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. 2010 Oct 20;151(12):5882–5892. doi: 10.1210/en.2010-0484

Energetic Metabolism and Human Sperm Motility: Impact of CB1 Receptor Activation

A Barbonetti 1, M R C Vassallo 1, D Fortunato 1, S Francavilla 1, M Maccarrone 1,a, F Francavilla 1,a
PMCID: PMC2999496  PMID: 20962050

Abstract

It has been reported that the endocannabinoid anandamide (AEA) exerts an adverse effect on human sperm motility, which has been ascribed to inhibition of mitochondrial activity. This seems to be at variance with evidence suggesting a major role of glycolysis in supplying ATP for sperm motility; furthermore, the role of AEA-binding receptors in mediating mitochondrial inhibition has not yet been explored. In this study, human sperm exposure to Met-AEA (methanandamide, nonhydrolyzable analog of AEA) in the micromolar range significantly decreased mitochondrial transmembrane potential (ΔΨm), similarly to rotenone, mitochondrial complex I inhibitor. The effect of Met-AEA (1 μm) was prevented by SR141716, CB1 cannabinoid receptor antagonist, but not by SR144528, CB2 antagonist, nor by iodoresiniferatoxin, vanilloid receptor antagonist. The effect of Met-AEA did not involve activation of caspase-9 or caspase-3 and was reverted by washing. In the presence of glucose, sperm exposure either to Met-AEA up to 1 μm or to rotenone for up to 18 h did not affect sperm motility. At higher doses Met-AEA produced a CB1-independent poisoning of spermatozoa, reducing their viability. Under glycolysis blockage, 1 μm Met-AEA, similarly to rotenone, dramatically abolished sperm motility, an effect that was prevented by SR1 and reverted by washing. In conclusion, CB1 activation induced a nonapoptotic decrease of ΔΨm, the detrimental reflection on sperm motility of which could be revealed only under glycolysis blockage, unless very high doses of Met-AEA, producing CB1-independent sperm toxicity, were used. The effects of CB1 activation reported here contribute to elucidate the relationship between energetic metabolism and human sperm motility.

CB1 activation induces a reversible, nonapoptotic decrease of mitochondrial potential, whose detrimental reflection on sperm motility is revealed under glycolysis blockage.

N-Arachidonoylethanolamine (anandamide, AEA) and 2-arachidonoylglycerol (2-AG) are the best characterized prototype members of two families of endocannabinoids, the fatty acid amides and the monoacylglycerols, respectively (1). These bioactive lipids owe their name to the ability to act primarily at cannabinoid receptors, CB1 and CB2, thus reproducing some of the biological actions of natural Cannabis sativa components (the phytocannabinoids), of which Δ9-tetrahydrocannabinol (THC) is the most prominent member. CB1 is highly expressed in the central nervous system and is also present in peripheral and extraneural sites. By contrast, CB2 is mainly present in the immune system (2,3,4) but has been detected also in neuronal cells (5,6).

Biochemical studies have revealed that the biological activity of AEA at its receptors is under a metabolic control (7). AEA is released from membrane phospholipid precursors, the N-acylphosphatidylthanolamines (NAPEs), through the activity of NAPE-hydrolyzing phospholipase D (8). Furthermore, a purported bi-directional endocannabinoid membrane transporter is responsible for the uptake of extracellular AEA (9,10,11). Once inside the cell, AEA is degraded to ethanolamine and arachidonic acid by the fatty acid amide hydrolase (12,13). Unlike 2-AG, AEA also acts at the intracellular binding site of the transient receptor potential vanilloid type 1 (TRPV1) channel, naturally activated by the pungent compound of hot chilli pepper, capsaicin, as well as by the irritant extract of the Euphorbia resinifera, resiniferatoxin (14,15).

Evidence has been accumulated that (endo)cannabinoids may affect male fertility in a number of species, both in vitro and in vivo (16,17,18). Chronic administration of THC to animals reduces the production, motility, and viability of spermatozoa (19,20). In in vitro experiments, both THC and AEA inhibit egg jelly-stimulated acrosome reaction in sea urchin spermatozoa (21,22,23). Furthermore, activation of CB1 by a metabolically stable analog of AEA, methanandamide (Met-AEA), was shown to inhibit sperm capacitation in the boar, where a fully functional endocannabinoid system has been characterized (24).

