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 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)

Effects of transforming growth factors-alpha and -beta on proliferation and apoptosis of rat theca-interstitial cells

“…Most reports focused on granulosa cells, in which effects of TGFs on proliferation seemed to be species-dependent. TGF-(and/or EGF) stimulated proliferation in the majority of studied species including bovine, porcine, and human granulosa cells (Gospodarowicz & Bialecki 1979, May et al 1988, Skinner & Coffey 1988, but had no effect on rat cells (Bendell & Dorrington 1990). In contrast, TGFstimulated proliferation of rat granulosa cells (Dorrington et al 1988), but had no mitogenic activity of cells from cow or pig (May et al 1988, Skinner et al 1987.…”

“…TGF-(and/or EGF) stimulated proliferation in the majority of studied species including bovine, porcine, and human granulosa cells (Gospodarowicz & Bialecki 1979, May et al 1988, Skinner & Coffey 1988, but had no effect on rat cells (Bendell & Dorrington 1990). In contrast, TGFstimulated proliferation of rat granulosa cells (Dorrington et al 1988), but had no mitogenic activity of cells from cow or pig (May et al 1988, Skinner et al 1987. In theca cells, TGF-/EGF stimulated proliferation in hen, cow and sheep (Roberts & Skinner 1991, Onagbesan et al 1994b, Campbell 1999, but had no effect on the cell number in rat (Foghi et al 1997).…”

“…Thus, in bovine granulosa cell cultures, TGF-inhibited stimulatory effect of EGF on growth (Skinner et al 1987) but in porcine granulosa cells TGF-potentiated EGF's stimulatory effect on cell division (May et al 1988). Outgrowth of chicken erythrocytic progenitor cells from various sources invariably required both TGF-and TGF- (Gandrillon et al 1999).…”

Modulatory Role of Epidermal Growth Factor in Follicle-Stimulating Hormone-lnduced DNA Synthesis in Cultured Rat Granulosa Cells.

“…Previous studies have shown that EGF stimulates the proliferation of granulosa cells isolated from man [10], cattle [11,12] and pigs [10,13,14]. However, so far no reports have been found documenting the mitogenic effect of EGF in rat.…”

Expression and actions of transforming growth factors during human follicular development

“…It is mitogenic for GCs in many large mammals (2,3) and promotes growth in long-term cultures of human GC-luteal cells (4). Although promoting GC division, TGF-a attenuates differentiated functions in these cells such as FSH-induced aromatase activity and E 2 synthesis (1,4).…”