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a Department of Molecular and Integrative Physiology, Ralph L. Smith Research Center, University of Kansas Medical Center, Kansas City, Kansas 66160-7338
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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9-tetrahydrocannabinol [(-)THC], a psychoactive derivative of marijuana [1, 4–9]. The CB1-R and CB2-R genes are expressed in preimplantation mouse embryos, and the levels of CB1-R in the embryo are much higher than those in the brain [10, 11]. Furthermore, the mouse uterus contains the highest levels of anandamide yet discovered in a mammalian tissue; the levels are lower at the implantation sites but higher at the interimplantation sites [3]. Taken together, the above findings suggested that preimplantation mouse embryos are targets for cannabinoid ligand-receptor signaling. Indeed, natural and synthetic cannabinoid agonists adversely affect preimplantation embryo development from the two-cell to the blastocyst stages in vitro, and these effects are mediated by CB1-R [10, 11]. Thus, an aberrant synthesis/metabolism of anandamide or an aberrant expression of the cannabinoid receptors in the uterus and/or embryo could contribute to early pregnancy failure. The turnover rate and fate of anandamide in the brain and other tissues are not yet well-defined, but recent evidence demonstrates that a tissue amidohydrolase, called anandamide amidase (E.C.3.5.1.4), can hydrolyze anandamide [12–16]. This membrane-associated enzyme is inhibited by PMSF, a serine protease inhibitor, and by several analogues of anandamide [8, 17–20]. Recently, human, rat, and mouse homologues of a fatty-acid amide hydrolase (FAAH) gene have been cloned [21, 22]. This enzyme rapidly hydrolyses anandamide and oleamide, a sleep-inducing lipid. In addition, cyclooxygenase-2 and lipoxygenases are also known to metabolize anandamide [23, 24]; the significance of this is not yet clear.
Synchronized development of the embryo to the blastocyst stage and differentiation of the uterus to the receptive state are essential to the process of implantation [25, 26]. In the mouse, one of the earliest conspicuous signs of the implantation process is an increased endometrial vascular permeability at the sites of blastocyst apposition [25]. This event coincides with the initial attachment reaction between the blastocyst trophectoderm and uterine luminal epithelium [27]. In the mouse, the attachment reaction occurs at 2200–2300 h on Day 4 [28] and is followed by localized stromal decidualization at the sites of blastocysts [27]. These events are regulated by coordinated effects of estrogen and progesterone (P4). However, the mechanisms by which estrogen transforms the P4-primed uterus to the receptive state, activates the blastocyst, and initiates the implantation process on Day 4 are not clearly defined.
Because anandamide levels fluctuate in the periimplantation mouse uterus and because this lipid mediator is detrimental for early preimplantation embryo development, it is important that the local levels of anandamide are tightly regulated during early pregnancy. Thus, we examined the expression of FAAH, the enzyme involved in the hydrolysis of anandamide, in the mouse embryo and uterus during early pregnancy.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CD-1 mice (Charles River Laboratory, Raleigh, NC) were housed in the animal care facility at the University of Kansas Medical Center according to NIH and institutional guidelines. Virgin females (48–60 days old, 20–25 g) were mated with fertile males of the same strain. The morning of finding a vaginal plug was designated Day 1 of pregnancy. Mice on Days 1–4 were killed at 0830–0930 h, and embryos were recovered from the reproductive tract to confirm pregnancy. On Days 5–8, mice were killed at 0900 h. Implantation sites on Days 5 and 6 were visualized by i.v. injection (0.1 ml/mouse) of a Chicago blue dye solution (0.1% in saline). Implantation sites were demarcated by discrete blue bands along the uterus [26]. On Days 7–8, implantation sites are distinct and blue dye injection is not required. Preimplantation embryos at the one-cell (Day 1), two-cell (Day 2), eight-cell/morula (Day 3), and blastocyst (Day 4) stages were collected by flushing the reproductive tracts. Embryos at these stages were pooled from several mice and were stored in a small volume of PBS at -70°C until used for reverse transcription (RT)-polymerase chain reaction (PCR).
