Inositol 1,4,5-trisphosphate can regulate growth and differentiation by modulating the release of intracellular Ca2+ in a variety of cellular systems, and it is involved in oocyte activation. Recent studies suggest that mammalian preimplantation development may also be regulated by the release of Ca2+ from intracellular stores. The rate of cavitation and cell division was accelerated after a transient elevation of intracellular Ca2+ levels was induced in morulae by exposure to ethanol or ionomycin. Embryos exposed to BAPTA-AM, a chelator of intracellular Ca2+, exhibited a brief dose-dependent reduction in basal Ca2+ levels, a temporal inhibition of ionophore-induced Ca2+ signalling and a subsequent delay in blastocoel formation. BAPTA-AM at 0.5 microM did not significantly alter the basal intracellular calcium level, but chelated Ca2+ that was released after ethanol exposure and thereby attenuated the ethanol-induced acceleration of cavitation. BAPTA-AM also inhibited cell division to the 16-cell stage in a dose-dependent manner, which correlated with the inhibition of cavitation. Thimerosal and inositol 1,4,5-trisphosphate significantly elevated the intracellular Ca2+ concentration in mouse morula-stage embryos, providing evidence for the existence of inositol 1,4,5-trisphosphate-sensitive Ca2+ stores. Although caffeine failed to release intracellular Ca2+, ryanodine induced a small biphasic release of Ca2+, suggesting that ryanodine-sensitive Ca2+ stores may also exist in mouse embryos. Morulae exposed to the calmodulin inhibitor W-7 exhibited a dose-dependent delay in blastocoel formation. A 4 hour exposure to 10 microM W-7 did not significantly alter cavitation, but attenuated the ionophore-induced stimulation of blastocoel formation. This finding suggests that the developmental effects produced through Ca2+ signalling are mediated by calmodulin. Our results demonstrate that Ca2+ release in mouse morulae occurs predominantly through the inositol 1,4,5-trisphosphate receptor, and that alteration of intracellular Ca2+ levels can accelerate or delay embryonic growth and differentiation, providing a mechanistic link between the regulation of oocyte and embryonic development.

Reference

Allbritton
N. L.
,
Meyer
T.
(
1993
)
Localized calcium spikes and propagating calcium waves.
Cell Calcium
14
,
691
697
Amundson
J.
,
Clapham
D.
(
1993
)
Calcium waves.
Curr. Opin. Neurobiol
3
,
375
382
Ayabe
T.
,
Kopf
G. S.
,
Schultz
R. M.
(
1995
)
Regulation of mouse egg activation: presence of ryanodine receptors and effects of microinjectedryanodine and cyclic ADP ribose on uninseminated and inseminated eggs.
Development
121
,
2233
2244
Baitinger
C.
,
Alderton
J.
,
Poenie
M.
,
Schulman
H.
,
Steinhardt
R. A.
(
1990
)
Multifunctional Ca2+/calmodulin-dependent protein kinase is necessary for nuclear envelope breakdown.
J. Cell Biol
111
,
1763
1773
Bement
W. M.
(
1992
)
Signal transduction by calcium and protein kinase C during egg activation.
J. Exp. Zool
263
,
382
397
Berridge
M. J.
(
1991
)
Cytoplasmic calcium oscillations: A two pool model.
Cell Calcium
12
,
63
72
Berridge
M. J.
(
1993
)
Inositol trisphosphate and calcium signalling.
Nature
361
,
315
325
Berridge
M. J.
,
Galione
A.
(
1988
)
Cytosolic calcium oscillators.
FASEB J
2
,
3074
3082
Berridge
M. J.
,
Dupont
G.
(
1994
)
Spatial and temporal signalling by calcium.
Curr. Opin. Cell Biol
6
,
267
274
Boitano
S.
,
Dirksen
E. R.
,
Sanderson
M. J.
(
1992
)
Intercellular propagation of calcium waves mediated by inositol trisphosphate.
Science
258
,
292
295
Bootman
M. D.
,
Taylor
C. W.
,
Berridge
M. J.
(
1992
)
The thiol reagent, thimerosal, evokes Ca2+spikes in HeLa cells by sensitizing the inositol 1,4,5-trisphosphate receptor.
