Marta Almada

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.

Modulation of the novel cannabinoid receptor - GPR55 - during rat fetoplacental development.

Decidualization process involves the morphological and functional transformation of endometrial stromal cells into decidual cells. This is a finely regulated process, which involves proliferation and differentiation of stromal cells into decidual cells, which is followed by regression of the decidual tissue, mainly by apoptosis, necessary to accommodate the growing embryo. Together with the endogenous cannabinoids (ECs) and the respective metabolizing-enzymes, the cannabinoid receptors complete the endocannabinoid system (ECS). Two cannabinoid receptors have been described so far, CB1 and CB2, though a third has been suggested, CB3. Although the ECS role in several biological functions, including reproductive processes, is now well documented, the current state of knowledge about this system is still incomplete. In order to investigate the expression of GPR55, referred as the novel cannabinoid receptor 3 (CB3), in the uterine maternal tissues during normal pregnancy we analysed its expression by Q-PCR, Western blot and immunohistochemistry during fetoplacental period. We found higher protein levels on day 14, after full development of mesometrial decidua. In addition, GPR55 was found in uterine natural killer (uNK) cells pointing to an involvement in the immunological reactions that occur during pregnancy. The prominent expression of GPR55 in decidual cells suggests a role in mediating cannabinoid signalling during fetoplacental development. Additionally, we have studied the effects resulting from its activation in primary decidual cell cultures, which revealed a potential modulation of cell viability through GPR55. The data presented here may clarify the role of GPR55 in fetoplacental development and highlights the presence of a new target for cannabinoid signalling during pregnancy.

Anandamide and decidual remodelling: COX-2 oxidative metabolism as a key regulator.

Recently, endocannabinoids have emerged as signalling mediators in reproduction. It is widely accepted that anandamide (AEA) levels must be tightly regulated, and that a disturbance in AEA levels may impact decidual stability and regression. We have previously characterized the endocannabinoid machinery in rat decidual tissue and reported the pro-apoptotic action of AEA on rat decidual cells. Cyclooxygenase-2 (COX-2) is an inducible enzyme that plays a crucial role in early pregnancy, and is also a key modulator in the crosstalk between endocannabinoids and prostaglandins. On the other hand, AEA-oxidative metabolism by COX-2 is not merely a mean to inactivate its action, but it yields the formation of a new class of mediators, named prostaglandin-ethanolamides, or prostamides. In this study we found that AEA-induced apoptosis in decidual cells involves COX-2 metabolic pathway. AEA induced COX-2 expression through p38 MAPK, resulting in the formation of prostamide E2 (PME2). Our findings also suggest that AEA-induced effect is associated with NF-kB activation. Finally, we describe the involvement of PME2 in the induction of the intrinsic apoptotic pathway in rat decidual cells. Altogether, our findings highlight the role of COX-2 as a gatekeeper in the uterine environment and clarify the impact of the deregulation of AEA levels on the decidual remodelling process.

The endocannabinoid anandamide impairs in vitro decidualization of human cells

Endocannabinoids (eCBs) are endogenous mediators that along with the cannabinoid receptors (CB1 and CB2), a membrane transporter and metabolic enzymes form the endocannabinoid system (ECS). Several eCBs have been discovered with emphasis on anandamide (AEA). They are involved in several biological processes such as energy balance, immune response and reproduction. Decidualization occurs during the secretory phase of human menstrual cycle, which involves proliferation and differentiation of endometrial stromal cells into decidual cells and is crucial for the establishment and progression of pregnancy. In this study, a telomerase-immortalized human endometrial stromal cell line (St-T1b) and non-differentiated primary cultures of human decidual fibroblasts from term placenta were used to characterize the ECS using immunoblotting and qRT-PCR techniques. It was shown that St-T1b cells express CB1, but not CB2, and that both receptors are expressed in HdF cells. Furthermore, the expression of fatty acid amide hydrolase (FAAH), the main degrading enzyme of AEA, increased during stromal cell differentiation. AEA inhibited cell proliferation, through deregulation of cell cycle progression and induced polyploidy. Moreover, through CB1 binding receptor, AEA also impaired cell differentiation. Therefore, AEA is proposed as a modulator of human decidualization. Our findings may provide wider implications, as deregulated levels of AEA, due to Cannabis sativa consumption or altered expression of the metabolic enzymes, may negatively regulate human endometrial stromal cell decidualization with an impact on human (in)fertility.Free Portuguese abstract: A Portuguese translation of this abstract is freely available at http://www.reproduction-online.org/content/152/4/351/suppl/DC1.

Anandamide interferes with human endometrial stromal-derived cell differentiation: An effect dependent on inhibition of cyclooxygenase-2 expression and prostaglandin E2 release.

