B.M. Fonseca

The rat as an animal model for fetoplacental development: a reappraisal of the post-implantation period

Following implantation in rodents, the uterine stromal fibroblasts differentiate into densely packed decidual cells. This process, called decidualization, is well-orchestrated and progresses both antimesometrially and mesometrially, creating two regions with distinctive cellular morphologies. In addition, subsequent placental development is dependent on the invasion of the trophoblast, the process intimately linked to the endometrial tissue remodelling and depending largely on the environment created by the decidua; this phenomenon is crucial for the establishment and maintenance of pregnancy. The key mechanisms underlying the maternal tissue remodelling and trophoblast invasion remain poorly understood. The rat, just like human beings, exhibits a highly invasive type of placental development, the haemochorial placentation. For obvious ethical reasons, the studies of endometrial tissue remodelling throughout pregnancy in humans are greatly limited. Although the rat differs somewhat from humans with regards to the implantation process, it is an appropriate model for studying the mechanisms of decidualization as well as subsequent remodelling of the uterine tissues and fetoplacental development. As decidual remodelling is very closely linked to placentation and the maternal-fetal interactions in the rat show several important similarities to human placentation, the morphological alterations occurring during the post-implantation period in the rat have been addressed in the present review.

N-Acylethanolamine Levels and Expression of Their Metabolizing Enzymes during Pregnancy

Decidualization is essential for a successful pregnancy and is a tightly regulated process influenced by the local microenvironment. Lipid-based mediators, such as the endocannabinoid anandamide, and other compounds that have cannabimimetic actions may act on the decidua during early pregnancy. In this study, the levels of N-arachidonoylethanolamine (anandamide) and two other N-acylethanolamines, N-oleoylethanolamine and N-palmitoylethanolamine, were measured in rat plasma and maternal tissues between d 8 and 19 of pregnancy by ultraperformance liquid chromatography tandem mass spectrometry. The spatiotemporal expression of N-acylethanolamine metabolizing enzymes in implantation units were also determined by quantitative PCR, Western blot, and immunohistochemistry and shown to vary with gestation being mainly localized in decidual cells. The data also indicated that plasma and tissues levels of all three N-acylethanolamines fluctuate throughout pregnancy. Tissue levels of endocannabinoids did not correlate with plasma, suggesting that during pregnancy, maternal tissue levels of endocannabinoids are primarily regulated by in situ production and degradation to create endocannabinoid gradients conducive to successful pregnancy.

Spatio-temporal expression patterns of anandamide-binding receptors in rat implantation sites: evidence for a role of the endocannabinoid system during the period of placental development.

BackgroundAlthough there is growing evidence that endocannabinoids play a critical role in early pregnancy, there are no studies describing the possible targets for this system after implantation. The endometrial stroma, which undergoes extensive proliferation and differentiation giving rise to the decidua and the trophoblast cells that invade after the initial stages of implantation, are potential targets. Since high anandamide (AEA) levels, the main endocannabinoid, are detrimental to implantation and in order to gain insight into the role of the endocannabinoid system in the development of the fetoplacental unit, the spatio-temporal pattern of expression of the anandamide-binding receptors, CB1, CB2 and the vanilloid receptor (TRPV1), were investigated by quantitative RT-PCR, western blot and immunohistochemistry.MethodsRat uterine maternal tissues from different days of pregnancy were used to investigate the expression of CB1, CB2 and vanilloid receptors by quantitative RT-PCR, western blot and immunohistochemistry.ResultsThe data indicate that all the three receptors were expressed in decidualized cells and placenta. Interestingly, CB1 and CB2 were also expressed in smooth muscle cells of maternal blood vessels and in endovascular trophoblast cells, whereas TRPV1 was mainly expressed in uterine natural killer (uNK) cells and in the longitudinal muscle layer throughout pregnancy. In all tissues, CB2 protein was present at a lower level than CB1.ConclusionThese observations support a role for the endocannabinoid system during the period of decidualization and placental development.

The endocannabinoid 2-arachidonoylglycerol (2-AG) and metabolizing enzymes during rat fetoplacental development: A role in uterine remodelling

The main endocannabinoids (EC) identified in mammalian tissues are N-arachidonoylethanolamide (AEA, anandamide), and 2-arachidonoylglycerol (2-AG). AEA levels are critical in pregnancy, especially during implantation, decidualization, and placental development. As 2-AG functions in pregnancy are still largely undefined, we hypothesized that it may also have a role during fetoplacental development. We showed that 2-AG is not only present in the rat mesometrial decidua and plasma during fetoplacental development, but that both 2-AG synthesizing (diacylglycerol lipase) and degradation (monoacylglycerol lipase) enzymes are expressed by decidual cells. While lower concentrations of 2-AG induced apoptosis of rat primary decidual cells, via the CB1 receptor, higher concentrations induced a dramatic effect on cell morphology, cell viability and lactate dehydrogenase release, triggered through a mechanism independent of CB1. This study provides evidences that 2-AG fluctuation in maternal tissues throughout normal pregnancy is primarily regulated by its metabolizing enzymes. Together, these data supports the hypothesis that a deregulation of the endocannabinoid system through aberrant cannabinoid signalling may impact normal uterine remodelling process and consequently normal pregnancy.

