1295
Development 110, 1295-1302 (1990)
Printed in Great Britain © The Company of Biologists Limited 1990
A cytosolic sperm factor stimulates repetitive calcium increases and
mimics fertilization in hamster eggs
KARL SWANN
MRC Experimental Embryology and Teratology Unit, St George's Hospital Medical School, Cranmer Terrace, London SW17 ORE, UK
Summary
Microinjection of cytosolic sperm extracts into unfertilized golden hamster eggs caused a series of increases in
cytoplasmic free calcium, Ca 2+ j, and membrane hyperpolarizing responses, HRs. These HRs and Ca 2+ j
transients are similar to those seen during in vitro
fertilization of hamster eggs. The sperm factor that is
responsible for causing these effects appears to be of high
molecular weight and protein based. Injection of sperm
factor activated eggs and mimicked fertilization in
causing repetitive HRs in the presence of phorbol esters
and in sensitizing the egg to calcium-induced calcium
release. Since these effects cannot be mimicked by
injecting G-protein agonists or calcium-containing solutions, it seems unlikely that a receptor-G-protein
signalling system is involved at fertilization. These data
instead suggest a novel signal transduction system
operates during mammalian fertilization in which a
protein factor is transferred from the sperm into the egg
cytoplasm after gamete membrane fusion.
Introduction
(Gould and Stephano, 1987; Turner et al. 1986; Jaffe et
al. 1988). These may in turn activate a phospholipase C
that produces the intracellular calcium-releasing second
messenger inositol 1,4,5-trisphosphate (Jaffe etal. 1988;
Whitaker, 1989). There is evidence that this messenger
system exists in hamster eggs. Activating G-proteins by
injecting GTP analogues or by applying serotonin
externally both trigger internal calcium release and
HRs (Miyazaki, 1988; Swann etal. 1989; Miyazaki etal.
1990). However, these stimuli do not cause the
persistent series of HRs seen at fertilization. Furthermore, there have been no reports in mammals of sperm
bound agonists that might stimulate G-proteins in eggs.
The relevance of a receptor-G-protein mechanism to
fertilization has remained unclear.
An alternative hypothesis for fertilization is that the
sperm triggers calcium release by first fusing with the
egg and then introducing a soluble factor into the egg
cytoplasm (Dale, 1988; Swann and Whitaker, 1990).
This has been suggested from the finding that injecting
extracts from sperm activates sea urchin, mouse and
rabbit eggs (Dale et al. 1985; Stice and Robl, 1990). It is
also feasible that the sperm could act in this way
because gamete membrane fusion occurs before the
start of the Ca2+, transient in sea urchin eggs and at
least around the same time as the first HR in hamster
eggs (McCulloh and Chambers, 1986; Whitaker et al.
1989; Miyazaki and Igusa, 1981). In this paper, I show
that injection of a cytosolic protein factor from
mammalian sperm causes a series of HRs and Ca2+j
transients with the same characteristics as those seen at
At fertilization in all species studied, sperm activate
eggs by causing transient increases in the intracellular
free calcium ion concentration, Ca2+i (Jaffe, 1983;
Whittingham, 1980; Whitaker and Steinhardt, 1985).
The Ca j changes that occur during fertilization of
mammalian eggs consist of a series of repetitive and
transient rises in Ca2+, that persist for more than one
hour after gamete membrane fusion (Cuthbertson and
Cobbold, 1985; Miyazaki 19886). Although treatments
that cause monotonic increases in Ca2+i activate
mammalian eggs, recent experiments in rabbit eggs
suggest that repetitive increases in Ca2+j are the more
physiological and effective stimulus to development
(Ozil, 1990). In hamster eggs each Ca2+j transient
causes an increase in plasma membrane potassium
conductance which generates a hyperpolarizing membrane potential response, an HR (Miyazaki and Igusa,
1981; Miyazaki, 19886). Consequently, at fertilization,
hamster eggs show a long lasting series of repetitive
HRs that are coincident with the repetitive Ca2+j
transients (Igusa and Miyazaki, 1983, 1986). Despite
the extensive characterization of HRs and repetitive
Ca2+j transients in hamster fertilization, it is not yet
known how sperm triggers these responses and hence
how the sperm activates the egg (Miyazaki, 19886).
There are two fundamentally different ideas on how
sperm generate Ca2+i transients in eggs. One hypothesis is that sperm act by binding to external receptors
that are linked to GTP-binding proteins in the egg
Key words: fertilization, calcium, spermatozoa, oocyte,
membrane potential.
