High levels of anandamide, an endogenous cannabinoid, block the growth of sheep preimplantation embryos by inducing apoptosis and reversible arrest of cell proliferation

High levels of anandamide, an endogenous cannabinoid, blockthe growth of sheep preimplantation embryos by inducingapoptosis and reversible arrest of cell proliferationM.Y. Turco1, K. Matsukawa1, M. Czernik1, V. Gasperi1, N. Battista1, L. Della Salda1,P.A. Scapolo1, P. Loi1, M. Maccarrone1,2,3† and G. Ptak1,3†1Department of Comparative Biomedical Sciences, University of Teramo 64100, Teramo, Italy; 2European Centre for Brain Research(CERC)/IRCCS S. Lucia Foundation, 00143 Rome, Italy3Correspondence address. Tel: þ39-0861-266837; Fax: þ39-0861-411285; E-mail: gptak@unite.it (G.P.)/Tel: þ39-0861-266875;Fax: þ39-0861-266877; E-mail: mmaccarrone@unite.it (M.M.)BACKGROUND: The process of implantation is mediated by various molecules, one of which is anandamide (AEA), alipid signalling ligand belonging to the family of endocannabinoids. AEA exerts its effects on implantation by bindingto the Type 1 Cannabinoid Receptor (CB1-R), expressed in both blastocysts and uterus. We wanted to know whetherthe endocannabinoid signalling system was present also in the sheep reproductive tract and which kind of effect(s)AEA had on the development of sheep blastocysts in vitro. METHODS: We analysed the expression and activity ofthe endocannabinoid system in sheep reproductive tracts and blastocysts. Hatched sheep blastocysts were thenexposed to AEA and its effect(s) were determined by TUNEL assay and by measuring the rate of necrosis and5-bromo-deoxyuridine incorporation. RESULTS: We show that the AEA signalling system is present in sheep andthat high concentrations of AEA induce apoptosis and inhibit cell proliferation via a CB1-R-dependent mechanism.Indeed, AEA effects were blocked when sheep blastocysts were cultured in the presence of the CB1-R antagonistSR161417A. Moreover, AEA inhibition of cell proliferation was reversible, as arrested embryos resumed a normalgrowth rate upon AEA removal from the medium. CONCLUSIONS: Our results suggest that disturbed regulationof AEA signalling via CB1-R may be associated with pregnancy failure. AEA could lower the quality of blastocystsby inducing apoptosis and inhibiting cell proliferation, thus making them incompetent for implantation.Keywords: anandamide; endocannabinoids; embryo development; implantation; sheepIntroductionSuccessful cross-talk between the embryo and receptive uterusmust take place to allow implantation and establish pregnancy.This process involves a sequence of finely regulated events thatultimately lead to adhesion and intimate contact between theblastocyst trophectoderm (TE) and the maternal endometrium.The cross-talk is mediated by many factors, whose exact rolesand pathways have not yet been fully determined. One of thesefactors is N-arachidonoyl-ethanolamine (anandamide, AEA), along-chain, unsaturated fatty acid-derivative that belong to afamily of lipid mediators described as the endocannabinoidsystem (Mechoulam, 2002; De Petrocellis et al., 2004; Bariet al., 2006). This endogenous ligand was identified as aresult of studies on the effects of D9-tetrahydrocannabinol(THC), the active constituent of marijuana, as AEA and THCshare their main target, i.e. the G-protein-coupled receptor,Type 1 Cannabinoid Receptor (CB1-R) (Mastuda et al., 1990).The use of illicit drugs, particularly marijuana, amongwomen has raised serious concerns about their effects on pregnancy(Sherwood et al., 1999; Fergusson et al., 2002). A significantnumber of females exposed to THC have reportedunsuccessful pregnancies due to failure of implantation orspontaneous abortion (Maccarrone et al., 2000a,b), lowerinfant birth weight (Fergusson et al., 2002) and cognitive deficitsof the offspring (Fried et al., 2003). These data and otherssuggest that the endocannabinoid system is operative in thereproductive apparatus (fo[...]