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Fig. 3. Localization of pY416 in non-capacitating and capacitating spermatozoon. (A-F) Spermatozoa were pre-incubated for 5 minutes with either 10 µM H89 (C,D) or the vehicle (A,B,E,F) before addition of reagents (1 mM dbcAMP and 1 mM PTX) to drive sperm capacitation. After a further 40-minute incubation, the cells were assessed for hyperactivated motility as described in Materials and Methods. Mouse spermatozoa from the cauda epididymides were fixed, washed and subjected to immunocytochemistry using anti-pY416 antibody in sperm cell populations having less than 5% total hyperactivation (C,D) or at least 95% hyperactivation (E,F). The secondary antibody only controls are shown in panels A and B. The induction of a hyperactivated state is clearly associated with phosphorylated Tyr416 (Y416-P) on the sperm tail (E,F). In non-capacitated cells, in which PKA had been blocked with H89, cAMP failed to elicit this response. Corresponding phase-contrast (lower panels) and FITC images (upper panels) are displayed.
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