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Dysregulated Cannabinoid Signaling Disrupts Uterine Receptivity for Embryo Implantation

2001; Elsevier BV; Volume: 276; Issue: 23 Linguagem: Inglês

10.1074/jbc.m100679200

1083-351X

Bibhash C. Paria, Haengseok Song, X. Wang, Patricia C. Schmid, Randy J. Krebsbach, Harald H.O. Schmid, Tom I. Bonner, Andreas Zimmer, Sudhansu K. Dey,

Dietary Effects on Health

The mechanisms by which synchronized embryonic development to the blastocyst stage, preparation of the uterus for the receptive state, and reciprocal embryo-uterine interactions for implantation are coordinated are still unclear. We show in this study that preimplantation embryo development became asynchronous in mice that are deficient in brain-type (CB1) and/or spleen-type (CB2) cannabinoid receptor genes. Furthermore, whereas the levels of uterine anandamide (endocannabinoid) and blastocyst CB1 are coordinately down-regulated with the onset of uterine receptivity and blastocyst activation prior to implantation, these levels remained high in the nonreceptive uterus and in dormant blastocysts during delayed implantation and in pregnant, leukemia inhibitory factor (LIF)-deficient mice with implantation failure. These results suggest that a tight regulation of endocannabinoid signaling is important for synchronizing embryo development with uterine receptivity for implantation. Indeed this is consistent with our finding that while an experimentally induced, sustained level of an exogenously administered, natural cannabinoid inhibited implantation in wild-type mice, it failed to do so inCB1 −/− /CB2 −/−double mutant mice. The present study is clinically important because of the widely debated medicinal use of cannabinoids and their reported adverse effects on pregnancy. The mechanisms by which synchronized embryonic development to the blastocyst stage, preparation of the uterus for the receptive state, and reciprocal embryo-uterine interactions for implantation are coordinated are still unclear. We show in this study that preimplantation embryo development became asynchronous in mice that are deficient in brain-type (CB1) and/or spleen-type (CB2) cannabinoid receptor genes. Furthermore, whereas the levels of uterine anandamide (endocannabinoid) and blastocyst CB1 are coordinately down-regulated with the onset of uterine receptivity and blastocyst activation prior to implantation, these levels remained high in the nonreceptive uterus and in dormant blastocysts during delayed implantation and in pregnant, leukemia inhibitory factor (LIF)-deficient mice with implantation failure. These results suggest that a tight regulation of endocannabinoid signaling is important for synchronizing embryo development with uterine receptivity for implantation. Indeed this is consistent with our finding that while an experimentally induced, sustained level of an exogenously administered, natural cannabinoid inhibited implantation in wild-type mice, it failed to do so inCB1 −/− /CB2 −/−double mutant mice. The present study is clinically important because of the widely debated medicinal use of cannabinoids and their reported adverse effects on pregnancy. leukemia inhibitory factor N-acylethanolamine Δ9-tetrahydrocannabinol Previous investigation suggested that cannabinoid exposure has adverse effects on pregnancy outcome (reviewed in Refs. 1Paria B.C. Das S.K. Dey S.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9460-9464Crossref PubMed Scopus (173) Google Scholar, 2Yang Z.M. Paria B.C. Dey S.K. Biol. Reprod. 1996; 55: 756-761Crossref PubMed Scopus (90) Google Scholar, 3Schmid P.C. Paria B.C. Krebsbach R.J. Schmid H.H.O. Dey S.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4188-4192Crossref PubMed Scopus (223) Google Scholar, 4Paria B.C. Ma W. Andrenyak D.M. Schmid P.C. Schmid H.H.O. Moody D.E. Deng H. Makriyannis A. Dey S.K. Biol. Reprod. 1998; 58: 1490-1495Crossref PubMed Scopus (96) Google Scholar, 5Wang J. Paria B.C. Dey S.K. Armant D.R. Biol. Reprod. 1999; 60: 839-844Crossref PubMed Scopus (74) Google Scholar). Cannabinomimetic drugs interact with two types of cannabinoid receptors, brain-type (CB1) and spleen-type (CB2) (6Howlett A.C. Annu. Rev. Pharmacol. Toxicol. 