Abstract
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.
Highlights
Introduction of Recombinant Proteins andAntisera into Streptolysin O-permeabilized Mouse Sperm—Sperm were collected from cauda epididymides of 12–15-week-old CD-1 mice and allowed to swim-up for 15 min in Krebs-Ringer buffer (KRB), pH 7.4, media containing 1.7 mM CaCl2 and 3 mg/ml BSA (37 °C, 5% CO2)
There is increasing evidence that the acrosome reaction (AR) in mammalian sperm is mediated by a SNARE complex acting in concert with the Ca2ϩ sensor, Syt [25,26,27, 57, 58, 64], similar to stimulated secretion in other secretory cells such as neurons
The AR is a singular, irreversible event, in contrast to the exocytosis and recycling that occurs in other systems
Summary
Introduction of Recombinant Proteins andAntisera into Streptolysin O-permeabilized Mouse Sperm—Sperm were collected from cauda epididymides of 12–15-week-old CD-1 mice and allowed to swim-up for 15 min in KRB, pH 7.4, media containing 1.7 mM CaCl2 and 3 mg/ml BSA (37 °C, 5% CO2). Sperm were diluted to a concentration of 5.0 ϫ 106 sperm/ml with KRB (ϪCa2ϩ) and permeabilized with 0.6 units/ml (Sigma) or 5 g/ml recombinant SLO for 5 min at 37 °C in 5% CO2 and subsequently supplemented with CaCl2 to a final concentration of 1 mM. Recombinant proteins or antibodies were added at the permeabilization step from a concentrated stock in KRB to the indicated final concentrations and maintained throughout the subsequent incubation. Assessment of Acrosomal Status—Fixed sperm were harvested by centrifugation at 800 ϫ g for 5 min and washed 2 times with 0.5 ml of 0.1 M ammonium acetate, pH 9.0. The sperm were subsequently resuspended in 0.1 M ammonium acetate, pH 9.0, and air-dried on glass slides. The slides were washed with water, methanol, and water for 5 min each. Sperm were imaged by bright-field microscopy using a Zeiss AxioPhot microscope and scored for acrosomal staining
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