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Department of Medical and Surgical Sciences, Clinica Medica 3 (M.R.); Department of Biological Chemistry (F.I.P., G.C.); and Department of Histology, Microbiology, and Medical Biotechnologies, Center of Male Gamete Cryopreservation (M.F., C.F.), University of Padova, Padova, Italy
Address all correspondence and requests for reprints to: Dr. M. Rossato, Department of Medical and Surgical Sciences, Clinica Medica 3, University of Padova, Via Ospedale 105, 35128 Padova, Italy. E-mail: marco.rossato{at}unipd.it.
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Introduction |
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-9-tetrahydrocannabinol, the primary psychoactive cannabinoid in marijuana, has pronounced adverse effects on male and female reproductive systems (2). Beside cannabinoids, a family of unsaturated fatty acid derivatives with cannabinoid-like actions have been identified in a number of different biological systems and are known as endocannabinoids; the main ones are anandamide [N-arachidonoylethanolamine (AEA)], 2-arachidonoylglycerol, and 2-arachidonoylglycerylether (noladin ether) (3, 4, 5). In the various cellular systems studied to date, cannabinoids and endocannabinoids exert their effects through the activation of specific cannabinoid receptors (CB-R) located on the surface of the target cells. Two different CB-R subtypes have been identified and cloned, CB1-R and CB2-R (6). CB1-R was first isolated in the brain (and then it was known as the central CB-R), but it is now clear that it is also expressed in many other tissues as well as peripheral nerves, spleen, peripheral leukocytes, uterus, testis, vascular endothelial and muscle cells, eye, and placenta (6). CB2-R was originally described in spleen and in cells of the immune system (6). The protein sequences of human CB1-R and CB2-R show the seven highly hydrophobic regions typical of the G protein-coupled receptor superfamily (6, 7). Both CB1-R and CB2-R are joined to a number of transduction pathways coupled to pertussis toxin-sensitive Gi/Go proteins: inhibition of adenylate cyclase activity and cAMP production; inhibition of voltage-gated Ca2+ channels of the L, N, P, and Q types; and activation of K+ channels; phospholipase C, and various protein kinases (6, 7). During the past years a great deal of data have been accumulating demonstrating that cannabinoids/endocannabinoids have important actions in the reproductive system. It has been shown that marijuana smoking is associated with the suppression of LH secretion and shortened luteal phase in women (8, 9) and to reductions of LH and testosterone plasma levels, leading to impotence and reduced sperm count, in men (10). Several experimental and clinical studies have shown adverse effects of marijuana exposure on embryo development and in early pregnancy (11, 12). Finally, the demonstration that female and male genital tract fluids contain significant concentrations of endocannabinoids suggests that these substances may influence important processes controlling sperm/egg functions and gamete interactions (13, 14). It is well known that cannabinoids inhibit fertilization in the sea urchin by reducing the fertilizing ability of sperm (15, 16), leaving egg receptivity unmodified and interfering only with the physiological mechanisms leading to sperm acrosome reaction (16), an event necessary for egg fertilization that occurs as sperm approach the egg (17).
Previous studies have shown that the human testis expresses the CB1-R subtype, but the identity of the specific testicular cell type(s) expressing this receptor has not been fully elucidated. Using an immunohistochemical technique, Wenger et al. (18) demonstrated the presence of CB1-R in mouse Leydig cells; other researchers have shown the presence of cannabinoid-binding sites in rat Sertoli cells that have been shown to express CB2-R and not CB1-R (19). In human sperm, specific binding of [3H]CP-55,940 to human sperm has been reported, suggesting the presence of cannabinoid receptors, but the specific CB-R subtype expressed in human sperm has not yet been characterized (16).
In the present study we evaluated the presence and localization of CB-Rs in human sperm, investigating the effects of their activation in human sperm functions and the signal transduction mechanisms involved in cannabinoid action.
