Peter Freed
ABSTRACT
Crude homogenates of the algae Fucus vesiculosus contain a factor(s) capable of
inhibiting the fertilization of Strongylocentrotus purpuratus (purple sea urchin) eggs. Previous
studies indicated that this effect might have been due to the polysaccharide fucoidan. I here
examined the effect of the homogenate at a molecular, cellular and ecological level. Inhibitory
activity was present in seawater homogenates of the algae at a concentration of approximately 2
mg fresh weight/ml. The inhibitory factor was heat stable and molecular sieve centrifuge
filtration showed the active fraction had a molecular weight above 10,000. Comparison of the
»10,000 MW fraction with fucoidan indicates that the inhibitor in the homogenate is not
fucoidan or is not fuciodan alone. Sperm binding experiments showed that the factor(s) prevents
sperm from penetrating the jelly layer of the eggs and possibly from penetrating the vitelline
layer. This suggests that the factor(s) inhibit a sperm enzyme that allows sperm to penetrate the
jelly layer and perhaps the vitelline layer. Studies assessing whether intact F. vesiculosus might
leak the inhibitory factor into the surrounding seawater-as in a tide pool- and thereby affect
fertilization were inconclusive. Characterization of this potent inhibitor could provide new
insights into the sperm-egg interactions leading to fertilization.
Peter Freed
INTRODUCTION
In the 1950’s a small literature showed that homogenates of the fucoid algae Fucus
vesiculosus inhibited the fertilization of the purple sea urchin Strongylocentrotus purpuratus
(Harding 1951, Runnström et al. 1959). Further work suggested that fucoidan in the algae might
be the inhibitory factor. However, this work neither indicated the mode of action of the
inhibitory factor nor did it speculate on whether this effect could be documented in situ. The
purpose of this project was to reexamine these older findings in light of our current knowledge of
fertilization. I also hoped to be able to determine if fucoidan was indeed the inhibitory factor in
the homogenates. Finally, I wanted to see if the effect seen in the laboratory might occur in
some form in nature.
There have been many significant scientific insights into the mechanisms of fertilization
since the 1950’s. The inhibitory factor(s) in the homogenate may have had one (or more) of
several cellular targets not yet discovered in the 1950’s. Two candidate targets that my data
seem to support are sperm-egg binding and sperm factors that degrade the outer egg coats. The
sperm has been shown to bind tightly to the egg surface during fertilization (Vacquier and Payne
1973). My data shows that the sperm do not bind to egg surface while exposed to the fucus
homogenate, but rather become embedded in the jelly layer. We also know that there are
species-specific sperm factors that are used to disperse the jelly coat of eggs prior to fertilization
Peter Freed
(Yamada and Aketa 1983). This knowledge combined with the fact that sperm cannot penetrate
the egg jelly indicates that the inhibitory factor may act on these sperm dispersal factors.
The action of sperm dispersal factors is inhibited by sulfated polysaccharides such as
heparin and glucose-6-sulfate (Yamada and Aketa 1983), making fucoidan a likely candidate for
the inhibitory factor. However, comparison of the action of fucoidan and the fucus homogenate
on normal urchin gametes as well as spectrophotometric analysis indicates that the inhibitory
factor in the homogenate is not fucoidan alone.
Fucoid algae living in the field may have similar effects as the homogenate does in the
laboratory. Fucus and purple urchins have some overlap in their vertical ranges (Russell 1987).
This results in a number of possible interactions that could cause the fucus to have a mechanism
for inhibiting urchin fertilization. Urchins may feed on fucus and, as a result, the algae produce a
factor that is capable of reducing urchin numbers. It is also possible that the algae and the
urchins compete for space and the algae tries to get more space by keeping urchin populations
low. However, preliminary trials show that the inhibitory factor is not simply leaked into
seawater by the algae.
Our current knowledge provides many new insights into how this inhibitory factor might
work. This project was designed to look for a cellular target of the inhibitory factor, to explore
the role of fucoidan as the inhibitory factor and to see if these inhibitory effects were evident in
nature.
Peter Freed
METHODS
Homogenate preparation and concentration approximation
Fucus gardneri (heretofore referred to as fucus) was collected from the rocky intertidal at
Hopkins Marine Station in Pacific Grove, California. Two pieces were cut to the same wet
weight (1-2 g). One of the samples was then homogenized in a 20ml glass homogenizer with a
teflon pestle in eight to ten ml seawater. Homogenization was stopped after approximately 50
strokes when there was no appreciable change in the appearance of the homogenate. The extract
was centrifuged for 5 minutes at 13,000 rpm in a microfuge and the supernatant used for
subsequent experiments. 500 ul aliquots of supernatant were frozen at -20’c and thawed
immediately prior to use.
