Abstract
Cortical granule exocytosis (CGE), also known as cortical reaction, is a calcium- regulated secretion that represents a membrane fusion process during meiotic cell division of oocytes. The molecular mechanism of membrane fusion during CGE is still poorly understood and is thought to be mediated by the SNARE pathway; nevertheless, it is unkown if SNAP (acronym for soluble NSF attachment protein) and NSF (acronym for N-ethilmaleimide sensitive factor), two key proteins in the SNARE pathway, mediate CGE in any oocyte model. In this paper, we documented the gene expression of α-SNAP, γ-SNAP and NSF in mouse oocytes. Western blot analysis showed that the expression of these proteins maintains a similar level during oocyte maturation and early activation. Their localization was mainly observed at the cortical region of metaphase II oocytes, which is enriched in cortical granules. To evaluate the function of these proteins in CGE we set up a functional assay based on the quantification of cortical granules metaphase II oocytes activated parthenogenetically with strontium. Endogenous α-SNAP and NSF proteins were perturbed by microinjection of recombinant proteins or antibodies prior to CGE activation. The microinjection of wild type α-SNAP and the negative mutant of α-SNAP L294A in metaphase II oocytes inhibited CGE stimulated by strontium. NEM, an irreversibly inhibitor of NSF, and the microinjection of the negative mutant NSF D1EQ inhibited cortical reaction. The microinjection of anti-α-SNAP and anti-NSF antibodies was able to abolish CGE in activated metaphase II oocytes. The microinjection of anti-γ SNAP antibody had no effect on CGE. Our findings indicate, for the first time in any oocyte model, that α-SNAP, γ-SNAP, and NSF are expressed in mouse oocytes. We demonstrate that α-SNAP and NSF have an active role in CGE and propose a working model.
Highlights
Mammalian fertilization is a process of fusion between a spermatozoon and an oocyte to create a zygote
We showed that that α-SNAP, γ-SNAP, and NSF are expressed in mouse oocytes, and that α-SNAP and NSF are required for cortical granule exocytosis
To determine which of these proteins is expressed in mouse oocytes, the presence of mRNA for the three SNAP isoforms andNSF was assessed by room temperature (RT)-PCR
Summary
Mammalian fertilization is a process of fusion between a spermatozoon and an oocyte to create a zygote. To guarantee the success of fertilization and embryo development a definitive block to polyspermy is necessary since polyspermy is embryonic lethal. The first two occur rapidly and their molecular basis remains largely unknown, and the third, slow and definitive, correlates with the exocytosis of cortical granules in Metaphase II (MII) oocytes [1]. Cortical granules exocytosis in mouse oocytes, known as cortical reaction, is a calcium regulated exocytosis. The cortical reaction differs from other exocytotic events in that cortical granules release occurs only once after oocytesfertilization and they are not renewed. The membrane fusion during this particular secretory process is thought to be mediated by SNAREs. The membrane fusion during this particular secretory process is thought to be mediated by SNAREs It is unknown if two essential proteins of membrane fusion, SNAP (acronym for soluble NSF attachment protein) and NSF (acronym for N-ethilmaleimide sensitive factor), are involved in the molecular mechanism of membrane fusion during cortical granule exoytosis (CGE)
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