Human spermatozoa, much like boar spermatozoa, exhibit the functional biochemical machinery needed to synthesize (NAPE-hydrolyzing phospholipase D) and degrade (endocannabinoid membrane transporter and fatty acid amide hydrolase) AEA (25) and express CB1 (25,26) and CB2 (27) receptors. AEA has been detected in fluids of male and female reproductive tracts (28,29), and, in human spermatozoa, (endo)cannabinoids have been reported to negatively affect acrosome reaction (26,30) and zona pellucida binding (28). Actually, the endocannabinoid system would exert a complex role in modulating human sperm functions involved in the acquisition of sperm-fertilizing ability. In fact, in addition to expressing a functional CB1 receptor that binds (endo)cannabinoids, human spermatozoa also express a functional TRPV1 receptor, the activation of which by intracellular AEA appears to be involved in the prevention of spontaneous acrosome exocytosis during capacitation, thereby preserving sperm-fertilizing potential (25).

It has been also claimed that (endo)cannabinoids negatively affect human sperm motility (26,28,30,31) and that this effect would be mediated by CB1 receptors (26,31). Because AEA and congeners have the potential to affect isolated mitochondria physiology (32,33) and AEA reduces mitochondrial activity in human spermatozoa (26), as do exogenous cannabinoids (34), it has been suggested that the effect of these substances on sperm motility could depend on a mechanism of energy depletion (35). This interpretation would implicitly ascribe a key role to mitochondrial oxidative phosphorylation in providing energy to the axoneme. This assumption should be critically revised, in the light of some interesting experimental data, suggesting a major role of glycolysis in supplying energy for sperm motility. Elegant experiments in the mouse have clearly demonstrated that glycolysis compensates for any lack of ATP production by mitochondria in maintaining sperm motility, and that mitochondrial inhibition can depress sperm motility only in the presence of glycolysis blockage (36). An obligatory role for glycolysis is supported by the lack of progressive motility in sperm from mice in which the gene for sperm-specific glyceraldehyde-3-phosphate dehydrogenases had been knocked out (37). However, it is unclear whether this glycolysis model is suitable for other mammalian spermatozoa, and there is still debate concerning whether glycolysis or oxidative phosphorylation should be considered as the major biochemical pathway for the supply of energy that supports human sperm motility (38,39,40).

In this study we further characterized the effect of the activation of AEA-binding receptors (CB1, CB2, and TRPV1), on human sperm mitochondrial function and investigated its reflection on sperm motility, providing interesting observations on the relationship between energy metabolism and motility in human spermatozoa.

Materials and Methods

Chemicals

R(+)-Methanandamide (Met-AEA), rotenone, 2-deoxy-d-glucose (DOG), and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenimidazolyl carbocyanine iodide (JC-1) were purchased from Sigma Chemical Co. (St. Louis, MO). Iodoresiniferatoxin (IRTX) was obtain from Tocris Cookson (Bristol, UK). SR141716 (SR1) and SR144528 (SR2) were kind gifts of Sanofi-Aventis Recherche (Montpellier, France). CaspGLOW Fluorescein Active Kits for caspase-9 and caspase-3 were obtained from BioVision (Mountain View, CA). Met-AEA is supplied as a 5 mg/ml solution in absolute ethanol; thus the solvent was changed by evaporating ethanol under a gentle stream of nitrogen before the addition of dimethyl sulfoxide (DMSO), purged with inert gas to prevent oxidation of Met-AEA. Stock solutions of Met-AEA, SR1, SR2, IRTX, rotenone, and JC-1 in DMSO were diluted in Biggers, Whitten, and Wittingham (BWW) medium to obtain the working concentrations just before use.

Medium

BWW medium consisted of 5.6 mm d-glucose, 44 mm sodium lactate, 0.27 mm sodium pyruvate, 95 mm NaCl, 25 mm NaHCO3, 4.6 mm KCl, 1.7 mm CaCl2, 1.2 mm KH2PO4, 1.2 mm MgSO4, 5 U/ml penicillin, 5 mg/ml streptomycin, pH 7.4. BSA was omitted, because it is known to bind endocannabinoids (9,41), thus impairing biochemical and functional assays. BWW minus glucose (BWW−Gluc) consisted of normal BWW lacking d-glucose. In BWW−Gluc containing the glucose analog DOG, 5.6 mm DOG replaced d-glucose.

