180. CHARACTERIZATION AND FUNCTION OF THE BULL SPERM PROTEIN SPAM1: TWO DISTINCT ISOFORMS WITH DISTINCT ROLES

Abstract

We identified an 80 kDa bull sperm protein (p80) that possesses homology with the Sperm adhesion molecule 1 (Spam1), a GPI-anchored glycoprotein conserved amongst mammals that is required for fertilization. Since bovine Spam1 had not been identified, the aim of this project was to determine if p80 is the bovine Spam1 and to test the hypothesis that it plays a role in gamete interaction during bovine fertilization. Amino acid sequence deduced from 3`/5`Race confirmed that homology between p80 and Spam1 in various species ranged from 47 to 61%. It also revealed specific differences including the absence of a GPI-anchor, the presence of a transmembrane domain, and N- and O- glycosylation sites. By generating and characterising antibodies against p80 N- and C-terminal domains, the protein orientation in the sperm membrane was evaluated. We identified two populations of p80 on the sperm head: one internalised in the anterior region and the second localised on the post-acrosomal region with its hyaluronidase domain exposed to the extracellular environment. Proteomic and immunologic analyses revealed that the p80 post-acrosomal population is a shorter isoform originating from the epididymis while the full length p80 located on the anterior region originates from the testis. Finally, the potential function of p80 during the sperm/zp interaction was evaluated by sperm/zona pellucida (zp) binding assay. The C-terminal extremity of p80 was implicated in sperm binding to the zp by antibody and native protein competition. Furthermore, glycosylation was not required during this interaction since deglycosylated p80 in the incubation medium had the same inhibitory effect on zona binding as the native p80. Collectively, the results demonstrated that p80 is the bovine Spam1, and that two isoforms are present on bull sperm. The hyaluronidase activity of the post-acrosomal isoform is required for cumulus penetration, while the other one participates in sperm/zp binding during fertilization.