Dynamics of cortical granule exocytosis at fertilization in living
mouse eggs.
Tahara, Masahiro, Keiichi Tasaka, Nobuyuki Masumoto, Akiko Mammoto,
Yoshihide Ikebuchi, and Akira Miyake.
Department of Obstetrics and Gynecology, Osaka University Medical
School, 2-2, Yamadaoka, Suita, Osaka 565, Japan
APStracts 2:0409C, 1995.
Sperm-egg fusion induces an intracellular free calcium concentration
([Ca2+]i) increase and exocytosis of cortical granules (CGs).
Recently, we developed a method to evaluate the kinetics of
exocytosis in single living cells, using an impermeable fluorescent
membrane probe, TMA-DPH. In this study, we evaluated cortical granule
(CG) exocytosis in living mouse eggs with TMA-DPH using digital
imaging and confocal laser scanning microscopy. Time-related changes
of CG exocytosis were estimated as the % increase of TMA-DPH
fluorescence. The increase of fluorescence in the egg started after
sperm attachment, continued at an almost uniform rate and ceased at
45-60 min. While the [Ca2+]i increase at fertilization was transient
or oscillatory, exocytosis was not always induced concomitantly with
each [Ca2+]i peak. Next, we determined some intracellular mediators
of exocytosis in the egg using this method. An intracellular calcium
chelator, BAPTA-AM, and a microfilament inhibitor, cytochalasin B,
blocked sperm-induced exocytosis. A GTP-binding protein activator,
AlF4-, induced exocytosis. These results suggest that [Ca2+]i,
microfilament and GTP-binding proteins may be involved in CG
exocytosis. In conclusion, this method has significant advantages for
studying of exocytosis in living eggs.
Received 3 August 1995; accepted in final form 10 November 1995.
APS Manuscript Number C478-5.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 8 December 95