Extracellular localization of proteasomes in human sperm

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Abstract
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 β‐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. Mol. Reprod. Dev. 68: 115–124, 2004.