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Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, I-00133 Italy; and Department of Biomedical Sciences and Technologies, University of L’Aquila (S.C., G.R.), I-67100 Italy
Address all correspondence and requests for reprints to: Dr. Mauro Maccarrone, Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Via Montpellier 1, I-00133 Rome, Italy. E-mail: maccarrone{at}med.uniroma2.it.
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Introduction |
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9-tetrahydrocannabinol (THC), the psychoactive principal of hashish and marijuana (5). Recently, AEA has been shown to also activate vanilloid receptors (6). In the periphery, AEA and 2-AG act as cardiovascular (7) and immune (8) modulators and show antiinflammatory activity (9). Moreover, endogenous cannabinoids have been involved in the inhibition of human breast and prostate cancer cell proliferation (10). Also, N-palmitoylethanolamine (PEA) is a biologically active endocannabinoid, reported to have antiinflammatory activity (11). However, its ability to bind to CB receptors is still controversial (12). The effect of AEA via CB1 and CB2 receptors depends on its extracellular concentration, which is controlled by 1) cellular uptake by a specific AEA membrane transporter (AMT), and 2) intracellular degradation by the AEA-hydrolyzing enzyme fatty acid amide hydrolase (FAAH). AMT (13) and FAAH (14) have been characterized in several mammalian cells and tissues, and together with AEA and congeners these proteins form the endocannabinoid system.
Growing evidence is accumulating showing that endocannabinoids modulate embryo-uterine interactions (15) and impair pregnancy and embryo development in mice (16), thus resembling the adverse effects of THC on reproduction (17). More recently, progesterone has been implicated in THC modulation of sexual receptivity in female rats (18), and dysregulation of cannabinoid signaling has been shown to disrupt uterus receptivity to the embryo implantation in mice (19). Along this line, we reported the association between decreased FAAH activity and expression in maternal peripheral lymphocytes and early pregnancy failure in humans (20), demonstrating that a dysregulation of AEA degradation might impair fertility. Despite the knowledge that chronic administration of THC to animals lowers testosterone secretion and reduces the production, motility, and viability of sperm (17), a role for the endocannabinoid system in controlling male fertility in mammals remains unknown. The binding of AEA to a CB receptor present on spermatozoa has been shown to reduce their fertilizing capacity in the sea urchin (21, 22). On the other hand, rat testis is able to synthesize AEA (23), and indeed this compound has been detected in human seminal plasma at nanomolar (
10 nM) concentrations (24). More recently, the presence of CB1 receptors in Leydig cells and their involvement in testosterone secretion have been demonstrated in mice (25). Also, the function of Sertoli cells has been shown to be altered by THC, although the molecular basis for this alteration has not been established (26). As Sertoli cells of the mammalian seminiferous epithelium are involved in the regulation of germ cell development by providing nutrients and hormonal signals needed for spermatogenesis (27), here we sought to investigate whether Sertoli cells were able to bind and degrade AEA, and whether this endocannabinoid might induce apoptosis in these cells, in view of its well documented proapoptotic activity (28, 29). In this context, the effect of FSH was also checked, because it dramatically impacts fetal and early neonatal Sertoli cell proliferation and is critical in determining spermatogenic capacity in adult mammals (30).
