The Epigenetics of the Endocannabinoid System

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.


Introduction
The endocannabinoid system (ES) is a complex cell-signalling system identified in the early 1990s following studies on the phytocannabinoid ∆ 9 -tetrahydrocannabinol (∆ 9 THC), the main psychoactive constituent of the marijuana plant Cannabis sativa [1]. It is widely distributed in biological tissues and is involved in many physiological activities such as pain control, motor functions, thermogenesis, sleep/wake cycle, learning and memory, synaptic plasticity, emotional (mood) regulation, stress response, food intake, inflammatory response, lipid and glucose metabolism, heart function, successful gametogenesis, and reproduction, amongst others [2]. The impairment of ES activity has been linked to several pathological conditions, from behavioural, neurological, and metabolic disorders to infertility and cancer, emphasizing the relevance of the pharmacological modulation of this system for the preservation of health status and the treatment of diseases [2,3]. Indeed, the high expression of the ES in brain areas playing a key role in conditioning processes such as drug-seeking behaviour, smoking, and alcohol addiction, emphasises that the ES is widely sensible to environmental epigenetic cues [4][5][6].
Currently, the ES represents a key signalling pathway involved in the modulation of most biological functions [1,2,18]. As a consequence, the pharmacological intervention of the ES components may represent a promising strategy for the management and the treatment of diseases, whereas the interference in ES signalling following phytocannabinoid abuse or the impairment of the system may represent a threat for the maintenance of health status.

Epigenetic Mechanisms: A Brief Overview
The environmental-dependent modulation of gene expression usually occurs at transcriptional, post-transcriptional, and translational levels. In general, the main epigenetic mechanisms involve the chemical modification of DNA and histone tails with consequences on chromatin architecture and accessibility to transcriptional factors and the production of specific regulatory ncRNA [8].
The main chemical modification of DNA is the covalent transfer of the methyl group (-CH 3 ) to cytosine located within cytosine-phosphate-guanine (CpG) islets in the gene promoter region, thus forming 5-methylcytosine (5mC). This modification changes the chromatin structure from an opened (transcriptionally active) to closed (transcriptionally inactive) state [24]. DNA methylation is typically erased during zygote formation to be newly established in the developing embryo in order to address proper embryo development and to drive gene imprinting, the process causing genes to be expressed from a parent of origin-specific manner [25]. Different DNA methyltransferases (DNMTs), the enzymes involved in this epigenetic modification, are classically responsible for de novo and maintaining methylation, but cooperative activity has also been reported and reviewed [26]. Classically, de novo methylation is established by DNMT3A and DNMT3B in participation with DNMT3L, a DNMT devoid of catalytic activity, but capable of assisting de novo methylation, increasing the ability of DNMTs to bind to the methyl group donor, S-adenosyl-L-methionine (SAM). Once established, DNA methylation status is maintained by DNMT1. Conversely, the Ten-eleven translocation methylcytosine dioxygenases (TET1-3) catalyse the oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) [27].
NcRNAs virtually influence every aspect of gene expression, thus representing emerging epigenetic marks detectable in both tissues and biological fluids with upcoming relevance in the regulation of biological functions, impact on health and disease status and possible employment for the prognosis, diagnosis and treatment of diseases [31,32]. They include microRNA (miRNA), transfer RNA (tRNA) fragments (tiRNA and tRF), long non-coding RNA (lncRNA), P-element induced wimpy testis (PIWI)-interacting RNAs (piRNAs), short interfering RNA (siRNA), and circular RNA (circRNA) [32,33].
MiRNAs (20-22 nt long) are endogenous small ncRNA classically involved in RNA interference and their exogenous counterparts are siRNAs. In most cases, they bind the 3' untranslated region of target mRNA inhibiting their translation into protein and inducing their degradation; however in the nucleus miRNAs may target mRNA co-transcriptionally, recruiting chromatin-modifying enzymes and inducing epigenetic regulation via DNA methylation or histone tails modifications [34].
Transfer RNAs (tRNAs) produce several fragments involved in repression of translation. Some of them, called 5'-(3'-) tRNA halves (tiRNA, 30-40 nt long), are stress-induced and are produced in humans by the endonuclease angiogenin that cleaves within the anticodon loops of mature tRNAs. Another group (17-26 nt long), usually referred as tRFs, is produced by the processing at the 5'-or 3'-end of mature or precursor tRNAs [35,36].
