The herbs were purchased from Globalherb Inc. (Andong, Korea). A fresh sample of Angelica gigas Nakai was collected at Bonghwa-gun, Gyeongbuk province, Korea. Epimedium koreanum Nakai was imported from China. The plants were identified by an Oriental medical doctor and phytology expert at the Korea Bio Medical Science Institute Co. Ltd. (Seoul, Korea). The major ingredients in the herbal formula of KH-465 are described in Table 1. Epimedium koreanum Nakai (1,500 g) and Angelica gigas Nakai (1,500 g) were extracted with 12,000 mL of 30% ethanol for 3 hours at 60℃ and 20% ethanol for 4 hours at 60℃, respectively. The solutions were filtered twice through a 50-µm and a 1-µm filter, and concentrated in a vacuum evaporator (60℃) to 52 degrees Brix and 43 degrees Brix, respectively. The residual solvent in each extract was removed in a spray dryer (SD-1000, EYELA; Tokyo Rikakikai Co., Ltd., Tokyo, Japan) for 48 hours at 60℃ in a vacuum. The resulting powders were mixed at a 6:4 weight ratio and stored in a plastic bag until use. The major peak of KH-465, icariin from Epimedium koreanum Nakai and decursin from Angelica gigas Nakai, were analyzed by high-performance liquid chromatography using a Dionex Ultimate 3000 series dual low-pressure ternary gradient pump (Dionex Softron GmbH, Germering, Germany). Three principal icariin and decursin peaks were detected in the chromatogram by diode array detection at 530 nm.

The animal studies were performed after receiving approval of the Institutional Animal Care and Use Committee (IACUC) in Pusan National University Hospital (IACUC approval No. PNUH-2014-060) and were handled according to Guide for the Care and Use of Laboratory Animals.

Twelve-week-old male Sprague-Dawley rats were divided into 5 groups: a normal control (NC) group, which received an intramuscular injection of normal saline, and experimental groups I, II, III, and IV, which received a subcutaneous injection of an LHRH agonist (leuprolide acetate, Leuplin®; Takeda Pharmaceutical Co., Osaka, Japan) for 4 weeks to induce spermatogenic failure. Group I (control) received oral administration of distilled water once a day. Groups II and III received 200 mg/kg and 400 mg/kg of KH-465 for 4 weeks, respectively. Group IV received testosterone enanthate (Jenasteron®; Jaytech Biogen, Seoul, Korea) for 4 weeks as a positive control. KH-465 was dissolved in distilled water and administered orally through an 8 F red Rob-Nel catheter. The dosage of KH-465 and duration of treatment were selected based on results from a preliminary experimental study (data not shown).

The cauda epididymides were dissected and washed thoroughly in normal saline. The epididymides were finely minced in normal saline containing 0.5% bovine serum albumin at 37℃. Sperm suspensions were placed on prewarmed glass slides and observed under a light microscope in a Neubauer hemocytometer. Sperm motility was expressed at the percentage of sperm that showed any movement. Three counts per sample were averaged.

Testosterone (ALPCO, Salem, NH, USA), free testosterone (Cusabio, Wuhan, China), luteinizing hormone (LH) (Enzo, Washington, DC, USA), and follicle-stimulating hormone (FSH) (Cusabio) levels in serum were measured using an enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's protocol. Briefly, 96-well plates were coated with primary antibody, and 50 µL of standard, control, or samples was added into each well of the plate and incubated following the manufacturer's instructions. After washing, 100 µL of the enzyme-conjugated solution was added and incubated. After the substrate was added, the maximum colorimetric response was determined. The absorbance of the sample was read at 450 nm.

Testicular tissues were quickly excised and then fixed in Bouin's solution (Sigma, Louisville, KY, USA) for 24 hours. The fixed testicular tissues were dehydrated and embedded in paraffin wax, sectioned to a 4 µm thickness with a microtome, stained with H&E, and examined using light microscopy at ×400 magnification. Ten characteristic sites in the seminiferous tubules were selected randomly and spermatogenic cell density was measured.

