Cortical Granule Exocytosis is Mediated by alpha-SNAP and N-Ethilmaleimide Sensitive Factor in Mouse Eggs

DE PAOLA, M. MATILDE; BELLO, OSCAR D.; MICHAUT, MARCELA A.

PLOS ONE

PUBLIC LIBRARY SCIENCE

Lugar: San Francisco; Año: 2015

1932-6203

Cortical granule exocytosis (CGE), also known as cortical reaction, is a calcium-regulated secretion that represents a membrane fusion process during meiotic celldivision of eggs. The molecular mechanism of membrane fusion during CGE is stillpoorly understood and is thought to be mediated by the SNARE pathway;nevertheless, it is unkown if SNAP (acronym for soluble NSF attachment protein) andNSF (acronym for N-ethilmaleimide sensitive factor), two key proteins in the SNAREpathway, mediate CGE in any egg model. In this paper, we documented the geneexpression of α-SNAP, γ-SNAP and NSF in mouse oocytes. Western blot analysisshowed that the expression of these proteins maintains a similar level during oocytematuration and egg activation. Their localization was mainly observed at the corticalregion of eggs, which is enriched in cortical granules. To evaluate the function of theseproteins in CGE we set up a functional assay based on the quantification of corticalgranules. Endogenous α-SNAP and NSF proteins were perturbed by microinjection ofrecombinant proteins or antibodies prior to CGE activation. The microinjection of wildtype α-SNAP and the negative mutant of α-SNAP L294A in eggs inhibited CGEstimulated by strontium. NEM, an irreversibly inhibitor of NSF, and the microinjection ofthe negative mutant NSF D1EQ inhibited cortical reaction. The microinjection of anti-α-SNAP and anti-NSF antibodies was able to abolish CGE in activated eggs. Themicroinjection of anti-γ SNAP antibody had no effect on CGE. Our findings indicate, forthe first time in any oocyte model, that α-SNAP, γ-SNAP, and NSF are expressed inmouse oocytes. We demonstrate that α-SNAP and NSF have an active role in CGEand propose a working model.