LncRNAs are bidirectional, antisense, intronic, intergenic, or overlapping transcripts capable of modulating the transcription of neighbouring protein-coding genes with remarkable tissue specificity. They also remodel chromatin and genome architecture or stabilize RNA through the recruitment of chromatin-modifying enzymes or directly acting as cis/trans scaffolding factors [37].
Originally characterized in germ cells, piRNAs (26-31 nt long) target heterochromatic regions through the formation of a PIWI-piRNA complex which usually is associated with the repressive histone/lysine methylation marks, but may also recruit different chromatin-modifying enzymes or facilitate transcription [33].
Lastly, circRNAs, a novel class of ncRNAs, are the result of back-splicing and usually are characterized by a covalently closed continuous loop without 5 or 3 polarities structure. Highly stable and widely expressed in mammalian cells, including spermatozoa [38], they usually modulate gene expression by acting as miRNA sponges [39].
Once established at embryo stage to define cell fate through the restriction of developmental potential [8], during the course of life genome activity is dynamically modulated under exogenous influence with gene activation or silencing during the life. Therefore, the continuous interaction between the internal and external environment addresses physiological development and health maintenance influencing both disease load and resistance [40]. Hence, there is hypothesis of an epigenetic "clock" phenomenon, a potential tracker of biological age, in which the aging-dependent genome-wide DNA hypomethylation leads to genome instability and occurrence of disease [41]. As a consequence, various epigenetic writers, readers, and erasers like maintenance and de novo DNMTs, TET proteins, histone acetyltransferases (HATs), deacetylases (HDACs), methyltransferases (HMT), and demethylases (amino oxidase homolog lysine demethylase 1 (KDM1) and JmjC domain-containing histone demethylases), or the ncRNA biosynthetic pathways have been identified in living organisms [32] and their activity is strongly related to the preservation of health status. Epigenetic modifications are linked to changes in development and behaviour, cancer, aging-related diseases, infertility, cardiovascular, neurological and metabolic disorders, or drug addiction, among others [42]. Hence, the development of drugs targeting epigenetic machinery represents the first step for the possible employment of personalized epigenetic therapy in the treatment of diseases [43].
Nevertheless, phytocannabinoids, endocannabinoids, and endocannabinoid receptor agonist/antagonists all affect epigenetic mechanisms in cell lines, animal models, and humans as well  (details in Table 2), with a long-term impact on health status and the possibility of transmission to the offspring through gametes, leading to trans-generational epigenetic inheritance ( Figure 1). Thus, the ES may represent a potential epigenetic target for the assessment of health/disease status, the treatment of disease, and the development of possible epigenetic therapies. Promotion of proliferation, migration, and invasion [59] miR-29a ↓CB1 ↑PPAR-γ Gain-of-function transgenic mice Block of the expressions of proinflammatory and profibrogenic mediators; attenuation of renal hypertrophy [60] miR-494 CB1 Myocardial biopsy specimens (n = 12 chronic heart failure (CHF) and n = 4 healthy controls) In CHF miR-494 is slightly increased leading to a compensatory response of the diseased myocardium. [61] miR-665 CB2 Myocardial biopsy specimens (n = 12 CHF and n = 4 healthy controls) In CHF miR-665 expression is significantly decreased leading to a compensatory response of the diseased myocardium. [61] hsa-miR-29b-3p CB1 Paediatric low-grade gliomas (P-LGG) (n = 33) and control brains (n = 6) Spontaneous involution of P-LGG may be induced by endocannabinoids [62] CB1 hsa-let-7d ↑ hsa-let-7d and other miRNA ↓CB1

Cannabis
Changes in DNA methylation Blood from schizophrenia patients (n = 98) and healthy controls (n = 108) Modulation of the immune response and protection against the neurobiological substrate of reality distortion symptoms in schizophrenia patients [65] Cannabidiol  JWH-133 Altered DNA methylation and hydroxymethylation at specific imprinted genes in sperm and placenta Paternal exposure in mouse Reduced sperm count in exposed animals and defects in placental and embryonic development [93]

Effects on Peripheral Tissues, Brain Functions, and Disease State
Peripherally, the activity of ES may be epigenetically modulated by diet. In fact, extra-virgin olive oil (EVOO), a typical lipid source in the Mediterranean diet which is rich in phenolic compounds, epigenetically modulates the expression rate of CNR1 in vivo and in vitro [51]. Thus, dietary EVOO administration reduces the methylation status of rat CNR1 promoter and the expression of miR23A and miR-301a-two modulators of CB1 in the pathogenesis of colorectal cancer, thus inducing the selective expression of CB1 in rat colon. Accordingly, in Caco-2 cells CB1 is less expressed than normal colon mucosa due to the hypermethylation of DNA at CNR1 promoter; in vitro EVOO, its phenolic extract (OPE), and authentic hydroxytyrosol (HT) upregulate CB1 expression with mechanisms for OPE and HT involving the reduction of CNR1 methylation at promoter level and leading to inhibition of cell proliferation [51].
