ABSTRACT
It was demonstrated that the addition of 10 nM speract to gametes of
Strongylocentrotus purpuratus dramatically increases fertilization in standard
laboratory conditions and under the effect of turbulence. Such an increase in
fertilization has not been observed by other researchers; the difference in results
is likely due to variations in experiment conditions, in particular egg suspension
(personal communications from D. Garbers, N. Suzuki, and V.D. Vacquier).
Speract's mechanism of action remains unresolved; this paper suggests that there
is a low-affinity receptor on the sperm tail to which speract binds, possibly
causing changes in sperm metabolism.
INTRODUCTION
Adult urchins, such as Strongylocentrotus purpuratus, shed gametes
simultaneously into the ocean, but gamete interactions are a rare event
(Pennington, 1985). In some instances, chemotaxis assists sperm in finding eggs;
no such activity has been demonstrated in S. purpuratus (Ward et. al. ,1985).
Yet, the jelly of this species contains peptides which can affect sperm motility and
metabolism. This peptide, speract, has an amino-acid sequence of Gly-Phe-Asp
Leu-Asn-Gly-Gly-Gly-Val-Gly but has no chemotactic effect on sperm of the
same species (Garbers et al., 1982).
Although speract does not appear to be chemotactic, Suzuki et. al. have
demonstrated that, at pH 6.6, speract can stimulate respiration and motility to the
maximal values observed in normal seawater at pH 8.0 (Suzuki et. al ., 1980;
Repaske & Garbers, 1983). Such activity is normally repressed at pH values
below 7.3 due to an extreme sensitivity of the ATPase in the axonemal dynein in
sperm (Vacquier & Trimmer, 1986). When assayed at a pH of 7.8-8.0, 4.4 nM
speract doubles sperm respiration (see discussion; Repaske & Garbers, 1983).
Speract has also been shown to increase the levels of both cyclic AMP and cyclic
GMP along with inducing net H+ efflux from sperm cells (Garbers et al.., 1982).
Yamaguchi et. al. has observed that speract augments the effect of the fucose
sulfate polysaccharide observed by Garbers to induce the acrosome reaction in H.
pulcherrimus (Yamaguchi et. al.., 1987).
This study was designed to investigate the role of speract on fertilization at
the physiological pH of seawater, 8.0, in S. purpuratus.. I examined speract's
effects at a low egg suspension and at a small sperm/egg ratio and found that
under these conditions, there is a significant enhancement of fertilization.
Speract'’s mechanism of action in improving fertilization remains unknown, but
the results of this paper suggest that there is also a low-affinity receptor for
speract; the potential receptor binds at nM concentrations perhaps causing
changes in sperm metabolism which increase fertilization rates.
MATERIALS AND METHODS
Experimental animals ... S. purpuratus were collected from Point Arena, CA
and maintained at 13-15 °C. Intracoelemic injections of 5M KCl induced gamete
spawning by adult S. purpuratus. Sperm was collected dry and stored at 4°C.
Eggs shed into seawater were washed by allowing them to settle and aspirating
off seawater. Egg concentration was determined by sedimentation in a Bauer-
Schenle tube. Äfter dilution to a 1.2% suspension, they were stirred at 16°C and
used within several hours after collection. When necessary, eggs were dejellied
by filtering them 12 times through a 90 micron NITEX; this was followed by
hand centrifugation and removal of the supernatant containing the jelly.
Dejellying was confirmed by staining the eggs with Sumi dye.
Chemicals ... Dr. David Garbers of Vanderbilt University provided the peptide,
speract, isolated from the egg jelly of S. purpuratus.. Solutions of .0001 M
speract were stored at -10° C; speract appeared to lose its effectiveness after
seven days in solution and thus fresh solutions were made weekly. A solution of
500 mM KCI, pH 8, was employed to inhibit fertilization after 60 seconds (final
molar concentration was 250 mM; Figure One). Sumi dye was used to visualize
jelly layers.
Preparation of sperm suspension ... The number of sperm per microliter of
semen was found by measuring the turbidity of a 1 microliter semen/ 1 ml
seawater solution at 340 nm; spectrophotometer results were compared to a
standard curve determined from hemocytometer counts (Vacquier & Payne,
1973; Figure Two).
