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a C.S. Mott Center for Human Growth & Development, Department of Obstetrics & Gynecology and Department of Anatomy & Cell Biology, Wayne State University School of Medicine, Detroit, Michigan 48201-1415
b Department of Molecular & Integrative Physiology, Ralph L. Smith Research Center, University of Kansas Medical Center, Kansas City, Kansas 66160-7336
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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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5ß1 integrin on the apical surface, and initiation of trophoblast outgrowth on fibronectin [1, 2]. This phenotypic transformation of the trophoblast cells is influenced by the maternal environment, which provides biologically active factors that regulate embryonic differentiation and uterine receptivity for implantation [3]. Anandamide (N-arachidonoylethanolamine) is a derivative of arachidonic acid that is synthesized in the pregnant mouse uterus at concentrations much higher than those found in the central nervous system [4–6]. Anandamide is one of the endogenous ligands for cannabinoid receptors, which are members of the G protein (Gi)-coupled receptor family [7–9]. Activation of cannabinoid receptors influences multiple intracellular signaling pathways by inhibiting adenylate cyclase activity [10, 11] and attenuating Ca2+ influx [10, 12]. Anandamide also activates focal adhesion kinase [13] and mitogen-activated protein kinase [14].
Two types of cannabinoid receptors have been identified. The brain-type cannabinoid receptor (CB1-R) is expressed primarily within the nervous system, and the spleen-type receptor (CB2-R) is expressed in many cells of the immune system [7, 8]. These two receptor isotypes exhibit similar ligand affinity, and the activation of both receptors inhibits cAMP accumulation. However, the activation of CB1-R inhibits N- and Q-type Ca2+ channels and activates K+ channels, while that of CB2-R appears to have no effect on cytosolic levels of either Ca2+ or K+ [10, 12]. Both CB1-R and CB2-R mRNAs are expressed in mouse preimplantation embryos [5], indicating that mouse preimplantation development could be influenced by ligand-receptor signaling mediated by endogenous cannabinoids.
Cannabinoids adversely affect fertilizing capacity of sperm and other reproductive activities [15–18]. Exposure of 2-cell mouse embryos to active cannabinoids is inhibitory to blastocyst cavitation, trophoblast proliferation, and hatching from the zona pellucida, and these effects are mediated by embryonic CB1-R [5, 6, 19]. The levels of uterine anandamide are inversely related to uterine receptivity for implantation; down-regulation is associated with uterine receptivity, while up-regulation is correlated with uterine refractoriness to blastocyst implantation. Further, anandamide levels are highest in the interimplantation sites and lowest at the sites of implantation [6]. Thus, the presence of cannabinoid receptors in embryos and anandamide in the uterus suggests that intracellular signaling initiated by cannabinoid-like ligands plays an important role in regulating periimplantation trophoblast development [5, 19]. Indeed, low levels of anandamide may be requisite for blastocyst growth and differentiation during implantation. Another endogenous cannabinoid-like ligand, sn-2 arachinonyl glycerol (2-AG), is produced by the neuronal cells [20]. Whether this lipid mediator is produced by the uterus is not yet known.
To examine the effects of cannabinoids on trophoblast differentiation, blastocysts were exposed during culture to the endogenous cannabinoids, anandamide and 2-AG, and a synthetic cannabinoid receptor agonist, Win55212-2. Working specifically through CB1-R, these agonists promoted blastocyst differentiation and trophoblast outgrowth within a narrow concentration range, as opposed to their inhibitory effects on 2-cell embryos. Our results have physiological implications for the anandamide concentration gradients observed between implantation and interimplantation sites [6].
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B6CBAF1/J mice (Jackson Laboratory, Bar Harbor, ME) were housed in the C.S. Mott Center vivarium at Wayne State University according to National Institutes of Health and institutional guidelines on the care and use of laboratory animals. Females were superovulated at 5–8 wk of age by i.p. injection of 5 IU eCG (Sigma Chemical Co., St. Louis, MO), followed 48 h later with 5 IU of hCG (Sigma). After hCG injection, they were mated overnight with males of the same strain. Blastocysts were recovered 90 h post-hCG by flushing uterine horns with M2 medium (Sigma). All embryos were cultured in Ham's F-10 medium (Sigma) supplemented with 4 mg/ml BSA, 100 U/ml penicillin, and 0.1 mg/ml streptomycin (all from Sigma) using a 5% CO2 incubator at 37°C.