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Materials and Methods |
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Anandamide, tetraethylammonium, rhodamine 123 (R-123), propidium iodide, and fluorescein isothiocyanate (FITC)-conjugated pisum sativum lectin were purchased from Sigma-Aldrich Corp. (Milan, Italy). SR-141716 was a gift from Dr. U. Pagotto (University of Bologna, Bologna, Italy). Anti-P-Y monoclonal antibodies were purchased from ICN Biotechnology (Irvine, CA). Fura-2/AM and bis-oxonol were purchased from Molecular Probes (Eugene, OR). Rabbit anti-CB1 polyclonal antibody and goat anti-CB2 polyclonal antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Goat antirabbit IgG-rhodamine-coupled antibodies were a gift from Prof. R. Rizzuto (University of Ferrara, Ferrara, Italy). Protease inhibitor mixture was obtained from Calbiochem (Darmstadt, Germany). BWW medium and Isolate (density gradients) were purchased from Irvine Scientific (Santa Ana, CA). All other chemicals were of analytical grade.
Semen collection
We evaluated five normozoospermic healthy men referred to our center for semen donation. Semen culture was negative, and antisperm antibodies were absent in all subjects. No subject had a history of previous cryptorchidism, testicular torsion, or genital tract infections. Each patient gave written informed consent for the use of his sperm sample.
Experimental protocol
Semen samples were collected after 3 d of sexual abstinence in sterile containers. After fluidification at room temperature for 30 min, standard seminal parameters were examined according to the WHO laboratory manual (20). All experiments were performed using motile sperm isolated by density gradients (Isolate, Irvine Scientific) following the manufacturer’s instructions. Sperm isolated by this method were collected, washed, and resuspended in BWW medium, adjusting the sperm concentration to 10 x 106/ml. Sperm samples were then incubated in the presence and absence of anandamide at different concentrations (0.1, 1.0, and 10.0 µM) for up to 60 min. Sperm viability and motility were evaluated after 0, 15, 30, and 60 min of incubation in the different experimental conditions described above. Sperm acrosome reaction was evaluated after 60-min incubation in the presence and absence of anandamide (1.0 µM). In separate experiments we evaluated the effects of anandamide (1.0 µM) on sperm acrosome reaction induced by the calcium ionophore ionomycin (1.0 µM). The effects of anandamide on sperm mitochondrial activity was evaluated after incubation with this endocannabinoid at different concentrations for 15 min.
When evaluating the effects of CB1-R activation by anandamide, some experiments were preceded by sperm preincubation with the specific CB1-R antagonist SR-141716 for 45 min before anandamide addition. Parallel control experiments were performed to evaluate the effects of SR141716 alone on sperm motility and viability.
CB1-R identification by Western blot
Sperm isolated as described above were resuspended in BWW medium (40 x 106 ml–1 for each sample) and centrifuged for 6 min at 6,000 rpm, and the packed cells were subjected to lysis in 200 µl buffer containing 25 mM Tris (pH 8), 0.02% NaN3, 1 mM EDTA, 1 mM sodium orthovanadate, and protease inhibitor cocktail and submitted three times to sonication for 6 sec each time at 35 mW. Membranes were separated from the cytosol by centrifugation (16,000 rpm for 40 min at 4 C) and washed once in lysis medium. Aliquots of membranes (15 µg) and diluted supernatants (15 µl) were subjected to SDS-PAGE (10% gels), transferred to nitrocellulose membranes, and immunostained with the appropriate antibodies for CB1-R and CB2-R subtypes. For immunostaining, the proteins transferred to nitrocellulose membranes were incubated with the appropriate antibody, followed by the biotinylated second antibody, and developed using an enhanced chemiluminescence detection system (ECL, Amersham Biosciences, Arlington Heights, IL).
