A total of 90 male Kunming mice (8-weeks-old; 35–40 g) were provided by the Changchun Institute of Biological Products Co., Ltd. (Changchun, China). The present study was conducted with approval from the Ethics Committee of Jilin Medical University (Changchun, China). Mice were housed at 21±3°C and 55–65% relative humidity under a 12 h dark-light cycle. Mice were given free access to a standard laboratory mouse diet and sterile water.

The 15 Chinese medicine components of WSSJD, including Cornu Cervi Nippon Parvum, Panax ginseng, Cynomorium songaricum, Cistanche deserticola, Radix Astragali, Epimedium brevicornum and Angelica sinensis (11), were purchased from Tongrentang (Beijing, China). These materials were decocted according to the traditional method of Chinese medicinal decoction (18). In brief, the ingredients were weighed according to the recipe, and a total of 250 ml distilled water was added to submerge the herbs. Herbs were soaked for 30 min and subsequently heated. The herbs were first boiled for 10 min and then the heat was reduced to a simmer for 20 min. Following 30 min of heating, the liquid decoction was separated from the herbs, which were further decocted with 200 ml distilled water at 80°C for another 30 min. This process was repeated and the final volume of decoction liquid was filtered through 4–5 layers of gauze. Liquid was also squeezed from the herbs into the filtered decoction. The decoction was subsequently heated at 80°C in a water bath for 6–7 h until the concentration reached 2 g crude drug/ml, and the decoction was stored at 4°C prior to use. new WSSJD was prepared based on the formulation of WSSJD with minor adjustments to the dosages of Panax ginseng, Cynomorium songaricum, Radix Astragali and Epimedium brevicornum. The decocting method was the same as that of WSSJD. new WSSJD comprised of the following: Panax ginseng, 6 g; Cynomorium songaricum, 9 g; Radix Astragali, 12 g; Epimedium brevicornum, 6 g; Cornu Cervi Nippon Parvum, 1 g; Cistanche deserticola, 9 g; Angelica sinensis, 6 g; Flatstem Milkvetch Seed, 9 g; Rhizoma Dioscoreae, 15 g; Largehead Atractylodes Rhizome, 6 g; Ligusticum wallichii, 3 g; Radix Paeoniae Alba, 6 g; Cinnamomum cassia, 1 g; Costustoot, 1.5 g and Fructus Foeniculi, 3 g.

The concentrations of crude drug extracts were determined as follows: Weight of the crude material/final volume. This calculation method is widely used in the study of traditional Chinese medicine (19–21).

Mice were randomly divided into 6 groups (15 mice per group) and were administrated with medicine intragastrically. The groups were as follows: Control (normal saline); dimethylsulfoxide (DMSO); CsA; CC; WSSJD; and new WSSJD. Mice in the CsA, CC, WSSJD and new WSSJD groups were intraperitoneally (i.p.) injected with 15 mg/kg/day CsA (Shanxi Powerdone Pharmaceutics Co., Ltd., Datong, China) for 30 days, as described previously (8). Mice in the control and DMSO groups underwent a daily i.p. injection with an equal volume of normal saline or DMSO solvent, respectively, throughout the 30-day experimental period. The concentration of DMSO diluted in normal saline was 3.25% (v/v). The CC group was administered with 21.6 mg/kg/day CC (GKH Pharmaceutical, Ltd., Guangzhou, China) as described previously (13), which was also diluted in 3.25% (v/v) DMSO (pH=7.2). Mice in the WSSJD and new WSSJD groups were administered with 12 g crude drug/kg/day of WSSJD and new WSSJD, respectively.

At the end of the experimental period (at day 31), mice were placed in sealed cages, which were subsequently infused with 10–30% CO₂. When mice ceased to move for 5 min, it was confirmed that mice had succumbed to CO₂ exposure.

Mice testes were harvested and immediately fixed in 4% paraformaldehyde (pH=7.2) at room temperature for 24 h, followed by ethanol dehydration, xylene treatment to remove the ethanol, wax embedding and sectioning. Histological sections (5 µm) were obtained using a rotary microtome. These were affixed to glass slides for H&E staining. Testicular sections were observed using highlight histopathological microscopy to evaluate the development of seminiferous tubules and obtain histometric data. The development of seminiferous tubules was assessed using the Johnsen scoring system (22).

