Introduction Anandamide (arachidonoylethanolamide, AEA) belongs to a group of endogenous lipids, which include amides, esters and ethers of long chain polyunsaturated fatty acids, collectively termed ‘endocannabinoids’ (Mechoulam, 2002; De Petrocellis et al., 2004). It binds to type-1 and type-2 cannabinoid receptors (CB1R and CB2R), thus having many actions in the central (Fride, 2002) and peripheral (Parolaro et al., 2002) nervous systems. These activities of AEA are terminated by cellular uptake through an AEA membrane transporter (AMT) (Hillard and Jarrahian, 2003), followed by degradation to ethanolamine and arachidonic acid by the fatty acid amide hydrolase (FAAH) (Bisogno et al., 2002; Bracey et al., 2002). The checkpoint in AEA synthesis is the N-acyl-phosphatidylethanolamines (NAPE)-hydrolyzing phospholipase D (NAPE-PLD), which releases, on demand, AEA from membrane NAPEs (Okamoto et al., 2004). Together with AEA and congeners such as 2- arachidonoylglycerol (2-AG), N-arachidonoyldopamine, noladin ether and virodhamine, these proteins form the ‘endocannabinoid system’ (De Petrocellis et al., 2004). In addition, type 1 vanilloid receptors (now called transient receptor potential channel vanilloid receptor subunit 1, TRPV1), that are six trans-membrane spanning proteins with intracellular N and C termini (Jordt and Julius, 2002), have been shown to be activated by AEA (De Petrocellis et al., 2001). Therefore, this lipid is also a true ‘endovanilloid’ (Van der Stelt and Di Marzo, 2004). Among the peripheral activities of AEA, regulation of fertility has attracted growing interest (Maccarrone and Finazzi-Agrò, 2004), adding the well-known effects of exogenous cannabinoids to its already established function as an endogenous ligand of CB receptors (reviewed by Park et al., 2003; Park et al., 2004). For example, low FAAH in circulating maternal lymphocytes has been shown to be an early (<8 weeks of gestation) predictor of spontaneous abortion in humans (Maccarrone and Finazzi-Agrò, 2004), and consistently FAAH expression is under control of fertility signals like progesterone and leptin (Maccarrone et al., 2003a). By contrast, mouse uterus contains the highest amounts of AEA as yet measured in any tissue (Paria and Dey, 2000), and uterine AEA can activate CB1 receptors in this organ, thus allowing epithelial changes needed for reproduction (Maccarrone and Finazzi-Agrò, 2004). Additionally, within a very narrow concentration range AEA regulates blastocyst function and implantation by differentially modulating mitogen-activated protein kinase signaling and calcium channel activity via CB1 receptors (Wang et al., 2003). Recently, it has been demonstrated that rat testis is able to synthesize AEA (Sugiura et al., 1996), and this compound has been detected also in human seminal plasma at nanomolar 4393 Anandamide (AEA) is the endogenous ligand of cannabinoid (CB) receptors, and as such it plays several central and peripheral activities. Regulation of female fertility by AEA has attracted growing interest, yet a role for this endocannabinoid in controlling sperm function and male fertility in mammals has been scarcely investigated. In this study we report unprecedented evidence that boar sperm cells have the biochemical machinery to bind and degrade AEA, i.e. type-1 cannabinoid receptors (CB1R), vanilloid receptors (TRPV1), AEA-synthesizing phospholipase D (NAPE-PLD), AEA transporter (AMT) and AEA hydrolase (FAAH). We also show that the non- hydrolyzable AEA analogue methanandamide reduces sperm capacitation and, as a consequence, inhibits the process of acrosome reaction (AR) triggered by the zona pellucida, according to a cyclic AMP-dependent pathway triggered by CB1R activation. Furthermore, activation of TRPV1 receptors seems to play a role of stabilization of the plasma membranes in capacitated sperm, as demonstrated by the high incidence of spontaneous AR occurring during the cultural period when TRPV1 activity was antagonized by capsazepine. We show that sperm cells have a complete and efficient endocannabinoid system, and that activation of cannabinoid or vanilloid receptors controls, at different time-points, sperm functions required for fertilization. These observations open new perspectives on the understanding and treatment of male fertility problems. Key words: Anandamide, Cannabinoid receptor, Cyclic AMP, Metabolism, Vanilloid receptor, Zona pellucida Summary Characterization of the endocannabinoid system in boar spermatozoa and implications for sperm capacitation and acrosome reaction Mauro Maccarrone 1, *, Barbara Barboni 1 , Andrea Paradisi 1 , Nicola Bernabò 1 , Valeria Gasperi 1,2 , Maria Gabriella Pistilli 1 , Filomena Fezza 1,2 , Pia Lucidi 1 and Mauro Mattioli 1 1 Department of Biomedical Sciences, University of Teramo, Pizza A. Moro 45, 64100 Teramo, Italy 2 Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Via Montpellier 1, 00133 Rome, Italy *Author for correspondence (e-mail: mmaccarrone@unite.it) Accepted 9 June 2005 Journal of Cell Science 118, 4393-4404 Published by The Company of Biologists 2005 doi:10.1242/jcs.02536 Research Article Journal฀of฀Cell฀Science