1995; 35: 607-634Crossref PubMed Scopus (444) Google Scholar, 7Pertwee R.G. Pharmacol. Ther. 1997; 74: 129-180Crossref PubMed Scopus (1282) Google Scholar, 8Matsuda L.A. Lolait S.J. Brownstein M.J. Young A.C. Bonner T.I. Nature. 1990; 346: 561-564Crossref PubMed Scopus (4162) Google Scholar, 9Munro S. Thomas K.L. Abu-sharr M. Nature. 1993; 365: 61-65Crossref PubMed Scopus (4079) Google Scholar). These receptor subtypes are negatively coupled to adenylate cyclase and to N-type and P/Q-type calcium channels and positively coupled to mitogen-activated protein kinase and to A-type potassium channels through Gi/o proteins (7Pertwee R.G. Pharmacol. Ther. 1997; 74: 129-180Crossref PubMed Scopus (1282) Google Scholar, 10Bouaboula M. Poinot-Chazel C. Bourrie B. Canat X. Calandra B. Rinaldi-Carmona M. Le Fur G. Casellas P. Biochem. J. 1995; 312: 637-641Crossref PubMed Scopus (457) Google Scholar). CB1 is expressed in brain and other peripheral tissues (7Pertwee R.G. Pharmacol. Ther. 1997; 74: 129-180Crossref PubMed Scopus (1282) Google Scholar, 8Matsuda L.A. Lolait S.J. Brownstein M.J. Young A.C. Bonner T.I. Nature. 1990; 346: 561-564Crossref PubMed Scopus (4162) Google Scholar, 9Munro S. Thomas K.L. Abu-sharr M. Nature. 1993; 365: 61-65Crossref PubMed Scopus (4079) Google Scholar, 10Bouaboula M. Poinot-Chazel C. Bourrie B. Canat X. Calandra B. Rinaldi-Carmona M. Le Fur G. Casellas P. Biochem. J. 1995; 312: 637-641Crossref PubMed Scopus (457) Google Scholar, 11Paria B.C. Das S.K. Dey S.K. Nahas G.G. Sutin K.M. Harvey D.J. Agurell S. Marihuana and Medicine. Humana Press, Totowa, NJ1999: 393-410Crossref Google Scholar). CB2 is expressed primarily in immune tissues including the spleen, leukocytes, and tonsils (12Galiege S. Mary S. Marchand J. Dussossoy D. Carriere D. Carayon P. Bouaboula M. Shire D. LeFur G. Casellas P. Eur. J. Biochem. 1995; 232: 54-61Crossref PubMed Scopus (1337) Google Scholar). We have previously shown that CB1 is expressed in the preimplantation mouse embryo at much higher levels than in the brain (1Paria B.C. Das S.K. Dey S.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9460-9464Crossref PubMed Scopus (173) Google Scholar, 2Yang Z.M. Paria B.C. Dey S.K. Biol. Reprod. 1996; 55: 756-761Crossref PubMed Scopus (90) Google Scholar). The discovery of cannabinoid receptors led to the identification of endogenous cannabinoid ligands, N-arachidonoylethanolamine (anandamide), and 2-arachidonoylglycerol (13Devane W.A. Hanus L. Breuer A. Pertwee R.G. Stevenson L.A. Griffin G. Gibson D. Mandelbaum A. Etinger A. Machoulam R. Science. 1992; 258: 1946-1949Crossref PubMed Scopus (4618) Google Scholar, 14Felder C.C. Veluz J.S. Williams H.L. Briley E.M. Matsuda L.A. Mol. Pharmacol. 1992; 42: 838-845PubMed Google Scholar, 15Mechoulam R. Ben Shabat S. Hanus L. Ligumsky M. Kaminski N.E. Schatz A.R. Gopher A. Almog S. Martin B.R. Compton D.R. Pertwee R.G. Griffin G. Bayewitch M. Barg J. Vogel Z. Biochem. Pharmacol. 1995; 50: 83-90Crossref PubMed Scopus (2315) Google Scholar, 16Sugiura T. Kondo S. Sukagawa A. Nakane S. Shinoda A. Itoh K. Yamashita A. Waku K. J. Biol. Chem. 1999; 274: 2794-2801Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 17Stella N. Schweitzer P. Piomelli D. Nature. 1997; 388: 773-777Crossref PubMed Scopus (1227) Google Scholar). We observed that anandamide is synthesized in the pregnant mouse oviduct and uterus (18Paria B.C. Deutsch D.D. Dey S.K. Mol. Reprod. Dev. 1996; 45: 183-192Crossref PubMed Scopus (128) Google Scholar) and that its levels are far higher in the uterus than in any other mammalian tissue examined (3Schmid P.C. Paria B.C. Krebsbach R.J. Schmid H.H.O. Dey S.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4188-4192Crossref PubMed Scopus (223) Google Scholar). However, anandamide levels are significantly lower at implantation sites than at interimplantation sites, suggesting endocannabinoid ligand-receptor signaling during implantation (3Schmid P.C. Paria B.C. Krebsbach R.J. Schmid H.H.O. Dey S.