No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
The Endocannabinoid System: An Ancient Signaling Involved in the Control of Male Fertility
Abstract
The effects of cannabinoids on human health have been known since the antiquities when the extract of the plant Cannabis sativa was used because of its psychoactivity. The scientific story of the cannabinoids started in the 1960s with the isolation and characterization of the active component of the plant. After the synthesis of cannabinoid analogues, the analysis of structure-effect relationships was implemented, and this had a similar effect to a positive "Pandora's box" opening. To date, numerous roles have been ascribed to the "endocannabinoid system." Here we describe its involvement in the control of male reproduction, taking into consideration possible evolutionary speculations. Indeed, the endocannabinoid system is a very ancient signaling system, being clearly present from the divergence of the protostomian/deuterostomian.
... Upon consumption, it acts via releasing of cannabinoid compounds that bind to cannabinoid receptors which form part of the endocannabinoid system (ECS). Numerous roles have been ascribed to the ECS, but it is known to also play a very important and specific role in the control of male reproduction [8]. An understanding of this system is therefore fundamental to be able to fully grasp the effect of exogenous cannabinoids (phytocannabinoids) on male reproductive function. ...
... Endocannabinoids are found to be widely dispersed in human tissues such as the central nervous system, peripheral nerves, leukocytes, spleen, uterus, and testicles [17]. It must therefore play a role in a number of physiological processes and appears to be deeply involved in the control of reproductive function [8,18]. Please refer to Fasano et al. [8] for a comprehensive overview of the ECS. ...
... It must therefore play a role in a number of physiological processes and appears to be deeply involved in the control of reproductive function [8,18]. Please refer to Fasano et al. [8] for a comprehensive overview of the ECS. ...
Marijuana has the highest consumption rate among all of the illicit drugs used in the USA, and its popularity as both a recreational and medicinal drug is increasing especially among men of reproductive age. Male factor infertility is on the increase, and the exposure to the cannabinoid compounds released by marijuana could be a contributing cause. The endocannabinoid system (ECS) is deeply involved in the complex regulation of male reproduction through the endogenous release of endocannabinoids and binding to cannabinoid receptors. Disturbing the delicate balance of the ECS due to marijuana use can negatively impact reproductive potential. Various in vivo and in vitro studies have reported on the empirical role that marijuana plays in disrupting the hypothalamus-pituitary-gonadal axis, spermatogenesis, and sperm function such as motility, capacitation, and the acrosome reaction. In this review, we highlight the latest evidence regarding the effect of marijuana use on male fertility and also provide a detailed insight into the ECS and its significance in the male reproductive system.
... The ECS is an ancient system, well-characterized in mammalian and non-mammalian vertebrates. Nevertheless there is evidence of ECS activity also occurs in invertebrates (Buznikov et al., 2010; Fasano et al., 2009; McPartland et al., 2006). Since the discovery of D 9 -tetrahydrocannabinol (D 9 -THC), many groups have reported on the effects of this compound in several invertebrate animal models. ...
... For instance, in the protozoan Tetrahymena pyriformis, D 9 -THC affects cell division; in the lobster it affects neurotransmitter release and in the sea urchin, an Echinodermata, it inhibits fertilizing ability of sperm cells (Fasano et al., 2009 and references inside; Schuel et al., 1991). Recently, evidence has emerged for eCBs production in molluscs, sea urchins and starfish (Buznikov et al., 2010; Fasano et al., 2009 and references inside). By contrast, the search for CB receptor orthologs in invertebrate genomes has been carried out without any success in both insects and nematodes ; similarly, data obtained in molluscs are still questionable. ...
... Although the search of CB 1 /CB 2 in sea urchins has been negative, candidate TRPV1 orthologs have recently been identified in S. purpuratus genome (Buznikov et al., 2010). Molecular cloning of CB receptors orthologs has produced positive results in the sea squirt, Ciona intestinalis, an urochordates, and in the amphioxus, Branchiostoma floridae, a cephalochordate; in vertebrates, a partial or complete molecular characterization of the ECS has been provided from fishes to birds and mammals, with duplication of CB 1 or CB 2 receptor genes found in fishes (Fasano et al., 2009 and references inside). To date, an elegant approach to assess the phylogenetic history of the ECS, has been carried out by McPartland and coworkers (2006). ...
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.
... Despite these conflicting data, an inhibitory action of THC at central level is plausible since it has been demonstrated that CB1 receptors are present both at pituitary (expecially in lactotrophs and gonadotrophs) and hypothalamic (in GnRH neurons) level. Endocannabinoids depress the pituitary secretion of thyroid-stimulating hormone (TSH), LH, growth hormone (GH), and prolactin, and the hypothalamic GnRH release in rats [81]. Specifically, the CB1 receptor agonist anandamide suppresses LH and testosterone secretion [82]. ...
... Alcohol ↓ Suppression of β-LH gene expression [14] Prolactin increase after acute ingestion [9] Inhibition of 3β-hydroxysteroid dehydrogenase and 17-ketosteroid reductase [9] Suppressed expression of StAR via ROS [10] Induction of the enzyme aromatase [11] ↓ Induction of apoptosis [10] Pro-oxidant effect [29] Cigarette smoking ↑ Enhanced GnRH or LH release [38,41] Inhibition of prolactin release [38] Competitive inhibition of testosterone glucuronidation [43] ↓ Induction of apoptosis [52,53] Pro-oxidant effect [56][57][58] Caffeine ↑ Induction of a stress-like hormonal pattern [64] ↔ Pro-oxidant effect at very high doses [69] Cannabis ↔ Inhibition of GnRH and LH in animal models [81,83] Reduced expression of LH receptor on testis in animal models [86] Reduced activity of testicular 3β-hydroxysteroid dehydrogenase in animal models [86] ↓ Induction of apoptosis [71] Cocaine ↔ Panhypopituitarism for pituitary infarction or inflammation (case reports) [96,97] ↓ Testicular vasoconstriction and ischemia [99] Induction of apoptosis [102] Pro-oxidant effect (reperfusion injury) [103] Amphetamines ↓ Decreased expression of GnRH mRNA [112] Activation of adenylate cyclase [106] Inhibition of 3b-hydroxysteroid dehydrogenase, P450c17, and 17-ketosteroid reductase [107] Reduced Ca 2 + influx [107] Increased testicular GABA concentration [111] ↓ Induction of apoptosis [114][115][116] Pro-oxidant effect [110] Testicular thermic damage [119] Increased testicular serotonin concentration [115] Increased testicular GABA concentration [111] Reduced testicular expression of progesterone and estrogen receptors [116] Opioids ↓ Inhibition of GnRH secretion [120,134] Hyperprolactinemia [120] ↓ Induction of apoptosis [128,140] Pro-oxidant effect [128][129][130][131][132][133][134][135][136][137][138][139][140] Anabolic-androgenic steroids (AAS) ↓ ...
Progressive deterioration of male reproductive function is occurring in Western countries. Environmental factors and unhealthy lifestyles have been implicated in the decline of testosterone levels and sperm production observed in the last fifty years. Among unhealthy lifestyles, substance and drug abuse is a recognized cause of possible alterations of steroidogenesis and spermatogenesis. Alcohol, opioids and anabolic-androgenic steroids are capable to reduce testosterone production in male interfering with testicular and/or hypothalamic-pituitary function. Other substances such as nicotine, cannabis, and amphetamines alter spermatogenesis inducing oxidative stress and subsequent apoptosis in testicular tissue. Substance and drug abuse is a potentially reversible cause of hypogonadism, defined as the failure of the testis to produce physiological concentrations of testosterone and/or a normal number of spermatozoa. The identification of the abuse is important because the withdrawal of substance intake can reverse the clinical syndrome. This review summarizes the most important clinical and experimental evidence on the effect of substance abuse on testosterone and sperm production.
... These are lipid mediators that mainly act via type 1 and type 2 cannabinoid receptors (CB1 and CB2 respectively), which are Gi/o protein-coupled receptors. The eCB system is one of the most ancient signaling system of vertebrates [9,10] with numerous pathways in the brain [11,12]. The main eCBs are anandamide (AEA) and 2arachidonoylglycerol (2-AG), which are the N-ethanolamide and the glyceryl ester of n-6 PUFA ARA respectively. ...
... Kisspeptin and GnRH) finds in the n-6 PUFA ARA derived eCBs key signaling intermediates. [10,13,16,17,18,141,143]. ...
Background:
Adequate dietary intake and nutritional status have important effects on brain functions and on brain health. Energy intake and specific nutrients excess or deficiency from diet differently affect cognitive processes, emotions, behaviour, neuroendocrine functions and synaptic plasticity with possible protective or detrimental effects on neuronal physiology. Lipids, in particular, play structural and functional roles in neurons. Here the importance of dietary fats and the need to understand the brain mechanisms activated by peripheral and central metabolic sensors. Thus, the manipulation of lifestyle factors such as dietary interventions may represent a successful therapeutic approach to maintain and preserve brain health along lifespan.
Methods:
This review aims at summarizing the impact of dietary fats on brain functions.
Results:
Starting from fat consumption, nutrient sensing and food-related reward, the impact of gut-brain communications will be discussed in brain health and disease. A specific focus will be on the impact of fats on the molecular pathways within the hypothalamus involved in the control of reproduction via the expression and the release of Gonadotropin-Releasing Hormone. Lastly, the effects of specific lipid classes such as polyunsaturated fatty acids and of the "fattest" of all diets, commonly known as "ketogenic diets", on brain functions will also be discussed.
Conclusion:
Despite the knowledge of the molecular mechanisms is still a work in progress, the clinical relevance of the manipulation of dietary fats is well acknowledged and such manipulations are in fact currently in use for the treatment of brain diseases.
... During the course of the years, the eCS has been characterized and studied from a functional point of view in many species [14][15][16][17]. In this regard, the use of nonmammalian animal models has contributed to a better comprehension about the eCS actions, especially in several reproductive events [16,[18][19][20]. In fact, nonmammalian vertebrates offer a broad spectrum of potentialities, besides, to allow evolutionary speculations. ...
... As indicated above, endocannabinoid activity requires multiple receptors, and this issue is stressed by the discovery of duplicated genes in fish [30,31], by the detection of several cannabinoid receptor splicing forms [32][33][34] as well as by the discussed existence of receptors other than CB1/CB2 [5]. In frog, the characterization of cb1 did not revealed any splicing form but nucleotide differences among brain/testis cDNA and genomic sequences together with the corresponding amino acidic variations [18,19,29] as a consequence of a possible editing process. Such a phenomenon seems to occur in other vertebrates and to affect RNA folding, stability and turnover. ...
Reproductive functions are regulated both at central (brain) and gonadal levels. In this respect, the endocannabinoid system (eCS) has a very influential role. Interestingly, the characterization of eCS has taken many advantages from the usage of animal models different from mammals. Therefore, this review is oriented to summarize the main pieces of evidence regarding eCS coming from the anuran amphibian Rana esculenta, with particular interest to the morphofunctional relationship between eCS and gonadotropin releasing hormone (GnRH). Furthermore, a novel role for endovanilloids in the regulation of a testicular GnRH system will be also discussed.
... During the course of the years, the eCS has been characterized and studied from a functional point of view in many species [14][15][16][17]. In this regard, the use of nonmammalian animal models has contributed to a better comprehension about the eCS actions, especially in several reproductive events [16,[18][19][20]. In fact, nonmammalian vertebrates offer a broad spectrum of potentialities, besides, to allow evolutionary speculations. ...
... As indicated above, endocannabinoid activity requires multiple receptors, and this issue is stressed by the discovery of duplicated genes in fish [30,31], by the detection of several cannabinoid receptor splicing forms [32][33][34] as well as by the discussed existence of receptors other than CB1/CB2 [5]. In frog, the characterization of cb1 did not revealed any splicing form but nucleotide differences among brain/testis cDNA and genomic sequences together with the corresponding amino acidic variations [18,19,29] as a consequence of a possible editing process. Such a phenomenon seems to occur in other vertebrates and to affect RNA folding, stability and turnover. ...