CB1-R gene expression
To evaluate the expression of the CB1-R gene in human sperm, total RNA was isolated using RNA-Bee (Tel-Test, Inc., Friendswood, TX), according to the manufacturer’s instructions. The RT reaction was carried out using a commercially available kit (Sensiscript Reverse Transcriptase, Qiagen, Chatsworth, CA). Briefly, 50 ng total RNA were primed with 20 pM random hexamers and incubated for 60 min at 37 C. For each sample, 1x RT buffer [10 mM Tris-HCl (pH 8.8), 50 mM KCl, and 0.1% Triton X-100], 0.5 mM of each deoxy-NTP, 10 U ribonuclease inhibitor, and 1 µl Sensiscript reverse transcriptase in a volume of 20 µl were combined. Oligonucleotide primers for CB1-R amplification had the following sequences: sense primer, 5'-CCAGTGTTCACAGGGCCGCAG-3'; and antisense primer, 5'-GGGTTCTTAGACTTCCAATTGTGTAGCC-3', and amplified a 398-bp fragment for the CB1-R sequence. PCR was carried out using 12 pmol of each primer in a final volume of 25 µl containing 1x PCR buffer [20 mM Tris-Cl, 50 mM KCl (pH 8.4), and Triton X-100], 2.5 mM MgCl2, 2.5 U Taq DNA polymerase, 0.2 mM deoxy-NTPs, and 2 µl cDNA for 37 cycles at 94 C for 1 min, 58 C for 1 min, and 72 C for 1 min. The mixture was electrophoretically separated on a 2% agarose gel. The band was excised, purified by Sephaglas BandPrep Kit (Amersham Biosciences), and sequenced (BMR, Sequencing Service C.R.I.B.I., University of Padova, Padova, Italy). To exclude the contamination with genomic DNA, before any cDNA experiment we performed a PCR with intronic primers to assess the presence of genomic DNA.
Localization of CB1-R in human sperm by immunocytochemistry
For immunostaining of sperm to localize the CB1-R, sperm were isolated as described above, suspended in BWW, smeared on a cleaned slide, allowed to air-dry, fixed in cold methanol (–20 C) for 5 min, washed in three changes of PBS, and then allowed to air-dry. For indirect immunofluorescence staining, slides were incubated with anti-CB1-R antibody at a dilution of 1:200 (1 µg/ml) for 60 min at 37 C. Slides were then washed in PBS three times, incubated with rhodamine-conjugated goat antirabbit IgG secondary antibody for 2 h at 37 C in the dark, washed in PBS three times, coverslipped, and examined by fluorescence microscope. Images were recorded using a Nikon digital camera (Coolpix 900, Nikon Corp., Tokyo, Japan). In addition, control experiments were carried out to check the specificity of the CB1-R antibody binding by omitting the primary antibody before anti-CB1-R antibody incubation. The specificity of the CB1-R binding was also confirmed by preincubating sperm in the presence of high concentrations of the CB1-R agonist anandamide (100 µM) for 1 h at room temperature (to saturate anandamide-binding sites) before incubation with the CB1-R primary antibody.
Evaluation of sperm motility and viability
Sperm motility was assessed by means of light microscopy, examining an aliquot of each sperm sample at the different times of incubation in the presence and absence of anandamide at different concentrations. Sperm motility was expressed as the percentage of total motile sperm. Sperm viability was determined by red-eosin exclusion test and expressed as the percentage of viable sperm.
