Alterations in the pro-resolving lipid mediator machinery within first trimester maternal tissue: Implications in decidualization and miscarriage risk.

A pivotal event in uterine receptivity and human reproduction is the differentiation of endometrial stromal cells into decidual cells, known as decidualization. Decidualization is interlinked with its inflammatory environment. Our study aimed to investigate the presence and role of pro-resolving lipid mediators in first trimester maternal tissue. We assessed the levels of LXA4 and RvD1, along with their metabolic LOX enzymes, in elective (control) and sporadic miscarriage samples. We investigated the effects of LXA4 and RvD1 on decidualization using primary endometrial stromal cells and the immortalized endometrial stromal St-T1b cell line. The upregulation of 12- and 15-LOX expression was observed in pregnancy tissue after sporadic miscarriage, suggesting an inflammatory imbalance. Furthermore, incubation with these lipid mediators led to a decrease in decidualization biomarkers PRL and IGFBP-1, accompanied by morphological changes indicative of aberrant differentiation. The expression of LOX enzymes in decidual natural killer cells suggests their involvement in regulating the inflammatory surroundings and the extent of decidualization.

Protein Interactions in Rhodopseudomonas palustris TIE-1 Reveal the Molecular Basis for Resilient Photoferrotrophic Iron Oxidation.

Rhodopseudomonas palustris is an alphaproteobacterium with impressive metabolic versatility, capable of oxidizing ferrous iron to fix carbon dioxide using light energy. Photoferrotrophic iron oxidation is one of the most ancient metabolisms, sustained by the pio operon coding for three proteins: PioB and PioA, which form an outer-membrane porin-cytochrome complex that oxidizes iron outside of the cell and transfers the electrons to the periplasmic high potential iron-sulfur protein (HIPIP) PioC, which delivers them to the light-harvesting reaction center (LH-RC). Previous studies have shown that PioA deletion is the most detrimental for iron oxidation, while, the deletion of PioC resulted in only a partial loss. The expression of another periplasmic HiPIP, designated Rpal_4085, is strongly upregulated in photoferrotrophic conditions, making it a strong candidate for a PioC substitute. However, it is unable to reduce the LH-RC. In this work we used NMR spectroscopy to map the interactions between PioC, PioA, and the LH-RC, identifying the key amino acid residues involved. We also observed that PioA directly reduces the LH-RC, and this is the most likely substitute upon PioC deletion. By contrast, Rpal_4085 demontrated significant electronic and structural differences from PioC. These differences likely explain its inability to reduce the LH-RC and highlight its distinct functional role. Overall, this work reveals the functional resilience of the pio operon pathway and further highlights the use of paramagnetic NMR for understanding key biological processes.

Long-Term Tamoxifen Effects in the Cyclic Interaction of the Endocannabinoid and Endocrine System in the Rat Central Nervous System.

Steroid hormones can modulate the endocannabinoid system (ECS). Within the female reproductive tract, estrogen increases the expression of the cannabinoid receptors CB1 and CB2, and modifies the levels of anandamide (AEA), the major endocannabinoid, by altering the expression of both AEA synthesis (NAPE-PLD) and catabolic enzymes (FAAH). Here, we addressed the mechanisms involved in ECS fluctuations within the central nervous system and evaluated the effects of tamoxifen (TAM), a selective estrogen receptor modulator, in central AEA regulation. The current results suggest that the hypothalamic and pituitary AEA levels change differently according to the brain area and phase of the estrous cycle. In TAM-treated rats, there is a disruption of the cyclic fluctuation and reduction of the AEA levels in all brain areas. In the pituitary gland, NAPE-PLD expression increases in the metestrus phase, whereas throughout the rat cycle their expression remained constant, even upon TAM treatment. The fluctuations of pituitary AEA levels result from altered FAAH and NAPE-LPD expression. In contrast, no differences in FAAH or NAPE-PLD hypothalamic expression were observed. Overall, this study presents a broad view of the distribution and expression of ECS elements in the central nervous system and a way to suggest possible brain areas involved in the interaction of the endocannabinoid and neuroendocrine systems to induce several behavioral responses.