Reproductive functions are regulated both at central (brain) and gonadal levels. In this respect, the endocannabinoid system (eCS) has a very influential role. Interestingly, the characterization of eCS has taken many advantages from the usage of animal models different from mammals. Therefore, this review is oriented to summarize the main pieces of evidence regarding eCS coming from the anuran amphibian Rana esculenta, with particular interest to the morphofunctional relationship between eCS and gonadotropin releasing hormone (GnRH). Furthermore, a novel role for endovanilloids in the regulation of a testicular GnRH system will be also discussed.
... The ECS is an ancient system, well-characterized in mammalian and non-mammalian vertebrates. Nevertheless there is evidence of ECS activity also occurs in invertebrates (Buznikov et al., 2010;Fasano et al., 2009;McPartland et al., 2006). Since the discovery of D 9 -tetrahydrocannabinol (D 9 -THC), many groups have reported on the effects of this compound in several invertebrate animal models. ...
... For instance, in the protozoan Tetrahymena pyriformis, D 9 -THC affects cell division; in the lobster it affects neurotransmitter release and in the sea urchin, an Echinodermata, it inhibits fertilizing ability of sperm cells (Fasano et al., 2009 and references inside;Schuel et al., 1991). Recently, evidence has emerged for eCBs production in molluscs, sea urchins and starfish (Buznikov et al., 2010;Fasano et al., 2009 and references inside). By contrast, the search for CB receptor orthologs in invertebrate genomes has been carried out without any success in both insects and nematodes; similarly, data obtained in molluscs are still questionable. ...
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.
... FAAH gene product is one of the key components in the endocannabinoid system. This system plays an important role in regulating a variety of physiological and cognitive processes including fertility [30,31], pregnancy [32], appetite [17], pain-sensation [33], neural plasticity [34], stress response [35], learning and memory [36], and in mediating the pharmacological effects of cannabis [33]. Cannabis is the most commonly used illicit substance among individuals with ADHD [37,38]. ...
... In previous research, cannabinoids or their derivatives were found to bind to endocannabinoid receptors, which are part of the endocannabinoid system. The dynamic effect of cannabinoids on binding to endocannabinoid receptors have been reported to play a strategic role in male reproductive function and other biological processes related to nervous system development, synaptic plasticity, as well as a general response to environmental insults [9]. Furthermore, alcohol has been shown to block the hypothalamuspituitary-gonadal axis' feedback mechanisms, causing damage to the proteins required for spermatogenesis and a decline in blood levels of testosterone [10]. ...
Alcoholism has been linked to problems with male reproductive function. The combined effects of alcohol, cannabis, and tobacco were compared in this study. A total of 35 rats were assigned randomly into seven groups A–G: animals in A were administered distilled water. Animals in B–G were either administered alcohol orally (30 ml 40% alcohol) or exposed to smoke from ignited tobacco (exposure to smoke from 0.7 g tobacco for 5 min) or cannabis (exposure to smoke from 0.7 g tobacco and cannabis for 5 min): B (orally administered alcohol), C (exposed to the smoke from tobacco), D (exposed to smoke from cannabis), E (treated with alcohol and exposed to smoke from tobacco), F (treated with alcohol and exposed to smoke from cannabis), G (treated with alcohol and exposed to smokes from tobacco and cannabis). Assays were carried on the testicular homogenate after a 14-day treatment. There was a significant increase in activity of alkaline phosphatase (P ≤ 0.05), concentrations of cholesterol, glutathione reductase, and malondialdehyde in treated rats by the co-administration of alcohol with cannabis and tobacco compared with the control group. The combined treatment also caused degeneration and morphological distortions of testicular cells. The biochemical and histoarchitectural change was due to oxidative damage attributable to the synergistic effects. The high binding energy of tetrahydrocannabinol ligand to prostate acid phosphatase may be a prediction that the ligand can have an inhibitory effect on the function of enzymes in the prostate.
... The endocannabinoid system (ECS) is a lipid cell signaling system required to maintain homeostasis. It is expressed from lower organisms to humans (except for insects) and this suggests a pivotal role of ECS for essential functions in animals [1][2][3]. The ECS system takes its name from the Cannabis sativa plant since some phytocannabinoids, including the main psychoactive principle ∆ 9 -tetrahydrocannabinol (∆ 9 -THC), can mimic the effects of endocannabinoids by binding to their endogenous receptors [4]. ...
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.
... Indeed, due to the differential pattern of expression observed, circRNAs have been suggested as a possible sperm quality markers [105,111]. Interestingly, paternal circRNAs may be transferred from SPZ into the oocyte during fertilization as in the case of SPZ-derived circNAPEPLDiso1, a circRNA of NAPEPLD, the enzyme involved in the biosynthesis of the endocannabinoid anandamide (AEA) [112], which has key roles in reproductive functions [113][114][115][116][117][118]. In comparison to unfertilized murine oocytes, the expression of circNAPEPLDiso1 significantly increases in fertilized oocytes, suggesting a paternal contribution [106]. ...
In the last 40 years, male reproductive health—which is very sensitive to both environmental exposure and metabolic status—has deteriorated and the poor sperm quality observed has been suggested to affect offspring development and its health in adult life. In this scenario, evidence now suggests that epigenetics shapes endocrine functions, linking genetics and environment. During fertilization, spermatozoa share with the oocyte their epigenome, along with their haploid genome, in order to orchestrate embryo development. The epigenetic signature of spermatozoa is the result of a dynamic modulation of the epigenetic marks occurring, firstly, in the testis—during germ cell progression—then, along the epididymis, where spermatozoa still receive molecules, conveyed by epididymosomes. Paternal lifestyle, including nutrition and exposure to hazardous substances, alters the phenotype of the next generations, through the remodeling of a sperm epigenetic blueprint that dynamically reacts to a wide range of environmental and lifestyle stressors. With that in mind, this review will summarize and discuss insights into germline epigenetic plasticity caused by environmental stimuli and diet and how spermatozoa may be carriers of induced epimutations across generations through a mechanism known as paternal transgenerational epigenetic inheritance.
... The endocannabinoid (ECB) system is a known and well-characterized endogenous system in mammalian and non-mammalian vertebrates [8,9]. Endocannabinoids are synthesized and released in response to increased intracellular Ca 2+ concentration [10]. ...
Cannabinoids (CBs) are involved in the neuroendocrine control of reproductive processes by affecting GnRH and gonadotropins secretion. The presence of cannabinoid receptors (CBR) in the pituitary raises a presumption that anandamide (AEA), the endogenous cannabinoid, may act on gonadotrophic hormones secretion directly at the level of the anterior pituitary (AP). Thus, the aim of the study was to investigate the influence of AEA on gonadotropins secretions from AP explants taken from anestrous ewes. It was demonstrated that AEA inhibited GnRH stimulated luteinizing hormone (LH) and follicle stimulating hormone (FSH) secretion from the AP explants. Anandamide influences both LH and FSH gene expressions as well as their release. AEA also affected gonadoliberin receptor (GnRHR) synthesis and expression. The presence of CB1R transcript in AP explants were also demonstrated. It could be suggested that some known negative effects of cannabinoids on the hypothalamic-pituitary-gonadal axis activity may be caused by the direct action of these compounds at the pituitary level.
... Virtually all steps of reproduction are affected by one or more elements of the ES. In fact, this signalling system is deeply involved in the central and local control of reproduction in both sexes, with functions related to the modulation of the hypothalamus-pituitary-gonad (HPG) axis, germ cell development, successful gametogenesis, production of high-quality gametes, fertilization, embryo implantation and growth, pregnancy, and delivery [18,[102][103][104][105][106][107][108][109]. Centrally, ES regulates the hypothalamic release of gonadotropin releasing hormone (GnRH) which in turn mediates the discharge of pituitary gonadotropins (follicle stimulating hormone (FSH) and luteinizing hormone (LH)), the hormones responsible for sex steroid biosynthesis in the gonads [105][106][107][108]. Nevertheless, the full ES has been characterized in mammalian and non-mammalian vertebrates. ...
The endocannabinoid system (ES) is a cell-signalling system widely distributed in biological tissues that includes endogenous ligands, receptors, and biosynthetic and hydrolysing machineries. The impairment of the ES has been associated to several pathological conditions like behavioural, neurological, or metabolic disorders and infertility, suggesting that the modulation of this system may be critical for the maintenance of health status and disease treatment. Lifestyle and environmental factors can exert long-term effects on gene expression without any change in the nucleotide sequence of DNA, affecting health maintenance and influencing both disease load and resistance. This potentially reversible “epigenetic” modulation of gene expression occurs through the chemical modification of DNA and histone protein tails or the specific production of regulatory non coding RNA (ncRNA). Recent findings demonstrate the epigenetic modulation of the ES in biological tissues; in the same way, endocannabinoids, phytocannabinoids, and cannabinoid receptor agonists and antagonists induce widespread or gene-specific epigenetic changes with the possibility of trans-generational epigenetic inheritance in the offspring explained by the transmission of deregulated epigenetic marks in the gametes. Therefore, this review provides an update on the epigenetics of the ES, with particular attention on the emerging role in reproduction and fertility.
... Virtually all steps of reproduction are affected by one or more elements of the ES. In fact, this signalling system is deeply involved in the central and local control of reproduction in both sexes, with functions related to the modulation of the hypothalamus-pituitary-gonad (HPG) axis, germ cell development, successful gametogenesis, production of high-quality gametes, fertilization, embryo implantation and growth, pregnancy, and delivery [18,[102][103][104][105][106][107][108][109]. Centrally, ES regulates the hypothalamic release of gonadotropin releasing hormone (GnRH) which in turn mediates the discharge of pituitary gonadotropins (follicle stimulating hormone (FSH) and luteinizing hormone (LH)), the hormones responsible for sex steroid biosynthesis in the gonads [105][106][107][108]. Nevertheless, the full ES has been characterized in mammalian and non-mammalian vertebrates. ...
The endocannabinoid system (ES) is a cell-signalling system widely distributed in biological tissues that includes endogenous ligands, receptors, and biosynthetic and hydrolysing machineries. The impairment of the ES has been associated to several pathological conditions like behavioural, neurological, or metabolic disorders and infertility, suggesting that the modulation of this system may be critical for the maintenance of health status and disease treatment. Lifestyle and environmental factors can exert long-term effects on gene expression without any change in the nucleotide sequence of DNA, affecting health maintenance and influencing both disease load and resistance. This potentially reversible “epigenetic” modulation of gene expression occurs through the chemical modification of DNA and histone protein tails or the specific production of regulatory non-coding RNA (ncRNA). Recent findings demonstrate the epigenetic modulation of the ES in biological tissues; in the same way, endocannabinoids, phytocannabinoids, and cannabinoid receptor agonists and antagonists induce widespread or gene-specific epigenetic changes with the possibility of trans-generational epigenetic inheritance in the offspring explained by the transmission of deregulated epigenetic marks in the gametes. Therefore, this review provides an update on the epigenetics of the ES, with particular attention on the emerging role in reproduction and fertility.
... FAAH gene product is one of the key components in the endocannabinoid system. This system plays an important role in regulating a variety of physiological and cognitive processes including fertility [30,31], pregnancy [32], appetite [17], pain-sensation [33], neural plasticity [34], stress response [35], learning and memory [36], and in mediating the pharmacological effects of cannabis [33]. Cannabis is the most commonly used illicit substance among individuals with ADHD [37,38]. ...
