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ABSTRACT: 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. The mammalian oviduct acts as a functional sperm reservoir providing a suitable environment that allows the maintenance of sperm fertilization competence until ovulation occurs. After ovulation, spermatozoa are gradually released from the oviductal reservoir in the caudal isthmus and ascend to the site of fertilization. Capacitating-related changes in sperm plasma membrane seem to be responsible for sperm release from oviductal epithelium. Anandamide is a lipid mediator that participates in the regulation of several female and male reproductive functions. Previously we have demonstrated that anandamide was capable to release spermatozoa from oviductal epithelia by induction of sperm capacitation in bovines. In the present work we studied whether anandamide might exert its effect by activating the nitric oxide (NO) pathway since this molecule has been described as a capacitating agent in spermatozoa from different species. First, we demonstrated that 1 µM NOC-18, a NO donor, and 10 mM L-Arginine, NO synthase substrate, induced the release of spermatozoa from the oviductal epithelia. Then, we observed that the anandamide effect on sperm oviduct interaction was reversed by the addition of 1 µM L-NAME, a NO synthase inhibitor, or 30 µg/ml Hemoglobin, a NO scavenger. We also demonstrated that the induction of bull sperm capacitation by nanomolar concentrations of R(+)-methanandamide or anandamide was inhibited by adding L-NAME or Hemoglobin. To study whether anandamide is able to produce NO, we measured this compound in both sperm and oviductal cells. We observed that anandamide increased the levels of NO in spermatozoa, but not in oviductal cells. These findings suggest that anandamide regulates the sperm release from oviductal epithelia probably by activating the NO pathway during sperm capacitation.
PLoS ONE 01/2012; 7(2):e30671. · 4.09 Impact Factor
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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.
PLoS ONE 01/2011; 6(2):e16993. · 4.09 Impact Factor
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ABSTRACT: We have previously shown that protein kinase A of the medically important zygomycete Mucor rouxii participates in fungal morphology through cytoskeletal organization. As a first step towards finding the link between protein kinase A and cytoskeletal organization we here demonstrate the cloning of the Rho1 gene and the characterization of its protein product. The RHO1 protein primary sequence shows 70-85% identity with fungal RHO1 or mammalian RhoA. Two protein kinase A phosphorylation sequences in adequate context are predicted, Ser73 and Ser135. The peptide IRRNSQKFV, containing Ser135 proved to be a good substrate for M. rouxii protein kinase A catalytic subunit. The over-expressed Rho1 fully complements a Saccharomyces cerevisiae null mutant. The endogenous protein was identified by western blot against a developed antibody and by ADP-ribosylation. Localization in germlings was visualized by immunofluorescence; the protein was localized in patches in the mother cell surface and excluded from the germ tube. Measurement of Rho1 expression during germination indicates that Rho1, at both the mRNA and protein levels, correlates with differentiation and not with growth. Rho1 has been shown to be the regulatory protein of the beta-1,3-glucan synthase complex in fungi in which beta-1,3-glucans are major components of the cell wall. Even though glucans have not been detected in zygomycetes, caspofungin, an echinochandin known to be an inhibitor of beta-1,3-glucan synthase complex, is shown here to have a negative effect on growth and to produce an alteration on morphology when added to M. rouxii growth culture medium. This result has an important impact on the possible participation of beta-1,3-glucans on the regulation of morphology of zygomycetes.
Antonie van Leeuwenhoek 05/2007; 91(3):237-51. · 2.09 Impact Factor
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ABSTRACT: The tripeptide Arg-Gly-Asp (RGD) (1 mM) as well as the polymer ProNectin F (20 nM) added to culture medium of the fungus Mucor rouxii (defined medium) produced a delay in the switch from isodiametric growth to tip growth; at the time of germination the mother cell had a 4.6 times larger volume with 3.6 times more germ tubes per cell than control germinating sporangiospores. Disruption of the actin network with 2 micro g of cytochalasin A per ml blocked the switch to tip growth; the effect was analogous to the one of 150 micro M dibutyryl-cyclic AMP (cAMP), which we previously described to promote isodiametric growth via protein kinase A. 150 micro M dibutyryl-cAMP antagonises partially the effect of 1 mM RGD; the cells still emit several germ tubes per mother cell but their number is smaller and the volume of the cell at germ tube emission is larger than with RGD alone. At higher concentrations the dibutyryl-cAMP overrides completely the effect of RGD. Our results suggest that M. rouxii has an RGD recognition system and demonstrate that RGD-containing peptides have a profound effect on the isotropic stage of growth and on the establishment of cell polarity and that cAMP analogues can override this effect.
Protoplasma 10/2003; 222(1-2):23-30. · 1.92 Impact Factor
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ABSTRACT: Dibutyryl-cAMP (150 μM) added to culture medium ofMucor rouxii cultures (106 spores/ml) affects its morphology by a reversible maintenance of isodiametric growth without significant alterations of growth parameters such as dry weight, number of nuclei, DNA content, and protein content. In cultures containing 104 spores/ml, the effect on morphology is accompanied by a more severe impairment of general growth and is obtained at higher dibutyryl-cAMP concentrations (500 μM). In cultures containing 106 spores/ml, rounded cells, 19 μm in diameter, with 70 nuclei are obtained, while in cultures containing 104 spores/ml, gigantic rounded cells, 70 μm in diameter, with up to 1000 nuclei are obtained.
Experimental Mycology.
