In total, 60 male Kunming mice (age, 4 weeks; weight, 25 g) were purchased from the Laboratory Animal Center of the Third Military Medical University (Chongqing, China). The mice were raised in specific pathogen-free animal rooms with free access to a rodent diet and water. Housing conditions comprised a temperature-controlled polysulfone cage (23–25°C) under a 12-h light/dark cycle.

All the mice were randomly divided into a control group and 2 treatment groups (20 animals/group). The treatment groups were intramuscularly injected with EB (Hangzhou Pharmaceutical Factory, Hangzhou, Zhejiang, China) at a concentration of 5 or 10 mg/kg body weight. This method was described in a previous study (17). The control group was injected with an equal quantity (150 µl) of corn oil (COFCO Grain Factory, Chongqing, China). The injection was performed every other day for 4 weeks. The testes were immediately excised from euthanised mice at the end of the experiments and then trimmed of fat and connective tissue. One of the testes from each group was fixed in Bouin's or 2.5% glutaraldehyde for histological analysis. The other testis was frozen in liquid nitrogen for biochemical and RNA detection. Cauda epididymis samples were collected for sperm counts. The present study was approved and monitored by the Animal Experiments Ethical Review Committee of Zunyi Medical University (Zunyi, China) (no. ZMU210500326). All procedures strictly adhered to the National Institutes of Health guidelines for the care and use of animals (18).

Sperm counts were obtained as previously described (19). The caudal epididymis was dissected and placed in 500 µl of saline media heated to 37°C, and then splayed openly using a back-cutting method. To give sperm time to escape into liquid, the caudal epididymis was left undisturbed for 30 sec. Subsequently, the number of sperm was assessed using a haemocytometer under a light microscope (Olympus, Tokyo, Japan) and expressed as 10⁶/100 mg of epididymal weight.

Testes were stained with haematoxylin and eosin (H&E) and haematoxylin-periodic acid-Schiff (PAS). Germ cells at stage VII of the seminiferous cycle, including spermatogonia, spermatocytes, and step 7 spermatids in 30 round seminiferous tubules per testicle (20), were counted using a conventional light microscope (Leica Microsystems GmbH, Wetzlar, Germany).

For TEM, the testis samples were cut into pieces (2×2 mm) and fixed in 2.5% glutaraldehyde (pH=7.4) for 6–8 h at 4°C. Next, they were washed and fixed in 2% OSO₄ for 1 h at 4°C. The tissue was finally dehydrated with alcohol solutions ascending in concentration gradient and embedded in araldite CY212. Semithin sections (1 µm) were cut and stained with toluidine blue. Ultrathin sections (60–70 nm) were cut and stained with uranyl acetate and alkaline lead citrate.

Oxidative stress in the testis was detected as previously described (21). In brief, the testis was homogenized in 0.8% sodium chloride solution (pH 7.4) containing 0.01 mol/l Tris-HCl, 1 mmol/l EDTA-2Na, and 0.01 mol/l sucrose, with a glass homogenizer according to the weight-to-volume ratio of 1 g:9 ml. After centrifugation of the homogenate at 839 × g for 15 min at 4°C, the supernatant was preserved. The activities of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), nitric oxide synthase (NOS), malondialdehyde (MDA) and nitric oxide (NO) content were specifically detected by colorimetric assay at different absorbable wavelengths as the purchased kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) indicated.

Testis tissue was rapidly frozen in liquid nitrogen and homogenized into powder. Adenosine phosphates were extracted from the powder with 10 µl/mg of 0.1 M perchloric acid in an ice bath for 1 min. The extraction mixture was centrifuged for 20 min at 5,031 × g, and the supernatant was removed by paper filtration. The filtrate solution was filtered again through a 0.45-mm filter. HPLC separation was achieved using continuous gradient elution as follows: Solution A, methanol; solution B, 0.1 M NaH₂PO₄ and 0.1 M Na₂HPO₄ mixed at a volume ratio of 9:1 and 10 mM tetrabutylammonium sulphate; solution A/B=12/88. The flow rate of the mobile phase was 0.6 ml/min, while the injection volume was 5 µl. Total retention time was ~5 min, and the gradient was run for 6 min to ensure full separation. The concentration of ATP was determined using the external standard method. Data were expressed as means of three repeat determinations.

In the testis homogenate, hexokinase (HK), pyruvate kinase (PK), and lactate dehydrogenase (LDH) activities and lactate and pyruvate content were specifically detected by colorimetric assay at different absorbable wavelengths as the purchased kit (Nanjing Jiancheng Bioengineering Institute) indicated.

As reported previously (22), total RNA was isolated from the testis and its quality was assessed by spectroscopy. Synthesis of cDNA was performed with the TransScript II One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (Beijing Transgen Biotech Co., Ltd., Beijing, China), according to the manufacturer's protocols. Quantitative PCR analysis for gene expression level was performed by the SYBR-Green Assay System with the Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The primers used in the present study are presented in Table I. PCR cycle parameters were 95°C for 15 sec and 55°C for 1 min, lasting for 40 cycles. The threshold line was set in the linear region of the plots above the baseline noise, and the value of the threshold cycle (C_T) was determined as the cycle number at which the threshold line crossed the amplification curve. PCR without template or with template substituted with total RNA was used as a negative control to verify experimental results. After amplification, the specificity of the PCR was determined by both melting curve analysis and gel electrophoresis to verify that only a single product of the correct size was present. Data were normalized against GAPDH, and all samples were amplified for 3 replications. Data are presented as the average fold increase ± standard error of mean (SEM).