Several single nucleotide polymorphisms (SNPs) of the fatty acid amide hydrolase (FAAH), the degrading enzyme of the endocannabinoids, have been shown to be associated with many neuropsychiatric disorders. Here, FAAH rs2295633 was studied in ADHD and case-control healthy children. There was a significant difference in the allele frequency (P = .04) and genotype distribution (P = .04) of the FAAH rs2295633 between ADHD cases and controls. The ADHD children appeared to have less of TT genotype (OR 0.396, 95% CI 0.178-0.884, p = .024) and T allele (OR 0.658, 95% CI 0.440-0.982, p = .04). To our best knowledge, this is the first statistical significant association between FAAH rs2295633 genotype and ADHD disorder. Larger sample sizes and functional studies are warranted to explore the clinical utility of FAAH genotyping as a possible marker for increased ADHD risk in children.
... NAPEPLD is one of the main actors of the endocannabinoid system, involved in the biosynthesis of the endocannabinoid anandamide (AEA) [26], with important roles in reproduction [27][28][29][30][31]. AEA levels are spatio-temporally regulated in the uterus during early pregnancy, so that they may mediate reciprocal interaction between blastocysts and uteruswith lower levels in the receptive uterus and at the implantation site [32,33]. ...
Circular RNAs (circRNAs) have a critical role in the control of gene expression. Their function in spermatozoa (SPZ) is unknown to date. Twenty-eight genes, involved in SPZ/testicular and epididymal physiology, were given in circBase database to find which of them may generate circular transcripts. We focused on circNAPEPLDiso1, one of the circular RNA isoforms of NAPEPLD transcript, because expressed in human and murine SPZ. In order to functionally characterize circNAPEPLDiso1 as potential microRNA (miRNA) sponge, we performed circNAPEPLDiso1-miR-CATCH and then profiled the expression of 754 miRNAs, by using TaqMan® Low Density Arrays. Among them, miRNAs 146a-5p, 203a-3p, 302c-3p, 766-3p and 1260a 1260a (some of them previously shown to be expressed in the oocyte), resulted enriched in circNAPEPLDiso1-miR-CATCHed cell lysate: the network of interactions generated from their validated targets was centered on a core of genes involved in the control of cell cycle. Moreover, computational analysis of circNAPEPLDiso1 sequence also showed its potential translation in a short form of NAPEPLD protein.
Interestingly, the expression analysis in murine unfertilized oocytes revealed low and high levels of circNAPEPLDiso1 and circNAPEPLDiso2, respectively. After fertilization, circNAPEPLDiso1 expression significantly increased, instead circNAPEPLDiso2 expression appeared constant.
Based on these data, we suggest that SPZ-derived circNAPEPLDiso1 physically interacts with miRNAs primarily involved in the control of cell cycle; we hypothesize that it may represent a paternal cytoplasmic contribution to the zygote and function as a miRNA decoy inside the fertilized oocytes to regulate the first stages of embryo development. This role is proposed here for the first time.
... The endocannabinoid system likely emerged early in evolution, as its elements are present in both invertebrates and vertebrates (Fasano et al. , 2009). All but one of the genes implicated in cannabinoid signaling in mammals have at least one homolog in zebrafish (Table 3, Supplementary Table 1S). ...
The endocannabinoid and opioid systems are two interplaying neurotransmitter systems that modulate drug abuse, anxiety, pain, cognition, neurogenesis and immune activity. Although they are involved in such critical functions, our understanding of endocannabinoid and opioid physiology remains limited, necessitating further studies, novel models and new model organisms. Zebrafish (Danio rerio) is rapidly emerging as one of the most effective translational models in neuroscience and biological psychiatry. Due to their high physiological and genetic homology to humans, zebrafish may be effectively used to study the endocannabinoid and opioid systems. Here, we discuss current models used to target the endocannabinoid and opioid systems in zebrafish, and their potential use in future translational research and high-throughput drug screening. Emphasizing the high degree of conservation of the endocannabinoid and opioid systems in zebrafish and mammals, we suggest zebrafish as an excellent model organism to study these systems and to search for the new drugs and therapies targeting their evolutionarily conserved mechanisms.
... Weekly cannabis use has been associated with a 28-29% reduction in sperm concentration and count. 63 The endocannabinoid system actively participates in the regulation of male fertility, 64 including by promoting meiosis via CB2 activation. 65 Therefore, the possibility of shared genetic pathways to male fertility and heavy cannabis use might provide a plausible alternative to more causal explanations. ...
Despite moderate heritability, only one study has identified genome-wide significant loci for cannabis-related phenotypes. We conducted meta-analyses of genome-wide association study data on 2080 cannabis-dependent cases and 6435 cannabis-exposed controls of European descent. A cluster of correlated single-nucleotide polymorphisms (SNPs) in a novel region on chromosome 10 was genome-wide significant (lowest P=1.3E−8). Among the SNPs, rs1409568 showed enrichment for H3K4me1 and H3K427ac marks, suggesting its role as an enhancer in addiction-relevant brain regions, such as the dorsolateral prefrontal cortex and the angular and cingulate gyri. This SNP is also predicted to modify binding scores for several transcription factors. We found modest evidence for replication for rs1409568 in an independent cohort of African American (896 cases and 1591 controls; P=0.03) but not European American (EA; 781 cases and 1905 controls) participants. The combined meta-analysis (3757 cases and 9931 controls) indicated trend-level significance for rs1409568 (P=2.85E−7). No genome-wide significant loci emerged for cannabis dependence criterion count (n=8050). There was also evidence that the minor allele of rs1409568 was associated with a 2.1% increase in right hippocampal volume in an independent sample of 430 EA college students (fwe-P=0.008). The identification and characterization of genome-wide significant loci for cannabis dependence is among the first steps toward understanding the biological contributions to the etiology of this psychiatric disorder, which appears to be rising in some developed nations.
... The proper coordination between mitosis, meiosis and differentiation is vital for successful spermatogenesis. Endocrine, paracrine, and autocrine control by sex hormones and locally produced mediators ensures the progression of spermatogenesis and the production of high quality spermatozoa for fertilization [116][117][118][119][120] with additional remodelling steps in membrane structure and epigenome occurring during the transit of spermatozoa in male reproductive tract [121][122][123][124]. A bimodal pattern of DNA methylation has been reported in male germ cells during the processes of specification and maturation, since the epigenetic marks are first erased in PGCs and then re-established from pro-spermato-gonia to entering meiosis, due to the coordinated activity of de-novo and maintenance DNMTs [125]. ...
Background:
Bisphenol A (BPA) is an endocrine disrupting chemical widely used in the manufacture of polycarbonate plastic and epoxy resin to produce a multitude of consumer products, food and drink containers, and medical devices. BPA is similar to estradiol in structure and thus interferes in steroid signalling with different outcomes on reproductive health depending on doses, life stage, mode, and timing of exposure. In this respect, it has an emerging and controversial role as a "reproductive toxicant" capable of inducing short and long-term effects including the modulation of gene expression through epigenetic modification (i.e. methylation of CpG islands, histone modifications and production of non-coding RNA) with direct and trans-generational effects on exposed organisms and their offspring, respectively.
Objective:
This review provides an overview about BPA effects on reproductive health and aims to summarize the epigenetic effects of BPA in male and female reproduction.
Results:
BPA exerts epigenetic effects in both male and female reproduction. In males, BPA affects spermatogenesis and sperm quality and possible trans-generational effects on the reproductive ability of the offspring. In females, BPA affects ovary, embryo development, and gamete quality for successful in vivo and in vitro fertilization (IVF).
Conclusion:
The exact mechanisms of BPA - mediated effects in reproduction are not fully understood; however, the environmental exposure to BPA - especially in fetal and neonatal period - deserves attention to preserve the reproductive ability in both sexes and to reduce the epigenetic risk for the offspring.
... The endocannabinoid system (ECS) is an ancient, evolutionarily conserved system, wellcharacterized in mammalian and non-mammalian vertebrates (Buznikov et al., 2010; Fasano et al., 2009; McPartland et al., 2006). Such a system comprises ECBs, several ECB receptors (CBs), many enzymatic machineries responsible for ECB degradation and biosynthesis and ECB transporters (EMT) (Pierantoni et al., 2009). ...
... There is also some evidence of a direct effect of cannabinoids on the testis [122][123][124][125][126]. Recent interest in the localization of the endocannabinoid system within male reproductive tissues and measurement of endocannabinoids in fluids of several mammalian species has been the subject of intense interest, as well as provoking controversy. ...
During pregnancy, a woman undergoes a multitude of normal physiochemical changes that are specific to pregnancy and with increasing frequency, challenged with pregnancy-related conditions. Additionally, there are a number of problems affecting the unborn fetus that, in turn, affect normal maternal biochemistry, endocrinology and physiology. Furthermore, normal biochemical, hormonal and clinically relevant measurements for the nonpregnant state often no longer apply to the pregnant woman. This makes clinical laboratory measurement difficult and normal clinical diagnosis using nonpregnant values potentially dangerous, with inappropriate treatment (when it is not required) and no treatment (when it is required) being offered. Problems within early pregnancy, for example, spontaneous and recurrent miscarriage, thus become very complicated to predict or treat. Therefore, reexamining what clinical chemical tests are available and what is just emerging from the experimental laboratory (and thus becoming available in the standard clinical laboratory), should inform the unwary clinician.
... Several findings underscore the critical role played by the endocannabinoids (eCBs) in the control of reproductive functions (Battista et al., 2012;Chianese et al., 2011a;Fasano et al., 2009;Meccariello et al., 2014;Pierantoni et al., 2009a). ...
... In this respect, enzymes involved in endocannabinoid biosynthesis and/or degradation occur throughout the animal kingdom including deuterostomian (i.e., sea urchin), protostomian (i.e., crustaceans and nematodes), and basal (i.e., cnidarians and placozoans) invertebrates [49]. Conversely, molecular cloning of CB 1 /CB 2 receptor orthologs has produced positive results only in urochordates (the sea squirt, Ciona intestinalis), in cephalochordata (the amphioxus, Branchiostoma floridae), in nonmammalian vertebrates (fish, amphibians, reptiles, and birds), and in mammals, with duplication of CB 1 or CB 2 genes found in fish [49][50][51]. Thus, given that CB 1 /CB 2 are unique to chordates, the molecular nature of endocannabinoid signaling in noncordate invertebrate is currently under investigation and may be related to primitive neuronal functions; conversely, the appearance of multiple receptors and receptor splicing forms coming from invertebrates to humans may indicate the subsequent occurrence of functional partitioning. ...
The endocannabinoid system (ECS) is an evolutionarily conserved master system deeply involved in the central and local control of reproductive functions in both sexes. The tone of these lipid mediators-deeply modulated by the activity of biosynthetic and hydrolyzing machineries-regulates reproductive functions from gonadotropin discharge and steroid biosynthesis to the formation of high quality gametes and successful pregnancy. This review provides an overview on ECS and reproduction and focuses on the insights in the regulation of endocannabinoid production by steroids, in the regulation of male reproductive activity, and in placentation and parturition. Taken all together, evidences emerge that the activity of the ECS is crucial for procreation and may represent a target for the therapeutic exploitation of infertility.
... However, much remains to be clarified about a direct and/or indirect role. In vivo studies, using nonmammalian and mammalian animal models, show that CNR1 acts at both central and local level [155,156]. In frog, an intriguing CNR1-GnRH (gonadotropin-releasing hormone) interplay occurs at a central level, with CNR1 regulating GnRH synthesis [16]; at testicular level, CNR1 also regulates GnRH1/2 and GnRH-R (GnRH-receptor) expression [157]. ...
