The monomeric GTP binding protein, rab3a, is associated with the acrosome in mouse sperm

Exocytosis of the sperm acrosome is an obligate precursor to successful egg penetration and subsequent fertilization. In most mammals, acrosomal exocy tosis occurs at a precise time, after sperm binding to the zona pellucida o f the egg, and is induced by a specific component of the zona pellucida. It may be considered an example of regulated secretion with the acrosome of t he sperm analogous to a single secretory vesicle. Monomeric G proteins of t he rab3 subfamily, specifically rab3a, have been shown to be important regu lators of exocytosis in secretory cells, and we hypothesized that these pro teins may regulate acrosomal exocytosis. Using alpha[P-32] GTP binding to I mmobilon blotted mouse sperm proteins, the presence of three or more monome ric GTP binding proteins was identified with M-r = 22, 24, and 26 x 10(3). Alpha[P-32] GTP binding could be competed by GTP and GDP, but not GMP, ATP, or ADP. Anti-peptide antibodies specific for rab3a were used to identify t he 24 kDa G protein as rab3a, Using immunocytochemistry, rab3a was localize d to the head of acrosome-intact sperm and was lost during acrosomal exocyt osis. It was identified in membrane and cytosolic fractions of sperm with t he predominant form being membrane-bound, and its membrane association did not change upon capacitation. Immunogold labeling and electron microscopy d emonstrated a subcellular localization in clusters to the periacrosomal mem branes and cytoplasm, These data identify the presence of rab3a in acrosoma l membranes of mouse sperm and suggest that rab3a plays a role in the regul ation of zona pellucida-induced acrosomal exocytosis. (C) 1999 Wiley-Liss, Inc.