Adult male Kunming mice aged 6 weeks were housed in groups of six per cage at a temperature of 22 ± 1 °C with a 12 h light–dark cycle (light on 7 a.m.–7 p.m.), and had free access to the food and water for 7 days prior to irradiation. All the experimental and animal handling procedures were approved by the Faculty Committee on the Use of Live Animals in Teaching and Research in the Shanxi province academy of traditional Chinese medicine (Taiyuan, China).
WuziYanzong prescription is composed of five different Chinese medicinal herbs (Table 1). In this study, all traditional Chinese herbs were purchased from Tong Ren Tang Chinese Medicine Co., Ltd. (Beijing, China) and ground to a fine powder. WZYZP powder was dissolved in 0.5% CMC and administrated p.o. at a volume of 20 ml/kg.
Testicular x-ray irradiation
Testicular irradiation was performed according to the previous report . Initially, mice were anesthetized with chloral hydrate (350 mg/kg, ip) and immobilized in the supine position. Only the testes region was irradiated with a single dose of 4 Gy x-rays while the body was shielded with 1-mm thick lead shields. Irradiation was carried out with a Gilardoni x-ray machine (15 mA, 250 kV; dose rate 0.96 Gy/min). Mice in control group were underwent the same anesthetic procedure but no irradiation. After irradiation all animals were returned to the animal facility.
The irradiated mice were randomly divided into 3 groups: (1) irradiated mice treated with distilled water; (2) irradiated mice treated with WZYZP 0.25 g/kg; (3) irradiated mice treated with WZYZP 1.0 g/kg. The control mice treated with distilled water were served as control group. Each group consisted of 12 mice with identical mean body weights. Daily oral administration of WZYZP for 21 days started from the day after irradiation. On day 21, blood samples were collected by orbital venous phlebotom for hormone determination. Then the mice were killed by cervical dislocation. The testicular, epididymal, prostatic and seminal vesicle tissues were collected and weighted. Then the epididymides were used for sperm count, motility and abnormal rate examination immediately. Meanwhile, one part of testes was homogenized for biochemical analysis and others were fixed in 10% formalin for histological analysis.
The testicular tissue was homogenized with ice-cold saline to be 10% (w/v) homogenates. Protein concentration was determined by the Coomassie blue protein-binding using bovine serum albumin as a standard. Malondialdehyde (MDA) in homogenized testis tissue was determined using an assay kit (Beyotime Biotechnology, China). Total oxidant status (TOS) and Total Antioxidant Status (TAS) of the tissues were measured by the colorimetric methods described by Erel . TAS results are expressed as μmol Trolox Equivalence per gram of tissue and TOS results are expressed as μmol H2O2 Equivalence per gram of tissue. Oxidative stress index (OSI) was calculated according to the following formula: OSI (Arbitrary Unit) = TOS/TAS .
Sperm count, motility and abnormal rate assays
The epididymal sperm count and abnormal sperm rate was determined by a modified method . Briefly, a 5 μl sperm sample was diluted with 95 μl diluent (5 g sodium bicarbonate, 1 ml 35% formalin and 25 mg eosin per 100 ml of distilled water). 10 μl of the thoroughly mixed diluted sperm suspension was transferred to each counting chamber of the neubauer hemocytometer. For determining the abnormal sperm rate, the fixed sperm suspension was placed on the slide and covered with a cover-slip. Approximately 200 sperm cells of each sample were examined under an optical microscope. The sperm were classified as normal, head abnormal or tail abnormal.
The progressive sperm motility was evaluated by a modified method . In brief, a 5 μl sperm sample was diluted with 95 μl Hank’s balanced salt solution. An aliquot of the sperm suspension was placed on the slide and covered with a cover-slip. For each sample, approximately 200 sperm cells were examined under an optical microscope. The sperm were classified as motile or immotile.
Serum hormone level assay
Blood samples were collected by orbital venous phlebotomy, and then centrifuged at 1000 × g at 4 °C for 10 min for serum separation. The levels of serum testosterone (T), luteinizing hormone (LH) and follicule-stimulating hormone (FSH) were determined using traditional radioimmunoassay methods and were performed according to the manufacturer’s instructions (Diagnostic System Laboratories, Webster, TX, USA).
Formalin-fixed tissue samples were dehydrated through an upgraded ethanol series, embedded in paraffin blocks and sectioned at 3 mm. Ultrathin sections of 5 μm were dewaxed by xylene, hydrated through a degraded ethanol series, and stained with hematoxylin-eosin. Then they were examined by a pathologist blinded to the treatments under an optical microscopy (Olympus, BX-51).
Western blot analysis
The testes were dissected out and homogenized in pre-chilled RIPA buffer (50 mM Tris–HCl, 150 mM NaCl, 0.5% Sodium deoxicholate, 0.1% SDS, 1% Tween-100, 5 mM EDTA, 1 mM EGTA, 1 mM PMSF). The lysate were centrifuged at 12,000 g for 15 min at 4 °C to pellet the debris. Supernatant solutions were collected as whole cytoplasm protein for analyzing PCNA. Protein concentrations were determined by the Coomassie blue protein binding method using bovine serum albumin as standard. Then samples containing equal amounts of protein (50 μg) were boiled in protein loading buffer for 10 min, separated on 12% SDS-polyacrylamide gels and transferred to PVDF membranes. The membranes were blocked with 3% BSA in Tris-buffer saline. Then the membranes were incubated at 4 °C overnight with the PCNA antibody (1:1000) and β-actin antibody (1:1000), respectively (Santa Cruz Biotechnologies, Santa Cruz, CA). After washing, the membranes were incubated with a horseradish peroxidase conjugated secondary antibody (mouse anti-rabbit IgG, 1:5000; rabbit anti-goat IgG, 1:5000) (Santa Cruz Biotechnologies, Santa Cruz, CA) for 1 h at room temperature and were visualized using an enzyme-linked chemiluminescence reaction by imager instrument (Fujifilm, Las-3000). Relative intensities of the bands were quantified by Image-Pro Express 6.0 software.
All the data were expressed as means ± standard deviation (SD). Differences between groups were assessed by one-way ANOVA and Tukey’s HSD test. A value of p < 0.05 was considered statistically significant.