The human endometrium undergoes cyclical growth, differentiation, and regression periods throughout the reproductive life. The process in which endometrial stromal cells proliferate and differentiate into decidual cells, named decidualization, prepares a receptive endometrium for implantation. Prostaglandins (PGs) and endocannabinoids (eCBs) are crucial mediators of this process. We have recently reported that the eCB anandamide (AEA) interferes with rat stromal cell differentiation, and on the other hand, PGs are also crucial for decidualization. Therefore, in this study, we analyzed the AEA levels, both in nondifferentiated and in decidualizing human endometrial stromal cells by liquid chromatography-mass spectrometry, and investigated the impact of AEA on PG release and cyclooxygenase-2 (COX-2) expression in human endometrial stromal-derived cell differentiation. For that, an ultra-performance liquid chromatography-mass spectrometry/mass spectrometry method to measure prostaglandin E2 (PGE2 ) and prostaglandin F2α in biological samples was developed and validated. We demonstrate that AEA levels in decidualizing cells are lower than those in nondifferentiated cells, whereas PGE2 levels and COX-2 expression are up-regulated. Thus, low AEA levels may be essential for the onset of decidualization. On the contrary, in AEA-treated cells undergoing decidualization, a decrease of COX-2 protein levels and PGE2 production, in a manner dependent on cannabinoid receptor 1 activation, was observed. Overall, these findings suggest that a deregulation of the intricate network that drives cell differentiation may compromise pregnancy and fertility. It is clinically relevant to understand the mechanisms that influence eCB and PG levels in the endometrium because they may shed light on the sequence of events that lead to a successful pregnancy.

Anandamide oxidative metabolism-induced endoplasmic reticulum stress and apoptosis

The Endocannabinoid System (ECS) has been recognized as a crucial player in human reproduction. Changes in the levels of anandamide (AEA), the main endocannabinoid (eCB), negatively affect reproductive events, such as implantation, decidualization and placentation. Cyclooxygenase-2 (COX-2) is a major enzyme expressed in the endometrium and its involvement in female reproductive system has evolved over the last few years. Currently, COX-2 oxidative metabolism is emerging as a key mediator of AEA-induced actions. In this study, we aimed to disclose the mechanisms underlying the effects of AEA in human endometrial stromal cell fate, using a human-derived endometrial cell line (St-T1b). We found that AEA has an anti-proliferative activity through a direct effect on cell cycle progression by inducing G2/M arrest. Moreover, high levels of AEA increased COX-2 activity, triggering apoptotic cell death, with loss of mitochondrial membrane potential, induction of caspase -9 and -3/-7 activities, and cleavage of poly (ADP-ribose) polymerase (PARP). In addition, the involvement of intracellular reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress was verified. These effects were prevented by pre-incubation with a selective COX-2 inhibitor. Therefore, we hypothesize that, in response to altered levels of this eCB, COX-2 oxidative metabolism of AEA may deregulate endometrial cell turnover and, consequently, interfere with cellular events crucial for implantation and decidualization, with a negative impact on human fertility.

The endocannabinoid system expression in the female reproductive tract is modulated by estrogen

The endocannabinoid system (ECS) is involved in several physiological events that resulted in a growing interest in its modulation. Moreover, the uterine levels of anandamide (AEA), the major endocannabinoid, must be tightly regulated to create proper embryo implantation conditions. However, there are no evidences about the regulation of AEA in uterus by estrogen. Thus, the aim of this study is to elucidate whether estradiol benzoate (EB) and tamoxifen (TAM) administration to ovariectomized (OVX) rats can induce changes in the expression of cannabinoid receptors and AEA-metabolic enzymes in uterus by evaluating gene transcription and protein levels by qPCR, Western blot and immunohistochemistry. Moreover, the plasmatic and uterine levels of AEA and of prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α), the major cyclooxygenase-2 (COX-2) products, were determined by UPLC-MS/MS. The immunohistochemistry showed that cannabinoid receptors, as well as AEA-metabolic enzymes are mainly located in the epithelial cells of both lumen and glands and, to a lesser extent, in the muscle cells. Moreover, EB administration to OVX rats significantly increased CB1, CB2, NAPE-PLD, FAAH and COX-2 expression and transcription. These effects were absent in TAM and TAM+EB treatments showing that this response is estrogen receptor dependent. Additionally, although uterine levels of AEA remained unchanged in EB or TAM treated animals, they showed a rise with EB treatment in plasma. The latter also produced a decrease in uterine PGE2 levels. In summary, these data collectively indicate that the expression of ECS components, as well as, the AEA and PGE2 levels in rat uterus is modulated by EB. Thus, estradiol may have a direct regulatory role in the modulation of ECS in female reproductive tissues.

The synthetic cannabinoid WIN-55,212 induced-apoptosis in cytotrophoblasts cells by a mechanism dependent on CB1 receptor

The endocannabinoid system has evolved as a key regulator in several pathological and physiological processes, including placentation, decidualization and implantation. In addition, it is known that Cannabis and cannabinoids negatively affect female reproduction. Although, the biological action of synthetic cannabinoids, such as WIN-55,212, in human fertility and pregnancy outcome remain to be unveiled. A tight balance between proliferation, differentiation and apoptosis of trophoblast cells is required for placental development and pregnancy outcome. Therefore, in this work, the effects of the synthetic cannabinoid WIN-55,212 in placental cytotrophoblast cells were explored. For that, it was used a human choriocarcinoma cell line, BeWo cells, and primary cultures of human cytotrophoblasts isolated from term placentas. Results demonstrate that this synthetic cannabinoid induces cell cycle arrest. We also observed that cell viability loss was associated with a disruption of mitochondrial membrane potential and activation of caspases -9 and -3/-7 independently of reactive oxygen species (ROS) production or recruitment of the endoplasmic reticulum stress marker CHOP. Moreover, these effects were prevented by pre-incubation with a selective cannabinoid receptor 1 (CBR1) antagonist (AM281). Thus, our results provide strong evidences of the apoptotic process induced by WIN-55,212 through the activation of the CBR1, which may reveal the impact of cannabinoids consumption during placental development.