Is tyrosine kinase activity necessary for fertilization-induced glucose-6¬
phosphate dehydrogenase release in Strongylocentrotus purpuratus eggs?
Geetika Agrawala
Hopkins Marine Station
Stanford University
Abstract
The activity of the pentose phosphate shunt increases soon after fertilization of
Strongylocentrotus purpuratus eggs, and this increase is correlated with a change in the
intracellular distribution of glucose-6-phosphate dehydrogenase (G6PD), the first and
rate-limiting enzyme in the pentose shunt. This enzyme is found in a bound, inactive
state in unfertilized eggs and in a soluble, active state in newly fertilized zygotes. The
signaling pathway that links fertilization to the release of G6PD from the bound state is
not known. In this study, I have investigated whether tyrosine kinase activity is
involved in fertilization-induced G6PD release in sea urchin eggs.
I determined that G6PD release does not occur in unfertilized eggs treated with
bindin or antibody against the sperm receptor. These two treatments activate the
specific tyrosine kinase that phosphorylates the sperm receptor. I also found that
inhibition of general tyrosine kinase activity using the tyrosine kinase inhibitors
genistein and tyrphostin 51 does not block G6PD release in sea urchin zygotes. As
deduced by immunoprecipitation of phosphotyrosines, genistein is inhibiting tyrosine
kinase activity while tyrphostin 51 is not. These preliminary data show that those
tyrosine kinases which are inhibited by genistein are not involved with G6PD release
after fertilization. I also discuss the possible reasons why tyrphostin 51, bindin, and
anti-receptor antibody treatment may have given inconclusive data.
Introduction
The study of fertilization in sea urchin eggs has brought about many insights into
the mechanisms cells can use to change from a dormant state to an active one (Epel,
1990). Currently we know much about the primary events of signal transduction at
fertilization, however we do not know as much about what happens downstream.
The pentose phosphate shunt is an alternative pathway to glycolysis. Through
this pathway, glucose is turned into various biosynthetic products necessary for cell
growth and division including NADPH which is necessary for DNA synthesis. Swezey
and Epel (1995) showed that the shunt is activated shortly after fertilization, however
the mechanism of this activation remains unclear.
The first and rate-limiting enzyme in the pentose shunt is glucose-6-phosphate
dehydrogenase (G6PD). Activity of this enzyme is a good indicator of activity of the
entire pentose shunt pathway. G6PD is in an inactive form bound to particulate matter
in the egg before fertilization and in an active, free, soluble form after fertilization
(Swezey and Epel, 1986). While release of G6PD at fertilization does not seem to
involve any covalent changes of the enzyme itself (Swezey and Epel, 1986), its release
may be an important part of egg activation after fertilization.
Another example of cell activation leading to an increase in shunt activity is
growth factor stimulation of mammalian rat kidney cells (Stanton and Seifter, 1988).
Interestingly, in these cells G6PD is in a bound form prior to stimulation by treatment
with growth factors, and treatment causes the rapid release of G6PD (Stanton et al.,
1991). Recently, Tian et al. (1994) discovered that cells transfected with platelet-derived
growth factor receptor (PDGFR) mutants that were tyrosine kinase deficient would not
release G6PD in response to platelet-derived growth factor (PDGF), the stimulating
ligand of the receptor. Thus, in these cells G6PD release seems to require the activation
of cellular tyrosine kinases.
While tyrosine kinase activity is necessary for G6PD release in somatic
mammalian cells, the correlation has not yet been made in sea urchin eggs at
fertilization. It is known, however, that in sea urchin eggs two- to fourfold increases in
tyrosine kinase activity (Satoh and Garbers, 1985) and the production of newly tyrosine-
phosphorylated proteins (Ciapa and Epel, 1991) occur minutes after fertilization. One
protein that is found to be tyrosine-phosphorylated soon after fertilization is the 350
kDa sperm receptor (Abassi and Foltz, 1994). This protein becomes phosphorylated in
response to sperm as well as bindin, the purified ligand of the receptor (Abassi and
Foltz, 1994). Hence, there must be activation of at least one tyrosine kinase by the
sperm receptor binding its ligand. Bindin is the species-specific protein isolated from
sperm that helps sperm to attach to eggs during fertilization (Lopez et al., 1993).
Therefore, if the signal for G6PD release is downstream of the activation of this tyrosine
kinase, treatment with bindin would be a direct and simple way to test whether GéPD
release involves tyrosine kinase activity.