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Materials and Methods |
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Chemicals
Chemicals were of the purest analytical grade. AEA, sodium nitroprusside (SNP), actinomycin D (ACTD), cycloheximide (CHX), MEM, collagenase, trypsin, hyaluronidase, and N,O'-dibutyryl cAMP [(Bu)2cAMP] were purchased from Sigma (St. Louis, MO). N-(4-Hydroxyphenyl)-arachidonoylamide (AM404), arachidonoyl-trifluoromethyl-ketone, and 2-AG were obtained from Research Biochemicals International (Natick, MA). 3-Morpholinosydnonimine (SIN-1) was purchased from Alexis Corp. (Läufelfingen, Switzerland). PEA was synthesized and characterized (purity, >96% by gas-liquid chromatography) as previously described (31). R+-Methanandamide (Met-AEA) and capsazepine (CAPS) were obtained from Calbiochem (La Jolla, CA). Cannabidiol (CBD) and 
-linolenoyl-vanillyl-amide (linvanil) were gifts from Dr. M. van der Stelt (Utrecht University, Utrecht, The Netherlands) and Dr. V. Di Marzo (Consiglio Nazionale delle Ricerche, Pozzuoli, Italy), respectively. N-Piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide (SR141716) and N-[1(S)-endo-1,3,3-trimethyl-bicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methyl-benzyl)-pyrazole-3-carboxamide (SR144528) were gifts from Sanofi Pharmaceuticals, Inc. (Montpellier, France). FSH (o-FSH-16) was obtained through the Hormone Distribution Program, NIDDK (Dr. Parlow). [3H]AEA (223 Ci/mmol), [3H]5-(1,1'-dimethyheptyl)-2-[1R,5R-hydroxy-2R-(3-hydroxypropyl) cyclohexyl]-phenol ([3H]CP55.940; 126 Ci/mmol), and [33P]orthophosphoric acid (100 Ci/mg) were obtained from NEN Life Science Products (Köln, Germany). [3H]2-AG was synthesized from 1,3-dibenzyloxy-2-propanol and [3H]arachidonic acid (200 Ci/mmol; ARC, Inc., St. Louis, MO) as previously reported (32). Anti-FAAH polyclonal antibodies were elicited in rabbits against the conserved FAAH sequence VGYYETDNYTMPSPAMR (33) conjugated to ovalbumin and were prepared by Primm S.r.l. (Milan, Italy). Rabbit anti-CB1 and anti-CB2 polyclonal antibodies were purchased from Cayman Chemicals (Ann Arbor, MI), and mouse antiactin monoclonal antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Goat antirabbit (GAR-AP) and goat antimouse antibodies conjugated to alkaline phosphatase were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA).
Cell culture and treatment
Purified Sertoli cells were isolated from decapsulated testes of 2- to 24-d-old mice by sequential enzymatic digestion (34). Fragments of seminiferous tubules were incubated in PBS containing 0.001% collagenase plus 0.02% deoxyribonuclease I for 1 h at 32 C in a shaking water bath (80 cycles/min), and released Leydig and interstitial cells were discarded with the supernatant after sedimentation at unit gravity. To remove peritubular cells, fragments were further digested with 1 mg/ml hyaluronidase and 0.02% deoxyribonuclase I for 1 h at 32 C in a shaking water bath. Finally, cell aggregates were passed through a nylon mesh (100-µm pore size) and were washed three times by centrifugation (100 x g for 5 min). The purity of Sertoli cell preparations was assessed by staining for peritubular cells with alkaline phosphatase (35) and for Leydig cells with 3ß-hydroxysteroid dehydrogenase (36). Contaminating cells were always less than 5%, in keeping with previous reports (34, 35, 36). Isolated Sertoli cells were either used immediately or plated in serum-free MEM supplemented with 2 mM glutamine and penicillin-streptomycin solution in 24-well culture plates (1 x 106 cells/well, 16 mm; Costar multiwell, Cambridge, MA) and cultured at 34 C in 5% CO2 for 24–48 h. Depending on the experimental conditions, Sertoli cells were treated with different doses or for various periods of time with endocannabinoids and related agonists/antagonists/inhibitors, with FSH, or with (Bu)2cAMP (0.2 mM). 33P labeling of proteins was performed by incubating Sertoli cells (2 x 106 cells/test) with the phosphatase inhibitor pervanadate (1 mM) and [33P]orthophosphate (250 µCi) as previously described (37). Then cell homogenates were subjected to electrophoretic analysis of phosphorylated proteins as previously reported (37). Viability was estimated by trypan blue dye exclusion test, which determined that cell number and density did not change during culture in the absence or presence of treatments.
