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Endocrinology Vol. 148, No. 3 1431-1439
Copyright © 2007 by The Endocrine Society

Follicle-Stimulating Hormone Activates Fatty Acid Amide Hydrolase by Protein Kinase A and Aromatase-Dependent Pathways in Mouse Primary Sertoli Cells

Gianna Rossi1, Valeria Gasperi1, Rita Paro, Daniela Barsacchi, Sandra Cecconi2 and Mauro Maccarrone2

Department of Biomedical Sciences and Technologies (G.R., R.P., S.C.), University of L’Aquila, 67100 L’Aquila, Italy; Department of Biomedical Sciences (V.G., D.B., M.M.), University of Teramo, 64100 Teramo, Italy; and European Center for Brain Research (CERC)/IRCCS S. Lucia Foundation (V.G., M.M.), 00143 Rome, Italy

Address all correspondence and requests for reprints to: Professor Mauro Maccarrone, Department of Biomedical Sciences, University of Teramo, Piazza A. Moro 45, 64100 Teramo, Italy. E-mail: mmaccarrone{at}unite.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Among the biological activities of the endocannabinoid anandamide (N-arachidonoylethanolamine) (AEA), growing interest has been attracted by the regulation of mammalian fertility. Recently we have shown that treatment of mouse primary Sertoli cells with FSH enhances the activity of the AEA hydrolase [fatty acid amide hydrolase (FAAH)], though the molecular details were not elucidated. Here, we investigated whether FSH was also able to affect the enzymes that synthesize AEA (N-acyltransferase and N-acyl-phosphatidyl-ethanolamine-phospholipase D), the endogenous content of this endocannabinoid, and the level of the AEA-binding vanilloid receptor 1 (transient receptor potential channel vanilloid receptor subunit 1). We show that FSH enhanced FAAH activity (up to ~500% of the controls) and expression (up to ~300%), leading to a marked reduction (down to ~15%) of AEA content. However N-acyltransferase and N-acyl-phosphatidyl-ethanolamine-phospholipase D activity, and transient receptor potential channel vanilloid receptor subunit 1 binding were not affected. We also show that diacylglycerol lipase and monoacylglycerol lipase, which respectively synthesize and degrade 2-arachidonoyl-glycerol, were not regulated by FSH, neither was the membrane transport of this endocannabinoid. In addition, we show that FAAH stimulation by FSH was abrogated by inhibitors of protein kinase A (PKA) and cytochrome-P450 aromatase, and was conversely mimicked by N,O’-dibutyryl cAMP and estrogen. Finally, we demonstrate that FSH protects Sertoli cells against the pro-apoptotic activity of AEA, through PKA and aromatase-dependent activation of FAAH. Altogether these data suggest that FAAH is the only target of FSH among the elements of the endocannabinoid system, and that its regulation by PKA and aromatase-dependent pathways impacts Sertoli cell proliferation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FSH IS A member of the glycoprotein hormone family that includes LH, human chorionic gonadotropin, and thyroid-stimulating hormone (1). It transmits its signals via the 75-kDa FSH receptor, a G protein-coupled receptor that activates adenylate cyclase and increases cAMP levels (2). Subsequently, cAMP activates protein kinase A (PKA), which phosphorylates several cellular proteins, including the cAMP response element (CRE)-binding protein, a transcription factor with multiple target genes (2, 3). Also, FSH-dependent stimulation of Sertoli cell proliferation is mediated by cAMP/PKA, and, in general, this pathway is believed to mediate most biological activities of this gonadotropin (3, 4). The binding of FSH to its receptor can also trigger a phosphatidylinositol 3-kinase signaling (5), which is required for the stimulatory actions of FSH on cytochrome P450-aromatase (ARO) expression (6). ARO is a terminal enzyme that transforms irreversibly androgens into estrogens and plays a key role in male reproduction (7). Indeed, spermatogenesis is under estrogen [17ß-estradiol (E2)] control (8), and E2 is considered a survival factor for germ cells (9). Sertoli cells are the major source of testicular estrogens (10), and FSH can increase E2 production by enhancing the promoter of the aro gene (11). In addition, Sertoli cells are induced by FSH to divide up to the establishment of the blood-testis barrier, which is completed in rats within 19 d of postnatal life (12). This seems noteworthy because the final number of Sertoli cells determines testicular size and the total spermatogenic output (13). In the same line, although FSH is not considered essential for a qualitatively normal spermatogenesis, its absence causes a dramatic reduction in the number of spermatozoa produced during adult life (14).