J. Biol. Chem
267
,
25113
25119
Carroll
J.
,
Swann
K.
(
1992
)
Spontaneous cytosolic calcium oscillations driven by inositol trisphosphate occur during in vitro maturation of mouse oocytes.
J. Biol. Chem
267
,
11196
11201
Charbonneau
M.
,
Grandin
N.
(
1992
)
A hypothesis on p34cdc2sequestration based on the existence of Ca2+-coordinated changes in H+and MPF activities during pus egg activation.
Biol. Cell
75
,
165
172
Chafouleas
J. G.
,
Bolton
W. E.
,
Hidaka
H.
,
Boyd
A. E.
III
,
Means
A.
(
1982
)
Calmodulin and the cell cycle: involvement in regulation of cell-cycle progression.
Cell
28
,
41
50
Cheung
W. Y.
(
1980
)
Calmodulin plays a pivitol role in cellular regulation.
Science
207
,
19
27
Clapper
D. L.
,
Walseth
T. F.
,
Dargie
P. J.
,
Lee
H. C.
(
1987
)
Pyridine nucleotide metabolism stimulates calcium release from sea urchin egg microsomes desensitized to inositol trisphosphate.
J. Biol. Chem
262
,
9561
9568
Colonna
R.
,
Tatone
C.
,
Malgaroli
A.
,
Eusebi
L.
,
Mangia
F.
(
1989
)
Effects of protein kinase C stimulation and free Ca2+rise in mammalian egg activation.
Gamete Res
24
,
171
183
Cuthbertson
K. S. R.
,
Cobbold
P. H.
(
1985
)
Phorbol ester and sperm activate mouse oocytes by inducing sustained oscillations in cell Ca2+.
Nature
316
,
541
542
Dardik
A.
,
Schultz
R. M.
(
1991
)
Blastocoel expansion in the preimplantation mouse embryo: stimulatory effect of TGF-alpha and EGF.
Development
113
,
919
930
Dargie
P. J.
,
Agre
M. C.
,
Cheung
L. H.
(
1990
)
Comparison of Ca2+mobilizing activities of cyclic ADP-ribose and inositol trisphosphate.
Cell. Reg
1
,
279
290
De Young
G. W.
,
Kiezer
J.
(
1992
)
A single-pool inositol 1,4,5-trisphosphate-receptor-based model for agonist-stimulated oscillations in Ca2+concentration.
Proc. Natl Acad. Sci. USA
89
,
9895
9899
Ducibella
T.
,
Albertini
D. F.
,
Anderson
E.
,
Biggers
J. D.
(
1975
)
The preimplantation mammalian embryo: characterization of intercellular junctions and their appearance during development.
Dev. Biol
45
,
231
250
Epel
D.
(
1990
)
The initiation of development at fertilization.
Cell. Diff. Devel
29
,
1
12
Erbach
G. T.
,
Lawitts
J. A.
,
Papaioannou
V. E.
,
Biggers
J. D.
(
1994
)
Differential growth of the mouse preimplantation embryo in chemically defined media.
Biol. Reprod
50
,
1027
1033
Felici
M. D.
,
Dolci
S.
,
Siracusa
G.
(
1991
)
An increase of intracellular free Ca2+is essential for spontaneous meiotic resumption by mouse oocytes.
J. Exp. Zool
260
,
401
405
Feng
L.
,
Pereira
B.
,
Kraus-Friedmann
N.
(
1992
)
Different localization of inositol 1,4,5-trisphosphate and ryanodine binding sites in rat liver.
Cell Calcium
13
,
79
87
Fissore
R. A.
,
Dobrinsky
J. R.
,
Balise
J. J.
,
Duby
R. T.
,
Robl
J. M.
(
1992
)
Patterns of intracellular Ca2+concentration in fertilized bovine eggs.
Biol. Reprod
47
,
960
969
Fissore
R. A.
,
Robl
J. M.
(
1993
)
Sperm, inositol trisphosphate, and thimerosal-induced intracellular Ca2+elevations in rabbit eggs.
Dev. Biol
159
,
122
130
Fu
T.
,
Sugimoto
Y.
,
Oki
T.
,
Murakami
S.