1296
K. Swann
fertilization in hamster eggs. The effects are less open to
artifactual imitation than activation experiments in
other eggs. These data represent clear evidence for the
existence of a factor in sperm that triggers the
development of the egg. They argue against the
receptor-G-protein hypothesis and suggest that a novel
signal transduction mechanism operates during mammalian fertilization.
Materials and methods
Gametes and media
Eggs were collected 15-16 h after HCG injection of 4- to 10week-old superovulated female Syrian hamsters (Bavister,
1989). Cumulus cells were removed by treating eggs with
ISOi.u.ml"1 hyaluronidase and the zona pellucidas were
removed by briefly treating eggs with 2.5mgml~1 trypsin
(Igusa and Miyazaki, 1983). Some of the zona-free eggs were
then placed immediately in drops of M2 solution containing
4 m g m r ' BSA (Fulton and Whittingham, 1978) under
paraffin oil on the heated stage of an epifluorescence
microscope (31-33°C). The bottom of the drops that the eggs
were in was made from a coverslip that had been coated with
polylysine (100 ^g ml"1) in order to attach the eggs to the
bottom of the drop. Eggs stored for later use were kept in
drops of M2 at 37 °C. All eggs were injected or fertilized
within 2 h of collection and assayed for activation by placing
them in an incubator for 2 h and examining them for meiotic
status using the vital fluorochrome Hoechst 33342 (Luttmer
and Longo, 1986).
Preparation and treatment of sperm extracts
Cytosolic sperm extracts were prepared from either hamster
or boar sperm. Hamster sperm was taken from the cauda
epididymis of 10- to 20-week-old males, and allowed to swim
out in m-TALP medium (Bavister, 1989). Ejaculated boar
sperm was suspended in an extender medium and washed into
Tyrodes T6 medium (Quinn et al. 1982) on the day after
ejaculation for use in extract preparation. For preparation of
extracts, suspensions of either sperm type were washed
extensively into an intracellular-like medium (ICM; 120mM
KC1, 20 mM Hepes, 100 ^M ethyleneglycol-bis-(/3-aminoethyl
ether)N,A',A'/,A''-tetraacetic acid (EGTA), 10mM Na-glycerophosphate, 200^M phenylmethylsulfonyl fluoride (PMSF),
lmM dithiothreitol (DTT), pH7.5 (all chemicals from Sigma,
UK). A final dense suspension (5-10xl0 8 spermml"1) was
lysed by either freeze-thaw cycles or by sonication. Homogenates were then spun at 100000 g for lh at 4°C and the
clear supernatant collected as the cytosolic fraction. The
protein concentration in these cytosolic extracts was about
lOmgml""1. Size fractionation was achieved by concentration
of extracts on centricon-100 ultrafiltration membranes (centricon-100, Amicon, UK), followed by dilution into the
intracellular-like medium, followed by reconcentration. This
fraction (protein cone. 50-100 mg ml"1) was labelled as the
> 100x10 (Mr) fraction and had a free calcium concentration
(measured with indo-1) of 60-110 nM. The filtrates of the first
ultrafiltration was the <100xl0 3 fraction. Heat-treatment
experiments were carried out on a single batch of the
>100xl0 3 fractions. Trypsin (type XI, Sigma, UK) treatment
was similarly carried out on a single 50;J1 batch of >100xl0 3
extract that was diluted 100-fold and divided into two, one
half treated with lOO^gmP1 of trypsin for 30min at room
temperature (24°C), the control half kept at room temperature for 30min. Trypsin-treated and control extracts then
went through a series of concentration and dilution cycles (to
dilute out the trypsin >100 fold) on centricon-100 membranes
before microinjection.
Microinjection, electrophysiology and calcium
measurements
Eggs were microinjected with broken tipped micropipettes
that were also used to record membrane potential (Swann et
al. 1989). Micropipettes were inserted into eggs by overcompensation of the negative capacitance on the preamplifier that
was being used to monitor membrane voltages and which was
in electrical contact with the pipette solution. To inject
solutions, pressure was applied to the back of the pipette
through a 50 ml hand-held syringe connected via plastic tubing
to a pipette holder with a side port. In some cases lOmgml
fluorescein dextran (35000 Mr, Sigma) was added to the
sperm extract and the cell fluorescence was measured after
injection with a photomultiplier tube to quantify the injection
volume (Lee, 1989). For experiments with indo-1, the
injection volume was estimated by measuring the displacement of cytoplasm by the injected solution with a graticule on
the eyepiece of the microscope and comparing this with
diameter of the egg. Injection volumes are generally
expressed as the percentage of the egg volume which is taken
as 200pi. Membrane voltages were recorded, via an amplifier
(Dagan 8700), on an oscilloscope and chart recorder.
