Abstract
Two competing models have been proposed to explain the
mechanism by which a sperm activates an egg at fertilization. One
postulates a receptor while the other relies on fusion as the primary means
of signal transduction. This study looked for a soluble activation factor
which could act as a diffusible messenger between the fused sperm and
egg. S purpuratus sperm were lysed with 1% Triton to bring all
solubilizable proteins and factors into solution. In a second set of
experiments, sperm were permeabilized with hypotonic solution and then
suspended in an extraction buffer of high salt and pH 8, designed to bring
only soluble cytoplasmic elements into solution. Both sets of extracts were
microinjected into eggs and activation was monitored by looking for the
rise of the fertilization membrane. Under the various conditions used in
this study, no soluble fraction from sperm activated the eggs when
microinjected.
Introduction
We tend to think of fertilization as the beginning of the development
of a new organism, despite the fact that reproduction is a cyclical process,
as that old adage about the chicken and the egg illustrates so aptly. But
fertilization does give the impression of a beginning, even on the molecular
level. This is especially true when we look at sea urchins. Eggs from these
animals remain in a relatively quiescent metabolic state until fertilization.
Fusion of the two gametes and the transfer of paternal genetic material is
accompanied by the reinitiation of the cell cycle and an increase in egg
metabolism, DNA and protein synthesis.
As a problem of signal transduction, this activation of the egg is
compelling, since so many aspects of the cell are affected. Two very early
events which are thought to be central to reinitiating protein synthesis and
metabolism are the wave of intracellular calcium release which crosses the
egg and the increase in cytoplasmic pH. Calcium especially has been
manipulated in experiments with fertilization or parthenogenic activation
(Whitaker and Steinhart, 1985; Swann and Whitaker, 1986; Turner et al,
1986; Steinhart and Epel, 1974; Steinhart et al, 1977; Brandriff et al, 1975).
These seem to demonstrate its importance as a fundamental component of
egg activation. But the calcium rise itself is not the initiating signal;
inositol trisphosphate (IP3) seems to be the messenger responsible for
releasing calcium from internal stores (Swann and Whitaker, 1986, Swann
et al, 1987; Busa et al, 1985). There is one report claiming that IP3 itself
could be released from the sperm after fusion and so activate the egg
directly (Iwasa et al, unpublished). Others have observed a turnover of
egg plasma membrane inositol lipids during early fertilization (Ciapa and
Whitaker, 1986) and propose that IP3 is a second messenger. If this is
true the question becomes how the sperm initiates IPa generation. In any
case, the question which is still to be answered asks about the nature of
the initiating signal of activation.
Two models are generally presented to account for the mechanism of
egg activation. The first postulates a receptor-mediated signal between
the sperm and the egg. The second proposes a diffusible activating factor
which enters the egg after fusion establishes cytoplasmic continuity. In its
most common version, this model draws an analogy with the IP second
messenger system in other cells (Berridge and Irvine, 1984). The
proposed receptor would activate a G-protein which in turn activates
phospholipase C to cleave membrane phosphatidyl inositols into IP3 and
diacylglycerol (figure 1). Experiments in sea urchins (Swann and
Whitaker, 1986; Turner et al, 1986) and Xenopus (Busa et al, 1985) have
demonstrated that microinjected IPa will cause calcium release and the
calcium-dependent cortical granule exocytosis. In addition, the
cytoplasmic concentration of IPa increases during fertilization (Ciapa and
Whitaker, 1986), suggesting that IPa is a relevant messenger in vivo.
The existence of a receptor and G-protein are much more dubious.
Kline et al (1985) found that Xenopus oocytes expressing muscarinic
acetylcholine or serotonin receptors could be artificially activated by
neurotransmitter. Since the receptors couple to G-proteins, their ability to
trigger activation indicates the existence of endogenous G-proteins with
the ability to cause calcium release. However, the physiological relevance
of the experiment is unclear. Experiments which tried to manipulate the
G-protein with toxins or GTP analogs have given ambiguous results.
