INHIBITED PROTEIN TYROSINE PHOSPHORYLATION IN SPERM FROM CRISP1 KNOCK OUT MICE

BATTISTONE MA; JULIETA MALDERA; ROMINA PAGOTTO; OMAR P. PIGNATARO; DÉBORA J. COHEN; PATRICIA S. CUASNICU

Aguas de Sao Pedro. Sao Pablo

Congreso; Epididymis V; 2010

Introduction: Mammalian sperm become competent to fertilize only after undergoing a series of changes that occur during their transit through the male and female reproductive tracts known as maturation and capacitation, respectively. Epididymal protein CRISP1 associates with the dorsal region of rat sperm during epididymal maturation. While a substantial amount of CRISP1 is released during capacitation suggesting its role as a decapacitating factor, part of the protein remains on sperm surface and participates in both sperm-zona pellucida interaction and gamete fusion by binding to egg-complementary sites. In agreement with this, capacitated sperm from Crisp1-/- mice recently generated in our laboratory exhibited a significantly lower ability to interact with the zona pellucida and the oolema. However, contrary to what it is expected for a decapacitating factor, tyrosine phosphorylation levels were significantly lower than in controls suggesting that CRISP1 could play a regulatory role during capacitation different from that originally expected. Aim: In view of previous reports indicating that the presence of CRISP1 during rat sperm capacitation inhibits protein tyrosine phosphorylation, we studied the effects of CRISP1 on Crisp1+/-and Crisp1-/- sperm capacitation. Methods: Sperm were incubated for 90–120 min under capacitated conditions; proteins were separated by SDS–PAGE and analyzed by Western blot with the anti-phosphotyrosine monoclonal antibody (1:1,000; clone 4G10; Upstate, Lake Placid, NY). Sperm protein tyrosine phosphorylation levels were detected by indirect immunofluorescence (IFI). Results: the presence of the protein did not modify protein tyrosine phosphorylation levels in any of these populations. The decrease in tyrosine phosphorylation levels in CRISP1 mutant sperm were also detected by IFI as judged by the faint fluorescent labeling in both midpiece and principal piece of Crisp1-/- sperm compared with the strong staining observed in Crisp1+/- sperm. Since it has been demonstrated that tyrosine phosphorylation is downstream a cAMP/PKA pathway, we investigated whether cAMP is involved in the observed reduction of tyrosine phopshorylation by exposing Crisp1-/- sperm to a cAMP analog (db-cAMP) and a phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine, IBMX). Results showed that, under these conditions, there was a reversion in the phosphorylation pattern of Crisp1-/- sperm supporting the existence of a lower content of cAMP in mutant sperm. This possibility was confirmed by the significant reduced levels of cAMP determined by radioimmunoassay in both Crisp1+/- and Crisp1-/- sperm. Discussion: Together, the results indicated that the inhibition of tyrosine phosphorylation in sperm lacking CRISP1 could be attributed to the lower levels of intracellular cAMP generated during capacitation. Although the molecular mechanisms underlying cAMP reduction are still unknown, at present we are investigating whether these observations are related to the reported ion channel regulating ability of CRISP proteins. Conclusion: Considering that sperm acquire both CRISP1 and the ability to undergo capacitation-induced tyrosine phosphorylation during their transit through the epididymis, these studies suggest that the lower levels of tyrosine phosphorylation in capacitated Crisp1-/-sperm might be due to the lack of association of CRISP1 during epididymal maturation.   Financial Support: National Research Council (CONICET), Agency for Scientific and Technological Promotion (ANPCyT) and World Heatlh Organization (WHO).