,
Okano
Y.
,
Nozawa
Y.
(
1991
)
Calcium oscillation associated with reduced protein kinase C activities in ras-transformed NIH 3T3 cells.
FEBS Lett
281
,
263
266
Galione
A.
,
Lee
H. C.
,
Busa
W. B.
(
1991
)
Ca2+-induced Ca2+release in sea urchin egg homogenates: modulation by cyclic ADP-ribose.
Science
253
,
1143
1146
Galione
A.
,
McDougall
A.
,
Busa
W. B.
,
Willmott
N.
,
Gillot
I.
,
Whitaker
M.
(
1993
)
Redundant mechanisms of calcium-induced calcium release underlying calcium waves during fertilization of sea urchin eggs.
Science
261
,
348
352
Gardiner
C. S.
,
Williams
J. S.
,
Menino
A. R. J.
(
1990
)
Sodium/potassium adenosine triphosphatase-and -subunit mRNA levels during mouse embryo development in vitro.
Biol. Reprod
43
,
788
794
Goodall
H.
(
1986
)
Manipulation of gap junctional communication during compaction of the mouse early embryo.
J. Embryol. Exp. Morph
91
,
283
296
Goodall
H.
,
Johnson
M. H.
(
1982
)
Use of carboxyfluorescein diacetate to study formation of permeable channels between mouse blastomeres.
Nature
295
,
524
526
Goodall
H.
,
Johnson
M. H.
(
1984
)
The nature of intercellular coupling within the preimplantation mouse embryo.
J. Embryol. Exp. Morph
79
,
53
76
Grandin
N.
,
Charbonneau
M.
(
1991
)
Cycling of intracellular free calcium and intracellular pH in Xenopus embryos: a possible role in the control of the cell cycle.
J. Cell Sci
99
,
5
11
Hepler
P. K.
(
1989
)
Calcium transients during mitosis: observations in flux.
J. Cell Biol
109
,
2567
2573
Hess
S. D.
,
Oortgeiessen
M.
,
Cahalan
M. D.
(
1993
)
Calcium oscillations in human T and natural killer cells depend upon membrane potential and calcium influx.
J. Immunol
150
,
2620
2633
Hoek
J. B.
,
Thomas
A. P.
,
Rubin
R.
,
Rubin
E.
(
1987
)
Ethanol-induced mobilization of calcium by activation of phosphoinostitide-specific phospholipase C in intact hepatocytes.
J. Biol. Chem
262
,
682
691
Homa
S. T.
(
1991
)
Neomycin, an inhibitor of phosphoinositide hydrolysis, inhibits the resumption of bovine oocyte spontaneous meiotic maturation.
J. Exp. Zool
258
,
95
103
Kao
J. P. Y.
,
Alderton
J. M.
,
Tsien
R. Y.
,
Steinhardt
R. A.
(
1990
)
Active involvement of calcium ion in mitotic progresion in Swiss 3T3 fibroblasts.
J. Cell Biol
111
,
183
196
Keating
T. J.
,
Cork
R. J.
,
Robinson
K. R.
(
1994
)
Intracellular free calcium oscillations in normal and cleavage-blocked embryos and artificially activated eggs of Xenopus laevis.
J. Cell Sci
107
,
2229
2237
Khidir
M. A.
,
Stachecki
J. J.
,
Krawetz
S. A.
,
Armant
D. R.
(
1995
)
Rapid inhibition of mRNA synthesis during preimplantation embryo development: vital permeabilization using lysolecithin potentiates the action of-amanitin.
Exp. Cell Res
219
,
619
625
Kimber
S. J.
,
Surani
M. A. H.
(
1981
)
Morphogenetic analysis of changing cell associations following release of 2-cell and 4-cell mouse embryos from cleavage arrest.
J. Embryol. Exp. Morph
61
,
331
345
Klee
C. B.
,
Crouch
T. H.
,
Krinks
M. H.
(
1979
)
Calcineurin: a calcium-and calmodulin-binding protein of nervous system.
Proc. Natl Acad. Sci. USA
76
,
6270
6273
Kline
D.
(
1988
)
Calcium-dependent events at fertilization of the frog egg: Injection of a calcium buffer blocks ion channel openning, exocytosis, and formation of pronuclei.
