CERAMIDE DISRUPTS SPERM MOTILITY AND INHIBITS FERTILIZATION IN
THE PURPLE SEA URCHIN, STRONGYLOCENTROTUS PURPURATUS
Nikolai Kaestnei
Sphingolipid signaling cascades play a role in regulating egg maturation and differentiation in a
variety of organisms. To determine if a similar mechanism is involved in fertilization, S.
purpuratus gametes were treated with cell-permeable ceramide. It was found to reduce sperm
motility, decrease sperm-egg binding, and inhibit fertilization. This effect was stereospecific and
could not be mimicked by a ceramide analog differing from ceramide by only a double bond.
These results suggest a role for ceramide signaling in regulating sea urchin fertilization
Introduction
The role of phospholipid metabolites as second messengers in important signal
transduction cascades has been firmly established (1), the paradigm being the phosphoinositide
signaling cascade. This transduction cascade is initiated when phospholipase C(PLC) cleaves
phosphatidyl inositol bisphosphate (PIP2) into diacylglycerol (DAG) and inositol triphosphate
(IP3). These two metabolic products serve as second messengers to respectively activate protein
kinase C(PKC) and to effect the release of calcium from the endoplasmic reticulum (2). An
analogous signal transduction cascade involving the degradation of sphingomyelin has gained
recent attention due to its role in antiproliferative cell responses. Most thoroughly characterized in
yeast and mammalian cells, it begins with the sphingomyelinase-mediated breakdown of
sphingomyelin into its choline head group and ceramide (3). A number of sphingomyelinase
inducers have been identified, including vitamin Dz, tumor necrosis factor-o (TNF-o), retinoic
acid, heat, ionizing irradiation, chemotherapeutic agents, and human immunodeficiency virus (4)
All lead to an increase in intracellular ceramide concentrations and result in either terminal
differentiation, apoptosis, or cell cycle arrest. These actions appear to be mediated by a ceramide¬
activated protein phospohatase (CAPP) that, in different cell types, can dephosphorylate the
retinoblastoma gene product, downregulate the c-myc protooncogene, and antagonize PKC (5). A
ceramide-activated protein kinase (CAPK) has also been discovered, but remains poorly
characterized. Recently, ceramide, as well as an exogenously-applied bacterial sphingomyelinase,
were found to induce the maturation of Xenopus oocytes (6). This suggests that meiotic cell cycle
progression in these animals (normally induced by progesterone) may act via a ceramide signal
cascade.
The discovery of ceramide’s role in frog oocyte maturation and the lack of studies
examining sphingolipid signaling in other aspects of early development prompted this closer look
at the possible use of ceramide as a second messenger during fertilization of the purple sea urchin.
Strongylocentrotus purpuratus. Early development has been well characterized in these intertidal
and subtidal inhabitants, and the large quantities of gametes released by each animal make them an
ideal model for studying many aspects of fertilization. Furthermore many of the events that follow
sperm-egg binding are induced by a phosphoinositide signaling cascade that generates DAG and
IPz as previously described. DAG activates PKC, which phosphorylates a Na/H exchange protein
and thereby instigates an increase in intracellular pH required to stimulate the protein synthesis,
DNA replication, and morphological changes required after fertilization (7). Ceramide’s putative
antagonism of PKC activity raises the possibility that it may modulate post- fertilization events in
the eggs of these animals. Indeed, this study was able to show that ceramide inhibited sea urchin
fertilization by affecting sperm motility and sperm-egg binding. Further proof of such a role may
help to establish an understanding of the mode of ceramide action and its role in regulating
cellular processes as well as providing a novel model system for dissecting the ceramide pathway.
Methods
Purple sea urchins (Strongylocentrotus purpuratus) were collected at low tide in Half
Moon Bay, California and maintained in covered, flow-through tanks at Hopkins Marine Station.
Urchins were spawned by immersing an animal halfway in seawater and applying a 50 V current
or injection with 2 ml 0.5 MKCl. Female gametes were collected by inverting the spawning
urchin on a beaker filled with filtered seawater, whereas male gametes were pipetted into a 1.5 ml
Eppendorf tube and stored dry at 4°C. All gametes were pipetted using blunted pipette tips, and
eggs were never dejellied. The density of an egg suspension was determined by centrifuging a 1
ml sample in a Bauer-Schenk tube and recording the percent volume of eggs. Sperm suspensions
were obtained by diluting the dry sperm with filtered seawater. The concentration of sperm used
for a given experiment was based on the maximal sperm dilution that could be used to produce
near 100% fertilization in seawater (in most experiments this was a 1:50,000 dilution achieved by
adding 20 ul of a 1:1,000 dilution to 1 ml of seawater). Fertilization was assessed by placing two
drops of an unfixed sperm-egg mixture on a microscope slide and determining the percentage of
eggs with a clearly defined fertilization envelope at lOX under darkfield conditions. Co ceramide
and C, dihydroceramide were obtained from Calbiochem and dissolved in DMSO to create 10
mM stock solutions. Both solutions were stored at -20 °C when not in use.
