Capacitation Is Associated with Tyrosine Phosphorylation and Tyrosine Kinase-Like Activity of Pig Sperm Proteins1

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 M(r) 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 M(r) 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.

Changes in content and localization of proteins phosphorylated at tyrosine, serine and threonine residues during ram sperm capacitation and acrosome reaction

“…The situation of phosphorylated proteins at the principal piece of the flagellum has been related to motility hyperactivation and to the presence of A-kinase anchoring proteins (which anchor PKA) located in the fibrous sheath of the sperm flagellum in several species (Carrera et al 1996, Mandal et al 1999, Jha & Shivaji 2002a, Pommer et al 2003. Conversely, a capacitation-related redistribution of phosphotyrosine residues to the acrosome has been reported in humans (Naz et al 1991), boars (Tardif et al 2001, Dube et al 2005, bulls (Cormier & Bailey 2003) and buffalo (Roy & Atreja 2008). Moreover, other studies have revealed that certain tyrosine phosphoproteins become localized to the sperm surface overlying the acrosome during capacitation suggesting a key role of these proteins in sperm-zona binding (Asquith et al 2004, Piehler et al 2006.…”

“…In addition, a capacitation-associated redistribution of phosphotyrosine residues to the acrosome has been reported in boars (Petrunkina et al 2001, Tardif et al 2001, bulls (Cormier & Bailey 2003) and buffalo (Roy & Atreja 2008), and specific phosphotyrosine proteins localized over the acrosomal region have been postulated to be involved in the zona pellucida interaction and/or fusion events (Leyton & Saling 1989, Naz et al 1991, Ficarro et al 2003, Dube et al 2005.…”

Porcine sperm capacitation involves tyrosine phosphorylation and activation of aldose reductase

“…Only sperm samples with over 95% of the sperm showing progressive motility were used for the experiments. Sperm capacitation was induced according to the method reported by of Tardif et al (2001), with slight modifications. The washed sperm were incubated either in 15 ml of Non-cap medium or in Cap medium (5!10 6 cells/ml) at 37 8C for 3 h in a 5% CO 2 atmosphere.…”

Effects of Seminal Plasma on Cooling‐Induced Capacitative Changes in Boar Sperm

“…The noncapacitating medium (NCM) contained 2.7 mM KCl, 1.5 mM KH 2 PO 4 , 8.1 mM Na 2 HPO 4 , 137 mM NaCl, 5.55 mM glucose, and 2 mM pyruvate (pH 7.4). Both media have been described previously (Tardif, 2001). …”