertility.
abstract: Are ten-eleven-translocation (TET) 1-3 family enzymes involved in human spermatogenesis and do they impact male fertility?
  
  
  
  TET1, TET2 and TET3 are successively expressed at different stages of human spermatogenesis, and their expression levels associate with male fertility.
  
  
  
  Spermatogenesis is a complex cell differentiation process accompanied by a drastic epigenetic remodeling. TET1-3 dioxygenases are essential for active DNA demethylation in the paternal pronucleus and in embryonic stem cells.
  
  
  
  Expression of TET1-3 mRNAs and proteinss and 5-hydroxymethylcytosine (5-hmC) proteins were analyzed in human testis tissues from men with obstructive azoospermia and exhibiting histologically normal spermatogenesis. Ejaculated spermatozoa from normozoospermic healthy volunteers, the 'controls' (TET1: n = 58; TET2-3: n = 63), and subfertile men who participated with their female partners in an ICSI-program, the 'patients' (TET1: n = 66; TET2-3: n = 64), were analyzed concerning the stored TET1-3 mRNAs, and the values were correlated to semen parameters and ICSI-outcomes.
  
  
  
  Testis sections were used for in situ hybridization (ISH) and immunohistochemical (IHC) studies to determine TET1-3 mRNA and protein expression, and for immunofluorescence (IF) detection of 5-hmC. Sperm samples from controls were analyzed by western blot, immunocytochemistry (ICC) and RT-PCR concerning the presence of non-degraded TET1-3 protein and mRNA. Sperm samples from controls and patients were used for quantitative TET1-3 mRNA analyses (reverse transcription-polymerase chain reaction) and for comparative statistical evaluations under consideration of semen parameters and ICSI-outcome (pregnancy).
  
  
  
  During human spermatogenesis TET1-3 proteins are successively expressed: TET2 is expressed in the cytoplasm of late pachytene spermatocytes of Stage V, TET1 starts to be expressed in the nuclei of Step 1 round spermatids at Stage I, and TET3 starts to be expressed in the nuclei of Step 3 round spermatids at Stage III. Five-hmC appears only in Step 5 elongated spermatids. All three TETs are still detectable at the mRNA and protein level in sperm cells in considerable amounts. Control men generally exhibited higher levels of TET1-3 in sperm. TET1- and TET3-mRNA levels in sperm were significantly negatively correlated with age (P = 0.0025 and P = 0.0343) and positively correlated with progressive sperm motility (P = 0.0007 and P = 0.018). All TETs showed a significant association with sperm concentration (P < 0.03). Patients diagnosed with oligozoospermia and/or asthenozoospermia (TET1: n = 35; TET2-3: n = 32) showed significantly reduced TET1-3 in sperm in comparison to controls (P = 0.003, P = 0.041 and P = 0.028), but not compared with normozoospermic patients. Levels of TET3 in sperm was significantly associated with high-fertilization rates (P = 0.009). Concerning ICSI-outcome, the lowest levels of TET1-3 mRNAs in sperm were found in the non-pregnant group. Increased TET2 in sperm was significantly associated with pregnancy (P = 0.006).
  
  
  
  Our results concerning the association of the mRNA level of TETs in ejaculated sperm cells to different fertility parameters are descriptive. Further studies clarifying the reasons for decreased TET1-3 levels in subfertile men and their effect on their sperm methylome are essential.
  
  
  
  The study gives a substantial indication that in human spermiogenesis, an active DNA demethylation process occurs with an involvement of TET enzymes, and that the level of TET1-3 expression is pivotal for male fertility.
  
  
  
  Research grant from the German Research Foundation (DFG) to U.S. (SCHA1531/1-1 and SCHA1531/2-1).
  
  
  
  None.
authors.affiliation: Department of Urology, Pediatric Urology and Andrology, Justus Liebig University Giessen, Rudolf-Buchheim Str. 7, 35392 Giessen, Germany.
authors.forename: Kai
authors.initials: K
authors.lastname: Ni
authors.affiliation: Department of Urology, Pediatric Urology and Andrology, Justus Liebig University Giessen, Rudolf-Buchheim Str. 7, 35392 Giessen, Germany.
authors.forename: Temuujin
authors.initials: T
authors.lastname: Dansranjavin
authors.affiliation: Division of Gynecological Endocrinology and Reproductive Medicine, Department of Gynecology and Obstetrics, Clinical Centre of Ludwig Maximilians University, Campus Grosshadern, Marchioninistr. 15, 81377 Munich, Germany.
authors.forename: Nina
authors.initials: N
authors.lastname: Rogenhofer
authors.affiliation: Department of Urology, Pediatric Urology and Andrology, Justus Liebig University Giessen, Rudolf-Buchheim Str. 7, 35392 Giessen, Germany.
authors.forename: Nihan
authors.initials: N
authors.lastname: Oeztuerk
authors.affiliation: Division of Pulmonary Pharmacotherapy, Universities of Giessen and Marburg Lung Center (UGMLC), Justus Liebig University Giessen, Aulweg 130, 35392 Giessen, Germany.
authors.forename: Johanna
authors.initials: J
authors.lastname: Deuker
authors.affiliation: Institute of Veterinary Anatomy, Histology and Embryology, Justus Liebig University Giessen, Frankfurter Str. 94, 35392 Giessen, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Bergmann
authors.affiliation: Department of Urology, Pediatric Urology and Andrology, Justus Liebig University Giessen, Rudolf-Buchheim Str. 7, 35392 Giessen, Germany.
authors.forename: Hans-Christian
authors.initials: HC
authors.lastname: Schuppe
authors.affiliation: Department of Urology, Pediatric Urology and Andrology, Justus Liebig University Giessen, Rudolf-Buchheim Str. 7, 35392 Giessen, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Wagenlehner
authors.affiliation: Department of Urology, Pediatric Urology and Andrology, Justus Liebig University Giessen, Rudolf-Buchheim Str. 7, 35392 Giessen, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Weidner
authors.affiliation: Department of Urology, Pediatric Urology and Andrology, Justus Liebig University Giessen, Rudolf-Buchheim Str. 7, 35392 Giessen, Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Steger
authors.affiliation: Department of Urology, Pediatric Urology and Andrology, Justus Liebig University Giessen, Rudolf-Buchheim Str. 7, 35392 Giessen, Germany undraga.schagdarsurengin@chiru.med.uni-giessen.de.
authors.forename: Undraga
authors.initials: U
authors.lastname: Schagdarsurengin
mesh_terms: D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D049308:Dioxygenases / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D007248:Infertility, Male / Q000235:genetics*; D008297:Male; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D011247:Pregnancy; D011256:Pregnancy Outcome; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012661:Semen / Q000378:metabolism; D055101:Semen Analysis; D020554:Sperm Injections, Intracytoplasmic; D013091:Spermatogenesis / Q000235:genetics*; D013737:Testis / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D006899:Mixed Function Oxygenases; C462539:TET1 protein, human; C578792:TET3 protein, human; D049308:Dioxygenases; C541081:TET2 protein, human
keywords: 5-hmC; ICSI; human spermatogenesis; male infertility; ten-eleven-translocation proteins