AbstractBackground: Macrophages and T lymphocytes have been identified in the regressing corpus luteum, and they are thought to participate in structural luteolysis (destruction and removal of luteal cells). Since these cells produce cytokines such as tumor necrosis factor‐α (TNF‐α) and interferon‐γ (IFN‐γ), we investigated the effects of these two cytokines on death of luteal cells in vitro.Methods: Mouse luteal cells were cultured in serum‐free medium with TNF‐α at 0,500,1,000,3,000, or 5,000 U/ml in the presence or absence of IFN‐γ at 1,000 U/ml for 3 or 6 days. Then, for estimation of the actions of these cytokines on induction of luteal cell death, we determined the number of viable cells, the percentage of fragmented DNA in total DNA extracted from cultured cells, and the percentage of cells with fragmented DNA in their nuclei by the trypan blue exclusion test, the sensitive micromethod for DNA assay, and the in situ DNA 3′ end labeling method, respectively. DNA fragmentation was also analysed by agarose gel electrophoresis, and cultured cells were examined by electron microscopy.Results:On day 3 of culture, IFN‐γ alone at 1,000 U/ml or TNF‐α alone at 500–5,000 U/ml did not decrease the number of viable cells, but a combination of IFN‐γ (1,000 U/ml) and TNF‐α (5,000 U/ml) did. On day 6, IFN‐γ alone at 1,000 U/ml or TNF‐α alone at 500, 1,000 and 3,000 U/ml did not decrease the number of viable cells, whereas TNF‐α alone at 5,000 U/ml did, and combinations of IFN‐γ and TNF‐α at 1,000, 3,000, and 5,000 U/ml decreased the number of viable cells in proportion to the concentration of TNF‐α. On days 3–6 of culture, combinations of IFN‐γ and TNF‐α that decreased the number of viable cells also increased the percentages of fragmented DNA in total DNA of cultured luteal cells and the percentages of luteal cells with fragmented DNA in their nuclei. Agarose gel electrophoresis of fragmented DNA showed a ladder‐like pattern, and electron microscopic examination showed luteal cells with the characteristics of apoptosis.Conclusions: The presence of IFN‐γ modulates the ability of TNF‐α to induce a reduction in the number of viable cells, although TNF‐α alone at high concentrations can