Male Sprague-Dawley rats (7–8 weeks old, weighing 250–300 g) were purchased from the Guangdong Medical Laboratory Animal Center of China. The animals were kept in a temperature-controlled room (20°C) with a 12 h-light/12 h-dark photoperiod and were give access to food and water ad libitum. All animal experiments were performed in accordance with the guidelines of the Guangdong Ocean University Animal Use Committee, and the protocols were approved by the Animal Ethics and Welfare Committee of Guangdong Ocean University (Approval No. 2014031903).

Dulbecco's modified Eagle's medium/nutrient mixture F-12 (DMEM/F12), fetal bovine serum (FBS), Hanks and penicillin/streptomycin were purchased from Gibco Laboratories (Grand Island, NY, USA). Bovine serum albumin (BSA), tauroursodeoxycholic acid (TUDCA), N-acetyl-L-cysteine (NAC) and collagenase were obtained from Sigma-Aldrich (St. Louis, MO, USA). Percoll™ (Sterile) was obtianed from GE Healthcare (Pittsburgh, PA, USA). The testosterone ELISA kit was purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). Anti-steroidogenic acute regulatory protein (StAR; Cat. no. sc-25806), anti-cholesterol side-chain cleavage enzyme (P450scc; Cat. no. sc-292456), anti-3β-hydroxysteroid dehydrogenase (3β-HSD; Cat. no. sc-30820), anti-glucose-regulated protein 78 (GRP78; Cat. no. sc-13968), and anti-C/EBP homologous protein (CHOP; Cat. no. sc-166682) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-actin (Cat. no. #4970) and anti-rabbit IgG (Cat. no. #7074) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). The endotoxin assay kit was purchased from Jinruisi Inc. (Nanjing, China). The bicinchoninic acid (BCA) protein assay kit was obtained from Shenenergy Inc. (Shanghai, China). TRIzol was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). M-MLV reverse transcriptase was obtained from Promega (Madison, WI, USA). SYBR Premix Ex Taq™ was obtained from Takara Biot echnology Co., Ltd. (Dalian, China). ECL chemiluminescent substrate was obtained from Millipore (Billerica, MA, USA).

The AGEs were prepared as previously described (28). Briefly, fatty acid-free BSA was incubated with 50 mM D-glucose in phosphate-buffered saline (PBS) solution in the dark and under ysterile conditions for 7 weeks at 37°C. Unincorporated glucose was removed by dialysis with PBS. Control non-glycated BSA was incubated in the absence of glucose under the same conditions. AGE-BSA solutions were tested for endotoxin concentrations and confirmed to be endotoxin free (<2.5 U/ml of endotoxin).

Rat Leydig cells were isolated from the testes of mature rats as previously described (29,30) with some modifications. Briefly, male Sprague-Dawley rats were sacrificed by CO₂ inhalation. The testes were quickly removed, decapsulated and placed in a 50-ml plastic tube containing 3 ml of collagenase solution (0.5 mg/ml) and incubated in an oscillating incubator (100 r/min, 34°C) for 30 min. The cell suspension was transferred to a 50-ml tube and kept on ice for 2 min to allow the tubules to settle. The supernatant containing Leydig cells was filtered through a 100-µm nylon cell strainer (BD Biosciences, San Jose, CA, USA). The cells were centrifuged at 1,500 rpm for 10 min at 4°C. The pellet was resuspended in 2 ml DMEM/F12 and loaded onto the top of the discontinuous Percoll gradient (21, 26, 37 and 60%) and centrifuged at 3,000 rpm for 30 min at 4°C. The cells in the interphase between 37 and 60% were collected and maintained in DMEM/F12 medium containing 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin at 34°C with 5% CO₂. The purity of the Leydig cells was examined by 3β-hydroxysteroid staining and >90% of the cells stained positive (data not shown).

The effects of AGEs on Leydig cell viability were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Rat Leydig cells were plated into 96-well culture plates. Following 48 h of incubation with various concentrations of AGEs (25, 50, 100 and 200 µg/ml), 100 µl MTT (5 mg/ml) were added to each well and the cells were incubated for 2 h at 34°C. The medium was discarded and 150 µl DMSO was then added to each well. The absorbance was measured at 490 nm using a microplate reader. The results were expressed as the percentage of MTT reduction, assuming that the absorbance of the control cells was 100%.

The rat Leydig cells cultured in 6-well plates were pre-incubated with various concentrations of AGEs or BSA for 12 h and the culture medium was replaced with fresh medium containing human chorionic gonadotropin (hCG; 4 ng/ml) with or without the same concentrations AGEs or BSA. Following treatment under different conditions for 12 h, the medium was collected and centrifugated 12,000 rpm for 5 min at 4°C, and the supernatant were collected and assayed for testosterone using ELISA kit. The sensitivity of the assay was 32 pg/ml. Intra- and inter-assay variations were below 6.6 and 7.5%, respectively.

Intracellular ROS levels were determined by measuring the probe, dichlorofluorescein diacetate (DCFH-DA). Briefly, cells treated with or without AGEs for 12 h were washed and incubated with fresh medium containing 10 µM DCFH-DA for 30 min in the dark. The medium was then removed and the cells were washed with PBS. Fluorescence at excitation, 485 nm and emission, 535 nm was measured using a microplate reader (TriStar LB941; Berthold Technologies, Oak Ridge, TN, USA).