Microinjection of GTP-B-S, a G-protein inhibitor, will inhibit cortical granule
exocytosis as a result of fertilization by sperm (Turner et al, 1986). The
same injections will also inhibit cortical granule exocytosis by
microinjected IP3. As the inhibitor of the G-protein must be acting after
the release of calcium, the G-protein itself cannot therefore be responsible
for the calcium wave (Whitaker and Crossley, in press; Turner et al,
communication). The receptor model is certainly attractive for its
elegance: a receptor mediated event sets off a self-propagating wave of
1P3 generation and calcium release while DAG activates protein kinase C,
raising pH and perhaps even causing membrane fusion. However, the
failure to demonstrate a critical role for G-proteins early in activation in
vivo seriously undermines the strength of this model.
In the alternative model, fusion of sperm and egg allows the
diffusion of an activating factor into the egg cytoplasm. This factor could
act in a number of ways, either acting locally to set off the IPa cascade or
to release some egg factor, or by diffusing itself to release calcium from the
egg’s internal stores (figure 2). An increase in membrane capacitance can
be measure almost simultaneously with the sperm-gated
hyperpolarization, that is to say from the initiation of sperm-egg contact
(McCulloh and Chambers, 1986). This implies that fusion of the plasma
membranes is the first event of fertilization. However, dye transfer and
EM experiments first detect fusion 4 to 8 seconds after the initial contact
(Hinkley et al, 1986; Longo et al, 1986) implying that cytoplasmic
continuity is not necessarily the first event. Whitaker et al (1989) try to
resolve the discrepancy which a complex model involving transient fusion
which becomes permanent later by the action of calcium, released locally
by the diffusible activating factor from the sperm. Despite the intricacies
of their model, it does not constitute evidence for the existence of the
putative activating factor. If an activating factor exists, its activity could
be found in and presumably purified from solubilized sperm cytoplasm.
Isolating an activity would be the best possible demonstration of the
model.
Several small molecules, including calcium, cGMP and IPz are known
to activate eggs when microinjected, but there are reasons to believe none
of these are the relevant diffusible messenger. The biggest problem with
using any of these in a model is the high concentration at which they
would need to be present to play the role of diffusible activator. For
example, Whitaker predicts that cGMP would have to be present in sperm
at approximately 1M to be effective (Whitaker and Crossley, in press).
There have been two reports of activity isolated in sperm extracts:
one in hamster (Swann, 1990) and the other in sea urchin (Dale et al,
1985). Swann has reported the isolation of a sperm protein which will
cause microinjected eggs to reproduce the occilations of calcium
characteristic of activated hamster eggs. The experiments described here
explored various protocols for solubilizing sperm in an attempt to describe
a sperm-derived activating factor. Based on the results discussed above,
this search focussed on a protein which could act as a diffusible messenger
from the sperm cytoplasm to the egg cytoplasm.
Materials and Methods
Gametes: Eggs were collected by shaking and then inverting S
purpuratus females over sea water. The eggs were stored in a 16° C water
bath with gentle stirring and were used in experiments within four hours
of collection. Sperm were collected dry from males microinjected with a
small amount of 5M KCl into the body cavity. Sperm were stored at 4° C
and were generally used within 24 hours of collection.
Extractions: Sea urchin sperm cannot be homogenized easily to
release cytoplasmic proteins. Instead, two methods of extraction were
used to bring soluble molecules into solution. The "brute force" method
employed a 1% Triton lysis buffer while the "finesse" method
permeabilized sperm in hypotonic but inert conditions and then extracted
presumably only cytoplasmic proteins in a high salt, high pH buffer.
I will
briefly outline the strategy of each method here and, as the primary
results of the experiments were modifications in procedure, the next
section will trace through the procedural variations tried along with the
effects of each experiment.
To begin all Triton extraction experiments, 4 to 8 ml sperm were
suspended in glycine rinse (see table 1 for composition of all buffers) and
then centrifuged. The rinse was repeated three times to remove sea
water, semen proteins and salts which might contaminate the extraction
buffer. Lysis buffer was then added to the sperm and the suspension was
mixed slowly for 2 hrs at 4 C. Following lysis, the sperm suspensions were
centrifuged at 100,000g for 1 hr at 4° C to remove DNA and undissolved
components. The supernatant was collected and became the crude extract
for microinjection.
