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Regular Article |
a Centre de Recherche en Biologie de la Reproduction, Département des Sciences Animales, Université Laval, Sainte-Foy, Quebec, Canada G1K 7P4
b Montreal General Hospital Research Institute, Surgery (Urology Division) and Department of Experimental Medicine, McGill University, Montreal, Quebec, Canada H3G 1A4
ABSTRACT
Capacitation represents the final maturational steps that render mammalian sperm competent to fertilize, either in vivo or in vitro. Capacitation is defined as a series of events that enables sperm to bind the oocyte and undergo the acrosome reaction in response to the zona pellucida. Although the molecular mechanisms involved are not fully understood, sperm protein phosphorylation is associated with capacitation. The hypothesis of this study is that protein tyrosine phosphorylation and kinase activity mediate capacitation of porcine sperm. Fresh sperm were incubated in noncapacitating or capacitating media for various times. Proteins were extracted with SDS, subjected to SDS-PAGE, and immunoblotted with an antiphosphotyrosine antibody. An Mr 32 000 tyrosine-phosphorylated protein (designated as p32) appeared only when the sperm were incubated in capacitating medium and concomitant with capacitation as assessed by the ionophore-induced acrosome reaction. The p32 was soluble in Triton X-100. Fractionation of sperm proteins with Triton X-114 demonstrated that after capacitation, this tyrosine phosphoprotein is located in both the cytosol and the membrane. Enzyme renaturation of sperm proteins was conducted in gels with or without either poly glu:tyr (a tyrosine kinase substrate) or kemptide (a protein kinase A substrate). An Mr 32 000 enzyme with kinase behavior was observed in all gels but was preferentially phosphorylated on tyrosine, as assessed by phosphorimagery and by thin layer chromotography to identify the phosphoamino acids. Indirect immunolocalization showed that the phosphotyrosine residues redistribute to the acrosome during capacitation, which is an appropriate location for a protein involved in the acquisition of fertility.
First decision: 19 March 2001.
1 This research was funded by the Natural Sciences and Engineering Research Council of Canada and the Fonds pour la Formation de Chercheurs et l'Aide à la Recherche.
2 Correspondence: Janice L. Bailey, Centre de Recherche en Biologie de la Reproduction, Département des Sciences Animales, Pavillon Paul-Comtois, Université Laval, Sainte-Foy, PQ, Canada G1K 7P4. FAX: 481 656 3766; janice.bailey{at}crbr.ulaval.ca
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