Treatment of animals
The experimental protocols were approved by the Ethical Committee for the Use of Laboratory Animals of the UNESP—Univ Estadual Paulista, Campus de Dracena, SP, Brazil (Protocol number 19/2012). Male Wistar rats weighing approximately 200 g were used in this study. The animals were obtained from the Central Bioterium of UNESP—Univ Estadual Paulista, Campus de Botucatu, SP, Brazil, and were maintained with a maximum of four rats per cage under standard laboratory conditions with water and food provided ad libitum.
The rats were randomly divided into four groups of ten animals each, according to the following treatments: Group 1 (G1) received corn oil by gastric gavage and a mixture of dimethyl sulfoxide (DMSO) and saline (0.9% NaCl) i.p.; Group 2 (G2) received vitamin E (100 mg/kg BW) dissolved in corn oil by gastric gavage and a mixture of DMSO and 0.9% NaCl i.p.; Group 3 (G3) received Gossypol Acetic Acid (5 mg/kg BW) dissolved in a mixture of DMSO and 0.9% NaCl i.p. and corn oil by gastric gavage and Group 4 (G4) received Gossypol Acetic Acid (5 mg/kg BW) dissolved in a mixture of DMSO and 0.9% NaCl i.p. and vitamin E (100 mg/kg BW) dissolved in corn oil by gastric gavage. Gossypol and vitamin E dose selection was based on previous reports [13, 14].
Fertility test
Fertility was estimated in four male rats of each group. After 14 days of treatment, each male was placed in an individual cage with two virgin untreated females of the same strain. They were left together for 10 days during which two estrus cycles should have elapsed [15]. After this period, the male rats were removed and the females kept in separate boxes until delivery. Once the rats calved, the quantity and the weight of offspring in each group were assessed.
Sperm counting
After 14 days of treatment six of the animals of each group were euthanized by decapitation and the testes and epididymides were collected.
The tail of the epididymides, previously cut into small pieces with scissors, was used for semen collection and the subsequent counting of sperm.
For the analysis of the total number of epididymal sperm, the epididymal tail of each animal was placed in 10 mL of normal saline (0.9% NaCl) and homogenized under cooling. One hundred microliters of the resulting mash of each epididymis was placed in an individual “eppendorf” type tube and 900 µL of 0.9% NaCl was added to a final volume of 1 mL. The number of sperm in this obtained solution was counted in 128 small squares of a Neubauer chamber. Counting was performed in an optical microscope with 40× magnification. The number of spermatozoa was determined using the following formula:
$${\text{S}} = {\text{C }} \times {\text{ V }} \times {\text{ CF}}$$
where S = Sum total per animal; C = number of counted spermatozoa; FC = factor of the camera (1.25) and V = dilution (106).
Homogenate preparation
The tunica albuginea and the main vessels were removed and each testis was placed in 25 mL of medium containing 250 mM sucrose, 0.2 mM EGTA, 0.1 mM EDTA, 5 mM HEPES–KOH (pH 7.4) and 0.1% bovine serum albumin (BSA), maintained at 4°C and then sliced and homogenized with a Potter-Elvehjem homogenizer. The protein concentration of the homogenate was determined using the biuret reaction with BSA as a standard [16].
Glutathione peroxidase activity
The activity of glutathione peroxidase (GPx) was determined by an indirect method based on the oxidation of GSH to GSSG, with the consequent oxidation of NADPH catalyzed by glutathione peroxidase [17]. One milliliter of 0.1 mM sodium phosphate buffer, pH 7.6, with 0.5 mM EDTA, 10 µL of 10% Triton X-100, testis homogenate (1 mg of protein) and 10 µL of 100 mM GSH and 10 µL of 25 mM H2O2 were added to 4 mL quartz cuvettes. After incubating the samples at 30°C for 5 min, 10 µL of 20 mM NADPH was added, and the variation in absorbance was determined at a wavelength of 340 nm in a spectrophotometer (Beckman-Coulter model DU-800, Fullerton, CA, USA). The oxidation of 1 µmol NADPH/min was used as a unit of GR. The specific activity was expressed as unit per mg of protein.