The endocannabinoid anandamide induces apoptosis in cytotrophoblast cells: Involvement of both mitochondrial and death receptor pathways

INTRODUCTION A balanced proliferation, apoptosis and differentiation in trophoblast cells of the human placenta is crucial for a proper placental development. Alteration in trophoblast apoptosis and differentiation are associated with gestational-related complications, such as preeclampsia, intrauterine growth restriction or miscarriages. The endocannabinoids (eCBs) have been recognized as new interveners in pregnancy events such as implantation and decidualization. However, their importance in placentation is poorly understood. We hypothesise that these novel lipid mediators may intervene in cytotrophoblast apoptosis and, concomitantly, have a role during placental development. METHODS primary human cytotrophoblasts (hCTs) and the human trophoblast-like choriocarcinoma cell line BeWo cells were exposed to Anandamide (AEA). It was investigated the cellular pathways involved in cell death, by the assessment of cell morphology, caspases activity, mitochondrial membrane potential (Δψm), reactive oxygen/nitrogen species (ROS/RNS) and western blot of cleaved Poly (ADP-ribose) polymerase 1 (PARP-1), truncated Bid (t-Bid) and IκB-α. RESULTS AEA decreased hCTs viability and induced morphological features of apoptosis (chromatin condensation and fragmentation), caspase-3/7 activation and PARP-1 cleavage. In BeWo, AEA also increased the activities of caspase-3/7 and 9, induced loss in Δψm and production of ROS/RNS. These effects were reversed by either CB1 or CB2 antagonists, whereas the increase in caspase-3/7 activity was only reversed with CB2 blockage. AEA-treated cells showed increased caspase-8 activation and formation of t-Bid, suggesting the interplay between intrinsic and extrinsic apoptotic pathways. AEA also increased IκB-α expression, a NF-κB regulatory protein. CONCLUSION Our results highlight the importance of eCBs in cytotrophoblast cell apoptosis and indicate that a crosstalk between intrinsic and extrinsic apoptotic pathways is involved in AEA-induced effects.

The endocannabinoid anandamide induces apoptosis of rat decidual cells through a mechanism involving ceramide synthesis and p38 MAPK activation

Anandamide (AEA) belongs to an endogenous family of lipid messengers, called endocannabinoids (ECs), which exert pharmacological effects by binding to selective membrane receptors, the CB1 and CB2 receptors. Increasing evidence suggests that AEA is involved in the regulation of a variety of cell signalling pathways both in experimental models and humans. We have previously demonstrated that ECs machinery operates in decidual cells and found that AEA, the principal EC, induced apoptosis in decidual cells through CB1. Here, we investigated in rat primary decidual cells the signal transduction pathways activated upon AEA binding to CB1. We found that AEA induces a significant increase in the level of intracellular ceramide. These effects were reversed by inhibiting CB1 receptor activation with AM251. The ceramide analogue, C6-ceramide, induced a decrease in decidual cell viability and of p38 MAPK phosphorylation. Additionally, the pharmacologic inhibition of de novo ceramide biosynthesis with l-cycloserine and fumonisin B reduced the AEA-effects on cell viability and p38 MAPK phosphorylation. Furthermore, AEA and C6-ceramide induced a drop in ΔΨm, an increase in ROS production and caspase-3/-7 activation, effects partially reverted by inhibitors of ceramide synthesis and of p38 MAPK. Taken together, we showed that AEA induces a reduction in decidual cell viability by a mechanism involving CB1 activation, which results in ceramide synthesis de novo and p38 phosphorylation, followed by mitochondrial stress and ROS production, leading to apoptosis.

The Endocannabinoid System in the Postimplantation Period: A Role during Decidualization and Placentation.

Although the detrimental effects of cannabis consumption during gestation are known for years, the vast majority of studies established a link between cannabis consumption and foetal development. The complex maternal-foetal interrelationships within the placental bed are essential for normal pregnancy, and decidua definitively contributes to the success of this process. Nevertheless, the molecular signalling network that coordinates strategies for successful decidualization and placentation are not well understood. The discovery of the endocannabinoid system highlighted new signalling mediators in various physiological processes, including reproduction. It is known that endocannabinoids present regulatory functions during blastocyst development, oviductal transport, and implantation. In addition, all the endocannabinoid machinery was found to be expressed in decidual and placental tissues. Additionally, endocannabinoids plasmatic levels were found to fluctuate during normal gestation and to induce decidual cell death and disturb normal placental development. Moreover, aberrant endocannabinoid signalling during the period of placental development has been associated with pregnancy disorders. It indicates the existence of a possible regulatory role for these molecules during decidualization and placentation processes, which are known to be particularly vulnerable. In this review, the influence of the endocannabinoid system in these critical processes is explored and discussed.