Membrane potentials during fertilization were recorded with
micropipettes filled with 3 M KC1 (40-70MQ resistance).
Hyperpolarizing current pulses were injected into eggs to
monitor membrane resistance or to elicit action potentials
(with the preamplifier in bridge balance mode).
For intracellular calcium measurements, eggs were injected
with 1-2 pi of a solution containing 10 mM indo-1 potassium
salt (Molecular Probes, USA) and 0.12 M KC1 and then left
for 5-10 min before the start of recording. Fluorescence was
measured with a Leitz Diavert inverted epifluorescence
microscope (360 nm excitation, 420 nm and 490 nm emission)
and two 9924B photomultiplier tubes connected to current-tovoltage converters (Thorn EMI, UK). Analogue voltage
signals were collected, stored and analyzed on computer using
the UMANS system (C. Regen, PO Box 361, Urbana, IL
61801, USA). Ratios (420nm/490nm) of 0.13 and 0.31 were
estimated to correspond to Ca2+; concentrations of 100 nM
and 1 iiM respectively.
Results
Cytosolic sperm extracts trigger repetitive HRs
Fig. 1A and B show that the microinjection of cytosolic
extracts prepared from epididymal hamster sperm or
ejaculated boar sperm induced a series of repetitive
HRs in hamster eggs similar to those seen at fertilization (Miyazaki, 19886). HRs induced by cytosolic
extract injection began during the injection period and
continued at regular intervals thereafter. Where
lengthy recordings were made the series of HRs
persisted for over one hour after injection. The factor in
sperm that triggered these repetitive HRs has a high
molecular weight. When sperm extracts were fractionated using ultrafiltration membranes, injection of the
high molecular weight fraction (molecules >100xl0 3 )
consistently triggered repetitive HRs with a delay of
5-60s (Fig. 1C and Table 1). In contrast, the injection
of the smaller molecular weight cytosolic fraction
Sperm factor mimics fertilization 1297
OT
-60mV-L
lmin
B
Or
-6OmV-llmin
OT
-5Omv-L
. prm
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v
lmin
OT
-5Omvi
Fig. 1. Repetitive hyperpolarizing membrane responses (HRs) in hamster eggs caused by microinjecting cytosolic sperm
fractions from either (A) epididymal hamster sperm, or (B) ejaculated boar sperm. (C) is a membrane potential recording
from an egg that showed repetitive HRs after injecting >100xl0 3 fraction of boar sperm extract, and (D) is a recording
from an egg that showed a single HR after injecting <100xl0 3 fraction from the same batch of extract as in (C).
Injections, made during the horizontal line, were 3 % , 9%, 3 % and 10% of egg volume in A-D, respectively. Small
hyperpolarizing current pulses, used to monitor membrane resistance in B-D, were 0.1 nA every 10s.
Table 1. The effects of injecting different sperm extracts upon the hyperpolarizing responses (HRs) in
unfertilized hamster eggs
Extract
>100xl0 3 fraction
<100xl0 3 fraction
100 ^<g ml"1 trypsin treated
control for trypsin
60°C heat treated
control for heat treatment
Number of eggs
injected
Mean inj. vol.
(% of egg vol.)
Number of HRs/
lOmin
9
9
8
5
8
7
3.8±1.6%
4.9±2.8%
6.613.8 %
4.7±1.0%
3.8±1.6%
3.4±1.0%
4.9±1.3
0.3±0.5*
0
5.0±1.0
0
5.1 + 1.8
Trypsin and heat treatments were on >100xl0 3 fractions of boar sperm extract. * Three eggs showed a single HR at the time of
injection, six others did not show any HRs. Data are the means±s.D.
(molecules <100x 103) only caused at most a single HR
immediately upon injection (Fig. ID and Table 1), and
repetitive HRs were never seen. The frequency of HRs
triggered by a given injection volume varied with
different batches of extract that were prepared.
However, in all cases for each preparation, or batch, of
extract the frequency of HRs was greater for larger
injection volumes (Fig. 2). The factor in the sperm that
triggered HRs appeared to be sperm specific and to act
only from inside the cell. Injection of foreign cytosol
obtained from brain or liver extracts failed to trigger
HRs (data not shown). Perfusing sperm extract on the
external surface of eggs, in a manner similar to that
used for serotonin (Miyazaki et al. 1990), failed to
trigger HRs in 8/8 eggs.
