BIOLOGY OF REPRODUCTION 58, 1490-1495 (1998)
Effects of Cannabinoids on Preimplantation Mouse Embryo Development and Implantation Are Mediated by Brain-Type Cannabinoid Receptors1 B.C. Paria,2,3 W. Ma,3 D.M. Andrenyak, 4 P.C. Schmid, 5 H.H.O. Schmid, 5 D.E. Moody, 4 H. Deng, 6 A. Makriyannis,6 and S.K. Dey 2,3 Department of Molecular and Integrative Physiology, 3 Ralph L. Smith Research Center, University of Kansas Medical Center, Kansas City, Kansas 66160-7338 Center for Human Toxicology,4 University of Utah, Salt Lake City, Utah 84112 The Hormel Institute,5 University of Minnesota, Austin, Minnesota 55912 Departments of Pharmaceutical Sciences and Molecular & Cell Biology,6 School of Pharmacy, University of Connecticut, Storrs, Connecticut 06269-2092 ABSTRACT We examined the relative importance of G (Gi) protein-coupled brain-type (CB1-R) and spleen-type (CB2-R) cannabinoid receptors in preimplantation embryo development using agonists and antagonists specific to CB1-R and CB2-R. The results establish that endogenous cannabinoid ligands, anandamide and sn-2 arachidonoylglycerol, arrest embryo development in vitro, and this effect is reversed by CB1-R antagonists SR141716A or AM 251, but not by SR144528, a CB2-R antagonist. A CB2-R selective agonist AM 663 failed to affect embryo development. These results suggest that cannabinoid effects on embryo development are mediated by CB1-R. We also observed that A9-tetrahydrocannabinol ([-]THC) infused in the presence of cytochrome P450 inhibitors interfered with blastocyst implantation. This adverse effect was reversed by coinfusion of SR141716A. The less active stereoisomer (+)THC plus the inhibitors failed to affect implantation. Analysis of tissue levels demonstrated that uterine accumulation of (-)THC occurred when it was infused in the presence of the P450 inhibitors. These results demonstrate that the uterus and perhaps the embryo have the cytochrome P450 enzymes to metabolize (-)THC and neutralize its adverse effects on implantation. Collectively, the present study demonstrates that cannabinoid effects on embryo development and implantation are mediated by embryonic and/or uterine CB1-R, but not CB2-R. INTRODUCTION The establishment of a receptive uterus for supporting embryo implantation is primarily regulated by coordinated effects of progesterone (P4 ) and estrogen [1, 2]. In the rodent, initiation of implantation is indicated by an increased endometrial vascular permeability at the sites of blastocysts [1]. This event coincides with the initial attachment between the luminal epithelium and blastocyst [3]. In the mouse, the attachment reaction occurs at 2200-2300 h on Day 4 of pregnancy [4]. This is followed by subsequent adherence and penetration by trophoblast cells through the underlying basement membrane and stromal decidualizaAccepted January 29, 1998. Received December 29, 1997. 'Supported in part by the National Institute on Drug Abuse (DA06668), the National Institute of Child Health and Human Development (HD12304), and the National Institute of General Medical Sciences (GM 45741). The center grants (HD02528 and HD33994) provided access to core facilities. Analysis of tissue levels of THC was performed by NIDA (DA-6-7205). 2 Correspondence: B.C. Paria or 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; e-mail:
[email protected]
tion [5, 6]. Uterine receptivity occurs for only a limited period during pregnancy or pseudopregnancy in this species [2, 7]. Thus, the prereceptive uterus on Day 3 becomes receptive on Day 4 (the day of implantation), whereas by Day 5 the uterus becomes refractory to blastocysts [2]. Ovariectomy on the morning of Day 4 before preimplantation ovarian estrogen secretion results in blastocyst dormancy and failure in implantation. This condition, known as delayed implantation, can be maintained by continued P 4 treatment and terminated by an injection of estrogen initiating blastocyst activation and implantation [2, 8, 9]. Cannabinoids exert a wide spectrum of central and peripheral effects [10, 11]. One of the concerns about exposure to cannabinoids is their reported adverse effects on reproductive functions, including retarded embryo development, fetal loss, pregnancy failure, and reduced fertilizing capacity of sperm (reviewed in [12-15]). Many of the central and peripheral cannabinoid effects are mediated by recently identified G (Gi) protein-coupled brain-type (CB 1-R) and spleentype (CB2-R) cannabinoid receptors [16-18]. Furthermore, two endogenous cannabinomimetic lipid derivatives, N-arachidonoyl ethanolamide (anandamide) and sn-2 arachidonoylglycerol (2-AG), have been isolated from brain and other tissues [19-24]. These compounds bind with high affinity to brain-type and spleen-type cannabinoid receptors and mimic most of the effects of (-)A 9 -tetrahydrocannabinol ([-]THC), a psychoactive derivative of marijuana. 