Cannabinoid Treatment
Anandamide, Win55212-2 (Research Biochemicals, Natick, MA), and 2-AG (kindly provided by Dr. P. Schmid, The Hormel Institute, University of Minnesota, Austin, MN) were dissolved in ethanol and diluted at least 106-fold in culture medium. Blastocysts were cultured continuously at 10 embryos per drop in 10-µl drops of medium containing the vehicle (10-6 dilution of ethanol) or the indicated concentration of cannabinoids. In some experiments, embryos were washed free of cannabinoids or vehicle after 30 min of incubation and then cultured without further addition of the ligands. For reversal experiments, blastocysts were cultured for 1 h with 8 nM SR141716A, a CB1-R antagonist, or SR144528, a CB2-R antagonist (Sanofi Recherche, Montpellier, France), and then in medium containing 8 nM of antagonist, with or without 7 nM cannabinoid ligands.
Fibronectin-Binding Assay
Fluorescent-green, 1.0-µm-diameter polystyrene microspheres (Bang's Laboratories, Carmel, IN) were coated with FN-120 (Life Technologies, Inc., Gaithersburg, MD), a 120-kDa fragment of fibronectin containing the central cell-binding domain [21], and blocked using 1-acid glycoprotein, as described previously [1]. Blastocysts were cultured in the absence of an adhesive substratum for 48 or 72 h. FBA up-regulation was achieved by incubating blastocysts for 1 h with 50 µg/ml FN-120 in culture medium at 37°C. Blastocysts were then incubated with 1 U/ml each of heparinase I, II, and III (Sigma) at 37°C for 30 min and rinsed through three drops of PBS containing 10 mg/ml BSA (PBS/BSA). The binding assay was conducted by incubating embryos at 4°C for 30 min in a 0.2% suspension of FN-120-coated microspheres prepared in PBS/BSA. Blastocysts were then washed free of unbound beads by transfer through four drops of PBS/BSA and fixed overnight in 2% paraformaldehyde (Polysciences, Warrington, PA) at 4°C. FBA was directly related to the fluorescence intensity of bound microspheres, which was quantified over the abembryonic pole of each blastocyst using computer-based image analysis described previously by Schultz and Armant [1].
Trophoblast Outgrowth Assay
As previously described [1, 22], outgrowth assays were conducted on plastic Petri dishes precoated overnight at 37°C with 50 µg/ml FN-120 [21]. The plastic surface was then rinsed three times with fresh culture medium. Blastocysts were drawn carefully through a narrow micropipette to mechanically remove their zonae pellucidae; they were then placed in individual microdrops on the FN-120-coated plate for an additional 96 h of culture. Trophoblast outgrowth was observed using a Leitz (Wetzlar, Germany) Fluovert FU inverted microscope and Hoffman modulation contrast optics at x200 magnification. The number of blastocysts exhibiting outgrowth was recorded at appropriate intervals between 24 and 120 h of culture and expressed as percentage outgrowth or as the time at which 50% of the blastocysts had commenced outgrowth (T50), as previously described [23]. Trophoblast outgrowth area was measured after 96 h of culture using an image analysis system, as previously described [22–24].
Statistical Analysis
All experiments were repeated at least three times. FBA values were obtained using at least 15 blastocysts, and percentage outgrowth values were based on a minimum of 90 blastocysts, or as indicated in Table 1. Differences in the temporal FBA profiles between treatment groups were tested for significance using a factorial ANOVA. This test indicated whether a shift had occurred in the relative amount of FBA at 48 h and 72 h of culture. Probit analysis was used to compute T50 values and statistically compare outgrowth rate [23].
