SEARCH

SEARCH BY CITATION

Keywords:

  • anandamide hydrolase;
  • apoptosis;
  • development;
  • endocannabinoids;
  • sex hormones.

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS and METHODS
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

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.

Abbreviations
FAAH

fatty acid amide hydrolase

CB1/2-R

type 1/2 cannabinoid receptor

AACOCF3

arachidonoyltrifluoromethylketone

AM404

N-(4-hydroxyphenyl)-arachidonoylamide.

Endocannabinoids are an emerging class of lipid mediators, isolated from brain and peripheral tissues [1,2] and found also in chocolate [3] and milk [4]. 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 [9]. Down-regulation of anandamide levels in mouse uterus has been associated with uterine receptivity, while up-regulation correlated with uterine refractoriness to embryo implantation [10]. 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 [10]. 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 [11]. 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 [13].

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 [17]. FAAH activity has also been demonstrated in mouse uterus [9], 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 [18]. Using a very sensitive radiochromatographic method [19], 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.

MATERIALS and METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS and METHODS
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Materials

Chemicals were of the purest analytical grade. Anandamide, arachidonic acid, ethanolamine, minimal essential medium and M2 culture medium, phenylmethanesulfonyl fluoride, 33258 Hoechst, arachidonoyltrifluoromethylketone (AACOCF3) and N-(4-hydroxyphenyl)arachidonoylamide (AM404) were purchased from Sigma. N-Piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide (SR 141716) and N-{1(S)-endo-1,3,3-trimethylbicyclo[2.2.1] heptan-2-yl}-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)pyrazole-3-carboxamide (SR 144528) were gifts from Sanofi Recherche (Montpelier, France). [3H]Anandamide (223 Ci·mmol−1 = 8251 GBq·mmol−1) and [3H]arachidonic acid (100 Ci·mmol−1= 3700 GBq·mmol−1) were purchased from NEN DuPont de Nemours (Koln, Germany).

Biological material

CD-1 mice were purchased from Charles River Laboratories. Females were superovulated at 5–8 weeks of age by intraperitoneal injection of 5 U pregnant mare's serum gonadotropin (Folligon, Intervet, Italy), followed 48 h later by 5 U human chorionic gonadotropin (Corulon, Intervet, Italy). They were then mated overnight, and the presence of a vaginal plug the next morning was taken to indicate day 0.5 of pregnancy. FAAH activity and content (see below) were determined in samples of uteri obtained from nonpregnant, pregnant, pseudopregnant and ovariectomized pregnant mice. To obtain pseudopregnancy, normal females were mated with vasectomized males. Delayed implantation was obtained by surgically removing the ovaries from pregnant mice on day 2.5 of pregnancy; ovariectomized mice were postoperatively subcutaneously injected with 1 mg per 0.1 mL progesterone (Depo-provera; Upjohn, Puurs, Belgium). On day 5.5 of pregnancy some females were killed, and others were injected with 0.05 µg per 0.1 mL 17β-estradiol (Benztrone, Amsa, Rome, Italy) to induce implantation and killed the day after [20]. FAAH activity and content were also evaluated in virgin females treated with the same concentrations of progesterone or estradiol and 3 days later uterine horns were excised and tested. Preimplantation embryos at the two-cell (1.5 days post coitum) and blastocyst (3.5 days post coitum) stages were collected by flushing the oviducts and uterus with M2 medium. Embryos were cultured in 0.5 mL minimal essential medium supplemented with 4 mg·mL−1 crystallized BSA (Bayer, Milan, Italy) in four-well Nunclon dishes, in 5% CO2 at 37 °C. All procedures adopted were approved by the Animal Care Committee of University of Rome ‘Tor Vergata’. Human tissues were obtained from uteri of three normally cycling premenopausal women who underwent endometrial biopsy as part of their infertility evaluation. These patients gave informed consent to the study. Biopsy samples were taken using a pipette endometrial suction cuvette (Veriurar Inc., Wilton, Wilts., UK), as described [21]. Tissues were removed and donated by M. Sbragia (Endocrinology and Reproduction Medical Center, Rome, Italy). The adenocarcinoma and the surrounding endometrial tissue (0.1 g each fresh tissue) were homogenized and used to compare FAAH activity and expression in tumor and healthy uterine epithelial cells.