In the brain, ES is involved in the homeostatic regulation of food intake, through the interplay with peripheral nutrient-sensors and the orexigenic and anorexigenic peptides produced within the arcuate nucleus of the hypothalamus, the brain region capable of capturing and integrating environmental cues with outcomes on feeding behaviour and reproduction [97]. To date, endocannabinoids act as orexigenic factors, stimulating food intake and fat deposition [97]; consistently, rimonabant (SR141716, Acomplia), the selective CB1 antagonist used in clinical trials for the treatment of obesity, reduces body weight, but due to severe psychiatric side effects, its use in patients has been discontinued [98].
Endocannabinoid are n-6 PUFA derivatives and an ideal ratio of 5 (n-6): 1 (n-3) has been suggested in order to preserve brain functions [97]. The evaluation of the plasma n6:n3 fatty acid ratio is therefore a possible risk factor to metabolic disease and might indicate an over activation of endocannabinoid signalling. Leptin, an anorexigenic peptide produced by white adipose tissue, inhibits hypothalamic ES [99], and Ob/Ob mice, lacking leptin, over-activate the hypothalamic endocannabinoid signalling [99] and are affected by infertility due to hypogonadotropic hypogonadism [100]. Consistently, leptin resistance has been associated to the over-activation of endocannabinoid signalling, with alterations in food intake and obesity development by a molecular mechanism involving the activation of CB1 [99]. Recently, sex-specific epigenetic changes related to leptin and endocannabinoid signalling have been reported in the hypothalamus of newborn rats following maternal high-fat diet HFD [52]. Prior obesity development, maternal HFD selectively induces the expression of CB1 in the hypothalamus of males, and of CB2 in females, with the former involved in the control of food intake and the latter mainly exerting a neuromodulatory role. Following maternal HFD, the hypothalamic expression of the transcriptional factor STAT3-a signalling intermediate in the leptin-dependent downregulation of the central ES-is down regulated in all newborns, but Almeida and co-workers reported sex-specific mechanisms in the leptin/ES interplay. In fact, while hypoleptinaemia occurred in newborn male rats only, in female rats only a decreased phosphorylation of STAT3 was observed. Thus, two complementary mechanisms impair leptin signalling, leading to the over expression of CB1. Furthermore, in male offspring maternal HFD causes CNR1 overexpression, with mechanisms involving chromatin remodelling at CNR1 promoter region by means of increased histone acetylation rate and increased binding of androgen receptor at CNR1 promoter as a consequence [52].
At present, the FAAH gene is the only component of ES epigenetically modulated in the hypothalamus by binge-eating, a recurrent process potentially influencing the development of eating disorders. An epigenetic mechanism consisting in the reduction of H3K4ac, without any change in DNA methylation and H3K27met3 at FAAH promoter region have been reported [45].
Interestingly, a highly conserved regulatory sequence in CNR1 intron 2 is responsible for the differential transcriptional activation of CB1 in brain regions like the hippocampus and hypothalamus, with effects on the sex-specific anxiety-related behavioural profile, ethanol intake, and hypothermic response following CB1 agonism, but without any significant changes in feeding patterns [101].
