Beside the role exerted at hypothalamic level to control reproductive activity in both sexes, the discovery of eCBs in gonads and reproductive fluids – from seminal plasma in males to oviductal fluid and milk in females – (50–52) pointed out the importance of eCB signaling in the gonads. Gonads have the ability to synthesize eCBs which in turn exert differential effects activating both different types of receptors or tissue-/cell-specific receptor subtypes, the latter obtained by both alternative splicing or transcription sites (53–55). The content of eCBs is regulated by biosynthetic/hydrolyzing enzyme balance, and the appropriate “eCBs tone” in loco, is critical for spermatogenesis progression in male and follicle maturation in female, for sperm quality and the acquisition of sperm functions related to fertilization (motility and capacitation), for fertilization, early-embryo migration, implantation and placentation, for parturition onset and labor as well (15, 17, 56–63). Focusing on males, evidence of eCB direct action into the testis has been provided in most vertebrates [fish (8, 64), frogs (32, 57, 65–68), mammals (21, 69–74)), whereas an ECS has also been described in spermatozoa (SPZ) collected from sea urchin (75), amphibians (65), rodents (76–79), bull (80), boar (81), and human (82–85). A specific and significant association between the use of marijuana and the occurrence of non-seminomatous and mixed testicular germ cell tumors (TGCT) has been recently reported in humans (86–88); although a deep characterization of ECS has never been provided in TGCT patients yet, these data may suggest that the recreational and therapeutic use of cannabinoids may represent a risk factor for TGCT. In general, a relationship between the expression of cannabinoid receptors and the outcome of sex-steroid-dependent cancer has been documented, thus the imbalance in the ECS and its interaction with sex-steroid hormone homeostasis may promote cancer development, proliferation, and migration [for recent review, see Ref. (89)]. Defects in eCB signaling or eCB tone have recently been reported in rat treated with HU210 – a synthetic analog of THC – (90) as well as in clinical cases of male infertility in humans (85, 91). Consistently, genetic inactivation of the AEA-hydrolyzing enzyme, Faah (Fatty acid amide hydrolase) results in increased levels of AEA in the male reproductive system that negatively affect sperm motility and impair sperm fertilizing ability (92), whereas defects in the acquisition of sperm motility during the epididymal transit have been reported in Cb1^−/− mice (76, 77). Thus, ECS is nowadays considered a potential therapeutic target in male infertility. ECS is widely expressed in testis in both germ and somatic cells, and a map of ECS localization in several species is provided in Table 1. The first intratesticular targets of eCBs to be identified were the Leydig cells (21, 93), consistent with the low basal testosterone production observed in both Cb1^−/− mice and AEA-treated controls, providing evidence of mechanisms other than the AEA/THC-dependent downregulation exerted at hypothalamic/pituitary levels. The direct effect of Bhang (cannabis) on 3β-HSD, a well-known marker of Leydig cell activity, also confirmed this issue (79). The involvement of CB1 signaling in the control of Leydig cell activity is not restricted to steroid (both testosterone/estradiol) production (21, 22, 93), but also extends to Leydig cells ontogenesis. In fact, as reported by Cacciola et al. (72), CB1 expression in differentiating adult Leydig cells negatively correlates with cell division and the characterization of Cb1^−/− mice phenotype revealed a 30% decrease in Leydig cells number (72), as well as low circulating estradiol level (22) [for recent review, see Ref. (94)].

In the germinal compartment, AEA reduces the spermatogenic output inducing the apoptosis of Sertoli cells (70) in a mechanism reversed by FSH-dependent activation of aromatase and by estradiol-dependent upregulation of Faah (71). Recent studies carried out by Grimaldi et al. (95) demonstrated that in mature Sertoli cells Faah gene is a direct target of estradiol whose promoter contains two proximal estrogen-responsive element (ERE) sequences named ERE2/3. In vivo, a mechanism involving the binding of ERβ to ERE 2/3 and the epigenetic modifications of Faah gene proximal promoter (demethylation of both DNA at CpG site and histone H3 at lysine 9) has been demonstrated (95); consistently FAAH silencing abolished estrogen protection against AEA-dependent apoptosis (95). Thus, AEA content finely toned by its hydrolyzing enzyme FAAH is a fundamental tool to prevent the apoptosis in Sertoli cells.

Beside the activity exerted on Sertoli cells, eCBs are critical for the progression of spermatogenesis from mitotic stages throughout the meiotic stages and spermiogenesis events. In such a context, the FAAH-dependent modulation of eCB tone and the cell-specific expression of CB1, CB2, and TRVP1 provide evidence of multiple, differential eCB-dependent signaling involved in the spermatogenetic events. In mouse, decreasing levels of 2-AG have been detected from spermatogonia (SPG) to spermatocytes (SPC) and spermatids (SPT), suggesting that 2-AG, through CB2 – the receptor highly expressed just in mitotic and meiotic stages, but retained in residual body during the spermiogenesis – may act as an autocrine/paracrine mediator during spermatogenesis (73). Conversely, the high expression of Trpv1 observed in meiotic stages (73) and the massive germ cell depletion detected in Trpv1 null mice (96) candidate TRPV1 as a controller of meiotic stages. Very recently, the involvement of both CB1 and TRPV1 in the opposite modulation of testicular GnRH signaling (15, 68, 97) – a master system involved in the control of both spermatogenesis progression and steroidogenetic activity – has been reported in the anuran amphibian, the frog R. esculenta (97), a seasonal breeder in which two GnRH molecular forms (GnRH-I and GnRH-II) and three GnRH receptors (GnRH-RI, -RII and -RIII) have been characterized in testis (68). In such a context, AEA might act as an autocrine/paracrine factor via CB1 and as an intracrine signal via TRPV1; thus, it might be hypothesized that AEA, through the activation of specific receptors, switches on/off testicular GnRH signaling, leading to germ cell progression (Figure 1).