Growing evidence has accumulated in recent years demonstrating that endogenous cannabinoids ("endocannabinods"), much like plant-derived cannabinoids (15), interfere with critical functions of mammalian and nonmammalian reproduction (16, 17). Endocannabinoids bind to and activate type-1 (CB1) and type-2 (CB2) cannabinoid receptors (18), which have been key regulators of embryo development, oviductal transport, and implantation (17). In line with this, it was observed that CB1–/–, CB2–/–, or CB1–/–xCB2–/– double mutant embryos show asynchronous development, compared with wild-type embryos (19, 20). In addition, a substantial number of CB1–/– and CB1–/–xCB2–/– mice show impaired oviductal transport with retention of embryos at the morula and blastocyst stages within the oviduct on d 4 (20), and approximately 40% of CB1–/– mice show pregnancy loss (19, 20). On the other hand, the impact of the deletion of CB1, CB2, or both on male fertility remains to be elucidated (16, 17). The endogenous ligands of CB1 and CB2 receptors are bioactive lipids that include anandamide (N-arachidonoylethanolamine) (AEA), the ethanolamide of arachidonic acid, 2-arachidonoylglycerol (2-AG), and some other amides, esters, and ethers of long chain polyunsaturated fatty acids (21). Endocannabinoids are synthesized on demand, and in the case of AEA the synthesis occurs by the sequential action of N-acyltransferase (NAT) (22) and N-acyl-phosphatidyl-ethanolamine (NAPE)-hydrolyzing phospholipase D (PLD) (23). By contrast, 2-AG is biosynthesized from membrane lipids by an sn-1-specific diacylglycerol lipase (DAGL) (24). In addition to CB1 and CB2 receptors (18), AEA can also activate type-1 vanilloid receptor [now called transient receptor potential channel vanilloid receptor subunit 1 (TRPV1)], a ligand-gated and nonselective cationic channel (25). A two-step mechanism consisting of cellular uptake followed by intracellular degradation terminates the biological actions of AEA and 2-AG. A purported endocannabinoid membrane transporter (EMT) seems to mediate the uptake of AEA and 2-AG according to a saturable, selective, and temperature-dependent process (26). Fatty acid amide hydrolase (FAAH) then cleaves intracellularly AEA (27), whereas monoacylglycerol lipase (MAGL) is mainly responsible for 2-AG hydrolysis (28). Altogether AEA and 2-AG, with other congeners like N-arachidonoyldopamine, noladin ether, and virodhamine, and the proteins that bind, transport, synthesize, and hydrolyze these lipids, form the "endocannabinoid system" (21).

AEA signaling is implicated in regulating sperm functions required for fertilization in invertebrates and mammals, including human beings (16, 17, 29). Recently, AEA has reduced human sperm motility and inhibited capacitation-induced acrosome reaction (30). In line with this, sperm cells of boar (Sus scropha), whose biology in some aspects resembles that of human beings, have possessed the biochemical machinery to bind (CB1 and TRPV1), synthesize (NAPE-PLD), and degrade (EMT and FAAH) AEA (31). Also, mouse Sertoli cells have been shown to express CB2 receptors, and the tools to transport (EMT) and hydrolyze (FAAH) AEA (32). Interestingly, FSH has increased FAAH activity in mouse Sertoli cells, thus reducing the pro-apoptotic potential of AEA toward these cells. However, the molecular mechanism of the effect of FSH on FAAH was not clarified. With this background, here we sought to investigate whether FAAH was the only target of FSH among the components of the endocannabinoid system and through which pathways FSH-dependent regulation could occur. To this end, we checked the effect of FSH on the AEA-synthesizing enzymes NAT and NAPE-PLD, and on the AEA-binding receptor TRPV1. We further extended the analysis to the proteins that synthesize, transport, and degrade the other major endocannabinoid 2-AG. We also checked whether two key pathways triggered by FSH, like those dependent on PKA and ARO (1, 2, 3), were implicated in the regulation of FAAH, and how these pathways might impact apoptosis of Sertoli cells induced by AEA.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals
Random-bred Swiss CD1 mice (Charles River, Como, Italy) were reared in our facilities. All animal experimentation described in this article was conducted in accordance with accepted standards of humane animal care. The local committees on animal care and use approved all experimental protocols that were applied according to accepted veterinary medical practice.

Chemicals
Chemicals were of the purest analytical grade. Myristoylated PKA inhibitor amide 14–22, capsazepine (N-[2-(4-chlorophenyl)ethyl]-1,3,4,5-tetrahydro-7,8-dihydroxy-2H-2-benzazepine-2-carbothioamide) (CPZ), and capsaicin were from Calbiochem (San Diego, CA). Resinferatoxin (RTX), 4-androsten-4-ol-3,17-dione, FSH, N,O’-dibutyryl cyclic AMP ((Bu)2cAMP), E2, actinomycin D, and cycloheximide were purchased from Sigma Aldrich Co. (St. Louis, MO). Cyclohexylcarbamic acid 3’-carbamoyl-biphenyl-3-yl ester (URB597) was from Cayman Chemicals (Ann Arbor, MI). JWH015 (2-methyl-1-propyl-1H-indol-3-yl)-1-naphthalenylmethanone) was from Tocris-Cookson (Bristol, UK). [3H]AEA (223 Ci/mmol), [3H]5-(1,1'-dimethyheptyl)-2-[1R,5R-hydroxy-2R-(3-hydroxypropyl) cyclohexyl]-phenol (126 Ci/mmol), [3H]RTX (43 Ci/mmol), sn-1-stearoyl-2-[14C]arachidonoyl-glycerol (56 mCi/mmol), and adenosine 5'-[{gamma}-32P] triphosphate (3000 Ci/mmol) were purchased from PerkinElmer Life Sciences (Boston, MA). [1,2,6,7-3H]Androst-4-ene-3,17ß-dione (105 Ci/mmol) and 1,2-di[1-14C] palmitoyl-phosphatidylcholine (111 mCi/mmol) were from Amersham Pharmacia Biotech (Uppsala, Sweden). N-[3H]Arachidonoyl-phosphatidylethanolamine ([3H]N-arachidonoyl-phosphatidylethanolamine, 200 Ci/mmol) and 2-oleoyl-[3H]glycerol (20 Ci/mmol) were from ARC (St. Louis, MO). 2-[3H]AG was synthesized from 1,3-dibenzyloxy-2-propanol and [3H]arachidonic acid (200 Ci/mmol; ARC), as reported (33). Primm S.r.l. (Milan, Italy) was used to prepare Rabbit anti-FAAH polyclonal antibodies (32), and goat antirabbit alkaline phosphatase conjugates were from Bio-Rad (Hercules, CA). 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 (SR144528) was a kind gift of Sanofi-Aventis Recherche (Montpellier, France).