Assay of FAAH

Mouse uterus, blastocysts and human specimens were washed in NaCl/Pi, homogenized and assayed for FAAH activity by RP-HPLC [19]. FAAH activity was expressed as pmol arachidonate released·min−1·(mg protein)−1. The apparent Km and Vmax of anandamide hydrolysis by FAAH were calculated by Lineweaver–Burk analysis, using [3H]anandamide in the concentration range 0–25 µm. Anandamide synthase activity was measured by following the formation of [3H]anandamide from [3H]arachidonic acid and ethanolamine, as reported [17,22], and was expressed as pmol anandamide formed· min−1·(mg protein)−1.

Immunochemical analysis

SDS/PAGE (12% gel) was performed under reducing conditions in a Mini Protean II apparatus (Bio-Rad), with 0.75-mm spacer arms [23]. Rainbow molecular-mass markers (Amersham International) were phosphorylase b, BSA and ovalbumin (97.4, 66.0 and 46.0 kDa, respectively). Mouse uterus homogenates (20 µg per lane) were subjected to SDS/PAGE, then slab gels were electroblotted on to 0.45-µm nitrocellulose filters (Bio-Rad), using a Mini Trans Blot apparatus (Bio-Rad) as reported [23]. Immunodetection of FAAH on nitrocellulose filters was performed with specific FAAH polyclonal antibodies (diluted 1 : 200), elicited in rabbits against the conserved FAAH sequence VGYYETDNYTMPSPAMR [24], conjugated to ovalbumin. This peptide antigen and the FAAH polyclonal antibodies were prepared by Primm (Milan, Italy). Goat anti-rabbit alkaline phosphatase conjugate (Bio-Rad), diluted 1 : 2000, was used as secondary antibody, and immunoreactive bands were stained with the alkaline phosphatase staining solution according to the manufacturer's instructions (Bio-Rad). ELISA was performed by coating the plate with mouse uterus homogenates (20 µg per well), allowed to react with FAAH polyclonal antibodies (diluted 1 : 300), and then with goat anti-rabbit alkaline phosphatase conjugate (diluted 1 : 2000). Color development of the alkaline phosphatase reaction was measured at 405 nm, using p-nitrophenyl phosphate as substrate. For peptide competition experiments, the peptide antigen was preincubated with a 1000-fold molar excess of FAAH polyclonal antibodies for 30 min at room temperature, before the addition of the antibodies to the wells [17]. Controls were carried out by using non-immune rabbit serum and included wells coated with different amounts of BSA. For immunohistochemistry, uterine horns were dissected from pregnant (5.5 days post coitum) and nonpregnant mice and fixed in 4% paraformaldehyde overnight. Tissues were repeatedly washed in NaCl/Pi containing 10 µg·mL−1 BSA, kept overnight in NaCl/Pi containing 30% sucrose, incorporated in OCT compound (Sakura Finetek Inc., Torrance, CA, USA) and frozen in liquid nitrogen. Cryosections (8–10 µm) were collected on poly-l-lysine-coated slides and incubated with 1 : 200 diluted FAAH polyclonal antibodies for 2 h at 37 °C. Samples were washed and labeled with 1 : 400 diluted Cy3-conjugated goat anti-(rabbit IgG) Ig (Chemicon International Inc., Ternecula, CA, USA) for 30 min at room temperature. Negative controls included sections in which the primary antibody was replaced by non-immune rabbit serum.

Determination of anandamide uptake by blastocysts

The uptake of [3H]anandamide (223 Ci·mmol−1) by mouse blastocysts (3.5 days post coitum) was studied essentially as described previously [17]. Blastocysts (16/test) were collected in M2 medium, washed in NaCl/Pi and transferred to 100 µL drops of the same solution. They were then incubated for 30 min at 37 °C with different amounts of [3H]anandamide, in the range 0–1000 nm. Blastocysts were washed five times in 100 µL drops of NaCl/Pi containing 10 µg·mL−1 BSA and finally resuspended in 200 µL NaCl/Pi. Membrane lipids were extracted in methanol/chloroform (2 : 1, v/v) as reported [19], resuspended in 0.5 mL methanol, mixed with 3.5 mL Sigma-Fluor liquid-scintillation cocktail for non-aqueous samples (Sigma), and radioactivity was measured in an LKB 1214 Rackbeta scintillation counter (LKB, Stockholm, Sweden). To distinguish non-protein-mediated from protein-mediated transport of anandamide into cell membranes, control experiments were carried out at 4 °C [17]. The apparent Km and Vmax of the uptake were calculated by Lineweaver–Burk analysis (in this case, the uptake at 4 °C was subtracted from that at 37 °C). The Q10 value was calculated as the ratio of anandamide uptake at 30 °C and 20 °C [16]. Anandamide uptake was expressed as pmol·min−1·(mg protein)−1, and the effect of AM404 determined by adding it directly to the incubation medium.

