title: Ex vivo culture of human fetal gonads: manipulation of meiosis signalling by retinoic acid treatment disrupts testis development.
abstract: OBJECTIVE
  What are the effects of experimentally manipulating meiosis signalling by addition of retinoic acid (RA) in cultured human fetal gonads?
  
  
  CONCLUSIONS
  RA-treatment accelerated meiotic entry in cultured fetal ovary samples, while addition of RA resulted in a dysgenetic gonadal phenotype in fetal testis cultures.
  
  
  BACKGROUND
  One of the first manifestations of sex differentiation is the initiation of meiosis in fetal ovaries. In contrast, meiotic entry is actively prevented in the fetal testis at this developmental time-point. It has previously been shown that RA-treatment mediates initiation of meiosis in human fetal ovary ex vivo.
  
  
  METHODS
  This was a controlled ex vivo study of human fetal gonads treated with RA in 'hanging-drop' tissue cultures. The applied experimental set-up preserves germ cell-somatic niche interactions and the investigated outcomes included tissue integrity and morphology, cell proliferation and survival and the expression of markers of meiosis and sex differentiation.
  
  
  METHODS
  Tissue from 24 first trimester human fetuses was included in this study, all from elective terminations at gestational week (GW) 7-12. Gonads were cultured for 2 weeks with and without addition of 1 µM RA. Samples were subsequently formalin-fixed and investigated by immunohistochemistry and cell counting. Proteins investigated and quantified included; octamer-binding transcription factor 4 (OCT4), transcription factor AP-2 gamma (AP2γ) (embryonic germ cell markers), SRY (sex determining region Y)-box 9 (SOX9), anti-Müllerian hormone (AMH) (immature Sertoli cell markers), COUP transcription factor 2 (COUP-TFII) (marker of interstitial cells), forkhead box L2 (FOXL2) (granulosa cell marker), H2A histone family, member X (γH2AX) (meiosis marker), doublesex and mab-3 related transcription factor 1 (DMRT1) (meiosis regulator), cleaved poly ADP ribose polymerase (PARP), cleaved Caspase 3 (apoptosis markers) and Ki-67 antigen (Ki-67) (proliferation marker). Also, proliferation was determined using a 5'-bromo-2'-deoxyuridine (BrdU) incorporation assay.
  
  
  RESULTS
  A novel ex vivo 'hanging-drop' culture model for human fetal gonads was successfully established. Continued proliferation of cells without signs of increased apoptosis was observed after 2 weeks of culture. In cultured fetal ovaries treated with RA, an increased number of meiotic germ cells (P < 0.05) and DMRT1-positive oogonia initiating meiosis (P < 0.05) was observed, which is in agreement with a previous study. In fetal testes, RA-treatment resulted in a decreased number of gonocytes (P < 0.05), a reduced percentage of proliferating gonocytes (P < 0.05), altered expression pattern of the somatic cell markers AMH and COUP-TFII, as well as disrupted seminiferous cord structure and testis morphology.
  
  
  CONCLUSIONS
  The number of samples included in this study was relatively small due to the limited availability of human fetal tissue.
  
  
  CONCLUSIONS
  The hanging-drop culture, similarly to other organ culture approaches, allows studies of germ cell-somatic niche interactions and determination of effects after manipulating specific signalling pathways. Our novel finding of disrupted fetal testis development after treatment with RA indicates that abnormal meiosis regulation can potentially cause gonadal dysgenesis. Further studies will elucidate the exact mechanisms and timing of observed effects.
  
  
  BACKGROUND
  This work was supported in part by an ESPE Research Fellowship, sponsored by Novo Nordisk A/S to A.Jø. Additional funding for this project was obtained from The Research Council of the Capital Region of Denmark (E.R.-D.M.), The Research Fund at Rigshospitalet (A.Ju. and J.E.N.), Familien Erichssens Fund (A.Jø.), Dagmar Marshalls Fund (A.Jø.) and Aase & Ejnar Danielsens Fund (A.Jø.). The authors have no conflicts of interest.
authors.affiliation: University Department of Growth and Reproduction and EDMaRC, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark aj@rh.regionh.dk.
authors.forename: A
authors.initials: A
authors.lastname: Jørgensen
authors.affiliation: University Department of Growth and Reproduction and EDMaRC, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark.
authors.forename: J E
authors.initials: JE
authors.lastname: Nielsen
authors.affiliation: Department of Gynaecology, Rigshospitalet, University of Copenhagen, Blegdamsvej 9, Copenhagen DK-2100, Denmark.
authors.forename: S
authors.initials: S
authors.lastname: Perlman
authors.affiliation: Department of Gynaecology, Rigshospitalet, University of Copenhagen, Blegdamsvej 9, Copenhagen DK-2100, Denmark.
authors.forename: L
authors.initials: L
authors.lastname: Lundvall
authors.affiliation: MRC Centre for Reproductive Health, The University of Edinburgh, The Queen's Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, UK Edinburgh Royal Hospital for Sick Children, 9 Sciennes Road, Edinburgh EH9 1LF, UK.
authors.forename: R T
authors.initials: RT
authors.lastname: Mitchell
authors.affiliation: University Department of Growth and Reproduction and EDMaRC, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark.
authors.forename: A
authors.initials: A
authors.lastname: Juul
authors.affiliation: University Department of Growth and Reproduction and EDMaRC, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark.
authors.forename: E
authors.initials: E
authors.lastname: Rajpert-De Meyts
mesh_terms: D054304:Anti-Mullerian Hormone / Q000378:metabolism; D017209:Apoptosis; D051837:COUP Transcription Factor II / Q000378:metabolism; D049109:Cell Proliferation; D046149:Embryo Culture Techniques; D005260:Female; D005333:Fetus / Q000473:pathology; D005854:Germ Cells / Q000166:cytology; D006107:Granulosa Cells / Q000166:cytology; D006801:Humans; D007150:Immunohistochemistry; D019394:Ki-67 Antigen / Q000378:metabolism; D007985:Leydig Cells / Q000378:metabolism; D008297:Male; D008540:Meiosis / Q000187:drug effects*; D009865:Oocytes / Q000166:cytology; D009867:Oogonia / Q000473:pathology; D009924:Organ Culture Techniques / Q000379:methods*; D010053:Ovary / Q000187:drug effects / Q000196:embryology; D010641:Phenotype; D012733:Sex Differentiation; D015398:Signal Transduction; D013737:Testis / Q000187:drug effects* / Q000196:embryology* / Q000473:pathology; D014157:Transcription Factors / Q000378:metabolism; D014212:Tretinoin / Q000737:chemistry*
chemical_list: D054304:Anti-Mullerian Hormone; D051837:COUP Transcription Factor II; D019394:Ki-67 Antigen; D014157:Transcription Factors; D014212:Tretinoin; C110547:DMRT1 protein; C095569:NR2F2 protein, human
keywords: germ cell differentiation; gonad development; gonadal dysgenesis; human fetal gonad culture; meiosis; retinoic acid