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

Interaction between lysophosphatidic acid, prostaglandins and the endocannabinoid system during the window of implantation in the rat uterus.

Bioactive lipid molecules as lysophosphatidic acid (LPA), prostaglandins (PG) and endocannabinoids are important mediators of embryo implantation. Based on previous published data we became interested in studying the interaction between these three groups of lipid derivatives in the rat uterus during the window of implantation. Thus, we adopted a pharmacological approach in vitro using LPA, DGPP (a selective antagonist of LPA3, an LPA receptor), endocannabinoids' receptor selective antagonists (AM251 and AM630) and non selective (indomethacin) and selective (NS-398) inhibitors of cyclooxygenase-1 and 2 enzymes. Cyclooxygenase isoforms participate in prostaglandins' synthesis. The incubation of the uterus from rats pregnant on day 5 of gestation (implantation window) with LPA augmented the activity and the expression of fatty acid amide hydrolase, the main enzyme involved in the degradation of endocannabinoids in the rodent uteri, suggesting that LPA decreased endocannabinoids' levels during embryo implantation. It has been reported that high endocannabinoids are deleterious for implantation. Also, LPA increased PGE2 production and cyclooxygenase-2 expression. The incubation of LPA with indomethacin or NS-398 reversed the increment in PGE2 production, suggesting that cyclooxygenase-2 was the isoform involved in LPA effect. PGs are important mediators of decidualization and vascularization at the implantation sites. All these effects were mediated by LPA3, as the incubation with DGPP completely reversed LPA stimulatory actions. Besides, we also observed that endocannabinoids mediated the stimulatory effect of LPA on cyclooxygenase-2 derived PGE2 production, as the incubation of LPA with AM251 or AM630 completely reversed LPA effect. Also, LPA augmented via LPA3 decidualization and vascularization markers. Overall, the results presented here demonstrate the participation of LPA3 in the process of implantation through the interaction with other groups of lipid molecules, prostaglandins and endocannabinoids, which prepare the uterine milieu for embryo invasion during the window of implantation

The role of anandamide during pregnancy : A short tale about the endocannabinoid system

The success of any species depends on its reproductive efficiency. Sexual procreation is initiated by interactions between a sperm and an egg leading to fertilization. The fertilized egg (embryo) undergoes several mitotic cell divisions, ultimately producing the blastocyst. The nurturing of an offspring within the body and production of a live birth is an enduring task, requiring safeguard regulatory systems at various critical steps. At the moment, there is still a significant knowledge gap in understanding the mechanisms by which a successful pregnancy is achieved. It is difficult to define the hierarchical landscape of the molecular pathways during human pregnancy, because of experimental difficulties and ethical restrictions on research with human embryos. It is hoped that experiments on mice and other animal models that bear certain reproductive similarities with humans combined with those feasible experiments in humans would generate meaningful information to address this critical issue. A deeper insight into these processes will help to generate new ideas and concepts for improving fertility and pregnancy-associated health issues in humans. During the last years, several studies have provided evidence that lipid mediators are important signaling molecules in coordinating a series of events during pregnancy. Increasing evidence points toward the pathophysiological significance of endocannabinoids, a group of bioactive lipid-signaling molecules, in both female and male fertility.Sociedad Argentina de Fisiologí

Localization of cannabinoid receptor type 2 during implantation.

<p>Immunolocalization of cannabinoid receptor and type-2 (CB2) in uteri from day 4 (A: luminal and B: glandular), day 5 (C), day 6 implantation sites (D) and day 6 inter-implantation sites (E: luminal, F: glandular). Tissue sections were processed by the immunoperoxidase technique using a polyclonal antibody directed against CB2. No staining was observed in the luminal and glandular epithelium when the first antibody was omitted (G). Black arrows denote specific staining. The scale bar indicates 20 µm.</p

Expression of cannabinoid receptor type-2 at implantation and pseudopregnancy.

<p>Cannabinoid receptor type-2 (CB2) messenger (A and C) and protein (B and D) were detected during peri-implantation (A and B) and on day 5 of pseudopregnancy (C and D). Results are shown as means ± S.E.M. N = 4–6 for each point. a: p<0.01 vs day 5, b: p<0.05 vs day 4. d4: day 4, d5: day 5, d6: day 6, IM: implantation sites, II: inter-implantation sites, psp: pseudopregnancy.</p

Effect of cannabinoid receptors selective antagonists on NOS activity.

<p>Cannabinoid receptors selective antagonists SR141716A (type 1, CB1) or SR144528 (type 2, CB2) were incubated alone for 30 min with day 5 pseudopregnant rat uterus and NOS activity was determined. Results are expressed as pmoles citrulline mg prot⁻¹ h⁻¹. N = 4–6 for each point.</p