Along with bindin, antibodies against the sperm receptor have been shown to
prevent fertilization (Foltz et al., 1993). Assuming this means that the antibodies are
binding to the receptor, they may also be causing tyrosine phosphorylation of the
receptor. If this is true, then anti-receptor antibody treatment would also be a simple
way to determine if G6PD release requires the activation of this particular tyrosine
kinase.
In an effort to determine the general role of tyrosine kinases in the activation of
the egg after fertilization, Moore and Kinsey (1995) have studied the effects of tyrosine
kinase inhibitors including genistein and tyrphostin B42 on sea urchin eggs. Genistein,
a flavinoid, competitively inhibits tyrosine kinases at their ATP-binding sites (see
reference in Edgecombe et al., 1991), and the family of tyrphostins competitively inhibit
tyrosine kinases at their substrate binding sites (Moore and Kinsey, 1995). While
genistein and tyrphostin B42 inhibit overall fertilization-dependent tyrosine kinase
activity, they do not prevent the early events of fertilization including elevation of the
fertilization membrane; however, they do inhibit later events including DNA synthesis,
cell division, and pronuclear migration (Moore and Kinsey, 1995). While these effects
are known, the effects of general tyrosine kinase inhibition on fertilization-induced
G6PD release by genistein and tyrphostin are not known.
The purpose of this study is to determine whether a tyrosine kinase is involved
in the increased activity of the pentose shunt at fertilization of sea urchin eggs by
addressing the following questions. (1) Is activity of the specific tyrosine kinase that
phosphorylates the sperm receptor at fertilization involved with G6PD release? (2)
Does inhibition of general tyrosine kinase activity block fertilization-induced G6PD
release?
Materials and Methods
Handling of Gametes
Tinduced the spawning of Strongylocentrotus purpuratus by injecting 1-2 ml of 0.5
MKCl into the coelomic cavity, and collected eggs by placing the females with their oral
sides up onto beakers filled with sea water such that their eggs shed directly into the sea
water. Egg jelly was removed by passing the egg suspension 5-6x through an 80 um
mesh and washing 2-3x with filtered sea water (FSW). To ascertain that this method
actually removed egg jelly, I examined the dejellied eggs with colloidal ink and
toluidine blue stain. The eggs were then resuspended to a 1% suspension, which I
determined using a Bauer-Schenk graduated centrifuge tube, and kept at 16°C. I
collected undiluted sperm by pipetting them directly from the surface of the urchin.
Undiluted sperm were stored at 4°C.
Typically, the eggs were fertilized by diluting dry sperm 1:100 in FSW and then
again 1:100 into the egg suspension for a total dilution of 1:10,000.
G6PD Assay
The amount of G6PD bound to the particulate fraction was determined as
described by Swezey and Epel (1986). I hand centrifuged 1 ml of egg or zygote
suspension, resuspended it to 1 ml in ice-cold 50 mM 2-(N-morpholino)ethane sulfonic
acid (pH 6.5 at 4°0), 6 mM EGTA, and 20 mM KC, and homogenized it using an
average of 12-20 strokes of a tight-fitting dounce homogenizer. Homogenate was then
spun for 5 minutes in an Eppendorf centrifuge (-12,000 g), and the supernatant (S1) was
removed and stored at 4°C. I resuspended the pellet in ice-cold 20 mM Tris (pH 8.0 at
4°C) containing 0.1 MKCI (KC buffer) with a volume equaling S1. The high ionic
strength of the KCl buffer caused the G6PD bound to the particulate fraction to become
soluble. The suspension was then spun again for 5 minutes in an Eppendorf centrifuge
(-12,000 g) and the supernatant (S2) was removed and stored at 4°C.
1determined the G6PD activity of S1 and S2 with a spectrophotometric assay.
The assay contained 35 mM Tris-HCl (pH 8.5), 7 mM MgCl2, 1 mM G6P, 0.4 mM NADP,
and 20-50 ul of sample in a total volume of 2 ml. G6PD activity was measured as the
rate of increase in absorbance at 340 nm due to the production of NADPH. One unit of
G6PD is defined as the amount necessary to catalyze the conversion of 1 umol NADP to
NADPH per minute at 20°C.
S1 contained G6PD that was unbound in the egg cells, and S2 contained G6PD
that was released from the particulate fraction. The total amount of G6PD in the
homogenate was the sum of the two, and the percentage of bound G6PD was equal to
the percent of the total activity (activity in S1 + activity in S2) contained in S2. I assayed
each Sl and S2 twice, and calculated percentages using mean values.
