Regulation of SNARE function during sperm capacitation to promote the acrosome reaction

Capacitation;Acrosome Reaction;Infertility;Fertilization;Membrane fusion;Phosphorylation;soluble N-ethylmaleimide-sensitive factor attachment protein receptor;Lipid rafts;SNARE Proteins

Shah, Syed Tahir Abbas

关键词: Capacitation;    Acrosome Reaction;    Infertility;    Fertilization;    Membrane fusion;    Phosphorylation;    soluble N-ethylmaleimide-sensitive factor attachment protein receptor;    Lipid rafts;    SNARE Proteins;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/45667/Syed%20Tahir%20Abbas_Shah.pdf?sequence=1&isAllowed=y

美国|英语

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

【 摘 要 】

Infertility is a tragedy for millions of couples worldwide. According to the World Health Organization, the male factor alone accounts for over one third of total infertility. There are various causes of male infertility but defective acrosomal exocytosis is perhaps one of the biggest contributors towards male infertility. Unfortunately there is little information available on the most frequent molecular causes of the inability of sperm to undergo acrosomal exocytosis. In the current study, my goal was to develop a clearer understanding of how the acrosome reaction is regulated during capacitation so that the molecular defects that cause infertility can be identified. I hypothesized that capacitation alters sperm protein phosphorylation promoting soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation and movement into membrane rafts in preparation for the acrosome reaction. Two t-SNAREs and a v-SNARE form a trans-SNARE complex, which is required for exocytosis. The core complex along with other regulatory proteins exerts an inward force by zippering from the N-termini. Ultimately hundreds of fusion pores are formed and hybrid vesicles are released.To test this hypothesis, I incubated mouse sperm in a capacitating (dmKRBT) or non-capacitating (dmKRBT without BSA or without HCO3-) medium and detergent-extracted sperm protein. To assess phosphorylation of proteins in SNARE complexes, syntaxin and associated proteins were immunoprecipitated with a syntaxin antibody and subsequently separated by SDS-PAGE. Samples were not boiled prior to SDS-PAGE to maintain the integrity of the SNARE complex. To detect changes in total phosphorylation of syntaxin-containing (SNARE) complexes, immunoprecipitated complexes were separated by SDS-PAGE and stained with Pro-Q Diamond. I observed phosphoprotein staining in 75, 100, and 150 and 230 KD protein complexes. Immunoblotting with a syntaxin antibody demonstrated that each complex contained syntaxin and was presumably a SNARE complex. After 30 min of capacitation, overall phosphorylation of SNARE complexes was higher in the 75, 100 and 150 KD complexes. To detect tyrosine phosphorylation of syntaxin and associated proteins, the anti-syntaxin immunoprecipitates were blotted with a phosphotyrosine antibody. Tyrosine phosphorylation of SNARE complexes decreased after 15 min of capacitation time. These data demonstrate SNARE complex phosphorylation is a dynamic process during capacitation and suggest that phosphorylation may regulate SNARE complex formation during capacitation in preparation for the acrosome reaction. Movement of SNAREs into membrane rafts was assessed using a detergent-free sucrose density gradient centrifugation method followed by immunoblotting with syntaxin and synaptobrevin antibodies. I observed movement of syntaxin and synaptobrevin into rafts in sperm incubated with capacitating dmKRBT and was delayed in dmKRBT lacking bicarbonate. No movement of syntaxin and synaptobrevin in sperm incubated with non-capacitating dmKRBT or dmKRBT lacking BSA was observed.Taken together, these data show that during capacitation, SNARE complex serine-threonine phosphorylation decreases whereas tyrosine phosphorylation increases. Syntaxin and synaptobrevin moved from non-raft fractions into rafts, and this movement was dependent on BSA and delayed in HCO3- free medium. These results suggest that capacitation shifts SNAREs into rafts, allowing trans-SNARE complex formation, which may ultimately promote membrane fusion at those sites. Results of these fundamental studies may be helpful to develop more accurate laboratory fertility assessments, to reduce fertility, when contraception is desired, and to diagnose infertility and develop corrective therapies, when conception is needed.