Identification of the bull sperm p80 protein as a PH-20 ortholog and its modification during the epididymal transit

We have identified an 80 kDa protein in ejaculated bull spermatozoa (p80) which is found in acrosomal and post-acrosomal areas of the head. It has a hyaluronidase activity and shares homologies with PH-20, a sperm surface glycoprotein involved in sperm-egg interaction. The aim of the present study was to characterize bull sperm p80 protein at the nucleic and amino acid levels to determine whether it is the bovine PH-20 ortholog. The complete nucleotide sequence determined by RT-PCR, 3' and 5' RACE show that bull p80, displays identity with the PH-20 nucleotide and amino acid sequences. Messenger RNA and protein expressions determined by Northern blot and immunohistochemistry revealed that the protein is testicular (expressed in spermatocytes and spermatids). The localization of p80 on spermatozoa, determined by indirect immunofluorescence using a monoclonal antibody, shows the protein in acrosomal and post acrosomal areas of the head with an increase in the signal intensity as sperm progress through the epididymis. Post-translational modifications of the protein were investigated during the epididymal maturation by Western blot on protein extracts from sperm collected in the caput, corpus and cauda portions of bull epididymis. Glycolysation status of sperm p80 protein on proteins from ejaculated and epididymidal sperm was investigated. Result show that the glycosylation status is modified as spermatozoa migrate through the epididymis. Hyaluronidase activity evaluated in protein extracts from spermatozoa of the three different epididymal sections revealed that the activity is higher at pH 7 than 4 and is not affected by epididymal maturation. These data strongly suggest that p80 is the bovine PH-20.