Fertilization. 1 ml of a 0.3% egg suspension was added to each of twelve 3 ml multiwell
Falcon dishes in a 15 °C cold room. Exposure to 5, 10, and 20 uM concentrations of ceramide and
dihydroceramide was achieved by adding appropriate volumes of stock solutions to each of the
egg suspensions, and the eggs were incubated for 5 minutes. A DMSO control contained the
equivalent amount of DMSO as used to make the ceramide solutions, while a seawater control
contained the same volume of seawater as total volume of solution used in the ceramide
treatment. Finally, 20 ul of a 1:1,000 sperm suspension was added to each Falcon dish and
fertilization was assessed under the light microscope as described above.
Egg Wash Experiment. Four 5 ml aliquots of a 0.3% egg suspension were placed in
separate 15 ml test tubes and each tube was incubated for 5 minutes in 20uM final concentration
of one of the above treatments. The tubes were then inverted three times and a 1 ml sample of
each was placed in a Falcon dish. The remaining egg suspensions were washed twice with filtered
seawater and then placed in separate Falcon dishes. All suspensions were fertilized with a
1:125,000 sperm dilution and fertilization was assayed as above after 5 minutes
Sperm Inhibition. Four 1 ml aliquots of a 1:1,000 sperm dilution were placed in
Eppendorf tubes and treated with 10 uM ceramide or dihydroceramide, and an equivalent DMSO
concentration or seawater. 1, 5, 10, and 20 ul from each sperm treatment was then added to
separate 1 ml 0.2% egg suspensions and fertilization was measured after 5 minutes by light
microscopy
Sperm-Egg Binding. Four 1 ml 1:1,000 sperm dilutions were placed in Eppendorf tubes
and each one incubated for 5 minutes with 20 uM of ceramide or dihydroceramide, or an
equivalent concentration of DMSO or seawater. 50 ul of each dilution were then placed in a
second set of Eppendorf tubes and 150 ul of a 1.2% egg suspension added to each. After 20
seconds, the eggs were fixed with 200 ul of 4% gluteraldehyde in 100 mM HEPES seawater at
pH 7.2. The suspensions were washed with filtered seawater after five minutes, and the number of
sperm bound to the maximum circumference of each egg was scored blindly at 40X under
darkfield conditions. T-statistics were calculated using SigmaPlot.
Acrosome Reaction (AR) Assay. To induce the acrosome reaction, egg jelly was prepared
by bringing a suspension of 25% eggs in 10 mM HEPES seawater (pH 8.2) down to pH 5 with
O.1 M HCl and swirling it for 2 minutes. The eggs were then returned to pH 8-8.2, centrifuged,
and the supernatant containing the egg jelly collected. The egg jelly was kept on ice while four
100 ul aliquots of 10 mM HEPES seawater (pH 7.9) were incubated with 80 uM of ceramide or
dihydroceramide, or equivalent amounts of seawater or DMSO as described above. 100 ul of a
1:100 sperm dilution was added and mixed gently by pipetting up and down. Aster 5 minutes, 200
ul of egg jelly were added using this same technique. After another 5 minutes, 400 ul of 5%
gluteraldehyde was added to each tube and the contents fixed for 15 minutes. A sperm squash was
prepared as described elsewhere (8) and assayed for the acrosomal reaction via 1000X phase-
contrast microscopy. Slides were moistened by exhaling warm air onto them and then cleaned
with a Kimwipe before placing 10 ul of sperm suspension on one side The fraction of acrosome
reacted sperm was determined by noting the loss of the phase-dense acrosomal granule at the tip
of a sperm and the presence of a faint process projecting from its apex.
Motility Comparisons. Previous studies showed that actively swimming sperm travel
down a centrifugal gravity gradient while immotile sperm remain largely suspended (9). Four 1 ml
1:1,000 sperm dilutions were placed in separate Eppendorf tubes and treated with 20 uM
ceramide or dihydroceramide, or equivalent DMSO or seawater concentrations. A fisth sperm
dilution was incubated in 1% formaldehyde to kill the sperm completely. All five dilutions were
transferred to glass cuvettes and their absorbance was measured at 540 nm in a
spectrophotometer. The cuvettes were then centrifuged inside 50 ml plastic test tubes at 120xg
for five minutes before recording absorbance a second time. The change in absorbance was
plotted as a percent of the original OD readings. A greater change in absorbance was correlated
with more actively swimming sperm.
