IBYME   02675
INSTITUTO DE BIOLOGIA Y MEDICINA EXPERIMENTAL
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Inhibited protein tyrosine phosphorylation in sperm from CRISP1 knockout mice
Autor/es:
BATTISTONE MA, MALDERA JA, PAGOTTO R; PIGNATARO OP, COHEN DJ, CUASNICU PS
Reunión:
Congreso; 5th International Conference on The Epididymis (Epididymis V); 2010
Resumen:
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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 regionof 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
oolemma. 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. 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.
Results indicated that 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
indirect immunofluorescence 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 in 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 evaluated by Western
blot, 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. 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. Considering
that sperm acquire both CRISP1 and the ability to undergo capacitation-induced
tyrosine phosphorylationduring 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.