In vitro evaluation of an anti-apoptotic drug, Z-VAD-FMK, for further use in ovarian tissue transplantation

Study question: In a model reproducing early ischemia after ovarian tissue transplantation, does the pan-caspase inhibitor Z-VAD-FMK could prevent granulosa cell apoptosis?
Summary answer: Results obtained with HGL5 granulosa cell line suggest that Z-VAD-FMK is efficient to protect granulosa cells from etoposide or CoCl2 induced apoptosis.
What is known already: Removal, cryopreservation and subsequent graft of ovarian strips after cancer treatment have been successfully used to re-establish female fertility. However, the pregnancy rate after autografting of cryopreserved tissue is about 30%. Indeed, the major problem after transplantation is follicular loss due to ischemic reperfusion injury.
Study design, size, duration: Three human granulosa cell lines (GC1a, HGL5 and COV434) were cultured during 48h with Z-VAD-FMK with or without etoposide to induce apoptosis. To reproduce the ischemic phase of the graft, cells were cultured without serum under reduced O2 (1%) or with CoCl2 (chemical hypoxia).
Participants/materials, setting, methods: Granulosa cells were used as a model since they are essential for oocyte survival. Metabolic cell activity was evaluated by the WST-1 assay. Cell apoptosis was analyzed by flow cytometry after annexin V-FITC and propidium iodide double staining. The mRNA levels and protein expression of apoptotic markers were evaluated using RT-qPCR and western blot analysis.
Main results and the role of chance: Flow cytometry showed that cells co-treated with etoposide and Z-VAD-FMK displayed a higher percentage of viable cells as compared to etoposide alone. When in vivo ischemic stage was mimicked (1% O2), no beneficial effect of the Z-VAD-FMK was detected. However, a significant decrease of the number of early apoptotic cells was evidenced by flow cytometry for HGL5 cells treated with Z-VAD-FMK. RT-qPCR and western blot analysis revealed that apoptotic molecules were not modulated. Metabolic activity of the 3 cell lines was reduced by CoCl2. For HGL5 cells, this decrease was partially reversed by Z-VAD-FMK. The number of viable cells was reduced by CoCl2 in HGL5 cells but Z-VAD-FMK allowed to preserve a similar number of viable and apoptotic cells than in control condition.
Limitations, reasons for caution: In this study we used 3 different cell lines but granulosa cells represent only a part of the cell types present in ovarian tissue biopsies. Experiences on the effect of Z-VAD-FMK on primary culture of granulosa cells have not yet been realized.
Wider implications of the findings: This study suggests that the use of an antiapoptotic drug could be efficient to improve ovarian tissue transplantation outcomes. Ovarian tissue grafting studies using our xenograft murine model will be performed to test the potential efficacy of this drug to improve tissue viability and primordial follicles preservation after transplantation.