Semen samples and sperm processing

Semen samples were collected according to the World Health Organization (WHO)-recommended procedure (42) by masturbation, from healthy normozoospermic postgraduate medical students.

The study was approved by the local Institutional Review Board, and all subjects signed an informed consent statement.

All samples were produced into sterile containers and left for at least 30 min to liquefy before processing. Motile sperm suspensions were obtained by swim up procedure. Briefly, spermatozoa were washed twice (700 × g for 7 min) in BWW medium. After the second centrifugation, supernatants were removed by aspiration, leaving 0.5 ml on the pellet, and after 30 min of incubation time, supernatants containing highly concentrated motile sperm were carefully aspirated. Sperm concentration was adjusted as needed for subsequent analysis.

Flow cytometric evaluation of mitochondrial membrane potential (Δψm)

The fluorescent lipophilic cationic dye JC-1 was used to evaluate transmembrane potential of sperm mitochondria. JC-1 possesses the unique ability to differentially label mitochondria with low and high Δψm. In mitochondria with high Δψm, JC-1 forms multimeric aggregates that emit orange light (wavelength of 590 nm) when excited at 488 nm. In mitochondria with low Δψm, JC-1 forms monomers that emit green light (525–530 nm) when excited at 488 nm. Sperm suspensions (1 ml) containing 5 × 106 spermatozoa were stained with 0.5 μl of JC-1 stock solution (3 mm in DMSO). Samples were incubated at 37 C in the dark for 60 min and then analyzed using a flow cytometer (Beckman-Coulter Epics XL-4; Beckman Coulter, Inc., Fullerton, CA) equipped with a 15 mW argon-ion laser for excitation. For each sample 10,000 events were recorded at a flow rate of 200–300 cells/sec. Based on the light scatter characteristics of swim up selected spermatozoa, debris and aggregates were gated out by establishing a region around the population of interest in the forward scatter/side scatter dot plot on a log scale.

Compensation between FL1 and FL2 was carefully adjusted according to the manufacturer’s instructions. Green fluorescence (480–530 nm) was measured in the FL-1 channel and orange-red fluorescence (580–630 nm) was measured in the FL-2 channel. The percentage of positive cells was evaluated on a 1023-channel scale, using the flow cytometer System II Version 3.0 software (Beckman Coulter, Inc.).

Flow cytometric assessment of activated caspases

Activation of caspase-9 and caspase-3 was evaluated by using ApoptosisCaspGLOW Fluorescein Active Caspase-3 and 9 Staining Kits (Biovision, Inc.), according to the manufacturer’s instructions. These assays use the caspase-9 or caspase-3 inhibitors, LEHD-FMK or DEVD-FMK, respectively, conjugated to fluorescein isothiocyanate (FITC), as fluorescent markers. These cell-permeable, nontoxic peptides covalently bind to activated caspases, serving as in situ markers for apoptosis (43,44,45). Briefly, in 300 μl of sperm suspension containing 0.3 × 106 spermatozoa, 1 μl of FITC-LEHD-FMK or FITC-DEVD-FMK was added before incubation for 1 h at 37 C in an atmosphere of 5% CO2. After three centrifugations (1000 × g for 4 min), spermatozoa were resuspended in 1 ml of PBS before addition of 1 μl of propidium iodide (PI), a red nucleic acid dye that is excluded from viable cells. Flow cytometry was performed within 10 min. With this technique, four sperm subpopulations were detected. Events in the lower-left quadrant represented live (no apoptotic) spermatozoa (FITC−/PI−); events in the lower-right quadrant represented live spermatozoa with active caspases (FITC+/PI−), events in the upper-right quadrant represented dead spermatozoa showing caspase activation (FITC+/PI+), and events in the upper-left quadrant represented dead spermatozoa with no caspase activation (FITC−/PI+).

