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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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Effect of R(+)methanandamide (Met-AEA) and anandamide (AEA) on bull sperm capacitation.Spermatozoa were incubated for 45 min at 38.5°C in sp-TALP medium with Met-AEA or AEA. Bars indicate the percentage of capacitated spermatozoa (A and B: % pattern B of CTC; C: % acrosome reacted spermatozoa). Data are expressed as mean±SEM. A, B: Assessment of sperm capacitation by CTC assay. A: Effect of increased concentrations of Met-AEA (0.14 nM-1.4 µM); (—) t = 0 and t = 45, sp-TALP at 0 and 45 min (control) incubation respectively (n = 5). B: Effect of AEA (1 nM); t = 0 and t = 45 (controls) (n  =  6). C: Assessment of sperm capacitation by LPC-induced acrosome reaction (AR)-PSA-FITC. Heparin (positive control) or (—) sp-TALP (control). Spermatozoa were incubated for 45 min at 38.5°C in sp-TALP medium with Met-AEA (1.4 nM) or AEA (1 nM) and then were incubated for 15 min either with or without LPC to induce AR. Bars show percentage of spermatozoa that underwent LPC-induced AR minus the percentage of spermatozoa that underwent spontaneous AR (n = 4). D: Evaluation of protein tyrosine phosphorylation. Sperm proteins were extracted and subjected to SDS-PAGE immunoblotting with a specific monoclonal antibody against phosphotyrosine (clone 4G10). Antibody against β-tubulin (50 kDa) was used as loading control. A representative experiment is shown. Numbers on the left-hand side of the gel represent the position of the relative molecular mass standards. Lane 1: sp-TALP (control), lane 2: Met-AEA (1.4 nM), lane 3: Heparin (60 µg/ml), lane 4: AEA (1 nM), (n = 3). E: Immunolocalization of tyrosine phosphorylated-proteins in bovine spermatozoa. Panels i and iii: sp-TALP (control), panel v: Met-AEA (1.4 nM), panel vii: Heparin (60 µg/ml), panels ii, iv, vi, and viii: phase contrast (scale bar = 10 µm). Specificity of the reaction was tested omitting the first antibody (Magnification, X400). *p<0.05 vs control; **p<0.01 vs. control; ***p<0.001 vs. control.
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pone-0016993-g001: Effect of R(+)methanandamide (Met-AEA) and anandamide (AEA) on bull sperm capacitation.Spermatozoa were incubated for 45 min at 38.5°C in sp-TALP medium with Met-AEA or AEA. Bars indicate the percentage of capacitated spermatozoa (A and B: % pattern B of CTC; C: % acrosome reacted spermatozoa). Data are expressed as mean±SEM. A, B: Assessment of sperm capacitation by CTC assay. A: Effect of increased concentrations of Met-AEA (0.14 nM-1.4 µM); (—) t = 0 and t = 45, sp-TALP at 0 and 45 min (control) incubation respectively (n = 5). B: Effect of AEA (1 nM); t = 0 and t = 45 (controls) (n  =  6). C: Assessment of sperm capacitation by LPC-induced acrosome reaction (AR)-PSA-FITC. Heparin (positive control) or (—) sp-TALP (control). Spermatozoa were incubated for 45 min at 38.5°C in sp-TALP medium with Met-AEA (1.4 nM) or AEA (1 nM) and then were incubated for 15 min either with or without LPC to induce AR. Bars show percentage of spermatozoa that underwent LPC-induced AR minus the percentage of spermatozoa that underwent spontaneous AR (n = 4). D: Evaluation of protein tyrosine phosphorylation. Sperm proteins were extracted and subjected to SDS-PAGE immunoblotting with a specific monoclonal antibody against phosphotyrosine (clone 4G10). Antibody against β-tubulin (50 kDa) was used as loading control. A representative experiment is shown. Numbers on the left-hand side of the gel represent the position of the relative molecular mass standards. Lane 1: sp-TALP (control), lane 2: Met-AEA (1.4 nM), lane 3: Heparin (60 µg/ml), lane 4: AEA (1 nM), (n = 3). E: Immunolocalization of tyrosine phosphorylated-proteins in bovine spermatozoa. Panels i and iii: sp-TALP (control), panel v: Met-AEA (1.4 nM), panel vii: Heparin (60 µg/ml), panels ii, iv, vi, and viii: phase contrast (scale bar = 10 µm). Specificity of the reaction was tested omitting the first antibody (Magnification, X400). *p<0.05 vs control; **p<0.01 vs. control; ***p<0.001 vs. control.

Mentions: CTC analysis provides a useful method for assessing intracellular calcium mobilisation in mammalian spermatozoa [30]. In vitro sperm capacitation experiments were performed with different concentrations of R(+)-methanandamide (Met-AEA), a non-hydrolysable AEA analogue [31]. Spermatozoa incubated in sp-TALP medium alone for 45 min were used for comparison. It was observed that Met-AEA promoted sperm capacitation at 1.4 and 14 nM concentrations compared to the control sample. The extent of capacitated spermatozoa is about twofold higher (23% at 1.4 nM Met-AEA concentration) compared to the control sample (8%) (Fig. 1A). Interestingly, Met-AEA, at either lower or higher concentrations, did not induce sperm capacitation (Fig. 1A). Anandamide (1 nM) also produced a significant increase in pattern B (Fig. 1B).


