Cyclic AMP-dependent phosphorylation of epididymal mouse sperm proteins during capacitation in vitro: identification of an Mr 95 000 phosphotyrosine-containing protein

in Reproduction

Cyclic AMP-dependent changes in phosphorylation of epididymal mouse sperm suspensions were examined in media designed to manipulate capacitation and the expression of parameters associated with full fertilizing ability, i.e. hyperactivated motility and the acrosome reaction. After initial assessment of cAMP-dependent protein kinase activity in frozen–thawed and lyophilized sperm suspensions using exogenous substrate, phosphorylation of endogenous sperm phosphoproteins was examined using sodium dodecyl sulfate poly-acrylamide gel electrophoresis followed by autoradiography or immunoblotting. Numerous phosphoproteins were detected in both uncapacitated and capacitated suspensions, the majority of which were probably concerned with motility; full expression of fertilizing ability appeared to involve an increase in the amount of endogenous phosphorylation as deduced from the decreased amount of 32P incorporation in these suspensions. The addition of the cAMP-dependent protein kinase inhibitors, H8 and PKI (6–22) amide, demonstrated that most of the phosphoproteins detected were phosphorylated in a cAMP-dependent manner. Of particular interest was a phosphoprotein with an Mr of about 95 000 which was consistently observed in capacitated suspensions. Evidence suggests that this may be phosphorylated on tyrosine residues, since the inclusion of orthovanadate, a phosphoryltyrosine phosphatase inhibitor, altered phosphorylation of this protein. Furthermore, immunodetection using the antiphosphotyrosine antibody, PY-20, identified five proteins with approximate Mr 116 000, 105 000, 95 000, 86 000, and 76 000, and possibly a sixth at 54 000. The 95 000 protein was consistently diminished in ionophore-treated spermatozoa, indicating that the protein was located in the acrosomal cap region. These results suggest that the protein may be the same phosphotyrosine-containing protein as that described by Leyton and Saling (1989) which has been proposed to play a role in acrosomal exocytosis.

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