Expression of cytochrome P-45017α, 3β-hydroxysteroid dehydrogenase/Δ5 → 4-isomerase, and steroid 5α-reductase in rat H540 Leydig tumor cells

The rat H540 Leydig tumor cell is established as a model for acute lutropin action on the initial step of steroidogenesis, namely the conversion of cholesterol to pregnenolone. Herein, we demonstrate that H540 cells express high levels of three steroid-metabolizing enzymes which are involved in the further processing of pregnenolone in the endoplasmic reticulum of the steroidogenic cell. In particular, in addition to expressing 17α-hydroxylase cytochrome P-450 (P-45017α) and β-hydroxysteroid dehydrogenase/Δ5 → 4-isomerase (3β-HSD), H540 cells also showed high levels of steroid 5α-reductase mRNA and activity. The H540 cells therefore exhibit similarity to Leydig cells from sexually immature animals which also demonstrate high 5α-reductase activity. Thus, after 3β-HSD-catalyzed formation from pregnenolone, progesterone was efficiently converted to 5α-pregnan-3,20-dione (5α-dihydroprogesterone) and subsequent metabolism to the corresponding 17α-hydroxylated derivative and 5α-androstan-3,17-dione in a reaction catalyzed by P-45017α. H540 cells have apparently very low 17-ketosteroid reductase activity and, therefore, a principal end-product of the steroidogenic pathway in these cells was 5α-androstan-3,17-dione. H540 cells maintained in primary culture under serum-free conditions accumulated demonstrable levels of mRNA species for P-54017α (1.7 kb), 3β-HSD (1.6 kb) and 5α-reductase (2.7 kb). This finding suggests that the H540 tumor cell model will not only be of utility in the study of acute lutropin action but also in the elucidation of mechanisms involved in the regulation of expression of various families of microsomal steroid-metabolizing enzymes.