The effects of delta-9-tetrahydrocannabinol administration in the regulation of female rat sociosexual behaviour.

By targeting the endocannabinoid system, delta-9-tetrahydrocannabinol (THC) modulates female motivated behaviours, influenced by sex hormones. Both medial preoptic nucleus (MPN) and ventromedial nucleus of the hypothalamus (VMN) are involved in the modulation of female sexual responses. The first triggers proceptivity, whereas the ventrolateral division of the latter (VMNvl) triggers receptivity. These nuclei are modulated by glutamate, which inhibits female receptivity, and GABA, which has a dichotomous action in female sexual motivation. Here, we evaluated the action of THC on the modulation of social and sexual behaviours, on signalling pathways of MPN and VMNvl and how sex hormones influence these parameters. Young ovariectomized female rats, given sex hormones (oestradiol benzoate, EB, and progesterone, P) and THC were used for behavioural testing and for immunofluorescence analyses of vesicular glutamate transporter 2 (VGlut2) and GAD (glutamic acid decarboxylase)67 expression. Results showed that females given EB + P exhibited a higher preference for male partner, as well as higher proceptivity and a higher receptivity than control or females given only EB. Females treated with THC presented similar responses in control or EB + P female rats and even more facilitated behavioural responses in EB females than the ones that did not receive THC. Immunofluorescence results in the MPN exhibited a decreased expression of GAD67 and VGlut2 in EB + THC-treated female rats. Within VMNvl of EB-primed rats no changes in the expression of both proteins were observed after THC exposure. This study demonstrates how the possible outcomes of endocannabinoid system instability within hypothalamic neuron connectivity can modify female rat sociosexual behaviour.

Retinoic acid (all-trans) presents antioxidant properties within human ovary and reduces progesterone production by human granulosa cells.

Both vitamin A and E support female reproduction and embryonic development. These vitamins have been associated with decreased fertility or failure to end the pregnancy in animals. An observational study was conducted on follicular fluid (FF) samples to determine the concentrations of fat-soluble vitamins of women undergoing in vitro fertilization and its correlation with assisted reproductive technology characteristics and pregnancy outcomes. Moreover, the effects of all-trans-retinoic acid (atRA) and alpha-tocopherol on granulosa cell viability, apoptosis, autophagy and hormonal production were evaluated. No association was identified between fat-soluble vitamin concentrations in FF and infertility aetiology, body mass index or woman's age. There were differences in follicular antioxidant profiles and ovarian response stimulation. In vitro evaluation of atRA and alpha-tocopherol reveals that, at physiological concentrations, both compounds may affect the viability of granulosa cells. In addition, these compounds are able to protect granulosa cells from oxidative stress, as well as to affect estradiol and progesterone production. Our data suggest that atRA and alpha-tocopherol levels should be well controlled as they may have implications in the function and viability of granulosa cells and highlights retinol as a marker of the oxidative defenses within ovary environment.

Increased expression of NLRP3 inflammasome components in granulosa cells and follicular fluid interleukin(IL)-1beta and IL-18 levels in fresh IVF/ICSI cycles in women with endometriosis.

The inflammasomes are a family of recently described multi-protein cytoplasmic sensors that orchestrate the inflammatory response and participate in a variety of inflammatory conditions. We hypothesized that the activation of pyrin domain­containing protein 3 (NLRP3) inflammasome by granulosa cells (hGCs) may be activated in women with endometriosis and influence oocyte maturation and IVF outcomes. We performed a cross-sectional study to investigate the NLRP3 inflammasome status in follicular fluid (FF) and in hGCs from 44 women undergoing controlled ovarian stimulation for IVF/ICSI. Study subjects were divided into two groups according to the infertility etiology: group with tubal or male factor (control, n = 22) vs. group with endometriosis (n = 22). The FF IL-1beta and IL-18 levels in the endometriosis group were significantly higher than those in the non-endometriosis group, i.e., 5010 pg/mL and 2738 pg/mL, respectively (p < 0.05). No correlation was found between clinical pregnancy and live birth rate and analyzed inflammasome component levels (p > 0.05). In addition, the hGCs from endometriosis women demonstrated high expression of NLRP3 inflammasome at both protein and mRNA levels. Higher expression of inflammasome components within the ovary compartment may result from the exaggerated inflammatory state associated with endometriosis and thus impact the fertility of these women.

