Article

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

Authors:
  • Ucibio Requimte, Faculty of Pharmacy University of Porto. Porto4Ageing
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Abstract

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.

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... The 5 µm-thick sections were mounted on poly-L-lysine-coated slides. From each rat, one set of slides was stained with hematoxylin & eosin, and another was processed for immunohistochemistry, as previously described [10]. Briefly, after dewaxing and rehydration, antigen recovery was performed with citrate buffer. ...
... The blocks were mounted on a Vibratome, sectioned at 40 µm, and sampled at regular intervals of 120 µm (one out of three sections), throughout the rostro-caudal extent pre-optic-hypothalamus area. Immunostaining was carried out as previously described in detail [10,29,30] using the primary antibodies against CB1, CB2, NAPE-PLD and FAAH (Table 1), all from St. Cruz Biotechnology (CA, USA) and incubated for 72 h, at 4 • C. After washing, the sections were incubated with the respective biotinylated secondary antibody (Table 1) followed by the ABC Kit. ...
... The upper phase was collected, dried at constant nitrogen stream, reconstituted in phase B following UPLC-MS/MS analysis. Chromatographic and detection details were reported in a previous work [10]. ...
Article
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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.
... Due to the important action of TAM in breast cancer treatment, it is essential to understand the morphological and biochemical side-effects of this drug in the. In this way, we have used an established model of ovariectomy followed by hormonal supplementation, in order to mimic only E 2 secretion along one cycle, preventing, in this way, the action of progesterone (Maia et al., 2017;Sá et al., 2015Sá et al., , 2016Sá et al., , 2018. The use of an ovariectomized animal with hormonal therapy is useful in this, since the endocrine uterine environment may be controlled by hormonal dose and schedule of administration. ...
... The use of an ovariectomized animal with hormonal therapy is useful in this, since the endocrine uterine environment may be controlled by hormonal dose and schedule of administration. Using this model and the administration of estradiol benzoate (EB), we have previously simulated one estrous cycle and studied the effects of hormonal action in the central mechanisms controlling the female sexual behavior (Maia et al., 2017;Sá et al., 2015Sá et al., , 2016Sá et al., , 2018 and in the uterine endocannabinoid system (Maia et al., 2017). ...
... The use of an ovariectomized animal with hormonal therapy is useful in this, since the endocrine uterine environment may be controlled by hormonal dose and schedule of administration. Using this model and the administration of estradiol benzoate (EB), we have previously simulated one estrous cycle and studied the effects of hormonal action in the central mechanisms controlling the female sexual behavior (Maia et al., 2017;Sá et al., 2015Sá et al., , 2016Sá et al., , 2018 and in the uterine endocannabinoid system (Maia et al., 2017). ...
Article
The endometrium is a particular sensitive target tissue for estradiol that is able to promptly modify its structure. Tamoxifen (TAM), a selective estrogen receptor modulator, was shown to promote a spectrum of uterine abnormalities, though the morphological and stereological effects of this drug in uterus is not clear. In this way, we have used an established model of ovariectomy followed by estradiol benzoate (EB) or TAM treatment and analyzed their effects in uterine histopathology and proliferation. Administration of EB promotes the unfolding and proliferation of the endometrium stroma, increasing uterine volume. No changes were found in uterine histomorphometric analysis upon TAM administration, except in the thickness of the luminal epithelium and endometrium layer. The latter may result from increased complexity and glandular volume density also observed in TAM treatment. In addition, EB induced PAX2 expression, an oncogene commonly found in epithelial tumors of the female genital tract, an effect that was weakened by previous TAM administration. Although treatments did not affect stroma cells proliferating index, in epithelial cells and, contrary to TAM, EB increased PCNA and not Ki67 expression. Collectively, our data suggest that the acute administration of TAM induces ERα-dependent atrophy of the uterine tissue and decreased the expression of proliferating cellular markers. On the contrary, if administered prior to EB, TAM is able to attenuate the action of the hormone in uterine morphology and biochemistry.
... The synthesis of AEA was significantly higher in the hypothalamus of OVX-E-primed rats compared to OVX rats or males [33]. Moreover, Maia and colleagues (2017) [37] reported a significant increase in NAPE-PLD and cyclooxygenase-2 (COX-2) at both the gene and protein levels following E2 administration in the uterus of OVX rats. It is noteworthy that the ECS activity and CB1 density in the central nervous system (CNS) are not static and fluctuate throughout the estrous cycle, with findings differing between humans and other species (reviewed in [20]). ...
... It is noteworthy that the ECS activity and CB1 density in the central nervous system (CNS) are not static and fluctuate throughout the estrous cycle, with findings differing between humans and other species (reviewed in [20]). In rodents, the expression of specific elements of the ECS is modulated by estrogen and other sex hormones in reproductive tissues [4,37]. However, E2 did not modify CB1 protein levels in OVX and OVX-E rats, although males showed lower CB1 expression than females [33]. ...
Article
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The endocannabinoid system (ECS) is a lipid signaling system involved in numerous physiological processes, such as endocrine homeostasis, appetite control, energy balance, and metabolism. The ECS comprises endocannabinoids, their cognate receptors, and the enzymatic machinery that tightly regulates their levels within tissues. This system has been identified in various organs, including the brain and liver, in multiple mammalian and non-mammalian species. However, information regarding the sex-specific regulation of the ECS remains limited, even though increasing evidence suggests that interactions between sex steroid hormones and the ECS may ultimately modulate hepatic metabolism and energy homeostasis. Within this framework, we will review the sexual dimorphism of the ECS in various animal models, providing evidence of the crosstalk between endocannabinoids and sex hormones via different metabolic pathways. Additionally, we will underscore the importance of understanding how endocrine-disrupting chemicals and exogenous cannabinoids influence ECS-dependent metabolic pathways in a sex-specific manner.
... Specifically, E2 and P4 down-regulated uterine NAPE-PLD expression via their nuclear receptors (Guo et al., 2005). However, a recent study revealed that the cannabinoid receptors, alongside NAPE-PLD and FAAH, had been up-regulated by E2 administration, and this effect could be blocked by previous tamoxifen (Maia et al., 2017). In addition, no changes in uterine AEA turnover, but instead an increase of plasma levels, were induced by E2 (Maia et al., 2017). ...
... However, a recent study revealed that the cannabinoid receptors, alongside NAPE-PLD and FAAH, had been up-regulated by E2 administration, and this effect could be blocked by previous tamoxifen (Maia et al., 2017). In addition, no changes in uterine AEA turnover, but instead an increase of plasma levels, were induced by E2 (Maia et al., 2017). A suggested peripheral contributor to this may be the endothelial cells, since E2 activates NAPE-PLD and inhibits FAAH and, as a consequence, AEA is released into the circulation (Maccarrone et al., 2002a). ...
Article
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BACKGROUND Obesity has now been recognized as a high-risk factor for reproductive health. Although remarkable advancements have been made in ART, a considerable number of infertile obese women still suffer from serial implantation failure, despite the high quality of embryos transferred. Although obesity has long been known to exert various deleterious effects on female fertility, the underlying mechanisms, especially the roles of lipid metabolism in endometrial receptivity, remain largely elusive. OBJECTIVE AND RATIONALE This review summarizes current evidence on the impacts of several major lipids and lipid-derived mediators on the embryonic implantation process. Emerging methods for evaluating endometrial receptivity, for example transcriptomic and lipidomic analysis, are also discussed. SEARCH METHODS The PubMed and Embase databases were searched using the following keywords: (lipid or fatty acid or prostaglandin or phospholipid or sphingolipid or endocannabinoid or lysophosphatidic acid or cholesterol or progesterone or estrogen or transcriptomic or lipidomic or obesity or dyslipidemia or polycystic ovary syndrome) AND (endometrial receptivity or uterine receptivity or embryo implantation or assisted reproductive technology or in vitro fertilization or embryo transfer). A comprehensive literature search was performed on the roles of lipid-related metabolic pathways in embryo implantation published between January 1970 and March 2022. Only studies with original data and reviews published in English were included in this review. Additional information was obtained from references cited in the articles resulting from the literature search. OUTCOMES Recent studies have shown that a fatty acids-related pro-inflammatory response in the embryo-endometrium boundary facilitates pregnancy via mediation of prostaglandin signaling. Phospholipid-derived mediators, for example endocannabinoids, lysophosphatidic acid and sphingosine-1-phosphate, are associated with endometrial receptivity, embryo spacing and decidualization based on evidence from both animal and human studies. Progesterone and estrogen are two cholesterol-derived steroid hormones that synergistically mediate the structural and functional alterations in the uterus ready for blastocyst implantation. Variations in serum cholesterol profiles throughout the menstrual cycle imply a demand for steroidogenesis at the time of window of implantation (WOI). Since 2002, endometrial transcriptomic analysis has been serving as a diagnostic tool for WOI dating. Numerous genes that govern lipid homeostasis have been identified and, based on specific alterations of lipidomic signatures differentially expressed in WOI, lipidomic analysis of endometrial fluid provides a possibility for non-invasive diagnosis of lipids alterations during the WOI. WIDER IMPLICATIONS Given that lipid metabolic dysregulation potentially plays a role in infertility, a better understanding of lipid metabolism could have significant clinical implications for the diagnosis and treatment of female reproductive disorders.
... In the female reproductive tract, the expression of some components of the ECS is directly modulated by estrogen. Maia and co-workers recently investigated the estradiol benzoate (EB) and tamoxifen dependent modulation of ECS in ovariectomized rats revealing that the expression of CB1 and CB2 receptors, cyclooxygenase-2 (COX-2), and AEA-metabolic enzymes (i.e., NAPE-PLD, FAAH) were higher in the uterus following EB administration; in parallel, the levels of AEA were higher in plasma, but not in the uterus whereas the production of prostaglandin (PG)E 2 -one of the major products of COX-2were higher in the uterus [128]. ...
... Accordingly, in mouse primary Sertoli cells, estradiol via the nuclear ERβ binds two EREs within the FAAH promoter and activates FAAH transcription [132]. An epigenetic mechanism involving the histone demethylase LSD1, that acts decreasing methylation of both DNA at CpG site and histone H3 at lysine 9, has been reported [128]. Interestingly, the stimulation of FAAH expression causes the increase in FAAH protein activity, decreasing AEA levels, and lastly protecting Sertoli cells from AEA-induced apoptosis [133]. ...
Article
Full-text available
The endocannabinoid system (ECS) is a lipid cell signaling system involved in the physiology and homeostasis of the brain and peripheral tissues. Synaptic plasticity, neuroendocrine functions, reproduction, and immune response among others all require the activity of functional ECS, with the onset of disease in case of ECS impairment. Estrogens, classically considered as female steroid hormones, regulate growth, differentiation, and many other functions in a broad range of target tissues and both sexes through the activation of nuclear and membrane estrogen receptors (ERs), which leads to genomic and non-genomic cell responses. Since ECS function overlaps or integrates with many other cell signaling systems, this review aims at updating the knowledge about the possible crosstalk between ECS and estrogen system (ES) at both central and peripheral level, with focuses on the central nervous system, reproduction, and cancer.
... AEA was quantified using liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) as described previously (Maia et al., 2017). In brief, the conditioned medium (0.5 mL) and maternal tissues (about 30 mg) were spiked with 2 μL of internal standard AEA-d4 (2.8 μM; Cayman Chemicals, USA) to estimate the efficiency of the lipid extraction procedure. ...
... B.02.03) was used for data processing. Chromatographic and detection specifications were already reported elsewhere (Maia et al., 2017). ...