Lastly, Jiang and co-workers proposed a central adiponectin-dependent mechanism to promote the peripheral bone formation through the epigenetic regulation of the hypothalamic expression of CB1, requiring HDAC5 binding to the transcription start site 2 (TSS2) region of the CNR1 gene in embryonic mouse hypothalamus cell line N1 [83].
Reproduction is a process highly sensitive to environmental factors like diet, stress, or endocrine disruptor exposure among others [32,126]. Thus its epigenetic modulation has been reported and reviewed elsewhere [32,[126][127][128]. At present, there is a knowledge gap on the possible epigenetic regulation of ES in the modulation of GnRH pulse and reproductive hormones, but, as reported in Section 4.1, the epigenetic modulation of hypothalamic ES has been recently reported in relationship to diet and nutritional status, conditions notably affecting reproductive ability [129]. On the contrary, the epigenetic modulation of ES has been reported in male gonads, with emerging roles in spermatozoa and consequences for fertility and embryo development ( Figure 2). Therefore, we will deeply analyse the epigenetic modulation of the ES in the testis. The first evidence of the epigenetic modulation of ES in the gonad concerns the expression of FAAH1 in Sertoli cells, the nurse cells within the testis whose survival depends on AEA tone, FSH, and oestradiol activity [130]. The FAAH1 gene promoter contains an oestrogen-responsive element (ERE) and is notably expressed under oestradiol control in primary mouse Sertoli cells [131]. However, the oestradiol-dependent transcriptional activation of FAAH1 requires not only the binding of ERβ to proximal ERE sequences (ERE2/3), but also the involvement of the histone demethylase LSD1, and decreased methylation of both DNA at the CpG site and H3K9 in the proximal promoter region [132].
The ES deeply affects the development and the activity of Leydig cells [111], but at present there is no evidence of the possible epigenetic modulation of ES in Leydig cells.
In spite of the lack of data on the possible epigenetic modulation of testicular somatic cells, new insights concern the possible ES-dependent modulation of spermatogenesis through epigenetic mechanisms. Germ cell development is deeply modulated by epigenetic mechanisms which first erase the epigenetic signature (DNA methylation and histone tail remodelling) in primordial germ cells (PGCs) from specifications to migration and proliferation, and subsequently resettle DNA methylation status with de novo development of epigenetic marks and gene imprinting in pro-spermatogonia [9,10]. During post-natal testis development, additional reprogramming of epigenetic marks occurs in two particular time frames: at the entry in meiosis-a process requiring CB2 activity [112,122]-and in post meiotic stages, notably under CB1 control [117,120]. Lastly, during the transit in male and female reproductive tracts, spermatozoa need ES activity for the acquisition of motility and capacitation, respectively [118,125], and this occurs through a deep remodelling process which includes, among others, the exchange/acquisition of such epigenetic marks as ncRNA through epidydymosomes, prostasomes, or oviductosomes [133][134][135][136] that integrates the marks already available in spermatozoa [38,137,138].
In detail, during the spermatogenesis in rodents, CB2 exerts a pivotal role in meiosis entry [112,122] and its hyper-or hypostimulation disrupts the temporal dynamics of the spermatogenesis with possible epigenetic mechanisms [92]. In fact, JWH-133, a CB2 specific agonist, stimulates the expression of the meiotic genes c-Kit and Stra8 by increasing and decreasing the levels of H3K4m3 and H3K9m2, respectively, in genomic regions flanking the transcription start sites. Interestingly, the global increase in H3K4m3 occurs through the JWH-133-dependent transcriptional activation of Prdm9, the gene encoding for a zinc finger protein with HMT activity that catalyses H3K4me3 during the meiotic prophase. As a consequence, prolonged exposure to JWH-133 or administration of the specific CB2 antagonist AM630 accelerates or delays spermatogenesis onset in immature mice, pointing out the importance of correct endocannabinoid signalling for proper spermatogenesis and the deleterious effect of exogenous cannabinoids on male fertility [92].
In this respect, the modulation or the interference in the endogenous ES may affect gamete quality or impact the epigenetic mark of gametes, both critical for pregnancy and embryo development, with the (remote, but real) possibility of trans-generational inheritance in the offspring.
