title: Involvement of cannabinoid receptor-1 activation in mitochondrial depolarizing effect of lipopolysaccharide in human spermatozoa.
abstract: Gram-negative bacteria frequently involved in urogenital tract infections release the endotoxin lipopolysaccharide (LPS); its receptor, toll-like receptor-4 (TLR4), has been recently identified in human spermatozoa, and its direct activation has been suggested in mediating adverse effects of LPS on human spermatozoa. However, the underlying signal transduction remains to be clarified. In other cell types, LPS induces the generation of endocannabinoids, which are involved in mediating endotoxin effects. In human spermatozoa, which exhibit a completely functional endocannabinoid system, the activation of cannabinoid receptor-1 (CB1) inhibited sperm mitochondrial membrane potential (ΔΨm). In this study, we tested the hypothesis of a contribution of CB1 activation by sperm-generated endocannabinoids in the adverse effects exerted by LPS on human spermatozoa. The exposure of motile sperm suspensions to E. coli LPS produced a significant decrease in sperm ΔΨm, assessed at flow cytometry with JC-1, similar to that induced by Metanandamide (Met-AEA), a non-hydrolyzable analogue of the endocannabinoid AEA. The LPS-induced inhibition of ΔΨm was prevented by the selective CB1 cannabinoid receptor antagonist, SR141716. However, the inhibition of ΔΨm induced by either LPS or Met-AEA did not affect sperm motility. Consistent with this finding, the CB1-mediated inhibition of ΔΨm was neither associated to mitochondrial generation of reactive oxygen species as evaluated by flow cytometry with MytoSox Red nor to apoptosis pathway activation as evaluated with cytoflorimetric assay for activated caspase-9 and caspase-3. Any oxidative genomic damage was also ruled out with the cytoflorimetric quantification of the oxidized base adduct 8-hydroxy-2'-deoxyguanosine. In conclusion, E. coli LPS inhibited sperm ΔΨm through the activation of CB1, but this effect was not accompanied to the activation of mitochondrial dysfunction-related apoptotic/oxidative mechanisms, which could affect sperm motility and genomic integrity.
authors.affiliation: Andrology Unit, Department of Life, Health and Environment Sciences, University of L'Aquila, L'Aquila, Italy; San Raffaele Sulmona Institute, Sulmona, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Barbonetti
authors.forename: M R C
authors.initials: MR
authors.lastname: Vassallo
authors.forename: M
authors.initials: M
authors.lastname: Costanzo
authors.forename: N
authors.initials: N
authors.lastname: Battista
authors.forename: M
authors.initials: M
authors.lastname: Maccarrone
authors.forename: S
authors.initials: S
authors.lastname: Francavilla
authors.forename: F
authors.initials: F
authors.lastname: Francavilla
mesh_terms: D000080242:8-Hydroxy-2'-Deoxyguanosine; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects*; D008928:Mitochondria / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D043884:Receptor, Cannabinoid, CB1 / Q000502:physiology*; D013081:Sperm Motility / Q000187:drug effects / Q000502:physiology; D013094:Spermatozoa / Q000187:drug effects* / Q000378:metabolism
chemical_list: D008070:Lipopolysaccharides; D017382:Reactive Oxygen Species; D043884:Receptor, Cannabinoid, CB1; D000080242:8-Hydroxy-2'-Deoxyguanosine; D003849:Deoxyguanosine
keywords: CB1 receptor; E. coli lipopolysaccharide; endocannabinoids; human spermatozoa; mitochondrial membrane potential