Later experiments tried to purify and concentrate the extract for
reasons which will be discussed below. The first approach to concentrating
the extract was to separate out the small molecules on a Sephadex G-25
desalting column eluted with ammonium bicarbonate, and then to
evaporate the elutant to leave a protein pellet to be redissolved in a few
hundred microliters of microinjection buffer. Twenty drop fractions were
collected and a spot assay (5ul from each tube spotted on Whatman filter
paper and developed five minutes in Comassie blue) performed to idetify
tubes with proteins. Those with protein were pooled, dried and
redissolved in a few hundred microliters of fluorescent microinjection
buffer. The second method concentrated extracts by adding ammonium
sulfate to 90% saturation to precipitated proteins. The precipitate was
collected, redissolved in microinjection buffer and then dialysed overnight
to remove the excess ammonium sulfate.
To permeabilize sperm without detergent, 1 ml dry sperm was
diluted into 12 ml of 25% sea water (75% distilled water) and then 8 or 10
mi 1 mM MgCl, 10 mM phosphate, pH 6.5, was added to completely swell
at least 90% of the sperm. Suspensions were centrifuged at 3000g for 5
minutes in a Sorvall centrifuge and the supernatant removed. The sperm
were re-suspended in a high salt, pH 8 buffer (table 1, trial 2) to solubilize
proteins. Sperm were mixed slowly at 4 C for 30 minutes and then spun at
3000g in an Eppendorf variable centrifuge. Supernatant was collected and
carboxyfluoresceine added to aid visualization during microinjection.
Microinjections: Jelly coats were first removed from the eggs by
passing them through a 90 micrometer Nitex mesh filter three times. De¬
jellied eggs were stuck to plastic Petri dishes coated with protamine sulfate
(3 or 5 mg/ml in distilled water). Dishes were then placed on a water-
cooled microscope stage (16° C). The sample to be microinjected was
loaded into a pipet pulled from 1 mm capillary tubes at 15 amps on à
Nargishe micropipet puller (Scientific Instruments Inc). Pipets were
mounted on a micromanipulator and pressure was delivered using a hand¬
built air valve at 35 psi for approximately 100ms. This generally resulted
in a delivered volume equal to about 1% of the egg's volume. Eggs were
viewed on a Zeiss epifluorescence microscope and successful
microinjections were monitored using the fluorescein filter set. The assay
for activation in the experiments presented here was the rise of the
fertilization membrane within about a minute of injection. Only eggs in
which the pipet entry did not itself cause visible damage were counted in
these experiments. Every experiment included the control injection of the
relevant buffer without sperm proteins. As a positive control for
fertilizability of the eggs, including those which had been microinjected,
each plate of eggs was fertilized by viable sperm after microinjections
were completed with that batch. In general, no batch of eggs remained
under the microscope for longer than 45 minutes.
Results
None of the extraction procedures described above resulted in a
solution which activated microinjected eggs. In this section I want to
explain the changes made in the protocol as the study went along and at
the same time look at the actual numbers of injections versus activations,
which are summarized in table 2.
Triton Extacts The composition of the original 1% Triton buffer
contained glycerol and inhibitors of proteases and phophatases to protect
proteins from degradation due to enzymes released during lysis and to
increase stability of proteins. Seven microinjections of extract resulted in
10
one partial activation, while five injections of the buffer alone, raised no
membranes. The presence of a partial activation indicated that a higher
concentration might be necessary to find the activity, since low
concentration injections of IPa and cGMP result in partial membranes also
(Ciapa, personal communication).
The first concentration was performed in the speed-vac. Glycerol
was left out of the lysis buffer (Triton 2 in table 1). The extract was
desalted on a Sephadex G-25 column eluted with 10 mM ammonium
bicarbonate, which was expected to evaporate during drying. The dried
proteins were redissolved in the microinjection buffer. As table 2
illustrates, the lysis buffer resulted in low background partial activation,
while injections of dried G-25 fractions without proteins resulted in six
non-activated eggs. The extract injections had encouraging results: in one
set of 8 injections, three were partially and one was fully activated. In a
second set of injections, six of nine eggs had strangely flattened sides
where they had been round originally. This happened again later with one
of the hypotonic extractions and its interpretation is uncertain. The shape
of the eggs resembled that of partially activated eggs but they lacked the
membrane.