Evaluation of sperm motility and viability

Sperm motility was evaluated by Computer-Aided Semen Analysis using ATS20 (JCD, Gauville, France). Ten microliters of each sperm sample were placed into a prewarmed (37 C) Makler counting chamber (Sefi Medical Instruments, Haifa, Israel). At least 200 spermatozoa were evaluated for each sample. Setting parameters were: analysis duration of 1 sec (30 frames); minimum contrast, 80; minimum size, 3; low size gate, 0.7; high size gate, 2.6; low intensity gate, 0.34; light intensity gate, 1.40. Spermatozoa exhibiting an average pathway velocity >5 μm/sec were categorized by the software as motile spermatozoa.

Sperm viability was evaluated under light microscope by the eosin technique, according to WHO (42).

Statistical analysis

Statistical analysis was performed using the SAS statistical software (version 9.1, 2003; SAS Institute, Inc., Cary, NC). Data were analyzed by ANOVA and post hoc comparisons between pairs of groups were performed by the Tukey’s studentized range-honestly significant difference (HSD) test. Statistical significance was accepted when P ≤ 0.05. Data were expressed as mean ± sem.

Results

Effect of CB1 activation on sperm mitochondrial transmembrane potential (Δψm)

Aliquots of the same motile sperm suspensions were exposed to increasing concentrations of Met-AEA (0.1, 0.5, 1, and 10 μm). As shown in Fig. 1, the exposure to 1 and 10 μm Met-AEA for 1 h produced a significant decrease in sperm mitochondrial transmembrane potential (Δψm), as indicated by a decrease on the percentage of spermatozoa exhibiting red-orange fluorescence, with respect to controls exposed to a proper dilution of DMSO. Conversely, sperm Δψm was not affected by Met-AEA at lower concentrations. As expected, Δψm was suppressed by the mitochondrial respiratory chain complex I inhibitor rotenone (1 μm), used as a positive control.

Figure 1.

Figure 1

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A, Typical histograms of fluorescence from flow cytometric analysis of sperm mitochondrial transmembrane potential (Δψm), evaluated by JC-1 staining. In each setting, a negative control of high quality, with high Δψm (Control), was used to set a region (F) including all spermatozoa exhibiting red-orange fluorescence for JC-1 (high Δψm). FL2LOG, fluorescence detector 2 log; SR1, SR141716 (0.1 μm); SR2, SR144528 (0.1 μm); IRTX (1 μm). B, Percentage of spermatozoa exhibiting red-orange fluorescence for JC-1 (high Δψm) under different experimental conditions. Means ± sem of four independent experiments with different donors.

To explore the possible involvement of AEA-binding receptors in mediating the Met-AEA effect on sperm Δψm, cell suspensions were separately exposed to 0.1 μm SR1, 0.1 μm SR2, or 1 μm IRTX, selective antagonists of CB1, CB2 or TRPV1 respectively, for 15 min before addition of Met-AEA. Each antagonist was used at a concentration previously shown to block the corresponding target in sperm (24,25). SR1 prevented the inhibitory effect of 1 μm Met-AEA, whereas SR2 or IRTX were ineffective. On the other hand, SR1 did not block inhibition by 10 μm Met-AEA, and none of the antagonists affected sperm Δψm when used alone (Fig. 1). Because CB1 was the only AEA-binding receptor engaged in Δψm modulation, the involvement of CB2 and TRPV1 was not investigated in the subsequent experiments.

To further characterize the mechanism of sperm Δψm inhibition by Met-AEA, we checked if the effect of this substance could be reverted by a simple washing step. To this end, sperm suspensions exposed for 1 h to 1 μm Met-AEA were divided into two aliquots: one of them was directly stained with JC-1 for Δψm evaluation; the other one was washed twice in BWW (1000 × g, × 4 min) and, after 1 h incubation, was exposed to JC-1. Simple sperm washing completely abolished the inhibitory effect of 1 μm Met-AEA on Δψm (Fig. 1).

