Caput or cauda of the epididymis was homogenized in 1 ml of Trizol (Invitrogen). Total RNA was extracted according to the manufacturer’s recommendations, dissolved in RNA storage solution (Ambion), UV quantified by a Bio-Photometer (Eppendorf), and stored to —80°C. RNA aliquots (6 ig) were digested by RNAse-free DNAse I (Ambion deoxyribonucleic acid [DNA]-free kit) in a 20-il final volume reaction mixture to remove contaminating genomic DNA. After DNAse digestion, concentration and purity of RNA samples were evaluated by the RNA-6000-Nano microchip assay, using a 2100 Bioanalyzer equipped with a 2100-Expert-Software (Agilent), following the manufacturer’s instructions. For all samples tested, the RNA integrity number was greater than 6 (relatively to a 0-10 scale). Three micrograms of total RNA, as evaluated by the 2100 Bioanalyzer, were reverse transcribed in a 25-il reaction mixture containing 50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgC^, 10 mM dithiothreitol, 1 mM deoxyribonucleotide triphosphates, 20 U of RNAse inhibitor (Invitrogen), 0.125 A260 units of hexanucleotide mixture (Invitrogen) for random priming, and 200 U of MoMuLV Superscript III reverse transcriptase (Invitrogen).
The reaction mixture was incubated in a thermocycler iCycler-iQ for a 5 min at a 55°C step, followed by a rapid chilling for 2 min at 4°C. The protocol was stopped at this step, and the MoMuLV reverse transcriptase was added to the samples, except for the negative controls (-RT). The incubation was resumed by two thermal steps: 10 min at 20°C followed by 90 min at 50°C. Finally, the reaction was terminated by heating at 95°C for 10 min. Quantitative real-time PCR was performed by an iCycler-iQ in a 25-il reaction mixture containing 1X iQ-SYBR-Green-Supermix (Bio-Rad), 20 ng of complementary DNA (cDNA; calculated on the basis of the retrotranscribed RNA), and 330 nM for each primer. The amplification profile consisted of an initial denaturation of 2 min at 94°C and 40 cycles of 30 sec at 94°C, annealing for 30 sec at optimum annealing temperature (TaOpt; see the following discussion), and elongation for 45 sec at 68°C. Fluorescence data were collected during the elongation step. further
A final extension of 7 min was carried out at 72°C, followed by melt-curve data analysis. Optimized primers for SYBR-Green analysis (and relative TaOpt) were designed by the Beacon-Designer software 6.0 version (Biosoft International) and were synthesized (high-performance liquid chromatography purification grade) by MWG-Biotech AG. Assays were performed in quadruplicate (maximum DCt of replicate samples less than 0.5), and a standard curve from consecutive fivefold dilutions (100-0.16 ng) of a cDNA pool representative of all samples was included for PCR efficiency determination. Relative expression analysis, correct for PCR efficiency and normalized with respect to reference gene p-actin, was performed by GENEX software (Bio-Rad) for groupwise comparison and statistical analysis.
Student t-test or analysis of variance (ANOVA) followed by the Duncan test for multigroup comparison was performed, when appropriate, to evaluate the significance of differences for number of motile spermatozoa. Data were expressed as the mean 6 SEM from at least three or four independent experimental procedures.
ANOVA followed by the Bonferroni test was used to evaluate the significance of differences for endocannabinoid levels in spermatozoa and epididymis. Data were expressed as the mean 6 SEM from at least three or four independent assays.
Tukey and Student-Newman-Keuls tests were carried out to evaluate the significance of differences for endocannabinoid levels in spermatozoa and epididymis of animals treated with OMDM-1. Data were expressed as the mean 6 SEM from at least three or four independent assays.