Spermatogenesis is a complex mechanism which allows the production of male gametes; it consists of mitotic, meiotic, and differentiation phases. Spermiogenesis is the terminal differentiation process during which haploid round spermatids undergo several biochemical and morphological changes, including extensive remodelling of chromatin and nuclear shape. Spermiogenesis is under control of endocrine, paracrine, and autocrine factors, like gonadotropins and testosterone. More recently, emerging pieces of evidence are suggesting that, among these factors, estrogens may have a role. To date, this is a matter of debate and concern because of the agonistic and antagonistic estrogenic effects that environmental chemicals may have on animal and human with damaging outcome on fertility. In this review, we summarize data which fuel this debate, with a particular attention to our recent results, obtained using type 1 cannabinoid receptor knockout male mice as animal model.
... Several findings underscore the critical role played by the endocannabinoids (eCBs) in the control of reproductive functions (Battista et al., 2012;Chianese et al., 2011a;Fasano et al., 2009;Meccariello et al., 2014;Pierantoni et al., 2009a). ...
Endocannabinoids – primarily anandamide (AEA) and 2-arachidonoylglycerol (2-AG) – are lipophilic molecules that bind to cannabinoid receptors (CB1 and CB2). They affect neuroendocrine activity inhibiting gonadotropin releasing hormone (GnRH) secretion and testosterone production in rodents, through a molecular mechanism supposed to be hypothalamus dependent. In order to investigate such a role, we choose the seasonal breeder, the anuran amphibian Rana esculenta, an experimental model in which components of the endocannabinoid system have been characterized. In February, at the onset of a new spermatogenetic wave, we carried out in vitro incubations of frog tes-tis with AEA, at 10 À9 M dose. Such a treatment had no effect on the expression of cytochrome P450 17alpha hydroxylase/17,20 lyase (cyp17) nor 3-b-hydroxysteroid dehydrogenase/D-5–4 isomerase (3b-HSD), key enzymes of steroidogenesis. To understand whether or not the functionality of the hypothalamus–pituitary axis could be essential to support the role of endocannabinoids in steroidogenesis, frogs were injected with AEA, at 10 À8 M dose. Differently from in vitro experiment, the in vivo administration of AEA reduced the expression of cyp17 and 3b-HSD. Whereas the effect on 3b-HSD was counteracted by SR141716A (Rimonabant) – a selective antagonist of CB1, thus indicating a CB1 dependent modulation – the effect on cyp17 was not, suggesting a possible involvement of receptors other than CB1, probably the type-1 vanilloid receptor (TRPV1), since AEA works as an endocannabinoid and an endovanilloid as well. In conclusion our results indicate that endocannabinoids, via CB1, inhibit the expression of 3b-HSD in frog testis travelling along the hypothalamus–pituitary axis.
... Endocannabinoids are lipidic mediators identified in several tissues (brain, testis, epididymis) and biological fluids (follicular fluid, maternal milk, blood) (Cobellis et al., 2006Cobellis et al., , 2010 Devane et al., 1992; Habayeb et al., 2004; Schuel et al., 2002; Sugiura et al., 1996). They regulate reproduction, in both males (Battista et al., 2012; Cacciola et al., 2008a Cacciola et al., , 2010 Chianese et al., 2011 Chianese et al., , 2012 Fasano et al., 2009; Francavilla et al., 2009; Lewis and Maccarrone, 2009; Lewis et al., 2012a,b; Maccarrone et al., 2003 Maccarrone et al., , 2005a Meccariello et al., 2006 Meccariello et al., , 2008 Pierantoni et al., 2009a; Sun et al., 2009) and females (Acone et al., 2009; Cacciola et al., 2010; Lazzarin et al., 2004; Maccarrone et al., 2005b; Sun and Dey, 2012; Trabucco et al., 2009; Wang et al., 2007 ), and specific cannabinoid receptors (Cnr1 and Cnr2) have been localized in male and female reproductive tracts (Grimaldi et al., 2009; Karasu et al., 2011; Pertwee et al., 1996; Pierantoni et al., 2009b). In the testis, Cnr1 is present in somatic and germ cells including SPT and SPZ (Barbonetti et al., 2010; Barboni et al., 2011; Bernabò et al., 2012; Cacciola et al., 2008a; Catanzaro et al., 2011; Cobellis et al., 2006; Gye et al., 2005; Maccarrone et al., 2003; Rossato et al., 2005). ...
... Endocannabinoids regulate reproduction in male and female [6,[11][12][13][14] and they have been identified as actors in the intricate process of spermiogenesis [15]. In male, they act, directly and/or indirectly, at both central (hypothalamus and hypophysis) [16][17][18] and local levels (testis, epididymis and deferent ducts) via their cognate receptors [19][20][21][22][23][24][25][26]. At a central level, CNR1 regulates GnRH synthesis and release in frogs and rats, respectively [17,27]. ...
The type 1-cannabinoid receptor, CNR1, regulates differentiation of spermatids. Indeed, we have recently reported that the genetic inactivation of Cnr1 in mice influenced chromatin remodeling of spermatids, by reducing histone displacement and then sperm chromatin quality indices (chromatin condensation and DNA integrity). Herein, we have studied, at both central and testicular levels, the molecular signals potentially involved in histone displacement. In particular, investigation of the neuroendocrine axis involved in estrogen production demonstrated down-regulation of the axis supporting FSH/estrogen secretion in Cnr1-knockout male mice. Conversely, Cnr1-knockout male mice treated with 17beta-estradiol showed a weak increase of pituitary Fsh beta subunit mRNA levels and rescue of sperm chromatin quality indices demonstrating that estrogens, possibly in combination with FSH secretion, play an important role in regulating chromatin remodeling of spermatids.
... The endocannabinoid system (ECS) is an ancient, evolutionarily conserved system, wellcharacterized in mammalian and non-mammalian vertebrates ( Buznikov et al., 2010;Fasano et al., 2009;McPartland et al., 2006). Such a system comprises ECBs, several ECB receptors (CBs), many enzymatic machineries responsible for ECB degradation and biosynthesis and ECB transporters (EMT) ( Pierantoni et al., 2009). ...
... Additionally, they express a complete and functional biochemical machinery required to synthesize (AEA-synthesizing phospholipase D; NAPE-PLD), degrade (fatty acid amide hydrolase; FAAH), and transport (purported AEA membrane transporter; AMT) AEA, along with the binding receptors CB1R and transient receptor potential vanilloid-1 (TRPV1)[22][23][24]. When high extracellular levels of this bioactive lipid or its non-hydrolysable analogue methanandamide (Met-AEA) are maintained during in vitro capacitation of spermatozoa from different species, the acquisition of the fertilizing ability is strongly inhibited[25][26][27]. Combining these experimental results with the evidence of decreasing AEA levels within the female genital tract[28,29], and the infertility consequence of chronic use of esocannabinoid derivates[30][31][32], the hypothesis of a physiological and pathological role of AEA and analogues in sperm male activation has been proposed; yet, the underlying intracellular pathways remain to be clarified. ...
Mammalian spermatozoa acquire their full fertilizing ability (so called capacitation) within the female genital tract, where they are progressively exposed to inverse gradients of inhibiting and stimulating molecules.
In the present research, the effect on this process of anandamide, an endocannabinoid that can either activate or inhibit cannabinoid receptors depending on its concentration, and bicarbonate, an oviductal activatory molecule, was assessed, in order to study the role exerted by the type 1 cannabinoid receptor (CB1R) in the process of lipid membrane remodeling crucial to complete capacitation. To this aim, boar sperm were incubated in vitro under capacitating conditions (stimulated by bicarbonate) in the presence or in the absence of methanandamide (Met-AEA), a non-hydrolysable analogue of anandamide. The CB1R involvement was studied by using the specific inhibitor (SR141716) or mimicking its activation by adding a permeable cAMP analogue (8Br-cAMP). By an immunocytochemistry approach it was shown that the Met-AEA inhibits the bicarbonate-dependent translocation of CB1R from the post-equatorial to equatorial region of sperm head. In addition it was found that Met-AEA is able to prevent the bicarbonate-induced increase in membrane disorder and the cholesterol extraction, both preliminary to capacitation, acting through a CB1R-cAMP mediated pathway, as indicated by MC540 and filipin staining, EPR spectroscopy and biochemical analysis on whole membranes (CB1R activity) and on membrane enriched fraction (C/P content and anisotropy).
Altogether, these data demonstrate that the endocannabinoid system strongly inhibits the process of sperm capacitation, acting as membrane stabilizing agent, thus increasing the basic knowledge on capacitation-related signaling and potentially opening new perspectives in diagnostics and therapeutics of male infertility.
... Endocannabinoid signalling is an ancient, evolutionarily conserved system involved in the control of several biological functions such as reproduction, food intake, energy balance, stress, pain, cognitive functions and motor control [10,11,18,26]. The system consists of cannabinoid receptors (mainly the G coupled type 1 and type 2 cannabinoid receptor, CB1 and CB2), endocannabinoids, their biosynthetic and degrading enzymes as well as a putative endocannabinoid membrane transporter [for recent review see 1,20]. ...
In the hypothalamus, endocannabinoids affect neuroendocrine activity by means of Gonadotropin-Releasing-Hormone-I (GnRH-I) inhibition. Since most vertebrates, human included, possess at least two GnRH molecular forms, the aim of this work was to investigate the effect of endocannabinoids on GnRH molecular forms other than GnRH-I and on GnRHRs. Thus, we cloned GnRH precursors as well as GnRH receptors (GnRHR-I, GnRHR-II, GnRHR-III) from the diencephalons of the anuran amphibian, Rana esculenta. GnRH-II expression was evaluated in pituitary, whole brain, spinal cord, hindbrain, midbrain and forebrain during the annual sexual cycle. Then, in post-reproductive period (May), GnRH-I, GnRH-II and GnRHRs expression was evaluated by quantitative real time (qPCR) after incubation of diencephalons with the endocannabinoid anandamide (AEA). AEA significantly decreased GnRH-I and GnRH-II expression, up regulated GnRHR-I and GnRHR-II mRNA and it had no effect upon GnRHR-III expression. These effects were counteracted by SR141716A (Rimonabant), a selective antagonist of type I cannabinoid receptor (CB1). In conclusion our results demonstrate a CB1 receptor dependent modulation of GnRH system expression rate (both ligands and receptors) in frog diencephalons. In particular, we show that AEA, besides GnRH-I, also acts on GnRH-II expression.
... Successively, it has been demonstrated that gonads synthesize and degrade eCBs, because they express NAPE-PLD, DAGL, FAAH and MAGL [191,78]. In detail, FAAH has been immunolocalized in spermatocytes (SPC), elongated spermatids (eSPT) and SPZ of R. esculenta testis [32], as well as in rodent testis where FAAH is also expressed in SCs and Leydig cells (LCs) [110,194,15,66]. Among different species, the presence of CB 1 and FAAH in the same germ cells confirms the hypothesis that FAAH regulates physiological eCB levels to drive SPT morphogenesis [32]. ...
Many advances have been carried out on the estrogens, GnRH and endocannabinoid system that have impact in the reproductive field. Indeed, estrogens, the generally accepted female hormones, have performed an unsuspected role in male sexual functions thanks to studies on non-mammalian vertebrates. Similarly, these animal models have provided important contributions to the identification of several GnRH ligand and receptor variants and their possible involvement in sexual behavior and gonadal function regulation. Moreover, the use of non-mammalian animal models has contributed to a better comprehension about the endocannabinoid system action in several mammalian reproductive events. We wish to highlight here how non-mammalian vertebrate animal model research contributes to advancements with implications on human health as well as providing a phylogenetic perspective on the evolution of reproductive systems in vertebrates.
... Accordingly, in R. esculenta testis, the expression profile of CB 1 (mRNA and protein) during the annual sexual cycle shows higher levels in September-October period, when seminiferous epithelium presents a massive number of eSPT and newly formed SPZ [144,224,236]. In mouse isolated germ cells, CB 1 mRNA is expressed in SPG, but gradually increases in purified SPC and SPT, whereas low CB1 mRNA levels have been identified in purified SCs [69]. ...
