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In vitro evaluation of the anti-apoptotic drug Z-VAD-FMK on human ovarian granulosa cell lines for further use in ovarian tissue transplantation

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Abstract

Purpose

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 % O2) 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)).

Conclusion

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.

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Acknowledgments

The authors acknowledge Erika Konradowski and Nathalie Lefin for their excellent technical assistance. The authors also thank Dr. S. Ormenese and R. Stephan from the GIGA-Imaging and Flow Cytometry facility for their support with FACS analyzes.

M.F. is Televie granted PhD students (F.R.S.-FNRS, Belgium). C.M. is Research Associate from the F.R.S.-FNRS (Belgium). This work was supported by grants from the Fonds de la Recherche Scientifique Médicale, the Fonds de la Recherche Scientifique-FNRS (F.R.S.-FNRS, Belgium), the Foundation against Cancer (foundation of public interest, Belgium), the Fonds spéciaux de la Recherche (University of Liège), the Centre Anticancéreux près l’Université de Liège, the Fonds Léon Fredericq (University of Liège), the Direction Générale Opérationnelle de l’Economie, de l’Emploi et de la Recherche from the S.P.W. (Région Wallonne, Belgium), and the Plan National Cancer (Service Public Fédéral).

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Authors and Affiliations

  1. Laboratory of Tumor and Developmental Biology, GIGA-R, University of Liège, Tour de Pathologie (B23), Sart Tilman, B-4000, Liège, Belgium

    Maïté Fransolet, Laurie Henry, Agnès Noël, Michelle Nisolle & Carine Munaut

  2. Department of Obstetrics and Gynecology, Hôpital de la Citadelle, University of Liège, B-4000, Liège, Belgium

    Laurie Henry, Soraya Labied & Michelle Nisolle

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  1. Maïté Fransolet
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  2. Laurie Henry
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Correspondence to Carine Munaut.

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Capsule

Granulosa cell lines are resistant to low oxygen concentration (1 % O2) and the caspase inhibitor Z-VAD-FMK could ensure follicular maintenance after ovarian tissue auto-transplantation.

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Fig. S1

Doxorubicin and etoposide dose–response curves in granulosa cells. The metabolic activity of GC1a (a) and HGL5 (b) cells treated with the indicated dose of doxorubicin or etoposide for 24 hours. Similar levels of cell death were obtained for identical concentrations of etoposide within the two cell lines. Etoposide at 50 μg/ml induced slightly less than 50 % cell death. (PPTX 111 kb).

Fig. S2

Time- and dose–response curves for anti-apoptotic drugs in granulosa cells. The metabolic activity, measured using the WST-1 method, of GC1a (a) and HGL5 (b) cells treated with the indicated dose of imatinib, nilotinib, sphingosine-1-phosphate and Z-VAD-FMK for 24 and 48 hours. At low (10 μM) and high (50 μM) concentrations, Z-VAD-FMK did not decrease the metabolic activity of the granulosa cells. (PPTX 125 kb).

Fig. S3

Time-response curve of granulosa cell metabolic activity after treatment with etoposide and anti-apoptotic drugs. In combination with etoposide, Z-VAD-FMK preserved a greater percentage of the metabolic activity of GC1a (a) and HGL5 (b) cells. (PPTX 124 kb).

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Fransolet, M., Henry, L., Labied, S. et al. In vitro evaluation of the anti-apoptotic drug Z-VAD-FMK on human ovarian granulosa cell lines for further use in ovarian tissue transplantation. J Assist Reprod Genet 32, 1551–1559 (2015). https://doi.org/10.1007/s10815-015-0536-9

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