A role for phosphorylation of glycogen synthase kinase-3 alpha in bovine sperm motility regulation

The long-term goal of our work is to understand biochemical mechanisms unde rlying sperm motility and fertility. In a recent study we showed that tyros ine phosphorylation of a 55-kDa protein varied in direct proportion to moti lity. Tyrosine phosphorylation of the protein was low in immotile compared to motile epididymal sperm. Inhibition or stimulation of motility by high c alcium levels or cAMP, respectively, results in a corresponding decrease or increase in tyrosine phosphorylation of the 55-kDa protein. Here we report purification and identification of this motility-associated protein. Solub le extracts from bovine caudal epididymal sperm were subjected to DEAF-cell ulose, Affi-Gel blue, and cellulose phosphate chromatography. Tyrosine phos phate immunoreactive fractions contained glycogen synthase kinase-3 (GSK-3) activity, suggesting a possible correspondence between these proteins. Thi s suggestion was verified by Western blot analyses following one-dimensiona l and two-dimensional gel electrophoresis of the purified protein using mon oclonal and affinity-purified polyclonal antibodies against the catalytic a mino-terminus and carboxy-terminus regions of GSK-3. Further confirmation o f the identity of these proteins came from Western blot analysis using anti bodies specific to the tyrosine phosphorylated GSK-3. Using this antibody, we also showed that GSK-3 tyrosine phosphorylation was high in motile compa red to immotile sperm. Immunocytochemistry revealed that GSK-3 is present i n the flagellum and the anterior portion of the sperm head. These data sugg est that GSK-3, regulated by phosphorylation, could be a key element underl ying motility initiation in the epididymis and regulation of mature sperm f unction.