Sertoli cell culture
Purified Sertoli cells were isolated from decapsulated testes of 20-d-old CD1 mice as described (32). Depending on the experimental procedures, cultured Sertoli cells (>95% pure) were treated for 24 h with the indicated concentrations of FSH, (Bu)2cAMP, E2, or endocannabinoid agonists/antagonists/inhibitors (serum-free MEM was added to the controls). The trypan blue dye exclusion test estimated viability, and determined that cell number and density did not change during culture in the absence or presence of treatments.

Assays of AEA metabolism and endogenous levels, and of TRPV1 binding
The synthesis of AEA through the activity of NAT was assayed using 1,2-di[1-14C]palmitoyl-phosphatidylcholine (1 x 106 dpm/test) as substrate, as reported (34). NAT activity was expressed as pmol [14C]N-palmitoylphosphatidylethanolamine formed per min per mg protein. The activity of NAPE-hydrolyzing PLD was assayed in Sertoli cell homogenates (50 µg/test) using 100 µM [3H]N-arachidonoyl-phosphatidylethanolamine, as reported (35). NAPE-PLD activity was expressed aspmol [3H]AEA released per min per mg protein. The hydrolysis of [3H]AEA by the FAAH activity was assayed in Sertoli cell homogenates (20 µg/test) with 10 µM [3H]AEA (32). FAAH activity was expressed as pmol [3H]arachidonic acid released per min per mg protein.

FAAH protein content was quantified by ELISA, performed on Sertoli cell homogenates (20 µg per well) with anti-FAAH polyclonal antibody (diluted 1:200), as reported (32). This polyclonal antibody recognizes a single immunoreactive band in Sertoli cell extracts, of the molecular weight expected for FAAH. Nonimmune rabbit serum (Primm S.r.l.) was used as a control of specificity (32). The ELISA test was calibrated by coating wells with different amounts of cell homogenates and was found to depend linearly on protein concentration in the range 0–100 µg per well. Within this range, the absorbance values at 405 nm (Y) depended on protein concentration (X) according to the equation: Y = 6.44 x 10–3X + 2.35 x 10–3 (R = 0.999), with a coefficient of variation of 3.82% (n = 3). RT-PCR was performed using total RNA (100 ng) isolated from Sertoli cells (10 x 106 per test), as described (34). The following primers were used for FAAH: 5'-TGGAAGTCCTCCAAAAGCCCAG (forward); 5'-TGTCCATAGACACAGCCCTTCAG (reverse). Five microliters of the reaction mixture were electrophoresed on a 6% polyacrylamide gel, and the RT-PCR products were excised from the gel and counted in a LKB1214 Rackbeta scintillation counter (Amersham Biosciences, Uppsala, Sweden), as reported (34). Products were validated by size determination and sequencing. The RT-PCR was calibrated by amplifying different amounts of RNA and was found to be linear in the range 0–500 ng per test. Within this range, the radioactivity (Y) depended on the amount of RNA (X) according to the equation: Y = 42 x + 3327 (R = 0.994), with a coefficient of variation of 4.64% (n = 3).

For the evaluation of endogenous levels of AEA, Sertoli cells (50 x 106 per test) were homogenized with an Ultra Turrax T25 (IKA Werke GmbH & Co. KG, Staufen, Germany) in 50 mM Tris-HCl, 1 mM EDTA (pH 7.4), and 1 mM phenylmethanesulfonyl fluoride buffer, lipids were extracted, and the organic phase was dried under nitrogen (31). Dry pellet was resuspended in 20 µl of methanol, and was processed and analyzed by HPLC with fluorometric detection (36), as reported (31). AEA content was expressed as pmol per mg protein.

Binding of 200 pM [3H]RTX, a selective agonist of TRPV1 receptors (31, 37), to Sertoli cells (50 x 106 per test) was performed on membrane fractions by rapid filtration assays (37), and was expressed as fmol [3H]RTX bound per mg protein. Nonspecific binding was determined in the presence of 1 µM "cold" RTX (31, 37).