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As previously reported [1, 2], FBA is developmentally regulated during serum-free culture of blastocysts. The normal developmental profile is distinguished by low levels of FBA after 48 h, high levels at 72 h, and low levels again after 96 h of culture [2]. Accordingly, we observed that fluorescent microspheres coated with FN-120 bound poorly to blastocysts cultured for 48 h (Fig. 1A). To investigate the role of cannabinoid-like ligands in regulating trophoblast differentiation, blastocysts were cultured in medium containing 7 nM of the cannabinoid agonists anandamide, 2-AG, or Win55212-2, and the rate of trophoblast differentiation was monitored. When blastocysts were cultured for 48 h in medium supplemented with anandamide, the microsphere probes revealed an abundance of receptors for FN-120 on the mural trophoblast surface (Fig. 1B), reflecting precocious trophoblast differentiation. Quantification of the fluorescence intensity of the bound FN-120-coated microspheres at the abembryonic pole showed that FBA increased at 72 h in vehicle-treated embryos, while anandamide, 2-AG, and Win55212-2 each produced high FBA after only 48 h of culture (Fig. 2). Cannabinoid-treated embryos exhibited down-regulation of FBA at 72 h, consistent with their accelerated rate of differentiation. Each of the three agonists shifted the developmental profile by approximately 24 h as compared to the vehicle treatment (p < 0.01), demonstrating that trophoblast development was accelerated by the activation of embryonic cannabinoid receptors. Blastocysts exposed for only 30 min to cannabinoid agonists exhibited a similar shift in their developmental profile (data not shown), suggesting that even a brief activation of cannabinoid receptors is sufficient to stimulate trophoblast differentiation.
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Cannabinoids Functioned through CB1-R but Not through CB2-R
Both CB1-R and CB2-R mRNAs are expressed in preimplantation mouse embryos [5]. Work from other cell systems indicates that anandamide binds to the two receptors with similar affinity and inhibition of cAMP accumulation [10, 12]. To investigate the involvement of these receptors in cannabinoid-induced trophoblast differentiation, we used specific antagonists to CB1-R (SR141716A) or CB2-R (SR144528). SR141716A abrogated the ability of anandamide, 2-AG, or Win55212-2 to accelerate trophoblast differentiation (p < 0.01; Fig. 2). During these treatments, FBA did not increase until 72 h; this result was similar to findings in the vehicle-treated embryos. In contrast, SR144528 did not noticeably attenuate cannabinoid-induced acceleration of trophoblast differentiation. This CB2-R-specific antagonist at 10 nM fails to reverse the inhibition of preimplantation development by 7 nM cannabinoids, and a CB2-R-specific agonist was without effect at several-fold above the effective concentration of anandamide [25]. These results establish that the activation of CB1-R, but not CB2-R, stimulates trophoblast cell differentiation.
The treatment of blastocysts with either of the antagonists alone did not influence their differentiation (Fig. 2). However, FBA of blastocysts treated with SR141716A appeared somewhat, albeit not significantly (p > 0.2), lower than in vehicle-treated embryos at 72 h, suggesting that the activation of CB1-R through an autocrine mechanism may support trophoblast differentiation.
Cannabinoids Induced Precocious Trophoblast Outgrowth through CB1-R
Trophoblast outgrowth is a reliable marker for trophoblast differentiation and migratory activity on extracellular matrix substrates such as fibronectin and laminin [26]. Therefore, we examined the effects of cannabinoid exposure on trophoblast outgrowth activity on FN-120. Trophoblast cells of blastocysts treated with 7 nM anandamide for 30 min at the outset of culture frequently began to dissociate and form large outgrowths after only 48 h, whereas most vehicle-treated blastocysts remained intact or only exhibited early signs of trophoblast cell adhesion and spreading (Fig. 3, A and B). Anandamide and Win55212-2 treatments shifted (p < 0.05, by Probit analysis) the T50 for outgrowth from 68 h for vehicle-treated blastocysts to 60 h and 61 h, respectively (Fig. 3C). Similar results were obtained when the cannabinoids remained in the medium throughout the entire culture period (data not shown). Again, the application of CB1-R antagonist blocked the acceleration of trophoblast differentiation induced by cannabinoids (Table 1). These observations, consistent with our FBA experiments, strongly indicated that cannabinoids at a concentration of 7 nM effectively accelerated periimplantation trophoblast differentiation.
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To assess the effect of cannabinoid treatment on trophoblast migratory activity, the area of trophoblast outgrowth was measured. After 96 h of culture, no difference (p = 0.99) was observed in outgrowth area between the blastocysts treated with vehicle (35 312 ± 14, 01 µm2) and anandamide (33 919 ± 11 045 µm2). Interestingly, although cannabinoids accelerated the rate of trophoblast differentiation, the viability of trophoblast cells may have been compromised. After prolonged outgrowth culture, trophoblast cells are observed that become rounded, nonadherent, or necrotic. Outgrowing trophoblast cells derived from cannabinoid-treated blastocysts showed signs of such degeneration by 96 h, which was not observed for another 24–48 h in vehicle-treated blastocysts. The early loss of outgrowing trophoblast cells was observed whether cannabinoids were present for only 30 min or continuously.