Article
Study question: What are the effects of endocannabinoid anandamide (AEA) in uterine natural killer (unK) cells from miscarriage decidua, regarding their cytokine profile and endometrial stromal cell (ESC) crosstalk? Summary answer: uNK-conditioned media from miscarriage samples present high TNF-α levels which inhibit ESC decidualisation. What is known already: AEA plasma levels are higher in women who have suffered a miscarriage. Moreover, AEA inhibits ESC proliferation and differentiation, although the levels and impact on the uNK cell cytokine profile at the feto-maternal interface remain elusive. Study design, size, duration: This laboratory-based study used human primary uNK cells which were isolated from first-trimester decidua (gestational age, 5-12 weeks) derived from 8 women with elective pregnancy termination and 18 women who suffered a miscarriage. Participants/materials, setting, methods: The first-trimester placental tissues were assayed for AEA levels by UPLC-MS/MS and respective enzymatic profile by western blot. The uNK cells were isolated and maintained in culture. The expression of angiogenic markers in uNK cells was examined by quantitative PCR (qPCR). The uNK-conditioned medium was analysed for IFN-γ, TNF-α and IL-10 production by enzyme-linked immunosorbent assay, and the impact on ESC differentiation was assessed by measuring decidual markers Prl, Igfbp-1 and Fox01 mRNA expression using qPCR. Main results and the role of chance: AEA levels were higher in miscarriage decidua compared with decidua from elective terminations. The uNK cell-conditioned medium from the miscarriage samples exhibited high TNF-α levels and interfered with the decidualisation of ESCs. Exacerbated inflammation and elevated TNF-α levels at the feto-maternal interface may trigger AEA signalling pathways that, in turn, may impact decidualisation and the angiogenic ability of uNK cells. Large-scale data: N/A. Limitations, reasons for caution: Primary uNK cell responses are based on a simple in vitro model. Thus, in complex microenvironments, such as the feto-maternal interface, the mechanisms may not be exactly the same. Also, the inflammatory events of miscarriage that, in this study, have happened prior to processing of the samples may cause different responses to that observed. In addition, the magnitude of the inflammatory response, required to trigger the AEA pathways that impact decidualisation and the uNK angiogenic ability in vivo, is still unclear. Wider implications of the findings: The endocannabinoid AEA is a modulator of reproductive competence. AEA not only may contribute to neuroendocrine homeostasis but also can take part in uterine changes occurring during early pregnancy. Study funding/competing interest(s): The work was supported by UID/MULTI/04378/2019 with funding from Fundação para a Ciência e a Tecnologia (FCT)/MCTES through national funds and PORTUGAL 2020 Partnership Agreement, NORTE-01-0145-FEDER-000024. S.C. Cunha acknowledges FCT for the IF/01616/2015 contract. There are no conflicts of interest.
... We hypothesized that maternal HF diet consumption during perinatal phase would program hepatic dysfunctions associated with dysregulation of ECS and redox homeostasis in the liver of adult rat offspring. Because ECS is known to be regulated by sex steroids 24,25 , we further hypothesized that liver ECS programming would occur in a sex dependent manner and liver ECS would be differentially regulated by estradiol levels in female adult rats. ...
... A possible mechanism would be the modulation of estrogen receptor (ER) expression in the liver, rather than estradiol serum levels. It has been reported the presence of response elements to estrogen in Cnr1 and Faah promoter region 44,45 , and estrogen has a stimulatory effect on ECS components 25 . Here, we showed that maternal HF diet increased the ER levels in liver of male offspring but not in female offspring, which may contribute to the increased CB1, CB2 and FAAH in males. ...
Article
Full-text available
Maternal diet plays a critical role in health development. Perinatal overnutrition induces metabolic dysfunctions and obesity in the offspring. Obesity is associated with endocannabinoid system (ECS) over activation and oxidative stress. Liver ECS activation induces hepatic steatosis, inflammation and fibrosis while the antagonism of cannabinoid receptors ameliorates these alterations. Here, we investigated the effect of perinatal maternal high-fat diet (HF, 29% of calories as fat) on the ECS and antioxidant system in liver of male and female adult rat offspring (180 days old). Maternal HF diet increased hepatic cannabinoid receptors, ECS metabolizing enzymes and triglyceride content, with male offspring more affected. ECS changes are likely independent of estradiol serum levels but associated with increased hepatic content of estrogen receptor, which can stimulate the expression of ECS components. Differently, maternal HF diet decreased the activity of the antioxidant enzymes glutathione peroxidase, superoxide dismutase and catalase, and increased oxidative stress markers in both sexes. Alterations in the redox homeostasis were associated with mitochondria damage but not with liver fibrosis. Our data suggest that maternal HF diet induces ECS over activation in adulthood, and that male offspring are at higher risk to develop liver disease compared with female rats.
... Therefore, we hypothesised that early obesity and leptin resistance could also modulate the ECS's components in adipose tissue of the HF offspring. Because the presence of sex steroid hormone response elements in the promoter of the cannabinoid receptors and the ECS-metabolising enzymes, and an in vivo regulation of the ECS by oestrogen (30)(31)(32)(33)(34) , we further hypothesised that perinatal maternal HF diet would alter the protein abundance of the ECS's components in adipose tissue of rat offspring in a sex-specific manner, in parallel to adipose tissue morphological and molecular signature changes and early obesity development. ...
... Maternal HF diet reduced ERα and ERβ content only in VIS WAT of female pups in parallel to increased CB1. In a very recent paper, it was demonstrated that oestradiol increases cannabinoid receptor expression in the uterus of ovariectomised rats (33) , but the direct role of oestrogen on adipose tissue CB1 content is unknown, and, possibly, oestrogen regulates CB1 in a tissue-specific manner. ...
Article
Perinatal maternal high-fat (HF) diet programmes offspring obesity. Obesity is associated with overactivation of the endocannabinoid system (ECS) in adult subjects, but the role of the ECS in the developmental origins of obesity is mostly unknown. The ECS consists of endocannabinoids, cannabinoid receptors (cannabinoid type-1 receptor (CB1) and cannabinoid type-2 receptor (CB2)) and metabolising enzymes. We hypothesised that perinatal maternal HF diet would alter the ECS in a sex-dependent manner in white and brown adipose tissue of rat offspring at weaning in parallel to obesity development. Female rats received standard diet (9 % energy content from fat) or HF diet (29 % energy content from fat) before mating, during pregnancy and lactation. At weaning, male and female offspring were killed for tissue harvest. Maternal HF diet induced early obesity, white adipocyte hypertrophy and increased lipid accumulation in brown adipose tissue associated with sex-specific changes of the ECS’s components in weanling rats. In male pups, maternal HF diet decreased CB1 and CB2 protein in subcutaneous adipose tissue. In female pups, maternal HF diet increased visceral and decreased subcutaneous CB1. In brown adipose tissue, maternal HF diet increased CB1 regardless of pup sex. In addition, maternal HF diet differentially changed oestrogen receptor across the adipose depots in male and female pups. The ECS and oestrogen signalling play an important role in lipogenesis, adipogenesis and thermogenesis, and we observed early changes in their targets in adipose depots of the offspring. The present findings provide insights into the involvement of the ECS in the developmental origins of metabolic disease induced by inadequate maternal nutrition in early life.
... The variety of effects of CBD observed in this study highlights the complexity of its mechanisms of action and the multiple pathways through which it affects stress and depression. While it is not yet fully understood whether the endocannabinoid, neuroinflammatory, and estrogenic changes induced by CBD are independent, there is compelling evidence suggesting that 17β-Estradiol can induce the overexpression of both CB1 and CB2 receptors [90,91], with CB2 also playing a role in anti-inflammatory processes [92]. Given that CBD affects both CB1 and CB2 receptors [15], it is likely that its effects across these pathways are interconnected rather than independent. ...
Article
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Evidence indicates a bidirectional link between depressive symptoms and neuroinflammation. This study evaluated chronic cannabidiol (CBD) treatment effects in male and female rats subjected to the unpredictable chronic mild stress (UCMS) model of depression. We analyzed the gene expression related to neuroinflammation, cannabinoid signaling, estrogen receptors, and specific microRNAs in the ventromedial prefrontal cortex (vmPFC), CA1, and ventral subiculum (VS). UCMS influenced immobility in a sex-specific manner, increasing it in males and decreasing it in females, effects that were reversed by CBD. CBD also normalized the UCMS-induced upregulation of tumor necrosis factor α (TNF-α) in the CA1 and VS in males. In both sexes, UCMS induced the upregulation of the nuclear factor kappa B subunit 1 (NF-κB1) gene in the VS, which was unaffected by CBD. Additionally, CBD reversed CB1 downregulation in the VS of males but not in the vmPFC of either sex. In males, CBD restored the UCMS-induced downregulation of VS estrogen receptor genes ERα and ERβ. UCMS also altered miR-146a-5p expression, downregulating it in females (VS) and upregulating it in males (CA1), with no CBD effect. These findings highlight the sex-specific mechanisms of CBD’s antidepressant effect, with hippocampal neuroinflammatory and estrogenic pathways playing a key role in males.
... Nevertheless, sex hormones could be among the reasonable explanations. For example, FAAH was found to be a contributor to the changes in plasma lipid profiles (Rahman et al. 2021) and regulated by estrogen (Maia et al. 2017). Taken together, our results reflect the complexity of ECS itself as well as its complex interactions with the diet and gender on lipid profiles (Alexander 2015) since FAAH rs324420 was closely interacted with FAAH protein expression and enzymatic activity (Chiang et al. 2004). ...
Article
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The present study was to explore relationship between serum lipid profiles and the polymorphism of rs324420 at fatty acid amide hydrolase (FAAH) gene (FAAH rs324420) and its confounders in Chinese adolescents. Serum lipids, glucose, and insulin levels were assessed using routine methods in a cohort of 708 high school students. Dietary intake was investigated by 3‐day diet records, and intakes of protein, fat, and carbohydrate were calculated. FAAH rs324420 was genotyped by polymerase chain reaction‐restriction fragment length polymorphism technique and confirmed through DNA sequencing. In the whole study population, increased HDL‐C levels were observed in FAAH rs324420 CC homozygotes than those in A allele carriers. Body mass index (BMI), gender, intake of fat, and FAAH rs324420 were predictive factors of HDL‐C levels in the whole study population. Moreover, BMI and intake of fat were predictors of HDL‐C levels in male FAAH rs324420 A allele carriers and female CC homozygotes, but only BMI was predictor of HDL‐C in female A allele carriers and male CC homozygotes. These results demonstrate that there are mutual effects of dietary fat with gender, BMI, and FAAH rs324420 on HDL‐C levels, which pave a novel way to explore the heterogeneous relationship of serum lipid profiles with diets or FAAH rs324420 and provide a new perspective of precision dietary interventions of dyslipidemia, especially in adolescents.
... Estrogens especially estradiole (E2) modulate the ECS, with positive effects on fertility and other reproductive activities. In addition, E2 may also influence progesterone, which is closely associated with pregnancy, and their interaction with eCBs appears to be crucial for pregnancy [32]. These results have thus raised the hypothesis that ECS regulation could help to modify some fertility-related problems and even treat reproductive disorders that are related to the ECS. ...
Article
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The endocannabinoid system is among the most important regulators of human reproduction. It already applies at the level of the sperm and the egg, plays an important role in the fertilization of the egg, its implantation, regulates the function of the placenta and participates in childbirth. The aim of this work is to summarize the knowledge accumulated so far and to show that the endocannabinoid system must be perfectly regulated in order to maintain a physiological pregnancy from implantation to delivery. Only an exceptional interplay of enzymes such as NAPE-PDL or FAAH, endogenous cannabinoids and cannabinoid receptors CB1 and CB2 can ensure the proper functioning of the reproductive organs and thus lead to delivery on time. Changes in the endocannabinoid system can lead to a number of pathological conditions, e.g., during blastocyst implantation, retardation of embryo development, impaired placental function or miscarriage. Soon, we can expect not only an understanding of all the regulatory events associated with the endocannabinoid system and other regulatory systems that participate in reproduction, but also several possibilities for pharmacotherapeutic interventions that can modify the formation, degradation and effect of endocannabinoids. It cannot be ruled out that some components of the endocannabinoid system could become a marker for monitoring pregnancy and childbirth.
... Studies have shown a potential role of endogenous activation of the ECS in pregnancy and reproduction and that the ECS functions in vital physiological processes involved in reproduction, including spermatogenesis, Leydig cell physiology, folliculogenesis, oocyte maturation, endometrial stromal cells proliferation and differentiation (Maia et al., 2017(Maia et al., , 2020. If present in the fetal compartment, exogenously applied cannabinoids can pass through and easily access the fetal nervous system during the prenatal period because of their physicochemical properties (Cabral & Jamerson, 2014). ...