Effect of Met-AEA on caspase activation

Because the loss of Δψm could reflect an early apoptotic stage, preceding phosphatidylserine externalization and coinciding with caspase activation (46,47,48), and endocannabinoids and plant-derived cannabinoids have emerged as key promoters of CB receptor-dependent apoptosis in central and peripheral cells (49,50,51,52,53,54), we checked the activation of caspase-9 (induced by mitochondrial apoptotic pathway) and caspase-3 (the downstream effector caspase) upon exposure to 1 μm Met-AEA. As shown in Fig. 2, in samples exposed to 1 μm Met-AEA for 1 h, the percentage of viable spermatozoa exhibiting activated caspase-9 or caspase-3 was similar to that observed in controls exposed to proper dilutions of DMSO. Staurosporine, a protein kinase inhibitor, has been characterized as a strong inducer of caspase activity and apoptosis in different cell types (55,56,57). Thus, this substance was used as a positive control for caspase activation. As shown in Fig. 2, the exposure to staurosporine (1 μm) for 1 h negatively affected sperm viability while producing a significant increase in the proportion of viable spermatozoa with activated caspase-9 and caspase-3.

Figure 2.

Figure 2

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Effect of Met-AEA on caspase-9 (A) and caspase-3 (B) activation. Top, Flow cytometric dot plots of double staining with PI and FITC label that allows detection of activated caspase-9 (FITC-LEHD-FMK) and activated caspase-3 (FITC-DEVD-FMK). Staurosporine was used as positive control for caspase activation. Bottom, Percentage of viable (PI−) spermatozoa exhibiting green fluorescence (FITC+) of caspase activation. Means ± sem of three independent experiments with different donors.

Effect of Met-AEA on sperm motility

Because CB1 activation inhibited mitochondrial function, in the second part of the study we explored its reflection on sperm motility.

Effect on sperm motility in the presence of glucose

When a complete glucose-containing BWW medium was used, the exposure to scalar doses of Met-AEA up to 1 μm and for up to 18 h did not produce any change either in the percentage of motile spermatozoa (Fig. 3A) or in the quality of motility (Table 1) with respect to controls. On the contrary, sperm motility was significantly affected by higher doses of Met-AEA (5 and 10 μm), due to cell poisoning that reduced sperm viability (Fig. 3A). In a subsequent set of experiments, the detrimental effect of 10 μm Met-AEA was shown to be CB1 independent, because it was not prevented by SR1 after 30 min of treatment (16.2 ± 4.0% vs. 15.8 ± 4.0%).

Figure 3.

Figure 3

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Effect of Met-AEA (A) and rotenone (B) on motility (left) and viability (right) of human spermatozoa in the presence of glucose (complete BWW medium). Number of replicates = 6, with different donors. Overall significance for treatment variations: P < 0.001 with ANOVA; *, P < 0.05 vs. ≤1 μm Met-AEA (Tukey’s HSD test).

Table 1.

Effect of Met-AEA on the quality of sperm motility, evaluated by Computer-Aided Semen Analysis (CASA), in the presence of glucose

Met-AEA (μm) VCL VSL VAP
30 min 0 82.7 ± 0.5 66.9 ± 2.4 70.0 ± 1.6
0.1 79.1 ± 2.4 65.0 ± 1.9 67.1 ± 1.5
0.5 82.6 ± 2.5 60.0 ± 2.0 65.4 ± 1.9
1 79.3 ± 3 61.7 ± 1.6 67.0 ± 2.1
5 46.7 ± 3.5a 33.4 ± 3.1a 29.0 ± 3.0a
10 0.1 ± 0.1a 0.7 ± 0.2a 0.1 ± 0.1a
2 h 0 61.3 ± 2.5 48.0 ± 2.9 59.1 ± 3.4
0.1 62.9 ± 2.2 48.3 ± 2.5 52.8 ± 2.8
0.5 63.3 ± 4.6 49.7 ± 2.9 54.0 ± 2.7
1 60.7 ± 3.7 45.7 ± 2.2 51.3 ± 2.5
5 35.6 ± 2.8a 22.7 ± 0.8a 22.0 ± 1.4a
10 0.4 ± 0.2a 0.0 ± 0.0a 0.3 ± 0.2a
18 h 0 41.0 ± 3.8 29.3 ± 2.8 32.3 ± 2.3
0.1 40.0 ± 4.1 28.0 ± 2.1 31.0 ± 1.0
0.5 39.7 ± 4.4 22.7 ± 1.5 28.0 ± 4.1
1 39.0 ± 3.5 20.3 ± 0.3 30.0 ± 2.5
5 19.3 ± 3.4a 10.0 ± 1.0a 15.3 ± 3.2a
10 0.3 ± 0.3a 0.0 ± 0.0a 0.0 ± 0.0a
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Number of replicates = 6, with different donors. Overall significance for treatment variations: P < 0.001 with ANOVA; 

a

P < 0.05 vs. ≤1 μm Met-AEA (Tukey’s HSD test). VCL, Curvilinear velocity (μm/sec); VSL, straight line velocity (μm/sec); VAP, average pathway velocity (μm/sec). 