However, in mammalian and non-mammalian vertebrates, CB1 activity is linked to the control of post-meiotic stages (32, 65, 69, 73). In particular, it has been suggested that ECS controls different steps of spermiogenesis that is the phase of spermatogenesis consisting in the differentiation of SPT in SPZ. In particular, post-meiotic haploid round spermatids (rSPT) undergo biochemical and morphological changes becoming elongated cells (eSPT) and then SPZ. Sperm cells are differentially released from Sertoli cells by spermiation, a process characterized by species-specific features (65, 98). In mammals, SPZ undergo further transformations in the epididymis, which enables SPZ for fertilization (76, 77). These cellular modifications, and in particular some structural changes observed in SPT (i.e., acrosome development, nuclear shaping and chromatin condensation), seem to be related to ECS and in particular to CB1 activity.

A detailed immunolocalization of CB1 has been reported in rat SPT. CB1 appears in rSPT, around the nucleus, during acrosome development; the signal is retained in the head of elongating and condensing SPT, always close to the acrosome region, suggesting a role for CB1 in spermiogenesis, probably in chromatin packaging and in acrosome and/or cellular shape configuration (57, 72, 81). In agreement, several data demonstrate that CB1 regulates acrosome reaction, chromatin condensation, and nuclear size of SPZ (82, 99). Recent observations demonstrate that CB1 is involved in chromatin remodeling of SPT. In fact, during spermiogenesis, as the nucleus elongates and assumes a specie-specific shape, the chromatin condenses. It is worth noting that chromatin condensation differentially occurs, depending on the species. In mammals, chromatin condensation starts in eSPT producing condensing and then condensed SPT, which are mature elongated cells with strongly packaged chromatin (100). Many events characterize these chromatin cyto-architecture changes (101). Early during spermiogenesis, it is possible to observe the expression and storage of specific proteins involved in condensation and in DNA integrity maintenance, such as transition proteins (TNPs) and protamines (PRMs) (102). Others events concern the following: (i) displacement and degradation of the nucleosome structure; (ii) histone replacement by TNPs and then by PRMs; (iii) transcriptional silencing; (iv) DNA repair; and finally, (v) repackaging of the protaminated chromatin into toroidal structures (103, 104) [for recent review, see Ref. (94)]. These events strongly preserve DNA by damage and are involved in mechanism related to sperm maturation. Indeed, it is well known that inefficient expression or activity of TNPs/PRMs deranges histone displacement and causes production of SPZ with histone retention, incomplete chromatin condensation, and DNA damage (74, 105, 106). In both humans and rodents, abnormal levels of sperm DNA damage are associated with lower conception, implantation, and fecundity rates, and with higher miscarriage probability (95, 107, 108). In this context, Chioccarelli et al. (74) showed that Cb1 gene deletion negatively influences chromatin remodeling in SPT, by reducing either transition protein 2 (Tnp2) levels or histone displacement. Secondary effects, related to the inefficient histone displacement (i.e., histone retention, uncondensed chromatin, DNA damage, and nuclear size elongation) have been postulated (22, 74). In agreement, in vivo and in vitro experiments show that AEA is able to act locally and upregulate Tnp2 mRNA levels through CB1, via PKC/PKA pathways (17, 74). Furthermore, in caput epididymis from CB1^−/− mice, the percentage of SPZ retaining histones as well as the percentage of SPZ with uncondensed chromatin or with DNA damage, is higher as compared to normal mice. Interestingly, DNA damage increased during the epididymal transit, from caput to cauda, suggesting that CB1 preserve sperm DNA integrity of SPZ during epidydimal transit (74).

Recently, it has been demonstrated that estradiol, probably via stimulatory effects on FSH secretion and/or directly via paracrine actions within the testis, preserve chromatin condensation, and DNA integrity of SPZ, likely by promoting histone displacement in SPT (99). Indeed, it has been reported that CB1^−/− male mice show low levels of circulating E₂, and when treated with 17β-estradiol, they rescue sperm chromatin quality by restoring histone content, chromatin packaging, DNA integrity, and nuclear length of SPZ (22, 99). These results corroborate the intriguing findings that the small nucleus of SPZ, containing chromatin that did not retain histones, appear fully condensed and able to preserve DNA from damage. On the contrary, the longer nucleus of SPZ, containing chromatin that retained histones, is uncondensed and unable to avoid DNA damage. The emerging exciting idea is that sperm nuclear dimensions can be a good marker for SPZ chromatin quality useful to select the SPZ qualitatively suitable for intracytoplasmic sperm injection (99).