Protective effect of vascular endothelial growth factor (VEGF) in frozen-thawed granulosa cells is mediated by inhibition of apoptosis

ObjectivesThis study was conducted to evaluate the involvement of apoptosis in the freeze-thaw process and to investigate the anti-apoptotic effect of vascular endothelial growth factor (VEGF) in the frozen-thawed granulosa cells.Study designIsolated rat granulosa cells were cultured, frozen-thawed, and were cultured for 24 h. Cell viabilities (by Trypan blue exclusion test) and apoptotic patterns (by Annexin-V/propidium iodide (PI) Double-Staining) were determined at each step. Apoptotic cell death was confirmed by following DNA degradation and caspase-3 activity.ResultsAfter freeze-thaw process and 24 h of culture, reductions in the cellular viabilities and increases in the number of cells containing degraded DNA were lower in the VEGF pretreated group than in the control group (p < 0.05). In the VEGF pretreated group, increases in the proportions of late apoptotic cells [Annexin-V (+)/PI (+)] were significantly lower and caspase-3 expression was prevented immediate after thawing (p < 0.05). Furthermore, increases in the proportions of early apoptotic cells [Annexin-V (+)/PI (−)] and reductions in the proportions of viable cells [Annexin-V (−)/PI (−)] were significantly lower in the VEGF pretreated group after culture for 24 h (p < 0.05). Of the different doses of VEGF pretreated, 50 ng/ml was found to be most effective with respect to protecting frozen-thawed granulosa cells from cryoinjury.ConclusionGranulosa cell damage induced by cryopreservation is mediated, at least in part, by an apoptotic process. Our preliminary results suggest that VEGF treatment before freeze-thaw process reduces rat ovarian granulosa cell damage by inhibiting apoptosis.