Starting from an historical overview of lasting Cannabis use over the centuries, we will focus on a description of the cannabinergic system, with a comprehensive analysis of chemical and pharmacological properties of endogenous and synthetic cannabimimetic analogues. The metabolic pathways and the signal transduction mechanisms, activated by cannabinoid receptors stimulation, will also be discussed. In particular, we will point out the action of cannabinoids and endocannabinoids on the different neuronal networks involved in reproductive axis, and locally, on male and female reproductive tracts, by emphasizing the pivotal role played by this system in the control of fertility.
... ECS is involved in fertility not only in humans and mammalians but also in non mammalian vertebrates and invertebrates, being the system highly conserved from evolutionary view point [53]. Schuel et al. [54] were the first to show in the sea urchin Strongylocentrotus purpuratus that cannabinoids directly affect the process of fertilization by reducing the fertilizing capacity of sperm. ...
N-acylethanolamides (NAEs) are naturally occurring signaling lipids consisting of amides and esters of long-chain polyunsaturated fatty acids. Usually they are present in a very small amounts in many mammalian tissues and cells, including human reproductive tracts and fluids. Recently, the presence of N-arachidonoylethanolamide (anandamide, AEA), the most characterised member of endocannabinoids, and its congeners palmitoylethanolamide (PEA) and oleylethanolamide (OEA) in seminal plasma, oviductal fluid, and follicular fluids was demonstrated. AEA has been shown to bind not only type-1 (CB1) and type-2 (CB2) cannabinoid receptors, but also type-1 vanilloid receptor (TRPV1), while PEA and OEA are inactive with respect to classical cannabinoid CB1 and CB2 but activate TRPV1 or peroxisome proliferator activate receptors (PPARs). This review concerns the most recent experimental data on PEA and OEA, endocannabinoid-like molecules which appear to exert their action exclusively on sperm cells with altered features, such as membrane characteristics and kinematic parameters. Their beneficial effects on these cells could suggest a possible pharmacological use of PEA and OEA on patients affected by some forms of idiopathic infertility.
... There is also some evidence of a direct effect of cannabinoids on the testis [122][123][124][125][126]. Recent interest in the localization of the endocannabinoid system within male reproductive tissues and measurement of endocannabinoids in fluids of several mammalian species has been the subject of intense interest, as well as provoking controversy. ...
Acylethanolamides such as anandamide (AEA), and monoacylglycerols like 2-arachidonoylglycerol are endocannabinoids that bind to cannabinoid, vanilloid and peroxisome proliferator-activated receptors. These compounds, their various receptors, the purported membrane transporter(s), and related enzymes that synthesize and degrade them are collectively referred to as the "endocannabinoid system (ECS)". Poorly defined cellular and molecular mechanisms control the biological actions of the ECS. Over the last decade evidence has been emerging to suggest that the ECS plays a significant role in various aspects of human reproduction. In this review, we summarize our current understanding of this role especially the involvement of AEA and related ECS elements in regulating oogenesis, embryo oviductal transport, blastocyst implantation, placental development and pregnancy outcomes, and sperm survival, motility, capacitation and acrosome reaction. Additionally, the possibility that plasma and tissue AEA and other cannabinoids may represent reliable diagnostic markers of natural and assisted reproduction and pregnancy outcomes in women will be discussed.
... Distinct ECS elements have been identified in seminal plasma [39], male reproductive tissues [40], Leydig and Sertoli cells [19,[41][42], as well as in male germ cells [41][42][43][44][45][46][47], from spermatogonia to mature spermatozoa [44,[48][49][50]. Overall, the present evidence supports an ''evolutionary'' role of ECS (and in particular of CB 1 and FAAH) as check points in reproduction [3,[9][10][51][52][53]. ...
In this review, we shall summarize the current knowledge on the endocannabinoid system (ECS), and on its involvement in the multifaceted process of male reproduction. In particular, we shall discuss the role of ECS in sperm biology and Sertoli cell proliferation and death, showing how endocannabinoids may regulate spermatogenesis and reproductive potential.
The available evidence highlights the existence of a distinctive network, including endocannabinoids and sex hormones, that warrants a successful pregnancy in mammals. In particular, it appears that the endocannabinoid-degrading enzyme FAAH (fatty acid amide hydrolase) has a central role in this array of signals, because it controls several steps of sperm biology, from motility to capacitation and acrosome reaction. Since the regulation of FAAH activity and expression by autocrine and paracrine factors may occur through genomic or non-genomic mechanisms mediated by type-1 cannabinoid receptor (CB1R) signaling, we also raise concerns about the use of CB1R agonists (like marijuana) or antagonists (like the anti-obesity drug Acomplia in subjects of reproductive age.
Based on the present data, we point out that FAAH might be a novel and potentially important target for the development of next generation therapeutics against infertility. In particular, a reduced reproductive potential seems to be paralleled by defective FAAH, suggesting that therapeutics able to enhance, rather than inhibit, enzyme activity might be useful fertility enhancers.
This review discusses the effects and mechanisms of ketogenic diet on neurodegenerative diseases on the basis of available evidence. A ketogenic diet refers to a high-fat, medium-protein, and low-carbohydrate diet that leads to a metabolic shift to ketosis. This review systematically summarizes the scientific literature supporting this effective treatment approach for neurodegenerative diseases, including effects on mitochondrial function, oxidative stress, neuronal apoptosis, neuroinflammation, and the microbiota–gut–brain axis. It also highlights the clinical evidence for the effects of ketogenic diet in the treatment of Alzheimer's disease, Parkinson's disease, and motor neuron disease. Finally, it discusses the common adverse effects of ketogenic therapy. Although the complete mechanism of ketogenic diet in the treatment of neurodegenerative diseases remains to be elucidated, its clinical efficacy has attracted many new followers. The ketogenic diet is a good candidate for adjuvant therapy, but its specific applicability depends on the type and the degree of the disease.
The endocannabinoid system was discovered and named because of the extended historical use of Cannabis throughout history for fiber, medicinal, and psychoactive reasons by many different cultures. Millions of people worldwide today use Cannabis. Synthetic cannabinoid agonists that were designed and created eventually helped scientists discover the cannabinoid receptors of the endocannabinoid system. From there on, the endocannabinoid system became seriously studied and researched in multiple scientific fields. The major biosynthetic pathways of the endocannabinoid system produce anandamide and 2-arachidonoylglycerol (2-AG).
Fatty-Acid amide hydrolase (FAAH) is the main degrading/metabolizing enzyme of anandamide, and monoglyceride lipase (MGL) is the main degrading enzyme for 2-AG. Cannabinoid-1 (CB1) receptors are primarily concentrated in the central nervous system (CNS), while Cannabinoid-2 (CB2) receptors are mainly found in the peripheral nervous system (PNS). The endocannabinoid system acts in a retrograde manner. Once an action potential is generated from the nervous system, endocannabinoids are synthesized post-synaptically and travel retrogradely to act on the presynaptic cannabinoid receptors. Once the endogenous ligand bind, the receptors cause depolarization-induced suppression of inhibition (DSI) in the synaptic knob, blocking calcium (Ca2+) from entering and forcing potassium (K+) to exit the cell, and this restores the neurons to their resting membrane potential (RMP). The endocannabinoid system plays a role in many significant body functions, like memory, gastrointestinal motility, and pain management. Thus, this is an important system to study due to the pharmacological benefits that can come from it.
The expression of the main cannabinoid receptors (CBR1 and CBR2) was investigated to evaluate the possible association with the sperm maturation from fertile and infertile individuals. One hundred subjects were classified into fertile (n = 50) and infertile groups (n = 50). Fresh semen samples were collected. Computer‐assisted semen analysis and acrosin activity test were done. RNA was extracted from mature and immature sperm pellets. Reverse transcriptase reaction and real‐time PCR were done to assess the levels of both CBR1 and CBR2 genes expression in all samples. Mature spermatozoa from both groups showed significantly higher levels of both CBR1 and CBR2 compared with the immature spermatozoa (p < .05). This increment was significantly more important in the fertile group (p < .05). In mature spermatozoa, CBR1 expression was significantly related to variation in sperm morphology, and CBR2 was significantly related to both sperm morphology and linearity index. In conclusion, CBR1 and CBR2 mRNA expression may closely direct the sperm maturation at different steps of the reproductive process.
It is estimated that 12%–15% of sexually active couples are infertile. When broken down by gender, a male component can be identified approximately 50% of the time either in isolation or in combination with a female factor. Previous research in a US male fertility clinic analyzing 1430 patients identified causes of infertility from most to least common: varicocele, idiopathic, obstruction, female factor, cryptorchidism, immunologic, ejaculatory dysfunction, testicular failure, drug effects/radiation, endocrinology, and all others. However, despite recent technologic and diagnostic advances, idiopathic infertility remains a common diagnosis, with approximately 25% of patients not having an identifiable cause of infertility. Regardless, many recognizable causes of male infertility are treatable or preventable; thus, a keen understanding of these conditions is paramount. This chapter comprises an overview of etiologies of male infertility, divided into pre-testicular, testicular, and post-testicular causes.
Endocannabinoids control male reproduction acting at central and local level via cannabinoid receptors. The cannabinoid receptor CB1 has been characterized in the testis, in somatic and germ cells of mammalian and non-mammalian animal models, and its activity related to Leydig cell differentiation, steroidogenesis, spermiogenesis, sperm quality and maturation. In this short review, we provide a summary of the insights concerning neuroendocrine CB1 activity in male reproduction focusing on adult Leydig cell ontogenesis and steroid biosynthesis.
Of all sexually active couples, 12–15% are infertile. When broken down by gender, a male component can be identified 50% of the time either in isolation or in combination with a female factor. The majority of the causes of male infertility are treatable or preventable, so a keen understanding of these conditions is paramount. Despite advancements in assisted reproductive technologies, the goal of a male infertility specialist is not simply to retrieve sperm. Instead, the male infertility specialist attempts to optimize a male’s reproductive potential and thereby allow a couple to conceive successfully through utilization of less invasive reproductive techniques. Often, this involves the use of sperm or testicular tissue cryopreservation prior to fertility insult. At the same time, the male fertility specialist is wary of underlying or causal, potentially serious medical or genetic conditions that prompted reproductive evaluation. Previous research in a US male fertility clinic analyzing 1,430 patients identified causes of infertility from most to least common: varicocele, idiopathic, obstruction, female factor, cryptorchidism, immunologic, ejaculatory dysfunction, testicular failure, drug effects/radiation, endocrinology, and all others. The focus of this book on the role of reactive oxygen species (ROS) is easily applied to the majority of the listed conditions (described in detail in later chapters) which comprise this chapter’s overview of pre-testicular, testicular, and post-testicular causes of male infertility.
Male infertility is caused by a variety of factors including anatomical and genetic abnormalities, but it is estimated that over 30 % of male infertility is of an unknown cause. The most likely contributors to idiopathic male infertility are life style and environmental factors such as poor diet, insufficient physical activity, exposure to toxins, and tobacco, alcohol and other substance abuse. This chapter describes the effects of common recreational and performance enhancing substance abuse such as marijuana, opioids, methamphetamine and ecstasy, cocaine, and creatine and steroids on male fertility. These drugs affect the testes, spermatozoa, and male fertility by negatively interfering with the hormones that are responsible for the proper regulation of the male reproductive system such as gonadotropin releasing hormone, luteinizing hormone, follicle stimulating hormone, and testosterone. Some of these drugs also interfere with spermatogenesis via direct testicular influence. It is therefore important to know, especially to those of the reproductive age wishing to have children, that abuse of these drugs and substances contribute to male infertility.
