Stephanie Yeh
BISC 216 Lab
Lowered sodium levels in fertilization environment induces polyspermy in
Strongylocentrotus purpuratus
By: Stephanie Yeh, Christine He, Yetunde Oyenuga
(Wellesley College, Department of Biological Sciences)
Abstract
Polyspermy, the fertilization of a single egg by multiple sperm, is a problem faced by
both vertebrates and invertebrates alike. To address this problem, which is lethal for
embryos, many organisms have evolved mechanisms that enforce monospermy. Urchins
like Strongylocentrotus purpuratus utilize sodium to rapidly block additional sperm from
fusing to the egg. To determine the effectiveness of these mechanisms, S. purpuratus
eggs were cultured in artificial sea water of varying sodium concentrations, and the
appearance of a fertilization envelope, which denoted successful fertilization, was
observed. Our results suggest that lowered levels of sodium had successfully induced
polyspermy, though some sodium must have been present for successful cleavage, and
that elevated levels of sodium had caused the eggs to become unviable. These outcomes
indicate a heavy reliance on surrounding sodium levels as a mechanism for polyspermy
block in S. purpuratus.
Introduction
Since eggs of different organisms release jelly coat peptides to attract sperm and
promote chemotaxis towards the egg, a single egg is often surrounded by many –
sometimes hundreds – of sperm. All of these sperm cells are vying to fertilize the same
egg, thus producing the problem of polyspermy. Polyspermy, the simultaneous
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fertilization of an egg by multiple sperm, is a problem faced by many eukaryotes, as it
produces unviable embryos that ultimately die. Therefore, many organisms have
developed different mechanisms to prevent the occurrence of polyspermy.
When multiple sperm penetrate the oocyte cytoplasm, these embryos are not able
to develop normally, often resulting in unusual mitotic divisions early on. Polyspermy,
which is among one of the most common abnormalities to occur during fertilization,
causes the embryo to inherit multiple sets of paternal centrosomes, which is plays a
crucial role in chromosome dynamics. The centrosome, which is introduced into the
embryo by the sperm at fertilization, controls the microtubules in cells and is responsible
for the organization of the first mitotic spindle (Schatten et al., 1991). If multiple sperm
fuse, multipolar spindles form, dividing the egg into four irregular blastomeres with
randomly distributed chromosomes rather than two equivalent blastomeres. The abnormal
division leads to chromosome fragmentation, a condition that results in to embryonic
death (Snook et al., 2011).
Sea urchins are often used as a model system in studies regarding fertilization or
development due to the nature of their reproductive process. Since fertilization of these
marine invertebrates occur externally in their surrounding environment, their gametes are
easily studied in vitro, with relatively large eggs and sperm that are easily collected and
cultured. Their transparent embryos also allow researchers to observe cellular division
during the early stages of development.
In urchins, such as Strongylocentrotus purpuratus, there are two understood
strategies to block polyspermy, one fast and one slow. The slow block, which is
irreversible, occurs when a calcium influx into the egg causes cortical granules to be
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secreted. These cortical granules then fuse with the cellular membrane, leading to the
formation of the fertilization envelope (Schuel, 1984). However, previous studies show
that this cortical reaction does not occur rapidly enough to ensure monospermic
fertilization (Rothschild and Swann, 1951; Schuel and Schuel, 1981). As soon as the
plasma membrane of one sperm comes in contact with the plasma membrane of the egg, a
faster, though incomplete, block of polyspermy is initiated to prevent the fusion of
additional sperm. The fast block mechanism is mediated by sodium, which floods into the
egg via Na+ channels once the sperm and egg interact. The influx of external sodium
causes an electrical depolarization of the egg’s plasma membrane, changing the
membrane potential from its resting, negative potential of -70mV to a positive potential
(Jaffe, 1976). This rapid change of membrane potential prohibits other sperm from
interacting and fusing with the egg, thus giving the egg additional time to complete the
cortical reaction. Together, these two mechanisms are effective in preventing polyspermy
in S. purpuratus.
Since the fast block mechanism is so heavily reliant on sodium levels, we
investigated the fertilization of S. purpuratus in seawater containing varying
concentrations of sodium. Previous studies on the mussel Mytilus edulis show induction
of polyspermy when the embryos were fertilized in low-sodium sea water (Togo et al.,
1995). In addition, experiments with Arbacia punctulata, a different species of urchin,
demonstrate that sodium cations were the most effective in causing polyspermic
fertilization when compared to other salts such as potassium or magnesium (Clark, 1936).
Therefore, we propose that we could induce polyspermy in S. purpuratus by lowering
levels of sodium in the culture environment.
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Our experiment demonstrated that S. purpuratus urchin eggs fertilized in low
sodium environments showed evidence of induced polyspermy and that eggs were not
fertilized in high sodium environments.
Materials & Methods
The subjects used for the experiment were the gametes collected from the sea
urchin S. purpuratus.