Gamete Collection
Strongylocentrotus purpuratus gametes were collected by injection of O.5M KCl.
Females were placed upside down on a 50 ml beaker of filtered sea water into which gametes
were shed. Sperm were collected “dry" by pipette from males set upside down. Sperm was
suspended at 1% in sea water and stored on ice each day for experimental work.
Testing homogenate effects on gametes and fertilization
The effects of the fucus homogenate were tested on many combinations of sperm and
egg. Those combinations and volumes are represented below:
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Peter Freed
Effect
Volumes
Plain sperm
100 ul 1% sperm
100 ul homogenate
800 ul filtered seawater
Plain eggs
500 ul egg suspension
100 ul homogenate
400 ul filtered sea water
Homogenate added to egg suspension and ther
500 ul egg suspension
sperm added
100 ul homogenate
10-100 ul 1% sperm
300-400 ul filtered seawater
Homogenate added sperm then eg
100 ul 1% sperm
suspension added
100 ul homogenate
800 ul filtered seawater
These were combined and then 500 ul of this
solution was mixed with 500 ul egg suspension
Homogenate added to egg suspension then the
500 ul egg suspension
eggs were washed and sperm added
100 ul homogenate
—
—Wash
Re-suspend in 500 ul filtered sea water and
then add 10-100 ul sperm.
Homogenate was used in concentrations ranging from 100% to 0.01%. Fertilization was
quantified by counting the number of eggs with a raised fertilization membrane out of 100 eggs.
Peter Freed
Sperm binding experiments
This technique was modified from Vacquier and Payne (1973). 100 ul egg suspension
were combined with 100 ul sperm and then 200 ul 5.6% formaldehyde was added 15 seconds
later. To examine homogenate effects 100 ul homogenate was added to 100 ul egg suspension
and allowed to sit for several minutes, 100 ul sperm was then added and then 300 ul 5.6%
formaldehyde 15 seconds later. 50 ul of each sample was viewed at 200x by light microscopy.
Removal of the jelly layer and homogenate effects on jellyless eggs
Jelly layers were visualized by mixing an equal quantity of 6% sumi ink dissolved in
seawater with the egg suspension and then by looking for a "halo" by light microscopy at 100x.
Egg jelly layers were removed in one of two ways. The first was to pour the egg
suspension several times through a 90 um nitex mesh. The second was to take suspended eggs
and add O.IM HCl until the suspension reached pH 5. The eggs were then added to a large
quantity of filtered seawater and pH corrected to 8 with 0.1M NaOH (if not already at this pH).
To test the effects of homogenate on jellyless eggs, 500 ul egg suspension were
combined with 100 ul homogenate or 100 ul filtered sea water and let sit for several minutes.
Then 50 ul sperm was added. Binding experiments were also performed on jellyless eggs by the
method described above.
Peter Freed
Removal of the vitelline layer and jelly layer and homogenate effects on treated eggs
Egg vitelline layers were removed by exposure to strong protease for 5 minutes at a final
concentration of 0.5 mg/ml pronase. Eggs were then washed twice in a large quantity of filtered
seawater. Jelly layers were then removed from these eggs using nitex as described above.
Homogenate effects were tested on both protease-treated and protease-treated jellyless
eggs in a like manner as for jellyless eggs. In this case, eggs were allowed to sit for 10 minutes
after the addition of sperm and then fertilization was quantified by counting the number of eg
with hyaline membranes out of 100 eggs.
Test for the effects of Sigma brand fucoidan
The effect of fucoidan was assessed at logarithmic dilutions of a 1 mg/ml solution of
fucoidan from 0.001% to 100% and also a 10 mg/ml solution was tested at 100%. 500 ul
fucoidan was added to 500 ul egg suspension and then 10 ul sperm was added and fertilization
scored as described.
Homogenate analysis: heat stability
500 ul homogenate was exposed to room temperature (20-24°C), 60’C and 100°C for 10
minutes. 100 ul of each heat treated homogenates were added to 500 ul egg suspension and then
10 ul sperm was added and fertilization scored as above.