Similarly, sperm exposure to scalar concentrations (1, 10, 100, and 1000 μm) of rotenone for up to 18 h did not affect sperm motility (Fig. 3B).

Effect on sperm motility under glycolysis blockage

Because mitochondrial inhibition did not suppress sperm motility in the presence of glucose (Fig. 3), we checked experimental conditions under which the reflection of mitochondrial inhibition on sperm motility could be revealed.

As shown in Fig. 4A, motility remained unchanged in spermatozoa suspended for 1 h in BWW either depleted of glucose (−Gluc) or complete and containing 5.6 mm 2-deoxy-d-glucose (DOG), an inhibitor of glycolysis. It decreased significantly when spermatozoa were exposed to DOG in glucose-depleted BWW medium (−Gluc+DOG), and was completely abolished only when sperm suspensions were exposed also to rotenone (1 μm), in addition to glycolysis blockage (BWW-Gluc+DOG). Therefore, the latter experimental condition was singled out for revealing a possible negative effect of Met-AEA on sperm motility, reflecting its inhibition of mitochondrial activity. In BWW-Gluc+DOG medium, 1 μm Met-AEA dramatically suppressed sperm motility, without affecting sperm viability, when compared with control under the same experimental conditions (Fig. 4B). In keeping with the effects on mitochondrial activity (Fig. 1), sperm motility was not significantly affected by lower concentrations (0.1 μm and 0.5 μm) of Met-AEA (Fig. 4B). Furthermore, the effect of 1 μm Met-AEA was completely prevented by 0.1 μm SR1 (Fig. 4B). In another set of experiments, the inhibition of sperm motility by 1 μm Met-AEA was found to be completely reversed by the washing step described above: under glycolysis blockage (BWW-Gluc+DOG), the percentage of motile spermatozoa dropped from 42.0 ± 3.0% to 4.0 ± 1.0% in samples exposed to 1 μm Met-AEA (P < 0.05) and was restored (35.0 ± 5.0%) after washing in BWW-Gluc + DOG (P < 0.05).

Figure 4.

Figure 4

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Effect of rotenone (A) and Met-AEA (B) on sperm viability and motility, as evaluated with Computer-Aided Semen Analysis, during 1 h incubation under conditions of glycolysis blockage. Number of replicates = 4, with different donors. Overall significance for treatment variations: P < 0.001 with ANOVA; *. P < 0.05 vs. all the others (Tukey’s HSD test). −Gluc, glucose-free BWW (containing pyruvate and lactate); +Gluc, complete BWW (containing glucose); DOG, (5.6 mm); SR1, SR141716 (0.1 μm); VCL, curvilinear velocity (μm/sec); VSL, straight line velocity (μm/sec); VAP, average pathway velocity (μm/sec).

Discussion

The inhibitory effects of cannabinoids on mitochondrial respiration has been known since the 1960s–1970s, when it was demonstrated that THC, the main active phytocannabinoid compound contained in marijuana, inhibited oxygen consumption in rat brain homogenates and in liver mitochondria (58,59,60). Similar findings were more recently reported in a number of cell types, where mitochondrial inhibition appeared to be involved in biological actions of phytocannabinoids (34,61,62), as well as of their endogenous counterparts (32,33). Mechanisms mediating this inhibition are as yet unknown (35).