Dev. Biol
126
,
346
361
Kline
D.
,
Kline
J. T.
(
1992
)
Repetitive calcium transients and the role of calcium in exocytosis and cell cycle activation in the mouse egg.
Dev. Biol
149
,
80
89
Kline
D.
,
Stewart-Savage
J.
(
1994
)
The timing of cortical granule fusion, content dispersal, and endocytosis during fertilization of the hamster egg: An electrophysiological and histochemical study.
Dev. Biol
162
,
277
287
Kline
J. T.
,
Kline
D.
(
1994
)
Regulation of intracellular calcium in the mouse egg:evidence for inositol trisphosphate-induced calcium release, but not calcium-induced calcium release.
Biol. Reprod
50
,
193
203
Kubota
H. Y.
,
Yoshimoto
Y.
,
Hiramoto
Y.
(
1993
)
Oscillation of intracellular free calcium in cleaving and cleavage-arrested embryos of Xenopus laevis.
Dev. Biol
160
,
512
518
Leach
R. E.
,
Stachecki
J. J.
,
Armant
D. R.
(
1993
)
Development of invitro fertilized mouse embryos exposed to ethanol during the preimplantation period: accelerated embryogenesis at subtoxic levels.
Teratol
47
,
57
64
Lechleiter
J. D.
,
Clapham
D. E.
(
1992
)
Molecular mechanisms of intracellular calcium excitability in X. laevis oocytes.
Cell
69
,
283
294
Lee
H. C.
,
Aarhus
R.
,
Walseth
T. F.
(
1993
)
Calcium mobilization by dual receptors during fertilization of sea urchin eggs.
Science
261
,
352
355
Lo
C. W.
,
Gilula
N. B.
(
1979
)
Gap junctional communication in the post-implantation mouse embryo.
Cell
18
,
411
422
Lorca
T.
,
Cruzalegul
F. H.
,
Fesquet
D.
,
Cavadore
J.-C.
,
Mery
J.
,
Means
A.
,
Doree
M.
(
1993
)
Calmodulin-dependent protein kinase II mediates inactivation of MPF and CSF upon fertilization of Xenopus eggs.
Nature
366
,
270
273
Lu
K. P.
,
Means
A. R.
(
1993
)
Regulation of the cell cycle by calcium and calmodulin.
Endo. Rev
14
,
40
58
Malgaroli
A.
,
Fesce
R.
,
Meldolesi
J.
(
1990
)
Spontaneous [Ca2+]ifluctuations in rat chromaffin cells do not require inositol 1,4,5-trisphosphate elevations but are generated by a caffeine-and ryanodine-sensitive intracellular Ca2+store.
J. Biol. Chem
265
,
3005
3008
Manejwala
F. M.
,
Schultz
R. M.
(
1989
)
Blastocoel expansion in the preimplantation mouse embryo: Stimulation of sodium uptake by cAMP and possible involvement of cAMP-dependent protein kinase.
Dev. Biol
136
,
560
563
Manejwala
F. M.
,
Cragoe
E. J.
Jr.
,
Schultz
R.M.
(
1989
)
Blastocoel expansion in the preimplantation mouse embryo: role of extracellular sodium and chloride and possible apical routes of their entry.
Dev. Biol
133
,
210
220
Mayer
B.
,
John
M.
,
Bohme
E.
(
1990
)
Purification of a Ca2+/calmodulin-dependent nitric oxide synthase from porcine cerebellum: cofactor role of tetrahydrobiopterin.
FEBS Lett
277
,
215
219
Means
A.
,
Dedman
J. R.
(
1980
)
Calmodulin—an intracellular calcium receptor.
Nature
285
,
73
77
Means
A.
,
VanBerkum
M. F. A.
,
Bagchi
I. C.
,
Lu
K. P.
,
Rasmussen
C. D.
(
1991
)
Regulatory functions of calmodulin.
Pharmacol. Ther
50
,
255
270
Mignery
G. A.
,
Sudhof
T. C.
,
Takei
K.
,
DeCamilli
P.
(
1989
)
Putative receptor for inositol 1,4,5-trisphosphate similar to ryanodine receptor.
