Mouse sperm-egg fusion was examined using two-photon and confocal microscopy. A delay of several minutes occurred between the first observable event of fusion (which was the diffusion of Ca2+-sensitive dyes from egg into sperm) and any change in egg cytoplasmic Ca2+. When indo-1 dextran was used to obtain ratiometric two-photon images, there was no detectable local increase in egg cytoplasmic Ca2+ near the site of sperm fusion. However, the sperm underwent a Ca2+ transient which appeared to be coincident with the egg cytoplasm Ca2+ transient, which suggested that there was a high permeability pathway for Ca2+ between egg and sperm. To exclude this pathway from providing trigger Ca2+ for the egg transient, we reduced bathing [Ca2+] to approx. 18 microM and 13nM (with EGTA). In these conditions the first egg Ca2+ transient was not prevented, which makes an obligatory role for extracellular Ca2+ in the initiation of the egg Ca2+ transient unlikely. Both FITC-albumin (70 kDa) and 10 kDa dextran-linked Ca2+ indicators were able to diffuse into the sperm from the egg. In addition, phycoerythrin (240 kDa) rapidly diffused into the sperm shortly after fusion (but before any changes in Ca2+ occurred). This suggests that the ‘pore(s)’ that form during sperm-egg fusion must be at least 8 nm in diameter. These data are compatible with the idea that a diffusible sperm protein could trigger the observed changes in intracellular Ca2+ in the egg, but do not exclude the possibility that other second messengers are generated during sperm-egg fusion.

REFERENCES

Albanesi
C.
,
Geremia
R.
,
Giorgio
M.
,
Dolci
S.
,
Sette
C.
,
Rossi
P.
(
1996
)
A cell-and developmental stage-specific promoter drives the expression of a truncated c-kit during mouse spermatid elongation.
Development
122
,
1291
1302
Betz
W. J.
,
Angleson
J. K.
(
1998
)
The synaptic vesicle cycle.
Ann. Rev. Physiol
60
,
347
363
Blobel
C. P.
,
Wolfsberg
T. G.
,
Turck
C. W.
,
Myles
D. G.
,
Primakoff
P.
,
White
J. M.
(
1992
)
A potential fusion peptide and an integrin ligand domain in a protein active in sperm-egg fusion.
Nature
356
,
248
252
Calcagno
M.
,
Campos
P. J.
,
Mulliert
G.
,
Suastegui
J.
(
1984
)
Purification, molecular and kinetic properties of glucosamine-6-phosphate isomerase (deaminase) from Escherichia coli.
Biochim. Biophys. Acta
787
,
165
173
Cheek
T. R.
,
McGuinness
O. M.
,
Vincent
C.
,
Moreton
R. B.
,
Berridge
M. J.
,
Johnson
M. H.
(
1993
)
Fertilisation and thimerosal stimulate similar calcium spiking patterns in mouse oocytes but by separate mechanisms.
Development
119
,
179
189
Cheng
H.
,
Lederer
M. R.
,
Lederer
W. J.
,
Cannell
M. B.
(
1996
)
Calcium sparks and [Ca2+]iwaves in cardiac myocytes.
Am. J. Physiol
270
,
148
–.
Collas
P.
,
Fissore
R.
,
Robl
J. M.
,
Sullivan
E. J.
,
Barnes
F. L.
(
1993
)
Electrically induced calcium elevation, activation, and parthenogenetic development of bovine oocytes.
Mol. Reprod. Dev
34
,
212
23
Conover
J. C.
,
Gwatkin
R. B. L.
(
1988
)
Pre-loading of mouse oocytes with DNA-specific fluorochrome (Hoechst 33342) permits rapid detection of sperm-oocyte fusion.
J. Reprod. Fert
82
,
681
690
Creton
R.
,
Jaffe
L. F.
(
1995
)
Role of calcium influx during the latent period in sea urchin fertilization.
Dev. Growth. Differ
37
,
703
709
Cuthbertson
K. S.
,
Cobbold
P. H.
(
1985
)
Phorbol ester and sperm activate mouse oocytes by inducing sustained oscillations in cell Ca2+.
Nature
316
,
541
542
Dupont
G.
,
McGuinness
O. M.
,
Johnson
M. H.
,
Berridge
M. J.
,
Borgese
F.
(
1996
)
Phospholipase C in mouse oocytes: characterization of beta and gamma isoforms and their possible involvement in sperm-induced Ca2+spiking.
Biochem. J
316
,
583
591
Fabiato
A.
(
1985
)
Spontaneous versus triggered contractions of ‘calcium-tolerant’ cardiac cells from the adult rat ventricle.
Basic Res. Cardiol
80
,
83
87
Fissore
R. A.
,
Robl
J. M.
(
1992
)
Intracellular Ca2+response of rabbit oocytes to electrical stimulation.
