Type-1 Cannabinoid Receptors Reduce Membrane Fluidity of Capacitated Boar Sperm by Impairing Their Activation by Bicarbonate

Background Mammalian spermatozoa acquire their full fertilizing ability (so called capacitation) within the female genital tract, where they are progressively exposed to inverse gradients of inhibiting and stimulating molecules. Methodology/Principal Findings In the present research, the effect on this process of anandamide, an endocannabinoid that can either activate or inhibit cannabinoid receptors depending on its concentration, and bicarbonate, an oviductal activatory molecule, was assessed, in order to study the role exerted by the type 1 cannabinoid receptor (CB1R) in the process of lipid membrane remodeling crucial to complete capacitation. To this aim, boar sperm were incubated in vitro under capacitating conditions (stimulated by bicarbonate) in the presence or in the absence of methanandamide (Met-AEA), a non-hydrolysable analogue of anandamide. The CB1R involvement was studied by using the specific inhibitor (SR141716) or mimicking its activation by adding a permeable cAMP analogue (8Br-cAMP). By an immunocytochemistry approach it was shown that the Met-AEA inhibits the bicarbonate-dependent translocation of CB1R from the post-equatorial to equatorial region of sperm head. In addition it was found that Met-AEA is able to prevent the bicarbonate-induced increase in membrane disorder and the cholesterol extraction, both preliminary to capacitation, acting through a CB1R-cAMP mediated pathway, as indicated by MC540 and filipin staining, EPR spectroscopy and biochemical analysis on whole membranes (CB1R activity) and on membrane enriched fraction (C/P content and anisotropy). Conclusions/Significance Altogether, these data demonstrate that the endocannabinoid system strongly inhibits the process of sperm capacitation, acting as membrane stabilizing agent, thus increasing the basic knowledge on capacitation-related signaling and potentially opening new perspectives in diagnostics and therapeutics of male infertility

CYTOSKELETON OF TROUT SPERMATOZOA

The presence and localization of different cytoskeletal proteins in Salmo gairdneri spermatozoa have been analyzed by several methods, including immunofluorescence, immunoelectron microscopy and immunoblotting. Antibodies against actin, desmin, vimentin and tubulin were used to study the cytoskeletal organization of sperm heads and tails. Actin was found, although scarce, in heads and in tails; desmin immunoreactivity was confined to the sperm head; vimentin was negative. Tubulin, besides its obvious presence in the sperm tail, was also localized in the sperm head

ELECTRON-MICROSCOPIC LOCALIZATION OF F-ACTIN IN ACROSOME REACTED BOAR SPERMATOZOA BY MEANS OF A PHALLOIDIN-FITC COMPLEX

7. L. Castellani Ceresa, M. F. Brivio, G. Radaelli. Electron Microscopic Localization of F actin in Acrosome Reacted Boar Spermatozoa by Means of a Phalloidin FITC Complex. J. Submicrosc. Cytol. Phatol., 23, n\ub02, 347 349, 1991.An immunocytochemical study at the ultrastructural level has been performed in boar spermatozoa in order to clarify the aggregation state of actin before and after the acrosome reaction. A new phalloidin probe has been used to detect F-actin: a phalloidin derivative conjugated with FITC, followed by incubation with an anti-FITC antibody. The protein A-gold technique was then applied for the localization of the antigenic sites. Gold particles were localized on the sperm surface only after the acrosome reaction which was induced by the ionophore A23187

Actin polymerization in boar spermatozoa: Fertilization is reduced with use of cytochalasin D

The aggregational state of actin in boar spermatozoa after capacitation and the acrosome reaction has been examined by several methods. In vitro fertilization (IVF) experiments were conducted in the presence and absence of cytochalasin D (CD) to evaluate the role of actin polymerization in the events of fertilization. The fertilizing capacity was very high in controls, but, when CD (an inhibitor of the polymerization of actin) was added to the capacitation medium, there was a marked decrease in the fertilizing capacity of the boar spermatozoa. There was a further decrease when CD was present during both capacitation and fertilization processes. In addition to the IVF tests, biochemical and immunoelectron microscopic methods were used to analyze the state of aggregation of actin in boar spermatozoa after capacitation, and the acrosome reaction. By immunoelectron microscopy with a phalloidin probe, there were no gold particles, indicating the presence of F\u2010actin on boar sperm heads capacitated and acrosome\u2010reacted in media containing CD. By sodium dodecyl sulfate\u2010polyacrylamide gel electrophoresis there were differences in NP\u201040 solubility, reflecting actin polymerization, between CD\u2010treated and untreated sperm. These results suggest that actin polymerizes during capacitation and the acrosome reaction and that this polymerization is essential to the fertilization process. \ua9 1993 Wiley\u2010Liss, Inc. Copyright \ua9 1993 Wiley\u2010Liss, Inc

LOCALIZATION AND DISTRIBUTION OF ACTIN IN MAMMALIAN SPERM HEADS

Actin was identified in boar and mole spermatozoa by utilizing indirect immunofiuorescence, immunoelectron microscopy, and SDS-PAGE, followed by blot and screening with an anti-actin monoclonal antibody. Actin was detected in two places in the sperm head: the equatorial segment of the acrosome and the postacrosomal region. The protein was present in a nonfilamentous form and was localized under the plasma membrane. A small amount of actin was also detected in the sperm tail. The function of actin in the sperm head is discussed. \ua9 1986

Fluorescence and Electron Microscopic Localization of F-actin in the Ependymocytes

The organization of F-actin in the ventricular system has been reported to display pronounced regional differences with respect to shape, size, and development. However, the real roles played by F-actin in these cells cannot be understood unless the precise localization of F-actin is defined. In the present study, we used double-fluorescence labeling to further examine the localization of F-actin in the ependymocytes and its spatial relation to the other two cytoskeletal components, microtubules and intermediate filaments. Then we converted fluorescence signals for F-actin to peroxidase/DAB reaction products by use of a phalloidin-based FITC-anti-FITC system. This detection technique provided an overview of the distribution of F-actin in the ependymocytes at the ultrastructural level, and has been proven to be helpful in correlating light and electron microscopic investigations. (J Histochem Cytochem 57:741–751, 2009