In our previous paper [M. Fujinoki et al. (2001) BIOMED: Res. 22, 45-58], we reported that two types of 36-kDa protein, which were designated as 36K-A protein and 36K-B protein, obtained from hamster sperm flagella were phosphorylated at serine residues associated with the regulation of motility activation. In the present experiments, it was suggested that these two types of 36-kDa protein were phosphorylated in a cAMP-dependent manner associated with motility activation of hamster spermatozoa. Because the 36K-B protein was the most intensely phosphorylated in a cAMP-dependent manner, attempts were made to further characterize it. The 36K-B protein was assumed to be localized in the middle piece. The localization of the 36K-B protein was the same as that of the 36-kDa protein reported in our previous paper [Y. Si et al. (1999) Mol. Reprod. Dev. 52, 328-334]. In order to identify the 36K-B protein, it was analyzed by peptide mass finger printing and amino acid sequencing. The results suggested that the 36K-B protein was a pyruvate dehydrogenase E1 component beta subunit and a component of the mitochondrial sheath of the middle piece.

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Mammalian sperm activation and hyperactivation is regulated by protein phosphorylation. Although tyrosine phosphorylation is considered very important, several studies have investigated whether serine and threonine phosphorylation are also associated with sperm activation and hyperactivation, and that was also the aim of the present study. Protein phosphorylation of hamster spermatozoa was detected by Western blotting using antiphospho-amino acid monoclonal antibodies after tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid sequences were analyzed using a peptide sequencer. Four proteins were phosphorylated at serine residues during hyperactivation via activation and their approximate molecular weights were 90, 38, 32 and 10 kDa, respectively. Five proteins were phosphorylated or dephosphorylated at threonine residues and their approximate molecular weights were 90, 70, 65, 35 and 10 kDa, respectively. The 10-kDa protein corresponded to a previously reported 10-kDa tyrosine phosphoprotein. N-terminal sequences of the 10-kDa protein were similar to carcinustatin, which is a neuropeptide. During hyperactivation, four serine phosphorylation and five threonine phospho- or dephosphorylations occurred, which suggested that the 10-kDa protein was phosphorylated at tyrosine residues when spermatozoa were activated and then dual-phosphorylated at the serine and threonine residues during hyperactivation. (Reprod Med Biol 2004;: 223-230).