SNARE complex assembly is required for human sperm acrosome reaction.
Exocytosis of the acrosome (the acrosome reaction) is a terminal morphological alteration that sperm must undergo prior to penetration of the extracellular coat of the egg. Ca(2+) is an essential mediator of this regulated secretory event. Aided by a streptolysin-O permeabilization protocol developed in our laboratory, we have previously demonstrated requirements for Rab3A, NSF, and synaptotagmin VI in the human sperm acrosome reaction. Interestingly, Rab3A elicits an exocytotic response of comparable magnitude to that of Ca(2+). Here, we report a direct role for the SNARE complex in the acrosome reaction. First, the presence of SNARE proteins is demonstrated by Western blot. Second, the Ca(2+)-triggered acrosome reaction is inhibited by botulinum neurotoxins BoNT/A, -E, -C, and -F. Third, antibody inhibition studies show a requirement for SNAP-25, SNAP-23, syntaxins 1A, 1B, 4, and 6, and VAMP 2. Fourth, addition of bacterially expressed SNAP-25 and SNAP-23 abolishes exocytosis. Acrosome reaction elicited by Rab3-GTP is also inhibited by BoNT/A, -C, and -F. Taken together, these results demonstrate a requirement for members of all SNARE protein families in the Ca(2+)- and Rab3A-triggered acrosome reaction. Furthermore, they indicate that the onset of sperm exocytosis relies on the functional assembly of SNARE complexes.
Tomes
Claudia N
CN
Laboratorio de Biología Celular y Molecular, Instituto de Histología y Embriología (IHEM-CONICET), Facultad de Ciencias Médicas, CC 56, Universidad Nacional de Cuyo, 5500 Mendoza, Argentina. ctomes@fmed2.uncu.edu.ar
Michaut
Marcela
M
De Blas
Gerardo
G
Visconti
Pablo
P
Matti
Ulf
U
Mayorga
Luis S
LS
D020101:Acrosome Reaction / Q000187:drug effects / Q000502:physiology*; D000328:Adult; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D015153:Blotting, Western; D001905:Botulinum Toxins / Q000494:pharmacology; D020013:Calcium Signaling; D002352:Carrier Proteins / Q000502:physiology; D005089:Exocytosis / Q000502:physiology; D006801:Humans; D046911:Macromolecular Substances; D008297:Male; D008565:Membrane Proteins / Q000502:physiology*; D005810:Multigene Family; D009419:Nerve Tissue Proteins / Q000502:physiology; D025941:Protein Interaction Mapping; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D050683:R-SNARE Proteins; D050600:SNARE Proteins; D013094:Spermatozoa / Q000502:physiology*; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins; D020692:rab3 GTP-Binding Proteins / Q000502:physiology
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D050683:R-SNARE Proteins; C100493:SNAP23 protein, human; C494208:SNAP25 protein, human; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins; D001905:Botulinum Toxins; D020692:rab3 GTP-Binding Proteins