SIGNAL TRANSDUCTION PATHWAYS INVOLVED IN RAT SPERM CAPACITATION

BATTISTONE MA; WEIGEL MUÑOZ M; ERNESTO JI; MALDERA JA; KRAPF D; VISCONTI P; CUASNICU PS

Congreso; 37th Annual Meeting of American Society of Andrology; 2012

Sperm capacitation relies on an increase in protein tyrosine phosphorylation mediated by PKA activation. Recent studies have suggested that Src tyrosine kinase family (SKF) is involved in capacitation-associated protein tyrosine phosphorylation either direct or indirectly. In order to elucidate between these two possibilities, we investigated whether SFK is involved in the signaling events leading to rat sperm capacitation. Western blot results showed that sperm capacitated in the presence of SFK inhibitors (SU6656 and SKI606; 50 µM), exhibited low levels of both tyrosine and PKA-substrate phosphorylation. Based on the described inhibition of ser/thr phosphatases by SFK, sperm were exposed to a ser/thr phosphatase inhibitor (okadaic acid, OA 10 nM) observing a reversion of the impaired protein phosphorylation produced by SU6656 and SKI606. However, OA was unable to induce tyrosine phosphorylation in either non capacitated sperm or sperm capacitated in the presence of the PKA inhibitor H89. Addition of both a cAMP agonist (dibutyryl cAMP, 5 mM) and a phosphodiesterase inhibitor (Pentoxifiline, 3mM) did not overcome the inhibition produced by the SFK inhibitors. In addition to their effect on PKA and protein tyrosine phosphorylation, SU6656 and SKI606 significantly inhibited capacitated sperm motility (69,4% (control) vs 42,9% (SU6656, p<0,0001)), vs 34,8% (SKI606, p<0,0001)), acrosome reaction occurrence (32,0% (control) vs 25,2% (SU6656, p<0,0001, vs 29,4% (SKI606, p<0,009)) and the sperm ability to fuse with zona pellucida-free oocytes in vitro (96,0% (control) vs 24,3% (SU6656, p<0,0001) vs 20,2% (SKI606, p<0,001)). These inhibitions were not observed when sperm were exposed to SU6656 or SKI656 in the presence of OA, indicating that the effect of the SFK inhibitors involves an up-regulation of the ser/thr phosphatase activity. Altogether, these results support both the involvement of SFK in rat sperm capacitation and the existence of two parallel pathways leading to capacitation: one requiring cAMP/PKA activation, and the other involving the inactivation of ser/thr phosphatases.