1took all fertilized samples 4-5 minutes after fertilization.
NADPH Assay
The amount of NADPH produced by the egg in vivo was studied as described by
Epel (1964). 1 put 3 ml of 1% egg suspension into a cuvette and measured NADPH
production by monitoring light emission at 460 nm when the cell suspension was
excited at 365 nm. The cells were maintained in suspension with a stirrer, and the eggs
were kept at 20°C throughout measurements. I added 1 ul of undiluted sperm to the
egg suspension while it was in the fluorimeter to asses NADPH changes due to
fertilization.
Bindin
A purified S. purpuratus bindin sample was obtained from K. Foltz at UCB. The
bindin sample was kept at -80°C until needed. Prior to use, I thawed the sample at 4°C
for three days, and then spun it for 30 minutes at 10,000 rpm (12,000 g) in an SS34 rotor
at 4°C. The supernatant contained the soluble bindin whose concentration was
determined using the Bicinchoninic Acid (BCA) assay. (Smith et al., 1985). Based upon
those protein concentrations, I incubated unfertilized eggs in 0.06-60.0 ng/ ml of soluble
bindin. Ttook samples for the G6PD assay 4-5 minutes after treatment with bindin.
Antibodies Against the Sperm Receptor
Purified anti-receptor antibodies were also obtained from K. Foltz. The anti-exo
antibody was made to the extracellular region of the receptor, and the anti-cyto
antibody was made to the cytoplasmic region of the receptor. I used the anti-exo
antibody as the experimental primary antibody at a concentration of 6 ug/ml, and for
the controls lused goat immunoglobulins (Sigma) or anti-cyto antibodies as primary
antibodies, also at 6 ug/ml. Unfertilized eggs were incubated with primary antibody
on an orbit shaker for 30 to 60 minutes at 16°C, then washed twice with FSW. Goat anti¬
rabbit IgG conjugated to the fluorescein derivative DTAF at a 1:62 dilution or anti-rabbit
IgG conjugated to the rhodamine-based dye TRITC (Sigma) at a 1:200 dilution were
used as secondary antibodies. I incubated the eggs in secondary antibody for 30
minutes on an orbit shaker at 16°C, then washed them once with FSW and took
samples.
Tyrosine Kinase Inhibitors
1made up 100 mM stock solutions of genistein (ICN) and tyrphostin 51 (Sigma)
in DMSO. Ttreated unfertilized eggs with 100 uM genistein or 100 uM tyrphostin 51 for
up to 90 minutes before fertilization (see Results for incubation times for each
experiment). Controls were treated with 0.1% DMSO for equal amounts of time.
Samples of treated unfertilized eggs were taken 15 minutes before fertilization, and the
zygote samples were taken 4-5 minutes post-fertilization of the remaining eggs.
Immunoprecipitation
Immunoprecipitation was done using a protocol modified from Ciapa and Epel
(1991). I concentrated 40 ml samples of 1% suspension to 5% and treated them with 5
mM aminotriazol (ATA). ATA weakens the fertilization envelopes (FEs) and thus helps
the extraction buffer lyse the eggs. Äfter 5 minutes I fertilized the samples with a 1:2000
dilution of sperm. Thirty seconds after adding sperm, I checked the eggs under a
microscope for FE elevation. Thirty seconds later the zygote suspensions were passed
8x through an 80 um mesh, washed with 10 ml FSW, and twice with 10 ml 1 M glycine,
1mMEGTA, pH 8.0 adjusted with 1 MTris. I then resuspended the final pellet with 3.6
ml (10% suspension) of extraction buffer, made of 50 mM HEPES (pH 7.4), 150 mM
Nac, 10% glycerol, 10 mM EDTA, 1 mM MgC2, 1% Nonidet-P 40, including 2 mM
sodium vanadate (ortho), 10 mM Na4P2O7, 10 mM NaF, 10 ug/ ml aprotinin, 10 ug/ml
leupeptin, 10 ug/ml pepstatin, 50 ug/ml soy bean trypsin inhibitor, and 1 mM
phenylmethylsulfonyl fluoride. This suspension was rotated at 4°C for 30 minutes. I
spun the extracts for 10 minutes at 10,000g in an SS34 rotor at 4°C. One ml samples of
supernatant were incubated with 5 ug/ml of the anti-phosphotyrosine antibody, PY-20
(ICN) for 4 hours, rotating, at 4°C. I also incubated two controls, one using 5 ug/ ml of
non-specific mouse IgGpp (Sigma) with lysate, and the other using PY-20 with no lysate,
T added 100 ul of a 1:1 dilution of agarose bead-conjugated anti-mouse IgG antibody
(Sigma) in extraction buffer, and incubated the samples for 1 hour, rotating, at 4°C. The
samples were then washed twice with 1 ml extraction buffer, once with 1 ml 0.5 M LiC.
