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J. Biol. Chem., Vol. 275, Issue 29, 21905-21913, July 21, 2000
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From the Growth of preimplantation embryos is
influenced by autocrine trophic factors that need to act by the 2-cell
stage, but their mode of action is not yet described. This report shows
that late zygote and 2-cell stage mouse embryos responded to
embryo-derived platelet-activating factor (PAF) with transient
increases in intracellular calcium concentration
([Ca2+]i). [Ca2+]i
transients were single global events and were specifically induced by
embryo-derived PAF. They were blocked by inhibition of phospholipase C
(U 73122) and an inositol trisphosphate (IP3) receptor
antagonist (xestospongin C), indicating the release of calcium from
IP3-sensitive intracellular stores. Transients were also
inhibited by the absence of calcium from extracellular medium and
partially inhibited by treatment with dihydropyridine (nifedipine, 10 µM), but not pimozide (an inhibitor of an embryonic
T-type calcium channel). (±)BAY K8644 (an L-type channel agonist)
induced [Ca2+]i transients, yet these were
completely inhibited by nifedipine (10 µM). The complete
inhibition of BAY K8644, but only partial inhibition of PAF by
nifedipine shows that L-type channels were only partly responsible for
the calcium influx. Depolarization of 2-cell embryos by 50 mM K+ did not inhibit PAF-induced calcium
transients, showing that the influx channels were not
voltage-dependent. Depletion of intracellular calcium
stores by thapsigargin revealed the presence of store-operated channels. The interdependent requirement for IP3-sensitive
internal calcium stores and extracellular calcium in the generation of PAF-induced transients may be explained by a requirement for
capacitative calcium entry via store-operated channels. A functionally
important role for the PAF-induced transients is supported by the
observation that inhibition of [Ca2+]i transients
by a PAF-antagonist (WEB 2086) or an intracellular calcium chelator
(1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetrakis-acetoxymethyl ester; BAPTA-AM) caused marked inhibition of early embryo development. Growth inhibition by BAPTA-AM was relieved
by addition of exogenous PAF.
Characterization and Functional Significance of Calcium
Transients in the 2-Cell Mouse Embryo Induced by an Autocrine Growth
Factor*
§¶,§¶,§,§,, and§
Human Reproduction Unit,
§ Department of Physiology, University of Sydney, Royal
North Shore Hospital,
St. Leonards, New South Wales 2065, Australia
*
This work was supported by grants from the Northern Sydney
Area Health Service, University of Sydney Faculty of Medicine, and New
South Wales Government Employees Health Fund.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
61-2-9926-7148; Fax: 61-2-9926-6343; E-mail:
chriso@med.usyd.edu.au.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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