Preparation of egg suspension ... All experiments utilized a 0.1% (or 1%, as
indicated) egg suspension. Assuming 3 x 106 egg per packed cc, a 0.1%
suspension was equivalent to 3000 eggs per ml. Sperm egg ratios were
determined by dividing the number of sperm present in a 10 ml (or 20 ml, as
indicated).001 sperm suspension by the number of total eggs present (30,000
eggs in 10 ml or 60,000 eggs in 20 ml).
Equipment ... For experiments involving turbulence, construction of a
turbulence generator involved first hot-gluing plastic mesh (.25 inch holes) to a
600 ml beaker; the beaker was then inserted into a styrofoam block adhered
securely to a Belles Glen Inc. orbital shaker's rotating platform. The rotating
machine's adjustable speeds allowed for a distinction between high and low
turbulence. High turbulence is defined as speed 5 (4.4 rotations per second) and
low turbulence is defined as speed 2 (1.8 rotations per second). Assays were
performed as indicated below, but a 20 ml volume of egg and sperm suspension
was used.
Standard Speract Assay ... All assays, other than the turbulence assay above,
were done in 16 x 150 mm glass test tubes; the procedure involved the addition of
a 1 ml sperm suspension (+/- speract) to 9 ml of a 0.1 % egg suspension,
inverting the tube for adequate gamete interaction at 15 second intervals, and
adding 10 ml of 500 mM KCl at 60 seconds to prevent more fertilization.
Percent fertilization was determined by incubating the fertilized eggs at 16 °C for
20 minutes and then scoring the number of eggs with hyaline layers on the first
100 eggs observed under 10 X magnification in phase contrast microscopy.
Three replicates of each experiment were performed. Unless otherwise
indicated, all experiments were carried out at pH 8.0 and at an egg suspension of
0.1 %.
Acrosome Reaction ... Two methods were used to measure percent acrosome
reaction:
Method 1.) Addition of a one-fifth volume of 37% formaldehyde to each sample
fixed the sperm (final formaldehyde concentration was 7.4%). One drop of fixed
sample was placed on a coverslip which was then placed sample-side-down onto
Kimwipes. The coverslip was thumb-squashed onto the Kimwipe to remove
excess water without moving or splintering the coverslip. The sample was then
observed with oil immersion viewing under 100 x phase contrast microscopy.
Percent acrosome reaction was scored by counting the first 100 sperm observed.
Method 2.) Addition of a one-tenth volume of 37% formaldehyde to each sample
fixed the sperm (final formaldehyde concentration was 3.7%). Äfter a settling
time of six hours, 5 microliters from the bottom of the sample tube were pipetted
onto colloiden & carbon coated nickel grids; excess sample was blotted off after
five minutes. The grids were rinsed in distilled water; extra water was
immediately absorbed with filter paper. Percent acrosome reaction was scored
by counting the first 100 sperm observed with the electron microscope.
Statistical Analysis of Data
All data were analyzed by the T test to determine if observed differences in
means were sufficiently different from one another and not due to random
variation in the data collection.
t- deviation of sample mean from true mean
estimated standard deviation of the mean
All calculated values for ’t' were compared against a standard table to determine
significance of data (Snedecor, 1956).
RESULTS
KCl and Inhibition of Fertilization
1 wanted to assess gamete interactions for a fixed interval (60 seconds) and
therefore needed a convenient method to interrupt fertilization. High KCl
inhibits fertilization, and I therefore tested various KCl concentrations to
determine the lowest concentration required to achieve such an effect. I found
that addition of isotonic (0.5 M) KCl for a final concentration of 250 mM KCI
completely inhibits fertilization and allows fertilized eggs to develop normally
(Figure One).
Fertilization in Turbulence
No research has yet examined the success of S. purpuratus fertilization
during turbulence, and thus, the turbulence generator was constructed to simulate
natural turbulence under laboratory conditions. I found that fertilization in high
turbulence (speed 5) occurs only with much higher sperm/egg ratios than those
needed for the same percent fertilization in low turbulence (speed 2). Using eggs
from the same female for all trials, a sperm/egg ratio of 80:1 was needed to
achieve 74% fertilization in low turbulence (speed 2) while 70% fertilization in
high turbulence (speed 5) required a sperm/egg ratio of 13,200:1 (Figure Three).
