Pregnenolone 16-Alpha Carbonitrile, an Agonist of Rodent Pregnane X Receptor, Regulates Testosterone Biosynthesis in Rodent Leydig Cells

Abstract

Leydig cells (LCs) in the testes produce the male sex hormone testosterone (T). Several xenobiotics, including clinical drugs, supplements, and environmental chemicals, are known to disrupt T homeostasis. Notably, some of these xenobiotics are known to activate the pregnane X receptor (PXR), a ligand-dependent nuclear receptor. However, it is currently unknown whether PXR is expressed in LCs and whether PXR activation alters T synthesis in rodent LCs. Therefore, in this study, we sought to determine whether PXR is expressed in rodent LCs and whether pregnenolone 16-alpha carbonitrile (PCN), the prototype agonist of rodent PXR, regulates T biosynthesis in rodent LCs. Hormonal as well as protein and gene expression analyses were conducted in rat primary LCs and MA-10 mouse Leydig cells. Results showed that PXR was expressed at the mRNA and protein level in both rat primary LCs and MA-10 cells. Incubation of rat primary LCs with PCN resulted in a significant decrease in T secretion. This PCN-induced decrease in T secretion was associated with decreased protein expression of key steroidogenic enzymes such as 3β-HSD and CYP17A1. RNA-seq results from MA-10 cells showed that PCN down-regulated the transcripts of steroidogenic enzymes and proteins involved in the T synthesis pathway. Together, these results suggest that PCN, an agonist of rodent PXR, can regulate T biosynthesis in rodent LCs by down-regulating the expression of the steroidogenic enzymes involved in T biosynthesis. Our results are significant as they provide a potential novel mechanism for disruption of testosterone homeostasis by a variety of xenobiotics.

Keywords: Leydig cells; MA-10 cells; pregnane X receptor (PXR); pregnenolone 16-alpha carbonitrile (PCN); testis; testosterone; xenobiotics.

Figure 1

PXR protein expression in rodent LCs. (A). Protein expression of rPXR in rat primary LCs was determined by Western blotting analysis (n = 2). Image shown is from a representative experiment. Lane 1, dog liver; Lane 2, COS-7 cells transfected with pcDNA; Lane 3, COS-7 cells transfected with FLAG-pcDNA; Lane 4, COS-7 cells transfected with human PXR (hPXR); Lane 5, COS-7 cells transfected with FLAG-hPXR; Lane 6, COS-7 cells transfected with 3XFLAG-hPXR; and Lanes 7 to 9, rat primary LCs. Marker molecular weights represent KDa. (B). Protein expression of mPXR in mouse MA-10 cells was determined by Western blotting analysis (n = 2). Image shown is from a representative experiment. All Lanes (1 to 7), MA-10 cells. Marker molecular weights represent KDa.

Figure 2

Effect of pharmacological activation of rPXR on testosterone secretion by rat primary LCs. (A). LCs were isolated from 35-day-old male rats. Pooled LCs were incubated in the culture media and treated with either DMSO (n = 6) or 10 µM PCN (n = 6) for 24 h. The media was aspirated and collected after 24 h treatments to measure the T levels using RIA. Data represent mean ± SD from six independent experiments. Statistical significance was determined using an unpaired Students t test. (B). Both DMSO and PCN-treated rat primary LCs were incubated in the culture medium without (basal) (n = 3) or with 100 ng/mL LH (LH) (n = 3) for 3 h. The media aliquots were collected to determine T production using RIA. Results are shown as the mean ± S.D. Determined by ANOVA and a Tukey’s multiple comparisons test.

Figure 3

Effect of pharmacological activation of rPXR on protein expression of key enzymes involved in testosterone biosynthesis in rat primary LCs. Western blots (n = 2) showing the protein expression of 3β-HSD, CYP17A1, CYP19, StAR, and β-actin in rat primary LCs treated by DMSO or 10 µM PCN for 24 h. Data shown are from representative experiments.

Figure 4

RNA-seq analysis identifies 1552 DEGs in MA-10 cells. (A) Principal component analysis and (B) dendrogram showing the clustering pattern of four RNA-seq samples. (C) MA plot and (D) volcano plot provide an overview of PCN (10 µM)-induced DEGs in an MA-10 cell transcriptome. (E) Gene ontology (GO) analysis shows top GO terms linked to up- and down-regulated DEGs.

Figure 5

Heatmaps of genes associated with (A) steroidogenesis and (B) cholesterol metabolism in MA-10 cells. Heatmaps show diverse expression levels (log₂ fold-change in FPKM value). To reduce the influence of low-expression genes, 10 was added to each FPKM value before calculating the fold change. C, Control (DMSO); P, PCN (10 µM).