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4188-4192Crossref PubMed Scopus (223) Google Scholar). Indeed anandamide at higher levels adversely affects embryo development and implantation, whereas at lower levels it stimulates embryo growth and differentiation via CB1 (1Paria B.C. Das S.K. Dey S.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9460-9464Crossref PubMed Scopus (173) Google Scholar, 2Yang Z.M. Paria B.C. Dey S.K. Biol. Reprod. 1996; 55: 756-761Crossref PubMed Scopus (90) Google Scholar, 3Schmid P.C. Paria B.C. Krebsbach R.J. Schmid H.H.O. Dey S.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4188-4192Crossref PubMed Scopus (223) Google Scholar, 4Paria B.C. Ma W. Andrenyak D.M. Schmid P.C. Schmid H.H.O. Moody D.E. Deng H. Makriyannis A. Dey S.K. Biol. Reprod. 1998; 58: 1490-1495Crossref PubMed Scopus (96) Google Scholar, 5Wang J. Paria B.C. Dey S.K. Armant D.R. Biol. Reprod. 1999; 60: 839-844Crossref PubMed Scopus (74) Google Scholar). Interestingly, 2-arachidonoylglycerol is present in the mouse uterus at amounts similar to or lower than the lowest anandamide levels (1–5 nmol/g of tissue), and its level does not vary significantly between implantation and interimplantation sites. 1B. C. Paria, P. C. Schmid, R. J. Krebsbach, H. H. O. Schmid, and S. K. Dey, unpublished results.1B. C. Paria, P. C. Schmid, R. J. Krebsbach, H. H. O. Schmid, and S. K. Dey, unpublished results. Using cannabinoid receptor mutant mice (19Zimmer A. Zimmer A.M. Hohmann A.G. Herkenham M. Bonner T.I. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5780-5785Crossref PubMed Scopus (888) Google Scholar) and physiological approaches, we further defined the role of ligand-receptor signaling with cannabinoids in preimplantation embryo development and in implantation.DISCUSSIONThe mechanism(s) by which the development of preimplantation embryos into active blastocysts is synchronized with uterine receptivity for implantation is not clearly understood. Our present observations of the asynchronous development of embryos deficient in cannabinoid receptors during the preimplantation period and of the coordinated down-regulation of both uterine anandamide levels and blastocyst cannabinoid receptors prior to implantation in wild-type mice suggest that ligand-receptor signaling with endocannabinoids locally helps in regulating the "window" of implantation. Although the Mendelian frequency of offspring resulting from heterozygous crossings of CB1-null mice is skewed resulting in a somewhat reduced number of homozygous offspring (19Zimmer A. Zimmer A.M. Hohmann A.G. Herkenham M. Bonner T.I. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5780-5785Crossref PubMed Scopus (888) Google Scholar), and although the pregnancy rate in mutant mice resulting from homozygous mating is somewhat lower, the birth of viable CB1 −/−,CB2 −/−, orCB1 −/− /CB2 −/− double mutant offspring suggests that the absence of embryonic and/or uterine cannabinoid receptors is not indispensable for embryonic development or implantation. However, the observed increased mortality ofCB1 −/− offspring perhaps could be due to inferior fetal development resulting from the implantation of slowly developing embryos (19Zimmer A. Zimmer A.M. Hohmann A.G. Herkenham M. Bonner T.I. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5780-5785Crossref PubMed Scopus (888) Google Scholar). The major phenotypes ofCB1 −/− orCB1 −/− /CB2 −/− double mutant mice are their resistance to exogenous cannabinoid exposure with respect to embryo development and implantation in vitro andin vivo. This suggests that embryonic development and implantation are likely to be affected by aberrant levels of exogenous or endogenous cannabinoids in the uterus and/or aberrant embryonic expression of cannabinoid receptors during early pregnancy.On day 5 of pseudopregnancy when the uterus is still receptive,LIF expression persists in uterine glands. In contrast,LIF expression is undetectable or extremely low in the nonreceptive day 6-uterus.3 Moreover, estrogen is essential for the induction of uterine LIF in mice (20Song H. Lim H. Das S.K. Paria B.C. Dey S.K. Mol. Endocrinol. 2000; 14: 1147-1161Crossref PubMed Scopus (158) Google Scholar, 28Bhatt H. Brunet L.J. Stewart C.