Inhibited protein tyrosine phosphorylation in sperm from crisp1 knock out mice

BATTISTONE MA; MALDERA JA; WEIGEL MUÑOZ M; ERNESTO JI; PAGOTTO R; PIGNATARO O; COHEN DJ ; CUASNICU PS

New Hampshire

Conferencia; Gordon Research Conference; 2011

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. Considering these observations, the aim of the present work was to investigate the molecular mechanisms underlying the lower levels of protein tyrosine phosphorylation observed in CRISP1 knockout sperm. Based on previous reports indicating that the presence of CRISP1 during rat sperm capacitation inhibited protein tyrosine phosphorylation, Crisp1+/- and Crisp1-/- sperm were capacitated in the absence or presence of 6 µM CRISP1. Results revealed that CRISP1 did not affect protein tyrosine phosphorylation levels in any of these populations. 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 significantly lower  cAMP levels detected in Crisp1-/- sperm by radioimmunoassay. Together, these results indicated that the inhibition of protein 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.