The Leydig cells were treated under different conditions and total RNA was then isolated using TRIzol reagent following the manufacturer's instructions. cDNA was synthesized from 1 µg of total RNA using M-MLV reverse transcriptase in a total reaction volume of 20 µl. The PCR reaction mixtures contained 10 µl SYBR^® Premix Ex Taq™, 0.4 µl of each primer (10 µM), 2 µl template cDNA and dH₂O up to a final volume of 20 µl. The cycling conditions were 94°C for 40 sec, followed by 40 cycles of 94°C for 15 sec, 64°C for 15 sec, and 72°C for 15 sec. The following primers were used: StAR forward, 5′-ACCACATCTACCTGCACGCCAT-3′ and reverse, 5′-CCTCTCGTTGTCCTTGGCTGAA-3′; 3β-HSD forward, 5′-AGCAAAAAGATGGCCGAGAA-3′ and reverse, 5′-GGCACAAGTATGCAATGTGCC-3′; P450scc forward, 5′-TTCCCATGCTCAACATGCCTC-3′ and reverse, 5′-ACTGAAAATCACATCCCAGGCAG-3′; β-actin forward, 5′-GGAAATCGTGCGTGACATTAAAG-3′ and reverse, 5′-CGGCAGTGGCCATCTCTT-3′. The relative gene expression levels were normalized to β-actin using the ∆∆Ct method, where Ct was the cycle threshold.

After being subjected to the various treatments, the cells were washed twice with PBS and then lysed for 20 min in lysis buffer (50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 2 mM EDTA, 10% glycerol, 0.1% NP-40, 0.5 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM NaF, 0.1 mM Na₃VO₄ and 1 mM dithiothreitol). The protein concentration was measured by BCA protein assay. Protein (30 µg) was separated by SDS-polyacrylamide gel electrophoresis, and transferred onto PVDF membranes. The membranes were blocked in 5% non-fat milk powder in Tris-buffered saline (TBS)/0.1% Tween-20 for 1 h at room temperature and incubated with specific antibodies (3β-HSD, P450scc, StAR, GRP78, CHOP, β-actin) in 5% BSA in TBS at 4°C overnight. After washing, the membranes were incubated with HRP-conjugated second antibody in TBS for 1 h at room temperature. Finally, the labeled proteins were detected using the ECL kit. Densitometric analyses of the bands were performed using ImageJ software (obtained from the NIH websites, http://rsb.info.nih.gov/nih-image).

Statistical comparison was carried out using one-way analysis of variance (ANOVA). The data represent the means ± SEM. Values of P<0.05 were considered to indicate statistically significant differences.

To assess the effect of AGEs on the viability of rat primary Leydig cells, the cells were treated with AGEs or BSA for 48 h and MTT assay was then performed. The viability of the cells treated with AGEs is shown in Fig. 1. The viability of the cells treated with 200 µg/ml BSA, or with 25, 50, 100 and 200 µg/ml AGEs was 101.1±9.2, 99.0±7.7, 96.8±9.3, 96.0±10.3 and 94.7±8.0% of the control value, respectively. These data indicated that the viability of the Leydig cells treated with AGEs (concentrations ≤200 µg/ml) for 48 h was not significantly altered.

The influence of AGEs on hCG-stimulated testosterone production in Leydig cells is illustrated in Fig. 2. Exposure to hCG at 4 U/ml induced a significant increase in testosterone secretion by rat Leydig cells (P<0.01). Following treatment with AGEs for 24 h, testosterone secretion by the rat Leydig cells was reduced in a dose-dependent manner, with significant decreases being observed from the concentration of 50 µg/ml AGEs.

In order to examine the influence of AGEs on the transcriptional levels of genes related to the testosterone synthetic pathway in rat Leydig cells, the mRNA levels of StAR, P450scc and 3β-HSD in rat Leydig cells treated with AGEs for 24 h were measured. Generally, treatment with AGEs led to a significant decrease in the mRNA levels of StAR, P450scc and 3β-HSD (Fig. 3). Furthermore, the protein levels of StAR, P450scc and 3β-HSD in the cells treated with AGEs for 24 h were investigated. The results revealed that AGEs decreased the StAR, P450scc and 3β-HSD protein levels in a dose-dependent manner (Fig. 4).

Since oxidative stress plays an important role in AGE-induced dysfunction in other tissues or cells, the role of oxidative stress in the inhibition of testosterone secretion by Leydig cells induced by AGEs was investigated. The results revealed that the AGEs significantly increased the levels of ROS in the rat Leydig cells in a concentration-dependent manner (Fig. 5). Following pre-treatment with NAC, an antioxidant agent, the inhibitory effects on testosterone secretion induced by AGEs were significantly reversed (Fig. 6).

The Leydig cells were pre-treated with TUDCA, an endoplasmic reticulum stress inhibitor, and the levels of testosterone were measured. As shown in Fig. 6, we found that TUDCA significantly inhibited the AGE-induced decrease in the secretion of testosterone (P<0.01). The results of western blot analysis revealed that the expression levels of endoplasmic reticulum stress-related proteins (CHOP and GRP78) were increased (Fig. 7). In particular, following treatment with 200 µg/ml AGEs, the expression levels of CHOP and GRP78 were 2.40- and 2.51-fold of those of the control, respectively (P<0.01). These results indicated that AGEs inhibited the synthesis of testosterone partly by inducing endoplasmic reticulum stress.