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

MAYORGA, LUIS; ALTAMIRANO, KARINA; ZANNI RUIZ, EMILIA; PAVAROTTI, MARTIN

JOURNAL OF ANDROLOGY

AMER SOC ANDROLOGY, INC

Año: 2019

0196-3635

Background: Capacitation is not a well-defined process, required for the acrosome reaction triggered by physiological stimuli. Invitro, capacitation is achieved by sperm incubation in artificial media supplemented with HCO3, Ca2+, and albumin. The role ofcapacitation in the membrane fusion machinery required for acrosomal exocytosis is not well-known. SNARE proteins are fundamentalfor intracellular membrane fusion and acrosomal exocytosis. We have previously shown that in capacitated spermatozoa, thefusion machinery is maintained in an inactive state until the acrosome reaction is initiated. In particular, SNARE proteins are assembledin neurotoxin-resistant complexes.Objective: This work aimed to study the dynamic changes of SNARE complexes during capacitation.Materials and Methods: The light chain of tetanus and botulinum neurotoxin has been widely used to study the configuration ofSNARE 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 presentin a monomeric configuration.Results: 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 incubationin a capacitation medium containing albumin was sufficient to render VAMP2 insensitive to the toxin.Discussion: 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.Conclusion: We propose that capacitation promotes the stabilization of the membrane fusion machinery required for acrosomalexocytosis in preparation for the stimulus-triggered acrosome reaction.