REGULATION OF GRANULOSA-CELL PROLIFERATION - FACILITATIVE ROLES OF PLATELET-DERIVED GROWTH-FACTOR AND LOW-DENSITY-LIPOPROTEIN

Recent studies suggest that epidermal growth factor (EGF) and/or transforming growth factor-α (TGF-α) and insulin-like growth factor-I (IGF-I) act synergistically to promote granulosa cell proliferation in vitro suggesting a similar role in vivo. Using a serum-restricted, monolayer culture system containing very low levels of platelet-poor plasma-derived serum (PPPDS), the faciliatative roles of platelet-derived growth factor (PDGF) and low density lipoprotein (LDL) with respect to growth factor-stimulated granulosa cell proliferation were investigatedIn nutrient medium containing only 0.1% PPPDS, PDGF (1-25 ng/ml) had no effect upon granulosa cell proliferation. When combined with EGF, which alone does not stimulate granulosa cell proliferation, PDGF dose-dependently increased cell proliferation to levels obtained with 10% fetal calf serum (2.4-fold increase relative to controls, P < 0.05). when combined with EGF and IGF-I, a combination which does stimulate mitosis in granulosa cells, PDGF again dose-dependently enhanced proliferation (P < 0.05). The extent of proliferation obtained with EGF + IGF-I + PDGF was consistently greater than that obtained with 10% fetal calf serum (P < 0.05) but significantly less than that obtained with EGF + fetal calf serum, a treatment which stimulates rapid granulosa cell proliferationLDL has been shown to greatly enhance granulosa cell steroidogenesis by providing exogenous cholesterol. However, cho-lesterol is also required for plasma membrane biosynthesis and cell growth. LDL alone, had no effect upon porcine granulosa cell proliferation relative to media controls (0.1% PPPDS) nor did it synergize with any single growth factor to induce mitosisWhen combined with EGF + IGF-I, and EGF + PDGF, but not PDGF + IGF-I, LDL dose-dependently (1-25 μg/ml) enhanced proliferation (P < 0.05) to levels equivalent to that obtained with 10% fetal calf serum. When combined with EGF, IGF-I, and PDGF, LDL at 10 μg/ml enhanced proliferation to an extent equivalent with EGF + fetal calf serum (a 5.4-fold increase relative to media controls). High density lipoprotein did not itself stimulate proliferation nor did it facilitate proliferation mediated by growth factorsWhen maintained in medium alone (0.1% PPPDS), the cell population doubling time was 8.0 ± 0.5 days. In the presence of EGF, IGF-I, PDGF, and LDL (10, 10, and 5 ng/ml, and 10 μg/ml, respectively) the doubling time was reduced to 2.0 ±0.1 days. This was statistically equivalent to that observed with EGF + 10% fetal calf serum (1.9 ± 0.1 days) and 10% follicular fluid from small (1-3 mm) antral follicles (2.5 ± 0.1 days). The population doubling time observed in 10% follicular fluid from large (>6 mm) antral follicles was not statistically different from that obtained with 10% fetal calf serum alone (3.8 ± 0.2 and 3.3 ± 0.2 days, respectively)These results suggest that PDGF and LDL, although not themselves mitogenic, can greatly amplify growth factor-mediated granulosa cell proliferation. Of the growth factors tested, EGF and hence, TGF-α, appear to be the primary mitogenic factors since proliferation is minimal in their absence. The rate of granulosa cell proliferation in vivo may be regulated, in part, by the availability of growth-promoting factors such as EGF/TGF-α, PDGF, IGF-I, and the availability of cholesterol. © 1990 by The Endocrine Society.