Tuesday, 28 June 2016

The Effect of Anandamide on Uterine Nitric Oxide Synthase Activity Depends on the Presence of the Blastocyst



Nitric oxide production, catalyzed by nitric oxide synthase (NOS), should be strictly regulated to allow embryo implantation. Thus, our first aim was to study NOS activity during peri-implantation in the rat uterus. Day 6 inter-implantation sites showed lower NOS activity (0.19±0.01 pmoles L-citrulline mg prot−1 h−1) compared to days 4 (0.34±0.03) and 5 (0.35±0.02) of pregnancy and to day 6 implantation sites (0.33±0.01). This regulation was not observed in pseudopregnancy. Both dormant and active blastocysts maintained NOS activity at similar levels. Anandamide (AEA), an endocannabinoid, binds to cannabinoid receptors type 1 (CB1) and type 2 (CB2), and high concentrations are toxic for implantation and embryo development. Previously, we observed that AEA synthesis presents an inverted pattern compared to NOS activity described here. We adopted a pharmacological approach using AEA, URB-597 (a selective inhibitor of fatty acid amide hydrolase, the enzyme that degrades AEA) and receptor selective antagonists to investigate the effect of AEA on uterine NOS activity in vitro in rat models of implantation. While AEA (0.70±0.02 vs 0.40±0.04) and URB-597 (1.08±0.09 vs 0.83±0.06) inhibited NOS activity in the absence of a blastocyst (pseudopregnancy) through CB2 receptors, AEA did not modulate NOS on day 5 pregnant uterus. Once implantation begins, URB-597 decreased NOS activity on day 6 implantation sites via CB1 receptors (0.25±0.04 vs 0.40±0.05). While a CB1 antagonist augmented NOS activity on day 6 inter-implantation sites (0.17±0.02 vs 0.27±0.02), a CB2 antagonist decreased it (0.17±0.02 vs 0.12±0.01). Finally, we described the expression and localization of cannabinoid receptors during implantation. In conclusion, AEA levels close to and at implantation sites seems to modulate NOS activity and thus nitric oxide production, fundamental for implantation, via cannabinoid receptors. This modulation depends on the presence of the blastocyst. These data establish cannabinoid receptors as an interesting target for the treatment of implantation deficiencies.


About half of all conceptuses are lost before the expected term, thus making human reproduction a rather inefficient process [1]. In vitro fertilization techniques (IVF) also result in a high frequency of spontaneous abortions [1]. For IVF, the clinical pregnancy rate per aspiration and per transfer is 26.0 and 29.5%, respectively [2]. Implantation seems to be the bottle neck for the improvement of these techniques.
Several reports point to a role for nitric oxide (NO) and anandamide in implantation. NO is produced by conversion of oxygen and L-arginine to NO and L-citrulline. This reaction is catalyzed by nitric oxide synthase (NOS), of which there are three different isoforms: endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS) and neuronal nitric oxide synthase (nNOS) in almost all types of cells [3]. Purcell and colleagues described that at implantation labelled iNOS cells are within the decidua and the ectoplacental cone, while eNOS is localized in vessels of the primary decidual zone [4]. Both iNOS and eNOS expression are higher at implantation sites. Neuronal NOS is localized mainly in the mesometrium and myometrium and does not appear to change throughout peri-implantation. Moreover, in utero administration of L-NAME, a non selective NOS inhibitor, or of NO-donors during the pre-implantation phase reduces implantation rates [5], [6]. All together these evidences strongly suggest that optimal levels of NO are crucial for endometrial function and embryo implantation.
Anandamide (N-arachidonoylethanolamine, AEA) is an endogenously produced cannabinoid-like lipid mediator [7] that mimics several actions of the natural Cannabis sativa component Δ9-tetrahydrocannabinol, which accounts for the majority of the reproductive hazards in marijuana users [8], [9]. AEA binds to and activates two classical G protein (Gi/o)–coupled cell-surface cannabinoid receptors, cannabinoid receptor type 1 (CB1) and type 2 (CB2) [10], [11]. It has been described that high AEA levels are detrimental for implantation [12] and embryo development [13]. However, the fact that the uterus contains the highest concentrations of AEA yet discovered in mammalian tissues [12], [14] suggests that it might play a role in reproduction. The groups of Paria et al. and Guo et al. were the first ones to report that the mouse uterus has the capacity to synthesize AEA [14], [15]. We have recently found that the rat uterus also has an AEA-synthesizing capacity [16], and as previously described in mice [14], [15], AEA production in the rat uterus is spatiotemporally regulated during implantation, being lower in the receptive uterus and at implantation sites.
In other systems different from the uterus, it has been described that while CB1 activates NOS, CB2 inhibits it [17], [18]. Particularly, in human endothelial cells from various blood vessels, CB1 immunoreactive components are present as is its coupling to anandamide-stimulated NOS-derived NO production [19]. The modulation of vascular diameter and vascular tone by NO represents a crucial point of interest in implantation, and interactions between NO and AEA could be of importance. We have previously observed that AEA increases uterine and deciduas NO production through iNOS activation in a mouse model of fetal resorption [20], [21].
Based on the above evidence, the principal aim of the present work was to study in an in vitro system if AEA modulated NOS activity at implantation and if the blastocyst participates of this interaction. Pseudopregnancy and ovariectomy-induced delayed implantation models were used as tools to understand the relative roles played by the embryo. The fact that AEA differently regulated NOS activity through CB1 and CB2 receptors and that this modulation depended on the presence of the blastocyst during implantation, contributes to better understand the significance of ligand – receptor signalling with AEA and NO as possible effectors in coordinating the series of events that leads to a successful pregnancy.