Re-localization of SNARE proteins in mouse sperm prior to the acrosome reaction

Sperm;Soluble NSF Attachment Protein Receptor (SNARE) proteins;Capacitation;Acrosome Reaction

Sharif, Momal

关键词: Sperm;    Soluble NSF Attachment Protein Receptor (SNARE) proteins;    Capacitation;    Acrosome Reaction;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/78670/SHARIF-THESIS-2015.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

The principal role of SNARE proteins is to arbitrate vesicle fusion to a target membrane. Formation of tripartite SNARE protein complexes between SNARE proteins on opposing membranes is the minimal requirement for membrane fusion. The SNARE protein family is large, consisting of more than 60 members. A member of the SNARE family, syntaxin, is found on the sperm plasma membrane while synaptobrevin, is found on the outer acrosomal membrane.During the sperm acrosome reaction, the outer acrosomal membrane fuses at hundreds of points with the overlying plasma membrane, resulting in release of the acrosomal contents. We hypothesize that syntaxin and synaptobrevin re-localize within the sperm plasma membrane prior to the acrosome reaction to form SNARE complexes and promote membrane fusion at hundreds of specific points.Immunofluorescence was used to localize both syntaxin and synaptobrevin in mouse epididymal sperm before and after capacitation. Sperm were fixed and incubated with antibodies to syntaxin, synaptobrevin and then fluorescent secondary antibodies. Super resolution Structured Illumination Microscopy (SR-SIM) was used to examine samples collected at 0, 10, 30, 60, and 120 min of capacitation time, to obtain 3D images of SNARE localization. Quantification of the images was completed using western blotting and by image analysis, using IMARIS, which interpreted the total syntaxin and synaptobrevin positive volume. Results showed that syntaxin-positive volume and syntaxin content remained the same in capacitated and non-capacitated sperm but the location of syntaxin after capacitation was more restricted to the apical ridge of the plasma membrane overlying the acrosome in more than 90% of the sperm observed. The effect of bicarbonate (HCO3-) and BSA, agents necessary in the medium for capacitation, was also investigated. Bicarbonate (HCO3-), which activates soluble adenylate cyclase, was not necessary for re-localization of syntaxin. On the other hand, BSA, which promotes cholesterol efflux, was required for syntaxin re-localization. In the case of synaptobrevin, the volume of protein remained the same in both capacitated and non-capacitated sperm. Synaptobrevin was found at the apical ridge of the sperm head prior to and following capacitation in approximately 83% of the sperm observed. Our results demonstrate that, unlike syntaxin, synaptobrevin does not shift during the plasma membrane modifications that occur in sperm during capacitation. Syntaxin and synaptobrevin did not co-localize at any time during capacitating and non-capacitating conditions in sperm. Our results will help in identification of pathways that may regulate SNARE localization and function during capacitation, membrane fusion and the acrosome reaction.