PARENT SESSION

(W297) RELATIONSHIP BETWEEN PROTEASOME ACTIVITY, PROTEIN PHOSPHORYLATION AND THE ACROSOME REACTION IN HUMAN SPERM.

Kong, Milene¹, Morales, Patricio¹, Diaz, Silvina¹, ¹ University of Antofagasta, Antofagasta, Chile

ABSTRACT- In previous studies, we have shown that the proteasome is present in human sperm and that this multicatalytic cellular complex plays a role during several steps of mammalian fertilization. However, we do not know whether proteosomal activity is regulated by protein phosphorylation. The aim of this work is to study the relationship between protein phosphorylation and proteosome activity in human sperm. Aliquots of highly motile sperm, selected by percoll gradient, were incubated for 0, 5 and 18 hr at 37 °C, 5% CO₂, with different concentration of: a) a tyrosine kinase inhibitor (genistein 0 to 100 M); b) a protein kinase A inhibitor (H89 0 to 100 M); or c) a protein kinase C inhibitor (tamoxifen 0 to 100 M). Controls aliquots were treated with the inhibitor solvent. The motility and the viability of the sperm were evaluated at the different times of incubation and the chymotrypsin-like activity of proteasome was assayed using the fluorogenic substrate Suc-Leu-Leu-Val-Tyr-AMC. Furthermore, aliquots of sperm capacitated during 18 hr were incubated for 30 min with kinase inhibitors and then treated with 0.7 M progesterone. The percentage of viable acrosome reacted sperm was evaluated using FITC-labeled Pisum sativum agglutinin and the Hoechst 33258 dye. The results indicate that treatment of the sperm with different doses of genistein and tamoxifen did not modify the chymotrypsin-like activity of the proteasome. On the other hand, this activity was significantly decreased and in a dose dependent manner by incubation with H89. Sperm treatment with genistein, H89, and tamoxifen significantly inhibited the occurrence of the acrosome reaction stimulated by progesterone. Western blot analysis suggests that sperm treatment with the proteasome inhibitor, epoxomicin, inhibited serine protein phosphorylation. These results suggest that the chymotrypsin-like activity of proteasome may be modulated by protein kinase A, and that both enzymes are involved in the progesterone-induced acrosome reaction. PEI 1355-03; Fondecyt 1040295.

KEY WORDS: proteasome, acrosome reaction, protein phosphorylation