Evaluation of embryo development and apoptosis

Groups of 10–20 embryos were cultured in the presence of the indicated concentrations of freshly prepared anandamide, for 48 h (blastocyst) or 72 h (two-cells). Embryo development was monitored and recorded every 12 h. At the end of the culture time, the embryos were fixed in 4% paraformaldehyde and stained for 5 min with 33258 Hoechst (0.5 µg·mL−1) to determine the presence of apoptotic nuclei [25].

Statistical analysis

Data are reported as mean ± SD from at least three independent determinations, each performed in duplicate. Statistical analysis was performed by Student's t-test, corroborated by the non-parametric Mann–Whitney test. Experimental data were elaborated using the instat program (GraphPad Software), and differences with P < 0.05 were considered significant.

Results

  1. Top of page
  2. Abstract
  3. MATERIALS and METHODS
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

FAAH activity and expression in mouse uterus during early pregnancy

Pilot experiments indicated that mouse uterus FAAH activity was linearly dependent on the amount of tissue homogenate (in the range 0–30 µg protein) and the incubation time (in the range 0–30 min), whereas it depended on anandamide concentration according to Michaelis–Menten kinetics (Fig. 1A and data not shown). It exhibited an apparent Km of 7.0 ± 0.7 µm and Vmax of 320 ± 30 pmol·min−1·mg−1. Its activity was assayed in the pH range 5.0–11.0 and the temperature range 20–65 °C. Under these experimental conditions, optimum pH and temperature were 9.0 and 37 °C, respectively. Western blotting showed that FAAH polyclonal antibodies specifically recognized a single immunoreactive band in uterus homogenates, corresponding to a molecular mass of ≈ 67 kDa (Fig. 1B). The FAAH polyclonal antibodies were used to quantitate FAAH expression in mouse uterus by ELISA, validated by antigen competition experiments [17]. It was found that FAAH activity and expression decreased during early pregnancy in a time-dependent manner, down to 10% (activity) and 40% (content) of the control after 5.5 days (Fig. 1C). The observation that FAAH activity decreased much more than enzyme content might be explained by the formation of a soluble inhibitor of FAAH. Kinetic analysis showed that FAAH activity towards anandamide had apparent Vmax values of 320 ± 30, 280 ± 30, 150 ± 15, and 50 ± 5 pmol·min−1·mg−1, in homogenates of mouse uterus at 0, 1.5, 3.5, and 5.5 days of pregnancy, whereas apparent Km was unchanged (7.0 ± 0.7 µm in all cases).

image

Figure 1. FAAH activity and expression in mouse uterus. (A) FAAH activity depended on anandamide concentration. (B) Specific antibodies to FAAH reacted with a single band in mouse uterus homogenates (20 µg per lane). FAAH activity (open bars) and content (hatched bars) decreased during early pregnancy (C) or pseudopregnancy (D). In (C,D), 100% = 160 ± 15 pmol·min−1·mg−1 (activity) or 0.330 ± 0.030 A405 units (content). In (B), molecular masses (kDa) are indicated on the right.

Download figure to PowerPoint

FAAH activity and expression during pseudopregnancy and delayed implantation

FAAH protein decreased in the pseudopregnant uterus following the same trend as observed during normal pregnancy (Fig. 1D), while enzyme activity decreased but to a significantly (P < 0.05, n = 8) slower rate (45% at day 5.5 in the pseudopregnant uterus vs. 10% in the pregnant uterus). We found that the fall in FAAH activity and expression was significantly (P < 0.01, n = 8) lower in ovariectomized animals than controls, and that estrogen treatment reversed this effect (Fig. 2A). On the other hand, a decrease in FAAH activity and expression similar to that observed in normal pregnancy was present in sham-operated or semi-ovariectomized mice (Fig. 2A). Progesterone and estrogen significantly (P < 0.01, n = 8) decreased both specific and total activity of FAAH compared with untreated controls (Fig. 2B). Moreover, the decreased FAAH activity was paralleled by decreased enzyme content, which leveled off at 60% of the untreated control (P < 0.05, n = 8) on treatment of mouse uterus with either hormone (not shown). Anandamide synthase activity was also present in the uterus of virgin females (40 ± 4 pmol·min−1·mg−1), which was approximately halved by progesterone (20 ± 2 pmol·min−1·mg−1) or estrogen (25 ± 3 pmol·min−1· mg−1).