On day 30 of treatment, the mice were anesthetized with 10% chloral hydrate (Shanghai Guoyao Chemical Reagent Co., Ltd) at 0.004 ml/g of body weight i.p. prior to euthanasia with CO₂. Trunk blood was harvested and the serum was separated and stored at −20°C. Briefly, blood was collected into 1.5-ml tubes and stored at 4°C overnight. The tube was then centrifuged at 1,300 × g at 4°C for 6 min and the serum was collected. ELISA kits (Shanghai Elisa Biotech Co., Ltd., Shanghai, China) were used to determine the serum contents of testosterone (cat. no. EIA-2380) and LH (cat. no. EIA-2385) according to the manufacturer's protocol. The optical density (OD) was determined using a Model 680 microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and the levels of testosterone and LH were determined based on the standard curve.

Testicular tissues were fixed in 4% formaldehyde at room temperature for 24 h and embedded in paraffin. Paraffin sections were cut into 5 µm sections and were subsequently dewaxed. Antigen retrieval was achieved by incubating sections in 0.01 M citrate buffer (pH=6.0) at 95–98°C for 5 min. The sections were subsequently blocked with 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany; A3675) at room temperature for 1 h. Following blocking, sections were incubated with rabbit polyclonal antibodies against LHR (cat. no. L6792; Sigma-Aldrich; Merck KGaA; 1:200) or P450 side chain cleavage (P450scc; cat. no. ab75497; Abcam, Cambridge, UK; 1:200) at 4°C overnight in the dark. The streptavidin-biotin complex (SABC) method was used to detect the expression of LHR and P450scc using an SABC kit (SA2010; Boster Biological Technology, Pleasanton, CA, USA), according to the manufacturer's protocol. For the tissues isolated from negative control mice, the primary antibody was replaced with PBS. Leydig cells with yellow or brown staining on the membrane or plasma were considered to be LHR-positive cells. Five slides were obtained from each sample and five fields in the Leydig tissue areas were selected at random and assessed under a fluorescence microscope (magnification, ×400). The mean OD of positive cells in the Leydig tissue areas was obtained using Image Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA). The expressions of LHR and P450scc were proportional to the OD values, with higher OD values representing higher protein expressions.

Mice testes were dissected, embedded in wax and sectioned. The sections (5-µm thick) were then stained using the TUNEL assay kit (MK1024; Boster Biological Technology), according to the manufacturer's protocol. In brief, testicular tissues form control (normal saline), DMSO, CsA, CC, WSSJD and new WSSJD groups were stained using a 1:100 dilution of biotin labeled digoxin antibody for 30 min at 37°C. Stained rat interstitial epithelial tissue (provided in the kit) was used as a positive control, and samples incubated with PBS instead of biotin labeled digoxin antibody were used as a negative control. The frequency of TUNEL-positive cells exhibiting green nuclear staining was evaluated using a laser scanning confocal microscope. A total of 10 random fields were assessed under high-magnification (×400) and positive cells were counted. The mean number of positive cells per field was calculated.

The cauda epididymidis was harvested following sacrifice and the epidermis was cut with a blade to release the sperm from the epididymis into 2 ml PBS at 37°C to obtain epididymis suspensions. The suspensions were subsequently incubated at 37°C for 10 min to allow sperm to swim out. Aggregations of sperm were discarded and the remaining sperm samples were isolated and resuspended in PBS to give a concentration of 10⁶ cells/ml. Sperm apoptosis was analyzed using a Annexin V-FITC Apoptosis Detection kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China), according to the manufacturer's protocol. In brief, 1 ml of sperm suspension was stained with 10 µl PI or with 500 µl binding buffer and 5 µl Annexin V-FITC in the dark for 10 min at room temperature. Cells were immediately analyzed by Epies XL flow cytometry (Beckman Coulter, Inc.) and using a TetraONE^™ System (6915050; Beckman Coulter, Inc.). Cells in the early stages of apoptosis were identified by Annexin V-positive staining and necrotic cells were identified by PI-positive staining.

