Proteasome activity and its relationship with protein phosphorylation during capacitation and acrosome reaction in human spermatozoa.
We have shown that the proteasome is present in mammalian sperm and plays a role during fertilisation. In this work we studied the relationship between protein phosphorylation and proteasomal activity in human sperm. Aliquots of motile sperm were incubated for 0, 5 and 18 h at 37 degrees C, 5% CO2, with different concentration of the kinase inhibitors genistein, H89 or tamoxifen. Control aliquots were treated with the inhibitor solvent. The chymotrypsin-like activity of the proteasome was assayed using a fluorogenic substrate. Aliquots of spermatozoa capacitated during 18 h were incubated for 30 min with kinase inhibitors and then with 7 microM progesterone (P). The percentage of viable acrosome-reacted sperm was evaluated using FITC-labeled Pisum sativum agglutinin. The results indicate that spermatozoa treated with different concentrations of genistein and tamoxifen did not modify the chymotrypsin-like activity of the proteasome during capacitation. On the other hand, proteasome activity was significantly decreased by incubation with H89. Sperm treatment with genistein, H89 and tamoxifen significantly inhibited the P-induced acrosome reaction. Western blot analysis indicated that the proteasome inhibitor, epoxomicin, reduced serine protein phosphorylation. These results suggest that the enzymatic activity of the proteasome is modulated by protein kinase A, and that both enzymes are involved in the P-induced acrosome reaction.
Morales
Patricio
P
Unit of Reproductive Biology, Faculty of Health Sciences, University of Antofagasta, Antofagasta, Chile. pmorales@uantof.cl
Díaz
Emilce S
ES
Kong
Milene
M
D020101:Acrosome Reaction / Q000502:physiology; D005306:Fertilization / Q000502:physiology*; D006801:Humans; D008297:Male; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D013075:Sperm Capacitation / Q000502:physiology; D013094:Spermatozoa / Q000378:metabolism*
D011506:Proteins; D046988:Proteasome Endopeptidase Complex