0.1 MTris (pH7.4), and once with 1 ml 10 mM Tris (pH 8.0), 100 mM NaCl, 1 mM
EDTA. Agarose beads were collected by centrifugation for 5 minutes in the Eppendorf
centrifuge between each wash. I resuspended the final pellets in 80 ul each of
electrophoresis sample buffer made of 2% SDS, 60 mM Tris (pH 6.8), 10% glycerol, 100
mM DTT, and a few grains of bromphenol blue, and placed them in an 80-85°C water
bath for 10 minutes. The samples were cooled to room temperature and spun for 5
minutes in the Eppendorf centrifuge. The supernatants were removed to new tubes and
frozen at -35°
Tran these samples on a 10% SDS polyacrylamide gel (Laemmli, 1970), and the
phosphotyrosines were revealed using a Western immunoblot (Towbin et al., 1979). The
blot was blocked for an hour in 20 mM Tris (pH 7.6), 138 mM NaCl, 0.1% Tween-20, 3%
BSA. Tused PY-20 as the primary antibody in a 1:2000 dilution, incubating for 1 hour,
and goat anti-mouse IgG(H+L) HRPO (Southern Biotechnology Associates, Inc.) as the
secondary antibody in a 1:2000 dilution, incubating for 1 hour. Before and after each
antibody incubation, I washed the blots 4x 5 minutes with 20 mls of saline solution
made of 20 mM Tris (pH 7.6), 0.8% Nacl, 0.1% Tween. The blot was developed using
the chemiluminescence assay described by Thorpe et al., (1985).
Results
Effect of Bindin and the Antibody Against the Sperm Receptor on G6PD Release
Tused two different approaches to determine whether the specific tyrosine kinase
that phosphorylates the sperm receptor after fertilization is involved in G6PD release.
The first was by treating unfertilized eggs with two concentrations of bindin: 60 pg/ml.
(used by Abassi and Foltz (1994) to stimulate tyrosine phosphorylation of the sperm
receptor); or 60 ng/ml (1000x as concentrated). Both concentrations of soluble bindin
had little or no effect on G6PD release (Table 1). These eggs fertilized normally, (FE
elevation of close to 100% of the eggs, as in controls) indicating that bindin was not
preventing access of sperm to its receptor. While the BCA protein assay is generally
accurate, there may have been some contaminants which gave an inordinately high
concentration of soluble bindin. In this case, the amount of bindin I used may not have
been enough to stimulate the egg.
The second approach was to treat eggs with anti-receptor antibody. This
treatment also seems to have no effect on G6PD release (Table 1). The secondary
antibodies used in these experiments were conjugated to fluorescent dyes, and because
the eggs did not fluoresce after treatment, it seemed that the primary antibodies were
not binding to the cell surface. The eggs fertilized normally, again indicating that the
receptor remained accessible to sperm. The high ionic strength of sea water may have
been causing a disruption between the antibody-antigen interaction, yielding these
results.
Effect of Tyrosine Kinase Inhibitors on G6PD Release
The method l used to learn if general tyrosine kinase activity is involved in
causing the G6PD release after fertilization was to treat eggs with the tyrosine kinase
inhibitors genistein and tyrphostin 51. Incubation of unfertilized eggs with 100 uM
genistein for 1 hour pre-fertilization does not block fertilization-induced G6PD release
(Figure 1) as 1 determined using the G6PD assay. This concentration of genistein was
used because Moore and Kinsey (1995) have shown that at concentrations between 37
uM and 150 uM genistein effectively blocks tyrosine kinase activity without disrupting
serine and threonine kinase activity. Fertilization of genistein-treated eggs occurs
normally as evidenced by FE elevation 1-2 minutes after adding sperm, however
development is arrested. Four hours after fertilization, division of genistein-treated
zygotes to the 2-cell stage had not occurred.
