CORT, DHEA, and 7αOHD were purchased from Steraloids (Wilton, NH). [1,2-³H] Corticosterone (³H-CORT), specific activity 40 Ci/mmol, was purchased from Dupont-New England Nuclear (Boston, MA). [1,2-³H]11-dehydrocorticosterone (³H-DHC) was prepared from labeled ³H-CORT as described earlier (24).

Male Sprague-Dawley rats (from 2 to 90 days old) were purchased from Shanghai Laboratory Animal Co. Ltd. (Shanghai, China). Rats were sacrificed at postnatal days 2, 7, 14, 21, 28, 35, and 90 by asphyxiation with CO₂. One testis per rat was removed and frozen in liquid nitrogen for RNA extraction. The other testis was punched 5 holes using a needle and was fixed by Bouin's solution. The animal protocol was approved by the Institutional Animal Care and Use Committee of the Wenzhou Medical University.

Testes were processed for purification of Leydig cells. The progenitor (PLC), immature (ILC), and adult Leydig cells (ALC) were purified from rat testes at 21, 35, and 90 days postpartum, as described previously (25, 26). In brief, forty 21-day-old (for PLCs), eighteen 35-day-old (for ILCs), and six 90-day-old (for ALCs) rats were sacrificed, followed by removal and decapsulation of testes. Then, the testes were digested with collagenase and cells were separated by Percoll density gradient centrifugation. Purities of Leydig cell fractions were evaluated by histochemical staining of 3β-hydroxysteroid dehydrogenase (3β-HSD), with 0.4 mM etiocholanolone as an enzyme substrate (27). At least six isolations per cell types were performed.

The microsomes from ALCs were prepared as described previously (6). In brief, ALCs were homogenized in cold 0.01M PBS, pH 7.2, containing 0.25 M sucrose. Subsequently, the homogenates were centrifuged at 700 × g for 30 min and the supernatants were transferred to other tubes followed by centrifugation at 10,000 × g for 30 min. The resultant supernatants were centrifuged twice at 105,000 × g for 1 h. The pellets were resuspended and the protein contents were determined by Bio-Rad Protein Assay Kit (cat# 500–0006, Bio-Rad, Hercules, CA). Microsomes were used to assay 11β-HSD1 oxidoreductase activities.

All primers in this study were chosen using a sequence analysis software package (Primer 3, Whitehead Institute for Biomedical Research, Cambridge, MA) following guidelines for internal stability. Forward and reverse primers were in different exons to minimize the effects of possible DNA contamination. The primers for Cyp7b1 were: 5'-GAAGTCCTGCGTGACGAAAT-3' (forward); 5'-CCTCAGAACCTCAAGAATAGCG-3' (reverse); and the size of PCR product is 138 bp. For the internal standard, primers to ribosomal protein S16 (Rps16) were used, as described previously (28).

PCR was carried out in a 25 μl volume using a 96-well plate format using the SYBR Green PCR Core Reagents purchased from Applied Biosystems (Foster City, CA). Primer titration was performed and the concentration of 300 nM was selected. Fluorescence was detected in real-time on an ABI 5700 system (PE Applied Biosystems). The mRNA levels were measured by a standard curve method and the levels were normalized to Rps16, the internal control. Each sample was run in duplicate, in parallel with no template controls.

Immunofluoroscent staining of CYP7B1 was performed on isolated ALCs using cells grown on 12-well microscope cover glasses. Cells were fixed with 4% formaldehyde, washed with PBS, and permeabilized with 0.1% Triton X100 in PBS plus 10% normal serum. Nonspecific binding was blocked by incubation with 10% normal serum before addition of the primary antibody. Cells were incubated with goat polyclonal CYP7B1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature. Cells were then incubated with Alexa488-conjugated second antibody for 1 h. The cells were counterstained with DAPI and mounted onto glass microscope slides, and cover-slipped. The slides were examined under a Nikon fluorescence microscope with a filter suitable for detecting the fluorescence of green color.

Testes were dehydrated in ethanol and xylene and embedded in paraffin for immunological analysis. 6 μm-thick transverse sections were prepared and mounted on glass slides. Avidin–biotin immunostaining was performed using a Vector kit (Burlingame, CA) according to the manufacturer's instructions. Antigen retrieval was carried out by boiling for 10 min in 10 mM (pH 6.0) citrate buffer, and endogenous peroxidase was blocked with 0.5% H₂O₂ in methanol for 30 min. The sections were then incubated with goat polyclonal CYP7B1 antibody (1:200, Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature. The antibody-antigen complexes were visualized with diaminobenzidine alone, resulting in brown cytoplasmic staining in positively labeled cells. The sections were counterstained with Mayer hematoxylin, dehydrated in graded concentrations of alcohol, and cover-slipped with resin (Permount, SP15-100; Fisher Scientific).