Subjects were fertilized in artificial seawater (ASW) containing five different
molarities of sodium: 0 mM (0 g/L NaCl), 212.5 mM (12.3 g/L NaCl), 425 mM (24.6 g/L
NaCl), 637.5 mM (36.9 g/L NaCl), and 850 mM (49.2 g/L NaCl). Standard ASW
contains 425 mM of sodium chloride, which we used as our positive control. To balance
ion levels of solutions with decreased amounts of sodium,
choline chloride was
substituted in the place of sodium as described previously in similar experiments (Schuel
et al., 1982).
Since the gametes were originally maintained in standard ASW, egg washes with
the seawater of varying sodium concentrations were performed. In five separate 15 mL
conical tubes, the eggs were added for each condition and allowed the settle for five
minutes. After settling, the eggs were rinsed with the new ASW once again and the steps
listed above were repeated. Before transferring the eggs into wells, the egg-ASW solution
was aspirated with a glass pipette.
Four drops of eggs were placed in six-well plates in their respective ASW
solutions, then subsequently fertilized using six drops of sperm. Each sodium condition
had three replicates, with one additional negative control well for each condition, where
no sperm was added.
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Approximately five minutes post-fertilization, Vaseline wells were set up on glass
slides for each sample and a small amount of the culture was aliquotted into the well to
be observed under the compound microscope. A total of 20 embryos were counted for
each condition and the number of eggs with fertilization envelopes was recorded. Those
embryos with a visible fertilization envelope had been counted as successfully fertilized.
The percentage of fertilized embryos was calculated and the data was analyzed.
Samples were observed for two hours post-fertilization to monitor development.
Results
A
B
D
C
E
Figure 1. S. purpuratus eggs cultured in low sodium environments exhibit abnormal
fertilization envelope formation, while eggs cultured in high sodium environments
are not fertilized. (A) Egg fertilized in 0 mM Na+ ASW solution; (B) Egg fertilized in
212.5 mM Na+ ASW solution; (C) Egg fertilized in 425 mM (standard) Na+ ASW
solution; (D) Egg fertilized in 637.5 mM Na+ ASW solution; (E) Egg fertilized in 850
mM Na+ ASW solution. Arrow denotes fertilization envelope. Images captured at 400x
magnification 10 minutes post-fertilization.
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100
90
Percentage fertilized (%)
80
70
60
50
40
30
20
10
0
0
12.3
24.6
36.9
Concentration of NaCl in ASW (g/L)
49.2
Figure 2. S. purpuratus eggs cultured in above-standard sodium environments are
not fertilized, while eggs cultured in below-standard sodium environments are
successfully fertilized. Eggs were maintained in five different ASW solutions with
varying levels of sodium chloride, with 24.6 g/L as the positive control. 0 g/L and 12.3
g/L NaCl solutions were substituted with 24.6 g/L and 12.3 g/L choline chloride,
respectively, to balance the ions within solution. Twenty eggs were checked for
fertilization envelopes under each condition ad the percentage fertilized was calculated
by diving the number of those with envelopes with the total number of eggs counted.
Error bars were determine using standard deviation.
Table 1. Mean ± SE for a test of the hypothesis that concentration of sodium (Na+)
affects rate of fertilization in S. purpuratus urchins. ANOVA df 4,10, p = 0.0001.
Tukey levels not sharing the same letter are significantly different.
Concentration of NaCl in
Mean percentage
Tukey HSD level
ASW (g/L)
fertilized ± SE
(ɑ < .05)
0
90 ± 5
A
12.3
96.7 ± 2.9
A, B
24.6
100 ± 0
B
36.9
0±0
C
49.2
1.7 ± 2.9
C
7
A
B
C
Figure 3. Abnormal cleavage exhibited in S. purpuratus embryos fertilized in 12.3
g/L NaCl ASW at the two-cell stage 2 hours post-fertilization. (A) and (B) Embryos
of eggs fertilized in 12.3 g/L NaCl ASW. (C) Embryo of egg fertilized in 24.6 g/L NaCl
(standard) ASW. Images captured at 400x magnification.
A
B
Figure 4. Abnormal development in surviving S. purpuratus embryos fertilized in
12.3 g/L NaCl ASW one week post-fertilization. (A) Normal pluteus-stage larvae of S.
purpuratus cultured in 24.6 g/L (standard) ASW. (B) Abnormally shaped embryo of S.
purpuratus cultured in 12.3 g/L ASW. Images captured at 400x magnification.
To test the efficacy of S. purpuratus fast block mechanisms during fertilization, the
urchin eggs were fertilized in seawater containing varying levels of sodium and observed.
Eggs were exposed to solutions with five different molarities of sodium prior to and
during fertilization. Post-fertilization, embryos with visible fertilization envelopes were
counted to determine percentage fertilized and embryos with abnormal cleavage were
noted.
An unfertilized egg can easily be distinguished from a fertilized embryo based on the
appearance of a fertilization envelope (Fig. 1). As part of the embryo’s defense against
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polyspermy, the sperm’s interaction with the vitelline envelope causes the slow block to
induce a series of irreversible events, such as the lifting of the fertilization envelope
through osmosis. Thus, we can ascertain that those eggs with a surrounding fertilization
envelope are fertilized. The fertilization envelope provides a reliable way to differentiate
from those eggs which were not fertilized from those which were.
