The Laminin-Induced Acrosome Reaction in Human Sperm Is Mediated by Src Kinases and the Proteasome1

Abstract

The aim of this work was to determine whether laminin (Ln), an extracellular matrix protein, induces the intracellular events that may be involved in producing the acrosome reaction in human sperm. To this end, we evaluated the effect of Ln on tyrosine phosphorylation, intracellular calcium concentration, proteasome activity, and phosphorylation in human sperm. Aliquots of highly motile sperm selected with a Percoll gradient, were incubated with different concentrations of Ln (0-20 μg/ml) for different periods (0-18 h). The percentage of viable acrosome-reacted sperm was evaluated using fluorescein isothiocyanate-labeled Pisum sativum agglutinin and Hoechst 33258 DNA dye. Tyrosine phosphorylation was evaluated by Western blot analysis. The chymotrypsin-like activity of the proteasome was evaluated with a fluorogenic peptide, and intracellular calcium concentration was measured with fura-2. The results indicate that Ln stimulated the acrosome reaction of human sperm in a dose-dependent manner. This increase was drastically inhibited in the presence of herbimycin A, SU6656, and epoxomicin. In addition, Ln increased proteasome activity and phosphorylation; both events were inhibited by herbimycin A and SU6656. Finally, Ln induced an increase in intracellular calcium concentration, which was inhibited by SU6656 and epoxomicin. These results suggest that Ln is able to induce the acrosome reaction. This effect may be mediated by Src kinase and the proteasome, with the consequent induction of a calcium influx.

Evaluation of morphological criteria of sperm quality before in vitro fertilization and intracytoplasmic sperm injection

“…Apoptotic sperm are differentiated from necrotic using double staining with fluorescein isothiocyanate (FITC)-labeled annexin V and Hoechst 33258 (Collodel et al 2009b, de Vantery Arrighiet al 2009). The number of viable acrosome-reacted sperm can be evaluated by staining with Hoechst 33258 and fluorescein isothiocyanate-conjugated Pisum sativum agglutinin lectin (Vigil et al 2008, Tapia et al 2011, Montjean et al 2012, Vigil et al 2012.…”

The association between CDC42 and caveolin-1 is involved in the regulation of capacitation and acrosome reaction of guinea pig and mouse sperm

“…Lee et al 2000, Cao et al 2002, Banquet et al 2011. The presence of c-Src has been shown in mammalian sperm, and its inhibition by SU6656, an inhibitor of SFK, blocks sperm processes related to tyrosine phosphorylation such as capacitation, motility hyperactivation, and the AR (Baker et al 2006, Mitchell et al 2008, Krapf et al 2010, Tapia et al 2011. To determine whether c-Src kinase is related to CAV1 phosphorylation, guinea pig sperm were capacitated in the presence of SU6656, and CAV1 phosphorylation was analyzed by Wb using the anti-p-Cav1 antibody.…”

Protein-Tyrosine Kinase Signaling in the Biological Functions Associated with Sperm

“…In the former system, G-protein-mediated production of AMP promotes PKA activation, PKA up-regulates Src (as seen in capacitated sperm), Src activates the secretion of heparin-binding EGF-like growth factor via MMP activation, thereby activating EGFR/kinase, and Src also affects the activity of EGFR/kinase through direct phosphorylation on tyrosine 845 [178], an Src-dependent phosphorylation site, whose phosphorylation has been implicated in some types of cancer cells [180–182]. Src activation and its importance for AE have also been demonstrated in humans [183, 184]. Another line of evidence suggests that SCF is involved in the promotion of mouse sperm AE through the activation of its cognate receptor/PTK c-Kit, PLC γ 1, and phosphatidylinositol 3-kinase (PI3K) [185].…”

“…As described above, not only ZP3, but also other reagents or experimental conditions (e.g., PG) are reportedly inducible for AE in vitro. Additionally, possible oviductal substances such as sperm-binding glycoprotein [188], laminin [184], fibronectin [189], and follicular fluid [190] have been shown to induce AE accompanied by PTK signaling. Taking these findings together, further analysis focusing on the roles of the sperm microenvironment during capacitation and AE (before reaching the egg plasma membrane) in vivo should enable greater understanding of the physiological impact of PTK signaling.…”