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

Abstract

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.

Epididymal SPAM1 Is a Marker for Sperm Maturation in the Mouse1

“…Based on its widespread expression in the epididymis, it is likely to play a role in both sperm maturation and storage. In the bull and mouse, it has been shown to be secreted in the epididymal luminal fluid (ELF) [11][12][13]. Consistent with the presence of SPAM1 in the ELF is the finding that it is secreted in conditioned media of cultured murine epididymal epithelial cells [11].…”

“…Sperm adhesion molecule 1 (SPAM1), the major testicular hyaluronidase, is among the glycosyl phosphatidylinositol (GPI)-linked glycoproteins secreted by the epididymal epithelium [11][12][13]. Originally thought to be testis-specific [14][15][16], SPAM1 has now been shown to be synthesized in the epithelium in all three regions of the epididymis, and is conserved in at least five mammalian species, as recently reviewed [17][18][19].…”

A molecular analysis of the population of mRNA in bovine spermatozoa

“…These tissues were used for spermatids isolation as well as for controls in several experiments. The purification of spermatids was carried out as described by Morin et al (2005). Briefly, the haploid germ cells were isolated from a piece of fresh testis under the tunica albuginea.…”

Murine SPAM1 is secreted by the estrous uterus and oviduct in a form that can bind to sperm during capacitation: acquisition enhances hyaluronic acid-binding ability and cumulus dispersal efficiency

“…Both the quantity and localization pattern of SPAM1 on sperm are altered as they traverse the epididymis: more SPAM1 is present on caudal sperm than on those from the caput (Deng et al 1999, Rutllant & Meyers 2001, Morin et al 2005, Martin-DeLeon 2006 and its distribution changes from being uniform to localized, predominantly on the plasma membrane of the anterior head (Deng et al 1999). The quantitative change suggests that sperm acquire SPAM1 during epididymal transit.…”