Insulin-Like Growth Factors (IGF5) as Autocrine/Paracrine Regulators of Granulosa Cell Differentiation and Growth: Studies with a Neutralizing Monoclonal Antibody to IGF-I1

Evidence that granulosa cells secrete and respond to insulin-like growth factors (IGFs) suggests, but does not prove, the importance of IGFs as intraovarian regulators. To further assess the role of these peptides in ovarian function, a neutralizing monoclonal antibody to IGF-I was employed to block the actions of IGFs in porcine follicular fluid and in granulosa cell-conditioned medium. In one series of experiments, granulosa cells from immature porcine follicles were cultured in medium containing porcine follicular fluid that had been charcoal-treated to remove steroids. As noted before, fluid from large follicles (LFF) stimulated progesterone production in a dose-dependent manner. The stimulatory effect of LFF (30% v/v) could be inhibited by greater than 50% by the anti-IGF monoclonal antibody. This inhibitory action was specific for the anti-IGF antibody and could be overcome by the addition of excess exogenous IGFs. In another series of experiments, granulosa cells were made dependent on endogenously produced IGFs by culturing them in a serum-free medium without exogenous growth factors. The effects of follicle-stimulating hormone (FSH), estradiol (E2), growth hormone (GH), and combinations thereof on progesterone production were inhibited by approximately 50% by the anti-IGF antibody. The effects of IGFs on indices of cell growth (judged by the criterion of being inhibited by the anti-IGF antibody) were less dramatic. A modest 18% increase in cell number was observed with FSH and E2 treatment in serum-free medium; this effect was virtually abolished by the antibody. In contrast, cell numbers in cultures with LFF were reduced by only 11% by the antibody, and the stimulatory effect of follicular fluid on 3H-thymidine incorporation was reduced by only 16%. These results suggest that the stimulatory effects of follicular fluid, FSH, estradiol, and GH on progesterone production are mediated at least in part by IGFs. In general, ovarian IGFs appear to be more important to granulosa cell cytodfferentiation than to cell replication. These studies provide the most direct evidence available supporting the concept of IGFs as autocrine/paracrine regulators in the ovary.