Speract's Effects in Turbulence
Having found much difficulty in attaining fertilization in high turbulence
(speed 5), I asked whether the egg peptide, speract, would have any effect on
fertilization in a high turbulence situation. The addition of 10 nM speract to S.
purpuratus eggs and sperm in high turbulence (speed 5) produced 33% fertilized
eggs at a sperm/egg ratio of 3200:1; gametes of the same urchin at an identical
sperm/egg ratio gave fertilization rates of only 15.5% (Figure Three). The
addition of 10 nM speract to gametes in low turbulence (speed 2) produced
45.3% fertilization at a sperm/egg ratio of 8.8:1; gametes of the same urchin at
an identical sperm/egg ratio gave fertilization rates of only 27.3% (Figure Four).
Success of Fertilization in Presence of Speract
The above results showed that 10 nM speract increases fertilization in
turbulence at speeds 2 and 5; this was also the case in a test-tube or standard
laboratory fertilization procedure. The addition of 10 nM speract to gametes in
the test-tube assay increased fertilization to 21% at a sperm/egg ratio of 26.7:1.
Gametes from an identical urchin at the same sperm/egg ratio with no addition of
speract only resulted in 6.6% fertilization (Figure Six).
Speract Concentration and Success of Fertilization
The effects of different speract concentrations are shown in figure seven,
using a sperm/egg ratio of 26.7:1. I found the greatest effect by speract was
attained at 20 nM with a sperm/egg ratio of 26.7. At concentrations above 20
nM, fertilization rates reached a plateau; concentrations below 10 nM were less
effective at increasing fertilization than at 10 nM (Figure Seven).
Speract and the Jelly Layer
Speract is present in the jelly layer, and I wondered what effect removal of
the jelly layer would have, and also whether speract would have an effect.
Dejellying eggs in the standard test-tube assay had no effect on fertilization
(Figure Eight).
1 next examined the effects of jelly removal in low turbulence. At a
sperm/egg ratio of 9:1 in low turbulence, 24.5% of jellied eggs fertilized;
addition of 10 nM speract increased fertilization rates to 39% (Figure Nine).
Dejellying the eggs lowered fertilization rates to 7%; addition of 10 nM speract
to dejellied eggs increased fertilization rates to 24% (Figure Nine).
Speract's Effect on Acrosome Reaction
Yamaguchi et. al. has observed (with H. pulcherrimus gametes) that
speract acts as a cofactor in the acrosome reaction alongside fucose sulfate
polysaccharide (Yamaguchi et. al., 1987). Because it was not known if speract
was affecting motility, respiration, or the acrosome reaction at pH 8.0, I
measured speract's effects on spontaneous acrosome reactions. In samples
containing 1 microliter of sperm in seawater, 2% of sperm undergo spontaneous
acrosome reactions; addition of lOnM speract gives 1% acrosome reactions.
Therefore, it appears that, by itself, 10 nM speract has no effect on sperm
reactivity (Figure Ten; Figure Eleven). Öther studies have demonstrated
similarly low figures for spontaneous acrosome reactions (Collins & Epel, 1977;
Clapper et. al, 1985). In the presence of egg jelly, 64% of sperm undergo
acrosome reactions; addition of 10 nM speract in the presence of egg jelly results
in 56% acrosome reactions (no significant difference).
Egg Suspension and Effect of Speract
The effect that I observed with speract was puzzling because such
observations had not been seen by other researchers (personal communications
from D. Garbers, N. Suzuki, & V.D. Vacquier). One possible explanation was
that in the situations used by otbers a high concentration of eggs was used; if so,
the eggs might naturally secrete enough speract to make any effects of added
speract invisible. When I increased the egg suspension to 1.0, I also lowered the
sperm/egg ratio to 3:1 to keep low fertilization rates; I achieved 10.5%
fertilization with 10 nM speract. 11.5% fertilization can be obtained with
gametes from the same female at an identical egg suspension and sperm/egg ratio
with no added speract. This demonstrates that at high egg suspensions and at a
low sperm/egg ratio, there is no apparent effect of 10 nM speract.