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11408-11412Crossref PubMed Scopus (487) Google Scholar). This is consistent with the absence of LIF expression in the P4-primed delayed implant mouse uterus and its rapid induction after an estrogen injection (20Song H. Lim H. Das S.K. Paria B.C. Dey S.K. Mol. Endocrinol. 2000; 14: 1147-1161Crossref PubMed Scopus (158) Google Scholar). Thus, the virtual absence of uterine LIF on day 6 and during delayed implantation correlates with higher uterine anandamide levels and implantation failure. However, we do not know whether the absence of LIF is the cause of higher uterine anandamide levels or whether the higher levels are the consequence of implantation failure in the absence of LIF.The physiological significance of anandamide in the uterus and cannabinoid receptors in the blastocyst is still not fully understood. Although it is clear that higher uterine anandamide and blastocyst cannabinoid receptor levels are detrimental to the implantation process, uterine anandamide and blastocyst cannabinoid receptors still persist, albeit at lower levels, at the time of implantation. This suggests that lower levels of anandamide and cannabinoid receptors are beneficial to implantation. This suggestion is consistent with our recent observation that whereas higher anandamide levels are detrimental to blastocyst outgrowth in culture, lower levels stimulate this event (5Wang J. Paria B.C. Dey S.K. Armant D.R. Biol. Reprod. 1999; 60: 839-844Crossref PubMed Scopus (74) Google Scholar). Similar biphasic (inhibitory and stimulatory) effects of anandamide at high and low concentrations are evident for other neural and behavioral functions (29Sulcova E. Mechoulam R. Fride E. Pharmacol. Biochem. Behav. 1998; 59: 347-352Crossref PubMed Scopus (239) Google Scholar), although the definitive cause of these biphasic effects of anandamide is not yet clearly understood (29Sulcova E. Mechoulam R. Fride E. Pharmacol. Biochem. Behav. 1998; 59: 347-352Crossref PubMed Scopus (239) Google Scholar). More recently, a bidirectional regulation of airway responsiveness by endogenous cannabinoids has been documented (30Calignano A. Katona I. Desarnaud F. Giuffrida A. La Rana G. Mackie K. Freund T.F. Piomelli D. Nature. 2000; 408: 96-101Crossref PubMed Scopus (160) Google Scholar). Nonetheless, it is envisioned that a biphasic paracrine signaling via anandamide and cannabinoid receptors influences the fate of the embryo-uterine interactions during implantation and that aberrant levels of uterine anandamide and/or embryonic cannabinoid receptors are likely to adversely affect embryonic development and implantation. This could be a mechanism to prevent implantation of abnormal embryos resulting from exposure to aberrant levels of cannabinoids. In conclusion, the present study highlights the importance of the ligand-receptor signaling with cannabinoids in female fertility and places the embryo and/or the uterus as targets for this signaling. Previous investigation suggested that cannabinoid exposure has adverse effects on pregnancy outcome (reviewed in Refs. 1Paria B.C. Das S.K. Dey S.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9460-9464Crossref PubMed Scopus (173) Google Scholar, 2Yang Z.M. Paria B.C. Dey S.K. Biol. Reprod. 1996; 55: 756-761Crossref PubMed Scopus (90) Google Scholar, 3Schmid P.C. Paria B.C. Krebsbach R.J. Schmid H.H.O. Dey S.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4188-4192Crossref PubMed Scopus (223) Google Scholar, 4Paria B.C. Ma W. Andrenyak D.M. Schmid P.C. Schmid H.H.O. Moody D.E. Deng H. Makriyannis A. Dey S.K. Biol. Reprod. 1998; 58: 1490-1495Crossref PubMed Scopus (96) Google Scholar, 5Wang J. Paria B.C. Dey S.K. Armant D.R. Biol. Reprod. 1999; 60: 839-844Crossref PubMed Scopus (74) Google Scholar). Cannabinomimetic drugs interact with two types of cannabinoid receptors, brain-type (CB1) and spleen-type (CB2) (6Howlett A.C. Annu. Rev. Pharmacol. Toxicol. 1995; 35: 607-634Crossref PubMed Scopus (444) Google Scholar, 7Pertwee R.G. Pharmacol. Ther. 1997; 74: 129-180Crossref PubMed Scopus (1282) Google Scholar, 8Matsuda L.A. Lolait S.J. Brownstein M.J. Young A.C. Bonner T.I. Nature. 