Regulation by retinoids of luteinizing hormone/chorionic gonadotropin receptor, cholesterol side-chain cleavage cytochrome P-450, 3β-hydroxysteroid dehydrogenase/Δ 5−4-isomerase and 17α-hydroxylase/C 17–20 lyase cytochrome P-450 messenger ribonucleic acid levels in the K9 mouse Leydig cell line

Abstract

Vitamin A is a potent regulator of testicular function. We have reported that retinol (R) and retinoic acid (RA) induced a down regulation of luteinizing hormone/human chorionic gonadotropin (LH/CG) binding sites in K9 Leydig cells. In the present study we evaluated the effect of R and RA on LH/CG receptors, cholesterol side-chain cleavage cytochrome P-450 (P-450 scc), 17α-hydroxylase/C 17–20 lyase (P-450 17α) and 3β-hydroxysteroid dehydrogenase (3β HSD) mRNA levels in K9 mouse Leydig cells. To validate K9 cells as a model for studying Leydig cell steroidogenesis at the molecular level, we first investigated the effect of hCG on mRNA levels of the steroidogenic enzymes. P-450 scc, 3β HSD and P-450 17α were expressed constitutively. The addition of 10 ng/ml hCG enhanced mRNA levels for the three genes within 2 h. Maximal accumulation of P-450 scc, P-450 17α and 3β HSD mRNA in treated cells represents a 2.5-, 8.5- and 4-fold increase over control values, respectively. P-450 17α expression reached a maximum by 4 h and then declined rapidly to return to control value by 24 h. The pattern of LH/CG receptor mRNAs in K9 cells was very similar to that of MA10 Leydig cells and showed six transcripts of 1.1, 1.6, 1.9, 2.6, 4.2 and 7.0kb. Treatment of cells with R or RA resulted in a time- and dose-dependent decrease in all six species. Comparing the time course of the reduction in mRNA levels and in binding activity, we postulate that retinoids down regulate LH/CG receptors primarily by reducing their mRNAs. Addition of R or RA greatly enhanced P-450 17α mRNA levels in a time- and dose-dependent manner with maximal induction at 12 h (about a 5-fold increase) using 10 −6 M R or RA. The level of P-450 17α mRNA remained high throughout the treatment. Under the same conditions, 3β HSD mRNA levels were repressed but the effect was long term (70% decrease after 8 days with 10 −6 M R or RA). On the contrary, P-450 scc expression was not affected.