Amount of Speract Present in Egg Suspension
The above results suggested that endogenous speract might be sufficiently
high in heavy suspensions to mask any effects of exogenous speract. According
to Yamaguchi et. al., 5 x 10° of H. pulcherrimus eggs contain 40 nmoles speract.
No other studies have produced concrete values, or estimates, of the amount of
speract naturally secreted by S. purpuratus or H. pulcherrimus (Yamaguchi et.
al., 1987). Because both species of urchins are almost of identical size, the values
for the amount of speract secreted from H. pulcherrimus were used as the
amount secreted from S. purpuratus.. In a 0.1% suspension of S. purpuratus
eggs, there are 4,000 eggs per ml which produce a total concentration of 32 nM
speract; this number is increased ten-fold to 320 nM when the egg suspension is
raised to 1.0%.
DISCUSSION
Speract was first isolated and purified from the egg jelly of S. purpuratus
nearly a decade ago, and at non-physiological pH (pH 6.6), has been
demonstrated to have significant physiological effects on sperm such as increased
motility and respiration, activation of K channels in the flagellar membrane,
K+ efflux and membrane hyperpolarization, cyclic nucelotide turnover possibly
responsible for Na“ entry, pHj increase, and Ca+ entry and removal (Repaske &
Garbers,1983; Lee, 1988; Schackmann & Chock, 1986; Garbers, 1989). Results
of my study are the first to show that at a particularly low egg suspension, low
sperm/egg ratio, and physiological pH, 10 nM speract improves fertilization
rates.
Perhaps the speract-induced increase in fertilization has not been
previously seen because of the conditions under which these experiments were
done. The amount of sperm used has been a critical variable in this study, and
sperm differs in number per microliter of semen from male to male; much of the
literature does not concern itself with exact numbers of sperm used, but it
appears to be of extreme importance (Dinnel et. al., 1987). The other crucial
factor involved was egg titer; higher egg titers secrete large quantities of speract,
making any addition of speract insignificant to the amount naturally present.
Although speract has been shown to increase fertilization in this study, its
absolute role is unclear. Because speract has already been demonstrated to
influence several components of sperm metabolism, the mechanism by which it
increases fertilization is likely an indirect one (Garbers, 1989; Repaske &
Garbers, 1983; Lee ,1988; Schackmann & Chock, 1986). Speract's method of
action stands unresolved, but several postulates for it exist.
10
Caz entry into sperm is thought to be one of the primary signals for
induction of the acrosome reaction; speract is suspected to act in the acrosome
reaction by inducing a transient rise in intracellular Ca2+ levels within the sperm;
(Collins & Epel, 1977; Yamaguchi et. al., 1987). Along with causing a fleeting
rise in intracellular Ca2+, it has been hypothesized that speract can regulate Ca2
within the sperm, an event of extreme significance to the acrosome reaction
(Schackmann & Chock, 1986). While it is believed that speract may even affect
the acrosome reaction by its enhancement of cyclic AMP concentration within the
sperm, speract is not thought to be responsible for triggering the acrosome
reaction. Yamaguchi demonstrated that speract acts as a cofactor with fucose
sulfate polysaccharide (another component isolated from the egg jelly) in
inducing the acrosome reaction in H. pulcherrimus sperm; my data coincides
with his results that speract itself is incapable of inducing the acrosome reaction
(Yamaguchi et. al., 1987).
A second method through which speract could act is through receptor-
mediated processes which initiate changes in ion flux from the sperm; some of
speract's effects are already known to be caused by the binding of speract to its
receptor, a high-affinity, 10-11 M, 77- kDa protein (Dangott & Garbers, 1984).
An 1231-labeled Bolton-Hunter adduct of speract was initially used to identify
the receptor but failed due to the lack of a free amino group which negated the
potential cross-linking studies to identify the receptor; thus an analogue termed
GGGIY2] was employed at pH 6.6 (Garbers, 1989; Dangott and Garbers, 1984).
The nM concentrations I have used do not agree with the idea of a high-affinity
receptor, and so it is suspected that there is yet another receptor, one of low¬
affinity, which has yet to be identified.
