Participation of the sperm proteasome in human fertilization.
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 beta-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 Ca(2+) influx using fura-2AM.


RESULTS
Intact sperm and sperm extracts possessed proteasome activity, which was susceptible to inhibition by clasto-lactacystin beta-lactone. Sperm-zona binding was not inhibited by clasto-lactacystin beta-lactone. However, both zona pellucida- and progesterone-induced acrosome reactions were inhibited by clasto-lactacystin beta-lactone. The proteasome inhibitor also blocked the sustained phase of the Ca(2+) influx provoked by progesterone but not the peak.


CONCLUSIONS
The human sperm proteasome is involved in the exocytosis of the acrosome, perhaps in events upstream of the plateau phase of the Ca(2+) influx.
Morales
Patricio
P
Department of Biomedicine, Faculty of Health Sciences, University of Antofagasta, Avda, Angamos 601, P.O. Box 170, Antofagasta, Chile. pmorales@uantof.cl
Kong
Milene
M
Pizarro
Eduardo
E
Pasten
Consuelo
C
D020101:Acrosome Reaction / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000032:analysis / Q000502:physiology*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D005260:Female; D005306:Fertilization / Q000502:physiology*; D006801:Humans; D007783:Lactones / Q000494:pharmacology; D008297:Male; D009097:Multienzyme Complexes / Q000032:analysis / Q000037:antagonists & inhibitors / Q000502:physiology*; D011374:Progesterone / Q000502:physiology; D046988:Proteasome Endopeptidase Complex; D013084:Sperm-Ovum Interactions / Q000187:drug effects; D013094:Spermatozoa / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D014020:Tissue Extracts / Q000737:chemistry; D015044:Zona Pellucida / Q000502:physiology
D015853:Cysteine Proteinase Inhibitors; D007783:Lactones; D009097:Multienzyme Complexes; D014020:Tissue Extracts; C098869:clasto-lactacystin beta-lactone; D011374:Progesterone; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D002118:Calcium