Article
Epidemiological studies examining the influence of cannabis across the lifespan show that exposure to cannabis during gestation or during the perinatal period is associated with later-life mental health issues that manifest during childhood, adolescence, and adulthood. The risk of later-life negative outcomes following early exposure is particularly high in persons who have specific genetic variants, implying that cannabis usage interacts with genetics to heighten mental health risks. Prenatal and perinatal exposure to psychoactive components has been shown in animal research to be associated with long-term effects on neural systems relevant to psychiatric and substance use disorders. The long-term molecular, epigenetic, electrophysiological, and behavioral consequences of prenatal and perinatal exposure to cannabis are discussed in this article. Animal and human studies, as well as in vivo neuroimaging methods, are used to provide insights into the changes induced in the brain by cannabis. Here, based on the literature from both animal models and humans, it can be concluded that prenatal cannabis exposure alters the developmental route of several neuronal regions with correlated functional consequences evidenced as changes in social behavior and executive functions throughout life.
... In fact, sex steroids have an important influence on the levels of AEA. Supporting these growing evidences, E 2 administration to ovariectomized rats increased CB1, CB2, NAPE-PLD, FAAH, and COX2 in the uterus and elevated AEA tone in plasma [115]. In agreement with this, AEA inhibited LH release in ovariectomized rats (and males), effect reversed with E 2 administration [102,116]. ...
... In particular, the transition from the late follicular phase to the early secretory stage of the menstrual cycle, during which ovulation occurs, is characterized by a drastic reduction in plasma level of estradiol and by a significant increment of progesterone, whose level remains elevated up to mid-secretory stage [78]. Interestingly, plasma AEA levels have been correlated with the dynamic changes of sex steroid hormones during the menstrual cycle [79,80], suggesting that these might partially regulate fluctuations in the AEA tone over this period [81][82][83]. In effect, plasma AEA levels are lower in the luteal secretory phase than those in the follicular proliferative phase of the menstrual cycle in healthy women [79,80,82,84]. ...
Article
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In recent years, Cannabis use/misuse for treating pregnancy-related symptoms and other chronic conditions has increased among pregnant women, favored by decriminalization and/or legalization of its recreational uses in addition to its easy accessibility. However, there is evidence that prenatal Cannabis exposure might have adverse consequences on pregnancy progression and a deleterious impact on proper neurodevelopmental trajectories in the offspring. Maternal Cannabis use could interfere with the complex and finely controlled role performed by the endocannabinoid system in reproductive physiology, impairing multiple gestational processes from blastocyst implantation to parturition, with long-lasting intergenerational effects. In this review, we discuss current clinical and preclinical evidence regarding the role of endocannabinoids in development, function, and immunity of the maternal–fetal interface, focusing on the impact of Cannabis constituents on each of these gestational processes. We also discuss the intrinsic limitations of the available studies and the future perspectives in this challenging research field.
... ECS modulates the endometrium plasticity and the uterus receptivity. AEA is considered to be the main factor enabling the synchronization between the embryo development and the endometrium, thus allowing its implantation during the so-called "implantation window" (Fig. 2) (46)(47)(48)(49)(50)(51). AEA levels are high in non-receptive uteri and in the interimplantary areas in the receptive uterus, and low in receptive uteri and in implantary areas (low levels of NAPE-PLD and high FAAH activity and expression), thus leading to the idea that a balanced endocannabinoid system is essential for the synchronization between embryo implantation and endometrium preparation for it (37). ...
... ECS modulates the endometrium plasticity and the uterus receptivity. AEA is considered to be the main factor enabling the synchronization between the embryo development and the endometrium, thus allowing its implantation during the so-called "implantation window" (Fig. 2) (46)(47)(48)(49)(50)(51). AEA levels are high in non-receptive uteri and in the interimplantary areas in the receptive uterus, and low in receptive uteri and in implantary areas (low levels of NAPE-PLD and high FAAH activity and expression), thus leading to the idea that a balanced endocannabinoid system is essential for the synchronization between embryo implantation and endometrium preparation for it (37). ...
... ECS modulates the endometrium plasticity and the uterus receptivity. AEA is considered to be the main factor enabling the synchronization between the embryo development and the endometrium, thus allowing its implantation during the so-called "implantation window" (Fig. 2) (46)(47)(48)(49)(50)(51). AEA levels are high in non-receptive uteri and in the interimplantary areas in the receptive uterus, and low in receptive uteri and in implantary areas (low levels of NAPE-PLD and high FAAH activity and expression), thus leading to the idea that a balanced endocannabinoid system is essential for the synchronization between embryo implantation and endometrium preparation for it (37). ...
Article
The discovery of the cannabinoid receptors CB1 and CB2 in 1990 and 1993, respectively, as well as of the two main endocannabinoids, anandamide in 1992 and 2-arachidonylglycerol in 1995, was an important step in identifying the strongest homeostatic system in the human body, namely the endocannabinoid system. Ever since, research has highlighted the crucial part played by this system in all the reproduction stages: folliculogenesis, spermatogenesis, oogenesis, fecundation, transport of the egg through the fallopian tubes, blastocyte implantation and pregnancy progression, as well as its implications in the physiopathology of the reproductive system: in endometriosis, ectopic pregnancy, miscarriage, preeclampsia, endometrial cancer, polycystic ovary syndrome, ovarian cancer. A special attention must be paid to the phytocannabinoids, natural components originating especially from the Cannabis plant inflorescences, whose medical effects are well-established nowadays with also acting on the receptors of the endocannabinoid system. The most recent research mainly focuses on the reproductive dysfunctions and disorders of the reproductive tissues, respectively, through its action upon the endocannabinoid system. Medical cannabis is nowadays legalized in more and more countries all over the world. At the same time, recreational cannabis remains one of the most consumed drugs (in Romania the most consumed one by young adults). Therefore, it is mandatory for specialists in obstetrics and gynecology, endocrinology, public health, hygiene or for general practitioners, to permanently update their information on this subject.
... 10,11 Preclinical research using ovariectomy to simulate menopause demonstrated that estrogen administration significantly increased expression of cannabinoid receptors and plasma levels of anandamide; these responses were estrogen receptor dependent. 12 In addition, cannabinoid treatments, including administration of anandamide, as well as antagonists of cannabinoid degradative enzymes, improve postovariectomy complications and reduce anxiety. [13][14][15] Further, administration of cannabinoids typically results in vasorelaxation, 16 suggesting that cannabinoid-based therapies may be particularly salient for treating vasomotor symptoms of menopause. ...
Article
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Objective: Expanding access to legal cannabis has dovetailed with increased interest in medical cannabis (MC) use; however, there is a paucity of research examining MC use to alleviate menopause-related symptoms. This survey study assessed patterns of MC use in perimenopausal and postmenopausal individuals. Methods: Participants (perimenopausal, n = 131; postmenopausal, n = 127) completed assessments of menopause-related symptomatology and cannabis use, including modes of use, type of use, and menopause-related symptoms addressed by MC use. Results: Most participants reported current cannabis use (86.1%) and endorsed using MC for menopause-related symptoms (78.7%). The most common modes of use were smoking (84.3%) and edibles (78.3%), and the top menopause-related symptoms for MC use were sleep disturbance (67.4%) and mood/anxiety (46.1%). Relative to postmenopausal participants, perimenopausal participants reported significantly worse menopause-related symptomatology on the vasomotor and psychosocial subscales of the Menopause-Specific Quality of Life Questionnaire (Ps ≤ 0.04), including greater burden of anxiety (P = 0.01) and hot flash (P = 0.04) symptoms. In addition, perimenopausal participants reported higher incidence of depression (P = 0.03) and anxiety diagnoses (P < 0.01), as well as increased use of MC to treat menopause-related mood/anxiety symptoms relative to postmenopausal participants (P = 0.01). Conclusions: Results suggest that many individuals are currently using MC as an adjunctive treatment for menopause-related symptoms, particularly sleep disturbance and mood/anxiety. Future research should examine the impact of different MC use characteristics (e.g., cannabinoid profiles) on the efficacy of MC use for menopause-related symptoms. Increased severity and prevalence of mood and anxiety symptoms in perimenopausal participants suggest promising targets for clinical trials of cannabinoid-based therapies.
... Interestingly, there was no consistent effect of sex on either basal or stress-induced changes in endocannabinoid signaling across these studies. Previous work has suggested that endocannabinoid levels, particularly that of AEA, may be sensitive to reproductive hormones such as estrogen (Hill et al., 2007;Maccarrone et al., 2002;Maia et al., 2017;Tabatadze et al., 2015). In the cohort of rats used for the restraint study, we found that females had globally higher AEA then males, regardless of stress condition; however, this was not seen in either of the other two cohorts. ...
Article
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Research over the past few decades has established a role for the endocannabinoid system for contributing to the neural and endocrine responses to stress exposure. The two endocannabinoid ligands, anandamide (AEA) and 2-arachidonylyl glycerol (2-AG), both play roles in regulating the stress response and both exhibit dynamic changes in response to stress exposure. Most of this previous research, however, was conducted in male rodents. Given that, especially in rodents, the stress response is influenced by sex, an understanding of how these dynamic responses of endocannabinoids in response to stress is influenced by sex could provide insight into sex differences of the acute stress response. We exposed adult, Sprague Dawley rats to different commonly utilized acute stress modalities, specifically restraint, swim and foot shock stress. Thirty minutes following stress onset, we excised the amygdala, hippocampus and medial prefrontal cortex, corticolimbic brain regions involved in the stress response, to measure endocannabinoid levels. When AEA levels were altered in response to restraint and swim stress, they were reduced, whereas exposure to foot shock stress led to an increase in the amygdala. 2-AG levels, when they were altered by stress exposure were only increased, specifically in males in the amygdala following swim stress, and in the hippocampus and medial prefrontal cortex overall following foot shock stress. This increase in 2-AG levels following stress only in males was the only sex difference found in stress-induced changes in endocannabinoid levels. There were no consistent sex differences observed. Collectively, these data contribute to our further understanding of the interactions between stress and endocannabinoid function.
... Interestingly, there are several pieces of evidence pointing to a mutual regulation between ECS and sex hormones in the CNS (Castelli et al., 2014) and peripheral tissues such as the uterus (Maia et al., 2017), gut (Proto et al., 2012), and adipose tissue (de Almeida et al., 2021). In healthy individuals, functional imaging studies have shown that males have more CB1 than females in several brain areas, including the PFC (Laurikainen et al., 2019), a region importantly involved in the hedonic response to palatable foods (Petrovich et al., 2005;Coccurello and Maccarrone, 2018). ...
Article
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The endocannabinoid system (ECS) is an important brain modulatory network. ECS regulates brain homeostasis throughout development, from progenitor fate decision to neuro- and gliogenesis, synaptogenesis, brain plasticity and circuit repair, up to learning, memory, fear, protection, and death. It is a major player in the hypothalamic-peripheral system-adipose tissue in the regulation of food intake, energy storage, nutritional status, and adipose tissue mass, consequently affecting obesity. Loss of ECS control might affect mood disorders (anxiety, hyperactivity, psychosis, and depression), lead to drug abuse, and impact neurodegenerative (Alzheimer’s, Parkinson, Huntington, Multiple, and Amyotrophic Lateral Sclerosis) and neurodevelopmental (autism spectrum) disorders. Practice of regular physical and/or mind-body mindfulness and meditative activities have been shown to modulate endocannabinoid (eCB) levels, in addition to other players as brain-derived neurotrophic factor (BDNF). ECS is involved in pain, inflammation, metabolic and cardiovascular dysfunctions, general immune responses (asthma, allergy, and arthritis) and tumor expansion, both/either in the brain and/or in the periphery. The reason for such a vast impact is the fact that arachidonic acid, a precursor of eCBs, is present in every membrane cell of the body and on demand eCBs synthesis is regulated by electrical activity and calcium shifts. Novel lipid (lipoxins and resolvins) or peptide (hemopressin) players of the ECS also operate as regulators of physiological allostasis. Indeed, the presence of cannabinoid receptors in intracellular organelles as mitochondria or lysosomes, or in nuclear targets as PPARγ might impact energy consumption, metabolism and cell death. To live a better life implies in a vigilant ECS, through healthy diet selection (based on a balanced omega-3 and -6 polyunsaturated fatty acids), weekly exercises and meditation therapy, all of which regulating eCBs levels, surrounded by a constructive social network. Cannabidiol, a diet supplement has been a major player with anti-inflammatory, anxiolytic, antidepressant, and antioxidant activities. Cognitive challenges and emotional intelligence might strengthen the ECS, which is built on a variety of synapses that modify human behavior. As therapeutically concerned, the ECS is essential for maintaining homeostasis and cannabinoids are promising tools to control innumerous targets.
