In the present study, we have tested the direct effects of tumor necrosis factor-alpha (TNF-alpha) on basal and human (h)CG-stimulated testosterone secretion by cultured purified Leydig cells isolated from immature porcine testes. TNF-alpha reduced (as much as 90% decrease) hCG-stimulated, but not basal testosterone secretion in a dose- and time-dependent manner. The maximal and half-maximal effects were, respectively, 3.75 ng/ml (2.2 x 10(-10) M) and 0.66 ng/ml (3.9 x 10(-11) M) of TNF-alpha after 48 h treatment. TNF-alpha antagonizes the gonadotropin hormonal action by affecting at least two types of biochemical steps. First, TNF-alpha reduced LH/hCG binding to a maximal decrease of 45% obtained with 2 ng/ml of TNF-alpha after 48 h of treatment. TNF-alpha also inhibited (44% decrease) hCG-stimulated cAMP production in optimal conditions (20 ng/ml, 72 h). Second, TNF-alpha significantly (P less than 0.001) reduced testosterone secretion stimulated with 8-bromo-cAMP (3 x 10(-3) M) in a similar range (86% decrease) to that observed with the gonadotropin. Such an observation indicates that the antigonadotropic action of the cytokine is exerted in a predominant manner at a step(s) located beyond cAMP formation. Furthermore, incubation of Leydig cells with 22R-hydroxycholesterol (5 micrograms/ml, 2 h) reversed most of the inhibitory effect of TNF-alpha on androgen production. Indeed, the TNF-alpha (20 ng/ml, 72 h) inhibitory effect on testosterone production was limited to about 20% (P less than 0.03) in Leydig cells supplied with 22R-hydroxycholesterol. Such a moderate effect of the cytokine in the presence of 22R-hydroxycholesterol compared with that observed when androgen secretion was stimulated with the gonadotropin (up to 90% inhibition) indicate that TNF-alpha acts by dramatically reducing cholesterol substrate availability in the mitochondria. Such an effect of TNF-alpha is directly exerted on Leydig cells since TNF-alpha receptors (dissociation constant approximately 5.4 x 10(-10) M) are present in primary cultures of purified porcine Leydig cells. Together, the present findings show that in Leydig cells TNF-alpha antagonizes the gonadotropin action on testosterone formation predominantly through a decrease in the availability of cholesterol substrate in the mitochondria.