Acrosomal status evaluation
To evaluate the effects of anandamide on sperm acrosome reaction, sperm aliquots were incubated in the absence and presence of various anandamide concentrations for 60 min and then acrosomal status was evaluated using FITC-conjugated pisum sativum agglutinin and flow cytometric analysis. In brief, after incubation in the different experimental conditions described above, sperm aliquots were incubated with FITC-conjugated lectin (1.0 µg/ml) for 30 min at 37 C. Propidium iodide (10 µg/ml) was added to each sperm suspension 20 min after FITC-PSA addition and incubated for 10 min. After incubation, each aliquot was washed with prewarmed PBS (37 C) for 10 min at 600 x g. After washing, each sperm pellet was suspended in PBS, and flow cytometric analysis was performed by means of flow cytometry (FACSCAN, BD Biosciences, Milan, Italy). Nonviable sperm (propidium iodide positive) were detected using a fluorescence detector 3 (FL3; detecting photons with a wavelength >670 nm); acrosome-reacted sperm were FITC positive and were detected using fluorescence detector 1 (FL1; detecting photons with a wavelength in the range 515–545 nm). Nonsperm events were gated out of the fluorometric analysis as determined from the forward and side scatter analysis. Ten thousand gated events were recorded for each analysis.
Sperm capacitation
Motile sperm, isolated as described above, were incubated in capacitating medium (BWW and 0.3% human serum albumin) for 6 h at 37 C in a controlled atmosphere in the presence and absence of 1.0 µM anandamide, evaluating the percentage of acrosome-reacted sperm at the end of incubation in the different experimental conditions.
Intracellular Ca2+ concentration ([Ca2+]i) measurement in human sperm
[Ca2+]i was measured using the fluorescent probe fura-2/AM (21). Sperm isolated as described above were incubated for 30 min at 37 C in the presence of fura-2/AM (2 µM). After loading, sperm were washed by centrifugation at 800 x g for 10 min, resuspended in BWW medium, and maintained at room temperature until used. [Ca2+]i was measured in a LS50B PerkinElmer fluorometer (Norwalk, CT), equipped with a thermostated and magnetically stirred cuvette holder, using 1.0-ml sperm aliquots. The excitation wavelength was alternated between 350 and 380 nm, and emission fluorescence was continuously monitored at 505 nm.
Evaluation of sperm plasma membrane potential changes
Sperm plasma membrane changes were monitored using the potential sensitive fluorescent dye bis-oxonol as previously described (22). Briefly, 1.5 x 106 sperm were placed in a cuvette thermostated at 37 C containing the bis-oxonol solution (200 nM) in saline. After stabilization of the fluorescent signal, additions of anandamide (1.0 µM) were made. Excitation and emission wavelengths were 540 and 580 nm, respectively. In some experiments evaluating the roles of the CB1-R antagonist SR141716 and the K+ channel blocker tetraethylammonium in anandamide-induced plasma membrane variations, sperm suspensions were preincubated with each specific blocker for 45 min before anandamide addition.
Evaluation of mitochondrial activity
Mitochondrial activity was evaluated using the fluorescent probe R-123 (1.0 µg/ml) as previously described (23). R-123 was incubated with sperm suspensions for 20 min when propidium iodide was added to sperm suspensions (10 µg/ml, final concentration), and incubation was prolonged for an additional 10 min. Sperm samples were then used for mitochondrial functionality evaluation together with estimation of sperm viability by means of a flow cytometer (FACSCAN, BD Biosciences, Franklin Park, NJ). A total of 10,000 sperm were analyzed per sample using forward and side scatter profiles. For R-123 and propidium iodide frequency plots using green fluorescence (FL1; functional mitochondria) and red fluorescence (FL3; nonviable sperm) were used to assess mitochondrial activity. A 488-nm filter was used for excitation of fluorescent probes. Data obtained from flow cytometry were acquired and analyzed using CellQuest software (BD Biosciences, Milan, Italy).
Microscopic epifluorescence assessment
In addition to flow cytometry studies, after incubation with fluorescent dyes, R-123 sperm samples were washed and resuspended (1 x 106/ml) in BWW medium, smeared on a cleaned slide, covered with a coverslip, and viewed by fluorescence microscopy at x1250 (Nikon Eclipse 300, Nikon Corp.) to confirm and analyze the R-123 labeling pattern before and after incubation with anandamide.