Garbers relates that in the cross-linking experiments used to identify the
high-affinity speract receptor, high concentrations of GGGIY21 speract could not
11
be used. Because of this, a low-affinity receptor may not have been detected
(Garbers, 1989). He also suggests that receptor molecules not capable of covalent
coupling with GGGY2) due to lack of a necessary functional group at the binding
site may exist (Garbers, 1989; Dangott & Garbers, 1984). It therefore remains
extremely plausible that there is another receptor yet to be identified.
The isolation of the high-affinity speract receptor was carried out at pH 6.6
(Dangott & Garbers, 1984). The experiments were probably performed at this
non-physiological pH because the increase in sperm respiration and motility
appear more dramatic at a low pH of 6.6. It is possible that the isolated high¬
affinity receptor is a high-affinity receptor at low pH and a low-affinity receptor
at high pH. Thus, there may be just one receptor.
Supposing that there are two receptors, the function of the second, a low-
affinity receptor, may be to signal the sperm that it is very close to the egg and
hence has an excellence opportunity to fertilize; it's binding of such a low-affinity
receptor may also intensify changes activated by binding of the high-affinity
receptor. There is also the possibility that binding of the high-affinity receptor
acts to increase the sperm motility and respiration so that the sperm can reach the
egg, and that binding of the low-affinity receptor informs the sperm that it has
reached the egg and that it should prepare to fertilize.
Speract has now been demonstrated to improve fertilization rates, but
whether such increases are necessary for S. purpuratus fertilization in the marine
intertidal stand unresolved. Because speract has already been implicated in many
fundamental aspects of sperm metabolism, however, its biological role in
fertilization is likely to be quite significant.
12
ACKNOWLEDGEMENTS
1 am deeply indebted to Professor David Epel for his direction, patience,
and great enthusiasm in this research project; his advice and suggestions have
been extremely insightful and invaluable. Dr. David Garbers of Vanderbilt
University provided the peptide speract, and so to him and to Dr. Epel I am
grateful for giving me the opportunity to explore speract's capabilities. I also
thank Professor Mark Denny for his assistance with my original project ideas
involving turbulence. Also, I give a special thanks to members of Professor
Epel's lab - to Rob Swezy, for his willingness to be of assistance at any time; to
Denis Larochelle for his lessons in microscopy; and to Chris Patton for his expert
assistance with electron microscope techniques. Thanks also to Sam Wang who
was readily available throughout the quarter for discussion and did a fantastic job
with the slides.
FIGURE LEGEND
Figure One ... Percent Fertilization versus KCl Concentration. The indicated
KCl concentrations were added to gametes at 60 seconds to interrupt fertilization.
Three Replicates Completed. Standard deviation (SD) of 100 mM is 1.2; SD of
150 mM is 5.6; SD of 200 mM is 10.6; SD of 250 mM is 0.3.
Figure Two ... Turbidity versus sperm per ml sea water. Semen of S.
purpuratus was suspended in sea water and formaldehyde (see results). Turbidity
was measured at 340 nM. The number of sperm per ml was determined by
hemocytometer counts. Three replicates completed for each hemocytometer
count.
Figure Three .. .Percent Fertilization Versus Sperm/Egg Ratio in Turbulence.
Experiments done in turbulence of speeds 2 and 5. Three replicates completed.
No fertilization at sperm/egg ratio of 80:1 at speed 5. SD of speed 2 at
sperm/egg ratio of 80:1 is 2.3; SD of speed 2 at sperm/egg ratio of 13200:1 is
0.9; SD of speed 5 at sperm/egg ratio of 13200:1 is 3.6.
Figure Four . .. Percent Fertilization With & Without Speract at Speed 5.
Experiment done in turbulence at speed 5. Sperm/Egg Ratio of 3200:1. SD
without speract - 0.7; SD with speract - 2.8. 2.5-1.0% chance from same
population. Three replicates completed speed five (high turbulence).
Figure Five . . .Percent Fertilization With & Without Speract at Speed 2.
Experiment carried out in turbulence at speed 2. Sperm/Egg Ratio of 8.8:1. SD
without speract - 13.5;SD with speract -15.1. 10-20% chance from same
population. Three replicates completed at speed 2 (low turbulence).
