Rab3A, a possible marker of cortical granules, participates in cortical granule exocytosis in mouse eggs

Journal Article · Sat Sep 10 04:00:00 EDT 2016 · Experimental Cell Research

DOI:https://doi.org/10.1016/J.YEXCR.2016.07.005· OSTI ID:22649753

Bello, Oscar Daniel; Cappa, Andrea Isabel; Paola, Matilde de; Zanetti, María Natalia ^[1]; Fukuda, Mitsunori ^[2]; Fissore, Rafael A. ^[3]; Mayorga, Luis S. ^[1]

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1. Instituto de Histología y Embriología, CONICET – Universidad Nacional de Cuyo, Av. Libertador 80, 5500 Mendoza (Argentina)
2. Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi 980-8578 (Japan)
3. Department of Veterinary and Animal Sciences, University of Massachusetts Amherst, 661 North Pleasant Street, Amherst, MA 01003 (United States)

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. - Highlights: • Rab3A has a similar migration pattern to cortical granules in mouse oocytes. • Rab3A can be a marker of cortical granules. • Active Rab3A triggered cortical granule exocytosis. • Blocking endogenous Rab3A inhibits cortical granule exocytosis. • Rab3A participates in cortical reaction in mouse oocytes.

• OSTI ID: 22649753
• Journal Information: Experimental Cell Research, Journal Name: Experimental Cell Research Journal Issue: 1 Vol. 347; ISSN 0014-4827; ISSN ECREAL
• Country of Publication: United States
• Language: English

Related Subjects

60 APPLIED LIFE SCIENCES
ANTIBODIES
BLOOD PLASMA
CALCIUM
CORRELATIONS
EGGS
FUNCTIONS
MATURATION
MEMBRANES
MICE
OOCYTES
PHYSIOLOGY
PROTEINS