Cannabinoids are a class of compounds that are plant-derived (phytocannabinoids, i.e. from Cannabis sativa), made naturally within the body, and chemically manufactured. For the purpose of this review, where the term “cannabinoids” is used, it will refer to the general class of compounds, unless specified otherwise. Endocannabinoids (endogenous cannabinoids) are unsaturated fatty acid derivatives with wide distribution in the human body [1,2,3]. The two most extensively studied endocannabinoids are N-arachidonoylethanolamine (anandamide; AEA) and 2-arachidonoylglycerol (2-AG). AEA was the first endocannabinoid to be discovered  and is an important intermediate in lipid metabolism . Its name was derived from the Sanskrit word “ananda” which means inner bliss, describing the euphoric effects of this ligand [5, 6]. Physiologically, it is produced ubiquitously  with the greatest tissue concentrations found in the brain . Under most physiological conditions, 2-AG concentrations are much higher than that of AEA . Interestingly, these endocannabinoids are not stored within cells, but are thought to be manufactured “on demand” from membrane phospholipid precursors [7, 8]. However, recent reports challenge this view suggesting that they may be stored intracellularly within lipid droplets referred to as adiposomes, thus allowing for intracellular accumulation [5, 9]. Others suggest that catabolic enzymes at the surface of adiposomes quickly lead to endocannabinoid degradation . In light of this, sequestering of endocannabinoids in adiposomes may prolong their half-life (hours rather than minutes), allowing them time to trigger nuclear receptors .
AEA and 2-AG are known to bind to and activate two G-protein coupled receptors (GPCR): cannabinoid receptor 1 (CB1) and CB2. Both receptor isoforms are ubiquitously expressed, with CB1 found more predominantly in the central nervous system and CB2 found largely in cells of the immune system [5, 10, 11]. The receptor actions of AEA and 2-AG are mimicked by the exogenous cannabinoid Δ-9-tetrahydrocannabinol (THC), the primary psychoactive component of cannabis [4, 12]. In fact, it was the discovery in the 1980s that THC could bind to receptors in the brain that led researchers to discover AEA, the prototypical endocannabinoid .
AEA: Synthesis, transport, and degradation
AEA acts as a partial agonist at CB1 and as a weak/partial agonist at CB2 . Interestingly, AEA is reported to demonstrate promiscuous binding activity , as it can trigger various signaling pathways via a number of different receptors, both extracellularly at CB1and CB2, intracellularly at the transient receptor potential vanilloid-1 (TRPV1) channel, and in the nucleus via the peroxisome proliferator-activated receptors (PPARs) . More recently, studies have suggested that AEA may also act at the level of the mitochondria via CB1 receptors situated on the mitochondrial outer membrane .
The biosynthesis of AEA occurs in two steps. First, AEA is released from phospholipid precursors in the plasma membrane to a phosphatidylethanolamine, leading to the formation of N-acylphosphatidylethanolamine (NAPE). In the second step, a type D phospholipase (NAPE-PLD) catalyzes the formation of AEA from its NAPE precursor . AEA is then rapidly taken up by cells [8, 15]. Movement of AEA across the phospholipid bilayer is thought to occur by simple diffusion or endocytosis [8, 15] since AEA is uncharged and lipid soluble. Other studies strongly suggest the involvement of a putative endocannabinoid membrane transporter (EMT) [8, 16, 17] which allows AEA to be rapidly shuttled to its intracellular targets [8, 15]. Some of the intracellular targets for AEA identified to date include AEA intracellular binding proteins (AIBPs), namely albumin, heat shock protein 70 (Hsp70) and fatty acid binding protein-5 and -7 (FABP-5 and -7) . It has been proposed that FABPs are principally involved in AEA trafficking and breakdown, as the use of a novel reversible FABP inhibitor, BMS309403, partially reduced AEA uptake . Ultimately, AEA is broken down by intracellular hydrolases into ethanolamine and arachidonic acid [5, 7]. A key regulator of AEA activity is the serine hydrolase fatty acid amide hydrolase (FAAH) which is bound to intracellular membranes, particularly the endoplasmic reticulum (ER) and nuclear membrane .
2-AG: Synthesis, transport, and degradation
Belonging to the monoacylglycerol (MAG) family of endocannabinoids, 2-AG acts equally at CB1 and CB2 as a full potent agonist, but it has not been shown to act at the TRPV1 receptor [5, 11]. Tissue levels of 2-AG are markedly higher than that of AEA within the same tissue . Coupled with its full agonistic activity at cannabinoid receptors, it has been proposed as the primary endogenous agonist of both CB1 and CB2 .
The biosynthesis of 2-AG involves the combined action of two membrane-bound enzymes: phospholipase C (PLC) and diacylglycerol lipase (DAGL) . Unlike AEA, only a few studies have investigated the mechanisms underlying the rapid cellular uptake of 2-AG, which is surprising given that it is more abundant than AEA . While the mechanism(s) may be dependent upon the cell type [8, 16, 18], the most likely routes of entry for 2-AG may be via endocytosis, simple diffusion, the same EMT as AEA or other transporters [8, 16].