... Recently, progesterone and estradiol are found to induce human trophoblast tubulogenesis associated with the LPA/LPA3 pathway [50]. Moreover, Pla2g10 is revealed to be induced by progesterone receptors and exclusively localized in the apical region of mouse uterine luminal epithelium to contribute to the uterine receptivity [51], while the endocannabinoid system and PGE2 levels in the human uterus are clarified to be modulated by estrogen [52]. Therefore, there may be a plausible mechanism by which steroid hormones effectively affect the phospholipid metabolism and endocannabinoid system for uterine receptivity modulation in mammals, but further studies are needed to validate these hypotheses. ...
Article
Metabolic regulation plays important roles in embryo development and uterine receptivity during early pregnancy, ultimately influencing pregnancy efficiency in mammals. The important roles of lipid metabolism during early pregnancy have not been fully understood. Here, we described the regulatory roles of phospholipid, sphingolipid, and cholesterol metabolism on early embryo development, implantation, and uterine receptivity through production of cannabinoids, prostaglandins, lysophosphatidic acid, sphingosine-1-phosphate, and steroid hormones. Moreover, the impacts of lipids and fatty acids on embryo development potential and the related epigenetic modifications are also discussed. This review aims to elucidate the modulations of lipid metabolism on uterine receptivity and embryo development, contributing to novel strategies to establish dietary balanced lipids and fatty acids for reducing early embryo loss.
... The expression of uterine NAPE-PLD has been down-regulated in the mice model. The down-regulation of uterine NAPE-PLD modulates the reduction of AEA levels [197] in tissues and performs a function to down-regulate the FAAH activity in the uterus of a pregnant mouse [194,195]. However, when accounted for both together with a discreet expression of NAPE-PLD in mice, the FAAH ratio's activity can be significant to regulate AEA levels in the mouse uterus and thereby maintenance of pregnancy or pathologies of endometrial (uterine cancer). ...
Article
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In humans, various sites like cannabinoid receptors (CBR) having a binding affinity with cannabinoids are distributed on the surface of different cell types, where endocannabinoids (ECs) and derivatives of fatty acid can bind. The binding of these substance(s) triggers the activation of specific receptors required for various physiological functions, including pain sensation, memory, and appetite. The ECs and CBR perform multiple functions via the cannabinoid receptor 1 (CB 1); cannabinoid receptor 2 (CB 2), having a key effect in restraining neurotransmitters and the arrangement of cytokines. The role of cannabinoids in the immune system is illustrated because of their immunosuppressive characteristics. These characteristics include inhibition of leucocyte proliferation, T cells apoptosis, and induction of macrophages along with reduced pro-inflammatory cytokines secretion. The review seeks to discuss the functional relationship between the endocannabinoid system (ECS) and anti-tumor characteristics of cannabinoids in various cancers. The therapeutic potential of cannabinoids for cancer-both in vivo and in vitro clinical trials-has also been highlighted and reported to be effective in mice models in arthritis for the inflammation reduction, neuropathic pain, positive effect in multiple sclerosis and type-1 diabetes mellitus, and found beneficial for treating in various cancers. In human models, such studies are limited; thereby, further research is indispensable in this field to get a conclusive outcome. Therefore, in autoimmune disorders, therapeutic cannabinoids can serve as promising immunosuppressive and anti-fibrotic agents.
... Nevertheless, accumulating evidence demonstrates that cannabis effects in reproductive function results, at least partially, from disturbances in the ECS [87]. Sex steroid hormones and eCBs are closely associated [88] and in uterine tissues, estradiol and ECS may regulate each other's activities [89]. During the menstrual cycle, there is also a dynamic change of AEA plasmatic levels [90]. ...
Article
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.
... The endocannabinoid system (ECS) is present in multiple organ systems and is involved in smooth muscle regulation, immune function, neuroendocrine modulation, and metabolism of tissues. [6][7][8][9][10][11][12][13][14] It includes CB 1 and CB 2 cannabinoid receptors, the endocannabinoid agonists anandamide and 2-arachidonoylglycerol (2-AG), and the enzymes that synthesize and metabolize the endocannabinoid ligands. 14,15 The CB 1 cannabinoid receptor is a G protein-coupled receptor with cell-specific activity and is encoded by the CNR1 gene. ...
Article
Background: The endocannabinoid system is present in multiple organ systems and is involved in smooth muscle regulation, immune function, neuroendocrine modulation, and metabolism of tissues. Limited data are available regarding the presence and role of this system in reproductive tissues. Components of the endocannabinoid system have been identified in myometrial and placental tissues. However, no study has investigated differential expression of the endocannabinoid system in labor. Objectives: The purpose of this study was to identify and quantify two components of the endocannabinoid system, the CB1 cannabinoid receptor and cannabinoid receptor interacting protein 1a (CRIP1a) in uterine and placental tissues, and to determine if there is differential expression in tissues exposed to labor. We hypothesized that CB1 cannabinoid receptor concentration would be altered in uterine and placental tissue exposed to labor compared with tissues not exposed to labor. Study Design: Uterine and placental tissue samples were collected in nine laboring and 11 nonlaboring women undergoing cesarean delivery. CB1 cannabinoid receptor and CRIP1a presence and quantification were evaluated using western blot, immunohistochemistry, and real-time quantitative polymerase chain reaction. Statistical comparisons of laboring and nonlaboring subjects were made for uterine and placental tissue using a Mann-Whitney test. Results: Immunohistochemistry demonstrated positive staining for CB1 cannabinoid receptors and CRIP1a in uterine tissue. The protein abundance of CB1 cannabinoid receptor in uterine tissue was significantly lower in tissues exposed to labor (p=0.01). The protein abundance of CRIP1a was lower in uterine tissue exposed to labor but did not reach statistical significance (p=0.06). mRNA expression of CB1 cannabinoid receptor (p=0.20) and CRIP1a (p=0.63) did not differ in labored compared with nonlabored uterine tissues. Conclusions: Our findings of diminished protein density of CB1 cannabinoid receptor in uterine tissue exposed to labor support the hypothesis that the endocannabinoid system plays a role in parturition. Our data add to the growing body of evidence indicating the endocannabinoid system is of importance for successful reproduction and support the need for additional research investigating this complex system as it pertains to labor. ClinicalTrials.gov ID: NCT03752021.
... We speculate that low levels of Cnr1 mRNA in OVX rats might be a counter-regulatory mechanism involving post-transcriptional and/or post-translational mechanisms, including micro RNAs [34,48,49]. Unlike previous studies showing that the estradiol supplementation increases the expression of CB1 receptor in OVX rat uterus [50] and human colorectal cancer cell [45], we demonstrated that OVX is related to increased CB1 expression in SUB WAT, indicating a tissue-specific effect of estradiol levels on CB1 expression. It is already well established that the plasma estradiol deficit due to castration or menopause is associated with increased adiposity [51,52], and the increased CB1 expression in SUB WAT might contribute to this phenotype. ...
Article
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PurposeObesity and high-fat (HF) diet are associated with over activation of the endocannabinoid system (ECS). We have demonstrated that maternal HF diet induces early obesity and modulates cannabinoid signaling in visceral (VIS) and subcutaneous (SUB) white adipose tissue (WAT) in weanling rat offspring. We hypothesized that perinatal maternal HF diet would program the expression of ECS in adipose tissue in a long-term way in parallel to alterations in epigenetic markers and sex hormone signaling.Methods Progenitor female rats received control diet (C, 9% fat) or isocaloric high-fat diet (HF, 28% fat) for 8 weeks before mating, gestation, and lactation. All pups were weaned to C diet and they were euthanized at 180 days old.ResultsMaternal HF diet induced overweight and increased SUB WAT mass of male and female adult offspring. Maternal HF diet induced hypertrophy of VIS and SUB adipocytes only in female offspring associated with increased type 1 cannabinoid receptor protein (CB1) and mRNA (Cnr1) levels. These changes were associated with increased estrogen receptor α binding to Cnr1 promoter in SUB WAT of adult female offspring, which may contribute to higher expression of Cnr1.Conclusion Increased CB1 signaling in adipose tissue might contribute to higher adiposity programmed by maternal HF diet because endocannabinoids stimulate the accumulation of fat in the adipose tissue. Our findings provide molecular insights into sex-specific targets for anti-obesity therapies based on the endocannabinoid system.
... Steroid hormones exert their effects through intracellular receptors that can regulate the expression of target genes by binding to specific enhancer elements [113]. The role of estrogens in modulating cannabinoid receptor expression and endocannabinoids levels is widely known, both in physiological and in pathological conditions [114][115][116]. Studies have demonstrated that 17β-estradiol increases the expression of CB2 receptors in osteoclasts in vitro through the recruitment of an estrogen-responsive element in the CB2 gene [117]. ...
Article
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In late December 2019, a novel coronavirus (SARS-CoV-2 or CoV-19) appeared in Wuhan, China, causing a global pandemic. SARS-CoV-2 causes mild to severe respiratory tract inflammation, often developing into lung fibrosis with thrombosis in pulmonary small vessels and causing even death. COronaVIrus Disease (COVID-19) patients manifest exacerbated inflammatory and immune responses, cytokine storm, prevalence of pro-inflammatory M1 macrophages and increased levels of resident and circulating immune cells. Men show higher susceptibility to SARS-CoV-2 infection than women, likely due to estrogens production. The protective role of estrogens, as well as an immune-suppressive activity that limits the excessive inflammation, can be mediated by cannabinoid receptor type 2 (CB2). The role of this receptor in modulating inflammation and immune response is well documented in fact in several settings. The stimulation of CB2 receptors is known to limit the release of pro-inflammatory cytokines, shift the macrophage phenotype towards the anti-inflammatory M2 type and enhance the immune-modulating properties of mesenchymal stromal cells. For these reasons, we hypothesize that CB2 receptor can be a therapeutic target in COVID-19 pandemic emergency.
... The cannabinoid receptors, alongside AEA and 2-AG metabolic enzymes, have also been characterized in uterine tissues throughout the menstrual cycle (Taylor et al., 2010;Scotchie et al., 2015). The expression of the metabolic enzymes and of the cannabinoid receptors was assessed by western blot, immunohistochemistry and real-time PCR, showing that these elements undergo changes according to the fluctuations in steroid hormones (Ribeiro et al., 2009;Scotchie et al., 2015;Maia et al., 2017). These variations consequently lead to changes in the eCB levels throughout the menstrual cycle, with an increase in AEA levels in the ovary upon ovulation (El-Talatini et al., 2009) and higher plasma levels in the follicular phase when compared to those in the luteal phase (Habayeb et al., 2004). ...
