Osteopontin is the 55-kilodalton fertility-associated protein in Holstein bull seminal plasma

Previously we identified four proteins in seminal plasma that were associated with bull fertility. The purpose of this study was to identify the 55-kDa protein prevalent in seminal plasma of higher-fertility males. The 55-kDa protein was quantified by video densitometry in two-dimensional electrophoresis gels of seminal plasma from 26 bulls of known fertility. Relative density of the 55-kDa protein was positively correlated (r = 0.48) with bull fertility. The 55-kDa (pI 4.5) fertility-associated protein spot was isolated by electroelution after two-dimensional PAGE separation of seminal plasma of 36 bulls. N-terminal sequence analysis of the pure protein yielded a 15-amino acid sequence (VKPXSSGXSEEKQLN) that was 86% homologous to bovine osteopontin-k precursor. Polyclonal antiserum generated against the 55-kDa protein reacted with a single spot in two-dimensional PAGE Western blots of seminal plasma. Western blot analyses using polyclonal antisera generated against the amino terminus (LF123) and carboxyl terminus (LF124) of human recombinant osteopontin confirmed that the 55-kDa polypeptide was osteopontin. Partially purified 55-kDa protein was obtained by HPLC-MonoQ column chromatography. Protein characterization revealed that the 55-kDa protein was glycosylated, but not phosphorylated, consistent with the identity of the 55-kDa protein as osteopontin.