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

Abstract

Exocytosis of the sperm acrosome is an obligate precursor to successful egg penetration and subsequent fertilization. In most mammals, acrosomal exocytosis occurs at a precise time, after sperm binding to the zona pellucida of 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 the sperm analogous to a single secretory vesicle. Monomeric G proteins of the rab3 subfamily, specifically rab3a, have been shown to be important regulators of exocytosis in secretory cells, and we hypothesized that these proteins may regulate acrosomal exocytosis. Using alpha[32P] GTP binding to Immobilon blotted mouse sperm proteins, the presence of three or more monomeric GTP binding proteins was identified with Mr = 22, 24, and 26 x 10(3). Alpha[32P] 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 the 24 kDa G protein as rab3a. Using immunocytochemistry, rab3a was localized to the head of acrosome-intact sperm and was lost during acrosomal exocytosis. It was identified in membrane and cytosolic fractions of sperm with the predominant form being membrane-bound, and its membrane association did not change upon capacitation. Immunogold labeling and electron microscopy demonstrated a subcellular localization in clusters to the periacrosomal membranes and cytoplasm. These data identify the presence of rab3a in acrosomal membranes of mouse sperm and suggest that rab3a plays a role in the regulation of zona pellucida -induced acrosomal exocytosis.