Spermiogenesis is the process leading to the formation of spermatozoa and is characterized by round spermatid elongation, acrosome and tail formation, nuclear shaping, and DNA packaging with transcriptional silencing as a consequence. Chromatin remodelling and DNA packaging are therefore the main nuclear events in spermiogenesis, consisting of a double-step process that requires histone replacement, first by transition proteins (TP2 and TP1) and then by protamines (PRM1 and PRM2), a class of small basic proteins [139]. The cooperation between HATs, HDACs, molecular chaperones, ubiquitination, and DNA repair systems drives the shift from a nucleosomal-based to a mainly protamine-based chromatin configuration [139]. In this respect, data from CB1 -/mice revealed the requirement of ES signalling for proper chromatin remodelling during spermiogenesis [117], and production of high-quality spermatozoa [140]. In fact, the genetic ablation of the CNR1 negatively affects the chromatin packaging, by affecting the content of TP2 mRNA and reducing histone displacement, with consequences on chromatin condensation and DNA integrity in the spermatozoa [117], which exhibit nuclear size elongation [140]. Such a mechanism is reversed by oestradiol administration, a treatment promoting histone displacement and chromatin condensation rescue in epididymal sperm collected from knock down animals [120].
In line with the results reported above, recent data revealed that the chronic administration of JWH-133 reduces sperm count in mouse and affects the epigenome of spermatozoa. Interestingly, the sperm from JWH-133 treated mice maintains the ability to fertilize eggs from untreated females, but impairment of embryo growth and defects in placental size have been reported, suggesting a possible interference in paternal inheritance through epigenetic mechanisms [93]. Accordingly defects in DNA methylation/hydroxymethylation at paternally expressed imprinted genes (i.e., Peg10 and Plagl1) have been reported in the sperm of JWH-133 treated animals and are maintained in placental tissue following fertilization. Thus, CB2 signalling may be critical for the integrity of the epigenome in the sperm, with the possibility of paternal epigenetic inheritance in the embryo, a process in which spermatozoa act as vectors for the delivery of epigenetic marks into the developing embryo. Consistently, two isoforms of CircNAPE-PLD (CircNAPE-PLD1 and CircNAPE-PLD2) are expressed in human and murine spermatozoa, and CircNAPE-PLD1 physically interacts with oocyte miRNAs involved in the progression of cell cycle [38]. Therefore, a new role of ES in the zygote to regulate the first stages of embryo development, through epigenetic paternal inheritance aimed at miRNA decoy, has emerged.
The above observations point out the possible risk for epigenome integrity of spermatozoa following marijuana use. Spermatozoa contain a complete ES devoted to the control of sperm physiology; acrosome reaction, acquisition of motility, spermatozoa-oocyte interaction all require the physiological activity of the endogenous ES [105,106]. Classically marijuana smokers exhibit a large set of reproductive failures, from imbalanced hormonal milieu and poor sperm quality to impairment of menstrual cycle, poor oocyte retrieval rate, low pregnancy rate, pre-term delivery, and prematurity with low fetal birth weight [102]. As a consequence, a recent study from the group of Murphy has been focused on the possible epigenetic effects of marijuana smoking and ∆ 9 THC on reproductive health status. In humans and rats, ∆ 9 THC exposure lowers sperm concentration and alters DNA sperm methylome with substantial shifts in both hypo-and hyper-DNA methylation, with the latter predominating [80]. In particular, 10.3% of differentially methylated CpG sites (409/3979) significantly correlate with sperm count and 183 individual CpG sites representing 177 named genes have methylation levels significantly correlated with measured ∆ 9 THC levels [80]. Altered CpG sites associated with genes involved in the Hippo signalling pathway and in pathways in cancer are common in both cannabis users and ∆ 9 THC-exposed rats [80]. Interestingly, ∆ 9 THC target genes in rat sperm substantially overlap with genes having altered methylation rate in the brain of rat offspring born to parents both exposed to ∆ 9 THC during adolescence [79].