In order to see whether the extract could be concentrated by an
additional ten-fold, as Ciapa's experiments with IPz indicated might be
necessary, the procedure was repeated with greater volumes of sperm.
11
For these next two experiments extracts were lyophilized instead of
evaporated in the Speed Vac to prevent denaturation by heating.
Unfortunately, after this treatment, the proteins did not redissolve easily,
making microinjection impossible due to the particulate nature of the
sample.
The final approach to concentrating Triton extracts used ammonium
sulfate salt added to 90% to precipitate and thus separate proteins from
the lysis buffer. The precipitate was then resuspended in a small volume
of the microinjection buffer and dialyzed to remove excess salt. In nine
microinjections, one activated, which seemed due to the injection itself, not
the buffer.
Hypotonic Extraction The second extraction strategy did not result
in a very concentrated solution. Presumably, however, it was enriched in
the activating factor. These experiments were based on Tilney's (1979)
description of hypotonic solutions which swell the sperm without lysing
them, and his observation that actin in the acrosomal vesicle is inert if
swollen sperm are left at pH 6.5, but goes into solution at pH 8. The first
two experiments used 10 mM EGTA in the extraction buffer (Trial 2,
compl in table 1) which was added after removing the hypotonic solution.
While many eggs were injected without activation, the general rate of
fertilization when sperm were added was poor among the injected eggs.
The poor fertilization could be attributed to too high a concentration of
12
EGTA, which buffers calcium release during the fertilization events, and so
prevents the calcium-dependent cortical granule exocytosis. No
concentration of EGTA could serve its original function of effectively
buffering the extra calcium from hypotonic solution and sperm which
remained in the pellet and at the same time not be too high for
microinjection itself (in which case it would buffer the activation calcium
wave). Therefore the procedure was modified to remove excess calcium
by rinsing the sperm three times in the hypotonic solution (each
resuspension followed by centrifugation in the Sorvall) before adding the
extraction buffer, now at 0.1 mM EGTA (trial 2, comp 2 in table 1). Even
under these conditions microinjection did not seem to activate the eggs,
although the high background activity in controls made it difficult to know
if a low level of activation activity was present. Twenty one injections of
extract included six partial membranes, but even the microinjection buffer
itself gave 30% activations with this batch of eggs.
Discussion
There are at least three possible reasons why the procedures
described above have failed to demonstrate the existence of a sperm¬
derived, diffusible activating factor. Most obvious is that the activity may
not exist. However, the methods have potential weaknesses which may be
responsible for the negative results. First, it is possible that the
13
procedures used failed to bring the relevant factor into solution. Second,
the relevant factor may be in the extracts, only in concentrations too low
for the microinjected solution to imitate the effects of the sperm during
fertilization. Finally, the purification and concentration which was
attempted may have resulted in denaturation and loss of activity.
It is possible that the factor was not pulled into solution. If, for
example the factor is transferred between the two gametes plasma
membranes and so is lipophilic, not hydrophilic. These experiments
searched for proteins rather than lipids because Swann’s results in
hamster indicate that an activating protein can be isolated from the sperm
in that system. At the same time, it is important to remember that species
vary, so sea urchins may not use a protein.
There is ground to criticize the hypotonic permeabilization as likely
not to have brought the factor into solution. Although at least some
number of proteins were extracted, there was also loss of proteins in the
rinses which preceded the extraction buffer. It is interesting to note that
the two reports of isolated sperm factors in the literature use very simple
techniques. Swann (1990) extracts directly into hypotonic solution and
Dale et al (1985) isolate their product in distilled water. I did not repeat
the Dale et al procedure partly because proteins tend not to be stable in
distilled water but largely because it seems likely that there is enough
calcium in the final injection medium to cause the activation they
14
observed.