1990; 346: 561-564Crossref PubMed Scopus (4162) Google Scholar, 9Munro S. Thomas K.L. Abu-sharr M. Nature. 1993; 365: 61-65Crossref PubMed Scopus (4079) Google Scholar). These receptor subtypes are negatively coupled to adenylate cyclase and to N-type and P/Q-type calcium channels and positively coupled to mitogen-activated protein kinase and to A-type potassium channels through Gi/o proteins (7Pertwee R.G. Pharmacol. Ther. 1997; 74: 129-180Crossref PubMed Scopus (1282) Google Scholar, 10Bouaboula M. Poinot-Chazel C. Bourrie B. Canat X. Calandra B. Rinaldi-Carmona M. Le Fur G. Casellas P. Biochem. J. 1995; 312: 637-641Crossref PubMed Scopus (457) Google Scholar). CB1 is expressed in brain and other peripheral tissues (7Pertwee R.G. Pharmacol. Ther. 1997; 74: 129-180Crossref PubMed Scopus (1282) Google Scholar, 8Matsuda L.A. Lolait S.J. Brownstein M.J. Young A.C. Bonner T.I. Nature. 1990; 346: 561-564Crossref PubMed Scopus (4162) Google Scholar, 9Munro S. Thomas K.L. Abu-sharr M. Nature. 1993; 365: 61-65Crossref PubMed Scopus (4079) Google Scholar, 10Bouaboula M. Poinot-Chazel C. Bourrie B. Canat X. Calandra B. Rinaldi-Carmona M. Le Fur G. Casellas P. Biochem. J. 1995; 312: 637-641Crossref PubMed Scopus (457) Google Scholar, 11Paria B.C. Das S.K. Dey S.K. Nahas G.G. Sutin K.M. Harvey D.J. Agurell S. Marihuana and Medicine. Humana Press, Totowa, NJ1999: 393-410Crossref Google Scholar). CB2 is expressed primarily in immune tissues including the spleen, leukocytes, and tonsils (12Galiege S. Mary S. Marchand J. Dussossoy D. Carriere D. Carayon P. Bouaboula M. Shire D. LeFur G. Casellas P. Eur. J. Biochem. 1995; 232: 54-61Crossref PubMed Scopus (1337) Google Scholar). We have previously shown that CB1 is expressed in the preimplantation mouse embryo at much higher levels than in the brain (1Paria B.C. Das S.K. Dey S.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9460-9464Crossref PubMed Scopus (173) Google Scholar, 2Yang Z.M. Paria B.C. Dey S.K. Biol. Reprod. 1996; 55: 756-761Crossref PubMed Scopus (90) Google Scholar). The discovery of cannabinoid receptors led to the identification of endogenous cannabinoid ligands, N-arachidonoylethanolamine (anandamide), and 2-arachidonoylglycerol (13Devane W.A. Hanus L. Breuer A. Pertwee R.G. Stevenson L.A. Griffin G. Gibson D. Mandelbaum A. Etinger A. Machoulam R. Science. 1992; 258: 1946-1949Crossref PubMed Scopus (4618) Google Scholar, 14Felder C.C. Veluz J.S. Williams H.L. Briley E.M. Matsuda L.A. Mol. Pharmacol. 1992; 42: 838-845PubMed Google Scholar, 15Mechoulam R. Ben Shabat S. Hanus L. Ligumsky M. Kaminski N.E. Schatz A.R. Gopher A. Almog S. Martin B.R. Compton D.R. Pertwee R.G. Griffin G. Bayewitch M. Barg J. Vogel Z. Biochem. Pharmacol. 1995; 50: 83-90Crossref PubMed Scopus (2315) Google Scholar, 16Sugiura T. Kondo S. Sukagawa A. Nakane S. Shinoda A. Itoh K. Yamashita A. Waku K. J. Biol. Chem. 1999; 274: 2794-2801Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 17Stella N. Schweitzer P. Piomelli D. Nature. 1997; 388: 773-777Crossref PubMed Scopus (1227) Google Scholar). We observed that anandamide is synthesized in the pregnant mouse oviduct and uterus (18Paria B.C. Deutsch D.D. Dey S.K. Mol. Reprod. Dev. 1996; 45: 183-192Crossref PubMed Scopus (128) Google Scholar) and that its levels are far higher in the uterus than in any other mammalian tissue examined (3Schmid P.C. Paria B.C. Krebsbach R.J. Schmid H.H.O. Dey S.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4188-4192Crossref PubMed Scopus (223) Google Scholar). However, anandamide levels are significantly lower at implantation sites than at interimplantation sites, suggesting endocannabinoid ligand-receptor signaling during implantation (3Schmid P.C. Paria B.C. Krebsbach R.J. Schmid H.H.O. Dey S.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4188-4192Crossref PubMed Scopus (223) Google Scholar). Indeed anandamide at higher levels adversely affects embryo development and implantation, whereas at lower levels it stimulates embryo growth and differentiation via CB1 (1Paria B.C. Das S.K. Dey S.