Angiogenesis is a critical event in luteal development and recent evidence suggests that ovarian granulosa cells secrete the angiogenic factor, VEGF, in response to the midcycle gonadotropin surge. This study tested the hypothesis that gonadotropins act directly on primate granulosa cells to initiate and regulate VEGF production. Monkeys received a standard follicular stimulation regimen [i.e., 30 IU r-hFSH for 6 days followed by 3 days of r-hLH + r-hFSH (30 IU each; Ares Serono)] followed by either an ovulatory stimulus (1000 IU r-hCG; n=3) or none (n=3). Nonluteinized granulosa cells (NLGC; i.e., no ovulatory stimulus) and luteinized granulosa cells (LGC; i.e., r-hCG treated) were aspirated from follicles the day after the last treatment. Cells were cultured for 6 days in basal medium alone (DMEM/F12 + 25 fg/ml hLDL) and in the presence of varying dosages of r-hLH and r-hFSH (0.1, 1, 10 and 100 ng/ml) and hCG (100 ng/ml). Medium was changed at 24 hr intervals and frozen until assayed for VEGF by ELISA (R&D Systems, Minneapolis, MN). Basal VEGF production by NLGC on day 1 (mean + SEM; 140 q 34 pg/ml) was 3-fold lower (P<0.05) than LGC (339 q 124) and declined progressively to nondetectable levels (<15 pg) by day 5 of culture, whereas basal production by LGC remained constant through day 6. Higher doses of r-hFSH (10 and 100 ng) increased (P<0.05) VEGF production by NLGC 6-fold on day 1 and 20-fold on day 4 of culture over the day 1 basal level. Similarly, r-hLH (1, 10 and 100 ng) increased (P<0.05) VEGF production by 3, 6 and 7-fold on day 1 and 7, 16 and 14-fold on day 4. In contrast, LGC had less than a 2.5-fold increase over day 1 basal VEGF levels in response to the highest doses of r-hFSH and r-hLH on both day 1 and 4. Comparison of r-hFSH, r-hLH and hCG (100 ng) over time indicated that VEGF production by NLGC increased (P<0.05) through day 6 of culture, with r-hFSH stimulation (+3500 pg/ml over basal) being greater (P<0.05) than r-hLH (+2500 pg/ml) and hCG (+2300 pg/ml). By comparison the 3 gonadotropins caused only a modest increase in VEGF production by LGC over the first 3-4 days of culture (+750 pg/ml over basal) where it remained through day 6. This is the first demonstration of a direct effect of gonadotropins on VEGF production by nonluteinized and luteinized granulosa cells. Further, this study suggests that FSH, as well as LH/CG, can modulate granulosa cell VEGF production in primates.
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