Porcine Sperm Capacitation and Tyrosine Kinase Activity Are Dependent on Bicarbonate and Calcium but Protein Tyrosine Phosphorylation Is Only Associated with Calcium1

Abstract

Mammalian sperm undergo capacitation in the female reproductive tract or under defined conditions in vitro. Although capacitation is now considered to be mediated by intracellular signaling events, including protein phosphorylation, the regulation of the transduction mechanisms is poorly understood. The objective of the present study was to evaluate the importance of medium components on capacitation of porcine sperm, the appearance of an M(r) 32 000 sperm protein (p32), and activity of a tyrosine kinase (TK-32). As determined by the ability of the sperm to undergo the A23187-induced acrosome reaction, pig sperm require bicarbonate and calcium but not BSA for capacitation in vitro. The appearance of p32 was assessed by immunoblotting SDS-extracted and separated sperm proteins using an anti-phosphotyrosine antibody. The appearance of p32 requires calcium, although p32 appears even in the absence of bicarbonate in the incubation medium, demonstrating that the appearance of this tyrosine phosphoprotein is not a final end point of pig sperm capacitation. An in-gel tyrosine kinase renaturation assay showed that TK-32 activity depends on calcium and bicarbonate in the incubation medium. Immunoprecipitation experiments using an anti-phosphotyrosine antibody and inhibitor demonstrated that p32 and TK-32 are different proteins. These data indicate that the signal transduction mechanisms of capacitation in pig sperm are different from those in other mammals, suggesting that certain species specificity may exist with respect to this phenomenon.

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

“…Consistent with other studies, we have demonstrated increases in protein tyrosine phosphorylation in both incubated and cooled sperm, including an increase in the phosphorylation of a 32-kd protein band (Green et al, 2001;Kaneto et al, 2002;Tardif et al, 2003;Bravo et al, 2005). This 32-kd protein has recently been identified as proacrosin-binding protein, and its phosphorylation has been clearly shown to be associated with capacitation (Dube et al, 2005).…”

Expression and tyrosine phosphorylation of sp32 regulate the activation of the boar proacrosin/acrosin system

“…Sp32 was highly expressed in capacitated sperm or during prophase in the acrosomal reaction. This observation fits the expectation that sp32 is a special protein participating in sperm capacitation (Tardif et al, 2003), suggesting that the expression level of sp32 regulates acrosomal protein activation. Frozen-thawed sperms also displayed clear sp32 bands but had lower amounts of sp32 compared to those of capacitated sperms or during prophase of the acrosomal reaction.…”

“…This indicates that P32 is phosphorylated sp32, and the phosphorylation accompanies boar sperm capacitation. In addition, sp32 plays an important role in the maturation of acrosin (Tardif et al, 2003). Therefore, sp32 is involved in capacitation through tyrosine phosphorylation and binds with proacrosin.…”

Current Concepts of Molecular Events During Bovine and Porcine Spermatozoa Capacitation

“…Incubation of murine spermatozoa in media devoid of bicarbonate, calcium, or albumin prevents capacitation as assessed by the ability of these spermatozoa to undergo a zona pellucidainduced acrosome reaction and fertilize oocytes in vitro [Visconti et al 1995]. Similarly, porcine spermatozoa capacitation is dependent upon calcium and bicarbonate, but does not appear to require albumin, possibly due to the remarkably low cholesterol: phospholipid ratio of porcine spermatozoa [Althouse 1992;Tardif et al 2003]. However, porcine spermatozoa capacitation can be inhibited by an increase in membrane cholesterol mediated by cyclodextrin (Galantino-Homer et al 2006).…”

“…After 2 hours of incubation in a capacitation-supporting environment, the concentration of intracellular calcium increased from 70 to 250 nM in human spermatozoa [Baldi et al 1991]. Similarly, bovine and porcine sperm capacitation have been shown to be calcium-dependent and associated with increases in intracellular calcium [Handrow et al 1989;Tardif et al 2003]. Voltage-gated calcium channels, calciumATPase inactivation, and the sodium-calcium pump have been postulated to result in this calcium influx [Florman et al 1992].…”