Spermatozoa were collected from caput and cauda of epididymis as described previously. The packed cells were subjected to lyses in tripledetergent buffer (PBS pH 7.4 containing 0.02% sodium azide, 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 4 ig/ml of leupeptin, aprotinin, pepstatin A, chymostatin, and 5 ig/ml of TPCK) and then submitted three times to sonication for 5 min, each time at 25 mW. Proteins were separated using 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose filter (GHealth Care) at 280 mA for 2.5 h at 4°C in order to evaluate CNR1 immunoreactivity. Filters were treated for 3 h to prevent nonspecific binding with blocking solution (5% nonfat powdered milk, 0.25% Tween-20 in Tris-buffered saline [TBS], pH 7.6) and then incubated with the primary antibody (anti-CNR1 Ab diluted 1:1000) in PBS 3% nonfat powdered milk solution overnight at 4°C on an orbital shaker. read
We used a polyclonal antibody directed against N-terminus domain (first 77 residues) of rat CNR1 antigen. Specificity of these antibodies has been investigated extensively in several species and was confirmed in the present experiments using the antibody previously preabsorbed for 18 h at 4°C on orbital shaker with a large excess (2 ig) of the corresponding antigen (peptide). Filters were washed in TBS-0.25% Tween20, incubated with 1:1000 horseradish peroxidase-conjugated IgG (DAKO) in TBS-1% normal swine serum (NSS; DAKO), and then washed three times in TBS-0.25% Tween20. The immune complexes were detected using the ECL-Western blotting detection system (GHealth Care) following the manufacturer’s instructions. The membranes, stripped at 60°C for 30 min in stripping buffer (100 mM 2-mercaptoetanol, 2% SDS, and 62.5 mM Tris-HCl, pH 7.6), were reprobed with MAPK1 (housekeeping gene) antibody (sc-154; Santa Cruz Biotechnology) to quantify protein content.
In Vivo Treatments
WT mice were treated on alternate days with intraperitoneal (i.p.) injections of vehicle (diluted ethanol or DMSO according to the drug producer’s indication), AM281 (1 mg/kg), SR (1 mg/kg), AM404 (10 mg/kg), or OMDM-1 (1 mg/kg). The treatment lasted 7 d (the period needed to move from caput to cauda), and at the end of this period, spermatozoa from caput and cauda of epididymis were separately collected, and the number of motile and viable cells was evaluated as described previously.
In Vitro Treatments of Spermatozoa
Spermatozoa separately collected from caput and cauda of epididymis were washed to get rid of epididymal endocannabinoids and treated with vehicle (0.05% ethanol or 0.005% DMSO according to relative drug concentrations used for each experiment), AEA (0.1 iM, 1 iM, 10 iM), SR141716A (1 iM, 10 iM), AM281 (10 iM), CPS (1 iM), CPZ (10 iM), or IRTX (0.5 iM). Each treatment lasted 15 min and was carried out at room temperature. Afterward, the number of motile and viable cells was evaluated as described previously. Drugs were given alone or in combination as indicated in the figures.