Ovarian actions of tumor necrosis factor-α (TNFα): Pleiotropic effects of TNFα on differentiated functions of untransformed swine granulosa cells

We have examined interactions between tumor necrosis factor-α (TNFα), a product of the immune system, and ovarian cells using serum-free monolayer cultures of untransformed swine granulosa cells. Recombinant human TNFα, a potent cytoactive product of activated macrophages, bound specifically and with high affinity to intact granulosa cells. Binding sites had an apparent K_d of 0.17 nM (95% confidence interval, 0.065-0.31), and a binding capacity of 80 nmol/μg DNA (95% confidence interval, 52-110). The binding capacity of granulosa cells for TNFα (but not the binding affinity) was increased approximately 2-fold by treatment with FSH and insulin. The biological effects of TNFα on pig granulosa cells were expressed after 48 and 96 h in culture. At the latter time, TNFα significantly suppressed insulin- and insulin- plus FSH-stimulated progesterone accumulation, with respective ID₅₀ values of 0.08 ± 0.008 and 0.06 ± 0.014 nM, but did not affect basal progesterone accumulation or DNA content. TNFα also significantly attenuated the stimulatory effect of combined treatment with FSH and insulin on cAMP generation during 48-96 h of culture. TNFα inhibited the stimulatory effects of forskolin, cholera toxin, and the cAMP analog 8-bromo-cAMP on progesterone accumulation, indicating multiple sites of action of this immune modulator. Inhibition of progestin biosynthesis was observed even in the presence of 25-hydroxycholesterol, a soluble oxygenated sterol substrate for the cholesterol side-chain cleavage reaction, and was accompanied by decreased concentrations of specific cellular mRNA encoding cholesterol side-chain cleavage enzyme. There were no changes in the amounts of a constitutively expressed enzyme, phosphoglyceraldehyde dehydrogenase. Inhibitory actions of TNFα were specific to de novo steroid hormone biosynthesis, since nanomolar concentrations of this cytokine stimulated accumulation of prostaglandin E₂ and prostaglandin F_2α basally and during treatment with FSH, cholera toxin, or 8-bromo-cAMP. In contrast, prostaglandin accumulation was not enhanced by interferon-γ or interleukin-2. In summary, untransformed porcine granulosa cells exhibit specific, high affinity, low capacity saturable binding sites for TNFα, and the number of such binding sites can be regulated by combined treatment with insulin and FSH. Granulosa cells are susceptible to the inhibitory actions of TNFα on FSH- and insulin-supported progesterone biosynthesis and cAMP accumulation. One important locus of TNFα action is blockade of hormonally stimulated increases in specific mRNA encoding the cholesterol side-chain cleavage cytochrome P450 enzyme. Suppressive effects of TNFα on steroidogenesis are specific, since they occur concurrently with TNFα's stimulation of prostaglandin E₂ and F_2α accumulation. We conclude that an array of pleiotropic actions of TNFα can be identified in normal ovarian (granulosa) cells.