Glutathione reductase activity
The activity of glutathione reductase (GR) was determined based in the reduction of GSSG to GSH by monitoring the oxidation of NADPH [18]. One milliliter of 0.1 mM sodium phosphate buffer, pH 7.6, with 0.5 mM EDTA, 10 µL of 10% Triton X-100, testis homogenate (1 mg of protein) and 10 µL of 100 mM GSSG were added to 4 mL quartz cuvettes. After incubating the samples at 30°C for 5 min, 10 µL of 10 mM NADPH was added, and the variation in absorbance was determined at a wavelength of 340 nm in a spectrophotometer (Beckman-Coulter model DU-800, Fullerton, CA, USA). The oxidation of 1 µmol NADPH/min was used as a unit of GR. The specific activity was expressed as unit per mg of protein.
Glutathione assay
The levels of GSH and GSSG were determined by a fluorometric reaction with o-phthalaldialdehyde (OPT) [19]. Testis homogenate (1 mg of protein) was added to medium (125 mM sucrose, 65 mM KCl and 10 mM HEPES–KOH, pH 7.4) to a final volume of 1 mL and treated with 0.5 mL of 13% trichloroacetic acid. The mixture was stirred and then centrifuged at 9,000×g for 3 min. For GSH levels, aliquots (100 µL) of the supernatant were mixed with 2 mL of 100 mM NaH2PO4 buffer at pH 8.0 containing 5 mM EGTA. One hundred microliters of a OPT solution (1 mg/mL) was added, and the fluorescence was measured 15 min later in a spectrofluorometer (Shimadzu-RFPC 5301, Tokyo, Japan) using 350/420 nm as the excitation/emission wavelength pair.
For GSSG levels, the supernatant was treated with 20 mM N-ethylmaleimide, which reacts with free thiol groups. Aliquots (100 µL) of samples were mixed with 1 mL of 1 M NaOH followed by OPT. The data are expressed in nmol/mg protein estimated using a standard curve.
Determination of NADPH level
Testis homogenate (2.0 mg protein) was added to medium (125 mM sucrose, 65 mM KCl and 10 mM HEPES–KOH, pH 7.4) to a final volume of 2.0 mL and centrifuged at 8,000×g for 3 min. The supernatant was collected, and the fluorescence was measured in a spectrofluorometer (Shimadzu-RFPC 5301, Tokyo, Japan) using 366/450 nm as the excitation/emission wavelength pair. The data are expressed in relative fluorescence units.
Membrane lipid peroxidation (LPO) assay
The level of LPO was estimated by malondialdehyde (MDA) generation [20]. The testis homogenate (5 mg of protein) was added to a tube. Following the addition of 0.2 mL of 8.1% SDS, 1.5 mL of 20% acetic acid and 1.5 mL of 0.67% thiobarbituric acid (TBA, aqueous solution), glass-distilled deionized water was added to a final volume of 4 mL. The mixture was incubated for 60 min at 85°C. The MDA-TBA complex was extracted with 5 mL of n-butanol and the absorbance was measured at 535 nm in a Genesys 10 UV spectrophotometer (Thermo Spectronic, Rochester, NY, USA). The MDA concentration was calculated with ε = 1.56 × 105/M/cm.
Isolation of testicular mitochondria
Testis mitochondria were prepared according to the methodology of Amaral et al. [21]. Part of the homogenate was centrifuged at 2,500×g for 10 min, and the supernatant fluid centrifuged at 10,000×g for 10 min. The pellet (mitochondrial fraction) was resuspended and repelleted twice at 10,000×g for 10 min. EGTA, EDTA and defatted BSA were omitted from the washing medium. Mitochondrial protein content was determined by the biuret method.
ATP quantification
ATP levels were determined using the firefly luciferin–luciferase assay system [22]. The mitochondrial suspension (1 mg protein) was suspended in 1 mL of a medium containing 65 mM KCl, 125 mM sucrose and 10 mM HEPES–KOH, pH 7.2 and centrifuged at 12,000×g for 10 min at 4°C, and the pellet was treated with 1 mL ice-cold 1 M HClO4. After centrifugation at 12,000×g for 10 min at 4°C, 100 µL aliquots of the supernatants were neutralized with 5 M KOH, suspended in 100 mM TRIS–HCl, pH 7.8 (1 mL final volume), and centrifuged at 12,000×g for 10 min. The supernatant was processed with a Sigma/Aldrich assay kit (Catalog Number FLAA) according to the manufacturer’s instructions and measured using a SIRIUS luminometer (Berthold, Pforzheim, Germany).
Statistical analysis
Significant differences were calculated by one-way analysis of variance (ANOVA) followed by the Tukey test using the GraphPad Prism software, version 4.0 for Windows (GraphPad Software, San Diego, CA, USA). Values of P < 0.05 were considered significant.