Role of vascular endothelial growth factor (VEGF) in granulosa cell function: involvement of heterotrimeric G-protein signalling pathways.

Abstract

Vascular Endothelial Growth Factor (VEGF) has been shown to be an absolute requirement for ovarian follicle development. Although VEGF is commonly regarded primarily as an angiogenic factor, granulosa cells are a major site of VEGF synthesis in the follicle and they express VEGF receptors (VEGFR1 and VEGFR2). Further, the development of the dominant follicle is characterised by a substantial increase in granulosa cell expression of VEGF and its receptors. In spite of this, potential non-angiogenic effects of VEGF in these follicles have not been elucidated. The objective of the three studies described in this thesis was to use an in vitro bovine granulosa cell model to investigate the roles of VEGF during development of the dominant follicle. In addition, in light of evidence in other cell types, potential interactions between VEGF signalling and heterotrimeric protein signalling in these follicles were also investigated. In the first study, granulosa cells were obtained from healthy follicles with diameters of 4 to 8 mm (corresponding to just before the selection of a dominant follicle during a follicular wave) or 9 to 14 mm (encompassing all developmental stages of a dominant follicle) and exposed to a range of VEGF concentrations (1 to 100 ng/ml) encompassing concentrations found naturally in bovine dominant follicles. VEGF at 1 ng/ml, but not at higher concentrations (P > 0.1), induced significant proliferation of bovine granulosa cells from 4 to 8 mm follicles (P = 0.024) and increased the proliferative response of these cells to FSH (P = 0.045). VEGF also induced a dose-dependent increase in ERK1/2 activation by granulosa cells from 4 to 8 mm follicles (P < 0.03) but did not have any effect on expression of the steroidogenic enzyme, CYP11A1, by these cells (P > 0.1). VEGF, at a dose of 1 ng/ml (P = 0.003), but not at higher doses (P > 0.1), induced an increase in COX-2 expression by granulosa cells from 9 to 14 mm follicles. In addition, LH stimulation of both ERK phosphorylation (P < 0.05) and COX-2 expression (P < 0.05) in granulosa cells from 9 to 14 mm follicles were prevented (P > 0.1) by specific inhibition of VEGFR2, indicating that VEGF may mediate COX-2 responses to LH in these cells. The second study sought to examine the expression of heterotrimeric G-protein á subunits and PLCâ isoforms by real-time PCR and westen blotting in bovine granulosa cells throughout follicle development to identify specific molecular components of heterotrimeric G-protein pathways that may functionally interact with intracellular VEGF signals. Results showed that GNAS, GNA11 and GNAI2 were all expressed at significantly (P < 0.05) higher levels in granulosa cells of pre-ovulatorysize follicles (10.0 to 13.9 mm) than in cells from smaller follicles (2.0 to 5.9 mm and 6.0 to 9.9 mm). In addition, all PLCB isoforms except PLCB2 were expressed in bovine granulosa cells with PLCB3 being more abundant than PLCB1 and -4. Levels of PLCB3 in granulosa cells from pre-ovulatory-size follicles were much higher (>16-fold; P < 0.005) than in smaller follicles. Immunocytochemical analysis revealed that PLCB3 was located primarily in the cytoplasm, whereas PLCB1 was distributed primarily in the nucleus. These results identified Gs, Gq/11, Gi2 and PLCâ3 as candidates for cross-talk between VEGF and heterotrimeric G-protein signalling during the development of the dominant follicle. The potential involvement of these molecules on VEGF-induced responses in granulosa cells from 9-14 mm follicles was investigated in the third study by determining the effects of specific inhibitors of Gi (pertussis toxin, PTX) or Gq/11 (YM-25489) or PLCB3 siRNAs on VEGF-induced p-ERK. Results showed a 2.3 fold mean increase in p-ERK in response to VEGF in the absence of G protein inhibitors (P < 0.0001) but a VEGF response that was completely or partially abolished, respectively, in the presence of PTX (P > 0.8) or YM-25489 (1.6-fold mean increase relative to untreated controls; P = 0.039). LH induced a 1.6 fold increase in p-ERK1/2 (P < 0.02) and this response was prevented by pre-incubation with PTX (P > 0.4) or YM-25489 (P > 0.5). In contrast, similar EGF-induced phosphorylation of ERK (about 5-fold relative to controls) occurred in the absence (P < 0.003) or presence of PTX (P < 0.003) or YM-25489 (P < 0.003). Transfection of granulosa cells with 3 siRNAs targeting PLCB3 that had been previously validated by western blotting and immunocytochemistry had no effect (P = > 0.7) on phosphorylation of ERK in response to VEGF, LH or EGF in granulosa cells. In conclusion, taken together, these results suggest novel roles of VEGF in stimulating granulosa cell proliferation and expression of COX-2 in bovine dominant follicles and implicate VEGF in synergising and/or mediating the effects of gonadotrophins in these cells. In addition, these results indicate a requirement for Gi2 and Gq/11 in VEGF activation of ERK1/2 and induction of the above responses in granulosa cells.