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(407) THE MONOMERIC G-PROTEIN RhoA IS WIDELY EXPRESSED IN MAMMALIAN SPERM, BUT DOES NOT PLAY A ROLE IN ACTIN CYTOSKELETAL REORGANIZATION IN PORCINE SPERM. Ducummon, Carl1, Berger, Trish1, 1 Department of Animal Science, Davis, CA ABSTRACT- Members of the Rho family of GTPases have been implicated in exocytotic reactions in a number of cell types. In these systems, the Rho-family GTPases have also been implicated in reorganizing the cortical cytoskeleton to permit exocytosis. The acrosome reaction (AR) in sperm is a modified exocytotic reaction involving the fusion of a single lysosomally derived secretory body, the acrosome, with the plasma membrane of the sperm. A homolog of Rho has been immunolocalized to the acrosome of sea urchin sperm and hypothesized to mediate the formation of the filamentous actin process necessary for fertilization (Castellano et al. Eur J Cell Biol (1997) 74:329-335). Previously we have demonstrated that RhoA is expressed in the acrosomal cap of porcine sperm and that its artificial activation by the membrane-permeable bacterial toxin CNF increases the rate of spontaneous acrosome reactions. Recent work using another bacterially derived toxin has implicated Rho GTPases in the regulation of actin polymerization during capacitation and suggested a role for this process in the AR (Brener et al. Biol Reprod (2003) 68:837-45). Using fluorescently labeled phalloidin, actin polymerization does not occur in the acrosomal cap of porcine sperm during capacitation even when RhoA is artificially activated using CNF. However, RhoA effector proteins are present in mammalian sperm. The guanine nucleotide regulatory protein, RhoGDI, colocalizes with RhoA to the acrosomal cap of rat and porcine sperm. Rho-activated PI(4)P5K, which regulates cytoskeletal reorganization by generation of PIP2, is immunolocalized to the entire head of porcine sperm; after acrosomal exocytosis immunostaining of these proteins is lost. These results suggest a role for RhoA and its effectors in mediating acrosomal exocytosis by an actin-independent pathway in diverse mammalian species. KEY WORDS: acrosome reaction, mammalian sperm, rho GTPase, actin cytoskeleton |
