Mouse blastocysts release a lipid which activates anandamide hydrolase in intact uterus

M.Maccarrone 2 M.DeFelici 1 F.G.Klinger 1 N.Battista 0 F.Fezza 2 E.Dainese 2 G.Siracusa 1 A.Finazzi-Agro` 0 0 Experimental Medicine and Biochemical Sciences, University of Rome 'Tor Vergata' , Rome 00133 , Italy 1 Public Health and Cell Biology 2 Department of Biomedical Sciences, University of Teramo , Piazza A. Moro 45, Teramo 64100, Italy, and Departments of Anandamide (N-arachidonoylethanolamine, AEA) is a major endocannabinoid, known to impair mouse pregnancy and embryo development and to induce apoptosis in blastocysts. Here we show that mouse blastocysts rapidly (within 30 min of culture) release a soluble compound, that increases by ~2.5-fold the activity of AEA hydrolase (fatty acid amide hydrolase, FAAH) present in the mouse uterus, without affecting FAAH gene expression at the translational level. This 'FAAH activator' was produced by both trophoblast and inner cell mass cells, and its initial biochemical characterization showed that it was fully neutralized by adding lipase to the blastocyst-conditioned medium (BCM), and was potentiated by adding trypsin to BCM. Other proteases, phospholipases A2, C or D, DNAse I or RNAse A were ineffective. BCM did not affect the AEA-synthesizing phospholipase D, the AEA-binding cannabinoid receptors, or the selective AEA membrane transporter in mouse uterus. The FAAH activator was absent in uterine fluid from pregnant mice and could not be identified with any factor known to be released by blastocysts. In fact, platelet-activating factor inhibited non-competitively FAAH in mouse uterus extracts, but not in intact uterine horns, whereas leukotriene B4 or prostaglandins E2 and F2a had no effect. Overall, it can be suggested that blastocysts may protect themselves against the noxious effects of uterine endocannabinoids by locally releasing a lipid able to cross the cell membranes and to activate FAAH. The precise molecular identity of this activator, the first ever reported for FAAH, remains to be elucidated. - Endocannabinoids are amides, esters and ethers of long chain polyunsaturated fatty acids, found in several human tissues (Fowler et al., 2001; Hanus et al., 2001), and in human reproductive fluids (Schuel et al., 2002). Anandamide (N-arachidonoylethanolamine, AEA) and 2-arachidonoylglycerol are the main endocannabinoids described to date (Howlett and Mukhopadhyay, 2000). They bind to both type-1 (CB1) and type-2 (CB2) cannabinoid receptors, thus mimicking several central and peripheral effects of D9-tetrahydrocannabinol (THC), the main active ingredient of hashish and marijuana (Mechoulam and Hanus, 2000). At the periphery, AEA and 2-arachidonoylglycerol show cardiovascular (Kunos et al., 2000), immune (Parolaro et al., 2002) and anti-inflammatory activities (De Petrocellis et al., 2000). Moreover, endogenous cannabinoids have been involved in the inhibition of human breast and prostate cancer cell proliferation (Melck et al., 2000). The effect of AEA via CB1 and CB2 receptors depends on its extracellular concentration, which is controlled by (i) cellular uptake by a specific AEA membrane transporter (AMT), and (ii) intracellular degradation by the AEAhydrolysing enzyme fatty acid amide hydrolase (FAAH). AMT (Hillard and Jarrahian, 2000) and FAAH (Ueda et al., 2000) have been characterized in several mammalian cells and tissues, and together with AEA and congeners form the endocannabinoid system. Evidence is accumulating about endocannabinoid modulation of embryouterine interactions (Paria and Dey, 2000), and impairment of pregnancy and embryo development in mice (Yang et al., 1996), thus recalling the adverse effects of THC on reproduction (Hall and Solowij, 1998). More recently, progesterone has been shown to be involved in THC modulation of sexual receptivity in female rats (Mani et al., 2001), and dysregulation of cannabinoid signalling has been shown to disrupt uterus receptivity to embryo implantation in mice (Paria et al., 2001). Along this line, we reported the association between decreased FAAH activity and expression in maternal peripheral lymphocytes and early pregnancy failure in humans, demonstrating that an impairment of AEA degradation might be connected with reduced fertility (Maccarrone et al., 2000a). Consistently, down-regulation of AEA levels in mouse uterus has been associated with increased uterine receptivity, which instead decreases when AEA is up-regulated (Yang et al., 1996; Schmid et al., 1997). Mouse uterus contains the highest levels of AEA so far detected in mammalian tissues, and is the only tissue where AEA appears to be the main (up to 95%) N-acylethanolamine (Schmid et al., 1997). Furthermore, the developmental arrest that occurs in mouse blastocysts in a non-receptive uterine environment correlates well with its higher levels of AEA and with the in vitro observation that AEA inhibits embryo development and zona-hatching of blastocysts (Paria et al., 1996; Yang et al., 1996; Schmid et al., 1997). Mouse blastocysts express a biologically active CB1 receptor, together with specific AMT and FAAH (Maccarrone et al., 2000b). They are driven to apoptosis by AEA, suggesting that an efficient degradation of this compound might be instrumental in preventing its blastotoxic effects (Maccarrone et al., 2000b). As a matter of fact, recent observations on FAAH mRNA accumulation (Paria et al., 1999) and FAAH activity (Maccarrone et al., 2000b) in pregnant mice suggest that this enzyme may be the checkpoint for maintaining the steady levels of AEA. Indeed, FAAH activity has recently been shown to correlate inversely with AEA levels in various tissues of FAAH knockout mice versus wild-type animals (Cravatt et al., 2001) and in blood of women with normal gestation versus those who aborted (Maccarrone et al., 2002). Here, we report that blastocysts release a soluble factor able to activate uterine FAAH, thus reducing the level of AEA and its negative effects on blastocyst survival and implantation. Materials and methods Materials and enzymes All chemicals were of the purest analytical grade. Leukotriene B4 (LTB4) and prostaglandins E2 (PGE2) and F2a (PGF2a) were from Cayman Chemical Company (USA). Platelet activating factor-16 (1-O-palmityl-2-acetylsn-glycero-3-phosphocholine; PAF-16) and -18 (1-O-stearyl-2-acetyl-snglycero-3-phosphocholine; PAF-18), deoxyribonuclease I (from bovine pancreas; DNAse I), ribonuclease A (from bovine pancreas; RNAse A), phospholipases C (from Bacillus cereus) and D (from Streptomyces chromofuscus), and lipase (from Chromobacterium viscosum) were from Calbiochem (USA). Anandamide (N-arachidonoylethanolamine, AEA), cycloheximide, phospholipase A2 (from Apis mellifera), trypsin and achymotrypsin (from bovine pancreas), bromelain (from pineapple stem), papain (from Papaya latex), and pepsin A (from porcine stomach) were from Sigma Chemical Company (USA). Dulbeccos modified Eagles medium (DMEM) and fetal calf serum (FCS) were from Life Technologies (Italy). N-(4hydroxyphe (...truncated)