Article
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Background: The endocannabinoid system (ECS) consists of the cannabinoid receptors CB1 and CB2, the main endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) and their metabolic enzymes N-acylphosphatidylethanolamine-specific phospholipase D, fatty acid amide hydrolase, diacylglycerol lipase and monoacylglycerol lipase. This system is involved in the modulation of essential physiological processes. Its role in the reproductive system has become significantly important in recent years, given its major role in events such as gametogenesis, decidualisation, implantation and placentation. Objective and rationale: In this paper, we review the literature and summarize the role of the ECS elements in reproduction and their potential as early markers for diagnosis of reproductive disorders or as pharmacological targets for treatment. Search methods: Original research and review papers published from 1964 to June 2019 were selected in terms of relevance, reliability and quality by searching PubMed, MEDLINE and Web of Science, using the following search terms: endocannabinoid system and endometriosis; endocannabinoid system and ectopic pregnancy; endocannabinoid system and miscarriage; endocannabinoid system and pre-eclampsia; endocannabinoid system and endometrial cancer; endocannabinoid system and reproduction; endocannabinoid, endometrium; placenta; N-acylethanolamines; anandamide; 2-arachidonoylglycerol; and cannabinoids. Outcomes: This review demonstrates relevant information concerning ECS alterations in endometriosis, ectopic pregnancy, miscarriage, pre-eclampsia and endometrial cancer. We highlight the importance of the endocannabinoids in endometrial and placental physiology and pathophysiology, from studies in vitro and in vivo and in clinical observations. The most studied of the endogenous cannabinoids is AEA. The levels of AEA were increased in plasma of patients with endometriosis and miscarriage, as well as in the fallopian tube of women with ectopic pregnancy and in endometrial biopsies of endometrial cancer. Changes in the pattern of expression of the cannabinoid receptor CB1 were also observed in endometrial biopsies of endometriosis, fallopian tube and decidua of patients with ectopic pregnancy and pre-eclamptic placenta. Moreover, alterations in CB2 expression have been reported in association with endometrial cancer. In general, studies on the cannabinoid signalling through CB2 and on the biological activities of the other major endocannabinoid, namely 2-AG, as well as its metabolic enzymes are scarce and avidly required. Wider implications: The pathophysiological mechanisms involved in the described endometrial and placental pathologies are still unclear and lack the means for an early diagnosis. Based on current evidence, though alterations in ECS are demonstrated at tissue level, it is difficult to associate plasmatic changes in AEA with specific endometrial and placental diseases. Thus, pairing alterations in AEA levels with 2-AG and/or other endocannabinoid-like molecules may provide more accurate and early diagnoses. In addition, patients may benefit from new therapies that target the ECS and endocannabinoid signalling.
... 26 One metabolomic study demonstrated that estrogen/progestin, glycerophospholipid metabolism, and sphingolipid metabolism were disturbed in patients with PD. 25,27 Glycerophospholipid and sphingolipid metabolism is the key pathway that produces PGF2α and leukotrienes, which cause myometrial contraction, vasoconstriction, and hypersensitization of pain fibers. Abnormal levels of estrogen lead to an excess of PG. 28,29 The herbs of our HPM shared similar treatment principles with Shaofu Zhuyu decoction, and Shaofu Zhuyu formula restores the metabolites of glycerophospholipid and sphingolipid metabolism and returns the levels of estrone back to a control-like level. 27 A recent study showed that HPM upregulated dihydroprogesterone, pregnenolone, and PGE2; downregulated estrone; and could regulate production of PG and leukotrienes through arachidonic acid metabolism. ...
Article
Objective: To observe the effect of herb-partitioned moxibustion (HPM) for primary dysmenorrhea. Methods: Six hundred and forty patients were randomized assigned (1∶1) to HPM group and control group. Duration of treatment was 3 months with 3 month follow-up. The primary outcome was pain relief measured by visual analogue scale (VAS). The second outcomes were Cox Menstrual Symptom Scale (CMSS), menstrual pain duration and frequency of analgesics usage. The exploratory outcome included quality of life, RESULTS: After the 3-month treatment and follow-ups, the pain intensity measured by VAS was significantly reduced in both groups compared with baseline (P < 0.05), and it was significantly decreased in HPM group than that of control group (P < 0.001). The higher proportion of participants in the HPM group had a decrease of at least 50% in VAS at the end of treatment, as compared with the control group (P < 0.001). At the 3rd and 6th month, the menstrual pain duration, CMSS score and frequency of analgesics usage in HPM group were significantly lower than those of control group (P < 0.05). After 3 month treatment and follow-ups,the scores of physical, psychological, social and environmental domains were significantly increased than baseline in both groups (P < 0.05), and the sores of physical, psychological and environmental domains were significantly higher in HPM group than those of control group (P < 0.05) . Conclusion: Herb-partitioned moxibustion reduced menstrual pain and improved quality of life, these were sustained for up to 3 months after treatment. Further research is needed to understand long term effect and the mechanism of the intervention.
... The presence of ECS elements in several reproductive fluids, cells and tissues like human serum and follicular fluid, ovary, uterus and placenta, have been documented by several studies in animal models and humans [45][46][47][48][49][50]. Cannabis extracts, THC and modulators of the ECS clearly have a major impact on virtually all steps of female reproduction [41,51,52]. ...
Article
Cannabis extracts like marijuana have the highest consumption rate worldwide. Yet, their societal acceptance as recreational and therapeutic drugs could represent a serious hazard to female human reproduction, because cannabis ingredients [termed (phyto)cannabinoids] can perturb an endogenous system of lipid signals known as endocannabinoids. Accumulated evidence on animal models and humans has demonstrated a crucial role of these endogenous signals on different aspects of female reproduction, where they act through an ensamble of proteins that synthesize, transport, degrade and traffic them. Several reports have recently evidenced the potential role of endocannabinoids as biomarkers of female infertility for disease treatment and prevention, as well as their possible epigenetic effects on pregnancy. The purpose of this review is to provide an update of data collected in the last decade on the effects of cannabinoids and endocannabinoids on female reproductive events, from development and maturation of follicles and oocytes, to fertilization, oviductal transport, implantation and labor. In this context, a particular attention has ben devoted to the ovary and the production of fertilizable oocytes, because recent studies have addressed this hot topic with conflicting results among species.
... Mechanistically, this sexual dimorphism may be influenced by hormones, as CB 2 activity in females has been demonstrated to involve estrogen, which also protects against inflammation. 28 Of particular interest is a significant change in CB 2 expression in maternal rats fed a high LA diet during pregnancy. CB 2 receptor expression in non-pregnant females has been demonstrated to be cardioprotective, with up-regulation decreasing risk of cardiovascular diseases. ...
Article
The endocannabinoid system (ECS), modulated by metabolites of linoleic acid (LA), is important in regulating cardiovascular function. In pregnancy, LA is vital for foetal development. We investigated the effects of elevated LA in H9c2 cardiomyoblasts in vitro and of a high linoleic acid (HLA, 6.21%) or low linoleic acid (LLA, 1.44%) diet during pregnancy in maternal and offspring hearts. H9c2 cell viability was reduced following LA exposure at concentrations between 300 and 1000 µM. HLA diet decreased cannabinoid receptor type 2 (CB2) mRNA expression in foetal hearts from both sexes. However, HLA diet increased CB2 expression in maternal hearts. The mRNA expression of fatty acid amide hydrolase (FAAH) in foetal hearts was higher in females than in males irrespective of diet and N-acyl phosphatidylethanolamine-specific phospholipase D (NAPE-PLD) mRNA expression showed an interaction between diet and sex. Data indicate that a high LA diet alters cell viability and CB2 expression, potentially influencing cardiac function during pregnancy and development of the offspring’s heart.
... However, these findings were different from the results of Leconte et al. [22], who reported that the CB1 and CB2 were expressed equally in the epithelial and stromal cell lines originated from eutopic endometrium and endometriosis, and other scholars [23] reported that no difference was found in endometrial CB1 immunoreactivity throughout the menstrual cycle. Evidence from studies on the ECS in reproduction has suggested that steroid hormones may affect the ECS [24,25], plasma AEA is proved to fluctuate with the menstrual cycle and reach a peak at ovulation [26], and Di Blasio et al. [27] reviewed that the levels of FAAH, NAPE-PLD, CB1, and CB2 all change with sex hormones in female reproductive tissues. These are in accordance with the cyclic changes we observed in the expression of CB1 and CB2, and the cyclic changes of ECS have been considered vital in reproduction [28]. ...
Article
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Objective: Adenomyosis is a common gynecologic benign disease that may have a life-long negative impact on women. Previous studies have indicated that the endocannabinoid system may participate in the progress of endometriosis. Our research aims to analyze the expression patterns of the typical cannabinoid receptors (CB1 and CB2), the main constituents of the endocannabinoid system, in endometrial samples derived from patients diagnosed as adenomyosis or not. Methods: Eutopic and corresponding ectopic endometrium from 45 premenopausal women diagnosed as adenomyosis and normal endometrium from 34 age-matched women lacking evidence of adenomyosis were examined by immunohistochemistry and quantitative real-time polymerase chain reaction (qRT-PCR) to determine the CB1 and CB2 expression levels. Results: In either the proliferative or the secretory phase, CB1 and CB2 protein and mRNA levels were both significantly lower in the eutopic and ectopic endometrium of adenomyosis when compared with normal endometrium. For women with adenomyosis, CB1 and CB2 protein and mRNA levels were much lower in the ectopic endometrium than the eutopic in both phases of the cycle. Both CB1 and CB2 protein and mRNA levels were increased during the secretory phase in normal endometrium, while CB1 lost its cyclic variation in the eutopic and ectopic endometrium from patients diagnosed as adenomyosis. Conclusion: The decreased expression of CB1 and CB2 in the eutopic and ectopic endometrium from patients diagnosed as adenomyosis suggests that cannabinoid receptors may participate in the pathogenesis of adenomyosis.
... Several studies have explored the role of intricate endocannabinoids-sex hormone-cytokine regulatory axis during pregnancy [57,70]. Sex steroid hormones, progesterone and estrogen, are involved in the maintenance of endocannabinoid levels [71]. Progesterone promotes lymphocyte FAAH activity involving transcription factors Stat3 and Ikaros, which results in lower AEA levels [72][73][74]. ...
Article
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Cannabinoids are the most commonly abused illicit drugs worldwide. While cannabis can be beneficial for certain heath conditions, abuse of potent synthetic cannabinoids has been on the rise. Exposure to cannabinoids is also prevalent in women of child-bearing age and pregnant women. These compounds can cross the placental barrier and directly affect the fetus. They mediate their effects primarily through G-protein coupled cannabinoid receptors, CB1 and CB2. In addition to significant neurological effects, cannabinoids can trigger robust immunomodulation by altering cytokine levels, causing apoptosis of lymphoid cells and inducing suppressor cells of the immune system. Profound effects of cannabinoids on the immune system as discussed in this review, suggest that maternal exposure during pregnancy could lead to dysregulation of innate and adaptive immune system of developing fetus and offspring potentially leading to weakening of immune defenses against infections and cancer later in life. Emerging evidence also indicates the underlying role of epigenetic mechanisms causing long-lasting impact following cannabinoid exposure in utero.
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Background: Cannabinoids, as member of Cannabis sativa L. derivatives (such as marijuana, hashish) are able to activate the endocannabinoid system via two endogenous receptors CB1 and CB2. This system plays an important role in the regulating folliculogenesis and fertility and affects many of the processes of the reproductive system. This study was conducted to investigate the possible effects of cannabinoid agonists and antagonists on the ovarian health and function of female mice. Material & Method: A total of 80 NMRI mice were divided into 10 groups. Treatment groups received CB1 or CB2 agonist or antagonist or a combination of them for 5 days. Animals were sacrificed;ovaries were removed, measured to determine the weight and volume, total RNA from the left ovary was extracted for q-PCR, and the right ovary was fixed in Boin’s fixative to evaluate folliculogenesis. Results:Treatment of animals with CB1/CB2 agonist + CB1 antagonist (W102+AM251) decreased the level of NAPE-PLD and increased the level of FAAH gene compared to all groups. CB2 antagonist (AM630) increased the number of primary, preantral and antral follicles as well as the volume and weight of ovaries, and estrogen levels. While the CB1 antagonist (AM251) significantly increased the number of micro vessels in the ovary. Conclusion: Cannabinoid products affect the physiology of the ovaries and impair folliculogenesis. The CB2 receptor appears to play a major role in this process. Antagonism at CB2 appeared to differentially affect cannabinoid-metabolizing enzymes in ovarian follicles and also differentially affects their maturation.. However, our preliminary novel findings in mice require human studies before application in clinics
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The endocannabinoid system (ECS) is associated with several physiological processes including reproduction. This system consists of the cannabinoid receptors, endocannabinoid ligands and enzymes which metabolize and degrade these fatty acids. Recent evidence shows that cannabinoid receptors are expressed in cells of the reproductive system including endometrial stromal cells, ovaries, and sperm cells. Emerging and recent research suggests that the ECS may play a significant role in reproduction. The endocannabinoid ligands anandamide and 2-arachidonoyl-glycerol are crucial for successful endometrium decidualization, placental development and embryo implantation. Alteration in cannabinoid receptors expression or in endocannabinoid homeostasis by excessive intake of cannabis during pregnancy is associated with negative pregnancy outcomes including preterm birth. The use of medicinal cannabis is becoming more widespread in Western countries, especially in people of reproductive age. Cannabis contains phytocannabinoids which modulate the ECS, and emerging evidence suggests that phytocannabinoids, through their action on cannabinoid receptors, may have a negative impact on fertility, pregnancy outcome and fetal health. In this mini-review, we highlight the recent advances in the field, which explore the role of endocannabinoids in early pregnancy and the effects of excessive intake of phytocannabinoids in pregnancy outcomes.
