title: In vitro evaluation of the anti-apoptotic drug Z-VAD-FMK on human ovarian granulosa cell lines for further use in ovarian tissue transplantation.
abstract: OBJECTIVE
  Because ovarian granulosa cells are essential for oocyte survival, we examined three human granulosa cell lines as models to evaluate the ability of the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) to prevent primordial follicle loss after ovarian tissue transplantation.
  
  
  METHODS
  To validate the efficacy of Z-VAD-FMK, three human granulosa cell lines (GC1a, HGL5, COV434) were treated for 48 h with etoposide (50 μg/ml) and/or Z-VAD-FMK (50 μM) under normoxic conditions. To mimic the ischemic phase that occurs after ovarian fragment transplantation, cells were cultured without serum under hypoxia (1 % O(2)) and treated with Z-VAD-FMK. The metabolic activity of the cells was evaluated by WST-1 assay. Cell viability was determined by FACS analyses. The expression of apoptosis-related molecules was assessed by RT-qPCR and Western blot analyses.
  
  
  RESULTS
  Our assessment of metabolic activity and FACS analyses in the normoxic experiments indicate that Z-VAD-FMK protects granulosa cells from etoposide-induced cell death. When cells are exposed to hypoxia and serum starvation, their metabolic activity is reduced. However, Z-VAD-FMK does not provide a protective effect. In the hypoxic experiments, the number of viable cells was not modulated, and we did not observe any modifications in the expressions of apoptosis-related molecules (p53, Bax, Bcl-xl, and poly (ADP-ribose) polymerase (PARP)).
  
  
  CONCLUSIONS
  The death of granulosa cell lines was not induced in our ischemic model. Therefore, a protective effect of Z-VAD-FMK in vitro for further use in ovarian tissue transplantation could not be directly confirmed. It will be of interest to potentially use Z-VAD-FMK in vivo in xenograft models.
authors.affiliation: Laboratory of Tumor and Developmental Biology, GIGA-R, University of Liège, Tour de Pathologie (B23), Sart Tilman, B-4000, Liège, Belgium.
authors.forename: Maïté
authors.initials: M
authors.lastname: Fransolet
authors.affiliation: Laboratory of Tumor and Developmental Biology, GIGA-R, University of Liège, Tour de Pathologie (B23), Sart Tilman, B-4000, Liège, Belgium.|Department of Obstetrics and Gynecology, Hôpital de la Citadelle, University of Liège, B-4000, Liège, Belgium.
authors.forename: Laurie
authors.initials: L
authors.lastname: Henry
authors.affiliation: Department of Obstetrics and Gynecology, Hôpital de la Citadelle, University of Liège, B-4000, Liège, Belgium.
authors.forename: Soraya
authors.initials: S
authors.lastname: Labied
authors.affiliation: Laboratory of Tumor and Developmental Biology, GIGA-R, University of Liège, Tour de Pathologie (B23), Sart Tilman, B-4000, Liège, Belgium.
authors.forename: Agnès
authors.initials: A
authors.lastname: Noël
authors.affiliation: Laboratory of Tumor and Developmental Biology, GIGA-R, University of Liège, Tour de Pathologie (B23), Sart Tilman, B-4000, Liège, Belgium.|Department of Obstetrics and Gynecology, Hôpital de la Citadelle, University of Liège, B-4000, Liège, Belgium.
authors.forename: Michelle
authors.initials: M
authors.lastname: Nisolle
authors.affiliation: Laboratory of Tumor and Developmental Biology, GIGA-R, University of Liège, Tour de Pathologie (B23), Sart Tilman, B-4000, Liège, Belgium. c.munaut@ulg.ac.be.
authors.forename: Carine
authors.identifier: http://orcid.org/0000-0003-4952-9637
authors.initials: C
authors.lastname: Munaut
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D061945:Caspase Inhibitors / Q000494:pharmacology; D015687:Cell Hypoxia / Q000187:drug effects; D002460:Cell Line / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D005047:Etoposide / Q000494:pharmacology; D005260:Female; D006107:Granulosa Cells / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D010053:Ovary / Q000637:transplantation*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism; D051020:bcl-X Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D000590:Amino Acid Chloromethyl Ketones; C494826:BAX protein, human; C494811:BCL2L1 protein, human; D061945:Caspase Inhibitors; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D005047:Etoposide; D011065:Poly(ADP-ribose) Polymerases
keywords: Etoposide; Fertility preservation; Hypoxia; Ovarian granulosa cells; Ovarian transplantation; Z-VAD-FMK