SYNERGISTIC ACTIONS OF CYTOKINES AND GROWTH-FACTORS IN ENHANCING PORCINE GRANULOSA-CELL GROWTH

In our studies of the growth-promoting effect of a cytokine, interleukin-1 (IL-1), on cultured porcine granulosa cells, we found that the potency of IL-1 action correlated with the serum concentration in the culture medium and that IL-1 acted synergistically with insulin to increase the number of cells in the presence of low serum concentrations (0.1-1%). With granulosa cells maintained in a quiescent state under serum-free conditions, we therefore examined the effects of combined treatment with IL-1 and peptide growth factors, including insulin, on [H-3]thymidine incorporation by these cells. IL-1 by itself enhanced [H-3]thymidine incorporation in a concentration-dependent manner. Moreover, IL-1 acted synergistically with insulin, epidermal growth factor (EGF), or fibroblast growth factor (FGF) to enhance [H-3]thymidine incorporation. Combinations of maximally effective concentrations of insulin (1-mu-g/ml), EGF (1 ng/ml), or FGF (50 ng/ml) with the maximally effective concentration of IL-1 (10 ng/ml) increased the levels of [H-3]thymidine incorporation to 10-, 22-, and 20-fold, respectively, over the control values. Whereas IL-2 (0.1-100 ng/ml) did not affect [H-3]thymidine incorporation, tumor necrosis factor alpha (TNF-alpha) stimulated [H-3]thymidine incorporation by itself and reproduced the actions of IL-1 to act synergistically with insulin, EGF, or FGF. When IL-1 and TNF-alpha were added together in relatively low concentrations (1 ng/ml each), the combination had synergistic effects in enhancing [H-3]thymidine incorporation. The present study demonstrates that cytokines and peptide growth factors act synergistically to markedly enhance porcine granulosa cell growth in vitro.