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The myometrium, especially the junctional zone (JZ), is now well documented to have a role in the pathogenesis of adenomyosis. Cannabinoid receptors have been shown to participate in the establishment of endometriosis and its pain perception. However, its relation to adenomyosis has not been identified yet. The aim of this study was to investigate the expression of cannabinoid receptor type I (CB1) and type II (CB2) in myometrium of uteri with and without adenomyosis and determine the correlation between their levels and clinical parameters of adenomyosis. We collected tissue samples of JZ and the outer myometrium from 45 premenopausal women with adenomyosis and 34 women without adenomyosis. CB1 and CB2 messenger RNA (mRNA) and protein expression levels were evaluated by the use of Western blotting and real-time quantitative polymerase chain reaction from all samples. Clinical information on the severity of dysmenorrhea and other data were collected. We found both CB1 and CB2 mRNA and protein levels in women with adenomyosis were significantly higher than those of controls, and CB1 expression levels in JZ were positively correlated with the severity of dysmenorrhea. These data suggest that cannabinoid receptor CB1 may be involved in the pathogenesis of dysmenorrhea in adenomyosis and may be a potential therapeutic target.
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The endocannabinoid system is an increasingly recognised pharmacological target for treating stress and anxiety disorders, including post-traumatic stress disorder (PTSD). Recent preclinical developments have implicated the endocannabinoid system in stress responses, emotional memories and fear extinction, all critical to PTSD aetiology. However, preclinical research in endocannabinoid biology has neglected the influential role of sex hormone differences on PTSD symptomology, which is particularly important given that PTSD is twice as common in women as in men. In this review, we compile and consider the evidence that the endocannabinoid system is influenced by ovarian hormones, with application to stress disorders such as PTSD. It is clear that therapeutic modulation of the endocannabinoid system needs to be approached with awareness of ovarian hormonal influences, and knowledge of these influences may enhance treatment outcomes for female PTSD populations.
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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, endocannabinoid's 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.
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Background Cyclooxygenases (COXs) are the rate limiting enzymes in the process of prostaglandins (PGs) synthesis, which are critical regulators of a number of reproductive processes, including ovulation, implantation, decidualization and parturition. The aim of the present study was to investigate the expression and regulation of COX-1 and COX-2 and levels of prostaglandins during rat pregnancy, in a model of pseudopregnancy and estrous cycle. Methods Uteri were collected from the cyclic rats on each day of estrous cycle, after every two days for pregnant (days 2 to 22) and pseudopregnant rats (days 1 to 9). In vitro primary endometrial stromal cells were cultured in the presence of steroid hormones and their respective inhibitors for the possible modulation of COX-1 and COX-2. Endometrial protein extracts were used for western blot analysis and tissue sections were prepared for protein localization using immunofluorescence. Measurements of PGF2alpha and PGE2 metabolites in serum were performed by enzyme immunoassay (EIA). Results COX-1 expression was found to be elevated during implantation and parturition, however, the levels of COX-1 decreased during decidualization periods. COX-2 was detected during early pregnancy from day 2 to 5, increased during decidual regression, and was also expressed at the time of parturition. COX-2 protein expression was found to be increased at estrus phase in cyclic rats. Both enzymes were found to be modulated in the endometrium of pseudopregnant rats, suggesting that they are regulated by 17beta-estradiol and progesterone. A significant increase in PGE2 metabolite levels was observed on day 10, 12 and 14 of pregnancy. However, an increase in PGF2alpha metabolite levels was observed only on day 14. The concentration of both these metabolites changed during pseudopregnancy and maximum levels were observed at day 7. Significant increase in PGE2 metabolite was observed at proestrus phase, on the other hand, PGF2alpha metabolite was significantly increased at proestrus and metestrus phase. COX-2 protein was regulated by 17beta-estradiol in cultured endometrial stromal cells which was blocked in the presence of ICI-182,780. Conclusions Taken together, these results suggest that COX-1 and COX-2 could be differentially regulated by steroid hormones and might be the key factors involved in embryo implantation, decidualization, decidua basalis regression and parturition in rats.
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Endocannabinoids are natural lipids able to bind to cannabinoid and vanilloid receptors. Their biological actions at the central and peripheral level are under the tight control of the proteins responsible for their synthesis, transport and degradation. In the last few years, several reports have pointed out these lipid mediators as critical signals, together with sex hormones and cytokines, in various aspects of animal and human reproduction. The identification of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in reproductive cells and tissues of invertebrates, vertebrates and mammals highlights the key role played by these endogenous compounds along the evolutionary axis. Here, we review the main actions of endocannabinoids on female and male reproductive events, and discuss the interplay between them, steroid hormones and cytokines in regulating fertility. In addition, we discuss the involvement of endocannabinoid signalling in ensuring a correct chromatin remodeling, and hence a good DNA quality, in sperm cells.
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Uterine gland formation occurs postnatally in an ovary- and steroid-independent manner in many species, including humans. Uterine glands secrete substances that are essential for embryo survival. Disruption of gland development during the postnatal period prevents gland formation, resulting in infertility. Interestingly, stabilization of beta-catenin (CTNNB1) in the uterine stroma causes a delay in gland formation rather than a complete absence of uterine glands. Thus, to determine if a critical postnatal window for gland development exists in mice, we tested the effects of extending the endocrine environment of pregnancy on uterine gland formation by treating neonatal mice with estradiol, progesterone, or oil for 5 days. One uterine horn was removed before puberty, and the other was collected at maturity. Some mice were also ovariectomized before puberty. The hormone-treated mice exhibited a delay in uterine gland formation. Hormone-treatment increased the abundance of uterine CTNNB1 and estrogen receptor alpha (ESR1) before puberty, indicating possible mechanisms for delayed gland formation. Despite having fewer glands, progesterone-treated mice were fertile, suggesting that a threshold number of glands is required for pregnancy. Mice that were ovariectomized before puberty did not undergo further uterine growth or gland development. Finally, to establish the role of the ovary in postpartum uterine gland regeneration, mice were either ovariectomized or given a sham surgery after parturition, and uteri were evaluated 1 wk later. We found that the ovary is not required for uterine growth or gland development following parturition. Thus, uterine gland development occurs continuously in mice and requires the ovary after puberty, but not after parturition.
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Endogenous cannabinoids play an important role in the physiology and behavioral expression of stress responses. Activation of the hypothalamic-pituitary-adrenal (HPA) axis, including the release of glucocorticoids, is the fundamental hormonal response to stress. Endocannabinoid (eCB) signaling serves to maintain HPA-axis homeostasis, by buffering basal activity as well as by mediating glucocorticoid fast feedback mechanisms. Following chronic stressor exposure, eCBs are also involved in physiological and behavioral habituation processes. Behavioral consequences of stress include fear and stress-induced anxiety as well as memory formation in the context of stress, involving contextual fear conditioning and inhibitory avoidance learning. Chronic stress can also lead to depression-like symptoms. Prominent in these behavioral stress responses is the interaction between eCBs and the HPA-axis. Future directions may differentiate among eCB signaling within various brain structures/neuronal subpopulations as well as between the distinct roles of the endogenous cannabinoid ligands. Investigation into the role of the eCB system in allostatic states and recovery processes may give insight into possible therapeutic manipulations of the system in treating chronic stress-related conditions in humans.
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Low levels of plasma arachidonoylethanolamide (anandamide) (AEA) (<2 nM) are associated with a successful early pregnancy in the mouse, and are thought to be regulated by sex steroid hormones. A similar association in the human may exist, although it has never been studied. The objective of this study was to investigate plasma AEA concentrations from the time of ovulation to implantation in pregnant and non-pregnant women, and whether AEA is hormonally regulated. Women who had undergone IVF/ICSI-embryo transfer were divided into pregnant (n = 12) and non-pregnant (n = 12) groups, based on serum beta-hCG >5 IU at 4 weeks and a viable intrauterine singleton pregnancy confirmed by ultrasound at 6 weeks gestation. Blood samples for plasma AEA and sex steroid hormonal measurements were taken at the time of oocyte collection, embryo transfer and pregnancy test, and an extra sample was also taken from the pregnant group at the viability ultrasound scan. In pregnant women, there was a significant initial decrease in plasma AEA levels from the day of oocyte retrieval to that of embryo transfer. In addition, in the viable pregnancy group, plasma AEA was high at 4 and 5 weeks gestation, and a decline was observed at 6 weeks gestation (P = 0.003). No correlations were seen between plasma AEA and serum estradiol (E2), progesterone (P4) or beta-hCG in pregnant women; however, there was a significant correlation between plasma AEA and E2 (P = 0.022), but not between plasma AEA and serum P4, in non-pregnant women. Our observations suggest that in successful pregnancy, a higher plasma AEA level at ovulation and a significantly lower level during implantation are required. The drop in AEA levels could be used as a biomarker for the appropriate timing of embryo transfer.
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To further investigate the relationship between plasma anandamide (AEA), sex steroids, and gonadotrophins to improve our understanding of how AEA may be involved in human fertility. Cross-sectional and longitudinal study. University Hospital of Leicester NHS Trust, Leicester Royal Infirmary. Healthy premenopausal and postmenopausal volunteers. UPLC-MS/MS-measured plasma AEA and ELISA-measured serum FSH, LH, estradiol, and progesterone levels at five different phases of the menstrual cycle and postmenopause. Plasma AEA, serum steroids and gonadotrophins. Changes in AEA levels were similar in the two cohorts. The mean +/- SEM levels in the early follicular phase (0.89 +/- 0.06) for the cross-sectional cohort and the longitudinal cohort (0.73 +/- 0.03) were higher than those in the late follicular phase (0.77 +/- 0.09 cross-sectional; 0.63 +/- 0.08 longitudinal). The highest AEA levels were measured at ovulation (1.38 +/- 0.14 and 1.33 +/- 0.16) and the lowest level was measured in the late luteal phase (0.66 +/- 0.07 and 0.56 +/- 0.06). There was a statistically significant positive correlation between AEA, estradiol (P=0.0015), LH (P<0.0001) and FSH levels but not progesterone (P=0.022). Peak plasma AEA occurred at ovulation and positively correlated with estradiol and gonadotrophin levels suggesting that these may be involved in the regulation of AEA levels.