Mol. Reprod. Dev
32
,
9
16
Flanagan
J. M.
,
Wall
J. S.
,
Capel
M. S.
,
Schneider
D. K.
,
Shanklin
J.
(
1995
)
Scanning transmission electron microscopy and small-angle scattering provide evidence that native Escherichia coli ClpP is a tetradecamer with an axial pore.
Biochemistry
34
,
10910
10917
Florman
H.M.
,
Arnoult
C.
,
Kazam
I.G.
,
Li
C.
,
O'Toole
C. M. B.
(
1998
)
A perspective on the control of mammalian fertilization by egg-activated into channels in sperm: a tale of two channels.
Biol. Reprod
59
,
12
16
Fulton
B. P.
,
Whittingham
D. G.
(
1978
)
Activation of mammalian oocytes by intracellular injection of calcium.
Nature
273
,
149
151
Gilkey
J. C.
(
1983
)
Roles of calcium and pH in activation of eggs of the medaka fish, Oryzias latipes.
J. Cell. Biol
97
,
669
678
Heffner
L. J.
,
Storey
B. T.
(
1981
)
The role of calcium in maintaining motility in mouse spermatozoa.
J. Exp. Zool
218
,
427
434
Jaffe
L. F.
(
1980
)
Calcium explosions as triggers of development.
Ann. N. Y. Acad. Sci
339
,
86
101
Jaffe
L. F.
(
1983
)
Sources of calcium in egg activation: a review and hypotheses.
Dev. Biol
99
,
256
276
Jaffe
L. F.
(
1991
)
The path of calcium in cytosolic calcium oscillations: a unifying hypothesis.
Proc. Natl. Acad. Sci. USA
88
,
9883
9887
Jones
K. T.
,
Carroll
J.
,
Merriman
J. A.
,
Whittingham
D. G.
,
Kono
T.
(
1995
)
Repetitive sperm-induced Ca2+transients in mouse oocytes are cell cycle dependent.
Development
121
,
3259
3266
Jones
K. T.
,
Whittingham
D. G.
(
1996
)
A comparison of sperm-and IP3-induced Ca2+release in activated and aging mouse oocytes.
Dev. Biol
178
,
229
237
Kimura
Y.
,
Yanagimachi
R.
,
Kuretake
S.
,
Bortkiewicz
H.
,
Perry
A.C.F.
,
Yanagimachi
H.
(
1998
)
Analysis of mouse oocyte activation suggests the involvement of sperm perinuclear material.
Biol. Reprod
58
,
1407
1415
Kline
D.
(
1988
)
Calcium-dependent events at fertilization of the frog egg: injection of a calcium buffer blocks ion channel opening, 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
Kubota
H. Y.
,
Yoshimoto
Y.
,
Yoneda
M.
,
Hiramoto
Y.
(
1987
)
Free calcium wave upon activation in Xenopus eggs.
Dev. Biol
119
,
129
136
Lawrence
Y.
,
Whitaker
M.
,
Swann
K.
(
1997
)
Sperm-oocyte fusion is the prelude to the initial Ca2+increase at fertilization in the mouse.
Development
124
,
233
241
Longo
F. J.
,
Lynn
J. W.
,
McCulloh
D. H.
,
Chambers
E. L.
(
1986
)
Correlative ultrastructural and electrophysiological studies of sperm-egg interactions of the sea urchin, Lytechinus variegatus.
Dev. Biol
118
,
155
66
McDougall
A.
,
Sardet
C.
,
Lambert
C. C.
(
1995
)
Different calcium-dependent pathways control fertilisation-triggered glycoside release and the cortical contraction in ascidian eggs.
Zygote
3
,
251
258
McGuinness
O. M.
,
Moreton
R. B.
,
Johnson
M. H.
,
Berridge
M. J.
(
1996
)
A direct measurement of increased divalent cation influx in fertilised mouse oocytes.
Development
122
,
2199
2206
McNiven
A. I.
,
Yoshida
S.
,
Georgiou
P.
,
House
C. R.
(
1988
)
A delayed all-or-none hyperpolarisation induced by a single Ca action potential in hamster eggs.
Pflugers. Arch
412
,
451
453
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.
,
Yuzaki
M.
,
Nakada
K.
,
Shirakawa
H.
,
Nakanishi
S.
,
Nakade
S.
,
Mikoshiba
K.
(
1992
)
Block of Ca2+wave and Ca2+oscillation by antibody to the inositol 1,4,5-trisphosphate receptor in fertilized hamster eggs.
Science
257
,
251
255
Myles
D. G.
(
1993
)
Molecular mechanisms of sperm-egg membrane binding and fusion in mammals.
Dev. Biol
158
,
35
45
Ozil
J.-P.
,
Swann
K.
(
1995
)
Stimulation of repetitive calcium transients in mouse eggs.
