Anandamide Activity and Degradation Are Regulated by Early Postnatal Aging and Follicle-Stimulating Hormone in Mouse Sertoli Cells

0013-7227/03/$15.00/0 Printed in U.S.A. Endocrinology 144(1):20 –28 Copyright © 2003 by The Endocrine Society doi: 10.1210/en.2002-220544 Anandamide Activity and Degradation Are Regulated by Early Postnatal Aging and Follicle-Stimulating Hormone in Mouse Sertoli Cells MAURO MACCARRONE*, SANDRA CECCONI*, GIANNA ROSSI, NATALIA BATTISTA, RICCARDO PAUSELLI, AND ALESSANDRO FINAZZI-AGRÒ 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 Anandamide (AEA), a prominent member of the endogenous ligands of cannabinoid receptors (endocannabinoids), is known to adversely affect female fertility. However, a potential role of AEA in male reproductive functions is unknown. Here we report evidence that immature mouse Sertoli cells have the biochemical tools to bind and inactivate AEA, i.e. a functional type-2 cannabinoid receptor (CB2R), a selective AEA membrane transporter, and an AEA-degrading enzyme fatty acid amide hydrolase. We show that, unlike CB2R, the activity of AEA membrane transporter and the activity and expression of FAAH decrease, whereas the apoptosis-inducing activity of AEA increases with age during the neonatal period. We also show that FSH reduces the apoptotic potential of AEA, but not that of its nonhydrolyzable analog methanandamide. Concomitantly, FSH enhances FAAH activity in a manner dependent on mRNA transcription and protein synthesis and apparently involving cAMP. These data demonstrate that Sertoli cells partake in the peripheral endocannabinoid system, and that FSH reduces the apoptotic potential of AEA by activating FAAH. Taken together, it can be suggested that the endocannabinoid network plays a role in the hormonal regulation of male fertility. (Endocrinology 144: 20 –28, 2003) E NDOCANNABINOIDS are amides, esters, and ethers of long-chain polyunsaturated fatty acids found in several human tissues (1, 2). N-Arachidonoylethanolamine, anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are the main endocannabinoids described to date (3, 4). They bind to both brain (CB1) and peripheral (CB2) cannabinoid receptors, thus mimicking some of the central and peripheral effects of ⌬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 anti- inflammatory 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 de- Abbreviations: ACTD, Actinomycin D; AEA, anandamide (N-arachidonoylethanolamine); 2-AG, 2-arachidonoylglycerol; AM404, N-(4hydroxyphenyl)-arachidonoylamide; AMT, anandamide (N-arachidonoylethanolamine) membrane transporter; Bmax, binding capacity; (Bu)2cAMP, N,O⬘-dibutyryl cAMP; CAPS, capsazepine (N-[2-(4chlorophenyl)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-3methyl-phenyl)-1-(4-methyl-benzyl)-pyrazole-3-carboxamide; THC, ⌬9-tetrahydrocannabinol; Vmax, maximum velocity. 20 Maccarrone et al. • Endocannabinoids and Sertoli Cells tected 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). Materials and Methods Experimental animals Random-bred Swiss CD1 mice were reared in our facilities. All animal experimentation described in this article was conducted in accordance with accepted standards of humane animal care. All experime (...truncated)