Unhealthy Diets Induce Distinct and Regional Effects on Intestinal Inflammatory Signalling Pathways and Long-Lasting Metabolic Dysfunction in Rats.

The intestinal epithelium is a principal site for environmental agents' detection. Several inflammation- and stress-related signalling pathways have been identified as key players in these processes. However, it is still unclear how the chronic intake of inadequate nutrients triggers inflammatory signalling pathways in different intestinal regions. We aimed to evaluate the impact of unhealthy dietary patterns, starting at a younger age, and the association with metabolic dysfunction, intestinal inflammatory response, and obesity in adulthood. A rat model was used to evaluate the effects of the consumption of sugary beverages (HSD) and a Western diet (WD), composed of ultra-processed foods. Both diets showed a positive correlation with adiposity index, but a positive correlation was found between the HSD diet and the levels of blood glucose and triglycerides, whereas the WD diet correlated positively with triglyceride levels. Moreover, a distinct inflammatory response was associated with either the WD or HSD diets. The WD induced an increase in TLR2, TLR4, and nuclear factor-kappa B (NF-κB) intestinal gene expression, with higher levels in the colon and overexpression of the inducible nitric oxide synthase. In turn, the HSD diet induced activation of the TLR2-mediated NF-κB signalling pathway in the small intestine. Altogether, these findings support the concept that early intake of unhealthy foods and nutrients are a main exogenous signal for disturbances of intestinal immune mechanisms and in a region-specific manner, ultimately leading to obesity-related disorders in later life.

The role of macrophages phenotypes in the activation of resolution pathways within human granulosa cells.

BACKGROUND: Inflammatory state within the ovaries can disrupt normal follicular dynamics, leading to reduced oocyte quality and infertility. How the production of inflammatory mediators generated by macrophages with different gene expression profile (M1 and M2) might activate inflammatory pathways, such as cyclooxygenase-2 (COX-2) and 5-, 12-, and 15-lipoxygenase (LOX), in human granulosa cells (hGCs) remains unclear. METHODS: In this study, we evaluated how M1 and M2 macrophages found in the ovaries affect the functions of hGCs isolated from women undergoing assisted reproductive technology (ART) and human ovarian granulosa COV434 cells. For this purpose, a model of interaction between hGCs and COV434 cells and conditioned media (CMs) obtained from culture of M0, M1 and M2 macrophages was established. We used real-time PCR and western blotting to detect the expression of COX-2 and 5-, 12-, and 15-LOX as biomarkers of oocyte competence. RESULTS: Our data showed that M2 macrophages with anti-inflammatory characteristics were able to significantly increase the expression of COX-2 in hGCs. We also demonstrated that M1 macrophages with pro-inflammatory characteristics were able to significantly increase the expression of 12-LOX in hGCs. However, there was no observed expression of 5-LOX and no significant alteration in the expression of 15-LOX in hGCs. Regarding COV434 cells, we found that CM from M2 macrophage resulted in an increase in COX-2, 5-LOX and 15-LOX mRNA and protein levels. No expression of 12-LOX by COV434 cells was observed when exposed to CMs from M1 and M2 macrophages. CONCLUSIONS: Our research indicated that the production of pro-resolving mediators by hGCs can, at least in part, reverse the physiological inflammation present in the ovaries.

In Vitro Effects of Mitochondria-Targeted Antioxidants in a Small-Cell Carcinoma of the Ovary of Hypercalcemic Type and in Type 1 and Type 2 Endometrial Cancer.

