Vascular Endothelial Growth Factor (VEGF) Receptor 1 (VEGFR-1) and the VEGF Binding Protein (Extracellular Domain of VEGFR-1; s-Flt) are Involved in Regulating Ovarian Availability of VEGF for Folliculogenesis in the Rodent

Abstract

VEGF acting through VEGFR-2 is of critical importance for physiologic folliculogenesis by regulating intraovarian angiogenic events. The role of VEGFR-1 and its binding protein, soluble VEGFR-1, in ovarian folliculogenesis is less understood. This receptor and its binding protein might be involved in the regulation of VEGF availability locally in the ovary and in blood (“decoy function”). We tested for such a possibility by studying the effect of a blocking antibody on ovarian function. This antibody prevents binding of VEGF toVEGFR-1 and the binding protein s-Flt. We hypothesized that this antibody would increase VEGF availability, which in turn would enhance folliculogenesis. Prospective Follicles of interest were stained with an antibody against VEGFR-1 (R&D). Experiment 1: Hypophysectomized (HX) mouse were injected i.p. with an anti-VEGF-R1 blocking antibody (treatment group; N=5) (MF 1, ImClone Systems; 2 mg/animal) or with saline (control group; N=5), and sacrificed 72h later. Angiogenesis and folliculogenesis were evaluated by staining for endothelial cells with PECAM (Pharmingen) and proliferation with BrDU (Zymed), and by counting the total number of the most developed preantral follicles per ovary. Experiment 2: HX mouse were injected with 20 IU of PMSG, and sacrificed 72h later. Treatment group animals received 2 doses of MF1 24h before and 48 after PMSG, while control animals were given saline injections. The total number of preovulatory follicles per ovary was counted, and follicular vasculature was evaluated. VEGF plasma levels were measured in all animals at the time of sacrifice. Endothelial VEGFR-1 staining was present in preantral and antral follicles. Anti-VEGFR-1 antibody increased plasma VEGF levels four-fold from a baseline level of 53±2.1 pg/ml (p<0.01). In the absence of gonadotropin (GT) vascular endowment of the most advanced follicles increased by about 30% when compared to control (p<0.05). Proliferating endothelial cells were more prominent in treatment group follicles (control 1 to 2 cells; treatment 5-7 cells). The total number of advanced preantral follicles increased from 8.5± 1.3 to 12.4±2.1 (p<0.05). No formation of antral follicles was observed in the absence of GT. Treatment with PMSG in the presence of anti-VEGFR-1 antibody increased the number of preovulatory follicles from 9.1±1.5 to 11.8±2.1 (p<0.05). Vascular density and number of proliferating endothelial cells was similar in both groups. Administration of anti-VEGFR-1 antibody increased plasma VEGF levels indicating that this antibody through its action on the extracellular domain of the VGFR-1 molecule augments availability of VEGF in the ovary. Enhanced availability of VEGF locally in the ovary in turn might stimulate follicular angiogenesis, which allows the development of an increased number of advanced preantral follicles, which are responsive to GT stimulation. Enhanced vascular endowment of developing follicles, in turn increases the delivery of substances required for follicle development, which might also help in the maintenance of function. Stimulation of animals exposed to anti-VEGFR-1 antibody with GT allows an increased number of follicles to reach the preovulatory stage. Therefore strategies, which increase intra-ovarian availability of VEGF, might be helpful in increasing the number of follicles responsive to GT treatment.