Similarly, incubation of unfertilized eggs with 100 uM tyrphostin 51 for 90
minutes pre-fertilization does not seem to have an effect on G6PD release (Figure 2).
Again, Moore and Kinsey (1995) showed that this concentration would adequately
block tyrosine kinase activity in sea urchin eggs. Fertilization of tyrphostin 51-treated
eggs occurs normally. However, zygotes in this group appear to develop normally as
well. Both control and tyrphostin-treated zygotes divided to the 2-cell stage by 4 hours
post-fertilization.
To ensure that genistein and tyrphostin 51 were actually inhibiting tyrosine
phosphorylation, Iimmunoprecipitated zygotes after pre-treatment with genistein and
tyrphostin with the anti-phosphotyrosine antibody, PY-20. I incubated a 1% suspension
of eggs in 0.1% DMSO (controls) or in 100 uM inhibitor for 90 minutes, and then
fertilized them and took samples. As expected, the resulting Western immunoblot
shows that the unfertilized control (lane 1) does not show many bands in the 65-98 kDa
region, indicating that tyrosine kinase activity is low. Also as expected, the fertilized
10
control (lane 4) shows several bands in this region, indicating that phosphorylation of
tyrosine residues (tyrosine kinase activity) has increased after fertilization. The
genistein-treated sample (lane 2), does not show these bands, verifying that genistein is
inhibiting general tyrosine kinase activity. However, the tyrphostin 51-treated sample
(lane 3) shows these bands, suggesting that tyrphostin 51 is not blocking tyrosine kinase
activity. Lane 5 is the antibody control (no lysate), and lane 6 is the lysate control (non¬
specific primary antibody) in which no bands other than antibody were detected.
Although genistein may not be inhibiting G6PD release, I wanted to determine
its effect on activity of the pentose shunt. Using a fluorescence assay, I found that the
rise in NADPH after fertilization in genistein-treated eggs was much less than the rise in
controls after both 60 minute and 90 minute genistein incubations. There was a 77%
reduction in NADPH fluorescence in genistein-treated zygotes. I deduced that a
reduction of approximately 50% can be accounted for by non-enzymatic effects of
genistein by the following experiment; The fluorescence of NADPH in filtered sea
water, in the absence of eggs, was assayed, and after adding genistein the fluorescence
signal reduced immediately by approximately 50%. This suggests that genistein is
causing a 23% decrease in shunt activity.
Discussion
Fertilization activates the egg metabolism, causing the egg to restart the cell cycle
resulting in growth and division. The activation of the zygote metabolism results from
the stimulation of many different biochemical pathways including the pentose
phosphate shunt. One hypothesis for the activation of the zygote metabolism includes
signal transduction events involving tyrosine kinase activity (see reference in Moore
and Kinsey, 1995). It has been shown that soon after fertilization, tyrosine kinase
activity in the egg increases two- to fourfold (Satoh and Garbers, 1985), and various
different newly tyrosine-phosphorylated proteins appear (Ciapa and Epel, 1991)
including the 350 kDa sperm receptor (Abassi and Foltz, 1994). Although tyrosine
kinase activity is known to increase after fertilization, its involvement in overall egg
activation or in the activation of specific biochemical pathways is an area of continuing
study.
The pentose phosphate shunt is a very important pathway used by many cells to
produce compounds necessary for cell growth and division including NADPH and
ribose units. NADPH is required by various pathways in the cell, including lipid, DNA
and protein synthesis, and riboses are needed for nucleotide synthesis. In sea urchin
eggs, the pentose shunt has been shown to be activated soon after fertilization (Swezey
and Epel, 1995). This activation is correlated with the release and activation of the rate-
limiting enzyme in the pathway, G6PD. G6PD is in a bound, inactive state in
unfertilized eggs, and in a free, soluble, active state in fertilized eggs (Swezey and Epel,
1986). The mechanism for G6PD release and activation of the pentose shunt in sea
urchin eggs at fertilization is currently unknown. It has been shown however, that in
mammalian rat kidney cells, the mechanism that activates the shunt through the release
of G6PD requires tyrosine kinase activity (Tian et al., 1991). In this case, treatment of
cells with PDGF causes the tyrosine kinase in the PDGFR to become active, which is
necessary for G6PD release (Tian et al. 1991).
The purpose of my study was to determine whether the mechanism for G6PD
release in sea urchin eggs after fertilization is similar to the mechanism used by
mammalian somatic cells, requiring tyrosine kinase activity.
