Differential Effects of Epidermal Growth Factor, Somatomedin-C/Insulin-Like Growth Factor I, and Transforming Growth Factor-β on Porcine Granulosa Cell Deoxyribonucleic Acid Synthesis and Cell Proliferation*

Abstract
Recent studies have suggested that the mammalian ovary synthesizes epidermal growth factor (EGF), somatomedin-C/insulin-like growth factor I (Sm-C), and transforming growth factor-.beta. (TGFb) and that these growth factors may in part form a basis for intraovarian regulation of granulosa cell proliferation and differentiation. The studies described herein were initiated to determine to what extent EGF, Sm-C, and TGFb function to regulate DNA synthesis and granulosa cell proliferation during primary monolayer culture. EGF, but neither Sm-C nor TGFb, alone consistently stimulated, in a dose-dependent manner, [3H]thymidine incorporation by porcine granulosa cells under defined conditions (P < 0.01). Sm-C (10 ng/ml) and TGFb (1 ng/ml) both enhanced EGF-stimulated [3H]thymidine incorporation (56% and 300%, respectively; P<0.05). The levels of incorporation obtained with EGF plus TGFb were equal to or greater than those obtained using fetal bovine serum alone. When EGF, Sm-C, and TGFb were combined, [3H]thymidine incorporation was equivalent to that obtained with EGF plus 10% fetal bovine serum, heretofore the most potent stimulatory combination for [3H]thymidine incorporation. Thus, under defined conditions, EGF, Sm-C, and TGFb act synergistically to promote DNA synthesis in primary cultures of porcine granulosa cells. Although DNA synthesis is a requisite step for but is not an accurate measurement of cell proliferation per se, we investigated whether the observed effects of EGF, Sm-C, and TGFb on DNA synthesis were realized in terms of actural cell proliferation. This was accomplished using platelet-poor plasma-derived serum (PPPDS; 0.1-2.5%), which contains reduced levels of endogenous growth factors but not components needed for cell attachment. EGF (P < 0.05), but neither Sm-c nor TGFb, alone consistently stimulated, in a dose-dependent manner, granulosa cell proliferation, an effect directly related to the PPDS concentration. Sm-C consistently and significantly (P < 0.05) enhanced EGF-stimulated cell proliferation in a dose-dependent manner. The facilitative effect of Sm-C was inversely related to PPPDS concentration, ranging from a 76 .+-. 15% increase at 0.1% PPPDS to a 14% increase at 1.0% PPPDS. TGFb exhibited a bifunctional effect on granulosa cell proliferation. At low levels of PPPDS (0.1% and 0.25%) and in the absence of Sm-C, TGFb enhanced EGF-stimulated cell division, and effect which, although small and variable (24 .+-. 16%), was consistent. As a function of increasing concentrations of PPPDS (0.5-2.5%), TGFb increasingly inhibited cell proliferation stimulated by EGF with or without Sm-C (44%; P<0.05). These results suggest that growth control of granulosa cells is multifactorial and that EGF, Sm-C, and TGFb may be important contributors. EGF and Sm-C appear to actively promote DNA synthesis notwithstanding, can either marginally stimulate or markedly inhibit porcine granulosa cell proliferation depending upon the experimental conditions.