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Endocrinology, doi:10.1210/en.2007-1799
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Endocrinology Vol. 149, No. 10 5052-5060
Copyright © 2008 by The Endocrine Society

Expression of the Endocannabinoid System in Human First Trimester Placenta and Its Role in Trophoblast Proliferation

Osama M. H. Habayeb, Anthony H. Taylor, Stephen C. Bell, David J. Taylor and Justin C. Konje

Endocannabinoid Research Group, Reproductive Sciences Section, Department of Cancer Studies and Molecular Medicine, University of Leicester, Leicester LE2 7LX, United Kingdom

Address all correspondence and requests for reprints to: Professor Justin Konje, Endocannabinoid Research Group, Reproductive Sciences Section, Department of Cancer Studies and Molecular Medicine, University of Leicester, Leicester LE2 7LX, United Kingdom. E-mail: jck4{at}le.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Materials
 Results
 Discussion
 References
 
The endocannabinoid, anandamide, which binds to two major receptor proteins, the cannabinoid receptors (CBs) 1 and 2 (CB1 and CB2), has been shown to play a role in first trimester miscarriage possibly through impairment of the developing trophoblast. Although the precise molecular mechanisms underlying this are unknown, plasma anandamide levels are known to be regulated by the progesterone-induced enzyme, fatty acid amide hydrolase (FAAH). Here, we tested the hypothesis that temporal-spatial expression of FAAH, CB1, and CB2 is regulated during early pregnancy and that anandamide detrimentally alters trophoblast proliferation. Transcripts for CB1, CB2, and FAAH were demonstrated in first trimester trophoblast extracts with only the CB1 transcript being significantly regulated. The significant 4.7-fold increase in expression at wk 10 gestation was reduced to 8.9% of the peak value by wk 12. Transcripts for CB2 showed a similar pattern of expression but were not significantly induced. By contrast, FAAH transcript levels appeared to increase toward the end of the first trimester, but again did not reach significance. These observations were supported by immunohistochemical studies that demonstrated a similar pattern of expression at the protein level, with cellular localization for all three proteins concentrated within the syncytiotrophoblast layer. Anandamide also prevented BeWo trophoblast cell proliferation in a dose-dependent manner, with a 50–60% significant inhibition of cell proliferation with concentrations in excess of 3 µM. This effect was mediated through CB2. Together, these data provide insights into how elevated plasma anandamide levels increase the risk of first trimester miscarriage.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Materials
 Results
 Discussion
 References
 
ANANDAMIDE [N-arachidonoylethanolamine (AEA)] is an endogenous cannabinoid that is involved in human reproduction (1). AEA is released from phospholipid precursors by cells in response to stimulation by neurotransmitters and hormones via the action of N-arachidonylphosphatidyl ethanolamine selective phospholipase-D (NAPE-PLD) (1, 2, 3, 4), and possibly through the actions of phospholipase C and phosphatases (5).

AEA exerts its effects mainly by acting on two cannabinoid receptors (CBs), CB1 and CB2. Although AEA is an endogenous agonist for CB1 and CB2, it also is an endogenous agonist for type-1 vanilloid receptors, and can directly affect a variety of other signal systems (6, 7) AEA is rapidly removed by a putative transporter and metabolized by the enzyme, fatty acid amide hydrolase (FAAH) into arachidonic acid and ethanolamine (8). AEA, CB1, CB2, and FAAH constitute the major components of the endocannabinoid system (1, 4) and have been subjected to extensive study with regard to reproduction. Other less well-studied members of the cannabinoid system such as 2-arachidonylethanolamine, N-oleoylethanolamine, N-palmitoylethanolamine, and the enzyme NAPE-PLD (9, 10) are gaining more prominence as part of this system. A critical balance between AEA release and degradation creates an endogenous AEA tone that regulates oviductal transport of preimplantation mouse embryos (11, 12). A similar process may regulate implantation in humans. Studies in the mouse have demonstrated that the local concentrations of AEA in the uterus regulate its receptivity toward the developing blastocyst with nonimplantation sites containing higher concentrations of AEA than the implantation sites (13). This temporal-spatial distribution of AEA in the uterus is complemented by an inverse correlation with FAAH expression, with high levels at the implantation site and low levels at non-implantation sites (14). In human studies it has been shown that low levels of FAAH in peripheral blood mononuclear cells in early pregnancy predict spontaneous miscarriage (15). In addition, the levels and activity of FAAH in peripheral blood mononuclear cells in women who failed to achieve pregnancy after embryo transfer in in vitro fertilization pregnancies are lower than in those who conceived (16). We have previously measured plasma AEA levels in normal human pregnancy and demonstrated that these vary in a pattern, suggestive of the involvement of gonadal steroids in its regulation (17). Combined, these studies suggest that AEA plays an important role in the establishment and maintenance of early human pregnancy.

