Resident ovarian macrophages are recognized as potential regulators of ovarian function, and the majority of evidence suggests that such regulation is mediated through cytokine secretions, particularly interleukin-1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF alpha). In this study we examine the effects of co-cultured macrophages, stimulation and inhibition of the immune system, and IL-1 beta and TNF alpha on gonadotropin-induced progesterone secretion from rat granulosa cells. Granulosa cells were isolated from ovarian follicles of proestrous rats and were cultured for a period of 48 h. Peritoneal macrophages, obtained from untreated rats or from animals pretreated with either thioglycollate (TG) or lipopolysaccharide (LPS), were plated at concentrations of 10(5), 5 x 10(3), or 2 x 10(3) cells/ml. After their adherence to the base of the culture wells, 1 ml of serum-free McCoy's medium containing 3 x 10(5) granulosa cells/ml were added to the macrophages. Gonadotropin-induced progesterone secretion was markedly inhibited in the co-cultures, and the degree of inhibition was dependent on both the number of co-cultured macrophages and whether or not the macrophages had been pre-activated by either TG or LPS. TG-activated macrophages were most potent in this respect. Such effects were not due to cytotoxic effects of the macrophages on granulosa cells as determined by a colorimetric assay for cellular growth and survival. In fact, macrophages increased the number of viable granulosa cells after 48-h culture. Granulosa cells obtained from animals pretreated with LPS showed a reduced ability to respond to ovine LH, although suppression of the immune system with cyclosporin had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)