Even if the extraction procedure is successful, activation may not be
observed if the factor is not present in high enough concentrations. Sperm
are very small cells with little cytoplasm and would therefore be expected
to contain only a small amount of factor. However, sperm have an
advantage over microinjection in acting very locally. Fusion followed by
diffusion would result in a very high effective concentration immediately
adjacent to the plasma membrane. In contrast, the pipet tip goes farther
into the cell than adjacent to the membrane, and the injection pressure
sends the microinjected solution in towards the center of the cell. It
follows that a higher concentration of material would therefore have to be
microinjected in order to achieve the same effective concentration at the
membrane as a single fusing sperm achieves. Assuming that the entire
sperm head contains the activating factor and that our procedure resulted
in 100% recovery, we calculated that the microinjection in the first Triton
extraction could be equivalent to 10 to 30 sperm. Since neither
assumption is true, and even this estimate is fairly low, lack of
concentration is certainly a valid consideration.
A second reason for assuming that concentration could be an
important variable is the partial activations which were occasionally
observed. The meaning of a partial activation is hard to assess. If there is
nothing wrong with the egg, so that it is capable of raising a membrane
15
through cortical granule exocytosis, then elevation of a section of the
membrane would seem an abnormal event. The abnormality of the event
is increased if the model which proposes a self-propagating cascade is
correct (figure 1). One could explain partial activation consistently if a
diffusible factor is needed above a certain concentration in order to cause
calcium release from the entire egg. In this case, a partial activation could
mean that the activating factor is simply not present in a high enough
concentration to release calcium beyond a given distance from point of
fusion or microinjection. Microinjection of both IPa and cGMP will result in
primarily partial membrane elevation if the concentration is ten-fold less
than that giving all complete activation (Ciapa, personal communication).
Thus if we believe in a a diffusible activator, finding partial activation
urges that the extract be concentrated in the hopes of increasing the
amount of factor above the threshold.
A second explanation for partial activation is the existance of not
one but two or even several factors which act in different ways to trigger
activation. For example, there may be a locally acting factor which causes
the partial activation while a second factor is needed to begin the self-
propagating wave. In this case, our extract may contain only one of the
necessary components of full activation. Again concentrating the solution
might result in our finding full activity, especially as multiple factors could
exist in smaller amounts and achieve a result through amplification.
16
Desalting followed by lyophilization and ammonium sulfate
precipitation were used to concentrate the extract. Using lyophilization
resulted in concentrations of 19 and 23 mg/ml protein in the injection
buffer, a significant increase. However, this may not have been enough.
The hypotonic permeabilization resulted in a much more dilute extract at
77 mg/ml, but it probably did not contain nuclear proteins, such as
histones, or membrane proteins, making it perhaps more concentrated in
the relevant cytoplasmic factors. In order to concentrate these extracts
further, the procedure would have to be modified to avoid concentrating
KCl and the difficulties in lypohilization solved first.
Lyophilizing, while it avoided heat, presented additional technical
problems since the activity could be lost by denaturation with the loss of
salt. The desalting column was eluted with ammonium bicarbonate so that
the proteins could be dried and concentrated without also increasing salt
and buffer concentrations. Unfortunately, the dried proteins did not fully
redissolve in the buffer, indicating that in fact there could have been
significant denaturation. The ammonium sulfate was used as an
alternative method of concentrating without drying; yet that extraction
also failed to activate microinjected eggs.
Despite the lack of activation in the one ammonium sulfate
experiment, work could be done to improve the procedure. The extract
was made with a small volume of sperm and perhaps a greater volume,
17
followed by fractionation as opposed to mass precipitation, could give a
cleaner extract with more likelihood of activating eggs if the factor exists.
A second potential improvement could be the use of detergents other than
Triton. Not all detergents are alike in what they will solubilize and
perhaps octylglucoside or another detergent would successfully solubilize
the activity where Triton had failed. Such a detergent would have the
additional advantage of being removable through dialysis, a much gentler
way of removing small molecules than the desalting column. Given the
possibility that the model might be wrong, in future experiments, it would
be worthwhile to try applying extracts to the outside of eggs from which
the vitelline layer had been removed.
