Luteinizing hormone, insulin like growth factor-1, and epidermal growth factor stimulate vascular endothelial growth factor production in cultured bubaline granulosa cells

• LH, IGF1 and EGF treatment increased expression of VEGF in the cultured GCs.
• These factors increased PCNA expression and decreased BAX expression in cultured GC.
• To conclude these three factors (a) have cytoprotective effect.
• And (b) stimulate VEGF production from granulosa cells of bubaline ovarian follicles.

The objective of this study was to characterize in vitro expression and secretion of vascular endothelial growth factor (VEGF) in bubaline granulosa cells (GC), grown in serum containing media supplemented with luteinizing hormone (LH), insulin like growth factor-1 (IGF-1), and epidermal growth factor (EGF) at three different doses and time durations. GCs were collected from ovarian follicles of varying diameters [Gp-I (small), 4–6 mm; Gp-II (medium), 7–9 mm; Gp-III (large), 10–13 mm; Gp-IV (pre-ovulatory), >13 mm]. In general, each of the three treatments resulted in a dose as well as time dependent increase in the mRNA expression and secretion of VEGF in the cultured GCs of Gp-IV follicles. These results were well supported by our observations on immunocytochemistry in Gp IV granulosa cell culture (GCC). We also looked into the expression dynamics of an anti-apoptotic factor – proliferating cellular antigen (PCNA) and a pro-apoptotic factor – Bcl-2-associated X protein (BAX) in GCs of Gp IV follicles on treatments with LH, IGF-1, and EGF to evaluate their cytoprotective/anti-apoptotic property. Relative expressions of PCNA and BAX showed a mutually opposite trend with the PCNA expression increasing and BAX expression decreasing with increase in dose and time to reach the zenith (P < 0.05) and nadir (P < 0.05) at the highest dose(s) at the maximum time duration (72 h) for PCNA and BAX respectively on treatment with all the three factors. Thus, it can be concluded that LH, IGF-1, and EGF treatments have a cytoprotective/anti-apoptotic effect and stimulate VEGF production in granulosa cells of bubaline pre-ovulatory follicles.