The Cannabinoid System and Male Reproductive Functions
Abstract
Cannabinoids, the main active components of marijuana, have been shown to exert different adverse effects on male reproduction both in vertebrates and invertebrates. In vivo, cannabinoids exert negative effects on hypothalamic–hypophyseal reproductive hormone secretion and testicular endocrine and exocrine functions. Furthermore, a large amount of experimental data obtained in vitro have clearly shown that cannabinoids negatively influence important sperm functions, including motility and acrosome reaction, two fundamental processes necessary for oocyte fertilisation. These inhibitory effects are mediated by the direct action of cannabinoids on sperm through the activation of the cannabinoid receptor subtype CNR1 that has been shown to be expressed in mature sperm. In the present paper, we briefly review the effects of cannabinoids and endocannabinoids, a particular group of endogenously produced cannabinoids, on male reproductive function.
Marijuana, the most widely used recreational drug worldwide (1), contains a large number of different active compounds known as cannabinoids, and Δ9-tetrahydocannabinol (THC) has been recognised as the main active molecule (2). Beside natural cannabinoids, different tissues can synthesise a group of related unsaturated fatty acid derivatives named endocannabinoids that act as endogenous ligands for the cannabinoid receptors. This so called ‘endocannabinoid system’ regulates many different functions in all tissues via interaction with two specific receptors, cannabinoid receptor subtype 1 (CNR1) and cannabinoid receptor subtype 2 (CNR2), whose tissue distribution and function have been previously described (3–5). A number of different endocannabinoids have been isolated within biological fluids, anandamide (N-arachidonoylethanolamine, AEA) and 2-arachidonoyl glycerol (2-AG) being the main components (3). In particular, AEA is the most abundant in reproductive secretions, thus suggesting a possible role for the endocannabinoid system in reproduction (6). CNR receptors have been identified along male reproductive tract: CNR1 receptors have been detected in the testis, prostate and vas deferens (7–10). Furthermore, functional CNR1 receptors have been found in sperm in different vertebrate and invertebrate species (10–12). To date, unequivocal data have been reported showing that cannabinoids control male reproductive functions both directly and indirectly, depending of the expression of the CNR receptor subtypes along the tissues controlling the testicular function (Table 1) with important final effects on fertility in the male (10–12).
| Tissue | Cannabinoid receptor subtype | Reference |
|---|---|---|
| Hypothalamus | ||
| GnRH secreting cells | CNR1/CNR2 | (20) |
| Pituitary | ||
| Gonadotrophs | CNR1 | (21) |
| Testis | ||
| Germ cells | CNR1 | (10) |
| Sertoli cells | CNR2 | (24) |
| Leydig cells | CNR1 | (8) |
| Epididymis | n.d. | – |
| Vas deferens | CNR1 | (38) |
| Seminal vesicles | n.d. | – |
| Prostate | CNR1 | (37) |
| Corpus cavernosum | CNR1 | (39) |
- CNR1, cannabinoid receptor subtype 1; CNR2, cannabinoid receptor subtype 2; n.d., not determined.
Cannabinoids and spermatogenesis
Cannabinoids and hypothalamus-pituitary-testis axis
Spermatogenesis is a long a complex process controlled by a number of different mechanisms (13). Gonadotrophins (FSH [follicle stimulating hormone] and LH [luteinising hormone]) play a crucial role in the induction and maintenance of spermatogenesis in mammals including man (14), involving a stimulatory effect of Sertoli and Leydig cells and a complex series of secreting events that follow. It is known that in males, cannabis smoking decreases plasma LH levels, leading to decreased plasma testosterone (15), although contradictory results have been reported (16, and references therein). FSH secretion has also been shown to be modulated by cannabinoids (17) and the combined effects on the gonadotrophins lead to decreased sperm count in males that has been consistently observed in men who smoke cannabis (18).