Molecular weight estimate of the active factor
Approximately 10 ml crude homogenate was centrifuged at 12,365g for 15 minutes. This
solution was then passed through a 10 um nitex mesh and then filtered through a 0.45 Millipore
Peter Freed
filter. The filtrate was then separated by centrifuge sieve filtration using a Millipore Centricon
Plus-20 centrifugal filter. Both filtrate and retentate effects on fertilization were tested on
normal eggs by combining 500 ul egg suspension with 500 ul sample and adding 50 ul sperm.
A dilution series was run on the retentate using one-third serial dilutions.
Pentose composition of the active factor
Carbohydrates were determined by the orcinol reaction for pentoses as described in
Colowick and Kaplan (1957) for the following samples: sea water, commercially-available
fucoidan in concentrations of 0% through 100 %, retentate, filtrate and raw homogenate. The
sugars were quantified by absorption at 670 nm in a Hitachi U-2000 double-beam
spectrophotometer and scanned over 400-700 nm wavelength range. Samples were scanned
against distilled water and seawater.
Microhabitat tests
One gram of fucus was placed in 100 ml of filtered sea water and allowed to sit outside in
the sun for 10, 30, 60, 180 or 360 minutes. Also 100 ml of plain filtered seawater was allowed to
sit for the same amount of time. The fucus was removed and the seawater was then allowed to
cool to near room temperature (21-24°C). 2 ml egg suspension was then added to 10 ml sea
water and allowed to sit for several minutes, 100ul sperm was added and fertilization scored as
above.
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Peter Freed
RESULTS
Fucus homogenate strongly inhibits fertilization in S. purpuratus eggs
The effects of the fucus homogenate was tested in concentrations from 0.01% to 100%
crude homogenate on normal gametes and had no appreciable morphological effect as observable
by light microscopy up to 200x. However, the homogenate did have marked effects on egg
fertilization (all effects compared to a control of 500 ul egg suspension, 500 ul filtered seawater
and 10 ul sperm). When raw homogenate is added to eggs there is 0% fertilization whereas
control fertilization was 100%. Fifty percent inhibition was achieved at a homogenate
concentration of 0.5% (figure 1). The homogenate and sperm mixture added to eggs decreased
fertilization by 20% indicating that there may be a time factor involved. This effect was seen to
be partially reversible since eggs combined with fucus and then washed showed 30% fertilization
when 10 ul of sperm was added where as the control was 100%.
Homogenate causes sperm to become stuck in the jelly layer
Sperm binding assessed by examination of eggs fixed in formaldehyde showed that in the
control situation eggs had » 100 sperm bound at the egg surface (figure 2). However, when the
binding assessment was carried out in the presence of the homogenate sperm were visibly stuck
in the jelly layer and did not bind to the egg surface (figure 3).
Removal of the jelly layer or vitelline layer alone does not increase fertilization and removal of
both increases fertilization
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Peter Freed
Because the sperm were embedded in the jelly layer, I looked to see if removing the jelly
layer would allow fertilization to occur in the presence of the homogenate. One hundred percent
of the jellyless eggs fertilize in the control situation. However, in the presence of the
homogenate there was 0% fertilization. I then looked to see if the sperm were becoming stuck to
the secondary layer around the egg, the vitelline layer. The sperm did not bind to the secondary
layer. This being the case, I looked to see if the eggs would fertilize without a vitelline layer.
However, 100% of the protease treated eggs fertilize in the control situation but 0% in the
presence of the homogenate. I anticipated that the sperm should have been able to fertilize the
eggs in the absence of both layers but I then discovered that protease treated eggs still had some
jelly layer. When this layer was removed by passing through nitex mesh the control eggs
showed 52% fertilization and the same eggs in the presence of homogenate showed 37%
fertilization.
Properties of the Fucus homogenate
The inhibitory factor was found to be active after exposure to 100’C for 10 minutes, with
100% fertilization in the control and 0% in the presence of homogenate. After the fraction was
run through the centrifuge sieve filter both the filtrate and the retentate were tested for activity
and the retentate was found to be the active fraction again reducing fertilization to 0% compared
to a 100% control.
1 next assayed for the presence of pentose sugars in the retentate using the orcinol
reaction. This test represents a way to see if there are fucoidan like compounds in the retentate
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Peter Freed
and filtrate and acts as an approximation for the amount of fucoidan in the extracts. Figure four
is a standard curve of commercial fucoidan dilutions. Using a third degree polynomial curve fit
on the normal curve, the absorption of the retentate indicates that the retentate is a 0.22 mg/ml
equi valent of fucoidan.