Oxidative stress in testicular tissue was assessed quantitatively by measuring superoxide dismutase (SOD) activity and 8-hydroxy-2′-deoxyguanosine (8-OHdG) content. Using the DNeasy Blood & Tissue kit (Qiagen, Valencia, CA, USA), total DNA was extracted from the testes. The 8-OHdG levels were measured with a Highly Sensitive 8-OHdG Check ELISA (Cusabio). After the final color was developed with the addition of 3,3′,5,5′-tetramethylbenzidine, the absorbance of the sample was measured at 450 nm. The tissue sample concentration was calculated from a standard curve and was corrected for the DNA concentration. The level of SOD activity in the tissue was measured using a SOD Assay Kit-WST (MyBiosource, San Diego, CA, USA), monitoring the decrease in the rate of superoxide-mediated reduction of nitroblue tetrazolium at 450 nm using a spectrophotometer. Experiments were performed in triplicate.

All data were presented as the mean±standard deviation. Data were analyzed using SPSS ver. 12.0 (SPSS Inc., Chicago, IL, USA). Data were evaluated using analysis of variance, with group comparisons made using the Student t-test. p-values <0.05 were considered to indicate statistical significance.

The mean testis and epididymis weights are shown in Table 2. There were no significant differences in the body, testis, or epididymis weight between the NC group and the LHRH agonist-induced infertility groups.

The mean sperm concentration and motility in the LHRH agonist-induced infertility control group I were significantly lower than in the NC group. However, administration of KH-465 improved sperm concentration and motility compared with group I. In particular, administration of 400 mg of KH-465 (group III) led to marked improvements compared to the LHRH-agonist control group I. There was a significant decrease in serum levels of testosterone, free testosterone, FSH, and LH in the LHRH agonist-induced infertility group I compared with the NC group. There were no significant differences in sex hormone levels among the experimental groups (Table 3). LH showed a tendency to be higher in groups II and III than in the LHRH-agonist control group I, but this trend was not statistically significant.

In the NC group, testicular sections showed small and round seminiferous tubules with a normal arrangement and several layers of spermatocytes forming the germinal cell layer (Fig. 1A). In the samples obtained at 8 weeks, the testicular sections revealed compact and variably sized seminiferous tubules lined by a thick layer of spermatogenic cells in variable stages of maturation, beginning from the primary spermatogonia and extending until final sperm production filled the lumen. The basement membrane of all tubules was thin, with no thickening or peritubular fibrosis (Fig. 1B). In group I, the interstitium was scanty, slightly edematous, and included scattered small groups of Leydig cells with small round monotonous nuclei and prominent tiny nucleoli (Fig. 1C). Upon LHRH induction, in the samples obtained at 8 weeks from group I, a considerable number of spermatocytes showed incomplete spermatogenesis, with arrest at the secondary spermatogenic stage and a reduced number of spermatozoa at the center of the tubules compared with the NC group. The interstitium was edematous, vascular, and congested with normal Leydig cells. Progressive loss of Sertoli cells or arrest at the secondary spermatogenic stage of sperm production was observed in the LHRH-treated groups at 8 weeks (Fig. 1D). However, the most notable morphological changes were observed in group III (treated with KH-465) and group IV (treated with testosterone enanthate). These groups showed complete restoration of normal spermatogenesis in almost all seminiferous tubules, which exhibited satisfactory terminal sperm production by a thick layer of spermatogenic cells in variable stages of maturation and normal Leydig cells (Fig. 1E, 1F).

Greater SOD expression was observed in the groups treated with KH-465 than in the LHRH agonist-induced control group I, but this trend was not statistically significant. Less 8-OHdG expression was observed in the group that received 400 mg of KH-465 than in the LHRH agonist-induced control group I (Table 4).