Determination of 2-AG uptake, synthesis, and hydrolysis
The uptake of 2-AG by Sertoli cells (2 x 106/test) was assayed as described previously for the transport of AEA (32). Cells (2 x 106/test) were incubated for 15 min, at 37 C or 4 C, with 400 nM [3H]2-AG, then they were washed three times in 2 ml PBS containing 1% BSA and were finally resuspended in 200 µl PBS. Membrane lipids were then extracted, resuspended in 0.5 ml methanol, mixed with 3.5 ml Sigma-Fluor liquid scintillation cocktail for non-aqueous samples (Sigma), and radioactivity was measured in a LKB1214 Rackbeta scintillation counter (Amersham Biosciences). To discriminate noncarrier-mediated from carrier-mediated transport of 2-AG through cell membranes, [3H]2-AG uptake at 4 C was subtracted from that at 37 C (32). The transport activity was expressed as pmol [3H]2-AG taken up per min per mg protein. The activity of DAGL was assayed with 10 µM sn-1-stearoyl-2-[14C]arachidonoyl-glycerol as substrate (24), and that of MAGL was determined using as substrate 10 µM 2-oleoyl-[3H]-glycerol (28). Both DAGL and MAGL activities were expressed as pmol product per min per mg protein.

PKA activity assay
The PKA assay kit (Calbiochem) was used to measure PKA activity in Sertoli cells. This assay is based on the phosphorylation of the Kemptide peptide by PKA, via transfer of a labeled phosphate from [{gamma}-32P]ATP (38). Briefly, cells were washed with PBS and homogenized at 4 C with an Ultra Turrax T25 in 25 mM Tris-HCl (pH 7.4), 0.5 mM EDTA, 0.5 mM EGTA, 0.05% Triton X-100, 10 mM ß-mercaptoethanol, containing 1 µg/ml leupeptin and 1 µg/ml aprotinin. The homogenates were centrifuged at 14,000 x g for 5 min, and supernatants (20 µg/test) were incubated for 30 min at 37 C in a shaking water bath with [{gamma}-32P]ATP (0.2 µCi/µl, final concentration) alone or in the presence of the myristoylated PKA inhibitor amide 14–22 (39). The reaction was stopped by adding 50% trichloroacetic acid solution and 1% BSA, to reduce nonspecific phosphorylation. PKA activity was expressed as pmol phosphate incorporated per min per mg protein.

Aromatase activity assay
Tritiated water release was used to evaluate aromatase (ARO) activity in Sertoli cells, as reported (40). Briefly, cell homogenates (20 µg/test) were incubated for 90 min at 37 C in a shaking water bath with 0.5 µM [1,2,6,7-3H]androst-4-ene-3,17ß-dione alone or in presence of 10 µM 4-androsten-4-ol-3,17-dione, a specific ARO inhibitor (41). Adding chloroform stopped the reaction, and the unconverted substrate was extracted with 5 volumes of chloroform. The aqueous phase was recovered, and an equal volume of a mixture of 5% charcoal and 0.5% dextran T-70 further removed residual substrate. The enzymatic activity was expressed as fmol tritiated water released per min per mg protein.

Evaluation of cell death
Apoptosis was estimated in Sertoli cells 24 h after each treatment (vehicle was added to the controls) by the cell death detection ELISA kit (Roche Molecular Biochemicals, Mannheim, Germany). This method is based on the evaluation of DNA fragmentation by an immunoassay for histone-associated DNA fragments in the cell cytoplasm and has been recently validated by comparison with cytofluorometric analysis (32), performed in a FACS Calibur Flow Cytometer (BD Biosciences, Lincoln Park, NJ). The 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 performed by the nonparametric Mann-Whitney U test, elaborating experimental data by the InStat 3 program (GraphPad Software for Science, San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of FSH on AEA synthesis and endogenous levels, and on TRPV1 binding
In a previous report we have demonstrated that Sertoli cells isolated from 2- to 24-d-old mice expressed a functional endocannabinoid system, and that binding of AEA to CB2 receptors and its transport through EMT were not affected by 24-h treatment with FSH, up to a concentration of 100 mU/ml (32). By contrast, FAAH activity was increased up to approximately 5-fold over the controls under the same experimental conditions. Here, we extended our investigation to the enzymes that synthesize AEA (NAT and NAPE-PLD) to the endogenous levels of this substance, and to the AEA-binding vanilloid receptor (TRPV1). As shown in Fig. 1Go, Sertoli cells expressed a functional TRPV1, which had a binding activity of 120 ± 15 fmol/mg protein with 200 pM [3H]RTX as ligand. They were also able to synthesize de novo AEA by NAT activity (22 ± 4 pmol/min·mg protein) and NAPE-PLD activity (55 ± 8 pmol/min·mg protein), and consistently showed detectable levels of endogenous AEA (97 ± 12 pmol/mg protein). Treatment with FSH (100 mU/ml) did not affect the activity of NAT or NAPE-PLD; neither did it affect TRPV1 binding (Fig. 1Go). Yet under the same experimental conditions, FSH markedly reduced AEA content, down to approximately 15% of controls.