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The present investigation examined the spatiotemporal expression of estrogen receptors (ER-alpha and ER-beta) and progesterone receptor (PR) in the periimplantation mouse uterus (days 1-8). ER-alpha messenger RNA (mRNA) was detected at much higher levels in the periimplantation uterus compared with that of ER-beta mRNA, the levels of which were very low in all uterine cells during this period. Results of in situ hybridization demonstrated expression of ER-alpha mRNA primarily in the luminal and glandular epithelia on days 1 and 2 of pregnancy. On days 3 and 4, the accumulation was localized primarily in stromal cells in addition to its presence in the epithelium. Following implantation on day 5, the accumulation of this mRNA was more condensed in the luminal and glandular epithelia, but declined in the subluminal epithelial stroma at the sites of implanting embryos. On days 6-8, the accumulation of ER-alpha mRNA was primarily localized in the secondary decidual zone (SDZ) with more intense localization in the subepithelial cells at the mesometrial pole. In contrast, signals were very low to undetectable in the primary decidual zone (PDZ), and no signals were detected in implanting embryos. The undifferentiated stroma underneath the myometrium also showed positive signals. The immunolocalization of ER-alpha protein correlated with the mRNA localization. Western blot analysis showed down-regulation of ER-alpha in day 8 decidual cell extracts consistent with the down-regulation of ER-alpha mRNA in decidual cells immediately surrounding the embryo on this day. The expression pattern of PR was also dynamic in the periimplantation uterus. On day 1, the accumulation of PR mRNA was very low to undetectable, whereas only a modest level of accumulation in the epithelium was noted on day 2. On days 3 and 4, the accumulation of this mRNA was detected in both the epithelium and stroma. In contrast, the expression was restricted only to the stroma with increased signals at the sites of implantation on day 5. On days 6-8, PR mRNA accumulation increased dramatically throughout the deciduum. The localization of immunoreactive PR correlated with the mRNA distribution in the periimplantation uterus. Taken together, the results demonstrate that the expression of ER-alpha, ER-beta, and PR is differentially regulated in the periimplantation mouse uterus. This compartmentalized expression of ER and PR provides information regarding the sites of coordinated effects ofestrogen and progesterone in the preparation of the uterus for implantation and decidualization during early pregnancy.
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Recent studies have demonstrated that the endogenous cannabinoids are important modulators of fertility in mammals. In particular, a role of the endocannabinoid system in early stages of embryo development, oviductal transport of embryos, pregnancy maintenance and labour has been demonstrated in rodents and/or in humans. In the present paper, we report the analysis of FAAH activity and protein content in the mouse uterus as a function of the natural oestrus cycle stages. Variations of FAAH activity are discussed in relationship to changes in sex steroid levels and to the possible action of AEA on remodelling of uterine tissues.
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Estrogen replacement therapy has been associated with reduction of cardiovascular events in postmenopausal women, though the mechanism for this benefit remains unclear. Here we show that at physiological concentrations estrogen activates the anandamide membrane transporter of human endothelial cells and leads to rapid elevation of calcium (apparent within 5 minutes) and release of nitric oxide (within 15 minutes). These effects are mediated by estrogen binding to a surface receptor, which shows an apparent dissociation constant (K(d)) of 9.4 +/- 1.4 nM, a maximum binding (B(max)) of 356 +/- 12 fmol x mg protein(-1), and an apparent molecular mass of approximately 60 kDa. We also show that estrogen binding to surface receptors leads to stimulation of the anandamide-synthesizing enzyme phospholipase D and to inhibition of the anandamide-hydrolyzing enzyme fatty acid amide hydrolase, the latter effect mediated by 15-lipoxygenase activity. Because the endothelial transporter is shown to move anandamide across the cell membranes bidirectionally, taken together these data suggest that the physiological activity of estrogen is to stimulate the release, rather than the uptake, of anandamide from endothelial cells. Moreover, we show that anandamide released from estrogen-stimulated endothelial cells, unlike estrogen itself, inhibits the secretion of serotonin from adenosine diphosphate (ADP)-stimulated platelets. Therefore, it is suggested that the peripheral actions of anandamide could be part of the molecular events responsible for the beneficial effects of estrogen.
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The present investigation examined the spatiotemporal expression of estrogen receptors (ER-α and ER-β) and progesterone receptor (PR) in the periimplantation mouse uterus (days 1–8). ER-α messenger RNA (mRNA) was detected at much higher levels in the periimplantation uterus compared with that of ER-β mRNA, the levels of which were very low in all uterine cells during this period. Results of in situ hybridization demonstrated expression of ER-α mRNA primarily in the luminal and glandular epithelia on days 1 and 2 of pregnancy. On days 3 and 4, the accumulation was localized primarily in stromal cells in addition to its presence in the epithelium. Following implantation on day 5, the accumulation of this mRNA was more condensed in the luminal and glandular epithelia, but declined in the subluminal epithelial stroma at the sites of implanting embryos. On days 6–8, the accumulation of ER-α mRNA was primarily localized in the secondary decidual zone (SDZ) with more intense localization in the subepithelial cel...
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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. © 2016 BioFactors, 2016.
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Cannabinoids have shown to have a variety effects on body systems. Through CB1 and CB2 receptors, amongst other, they exert an effect by modulating neurotransmitter and cytokine release. Current research in the role of cannabinoids in the immune system shows that they possess immunosuppressive properties. They can inhibit proliferation of leucocytes, induce apoptosis of T cells and macrophages and reduce secretion of pro-inflammatory cytokines. In mice models, they are effective in reducing inflammation in arthritis, multiple sclerosis, have a positive effect on neuropathic pain and in type 1 diabetes mellitus. They are effective as treatment for fibromyalgia and have shown to have anti-fibrotic effect in scleroderma. Studies in human models are scarce and not conclusive and more research is required in this field. Cannabinoids can be therefore promising immunosuppressive and anti-fibrotic agents in the therapy of autoimmune disorders.
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The lasting research on the endocannabinoid system (ECS) has now provided solid and convincing evidence that proves the detrimental effects of recreational drug abuse (a growing habit among teenagers) on fertility. Endocannabinoids (eCBs) affect reproductive events from gametogenesis to fertilization, from embryo implantation to the final outcome of pregnancy and, thus, they have been proposed as suitable biomarkers to predict the reproductive potential of male and female gametes in clinical practice. Novel tools for reproductive medicine are highly sought after, and here we report the latest findings on the impact of the ECS on fertility, demonstrating how basic research can be translated into new medical strategies.
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Recently, endocannabinoids have emerged as signaling 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 remodeling process. Copyright © 2015. Published by Elsevier B.V.
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The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-DeltaDeltaCr) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-DeltaDeltaCr) method. In addition, we present the derivation and applications of two variations of the 2(-DeltaDeltaCr) method that may be useful in the analysis of real-time, quantitative PCR data. (C) 2001 Elsevier science.
Article
Endocannabinoids are an emerging class of lipid mediators, which mimic several effects of cannabinoids. Anandamide (arachidonoylethanolamide) is a major endocannabinoid, which has been shown to impair pregnancy and embryo development. The activity of anandamide is controlled by cellular uptake through a specific transporter and intracellular degradation by the enzyme anandamide hydrolase (fatty acid amide hydrolase, FAAH). We characterized FAAH in mouse uterus by radiochromatographic and immunochemical techniques, showing that the enzyme is confined to the epithelium and its activity decreases appreciably during pregnancy or pseudopregnancy because of lower gene expression at the translational level. Ovariectomy prevented the decrease in FAAH, and both progesterone and estrogen further reduced its basal levels, suggesting hormonal control of the enzyme. Anandamide was shown to induce programmed cell death in mouse blastocysts, through a pathway independent of type-1 cannabinoid receptor. Blastocysts, however, have a specific anandamide transporter and FAAH, which scavenge this lipid. Taken together, these results provide evidence of an interplay between endocannabinoids and sex hormones in pregnancy. These findings may also be relevant for human fertility, as epithelial cells from healthy human uterus showed FAAH activity and expression, which in adenocarcinoma cells was increased fivefold.
Article
Endocannabinoids act as a major neuromodulatory system in a variety of physiological and behavioral functions. Three major lines of evidence suggest that the endocannabinoid system interacts with gonadal hormones. First, the endocannabinoid system is implicated in behaviors and physiological functions that are known to be regulated in part by gonadal hormones. Second, receptors and metabolic enzymes of the endocannabinoid system are localized extensively on structures in the hypothalamic-pituitary-gonadal axis. Third, changes in levels of gonadal hormones alter endocannabinoid signaling. Here we reviewed and summarized the current evidence regarding the interaction between the endocannabinoid system and androgens, estrogens, and progesterone. Overall, it appears that bidirectional interactions characterize the relationship between endocannabinoids and gonadal hormones, with endocannabinoids down-regulating hypothalamic-pituitary-gonadal activity and gonadal hormones modulating protein expression in the endocannabinoid system. An understanding of these interactions will have implications for elucidating the neuroendocrine mechanisms underlying a number of behavioral and physiological functions as well as potential pharmaceutical treatments for disorders of these functions.
Article
In rodent uterus, both up- and down-regulation of estrogen receptor alpha (ERalpha) messenger ribonucleic acid (mRNA) and protein levels by estradiol has been demonstrated; however, it is not known which of the uterine compartments (endometrial epithelium, stroma, myometrium) respond to estradiol with autoregulation of ERalpha. The purpose of the present study was to investigate and compare the kinetics and cell type-specific effects of estradiol on uterine ERalpha expression in immature and adult rats. Ovariectomized female rats were injected s.c. with sesame oil or estradiol-17beta. Uteri were collected and analyzed for changes in ERalpha mRNA using RNase protection assays (RPA) and in situ hybridization using radiolabeled probes specific for ERalpha. Immunohistochemical analysis was performed with a polyclonal antibody specific to ERalpha. Expression of ERalpha in the uterine epithelial cells decreased at 3 and 6 h after estradiol administration to immature and adult rats, respectively. At 24 h, ERalpha mRNA levels in the immature and mature rat uterus were higher than pretreatment levels but returned to baseline by 72 h. Pretreatment with cycloheximide did not block the 3-h repressive effect of estradiol, suggesting that the estradiol-induced decrease in ERalpha mRNA occurs independent of new protein synthesis. A decrease in ERalpha mRNA and protein was also observed in uterine epithelia at 3 and 6 h after an estradiol injection to immature and adult rats, and intensity of both the in situ hybridization signal and the immunostaining in the epithelium increased at 24 and 72 h. However, the periluminal stromal cells in the adult uterus and the majority of stromal cells of the immature uterus appeared to have increased ERalpha expression. The results indicate that down-regulation of ERalpha in the epithelia and up-regulation of stromal ERalpha play a role in early events associated with estradiol-induced cell proliferation of the uterine epithelia.
Article
The majority of the studies on the actions of estrogens in the ventrolateral part of the hypothalamic ventromedial nucleus (VMNvl) concern the factors that modulate the receptive component of the feminine sexual behavior and the expression of molecular markers of neuronal activation. To further our understanding of the factors that regulate synaptic plasticity in the female VMNvl, we have examined the effects of estradiol and progesterone, and of estrogen receptor (ER) subtype selective ligands on the number of dendritic and spine synapses established by individual VMNvl neurons and on sexual behavior. In contrast to earlier studies that analyzed synapse densities, our results show that exogenous estradiol increases the number of spine as well as of dendritic synapses, irrespective of the dose and regimen of administration. They also reveal that an effective dose of estradiol administered as one single pulse induces the formation of more synapses than the same dose administered as two pulses on consecutive days. Our results further show that both ER subtypes are involved in the mediation of the synaptogenic effects of estrogens on VMNvl neurons since the administration of the selective ERalpha, propyl-pyrazole-triol (PPT), and ERbeta, diarylpropionitrile (DPN), agonists induced a significant increase in the number of synapses that, however, was more exuberant for PPT. Despite its relevant role in feminine sexual behavior, progesterone had no synaptogenic effect in the VMNvl as no changes in synapse numbers were noticed in rats treated with progesterone alone, with estradiol followed by progesterone or with the antiprogestin mifepristone (RU486). Except for the sequential administration of estradiol and progesterone, none of the regimens was associated with lordosis response to vaginocervical stimulation. Therefore, from the sex steroids that undergo cyclic variations over the estrous cycle, only estrogens, acting through both ERalpha and ERbeta, play a key role in the activation of the neural circuits involving the ventromedial nucleus of the hypothalamus.