The effects of cannabinoids on gonadotrophin secretion seem to be localised upstream to the pituitary, and, in particular, at the pre-optic area of the hypothalamus where CNR1 receptors have been localised and where LH releasing hormone (LHRH)-secreting neurones reside (19). At this level, cannabinoids seem to modulate negatively the activity of LHRH-secreting neurones by direct and indirect mechanisms (20). Beyond the well known effect of cannabinoids on gonadotrophin secretion at the hypoyhalamic level modulating gonadal function, previous observations have localised CNR1 in the anterior pituitary, suggesting a direct effect of cannabinoids on LH and FSH secretion at the pituitary level (21).
Cannabinoids and male germ cells
Evidence primarily from animal studies has shown that CNR1 is expressed in germ cells, from spermatogonia to mature sperm. For instance Gye et al. (8) demonstrated the presence of this receptor in spermatogonia, primary spermatocytes and sperm in mouse testis, and these observations have been replicated by Cobellis et al. (11) in the testis of invertebrates. Current knowledge clearly shows that cannabinoids negatively influence the spermatogenetic process, reducing germ cell proliferation and reproductive organ weight (11, 22). Sertoli cells constitute one of the main components of the seminiferous tubule, and their number – fixed at puberty – is closely related to the total sperm production. Sertoli cells surround and nurse germinal cells during their maturation. By mediating the actions of all hormonal stimuli regulating the spermatogenetic process they provide a finely tuned microenvironment leading to germ cell maturation from spermatogonium to mature sperm (23).
CNR2 has been recently described in Sertoli cells, modulating apoptosis in these cells (24). Moreover, Leydig cells have been shown to express CNR1, which, when activated, induces a reduction of testosterone production, thus further inhibiting the spermatogenetic process (because high local levels of testosterone are required to maintain spermatogenesis (8)). Furthermore, testosterone is necessary for epididymis and seminal vesicle activity, which influence important sperm functions such as motility (25). Thus, the complex events coordinating the male spermatogenesis and integrating brain and testicular messages are modulated by cannabinoids at both the hypothalamic–pituitary and the testicular level.
Cannabinoids and sperm functions
Cannabinoids and sperm motility
Since the first report published by Perez et al. (26) in 1981, experimental data have accumulated confirming that cannabinoids negatively influence the motility of sperm in different mammalian species. Apart from observations obtained in sea urchins showing no significant effects of cannabinoids on sperm motility (12), these substances inhibit sperm motility in different mammalian species, including man (10, 27). The observations on the negative effects of cannabinoids on sperm motility were reported in the mid 1970s and early 1980s; however, CNR1 and CNR2 were not identified until the early 1990s, when expression of CNR1 was demonstrated in the testis (28). Only recently, taking a pharmacological experimental approach, the inhibitory effects of cannabinoids on mammalian sperm motility have been attributed to CNR1 activation (10). Very recently, Cobellis et al. (11) replicated these results in sperm from Rana esculenta, taking the same pharmacological approach. Furthermore, the same authors extended these observations by means of a molecular genetic experimental design, showing that sperm from CNR1-knockout mice show a dramatic increase in motility percentages in the caput epididymis. This clearly demonstrates that the lack of CNR1 signalling increases the acquisition of sperm motility (9).
Although the studies evaluating the effects of cannabinoids on sperm motility are not abundant, it seems that in general the activation of CNR1 inhibits motility in invertebrate and vertebrate sperm, at least in vitro. Contradictory results have been reported, however, regarding the effects of cannabinoids ingestion, as well as marijuana smoking, on human sperm motility in vivo. Variation in these results may be owing to the experimental design or the small number of subjects participating to the different clinical studies (29).
Cannabinoids and sperm capacitation
After ejaculation, sperm have to undergo a complex – and still unclear – series of biochemical and morphological events collectively known as ‘capacitation’, before they are capable of fertilising an egg (30). This includes remodelling the lipid plasma membrane, which is required to improve the ability of sperm to respond to stimuli that induce the acrosome reaction, facilitating the interaction with the egg. These changes are activated by still not fully elucidated mechanisms involving the adenylate cyclase/cAMP/protein kinase A (PKA) signalling pathway, leading to tyrosine phosphorylation of different proteins (31). These processes are inhibited by cannabinoids and CNR1 activation (27, 31). These effects are specific: they are counteracted by sperm pre-incubation with the CNR1 antagonist SR141716.
