Leukemia inhibitory factor antagonizes gonadotropin induced-testosterone synthesis in cultured porcine Leydig cells: Sites of action

In the present report, the action of leukemia inhibitory factor (LIF) on te sticular steroid hormone formation was studied. For this purpose, the direc t effects of LIF were evaluated on basal and human (h)CG-stimulated testost erone synthesis by cultured, purified Leydig cells isolated from porcine te stes. LIF reduced (more than 60%) hCG-stimulated testosterone synthesis. Th is inhibitory effect was exerted in a dose- and time-dependent manner. The maximal and half-maximal effects were obtained with, respectively, 10 ng/ml (0.5 nM) and 2.5 ng/ml (0.125 nM) of LIF after a 48-h treatment of the Ley dig cells. Such an effect of the cytokine was not a cytotoxic effect, becau se it was reversible and Leydig cells recovered most of their steroidogenic activity after the removal of LIF. Considering the sites of action of LIF in inhibiting gonadotropin-stimulated testosterone formation, it was shown that LIF significantly (P < 0.002) reduced, in a comparable range (about 60 % decrease), testosterone synthesis stimulated with LH/hCG or with pharmaco logical agents that enhance cAMP levels (cholera toxin, forskolin, and PG E 2), and testosterone synthesis stimulated with 8-bromo-cAMP. Such an observ ation indicates that the antigonadotropic action of the cytokine is exerted in a predominant manner at a step (or steps) located beyond cAMP formation . Furthermore, incubation of Leydig cells with 22R-hydroxy-cholesterol (5 m ug/ml, 2 h), a cholesterol substrate derivative that does not need an assis ted process to be delivered to the inner mitochondrial membrane, reversed m ost of the inhibitory effect of LIF on the steroid hormone formation. Such results indicate that LIF acts by reducing cholesterol substrate availabili ty in the mitochondria. Consequently, LIF action was tested on steroidogeni c acute regulatory protein and PER (peripheral benzodiazepine receptor) sho wn to be potentially involved in such a cholesterol transfer. LIF reduced, in a dose- and time-dependent manner, LH/hCG-induced steroidogenic acute re gulatory protein messenger RNA levels. The maximal inhibitory effect was ob tained with 6.6 ng/ml of LIF after 48 h of treatment. In contrast, LIF had no effect on PER messenger RNA expression or PER binding. This inhibitory e ffect of LIF on Leydig cell steroidogenesis is probably exerted via an auto /paracrine action of the cytokine. Indeed, by immunohistochemistry, LIF and LIF receptor proteins were identified in Leydig and Sertoli cells but not in other testicular cell types, except for LIF receptor in spermatogonia. F urthermore, the presence of LIF and its receptor in Leydig cells at the neo natal and adult periods suggests that the inhibitory effect of LIF on andro gen formation reported here probably occurs in both the fetal and the adult Leydig cell populations during testicular development.