The homogenate is not fucoidan alone
Comparison of the absorption curves of fucoidan and the retentate show similar but not
identical curves suggesting that the sugar composition differs from fucoidan. As seen, the
absorption in the 600-700 nm range is very similar however; the peaks are not the same in the
400-500 nm range.
Comparison of the effects of fucoidan and the homogenate on fertilization also indicate
that the inhibitory action on fertilization is not due to fucoidan as seen in figures seven and eight.
Fucoidan does not inhibit fertilization of eggs in concentrations up to 10 mg/ml (figure 7).
Additionally, fucoidan causes what appears to be a secondary fertilization membrane to rise
(figure 7) and then causes cells to lyse (figure 8). None of these effects were seen with the
homogenate.
Can intact fucus exude the inhibitory activity?
The microhabitat experiments were set up to determine if the inhibitory factor in whole
fucus plants was leaked into seawater. The water in the microhabitat experiments had no
appreciable effect on the level of fertilization of eggs. In each case there was a -15% decrease in
fertilization between the control situation and the fucus treated water.
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Peter Freed
DISCUSSION
My results confirm and considerably extend the earlier observations on the inhibitory
effects of algal homogenates on urchin fertilization. The fucus homogenate contains a potent
inhibitor of purple urchin fertilization, which eliminates the sperm’s ability to pass through the
jelly layer. The inhibitory factor is a heat stable macromolecule(s) with a molecular weight
greater than 10,000, but does not appear to be fucoidan as was previously suggested. Finally,
while there is reason to suspect that there might be in situ effects of the inhibitory factor, my data
showed no such effects.
The fucus homogenate contains a potent inhibitor of purple urchin fertilization. The
interaction with the eggs is time dependent. If the factor is allowed to interact with the eggs for
several minutes before the addition of sperm, fertilization is completely blocked; however, if
sperm and homogenate are added directly to the eggs, without giving the homogenate time to
interact with the eggs prior to addition of the sperm, there will be 80% fertilization. Finally, if
the homogenate is washed off of the eggs there is 30% fertilization, which indicates that the
activity of the homogenate on eggs is at least partially reversible. The factor is effective at very
low levels, producing 50% inhibition even at a 1:500 dilution, which is equivalent to 4 ug/ml of
fucoidan.
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Peter Freed
The inhibitory factor caused sperm to become embedded in the jelly layer, suggesting
that passage through this layer is the cellular target of the inhibitory factor. I then looked to see
if binding occurred after the jelly layer had been removed; sperm-egg binding did not occur, nor
were the eggs fertilized. These data led me to think that perhaps the sperm could not penetrate
the vitelline layer of the egg. When both the jelly layer and the vitelline layer of the eggs were
removed, the inhibitory action was significantly decreased.
These findings lead me to posit that the active component of the fucus homogenate is an
inhibitor of a sperm factor responsible for dispersing the jelly layer and perhaps the vitelline
layer of the eggs. The work of Yamada and Aketa (1983) showed that there are indeed species-
specific sperm dispersal factors. It is possible that the homogenate is blocking the action of one
of these and thereby causing the sperm to become stuck in the jelly layer. This would be a good
starting point for future research.
Analysis of the chemical composition of the homogenate proved to be difficult at more
than a crude level. However, it is also apparent that the active compound (or perhaps
compounds) has a molecular weight greater than 10,000. This rules out simple molecules as the
active compound(s). Furthermore, the heat stability rules out most proteins. Combined, these
observations likely indicate a complex macromolecule(s) as the active component(s), perhaps a
complex polysaccharide or polyphenol, two families of compounds common in fucoid algae.
Fucoidan is a large, sulfated, polysaccharide and was the candidate for this active molecule based
on previous studies (Patankar et al. 1993). This is consistent with studies showing that other
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Peter Freed
sulfated polysaccharides (heparin) or sugar sulfates (glucose-6-sulfate) block the action of sperm
dispersal factors (Yamada and Aketa 1983). However, the spectrophotometric analysis for
fucoidan in the homogenate, combined with the bioassay, indicate that fucoidan alone is not
responsible for the inhibition of fertilization. As was presented in the absorption data (Figures 4
and 5), the peaks at 600-700 nm show similarities between the two samples, but the increased
absorption in the retentate at 400-500 nm is indicative of other unidentified carbohydrates in the
extract, many of which may have some action on fertilization. However, the orcinol reaction
only detects pentoses and therefore is at best an estimate of relative fucoidan concentration in the
homogenate. In the future, more extensive analyses that could give quantitative assessments of
the various types of sugars in the homogenate would allow better understanding of its action.
