VESICLE-ASSOCIATED MEMBRANE PROTEIN 1 AND 3 PARTICIPATE IN CORTICAL GRANULE EXOCYTOSIS IN METAPHASE II OOCYTES

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

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017

Sociedad Argentica de Investigación Bioquímica y Biología Molecular

Followingfertilization, cortical granules (CG) undergo exocytosis (CGE) to release theircontent into the perivitelline space, avoiding polyspermy and ensuring normalembryonic development. CGE, also known as cortical reaction, is acalcium-regulated secretion that represents a membrane fusion process duringmeiotic cell division of oocytes. Several studies suggest that CGE is a SNAREprotein-mediated pathway; however, it is still unknown if VAMP (acronym forvesicle-associated membrane protein) is present and mediates CGE in mouseoocyte. We hypothesized that oocyte uses the same conserved membrane fusionmachinery as neurons and human sperm, and that VAMP, one of the SNAREproteins required for membrane fusion, is present in mouse oocyte. Our aim wasto identify and characterize the main VAMP isoforms related to regulatedexocytosis in other secretory models: VAMP1, VAMP2 and VAMP3. We firstinvestigated the expression of VAMP isoforms by RT-PCR. The results revealedthat VAMP1, 2 and 3 are expressed in mouse oocyte. Western blot analysisindicated that VAMP1 and VAMP3 (but not VAMP 2) were present in mature mouseoocytes. Indirect immunofluorescence experiments revealed that VAMP1 and VAMP3are predominantly observed in the CG-enriched cortical region during eggmaturation. To evaluate the function of these proteins in CGE, endogenous VAMPswere perturbed by microinjection of antibodies prior to CGE activation. Themicroinjection of specific antibodies against either VAMP1 or VAMP3 inmetaphase II oocytes inhibited CGE stimulated by SrCl2. Nevertheless, themicroinjection of anti-VAMP2 antibody had no effect on CGE. Furthermore, recombinanttetanus toxin light chain (which cleaves VAMP) microinjection experimentsshowed that the microinjection of tetanus toxin was able to abolish CGE inactivated metaphase II oocytes and that SNARE complex is in cis configuration.Altogether, our findings indicate that VAMP1 and VAMP3 have an active role inCGE process.