J. Physiol. (Lond.)
483
,
331
346
Parrington
J.
,
Swann
K.
,
Shevchenko
V. I.
,
Sesay
A. K.
,
Lai
F. A.
(
1996
)
Calcium oscillations in mammalian eggs triggered by a soluble sperm protein.
Nature
379
,
364
368
Primakoff
P.
,
Hyatt
H.
,
Tredick-Kline
J.
(
1987
)
Identification and purification of a sperm surface protein with a potential role in sperm-egg membrane fusion.
J. Cell. Biol
104
,
141
149
Quinn
P.
,
Barros
C.
,
Whittingham
D. G.
(
1982
)
Preservation of hamster oocytes to assay the fertilizing capacity of human spermatozoa.
J. Reprod. Fert
66
,
161
168
Rochwerger
L.
,
Cohen
D. J.
,
Cuasnicu
P. S.
(
1992
)
Mammalian sperm-egg fusion: the rat egg has complementary sites for a sperm protein that mediates gamete fusion.
Dev. Biol
153
,
83
90
Sauer
H.
,
Pratsch
L.
,
Tschopp
J.
,
Bhakdi
S.
,
Peters
R.
(
1991
)
Functional size of complement and perforin pores compared by confocal laser scanning microscopy and fluorescence microphotolysis.
Biochim. Biophys. Acta
1063
,
137
146
Schmidt
T.
,
Patton
C.
,
Epel
D.
(
1982
)
Is there a role for the Ca2+influx during fertilization of the sea urchin egg?.
Dev. Biol
90
,
284
90
Schultz
R. M.
,
Kopf
G. S.
(
1995
)
Molecular basis of mammalian egg activation.
Curr. Top. Dev. Biol
30
,
21
62
Sette
C.
,
Bevilacqua
A.
,
Bianchini
A.
,
Mangia
F.
,
Geremia
R.
,
Rossi
P.
(
1997
)
Parthenogenetic activation of mouse eggs by microinjection of a truncated c-kit tyrosine kinase present in spermatozoa.
Development
124
,
2267
2274
Soeller
C.
,
Cannell
M. B.
(
1996
)
Construction of a two-photon microscope and optimisation of illumination pulse duration.
Pflugers Arch
432
,
555
561
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
Swann
K.
,
Whitaker
M.
(
1986
)
The part played by inositol trisphosphate and calcium in the propagation of the fertilization wave in sea urchin eggs.
J. Cell. Biol
103
,
2333
2342
Swann
K.
,
Lai
F. A.
(
1997
)
A novel signalling mechanism forgenerating Ca2+ oscillations at fertilization in mammals.
BioEssays
19
,
371
378
Swann
K.
,
Igusa
Y.
,
Miyazaki
S.
(
1989
)
Evidence for an inhibitory effect of protein kinase C on G-protein-mediated repetitive calcium transients in hamster eggs.
EMBO J
8
,
3711
3718
Takamatsu
T.
,
Wier
W. G.
(
1990
)
High temporal resolution video imaging of intracellular calcium.
Cell Calcium
11
,
111
120
Visconti
P.E.
,
Kopf
G.S.
(
1998
)
Regulation of protein phosphorylation during sperm capacitation.
Biol. Reprod
59
,
1
6
Whitaker
M.
,
Swann
K.
(
1993
)
Lighting the fuse at fertilization.
Development
117
,
1
12
Wier
W. G.
,
Blatter
L. A.
(
1991
)
Ca(2+)-oscillations and Ca(2+)-waves in mammalian cardiac and vascular smooth muscle cells.
Cell Calcium
12
,
241
254
Wolfsberg
T. G.
,
White
J. M.
(
1996
)
ADAMs in fertilization and development.
Dev. Biol
180
,
389
401
Wolosker
H.
,
Kline
D.
,
Bian
Y.
,
Blackshaw
S.
,
Cameron
A. M.
,
Fralich
T. J.
,
Schnaar
R. L.
,
Snyder
S. H.
(
1998
)
Molecularly cloned mammalian glucosamine-6-phosphate deaminase localizes to transporting epithelium and lacks oscillin activity.
FASEB J
12
,
91
99
Wyke
A.M.
,
Impraim
C.C.
,
Knutton
S.
,
Pasternak
C.A.
(
1980
)
Components involved in virally mediated membrane fusion and permeability changes.
Biochem. J
190
,
625
638
Yanagimachi
R.
(
1978
)
Calcium requirement for sperm-egg fusion in mammals.
Biol. Reprod
19
,
949
958
Zucker
R. S.
,
Steinhardt
R. A.
,
Winkler
M. M.
(
1978
)
Intracellular calcium release and the mechanisms of parthenogenetic activation of the sea urchin egg.
Dev. Biol
65
,
285
295
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