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9460-9464Crossref PubMed Scopus (173) Google Scholar, 2Yang Z.M. Paria B.C. Dey S.K. Biol. Reprod. 1996; 55: 756-761Crossref PubMed Scopus (90) Google Scholar, 3Schmid P.C. Paria B.C. Krebsbach R.J. Schmid H.H.O. Dey S.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4188-4192Crossref PubMed Scopus (223) Google Scholar, 4Paria B.C. Ma W. Andrenyak D.M. Schmid P.C. Schmid H.H.O. Moody D.E. Deng H. Makriyannis A. Dey S.K. Biol. Reprod. 1998; 58: 1490-1495Crossref PubMed Scopus (96) Google Scholar, 5Wang J. Paria B.C. Dey S.K. Armant D.R. Biol. Reprod. 1999; 60: 839-844Crossref PubMed Scopus (74) Google Scholar). Interestingly, 2-arachidonoylglycerol is present in the mouse uterus at amounts similar to or lower than the lowest anandamide levels (1–5 nmol/g of tissue), and its level does not vary significantly between implantation and interimplantation sites. 1B. C. Paria, P. C. Schmid, R. J. Krebsbach, H. H. O. Schmid, and S. K. Dey, unpublished results.1B. C. Paria, P. C. Schmid, R. J. Krebsbach, H. H. O. Schmid, and S. K. Dey, unpublished results. Using cannabinoid receptor mutant mice (19Zimmer A. Zimmer A.M. Hohmann A.G. Herkenham M. Bonner T.I. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5780-5785Crossref PubMed Scopus (888) Google Scholar) and physiological approaches, we further defined the role of ligand-receptor signaling with cannabinoids in preimplantation embryo development and in implantation. DISCUSSIONThe mechanism(s) by which the development of preimplantation embryos into active blastocysts is synchronized with uterine receptivity for implantation is not clearly understood. Our present observations of the asynchronous development of embryos deficient in cannabinoid receptors during the preimplantation period and of the coordinated down-regulation of both uterine anandamide levels and blastocyst cannabinoid receptors prior to implantation in wild-type mice suggest that ligand-receptor signaling with endocannabinoids locally helps in regulating the "window" of implantation. Although the Mendelian frequency of offspring resulting from heterozygous crossings of CB1-null mice is skewed resulting in a somewhat reduced number of homozygous offspring (19Zimmer A. Zimmer A.M. Hohmann A.G. Herkenham M. Bonner T.I. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5780-5785Crossref PubMed Scopus (888) Google Scholar), and although the pregnancy rate in mutant mice resulting from homozygous mating is somewhat lower, the birth of viable CB1 −/−,CB2 −/−, orCB1 −/− /CB2 −/− double mutant offspring suggests that the absence of embryonic and/or uterine cannabinoid receptors is not indispensable for embryonic development or implantation. However, the observed increased mortality ofCB1 −/− offspring perhaps could be due to inferior fetal development resulting from the implantation of slowly developing embryos (19Zimmer A. Zimmer A.M. Hohmann A.G. Herkenham M. Bonner T.I. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5780-5785Crossref PubMed Scopus (888) Google Scholar). The major phenotypes ofCB1 −/− orCB1 −/− /CB2 −/− double mutant mice are their resistance to exogenous cannabinoid exposure with respect to embryo development and implantation in vitro andin vivo. This suggests that embryonic development and implantation are likely to be affected by aberrant levels of exogenous or endogenous cannabinoids in the uterus and/or aberrant embryonic expression of cannabinoid receptors during early pregnancy.On day 5 of pseudopregnancy when the uterus is still receptive,LIF expression persists in uterine glands. In contrast,LIF expression is undetectable or extremely low in the nonreceptive day 6-uterus.3 Moreover, estrogen is essential for the induction of uterine LIF in mice (20Song H. Lim H. Das S.K. Paria B.C. Dey S.K. Mol. Endocrinol. 2000; 14: 1147-1161Crossref PubMed Scopus (158) Google Scholar, 28Bhatt H. Brunet L.J. Stewart C.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11408-11412Crossref PubMed Scopus (487) Google Scholar). This is consistent with the absence of LIF expression in the P4-primed delayed implant mouse uterus and its rapid induction after an estrogen injection (20Song H. Lim H. Das S.K. Paria B.C. Dey S.K. Mol. Endocrinol. 