image

Figure 2. Effect of sex hormones on mouse uterus FAAH. (A) The decrease in FAAH activity (open bars) and content (hatched bars) from day 0 to day 5.5 of pregnancy was smaller in ovariectomized mice (ovari.) and larger in the same animals treated with estrogen (ovari. + estro.), 100% = 25 ± 3 pmol·min−1·mg−1 (activity) or 0.130 ± 0.014 A405 units (content) or semi-ovariectomized (Emi-ovari). (B) Treatment of virgin females with sex hormones decreased FAAH specific activity (open bars) and total activity (hatched bars). 100% = 180 ± 20 pmol·min−1·mg−1 (specific activity) or 35 000 ± 4000 pmol·min−1·mg−1 (total activity). In (A), *P < 0.01 compared with 5.5 day control (n = 8), **P < 0.01 compared with ovariectomized (n = 8). In (B), *P < 0.01 compared with control (n = 8).

Download figure to PowerPoint

FAAH localization in mouse uterus and FAAH activity in human uterus

Antibodies to FAAH were used to immunolocalize FAAH in the luminal (Fig. 3A) and glandular (Fig. 3B) epithelia of nonpregnant mouse uterus. In keeping with the activity and ELISA data, FAAH was hardly detectable in the uterus of 5.5-day-pregnant mice (not shown).

image

Figure 3. Immunolocalization of FAAH and anandamide-induced apoptosis. FAAH was immunolocalized in the luminal (A) and glandular (B) epithelia of nonpregnant mouse uterus. Control mouse blastocysts (C) showed increased numbers of apoptotic cells on treatment with 40 nm anandamide (D). Original magnification: (A) (B) = ×350; (C,D) = ×175. Bar represents 10 µm (A,B) or 5 µm (C,D). Arrowheads indicate autofluorescence of unidentified cells (A,B) or apoptotic cells (D).

Download figure to PowerPoint

FAAH activity was also analyzed in human uterine epithelial cells, where it showed an apparent Km and a Vmax for anandamide of 4.0 ± 0.5 µm and 1000 ± 100 pmol·min−1·mg−1, respectively. Remarkably, FAAH activity in human adenocarcinoma cells was more than five times higher than in healthy controls (Fig. 4). Quantitation of FAAH by ELISA, validated by antigen competition experiments, showed that FAAH expression paralleled the activity (Fig. 4).

image

Figure 4. FAAH activity and expression in human uterus. FAAH activity (open bars) and content (hatched bars) was significantly increased in adenocarcinoma cells compared with healthy epithelial cells. Antigen competition ELISA (closed bars) validated the specificity of FAAH quantitation. *P < 0.01 compared with control (n = 3). 100% = 600 ± 50 pmol·min−1·mg−1 (activity) or 0.500 ± 0.050 A405 units (content).

Download figure to PowerPoint

FAAH activity and anandamide uptake by mouse blastocysts

FAAH activity showed Michaelis–Menten kinetics with apparent Km and Vmax towards anandamide of 4.0 ± 0.4 µm and 200 ± 20 pmol·min−1·mg−1, respectively (Fig. 5A). Specific inhibitors of FAAH activity, such as phenylmethanesulfonyl fluoride [14,17] and AACOCF3[26], almost completely inhibited the hydrolysis of anandamide by mouse blastocysts (10% and 5% residual activity), when used at 100 µm and 10 µm, respectively. Blastocysts also showed anandamide synthase activity (25 ± 3 pmol·min−1·mg−1), which was reduced to 15% and 10% of the control by 100 µm phenylmethanesulfonyl fluoride and 10 µm AACOCF3, respectively.

image

Figure 5. FAAH activity and anandamide transporter in mouse blastocysts. FAAH activity (A) and anandamide uptake (B) depended on anandamide concentration. In (B), anandamide uptake by blastocysts was measured at 37 °C, in the absence (•) or presence (asterisk) of 10 µm AM404, or at 4 °C (▴). (C) Anandamide impaired embryo development from two cells to blastocyst (open bars) and blastocyst hatching (hatched bars). (D) Anandamide induced programmed cell death in mouse blastocysts.

