Vascular endothelial growth Factor A (VEGF): novel survival role in rat ovarian follicular cells.

TESONE M; IRUSTA G; ABRAMOVICH D; PARBORELL F

Pittsburgh, Pennsylvania, eeuu

Congreso; SSR Annual Meeting; 2009

Society for the Study of Reproduction (SSR)

<!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman"; mso-ansi-language:EN-US;} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> VEGF plays a key role in the regulation of angiogenesis in the ovary. This protein and its receptors VEGFR1 and VEGFR2 are expressed in ovarian cells from many species. It has been demonstrated that VEGF is a survival factor in other cell types as endotelial cells, myocytes and neurons. The objective of this study was to determine the in vitro effect of VEGF on proliferation and apoptosis of ovarian follicular cells. Female immature rats were injected with diethylstilbestrol (DES) during 3 days. Isolated early antral follicles were cultured during 24-48 h with FSH (20 ng/ml) or VEGF (50-100 ng/ml, VEGF50 and VEGF100 respectively). Protein extraction was performed to measure PCNA (Proliferating Cell Nuclear Antigen) and caspase-3 levels by Western blot. Follicular DNA was isolated to evaluate apoptotic DNA fragmentation (ladder) in agarose gels. VEGF increased PCNA follicular levels (Basal: 0.38±0.09 vs VEGF100: 1.04±0.22 arbitrary units, p<0.05). FSH and VEGF decreased the p17 active fragment of caspase 3 (Basal: 0.58±0.05; FSH: 0.27±0.05; VEGF50: 0.26±0.04 arbitrary units, p<0.05). Besides, FSH and VEGF significantly decreased the apoptotic DNA fragmentation (Basal: 216.7±17.5; FSH: 107.1±7.2; VEGFA50: 139.5±12.4; VEGFA100:118.0±24.7 arbitrary units). Granulosa cells were isolated by follicular punction and cultured in the presence of FSH (20 ng/ml) and Estradiol (E, 50 ng/ml) with or without VEGF (50-100 ng/ml, VEGF50 andVEGF100 respectively). Cell proliferation was measured by tritiated Thymidine incorporation and apoptosis by Tunel assay. VEGF produced a significant increase in DNA synthesis measured in FSH+E stimulated cell cultures (Basal: 1506±228; FSH+E: 4169±213; FSH+E+VEGF50: 5540±289; FSH+E+VEGF100: 6041±188 cpm/well). The presence of FSH+E in the cultures prevented the spontaneous granulosa cell apoptosis and VEGF amplified this effect (Basal: 68.3±0.9; FSH+E: 50.4±2.3; FSH+E+VEGF50: 33.8±4.8; FSH+E+VEGF100: 39.1±4 % of apoptotic cells). In conclusion, VEGF decrease apoptosis and increase follicular cell proliferation, suggesting a survival role in rat ovarian cells. The mechanism could take place through a direct effect mediated by the VEGF receptors previously described in our laboratory in granulosa and theca cells from early antral follicles. Supported by: UBA, FONCYT, CONICET and Roemmers Foundation.