Animals and experimental design
Sprague-Dawley male rats (7-weeks old) were purchased from Shizuoka Laboratory Animal Center (Hamamatsu, Japan), housed in a light (14-h light cycle; lights on at 06:00 h) and temperature (23–27 °C)-controlled room, and fed standard food pellets and water ad libitum. All procedures were performed according to the guidelines described in the Shizuoka University of Health Guidelines for the Care and Use of Experimental Animals.
Rats were divided into three groups: sham control (n = 6), CP (n = 7), and CP + RLN (n = 7) groups, because a preliminary study found that control animals treated with RLN were not significantly different from controls not treated with RLN in terms of testicular function, including morphology, spermatogenic status, and apoptosis. CP (cis-Diammineplatinum (II) dichloride; Sigma-Aldrich, St. Louis, MO, USA) was intraperitoneal injected at a single dose (6 mg/kg). The dose of CP was designed according to our pilot study and previous report  that demonstrated significant testicular as well as renal damages in rats. RLN or saline infusion started immediately after CP treatment. The RLN used here was purified from pregnant sow ovaries . For continuous infusion of porcine RLN (500 ng/h) or saline for 5 days, animals were anesthetized by intraperitoneal injection of xylazine (Ceractal® 2% solution; Bayer, Osaka, Japan) and pentobarbital sodium (Nembutal® Sodium Solution; Sumitomo Dainippon Pharma, Osaka, Japan) and surgically implanted with an ALZET mini-osmotic pump (Model 2001; DURECT, Cupertino, CA, USA) under the skin. RLN dose was chosen based on a previous study showing that injection of this dose resulted in serum RLN levels comparable to those recorded in mid-pregnant rats .
At 5 days post-CP administration, rats were anesthetized by intraperitoneal injection of xylazine and pentobarbital sodium, and the testes, epididymides, and accessory sex glands were removed and weighed after blood was taken directly from the heart. Blood was centrifuged at 1500 g for 15 min at 4 °C, and serum was stored at − 80 °C. One of the testes was rapidly frozen and stored in liquid nitrogen for biochemical analysis, and the other was prepared for histological examination by fixation with 10% neutral-buffered formalin and embedded in paraffin. Epididymal sperm was also collected from cauda epididymides.
Serum testosterone concentrations were measured using a TR-FIA kit (PerkinElmer, Waltham, MA, USA), with an assay detection limit of 0.15 ng/ml and the intra- and inter-assay coefficients of variability of 2.3 and 6.6%, respectively.
Testicular specimens were cut at 4-μm sections, stained with hematoxylin and eosin (HE), and examined under a BX50 microscope equipped with a CCD camera (Olympus, Tokyo, Japan) and Instudio V105 software (Pixera, Kanagawa, Japan). The diameters of seminiferous tubules were morphometrically determined using transverse sections of each seminiferous tubule from the experimental groups according to a previously described method . Johnsen’s score was used to categorize spermatogenesis . Each tubular section was given a score ranging from 10 to one according to the presence or absence of the main cell types arranged in the order of maturity. Furthermore, the histopathological parameters by Cosentino et al. , including degeneration of the germ cell layer, disarray of germ cell layers, loss of spermatozoa/spermatids, arrested germ cells in different stages of division, and necrotic germ cells, were used to assess overall histological damage, with each testis scored zero to four.
Quantitative real-time RT-PCR (qPCR)
Total RNA from testes was extracted using ISOGEN reagent (Nippon Gene, Tokyo, Japan), treated with RNase-free DNase I (Qiagen, Hilden, Germany), and reverse transcribed using an oligo(dT)18 primer with ReverTra Ace (Toyobo, Tokyo, Japan). Quantification of complementary DNA (cDNA) from each testis was measured using Bio Photometer (Eppendorf, Hamburg, Germany), followed by dilution with nuclease-free water. Aliquots of diluted cDNA were stored at − 20 °C.
Levels of target gene expression were determined using 100 ng of template cDNA per reaction, and quantified on either a Prism 7000 Real Time PCR System (Life Technologies, Carlsbad, CA, USA) or a Thermal Cycler Dice Real-Time System III TP950 (Takara Bio, Shiga, Japan) with appropriate probes or primers. The expression of each gene was normalized to glyceraldehyde 3-phosphate dehydrogenase (Gapdh) levels and quantified by using the 2−ΔΔCq method .
Apoptotic germ cells in deparaffinized sections were detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end-la beling (TUNEL) assay using an ApoTag peroxidase in situ apoptosis detection kit (EMD Millipore, Temecula, CA, USA). The number of TUNEL-positive cells was counted in each seminiferous tubule. An apoptotic index was calculated by counting the number of round seminiferous tubules expressing greater than three TUNEL-positive cells, dividing by the total number of essentially round seminiferous tubule, which ranged from 102 to 182 in each animal, and multiplying the product by 100, as described previously . Additionally, the expression of the proapoptotic caspase 3 (Casp3) and the antiapoptotic B-cell lymphoma 2 (Bcl2), as well as that of Rxfp1 and the pro-inflammatory cytokine interleukin 6 (Il6), was detected by qPCR. Cycling conditions were as follows: 95 °C for 2 min, and 45 cycles of 15 s at 95 °C and 1 min at 60 °C; reactions were performed in triplicate on a Prism 7000 Real Time PCR System (Life Technologies). TaqMan Fast Advanced Master Mix (Life Technologies) was used in a final volume of 20 μl containing Casp3, Bcl2, and Gapdh probes designated Rn00563902_m1, Rn99999125_m1, and Rn99999916_s1, respectively (TaqMan Gene Expression Assay; Life Technologies). PowerSYBR Green PCR Master Mix (Life Technologies) was used in the same volume and conditions with primers specific for Il6, and Rxfp1 (Additional file 1: Table S1).
