All drugs (Table 1)—Anandamide (AEA; Sigma Aldrich); SR141716A (SR; Sanofy) or AM281 (Sigma Aldrich) selective CNR1 (CB1) antagonists; AM404 (Cayman Chemical) or OMDM-1 (Cayman Chemical) EMT inhibitors; Capsaicin (CPS), a TRPV1 agonist (Tocris Biosciences); Capsazepin (CPZ; Tocris Biosciences) or Iodoresiniferotoxin (IRTX; LC Laboratories) selective TRPV1 antagonists—had the purest analytical grade and were solved in ethanol or dimethyl sulfoxide (DMSO) according to the manufacturer’s instructions. canadian health mall
Wild-type (WT) (CD1) and Cnr1 knockout (KO) mice were purchased by Charles River (Charles River Laboratories) and maintained under constant temperature (22°C) and lighting (12L:12LD), with food and water provided ad libitum.
All animal studies were carried out in accordance with the principles and procedures outlined in the National Institute of Health Guide for Care and Use of Laboratory Animals and approved by the Italian Ministry of Research and the Italian Ministry of Health.
WT and/or CnrlKO mice were killed by CO2 asphyxia, and the removed epididymis were cut in caput and cauda for spermatozoa collection. Tissues (caput and cauda) were immersed separately in phosphate-buffered saline (PBS; 13.6 mM NaCl; 2.68 mM KCl; 8.08 mM Na2HPO^ 18.4 mM KH2PO^ 0.9 mM CaCl2; 0.5 mM MgCl2; pH 7.6) and cu2t into4 a few pieces 2to le4t spermatozoa flow out from the ducts. Then spermatozoa samples were filtered, and the eluted solution (approximately 1 X 106 sperm/ml, 4 ml for caput and 6 ml for cauda) was analyzed to evaluate the number of viable and motile cells.
Spermatozoa samples used for lipid extraction were sequentially centrifuged to time at 7000 X g at 4°C to accurately get rid from supernatant. Weighed pellets were immediately frozen and stored at —80°C. Sampling was carried out within 5 min. Preliminarily, we have verified that sperm motility and viability are not affected by sampling procedure or by in vitro incubation.
Analysis of WT and Cnr1 KO Spermatozoa
We used WT (n = 8 animals) and Cnr1 KO (n = 6 animals) mice to analyze number of viable and motile spermatozoa from caput and cauda of epididymis. Number of viable spermatozoa was evaluated using propidium iodide and flow cytometric analysis (FACS-Calibur Flow Cytometer). Number of motile spermatozoa was evaluated by two observers under a light microscope using hemocytometer (Burker Chamber). This procedure was validated using doubleblind test.
In WT control animals, immotile sperm and sluggish, irregular, as well as circular (non progressive) motion was detected in caput, while vigorous progressive motility was detected in cauda spermatozoa. Data were plotted as the percentage of motile/live spermatozoa.