Fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor A (VEGFA) synergistically promote steroidogenesis and survival of cultured buffalo granulosa cells

The present study investigated the combined effect of fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor A (VEGF-A) on estradiol (E2) secretion and relative abundance of mRNA for aromatase enzyme (CYP19A1), proliferating cell nuclear antigen (PCNA) and BCL-2 associated X protein (BAX) in cultured buffalo granulosa cells (GCs). Follicles were isolated and classified into four groups based on size and E2 concentration in follicular fluid (FF): Small, 4-6 mm diameter, E2 < 0.5 ng/ml; Medium, 7-9 mm, E2 = 0.5-5 ng/ml; Large, 10-13 mm, E2 = 5-40 ng/ml; Preovulatory (PFs), >14 mm, E2 > 180 ng/ml. The GCs of PF were cultured in 24 well cell culture plates and allowed to become 75-80% confluent. Then cultured GCs were treated with FGF2 (200 ng/ml) and VEGF-A (100 ng/ml) separately and in combination for three incubation periods (24, 48 and 72 h). Estradiol secretion was greater in GCs treated with FGF2 + VEGF-A compared to FGF2 or VEGF-A at all incubation periods and was greatest (P < 0.05) at 72 h of incubation. The relative abundance of CYP19A1 and PCNA mRNA were relatively consistent with the amount E2 secretion. In contrast, the relative abundance of Bax mRNA was less in GCs treated with the combination of FGF2 and VEGF-A as compared to either FGF2 or VEGF-A alone and the least concentration (P < 0.05) was at 72 h of incubation. Findings with use of immunocytochemistry of cells treated with these factors were consistent to the relative abundance of mRNA transcript for the factor. The present findings indicate that FGF2 and VEGF-A may function in a synergistic manner to promote steroidogenesis and survival of cultured buffalo GCs.