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

BACKGROUND: 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. 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 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. 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 incubation in 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 acrosomal exocytosis in preparation for the stimulus-triggered acrosome reaction.

Kinetics of human sperm acrosomal exocytosis.

The acrosome reaction is a unique event in the lifespan of sperm characterized by the exocytosis of the acrosomal content and the release of hybrid vesicles formed by patches of the outer acrosomal membrane and the plasma membrane. This unique regulated exocytosis is mediated by essentially the same membrane fusion machinery present in neuroendocrine cells. However, whereas secretion in neuroendocrine cells occurs in less than a second, the acrosome reaction is normally assessed after several minutes of incubation with inducers. In this report, we measured the kinetics of human sperm exocytosis triggered by two stimuli (calcium ionophore and progesterone) by using electron microscopy and three different approaches based on the incorporation of fluorescent Pisum sativum agglutinin into the acrosome upon opening of fusion pores connecting the extracellular medium with the acrosomal lumen. The results with the different methods are consistent with a slow kinetics (t½ = 14 min). We also manipulated the system to measure different steps of the process. We observed that cytosolic calcium increased with a relatively fast kinetics (t½ = 0.1 min). In contrast, the swelling of the acrosomal granule that precedes exocytosis was a slow process (t½ = 13 min). When swelling was completed, the fusion pore opening was fast (t½ = 0.2 min). The results indicate that acrosomal swelling is the slowest step and it determines the kinetics of the acrosome reaction. After the swelling is completed, the efflux of calcium from intracellular stores triggers fusion pores opening and the release of hybrid vesicles in seconds.