Statistical analysis
Data are expressed as the mean ± SD and were analyzed by t test and ANOVA (StatView, Abacus Concepts, Inc., Berkeley, CA). P < 0.05 was chosen as the limit for statistical significance.
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Figure 1
shows a representative immunoblot from Western blot analysis of human sperm extracts from a typical experiment using sperm sample from a single donor as representative of the others that gave similar results. The specific anti-CB1-R monoclonal antibody recognized a band of the molecular size expected for CB1-R and corresponding to a single band of immunoreactivity with the same molecular weight identified in lysates from human brain (Fig. 1A). The anti-CB2-R antibody did not recognize any band in the blot, whereas a single band of immunoreactivity with the expected molecular weight for CB2-R was identified in lysates from human spleen (Fig. 1B).
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Considering the results of the Western blot experiments we investigated the expression of CB1 mRNA in human sperm. Figure 2 shows the PCR amplification from total RNA isolated from human sperm using the forward and reverse primer pair designed from the human CB1 cDNA sequence. The single band observed corresponds to the expected size of the coding region of the CB1 cDNA. No amplification was obtained from the negative control.
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A set of experiments was performed to localize CB1-R in human sperm using an immunofluorescent technique and a polyclonal antibody raised against the carboxyl-terminal region of the human CB1-R. In Fig. 3A, a typical image is shown of fluorescence localized predominantly to the sperm head and middle piece, leaving the tail almost completely unstained. Competition experiments to determine the binding specificity showed that the fluorescent signal was substantially undetectable when sperm were preincubated with excess anandamide (100 µM, as reported in Materials and Methods) before anti-CB1-R antibody incubation (Fig. 3B). No fluorescent signal was obtained when primary antibody was omitted, thus confirming the specificity of the antibody binding (Fig. 3C).
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Fig. 4 shows the effects of anandamide on sperm viability and motility. Anandamide inhibited significantly sperm motility in a dose dependent manner with a IC50 of about 0.1 µM. At the dose of 1.0 µM anandamide reduced sperm total motility to very low levels (as high as 10% of the total motile sperm), with 100% immotile sperm at a dose of 10.0 µM. The effects of anandamide on sperm motility were rapid, reaching maximal activity after 15 min of incubation (data not shown). On the contrary, sperm viability was not significantly affected by anandamide concentrations up to 1.0 µM, whereas at higher concentrations, sperm viability decreased. Thus, the reduction of sperm motility is due to toxic effects on sperm viability. The inhibitory effects of anandamide on sperm motility and viability were largely antagonized by the specific CB1-R antagonist SR141716, whereas the antagonist alone had no significant effects on sperm motility and viability up to a dose of 10 µM (Fig. 4). Treatment of sperm with increasing doses of anandamide did not induce any effect on acrosomal exocytosis and did not interfere with the acrosome reaction induced by the ionophore ionomycin (Fig. 5). On the contrary, anandamide (1.0 µM) inhibited the sperm acrosome reaction induced by incubation in capacitating medium (Fig. 5), confirming previous results obtained in human sperm (16).
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To investigate the possible signaling pathway initiated by CB1-R activation by anandamide, we evaluated its effects on sperm [Ca2+]i and plasma membrane potential. As shown in Fig. 6, anandamide did not induce any variation of sperm [Ca2+]i, whereas ionomycin was able to strongly increase [Ca2+]i. The effects of anandamide on sperm plasma membrane potential were also evaluated using the plasma membrane potential-sensitive fluorescent dye bis-oxonol as previously described (22). As shown in Fig. 7, anandamide induced a rapid plasma membrane hyperpolarization (Fig. 7, trace a) that was completely inhibited by sperm preincubation with the specific CB1-R antagonist SR141716 (Fig. 7, trace b). The addition to sperm suspension of the CB1-R antagonist SR141716 did not induce any modification of sperm [Ca2+]i (data not shown) and plasma membrane potential at a dose of 1.0 µM (Fig. 7, trace b). Sperm preincubation with tetraethylammonium (20 mM), a well known K+ channel inhibitor, completely reduced the hyperpolarizing effects of anandaminde in human sperm (Fig. 7, trace c), thus demonstrating the role of K+ flux in the hyperpolarizing effects of anandamide in human sperm.