Nature
342
,
192
195
Missiaen
L.
,
Taylor
C. W.
,
Berridge
M. J.
(
1991
)
Spontaneous calcium release from inositol trisphosphate-sensitive calcium stores.
Nature
352
,
241
244
Missiaen
L.
,
Taylor
C. W.
,
Berridge
M. J.
(
1992
)
Luminal Ca2+promoting spontaneous Ca2+release from inositol trisphosphate-sensitive stores in rat hepatocytes.
J. Physiol
455
,
623
640
Miyazaki
S.
,
Hashimoto
N.
,
Yoshimoto
Y.
,
Kishimoto
T.
,
Igusa
Y.
,
Hiramoto
Y.
(
1986
)
Temporal and spatial dynamics of the periodic increase in intracellular free calcium at fertilization of golden hamster eggs.
Dev. Biol
118
,
259
267
Miyazaki
S.
,
Shirakawa
H.
,
Nakada
K.
,
Honda
Y.
,
Yuzaki
M.
,
Nakade
S.
,
Mikoshiba
K.
(
1992
)
Antibody to the inositol trisphosphate receptor blocks thimerosal-enhanced Ca2+-induced Ca2+release and Ca2+oscillations in hamster eggs.
FEBS Lett
309
,
180
184
Miyazaki
S.
,
Shirakawa
H.
,
Nakada
K.
,
Honda
Y.
(
1993
)
Essential role of the inositol 1,4,5-trisphosphate receptor/Ca2+release channel in Ca2+waves and Ca2+oscillations at fertilization of mammalian eggs.
Dev. Biol
158
,
62
78
Nuccitelli
R.
(
1991
)
How do sperm activate eggs?.
Dev. Biol
25
,
1
16
Osipchuk
Y.
,
Cahalan
M.
(
1992
)
Cell-to-cell spread of calcium signals mediated by ATP receptors in mast cells.
Nature
359
,
241
244
Ozil
J. P.
(
1990
)
The parthenogenetic development of rabbit oocytes after repetitive pulsatile electrical stimulation.
Development
109
,
117
127
Paria
B. C.
,
Dey
S. K.
(
1990
)
Preimplantation embryo development in vitro: Cooperative interactions among embryos and role of growth factors.
Proc. Natl Acad. Sci. USA
87
,
4756
4760
Poenie
M.
,
Alderton
J.
,
Tsien
R. Y.
,
Steinhardt
R. A.
(
1985
)
Changes of free calcium levels with stages of the cell division cycle.
Nature
315
,
147
149
Pratt
H. P.
,
Chakraborty
J.
,
Surani
M. A.
(
1981
)
Molecular and morphological differentiation of the mouse blastocyst after manipulations of compaction with cytochalasin D.
Cell
26
,
279
292
Rasmussen
C. D.
,
Means
A. R.
(
1987
)
Calmodulin is involved in regulation of cell proliferation.
EMBO J
6
,
3961
3968
Rasmussen
C. D.
,
Means
A. R.
(
1989
)
Calmodulin is required for cell cycle progression during G1 and mitosis.
EMBO J
8
,
73
82
Sasaki
Y.
,
Hidaka
H.
(
1982
)
Calmodulin and cell proliferation.
Biochem. Biophys. Res. Commun
104
,
451
456
Silver
R. B.
(
1990
)
Calcium and cellular clocks orchestrate cell division.
Ann. NY Acad. Sci
582
,
207
221
Spindle
A.
,
Nagano
H.
,
Pedersen
R. A.
(
1985
)
Inhibition of DNA replication in preimplantation mouse embryos by aphidicolin.
J. Exp. Zool
235
,
289
295
Stachecki
J. J.
,
Yelian
F. D.
,
Schultz
J. F.
,
Leach
R. E.
,
Armant
D. R.
(
1994
)
Blastocyst cavitation is accelerated by ethanol-or ionophore-induced elevation of intracellular calcium.
Biol. Reprod
50
,
1
9
Stachecki
J. J.
,
Yelian
F. D.
,
Leach
R. E.
,
Armant
D. R.
(
1994
)
Murine blastocyst outgrowth and implantation rates following exposure to ethanol or A23187 during in vitro culture.