Small-cell carcinoma of the ovary of hypercalcemic type (SCCOHT) and endometrial cancer from type 1 and type 2 are gynecological tumors that affect women worldwide. The treatment encompasses the use of cytotoxic drugs that are nonspecific and inefficient. "Mitocans", a family of drugs that specifically target tumor cells' mitochondria, might be a solution, as they conjugate compounds, such as antioxidants, with carriers, such as lipophilic cations, that direct them to the mitochondria. In this study, caffeic acid was conjugated with triphenylphosphonium (TPP), 4-picolinium, or isoquinolinium, forming 3 new compounds (Mito6_TPP, Mito6_picol., and Mito6_isoq.) that were tested on ovarian (COV434) and endometrial (Hec50co and Ishikawa) cancer cells. The results of MTT and neutral red assays suggested a time- and concentration-dependent decrease in cell viability in all tumor cell lines. The presence of apoptosis was indicated by the Giemsa and Höechst staining and by the decrease in mitochondrial membrane potential. The measurement of intracellular reactive oxygen species demonstrated the antioxidant properties of these compounds, which might be related to cell death. Generally, Mito6_TPP was more active at lower concentrations than Mito6_picol. or Mito6_isoq., but was accompanied by more cytotoxic effects, as shown by the lactate dehydrogenase release. Non-tumorous cells (HFF-1) showed no changes after treatment. This study assessed the potential of these compounds as anticancer agents, although further investigation is needed.

Early unhealthy eating habits underlie morpho-functional changes in the liver and adipose tissue in male rats.

Early-life consumption of high-fat and sugar-rich foods is recognized as a major contributor for the onset of metabolic dysfunction and its related disorders, including diabetes and nonalcoholic fatty liver disease. The lifelong impact of early unhealthy eating habits that start at younger ages remains unclear. Therefore, to better understand the effects of diet, it is essential to evaluate the structural and functional changes induced in metabolic organs and potential mechanisms underlying those changes. To investigate the long-term effects of eating habits, young male rats were exposed to high-sugar and high-energy diets. After 14 weeks, body composition was assessed, and histopathological changes were analyzed in the liver and adipose tissue. Serum biochemical parameters were also determined. Expression of inflammatory markers in the liver was evaluated by immunohistochemistry. Our results revealed that serum levels of glucose, creatinine, aspartate transaminase (AST), alanine transaminase (ALT), and lipid profile were increased in rats red high-sugar and high-energy diets. Histopathological alterations were observed, including abnormal hepatocyte organization and lipid droplet accumulation in the liver, and abnormal structure of adipocytes. In both unhealthy diet groups, hepatic expression of Toll-like receptor 4 (TLR4), cyclooxygenase 2 (COX-2), and E-selectin were increased, as well as a biomarker of oxidative stress. Together, our data demonstrated that unhealthy diets induced functional and structural changes in the metabolic organs, suggesting that proinflammatory and oxidative stress mechanisms trigger the hepatic alterations and metabolic dysfunction.

Deciphering Molecular Factors That Affect Electron Transfer at the Cell Surface of Electroactive Bacteria: The Case of OmcA from Shewanella oneidensis MR-1.

Multiheme cytochromes play a central role in extracellular electron transfer, a process that allows microorganisms to sustain their metabolism with external electron acceptors or donors. In Shewanella oneidensis MR-1, the decaheme cytochromes OmcA and MtrC show functional specificity for interaction with soluble and insoluble redox partners. In this work, the capacity of extracellular electron transfer by mutant variants of S. oneidensis MR-1 OmcA was investigated. The results show that amino acid mutations can affect protein stability and alter the redox properties of the protein, without affecting the ability to perform extracellular electron transfer to methyl orange dye or a poised electrode. The results also show that there is a good correlation between the reduction of the dye and the current generated at the electrode for most but not all mutants. This observation opens the door for investigations of the molecular mechanisms of interaction with different electron acceptors to tailor these surface exposed cytochromes towards specific bio-based applications.

Effects of chronic tamoxifen treatment in female rat sexual behaviour.

