Abstract
Anandamide (AEA), a major endocannabinoid, binds to cannabinoid and vanilloid receptors (CB1, CB2 and TRPV1) and affects many reproductive functions. Nanomolar levels of anandamide are found in reproductive fluids including mid-cycle oviductal fluid. Previously, we found that R(+)-methanandamide, an anandamide analogue, induces sperm releasing from bovine oviductal epithelium and the CB1 antagonist, SR141716A, reversed this effect. Since sperm detachment may be due to surface remodeling brought about by capacitation, the aim of this paper was to investigate whether anandamide at physiological concentrations could act as a capacitating agent in bull spermatozoa. We demonstrated that at nanomolar concentrations R(+)-methanandamide or anandamide induced bull sperm capacitation, whereas SR141716A and capsazepine (a TRPV1 antagonist) inhibited this induction. Previous studies indicate that mammalian spermatozoa possess the enzymatic machinery to produce and degrade their own AEA via the actions of the AEA-synthesizing phospholipase D and the fatty acid amide hydrolase (FAAH) respectively. Our results indicated that, URB597, a potent inhibitor of the FAAH, produced effects on bovine sperm capacitation similar to those elicited by exogenous AEA suggesting that this process is normally regulated by an endogenous tone. We also investigated whether anandamide is involved in bovine heparin-capacitated spermatozoa, since heparin is a known capacitating agent of bovine sperm. When the spermatozoa were incubated in the presence of R(+)-methanandamide and heparin, the percentage of capacitated spermatozoa was similar to that in the presence of R(+)-methanandamide alone. The pre-incubation with CB1 or TRPV1 antagonists inhibited heparin-induced sperm capacitation; moreover the activity of FAAH was 30% lower in heparin-capacitated spermatozoa as compared to control conditions. This suggests that heparin may increase endogenous anandamide levels. Our findings indicate that anandamide induces sperm capacitation through the activation of CB1 and TRPV1 receptors and could be involved in the same molecular pathway as heparin in bovines.
Highlights
Mammalian spermatozoa are not able to fertilize an egg immediately upon ejaculation
We have recently demonstrated that bull spermatozoa express cannabinoid receptor 1 (CB1), cannabinoid receptor 2 (CB2) and fatty acid amide hydrolase (FAAH), the enzyme that degrades AEA and regulates its endogenous levels
Sperm capacitation related-events were evaluated by chlortetracycline (CTC) assay, induction of protein tyrosine phosphorylation patterns and L-a-lysophosphatidylcholine (LPC)-induced acrosome reaction assessed by Pissum sativum agglutinin-FITC staining (PSA-FITC)
Summary
Mammalian spermatozoa are not able to fertilize an egg immediately upon ejaculation. They acquire this ability during their transit through the female genital tract in a process known as capacitation, where they undergo a large number of membrane and metabolic modifications such as an increase in intracellular ions and protein tyrosine phosphorylation, generation of reactive oxygen species and changes in metabolism, motility and plasma membrane fluidity [1,2,3,4,5].The mammalian oviduct acts as a functional sperm reservoir providing a suitable environment that allows the maintenance of sperm fertilization competence until ovulation occurs [6]. Mammalian spermatozoa are not able to fertilize an egg immediately upon ejaculation. They acquire this ability during their transit through the female genital tract in a process known as capacitation, where they undergo a large number of membrane and metabolic modifications such as an increase in intracellular ions and protein tyrosine phosphorylation, generation of reactive oxygen species and changes in metabolism, motility and plasma membrane fluidity [1,2,3,4,5]. The interaction between oviductal epithelial cells and spermatozoa is thought to prolong sperm life by delaying capacitation until ovulation-associated signals [7], induce the release of adhering sperm subpopulations [7,8]. Bull spermatozoa are capacitated in vitro by exposure to different glycosaminoglycans such as heparin, hyaluronan and heparan sulphate [12,16]
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