Previous studies demonstrating that both FAAH and CB1 are expressed in term human placenta and fetal membranes (18) suggested that the endocannabinoid system is likely to be present from early in gestation, however, this has not been extensively investigated, and there are no data on the expression of CB2 in early gestational tissues. In addition, the effect of AEA on human trophoblast growth and survival has not been examined. However, AEA is known to have a biphasic growth/survival effect on the mouse blastocyst with low (7–14 nM) levels stimulating trophoblast outgrowth and high (28–56 nM) levels inhibiting growth (6, 19, 20). These data suggest that a similar effect may be observed in the human trophoblast. Therefore, the aims of this study were to: 1) determine the expression of FAAH and the CBs, CB1 and CB2, in the trophoblast during the first trimester of pregnancy; 2) describe any changes that might occur during gestation; and 3) determine whether AEA would have any effect on the growth of a human cellular model of the first trimester trophoblast.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Materials
 Results
 Discussion
 References
 
Tissue collection
The local Research Ethics Committee approved this study, and all volunteers gave signed informed consent. Products of conception were collected between 7 and 12 wk gestation from women undergoing surgical termination of pregnancy. All had an ultrasound scan examination to date accurately the pregnancies before pregnancy termination; standard practice in the gynecology unit. Collected samples were transported on ice to the laboratory, where blood was washed away with sterile PBS and two pieces of trophoblast dissected free with the aid of a dissecting microscope. One piece was fixed in 10% neutral-buffered formalin for 4 d before embedding in paraffin wax. A second portion of tissue (~100 mg) was placed into a sterile polypropylene tube and snap frozen in liquid nitrogen for 5 min. The latter samples were stored at –80 C for later RNA analysis. Archival term placental tissues from women undergoing elective cesarean section acted as positive controls (21).


   Materials
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 Abstract
 Introduction
 Materials and Methods
 Materials
Results
 Discussion
 References
 
Anti-CB1 and anti-CB2 rabbit polyclonal antibodies were obtained from Sigma-Aldrich Ltd. (Poole, Dorset, UK), whereas anti-FAAH rabbit polyclonal antibody was obtained from (Alpha Diagnostics International, Inc., San Antonio, TX). Microwave antigen retrieval (22) was performed for both CB1 and CB2 studies but was not required for the FAAH studies. The secondary antirabbit horseradish peroxidase conjugates were from Dako (Glostrup, Denmark), and the tyramide amplification and detection system was from PerkinElmer LAS (Beaconsfield, Buckinghamshire, UK). An ABC detection system (ABC Elite; Vector Laboratories, Peterborough, UK) was used in conjunction with 3,3'-diaminobenzidine (Vector laboratories) to detect the presence of immunoreactive complexes for the anti-CB2 and anti-FAAH antibodies, whereas the tyramide amplification system was substituted for ABC Elite for the detection of anti-CB1 complexes. The anti-FAAH antibodies were used at an optimal dilution of 1:2000 in PBS [137 mM NaCl, 9.2 mM Na2HPO4, and 1.46 mM KH2PO4 (pH 7.6)], CB1 at 1:4000, and CB2 at 1:500 dilutions. For each antibody used, negative controls were performed using the same concentrations of rabbit IgG (for CB1 and CB2; Vector Laboratories) and normal rabbit serum (for FAAH; Dako).

Expression of the endocannabinoid system in the first trimester placenta
Total cellular RNA was extracted from frozen placenta samples using TRIZOL reagent (Invitrogen, Paisley, UK) according to the manufacturer’s instructions, and the concentration, purity, and integrity of the resulting RNA were determined spectrophotometrically before the presence of mRNA for CB1, CB2, and FAAH, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was determined using end-point RT-PCR.