Binding to cannabinoid receptors
For cannabinoid receptor studies, membrane fractions were prepared from freshly isolated Sertoli cells (25 x 106/test) as previously reported (29), and were used in rapid filtration assays with the synthetic cannabinoid [3H]CP55.940 at 400 pM (29). Apparent dissociation constant (Kd) and maximum binding (Bmax) values of [3H]CP55.940 were calculated from saturation curves through nonlinear regression analysis with the PRISM 3 program (GraphPad Software, Inc., San Diego, CA) (29). Unspecific binding was determined in the presence of 10 µM AEA, and the inhibition constant of [3H]CP55.940 binding by AEA was determined as reported previously (38). Binding of [3H]AEA to Sertoli cell membrane was evaluated with the same filtration assays as those used for [3H]CP55.940 (29). The expression of CB1 and CB2 receptors in 4-d-old Sertoli cells was assessed by Western blot analysis, performed as detailed below for FAAH, using anti-CB1 or anti-CB2 polyclonal antibodies (each diluted 1:250) and GAR-AP (diluted 1:2000) as second antibody (31). Saturation curves of [3H]CP55.940 binding and Western blot analysis of CB1 and CB2 receptors were performed under the same experimental conditions on mouse brain and mouse spleen extracts.
FAAH activity and expression
FAAH (EC 3.5.1.4) activity and its apparent Michaelis-Menten constant (Km) and maximum velocity (Vmax) were determined in freshly isolated or cultured Sertoli cells as previously reported (31). Cell homogenates from freshly isolated cells (20 µg/lane) were prepared as previously described (31) and subjected to SDS-PAGE (12%), under reducing conditions. The Rainbow molecular weight markers (Amersham Pharmacia Biotech, Little Chalfont, UK) were phosphorylase b (97.4 kDa), BSA (66.0 kDa), ovalbumin (46.0 kDa), and soybean trypsin inhibitor (27.0 kDa). For immunochemical analysis, gels were electroblotted onto 0.45-µm pore size nitrocellulose filters (Bio-Rad Laboratories, Inc.) and immunoreacted with anti-FAAH polyclonal (1:200) or anti-actin monoclonal (1:1000) antibodies using GAR-AP or goat antimouse antibodies conjugated to alkaline phosphatase (diluted 1:2000) as second antibody, respectively (31). Densitometric analysis of filters was performed by means of a Floor-S Multi-Imager equipped with a Quantity One software (Bio-Rad Laboratories, Inc.). The same anti-FAAH antibodies (diluted 1:300) were also used to determine FAAH protein content by ELISA, coating wells with cell homogenates (20 µg/well) as previously reported (31). RT-PCR was performed using total RNA isolated from Sertoli cells (10 x 106 cells) by means of the S.N.A.P. Total RNA Isolation Kit (Invitrogen, Carlsbad, CA) as previously described (31). The primers were as follows: for FAAH: forward, 5'-TGGAAGTCCTCCAAAAGCCCAG; reverse, 5'-TGTCCATAGACACAGCCCTTCAG; and for 18S rRNA: forward, 5'-AGTTGCTGCAGTTAAAAAGC; reverse, 5'-CCTCAGTTCCGAAAA CCAAC.
Five microliters of the reaction mixture were electrophoresed on a 6% polyacrylamide gel, which was then dried and subjected to autoradiography (31). The autoradiographic films were subjected to densitometric analysis using a Floor-S Multi-Imager equipped with a Quantity One software (Bio-Rad Laboratories, Inc.). Products were validated by size determination and sequencing (31).
Analysis of anandamide uptake
The uptake of [3H]AEA by the AMT of intact Sertoli cells (2 x 106/test) was performed as described previously (39). To discriminate noncarrier-mediated from carrier-mediated transport of AEA through cell membranes, [3H]AEA uptake at 4 C was subtracted from that at 34 C (39). The Q10 value of AMT was calculated as the ratio of AEA uptake at 30 C to that at 20 C (40). Incubations (15 min) were also carried out with different concentrations of [3H]AEA (0–800 nM) to determine the apparent Km and Vmax of AMT by Lineweaver-Burk analysis (also in this case the uptake at 4 C was subtracted from that at 34 C). The effects of different compounds on the uptake (15 min) of 200 nM [3H]AEA by AMT were determined by adding each substance directly to the incubation medium at the indicated concentrations. Cell viability after each treatment was greater than 90% in all cases.