The expression of genes encoding the antioxidant enzymes superoxide dismutase 1 (Sod1), catalase (Cat), and glutathione (GSH) peroxidase 1 (Gpx1), as well as enzymes involved in GSH synthesis, namely GSH reductase (Gsr) and GSH synthase (Gss), was detected by qPCR. Additionally, we examined gene expression of Cyp11a1 encoding the rate-limiting enzyme P450scc for steroid biosynthesis. Cycling conditions were as follows: 95 °C for 2 min, and 40 cycles of 5 s at 95 °C and 30 s at 60 °C. Reactions were performed in triplicate on a Thermal Cycler Dice Real-Time System III TP950 (Takara Bio). TaqMan Fast Advanced Master Mix (Life Technologies) was used in a final volume of 20 μl containing Sod1, Cat, Gpx1, Gsr, Gss, Cyp11a1 and Gapdh probes designated as Rn00566938_m1, Rn00560930_m1, Rn00577994_g1, Rn01482159_m1, Rn00564188_m1, Rn00568733_m1, and Rn99999916_s1, respectively (TaqMan Gene Expression Assay; Life Technologies).
Testicular samples were homogenized in ice-cold 5 mM phosphate buffer (PB; pH 7.0) with an ULTRA-TURRAX T8 (IKA, Staufen, Germany), and centrifuged at 10,000 g for 20 min at 4 °C, after which the supernatant was subjected to the following assays. Protein concentrations were determined by the method of Lowry-Folin  with BSA as a standard.
Superoxide dismutase (SOD) was measured using a commercially available assay kit (Dojindo Laboratories, Kumamoto, Japan). The SOD assay was based on the generation of superoxide radicals produced by the xanthine–xanthine oxidase system, which reacts with nitroblue tetrazolium (NBT) to form a red formazan dye. SOD activity is then measured at 450 nm to determine the degree of inhibition of this reaction. One unit of SOD was defined as the enzyme activity causing 50% inhibition of the NBT reduction rate. SOD-like activity was expressed as U/mg protein.
CAT was measured according to the method of Johansson and Borg  and based on the reaction of CAT with methanol in the presence of a known concentration of H2O2. The formaldehyde produced was measured at 510 nm using Purpald solution (Sigma-Aldrich) as a chromogen. CAT-like activity was expressed as nmol/min/mg protein.
GPx was assayed according to the method of Yazdanparast et al. , with the activity measured based on the principle that oxidized GSH produced by GPx is reduced at a constant rate by GSH reductase using NADPH (Wako Pure Chemicals, Osaka, Japan) as a cofactor, which allows maintenance of predictable levels of reduced GSH. The oxidative rate of NADPH was monitored at 340 nm, and GPx activity was measured as nmol NADPH/min/mg protein.
Antioxidant GSH level was estimated according to the method of Ellman , which is not sensitive. Absorbance was noted spectrophotometrically at 412 nm, and GSH concentration was expressed as μmol/mg protein.
The lipid peroxidation marker and indirect indicator of ROS, malondialdehyde (MDA) was measured using a commercially available assay kit (BIOXYTECH MDA-586; OxisResearch, Portland, OR, USA), and MDA concentration was measured as nmol/mg protein.
Analyses of sperm morphology, movement and MDA levels
Epididymal sperm was collected from cauda epididymides as described by others . Briefly, the cauda epididymis was placed in modified Krebs−Ringer bicarbonate medium (m-KRB; pH 7.8) , minced with scissors to release spermatozoa and incubated for 30 min at 37 °C in an atmosphere of 5% CO2, after which the spermatozoa were used to evaluate sperm count, quality, motility and MDA levels.
It should be noted that sperm count results are relative, given that collection of sperm from different males can give significantly different results. The quality of epididymal sperm was assessed based on sperm morphology, as described previously . To assess sperm morphology, the percentages of dead, normal sperm, as well as abnormalities in the sperm head and tail were estimated by eosin-nigrosine staining [38, 39]. Sperm movement was analyzed using a Cellsoft CASA (Cryo Resources, Montgomery, NY, USA) [37, 38]. Sperm samples were diluted to 8 × 106/ml with m-KRB, introduced into the 2X-CEL chamber (Hamilton Thorne, Beverly, MA, USA) on a 37 °C heated microscope stage (Microwarm Plate 30; Kitazato, Tokyo, Japan), and videotaped multiple viewing areas. The percentage of motile sperm, curvilinear velocity (μm/s), linearity, beat-cross frequency (Hz), the mean amplitude of lateral head distance (ALH; μm) and the percentage of circular cells were determined for at least 1000 spermatozoa in each sample. For MDA measurement, spermatozoa were extracted by sonication in ice-cold 5 mM PB, and centrifuged at 10,000 g for 5 min at 4 °C, after which the supernatant was subjected to MDA assay.
Values were presented as means ± S.E.M. Data were analyzed by one-way ANOVA, together with Fisher’s Protected Least Significant Difference multiple range test using JMP 10 software (SAS Institute, Cary, NC, USA) to compare the means of different groups. P < 0.05 was considered statistically significant.