End-point RT-PCR
One microgram of total RNA was reversed transcribed with avian myeloblastosis virus (AMV)-reverse transcriptase (RT) (Promega, Southampton, UK) at 42 C for 1 h in the presence of 5 U ribonuclease inhibitor (RNasin; Promega). A minus RT reaction was obtained by omitting the AMV-RT enzyme. At the end of the reaction, the enzymes were denatured by heating at 95 C for 5 min and the cDNA stored at –20 C. One microliter of cDNA was subject to PCR using 10 pmol/µl specific primers for CB1, CB2, FAAH, and GAPDH using the annealing temperatures given in Table 1Go. PCR products were resolved on 3% agarose gels and stained with ethidium bromide (2 µg/ml; ICN Biomedicals, Basingstoke, UK) for 15 min, before being destained with distilled water for 30 min. Gel images were captured using a Syngene GeneGenius system (Syngene, Cambridge, UK) equipped with GeneSnap version 6 gel documentation software. For quantification of relative transcript levels, samples were subjected to quantitative real-time RT-PCR.


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  		TABLE 1. Primer design and expected amplicons sizes

 
Quantitative real-time RT-PCR
Amplification of CB1, CB2, FAAH, and GAPDH cDNA was performed in a Roche LightCycler (Roche Diagnostics Ltd., Lewes, UK) using 1 µl cDNA for CB2 and GAPDH, and 2 µl cDNA for CB1 and FAAH, with the LightCycler FastStart DNA master SYBR Green I kit (Roche Diagnostics). Mastermix reactions were prepared as per the manufacturer’s instructions on ice with 10 pmol each primer pair (Table 1Go), and the cDNA was added last. Controls included a water blank, minus RT control for each trophoblast sample, a series of diluted cloned human CB1, CB2 (CNR01LTN00 and CNR020TN00, respectively; University of Missouri-Rolla cDNA Resource Center, Rolla, MO), FAAH (12), and GAPDH (BioChain Institute Inc., Hayward, CA) cDNA targets adjusted so that a standard curve from 1–10,000,000 pmol cDNA could be constructed. In addition, a series of diluted pooled trophoblast cDNA (1:10 to 1:10,000 dilutions) were used to calculate amplification efficiencies for each gene amplified. The cycle conditions were 95 C for 10 min, followed by 45 cycles of 95 C for 10 sec, 60 C for 5 sec, 72 C for 15 sec for GAPDH, 95 C for 12 sec, 62 C for 7 sec, 72 C for 15 sec for CB1 and CB2; and 95 C for 12 sec, 59 C for 9 sec, and 72 C for 12 sec for FAAH. The crossing points were then used to measure the transcript concentrations from the standard curves and the predicted melting temperatures of the amplified products used to confirm that only the target DNA was amplified.

Detection of immunoreactive CB1, CB2, and FAAH
Tissues sections (4 µm) were mounted onto silanized glass microscope slides and dried for 7 d at 37 C before use. Slides were dewaxed in xylene three times for 3 min and rehydrated in graded alcohol for 3 min, followed by incubation in distilled water for 3 min. Microwave antigen retrieval was performed for CB1 and CB2 only by incubating the slides in 10 mM citric acid buffer (pH 6.0) heated at 700 W for 10 min (23). Endogenous peroxidase activity was then blocked by incubation in 6% H2O2 in water for 10 min. Blocking of nonspecific protein binding sites was performed by incubation in 10% normal goat serum for 10 min at room temperature. Endogenous avidin and biotin sites were blocked using the Avidin-Biotin Blocking Kit (Vector Laboratories) as recommended by the manufacturer. Primary antibodies diluted in Tris-buffered saline [0.5 M Trizma, 1.5 M NaCl, and 2 mM MgCl2 (pH 7.6); 100 µl/slide] were added, and the slides were incubated in a humid chamber overnight at 4 C. Slides were then washed in [Tris-buffered saline containing 0.1% BSA (Fraction V; Sigma-Aldrich)] for 30 min. After washing the slides for 30 min in Tris-buffered saline, biotinylated goat antirabbit antibody (Vector Laboratories) diluted to 1:400 in TBA was applied for 30 min at room temperature. After an additional wash in Tris-buffered saline, ABC Elite reagent (for CB2 and FAAH) and tyramide amplification reagent (for CB1) were applied according to the manufacturer’s detailed instructions. After additional washing in Tris-buffered saline for 20 min, 3,3'-diaminobenzidine was added to each slide (100 µl/slide) for 5 min. Slides were then washed in distilled water for 5 min before counterstaining in Mayer’s hematoxylin for 15 sec. After washing in running tap water for 5 min, slides were dehydrated in graded alcohols, cleared in xylene twice for 6 min before mounting with di-n-butylphthalate xylene (DPX) mounting medium (BDH, Poole, Dorset, UK).