Download figure to PowerPoint

Blastocysts were able to accumulate [3H]anandamide in a temperature-dependent (Q10 = 1.5), time-dependent (t1/2 = 5 min) and concentration-dependent way (Fig. 5B and data not shown). [3H]Anandamide uptake was saturable (apparent Km = 160 ± 20 nm and Vmax = 0.32 ± 0.04 pmol·min−1· mg−1, respectively) and was almost completely inhibited by 10 µm AM404 (Fig. 5B), a selective inhibitor of the anandamide transporter [15,27].

Impairment of hatching and development of mouse blastocysts and induction of apoptosis by anandamide

Nanomolar concentrations of anandamide inhibited development of two-cell embryos to blastocysts and zona-hatching of blastocysts developed from eight-cell embryos in vitro (Fig. 5C). Both detrimental effects of 40 nm anandamide could be reversed by equimolar concentrations of SR 141716, a CB1-R antagonist, but not by the CB2-R antagonist SR 144528 (up to 1 µm). At the same concentration, anandamide was found to induce programmed cell death in mouse blastocysts, 40 nm anandamide yielding a fourfold increase in apoptotic cells as compared with the untreated control (Figs 5D and 3C,D). Anandamide-induced apoptosis could not be prevented by coadministration of either SR 141716 or SR 144528 (up to 1 µm).

Discussion

  1. Top of page
  2. Abstract
  3. MATERIALS and METHODS
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

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 [9], 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 [18]. 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 [18].

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 [28]. 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 [29]. 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 [18]. 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 [17]. 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 [24] 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 [10]. 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 [35], 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 [15] and human neuronal and immune cells [17] 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) [15] and human cells (Km = 130–200 nm) [17]. 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 [10]. 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 [38], and another gave preliminary evidence on its ability to induce apoptosis in lymphocytes [39]. It is noteworthy that Δ9-tetrahydrocannabinol has been recently shown to promote apoptosis in glioma cells, through a CB1-R independent mechanism [40].

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.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS and METHODS
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The authors wish to thank Dr M. Sbragia (Endocrinology and Reproductive Medical Center, Rome, Italy) for kindly donating human specimens, and Drs M. Bari and R. Agostinetto for their skilful assistance. This investigation was supported by Istituto Superiore di Sanità (II AIDS Programme) and Ministero dell’Università e della Ricerca Scientifica e Tecnologica (MURST-PRIN 1997 and 1998).