Western blotting was performed as previously described (29). Briefly, rat PLC, ILC, and ALC homogenates (30 μg protein each sample) were electrophoresed on 10% polyacrylamide gel containing sodium dodecyl sulfate. After electrophoresis, proteins were transferred onto a nitrocellulose membrane, and the membrane was incubated with 5% non-fat milk for 1 h to block the non-specific binding. Then, the membrane was incubated with primary antibodies against the following antigens: CYP7B1 (Santa Cruz Biotechnology) and β-actin (ACTB, Cell Signaling Technology, Danvers, MA), which serves the internal control. The membrane was washed and incubated with a 1:5,000 dilution of second antibody conjugated to horseradish peroxidase. Immunoreactive bands were visualized by an ECL kit (Amersham, Arlington Heights, IL). The intensity of the protein was quantified using Image J software. The CYP7B1 was adjusted to ACTB.

11β-HSD1 oxidase and reductase in intact ALCs were performed as previously described (15).11β-HSD1 oxidase activity assay tubes contained 25 nM CORT spiked with 60,000 dpm [³H]-CORT (a concentration within the range of physiological levels of CORT). 11β-HSD1 reductase activity assay tubes contained 25 nM DHC spiked with 60,000 dpm [³H]-DHC. 2.5 × 10⁴ intact ALCs were added to start the reaction and the reaction was maintained in 34°C and 75 rpm shaking water bath for 30 min. The reactions were stopped by adding 2 ml ice-cold ether. The steroids were extracted, and the organic layer was dried under nitrogen. The steroids were separated chromatographically on thin layer plates in chloroform and methanol (90:10, v/v), and the radioactivity was measured using a scanning radiometer (System AR2000, Bioscan Inc., Washington, DC). The percentage conversion of CORT to DHC and DHC to CORT was calculated by dividing the radioactive counts identified as DHC (or CORT, respectively) by the total counts associated with CORT plus DHC.

11β-HSD1 oxidase and reductase in microsomes were performed as previously described (15). For measurements in microsomal preparations, microsomes were incubated with 25 nM [³H]-CORT and 0.2 mM NADP⁺ for oxidase or 25 nM [³H]-DHC and 0.2 mM NADPH for reductase activity in presence of different concentrations of DHEA or 7αOHD. For testing mode of action, microsomes were incubated with 2 nM to 10 μM [³H]-CORT and 0.2 mM NADP⁺ for oxidase or 2 nM to 10 μM [³H]-DHC and 0.2 mM NADPH for reductase activity. 1.5 μg of ALC microsomes were added to initiate the reaction. The reactions were stopped by adding 2 ml ice-cold ether. The steroids were extracted, and the organic layer was dried under nitrogen. The steroids were separated chromatographically on thin layer plates in chloroform and methanol (90:10, v/v), and the radioactivity was measured using a scanning radiometer (System AR2000, Bioscan Inc., Washington, DC). The percentage conversion of CORT to DHC and DHC to CORT was calculated by dividing the radioactive counts identified as DHC (or CORT, respectively) by the total counts associated with CORT plus DHC.

Experiments were repeated three to four times. Data were subjected to analysis by one-way ANOVA followed by ad hoc DUNCAN multiple comparisons testing to identify significant differences between groups when three and more groups were calculated, or by the student t-test when two groups were calculated. All data are expressed as means SEM. Differences were regarded as significant at ^*P < 0.05 or ^**P < 0.01 or ^***P < 0.001.

11β-HSD1 reductase converts DHC into CORT. We measured 11β-HSD1 reductase activity in intact ALCs in presence of DHEA (10 nM−10 μM). DHEA inhibited 11β-HSD1 reductase activity with the lowest observable effective level (LOEL) of 100 nM and it lowered 11β-HSD1 reductase activity to 12% of control at 10 μM (Figure 1A). The IC₅₀ value of DHEA of inhibiting 11β-HSD1 reductase activity was 1.04 μM (Table 1). This indicates that DHEA is a potent inhibitor of 11β-HSD1 reductase in intact ALCs.

11β-HSD1 oxidase converts CORT into DHC. We measured 11β-HSD1 oxidase activity in intact ALCs in presence of DHEA (10 nM-10 μM). DHEA stimulated 11β-HSD1 oxidase activity with LOEL of 100 nM (Figure 1B). DHEA increased 11β-HSD1 oxidase activity to 300% of control at 10 μM. The EC₅₀ value of DHEA of stimulating 11β-HSD1 oxidase activity was 0.97 μM (Table 1). We calculated 11β-HSD1 oxidase/reductase ratio (Figure 1C) and found that 11β-HSD1 oxidase/reductase ratio in the control was 1.6-fold conforming to the previous report (17). DHEA increased this ratio by 26-folds at 10 μM, indicating that DHEA makes 11β-HSD1 more oxidative.

In order to separate the potential contribution of the pentose phosphate pathway that is located in the cytosol, 11β-HSD1 reductase activity was measured in ALC microsomes in presence of DHEA (10 nM-10 μM). DHEA decreased ALC microsomal 11β-HSD1 reductase activity with LOEL of 100 nM and it lowered 11β-HSD1 reductase activity to 25% of control at 10 μM (Figure 2A). The IC₅₀ value of DHEA of inhibiting 11β-HSD1 reductase activity in ALC microsomes was 1.29 μM (Table 1). This is similar to what was found in intact ALCs.