Images were taken on an Axioplan transmission microscope equipped with a Sony DXC-151P analog camera (Sony Corp., Kanagawaken, Japan) (24) connected to a computer running Axiovision image capture and processing software (Axiovision version 4.4; Carl Zeiss Ltd., Welwyn Garden City, Hertfordshire, UK). Images were captured at either x10 or x40 magnification in the presence of daylight and medium value neutral density filters with the lamp set at 6400 K. Image backgrounds were color corrected to neutral gray with the use of ColorPilot software (version 4.62; www.colorpilot.com).

Anandamide on BeWo cell growth
The human choriocarcinoma cell line BeWo (86082803; European Collection of Cell Cultures, Salisbury, Wiltshire, UK), chosen because it is a good model for the human first trimester trophoblast (25) and because the cells contain the CBs (21) and respond to cannabinoid stimulation (26), was maintained in Ham’s F12 medium (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen) and cultured at 37 C in a humidified atmosphere of 5% CO2 in air. BeWo cells were plated onto Nunc six-well plates in triplicate for each data point (Fisher Scientific, Loughborough, Leicestershire, UK) at a density of 4 x 105 cells per well (27). After 48 h culture, the medium was exchanged with one that contained 5% fetal calf serum and up to 30 µM anandamide [final concentration of 0.1% (vol/vol) ethanol]. The control consisted of culture medium containing 0.1% (vol/vol) ethanol. Cells were cultured with media plus additives for 1 h to allow nonspecific binding of the anandamide to plastic to reach equilibrium. The medium was then replaced and culture continued for an additional 48 h, with fresh medium replaced at 24 h. Photomicrographs were obtained using a Nikon Eclipse TE2000-U inverted microscope equipped with a DN-100 digital camera image capture system (Nikon Corp., Tokyo, Japan).

To examine the effect of anandamide on cell survival and cell proliferation indices, we plated BeWo cells onto Nunc 96-well plates (Fisher Scientific) at 1 x 104 cells per well, in 200 µl normal growth medium and allowed them to proliferate for 48 h. The medium was then changed to one that contained anandamide for 1 h to allow for nonspecific binding of the endocannabinoid to the plasticware. The medium was then replaced with the same anandamide-containing media and culture continued for an additional 23 h, when the media was changed again and the cell reincubated for an additional 24 h.

In a separate series of experiments to determine which of the CBs could be mediating the effect of anandamide, BeWo cells were treated with 10 µM anandamide in the presence of a receptor blocking concentration (300 nM) of the CB1-selective antagonist SR141716A or the CB2-selective antagonist SR144528 that does not activate other receptors (28).

After 48 h culture in the presence and absence of anandamide, cell numbers were assessed using the Cell Proliferation and Apoptosis Kit II (Roche Diagnostics) as per the manufacturer’s instructions with measurements taken on a Multiskan Ascent ELISA plate reader (Labsystems Oy, Helsinki, Finland), with the detection filter set at 420 nm and the reference set at 620 nm. Cell numbers were obtained by calibration against a standard curve of untreated BeWo cell numbers grown in parallel (26, 29). To make direct comparisons between cultures, we then converted the cell numbers to a percentage of the untreated control.

