Insulin and insulin-like growth factors (IGFs) promote vascular endothelial growth factor (VEGF) secretion by macaque granulosa cells from preovulatory follicles.

Abstract

Objective: To investigate the role of insulin−related factors in the regulation of VEGF production by nonluteinized granulosa cells from preovulatory follicles in monkeys. Design: Prospective, comparative, experimental study. Materials/Methods: Beginning at menses, female rhesus monkeys received recombinant human gonadotropins [30 IU FSH twice daily for 6 days, followed by 30 IU FSH and 30 IU LH twice daily for 2-3 days; Ares Advanced Technologies, Inc]. Granulosa cells were obtained by follicle aspiration from anesthetized animals the morning after the last FSH + LH treatment (nonluteinized granulosa cells; NLGC). Enriched preparations of NLGCs from individual monkeys were plated on fibronectin at 5 × 104 cells/well in 48-well plates in DMEM-Ham’s F-12 medium containing 5 μg/ml transferrin, 5 ng/ml H2SeO3, 10 μg/ml aprotinin and 25 μg/ml human low-density lipoprotein. Cells were incubated at 37°C in 5% CO2 -95% air environment for 3 days in the presence and absence of 100 ng/ml hCG (CR 123) and 1, 5, 10, 20, 50, 100 ng/ml of r-h IGF-1 or IGF-2 (R&D Systems, Inc.) or r-h Insulin (ICN Biomedicals Inc.). Media were collected daily and assayed for VEGF (Quantikine VEGF ELISA, R&D Systems; validated for macaque, which is identical to human, VEGF). Cells were cultured in triplicate for each treatment group, and each experiment was replicated 3–5 times. Differences in VEGF levels were determined by one-way ANOVA. Results: NLGCs cultured in chemically-defined conditions for 24 hours secreted low, but detectable, levels of VEGF (3–61 pg/ml) into the media. There was a dose-dependent increase in VEGF levels with addition of 1 to 100 ng/ml IGF-1 (48 to 238 pg/ml; p < 0.001), IGF-2 (69 to 140 pg/ml; p < 0.001), or insulin (30 to 135 pg/ml; p < 0.001). Maximal stimulation was observed with ≥ 20 ng/ml IGF-1 or IGF-2 or 100 ng/ml insulin. Exposure to surge levels of gonadotropin (100 ng/ml hCG) also increased (p < 0.001) VEGF levels compared to controls. However, hCG plus 50 ng/ml IGF-1, IGF-2 or insulin further increased VEGF levels compared to hCG alone (449 vs 285; 503 vs 435; 651 vs 278 pg/ml, respectively; p < 0.001 for IGF-1 and insulin). VEGF levels became nondetectable by 2-3 days in the absence of insulin/IGF or hCG. However, the presence both hCG and IGF-1/-2 or insulin, but neither factor alone, sustained VEGF levels at 530 ± 152, 779 ± 369, 509 ± 210 pg/ml respectively. Conclusions: We reported previously that, unlike in other tissues, gonadotropins (LH/CG) and not hypoxia are major regulators of VEGF secretion by macaque NLGCs. The current data are consistent with the concept that IGFs/insulin act in concert with the midcycle gonadotropin surge to promote VEGF production by granulosa cells in the preovulatory follicle. Supported By: SCCPRR HD18185, RR00163 and Fogarty TWO 00668.