Vesicle-Associated Membrane Protein 1 and 3 participatein cortical granule exocytosis in metaphase II oocytes

MICHAUT, MARCELA A.; GARRIDO, FACUNDO M; DE PAOLA, M. MATILDE

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias. LIII Reunión anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2017

Sociedades de Biociencias

Following fertilization, cortical granules (CG) undergo exocytosis (CGE) to release their content into the perivitelline space, avoiding polyspermy and ensuring normal embryonic development. CGE, also known as cortical reaction, is a calcium-regulated secretion that represents a membrane fusion process during meiotic cell division of oocytes. Several studies suggest that CGE is a SNARE protein-mediated pathway; however, it is still unknown if VAMP (acronym for vesicle-associated membrane protein) is present and mediates CGE in mouse oocyte. We hypothesized that oocyte uses the same conserved membrane fusion machinery as neurons and human sperm, and that VAMP, one of the SNARE proteins required for membrane fusion, is present in mouse oocyte. Our aim was to identify and characterize the main VAMP isoforms related to regulated exocytosis in other secretory models: VAMP1, VAMP2 and VAMP3. We first investigated the expression of VAMP isoforms by RT-PCR. The results revealed that VAMP1, 2 and 3 are expressed in mouse oocyte. Western blot analysis indicated that VAMP1 and VAMP3 (but not VAMP 2) were present in mature mouse oocytes. Indirect immunofluorescence experiments revealed that VAMP1 and VAMP3 are predominantly observed in the CG-enriched cortical region during egg maturation. To evaluate the function of these proteins in CGE, endogenous VAMPs were perturbed by microinjection of antibodies prior to CGE activation. The microinjection of specific antibodies against either VAMP1 or VAMP3 in metaphase II oocytes inhibited CGE stimulated by SrCl2. Nevertheless, the microinjection of anti-VAMP2 antibody had no effect on CGE. Furthermore, recombinant tetanus toxin light chain (which cleaves VAMP) microinjection experiments showed that the microinjection of tetanus toxin was able to abolish CGE in activated metaphase II oocytes and that SNARE complex is in cis configuration. Altogether, our findings indicate that VAMP1 and VAMP3 have an active role in CGE process.