Data analysis
The absolute levels of CB1, CB2, FAAH, and GAPDH transcript were calculated using Pfaffl’s method (30), data normalized to the wk 7 samples, and expressed as the mean ± SD relative to those of controls. All data were then analyzed for differences using one-way ANOVA with Tukey’s honestly significant difference test within the InStat version 3.0 software package (GraphPad Software Inc., San Diego, CA; http://www.graphpad.com). Statistical significance was accepted when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Materials
 Results
Discussion
 References
 
Expression of the endocannabinoid system in the first trimester placenta
RT-PCR of RNA extracts taken from placentas between 7 and 12 wk gestation indicated the presence of amplified DNA, consistent with the expected amplicon sizes of 853 bp for CB1, 851 bp for CB2, 249 bp for FAAH, and 347 bp for GAPDH (Fig. 1Go). In addition, there was an absence of products in the lanes obtained for the –RT controls, indicating that the amplified products were obtained from mRNA and not contaminating genomic DNA. A detailed analysis of the expression patterns for the CB1 transcript indicated that there was a significant decrease in the expression of CB1 transcript from 4.77 ± 1.28 at wk 10 gestation to 0.40 ± 0.15 arbitrary units at wk 12: a significant 91% decrease in expression (P < 0.01; Student’s t test). Although there appeared to be a differential expression of both CB2 and FAAH transcripts in the trophoblast during the first trimester, these data did not reach statistical significance (Fig. 1BGo).


Figure 1
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  		FIG. 1. Expression of the endocannabinoid system in first trimester trophoblast biopsies. A, Ethidium bromide-stained agarose gels of RT-PCR products for CB1, CB2, FAAH, and GAPDH for placenta samples taken from wk 7 of the first trimester. +, Indicates the presence of AMV-RT in the RT reaction; –, indicates the absence of enzyme. The relative size markers from the 100-bp DNA ladder are shown on the left. B, Relative levels of CB1, CB2, and FAAH transcripts compared with wk 7 gestation using quantitative real-time RT-PCR. Data are the mean values ± SD after normalization to the relative levels of GAPDH according to the method of Pfaffl (30 ) and are from three experiments performed in triplicate for each sample. Each week of gestation is represented by four independent samples (n = 4). *, P < 0.05; one-way ANOVA with Tukey’s honestly significant difference test compared with wk 7; {theta}, P < 0.001 compared with wk 10. C, Ethidium bromide-stained agarose gels of RT-PCR products for CB1 (left panel) and CB2 (right panel) from the batch of BeWo cells used in this study. +, Indicates the presence of AMV-RT in the RT reaction; –, indicates the absence of enzyme. The relative size markers (M) from the 100-bp DNA ladder are shown on the left.

 
Detection of immunoreactive CB1, CB2, and FAAH
Using term fetal membranes as a positive control tissue (18), the optimal conditions for the immunohistochemistry studies were obtained and indicated that the primary antibody dilutions for FAAH, CB1, and CB2 were 1:2000, 1:4000, and 1:500, respectively. Rabbit serum or isotype IgG diluted to the same concentrations were nonreactive (Fig. 2Go). Using these dilutions of primary reagents and a common secondary detection system, there was also clear specific immunostaining for FAAH, CB1, and CB2 on first trimester trophoblast samples.


Figure 2
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  		FIG. 2. CB1, CB2, and FAAH immunohistochemistry. Demonstration of specific immunostaining for CB1 (B and D), CB2 (F and H), and FAAH (J and L), and IgG controls (A and C, E and G, and I and K) in term fetal membranes (A, B, E, F, I, and J; control tissues) and first trimester trophoblast (C, D, G, H, K, and L). Immunoreactive CB1, CB2, and FAAH are found in the amnion (a), chorion (c), and decidua (d) of the term fetal membranes, as expected. In first trimester trophoblast samples, specific CB1, CB2, and FAAH immunostaining was mainly confined to the cytotrophoblast and syncytiotrophoblast layers (arrows) and the mesenchymal cores of villi at all stages of development.The images are taken from a representative tissue obtained at 8 wk gestation at x10 magnification. Bar, 50 µm.

 
Immunoreactive FAAH staining patterns
Immunoreactive FAAH was detected in all 1st trimester trophoblast tissues between wk 7 and 12 gestation. FAAH immunoreactivity was detected in both the cytotrophoblast and the syncytiotrophoblast layers (Fig. 3Go). FAAH immunoreactivity in the syncytiotrophoblast layer increased gradually between the 7th and the 10th gestational week. By the 11th week of gestation, FAAH immunoreactivity at the syncytiotrophoblast layer diminished to the point where it was barely detectable within large parts of the trophoblast, an effect that persisted into the 12th week (Fig. 3Go). FAAH immunoreactivity was also demonstrated in the mesenchymal core of the developing villi. Immunoreactive FAAH was predominately noted throughout the cytoplasm of the cells with little or no nuclear staining (Fig. 3Go), in keeping with the membranous and intracytoplasmic vesicular localization of FAAH (8).


