title: Rab3A, a possible marker of cortical granules, participates in cortical granule exocytosis in mouse eggs.
abstract: Fusion of cortical granules with the oocyte plasma membrane is the most significant event to prevent polyspermy. This particular exocytosis, also known as cortical reaction, is regulated by calcium and its molecular mechanism is still not known. Rab3A, a member of the small GTP-binding protein superfamily, has been implicated in calcium-dependent exocytosis and is not yet clear whether Rab3A participates in cortical granules exocytosis. Here, we examine the involvement of Rab3A in the physiology of cortical granules, particularly, in their distribution during oocyte maturation and activation, and their participation in membrane fusion during cortical granule exocytosis. Immunofluorescence and Western blot analysis showed that Rab3A and cortical granules have a similar migration pattern during oocyte maturation, and that Rab3A is no longer detected after cortical granule exocytosis. These results suggested that Rab3A might be a marker of cortical granules. Overexpression of EGFP-Rab3A colocalized with cortical granules with a Pearson correlation coefficient of +0.967, indicating that Rab3A and cortical granules have almost a perfect colocalization in the egg cortical region. Using a functional assay, we demonstrated that microinjection of recombinant, prenylated and active GST-Rab3A triggered cortical granule exocytosis, indicating that Rab3A has an active role in this secretory pathway. To confirm this active role, we inhibited the function of endogenous Rab3A by microinjecting a polyclonal antibody raised against Rab3A prior to parthenogenetic activation. Our results showed that Rab3A antibody microinjection abolished cortical granule exocytosis in parthenogenetically activated oocytes. Altogether, our findings confirm that Rab3A might function as a marker of cortical granules and participates in cortical granule exocytosis in mouse eggs.
authors.affiliation: Instituto de Histología y Embriología, CONICET - Universidad Nacional de Cuyo, Av. Libertador 80, 5500 Mendoza, Argentina.
authors.forename: Oscar Daniel
authors.initials: OD
authors.lastname: Bello
authors.affiliation: Instituto de Histología y Embriología, CONICET - Universidad Nacional de Cuyo, Av. Libertador 80, 5500 Mendoza, Argentina.
authors.forename: Andrea Isabel
authors.initials: AI
authors.lastname: Cappa
authors.affiliation: Instituto de Histología y Embriología, CONICET - Universidad Nacional de Cuyo, Av. Libertador 80, 5500 Mendoza, Argentina.
authors.forename: Matilde
authors.initials: M
authors.lastname: de Paola
authors.affiliation: Instituto de Histología y Embriología, CONICET - Universidad Nacional de Cuyo, Av. Libertador 80, 5500 Mendoza, Argentina.
authors.forename: María Natalia
authors.initials: MN
authors.lastname: Zanetti
authors.affiliation: Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan.
authors.forename: Mitsunori
authors.initials: M
authors.lastname: Fukuda
authors.affiliation: Department of Veterinary and Animal Sciences, University of Massachusetts Amherst, 661 North Pleasant Street, Amherst, MA 01003, USA.
authors.forename: Rafael A
authors.initials: RA
authors.lastname: Fissore
authors.affiliation: Instituto de Histología y Embriología, CONICET - Universidad Nacional de Cuyo, Av. Libertador 80, 5500 Mendoza, Argentina.
authors.forename: Luis S
authors.initials: LS
authors.lastname: Mayorga
authors.affiliation: Instituto de Histología y Embriología, CONICET - Universidad Nacional de Cuyo, Av. Libertador 80, 5500 Mendoza, Argentina; Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Cuyo, Argentina. Electronic address: mmichaut@gmail.com.
authors.forename: Marcela A
authors.initials: MA
authors.lastname: Michaut
mesh_terms: D000818:Animals; D003594:Cytoplasmic Granules / Q000378:metabolism*; D005089:Exocytosis; D005260:Female; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006736:Horses; D006801:Humans; D008677:Metaphase; D051379:Mice; D008845:Microinjections; D009865:Oocytes / Q000166:cytology* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020826:rab3A GTP-Binding Protein / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D020826:rab3A GTP-Binding Protein
keywords: Cortical granule exocytosis; Cortical reaction; Mouse egg; Mouse oocyte; Rab3A