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Examination of R-123-stained sperm revealed green fluorescence localized in sperm mitochondria in the middle piece, as expected (Fig. 8A). In control sperm (vehicle only), the large majority (86%) showed bright fluorescence localized in their mitochondria. Anandamide induced a dose-dependent decrease in the number and intensity of bright fluorescent mitochondria in sperm, indicating a progressive decline in sperm mitochondrial activity after incubation with this endogenous cannabinoid (Fig. 8C). The effects of various concentrations of anandamide on sperm mitochondrial activity, motility, and viability are reported in Fig. 9.
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Discussion |
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Previous studies have shown that human testis express transcripts of the CB1-R subtype (24) and that human sperm possess functional CB-Rs (16), but to date no direct evidence has been provided for their expression in human sperm. The results of the present study show that CB1-R is expressed in human sperm, as demonstrated by the presence of both mRNA and protein for this receptor. No evidence has been demonstrated for the presence of CB2-R subtype at protein level, confirming the observations made in pharmacological studies (16). The experimental data concerning the effects of cannabinoids on human sperm have clearly shown that these substances inhibit sperm motility in a dose-dependent manner, and the results of the present study confirm those previous observations (14, 15, 16). Involvement of the CB1-R in the actions of anandamide is also demonstrated by the antagonizing effects of SR141716, a specific antagonist of the CB1-R. No direct effects were observed in the induction of the acrosome reaction by anandamide in human sperm, in agreement with previous observations (16). Furthermore, the results of the present study confirm the inhibitory effects of anandamide on acrosome reaction induction after incubation in capacitating medium. On the contrary, anandamide did not inhibit the acrosomal loss induced by the Ca2+ ionophore ionomycin.
The mechanisms underlying the effects of anandamide in human sperm have not yet been elucidated, although influences of cannabinoids on ion channels, adenylate cyclase activity, and protein phosphorylation might be involved. The activation of the CB1-R in various cell types has been coupled to ion channel modulation (6, 7, 25). We did not observe any stimulatory effect of anandamide on sperm [Ca2+]i, thus excluding any role of calcium in the effects of this endocannabinoid in human sperm after CB1-R activation. The monitoring of sperm plasma membrane potential revealed that anandamide induced a rapid plasma membrane hyperpolarization that was reduced by sperm preincubation with tetraethylammonium, a well known inhibitor of K+ channels (26). These results show that anandamide activates an efflux of K+ from sperm cytoplasm, leading to plasma membrane hyperpolarization. These effects were fully antagonized by the CB1-R antagonist SR141716, demonstrating the specificity of anandamide action. It is still unclear how these biological phenomena interfere with sperm activities, but the role of plasma membrane hyperpolarization in the regulation of sperm functions has been previously suggested (27).
It has been reported that cannabinoids alter mitochondrial function in living cells (28, 29), and it has recently been demonstrated that tetrahydrocannabinol, the main active compound of marijuana, inhibits mitochondrial activity of epithelial cells from a lung cancer cell line, with inhibitory effects starting at concentrations less than 1–3 µM (28). We evaluated the effects of cannabinoids on mitochondrial function in human sperm, observing that anandamide inhibits sperm mitochondrial activity in a dose-dependent manner. These inhibitory effects on mitochondrial activity were rapid, starting after 15 min of incubation in the presence of anandamide. At concentrations up to 1.0 µM, anandamide significantly inhibited sperm motility, leaving sperm viability only slightly altered, thus indicating that the effects of anandamide on sperm motility are specific and not due to toxic effects up to these concentrations. It is well known that the kinetic activity of sperm results from the various components involved in motility activation as well as the integrity of all flagellar structural proteins, oxidation of energetic substrates and ATP production, transformation of chemical energy into mechanical movement, and the activity of all enzymes involved in the flagellar beating (30, 31). The decline in mitochondrial activity would be expected to decrease energy supply, thus affecting various sperm functions as well as motility. Different hypotheses may explain the negative effects of cannabinoids on mitochondria: 1) cannabinoids may interfere with mitochondrial electron transport (32, 33); 2) cannabinoids could impair mitochondrial activity via depletion of NADH, as previously shown (28); or 3) cannabinoids may interfere with the mitochondrial permeability transition pore complex, as recently suggested (28).
