Hum. Reprod. Advance Access published online on July 9, 2008
Human Reproduction, doi:10.1093/humrep/den258
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High levels of anandamide, an endogenous cannabinoid, block the growth of sheep preimplantation embryos by inducing apoptosis and reversible arrest of cell proliferation
1 Department of Comparative Biomedical Sciences, University of Teramo 64100, Teramo, Italy 2 European Centre for Brain Research (CERC)/IRCCS S. Lucia Foundation, 00143 Rome, Italy
3 Correspondence address. Tel: +39-0861-266837; Fax: +39-0861-411285; E-mail: gptak{at}unite.it (G.P.)/Tel: +39-0861-266875; Fax: +39-0861-266877; E-mail: mmaccarrone{at}unite.it (M.M.)
BACKGROUND: The process of implantation is mediated by various molecules, one of which is anandamide (AEA), a lipid signalling ligand belonging to the family of endocannabinoids. AEA exerts its effects on implantation by binding to the Type 1 Cannabinoid Receptor (CB1-R), expressed in both blastocysts and uterus. We wanted to know whether the 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 of the endocannabinoid system in sheep reproductive tracts and blastocysts. Hatched sheep blastocysts were then exposed to AEA and its effect(s) were determined by TUNEL assay and by measuring the rate of necrosis and 5-bromo-deoxyuridine incorporation.
RESULTS: We show that the AEA signalling system is present in sheep and that 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 antagonist SR161417A. Moreover, AEA inhibition of cell proliferation was reversible, as arrested embryos resumed a normal growth rate upon AEA removal from the medium.
CONCLUSIONS: Our results suggest that disturbed regulation of AEA signalling via CB1-R may be associated with pregnancy failure. AEA could lower the quality of blastocysts by inducing apoptosis and inhibiting cell proliferation, thus making them incompetent for implantation.
Key words: anandamide/endocannabinoids/embryo development/implantation/sheep
Equally senior authors. Submitted on June 11, 2007; resubmitted on May 16, 2008; accepted on May 28, 2008.