Once within the cytosol, 2-AG becomes a substrate for the chief catalytic enzyme monoacylglycerol lipase (MAGL), associated with the inner membrane, which degrades 2-AG to glycerol and arachidonic acid . In addition to MAGL, two other 2-AG hydrolases have been identified: α,β-hydrolase-6 and -12 (ABHD-6 and -12), which are integral cell membrane proteins and thought to share the catalytic triad with MAGL . In addition to the consensus regarding the putative degradative enzymes, these prototypical endocannabinoids may also be degraded via oxidation by cyclooxygenase (COX), lipoxygenase (LOX), or cytochrome P450 . These and other biosynthetic and degradative pathways for AEA and 2-AG are discussed at length in Fezza et al .
THC and other ligands
THC was the first exogenous ligand of cannabinoid receptors to be discovered . The physiological effects of THC are clinically concerning and need to be well delineated. Since this compound is highly lipophilic [5, 12, 19], it can be readily sequestered into adipose tissue, resulting in a rapid decrease in plasma concentrations , followed by a slow release into circulation over extended periods of time . This tissue distribution permits longer-lasting stimulation of the cannabinoid receptors. This is unlike that of the locally released endocannabinoids AEA and 2-AG, which are rapidly inactivated by their transporters and hydrolases .
Endogenously, other fatty acid derived compounds, including N-arachidonoyldopamine (NADA), 2-arachidonoylglycerylether (noladin ether) and O-arachidonoyl ethanolamine (virodhamine) have also been identified as endocannabinoids, although information on their function and biological relevance is somewhat limited [4, 5, 10]. Interestingly, a novel group of ligands has been identified, referred to as retro-anandamides, which are characterized by a reversal in the positions of the carbonyl and the amido groups . While these compounds demonstrate reduced affinity for CB1 and CB2 receptors as compared to AEA, they are resistant to FAAH catabolism resulting in increased stability relative to AEA . These same authors, in an earlier report, identified the first metabolically stable AEA analogue, (R)-methanandamide, which exhibited significantly greater affinity for CB1 and resistance to FAAH degradation when compared to AEA . Despite their presence, these and other ligands have received less scientific attention than AEA and 2-AG, perhaps due to the difficulty involved in isolating them from biological tissues .
CB1 and CB2 belong to a large superfamily of seven-transmembrane spanning GPCRs . AEA or 2-AG ligand binding to either CB1 or CB2 leads to multiple signal transduction mechanisms, including the inhibition of adenylate cyclase , a common target for activated G proteins, and consequent decrease in intracellular cAMP levels, increased potassium influx, and/or inhibition of certain calcium channels, thus reducing calcium influx [21, 22]. The intracellular signals arising from these cascades subsequently leads to the regulation of growth, proliferation, and/or differentiation .
The CB1 receptor
CB1 receptors are found mostly within the central nervous system . Peripherally, CB1 receptors have been identified in the spleen, heart, adrenal gland, ovaries, endometrium, testes, among others [1, 22]. Furthermore CB1 receptors have also been localized intracellularly on the mitochondrial outer membrane . Mitochondria regulate the energy demands of the cell, thus compromises in its function, from aberrant cannabinoid signaling [23, 24], will deregulate energy metabolism . For example, THC induced mitochondrial dysfunction has been associated with pathologies such as stroke . Mechanistically, mitochondrial CB1 receptors are thought to modulate complex I activity via a process involving soluble adenylyl cyclase . Since mitochondrial function controls apoptosis, disruption of this role can impact the process of producing quality gametes, subsequently interfering with embryogenesis and lead to the production poor quality embryonic stem cells . Furthermore, ovarian ageing is also associated with increased accumulation of mitochondrial DNA mutations which are likely to affect mitochondrial biogenesis and impact oocyte quality . Additionally, placental oxidative stress is also linked to mitochondrial dysfunction  and may impact vascular remodeling in the placenta  which is important for tissue oxygenation and organ function. Dysregulation of placental vascular development can result in a number of adverse pregnancy outcomes such as intrauterine growth restriction and preeclampsia [32, 33]. CB1 receptors are also found in the hypothalamus, the central regulator of energy homeostasis, further implicating a role for the ECS in energy balance. Moreover, the preoptic area of the hypothalamus contain CB1 receptors, from which secretory neurons for gonadotropin-releasing hormone (GnRH) are located .
The CB2 receptor
Like the CB1 receptor, CB2 is also a G protein coupled receptor demonstrating 44% sequence homology to CB1 [6, 22]. CB2 receptors are predominantly found peripherally within cells of the immune system, such as lymphocytes and macrophages . El-Talatini et al confirmed the presence of CB2 receptors in the ovarian cortex, ovarian medulla, and ovarian follicles from human samples , which followed a similar staining pattern as CB1 in the same tissues . However, Wang et al found that in murine oocytes, the action of endocannabinoids were mediated by CB1 receptor activation, not that of CB2. . This interspecies difference highlights the importance of utilizing human reproductive tissues as a means to study the impact of the ECS on its function/dysregulation .
Endocannabinoids are thought to also target a number of other orphan receptors. These include the GPR55 (also known as CB3) and GPR119 receptors which have signaling mechanisms distinct from CB1/CB2 [3, 16, 36]. Other receptors targeted by endocannabinoids include TRPV1, cytosolic target for AEA; and nuclear PPAR. Pertwee et al provide a detailed review of these other receptors and their pharmacology .