The factor in the large molecular weight fraction that
was responsible for triggering the series of HRs appears
to be protein based. Heating the extracts for 30min at
60°C abolished HR-inducing activity (Fig. 3A and
Table 1). Extracts that were treated with lOOjUgml"1
trypsin also failed to induce HRs after injection
(Fig. 3C and Table 1). Control injections of untreated
extracts from the same batch caused repetitive HRs in
all cases (Fig. 3B and D and Table 1). As well as
causing membrane potential changes, the injection of
these large molecular weight fractions of sperm extracts
1298 K. Swann
O
7 •
6
NUMBER
OF HRs
IN 5min
that completed meiosis 2 h after injection of the ICM
alone (which failed to trigger HRs).
Fig. 2. The
effect of injecting different volumes of the
>100xl03 fraction of boar sperm extract upon the
frequency of HRs in unfertilized hamster eggs. Data are
plotted for two different batches of extract distinguished by
the open and closed circles.
Free calcium in eggs at fertilization or after sperm
factor injection
The HRs at fertilization are a result of corresponding
repetitive rises in Ca2+i (Miyazaki, 19886). Fig. 4 shows
Ca2+j measurements in single hamster eggs that had
been injected with the fluorescent calcium indicator dye
indo-1 (Grykiewicz et al. 1985). Injection of the
>100xl0 3 fraction of boar sperm cytosol causes a series
of repetitive Ca2+j transients in indo-1-loaded eggs
(Fig. 4A). These repetitive Ca2+j increases are similar
to those occurring during in vitro fertilization with
hamster sperm (Fig. 4B). Similar changes in free
calcium as measured by indo-1 fluorescence were seen
in four other sperm extract injected eggs and three
other fertilized eggs. These data confirm that the
injection of sperm extracts causes repetitive rises in
Ca2+j similar to that seen during fertilization.
activated hamster eggs. The earliest signs of activation
of mammalian eggs are the resumption of meiosis and
the emission of the 2nd polar body (Whittingham,
1980). Many eggs did not survive after withdrawal of the
blunt pipettes used to inject sperm extracts. Nevertheless, when eggs were stained with Hoechst 33342 and
examined 2h after injecting 1-10 pi of boar sperm
extract, 12/14 eggs that survived the injection procedure had completed meiosis and emitted a second
polar body. This compared with only 1/11 control eggs
Characteristics of HRs triggered by the sperm factor
compared with fertilization
One characteristic of HRs at fertilization is their
resistance to inhibition by phorbol esters. It has been
shown that although injecting GTP analogues, or
applying serotonin externally, triggers several repetitive
HRs in hamster eggs (Miyazaki, 1988a; Miyazaki et al.
1990), such G-protein-mediated HRs are blocked by
incubating eggs in the phorbol ester, tetradecanoyl
phorbol 13-acetate (TPA) (Swann et al. 1989). In
o
5
o
4
o
o
3
o
2
1
2
3
%
4
5
EGG VOWME
6
7
8
10
INJECTED
OT
Imin
B
OT
•y-"T"~T
-50mV
Imin
OT
-50mV..
Imin
OT
r|T
-50mvl
T'"|
TTrmrrrm
Fig. 3. Repetitive HRs in eggs
injected with >100xl0 3
fraction of boar sperm extract.
(A) Recording from an egg
that failed to show HRs after
heat treatment after a 2 % egg
volume injection and
(B) Recording from an egg
that showed HRs after being
injected with a 3 % injection of
extract from the same batch as
in A. (C) An egg that failed to
show HRs after being injected
with 5 % egg volume of the
trypsin-treated extract and
(D) a control egg that showed
repetitive HRs after 5 %
injection of control extract.
Sperm factor mimics fertilization
1299
0-45
01
t
o
o
»—
<
0 45
B
0
mm
contrast, HRs at fertilization are not blocked by TPA.
Fig. 5A shows that sperm factor injection triggered
repetitive HRs in the presence of 100 nM TPA. The
batch of sperm extract used in these experiments caused
8.3±2.3 HRs/lOmin (n=4) in control eggs and 9.4±2.6
HRs/lOmin (n=5) in TPA-treated eggs (means and
S.D.). Since the same stock of TPA blocked HRs in
GTPy-injected eggs (Swann et al. 1989), these data
again suggest an identity between the HRs at fertilization and those after sperm extract injection.