Hybridization Probes
A 287-base pair (bp) fragment (nucleotides [nt] 1360–1646) of murine FAAH [22] was amplified by RT-PCR from mouse liver RNA and inserted into the multiple cloning site of pCR II-TOPO vector using a TOPO TA cloning kit (Invitrogen Corp., Carlsbad, CA). The authenticity of the clone was determined by sequence analysis. For Northern hybridization, 32P-labeled antisense cRNA probes for FAAH were generated using SP6 polymerase. For in situ hybridization, sense and antisense 35S-labeled probes were generated using T7 and SP6 polymerases, respectively. A part of the ribosomal protein L-7 (rpL7, a house-keeping gene) cDNA (246 bp) was subcloned into pCR-Script vector and used as a template for synthesis of 32P-labeled antisense rpL7 probe with T7 polymerase [29]. The probes had specific activities of 2 x 109 dpm/µg.
Analysis of Uterine FAAH mRNA by Northern Hybridization
Total RNA was extracted from whole uteri by a modified guanidine thiocyanate procedure [28]. Poly(A)+ RNA was isolated by oligo(dT)-cellulose column chromatography [30]. Poly(A)+ RNA (2 µg) was denatured, separated by formaldehyde-agarose gel electrophoresis, and transferred and cross-linked to a membrane by UV irradiation (Spectrolinker; Spectronics Corp., Westbury, NY). Northern blots were prehybridized, hybridized, and washed as described previously [28]. The same blots were sequentially hybridized to FAAH and rpL7 probes, and the hybrids were detected by autoradiography.
Analysis of Uterine Cell-Specific Expression of FAAH mRNA by In Situ Hybridization
In situ hybridization was performed as described previously [28]. On specific days of pregnancy, uterine horns were excised and cut into small pieces or separated into implantation and interimplantation sites. Frozen sections (10 µm) were mounted onto poly-L-lysine-coated slides. When required, frozen sections were cut serially to detect the sites of blastocysts. Sections were fixed in 4% paraformaldehyde in PBS for 15 min at 4°C. After prehybridization, uterine sections were hybridized to 35S-labeled FAAH antisense cRNA probe for 4 h at 45°C. Uterine sections were also hybridized to the labeled sense probe (negative control). After hybridization and washing, the slides were incubated with ribonuclease (RNase) A (20 µg/ml) at 37°C for 15 min. RNase A-resistant hybrids were detected after 1–3 days of autoradiography using Kodak NTB-2 liquid emulsion (Eastman Kodak, Rochester, NY). The slides were post-stained with hematoxylin and eosin.
Analysis of Uterine FAAH Protein by Western Blotting
The method essentially followed the protocol as described previously [29]. In brief, Day 1 and Day 4 mouse uteri were collected into buffer A (10 mM Tris-HCl [pH 7.4], 250 mM sucrose, 2 mM EGTA, 10 µg/ml leupeptin, 20 µg/ml PMSF, 10 µg/ml aprotinin). Tissues were homogenized in the same buffer and centrifuged at 900 x g for 10 min at 4°C. The supernatants were recentrifuged at 110 000 x g for 1 h at 4°C. The pellets were resuspended in the same buffer and spun again for 1 h at 110 000 x g at 4°C. The pellets (total cell membrane fraction) were then dissolved in buffer B (10 mM Tris-HCl [pH 7.4] 0.15 mM NaCl, 1 mM EGTA, 10 µg/ml leupeptin, 20 µg/ml PMSF, 10 µg/ml aprotinin), and protein concentrations were measured. Aliquots of protein (60 µg) were mixed with sample buffer and boiled for 5 min. The samples were run on a 7.5% SDS-polyacrylamide gel under reducing conditions. The separated proteins on the gel were transferred onto a nitrocellulose membrane. The membrane was preincubated with 5% nonfat dry milk in Tris-buffered saline (TBS: 10 mM Tris-HCl [pH 8.0], 150 mM NaCl) for 2 h to block nonspecific binding. The membrane was incubated in rabbit antipeptide antibody to FAAH [21] for 18 h at 4°C. This antibody was kindly provided by Dr. Benjamin F. Cravatt (The Scripps Research Institute, La Jolla, CA). The membrane was washed 3 times for 10 min each in 5% nonfat dry milk in TBS and incubated with goat anti-rabbit IgG conjugated with horseradish peroxidase (1:15 000) for 1 h. The membrane was again washed 3 times (10 min each) in 5% nonfat dry milk in TBST (TBS plus 0.05% Tween-20) and 3 times in TBS. Signals were detected with an ECL kit (Amersham, Arlington Heights, IL).
