Synaptotagmin VI and VIII and syntaxin 2 are essential for the mouse sperm acrosome reaction.
The sperm acrosome is a large secretory granule that undergoes calcium-stimulated exocytosis by a mechanism analogous to neuronal secretion. In neurons the core SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex, composed of syntaxin (Stx), SNAP-25, and VAMP2, mediates vesicle fusion, whereas calcium regulation is thought to be accomplished by the synaptotagmin (Syt) family, some of which exhibit calcium-dependent binding to syntaxin and SNAP-25. Sperm express Syt VI and VIII and Stx2, which are co-localized to the acrosomal compartment where they might mediate exocytosis in response to calcium influx. Therefore, we examined the calcium dependence and isoform-specific interaction of Syt and Stx. We found that Stx2 binds to Syt I, VI, and VIII in a calcium-dependent manner with EC(50) values of 175, 233, and 96 mum calcium, respectively. We also determined that the EC(50) for calcium of the acrosome reaction in streptolysin O-permeabilized sperm is 87 mum, which closely coincides with the calcium sensitivity of Stx2 and Syt VIII interaction. Consistent with this is the greater potency of recombinant Syt VIII, VI, and Stx2 compared with other isoforms in inhibiting the acrosome reaction in streptolysin O-permeabilized sperm. Similarly, introduction of Syt VIII-specific antibodies was equally effective in inhibiting the acrosome fusion. Taken together, our data suggest a critical role for Syt VIII and Stx2 in membrane fusion and acrosome reaction in the sperm.
Hutt
Darren M
DM
Ottawa Health Research Institute, Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario K1Y 4E9, Canada.
Baltz
Jay M
JM
Ngsee
Johnny K
JK
D020101:Acrosome Reaction / Q000187:drug effects / Q000502:physiology*; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D000954:Antigens, Surface / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D002463:Cell Membrane Permeability; D003001:Cloning, Molecular; D005982:Glutathione Transferase / Q000235:genetics; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins / Q000494:pharmacology; D013094:Spermatozoa / Q000737:chemistry; D013301:Streptolysins; D050857:Synaptotagmins; D050827:Syntaxin 1
D000906:Antibodies; D000954:Antigens, Surface; D001426:Bacterial Proteins; D002135:Calcium-Binding Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013301:Streptolysins; D050827:Syntaxin 1; C494300:Syt6 protein, mouse; C494306:Syt8 protein, mouse; C025964:streptolysin O; D050857:Synaptotagmins; D005982:Glutathione Transferase; D002118:Calcium