Effects of Fibroblast and Epidermal Growth Factors on Ovarian Cell Proliferationin Vitro. I. Characterization of the Response of Granulosa Cells to FGF and EGF

Despite numerous studies on the effects of gonadotropins on ovarian cells in tissue culture, the factors controlling the proliferation of granulosa cells in vitro remain unknown. We have examined the effect of fibroblast growth factor (FGF) and epidermal growth factor (EGF) on granulosa cell proliferation in vitro in an attempt to clarify their possible roles in the control of ovarian development. FGF and EGF both stimulate DNA synthesis in resting populations of granulosa cells. The halfmaximal response for this effect with FGF was observedat4 x lO-11MandwithEGFat 1.5 x 10-13M. Autoradiography demonstrated that the whole cell population initiated DNA synthesis in the presence of either EGF or FGF, thus precluding an additive effect of the two mitogens. When cells were maintained at low density (100 cells/cm2) in the presence of low serum (1%) they divided with a doubling time of 48-72 h, but addition of either EGF or FGF accelerated their proliferation. The doubling time observed in the presence of FGF was 16 h versus 20 h with EGF and the final cell density reached in the presence of EGF or FGF was 20 times that of cells maintained in the presence of 1% calf serum alone. In the presence of 10% serum, granulosa cells had a doubling time of 24 h and the final density reached was similar to that observed in 1% serum with EGF and FGF. Addition of EGF or FGF to 10% serum resulted in a final density 3 to 4-fold higher than that observed with 10% serum alone. The ultrastructure of the granulosa cells grown in the presence of EGF or FGF was similar to that of cells maintained in the absence of added mitogens. The only marked difference was that cells grown in the presence of FGF or EGF had a high lipid granule content while cells grown in their absence had a low lipid granule content. The effect of various concentrations of FGF and EGF on the proliferation of granulosa cells has been analyzed. The minimal effective dose of EGF was 3 x 10-14M and saturation was observed at 3 x 10-11M, with a half-maximal response at 6 x 10-13M. With FGF the minimal dose stimulating proliferation was 1.5 x 10-12M and saturation was achieved at 1.5 x 10-10M, with a half-maximal response at 3 x 10-11M. Our results show that EGF and FGF are the most potent mitogens ever observed and are mitogenic for granulosa cells at 300 to 3000-fold lower concentrations than for other cell types which have been studied, such as fibroblasts or lens epithelial cells.