Human sperm capacitation is necessary for SNARE assembly in neurotoxin-resistant complexes

Capacitation is not a well-defined process, required for the acrosome reaction triggered by physiological stimuli. In vitro, capacitation is achieved by sperm incubation in artificial media supplemented with HCO₃^- , Ca²⁺ , and albumin. The role of capacitation in the membrane fusion machinery required for acrosomal exocytosis is not well-known. SNARE proteins are fundamental for intracellular membrane fusion and acrosomal exocytosis. We have previously shown that in capacitated spermatozoa, the fusion machinery is maintained in an inactive state until the acrosome reaction is initiated. In particular, SNARE proteins are assembled in neurotoxin-resistant complexes.

This work aimed to study the dynamic changes of SNARE complexes during capacitation.

The light chain of tetanus and botulinum neurotoxin has been widely used to study the configuration of SNARE proteins. For this purpose, we developed a recombinant light chain of tetanus neurotoxin linked to a polyarginine peptide. This membrane-permeant protein was able to cleave cytosolic VAMP2 (a SNARE protein required for acrosome reaction) when present in a monomeric configuration.

The results show that the VAMP2 is cleaved by the membrane-permeant tetanus neurotoxin in non-capacitated spermatozoa, indicating that, before capacitation, SNAREs are not assembled in stable toxin-resistant complexes. However, 2 h of incubation in a capacitation medium containing albumin was sufficient to render VAMP2 insensitive to the toxin.

We conclude that during capacitation, the SNARE proteins become engaged in stable fully assembled cis-SNARE complexes. This step is likely essential to prevent untimely activation of the membrane fusion machinery.

We propose that capacitation promotes the stabilization of the membrane fusion machinery required for acrosomal exocytosis in preparation for the stimulus-triggered acrosome reaction.