Another characteristic of a fertilized hamster egg is
the increased sensitivity of the egg to calcium-induced
calcium release (CICR) (Igusa and Miyazaki, 1983).
This can be demonstrated in hamster eggs by artificially
generating action potentials that cause calcium influx
(McNiven et al. 1988). This method is analogous to that
used to show increased CICR in caffeine-treated
sympathetic neurons (Kuba, 1980). Fig. 5B shows that,
after injecting a small amount of sperm factor, 5-10
action potentials triggered a premature HR in a
medium containing 5 mM external calcium. Under these
conditions, 5-10 action potentials also triggered premature HRs in fertilized eggs (Fig. 5C). However, up to 30
Fig. 4. Indo-1 fluorescence
(emission ratio 420 nm/490 nm)
as a measure of intracellular
calcium in single hamster eggs.
In A, the egg was injected, at
the arrow, with 1-2 pi of the
>100xl0 3 boar sperm extract
and, in B, the egg was
inseminated 2-3 min before the
start of the record. Similar
repetitive changes were seen in
four other sperm-extractinjected eggs and three other
fertilized eggs.
action potentials never caused HRs in unfertilized eggs
(Fig. 5D), or in caffeine-treated eggs (Swann, unpublished observations). These data suggest that sperm
factor injection, like fertilization, increases CICR
within the egg (Igusa and Miyazaki, 1983).
Discussion
The present results show that sperm cytosol contains a
cytosolic protein factor that induces repetitive rises in
Ca2+; and HRs in hamster eggs that imitate fertilization
and lead to egg activation. Previous microinjection
experiments in marine invertebrate and mammalian
eggs have suggested that sperm cytosol contains an eggactivating factor (Dale et al. 1985; Stice and Robl,
1990). The experiments in rabbit and mouse eggs also
suggested that the sperm factor was protein based (Stice
and Robl, 1990). However, in these activation experiments, the solutions that were injected contained
significant levels of calcium. Calcium can itself cause
activation and may have contributed to the observed
effects in combination with proteins from the sperm
(Stice and Robl, 1990). This problem has made it
1300 K. Swann
Imin
OT
B
\J
Imin
D
OT
10
20
30
V
Fig. 5. (A) Repetitive HRs after sperm factor injection (at the horizontal bar) into an egg bathed for 15 min before
injection in M2 medium containing 100 nM TPA. A slight hyperpolarization that developed later in the record appeared to
be due to a small increase in membrane resistance. (B) After injecting sperm extracts (~0.2% egg volume), generating 9
action potentials 102 s after the previous HR (with conditioning hyperpolarizing pulses) triggered a premature HR since
HRs otherwise occurred at regular intervals of 150-160 s. Generating action potentials (with 1-2 nA conditioning current
pulses) at least 30 s before the next expected HR, caused similar premature HRs in four other sperm-extract-injected eggs.
(C) A premature HR triggered in a fertilized egg by 6 action potentials at 40 s after the previous HR (within a series of
HRs with a 70-80s interval). Similar results were seen in five other fertilized eggs. (D) One of five unfertilized eggs that
failed to generate an HR after a series of 10, 20 or 30 action potentials. The time scale is the same for B, C and D, small
hyperpolarizing current pulses in A and C are 0.1 nA. Conditions were as in Fig. 1 except that in B-D eggs were bathed in
5mM calcium medium (Igusa and Miyazaki, 1983).
difficult to draw any clear conclusions about the
existence of a cytosolic sperm factor.
In the present experiments, calcium in the injected
solutions did not contribute to the imitation of
fertilization. Sperm homogenates were prepared in a
buffer that contained EGTA and effective batches had
free calcium concentrations of around 100 nM. The
>100xl0 3 fraction of sperm extracts also clearly
contained low calcium since it caused HRs with a delay
after injection; HRs occur immediately upon injection
of calcium-containing solutions with a threshold for free
; M (Igusa and Miyazaki, 1983,
calcium of less than 0.7 U
1986). In any case, in the present experiments, the
presence of calcium in the injection solution is
immaterial. Sustained repetitive HRs in hamster eggs
cannot be triggered by injecting calcium in single, or in
small repetitive pulses (Igusa and Miyazaki, 1983). The
ability of sperm extract injection to enhance CICR in
the egg is also impossible to mimic by calcium injection
since unfertilized eggs otherwise show a decrease in the
sensitivity of CICR after one or two HRs (Igusa and
Miyazaki, 1983; McNiven et al. 1988). The repetitive
HRs in hamster eggs, therefore, represents an assay of a
sperm factor that is not open to the potential artifacts of
calcium contamination.