FIGURE LEGEND
Figure 1: Model for activation of the egg through a G-protein-linked
receptor. Binding of sperm to the putative receptor activates a G¬
protein which diffuses through the membrane to activate
phospholipase C. This enzyme cleaves polyphosphatidyl inositol in
the membrane to generate IPa and DAG. IP releases calcium from
internal stores while DAG activates protein kinase C. Calcium feeds
back to increase PLC activity and, together with the rise in pH,
increases protein synthesis and causes the other events of activation.
Figure 2: Three models for the action of a diffusible factor in activating
the egg. Such a factor could trigger a self-propagating cascade, such
as the cascade proposed to accompany receptor mediated activation
(figure 1). Alternatively the factor itself could diffuse through out
the egg and cause calcium release directly. Finally, the factor might
act locally to release a greater amount of a diffusible factor from the
egg. For example, the sperm factor might cause an amplified local
production of IPz which would then diffuse through the cytoplasm
and activate the egg.
Table 1: Composition of all buffers used in the experiments described.
Trial 1 refers to the experiments with Triton as the primary means
of solubilizing. Trial 2 buffers are those into which sperm were
placed for extraction after having been swollen with 1 mM MgCl, 10
mM phosphate buffer, pH 6.5.
Table 2: Results from all experiments. The experiment labelled Triton 1
was performed using the first Triton buffer (see table 1) and without
spinning the sperm suspension at 100,000 g. The second triton
experiment (Triton 2) also used the first Triton buffer, this time with
the centrifugation step. The control in both of these experiments is
the lysis buffer diluted into the microinjection buffer at the same
ratio used with the sample. Triton 3 used the second triton buffer
(see table 1) and centrifugation, followed by desalting on the
Sephadex G-25 column and then drying using the Speed Vac.
Controls here include lysis buffer and the blanks of column fractions
without proteins also dried and then redisolved in microinjection
buffer. The fourth Triton experiment also used Triton buffer 2. This
time concentration was accomplished using ammonium sulfate
precipitation at 90% saturation with the salt.
The first three experiments with hypotonic treatment, followed
by extraction buffer all used the first composition of extraction
buffer as listed in table 1. Controls here are the extraction buffer
itself, made visible by adding carboxyfluorescein directly. For all
three of these experiments the fertilization rate when sperm were
added was very low among injected eggs. Thus for the last
experiment, a second extraction buffer composition was used with
lower EGTA, while excess calcium was removed by washing.
20
Receptor Model of Activation:
plasma membrane
C
receptor
Figure 1

G-protein
phospholpase C

P3
Calcium
—polyphosphatidyl inositol
PAG protein kinase C
pH increase
cortical granule exocytosis
protein and DNA synthesis
Ihree models for activation by a diffusable factor:
direct diffusion
self-propagating cascade
Figure 2
egg factor diffusion
Table 1
Trial 1
PIPES MgCl, Glycerol Triton inhibitorsa
Butfer
KC
EGTA
—
Triton
150
50
10%
1%
yes
Triton 2
150
10
50
1%
yes
—
—
Microinjection
480
20
O.1
Trial 2
EGTA TAPS Mgoia
Butfer
KC
480
Comp1
10
480
Comp1
O.1
a: 2 mM vanadate, 10 mM sodium pyrophosphate, 10 mg/mi aprotinin, 10 mg/mi
leupeptin, 1.4 mM bacitracine, 10 mM NaF, 1 mM DTT, 1 mM PMSE
Iable 2
Experiment
Triton 1
control

Triton 2
control
Triton 3
control (lysis
buffer)
control (blanks
Triton 4
control
Hypo/Ext 1
Hypo/Ext 2
control
Hypo/Ext 3
control
Hypo/Ext 4
control
total
injections
15
17
34
6
9
16
31
16
6
21
activations
full
partial
0
3 flattened
0
1
0
5 bubbles
7 flattened
2 flattened
1
2 flattened
3 flattened
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