The medial preoptic (MPN) and the ventromedial hypothalamic nuclei (VMN) modulate the estrogen receptor (ER)-dependent female sexual behavior, a response that is inhibited by tamoxifen (TAM), a modulator of the steroid receptor activation. With the objective to assess TAM action in the brain areas involved in the modulation sexual cues, an animal model on long-term TAM therapy to intact female rats, was used to mimic the 5-year prophylactic TAM therapy offered to women at higher risk of breast cancer. After three months treatment, female sexual behavior with a stud male rat was evaluated. Upon sacrifice, the brains were removed and the MPN and the ventrolateral division of the VMN were screened for the effects of TAM in the expression of ERα, ERß and progesterone receptor. Results show that TAM inhibited the receptive component of the female sexual behavior. Even though TAM decreased estrogen and progesterone levels to values similar to the ones of estrous and diestrus rats, the biochemical data failed to demonstrate such possible causation for the behavioral response. In fact, TAM administration induced a constant low level of ovarian hormones that changed the pattern of ER and PR expression as well as receptor co-expression in the brain areas regulating the behavioral response, dissimilar to the ones seen in the cycle phases with the same low hormone levels. Nevertheless, present data suggests that by affecting ER- and/or PR-dependent mechanisms, TAM may modulate the hypothalamus, a region known to participate in several social behaviors.

Cannabis and Cannabinoids in Reproduction and Fertility: Where We Stand.

Although cannabis use is increasing in general population, their prevalence among young adults is remarkably high. In recent years, their medical use gained a renewed interest. However, it can underline the reputation of cannabis being a harmless drug. Between cannabinoids, uniquely found on the cannabis plant, Δ9-tetrahydrocannabinol (THC) is the well-studied compound. It is responsible for the psychoactive effects via central cannabinoid receptors. Nevertheless, cannabinoids interact with other chemical signalling systems such as the hypothalamic-pituitary-gonadal axis. THC indirectly decreases gonadotropin-releasing hormone (GnRH) secretion by the hypothalamus. The consequences are diverse, and several key hormones are affected. THC disturbs important reproductive events like folliculogenesis, ovulation and sperm maturation and function. Although generally accepted that cannabinoid consumption impacts male and female fertility, prevailing evidence remains largely on pre-clinical studies. Here, we introduce cannabinoids and the endocannabinoid system, and we review the most prominent clinical evidence about cannabis consumption in reproductive potential and teratogenicity.

Bovine Colostrum Supplementation Improves Bone Metabolism in an Osteoporosis-Induced Animal Model.

Osteoporosis is characterized by bone loss. The present study aims to investigate the effects of bovine colostrum (BC) on bone metabolism using ovariectomized (OVX) and orchidectomized (ORX) rat models. Twenty-seven-week-old Wistar Han rats were randomly assigned as: (1) placebo control, (2) BC supplementation dose 1 (BC1: 0.5 g/day/OVX, 1 g/day/ORX), (3) BC supplementation dose 2 (BC2: 1 g/day/OVX, 1.5 g/day/ORX) and (4) BC supplementation dose 3 (BC3: 1.5 g/day/OVX, 2 g/day/ORX). Bone microarchitecture, strength, gene expression of VEGFA, FGF2, RANKL, RANK and OPG, and bone resorption/formation markers were assessed after four months of BC supplementation. Compared to the placebo, OVX rats in the BC1 group exhibited significantly higher cortical bone mineral content and trabecular bone mineral content (p < 0.01), while OVX rats in the BC3 group showed significantly higher trabecular bone mineral content (p < 0.05). ORX rats receiving BC dose 2 demonstrated significantly higher levels of trabecular bone mineral content (p < 0.05). Serum osteocalcin in the ORX was pointedly higher in all BC supplementation groups than the placebo (BC1: p < 0.05; BC2, BC3: p < 0.001). Higher doses of BC induced significantly higher relative mRNA expression of OPG, VEGFA, FGF2 and RANKL (p < 0.05). BC supplementation improves bone metabolism of OVX and ORX rats, which might be associated with the activation of the VEGFA, FGF2 and RANKL/RANK/OPG pathways.

Antioxidant Effects of Chalcones during the Inflammatory Response: An Overall Review.

