OBJECTIVES

This 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 DESIGN

Isolated rat granulosa cells were cultured, frozen-thawed, and were cultured for 24h. 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.

RESULTS

After freeze-thaw process and 24h 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 24h (p<0.05). Of the different doses of VEGF pretreated, 50ng/ml was found to be most effective with respect to protecting frozen-thawed granulosa cells from cryoinjury.

CONCLUSION

Granulosa 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.