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Anandamide capacitates bull spermatozoa through CB1 and TRPV1 activation.

Gervasi MG, Osycka-Salut C, Caballero J, Vazquez-Levin M, Pereyra E, Billi S, Franchi A, Perez-Martinez S - PLoS ONE (2011)

Bottom Line: Previously, we found that R(+)-methanandamide, an anandamide analogue, induces sperm releasing from bovine oviductal epithelium and the CB1 antagonist, SR141716A, reversed this effect.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.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.

View Article: PubMed Central - PubMed

Affiliation: Centro de Estudios Farmacológicos y Botánicos, Facultad de Medicina, Consejo Nacional de Investigaciones Científicas y Técnicas-Universidad de Buenos Aires, Buenos Aires, Argentina.

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.

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Expression and localization of TRPV1 in bovine spermatozoa.A: Sperm proteins were extracted and subjected to SDS-PAGE immunoblotting with a specific antibody against TRPV1. Lanes 1–2: antiTRPV1, lanes 3–4: antiTRPV1 plus blocking peptide, (n = 3). B: Immunolocalization of TRPV1; left panel: spermatozoa incubated with antiTRPV1; right panel: spermatozoa incubated with IgG fractions from non-immunized rabbits at the same concentration that the primary antibody (n = 3); Scale bar: 20 µm (Magnification, X600).
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pone-0016993-g003: Expression and localization of TRPV1 in bovine spermatozoa.A: Sperm proteins were extracted and subjected to SDS-PAGE immunoblotting with a specific antibody against TRPV1. Lanes 1–2: antiTRPV1, lanes 3–4: antiTRPV1 plus blocking peptide, (n = 3). B: Immunolocalization of TRPV1; left panel: spermatozoa incubated with antiTRPV1; right panel: spermatozoa incubated with IgG fractions from non-immunized rabbits at the same concentration that the primary antibody (n = 3); Scale bar: 20 µm (Magnification, X600).

Mentions: Since our results suggested that AEA induces sperm capacitation, we then investigated its possible activation pathway. We have previously found that bull spermatozoa express CB1 receptors [20]. In the present work, we studied the expression of TRPV1 in spermatozoa since this receptor is also a divalent ion channel and it is known that calcium is involved in sperm capacitation [13], [30]. Western blot analysis showed a single immunoreactive band of the molecular size expected for TRPV1 (∼100 kDa); this band was absent when the blocking peptide was added (Fig. 3A). Also, immunocytochemical analysis showed TRPV1 localization predominantly on the tail, the apical region of the acrosome and the post-acrosomal region in sperm head (Fig. 3B). Staining was specific because spermatozoa incubated with IgG fractions from non-immunized rabbits (Fig. 3B) or without the primary antibody (data not shown) did not show any signal.


Anandamide capacitates bull spermatozoa through CB1 and TRPV1 activation.

Gervasi MG, Osycka-Salut C, Caballero J, Vazquez-Levin M, Pereyra E, Billi S, Franchi A, Perez-Martinez S - PLoS ONE (2011)

Expression and localization of TRPV1 in bovine spermatozoa.A: Sperm proteins were extracted and subjected to SDS-PAGE immunoblotting with a specific antibody against TRPV1. Lanes 1–2: antiTRPV1, lanes 3–4: antiTRPV1 plus blocking peptide, (n = 3). B: Immunolocalization of TRPV1; left panel: spermatozoa incubated with antiTRPV1; right panel: spermatozoa incubated with IgG fractions from non-immunized rabbits at the same concentration that the primary antibody (n = 3); Scale bar: 20 µm (Magnification, X600).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3037938&req=5

pone-0016993-g003: Expression and localization of TRPV1 in bovine spermatozoa.A: Sperm proteins were extracted and subjected to SDS-PAGE immunoblotting with a specific antibody against TRPV1. Lanes 1–2: antiTRPV1, lanes 3–4: antiTRPV1 plus blocking peptide, (n = 3). B: Immunolocalization of TRPV1; left panel: spermatozoa incubated with antiTRPV1; right panel: spermatozoa incubated with IgG fractions from non-immunized rabbits at the same concentration that the primary antibody (n = 3); Scale bar: 20 µm (Magnification, X600).
Mentions: Since our results suggested that AEA induces sperm capacitation, we then investigated its possible activation pathway. We have previously found that bull spermatozoa express CB1 receptors [20]. In the present work, we studied the expression of TRPV1 in spermatozoa since this receptor is also a divalent ion channel and it is known that calcium is involved in sperm capacitation [13], [30]. Western blot analysis showed a single immunoreactive band of the molecular size expected for TRPV1 (∼100 kDa); this band was absent when the blocking peptide was added (Fig. 3A). Also, immunocytochemical analysis showed TRPV1 localization predominantly on the tail, the apical region of the acrosome and the post-acrosomal region in sperm head (Fig. 3B). Staining was specific because spermatozoa incubated with IgG fractions from non-immunized rabbits (Fig. 3B) or without the primary antibody (data not shown) did not show any signal.

Bottom Line: Previously, we found that R(+)-methanandamide, an anandamide analogue, induces sperm releasing from bovine oviductal epithelium and the CB1 antagonist, SR141716A, reversed this effect.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.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.

View Article: PubMed Central - PubMed

Affiliation: Centro de Estudios Farmacológicos y Botánicos, Facultad de Medicina, Consejo Nacional de Investigaciones Científicas y Técnicas-Universidad de Buenos Aires, Buenos Aires, Argentina.

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.

Show MeSH
Related in: MedlinePlus