Characterization of an 80-kilodalton bull sperm protein identified as PH-20

This paper presents the partial characterization and the identification of an 80-kDa protein detected in bull spermatozoa using a monoclonal antibody directed against a 16-amino acid long peptide from the N-terminal domain of the protooncogene p60(src) from the Rous Sarcoma Virus When subjected to t wo-dimensional electrophoresis, this 80-kDa protein migrated as several iso forms, with an isoelectric point ranging from 7.4 to 8.2. Amino acid sequen ce analysis of a peptide obtained following trypsin digestion of the bull s perm protein showed homology to the PH-20/hyaluronidase precursor sperm pro tein. As for PH-20, this bull sperm 80-kDa protein is located at the plasma membrane surface in the postacrosomal region of the head. An increased imm unolabeling in the anterior head region of fixed/permeabilized spermatozoa was observed when these cells were incubated under capacitating conditions, whereas most sperm cells challenged with the calcium ionophore A23187 to a crosome react lost their labeling almost completely. As for the PH-20 prote in, the 80-kDa bull sperm protein possesses a hyaluronidase activity that i s higher at pH 7.0 than at pH 4.0 in an ingel assay. Unlike what has been o bserved in the guinea pig, mouse, and human PH-20, this 80-kDa protein was not released from the surface of bull spermatozoa by treatment with phospha tidylinositol-specific phospholipase C or with trypsin. However, this prote in was not sedimented by a 100 000 x g centrifugation after nitrogen cavita tion, which suggests that the 80-kDa protein is loosely attached to the spe rm membrane by a yet-unknown mechanism. These results suggest that the 80-k Da bull sperm protein shares many homologies with the sperm PH-20 protein r eported in the literature and, most likely, is the bull sperm homologue of the PH-20.