Differential effects of epidermal growth factor, somatomedin-C/insulin-like growth factor I, and transforming growth factor-beta 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 less than 0.01). Sm-C (10 ng/ml) and TGFb (1 ng/ml) both enhanced EGF-stimulated [3H]thymidine incorporation (56% and 300%, respectively; P less than 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 less than 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 less than 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.(ABSTRACT TRUNCATED AT 400 WORDS)