OSTEOPONTIN IS THE 55-KILODALTON FERTILITY-ASSOCIATED PROTEIN IN HOLSTEIN BULL SEMINAL PLASMA

Previously we identified tour proteins in seminal plasma that were ass ociated with bull fertility. The purpose of this study was to identify the 55-kDa protein prevalent in seminal plasma of higher-fertility ma les. The 55-kDa protein was quantified by video densitometry in two-di mensional electrophoresis gels of seminal plasma from 26 bulls of know n fertility. Relative density of the 55-kDa protein was positively cor related (r = 0.48) with bull fertility. The 55-kDa (pI 4.5) fertility- associated protein spot was isolated by electroelution after two-dimen sional PAGE separation of seminal plasma of 36 bulls. N-terminal seque nce analysis of the pure protein yielded a 15-amino acid sequence (VKP XSSGXSEEKQLN) that was 86% homologous to bovine osteopontin-k precurso r. Polyclonal antiserum generated against the 55-kDa protein reacted w ith a single spot in two-dimensional PAGE Western blots of seminal pla sma. Western blot analyses using polyclonal antisera generated against the amino terminus (LF123) and carboxyl terminus (LF124) of human rec ombinant osteopontin confirmed that the 55-kDa polypeptide was osteopo ntin. Partially purified 55-kDa protein was obtained by HPLC-MonoQ col umn 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.