Minisymposium XI. New Insights Through Modern Approaches into Gamete Biology and Acquisition of Fertilization Competence
Chair(s): Evans, Janice1, 1 Johns Hopkins, Baltimore, MD
Location: CCQ 205ABC
(MS32) INSIGHT INTO SPERM CAPACITATION: TYROSINE PHOSPHORYLATION OF SP32, A PROACROSIN BINDING PROTEIN.
Bailey, Janice 1, Dubé, Charlotte1, Leclerc, Pierre 2, Guillemette, Christine1, Beaulieu, Martin1, 1 Université Laval, Quebec City, QC, Canada2 Centre Hospitalier de l'Université Laval, Quebec City, QC, Canada
ABSTRACT- Capacitation refers to the collection of cellular modifications that allow mammalian sperm to bind the zona pellucida of the oocyte and undergo the acrosome reaction. Therefore, it is necessary for successful fertilisation in vivo and in vitro, perhaps with the exception of intracytoplasmic sperm injection. Capacitation is a signal transduction-mediated phenomenon, triggered in the absence of a ligand on the sperm, and is regulated by cAMP, calcium, protein tyrosine phosphorylation and as yet unidentified tyrosine kinase(s). We have reported that in porcine sperm, the appearance of a tyrosine phosphorylated protein, p32, coincides with capacitation. Western blotting of pig sperm proteins separated successively under non-reducing then reducing conditions showed p32 only when sperm were incubated in capacitating conditions. Sequencing p32 by MS/MS revealed it to be sp32, a protein involved in acrosin maturation in pig sperm and binds the Mr 55,000 and 53,000 forms of proacrosin as well as the Mr 49,000 acrosin intermediate (Baba et al J Biol Chem 1994 269:10133-40). Our hypothesis, therefore, is that p32 is a tyrosine phosphorylated form of sp32, the acrosomal protein implicated in acrosin activation. Hybridising the same membranes with anti-sp32 antibody demonstrated that sp32 is present in both non-capacitating and capacitating conditions; anti-sp32 recognised the same spot as p32 in extracts from capacitated sperm. Immunoprecipitation with either anti-phosphotyrosine or anti-sp32 antibody corroborated these results. Including calcium chelators in the media demonstrated that the appearance of sp32 is calcium-dependent, similar to what we have previously established for p32. Indirect immunofluorescence with anti-phosphotyrosine antibody or anti-sp32 antibody show similar labelling of capacitated sperm, supporting the hypothesis that p32 is a tyrosine phosphorylated form of sp32. Both indirect immunofluorescent and electron microscopy showed that sp32 is lost from the sperm following the A23187-induced acrosome reaction. Despite these intriguing data, numerous questions remain regarding the regulation of sp32 tyrosine phosphorylation and its importance to sperm function. This project is funded by NSERC; CD and MB were recipients of FQRNT scholarships.
KEY WORDS: sperm, capacitation, phosphorylation, acrosome