PARENT SESSION

(T311) EFFECT OF FIBRONECTIN (FN) ON PROTEASOMAL ACTIVITY, ACROSOME REACTION (AR), AND TYROSINE PHOSPHORYLATION IN HUMAN SPERM.

Morales, Patricio¹, Diaz, Silvina¹, Kong, Milene¹, Perez, Boris¹, ¹ University of Antofagasta, Antofagasta, Chile

ABSTRACT- We previously showed that the human sperm proteasome plays a significant role during several steps of the fertilization process. The aim of this work was to study the effect of Fn, an extracellular matrix protein present in the oocyte′s cumulus oophorus, on the proteasome activity, the AR, and protein phosphorylation of human sperm. Aliquots of highly motile sperm, selected through a percoll gradient, were incubated for 0 hr (T0), 4.5 hr (T4.5) and 18 hr (T18), at 37 °C, 5% CO₂, with different concentrations of Fn (0 to 100 mg/ml). At all times, the chymotrypsin-like and trypsin-like activity of the proteasome was measured using the fluorogenic substrates, Suc-Leu-Leu-Val-Tyr-AMC and Boc-Gln-Ala-Arg-AMC, respectively. In addition, at T18 a sperm aliquot was incubated for 15 min with 0.5 M progesterone (P) in the presence or absence of 10 M epoxomicin (proteasome inhibitor). The percentage of viable acrosome reacted sperm was evaluated using FITC-labeled Pisum sativum agglutinin and the Hoechst 33258 dye. Tyrosine phosphorylation was evaluated by western blots. The results indicate that Fn stimulated the chymotrypsin-like and the trypsin-like activities of the sperm proteasome, in a dose dependent manner at all incubation times. For example, in the presence of 100 mg/ml Fn, the chymotrypsin-like activity of the proteasome increased in regard to the control 47% at T0, 69% at T4.5, and 100% at T18. In addition, Fn stimulated the AR of human sperm in a dose dependent manner (control= 16±2% vs. 100 mg/ml Fn= 34±1.8%, mean±sem. P<0.001). This increase in AR was drastically inhibited in the presence of 10 M epoxomicin (11±1.8%). On the other hand, progesterone potentiated the capacity of Fn to induce the human sperm AR (P alone= 33±1%; Fn alone=34±1.8%; P + Fn= 55±3%. P<0.001). Finally, western blot analysis revealed that Fn increased tyrosine protein phosphorylation, both at T0 and at T5. These results suggest that Fn, an extracellular matrix protein, is able to activate the sperm proteasome and to induce the AR in human sperm. This effect may involve binding with specific receptors (integrins) on the sperm surface and the activation of tyrosine kinases. Fondecyt 1040295.

KEY WORDS: extracellular matrix, cumulus oophorus, acrosome reaction, proteasome