Rab3A Is Required before, and SNAREs and Synaptotagmin VI after, Intra-Acrosomal Ca²⁺ Efflux (original) (raw)

2013

Abstract

<p>Permeabilized spermatozoa were loaded with 10 μM NP-EGTA-AM (NP) for 15 min at 37 °C to chelate intra-acrosomal Ca²⁺. AE was then initiated by adding 0.5 mM CaCl₂ (10 μM free Ca²⁺)(Ca²⁺). After further 15 min incubation at 37 °C to allow exocytosis to proceed up to the intra-acrosomal Ca²⁺-sensitive step, sperm were treated for 15 min at 37 °C with antibodies that recognize Rab3A (20 μg/ml, anti-Rab3A), SNAP25 (20 μg/ml, anti-SNAP25), syntaxin1A (1/25 dilution, anti-Stx1A), VAMP2 (20 μg/ml, anti-VAMP2), or synaptotagmin VI (30 μg/ml, anti-StgVI). All these procedures were carried out in the dark. UV flash photolysis of the chelator was induced at the end of the incubation period (hν), and the samples were incubated for 5 min to promote exocytosis (NP→Ca²⁺→antibody→hν, black bars; a diagram of the experiment is shown at the top of the figure). Sperm were then fixed and AE was measured as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030323#s4" target="_blank">Materials and Methods</a>. Several controls were included (grey bars): background AE in the absence of any stimulation (control); AE stimulated by 10 μM free Ca²⁺ (Ca²⁺), inhibitory effect of NP-EGTA-AM in the dark (NP→Ca²⁺→dark), and the recovery upon illumination (NP→Ca²⁺→hν); and inhibitory effect of the antibodies when present throughout the experiment (NP→antibody→Ca²⁺→hν). The data were normalized as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030323#s4" target="_blank">Materials and Methods</a> (mean ± SEM). Statistical analysis is provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030323#st002" target="_blank">Table S2</a>.</p

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