Figure Six ... Percent Fertilization With & Without Speract. Experiment
carried out by standard test-tube assay. Sperm/Egg Ratio of 26.7:1. SD without
speract - 2.5; SD with speract - 6.0. 0.5- 0.1% chance from same population.
Three replicates made.
Figure Seven .. .Percent Fertilization Versus Speract Concentration. Experiment
carried out by standard test-tube assay. O nM speract gave 15.3% fert; 5 nM
speract gave 22.3% fert.; 10 nM speract gave 27.6% fert.; 20 nM speract gave
37.3% fert.; 40 nM speract gave 32.0% fert.; 100 nM speract gave 29.0% fert.;
Sperm/Egg Ratio 26.7:1. Three replicates made. SD at 5 nM is 2.2; SD at 10 nM
is 2.3; SD at 20 nM is 1.2; SD at 40 nM is 5.2; SD at 100 nM is 6.7.
Figure Eight ... Percent Fertilization As A Function of Jelly Layer. Experiment
carried out in standard test-tube assay. Jelly removed by filtering eggs through
14
90 micron NITEX. Sperm/Egg Ratio 33. SD of jellied eggs-4.1; SD of dejellied
eggs - 2.4 and values are not significantly different.
Figure Nine ... Percent Fertilization With & Without Speract. Experiment
carried out in turbulence. Jelly removed by filtering eggs through 90 micron
NITEX Sperm/Egg Ratio 9. SD of jellied without speract - 2.1 SD of jellied with
speract - 1.4; SD of dejellied without speract - 2.8; SD of dejellied with speract :
2.8 1.0-2.5% jellied eggs with & without speract from same population; 2.5-5%
chance dejellied eggs with & without speract from same population. Three
replicates completed at speed 2 (low turbulence).
Figure Ten .. .Electron Microscope Photograph of the Acrosome Reaction in
Sperm. Sperm on the right has reacted; the sperm on the left is unreacted.
Magnified x 25,000.
Figure Eleven . . .Percent Acrosome Reaction With & Without Speract.
Experiment carried out with both methods; both gave same result (see methods &
materials). No sperm/egg ratio. SD without speract - 4.9; SD with speract - 4.9.
Two replicates completed.
Figure Twelve ... Percent Fertilization Versus Speract at 1% Eggs. Experiment
carried out by standard test-tube assay. Sperm/Egg Ratio 3:1. SD without
speract - 4.9. SD with speract - 2.1. Three replicates completed.
REFERENCES
Clapper, David L., Davis, James A., Lamothe, Paul J., Patton, Chris, and Epel,
David. Involvement of Zinc in the Regulation of pHj, Motility, and
Acrosome Reactions in Sea Urchin Sperm. J. Cell Biol. (1985) 100: 1817-
1823.
Collins, F. and Epel, D. The Role of Calcium lons in the Acrosome Reaction of
Sea Urchin Sperm. Exp. Cell. Res. 106: 211-222 (1977).
Dangott, Lawrence J. and Garbers, David L. Identification and Partial
Characterization of the Receptor for Speract. J. Biol. Chem. 259:
13712-13716 (1984).
Dinnel, Paul A., Link, Jeanne M. and Stober, Quentin J. Improved Methodology
for a Sea Urchin Sperm Cell Bioassay for Marine Waters. Arch. Environ.
Contam. Toxicol. 16: 23-32 (1987).
Garbers, David L., Watkins, Helen D., Hansbrough, J.R., Smith, Adaline, and
Misono, Kunio S. The Amino Acid Sequence and Chemical Synthesis of
Speract and of Speract Analogues. J. Biol. Chem. 257: 2734-2737
(1982).
Garbers, David L. Molecular Basis of Fertilization. Annu. Rev. Biochem. 58:
719-42 (1989).
Hagstrom, B.E. Further Experiments on Jelly-Free Sea Urchin Eggs. Exp. Cell.
Res. 17: 256-261 (1959).
Lee, Hon Cheung. Internal GTP Stimulates the Speract Receptor Mediated
Voltage Changes in Sea Urchin Spermatozoa Membrane Vesicles. Dey.