Spermatogenesis, a highly conserved process in vertebrates, is mainly under the hypothalamic-pituitary control, being regulated by the secretion of pituitary gonadotropins, follicle stimulating hormone, and luteinizing hormone, in response to stimulation exerted by gonadotropin releasing hormone from hypothalamic neurons. At testicular level, gonadotropins bind specific receptors located on the somatic cells regulating the production of steroids and factors necessary to ensure a correct spermatogenesis. Indeed, besides the endocrine route, a complex network of cell-to-cell communications regulates germ cell progression, and a combination of endocrine and intra-gonadal signals sustains the production of high quality mature spermatozoa. In this review, we focus on the recent advances in the area of the intra-gonadal signals supporting sperm development.
Male infertility is a major cause of problems for many couples in conceiving a child. Recently, lifestyle pastimes such as alcohol, tobacco and marijuana have been shown to have further negative effects on male reproduction. The endocannabinoid system (ECS), mainly through the action of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) at cannabinoid (CB(1), CB(2)) and vanilloid (TRPV1) receptors, plays a crucial role in controlling functionality of sperm, with a clear impact on male reproductive potential. Here, sperm from fertile and infertile men were used to investigate content (through LC-ESI-MS), mRNA (through quantitative RT-PCR), protein (through Western Blotting and ELISA) expression, and functionality (through activity and binding assays) of the main metabolic enzymes of AEA and 2-AG (NAPE-PLD and FAAH, for AEA; DAGL and MAGL for 2-AG), as well as of their binding receptors CB(1), CB(2) and TRPV1. Our findings show a marked reduction of AEA and 2-AG content in infertile seminal plasma, paralleled by increased degradation: biosynthesis ratios of both substances in sperm from infertile versus fertile men. In addition, TRPV1 binding was detected in fertile sperm but was undetectable in infertile sperm, whereas that of CB(1) and CB(2) receptors was not statistically different in the two groups. In conclusion, this study identified unprecedented alterations of the ECS in infertile sperm, that might impact on capacitation and acrosome reaction, and hence fertilization outcomes. These alterations might also point to new biomarkers to determine male reproductive defects, and identify distinct ECS elements as novel targets for therapeutic exploitation of ECS-oriented drugs to treat male fertility problems.
Endocannabinoids are fatty acid amides like anandamide (AEA), and monoacylglycerols like 2-arachidonoylglycerol, that bind to cannabinoid, vanilloid and peroxisome proliferator-activated receptors. Their biological actions are controlled through not yet fully characterized cellular mechanisms. These compounds, together with their related enzymes, that include key proteins for the synthesis and degradation of endocannabinoids, cannabinoid and non-cannabinoid receptors, and purported membrane transporter(s), form the “endocannabinoid system (ECS)”. In the past few years AEA and related ECS elements have emerged as essential players in various aspects of human reproduction, both for males and females. Here, the key features of the ECS and the potential of its components to direct human fertility towards a positive or negative end will be reviewed. In particular, the involvement of AEA and related ECS elements in regulating embryo oviductal transport, blastocyst implantation and placental development (in females), and sperm survival, motility, capacitation and acrosome reaction (in males) will be addressed, as well as the role of endocannabinoids in sperm–oviduct interactions. Additionally, the possibility that blood AEA and its hydrolase FAAH may represent reliable diagnostic markers of natural and assisted reproduction in humans will be discussed, along with the therapeutic exploitation of ECS-oriented drugs as useful fertility enhancers.
Epidemiological studies have highlighted the ever growing use of illegal drugs among teenagers. The negative effects of marijuana (a Cannabis sativa extract) on reproductive health are poorly known among young people, although chronic exposure to delta-9-tetrahydrocannabinol, the main psychoactive constituent of marijuana, impairs human reproductive potential by disrupting menstrual cycle, suppressing oogenesis and impairing embryo implantation and development, in women, and by increasing ejaculation problems, reducing sperm count and motility, and generating loss of libido and impotence, in men. Endocannabinoids, their metabolic enzymes and target receptors form the so called "endocannabinoid system" and they have been demonstrated to respond to fertility signals. In addition, they interfere with hormones, cytokines and other signalling molecules in both female and male reproductive events. In this review, we shall summarize the current knowledge on the endocannabinoid system, and on the multifaceted roles played by endocannabinoids in reproduction along the evolutionary axis from invertebrates to mammals. Furthermore, we shall discuss the potential use of distinct elements of the endocannabinoid system for the diagnosis and/or treatment of human infertility.
Testicular morphology of vertebrate testis indicates requirement of local control. In urodeles, the testis is organized in lobes of increasing maturity throughout the cephalocaudal axis. The anuran testis is organized in tubules. Spermatogenesis occurs in cysts composed by Sertoli cells enveloping germ cells at synchronous stages. Moreover, in numerous species germ cell progression lasts a year which defines the sexual cycle. Due to the above quoted features, research on factors regulating germ cell progression in amphibians may reach greater insight as compared with mammalian animal models. In particular, studies on endocrine and paracrine/autocrine factors involved in the regulation of germ cell functions reveal that fos activation and a J protein, previously specifically found in mouse testis, exert an important role in spermatogonial proliferation and maturation of post-meiotic stages, respectively.
Results of a first-stage Sea Urchin Genome Project are summarized here. The species chosen was Strongylocentrotus purpuratus, a research model of major importance in developmental and molecular biology. A virtual map of the genome was constructed
by sequencing the ends of 76,020 bacterial artificial chromosome (BAC) recombinants (average length, 125 kb). The BAC-end
sequence tag connectors (STCs) occur an average of 10 kb apart, and, together with restriction digest patterns recorded for
the same BAC clones, they provide immediate access to contigs of several hundred kilobases surrounding any gene of interest.
The STCs survey >5% of the genome and provide the estimate that this genome contains ≈27,350 protein-coding genes. The frequency
distribution and canonical sequences of all middle and highly repetitive sequence families in the genome were obtained from
the STCs as well. The 500-kb Hox gene complex of this species is being sequenced in its entirety. In addition, arrayed cDNA libraries of >105 clones each were constructed from every major stage of embryogenesis, several individual cell types, and adult tissues and
are available to the community. The accumulated STC data and an expanding expressed sequence tag database (at present including
>12,000 sequences) have been reported to GenBank and are accessible on public web sites.
The function of the central cannabinoid receptor (CB1) was investigated by invalidating its gene. Mutant mice did not respond to cannabinoid drugs, demonstrating the exclusive
role of the CB1 receptor in mediating analgesia, reinforcement, hypothermia, hypolocomotion, and hypotension. The acute effects of opiates
were unaffected, but the reinforcing properties of morphine and the severity of the withdrawal syndrome were strongly reduced.
These observations suggest that the CB1 receptor is involved in the motivational properties of opiates and in the development of physical dependence and extend the
concept of an interconnected role of CB1 and opiate receptors in the brain areas mediating addictive behavior.
Delta(9)-tetrahydrocannabinol, the psychoactive ingredient of cannabis, exerts effects in humans by binding to the G-protein-coupled cannabinoid receptors CB1 and CB2, which are ex pressed in the nervous and immune systems, respectively,. Genes encoding CB1 receptors have also been discovered in non-mammalian vertebrates, including the puffer fish Fugu rubripes. Here the identification of a Fugu gene that encodes an ortholog of mammalian CB2 receptors is reported. This is the first CB2 gene to be identified in a non-mammalian vertebrate, and it indicates that the gene duplication event that gave rise to CB1 and CB2 receptors occurred before the divergence of teleosts and tetrapods. Moreover, non-mammalian vertebrate species can now be considered as potential model systems in which the physiological roles of the CB2 receptor can be investigated.
Correction to: The EMBO Journal (2005) 24, 3026–3037. doi:10.1038/sj.emboj.7600784
Arachidonylethanolamide, an arachidonic acid derivative in porcine brain, was identified in a screen for endogenous ligands
for the cannabinoid receptor. The structure of this compound, which has been named "anandamide," was determined by mass spectrometry
and nuclear magnetic resonance spectroscopy and was confirmed by synthesis. Anandamide inhibited the specific binding of a
radiolabeled cannabinoid probe to synaptosomal membranes in a manner typical of competitive ligands and produced a concentration-dependent
inhibition of the electrically evoked twitch response to the mouse vas deferens, a characteristic effect of psychotropic cannabinoids.
These properties suggest that anandamide may function as a natural ligand for the cannabinoid receptor.
Marijuana and many of its constituent cannabinoids influence the central nervous system (CNS) in a complex and dose-dependent manner. Although CNS depression and analgesia are well documented effects of the cannabinoids, the mechanisms responsible for these and other cannabinoid-induced effects are not so far known. The hydrophobic nature of these substances has suggested that cannabinoids resemble anaesthetic agents in their action, that is, they nonspecifically disrupt cellular membranes. Recent evidence, however, has supported a mechanism involving a G protein-coupled receptor found in brain and neural cell lines, and which inhibits adenylate cyclase activity in a dose-dependent, stereoselective and pertussis toxin-sensitive manner. Also, the receptor is more responsive to psychoactive cannabinoids than to non-psychoactive cannabinoids. Here we report the cloning and expression of a complementary DNA that encodes a G protein-coupled receptor with all of these properties. Its messenger RNA is found in cell lines and regions of the brain that have cannabinoid receptors. These findings suggest that this protein is involved in cannabinoid-induced CNS effects (including alterations in mood and cognition) experienced by users of marijuana.
Cannabinoids are potent pharmacological substances derived from marihuana. The effects of delta 9-tetrahydrocannabinol (THC), cannabinol (CBN), and cannabidiol (CBD) on fertilization in the sea urchin Strongylocentrotus purpuratus were investigated. Insemination of THC-treated eggs (5-400 microM) with excess sperm did not result in polyspermic fertilization. At minimal sperm densities, THC (0.1-10 microM) inhibited fertilization in a dose-dependent manner. Pretreatment of eggs with THC did not reduce their receptivity to sperm. Pretreatment of sperm with THC reduced their fertilizing capacity. The concentration of THC required to reduce sperm fertility by 50% was 1.1 +/- 1.1 microM. The fertilizing capacity of THC-treated sperm depended on concentration of sperm and duration of pretreatment. The fertility of sperm at minimal densities was reduced by 50% at 129.3 +/- 43 s treatment with 10 microM THC. The adverse effect of THC on sperm fertility was reversible. CBN and CBD at comparable concentrations (0.1-10 microM) inhibited fertilization in a manner similar to THC. First division was not delayed in zygotes that were fertilized with sperm pretreated with 10 microM THC. These studies show that cannabinoids directly affect the process of fertilization in sea urchins by reducing the fertilizing capacity of sperm.
In this study, we report the isolation from canine intestines of 2-arachidonyl glycerol (2-Ara-Gl). Its structure was determined by mass spectrometry and by direct comparison with a synthetic sample. 2-Ara-Gl bound to membranes from cells transiently transfected with expression plasmids carrying DNA of either CB1 or CB2--the two cannabinoid receptors identified thus far--with Ki values of 472 +/- 55 and 1400 +/- 172 nM, respectively. In the presence of forskolin, 2-Ara-Gl inhibited adenylate cyclase in isolated mouse spleen cells, at the potency level of delta 9-tetrahydrocannabinol (delta 9-THC). Upon intravenous administration to mice, 2-Ara-Gl caused the typical tetrad of effects produced by THC: antinociception, immobility, reduction of spontaneous activity, and lowering of the rectal temperature. 2-Ara-Gl also shares the ability of delta 9-THC to inhibit electrically evoked contractions of mouse isolated vasa deferentia; however, it was less potent than delta 9-THC.