Data analysis was performed using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA) and expressed as the mean + or ± standard deviation as indicated. Differences between groups were evaluated for significance using one-way analysis of variance followed by a Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To investigate the potential protective effect of new WSSJD on the development of mouse testicular seminiferous tubules, the morphology of H&E-stained mouse testes were compared under light microscopy. The seminiferous tubules of mice from the CsA and CC groups exhibited shrinkage of the tubule fringe, decreased tubule diameters and reduced layers of testicular seminiferous epithelium compared with the control. In addition, spermatogenic cells exhibited a disordered arrangement, the number of spermatogenic cells was reduced and few mature sperm were visible in the lumen (Fig. 1). In mice from the DMSO, WSSJD and new WSSJD groups, more layers of testicular seminiferous epithelia were visible and the seminiferous tubule fringe was integrated without shrinkage or collapse (Fig. 1). The Johnsen scores did not differ significantly between the DMSO group and control groups, or between the CC and DMSO groups. However, the Johnsen score was significantly decreased in the CsA group and WSSJD group compared with the control group, and the scores of the WSSJD and new WSSJD groups were significantly increased compared with the CC or CsA group (P<0.05; Table I). These results indicated that new WSSJD and WSSJD promoted the development of seminiferous epithelium following CsA treatment.

The levels of testosterone and LH in the serum were subsequently measured. Serum levels of testosterone and LH were unaffected following DMSO administration. By contrast, serum testosterone was significantly downregulated and LH was significantly upregulated in CsA-treated mice, relative to controls (P<0.05; Fig. 2), and new WSSJD administration significantly restored testosterone to near control levels (P<0.05; Fig. 2). For serum LH, the protective effects of WSSJD was similar to that of CC; however, new WSSJD decreased serum testosterone to a significantly lower level than that observed with CC and WSSJD (P<0.05; Fig. 2), suggesting a superior protective effect of new WSSJD over CC or WSSJD.

The expressions of P450scc and LHR were assessed in testicular Leydig cells by immunohistochemistry (Figs. 3 and 4). In the control group, LHR was expressed on the outer membranes of Leydig cells between the seminiferous tubules (Fig. 3A), and P450cc was expressed in the cytoplasm of testicular Leydig cells (Fig. 4A). DMSO treatment had no significant effect on the expressions of LHR and P450scc. By contrast, CsA treatment significantly decreased the expressions of LHR and P450scc compared with control mice (P<0.05; Figs. 3B and 4B). In turn, the expressions of LHR and P450scc were significantly increased by treatment with CC, WSSJD or new WSSJD compared with the CsA group (P<0.05; Figs. 3B and 4B). In addition, levels of LHR and P450scc in testicular Leydig cells were significantly higher in WSSJD and new WSSJD mice compared with CC mice (P<0.05; Figs. 3B and 4B), and new WSSJD induced a significantly greater upregulation than WSSJD (P<0.05; Figs. 3B and 4B).

The apoptosis of spermatogenic cells in the mouse testes was analyzed by a TUNEL assay. The nuclei of apoptotic cells were principally observed in the spermatogonia and primary spermatocytes (Fig. 5A). Spermatogonia are larger than spermatocytes and are located closer to the basement membrane (23). DMSO treatment had no significant effect on the number of apoptotic spermatogenic cells in the testes. By contrast, treatment with CsA significantly increased the number of apoptotic spermatogenic cells compared with the control and DMSO groups (P<0.05; Fig. 5B). In turn, administration of CC, WSSJD or new WSSJD significantly reduced CsA-induced apoptosis (P<0.05; Fig. 5B). Furthermore, the number of apoptotic testicular spermatogenic cells in the WSSJD and new WSSJD groups was significantly reduced compared with the CC group (P<0.05), and new WSSJD was significantly more effective than WSSJD (P<0.05; Fig. 5B).

To verify the protective effects of new WSSJD against CsA-induced sperm apoptosis, the survival and early apoptosis of sperm in the epididymis were determined. Epididymal sperm were stained with PI or Annexin V and analyzed by flow cytometry (Figs. 6 and 7). In accordance with the aforementioned results, CsA treatment significantly reduced the percentage of live sperm and significantly increased the percentage of early apoptotic sperm (P<0.05; Figs. 6B and 7B), while DMSO treatment had no effect. WSSJD and new WSSJD significantly increased the percentage of live sperm (P<0.05; Fig. 6B) and reduced the percentage of early apoptosis sperm (P<0.05; Fig. 7B) compared with CsA treatment. The percentages of live and early apoptotic sperm in WSSJD and CC mice did not differ significantly, while new WSSJD induced a significantly greater increase in live sperm percentage compared with the CC group (P<0.05; Fig. 6B) and significantly decreased the percentage of early apoptotic sperm compared with the CC and WSSJD groups (P<0.05; Fig. 7B).