Figure 1
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  		FIG. 1. Effect of FSH on AEA synthesis and levels, and on TRPV1 receptors. Activity of the AEA-synthesizing enzymes NAT and NAPE-PLD, endogenous content of AEA and binding to TRPV1 receptors were assayed in Sertoli cells, treated or not with 100 mU/ml FSH for 24 h (100% = 22 ± 4 pmol/min·mg protein for NAT activity; 55 ± 8 pmol/min·mg protein for NAPE-PLD activity; 97 ± 12 pmol/mg protein for AEA content; or 120 ± 15 fmol/mg protein for TRPV1 binding). Vertical bars represent SD values (n = 4). *, P < 0.01 vs. controls.

 
Effect of FSH on 2-AG metabolism
We further extended our study to the metabolism of 2-AG, the most bioactive endocannabinoid together with AEA (21). The biosynthesis of 2-AG occurs via DAGL activity, whereas MAGL catalyzes the main hydrolytic pathway of 2-AG (42). Results reported in Table 1Go showed that treatment with FSH (100 mU/ml) for 24 h did not affect DAGL or MAGL activity, neither did it affect the transport of [3H]2-AG across the plasma membranes.


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  		TABLE 1. Effect of FSH treatment on 2-AG metabolism in Sertoli cells

 
Signaling pathways involved in the modulation of FAAH by FSH
Based on the evidence that AEA hydrolase was the only target of FSH among the proteins of the endocannabinoid system, we next sought to investigate which pathways might be involved in FSH-dependent enhancement of FAAH activity. In particular, critical pathways triggered by FSH to control Sertoli cells were studied, like those dependent on PKA and ARO (1, 3, 6). In a first set of experiments, increasing doses of FSH (from 0–100 mU/ml) were found to enhance significantly the activity of PKA and ARO, which reached a maximum of approximately 6-fold and approximately 3-fold over the unstimulated controls, respectively, upon exposure to a dose of 100 mU/ml (data not shown). Incidentally, these data extend to the activity of ARO a recent report showing that FSH stimulates its mRNA and protein content in Sertoli cells (6). The increase of PKA and ARO activity paralleled the dose-dependent increase of FAAH activity, which reached an approximate 5-fold maximum over the controls at 100 mU/ml FSH, in keeping with our previous report (32). The latter concentration of FSH was chosen for further experiments.

The hypothesis that PKA and ARO-dependent pathways could be instrumental in mediating the stimulatory effect of FSH on FAAH was further checked by co-incubating FSH-stimulated Sertoli cells with specific PKA and ARO inhibitors. In the presence of 50 µM myristoylated amide 14–22, a specific inhibitor of PKA (3, 39), a significant decrease of the activity of both PKA (down to ~20% of the controls), and FAAH (~35%) was recorded, whereas the addition of 10 µM 4-androsten-4-ol-3,17-dione, a specific ARO inhibitor (41, 43), minimized ARO (~15%) and again reduced FAAH (~30%) activity (Fig. 2Go). In addition, the combination of both PKA and ARO inhibitors decreased FAAH activity down to approximately 15% of the untreated controls, suggesting that PKA and ARO could act synergistically on FAAH. Finally, exposure of Sertoli cells to 100 nM URB597, a selective FAAH inhibitor (44), minimized FAAH activity (down to ~10% of the controls), without affecting the activity of PKA or ARO (Fig. 2Go).


Figure 2
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  		FIG. 2. Effect of inhibitors of PKA, ARO, or FAAH on enzyme activity in FSH-stimulated cells. Sertoli cells were treated for 24 h with 100 mU/ml FSH alone or in the presence of the PKA inhibitor myristoylated amide 14–22 (50 µM), the ARO inhibitor 4-androsten-4-ol-3,17-dione (10 µM), the FAAH inhibitor URB597 (100 nM), or their combination (100% = 6 ± 1 pmol/min·mg protein for PKA; 78 ± 10 fmol/min·mg protein for ARO; or 180 ± 20 pmol/min·mg protein for FAAH). *, P < 0.01 vs. FSH-treated cells. #, P < 0.05 vs. cells treated with PKA inhibitor or ARO inhibitor. Vertical bars represent SD values (n = 3).

 
We then investigated the modulation of FAAH by (Bu)2cAMP and E2, two substances that should mimic the effect of PKA or ARO, respectively. In fact, (Bu)2cAMP is a synthetic analog of cAMP that activates PKA (3), and E2 is the final product of ARO (7). Sertoli cells were treated for 24 h with concentration ranges of (Bu)2cAMP (0–200 µM; Ref. 32) and of E2 (0–200 nM; Ref. 45) already used for mimicking in vitro physiological conditions. As shown in Fig. 3AGo, (Bu)2cAMP induced a dose-dependent increase of FAAH activity that reached a maximum of approximately 4-fold over the untreated controls. Also, E2 dose dependently stimulated FAAH activity, with an approximate 2.5-fold maximum.


Figure 3
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  		FIG. 3. Effect of (Bu)2cAMP and E2 on FAAH activity and expression. A, Dose dependence of FAAH activity after 24 h of treatment with (Bu)2cAMP or E2 (100% = 180 ± 20 pmol/min·mg protein). B, Effect of FSH, (Bu)2cAMP, E2, or their combination on FAAH expression in Sertoli cells (100% = 0.130 ± 0.015 absorbance units at 405 nm, for the protein content; or 8000 ± 1000 cpm for the mRNA level). In both panels, vertical bars represent SD values (n = 4). *, P < 0.01. **, P < 0.05 vs. controls. #, P < 0.01 vs. cells treated with (Bu)2cAMP.