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)

Effect of R(+)methanandamide (Met-AEA) and anandamide (AEA) on bull sperm capacitation.Spermatozoa were incubated for 45 min at 38.5°C in sp-TALP medium with Met-AEA or AEA. Bars indicate the percentage of capacitated spermatozoa (A and B: % pattern B of CTC; C: % acrosome reacted spermatozoa). Data are expressed as mean±SEM. A, B: Assessment of sperm capacitation by CTC assay. A: Effect of increased concentrations of Met-AEA (0.14 nM-1.4 µM); (—) t = 0 and t = 45, sp-TALP at 0 and 45 min (control) incubation respectively (n = 5). B: Effect of AEA (1 nM); t = 0 and t = 45 (controls) (n  =  6). C: Assessment of sperm capacitation by LPC-induced acrosome reaction (AR)-PSA-FITC. Heparin (positive control) or (—) sp-TALP (control). Spermatozoa were incubated for 45 min at 38.5°C in sp-TALP medium with Met-AEA (1.4 nM) or AEA (1 nM) and then were incubated for 15 min either with or without LPC to induce AR. Bars show percentage of spermatozoa that underwent LPC-induced AR minus the percentage of spermatozoa that underwent spontaneous AR (n = 4). D: Evaluation of protein tyrosine phosphorylation. Sperm proteins were extracted and subjected to SDS-PAGE immunoblotting with a specific monoclonal antibody against phosphotyrosine (clone 4G10). Antibody against β-tubulin (50 kDa) was used as loading control. A representative experiment is shown. Numbers on the left-hand side of the gel represent the position of the relative molecular mass standards. Lane 1: sp-TALP (control), lane 2: Met-AEA (1.4 nM), lane 3: Heparin (60 µg/ml), lane 4: AEA (1 nM), (n = 3). E: Immunolocalization of tyrosine phosphorylated-proteins in bovine spermatozoa. Panels i and iii: sp-TALP (control), panel v: Met-AEA (1.4 nM), panel vii: Heparin (60 µg/ml), panels ii, iv, vi, and viii: phase contrast (scale bar = 10 µm). Specificity of the reaction was tested omitting the first antibody (Magnification, X400). *p<0.05 vs control; **p<0.01 vs. control; ***p<0.001 vs. control.
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Related In: Results  -  Collection

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

pone-0016993-g001: Effect of R(+)methanandamide (Met-AEA) and anandamide (AEA) on bull sperm capacitation.Spermatozoa were incubated for 45 min at 38.5°C in sp-TALP medium with Met-AEA or AEA. Bars indicate the percentage of capacitated spermatozoa (A and B: % pattern B of CTC; C: % acrosome reacted spermatozoa). Data are expressed as mean±SEM. A, B: Assessment of sperm capacitation by CTC assay. A: Effect of increased concentrations of Met-AEA (0.14 nM-1.4 µM); (—) t = 0 and t = 45, sp-TALP at 0 and 45 min (control) incubation respectively (n = 5). B: Effect of AEA (1 nM); t = 0 and t = 45 (controls) (n  =  6). C: Assessment of sperm capacitation by LPC-induced acrosome reaction (AR)-PSA-FITC. Heparin (positive control) or (—) sp-TALP (control). Spermatozoa were incubated for 45 min at 38.5°C in sp-TALP medium with Met-AEA (1.4 nM) or AEA (1 nM) and then were incubated for 15 min either with or without LPC to induce AR. Bars show percentage of spermatozoa that underwent LPC-induced AR minus the percentage of spermatozoa that underwent spontaneous AR (n = 4). D: Evaluation of protein tyrosine phosphorylation. Sperm proteins were extracted and subjected to SDS-PAGE immunoblotting with a specific monoclonal antibody against phosphotyrosine (clone 4G10). Antibody against β-tubulin (50 kDa) was used as loading control. A representative experiment is shown. Numbers on the left-hand side of the gel represent the position of the relative molecular mass standards. Lane 1: sp-TALP (control), lane 2: Met-AEA (1.4 nM), lane 3: Heparin (60 µg/ml), lane 4: AEA (1 nM), (n = 3). E: Immunolocalization of tyrosine phosphorylated-proteins in bovine spermatozoa. Panels i and iii: sp-TALP (control), panel v: Met-AEA (1.4 nM), panel vii: Heparin (60 µg/ml), panels ii, iv, vi, and viii: phase contrast (scale bar = 10 µm). Specificity of the reaction was tested omitting the first antibody (Magnification, X400). *p<0.05 vs control; **p<0.01 vs. control; ***p<0.001 vs. control.
Mentions: CTC analysis provides a useful method for assessing intracellular calcium mobilisation in mammalian spermatozoa [30]. In vitro sperm capacitation experiments were performed with different concentrations of R(+)-methanandamide (Met-AEA), a non-hydrolysable AEA analogue [31]. Spermatozoa incubated in sp-TALP medium alone for 45 min were used for comparison. It was observed that Met-AEA promoted sperm capacitation at 1.4 and 14 nM concentrations compared to the control sample. The extent of capacitated spermatozoa is about twofold higher (23% at 1.4 nM Met-AEA concentration) compared to the control sample (8%) (Fig. 1A). Interestingly, Met-AEA, at either lower or higher concentrations, did not induce sperm capacitation (Fig. 1A). Anandamide (1 nM) also produced a significant increase in pattern B (Fig. 1B).

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