title: Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) pre-exposure ensures follicle integrity during in vitro culture of ovarian tissue but not during cryopreservation in the domestic cat model.
abstract: OBJECTIVE
  Temporary and reversible downregulation of metabolism may improve the survival of tissues exposed to non-physiological conditions during transport, in vitro culture, and cryopreservation. The objectives of the study were to (1) optimize the concentration and duration of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP-a mitochondrial uncoupling agent) exposures for biopsies of domestic cat ovarian tissue and (2) examine the effects of FCCP pre-exposures on follicle integrity after tissue culture and/or cryopreservation.
  
  
  METHODS
  Biopsies of cat ovarian tissue were first treated with various concentrations of FCCP (0, 10, 40, or 200 nM) for 10 or 120 min to determine the most suitable pre-exposure conditions. Based on these results, tissues were pre-exposed to 200 nM FCCP for 120 min for the subsequent studies on culture and cryopreservation. In all experiments and for each treatment group, tissue activity and integrity were measured by mitochondrial membrane potential (relative optical density of rhodamine 123 fluorescence), follicular viability (calcein assay), follicular morphology (histology), granulosa cell proliferation (Ki-67 immunostaining), and follicular density.
  
  
  RESULTS
  Ovarian tissues incubated with 200 nM FCCP for 120 min led to the lowest mitochondrial activity (1.17 ± 0.09; P < 0.05) compared to control group (0 nM; 1.30 ± 0.12) while maintaining a constant percentage of viable follicles (75.3 ± 7.8 %) similar to the control group (71.8 ± 11.7 %; P > 0.05). After 2 days of in vitro culture, percentage of viable follicles (78.8 ± 8.9 %) in similar pre-exposure conditions was higher (P < 0.05) than in the absence of FCCP (61.2 ± 12.0 %) with percentages of morphologically normal follicles (57.6 ± 17.3 %) not different from the fresh tissue (70.2 ± 7.1 %; P > 0.05). Interestingly, percentages of cellular proliferation and follicular density were unaltered by the FCCP exposures. Based on the indicators mentioned above, the FCCP-treated tissue fragments did not have a better follicle integrity after freezing and thawing.
  
  
  CONCLUSIONS
  Pre-exposure to 200 nM FCCP during 120 min protects and enhances the follicle integrity in cat ovarian tissue during short-term in vitro culture. However, FCCP does not appear to exert a beneficial or detrimental effect during ovarian tissue cryopreservation.
authors.affiliation: Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, 10330, Thailand.
authors.forename: Nae
authors.initials: N
authors.lastname: Tanpradit
authors.affiliation: Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, 10330, Thailand.
authors.forename: Kaywalee
authors.initials: K
authors.lastname: Chatdarong
authors.affiliation: Smithsonian Conservation Biology Institute, National Zoological Park, Washington, DC, 20008, USA. comizzolip@si.edu.
authors.forename: Pierre
authors.initials: P
authors.lastname: Comizzoli
mesh_terms: D000818:Animals; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000008:administration & dosage*; D002415:Cats; D049109:Cell Proliferation / Q000187:drug effects; D015925:Cryopreservation; D005260:Female; D005615:Freezing; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D006080:Ovarian Follicle / Q000187:drug effects / Q000254:growth & development*; D010053:Ovary / Q000187:drug effects / Q000254:growth & development*; D046509:Tissue Culture Techniques / Q000379:methods
chemical_list: D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone
keywords: Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP); Domestic cat; Follicular viability; In vitro culture; Mitochondrial membrane potential; Ovarian tissue