Figure 3
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  		FIG. 3. Effect of gestation on the expression of immunoreactive FAAH in the first trimester trophoblast. Immunohistochemical staining for FAAH was found in the syncytiotrophoblast, cytotrophoblast, and mesenchymal fibroblastic core in all trophoblastic samples from wk 7–12. FAAH immunoreactivity in the syncytiotrophoblast (arrows), diminished by the wk 11 in the brush-border epithelium (predominantly blue cells), an effect that persists at wk 12. Representative images were taken at x40 magnification.

 
Immunoreactive CB1 staining patterns
Immunoreactive CB1 was also detected in first trimester trophoblast tissue. CB1 immunoreactivity was detected in all types of trophoblast through various stages of maturation (Fig. 2Go). CB1 immunoreactivity in the syncytiotrophoblast layer diminished in intensity but did not disappear in gestation (wk 10–12; Fig. 4Go) as immunoreactive FAAH had. There was no CB1 immunoreactivity in either fetal blood cells or infiltrating maternal plasma cells, but CB1 immunoreactivity was detected in the endothelial cells of the blood vessels but not in the vascular smooth muscle cells (Fig. 4Go).


Figure 4
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  		FIG. 4. Effect of gestation on CB1 expression in the 1st trimester trophoblast. Immunohistochemical staining for CB1 was found in the syncytiotrophoblast, cytotrophoblast, and mesenchymal fibroblastic core of all placental samples from wk 7–12. CB1 immunoreactivity in the syncytiotrophoblast layer decreased from wk 10 onward. Positive staining in the endothelial cells of blood vessels (black arrow) was countered by negative staining in the smooth muscle cells of the blood vessels (red arrow) and fetal erythrocytes (red asterisk) and infiltrating maternal plasma cells (P). By the 12th week of gestation, there was a small decrease in the expression of the syncytiotrophoblast (s) compared with the cytotrophoblast (c). Representative images were taken at x40 magnification. Bar, 50 µm.

 
Immunoreactive CB2 staining patterns
Immunoreactive CB2 was also detected in first trimester trophoblast tissue. CB2 immunoreactivity was detected in all types of trophoblasts through various stages of the first trimester of gestation (Fig. 2Go). The intensity of CB2 immunoreactivity in the syncytiotrophoblast remained constant throughout the first trimester (Fig. 5Go), which differed from the immunoreactive staining patterns for FAAH and CB1. There was no CB2 immunoreactivity in either fetal blood cells or infiltrating maternal plasma cells, but CB2 immunoreactivity was detected in the endothelial cells of the blood vessels but not in the vascular smooth muscle cells (Fig. 5Go).


Figure 5
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  		FIG. 5. Effect of gestation on CB2 expression in the first trimester trophoblast. Immunohistochemical staining for CB2 was found in the syncytiotrophoblast, cytotrophoblast, and mesenchymal fibroblastic core of all placental samples from wk 7–12. CB2 immunoreactivity in the syncytiotrophoblast layer remains constant throughout the first trimester. Positive staining in the endothelial cells of blood vessels (black arrow) and the negative staining in the smooth muscle cells of the blood vessels (red arrow). Representative images were taken at x40 magnification. Bar, 50 µm.

 
Effect of anandamide on BeWo cell growth
BeWo cells were plated such that cultures achieved approximately 70–80% confluency after 48 h, the point at which AEA treatments were initiated, and continued for an additional 48 h with a range of AEA concentrations (26). Under these conditions AEA demonstrated an inhibitory effect on the BeWo cell cultures (Fig. 6Go), but only at concentrations in excess of 3 µM, where confluency was significantly reduced from approximately 80% at 3 µM, to approximately 65–70% at 15 and 30 µM. Cultures treated with 15 and 30 µM AEA did not exhibit increased cell death or failure to attach to the substratum, as evidenced by the lack of increase of shedding of cells in the spent medium (Fig. 6AGo). The antiproliferative effects of high AEA concentrations were mediated via the CB2 receptor (Fig. 6CGo). BeWo cells treated with 10 µM AEA demonstrated a significant 40–50% reduction in cell density after 48 h treatment. In the presence of 300 nM of the CB1-selective antagonist SR141716A, the data were very similar to the control, whereas in the presence of 300 nM of the CB2-selective antagonist SR144528, the growth-inhibitory effect of AEA was abrogated (Fig. 6CGo).