Consistently with the above observation, ∆ 9 THC exposure-dependent changes in the DNA methylation of rat sperm [80] do not significantly impact the clinical health of the offspring (e.g., litter size, sex ratio, pup birth weight, survival, and growth) but cause long-lasting neurobehavioral effects in the offspring with impairment in attentional performance [78]. In addition, cannabis use in humans causes in the sperm the hypomethylation at 9 CpG sites located in intron 7 of the autism candidate gene Discs-Large Associated Protein 2 (DLGAP2), a gene involved in synapse organization and neuronal signalling [80,81]. Similarly, ∆ 9 THC exposure in adult rats differently methylated DLGAP2 gene in spermatozoa [81].
Lastly, IBN Lahmar Andaloussi et al. recently reported the behavioural and epigenetic effects of stress in male rats whose fathers were exposed to cannabinoids during adolescence [95] in the presence or absence of the synthetic CB1 agonist WIN55.212-2. Interestingly, stress exposure induced a significant anxiogenic-like effect but did not affect the episodic-like memory in the offspring of WIN55.212-2-exposed fathers only, with significant increases in global DNA methylation and DNMT1 and DNMTa3 transcription in the prefrontal cortex [95]. Thus, these results suggest that chronic exposure to cannabinoids during adolescence may lead to a trans-generational transfer of stress susceptibility to the offspring through the transfer of epigenetic marks in gametes.
Taken together, the use of cannabis for recreational use may represent a serious risk for both the fertility of marijuana smokers and the health of the offspring.

Conclusions
The ES is an almost ubiquitous cell signalling system regulating several processes inside cells that are not yet completely understood. From its discovery in 1990s, it was clear that this system could be modulated by both extrinsic (cannabis and its derivatives) and intrinsic (the endogenous ligands) signals. Subsequent studies pointed out that the ES is much more complex than was thought since it is able to cross-talk with many other transduction cell signalling pathways, therefore regulating key biological processes such as cell proliferation and differentiation, synaptic plasticity, gametogenesis, and fertility. From the data herein reported, it emerges that epigenetic modifications of the ES by means of DNA methylation, histone acetylation/deacetylation at the CNR1, and FAAH genes encoding the CB1 receptor and FAAH hydrolysing enzyme may play a relevant role both in physiological processes regulating (male) fertility and reproduction as well as in disease pathogenesis and progression including cancer. Interestingly, it has been documented that external epigenetic cues such as alcohol induce DNA methylation changes in the mouse model of foetal alcohol spectrum disorder, and the lack of a functional CNR1 gene protects against ethanol-induced impairments of DNMT1, DNMT3A, and DNA methylation [49]. This suggests that the ES itself may act as an epigenetic signal regulating gene expression. On the other hand, the altered DNA methylation of both GPR55 and CB1 encoding genes, resulting in increased expression of GPR55 and reduced levels of the CB1 in CRC patients [58], also supports the hypothesis that the ES receptors could behave either as tumour promoting or tumour suppressor genes depending on the kind of epigenetics changes they undergo. The ES has been recognized as a strong modulator in the central and local control of reproduction in both sexes. It is involved in the regulation of HPG axis, successful gametogenesis, fertilization, and embryo implantation and development. Therefore, it is conceivable that environmental factors, by epigenetically affecting the ES, could induce adverse effects on reproductive system functions per se or alternatively, change gene expression profile with a transgenerational inheritance in the offspring. Within this context, the emerging literature tags the ES signalling as critical for the integrity of the epigenome in the sperm suggesting the possibility of a paternal epigenetic inheritance in the embryo through a process in which spermatozoa act as vectors for delivering epigenetic marks into the developing embryo, and in our opinion this is a very interesting issue that has to be further studied. To date, most studies have been conducted in experimental models other than humans and the few studies in humans involve a limited number of subjects and do not extend to the offspring. However, the overlapping DNA methylation profiles detected in both human and rat sperm suggest that data in experimental models may be the basis for further investigations and confirmation in human subjects. Similarly, the use of phytocannabinoids or synthetic cannabinoids for therapeutic purposes point out the possible trans-generational transmission of epigenetic marks, revealing the need for particular caution and attention in the field. Finally, epigenetic modifiers of the ES could be also a promising tool to treat eating disorders or manage pathological conditions involving alterations of the ES system. However, the significance of ES epigenetics is still an open question needing deeper investigation to better characterize the real consequences of the epigenetic changes of this intriguing cell signalling system.