References

  1. Top of page
  2. Abstract
  3. MATERIALS and METHODS
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  • 1
    Devane, W.A., Hannus, L., Breuer, A., Pertwee, R.G., Stevenson, L.A., Griffin, G., Gibson, D., Mandelbaum, A., Etinger, A. & Mechoulam, R. (1992) Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science 258, 19461949.
  • 2
    Mechoulam, R., Fride, E. & Di Marzo, V. (1998) Endocannabinoids. Eur. J. Pharmacol. 359, 118.
  • 3
    di Tomaso, E., Beltramo, M. & Piomelli, D. (1996) Brain cannabinoids in chocolate. Nature (London) 382, 677678.
  • 4
    Di Marzo, V., Sepe, N., De Petrocellis, L., Berger, A., Crozier, G., Fride, E. & Mechoulam, R. (1998) Trick or treat from food endocannabinoids? Nature (London) 396, 636.
  • 5
    Calignano, A., La Rana, G., Giuffrida, A. & Piomelli, D. (1998) Control of pain initiation by endogenous cannabinoids. Nature (London) 394, 277281.
  • 6
    Meng, I.D., Manning, B.H., Martin, W.J. & Fields, H.L. (1998) An analgesia circuit activated by cannabinoids. Nature (London) 395, 381383.
  • 7
    Das, S.K., Paria, B.C., Chakraborty, I. & Dey, S.K. (1995) Cannabinoid ligand-receptor signaling in the mouse uterus. Proc. Natl Acad. Sci. USA 92, 43324336.
  • 8
    Ness, R.B., Grisso, J.A., Hirschinger, N., Markovic, N., Shaw, L.M., Day, N.L. & Kline, J. (1999) Cocaine and tobacco use and the risk of spontaneous abortion. N. Engl. J. Med. 340, 333339.
  • 9
    Paria, B.C., Deutsch, D.D. & Dey, S.K. (1996) The uterus is a potential site for anandamide synthesis and hydrolysis. differential profiles of anandamide synthase and hydrolase activities in the mouse uterus during the periimplantation period. Mol. Rep. Dev. 45, 183192.
  • 10
    Schmid, P.C., Paria, B.C., Krebsbach, R.J., Schmid, H.H.O. & Dey, S.K. (1997) Changes in anandamide levels in mouse uterus are associated with uterine receptivity for embryo implantation. Proc. Natl Acad. Sci. USA 94, 41884192.
  • 11
    Di Marzo, V. (1998) “Endocannabinoids” and other fatty acid derivatives with cannabimimetic properties. biochemistry and possible physiopathological relevance. Biochim. Biophys. Acta 1392, 153175.
  • 12
    Yang, Z.-M., Paria, B.C. & Dey, S.K. (1996) Activation of brain-type cannabinoid receptors interferes with preimplantation mouse embryo development. Biol. Reprod. 55, 756761.
  • 13
    Wang, J., Paria, B.C., Dey, S.K. & Armant, D.R. (1999) Stage-specific excitation of cannabinoid receptor exhibits differential effects on mouse embryonic development. Biol. Reprod. 60, 839844.
  • 14
    Di Marzo, V., Bisogno, T., De Petrocellis, L., Melck, D., Orlando, P., Wagner, J.A. & Kunos, G. (1999) Biosynthesis and inactivation of the endocannabinoid 2-arachido-noylglycerol in circulating and tumoral macrophages. Eur. J. Biochem. 264, 258267.
  • 15
    Beltramo, M., Stella, N., Calignano, A., Lin, S.Y., Makriyannis, A. & Piomelli, D. (1997)Functional role of high-affinity anandamide transport, as revealed by selective inhibition. Science 277, 10941097.
  • 16
    Hillard, C.J., Edgemond, W.S., Jarrahian, A. & Campbell, W.B. (1997) Accumulation of N-arachidonoylethanolamide (anandamide) into cerebellar granule cells occurs via facilitated diffusion. J. Neurochem. 69, 631638.
  • 17
    Maccarrone, M., van der Stelt, M., Rossi, A., Veldink, G.A., Vliegenthart, J.F.G. & Finazzi-Agrò, A. (1998) Anandamide hydrolysis by human cells in culture and brain. J. Biol. Chem. 273, 3233232339.
  • 18
    Paria, B.C., Zhao, X., Wang, J., Das, S.K. & Dey, S.K. (1999) Fatty-acid amide hydrolase is expressed in the mouse uterus and embryo during the periimplantation period. Biol. Reprod. 60, 11511157.
  • 19
    Maccarrone, M., Bari, M. & Finazzi-Agrò, A. (1999) A sensitive and specific radiochromatographic assay of fatty acid amide hydrolase. Anal. Biochem. 267, 314318.
  • 20
    Bergstrom, S. (1978) Experimentally delayed implantation. In Methods in Mammalian Reproduction(Daniel, J.C., ed.), pp. 419435. Academic Press, New York.
  • 21