 
We further ascertained whether the effect of FSH, (Bu)2cAMP and E2 on FAAH activity could be due to the modulation of faah gene expression. Anti-FAAH antibodies were used to quantify FAAH protein, whereas RT-PCR followed by liquid scintillation counting was used to quantify FAAH mRNA (32, 34). Treatment with FSH (100 mU/ml) for 24 h was found to induce an approximate 3-fold increase of FAAH transcription and translation, whereas 200 µM (Bu)2cAMP induced a small (~1.5-fold) but significant (P < 0.05) increase of both FAAH protein and mRNA, compared with controls (Fig. 3BGo). After the treatment of Sertoli cells with 200 nM E2, FAAH transcription and translation reached a level comparable to that induced by FSH alone. Furthermore, the combination of 200 µM (Bu)2cAMP and 200 nM E2 approximately doubled the effect of (Bu)2cAMP alone on FAAH mRNA and protein (P < 0.01 in both cases), while increasing only slightly (~20%; P > 0.05) the effect of E2 alone (Fig. 3BGo). In keeping with these data, the increase of FAAH activity upon treatment with 100-mU/ml FSH (900 ± 80 vs. 180 ± 20 pmol/min·mg protein of controls; P < 0.01) was fully inhibited by 0.1 µg/ml actinomycin D (down to 220 ± 25 pmol/min·mg protein; P < 0.01 vs. FSH), or by 0.1 µg/ml cycloheximide (down to 240 ± 25 pmol/min·mg protein; P < 0.01 vs. FSH), further demonstrating that the stimulatory effect of the gonadotropin required new gene transcription and translation, respectively.

Effect of FSH on the apoptotic potential of AEA
Treatment of Sertoli cells with 1 µM AEA for 24 h led to DNA fragmentation (~3-fold over the controls), and 0.1 µM SR144528, a selective antagonist of CB2 receptors (18), further increased this effect of AEA by approximately 50% (from ~2.8 to ~4.2-fold over the controls). However, 100 mU/ml FSH minimized AEA-induced apoptosis, which almost returned to the control level (Fig. 4Go). These data recapitulate our previous findings, showing that AEA dose dependently induce apoptosis in Sertoli cell, and that CB2R antagonist potentiates, whereas FSH treatment reduces, the apoptotic potential of AEA (32). In addition, here AEA-induced apoptosis was found inhibited by 10 µM CPZ, a selective antagonist of TRPV1 receptor (37), suggesting that activation of this receptor was instrumental in triggering the programmed death of Sertoli cells. In this context, it should be recalled that SR144528 can act as an inverse agonist of CB2 receptors (46) and that, as such, it can mimic the actions of agonists in certain cell systems (47). Therefore, we used JWH015, a selective CB2R agonist (48), to ascertain whether the potentiation of the effect of AEA by SR144528 could be due to an agonist, rather than antagonist, action. We found that 1 µM JWH015 did not induce per se any apoptosis of Sertoli cells under the same experimental conditions used for AEA. However, 100 nM capsaicin, a selective TRPV1 agonist that does not bind to CBR (37), was enough to increase apoptosis of Sertoli cells by approximately 3-fold over the controls (data not shown), much alike 1 µM AEA (Fig. 4Go). Together, these data suggest that activation of CB2 receptors protected Sertoli cells against AEA-induced apoptosis, whereas activation of TRPV1 receptors induced it. This dual modulation of apoptosis by AEA via CB2 or TRPV1 receptors resembles a general mechanism already observed in neuronal and non-neuronal cells (49). Interestingly, the protective effect of FSH against AEA-induced apoptosis was prevented by 50 µM myristoylated amide 14–22 or by 10 µM 4-androsten-4-ol-3,17-dione, selective inhibitors of PKA or ARO, respectively (Fig. 4Go). The effect of these inhibitors was additive, and, in fact, their combination fully abrogated the anti-apoptotic effect of FSH. On the other hand, 200 µM (Bu)2cAMP or 200 nM E2 mimicked the protection exerted by FSH against AEA-induced apoptosis when added to Sertoli cells together with 1 µM AEA (Fig. 4Go).