J. Reprod. Fertil
101
,
611
617
Streffer
C.
,
VanBeuningen
D.
,
Molls
M.
,
Zamboglou
N.
,
Schulz
S.
(
1980
)
Kinetics of cell proliferation in the preimplanted mouse embryo in vivo and in vitro.
Cell. Tissue Kinetics
13
,
135
143
Stricker
S. A.
(
1995
)
Time-lapse confocal imaging of calcium dynamics in starfish embryos.
Dev. Biol
170
,
496
518
Sun
F. Z.
,
Hoyland
J.
,
Huang
X.
,
Mason
W.
,
Moor
R. M.
(
1992
)
A comparison of intracellular changes in porcine eggs after fertilization and electroactivation.
Development
115
,
947
956
Surani
M. A. H.
,
Barton
S. C.
,
Burling
A.
(
1980
)
Differentiation of 2-cell and 8-cell mouse embryos arrested by cytoskeletal inhibitors.
Exp. Cell Res
125
,
275
286
Swann
K.
(
1991
)
Thimerosal causes calcium oscillations and sensitizes calcium-induced calcium release in unfertilized hamster eggs.
FEBS Lett
278
,
175
178
Tanaka
Y.
,
Hayashi
N.
,
Kaneko
A.
,
Ito
T.
,
Miyoshi
E.
,
Sasaki
Y.
,
Fusamoto
H.
,
Kamada
T.
(
1992
)
Epidermal growth factor induces dose-dependent calcium oscillations in single fura-2-loaded hepatocytes.
Hepatol
16
,
479
486
Tombes
R. M.
,
Simerly
C.
,
Borisy
G. G.
,
Schatten
G.
(
1992
)
Meiosis, egg activation, and nuclear envelope breakdown are differentially reliant on Ca2+, whereas germinal vesicle breakdown is Ca2+independent in the mouse oocyte.
J.Cell Biol
117
,
799
811
Tsien
R. W.
,
Tsien
R. Y.
(
1990
)
Calcium channels, stores, and oscillations.
Ann. Rev. Cell Biol
6
,
715
760
Vitullo
A. D.
,
Ozil
J. P.
(
1992
)
Repetitive calcium stimuli drive meiotic resumption and pronuclear development during mouse oocyte activation.
Dev. Biol
151
,
128
136
Watson
A. J.
,
Kidder
G. M.
(
1988
)
Immunofluorescence of the timing of appearance and cellular distribution of Na/K-ATPase during mouse embryogenesis.
Dev. Biol
126
,
80
90
Werb
Z.
(
1990
)
Expression of EGF and TGF-genes in early mammalian development.
Mol. Reprod. Dev
27
,
10
15
Whitaker
M.
,
Patel
R.
(
1990
)
Calcium and cell cycle control.
Development
108
,
525
542
Wiley
L. M.
,
Eglitis
M. A.
(
1981
)
Cell surface and cytoskeletal elements: cavitation in the mouse preimplantation embryo.
Dev. Biol
86
,
493
501
Wood
S. A.
,
Kaye
P. L.
(
1989
)
Effects of epidermal growth factor on preimplantation mouse embryos.
J. Reprod. Fertil
85
,
575
582
Worley
P. F.
,
Baraban
J. M.
,
Colvin
J. S.
,
Snyder
S. H.
(
1987
)
Characterization of inositol trisphosphate receptor binding in brain. Regulation by pH and calcium.
J. Biol. Chem
262
,
12132
12136
Xu
Z.
,
Kopf
G. S.
,
Schultz
R. M.
(
1994
)
Involvement of inositol 1,4,5-trisphosphate-mediated Ca2+release in early and late events of mouse egg activation.
Development
120
,
1851
1859
Yagi
K.
,
Yazama
M.
,
Kakiuchi
S.
,
Ohshima
M.
(
1978
)
Identification of an activator protein for myosin light chain kinase as the Ca2+-dependent modulator protein.
J. Biol. Chem
253
,
1338
1340
Ziomek
C. A.
,
Johnson
M. H.
(
1980
)
Cell surface interaction induces polarization of mouse 8-cell blastomeres at compaction.
Cell
21
,
935
942
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