Physiologically, sperm capacitation occurs within the female genital tract and it has been demonstrate that sperm leaving seminal plasma and reaching the uterus and oviduct swim in an external milieu presenting progressively reduced concentrations of endocannabinoids (6). As sperm approach the egg, they encounter the lowest concentrations of endocannabinoids present in the female genital tract, thus suggesting that the activation of the endocannabinoid system in sperm seems to maintain the sperm in an uncapacitated, quiescent condition before interacting with the egg (10).
Cannabinoids and sperm acrosome reaction
Sperm capacitation is a fundamental pre-requisite for exocytosis of the acrosome, a specialised secretory granule located at the apex of sperm head (32). Sperm acrosome reaction occurs as the sperm faces the egg, and leads to the release of proteolytic enzymes necessary for penetration of the egg’s coats, the exposure of specialised sperm membrane sites that allow attachment and fusion with the egg’s plasma membrane and then to fertilisation (33). Sperm that do not undergo the acrosome reaction are not able to bind to the egg’s zona pellucida and cannot fertilise it. Substantially homogeneous data exist indicating that CNR1 activation reduce the ability of sperm to undergo acrosome reaction both in vertebrates and invertebrates (10, 12, 27, 31). These data further confirm the inhibitory effects of cannabinoids on sperm fertilising ability.
Importantly, since cannabinoids do not affect fertility of eggs, these effects are directed only to sperm (27). The mechanisms involved in cannabinoid inhibition of the sperm acrosome reaction are still unknown. It is well known that ion channels and regulation of ion fluxes through the sperm plasma membrane are crucial for the acrosome reaction, and the most important physiological regulator appears to be Ca2+ (34). In this regard we have previously shown that the endocannabinoid AEA did not modify intracellular Ca2+ concentrations in human sperm, possibly ruling out any interfering effect of cannabinoids on Ca2+ signalling (10). CNR1 is a G-protein-coupled receptor that has been shown to inhibit adenylate cyclase activity (3). Intracellular levels of cAMP rise during acrosome reaction, cell-permeant cAMP analogues induce the acrosome reaction, and pharmacological inhibitors of cAMP-dependent PKA reduce this exocytotic event (35, 36): these observations suggest that the adenylate cyclase/cAMP/PKA system plays a primary role in the signalling pathway leading to sperm acrosome reaction and egg fertilisation. The inhibitory effect of cannabinoids on this fundamental signalling pathway, as recently described in mammalian sperm (31), might explain the negative effect these agents have, through CNR1 activation, on sperm capacitating and acrosome reaction.
Conclusions
Marijuana is the most commonly used recreational drug worldwide and it is now well recognised that the active components of marijuana – as well as endogenously synthesised endocannabinoids – negatively affect sperm functions, both in invertebrates and vertebrates, impairing the fertilisation process and thus reproductive function. This is important, given the prevalence of marijuana smoking particularly in the young. These observations, together with the demonstration that the male reproductive tract possesses the whole enzymatic machinery for the synthesis and metabolism of endocannabinoids, could suggest the existence of particular clinical situations, beyond marijuana smoking, characterised by a pathological increase of endocannabinoids within the male reproductive tract, that could lead to an impairment of sperm function.
Furthermore, if we consider that the use of cannabinoids has been legally authorised in some countries for the treatment of some pathological conditions (such as appetite stimulation in patients with AIDS, treatment of nausea and vomiting, reduction of symptoms in some neurological diseases, reducing intraocular pressure in glaucoma patients, treatment of depression and pain), the possible negative effects of these therapeutic cannabinoid agonists on human male fertility have to be taken into careful account.
Conflicts of interest
MR, CP and RV have received speaker's fees from Sanofi-Aventis.