Figure 4
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  		FIG. 4. Pro-apoptotic activity of AEA. Effect of 1 µM AEA alone or in the presence of 0.1 µM SR144528, 10 µM CPZ, 100 mU/ml FSH, 50 µM myristoylated amide 14–22 (PKA inhibitor), 10 µM 4-androsten-4-ol-3,17-dione (ARO inhibitor), 200 µM (Bu)2cAMP, 200 nM E2, or their combinations on DNA fragmentation after 24 h of treatment. Each compound added to Sertoli cells together with AEA had no effect on DNA fragmentation when used alone. Horizontal bars represent SD values (n = 3). *, P < 0.01. **, P < 0.05 vs. controls. #, P < 0.05. @, P < 0.01 vs. AEA-treated cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this investigation we report unprecedented evidence showing that FSH stimulates specifically the AEA-hydrolase FAAH, but not the AEA-synthesizing enzymes NAT and NAPE-PLD, the AEA-binding TRPV1 receptor, or the proteins that metabolize (DAGL and MAGL) or transport 2-AG. Together with a previous report demonstrating that AEA-binding CB2 receptors and AEA transporter of Sertoli cells are not modulated by FSH (32), these data strongly support the concept that FAAH is the only target of FSH among the elements of the endocannabinoid system in Sertoli cells. In this context, it seems noteworthy that the uptake of 2-AG is likely to occur through the same EMT that transports AEA (50, 51). Therefore, the lack of effect of FSH on 2-AG transport shown here corroborates the lack of effect on the uptake of AEA by the same cells (32). We also show that FSH markedly reduces endogenous levels of AEA, thus protecting Sertoli cells against the pro-apoptotic potential of AEA. In addition, we demonstrate that the stimulatory effect of FSH on FAAH and, thus, the reduction of endogenous AEA is mediated by PKA and ARO-dependent pathways, which affect to a different extent faah gene expression. Together, these data strongly suggest that AEA can contribute to the mitogenic and regulative functions of FSH on primary Sertoli cells, most notably cell proliferation (12). It is well demonstrated that Sertoli cells are critical for coordinating spermatogenesis in mammals. In fact, these somatic cells not only exert nutritional and structural roles but also are indispensable for male germ cell development and movement (52). The fact that Sertoli cells control the release of the mature spermatids from the seminiferous epithelium underlines their capacity to impact the total spermatogenic output in adult life (52). In line with this, the treatment of immature Sertoli cells with FSH has up-regulated or down-regulated 100–300 transcripts by 2-fold or greater (53). Yet, a major issue remains to understand the signaling pathways triggered by FSH to guide Sertoli cell functions, in particular those involved in the control of their growth and differentiation, and, hence, of spermatogenesis (52, 54). Therefore, a major outcome of this investigation seems the fact that FAAH is a specific target of FSH, and that its stimulation occurs via PKA and ARO-dependent pathways. Two types of experiments supported this hypothesis: 1) a pharmacological approach, whereby selective inhibitors of PKA or ARO were shown to inhibit also FAAH; and 2) the use of (Bu)2cAMP and E2, which mimicked the stimulatory effect of FSH. In this context, the observation that PKA and ARO inhibitors had an additive effect when used in combination (Fig. 2Go) seems to suggest that the PKA and ARO-dependent pathway were independent. Interestingly, stimulation of FAAH activity by FSH occurred through increased expression of the faah gene at both transcriptional and translational levels (Fig. 3BGo). Treatment with 200 µM (Bu)2cAMP enhanced FAAH expression by approximately 1.5-fold over the controls, while increasing FAAH activity up to approximately 4-fold, whereas 200 nM E2 stimulated FAAH activity (~2.5-fold) and expression (~3-fold) to similar extents (compare Fig. 3Go, A and B). These findings suggest that the PKA-dependent pathway stimulates FAAH mainly by acting on enzyme activity, rather than on faah gene expression. Because a direct phosphorylation of FAAH upon FSH treatment has already been excluded (32), it can be proposed that accessory proteins are expressed upon FSH treatment, which in turn may work as FAAH activators. It should be recalled that current knowledge of FSH biological activities suggests that the hormone exerts not only transcriptional effects but can also act post transcriptionally (3) [e.g. by regulating nuclear export (55) or stability (56) of mRNAs]. On the other hand, the human faah promoter contains a CRE-like site (34), and it is known that binding of FSH to its receptor can trigger the phosphorylation of the CRE-binding protein (53). Stimulation of FAAH by E2 seems to be due entirely to enhanced faah gene expression at the transcriptional and translational levels. This observation is consistent with the presence of an estrogen response element in the mouse faah promoter (34, 57). In this line, it should be recalled that in a previous report, we have found that in 16-d-old Sertoli cells, FSH up-regulates FAAH activity in a manner dependent on mRNA transcription and protein synthesis, but apparently not on FAAH protein expression (32). This is at variance with the present findings on 20-d-old Sertoli cells and might be due to the different age of cells, a condition known to affect the biological activity of FSH (1, 2, 5, 32). More importantly, the difference between 16 and 20-d-old Sertoli cells seems to support the hypothesis that increased FAAH activity does not depend solely on enhanced expression of faah gene but can also occur through enhanced expression of accessory proteins, as shown here for (Bu)2cAMP (Fig. 3Go, A and B). At any rate, the observation that FSH activates FAAH through PKA and ARO-dependent pathways seems a major finding of this study. The overall regulation of FAAH by FSH is schematically depicted in Fig. 5Go.


Figure 5
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  		FIG. 5. Regulation of FAAH by FSH via PKA and ARO-dependent pathways. The scheme suggests that FSH receptor triggers activation of PKA, which phosphorylates transcription factors and, more importantly, accessory proteins able to act as enzyme activators. Independently of this pathway, FSH can trigger an ARO-dependent route, which enhances faah gene expression by acting directly at the promoter level.

