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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 micromolar concentration of Met-AEA on bull heparin-induced sperm capacitation.Spermatozoa were incubated for 45 min at 38.5°C in sp-TALP medium with heparin, Met-AEA (1.4 µM) or Heparin + Met-AEA (1.4 µM). Bars indicate the percentage of capacitated spermatozoa (A: % pattern B of CTC; B: % acrosome reacted spermatozoa). Data are expressed as mean ± SEM. A: Assessment of sperm capacitation by CTC assay. (—) t = 0 and t = 45, sp-TALP at 0 and 45 min (control) incubation respectively (n = 4). *p<0.05 vs control and Heparin (60 µg/ml) + Met-AEA (1.4 µM). B: Assessment of sperm capacitation by acrosome reaction evaluated by PSA-FITC; (—) t = 45 (control) (n = 4). ***p<0.001 vs control; **p<0.01 vs Heparin (60 µg/ml) + Met-AEA (1.4 µM).
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pone-0016993-g002: Effect of micromolar concentration of Met-AEA on bull heparin-induced sperm capacitation.Spermatozoa were incubated for 45 min at 38.5°C in sp-TALP medium with heparin, Met-AEA (1.4 µM) or Heparin + Met-AEA (1.4 µM). Bars indicate the percentage of capacitated spermatozoa (A: % pattern B of CTC; B: % acrosome reacted spermatozoa). Data are expressed as mean ± SEM. A: Assessment of sperm capacitation by CTC assay. (—) t = 0 and t = 45, sp-TALP at 0 and 45 min (control) incubation respectively (n = 4). *p<0.05 vs control and Heparin (60 µg/ml) + Met-AEA (1.4 µM). B: Assessment of sperm capacitation by acrosome reaction evaluated by PSA-FITC; (—) t = 45 (control) (n = 4). ***p<0.001 vs control; **p<0.01 vs Heparin (60 µg/ml) + Met-AEA (1.4 µM).

Mentions: We also investigated whether bull sperm capacitation induced by heparin (60 µg/ml) is modified by the presence of micromolar concentrations of Met-AEA. We found, by CTC and LPC-induced acrosome reaction, that micromolar concentrations of Met-AEA inhibited heparin-induced sperm capacitation (Figures 2A, 2B) but those concentrations alone did not exert any inhibitory effect (Figures 2A, 2B).


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 micromolar concentration of Met-AEA on bull heparin-induced sperm capacitation.Spermatozoa were incubated for 45 min at 38.5°C in sp-TALP medium with heparin, Met-AEA (1.4 µM) or Heparin + Met-AEA (1.4 µM). Bars indicate the percentage of capacitated spermatozoa (A: % pattern B of CTC; B: % acrosome reacted spermatozoa). Data are expressed as mean ± SEM. A: Assessment of sperm capacitation by CTC assay. (—) t = 0 and t = 45, sp-TALP at 0 and 45 min (control) incubation respectively (n = 4). *p<0.05 vs control and Heparin (60 µg/ml) + Met-AEA (1.4 µM). B: Assessment of sperm capacitation by acrosome reaction evaluated by PSA-FITC; (—) t = 45 (control) (n = 4). ***p<0.001 vs control; **p<0.01 vs Heparin (60 µg/ml) + Met-AEA (1.4 µM).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3037938&req=5

pone-0016993-g002: Effect of micromolar concentration of Met-AEA on bull heparin-induced sperm capacitation.Spermatozoa were incubated for 45 min at 38.5°C in sp-TALP medium with heparin, Met-AEA (1.4 µM) or Heparin + Met-AEA (1.4 µM). Bars indicate the percentage of capacitated spermatozoa (A: % pattern B of CTC; B: % acrosome reacted spermatozoa). Data are expressed as mean ± SEM. A: Assessment of sperm capacitation by CTC assay. (—) t = 0 and t = 45, sp-TALP at 0 and 45 min (control) incubation respectively (n = 4). *p<0.05 vs control and Heparin (60 µg/ml) + Met-AEA (1.4 µM). B: Assessment of sperm capacitation by acrosome reaction evaluated by PSA-FITC; (—) t = 45 (control) (n = 4). ***p<0.001 vs control; **p<0.01 vs Heparin (60 µg/ml) + Met-AEA (1.4 µM).
Mentions: We also investigated whether bull sperm capacitation induced by heparin (60 µg/ml) is modified by the presence of micromolar concentrations of Met-AEA. We found, by CTC and LPC-induced acrosome reaction, that micromolar concentrations of Met-AEA inhibited heparin-induced sperm capacitation (Figures 2A, 2B) but those concentrations alone did not exert any inhibitory effect (Figures 2A, 2B).

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