Article
Anandamide is an endocannabinoid known to participate in reproductive processes. This study observed that 17beta-oestradiol and progesterone modulated the production of anandamide and its metabolizing enzymes in the rat uterus. Anandamide production was highest at the oestrous stage and 17beta-oestradiol and progesterone stimulated its synthesis in ovariectomized rats. During early pregnancy, anandamide production remained constant on days 1-5 of gestation and diminished towards day 6. On day 6, implantation sites showed lower synthesis compared with interimplantation sites. In the delayed implantation model, 17beta-oestradiol inhibited anandamide synthesis compared with progesterone. During pseudopregnancy, anandamide production did not decrease towards day 6 as occurred during normal gestation. The administration of 17beta-oestradiol augmented anandamide production in rats on day 5 of pseudopregnancy; the treatment with mifepristone did not produce any change in anandamide synthesis. Anandamide-metabolizing enzymes were regulated by progesterone and 17beta-oestradiol. The effect of ovarian hormones on the synthesis of anandamide depends on different physiological conditions, oestrous cycle and early pregnancy, and on the presence of the activated blastocyst. Thus, ovarian hormones, as signals that emanate from the mother, operate in conjunction with the blastocyst intrinsic programme, regulating the synthesis of anandamide in a specific manner during crucial reproductive events that may compromise pregnancy outcome.
Article
Endogenous uterine prostaglandin levels were monitored in the cycling rat and prostaglandins of the F-type were found to rise at proestrus when estrogen levels have been shown to be maximal. Evidence that this is an estrogen-induced event is furnished by the finding that estradiol-17beta caused a similar rise in prostaglandin F levels in uteri of ovariectomized rats, an action blocked by coadministration of progesterone. Examination of in vitro prostaglandin synthetase by uterine microsomal fractions from cycling rats and estrogen-treated ovariectomized rats revealed that the action of estrogen to control prostaglandin synthesis in a directional manner is accomplished, at least in part, by regulation of the prostaglandin synthetase complex, resulting in a patterned alteration in the ratio of prostaglandin F to prostaglandin E. These results demonstrate that prostaglandins are involved in the expression of estrogen action in the rat uterus.
Article
These experiments utilized the estrogen antagonists CI-628, nafoxidine, and tamoxifen as tools to investigate potential molecular mechanisms of estrogen activation of female rat sexual behavior. Adult female rats, ovariectomized 4–7 days previously and matched for body weight, were administered single sc injections of one of the three antiestrogens, and the ability of the antagonists to block estrogen-induced sexual behavior, to deplete and replenish hypothalamic estrogen receptors, and to inhibit the binding of estradiol by hypothalamic nuclei 2 hr, or 1, 2, 4, or 7 days later was assessed. All three compounds produced a dose- and time-dependent inhibition of estrogen-activated lordosis, with tamoxifen being the most potent inhibitor. The three antiestrogens also caused prolonged depletion of hypothalamic estrogen receptors, but there was no correlation between receptor levels and the degree of inhibition of lordosis behavior at any time point following antiestrogen treatment. Rats showed high levels of sexual receptivity when antiestrogens were injected 2, 4, or 7 days before estrogen; however, hypothalamic estrogen receptors were still markedly (up to 70%) reduced at some of these time points. In contrast, there was a large (r = 0.67), significant correlation between the ability of all three agents to reduce [3H]estradiol binding by brain cell nuclei and their ability to reduce the display of estrogen-induced female sexual behavior. Antiestrogen injections which inhibited lordosis always decreased the level of specific estradiol binding by hypothalamic nuclei. These data indicate that delayed receptor replenishment does not adequately explain the antagonism of lordosis behavior by antiestrogens. The results presented here strongly point to the cell nucleus as the critical locus of receptor-mediated interactions which underlie estrogen and antiestrogen regulation of female sexual behavior.
Article
Expression of the gene for prostaglandin synthase (PGS) was examined in whole endometrial tissue derived from ewes during the oestrous cycle (Days 4-14), on Day 15 of pregnancy and following ovariectomy and treatment with ovarian steroid hormones. Whilst no significant differences were seen in PGS mRNA concentrations analysed by Northern blot analysis in endometrial tissue during the oestrous cycle or in early pregnancy, treatment of ovariectomized (OVX) ewes with oestradiol-17 beta markedly reduced endometrial PGS mRNA concentration. There was no difference in PGS mRNA concentration in ewes treated with progesterone, either alone or in conjunction with oestrogen, from that in OVX controls. In contrast, differences in immunolocalization of PGS observed in uterine tissue from OVX-steroid-treated ewes were much more marked and reflected similar changes seen previously in the immunocytochemical distribution of endometrial PGS during the oestrous cycle. In OVX ewes and those treated with oestrogen, immunocytochemical staining for PGS was seen in stromal cells, but little immunoreactive PGS was located in the endometrial epithelial cells. However, in ewes treated with progesterone alone or with oestrogen plus progesterone, PGS was found in luminal and glandular epithelial cells and in stromal cells. Intensity of immunostaining for PGS in endothelial cells and myometrium did not differ between the treatments. Thus, whilst oestrogen lowers PGS mRNA in the endometrium, presumably in stroma, it may also increase the stability of the enzyme itself in the stromal cells. Although oestradiol-17 beta has no effect on PGS in endometrial epithelium, progesterone stimulates the production of PGS in endometrial epithelial cells without altering the overall abundance of PGS mRNA in the endometrium as a whole. Conceptus-induced changes in PGF-2 alpha release by ovine endometrium would not appear to be mediated via effects on PGS gene expression or protein synthesis.
Article
Responses of male and female rats during the initiation of lordosis by the female are described using observations with films taken from a side or ventral view. Where possible, behavioral events were described objectively, quantified and plotted as a function of time. Responses of receptive and unreceptive females to mounts by the male rat are compared.
Article
Cannabinoid effects on brain dopaminergic activity vary as a function of gonadal status. In this work, we examined whether these variations might be due to sex steroid-dependent differences in brain cannabinoid receptors (CNr). Four experiments were done: (i) male versus females; (ii) females at each stage of the ovarian cycle; (iii) estradiol (E2) and/or progesterone (P)-replaced ovariectomized (OVX) females; and (iv) testosterone (T)-replaced orchidectomized males. The density of CNr in the medial basal hypothalamus fluctuated in females during the estrous cycle. The density was higher in diestrus and decreased in estrus. This parameter did not change after ovariectomy and E2 replacement. However, P increased the density of CNr when administered to OVX rats acutely treated with E2, but not administered alone or after chronic E2 treatment. In the striatum, the affinity of CNr was slightly higher in males than females, with no changes in density. Ovariectomy increased the affinity of CNr, which normalized only after administration of acute E2. Interestingly, the high affinity values observed in this area after P alone or combined with E2, corresponded to low densities as compared with intact females. In the limbic forebrain, the affinity for the cannabinoid ligand was also higher in males than females with no changes in density. Affinity was also higher in diestrus and lower in estrus, whereas density was unchanged. Ovariectomy decreased CNr density. A normal situation was found after administration of acute E2 or P alone, whereas chronic E2 markedly increased the density of CNr as compared with both intact and OVX females. Interestingly, this latter increase was prevented by coadministration of P. Orchidectomy did not affect CNr density, but administration of T produced a marked decrease. In the mesencephalon, the density and affinity of CNr was higher in males than females. Administration of P to OVX rats produced opposite effects, increasing the density when administered alone and decreasing it when administered to acute E2-replaced OVX rats. In summary, these results reveal the existence of subtle, sometimes more pronounced, sex dimorphisms, fluctuations along the ovarian cycle and changes after gonadectomy and sex steroid replacement in CNr density and affinity in certain brain areas. This supports the hypothesis of possible sex steroid-dependent differences in the sensitivity of certain neuronal processes to cannabinoid treatment.
Article
We used RT-PCR to measure relative differences in cannabinoid receptor (CB) mRNAs in the rat eye, comparing CB1 or CB2 transcripts to that of the normalizing reference gene beta2 microglobulin (beta2m). Significantly higher levels of CB1 mRNA levels were found in the ciliary body (0.84+/-0.05% of beta2m) than in the iris, (0.34+/-0.04% of beta2m), retina (0.07+/-0.005% of beta2m) and choroid (0.06+/-0.005% of beta2m). CB2 mRNA was undetectable. This expression pattern supports a specific role for the CB1 receptor in controlling intraocular pressure, helping to explain the antiglaucoma property of cannabinoids.
Article
Recent studies have demonstrated the occurrence of endocannabinoid synthesis and of gene expression and immunoreactivity for the cannabinoid CB(1) receptor in the anterior pituitary gland. Since the activity of this gland is under the influence of circulating sex steroids, the present study was designed to elucidate whether expression of the CB(1) receptor gene in the anterior pituitary gland is also under the influence of these steroids. To this aim, we first examined the possible changes in the levels of CB(1) receptor-mRNA transcripts in the anterior pituitary gland of intact male rats and normal cycling female rats at the different stages of the ovarian cycle. We observed that males had higher levels of CB(1) receptor-mRNA transcripts than females. In addition, these transcripts fluctuated in females during the different phases of the ovarian cycle, with the highest values observed on the second day of diestrus and the lowest on estrus. In these animals, we also measured the content of endocannabinoids in the anterior pituitary gland and the hypothalamus. We observed that females had higher contents of anandamide than males in both cases. The content of anandamide in females also fluctuated during the ovarian cycle in both the anterior pituitary gland and the hypothalamus. The highest values in the anterior pituitary gland were found in the estrus and the lowest on the first day of diestrus and proestrus, whereas the inverse tendency was found in the hypothalamus. No changes were observed in the other major endocannabinoid, 2-arachidonoyl-glycerol, between males and females and during the ovarian cycle. To further explore the potential influence of circulating sex steroids on CB(1) receptor gene expression in the anterior pituitary gland, as a second objective, we examined the possible changes in the amount of transcripts for this receptor in gonadectomized and sex steroid-replaced gonadectomized rats of both sexes. We observed that orchidectomy (ORCHX) in males reduced CB(1) receptor-mRNA levels, whereas replacement with dihydrotestosterone also maintained low levels of this messenger. In females, estradiol-replaced ovariectomized (OVX) rats exhibited significantly lower CB(1) receptor-mRNA levels than OVX animals that had not been replaced with this estrogen. In this experiment, we also examined if the previously reported response of the CB(1) receptor gene in the anterior pituitary lobe to chronic administration of Delta(9)-tetrahydrocannabinol (Delta(9)-THC) is under sex steroid influence. We observed that chronic Delta(9)-THC treatment decreased CB(1) receptor-mRNA levels in intact and ORCHX males, but not in dihydrotestosterone-replaced ORCHX males. In females, Delta(9)-THC treatment produced no effect in both OVX- and estradiol-replaced OVX rats. In summary, these data collectively support that expression of the CB(1) receptor gene in the anterior pituitary gland is regulated by sex steroids in both males and females. Furthermore, gonadal steroids appear to affect the response of this gene to chronic cannabinoid administration. We have also observed that anandamide contents in the anterior pituitary gland and the hypothalamus might be controlled by circulating sex steroids. The functional implications of these data are discussed.
Article
Endocannabinoids are an emerging class of lipid mediators, which mimic several effects of cannabinoids. Anandamide (arachidonoylethanolamide) is a major endocannabinoid, which has been shown to impair pregnancy and embryo development. The activity of anandamide is controlled by cellular uptake through a specific transporter and intracellular degradation by the enzyme anandamide hydrolase (fatty acid amide hydrolase, FAAH). We characterized FAAH in mouse uterus by radiochromatographic and immunochemical techniques, showing that the enzyme is confined to the epithelium and its activity decreases appreciably during pregnancy or pseudopregnancy because of lower gene expression at the translational level. Ovariectomy prevented the decrease in FAAH, and both progesterone and estrogen further reduced its basal levels, suggesting hormonal control of the enzyme. Anandamide was shown to induce programmed cell death in mouse blastocysts, through a pathway independent of type-1 cannabinoid receptor. Blastocysts, however, have a specific anandamide transporter and FAAH, which scavenge this lipid. Taken together, these results provide evidence of an interplay between endocannabinoids and sex hormones in pregnancy. These findings may also be relevant for human fertility, as epithelial cells from healthy human uterus showed FAAH activity and expression, which in adenocarcinoma cells was increased fivefold.
Article
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data.