Evaluation of cell death
To evaluate apoptosis, Sertoli cells were incubated for 24 h in the absence (control) or presence of AEA (0.25–1 µM) and congeners (Met-AEA, 2-AG, and PEA, each used at 1 µM). In another series of experiments, Sertoli cells were cultured for 24 h in the presence of AEA (1 µM), alone or with CB receptor antagonists or AMT modulators, or with various concentrations of FSH (0–100 mU/ml). Apoptosis was estimated by the cell death detection ELISA kit (Roche Molecular Biochemicals, Mannheim, Germany), based on the evaluation of DNA fragmentation by an immunoassay for histone-associated DNA fragments in the cell cytoplasm. This method has been recently validated by comparison with cytofluorometric analysis (29), performed in a FACSCalibur Flow Cytometer (BD Biosciences, Lincoln Park, NJ). This latter technique quantifies apoptotic body formation in dead cells by staining with propidium iodide (50 µg/ml).
Statistical analysis
The data reported in this paper are the mean ± SD of at least three independent determinations, each performed in duplicate. Statistical analysis was determined by the nonparametric Mann-Whitney test, elaborating experimental data by means of the InStat 3 program (GraphPad Software, Inc.).
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). The displacement data allowed to calculate an inhibition constant of 1700 ± 200 nM for Sertoli cells of both ages, a value typical of the binding of AEA to CB2 receptors (42). Consistently, the selective CB2 antagonist SR144528, but not the selective CB1 antagonist SR141716 (41), dose-dependently displaced [3H]CP55.940, suggesting that only CB2 receptors were expressed on the Sertoli cell surface (Fig. 1B). To further confirm the presence of CB2 receptors in Sertoli cells, saturation curves of [3H]CP55.940 binding to cell membranes and Western blot analysis of cell extracts were performed and compared with the same analysis in mouse brain (a positive control for CB1) and in mouse spleen (a positive control for CB2) (41, 42). [3H]CP55.940 was found to bind to Sertoli cell membranes with saturation curves very close to those obtained with mouse spleen membranes (Fig. 2A). From these saturation curves, Kd values of 311 ± 50 and 245 ± 30 pM and Bmax values of 233 ± 12 and 276 ± 10 fmol/mg protein could be calculated for Sertoli cells and spleen, respectively. On the other hand, binding of [3H]CP55.940 to mouse brain (Fig. 2A) showed a Kd of 598 ± 86 pM and a Bmax of 1773 ± 115 fmol/mg protein. The Kd and Bmax values found here for mouse brain and spleen are in agreement with previous reports (reviewed in Ref. 41). Consistent with the binding data, Western blot analysis showed that specific anti-CB2, but not anti-CB1, antibodies recognized a single immunoreactive band in Sertoli cell extracts (Fig. 2, B and C), further corroborating that these cells express functional CB2 receptors only. Moreover, Sertoli cells were able to bind [3H]AEA, and 1 µM SR144528, but not 1 µM SR141716, fully displaced it, whereas 1 µM CBD, an antagonist of the endothelial type cannabinoid receptor (7), and 1 µM CAPS, an antagonist of vanilloid receptors (43), were ineffective (not shown).
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). Kinetic analysis showed that FAAH hydrolyzed AEA in 4- and 16-d-old cells with apparent Vmax values of 250 ± 25 and 150 ± 15 pmol/min·mg protein, whereas the apparent Km was 3.5 ± 0.4 µM in both cases (Fig. 3B). Western blot analysis of Sertoli cell extracts showed that specific anti-FAAH antibodies recognized a single immunoreactive band of the molecular size expected for FAAH, the intensity of which decreased age-dependently (Fig. 4A). Densitometric analysis of the filter shown in Fig. 4A indicated that FAAH protein content in 16-d-old cells decreased to 60% compared with that in 4-d-old cells (100% = 1000 ± 100 U/mm2). On the other hand, 4- and 16-d-old cells expressed the same levels of actin (Fig. 4A), ruling out that the different levels of FAAH in these cells might be due to unequal loading of proteins. The same anti-FAAH antibodies were used to quantify FAAH content by ELISA, showing that the decrease in enzymatic activity in Sertoli cells (Fig. 3A) was dependent on a lower FAAH expression. Moreover, FAAH activity in 4-d-old Sertoli cells was undetectable at pH 5.0 and was fully inhibited by 10 µM arachidonoyl-trifluoro-methyl-ketone, a specific inhibitor (14). The enzyme was also able to hydrolyze 2-AG with an apparent Km of 4.2 ± 0.4 µM and a Vmax of 300 ± 30 pmol/min·mg protein. RT-PCR amplification of Sertoli cell cDNA showed a single band of the expected molecular size for the FAAH gene, which decreased age-dependently (Fig. 4B). Densitometric analysis of the autoradiographic film shown in Fig. 4B indicated that in 16-d-old cells FAAH mRNA decreased to 50% of that in 4-d-old cells (100% = 2700 ± 300 U/mm2). Under the same experimental conditions, the expression of the 18S rRNA gene was unaffected (Fig. 4B).
