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- MATERIALS and METHODS
Endocannabinoids are an emerging class of lipid mediators, which mimic several effects of cannabinoids. Anandamide (arachidonoylethanolamide) is a major endocannabinoid, which has been shown to impair pregnancy and embryo development. The activity of anandamide is controlled by cellular uptake through a specific transporter and intracellular degradation by the enzyme anandamide hydrolase (fatty acid amide hydrolase, FAAH). We characterized FAAH in mouse uterus by radiochromatographic and immunochemical techniques, showing that the enzyme is confined to the epithelium and its activity decreases appreciably during pregnancy or pseudopregnancy because of lower gene expression at the translational level. Ovariectomy prevented the decrease in FAAH, and both progesterone and estrogen further reduced its basal levels, suggesting hormonal control of the enzyme. Anandamide was shown to induce programmed cell death in mouse blastocysts, through a pathway independent of type-1 cannabinoid receptor. Blastocysts, however, have a specific anandamide transporter and FAAH, which scavenge this lipid. Taken together, these results provide evidence of an interplay between endocannabinoids and sex hormones in pregnancy. These findings may also be relevant for human fertility, as epithelial cells from healthy human uterus showed FAAH activity and expression, which in adenocarcinoma cells was increased fivefold.
Endocannabinoids are an emerging class of lipid mediators, isolated from brain and peripheral tissues [1,2] and found also in chocolate  and milk . They mimic the psychotropic, hypnotic, tranquilizing, antiemetic, anticonvulsive and analgesic effects of cannabinoids [5,6]. These compounds, in particular Δ9-tetrahydrocannabinol, have been reported to have adverse effects on reproductive functions, including retarded embryo development, fetal loss and pregnancy failure [7,8]. A major endocannabinoid, anandamide (arachidonoylethanolamide), has been shown to impair pregnancy and embryo development . Down-regulation of anandamide levels in mouse uterus has been associated with uterine receptivity, while up-regulation correlated with uterine refractoriness to embryo implantation . Remarkably, mouse uterus contains the highest levels of anandamide detected so far in mammalian tissues, and is the only tissue in which anandamide is the main component (up to 95%) of N-acylethanolamines . Anandamide is an endogenous ligand for both the brain-type (CB1-R) and spleen-type (CB2-R) cannabinoid receptors, mimicking several actions of cannabinoids on the central nervous system and in peripheral tissues . Mouse embryos express both CB1-R and CB2-R mRNA, the levels of the former being much higher than those found in brain [7,9,10]. CB1-R activation is detrimental for mouse preimplantation development [10,12], but appears to accelerate trophoblast differentiation .
The effect of anandamide through CB1-R and CB2-R depends on its concentration in the extracellular space, which is controlled by a two-step process: (a) cellular uptake by a specific anandamide transporter, and (b) intracellular degradation by the enzyme anandamide hydrolase (fatty acid amide hydrolase, FAAH). Anandamide transporter and FAAH have been characterized in mammalian cell lines [14–16] and more recently in human cells in culture and brain . FAAH activity has also been demonstrated in mouse uterus , although the methodology used was not suitable for accurate kinetic analysis. FAAH mRNA level has been measured in both mouse uterus and embryos during the peri-implantation period . Using a very sensitive radiochromatographic method , in the present study we characterize FAAH activity in both mouse uterus and embryos during early pregnancy. FAAH expression was also measured at the protein level, showing that the enzyme is localized in the endometrial epithelium, and that sex hormones down-regulate FAAH activity and expression in early pregnancy. Anandamide transporter and FAAH were also demonstrated and characterized in mouse embryos, suggesting that anandamide degradation may be instrumental in preventing anandamide-induced programmed cell death (apoptosis) of developing embryos.
- Top of page
- MATERIALS and METHODS
In this study we characterized FAAH activity and expression in both mouse uterus and embryos during early pregnancy. FAAH was localized in the endometrial epithelium and was down-regulated by sex hormones. Under our experimental conditions, the optimum pH and temperature of mouse uterus FAAH were determined, and the kinetic constants of enzyme activity (Fig. 1A) were calculated. Remarkably, the observed apparent Km and Vmax were one order of magnitude smaller than those calculated with column filtration procedures , far from the resolution and reproducibility of HPLC. Moreover, the observed molecular mass of FAAH (Fig. 1B) was in agreement with that expected from the size of FAAH mRNA in mouse uterus . Activity and expression of FAAH decreased during early pregnancy (Fig. 1C), an observation that, together with kinetic analysis of the enzyme at the different stages of pregnancy, suggests lower expression of the same gene, rather than the presence of FAAH isozymes with different kinetic properties. Recent observations on FAAH mRNA accumulation in pregnant mice corroborate this concept .
