Immunocytochemical and biochemical characterization of guanine nucleotide-binding regulatory proteins in mammalian spermatozoa

Abstract

Polyclonal antisera directed against conserved and subtype-specific peptide sequences of the α-subunits of guanine nucleotide-binding regulatory proteins (G proteins) were used to characterize the nature of mammalian sperm G proteins and to determine whether their localization was consistent with their proposed roles in mediating ZP3-induced acrosomal exocytosis. Mouse and guinea pig sperm exhibit positive immunofluorescence in the acrosomal region using an antiserum directed against a peptide region common to all α-subunits of G proteins (G α). The immunofluorescence disappears after sperm have undergone the acrosome reaction, suggesting that the immunoreactive material is associated with the plasma membrane/outer acrosomal membrane region overlying the acrosome. The presence of G proteins in this region is confirmed by the presence of a M r 41,000 substrate for pertussis toxin (PT)-catalyzed [ 32P]ADP-ribosylation in purified plasma membrane/outer acrosomal membrane hybrid vesicles obtained from acrosome-reacted guinea pig sperm. Immunoprecipitation and polyacrylamide gel electrophoresis of PT-catalyzed [ 32P]ADP-ribosylated protein(s) using anti-peptide antisera generated against sequences unique to G iα1, G iα2, and G iα3 confirm the existence of all three G i subtypes in mouse sperm extracts. Indirect immunofluorescence using an antiserum directed against a peptide region present in G zα, a PT-insensitive G protein, demonstrates positive immunoreactivity in the postacrosomal/lateral face region of the mouse sperm head. This immunoreactivity is retained during acrosomal exocytosis in response to solubilized ZP and then disappears subsequent to this exocytotic event. These data demonstrate that G i protein α-subunits are present in the acrosomal region of mammalian sperm, consistent with their postulated role in regulating ZP3-mediated acrosomal exocytosis, and that PT-insensitive G zα is found in a region of the sperm head distinct from that of the G iα subunits.