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) typical of AMT (13, 39, 40). Apparent Vmax values of AMT were 86 ± 9 and 58 ± 6 pmol/min·mg protein in 4- and 16-d-old cells, respectively. The apparent Km was 160 ± 16 nM in both cases. The uptake of 200 nM [3H]AEA was almost completely inhibited by 10 µM AM404 or 10 µM linvanil, specific inhibitors of AMT (39, 44), whereas it was doubled by the nitric oxide (NO) donor SNP, used at 5 mM, and by the peroxynitrite-donor SIN-1 used at 1 mM (Fig. 5B).
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). In 4-d-old Sertoli cells, apoptosis was significant (P < 0.05) at 0.5 µM AEA (1.5-fold over the control) and reached a maximum (2-fold) at 1 µM AEA (Fig. 6A). Remarkably, in 16-d-old cells the proapoptotic activity of AEA was significant (P < 0.05) at 0.25 µM, and it remained significantly higher than that in 4-d-old cells at all of the concentrations used (Fig. 6A). FACS analysis showed that 4- and 16-d-old Sertoli cells treated for 24 h with 1 µM AEA had approximately 9% and 14% apoptotic bodies, compared with about 4% in control Sertoli cells. Met-AEA, a nonhydrolyzable analog of AEA (45), was more effective than AEA in inducing DNA fragmentation, again more in 16- than in 4-d-old cells, whereas AEA congeners 2-AG and PEA were ineffective (Fig. 6B). DNA fragmentation induced by 1 µM AEA in 16-d-old cells was significantly (P < 0.05) enhanced by 0.1 µM SR144528 or 10 µM AM404, whereas 5 mM SNP significantly decreased it, and SR141716, CBD, and CAPS (each used at 1 µM) were ineffective (Fig. 6C). Finally, FSH dose dependently inhibited DNA fragmentation induced by 1 µM AEA (Fig. 6C), although it was ineffective on apoptosis induced by 1 µM Met-AEA (not shown).
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). The effect of increasing concentrations of FSH (in the 0–100 mU/ml range) on FAAH activity was further tested in 16-d-old Sertoli cells, demonstrating that hydrolase activity increased with gonadotropin concentration, but was fully inhibited by 0.1 µg/ml ACTD or CHX (Fig. 7B). Treatment of cells with 0.2 mM (Bu)2cAMP for 24 h also enhanced FAAH activity up to 3.5-fold over that in untreated controls (Fig. 7B). Furthermore, 33P labeling failed to detect any band with the electrophoretic mobility of FAAH in either control cells or cells treated with 100 mU/ml FSH for 24 h (data not shown). Finally, FSH (100 mU/ml for 24 h) did not affect AMT activity or the binding of [3H]CP55.940, which were 94% or 90% of the control values, respectively (P > 0.05 in both cases).