The physiological significance of the present observations are still unclear. It has been demonstrated that seminal plasma contains a significant amount of anandamide, which decreases progressively in the uterus, oviduct, and follicular fluid (13). As sperm leave seminal plasma and approach the egg in the female reproductive tract, they are exposed to progressively decreasing anandamide concentrations. It has been speculated that the high anandamide concentrations observed in seminal plasma may contribute to maintain sperm in a quiescent metabolic condition to be fully activatable within the female reproductive tract (16). In this respect the decreasing anandamide concentrations of female reproductive tract secretions could reduce its inhibitory effect on sperm, thus rendering them suitable for capacitation and fertilizing ability acquisition. Furthermore, it is possible that anandamide, together with other endogenous cannabinoids, could be increased in different pathological conditions, thus leading to alterations in sperm function, as described recently in a group of women with recurrent abortion (12). This could be important in pathological conditions in which anandamide production and/or catabolism is altered within male and, especially, female reproductive tracts, where the inhibitory effects of endocannabinoids may alter sperm functions, thus reducing their fertilizing ability.
The results of the present study may have other significant clinical implications. It is well known that marijuana smokers show an alteration in fertility potential (2, 16). This could be due at least in part to a negative influence on the mechanisms regulating the hypothalamic-pituitary-testicular axis, but also to a direct inhibitory effect of cannabinoids on human sperm (16, 18). Furthermore, cannabinoids may alter sperm functions in vivo via indirect mechanisms due to alteration of the functionality of Leydig and Sertoli cells that express CB-Rs (18, 19, 34) and of which steroidal and protein secretions play a primary role in the regulation of germ cell maturation to produce fully competent mature sperm.
Because marijuana is widely used around the world, especially among the young (1, 35), the negative effects of cannabinoids on male reproductive functions should be carefully considered. Tetrahydrocannabinol, the major active constituent of marijuana, has a long half-time, and because this highly lipophilic substance is easily stored in fat tissue (36), it is possible that subjects addicted to chronic use and abuse of marijuana may experience elevated cannabinoid concentrations in their biological fluids, with important negative effects on fertility. In this respect it has been shown that after tetrahydrocannabinol administration, approximately 0.06% and 0.02% of the administered dose concentrates in brain and testis, respectively, thus confirming the efficacy of the blood-brain and blood-testis barriers (37, 38). In contrast, within epididymal fat, tetrahydrocannabinol concentrations are 8 times higher than those in the brain after a single administration of tetrahydrocannabinol, and concentrations are 40–80 times higher after multiple administrations (36). Sperm reside within the epididymis before being ejaculated; thus, the presence of very high concentrations of cannabinoids in this region may significantly alter sperm functions. Furthermore, tetrahydrocannabinol concentrations up to 0.8 µM have been detected in peripheral blood of subjects after marijuana cigarette smoking (37, 38). These systemic concentrations are very close to those that alter human sperm mitochondrial activity and motility. Thus, the results of this study may be significant from a physiopathological point of view. The male reproductive tract possesses the complete enzymatic machinery to synthesize and metabolize endocannabinoids, and there may be clinical pathological conditions leading to an increase in the endocannabinoid concentration within male reproductive tract secretions and to alterations in sperm function (2, 13, 19, 39). Finally, the possible use of cannabinoid receptor agonists in the treatment of some pathological conditions (40, 41) should be weighed against the possible adverse effects of these drugs on human fertility.