Figure 6
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  		FIG. 6. Effect of anandamide on BeWo cell growth. A, BeWo cells plated at 4 x 105 cells per well onto six-well plates were allowed to proliferate for 48 h before treatment with the indicated concentrations of anandamide (AEA) for an additional 48 h with the medium exchanged after 24 h. Note the decreased cell growth in the cultures treated with AEA (bar, 10 µm). B, BeWo cells plated at 1 x 104 cells per well in 96-well plates were allowed to proliferate for 48 h before treatment with the indicated concentrations of anandamide (AEA) for an additional 48 h with the medium exchanged after 24 h. The cells were incubated with XTT proliferation agent for 4 h, and the levels of color developed measured with absorbance at 492 nm and corrected for microplate imperfections at 620 nm, and then converted to the cell numbers relative to the untreated controls for each plate. The data are presented as mean ± SEM for four independent experiments performed in quadruplicate (n = 4). *, P < 0.05; ***, P < 0.001 Kruskal-Wallis one-way ANOVA with Dunn’s post test. C, BeWo cells plated at 1 x 104 cells per well onto 96-well plates were allowed to proliferate for 48 h before treatment with 10 µM anandamide (AEA) or 0.1% ethanol for an additional 48 h with the medium exchanged after 24 h in the presence or absence of 300 nM of the CB1-selective antagonist SR141716A or the CB2-selective antagonist SR144528. The antagonist diluent [0.1% dimethylsulfoxide (DMSO)] acted as the control. The data are presented as the mean ± SEM for three independent experiments performed in quadruplicate (n = 3). *, P < 0.05 Student’s paired t test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Materials
 Results
 Discussion
References
 
Our observations confirm that various components of the endocannabinoid system (CB1, CB2, and FAAH) are expressed in 1st trimester tissues. In addition, we have demonstrated that CB1 receptor transcript levels diminish after 9 wk gestation, immunoreactive FAAH disappears from the syncytiotrophoblast by the 11th week of gestation, and that CB2 expression, at the transcript and protein level, remains constant throughout the 1st trimester.

Because the syncytiotrophoblast is in direct contact with maternal blood, the presence of FAAH in this layer suggests that it could be acting to protect the growing embryo from the detrimental effects of AEA. The disappearance of FAAH at 11 wk when circulation in the placental unit is fully established is interesting because it suggests that the protective role of the syncytiotrophoblast is only necessary before the establishment of the placental unit. Whether this is due to the fact that AEA levels in maternal plasma decrease at this point of gestation (17) is unclear. The concurrent increase of FAAH expression in the mesenchymal core of villi in the fully established placental unit suggests that this area of the developing placenta then assumes the protective role of the developing syncytiotrophoblast. It may also explain why there is no concomitant decrease in the levels of FAAH mRNA from homogenized tissues (Fig. 1Go).

Our demonstration of CB1 immunoreactive protein and transcripts in first trimester human trophoblast is in accordance with the findings of Kenney et al. (21) but differs from those of Helliwell et al. (31). The reason for the discrepancy in these observations is unclear but may be related to methodological differences because we found that CB1 immunoreactivity was demonstrable after a process of antigen retrieval by microwave energy and the use of the tyramide amplification system to increase the immunoreactive signal (Fig. 4Go). The immunostaining pattern in term fetal membranes was consistent with that presented (18), suggesting that the methodology used here is appropriate for immunohistochemical detection of CB1. Methodological differences in these studies could be one explanation because we used an improved method that allowed the detection of very low levels of CB1 protein, when compared with that of FAAH and CB2 in the first trimester trophoblast.