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Immature Sertoli cells express functional type 2 cannabinoid (CB2) receptors on their surface, as suggested by 1) the displacement of [3H]CP55.940 by AEA and SR144528, 2) the Kd and Bmax values calculated from saturation binding curves, and 3) the cross-reactivity with specific anti-CB2 antibodies. The level of these CB2 receptors is constant for at least 16 d, whereas FAAH activity declines age-dependently, due to a lower gene expression. The similarity of kinetic data for AEA hydrolysis in Sertoli cells from 4- and 16-d-old mice indicates that lower amounts of the same enzyme, rather than different FAAH isozymes, are expressed during postnatal testis development. This is the first demonstration of modulation of the endocannabinoid system by early postnatal aging. The uptake of AEA through its specific carrier (AMT) also declines in aging Sertoli cells during the neonatal period. The molecular properties of AMT are not known, and no probes are yet available to measure its expression (13). However, the observation that AMT in 4- and 16-d-old Sertoli cells has the same Km, but different Vmax, values suggests an age-dependent down-regulation of the expression of the same carrier protein. As the affinity of Sertoli AMT for AEA is very close to those of human lymphocytes (20) and endothelial cells (39), it can be proposed that the same carrier is present on the surface of different peripheral cells. Moreover, AEA uptake by Sertoli AMT, like that of other human peripheral cells, was significantly increased by the NO donor SNP and even more by the peroxynitrite donor SIN-1 (20, 39). The up-regulation of AMT by NO might be relevant in vivo, because NO plays several roles in regulating male fertility (48, 49). In particular, NO regulates the contribution of Sertoli cells to fertility and inflammation-mediated infertility (49, 50), and a faster removal of AEA from the extracellular space, which leads to termination of its biological activity, might be the rationale for these effects of NO. However, further experiments are needed to corroborate this hypothesis and to unravel the possible interplay between the endocannabinoid system and NO in male reproductive endocrinology.
A major finding of this investigation is that AEA can induce DNA fragmentation in Sertoli cells, and that this process is more evident upon early postnatal aging. Growing evidence is being collected which suggests that AEA might have proapoptotic activity, both in vivo (28) and in vitro (29), yet the mechanism(s) of AEA-induced apoptosis remains to be elucidated. To date activation of CB1 (28), CB2 (51), or vanilloid (29) receptors has been implicated in different experimental systems, but new receptors (52) or as yet unidentified receptors (3, 4) might also be involved. In this context an interesting finding is that in some cellular models activation of CB1 (e.g. in neuroblastoma cells) or CB2 (e.g. in lymphoma cells) receptors significantly reduces AEA-induced apoptosis, implying a protective, rather than causative, role of these receptors against the apoptotic potential of AEA (29, 53). Here, it is shown that the proapoptotic effect of AEA in Sertoli cells was specific, as the congeners 2-AG and PEA were ineffective, and was not mediated by type 1, type 2, or endothelial-type cannabinoid receptors or by vanilloid receptors, as shown by the lack of effect of their specific antagonists. However, the blockade of AEA uptake by the AMT inhibitor AM404 enhances the proapoptotic activity of the endocannabinoid, whereas the opposite occurred when AMT activity was enhanced by SNP. Taken together, these data suggest that AEA acts at the membrane level,through either a nonreceptor-mediated mechanism or a nonclassical cannabinoid receptor (3, 4). Nevertheless, CB2 receptor inactivation by SR144528 further increased AEA-induced apoptosis, suggesting that CB2 receptors have a protective role against the cytotoxic effects of AEA, in keeping with recent observations with immune (29) and neuronal (53) cells. On the other hand, it is noteworthy that FSH dose-dependently inhibited apoptosis induced by AEA, but not that induced by its stable analog Met-AEA (this report and data not shown). Therefore, it could be speculated that degradation might be critical in the ability of AEA to induce apoptosis in the testis. Indeed, FSH does not affect the binding of AEA to CB2 nor AMT activity of Sertoli cells, but induces a remarkable (4- to 5-fold) increase in FAAH activity, but not FAAH gene expression. Several activities of FSH on prepubertal Sertoli cells are mediated by cAMP and can be mimicked by cAMP analogs (27). Along this line, a cAMP-dependent pathway appears to be involved in FAAH activation, as suggested by the fact that (Bu2)cAMP mimicked the effect of FSH. This effect is also dependent on mRNA transcription and protein synthesis, as shown by its inhibition by ACTD and CHX. Moreover, FAAH gene has a consensus sequence for SH3-binding domains (54); however, FSH does not lead to FAAH phosphorylation, as shown by 33P labeling experiments. Therefore, it can be proposed that FSH enhances FAAH activity by stimulating the cAMP-dependent synthesis of an activator of the enzyme. Also, an indirect regulation of FAAH by compounds generated by unrelated enzymes can be considered (37). At any rate, the observation that FSH activates FAAH adds a new player to the hormone/cytokine/endocannabinoid network regulating fertility in mammals, after the report on the effect of progesterone on the same enzyme (31). Along this line, taking into account that exogenous cannabinoids lower testosterone secretion and impair sperm function (17), the presence in Sertoli cells of a complete endocannabinoid system speaks in favor of a physiological role of AEA in controlling male fertility. Yet, the details and clinical implications of the action of this endogenous cannabinoid in the testis remain to be elucidated.