Overall, eggs in lower sodium levels (0 g/L and 12.3 g/L NaCl) were successfully
fertilized as the majority of eggs under these conditions had fertilization envelopes (Fig.
1; Fig. 2). Surprisingly, those embryos cultured without any sodium in the ASW (0 g/L
NaCl) had fertilization envelopes, which were abnormally thin in comparison to the
fertilization envelopes developed in the positive control (24.6 g/L NaCl). Eggs in
elevated sodium levels (36.9 g/L and 49.2 g/L NaCl) did not have any fertilization
envelopes appear, suggesting that the eggs became unviable at these sodium
concentrations. Our negative controls (eggs cultured in the five different conditions
without the addition of sperm) proved that our egg samples were not contaminated or
defective (data not shown).
Embryos cultured under the 0 g/L condition and embryos cultured under the 36.9
g/L and 49.2 g/L conditions were significantly different from those in the standard ASW
condition, suggesting that extremely high or low levels of sodium negatively impacted
fertilization as predicted (ANOVA, F = 973.8, df = 4,10, p = 0.0001; Tukey HSD test
(Table 1)).
On average, embryos in the 12.3 g/L condition were extremely efficient, with a
96.67% fertilization rate (Fig. 2). Embryos in the 0 g/L condition also displayed high
levels of success, with a 90.00% fertilization rate. However, these two conditions
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exhibited anomalies during development. The embryos fertilized in the no-sodium
condition failed to cleave 2-hours post-fertilization, suggesting that these embryos most
likely died. In comparison, embryos fertilized in the low-sodium (12.3 g/L NaCl)
condition did cleave after 2 hours, though the cleavage was exceptionally irregular,
suggesting polyspermy (Fig. 3).
The cleaved embryos were observed for a week following fertilization. The
embryos fertilized in standard ASW successfully developed into normal pluteus-stage
larvae, while the embryo fertilized in 12.3 g/L NaCl ASW developed abnormally (Fig. 4).
Eggs in cultured in the higher levels of sodium were also observed after a week, though
no eggs were found, suggesting that they had been lysed (not pictured). These results
suggest that low-sodium conditions leads to polyspermy and proves to be lethal to the
embryos.
Discussion
To test the possible effects of changing salinity on fertilization rates, we fertilized
S. purpuratus sea urchin eggs in solutions of various sodium concentrations. From our
results, we can conclude that lower-than-standard levels of sodium led to successful
fertilization, though not normal development, while high-than-standard levels of sodium
led to the eggs become unviable. The discrepancy between fertilization rates indicates
that fertilization is heavily dependent on sodium concentrations.
The appearance of an uneven number of blastomeres, all seemingly different
sizes, indicates polyspermy as previously described (Schatten et al., 1991; Snook et al.,
2011). The abnormal cleavage patterns exhibited in the 12.3 g/L condition suggests that
sodium does indeed play an essential role in the fast block mechanisms in sea urchins.
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From these results, we can conclude that lowered levels of sodium can induce
polyspermy in these organisms. This result is supported by many studies on other species,
including the sand dollar Enchinarachnius parma (Allen and Pechenik, 2010; Cross and
Elinson, 1980; Nishioka and Cross, 1978).
Surprisingly, the no-sodium condition led to unviable embryos which did not
cleave or develop past fertilization. Previous studies have shown that sodium ions are
essential in the hardening of the fertilization envelope and its subsequent development,
giving a possible explanation for why these embryos failed to cleave after two hours
(Schuel et al., 1982).
In addition, the higher-than-standard sodium conditions produced unviable eggs
which were not fertilized. One potential explanation for this result is that as sodium
rushed into the egg via the Na+ channels (due to the concentration gradient effect of the
hypertonic solution), the cells shriveled and subsequently and died. Their abnormal,
somewhat withered shape further suggests that even slightly increased levels of sodium
leads is lethal to these gametes, though these mechanisms are not fully understood (Fig.
1).
We can improve the experiment by keeping consistent fertilization times, as some
eggs were fertilized later than others, and were thus exposed to their sodium solutions for
longer periods of time. We also hope to perform the same experiment on another species,
possibly S. droebachiensis, as a comparison to see if the effects of different sodium
concentrations stays consistent across species, especially species which develop in ocean
regions with varying salinities. In addition, we can also increase the number of conditions
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of salt concentrations within our range to determine a precise sodium concentration
which induces polyspermy or produces egg death.
Due to the effects of global warming, the salinity of Earth’s oceans is changing,
an occurrence that may prove detrimental to populations of sea urchins or other marine
eukaryotes. Research shows that the salinity of many oceans are becoming diluted in
some regions, while more salt-concentrated in others (Curry and Mauritzen, 2005). Our
study establishes a direct correlation between non-standard sodium concentrations and
lowered fertilization in S. purpuratus. Those organisms inhabiting the regions where the
salinity is increased faces the issue of egg death, while those inhabiting the regions where
the salinity is decreased faces the problem of polyspermy, which is lethal for embryos.
Thus, global warming and climate change endangers not only sea urchin populations, but
also populations of marine vertebrates and invertebrates with salt-dependent fertilization
mechanisms.
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