Bindin and the Antibody Against the Sperm Receptor
As Abassi and Foltz (1994) have shown, when eggs are treated with bindin, the
sperm receptors within the egg membrane become tyrosine-phosphorylated, indicating
that a tyrosine kinase has been activated. Incubating eggs with anti-receptor antibodies
may also cause a similar response of tyrosine kinase activation. I studied the effects of
treating eggs with bindin and anti-receptor antibodies to see if a G6PD release-response
would be elicited due to the activation of this tyrosine kinase.
According to my results, bindin is not causing G6PD release, however this
conclusion may be particular to these experiments. Because bindin did not prevent
sperm from fertilizing the eggs, there may not have been enough bindin to interact with
its receptor. One way to ascertain whether bindin is interacting with the receptor would
be to determine whether bindin-treatment is causing the receptor phosphorylation as
described by Abassi and Foltz (1994).
Similarly, the anti-receptor antibody treatments show no effect on G6PD release.
However, in this case, the antibody-antigen interaction may be disrupted by the high
ion content of sea water. That the eggs were not fluorescing when treated with
fluorescent dye-conjugated secondary antibodies supports the idea that the antibody
did not successfully bind the receptor. Previously, experiments done with these
antibodies have been on in vitro membrane preparations and the jonic strength of
solutions has been kept low (Abassi and Foltz, 1994). Binding of the antibody to live
eggs can be enhanced by incubating the eggs in a solution that is isotonic to sea water.
but has a low ionic strength. The osmolarity of this solution can be maintained by
adding non-ionic compounds such as sucrose instead of ions. The effect of the anti
receptor antibody treatments on G6PD release must be studied further by first
ascertaining that these antibodies are, in fact, binding to the sperm receptor.
Tyrosine Kinase Inhibitors
Genistein and tyrphostin are both general tyrosine kinase inhibitors which
prevent fertilization-induced tyrosine kinase activity in sea urchin eggs (Moore and
Kinsey, 1995). In an effort to determine if general tyrosine kinase inhibition would
block fertilization-induced G6PD release, I studied the effects of treating eggs with
genistein and tyrphostin 51.
Genistein does not inhibit G6PD release in sea urchin eggs. In addition, the
Western immunoblot indicates that tyrosine phosphorylation is being inhibited under
these conditions. Taken together, these results show that tyrosine kinases may not be
involved in fertilization-induced G6PD release. Although genistein may not be
preventing G6PD release upon fertilization, it may be affecting pentose shunt activity,
Preliminary results from the in vivo NADPH assay show that there is a 77% reduction in
the amount of NADPH fluorescence in genistein-treated zygotes. However, because a
reduction of least 50% can be accounted for by non-enzymatic effects, it is unclear
whether genistein is reducing the activity of the pentose shunt, or the reduction in
fluorescence emission is due to some other factor. Further investigation of genistein
effects on the pentose shunt will have to be done.
Tyrphostin 51 also does not seem to inhibit G6PD release in sea urchin eggs
However, as evidenced by the Western immunoblot, this compound does not seem to
be inhibiting tyrosine phosphorylation. Moore and Kinsey (1995) studied the effects of
tyrphostin B42, a compound in the same family of inhibitors as tyrphostin 51. They
found that 100 uM tyrphostin B42 blocked tyrosine kinase activity. However, subtle
differences in structure between these two compounds may account for the lack of
inhibition by tyrphostin 51. For example, tyrphostin 51 may not be as permeant as
tyrphostin B42. It is possible that tyrphostin 51 was not getting into the cells during the
90 minute incubation. This could explain its lack of tyrosine kinase inhibition as well as
its lack of G6PD release and cell-division inhibition. Another possibility for the inability
of tyrphostin 51 to block tyrosine kinase activity is that tyrphostin 51 is not effective at a
100 uM concentration. However, because the ICso of tyrphostin 51 is 800 nM (Sigma
catalog, 1995), the 100 uM concentration should have been more than sufficient to cause
inhibition.
