Division of Anatomy and Cell Biology, Department of Pathology and Anatomical Sciences, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, New York, USA.
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First published August 1, 2006 - More info
Synchronized preimplantation embryo development and passage through the oviduct into the uterus are prerequisites for implantation, dysregulation of which often leads to pregnancy failure in women. Cannabinoid/endocannabinoid signaling via cannabinoid receptor CB1 is known to influence early pregnancy. Here we provide evidence that a critical balance between anandamide synthesis by N-acylphosphatidylethanolamine–selective phospholipase D (NAPE-PLD) and its degradation by fatty acid amide hydrolase (FAAH) in mouse embryos and oviducts creates locally an appropriate “anandamide tone” for normal development of embryos and their oviductal transport. FAAH inactivation yielding higher anandamide or experimentally induced higher cannabinoid [(-)-Δ9-tetrahydrocannabinol] levels constrain preimplantation embryo development with aberrant expression of Cdx2, Nanog, and Oct3/4, genes known to direct lineage specification. Defective oviductal embryo transport arising from aberrant endocannabinoid signaling also led to deferred on-time implantation and poor pregnancy outcome. Intercrossing between wild-type and Faah–/– mice rescued developmental defects, not oviductal transport, implying that embryonic and maternal FAAH plays differential roles in these processes. The results suggest that FAAH is a key metabolic gatekeeper, regulating on-site anandamide tone to direct preimplantation events that determine the fate of pregnancy. This study uncovers what we believe to be a novel regulation of preimplantation processes, which could be clinically relevant for fertility regulation in women.
Haibin Wang, Huirong Xie, Yong Guo, Hao Zhang, Toshifumi Takahashi, Philip J. Kingsley, Lawrence J. Marnett, Sanjoy K. Das, Benjamin F. Cravatt, Sudhansu K. Dey
Anandamide (N-arachidonoylethanolamide) is a lipid signal molecule that was the first endogenous agonist for cannabinoid receptors to be discovered. Cannabinoid receptor type 1 (CB1) is widely distributed in neurons and nonneuronal cells in brain and peripheral organs including sperm, eggs, and preimplantation embryos. A study by Wang and colleagues in this issue of the JCI demonstrates that a critical balance between anandamide synthesis by N-acylphosphatidylethanolamine–selective phospholipase D (NAPE-PLD) and its degradation by fatty acid amide hydrolase (FAAH) in mouse embryos and oviducts creates locally an appropriate “anandamide tone” required for normal embryo development, oviductal transport, implantation, and pregnancy (see the related article beginning on page 2122). Adverse effects of elevated levels of anandamide on these processes resulting from FAAH inactivation are mimicked by administration of (-)-Δ9-tetrahydrocannabinol (THC; the major psychoactive constituent of marijuana), due to enhanced signaling via CB1. These findings show that exogenous THC can swamp endogenous anandamide signaling systems, thereby affecting multiple physiological processes.
Attempts to understand the mechanisms responsible for the psychoactive properties of tetrahydrocannabinol (THC) in marijuana led to the discovery of cannabinoid receptors and their endogenous ligands, the endocannabinoids (1). Cannabinoid receptors are G protein–coupled receptors. Two subtypes have been cloned and characterized to date. Cannabinoid receptor type 1 (CB1) was originally cloned from rat and human brain, while CB2 was originally cloned from human promyelocytic leukemia HL-60 cells. Anandamide and related fatty acid derivatives that activate these receptors are endocannabinoids. Anandamide is released from membrane phospholipid precursors by N-acylphosphatidylethanolamine–selective phospholipase D (NAPE-PLD) when neurons and other cells are stimulated by neurotransmitters and hormones. Released anandamide is quickly eliminated by membrane-bound fatty acid amide hydrolase (FAAH), indicative of a role for this lipid in cell signaling.
Anandamide signaling via cannabinoid receptors is not restricted to the central nervous system. We now know that CB1, CB2, NAPE-PLD, and FAAH are widely distributed in nonneuronal somatic cells of peripheral organs including those of the reproductive system, as well as in germ cells of vertebrates and invertebrates (1–8). This may account for the effects of marijuana and THC on multiple aspects of reproductive physiology, including secretion of gonadotrophic hormones by the pituitary gland, secretion of sex steroids by gonads, sperm production, ovulation, mating behavior, sperm capacitation, fertilization, early embryonic development, implantation of blastocysts into the uterine endometrium, placental functions, fetal growth, number of pregnancies carried to term, lactation, suckling behavior of newborns, postnatal development, and growth of malignant breast and prostate cells.