In conclusion, we have found that altered levels of FSH can affect testis development through the control of the proapoptotic potential of AEA. This observation together with the well established relationship of Sertoli cell number to the total spermatogenic output of the testis can contribute to the negative effects exerted on testicular development by altered FSH concentrations as well as by mutations of the FSH receptor gene (55). In this context, the finding that Sertoli cells partake in the peripheral endocannabinoid system opens new perspectives to the understanding and treatment of male fertility problems.
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Acknowledgments |
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1 M.M. and S.C. contributed equally to this work. 
Abbreviations: ACTD, Actinomycin D; AEA, anandamide (N-arachidonoylethanolamine); 2-AG, 2-arachidonoylglycerol; AM404, N-(4- hydroxyphenyl)-arachidonoylamide; AMT, anandamide (N-arachidonoylethanolamine) membrane transporter; Bmax, binding capacity; (Bu)2cAMP, N,O'-dibutyryl cAMP; CAPS, capsazepine (N-[2-(4-chlorophenyl)ethyl]-1,3,4,5-tetrahydro-7,8-dihydroxy-2H-2-benzazepine-2-carbo-thioamide); CBD, cannabidiol; CB1/2R, type 1/2 cannabinoid receptors; CHX, cycloheximide; CP55.940, 5-(1,1'-dimethyheptyl)-2-[1R,5R-hydroxy-2R-(3-hydroxypropyl) cyclohexyl]-phenol; FAAH, fatty acid amide hydrolase; GAR-AP, goat antirabbit antibodies conjugated to alkaline phosphatase; Kd, dissociation constant; Km, Michaelis-Menten constant; NO, nitric oxide; PEA, N-palmitoylethanolamine; SIN-1,3-morpholinosydnonimine; SNP, sodium nitroprusside; SR141716, N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide; SR144528, N-[1(S)-endo-1,3,3-trimethyl-bicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methyl-phenyl)-1-(4-methyl-benzyl)-pyrazole-3-carboxamide; THC, 9-tetrahydrocannabinol; Vmax, maximum velocity.
Received May 23, 2002.
Accepted for publication August 22, 2002.
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 M. Bari, P. Spagnuolo, F. Fezza, S. Oddi, N. Pasquariello, A. Finazzi-Agro, and M. Maccarrone Effect of Lipid Rafts on Cb2 Receptor Signaling and 2-Arachidonoyl-Glycerol Metabolism in Human Immune Cells J. Immunol., October 15, 2006; 177(8): 4971 - 4980. [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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 C. C. Felder, A. K. Dickason-Chesterfield, and S. A. Moore Cannabinoids Biology: The Search for New Therapeutic Targets Mol. Interv., June 1, 2006; 6(3): 149 - 161. [Abstract] [Full Text] [PDF]
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 S. V. Siegmund, E. Seki, Y. Osawa, H. Uchinami, B. F. Cravatt, and R. F. Schwabe Fatty Acid Amide Hydrolase Determines Anandamide-induced Cell Death in the Liver J. Biol. Chem., April 14, 2006; 281(15): 10431 - 10438. [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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 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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 M. Rossato, F. Ion Popa, M. Ferigo, G. Clari, and C. Foresta Human Sperm Express Cannabinoid Receptor Cb1, the Activation of Which Inhibits Motility, Acrosome Reaction, and Mitochondrial Function J. Clin. Endocrinol. Metab., February 1, 2005; 90(2): 984 - 991. [Abstract] [Full Text] [PDF]
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, P < 0.05 (vs. AEA-treated cells).