Steroidogenesis of Cultured Purified Pig Leydig Cells: Effects of Lipoproteins and Human Chorionic Gonadotropin*

Abstract

Dispersed Leydig cells were prepared from pig testes and purified in a discontinuous Percoll gradient. About 95% of these cells stained for 3β-hydroxysteroid dehydrogenase. The cells were cultured in a chemically defined medium. Testosterone production was low (2 ± 0.4 ng/10⁶ cells/day) under basal conditions, but increased by 8- to 10-fold on the third day of daily human CG (hCG) treatment. Addition to the medium of both human and pig low density lipoprotein (LDL) produced a dramatic increase in both basal (8-fold) and acute hCG-stimulated (5-fold) testosterone production, whereas both human and pig high density lipoprotein were far less effective. Furthermore the effect of lipoproteins was synergistic with that of hCG. The effects of human LDL on both basal and hCG-stimulated testosterone productions were dose-dependent. Maximum effect was achieved at a protein concentration of 10–40 μg/ml with an ED₅₀ of about 4 μg/ml. Three days of pretreatment with hCG or (Bu)₂cAMP alone induced Leydig cell steroidogenic refractoriness to both hCG and (Bu)₂cAMP stimulation. Concomitant treatment with LDL restored the steroidogenic capacity, but only partially. Production of pregnenolone and testosterone of desensitized cells was significantly higher than that of control cells under basal conditions, but was 60% and 40% lower, respectively, after acute hCG stimulation. Moreover, the conversion of exogenous pregnenolone to testosterone by desensitized cells was only 60% of that of control cells. These results show that the de novo synthesis of cholesterol is able to account for only 25% of the maximal steroidogenic capacity of pig Leydig cells and that hCG-induced steroidogenic desensitization is only partially due to cholesterol depletion.