Differential Concentration-Dependent Effects of Cannabinoids on Trophoblast Differentiation
To investigate the concentration-dependent effects of cannabinoids on trophoblast differentiation, blastocysts were cultured in medium containing higher concentrations of anandamide, and FBA was estimated after 48 and 72 h of culture (Fig. 4). Acceleration of trophoblast differentiation was observed when anandamide was present at 7 or 14 nM (p < 0.05). However, 28 nM anandamide inhibited trophoblast differentiation, and FBA was not detected at either 48 h or 72 h. These results indicate that cannabinoids at higher concentrations are detrimental to trophoblast development and thus implantation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The differentiation-promoting effects of cannabinoids on trophoblast cells at low nanomolar levels are surprising, since similar concentrations were inhibitory at the earlier stages of embryonic development. This stage-specific effect may explain why the preimplantation uterine environment is normally hostile to premorula-stage embryos and why blastocysts fail to survive in the nonreceptive uterine environment. Thus, endogenous cannabinoid-like ligands in the uterus, such as anandamide, may regulate the window of implantation by synchronizing trophoblast differentiation with preparation of the uterus for the receptive state. High concentrations of anandamide in the interimplantation sites may limit trophoblast migrations beyond the implantation sites, as evidenced by the failure of anandamide to stimulate cell migration and the decreased longevity of trophoblast cells exposed to cannabinoids. Therefore, anandamide appears to possess dual functions that may serve to both promote trophoblast invasion at implantation sites and limit invasion beyond those sites.
Cyclooxygenase (COX) is the rate-limiting enzyme in the synthesis of prostaglandins (PGs). COX exists in two isoforms, the constitutive COX-1 and inducible COX-2. We have recently demonstrated that COX-2 is expressed in the uterus solely at the sites of blastocyst apposition during implantation and that targeted disruption of the COX-2 gene, but not COX-1, interferes with implantation in mouse [27]. Since COX-2, but not COX-1, is capable of converting anandamide to PGE2 ethanolamide [28], it is possible that the relatively lower levels of anandamide at the implantation sites compared with higher levels at the interimplantation sites are regulated by COX-2. COX-2 is able to compete with other enzymes for arachidonic acid, the precursor for synthesis of both PGs and anandamide, suggesting that COX-2 may be involved in regulating the tone of these two lipid mediators conducive to implantation. However, it should be noted that the activity of amidase, an anandamide-hydrolyzing enzyme, is also higher at the implantation sites as opposed to interimplantation sites [29].
The mechanism by which low levels of cannabinoids promote trophoblast differentiation is not yet clearly understood. A key attribute of cannabinoids is their impact on multiple intracellular signaling pathways. We have observed that mobilization of the second messenger, Ca2+, in morulae and blastocysts accelerates cavitation [30] and trophoblast differentiation [23, 30, 31]. Induction of Ca2+ signaling at the blastocyst stage alters the expression of over 3% of mRNAs within 1 h [32], which likely modifies the course of subsequent development. The ability of intracellular signaling through Ca2+ to stimulate preimplantation development is dependent upon activation of calmodulin [33], a Ca2+-dependent regulatory protein that influences multiple signaling pathways [34]. Reduced long-term survival of trophoblast cells and poor preimplantation development in the presence of low levels of cannabinoids may, therefore, be related to the inhibition of Ca2+ influx associated with cannabinoid receptor activation [10, 12]. In blastocysts, low cannabinoid concentrations may activate downstream pathways common to cannabinoid receptors and calmodulin without sufficient disruption of Ca2+ homeostasis to preclude the acceleration of trophoblast differentiation. For example, cannabinoids stimulate mitogen-activated protein kinase [14], which is involved in cell proliferation and differentiation; mitogen-activated protein kinase and calmodulin-dependent protein kinases may share downstream substrates that regulate trophoblast development. In summary, this investigation reveals an exciting observation of concentration- and stage-specific functions of endogenous cannabinoid-like molecules during preimplantation embryonic development.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: D. Randall Armant, C.S. Mott Center for Human Growth & Development, Department of Obstetrics & Gynecology, Wayne State University School of Medicine, 275 East Hancock, Detroit, MI 48201. FAX: 313 577 8554; d.armant{at}wayne.edu
Accepted: November 11, 1998.
Received: September 21, 1998.
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REFERENCES |
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