 
A further point of interest seems the observation that FSH, by stimulating FAAH, markedly reduces (down to ~15% of the controls) the endogenous tone of AEA (Fig. 1Go). The finding that enhanced FAAH activity corresponds to reduced AEA levels is in keeping with the observation that genetic ablation of FAAH leads to approximately 15-fold higher AEA levels in faah knockout mice, compared with wild-type littermates (58). More interestingly, we show that the down-regulation of the AEA level by FSH might be a method to control Sertoli cell number. In fact, we show that FSH reduces the apoptotic potential of AEA through PKA and ARO activity (Fig. 4Go). In support of this hypothesis: 1) PKA and ARO inhibitors reduced the protection exerted by FSH against AEA-induced apoptosis in an additive manner; and 2) the effect of FSH on DNA fragmentation was mimicked by (Bu)2cAMP and E2. It should be also stressed that the novel anti-apoptotic effect of E2 documented here in Sertoli cells gives a biochemical background to previous data demonstrating that aro-null mice, unable to convert testosterone to E2, show a drastic increase in Sertoli cell apoptosis (59). Together, the present observations also support the notion that FSH and E2 play a key role in determining Sertoli cell number (60), thus adding a new dimension to the signaling networks based on gonadotropins, steroid hormones, and endocannabinoids (61). Additionally, it seems noteworthy that the present data on the effect of AEA on Sertoli cells lend support to previous studies on the effect of the exogenous CBR agonist {Delta}9-tetrahydrocannabinol (THC), the primary psychoactive constituent of marijuana, on sperm production in human beings and experimental animals (16, 17, 62). In fact, THC has directly affected Sertoli cell functions by: 1) inhibiting FSH-stimulated cAMP production (63); 2) stimulating secretion of androgen binding protein (64); and 3) stimulating secretion of lactate (65). Furthermore, other potential targets for THC in testis are Leydig cells (66, 67) and remarkably developing spermatocytes (29, 68). Altogether, these observations point to (endo)cannabinoids as critical regulators of male fertility, unmasking the molecular targets implicated in the health damage by cannabis use and abuse, on the one hand, and the potential therapeutic exploitation of (endo)cannabinoid-oriented drugs for the treatment of infertility, on the other hand. On a final note, the concept that the control of the endogenous tone of AEA might be physiologically instrumental for the control of the Sertoli cell population by FSH is in keeping with the lack of effect of this gonadotropin on 2-AG metabolism (Table 1Go). In fact, whereas the latter endocannabinoid shares several biological activities with AEA, both centrally and peripherally (21), it does not induce apoptosis in neuronal or non-neuronal cells (49).

In conclusion, the present investigation suggests that AEA-dependent signaling, controlled by FSH through the modulation of FAAH, might be a method for the modulation of Sertoli cell number and, consequently, male fertility by this gonadotropin. In this context, it should be recalled that FAAH deficiency has limited early pregnancy events in human beings (69) as well as mice (70); therefore, the present investigation, showing that gonadotropins and steroids up-regulate FAAH activity and expression, seems to open a new perspective also for the treatment of female infertility.


   Acknowledgments
 
We thank Dr. Monica Bari, Filomena Fezza (University of Rome "Tor Vergata"), Natalia Battista, and Sergio Oddi (University of Teramo) for their valuable help with the biochemical assays, and Dr. Patrizia Falasca for her assistance with cell cultures.


   Footnotes
 
This work was supported by Fondazione della Cassa di Risparmio di Teramo (TERCAS 2004), Ministero della Salute (R.C. 2005), Istituto Superiore di Sanità (AIDS Project 2005), and Agenzia Spaziale Italiana (DCMC and MoMa projects, 2006).

Disclosure Statement: The authors have nothing to disclose.

First Published Online November 16, 2006

1 G.R. and V.G. contributed equally to the study. Back

2 S.C. and M.M. are equally senior authors. Back

Abbreviations: AEA, Anandamide (N-arachidonoylethanolamine); 2-AG, 2-arachidonoylglycerol; ARO, cytochrome P450-aromatase; (Bu)2cAMP, N,O’-dibutyryl cAMP; CPZ, capsazepine (N-[2-(4-chlorophenyl)ethyl]-1,3,4,5-tetrahydro-7,8-dihydroxy-2H-2-benzazepine-2-carbothioamide); CRE, cAMP response element; DAGL, diacylglycerol lipase; E2, 17ß-estradiol; EMT, endocannabinoid membrane transporter; FAAH, fatty acid amide hydrolase; JWH015, (2-methyl-1-propyl-1H-indol-3-yl)-1-naphthalenylmethanone; MAGL, monoacylglycerol lipase; NAT, N-acyltransferase; NAPE, N-acyl-phosphatidyl-ethanolamine; PKA, protein kinase A; PLD, phospholipase D; RTX, resinferatoxin; 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, {Delta}9-tetrahydrocannabinol; TRPV1, transient receptor potential channel vanilloid receptor subunit 1; URB597, cyclohexylcarbamic acid 3’-carbamoyl-biphenyl-3-yl ester.

Received July 20, 2006.

Accepted for publication November 8, 2006.


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