    Hill, G.A., Herbert, C.M., Parker, R.A. & Werz, A.C. (1989) Comparison of late luteal phase endometria utilizing the Nowak cuvette or pipette endometrial suction cuvette. Obstet. Gynecol. 73, 443450.
  • 22
    Ueda, N., Kurahashi, Y., Yamamoto, S. & Tokunaga, T.J. (1995) Partial purification and characterization of the porcine brain enzyme hydrolyzing and synthesizing anandamide. J. Biol. Chem. 270, 2382323827.
  • 23
    Maccarrone, M., Veldink, G.A. & Vliegenthart, J.F.G. (1991) An investigation on the quinoprotein nature of some fungal and plant oxidoreductases. J. Biol. Chem. 266, 2101421017.
  • 24
    Giang, D.K. & Cravatt, B.F. (1997) Molecular characterization of human and mouse fatty acid amide hydrolase. Proc. Natl Acad. Sci. USA 94, 22382242.
  • 25
    Brison, D.R. & Schultz, R.M. (1997) Apoptosis during mouse blastocyst formation. evidence for a role for survival factors including transforming growth factor α. Biol. Reprod. 56, 10881096.
  • 26
    Koutek, B., Prestwich, G.D., Howlett, A.C., Chin, S.A., Salehani, D., Akhavan, N. & Deutsch, D.G. (1994) Inhibitors of arachidonoyl ethanolamide hydrolysis. J. Biol. Chem. 269, 2293722940.
  • 27
    Piomelli, D., Beltramo, M., Glasnapp, S., Lin, S., Goutopoulos, A., Xie, X.Q. & Makriyannis, A. (1999) Structural determinants for recognition and translocation by the anandamide transporter. Proc. Natl Acad. Sci. USA 96, 58025807.
  • 28
    Everett, J.W. (1969) Neuroendocrine aspects of mammalian reproduction. Annu. Rev. Physiol. 31, 383416.
  • 29
    Flint, A.P.F., Renfree, M.B. & Weir, B.J. (1980) Embryonic diapause in mammals. J. Reprod. Fertil. S29.
  • 30
    Coulson, P.B. & Pavlik, E.J. (1977) Effects of estrogen and progesterone on cytoplasmic estrogen receptor and rates of protein synthesis in rat uterus. J. Steroid Biochem. 8, 205212.
  • 31
    DeSombre, E.R. & Kuivanen, P.C. (1985) Progestin modulation of estrogen-dependent marker protein synthesis in the endometrium. Semin. Oncol. 12, 611.
  • 32
    Kurahashi, Y., Ueda, N., Suzuki, H., Suzuki, M. & Yamamoto, S. (1997) Reversible hydrolysis and synthesis of anandamide demonstrated by recombinant rat fatty acid amide hydrolase. Biochem. Biophys. Res. Commun. 237, 512515.
  • 33
    Venance, L., Piomelli, D., Glowinski, J. & Giaume, C. (1995) Inhibition by anandamide of gap junctions and inercellular calcium signalling in striatal astrocytes. Nature (London) 376, 590594.
  • 34
    Boger, D.L., Patterson, J.E., Guan, X., Cravatt, B.F., Lerner, R.A. & Gilula, N.B. (1998) Chemical requirements for inhibition of gap junction communication by the biologically active lipid oleamide. Proc. Natl Acad. Sci. USA 95, 48104815.
  • 35
    Maccarrone, M., Bari, M., Menichelli, A., Del Principe, D. & Finazzi-Agrò, A. (1999) Anandamide activates human platelets through a pathway independent of the arachidonate cascade. FEBS Lett. 447, 277282.
  • 36
    Afford, S.C., Randhawa, S., Eliopoulos, A.G., Hubscher, S.G., Young, L.S. & Adams, D.H. (1999) CD40 activation induces apoptosis in cultured human hepatocytes via induction of cell surface fas ligand expression and amplifies fas-mediated hepatocyte death during allograft rejection. J. Exp. Med. 189, 441446.
  • 37
    Tonnetti, L., Veri, M.C., Bonvini, E. & D’Adamio, L. (1999) A role for neutral sphingomyelinase-mediated ceramide production in T cell receptor-induced apoptosis and mitogen-activated protein kinase-mediated signal transduction. J. Exp. Med. 189, 15811589.
  • 38
    De Petrocellis, L., Melck, D., Palmisano, A., Bisogno, T., Laezza, C., Bifulco, M. & Di Marzo, V. (1998) The endogenous cannabinoid anandamide inhibits human breast cancer cell proliferation. Proc. Natl Acad. Sci. USA 95, 83758380.
  • 39
    Schwarz, H., Blanco, F.J. & Lotz, M. (1994) Anandamide, an endogenous cannabinoid receptor agonist, inhibits lymphocyte proliferation and induces apoptosis. J. Neuroimmunol. 55, 107115.
  • 40
    Sànchez, C., Galve-Roperh, I., Canova, C., Brachet, P. & Guzman, M. (1998) Δ9-Tetrahydrocannabinol induces apoptosis in C6 glioma cells. FEBS Lett. 436, 610.
Footnotes
  1. Enzyme: anandamide hydrolase (EC 3.5.1.4).

  2. Note: a web page is available at http://WWW.UNIROMA2.IT