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Acknowledgments |
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Footnotes |
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First Published Online November 23, 2004
Abbreviations: AEA, N-Arachidonoylethanolamine; [Ca2+]i, intracellular Ca2+ concentration; CB-R, cannabinoid receptor; FITC, fluorescein isothiocyanate; R-123, rhodamine 123.
Received July 2, 2004.
Accepted November 12, 2004.
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References |
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9-Tetrahydrocannabinol: a potent inhibitor of episodic luteinizing hormone secretion. J Pharmacol Exp Ther 213:306–3089-tetrahydrocannabinol disrupts mitochondrial function and cell energetics. Am J Physiol 284:L298–L306
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 P. Pacher, S. Batkai, and G. Kunos The Endocannabinoid System as an Emerging Target of Pharmacotherapy Pharmacol. Rev., September 1, 2006; 58(3): 389 - 462. [Abstract] [Full Text] [PDF]
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 H. Wang, S. K. Dey, and M. Maccarrone Jekyll and Hyde: Two Faces of Cannabinoid Signaling in Male and Female Fertility Endocr. Rev., August 1, 2006; 27(5): 427 - 448. [Abstract] [Full Text] [PDF]
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G. Cobellis, G. Cacciola, D. Scarpa, R. Meccariello, R. Chianese, M. F. Franzoni, K. Mackie, R. Pierantoni, and S. Fasano Endocannabinoid System in Frog and Rodent Testis: Type-1 Cannabinoid Receptor and Fatty Acid Amide Hydrolase Activity in Male Germ Cells Biol Reprod, July 1, 2006; 75(1): 82 - 89. [Abstract] [Full Text] [PDF]
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A. Ambrosini, G. Zolese, S. Ambrosi, L. Ragni, L. Tiano, G. Littarru, E. Bertoli, F. Mantero, M. Boscaro, and G. Balercia Oleoylethanolamide Protects Human Sperm Cells from Oxidation Stress: Studies on Cases of Idiopathic Infertility Biol Reprod, April 1, 2006; 74(4): 659 - 665. [Abstract] [Full Text] [PDF]
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U. Pagotto, G. Marsicano, D. Cota, B. Lutz, and R. Pasquali The Emerging Role of the Endocannabinoid System in Endocrine Regulation and Energy Balance Endocr. Rev., February 1, 2006; 27(1): 73 - 100. [Abstract] [Full Text] [PDF]
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H. Schuel and L. J. Burkman A Tale of Two Cells: Endocannabinoid-Signaling Regulates Functions of Neurons and Sperm Biol Reprod, December 1, 2005; 73(6): 1078 - 1086. [Abstract] [Full Text] [PDF]
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 M. Maccarrone, B. Barboni, A. Paradisi, N. Bernabo, V. Gasperi, M. G. Pistilli, F. Fezza, P. Lucidi, and M. Mattioli Characterization of the endocannabinoid system in boar spermatozoa and implications for sperm capacitation and acrosome reaction J. Cell Sci., October 1, 2005; 118(19): 4393 - 4404. [Abstract] [Full Text] [PDF]
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) and the presence (
) of AEA at various concentrations. For evaluation of the effects of the CB1-R antagonist SR141716 on AEA-induced modifications of sperm motility, sperm were preincubated in the presence of equimolar SR141716 concentrations for 45 min before AEA addition (
). Incubation of sperm with SR141716 alone did not induce any significant modification of sperm motility (•). Sperm motility was evaluated as described in the text. Data are expressed as a percentage of the total motile sperm. *, P < 0.01; **, P < 0.001 (vs. control and SR141716-pretreated samples). B, Sperm were incubated in the absence (