The major active ingredient of marijuana, delta 9-tetrahydrocannabinol (delta 9-THC), has been used as a psychoactive agent for thousands of years. Marijuana, and delta 9-THC, also exert a wide range of other effects including analgesia, anti-inflammation, immunosuppression, anticonvulsion, alleviation of intraocular pressure in glaucoma, and attenuation of vomiting. The clinical application of cannabinoids has, however, been limited by their psychoactive effects, and this has led to interest in the biochemical bases of their action. Progress stemmed initially from the synthesis of potent derivatives of delta 9-THC, and more recently from the cloning of a gene encoding a G-protein-coupled receptor for cannabinoids. This receptor is expressed in the brain but not in the periphery, except for a low level in testes. It has been proposed that the nonpsychoactive effects of cannabinoids are either mediated centrally or through direct interaction with other, non-receptor proteins. Here we report the cloning of a receptor for cannabinoids that is not expressed in the brain but rather in macrophages in the marginal zone of spleen.
Anandamide (arachidonylethanolamide) is an endogenous cannabinoid receptor agonist in mammalian brain. Sea urchin sperm contain a high-affinity cannabinoid receptor similar to the cannabinoid receptor in mammalian brain. (-)-delta 9-Tetrahydrocannabinol (THC), the primary psychoactive cannabinoid in marihuana, reduces the fertilizing capacity of sea urchin sperm by blocking the acrosome reaction that normally is stimulated by a specific ligand in the egg's jelly coat. We now report that anandamide produces effects similar to those previously obtained with THC in Strongylocentrotus purpuratus in reducing sperm fertilizing capacity and inhibiting the egg jelly-stimulated acrosome reaction. Arachidonic acid does not inhibit the acrosome reaction under similar conditions. The adverse effects of anandamide on sperm fertilizing capacity and the acrosome reaction are reversible. The receptivity of unfertilized eggs to sperm and sperm motility are not impaired by anandamide. Under conditions where anandamide completely blocks the egg jelly-stimulated acrosome reaction, it does not inhibit the acrosome reaction artificially initiated by ionomycin, which promotes Ca2+ influx, and nigericin, which activates K+ channels in sperm. These findings provide additional evidence that the cannabinoid receptor in sperm plays a role in blocking the acrosome reaction, indicate that anandamide or a related molecule may be the natural ligand for the cannabinoid receptor in sea urchin sperm, and suggest that binding of anandamide to the cannabinoid receptor modulates stimulus-secretion-coupling in sperm by affecting an event prior to ion channel opening.
Endogenous neuromodulatory molecules are commonly coupled to specific metabolic enzymes to ensure rapid signal inactivation. Thus, acetylcholine is hydrolysed by acetylcholine esterase and tryptamine neurotransmitters like serotonin are degraded by monoamine oxidases. Previously, we reported the structure and sleep-inducing properties of cis-9-octadecenamide, a lipid isolated from the cerebrospinal fluid of sleep-deprived cats. cis-9-Octadecenamide, or oleamide, has since been shown to affect serotonergic systems and block gap-junction communication in glial cells (our unpublished results). We also identified a membrane-bound enzyme activity that hydrolyses oleamide to its inactive acid, oleic acid. We now report the mechanism-based isolation, cloning and expression of this enzyme activity, originally named oleamide hydrolase, from rat liver plasma membranes. We also show that oleamide hydrolase converts anandamide, a fatty-acid amide identified as the endogenous ligand for the cannabinoid receptor, to arachidonic acid, indicating that oleamide hydrolase may serve as the general inactivating enzyme for a growing family of bioactive signalling molecules, the fatty-acid amides. Therefore we will hereafter refer to oleamide hydrolase as fatty-acid amide hydrolase, in recognition of the plurality of fatty-acid amides that the enzyme can accept as substrates.
Anandamide, an endogenous ligand for central cannabinoid receptors, is released from neurons on depolarization and rapidly
inactivated. Anandamide inactivation is not completely understood, but it may occur by transport into cells or by enzymatic
hydrolysis. The compoundN-(4-hydroxyphenyl)arachidonylamide (AM404) was shown to inhibit high-affinity anandamide accumulation in rat neurons and astrocytes
in vitro, an indication that this accumulation resulted from carrier-mediated transport. Although AM404 did not activate cannabinoid
receptors or inhibit anandamide hydrolysis, it enhanced receptor-mediated anandamide responses in vitro and in vivo. The data
indicate that carrier-mediated transport may be essential for termination of the biological effects of anandamide, and may
represent a potential drug target.
Based on both binding and functional data, this study introduces SR 144528 as the first, highly potent, selective and orally active antagonist for the CB2 receptor. This compound which displays subnanomolar affinity (Ki = 0.6 nM) for both the rat spleen and cloned human CB2 receptors has a 700-fold lower affinity (Ki = 400 nM) for both the rat brain and cloned human CB1 receptors. Furthermore it shows no affinity for any of the more than 70 receptors, ion channels or enzymes investigated (IC50 > 10 microM). In vitro, SR 144528 antagonizes the inhibitory effects of the cannabinoid receptor agonist CP 55,940 on forskolin-stimulated adenylyl cyclase activity in cell lines permanently expressing the h CB2 receptor (EC50 = 10 nM) but not in cells expressing the h CB1 (no effect at 10 microM). Furthermore, SR 144528 is able to selectively block the mitogen-activated protein kinase activity induced by CP 55,940 in cell lines expressing h CB2 (IC50 = 39 nM) whereas in cells expressing h CB1 an IC50 value of more than 1 microM is found. In addition, SR 144528 is shown to antagonize the stimulating effects of CP 55,940 on human tonsillar B-cell activation evoked by cross-linking of surface Igs (IC50 = 20 nM). In vivo, after oral administration SR 144528 totally displaced the ex vivo [3H]-CP 55,940 binding to mouse spleen membranes (ED50 = 0.35 mg/kg) with a long duration of action. In contrast, after the oral route it does not interact with the cannabinoid receptor expressed in the mouse brain (CB1). It is expected that SR 144528 will provide a powerful tool to investigate the in vivo functions of the cannabinoid system in the immune response.
The function of the central cannabinoid receptor (CB1) was investigated by invalidating its gene. Mutant mice did not respond to cannabinoid drugs, demonstrating the exclusive role of the CB1 receptor in mediating analgesia, reinforcement, hypothermia, hypolocomotion, and hypotension. The acute effects of opiates were unaffected, but the reinforcing properties of morphine and the severity of the withdrawal syndrome were strongly reduced. These observations suggest that the CB1 receptor is involved in the motivational properties of opiates and in the development of physical dependence and extend the concept of an interconnected role of CB1 and opiate receptors in the brain areas mediating addictive behavior.
Herbalists claim that the polypharmacy of botanical remedies provides 2 advantages over single-ingredient drugs: (1) primary active ingredients in herbs are synergized by secondary compounds, and (2) secondary compounds mitigate the side effects caused by primary active ingredients. To examine this second claim, medical marijuana was compared with its primary active ingredient, tetrahydrocannabinol.
A search on MEDLINE (1966-1999), AGRICOLA (1990-1999), and Biological and Agricultural Index (1964-1999) using keywords "cannabinoids," "marijuana," "tetrahydrocannabinol," "Cannabis," and "hemp." Phytochemical and ethnobotanical data were searched with the Agricultural Research Service database. Unindexed botanical journals were scanned by hand.
Studies documenting the efficacy of secondary compounds mitigating side effects of tetrahydrocannabinol consisting of double-blind trials, unblinded studies, animal models, and in vitro experiments.
Data validity was assessed by consensus, weighted by source (peer-reviewed article vs popular press), identification methodology (analytical chemistry vs clinical history), and frequency of independent observations.
Good evidence suggests that some side effects of tetrahydrocannabinol are mitigated by other volatile compounds present in the essential oil of marijuana. Inhaling tetrahydrocannabinol, which avoids first-pass hepatic metabolism, has advantages over ingesting it. Other cannabinoids, terpenoids, and flavonoids can reduce tetrahydrocannabinol-induced anxiety, cholinergic deficits, and immunosuppression. Other compounds increase cerebral blood flow, enhance cortical activity, kill respiratory pathogens, and provide anti-inflammatory activity. The hazards of marijuana smoke can be reduced with appropriate technology. Proprietary Cannabis extracts containing a mixture of cannabinoids, terpenoids, and flavonoids are currently being developed and tested.
The present study was aimed to test whether the endogenous ligand for the cannabinoid receptor, anandamide (ANA) could produce direct effects on anterior pituitary (AP) hormone secretion in vitro. AP cells, dispersed in Krebs-Ringer, were treated with either (1) different concentrations of ANA, (2) with ANA after pre-treatment with the selective central cannabinoid receptor (CB1) antagonist, SR 141716 (SR) or (3) with SR alone. After 30 min of incubation the luteinizing hormone (LH), follicle stimulating hormone (FSH), prolactin (PRL), adrenocorticotroph hormone (ACTH) as well as growth hormone (GN), were measured in the culture media. All these hormone levels were affected with the exception of FSH, which did not change after the treatment. LH and PRL levels were decreased while ACTH and GH increased. The LH and PRL decrease were prevented by SR. SR did not antagonize the stimulatory effect exerted by ANA on ACTH and GH release from AP cells. We postulate that ANA inhibits or activates the release and/or synthesis of AP hormones. This activation may occur via CBI receptor in the case of LH and PRL; the other effects of the endogenous cannabinoids are probably mediated via another postulated (CBx) receptor for which SR is not an effective antagonist. The results indicate that ANA may influence AP hormone secretion, acting directly on the gland.
A tritiated form of CP-55,940, a Pfizer cannabinoid analog that is 20- to 100-fold more potent than {Delta}{sup 9}-tetrahydrocannabinol in various in vivo and in vitro models of cannabimimetric activity, was used as the tool with which to probe for a cannabinoid receptor in rat cortical membranes. The bound and free ligand were successfully separated using a centrifugation assay. Specific binding was saturable, rapidly attained, and completely reversible. The K{sub D}'s derived from kinetic analysis of binding agreed well with the K{sub D}'s derived from saturation and displacement analysis. The ({sup 3}H)CP-55,940 binding site exhibited high affinity with a K{sub D} of 68 pM as determined by LIGAND analysis of homologous displacement studies. The ability of other cannabinoid drugs to displace ({sup 3}H)CP-55,940 binding correlated well with the potency of these drugs in in vivo and in vitro models of cannabimimetic activity. The K{sub i} of {Delta}{sup 9}-THC was 1.6 nM. Cannabidiol and cannabigerol, which both lack psychoactivity in man, displaced specific binding by less than 50% at 1 {mu}M.
The stimulus-induced biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG) in intact mouse J774 macrophages and the inactivation of 2-AG by the same cells or by rat circulating macrophages was studied. By using gas chromatography-mass spectrometry, we found that ionomycin (5 µm) and lipopolysaccharide (LPS, 200 µg·mL−1) cause a 24-fold and 2.5-fold stimulation of 2-AG levels in J774 cells, respectively, thus providing unprecedented evidence that this cannabimimetic metabolite can be synthesized by macrophages. In J774 cells, LPS also induced a 7.8-fold increase of the levels of the other endocannabinoid, anandamide, and, in rat circulating macrophages, an almost twofold increase of 2-AG levels. Extracellular [3H]2-AG was cleared from the medium of intact J774 macrophages (t1/2 = 19–28 min) and esterified to phospholipids, diacylglycerols and triglycerides or hydrolyzed to [3H]arachidonic acid and glycerol. These catabolic processes were attenuated differentially by various enzyme inhibitors. Rat circulating macrophages were shown to contain enzymatic activities for the hydrolysis of 2-AG, including: (a) fatty acid amide hydrolase (FAAH), the enzyme responsible for anandamide breakdown and previously shown to catalyse also 2-AG hydrolysis, and (b) a 2-AG hydrolase activity different from FAAH and down-regulated by LPS. High levels of FAAH mRNA were found in circulating macrophages but not platelets, which, however, contain a 2-AG hydrolase. Both platelets and macrophages were shown to express the mRNA for the CB1 cannabinoid receptor. A macrophage 2-AG hydrolase with apparent Km = 110 µm and Vmax = 7.9 nmol·min−1·(mg protein)−1 was partially characterized in J774 cells and found to exhibit an optimal pH of 6–7 and little or no sensitivity to typical FAAH inhibitors. These findings demonstrate for the first time that macrophages participate in the homeostasis of the hypotensive and immunomodulatory endocannabinoid 2-AG through metabolic mechanisms that are subject to regulation.