Reactive oxygen/nitrogen species (ROS/RNS) are produced physiologically by several mechanisms, especially during the inflammatory response. However, their overproduction can lead to the evolution of conditions known as oxidative/nitrosative stress, resulting in the establishment of chronic inflammatory diseases. Chalcones are considered as a class of flavonoids having the molecular pattern 1,3-diaryl-2-propen-1-one. In the last few years, the antioxidant property of chalcones has been extensively studied, mainly due to their ability to inhibit the production or scavenging ROS/RNS. The antioxidant activity of chalcones, focusing on the production of ROS/RNS during the inflammatory response, is demonstrated and discussed in the present review. This literature revision was based on the modulatory effects of chalcones against different enzymes, such as superoxide dismutase (SOD), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, catalase (CAT), myeloperoxidase (MPO) and inducible nitric oxide synthase (iNOS), and, in the scavenging of ROS/RNS. Whenever possible, the structure-activity relationship (SAR) was established. Through the analysis accomplished in this review, it can be observed that the presence of substituents, e.g. hydroxyl, methoxyl, prenyl, and halogen atoms in the chalcones scaffold, often occurs and can improve their modulatory activities, namely, in the production of ROS/RNS during the inflammatory response.

Low Doses of Resveratrol Protect Human Granulosa Cells from Induced-Oxidative Stress.

Resveratrol is a phytoalexin present in plant-derived foods, including grape's skin, cocoa, and peanuts. Evidence suggests that it has beneficial effects on human health because of its antioxidant properties. However, there is limited knowledge about the part played by resveratrol in ovarian function. In this paper, the influence of resveratrol on granulosa cells (GC) was evaluated. In addition to being the main estradiol producers, GC are in direct contact with the oocyte, playing a fundamental role in its growth and development. The cell line COV434 and human granulosa cells (hGC), obtained from women undergoing assisted reproductive technology (ART), were used. GC were treated with resveratrol (0.001-20 µM) at different times (24-72 h). Low concentrations of this compound suggest a protective role, as they tend to reduce ROS/RNS formation after inducement of stress. On the contrary, high concentrations of resveratrol affect GC viability and steroidogenic function. As it may act as a direct modulator of GC oxidative balance, this work may help to clarify the impact of resveratrol on GC and the usefulness of this antioxidant as adjunct to infertility treatments.

Crossing the Wall: Characterization of the Multiheme Cytochromes Involved in the Extracellular Electron Transfer Pathway of Thermincola ferriacetica.

Bioelectrochemical systems (BES) are emerging as a suite of versatile sustainable technologies to produce electricity and added-value compounds from renewable and carbon-neutral sources using electroactive organisms. The incomplete knowledge on the molecular processes that allow electroactive organisms to exchange electrons with electrodes has prevented their real-world implementation. In this manuscript we investigate the extracellular electron transfer processes performed by the thermophilic Gram-positive bacteria belonging to the Thermincola genus, which were found to produce higher levels of current and tolerate higher temperatures in BES than mesophilic Gram-negative bacteria. In our study, three multiheme c-type cytochromes, Tfer_0070, Tfer_0075, and Tfer_1887, proposed to be involved in the extracellular electron transfer pathway of T. ferriacetica, were cloned and over-expressed in E. coli. Tfer_0070 (ImdcA) and Tfer_1887 (PdcA) were purified and biochemically characterized. The electrochemical characterization of these proteins supports a pathway of extracellular electron transfer via these two proteins. By contrast, Tfer_0075 (CwcA) could not be stabilized in solution, in agreement with its proposed insertion in the peptidoglycan wall. However, based on the homology with the outer-membrane cytochrome OmcS, a structural model for CwcA was developed, providing a molecular perspective into the mechanisms of electron transfer across the peptidoglycan layer in Thermincola.

Concentrations of the endocannabinoid N-arachidonoylethanolamine in the follicular fluid of women with endometriosis: the role of M1 polarised macrophages.

Although N-arachidonoylethanolamine (AEA; also known as anandamide) is present in human follicular fluid (FF), its regulation remains unknown. Therefore, the aims of the present study were to: (1) investigate the relationships between FF AEA concentrations in women undergoing assisted reproductive technology and their age, body mass index, ART characteristics and fertility treatment outcomes; and (2) assess how different inflammatory patterns may trigger AEA production by human granulosa cells (hGCs). FF AEA concentrations were higher in women undergoing IVF than in those undergoing intracytoplasmic sperm injection group. FF AEA median concentrations were lower in women undergoing ART because of male factor infertility than in women with endometriosis (1.6 vs 2.5nM respectively), but not women with tubal, hormonal or unexplained infertility (1.6, 2.4 and 1.9nM respectively). To evaluate the effects of macrophages on AEA production by hGCs, hGCs were cocultured with monocyte-derived macrophages. The conditioned medium from M1 polarised macrophages increased AEA production by hGCs. This was accompanied by an increase in AEA-metabolising enzymes, particularly N-acyl phosphatidylethanolamine-specific phospholipase D. The results of the present study show that high FF AEA concentrations in patients with endometriosis may be associated with the recruitment of inflammatory chemokines within the ovary, which together may contribute to the decreased reproductive potential of women with endometriosis. Collectively, these findings add a new player to the hormone and cytokine networks that regulate fertility in women.