Analysis of FAAH mRNA in the Preimplantation Embryo by RT-PCR
To examine FAAH mRNA expression in the preimplantation mouse embryo, RT-PCR was employed. Oligonucleotide primers for FAAH were synthesized on the basis of cloned mouse cDNA sequences [22]. The primers were 5'-GAGATGTATCGCCAGTCCGT-3' (nt 1360–1379; sense) and 5'-ACAGGCAGGCCTATACCCTT-3' (nt 1627–1646; antisense). The rpL7 sense and antisense primers designed from the mouse rpL7 cDNA [31] were (5'-TCAATGGAGTAAGCCCAAAG-3' (nt 359–378) and 5'-CAAGAGACCGAGCAATCAAG-3' (nt 585–604), respectively. The internal sense oligonucleotides, 5'-GTCACCACTGTGACCGCTGA-3' (nt 1531–1550) and 5'-GATTGCCTTGACAGATAATTC-3' (nt 564–584), were used for Southern hybridization of RT-PCR-amplified products for FAAH and rpL7, respectively. Total RNAs from mouse brain and uterus, and 80 mouse embryos at the one- or two-cell stage and 70 embryos at the eight-cell/morula or blastocyst stage were isolated [10, 28, 32]. Total RNA (2 µg) from brain and uterus or 25% of the embryonic RNA was reverse-transcribed by using specific antisense primers. One eighth of the RT products were subjected to PCR amplification using specific sense and antisense primers as described [32]. PCR cycle parameters were as follows: 94°C for 1.5 min, 55°C for 1 min, and 72°C for 1 min for the first two cycles, followed by 94°C for 0.5 min, 55°C for 0.5 min, and 72°C for 0.5 min for 40 cycles. One fifth of the amplified product was electrophoresed on agarose gels (1.5%), blotted, and analyzed by Southern hybridization [10, 32]. Experimental and controls were run simultaneously.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Steady-state levels of FAAH mRNA in the uterus on Days 1–8 of pregnancy were examined by Northern hybridization using a 32P-labeled cRNA probe (Fig. 1). Mouse liver and brain RNAs served as positive controls [21, 22]. A 2.5-kb transcript of FAAH mRNA, similar to that identified in rat brain and liver [21], was detected in the mouse uterus, liver, and brain (Fig. 1). Consistent with the previous report [21], a transcript larger than 2.5 kb was also detected in these samples. This transcript could be either unprocessed or alternatively spliced forms of the 2.5-kb transcript. Integrity and loading of RNA samples were monitored by rehybridizing the same blots to an rpL-7 probe, a house-keeping gene. The levels of FAAH mRNA were considerably higher in the brain and liver than in the uterus. The levels of this mRNA in whole uterine poly(A)+ RNA samples were higher on Days 1–4 but declined on later days (Days 5–8) of pregnancy. The uterus is composed of heterogeneous cell types that undergo rapid and dynamic changes during early pregnancy. Thus, cell-specific expression of FAAH mRNA was examined by in situ hybridization.
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In Situ Hybridization of FAAH mRNA in the Periimplantation Uterus
As shown in Figure 2, autoradiographic signals were primarily localized in the luminal and glandular epithelia on Days 1–4 of pregnancy. On Days 5–8, FAAH mRNA accumulation primarily occurred in the implanting blastocysts. Low levels of signals were also evident in the remaining luminal epithelium and peripheral glands at the implantation sites, as well as at the interimplantation sites on these days (Fig. 3).
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Western Blot Detection of FAAH Protein in the Uterus
As previously described [21], Western blotting using an antipeptide antibody to FAAH detected an ~60-kDa protein in uterine membrane preparations (Fig. 4). A protein of lower molecular weight was also detected. The levels of the high-molecular-weight protein in the uterus did not appear to show changes on Days 1 and 4 of pregnancy, while the levels of the low-molecular-weight protein apparently decreased on Day 4. The significance of this finding is not yet clear. No positive immunoreactive bands were detected with nonimmune serum (data not shown). The results of Northern and Western blots suggest that two forms of FAAH may be expressed in the mouse uterus. However, it is not known whether the low-molecular-weight protein possesses any enzymatic activity. Nonetheless, the result suggests that FAAH mRNA is translated in the uterus. The presence of anandamide-hydrolysing activity in the mouse uterus as observed previously [33] is consistent with the expression of FAAH in this tissue. This antibody was not suitable for cell-specific localization of FAAH by immunohistochemistry.
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RT-PCR Detection of FAAH mRNA in the Preimplantation Mouse Embryo and Uterus
RT-PCR detected FAAH mRNA in one-cell and two-cell mouse embryos, whereas this mRNA was not detected in embryos at the eight-cell/morula stage. It was again detected in Day 4 blastocysts (Fig. 5). The results suggest the presence of maternal message in earlier stages of embryos. In contrast, the detection of FAAH mRNA in blastocysts suggests embryonic gene activation. The higher levels of this mRNA in Day 1 uterus compared with Day 5 uterus is consistent with Northern hybridization results. The mouse brain RNA used as a positive control detected FAAH mRNA (Fig. 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The presence of the FAAH mRNA primarily in the epithelial cells throughout the periimplantation period suggests that this gene is not under the regulation of ovarian estrogen and P4, because this pattern does not reflect dynamic cellular changes that result from estrogen and P4 stimulation during the periimplantation period [36]. For example, the uterus comprises heterogeneous cell types that respond differentially to estrogen and P4. In the adult mouse uterus, estrogen stimulates proliferation of luminal and glandular epithelial cells, while proliferation in the stroma results from a combined action of P4 and estrogen. A similar steroid hormonal modulation of cell-specific proliferation occurs in the uterus during early pregnancy. Preovulatory ovarian estrogen directs epithelial cell proliferation on Days 1 and 2 of pregnancy. On Day 3, P4 from newly formed corpora lutea initiates stromal cell proliferation that is further potentiated by preimplantation estrogen secretion on Day 4. After initiation of implantation, stromal cells surrounding the blastocysts undergo extensive proliferation and differentiation into decidual cells (decidualization) [36]. Thus, the mechanism by which FAAH is regulated in the uterus will require further investigation.
Cannabinoid agonists including anandamide at a low level (7 nM) adversely affect development of two-cell embryos into blastocysts in vitro, and continuous infusion of CP-55940, a synthetic cannabinoid, interferes with blastocyst zona-hatching and implantation. These effects are mediated via CB1-R [3, 10, 11, 34]. In contrast, blastocysts exposed to the same low level of anandamide in vitro exhibit accelerated trophoblast differentiation and outgrowth, but this endogenous agonist at a higher concentration (28 nM) interferes with these events. Again, these effects are mediated via CB1-R [37]. These results suggest that while higher levels of cannabinoid agonists adversely affect blastocyst functions, lower levels are rather beneficial to blastocyst functions. This is consistent with our recent findings that while single injections of (-)THC fail to affect implantation, higher uterine levels of (-)THC resulting from its continuous infusion in the presence of cytochrome P450 inhibitors interfere with blastocyst functions and implantation [34]. Thus, the stages of embryonic development and levels of endogenous ligands perhaps determine the fate of embryonic development during early pregnancy.
Cyclooxygenase (COX) is the rate-limiting enzyme in the biosynthesis of prostaglandins (PGs). COX exists in two isoforms, the constitutive COX-1 and the inducible COX-2. We have demonstrated that COX-2 is expressed in the uterus solely at the sites of blastocyst apposition during implantation and that the targeted disruption of the COX-2 gene, but not the COX-1 gene, interferes with implantation and decidualization in the mouse [38]. Recently, it has also been shown that COX-2, but not COX-1, is capable of converting anandamide to PGE2 ethanolamide [23]. Thus, it is possible that embryonic FAAH and uterine COX-2 at the sites of implantation maintain the optimal levels of anandamide favorable to the initiation and progression of implantation. COX-2 may also compete for the substrate arachidonic acid, the precursor for the synthesis of both PGs and anandamide, at the implantation sites. Thus, COX-2 could be important in regulating the balance between the two lipid mediators favorable to implantation.
Recent investigations using partially purified porcine brain enzyme [14] and COS-7 cells transiently transfected with rat FAAH cDNA [39] suggest that anandamide amidohydrolase and synthase activities are attributable to a single protein [14]. However, the remarkable down-regulation of uterine FAAH mRNA from Day 5 of pregnancy but with sustained levels of anandamide suggests that the enzyme responsible for anandamide synthesis in the uterus is different from FAAH [3]. The isolation and cloning of the enzyme for anandamide synthesis is needed to better understand the synthesis and metabolism of anandamide in the uterus during early pregnancy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: S.K. Dey, Department of Molecular and Integrative Physiology, MRRC 37/3017, University of Kansas Medical Center, Kansas City, KS 66160–7338. FAX: 913 588 5677; sdey{at}kumc.edu
Accepted: December 15, 1998.
Received: October 1, 1998.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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