Biol. 126: 91-97 (1988).
Lee, Hon Cheung and Garbers, David L. Modulation of the Voltage-sensitive
Na+H+ Exchange in Sea Urchin Spermatozoa through Membrane
Potential Changes Induced by the Egg Peptide Speract. J. Biol. Chem.
261: 16026-16032 (1986).
Lillie, Frank. Problems of Fertilization.. University of Chicago Press: Chicage
1919.
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Kopf, G.S. and Garbers, D.L. Calcium and Fucose-sulfate-rich Polymer
Regulate Sperm Cyclic Nucleotide Metabolism and the Acrosome Reaction.
Biol. Reprod. 22: 1118-1126 (1980).
Pennington, J. Timothy. The Ecology of Fertilization of Echinoid Eggs: The
Consequences of Sperm Dilution , Adult Aggregation , and Synchronous
Spawning. Biol. Bull. 169: 417-430 (1985).
Repaske, David R. and Garbers, David L. A Hydrogen lon Flux Mediates
Stimulation of Respiratory Activity by Speract in Sea Urchin Spermatozoa.
J. Biol. Chem. 258: 6025-6029 (1983).
Schackmann, Robert W. and Chock, P. Boon. Alteration of Intracellular Ca 241
in Sea Urchin Sperm by the Egg Peptide Speract. J. Biol. Chem. 261:
8719-8728 (1986).
Snedecor, George W. Statistical Methods. lowa State University Press: Ames,
1956.
Suzuki, N. Nomura K., Ohtake, H. Sperm Activating Peptides Obtained From
Jelly Coat of Sea Urchin Eggs. Zool. Mag. 89:350 (1980).
Trimmer, James S. and Vacquier, Victor D. Activation of Sea Urchin Gametes.
Annu. Rev. Cell Biol.. 2:1-26 (1986).
Vacquier, V. and Payne, J. Methods for Quantitating Sea Urchin Sperm-Egg
Binding. Exp. Cell Res. 82: 227-235 (1973).
Ward, G.E., Brokaw, C.J., Garbers, D.L., and Vacquier, V.D. Chemotaxis of
Arbacia punctulata Spermatozoa to Resact, a Peptide from the Egg Jelly
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pulcherrimus. Develop. Growth & Differ. 30: 159-167 (1987).
% FERTILIZATION VERSUS KCI CONCENTRATION
100
2
80
60
40
20
80
100
200
300
KCI CONCENTRATION ImMI
Fig. 1
TURBIDITY VERSUS SPERM PER ML SEA WATER
0.5
0.4
0.3
0.2
0.1
10 2
50 60
SPERM X 100000/m
% FERTILIZATION VERSUS SPERM/EGG RATIO IN TURBULENCE
120
2
100
80
SPEED 2
60
ESPEED 5
40
20

13200
30
SPERM/EGG RATIO
Fig. 3
% FERTILIZATION WITH & WITHOUT SPERACT AT SPEED 5
40
30
20
10

PRESENCE OF SPERACT
Fig. 4
% FERTILIZATION WITH & WITHOUT SPERACT AT SPEED 2
60
50
40
30
20
E
LL
10
8
PRESENCE OF SPERACT
% FERTILIZATION WITH & WITHOUT SPERACT
30
20
10

8
PRESENCE OF SPERACT
g6
% FERTILIZATION VERSUS SPERACT CONCENTRATION
50
2
40

+ 30
20


10
20 40 60 80 100
SPERACT CONCENTRATION (nM)
Fig.
% FERTILIZATION AS A FUNCTION OF JELLY LAYER
40
30
20
10

PRESENCE OF JELLY
Fig. 8
% FERTILIZATION WITH & WITHOUT SPERACT
50
40
30
JELLIED
DEJELLIEL
20
10


PRESENCE OF SPERACT
Fig. 9
Fig. 10
% ACROSOME REACTION WITH & WITHOUT SPERACT
10

PRESENCE OF SPERACT
Fig. 11
% FERTILIZATION VERSUS SPERACT AT 1% EGGS
14
12
10


PRESENCE OF SPERACT
Fig. 12