Endocannabinoid signaling via CB1 regulates cleavage of mouse eggs, oviductal transport of preimplantation embryos, blastocyst hatching from the zona pellucida, and implantation of blastocysts into the uterine mucosa (3, 6, 7). In the study by Wang et al. in this issue of the JCI (9), NAPE-PLD was detected in mouse embryos at the stage of the fertilized egg through to the blastocyst stage, while FAAH first appeared in 2-cell embryos (Figure 1). FAAH expression was upregulated in the morula and blastocyst. FAAH was mostly expressed in trophectoderm of blastocysts, suggesting an active role of embryonic FAAH in reducing anandamide levels at potential implantation sites in the uterine mucosa (6, 9). An inverse distribution of these enzymes was present in mouse oviducts on days 1–4 of pregnancy, with higher levels of Nape-pld in epithelium of the isthmus than of the ampulla (9). In contrast, Faah was expressed at higher levels in ampulla than in isthmus epithelium. Suppression of FAAH activity in embryos and oviducts pharmacologically or by genetic ablation elevated anandamide levels in situ, which resulted in inhibition of embryonic development and expression of cell lineage genes required for differentiation, retention of embryos within the oviduct, impaired implantation, and reduced fertility. Administration of exogenous THC similarly affected early pregnancy events. Furthermore, the adverse effects on initiation of pregnancy resulting from inactivation of FAAH and by addition of THC were blocked by the CB1 selective antagonist SR141716. Unlike endogenous neurotransmitters/neuromodulators, which are rapidly released and degraded locally on demand in the body, exogenous drugs such as THC persist and swamp endogenous anandamide signaling systems, thereby exerting long-term effects on multiple physiological processes. The findings reported by Wang et al. (9) describe an extremely provocative model for the biological effects of THC and marijuana on functions of other peripheral organs as well as the brain.
FAAH is a key metabolic gatekeeper regulating anandamide tone in oviduct and preimplantation embryos. CB1, NAPE-PLD, and FAAH are expressed in the oviduct and embryo. A concentration gradient for anandamide in the oviduct is suggested by the expression of higher levels of Nape-pld in the oviduct epithelium of the isthmus, in contrast to higher levels of Faah in the ampulla (9). Elevating anandamide levels in the oviduct by administration of FAAH inhibitors or by mating Faah–/– mice inhibits cleavage, impairs oviductal transport of preimplantation embryos, and reduces fertility. NAPE-PLD was detected in mouse embryos at the stage of the fertilized egg through to the blastocyst stage, while FAAH first appeared in 2-cell embryos. Treatment of mice with THC causes retarded development of embryos and oviductal retention of embryos. The adverse effects of inactivating FAAH and of administering exogenous THC on these processes are prevented by administration of the CB1-selective antagonist SR141716, which suggests that these effects are mediated via CB1. Exposure to THC swamps the endogenous anandamide tone, leading to early pregnancy failure. Figure adapted from an image kindly provided by S.S. Gambhir and J. Strommer, Stanford University, Stanford, California, USA.
Aberrant functioning of anandamide signaling systems in embryos and oviducts in women may lead to ectopic pregnancy in the oviduct and/or impaired fertility (3, 7, 9). Similar adverse effects may be associated with abuse of marijuana by women of reproductive age. This possibility is consistent with studies showing that women who smoke tobacco show an increased incidence of ectopic pregnancy (10), which correlates with the direct inhibitory effects of tobacco smoke and nicotine on oviductal transport of embryos in hamsters (11). Faah-null female mice are less fertile than wild-type females (9). This observation is clinically relevant, since reduced peripheral FAAH activity is associated with spontaneous abortion in women (4). Oviductal transport of embryos in mice was shown to be regulated by crosstalk between anandamide signaling via CB1 and β-adrenergic receptors in the oviductal muscularis (7). Acetaminophen is a popular over-the-counter analgesic and antiinflammatory agent. Following deacylation, it is converted in the body to the bioactive N-acylphenolamine (also known as AM404) via FAAH-dependent arachidonic acid conjugation (12). AM404 is a ligand for CB1 receptors and is a potent inhibitor of anandamide reuptake, the inhibition of which leads to elevated anandamide levels in the body. Do women using β-adrenergic medications or acetaminophen show elevated incidences of ectopic pregnancy and impaired fertility?
