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J. Biol. Chem., Vol. 280, Issue 21, 20197-20203, May 27, 2005

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Synaptotagmin VI and VIII and Syntaxin 2 Are Essential for the Mouse Sperm Acrosome Reaction^*

Darren M. Hutt, Jay M. Baltz¶, and Johnny K. Ngsee||

From the Ottawa Health Research Institute, Department of Cellular and Molecular Medicine and ^¶Department of Obstetrics and Gynecology, Division of Reproductive Medicine, University of Ottawa, Ottawa, Ontario K1Y 4E9, Canada

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 EC₅₀ values of 175, 233, and 96 µM calcium, respectively. We also determined that the EC₅₀ for calcium of the acrosome reaction in streptolysin O-permeabilized sperm is 87 µM, 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.

Received for publication, November 15, 2004 , and in revised form, March 2, 2005.

^* This work was supported by a grant from the Natural Sciences and Engineering Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of an Ontario graduate scholarship.

^|| To whom correspondence should be addressed: Ottawa Health Research Institute, Dept. of Cellular and Molecular Medicine, University of Ottawa, 725 Parkdale Ave., Ottawa, ON K1Y 4E9, Canada. Tel.: 613-798-5555 (ext. 17079); Fax: 613-761-5365; E-mail: jngsee{at}ohri.ca.

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