Abstract : Investigation of cannabinoid pharmacology in a vertebrate with a phylogenetic history distinct from that of mammals may allow better understanding of the physiological significance of cannabinoid neurochemistry. Taricha granulosa, the roughskin newt, was used here to characterize an amphibian cannabinoid receptor. Behavioral experiments demonstrated that the cannabinoid agonist levonantradol inhibits both newt spontaneous locomotor activity and courtship clasping behavior. Inhibition of clasping was dose-dependent and potent (IC50 = 1.2 μg per animal). Radioligand binding studies using [3H]CP-55940 allowed identification of a specific binding site (KD = 6.5 nM, Bmax = 1,853 fmol/mg of protein) in brain membranes. Rank order of affinity of several ligands was consistent with that reported for mammalian species (KD, nM) : CP-55940 (3.8) > levonantradol (13.0) > WIN55212-2 (25.7) ▴ anandamide (1,665) >> anandamide + 100 μM phenylmethylsulfonyl fluoride (2,398). The cDNA encoding the newt CB1 cannabinoid receptor was cloned, and the corresponding mRNA of 5.9 kb was found to be highly expressed in brain. A nonclonal Chinese hamster ovary cell line stably expressing the newt CB1 cannabinoid receptor was prepared that allowed demonstration of cannabinoid-mediated inhibition of adenylate cyclase (EC 4.6.1.1) activity. This inhibition was dose-dependent and occurred at concentrations consistent with affinities determined through radioligand binding experiments. The behavioral, pharmacological, and molecular cloning results demonstrate that a CB1 cannabinoid receptor is expressed in the CNS of the roughskin newt. This amphibian CB1 is very similar in density, ligand binding affinity, ligand binding specificity, and amino acid sequence to mammalian CB1. The high degree of evolutionary conservation of cannabinoid signaling systems implies an important physiological role in vertebrate brain function.
Cannabinoid receptors have been described in sea urchin sperm and shown to mediate inhibition of sperm acrosome reaction. Anandamide (arachidonoyl-ethanolamide), the mammalian physiological ligand at the cannabinoid CB1 receptor, has been subsequently found to effect this inhibition. Here we present data showing that ovaries from the sea urchin Paracentrotus lividus contain anandamide and two related acyl-ethanolamides, as well as enzymatic activities potentially responsible for their biosynthesis and degradation. Pilot experiments carried out with either ovaries or spermatozoa, extracted from both P. lividus and Arbacea lixula and radiolabelled with [14C]ethanolamine, showed that in sexually mature ovaries of both species significant levels of radioactivity were incorporated into a lipid component with the same chromatographic behaviour as anandamide. Lipid extracts from P. lividus ovaries were purified and analysed by gas chromatography/mass spectrometry which showed the presence of low but measurable amounts of anandamide, palmitoyl- and stearoyl-ethanolamides. The extracts were also found to contain lipid components with the same chromatographic behaviour as the N-acyl-phosphatidyl-ethanolamines, the phospholipid precursors of acyl-ethanolamides in mammalian tissues, and capable of releasing anandamide, palmitoyl- and stearoyl-ethanolamides upon digestion with S. chromofuscus phospholipase D. Accordingly, whole homogenates from P. lividus contained an enzymatic activity capable of converting synthetic [3H]N-arachidonoyl-phosphatidyl-ethanolamine into [3H]anandamide. Finally, mature ovaries of P. lividus were shown also to contain an amidohydrolase activity which catalyses the hydrolysis of anandamide and palmitoyl-ethanolamide to ethanolamine. This enzyme displayed subcellular distribution, pH/temperature dependency profiles and sensitivity to inhibitors similar but not identical to those of the previously described `anandamide amidohydrolase' from mammalian tissues. These data support the hypothesis, formulated in previous studies, that anandamide or related metabolites may be oocyte-derived cannabimimetic regulators of sea urchin fertility. © 1997 Elsevier Science B.V. All rights reserved.
In this study, we report the isolation from canine intestines of 2-arachidonyl glycerol (2-Ara-Gl). Its structure was determined by mass spectrometry and by direct comparison with a synthetic sample. 2-Ara-Gl bound to membranes from cells transiently transfected with expression plasmids carrying DNA of either CB1 or CB2—the two cannabinoid receptors identified thus far—with Ki values of 472 ± 55 and 1400 ± 172 nM, respectively. In the presence of forskolin, 2-Ara-Gl inhibited adenylate cyclase in isolated mouse spleen cells, at the potency level of Δ9-tetrahydrocannabinol (Δ9-THC). Upon intravenous administration to mice, 2-Ara-Gl caused the typical tetrad of effects produced by THC: antinociception, immobility, reduction of spontaneous activity, and lowering of the rectal temperature. 2-Ara-Gl also shares the ability of Δ9-THC to inhibit electrically evoked contractions of mouse isolated vasa deferentia; however, it was less potent than Δ9-THC.
The function of central cannabinoid (CB1) receptor was investigated in the regulation of the pituitary–gonad axis in CB1 receptor knockout male mouse. Serum luteinizing hormone (LH) and testosterone (T) levels and basal T secretion in vitro of testes were significantly decreased in mutant (CB1−/−) mice. The receptor agonist, anandamide (ANA), suppressed LH and T secretion in wild type (CB1+/+) mice but had no effect in receptor inactivated animals. The results are the first descriptions indicating the direct action of CB1 receptors on LH and T secretion and the immunohistological demonstration of CB1 receptors in the Leydig cells. The results also indicate that CB1 receptors are responsible for the effects of exogenous cannabinoids on reproductive functions.
Endocannabinoids are endogenous ligands for plasma membrane receptors (CB1 and CB2), belonging to the superfamily of G-protein-coupled receptors. They mimic some of the effects played by D9-tetrahydrocannabinol (THC), the active principle isolated from Cannabis sativa. N-arachidonoylethanolamine (anandamide, AEA) is the main endocannabinoid described to date in the testis and in human seminal plasma. However, the activity of AEA in controlling male reproduction is still poorly understood. In this study we report on physiological activity of endocannabinoids in the male reproductive tract. Using wild type (WT) and CB1 knock out mice (CB1KO) we show that endocannabinoids act in the epididymus. Here, through CB1, they inhibit sperm motility measured as the percentage of motile spermatozoa (SPZ). In particular, while in WT mice, as expected, the percentage of motile SPZ (measured in caput and cauda of epididymus) was significantly lower in the caput as compared with the cauda, in CB1KO mice a strong increase of motile SPZ in the caput was measured.
Delta 9-Tetrahydrocannabinol (THC) elicited a dose-dependent (3.2-24 muM) response for form/movement, cellular growth and division in log growth phase and division-synchronized Tetrahymena pyriformis GL. Progressive dose-dependent action of THC on division delays in division-synchronized cell cultures was correlated with a concomitant reduction of division maxima and the percent of cells that completed division I. THC depressed the incorporation of 5-3H-uridine, 2-14C-thymidine and L-3-14C-phenylalanine into RNA, DNA and protein macromolecules respectively of division-synchronized Tetrahymena during division I. The depression of incorporation of 5-3H-uridine into nucleic acid macromolecules was correlated with a reduction of exogenous precursor in the cellular pool. The specific activity of radiolabeled mRNA and nascent polypeptides of polyribosomal fractions from synchronized cells was reduced by THC treatment. THC caused an inhibition of the incorporation of 5-3H-uridine into ribosomal RNA (17S and 25S RNA) and ribosomal precursor RNA (35S RNA) of synchronized cells.
Inhibitors of mitochondrial respiration, phosphorylation inhibitors, and uncoupling agents have been reported to delay or inhibit mitosis in cultured mammalian cells. Although the molecular mechanism by which mitosis is delayed in the presence of most respiratory inhibitors presumably involves lowered ATP production for mitotic requirements, one respiratory inhibitor, rotenone, was determined to arrest mitosis by an unrelated mechanism. Cell cycle kinetics studies, oxygen consumption measurements, and viscosity assays indicate that rotenone arrests cultured mammalian cells in mitosis by inhibiting spindle microtubule assembly by a mechanism analogous with colchicine, Colecemid and related antimitotic drugs. Amytal, which blocks electron transport at the same site as does rotenone, failed to arrest cell progression at mitosis. Rotenone delayed cell progression in all phases of the cell cycle, apparently as a direct result of respiration inhibition. Thus, rotenone appears to exert a dual function on events of the cell cycle.
The determination and characterization of a cannabinoid receptor from brain are reported. A biologically active bicyclic cannabinoid analgetic CP-55,940 was tritium-labeled to high specific activity. Conditions for binding to rat brain P2 membranes and synaptosomes were established. The pH optimum was between 7 and 8, and specific binding could be eliminated by heating the membranes to 60 degrees. Binding to the P2 membranes was linear within the range of 10 to 50 micrograms of protein/ml. Specific binding (defined as total binding displaced by 1 microM delta 9-tetrahydrocannabinol (delta 9-THC) or 100 nM desacetyllevonantradol) was saturable. The Kd determined from Scatchard analysis was 133 pM, and the Bmax for rat cortical P2 membranes was 1.85 pmol/mg of protein. The Hill coefficient for [3H]CP-55,940 approximated 1, indicating that, under the conditions of assay, a single class of binding sites was determined that did not exhibit cooperativity. The binding was rapid (kon approximately 2.6 x 10(-4) pM-1 min-1) and reversible (Koff approximately 0.016 min-1) and (koff' greater than 0.06 min-1). The two Kd values estimated from the kinetic constants approximately 55 pM and exceeded 200 pM, respectively. The binding of the agonist ligand [3H]CP-55,940 was decreased by the nonhydrolyzable GTP analog guanylylimidodiphosphate. The guanine nucleotide induced a more rapid dissociation of the ligand from the binding site, consistent with an allosteric regulation of the putative receptor by a G protein. The binding was also sensitive to MgCl2 and CaCl2. Binding of [3H]CP-55,940 was displaced by cannabinoid drugs in the following order of potency: CP-55,940 greater than or equal to desacetyllevonantradol greater than 11-OH-delta 9-THC = delta 9-THC greater than cannabinol. Cannabidiol and cannabigerol displaced [3H]CP-55,940 by less than 50% at 1 microM concentrations. The (-)-isomer of CP-55,940 displaced with 50-fold greater potency than the (+)-isomer. This pharmacology is comparable to both the inhibition of adenylate cyclase in vitro and the analgetic activity of these compounds in vivo. The criteria for a high affinity, stereoselective, pharmacologically distinct cannabinoid receptor in brain tissue have been fulfilled.
Twenty heterosexual men 18 to 28 years of age who used marihuana at least four days a week for a minimum of six months without use of other drugs during that interval were studied. Mean (± S.E.M.) plasma testosterone — 416 ± 34 ng per 100 ml — was significantly lower in this group than that in the control-group mean — 742 ± 29 ng per 100 ml — for age-matched men who had never used marihuana. Decreased testosterone was dose related. Abstention from marihuana use and stimulation with human chorionic gonadotropin during continued marihuana use produced marked increases in testosterone. Measurements of liver function, circulating gonadotropins, prolactin, Cortisol and thyroxine were within normal limits. Six of 17 men (35 per cent) showed oligospermia, and two men were impotent. The data suggest that chronic intensive use of marihuana may produce alterations in male reproductive physiology through central (hypothalamic or pituitary) action. (N Engl J Med 290:872–874, 1974)