A problem with attempts to demonstrate a sperm
factor is that it is difficult to conclude from activation
data alone that the sperm actually uses a given factor at
fertilization. Many early morphological signs of parthenogenetic activation are indistinguishable from
activation at fertilization (Whittingham, 1980; Jaffe,
1983). In many non-mammalian species of egg even the
electrical and Ca2+j changes are similar at fertilization
and during parthenogenetic activation (Hagiwara and
Jaffe, 1979; Jaffe, 1983). Any factor derived from sperm
could then fortuitously activate eggs but play no role in
fertilization (Swann and Whitaker, 1990). These reservations do not seem applicable to the present experiments. The sustained membrane potential and Ca2+j
changes during hamster fertilization represent a stereotypic response that is not mimicked during parthenogenetic activation, by injecting G-protein activators,
calcium solutions, or by applying calcium ionophores
(Igusa and Miyazaki, 1983; Eusebi and Siracusa, 1983;
Swann etal. 1989; Miyazaki etal. 1990). Thefindingthat
sperm extracts cause repetitive Ca -, increases and
Sperm factor mimics fertilization 1301
HRs in hamster eggs with characteristics that are
unique to the fertilization response, therefore, provides
the clearest evidence that during fertilization the egg is
activated by a sperm factor introduced into the egg
cytoplasm after gamete membrane fusion (Loeb, 1913).
The injection of sperm extracts triggered HRs with a
frequency that varied depending upon the amount
injected. In zona-free fertilization experiments in
hamster eggs, the number of fused sperm determines
the frequency of HRs (Miyazaki and Igusa, 1981). The
reason for this frequency dependence may be because
more penetrating sperm bring more sperm factor into
the egg. The frequency dependence of HRs can be used
to assess the amount of factor contained in sperm.
Different batches of extracts showed variable activity in
inducing HRs, for reasons that remain unclear.
Nevertheless, with injections equivalent to 5-10 sperm
and with the most active batches a frequency of HRs
was triggered (in Fig. 1A and B) that is comparable to
that seen during polyspermic fertilization with 5 or
more sperm (Miyazaki and Igusa, 1981). This suggests
that sperm cytosol does contain enough of this HRinducing factor to cause the response at fertilization.
The finding that sperm extracts from boars were as
effective in triggering HRs as those from hamster is
consistent with previous experiments. Zona-free hamster eggs can be fertilized by sperm from many different
mammalian species, including pigs, and the penetration
of foreign sperm has been shown to trigger HRs in eggs
similar to those seen during hamster sperm penetration
(Hanada and Chang, 1972; Pavlok, 1981; Igusa et al.
1983).
Important questions remain about the identity and
mechanism of action of the sperm factor, as well about
its localization in the sperm. Given that the current data
suggest it is protein-based, it may have a specific
enzyme activity. The factor could generate InsP3 from
within the egg cytoplasm because sustained InsP3
injection can cause repetitive HRs in hamster eggs
(Swann et al. 1989). However, the insensitivity of the
factor-induced HRs to phorbol esters makes the
involvement of GTP-binding proteins uncertain.
Another possibility is that, once inside the egg, the
factor affects calcium stores directly to enhance CICR.
The enhancement of CICR by caffeine can lead to
oscillations in Ca2+j in muscle and nerve cells that
resemble those seen in hamster eggs (Endo et al. 1970;
Kuba, 1980; Berridge and Gallione, 1989). Since
caffeine does not increase CICR in hamster eggs, it will
be important to identify how the sperm factor enhances
CICR and whether this involves InsP3 production.
Apart from the hypothesis for fertilization the
current data also suggest the existence of a novel signal
transduction mechanism involving the transfer of a
cytosolic protein factor from one cell to another. Since
the sperm factor does not appear to act upon cell
surface receptors, it may represent a new class of
intracellular calcium regulator that may play a role after
fertilization. Early embryos and some somatic cells
display Ca 2+ ; transients during the cell cycle with no
apparent external stimulation (Poenie et al. 1985;
Whitaker and Patel, 1990). The characterization of the
sperm factor should help explain both how a sperm
fertilizes an egg as well as provide new insights into
intracellular calcium regulation.
I thank David Whittingham and Michael Whitaker for
advice, support and criticism, Dave Gilbert and Melanie
Monteiro for technical assistance and Paula Baker of the
Cattle Breeding Centre, Shinfield, for supplies of boar semen.
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(Accepted 10 September 1990)