2000; 14: 1147-1161Crossref PubMed Scopus (158) Google Scholar). Thus, the virtual absence of uterine LIF on day 6 and during delayed implantation correlates with higher uterine anandamide levels and implantation failure. However, we do not know whether the absence of LIF is the cause of higher uterine anandamide levels or whether the higher levels are the consequence of implantation failure in the absence of LIF.The physiological significance of anandamide in the uterus and cannabinoid receptors in the blastocyst is still not fully understood. Although it is clear that higher uterine anandamide and blastocyst cannabinoid receptor levels are detrimental to the implantation process, uterine anandamide and blastocyst cannabinoid receptors still persist, albeit at lower levels, at the time of implantation. This suggests that lower levels of anandamide and cannabinoid receptors are beneficial to implantation. This suggestion is consistent with our recent observation that whereas higher anandamide levels are detrimental to blastocyst outgrowth in culture, lower levels stimulate this event (5Wang J. Paria B.C. Dey S.K. Armant D.R. Biol. Reprod. 1999; 60: 839-844Crossref PubMed Scopus (74) Google Scholar). Similar biphasic (inhibitory and stimulatory) effects of anandamide at high and low concentrations are evident for other neural and behavioral functions (29Sulcova E. Mechoulam R. Fride E. Pharmacol. Biochem. Behav. 1998; 59: 347-352Crossref PubMed Scopus (239) Google Scholar), although the definitive cause of these biphasic effects of anandamide is not yet clearly understood (29Sulcova E. Mechoulam R. Fride E. Pharmacol. Biochem. Behav. 1998; 59: 347-352Crossref PubMed Scopus (239) Google Scholar). More recently, a bidirectional regulation of airway responsiveness by endogenous cannabinoids has been documented (30Calignano A. Katona I. Desarnaud F. Giuffrida A. La Rana G. Mackie K. Freund T.F. Piomelli D. Nature. 2000; 408: 96-101Crossref PubMed Scopus (160) Google Scholar). Nonetheless, it is envisioned that a biphasic paracrine signaling via anandamide and cannabinoid receptors influences the fate of the embryo-uterine interactions during implantation and that aberrant levels of uterine anandamide and/or embryonic cannabinoid receptors are likely to adversely affect embryonic development and implantation. This could be a mechanism to prevent implantation of abnormal embryos resulting from exposure to aberrant levels of cannabinoids. In conclusion, the present study highlights the importance of the ligand-receptor signaling with cannabinoids in female fertility and places the embryo and/or the uterus as targets for this signaling. The mechanism(s) by which the development of preimplantation embryos into active blastocysts is synchronized with uterine receptivity for implantation is not clearly understood. Our present observations of the asynchronous development of embryos deficient in cannabinoid receptors during the preimplantation period and of the coordinated down-regulation of both uterine anandamide levels and blastocyst cannabinoid receptors prior to implantation in wild-type mice suggest that ligand-receptor signaling with endocannabinoids locally helps in regulating the "window" of implantation. Although the Mendelian frequency of offspring resulting from heterozygous crossings of CB1-null mice is skewed resulting in a somewhat reduced number of homozygous offspring (19Zimmer A. Zimmer A.M. Hohmann A.G. Herkenham M. Bonner T.I. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5780-5785Crossref PubMed Scopus (888) Google Scholar), and although the pregnancy rate in mutant mice resulting from homozygous mating is somewhat lower, the birth of viable CB1 −/−,CB2 −/−, orCB1 −/− /CB2 −/− double mutant offspring suggests that the absence of embryonic and/or uterine cannabinoid receptors is not indispensable for embryonic development or implantation. However, the observed increased mortality ofCB1 −/− offspring perhaps could be due to inferior fetal development resulting from the implantation of slowly developing embryos (19Zimmer A. Zimmer A.M. Hohmann A.G. Herkenham M. Bonner T.I. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5780-5785Crossref PubMed Scopus (888) Google Scholar). The major phenotypes ofCB1 −/− orCB1 −/− /CB2 −/− double mutant mice are their resistance to exogenous cannabinoid exposure with respect to embryo development and implantation in vitro andin vivo. This suggests that embryonic development and implantation are likely to be affected by aberrant levels of exogenous or endogenous cannabinoids in the uterus and/or aberrant embryonic expression of cannabinoid receptors during early pregnancy. On day 5 of pseudopregnancy when the uterus is still receptive,LIF expression persists in uterine glands. In contrast,LIF expression is undetectable or extremely low in the nonreceptive day 6-uterus.3 Moreover, estrogen is essential for the induction of uterine LIF in mice (20Song H. Lim H. Das S.K. Paria B.C. Dey S.K. Mol. Endocrinol. 2000; 14: 1147-1161Crossref PubMed Scopus (158) Google Scholar, 28Bhatt H. Brunet L.J. Stewart C.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11408-11412Crossref PubMed Scopus (487) Google Scholar). This is consistent with the absence of LIF expression in the P4-primed delayed implant mouse uterus and its rapid induction after an estrogen injection (20Song H. Lim H. Das S.K. Paria B.C. Dey S.K. Mol. Endocrinol. 2000; 14: 1147-1161Crossref PubMed Scopus (158) Google Scholar). Thus, the virtual absence of uterine LIF on day 6 and during delayed implantation correlates with higher uterine anandamide levels and implantation failure. However, we do not know whether the absence of LIF is the cause of higher uterine anandamide levels or whether the higher levels are the consequence of implantation failure in the absence of LIF. The physiological significance of anandamide in the uterus and cannabinoid receptors in the blastocyst is still not fully understood. Although it is clear that higher uterine anandamide and blastocyst cannabinoid receptor levels are detrimental to the implantation process, uterine anandamide and blastocyst cannabinoid receptors still persist, albeit at lower levels, at the time of implantation. This suggests that lower levels of anandamide and cannabinoid receptors are beneficial to implantation. This suggestion is consistent with our recent observation that whereas higher anandamide levels are detrimental to blastocyst outgrowth in culture, lower levels stimulate this event (5Wang J. Paria B.C. Dey S.K. Armant D.R. Biol. Reprod. 1999; 60: 839-844Crossref PubMed Scopus (74) Google Scholar). Similar biphasic (inhibitory and stimulatory) effects of anandamide at high and low concentrations are evident for other neural and behavioral functions (29Sulcova E. Mechoulam R. Fride E. Pharmacol. Biochem. Behav. 1998; 59: 347-352Crossref PubMed Scopus (239) Google Scholar), although the definitive cause of these biphasic effects of anandamide is not yet clearly understood (29Sulcova E. Mechoulam R. Fride E. Pharmacol. Biochem. Behav. 1998; 59: 347-352Crossref PubMed Scopus (239) Google Scholar). More recently, a bidirectional regulation of airway responsiveness by endogenous cannabinoids has been documented (30Calignano A. Katona I. Desarnaud F. Giuffrida A. La Rana G. Mackie K. Freund T.F. Piomelli D. Nature. 2000; 408: 96-101Crossref PubMed Scopus (160) Google Scholar). Nonetheless, it is envisioned that a biphasic paracrine signaling via anandamide and cannabinoid receptors influences the fate of the embryo-uterine interactions during implantation and that aberrant levels of uterine anandamide and/or embryonic cannabinoid receptors are likely to adversely affect embryonic development and implantation. This could be a mechanism to prevent implantation of abnormal embryos resulting from exposure to aberrant levels of cannabinoids. In conclusion, the present study highlights the importance of the ligand-receptor signaling with cannabinoids in female fertility and places the embryo and/or the uterus as targets for this signaling. We thank Dr. H. Lim for the critical and constructive discussions we had with her during the course of this work.