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 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. 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. Upon stimulation by direct contact with the zona pellucida or progesterone signaling via an unidentified pathway, the sperm undergoes the regulated exocytosis of its single secretory granule, the acrosome. This acrosome reaction (AR) 1The abbreviations used are: AR, acrosome reaction; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; Stx, syntaxin; SNAP, soluble N-ethylmaleimide; VAMP, vesicle-associated membrane protein; Syt, synaptotagmin; SLO, streptolysin O; HA, hemagglutinin; BSA, bovine serum albumin; KRB, Krebs-Ringer buffer; GST, glutathione S-transferase; Abs, Abstrakt. 1The abbreviations used are: AR, acrosome reaction; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; Stx, syntaxin; SNAP, soluble N-ethylmaleimide; VAMP, vesicle-associated membrane protein; Syt, synaptotagmin; SLO, streptolysin O; HA, hemagglutinin; BSA, bovine serum albumin; KRB, Krebs-Ringer buffer; GST, glutathione S-transferase; Abs, Abstrakt. must occur before the sperm can penetrate the zona pellucida, since the acrosome contains hydrolytic enzymes necessary for degradation of the surrounding zona. In addition, the newly exposed membrane surface, the inner acrosomal membrane, contains secondary binding sites that mediate continued binding to the zona pellucida during penetration. Therefore, the AR is a prerequisite for sperm-egg plasma membrane binding and fusion (1Wassarman P.M. Jovine L. Litscher E.S. Nat. Cell Biol. 2001; 3: 59-64Crossref PubMed Scopus (339) Google Scholar). It has been established that a rise in intracellular calcium (Ca2+i) subsequent to zona pellucida or progesterone binding triggers the AR (2Green D.P. J. Cell Sci. 1978; 32: 165-176Crossref PubMed Google Scholar, 3Florman H.M. Dev. Biol. 1994; 165: 152-164Crossref PubMed Scopus (172) Google Scholar, 4Arnoult C. Cardullo R.A. Lemos J.R. Florman H.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13004-13009Crossref PubMed Scopus (204) Google Scholar, 5Bailey J.L. Storey B.T. Mol. Reprod. Dev. 1994; 39: 297-308Crossref PubMed Scopus (74) Google Scholar, 6Kobori H. Miyazaki S. Kuwabara Y. Biol. 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The other that was two binding sites with and the and which the form where is the of the to with EC50 and the are in this to with the EC50 The the by the and was by an of the The in binding was to a with a the as with a of of the the calcium dependence of the AR in the calcium in the to and the of AR was in sperm plasma membranes been with to of calcium the of fusion with the calcium in a in AR in sperm which to as calcium the of the data a response as and an EC50 of 87 μm The determined was which that the AR exhibit A of data was and a of data are consistent with a dependence of AR with a of as a of the a of the and the EC50 We a two binding sites for a to the as was a in the data This EC50 values of and μm for the and The EC50 is of the as that with the the was the In addition, a of the by the by an of the that the a Therefore, a the and and was used for of and in dependence of binding the isoforms of Syt and to be the protein in Syt VI and VIII and Stx2 K. T. K. H. Mol. Reprod. Dev. 2000; PubMed Scopus Google Scholar, M. R. M. Dev. Biol. 2001; PubMed Scopus Google Scholar, D.M. Cardullo R.A. Biol. Reprod. PubMed Scopus Google Scholar), was determined by an in binding the of recombinant or was to I, VI, or VIII to in a in the of The of that to Syt was by The data on a binding with a of the and of the and for the binding with in the EC50 values the and We the binding of Syt to as a since this interaction has been and to exhibit an EC50 in the of μm E.R. S. Jahn R. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, M. K. M. 1993; Full Text PDF PubMed Scopus Google Scholar). EC50 of μm for this is the determined In addition, we have that Syt binds to Stx2 and determined the EC50 for this interaction to be that for μm We found that Syt VIII to Stx2 and that this binding was with an EC50 of 96 Syt VIII binding to was also with a EC50 of in to a C. B. N. Sudhof T.C. Nature. 1995; PubMed Scopus Google Scholar), we found that Syt VIII in a with isoforms with for We also found that Syt VI to isoforms and that the binding was binding of with an EC50 for binding to Stx2 of μm and for of μm of the by VIII data the isoforms to be in the interaction Syt VIII and occur as to its EC50 to Syt to Syt VIII has a role in the AR, we we recombinant VIII and a VIII peptide D.M. Cardullo R.A. Biol. Reprod. PubMed Scopus Google Scholar) into sperm and the of AR stimulation with We found that sperm with VIII in a of AR to sperm or sperm with Similarly, of sperm with VIII in an of the AR to the or sperm We also a a this in of the acrosome reaction of the by a role of Syt in the AR, we of the of Syt into sperm. The of the of Syt VIII in a of the the of recombinant to and in and of the AR, The of an to the for SNARE of an protein a with role in membrane U. M. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar) The of the of Syt VI to the sperm also in a of the AR that to the of the of Syt on the AR or in a of the AR the The of Syt I, VI, and VIII was the protein was by for before its to the sperm suggest that Syt is involved in the regulation of the AR and of a role for Syt VIII in the of the acrosome reaction in the of and recombinant synaptotagmin and sperm with or recombinant Syt I, VI, or VIII or or and the of AR was with The values the of and are as a of of the with the role of in the regulation of the AR, we of the of or into sperm and examined to with the of sperm with Stx2 in of AR to sperm with of The of in of AR to sperm. the to and in a and of AR, to sperm with Abs, where was with the the of or the with of the recombinant protein suggest that and be of the fusion involved in the There is evidence that the AR in sperm is mediated by a SNARE complex in with the Syt M. P. U. Dev. Biol. PubMed Scopus Google Scholar, J. P. L. Dev. Biol. 2000; PubMed Scopus Google Scholar, K. T. K. H. Mol. Reprod. Dev. 2000; PubMed Scopus Google Scholar, J. Cell 2001; PubMed Scopus Google Scholar, M. M. M. Biochem. PubMed Scopus Google Scholar, Mol. Reprod. Dev. 2000; PubMed Scopus Google Scholar), to in other secretory cells as The AR is a in to the exocytosis and that in other the secretory exhibit that in other cells to and the SNARE and Syt isoforms used be for this A number of isoforms of the and VAMP as as isoforms of Syt have been to be the protein in sperm M. P. U. Dev. Biol. PubMed Scopus Google Scholar, J. P. L. Dev. Biol. 2000; PubMed Scopus Google Scholar, M. R. M. Dev. Biol. 2001; PubMed Scopus Google Scholar, D.M. Cardullo R.A. Biol. Reprod. PubMed Scopus Google Scholar). in sperm are In addition, as of with the Syt isoforms to be in sperm to be C. B. N. Sudhof T.C. Nature. 1995; PubMed Scopus Google Scholar), whereas the AR is by was acrosomal exocytosis was regulated of the sensitivity of the AR, we that triggers the AR in the sperm with an EC50 of 87 μm, that the calcium involved in the regulation of the sperm AR is calcium or this This is with the EC50 of μm by the calcium dependence of fusion sperm plasma membrane and outer acrosomal membrane B. H. J. Cell Sci. 1995; PubMed Google Scholar), into the in that in of in sperm the values for of μm before AR, which to μm the AR was C. A. R.A. Florman H.M. Mol. Biol. 2000; PubMed Scopus Google Scholar). This of of be to the to the of fusion. has been in the in secretory cells the of fusion is of the in the to the of the and the plasma that mediate N. N. C. K. T. S. M. M. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). the to AR in sperm or might a of sensitivity of the We in other secretory or Syt isoforms as the in the sperm AR and the in to the SNARE core complex via binding to Therefore, we have the for exocytosis in Syt VI, Syt VIII, and Stx2, exhibit interaction. In a C. B. N. Sudhof T.C. Nature. 1995; PubMed Scopus Google Scholar), the of Syt VI was to to was In addition, the sensitivity of binding was on the of the the of each and the and Syt as in an in we that Syt VI and Syt VIII binding with and The for binding stimulation by was Syt Syt VIII Syt VI, that binding a for Syt and VIII for Syt Syt binding to the to be in Stx2, was the was Syt VIII Syt Syt VI, as Syt binding occur this to the in the of AR be Syt VIII, with Syt VI a secondary The stimulation of AR by in sperm with an EC50 that closely that which binding Syt VIII and This close the that isoforms have a role in the sperm data must be with the of data with sperm in sperm and the that binding of Syt and isoforms with the of the dependence of AR and Syt binding the we used is of a role in the We have to this interaction in by of Syt VIII or Stx2 sperm the VIII, the Syt VIII the Stx2 antibodies was effective in the role of inhibiting the of the sperm are and and as of and be we and have the SLO, to the plasma membrane of the sperm and to the M. P. U. Dev. Biol. PubMed Scopus Google Scholar, R. M. C. Biol. Reprod. 2000; PubMed Scopus Google Scholar, M. R. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). The we used in the of in the plasma membrane that are large to for the introduction of that the of proteins of which the acrosome. We two a Syt peptide that we have with other Syt isoforms D.M. Cardullo R.A. Biol. Reprod. PubMed Scopus Google Scholar) and a the of Syt The with other Syt we have its The of VIII peptide or Syt VIII to sperm in an and of the AR, to sperm or sperm with the of This by the that is Syt that Syt VIII is for the AR in sperm. We a role for Syt VI, as to We a of the AR in sperm by the of recombinant Syt VI or VIII recombinant Syt proteins a of whereas the of Syt the was to that Syt VI and Syt VIII be the isoforms to to the sperm SNARE complex. The of data is that the of AR by recombinant of Syt I, VI, or VIII is to as Syt binding to target sperm The was as the of of an Abs, on AR, and the with recombinant of Syt I, VI, and VIII was the proteins by In to our for Syt VIII the of a to sperm in of the is that Stx2 is critical for the an be that Stx2 is into the SNARE core complex with SNAP-25 and VAMP be to the binding to its which of that are involved in binding to other core SNARE proteins or Syt, occur with Stx2 In the of recombinant of Stx2 in of AR to sperm with a with its the of that we The of recombinant also in of AR to and a of to sperm with a the Stx2 was a of the AR was with the of recombinant Syt, the was as or The of data is that the interaction of the Stx2 with the Syt or other SNARE proteins in sperm is by the of of of the to the SNARE complex. The potency of Stx2 and its in sperm that is a for the in sperm. In we have established that two Syt Syt VI and Syt VIII, can exhibit binding to isoforms can mediate and our data suggest that Syt VIII has a role in the AR and that Syt VI also in the role of Syt The that binds to or of is to be Stx2 the We that this of SNARE proteins and for the of the sperm

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