Up-regulation of the endocannabinoid system in the uterus of leptin knockout (ob/ob) mice and implications for fertility

Molecular Human Reproduction Vol.11, No.1 pp. 21–28, 2004 Advance Access publication November 19, 2004 doi:10.1093/molehr/gah130 Up-regulation of the endocannabinoid system in the uterus of leptin knockout (ob/ob) mice and implications for fertility M.Maccarrone1,6, E.Fride2, T.Bisogno3, M.Bari1, M.G.Cascio3, N.Battista1, A.Finazzi Agrò4, R.Suris2, R.Mechoulam5 and V.Di Marzo3,7 1 6 To whom correspondence should be addressed at: Department of Biomedical Sciences, University of Teramo, Piazza A. Moro 45, 64100 Teramo, Italy. E-mail: 7 To whom correspondence should be addressed at: Endocannabinoid Research Group, Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Via Campi Flegrei 34, Comprensorio Olivetti, Fabbricato 70, 80078 Pozzuoli (Napoli), Italy. E-mail: The levels of the endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG) are under the negative control of leptin in the rodent hypothalamus. As leptin and endocannabinoids play opposite roles in the control of reproduction, we have investigated whether the impaired fertility typical of leptin-defective ob/ob mice is due, in part, to enhanced uterine endocannabinoid levels. We found that levels of both anandamide and 2-AG in the uterus of ob/ob mice are significantly elevated with respect to wild-type littermates, due to reduced hydrolase activity in the case of anandamide, and to reduced monoacylglycerol lipase and enhanced diacylglycerol lipase activity in the case of 2-AG. Furthermore, the process mediating endocannabinoid cellular uptake was also impaired in ob/ob mice, whereas the levels of cannabinoid and anandamide receptors were not modified. Although ineffective in wild-type mice, treatment of ob/ob mice with leptin re-established endocannabinoid levels and enzyme activities back to the values observed in wild-type littermates. Finally, treatment of ob/ob females with the CB1 receptor antagonist SR141716A did not improve their fertility, and inhibition of endocannabinoid inactivation with the endocannabinoid uptake inhibitor OMDM-1 in wild-type females did not result in impaired fertility. Key words: anandamide/2-arachidonoylglycerol/fertility/leptin/ob/ob mice Introduction Leptin is the 16kDa non-glycosylated product of the obese (ob/ob) gene, which is secreted by adipose cells, is released into the circulation and transported across the blood– brain barrier into the central nervous system, where it regulates energy homeostasis (Ahima and Flier, 2000). Leptin also serves systemic functions apart from those related to food intake and energy expenditure in mammals, including regulation of fertility (Cunningham et al., 1999; Ahima and Flier, 2000). Indeed the leptin receptor, which belongs to the class I cytokine superfamily, is present at all points along the hypothalamic – pituitary– gonadal axis (Cunningham et al., 1999). Furthermore, leptin has been found in preimplantation embryos (Antczak and Van Blerkom, 1997), but leptin mRNA was detected starting from the blastocyst stage (Kawamura et al., 2002). Leptin synthesis has been demonstrated in the placenta (Masuzaki et al., 1997) and uterine leptin levels are elevated in the pregnant mouse (Kawamura et al., 2002). Further, addition of leptin to embryo cultures promoted the development of preimplantation embryos (Kawamura et al., 2003). On the basis of these findings, it has been proposed that leptin secreted from the reproductive tract stimulates embryonal development at the preimplantation stage, until it is produced in sufficient amounts independently (Kawamura et al., 2003). In fact, mice genetically defective in leptin (ob/ob knockout) are obese and infertile, and administration of exogenous leptin can reverse both defects (Cunningham et al., 1999; Ahima and Flier, 2000). It appears, however, that leptin regulates fertility in these animals at the premating stage, but is not required for the normal course of pregnancy and birth (Chehab, 2000). Recently, leptin has been shown to reduce the levels of anandamide [arachidonoylethanolamide (AEA)] and the other endocannabinoid, 2-arachidonoylglycerol (2-AG), in the hypothalamus of ob/ob mice, suggesting that these compounds partake of the neural orexigenic circuitries down-regulated by leptin (Di Marzo et al., 2001; Kirkham et al., 2002). 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 nervous system (Fride, 2002a) and in the periphery (Parolaro et al., 2002). 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 (AA) by the enzyme AEA hydrolase (fatty acid amide hydrolase, Molecular Human Reproduction vol. 11 no. 1 q European Society of Human Reproduction and Embryology 2004; all rights reserved 21 Downloaded from http://molehr.oxfordjournals.org/ at Universidad del Republica on December 17, 2016 Department of Biomedical Sciences, University of Teramo, Piazza A. Moro 45, 64100 Teramo, Italy, 2Departments of Behavioral Sciences and of Molecular Biology, College of Judea and Samaria, 44837 Ariel, Israel, 3Endocannabinoid Research Group, Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Via Campi Flegrei 34, Comprensorio Olivetti, Fabbricato 70, 80078 Pozzuoli (Napoli), 4Department of Experimental Medicine and Biochemical Sciences, University of Rome ‘Tor Vergata’, Via Montpellier 1, 00133 Rome, Italy and 5Department of Natural Products, Hebrew University, 91120 Jerusalem, Israel M.Maccarrone et al. 22 Materials and methods Materials Chemicals were of the purest analytical grade. Leptin (mouse recombinant) and anandamide (AEA) were purchased from Sigma Chemical Co. (St Louis, MO). N-Piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3pyrazole-carboxamide (SR141716A) was a kind gift from Sanofi Recherche (Montpellier, France). The OMDM-1 was synthesized as described previously (Ortar et al., 2003). [3H]AEA (223 Ci/mmol) and [3H]CP55,940 (5-(1,10 -dimethylheptyl)-2-[1R,5R-hydroxy-2R-(3-hydroxypropyl) cyclohexyl]-phenol, 126 Ci/mmol) were from NEN Life Science Products, Inc. (Boston, MA). [3H]AA (200 Ci/mmol) and N-[3H]arachidonoyl-phosphatidylethanolamine ([3H]NArPE, 200 Ci/mmol) were from ARC (St Louis, MO). [3H]2-AG was prepared from [3H]AA, as described recently (Cartoni et al., 2004). The labelled substrates for DAG and MAG lipases, i.e. sn-1-stearoyl-2[3H]-arachidonoyl-glycerol (56 mCi/mmol) and 2[3H]-arachidonoyl-glycerol (1 mCi/mmol) were purchased from Amersham Biosciences and synthesized in-house as previously described (Bisogno et al., 2003), respectively. Animals and treatments Leptin-deficient (B6.V-Lepob, ‘ob/ob’) (...truncated)