According to previous results obtained by using AEA (26), in the present study 1 μm Met-AEA significantly inhibited Δψm of human spermatozoa. The first novel and interesting observation arising from this study is that the effect of Met-AEA on sperm Δψm occurred via activation of CB1, because it was prevented by the CB1-selective antagonist SR1. Instead, the two other AEA-binding receptors CB2 and TRPV1 were not involved, as demonstrated by using the selective antagonists SR2 and IRTX, respectively. Incidentally, it should be recalled that, whereas TRPV1 clearly impacts human sperm function (25), CB2 is expressed in these cells (27) but does not seem to be functional (25). Some evidence of a mitochondrial localization of CB1 receptors in human spermatozoa has been recently reported (63), emphasizing the need to examine their possible involvement in mediating the effect of cannabinoids on mitochondria.

Recently, evidence has been collected for the ability of (endo)cannabinoids to trigger apoptotic pathways through CB1 or CB2 (49,50,51,52,53,54). Although a decrease in Δψm, evaluated by JC-1 staining, could represent an early marker of apoptosis (46,47,48), here the possible involvement of apoptotic pathways in the effect of Met-AEA on sperm mitochondrial activity was ruled out by caspase activation assays. Activation of these cytosolic cysteine proteases is one of the earliest and most common features of programmed cell death (64). In particular, along the apoptotic cascade of events mitochondrial membrane damage/permeabilization leads to the release of cytochrome c, which activates caspase-9 and ultimately caspase-3, the downstream executioner of apoptosis (65). Treatment with 1 μm Met-AEA inhibited sperm Δψm (Fig. 1), yet it did not produce any detectable activation of caspase-9 or of caspase-3 (Fig. 2). Furthermore, consistent with the lack of apoptosis, a simple sperm washing completely abolished the inhibitory effect of 1 μm Met-AEA on Δψm. At higher concentrations (10 μm), this substance became toxic, and reduced sperm viability in a CB1-independent manner.

Another major outcome of this investigation is that, in the presence of extracellular glucose (i.e. standard BWW medium), exposure to Met-AEA at concentrations up to 1 μm and for up to 18 h did not affect sperm motility. Similarly, sperm motility was not affected by exposure for up to 18 h to rotenone, a well-known inhibitor of the respiratory chain complex I. Recently, short-term (4 h) exposure to rotenone failed to inhibit human sperm motility but significantly reduced it when the incubation period was extended to 24 h (66). This effect was a possible consequence of late lipoperoxidative damage induced by mitochondrial generation of reactive oxygen species (66). The observation that in the presence of glucose sperm motility was not affected by mitochondrial inhibition, caused by either Met-AEA or rotenone, indicates that ATP generated by glycolysis (rather than by mitochondrial respiration) fully supports flagellar movement, thus extending previous data from mouse models (36,37). Consistently, the inhibitory effect of Met-AEA and rotenone on sperm motility could be revealed only in the presence of glycolysis blockage. In a medium lacking glycolysable sugars, substrates for mitochondrial respiration present in BWW medium (e.g. pyruvate and lactate) could support high-motility levels, by at least two mechanisms: 1) ATP is synthesized in mitochondria and then is supplied to the entire flagellum by diffusion; 2) energy produced by mitochondrial respiration is used to drive gluconeogenesis and thus to provide glucose for glycolytic ATP production in the flagellum. The first hypothesis could be ruled out, because the addition of DOG significantly decreased motility supported by pyruvate and lactate in the absence of glucose. It should be recalled that, once inside the cell, DOG is phosphorylated by sperm hexokinase but is not metabolized any further (67); additionally, DOG inhibits glycolysis by competing with glucose for key enzymes, the affinity for glucose of which is much higher than that for DOG itself (36). This fact could explain the lack of effects of DOG on sperm motility in the presence of extracellular glucose (Fig. 4A). Furthermore, in the presence of pyruvate and lactate (BWW-Gluc), sperm motility was strongly impaired but not completely abolished by DOG, suggesting that the amount of glucose produced by mitochondrial gluconeogenesis could still compete to some extent with DOG, due to the higher affinity of glycolytic enzymes for glucose. This hypothesis has been challenged (38), because phosphorylation of DOC would consume most of phosphate in the sperm, making it unavailable for oxidative phosphorylation; consequently, sperm motility would be affected. As a matter of fact, Williams and Ford (68) reported that, in human spermatozoa incubated in BWW-Gluc+DOG, ATP content was significantly lower than in those incubated in sugar-free BWW. As postulated by Ford (38), the reduced availability of phosphate for oxidative phosphorylation should increase the mitochondrial membrane potential by preventing the discharge of the proton motive force (38). We evaluated the effect of DOG in glucose-free BWW on Δψm and on sperm motility at 1 h. In four experiments, the mean intensity of JC-1 red fluorescence in spermatozoa incubated in BWW-Gluc+DOG showed a nonsignificant approximately 5% increase compared with those kept in glucose-free BWW (data not shown). At any rate, whether or not DOG could affect mitochondrial respiration does not seem to represent a major issue in the context of the present study; in fact, a decreased mitochondrial ATP production could affect gluconeogenesis, and subsequently the glucose availability for glycolysis, rather than affecting motility through a reduced availability of mitochondrial ATP along the flagellum. Direct evidence for the occurrence of gluconeogenesis, glycogen storage, and subsequent decrease of glycogen levels has been provided in dog spermatozoa incubated in a sugar-free medium (69).