Despite the growing evidence that anandamide adversely affects uterine receptivity and embryo implantation [7,10,12,13] and that anandamide degradation by FAAH may have physiological significance in these processes [9,18], regulation of FAAH during early pregnancy is still obscure. In this study, we used two different methods of manipulating pregnancy-related uterine changes (pseudopregnancy and ovariectomy-induced delayed implantation) as tools to understand the relative roles played by the embryo and sex hormones in modulating the above changes in FAAH activity and expression in the pregnant mouse. Pseudopregnancy occurs in female mice and rats mated to a sterile male: the uterus undergoes all the normal changes that prepare it for implantation, but no embryos are present in the uterine lumen . Therefore, down-regulation of FAAH expression in pseudopregnant mice (Fig. 1D) was independent of the presence of embryos in the uterus. However, the decrease in FAAH activity was more marked in the presence of embryo (compare Fig. 1C and D), suggesting that the latter may release an enzyme inhibitor, the molecular identity of which is as yet unknown. On the other hand, delayed implantation is a phenomenon that occurs when a pregnant female mouse is ovariectomized before implantation: the preparation of the uterus for implantation is arrested and the blastocyst remains in a state of suspended development . Hormonal administration is rapidly followed by uterine changes that make it receptive to the implanting blastocyst. The observation that the fall in FAAH activity and expression was significantly lower in ovariectomized animals than controls, and that estrogen treatment reversed this effect (Fig. 2A), suggests that sex hormones may regulate FAAH activity by modulating gene expression at the translational level. The results of the treatment of virgin females with progesterone or estrogen (Fig. 2B) corroborated this hypothesis. It should be stressed that hormone treatment may increase protein synthesis in the uterus [30,31]. Thus, a change in FAAH specific activity (i.e. activity per mg protein) may be seen in hormone-treated animals rather than a true decrease. To check this possibility, we also measured the total activity of FAAH (i.e. activity per uterine horn). In Fig. 2B (hatched bars), total activity showed the same trend as specific activity, ruling out the possibility that the observed decrease was apparent. Therefore, it can be concluded that sex hormones down-regulate FAAH activity by reducing gene expression at the level of protein synthesis. This is a demonstration of a direct interplay between endocannabinoid degradation and sex hormones in mammals. Also anandamide synthase activity was measured in mouse uterus, and was found to respond to sex hormones in the same way as FAAH. Although it is still under debate whether or not anandamide hydrolase and synthase activities belong to the same [22,32] or different [9,18] enzymes, these data demonstrate that the two activities are under the same hormonal control.
FAAH was localized in the luminal (Fig. 3A) and glandular (Fig. 3B) epithelia of nonpregnant mouse uterus. In situ hybridization consistently detected FAAH mRNA primarily in uterine luminal and glandular epithelial cells . Human uterine epithelial cells also showed appreciable FAAH activity, which was more than five times higher in human adenocarcinoma cells (Fig. 4). These findings are consistent with an epithelial localization for FAAH in the human endometrium also. In this context, it is noteworthy that the Km values for FAAH from mouse and human uterus were comparable with those recently reported for human brain, human neuroblastoma and lymphoma cell lines, whereas Vmax values varied . Therefore, it can be proposed that the same enzyme is differently expressed in various species and in different tissues of the same species. Sequence homology between rat, mouse and human FAAH genes  suggests that the FAAH gene is indeed highly conserved. Therefore, the observations reported here on the hormonal regulation of FAAH in mouse uterus may also hold true for the human counterpart.
It can be proposed that down-regulation of FAAH during early pregnancy may allow higher local levels of anandamide, which indeed have been shown to increase with pregnancy in the mouse uterus . In turn, anandamide may play a role in the endometrial changes associated with pregnancy, for instance through inhibition of gap junctions and intercellular calcium signaling [33,34]. Interestingly, anandamide has been recently reported to mobilize intracellular calcium , thus also perturbing intracellular signaling.
FAAH activity was demonstrated and characterized in mouse blastocysts (Fig. 5A). To be hydrolyzed by FAAH, anandamide must be transported into the cell. Recent experiments performed on rat neuronal and leukemia cells  and human neuronal and immune cells  clearly showed the presence of a high-affinity anandamide transporter in the cell outer membranes. A similar transporter was found in mouse blastocysts (Fig. 5B). The affinity of this transporter was comparable with that of the anandamide carrier in rat astrocytes (Km = 320 nm)  and human cells (Km = 130–200 nm) . The anandamide transporter and FAAH of blastocysts may play a critical role, because nanomolar concentrations of anandamide inhibited embryo development and blastocysts hatching in vitro (Fig. 5C). Both detrimental effects of anandamide were inhibited by a CB1-R antagonist, in line with the hypothesis that they were mediated by the type-1 cannabinoid receptor . Interestingly, anandamide-induced apoptosis (Fig. 5D) was not prevented by CB1-R or CB2-R antagonists, ruling out the involvement of either cannabinoid receptor in the induction of programmed cell death. Therefore, the arrest of embryo development and blastocyst hatching by anandamide did not involve the deployment of apoptotic programs [36,37]. As yet, only one report has demonstrated the ability of anandamide to inhibit cancer cell proliferation , and another gave preliminary evidence on its ability to induce apoptosis in lymphocytes . It is noteworthy that Δ9-tetrahydrocannabinol has been recently shown to promote apoptosis in glioma cells, through a CB1-R independent mechanism .
Collectively, our findings lead to a general picture suggesting that decreased FAAH activity in the mouse uterus during early pregnancy may allow higher levels of anandamide to accumulate, which may be instrumental in modifying the endometrium during pregnancy. The detrimental effects of anandamide on the blastocysts are prevented by the presence of an efficient anandamide transporter and FAAH in these cells, which rapidly scavenge this lipid. These events are under hormonal control, showing an interplay between endocannabinoids and sex hormones in regulating fertility in mammals.