Extracellular localization of proteasomes in human sperm.
The proteasome, a multienzymatic protease complex is present in human sperm. Here we present evidence indicating that the proteasome has an extracellular localization, on the plasma membrane of the sperm head. Motile sperm (>90%) in PBS were incubated with the proteasome inhibitors clasto-lactacystin beta-lactone or epoxomicin. Then, the substrate Suc-Leu-Leu-Val-Tyr-AMC (SLLVY-AMC) was added and the enzyme activity evaluated in a spectrofluorometer. Other aliquots were resuspended in Tyrode's medium and incubated at different concentrations for various times with or without inhibitors in the presence of 0.4% azocasein. Hydrolysis of azocasein was evaluated at 440 nm. In addition, sperm membrane proteins were obtained incubating the sperm with Triton X-114 or with 0.5 M KCl plus Triton X-100 and removing insoluble material by centrifugation at 5,000g for 40 min. Proteasomal activity was evaluated with SLLVY-AMC and its presence corroborated by Western blotting. Formaldehyde fixed, unpermeabilized sperm were incubated with anti-proteasome monoclonal antibodies and evaluated using indirect immunofluorescence. The effect of proteasome inhibitors upon the progesterone-induced acrosome reaction was also evaluated. Results indicated that (a) whole, intact sperm were able to hydrolyze the proteasome substrates SLLVY-AMC and azocasein; this activity was inhibited by proteasome inhibitors; (b) proteasomal activity was detected in soluble sperm membrane protein preparations and Western blotting revealed the presence of the proteasome in these fractions; (c) indirect immunofluorescence revealed staining of the head region, particularly of the post acrosomal region; and (d) the proteasome plays an important role during the acrosome reaction.
Morales
Patricio
P
Department of Biomedicine, Faculty of Health Sciences, University of Antofagasta, Antofagasta, Chile. pmorales@uantof.cl
Pizarro
Eduardo
E
Kong
Milene
M
Jara
Marcos
M
D020101:Acrosome Reaction; D002364:Caseins / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D005089:Exocytosis; D006801:Humans; D006868:Hydrolysis; D008297:Male; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011374:Progesterone / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D013094:Spermatozoa / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism
D002364:Caseins; D009097:Multienzyme Complexes; C026018:azocasein; D011374:Progesterone; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex