BASIC FIBROBLAST GROWTH-FACTOR ENHANCES TESTOSTERONE SECRETION IN CULTURED PORCINE LEYDIG-CELLS - SITE(S) OF ACTION

The effect and mechanism of action of basic fibroblast growth factor (bFGF) on testicular steroidogenesis were investigated using as a model primary cultures of purified porcine Leydig cells from immature intact animals. Basic FGF increased basal and human chorionic gonadotrophin (hCG)-induced testosterone accumulation (with an ED50 of 0.64 ng/ml bFGF, 35 pM) in the medium following a long-term treatment. The effects of bFGF (10 ng/ml, 72 h) were found at all hCG concentrations tested (0.001-1 ng/ml), the growth factor affecting the maximal steroidogenic capacity of the Leydig cells but not their sensitivity to the gonadotrophin. In this context, we have therefore investigated whether the stimulatory effect of bFGF on testosterone formation was related to an increase of the steroidogenic enzyme activities. The data obtained indicate that the growth factor did not affect the gonadotrophin action on the formation of DELTA5-steroid hormone, namely dehydroepiandrosterone (DHEA) (evaluated in the presence of 10(-5) M WIN 24540, an inhibitor of 3beta-hydroxysteroid dehydrogenase/isomerase). By contrast, bFGF (10 ng/ml, 72 h) was found to increase in a comparable manner the conversion of pregnenolone, DHEA and DELTA4-androstenedione into testosterone, suggesting a stimulatory effect on 17beta-hydroxysteroid dehydrogenase activity. Indeed, bFGF enhanced in a dose-dependent manner (ED50 = 39 pM) this enzyme activity evaluated through the conversion of DELTA4-androstenedione to testosterone. These effects of bFGF on Leydig cell steroidogenic activity are probably exerted through specific membrane bFGF receptors. Scatchard analysis of the binding of bFGF to cultured purified Leydig cells revealed the presence of a high affinity (K(d) = 35.5 pM) binding site system for bFGF. Affinity labelling to these receptors by covalent attachment to I-125-FGF with disuccinimidyl suberate and subsequent electrophoretic analysis of the labelled complexes revealed the specific binding to two predominant molecules of 145 and 125 kDa. In conclusion, the present study demonstrates that bFGF enhanced steroidogenesis probably via an increase of 17beta-hydroxysteroid dehydrogenase activity. Such an effect is likely exerted directly on testicular Leydig cells as shown by the presence of specific membrane bFGF receptors on these testicular cells in primary cultures.