Conclusions
My study suggests that the activity of the tyrosine kinases blocked by genistein is
not necessary for G6PD release in S. purpuratus eggs at fertilization. These results lead
me to the following general questions which must be answered to get a better
understanding of the mechanisms involved in G6PD release and pentose shunt
activation at fertilization of sea urchin eggs, as well as the involvement of tyrosine
kinases in development. (1) Are there tyrosine kinases that are not inhibited by
genistein which lead to G6PD release? While genistein is a general tyrosine kinase
inhibitor, it most likely does not inhibit every tyrosine kinase in the cell. To be sure that
tyrosine kinase activity is not required for G6PD release, effects of various other
inhibitors would have to be studied. (2) Are any early developmental events blocked by
genistein-inhibition of tyrosine kinases? We know that some of the later events of egg
activation, including pronuclear migration, DNA synthesis, and cell division, are
blocked by genistein. We also know that the early event of fertilization membrane
elevation is not blocked (Moore and Kinsey, 1995), indicating that the early intracellulai
Caz rise is occurring. However, we do not know how genistein effects some of the
other early events of fertilization, including the significant pH rise needed for proper
development. (3) How would inhibiting tyrosine phosphatase activity affect
fertilization-induced G6PD release? By inhibiting tyrosine phosphatase activity, the
relative amount of tyrosine phosphorylation would rise over time and might cause a
release of G6PD if tyrosine phosphorylation is required for release in sea urchin eggs
Answers to these questions will help to elucidate the mechanism involved with
G6PD release at fertilization of sea urchin eggs, and will help to determine whether this
mechanism is similar to that used by somatic mammalian cells. These mammalian cells
differ substantially from sea urchin eggs, both in type of cell and event that causes
G6PD release. The rat kidney cells are somatic mammalian cells whereas the sea urchin
eggs are germ-line cells. G6PD release in the rat kidney cells occurs through hormone
treatment and requires tyrosine kinase activity, while sea urchin eggs release G6PD at
fertilization. The hormone treatment can occur numerous times in the life of the kidney
cell, whereas fertilization occurs only once in the life of the egg cell. Because of these
differences, the mechanism that releases G6PD may be quite different for these two
types of cells, and tyrosine kinase activity may not be necessary for G6PD release in sea
urchin eggs. If tyrosine kinases are not involved in G6PD release in sea urchin eggs at
fertilization, then the intriguing question is what is the mechanism of this activation.
and how else do the mammalian and urchin mechanisms differ?
Works Cited
Abassi, Y. A. and K. R. Foltz. 1994. Tyrosine phosphorylation of the egg receptor for
sperm at fertilization. Developmental Biology. 164: 430-443
Capa, B. and D. Epel. 1991. A rapid change in phosphorylation on tyrosine
accompanies fertilization of sea urchin eggs. FEBS. 295: 167-170.
Edgecombe, M., R. Patel, and M. Whitaker. 1991. A cyclin-abundance cycle
independent p34ede tyrosine phosphorylation cycle in early sea urchin embryos.
The EMBO Journal. 10: 3769-3775.
Epel, D. 1964. A primary metabolic change of fertilization: Interconversion of pyridine
nucleotides. Biochemical and Biophysical Research Communications. 17: 62-68.
Epel, D. 1990. The initiation of development at fertilization. Cell Differentiation and
Development. 29: 1-12.
Foltz, K. R., J. S. Partin, and W. J. Lennarz. 1993. Sea urchin egg receptor for sperm:
Sequence similarity of binding domain and hsp70. Science. 259: 1421-1425.
Laemmli, U. K. 1970. Ceavage of structural proteins during the assembly of the head
of bacteriophage T4. Nature. 227: 680-685.
Lopez, A., S. J. Miraglia, and C. G. Glabe. 1993. Structure/ function analysis of the sea
urchin sperm adhesive protein bindin. Developmental Biology. 156: 24-33.
Moore, K. L. and W. H. Kinsey. 1995. Effects of protein tyrosine kinase inhibitors on
egg activation and fertilization-dependent protein tyrosine kinase activity.
Developmental Biology. 168: 1-10.
Satoh, N. and D. L. Garbers. 1985. Protein tyrosine kinase activity of eggs of the sea
urchin Strongylocentrotus purpuratus: The regulation of its increase after fertilization.
Developmental Biology. 111: 515-519.
Sigma Chemical Company 1995 Catalog. 1031.
Smith, P. K., R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gartner, M. D.
Provenzano, E. K. Fujimoto, N. M. Goeke, B.J. Olson, and D. C. Klenk. 1985.
Measurement of protein using bicinchoninic acid. Analytical Chemistry. 150: 76-85
Stanton, R. C. and J. L. Seifter. 1988. Epidermal growth factor rapidly activates the
hexose monophosphate shunt in kidney cells. American Journal of Physiology. 253
(Cell Physiology 22): C267-C2
Stanton, R. C., J. L. Seifter, D. C. Boxer, E. Zimmerman, and L. C. Cantley. 1991. Rapid
release of bound glucose-6-phosphate dehydrogenase by growth factors. The Journal
of Biological Chemistry. 266: 12442-12448.
