Signalling pathways associated with the bicarbonate-dependent rat sperm capacitation process

CUASNICÚ, PS; DA ROS, VG; PIGNATARO, O; VISCONTI, PE

Madrid, Spain

Simposio; 10th International Symposium on Spermatology; 2006

International Symposium on Spermatology

Previous results from our group demonstrated that bicarbonate is required to support rat spermcapacitation by regulating protein tyrosine phosphorylation, migration of epididymal protein DE/CRISP-1 to the equatorial segment and expression of the sperm fusion ability. However, while phosphorylation could occur even when bicarbonate was replaced by a cAMP analogue, the other two parameters seem to require additional bicarbonate properties. To further study the pathway involve in the bicarbonate regulation of rat sperm capacitation, intracellular cAMP levels were measured (by RIA) in sperm incubated in the presence or absence of the anion. Results showed a time-dependent increase in cAMP levels (peak at 30 min) only in those sperm incubated in the presence of bicarbonate. To investigate whether this bicarbonate-dependent cAMP accumulation was induced by phorbol esters as observed in the hamster, rat sperm were incubated in the presence or absence of bicarbonate, with or without 10 µM PMA. Results indicated that sperm incubated for 30 min with Bicarbonate and PMA showed a significantincrease in cAMP contents not observed in the absence of PMA. This cAMP accumulation induced by PMA was dependent of the presence of bicarbonate in the medium. In order to test whether the cAMP produced was required for the three capacitation parameters previously studied, the involvement of PKA was studied using the inhibitor H89. Tyrosine phosphorylation was significant reduced by addition of 30 µM H89 but not by DMSO (Western blot and indirect immunofluorescence assays). However, migration of DE/CRISP-1 was not affected, and the expression of the fusion ability (fusion assays) could only be partially inhibited by H89 but not by DMSO, even in the presence of H89 during gamete co-incubation. Taken together, our results provide more information on the regulation of rat sperm capacitation by bicarbonate, confirming that some capacitation-associated events, although bicarbonate-dependent, would be cAMP/PKA-independent.