Under conditions that blocked glycolysis, inhibition of mitochondrial activity by Met-AEA, as well as by rotenone, led to sperm immobilization without affecting sperm viability (Fig. 4). Also this effect of Met-AEA, much like that on sperm Δψm, was mediated by CB1 receptors, although the underlying signal transduction remains to be clarified. At any rate, these effects mediated by CB1 receptors were exerted by Met-AEA doses higher than the physiological levels of AEA in the male and female reproductive tracts (38,39). It will be interesting to explore whether they could occur in the case of increased levels of endogenous cannabinoids (e.g. in obesity), or of exposure to exogenous cannabinoids. It will be also interesting to explore whether these effects could be exerted by physiological levels of 2-AG, which in a recent report (70) appeared to be involved in the suppression of progressive motility of mouse spermatozoa in the caput of epididymis, through CB1 activation.

In conclusion, the present study provides unprecedented evidence that in human sperm CB1 activation induces an apoptosis-independent decrease of mitochondrial membrane potential. This inhibition does not produce detectable effects on sperm motility in vitro, when standard glucose-containing media are used, because glycolysis can compensate for any loss of ATP produced by mitochondria. An adverse effect of Met-AEA on sperm motility can be revealed only in the presence of glycolysis blockage, or at poisoning doses of this compound. How and to what extend these in vitro findings could be extended to in vivo sperm exposure to (endo)cannabinoids is hard to predict. Both glycolysis and mitochondrial respiration participate in ATP production and depend on each other to control the functional state of sperm in environments with different energetic substrates (39). Notably, in fluids of the female genital tract of a number of species, the levels of lactate are higher than those of glucose and other glycolysable substrates (71,72,73,74), suggesting a possible major contribution of mitochondrial respiration to support sperm motility. This hypothesis could explain why a sperm cell that, during its differentiation, dramatically reduces its volume by removing any unnecessary structure must retain its mitochondria. In line with this, in the female genital tract sperm exposure to exogenous cannabinoids like THC, by impairing mitochondrial activity, could adversely affect sperm motility. Taken together, the effects of CB1 activation reported here represent a further improvement in understanding the relationship between energetic metabolism and motility in human sperm physiology, with a potential therapeutic exploitation for the treatment of human infertility.

Acknowledgments

We thank all staff members of the S. Liberatore Hospital of Atri: their generous hospitality after the 2009 earthquake of L’Aquila allowed us to complete this study.

Footnotes

This work was supported by a grant from Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR).

Disclosure Summary: The authors have nothing to disclose.

First Published Online October 20, 2010

Abbreviations: AEA, Anandamide; 2-AG, 2-arachidonoylglycerol; BWW, Biggers, Whitten, and Wittingham; DMSO, dimethyl sulfoxide; DOG, 2-deoxy-d-glucose; FITC, fluorescein isothiocyanate; HSD, honestly significant difference; IRTX, iodoresiniferatoxin; JC-1,5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenimidazolyl carbocyanine iodide; Met-AEA, methanandamide, nonhydrolyzable analog of AEA; NAPE, N-acylphosphatidylthanolamine; PI, propidium iodide; THC, Δ9-tetrahydrocannabinol; TRPV1, transient receptor potential vanilloid type 1.

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