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


METHODS
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.


CONCLUSIONS
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.
Shin
So-Young
SY
Infertility Clinic, Department of Obstetrics and Gynecology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea.
Lee
Jin-Young
JY
Lee
EunYoung
E
Choi
Jongyeob
J
Yoon
Byung-Koo
BK
Bae
DukSoo
D
Choi
DooSeok
D
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D018929:Cell Culture Techniques / Q000379:methods; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D015925:Cryopreservation; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D006107:Granulosa Cells / Q000187:drug effects* / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000494:pharmacology; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology*
D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; C467498:vascular endothelial growth factor A, rat