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 [25, 26]. 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 [21]. These findings suggest that preimplantation mouse embryos are possible targets for cannabinoid ligand-receptor signaling. Indeed, natural and synthetic cannabinoid agonists arrest preimplantation embryo development in vitro, and a specific CB 1-R antagonist reverses this effect [25, 26]. These studies suggested that embryonic arrest in response to cannabinoid agonists appeared to be mediated by CB 1-R. However, preimplantation embryos express both the CB1-R and CB2-R genes [25], and thus we used specific antagonists and agonists to CB 1R and CB2-R to determine whether embryonic CB2-R is also functional in the embryo. We speculated that cannabinoid agonists may also interfere with implantation. In this respect, single or repeated injections of (-)THC during the preimplantation period had no detrimental effects on implantation [27], although a
1490
CANNABOIDS EFFECTS ON EMBRYO DEVELOPMENT AND IMPLANTATION synthetic cannabinoid CP 55,940 infused at a constant rate via miniosmotic pumps during this period interfered with zona hatching and implantation [21]. This may imply that mechanisms exist which protect mice against the adverse effects of (-)THC during early pregnancy. One possible explanation is the rapid metabolism of (-)THC by the cytochrome P450 system [28]. Indeed, (-)THC elicits responses in the tuberoinfundibular dopaminergic neurones in female rats if cytochrome P450-linked monooxygenase enzymes are inhibited [29]. Thus, we examined whether inhibition of the cytochrome P450-linked monooxygenase system interferes with implantation in mice exposed to (-)THC. MATERIALS AND METHODS Animals Adult CD-1 female mice (48 days old; Charles River Laboratory, Raleigh, NC) were housed in the animal care facility at University of Kansas Medical Center according to the National Institutes of Health (NIH) and institutional guidelines on the care and use of laboratory animals. They were mated with fertile males of the same strain to induce pregnancy (Day 1 = vaginal plug). Chemicals and Reagents The active cannabinoid (-)THC and its nearly inactive stereoisomer (+)THC dissolved in ethanol were obtained from National Institute on Drug Abuse, Rockville, MD. Anandamide was purchased from Caymann Chemical Co. (Ann Arbor, MI), and SR141716A and SR144528, specific CB1-R and CB2-R antagonists [30-32], respectively, were generously provided by Sanofi Recherche (Montepellier, France). Two-AG was synthesized from triarachidonoylglycerol (Nu CheK Prep, Elysian, MN) by reaction with lipase (type XI) from Rhizopus arrhizus (Sigma Chemical, Co., St. Louis, MO) in 50 mM sodium acetate (pH 5.5), 0.1 M NaCl, and 10 mM CaC12, essentially as described previously [23]. AM 663, a preferential agonist to CB2-R (unpublished results) and AM 251, a specific antagonist to CB1-R [33, 34] were synthesized by Makriyannis and his group. AM 663 has inhibitor constant (Ki) values of 20.9 nM and 2.9 nM for the CB1-R and CB2-R, respectively, and behaves as a full agonist on the Gi-adenylyl cyclasecoupled system. Inhibitors of cytochrome P450-linked monooxygenase system, metyrapone and clotrimazole, were purchased from Aldrich Chemical Co. (Milwaukee, WI) and Sigma Chemical Co., respectively. THC-d 3 was obtained from Radian Corp (Austin, TX); acetonitrile, hexane, ethyl acetate, and heptane were purchased from Baxter (McGraw Park, IL). NaOH, HCI, and trifluoroacetic anhydride were purchased from Fisher (Fair Lawn, NJ), Mallinckrodt (St. Louis, MO), and Pierce (Rockford, IL), respectively. Culture of Preimplantation Embryos To study the effects of cannabinoid agonists and their reversal by CB1-R antagonists or CB2-R antagonists on embryo development, 2-cell embryos were recovered on Day 2 (0830-0900 h), and those from several mice were pooled in Whitten's medium containing 0.3% BSA. Embryos were cultured in groups of 5-10 in 25 ,ul of Whitten's medium under silicon oil in an atmosphere of 5% CO 2 and 95% air at 37°C for 72 h [35, 36] in the absence or presence of these agonists and/or antagonists. All test agents were