Characterisation of the endocannabinoid system in rat haemochorial placenta.

Trophoblast cells that comprise the placenta play a crucial role in the complex cross-talk between fetus and maternal tissues. Although anandamide and 2-arachidonoylglycerol, the best studied endocannabinoids, affect trophoblast attachment and outgrowth, the functional significance of the endocannabinoid system in the development of placenta has not been established. We investigated the correlation between endocannabinoid levels and the pattern of expression of the receptors and metabolic enzymes of the endocannabinoid system during rat placental development. Here, we showed that all the endocannabinoid machinery is dynamically expressed in the functionally distinct basal and labyrinth zones of the rat placenta. Indeed, endocannabinoid levels are shown to increase with the progression of pregnancy. Together, these data support a role for the endocannabinoid system in normal placental function and evidence for a potential novel cellular target for the deleterious action of cannabis-derived compounds during the second half of pregnancy.

Transient receptor potential vanilloid 1 is expressed in human cytotrophoblasts: induction of cell apoptosis and impairment of syncytialization.

The normal development of placenta relies essentially on a balanced proliferation, differentiation and apoptosis of cytotrophoblasts. These processes are tightly regulated by several hormones, cytokines, lipids and other molecules and anomalies in these events are associated with gestational complications. The cation channel transient receptor potential vanilloid 1 (TRPV1) is expressed in several organs and tissues and it participates in cellular events like nociception, inflammation and cell death. However, the expression and importance of this receptor in human placenta still remains unknown. In this work, we found that TRPV1 is expressed in human cytotrophoblasts and syncytiotrophoblasts. Furthermore, the TRPV1 agonists capsaicin and anandamide decreased cytotrophoblast viability and induced morphological alterations, such as chromatin condensation and fragmentation, which suggest the occurrence of apoptosis. Also, both TRPV1 agonists induced a loss of mitochondrial membrane potential and an increase of caspase 3/7 activity and production of reactive species of oxygen and nitrogen. Furthermore, capsaicin (10 μM) impaired the spontaneous in vitro differentiation of cytotrophoblasts into syncytiotrophoblasts by triggering TRPV1, as observed by the decrease in placental alkaline phosphatase activity and in human chorionic gonadotropin secretion. On the other hand, anandamide decreased placental alkaline phosphatase activity via a TRPV1-independent mechanism but did not influence the secretion of human chorionic gonadotropin. In conclusion, we showed that TRPV1 is expressed in human cytotrophoblasts and syncytiotrophoblasts and also reported the involvement of this receptor in cytotrophoblast apoptosis and differentiation.

2-arachidonoylglycerol effects in cytotrophoblasts: metabolic enzymes expression and apoptosis in BeWo cells.

The major endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) is a member of the endocannabinoid system (ECS) that participates in cell proliferation and apoptosis, important events for the homoeostasis of biological systems. The formation of placenta is one of the most important stages of pregnancy and its development requires highly regulated proliferation, differentiation and apoptosis of trophoblasts. Anomalies in these processes are associated with gestational pathologies. In this work, we aimed to study the involvement of 2-AG in cytotrophoblast cell turnover. We found that 2-AG biosynthetic (diacylglycerol lipase A) and degradative (monoacylglycerol lipase) enzymes are expressed in human cytotrophoblasts and in BeWo cells. We also found that 2-AG induces a decrease in cell viability in a time- and concentration-dependent manner and exerts antiproliferative effects. The loss of cell viability induced by a 48-h treatment with 2-AG (10 μM) was accompanied by chromatin fragmentation and condensation, morphological features of apoptosis. Additionally, 2-AG induced an increase in caspase 3/7 and 9 activities, a loss of mitochondrial membrane potential (Δψm) and an increase in reactive oxygen species (ROS)/reactive nitrogen species (RNS) generation, suggesting the activation of the mitochondrial pathway. Moreover, whereas Δψm loss and ROS/RNS generation were significantly attenuated by the antagonists of both the cannabinoid receptors 1 and 2 (CB1 and CB2), the increase in caspase 3/7 and 9 activities and loss of cell viability were reversed only by the antagonist of CB2 receptor; the blockage of the eCB membrane transporter and the depletion of cholesterol failed to reverse the effects of 2-AG. Therefore, this work supports the importance of cannabinoid signalling during cytotrophoblast cell turnover and that its deregulation may be responsible for altered placental development and poor pregnancy outcomes.