11β-HSD1 oxidase activity was measured in ALC microsomes in presence of DHEA (10 nM-10 μM). DEHA did not affect microsomal 11β-HSD1 oxidase activity. Actually, DHEA also inhibited 11β-HSD1 oxidase activity at 10 μM (Figure 2B). Although DHEA did not affect 11β-HSD1 oxidase, 11β-HSD1 oxidase/reductase ratios in ALC microsomes were still significantly higher than control at 1 and 10 μM DHEA (Figure 2C). These data indicate that the stimulation of 11β-HSD1 oxidase by DHEA requires intact ALCs.

We determined that the timing of Cyp7b1 expression in Leydig cells was developmentally relevant. Cyp7b1 mRNA levels detected by qPCR in templates prepared from rat testis on postnatal days 2–90 did not vary significantly (Figure 3). When Leydig cells were enriched in developmental stages (PLCs at 21 days, ILCs at 35 days, and ALCs at 90 days postpartum), Cyp7b1 mRNA was 3-folds more abundant in ILCs and 50-folds more abundant in ALCs than that in PLCs (Figure 3). Cyp7b1 mRNA levels were enriched by 33-folds in ALCs when compared to age-matched testes. This suggests that Cyp7b1 is primarily expressed in ALCs.

Previous studies have demonstrated that CYP7B1 (encoded by Cyp7b1) catalyzes DHEA into 7αOHD and that the epimerase activity of 11β-HSD1 converts 7αOHD into 7βOHD (20–22). Besides human, pig and rat 11β-HSD1 can catalyze 7αOHD using NADP+ into 7KD to generate NADPH (23). In order to investigate a potential metabolic coupling between CYP7B1 and 11β-HSD1, we first sought to show whether CYP7B1 was present in rat ALCs. CYP7B1 was detected in ALCs by fluorescent immunohistochemistry and CYP7B1 was present in the cytoplasmic membrane (Figures 4A–E) when compared to the negative control (Figure 4F). Western blotting also showed the highest expression of CYP7B1 in rat ALCs (Figure 4G). This suggests that CYP7B1 is primarily expressed in ALCs.

CYP7B1 converts DHEA into 7αOHD. 11β-HSD1 reductase activity was measured in intact ALCs in presence of 7αOHD (10 nM−10 μM). 7αOHD increased 11β-HSD1 reductase activity with LOEL of 100 nM (Figure 5A). 7αOHD increased 11β-HSD1 reductase activity to 200% of control at 1 and 10 μM (Figure 5A). The EC₅₀ value of 7αOHD of increasing 11β-HSD1 reductase activity was 0.7 μM (Table 1). This suggests that 7αOHD does the opposite action of DHEA on 11β-HSD1 reductase activity in intact ALCs.

11β-HSD1 oxidase activity was measured in intact ALCs in presence of 7αOHD (10 nM−10 μM). 7αOHD decreased 11β-HSD1 oxidase activity with LOEL of 100 nM (Figure 5B). 7αOHD decreased 11β-HSD1 oxidase activity to 60% of control at 1 μM and 25% of control at 10 μM (Figure 5B). The IC₅₀ value of 7αOHD of inhibiting oxidase activity was 1.17 μM (Table 1). We calculated 11β-HSD1 oxidase/reductase ratio (Figure 5C). 7αOHD decreased 11β-HSD1 oxidase/reductase ratio to 50% of control at 1 μM and to 20% of control at 10 μM. This indicates that 7αOHD makes 11β-HSD1 more reductive.

11β-HSD1 reductase activity was measured in ALC microsomes in presence of 7αOHD (10 nM-10 μM). 7αOHD increased 11β-HSD1 reductase activity with LOEL of 100 nM. However, at the highest concentration (100 nM), 7αOHD did not affect 11β-HSD1 reductase activity (Figure 6A). The EC₅₀ value of 7αOHD of increasing 11β-HSD1 reductase activity was 200 nM (Table 1).

11β-HSD1 oxidase activity was measured in ALC microsomes in presence of 7αOHD (10 nM−10 μM). 7αOHD decreased 11β-HSD1 oxidase activity with LOEL of 100 nM (Figure 6B). 7αOHD decreased 11β-HSD1 oxidase activity to 60% of control at 1 μM and to 25% of control at 10 μM (Figure 6B). The IC₅₀ value of 7αOHD for inhibiting oxidase activity was 0.93 μM (Table 1). 7αOHD significantly decreased 11β-HSD1 oxidase/reductase ratio in ALC microsomes at 0.1–10 μM (Figure 6C). The action of 7αOHD in ALC microsomes was similar to that in intact ALCs, indicating that 7αOHD acts on 11β-HSD1 within SER.

We measured the rates of reductase reactions in ALC microsomes in presence of different DHEA concentrations (0, 1, and 10 μM) in order to evaluate the mode of action. DEHA was a competitive inhibitor of 11β-HSD1 reductase (Figure 7A).

We measured the rates of oxidase reactions in ALC microsomes in presence of different 7αOHD concentrations (0, 1, and 10 μM) in order to evaluate the mode of action. 7αOHD was a competitive inhibitor of 11β-HSD1 oxidase (Figure 7B).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.