1491
dissolved in ethanol and diluted with the Whitten's medium. The final ethanol concentration was less than 0.1%. The control cultures contained the same concentration of ethanol. Cannabinoid-like ligands were added at the beginning of cultures. To examine the reversibility of ligandinduced arrest of embryo development by the CB1-R or CB2-R antagonist, embryos were precultured with a specific antagonist 1 h before the culture with a specific agonist and the antagonist. The embryos were observed every 12 h to monitor their development. After termination of culture, the number of embryos that had formed blastocysts was recorded, and those which had not formed blastocysts were examined to determine the stage at which their development was arrested. Experiments were repeated 3-6 times, and statistical analysis was performed using chisquare and Fisher's Exact Test. In Vivo Delivery of Drugs (-)THC or its less active stereoisomer (+)THC was delivered at a constant rate via miniosmotic pumps (Alzet Corporation, Palo Alto, CA) to study their effects on implantation. (-)THC or (+)THC was concentrated in a vacuum dryer and resuspended in propylene glycol to obtain a concentration of 4 mg/0.1 ml to render it compatible for miniosmotic pump [37]. SR 141716A (CB1-R antagonist) was dissolved in a minimum volume of ethanol and diluted in propylene glycol to the desired concentration. Miniosmotic pumps (mean fill volume 96 lI and pumping rate 0.52 il/h) were filled with (-)THC, (+)THC, (-)THC plus SR141716A, or SR141716A alone and placed s.c. under the back skin. Cytochrome P450 inhibitors metyrapone and/ or clotrimazole were dissolved in ethyl alcohol, diluted in sesame seed oil and injected i.p. Miniosmotic pumps containing the drugs were placed in mice on Day 2 of pregnancy and continued through Day 5. Metyrapone and/or clotrimazole were first injected 2 h before the installation of the pumps and injected twice daily until Day 4 of pregnancy. In a separate set of experiments, the pumps and injections of P450 inhibitors were withdrawn on Day 5 of pregnancy, and mice were killed on Day 8 of pregnancy to observe any delay in implantation. On Day 5 or 8 of pregnancy, implantation sites were determined by an i.v. injection of 0.1 ml of 1% Chicago blue B in saline 5 min before killing. Blue bands along the uterine horns indicated sites of implantation. Uteri without implantation sites were flushed with Whitten's medium to recover blastocysts. The stage of development and morphological appearance of embryos were recorded under a dissecting microscope. Mice without implantation sites or blastocysts were excluded from the experiments. Measurement of Plasma and Uterine Levels of (-)THC In a separate set of experiments, blood and uterine tissues were collected from those mice receiving (-)THC via miniosmotic pumps with or without the cytochrome P450 inhibitors. Mice received infusions of (-)THC (20 Rtg/h) via miniosmotic pumps from Days 2 to 5 of pregnancy with or without two daily injections of metyrapone and clotrimazole (50 mg/kg). Blood and uteri were collected on the morning (0900 h) of Day 5. These specimens were also collected from a group of mice 1 h and 4 h after an i.v. injection of (-)THC (10 mg/kg) on Day 4 of pregnancy. Blood was processed to generate plasma. The uterus from each mouse was homogenized in 1 ml of sterile water and centrifuged at 10 000 x g for 20 min, and the supernatant
1492 A
PARIA ET AL. Ar
o
o 75
w 0 IJ
50
0 CD U-
o
uo -J
w
0 U) (o
A
ri
100 a
o
8I-
0 I-
25
Ma 0
nM .
.
AN
.
.
AN +AM 251
2-AG (nM)
AM 251 B
10( 0
W
a0-J 75 w
B
W
0
0 U) I-
o,
U) 0
w QO LU 0
0I-
U)
W U)
50
25
I0 O >O0 (.3
O I-U) 4:
VEHICLE
10
20 AM 663
50
7
01
O3 +
nM
z
+ z