Tissues were lysed in RIPA buffer (Promega Corporation, Madison WI, USA) containing protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland). The concentrations of total protein in the whole-cell lysate were determined by the Bradford assay. An equal amount (20 µg) of protein in each sample was separated by 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride membrane (EMD Millipore, Bedford, MA, USA). The membrane was incubated with 5% (w/v) skim milk in phosphate-buffered saline (PBS; pH 7.4) containing 0.05% Tween-20 (PBS-T) at 20°C for 1 h and then probed with anti-5′ adenosine monophosphate-activated protein kinase (AMPK) antibody (1:800 dilution; cat. no. ab32047, Abcam, Cambridge, MA, USA), anti-p-AMPK antibody (1:1,500 dilution; cat. no. ab23875, Abcam), and anti-β-actin antibody (1:1,000 dilution; cat. no. ab8227, Abcam) for 2 h at room temperature. The membrane was washed in PBS-T and then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (dilution 1:1,500; cat. no. sc-2004; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for 1 h at room temperature. Reactive proteins were detected using enhanced chemiluminscent and SuperSignal Chemiluminescent Substrates (Pierce; Thermo Fisher Scientific, Inc.). The protein intensity was semi-quantified using the ChemiDoc MP Imaging System (version 5.1.2; Bio-Rad Laboratories, Inc.).

Data were analysed using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). All experimental data are presented as the mean ± SEM. One-way analysis of variance (ANOVA) and Dunnett's post hoc test were employed when comparisons were performed between a control group and >1 experimental group to identify their differences. A P-value <0.05 was considered to indicate a statistically significant result.

The weights of the cauda epididymides and sperm counts were recorded (Table II), which revealed that there was a significant decrease in caudal epididymis weight in EB-treated mice (both P<0.05). As indicated by sperm suspension analysis, the control mice exhibited normal sperm counts; however, no sperm were observed in EB-treated mice (both P<0.01 vs. control).

H&E and PAS staining were performed to reveal the morphology of spermatogenic cells at different developmental stages in the seminiferous tubules. Representative spermatogonium, primary spermatocyte, and step 7 spermatids were indicated by the black arrows (Fig. 1A, right). Through statistical analysis, it was determined that 5 and 10 mg/kg EB decreased all the number of spermatogonium (P<0.05), primary spermatocyte (P<0.05), and step 7 spermatids (P<0.05) in the seminiferous tubules (Fig. 1A, left).

The nuclei of SCs exhibited an irregular oval shape at the ultrastructural level, and the nuclei were prominent in most sections of the SCs (Fig. 1Ba-1). The electron-dense perinuclear heterochromatin clumps could also be observed in the SCs. The cytoplasm of SCs contained numerous mitochondria, which had a circular outline with an electron-dense matrix and complete cristae (Fig. 1Ba-2). The ultrastructure of SCs in EB-treated groups displayed irregular oval-shaped nuclei and electron-dense perinuclear heterochromatin clumps (Fig. 1Bb-1 and c-1). However, almost all SCs had marked alterations in mitochondria. In the 5 mg/kg EB-treated group, the mitochondria were transformed to vacuoles with irregular cristae (Fig. 1Bb-2); in the 10 mg/kg EB-treated group, the mitochondria became giant organelles with irregular cristae and vacuoles were clearly observable (Fig. 1Bc-2).

Compared with the control group, the activities of SOD, CAT, and GSH-Px were significantly decreased (P<0.05; Fig. 2B) by EB treatments, while the contents of MDA and NO as well as NOS activity (P<0.05 or P<0.01, Fig. 2B) were increased. In addition, the mRNA expression levels of SOD (P<0.05), CAT (P<0.05), and GSH-Px (P<0.01) in the EB-treated group were significantly reduced (Fig. 2A).

HPLC analysis confirmed that ATP content in EB-treated groups was significantly decreased compared with the control groups (P<0.05; Fig. 3A), while the testicular glucose and pyruvate were increased (P<0.01; Fig. 3B). The activity of AMPK is primarily assessed by the protein phosphorylation level. GLUT3, MCT2 and MCT4 are transporters. Their functions are mainly affected by their gene expression. Protein levels of AMPK and p-AMPK were decreased by EB in a dose-dependent manner (Fig. 3C). The p-AMPK/AMPK ratio was decreased by 5 mg/kg EB (P<0.05), but increased by 10 mg/kg EB (P<0.05). The relative mRNA expression of GLUT3, MCT2, and MCT4 is displayed in Fig. 3D. The mRNA expression level of GLUT3 was significantly upregulated by EB (P<0.05), whereas MCT2 and MCT4 expression in the testis was downregulated (P<0.05). There was no difference in the concentration of lactate between EB-treated and control groups (data not shown). The activities of HK and PK were significantly decreased by EB (P<0.05; Fig. 3E), however the activity of LDH was almost unchanged between the 3 groups (data not shown).