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*

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-β (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 EGFstimulated [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 actual 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 PPPDS 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 the 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, an 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 and proliferation, whereas TGFb, its facilitative effect on DNA synthesis notwithstanding, can either marginally stimulate or markedly inhibit porcine granulosa cell proliferation depending upon the experimental conditions. (Endocrinology123: 168–179,1988)