Our immunohistochemical data, which were supported by the presence of transcripts for FAAH, CB1, and CB2 (Fig. 1Go) were different from that previously reported (31). A careful Basic Local Alignment Search Tool (BLAST) search of the published primer sequences (31) indicated that the sequences used are unlikely to detect human CB1. Indeed, when we applied the previously used protocols, we were unable to detect CB1 (data not shown). However, using primer sets of Kenney et al. (21), which were previously validated for term placenta samples and BeWo cells, where cannabinoids prevented serotonin transport, it was clear that human first trimester trophoblast contain transcripts of both CB1 and CB2. Because we failed to detect human FAAH (data not shown) with the published FAAH primer sets under the experimental conditions set out in the paper of Helliwell et al. (31), we designed our own set of primers that are directed against exons 14 and 15 of the human FAAH gene (GenBank accession no. AH007340). As can be seen in Fig. 1Go, the predicted product from RT-PCR was generated in all trophoblast samples, and the levels were not modulated through the first trimester, suggesting that the use of a more sensitive amplification technique in the immunohistochemistry studies and of better gene-specific primers may explain the discrepancy between our study and that of Helliwell et al. (31).

Our observations suggest that trophoblastic FAAH levels and CB2 levels do not alter significantly during the first trimester but alter their cellular distribution from the syncytiotrophoblast to the mesenchymal core of the villus. The most significant change was the diminution of CB1 expression after the ninth week of gestation, a point of critical alteration in the developing placenta, where the maternal and fetal blood meet in a meaningful way for the first time (32, 33).

The placenta forms an efficient barrier between fetal and maternal blood. However, it is unlikely to effectively separate maternal endocannabinoids from the fetal compartment. This is because the exocannabinoid, {Delta}9-tetrahydrocannabinol, is known to cross freely the placenta and enter the fetal circulation, whereby it causes detrimental effects on fetal growth and development (34, 35). Therefore, because cannabinoids have a direct effect on the developing placenta (21, 36), it was our hypothesis that if high levels of maternal anandamide were detrimental to early placental and fetal development, FAAH expression would be high in the trophoblast during normal early pregnancy, as has been hypothesized before (31).

The fact that the protein levels of FAAH remained constant in early gestation and appeared to diminish after 9 wk gestation may reflect the protection afforded the fetus during this critical time. Although larger subject numbers would be required to confirm our findings, this study suggests that FAAH levels in the human trophoblast decline between the 10th and 12th week of gestation. These data are similar to a previous study that examined maternal plasma FAAH levels in 50 women who had normal pregnancies between gestational ages 7–12 wk (15). That study found that the levels of FAAH activity peaked at 9–10 wk gestation. An investigation into the factors regulating FAAH and AEA levels at the trophoblast level may provide an explanation for the observed changes.

There is growing evidence that a major transition in placental physiology occurs at approximately 10 wk gestation with the dissipation of trophoblast plugs from the spiral arteries, consequently allowing maternal blood to perfuse the placenta for the 1st time (37, 38). Thus, at about the 10th week of gestation, the human fetus must begin to protect itself against circulating maternal anandamide, but because the CB1 and CB2 receptors are expressed in the syncytiotrophoblast, endocannabinoids are likely to have a regulatory role in the placenta. One suggestion is that endocannabinoids may be involved in the removal of damaged cells by a process of apoptosis. Indeed, there is emerging evidence that endocannabinoids have a significant apoptotic potential in other organs (39). Our studies on the effect of anandamide on BeWo cell growth (Fig. 6Go) tend to support this suggestion because high concentrations of AEA correlated with lower cell numbers and reduced cell growth. Together, the data presented may explain, in part, how elevated plasma anandamide levels may lead to an increased risk of 1st trimester pregnancy loss.


   Acknowledgments
 
We thank Muna Abbas, Reshma Bharkhada, and Susan Spurling for their expert technical assistance. We thank Laurence Goldenberg and Frances Barth of Sanofi-Aventis, France for the generous gifts of SR141716A and SR144528, respectively.


   Footnotes
 
This work was funded by a generous grant from the University Hospitals of Leicester National Health Service Trust.

Disclosure Statement: The authors have nothing to declare.

First Published Online July 3, 2008

Abbreviations: AEA, N-arachidonoylethanolamine; AMV, avian myeloblastosis virus; CB, cannabinoid receptor; FAAH, fatty acid amide hydrolase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NAPE-PLD, N-arachidonylphosphatidyl-ethanolamine selective phospholipase-D; RT, reverse transcriptase.

Received December 28, 2007.

Accepted for publication June 24, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Materials
 Results
 Discussion
 References
 

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