Swezey, R. R. and D. Epel. 1986. Regulation of glucose-6-phosphate dehydrogenase
activity in sea urchin eggs by reversible association with cell structural elements.
The Journal of Cell Biology. 103: 1509-1515.
Swezey, R. R. and D. Epel. 1995. The in vivo rate of glucose-6-phosphate
dehydrogenase activity in sea urchin eggs determined with a photolabile caged
substrate. Developmental Biology. in press.
Thorpe, G.H. G., L. J. Kricka, S. B. Moseley, and T. P. Whitehead. 1985. Phenols as
enhancers of the chemiluminescent horseradish peroxidase-luminol-hydrogen
peroxide reaction: Application in luminescence-monitored enzyme immunoassays.
Clinical Chemistry. 31: 1335-1341.
Tian, W., J. N. Pignatare, and R. C. Stanton. 1994. Signal transduction proteins that
associate with the platelet-derived growth factor (PDGF) receptor mediate the
PDGF-induced release of glucose-6-phosphate dehydrogenase from permeabilized
cells. The Journal of Biological Chemistry. 269: 14798-14805.
Towbin, H., T. Staehlin, and J. Gordon. 1979. Electrophoretic transfer of proteins from
polyacrylamide gels to nitrocellulose sheets: Procedure and some applications.
Proceedings of the National Academy of Sciences, USA. 76: 4350-4354.
TABLE 1
Effects of Bindin and Anti-Bindin Receptor Antibodies on G6PD Release in
Unfertilized S. purpuratus Eggs
(Data expressed as percentage of bound G6PD)
Control
Bindin 60 ng/ml
Bindin 60 pg/ml
Experiment 1
52.0
45.0
52.0
Control
Anti-Exo IgG
Control IgG
Experiment 2
55.6
47.6
44.4
Experiment 3
68.5
66.3
56.2
Note: Unfertilized eggs were treated with either 60 ng/ ml or 60 pg/ ml soluble bindin
in experiment 1. Anti-exo receptor IgG antibodies were used in both experiments 2 and
3. The control IgG for experiment 2 was goat immunoglobulins, and the control IgG for
experiment 3 was anti-cyto receptor IgG antibody. In all experiments, G6PD bound
after fertilization of controls was less than or equal to 10%. Each value in the table was
calculated from the mean of two assays on each sample.
Figure Legends
Figure 1: 1% egg suspensions were incubated for 1 hr with 100 uM genistein before
fertilizing and assaying for G6PD activity. Controls were incubated in 0.1% DMSO.
Both unfertilized samples were taken 15 minutes before the fertilized samples. The
graph shows the means of two experiments, and bars represent ranges of these values.
Figure 2: 1% egg suspensions were incubated for 90 minutes with 100 uM tyrphostin 51
before fertilizing and assaying for G6PD activity. Controls were incubated in 0.1%
DMSO. Both unfertilized samples were taken 15 minutes before the fertilized samples.
Data are from one experiment.
Figure 3: Eggs were treated with 100 uM genistein and 100 uM tyrphostin 51 for 90
minutes before fertilization and subsequent sampling for immunoprecipitation.
Unfertilized and fertilized controls were treated with 0.1% DMSO for 90 minutes as
well. All samples were immunoprecipitated using the anti-phosphotyrosine antibody
PY-20. Unfertilized control, lane 1. Genistein, lane 2. Tyrphostin 51, lane 3. Fertilized
control, lane 4. An antibody control using no lysate and a control using non-specific
mouse IgG as a primary antibody were also done (lanes 5 and 6 respectively).
20
EFFECTS OF CENISTEIN ON GGPD RELEASE
100-
80
60
40
20

UNFERT
FERT
UNFERT
FERT
GENISTEIN
CONTROLS
FIGURE 1
a
EFFECTS OF TYRPHOSTIN 51 ON GGPD RELEASE
100-
80
60
40-
20
UNFERT
FERT
UNFERT
FERT
TRPHOSTIN 51
CONTROLS
FIGURE 2
Tyrosine Kinase Inhibition by
Genistein and Tyrphostin 51
KDa
200
97.4
69


46
30 *
21.5
Figure 3