Background: Fertilization in mammals comprises the sequential interactions of the sperm with the cumulus oophorus, zona pellucida, and oocyte plasma membrane. Here we investigate proteasome activity in human sperm and its possible involvement during the fertilization process. Methods: Proteasome activity was measured in intact sperm and in sperm extracts using the fluorogenic substrate Suc-Leu-Leu-Val-Tyr-AMC, in the presence or absence of the specific proteasome inhibitor, clasto-lactacystin β-lactone. The participation of the proteasome was evaluated during (i) sperm-zona binding using the hemizona assay; (ii) zona pellucida-induced acrosome reactions with a pulse and chase design; (iii) progesterone-induced acrosome reactions incubating overnight capacitated sperm with progesterone; and (iv) progesterone-induced Ca2+ influx using fura-2AM. Results: Intact sperm and sperm extracts possessed proteasome activity, which was susceptible to inhibition by clasto-lactacystin β-lactone. Sperm-zona binding was not inhibited by clasto-lactacystin β-lactone. However, both zona pellucida- and progester-one-induced acrosome reactions were inhibited by clasto-lactacystin β-lactone. The proteasome inhibitor also blocked the sustained phase of the Ca2+ influx provoked by progesterone but not the peak. Conclusion: The human sperm proteasome is involved in the exocytosis of the acrosome, perhaps in events upstream of the plateau phase of the Ca2+ influx.
Bibliographical noteFunding Information:
This work was financed by Fondecyt 1011051.
- Calcium influx
- Human zona pellucida
- Sperm-zona binding