There are provocative similarities between anandamide signaling in embryos and oviductal epithelia and signaling pathways associated with sperm capacitation in the female reproductive tract. Studies on human and porcine sperm suggested the possible existence of an anandamide concentration gradient in the oviduct that regulates sperm capacitation (2, 3, 8, 9, 13, 14). A mechanism underlyng this postulated anandamide gradient is indicated by the differences in the distribution of Nape-pld and Faah in mouse oviduct (9). Sperm and preimplantation mouse embryos express functional CB1 receptors as well as key enzymes (NAPE-PLD and FAAH) for the release of anandamide and its subsequent rapid removal (3, 6, 8, 9, 13, 14). Anandamide is present in human mid-cycle oviductal fluid (2, 3, 8, 14). With respect to mouse embryos, maternal NAPE-PLD and FAAH in the oviduct are primarily concerned with regulation of oviductal transport, while in the embryo these proteins regulate embryonic development (9). Does a similar division of labor regulate sperm capacitation and fertilizing potential within the oviduct?
As the first endocannabinoid to be discovered, anandamide has attracted the most attention from investigators (1). Similarly, the biological functions of CB1 have been studied much more extensively than those mediated by CB2, because CB1 receptors in the brain are directly involved in the psychoactive effects of THC. Significantly, porcine sperm and preimplantation mouse embryos express CB2 receptors (6, 8, 13). The function(s) of CB2 in sperm and embryos have yet to be examined. We also do not know whether other endocannabinoids (e.g., ethanolamides of other fatty acids, 2-arachidonoylglycerol [2-AG], noladin ether, virodhamine, etc.) are constituents of reproductive tract fluids. If so, how do these endocannabinoids affect sperm capacitation and fertilizing potential, as well as embryo development and transport? Anandamide mimics the effects of THC by serving as a partial agonist for CB1 and CB2 (15). Furthermore, 2-AG is found at much higher concentrations than anandamide in the brain and peripheral organs. 2-AG is a full agonist for both CB1 and CB2. Thus, 2-AG may be a more physiologically significant agonist for cannabinoid receptors than anandamide. Studies on neurons and other somatic cells suggest that endocannabinoids may elicit different responses within the same cell population by binding to different receptor subtypes or splice variants, differentially colocalizing receptors with signaling molecules, and differentially activating G proteins (16, 17). Hence, the presence of other endocannabinoids in the microenvironment surrounding sperm or preimplantation embryos might orchestrate subtle differences in their physiology.
Anandamide also can produce biological responses via mechanisms that do not involve cannabinoid receptors (8, 17). It is an agonist for transient receptor potential channel vanilloid receptor subunit 1 (TRPV1) and can directly inhibit certain Ca2+ and K+ channels, directly activate glutamate ionotropic receptors, and directly block activation of nicotinic α polypeptide 7 receptor by acetylcholine. Significantly, anandamide inhibits spontaneous acrosome reactions in boar sperm during capacitation by activating TRPV1 receptors (13). Thus, alternate pathways for the actions of anandamide and other endocannabinoids in reproductive physiology and other bodily functions warrant careful examination.
Anandamide signaling is directly involved in a myriad of physiological and pathological processes and presents many potential targets for the development of novel therapeutic drugs (18). Unfortunately, cannabinergic ligands affect almost every physiological system investigated (1). Thus, drugs acting on anandamide signaling may produce a wide variety of side effects that would limit their utility. For example, possible medicinal uses of THC are limited by its psychoactive properties. One strategy for drug development targets specific cannabinoid receptors. The CB1 antagonist SR141716 (rimonabant; trade name Acomplia) suppresses appetite (19). The European Medicines Agency recently recommended approval of Acomplia for use as a weight-loss drug. An alternate approach targets removal and degradation of anandamide (18). Basal levels of anandamide in the brain and peripheral organs are quite low (15). It is rapidly released and degraded locally. Thus, drugs that prevent anandamide reuptake and hydrolysis might be useful clinically (18). Given the results presented in the current report (9), such drugs may need to be carefully evaluated to judge their effects on women of reproductive age and those that are pregnant.
Nonstandard abbreviations used: 2-AG, 2-arachidonoylglycerol; CB1, cannabinoid receptor type 1; FAAH, fatty acid amide hydrolase; NAPE-PLD, N-acylphosphatidylethanolamine–selective phospholipase D; THC, (-)-Δ9-tetrahydrocannabinol.
Conflict of interest: The author has declared that no conflict of interest exists.
Reference information: J. Clin. Invest.116:2087–2090 (2006). doi:10.1172/JCI29424.
See the related article beginning on page 2122.