PARENT SESSION

(W345) THE ROLE OF ACTIN IN SPERM ACROSOMAL EXOCYTOSIS.

French, Stephanie ¹, Cruz, Aurora ¹, Shi, Xudong ¹, Miller, David¹, ¹ University of Illnois at Urbana-Champaign, Urbana, IL

ABSTRACT- The receptor -1,4galactosyltransferase-I (GalTase-I) is found in two locations, on the Golgi membrane and on the plasma membrane of some cells, including sperm. GalTase-I on sperm interacts with the glycoprotein ZP3 on the zona pellucida. This interaction activates a signal by the cytoplasmic domain and induces exocytosis of the acrosome, otherwise known as the acrosome reaction. GalTase-I signaling is G-protein dependent, but the precise signaling mechanism is unclear, although the cytoplasmic domain may bind and activate downstream signals. To identify testis genes encoding proteins with affinity for the GalTase-I cytoplasmic domain, we expressed a boar testis library as a fusion to a T7 phage coat protein on the phage surface. We found that the cytoplasmic domain of GalTase-I bound directly with a phage clone encoding actin. To confirm this interaction, a GalTase-I binding assay and a dot blot assay were performed with the immobilized phage encoding actin. Results indicated that GalTase-I interacts directly with actin. Previous studies have shown that actin may play an important role in acrosomal exocytosis. To determine the importance of actin polymerization, we incubated cauda epididymal mouse sperm with cytochalasins during capacitation and then induced the acrosome reaction with calcium ionophore A23187. Cytochalasin B and D (inhibitors of actin polymerization) blocked the calcium ionophore induced acrosome reaction in sperm. A more specific inhibitor of actin polymerization, latrunculin A, did not block the ionophore-induced acrosome reaction. The membrane-impermeable fungal toxin phalloidin, an inhibitor of actin depolymerization, inhibited the ionophore-induced acrosome reaction (about 82% of sperm remained acrosome-intact) when added to capacitated streptolysin O-permeablized sperm. These results indicate that actin depolymerization is required for the sperm acrosome reaction.

KEY WORDS: cytoskeleton, acrosome reaction, actin, zp3