451.full-2
FIG. 1. Modulation of the number of motile spermatozoa in caput and cauda epididymis. Sperm analysis on WT or Cnr1 KO mice (A). Sperm analysis after in vivo treatment with the CNR1 antagonist AM281 (B) or SR (C). Data are means 6 SEM. SPZ, spermatozoa. more

Lipid Extraction and Endocannabinoid Measurement

Tissues or cells were homogenized in 5 volumes of chloroform/methanol/ Tris HCl 50 mM (2:1:1) containing 10 pmol of d8-AEA and 50 pmol of d5-2AG. Homogenates were centrifuged at 13 000 X g for 16 min (4°C), and the aqueous phase plus debris were collected and extracted four times with 1 volume of chloroform. The lipid-containing organic phases were dried down and prepurified by open bed chromatography on silica columns eluted with increasing concentrations of methanol in chloroform. Fractions for AEA and 2-AG measurement were obtained by eluting the columns with 9:1 (by volume) chloroform/methanol and then directly analyzed by liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS). LC-APCI-MS analyses were carried out in the selected ion monitoring mode as described in Di Marzo et al., using m/z values of 356 and 348 (molecular ions +1 for deuterated and undeuterated AEA) and 384.35 and 379.35 (molecular ions +1 for deuterated and undeuterated 2-AG). AEA and 2-AG levels were therefore calculated on the basis of their area ratios with the internal deuterated standard signal areas, their amounts in pmol normalized per gram of cells or tissue.

DAG Lipase Enzymatic Assay

Membrane fractions obtained by differential centrifugation as described in Bisogno et al. were incubated in the presence of test compounds with sn-1-[14C]-oleoyl-2-arachidonoylglycerol in 50 mM Tris-HCl, pH 7, for 20 min at 37°C, synthesized in our laboratory as previously described. After the incubation, lipids were extracted with 2 volumes of CHCLj/MeOH 2:1 (by volume). The organic phases containing lipids were fractionated by TLC on silica on polypropylene plates using CHCl3/MeOH/NH4OH (85:15:1 by volume) as the eluting system. The bands corresponding to [14C]-oleic acid were cut, and radioactivity was measured with a p-counter.

MAG Lipase Enzymatic Assay

Cytosolic fraction obtained by differential centrifugation as described in Bisogno et al. were incubated in the presence of test compounds with 2-[3H]-arachidonoylglycerol, synthesized in our laboratory as previously described in 50 mM Tris-HCl, pH 7, for 20 min at 37°C. After the incubation, lipids were extracted with 2 volumes of CHCLj/MeOH 2:1 (by volume). The organic phases containing lipids were fractionated by TLC on silica on polypropylene plates using CHCl3/MeOH/NH4OH (85:15:1 by volume) as the eluting system. The band corresponding to [3H]-arachidonic acid were cut, and radioactivity was measured with a p-counter.

Category: Cell Start-Up / Tags: arachidonoyl-glycerol, intracellular sites, lipid mediators