An impetus for exploring the possible effects of the inhibitory factor in nature was to
examine the potential species interactions of the fucus and the urchins. However, the data in the
microhabitat experiments indicate that the inhibitory factor is not leaked into the seawater from
whole plants. These preliminary experiments only looked at the natural release from plants in
water incubated in sunlight. The algae is likely subject to tidal grinding, de- and rehydration and
warming in its life history and any one of these mechanisms could cause an inhibitory effect in
situ. Further studies should be done to test these mechanisms and their implications.
In conclusion, the inhibitory action of a factor(s) contained in fucus homogenates as
shown in work from the 1950’s has been confirmed. It is a complex macromolecule(s) that is
heat stable and has a molecular weight greater than 10,000 but is not fucoidan acting alone as
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Peter Freed
previously thought. The inhibitory factor appears to be an inhibitor of sperm dispersal factors
acting on the sperm’s ability to penetrate the jelly layer and perhaps the vitelline layer of the egg.
Such a potent inhibitor likely has a wide variety of applications. Future research should be
directed towards determining if it similarly acts on other animal models or if it might be
applicable in humans.
ACKNOWLEDGEMENTS
1 would like to thank Dr. David Epel for his invaluable time and insights and endless
enthusiasm. I would also like to thank all of the members of his lab for their technical support
and also all of the professors of Biology 175H.
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Peter Freed
REFERENCES
Colowick, S. P., and N. O. Kaplan. 1957. Methods in Enzymology. Academic Press, New York.
Harding, C. V. 1951. The action of certain polysaccharides on fertilization in the sea urchin egg.
Experimental Cell Research 2:403-415.
Patankar, M. S., S. Oehninger, T. Barnett, R. L. Williams, and G. F. Clark. 1993. A revised
structure of fucoidan may explain some of its biological activities. The Journal of
Biological Chemistry 268:21770-21776.
Runnström, J., B. E. Hagström, and P. Perlmann. 1959. Fertilization. Pages 358-362 in J. Brachet
and A. E. Mirsky, editors. The Cell: Biochemistry, Physiology, Morphology. Academic
Press, New York.
Russell, M. P. 1987. Life history traits and resource allocation in the purple sea urchin
Strongylocentrotus purpuratus (Stimpson). The Journal of Experimental Marine Biology
and Ecology 108:199-216.
Vacquier, V. D., and J. E. Payne. 1973. Methods for quantitating sea urchin sperm-egg binding.
Experimental Cell Research 82:227-235.
Yamada, Y., and K. Aketa. 1983. A species-specific sperm factor dispersing the jelly coat of the
egg of the sea urchin, Anthocidaris crassispina. Gamete Research 8:279-293.
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Peter Freed
FIGURE LEGENDS
1. The concentration curve showing the relative inhibitory action of the fucus homogenate over
à range of concentrations.
2. A normal egg, fixed in formaldehyde, with sperm bound to the egg surface.
3. A normal egg exposed to fucus homogenate, fixed in formaldehyde, with sperm embedded in
the jelly layer as shown by the arrows.
4. The absorption of commercially-available fucoidan treated with the orcinol reaction. The
solid line is a third degree polynomial curve fit and the dashed line is the absorption of the
retentate and its equivalent concentration.
5. A spectral scan of 0.3 mg/ml commercially-available fucoidan against seawater.
6. A spectral scan of the retentate against seawater.
7. Normal eggs fertilized in the presence of commercially-available fucoidan. All eggs
fertilized. Some were normal as seen on the left and some raised secondary membranes as
seen on the egg on the right.
8. Eventually, all eggs fertilized in the presence of fucoidan at a concentration of 1 mg/ml or
higher lysed.
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taakakakakakakakata-
%
Figure 2
Figure 3
0
ondosqy
" —
Fucoidan (0.3 mg/ml) Against Sea Water
3.000

-O.100
700
400
500
600
Wavelegnth (nm)
Figure 5
Retentate Against Sea Water
3.000
-O.100
400
700
500
600
Wavelegnth (nm)
Figure 6.
Figure 7
Figure 8