Impact of tetrahydrocannabinol on the endocannabinoid 2-arachidonoylglycerol metabolism: ABHD6 and ABHD12 as novel players in human placenta.

Cannabis use has been increasing worldwide for recreational and medical purposes. Consumption by pregnant women is associated with disturbances in pregnancy outcome, such as low birth weight, prematurity and intrauterine growth retardation, though the underlying biochemical mechanisms are unknown. The endocannabinoid system is involved in several reproductive events and the disruption of its homeostasis by ∆9-tetrahydrocannabinol (THC), the main psychoactive cannabinoid, may lead to a negative gestational outcome. In human placenta, THC impairs the levels of the endocannabinoid anandamide (AEA). The other major endocannabinoid, 2-arachidonoylglycerol (2-AG) also plays an important role on proper placentation and pregnancy success. However, THC impact on 2-AG homeostasis has never been addressed. Hence, the effects of THC in 2-AG levels and metabolic enzymes expression were explored. Long-term treatment impairs the expression of the main 2-AG synthetic and degradative enzymes. Curiously, with the highest concentration, despite the maintenance of diacylglycerol lipase alpha (DAGLα) and the decrease in monoacylglycerol lipase (MAGL) expression, 2-AG levels remain constant. Given the endocannabinoid signalling local tight regulation, we hypothesize the involvement of other 2-AG degradative enzymes. Indeed, THC increases the expression of the hydrolyzing enzymes alpha beta hydrolase domain-6 (ABHD6) and -12 (ABHD12), that we firstly describe in human placental tissues. The results show that THC, depending on time of exposure, induces alterations in 2-AG metabolic enzymes expression in placental explants, highlighting the importance of 2-AG regulation and endocannabinoid signalling in placental development. Alterations in this homeostasis may explain the negative pregnancy outcome related to cannabis consumption.

The Cannabinoid Delta-9-tetrahydrocannabinol Disrupts Estrogen Signaling in Human Placenta.

Cannabis consumption is increasing worldwide either for recreational or medical purposes. Its use during gestation is associated with negative pregnancy outcomes such as, intrauterine growth restriction, preterm birth, low birth weight, and increased risk of miscarriage, though the underlying molecular mechanisms are unknown. Cannabis sativa main psychoactive compound, Δ9-tetrahydrocannabinol (THC) is highly lipophilic, and as such, readily crosses the placenta. Consequently, THC may alter normal placental development and function. Here, we hypothesize alterations of placental steroidogenesis caused by THC exposure. The impact on placental estrogenic signaling was examined by studying THC effects upon the enzyme involved in estrogens production, aromatase and on estrogen receptor α (ERα), using placental explants, and the cytotrophoblast cell model BeWo. Aromatase expression was upregulated by THC, being this effect potentiated by estradiol. THC also increased ERα expression. Actions on aromatase were ERα-mediated, as were abolished by the selective ER downregulator ICI-182780 and dependent on the cannabinoid receptor CB1 activation. Furthermore, the presence of the aromatase inhibitor Exemestane did not affect THC-induced increase in ERα expression. However, THC effects on ERα levels were reversed by the antagonists of CB1 and CB2 receptors AM281 and AM630, respectively. Thus, we demonstrate major alterations in estrogen signaling caused by THC, providing new insight on how cannabis consumption leads to negative pregnancy outcomes, likely through placental endocrine alterations. Data presented in this study, together with our recently reported evidence on THC disruption of placental endocannabinoid homeostasis, represent a step forward into a deeper comprehension of the puzzling actions of THC.