Sperm Respiration. Sperm respiratory rates under the four treatment conditions were
qualitatively compared using an oxygen-sensing electrode. Seawater was placed in the 2 ml
respiration chamber, stirred continuously, and the electrode allowed to equilibrate until the
quantity of oxygen measured in the seawater was constant. 1 ul of sperm concentrate was then
added to the chamber to create a 1:2,000 final dilution. Once a constant sperm respiratory rate
was observed, 40 ul of each drug was added to the chamber through a small aperture and the
change in oxygen values noted and compared to the initial respiration rate (as determined
qualitatively by slope comparisons since technical problems made calibrations of the respirometer
impossible). All four trials were repeated once.
Results
Fertilization. The initial experiments established a concentration-dependent inhibition of
fertilization by ceramide but not by its analog, dihydroceramide, or by DMSO and seawater (Fig
1). Significant reductions in fertilization efficiency were observed at 5, 10, and 20 uM ceramide
compared to all three other treatments. Rates of fertilization ranged from 94 to 99% in untreated
controls while dropping to 68% with 20 uM ceramide
Egg Wash Experiment. As seen in figure 2, only 58% of eggs in O uM ceramide were
fertilized, in contrast to 91 to 94% of control eggs. However, after washing the treated eggs in
filtered seawater, normal levels of fertilization were achieved. Fertilization in the four washed
treatments varied from 95 to 97%. These results suggest that ceramide inhibition of sea urchin
fertilization may be reversible.
Sperm Inhibition. Treating the sperm with ceramide also resulted in inhibition of
fertilization, although the degree of inhibition was dependent on the volume of sperm used to
fertilize the eggs (Fig. 3). At 1 and 5 ul of sperm dilution, fertilization was significantly reduced to
45 and 80%, respectively. At 10 and 20 ul of added sperm, however, no inhibition was evident,
probably because excess sperm at these levels allowed fertilization to occur despite a large
number of inhibited sperm. Controls all fertilized at close to 100%. These results indicate that
sperm may be the major recipient of ceramide inhibition because fertilization is suppressed at
levels similar to those seen in initial fertilization experiments
Sperm-Egg Binding. Sperm-egg binding was severely disrupted in ceramide-treated
sperm, as seen in Figure 4. An average of only 0.5 sperm were bound to each egg in the presence
of ceramide, whereas sperm treated with dihydroceramide, DMSO, or seawater averaged 2.5, 3.2,
and 2.8 sperm per egg.
Acrosome Reaction Assay. Ceramide, dihydroceramide, and DMSO all significantly
inhibited the acrosome reaction relative to seawater controls (Fig. 5). Only 11 to 13% of sperm
reacted under these three conditions, in contrast to the 22% that reacted in seawater. The three
lower levels of reactivity did not differ significantly from each other.
Motility Comparisons. The change observed in the ODs4o was lowest in dead,
formaldehyde-treated sperm (19%) and highest in seawater and dihydroceramide-treated sperm
(around 50%) (Fig. 6). Sperm treated with ceramide had a 22% change in absorbance,
approximating the difference observed in formaldehyde-treated sperm. Oddly, DMSO-treated
sperm fell in between the extremes at 39%. This latter data suggests that despite the general
indications of reduced motility in ceramide-treated sperm, these results must be viewed with
caution because the DMSO treatment differed from other treatments containing similar amounts
of DMSO (see below).
Sperm Respiration. Before treatment, the rate of decrease in oxygen levels appeared
identical. However, treatments containing DMSO created an electrode artifact upon addition that
caused oxygen levels to rise rapidly and equilibrate only after 5 minutes (Fig. 7). At that time it
was possible to qualitatively compare oxygen levels before and after addition of these treatments.
Ceramide caused an apparent halt in respiration as indicated by the lack of a further reduction in
O2 levels. Dihydroceramide similarly showed a decrease in the rate of oxygen consumption,
though not as dramatically as in ceramide-treated sperm. On the other hand, addition of DMSO or
seawater caused oxygen levels to decrease at rates comparable to those before treatment.
Numbers next to respiration plots indicate a qualitative comparison of slopes between the
different treatments but are not actual measurements of oxygen consumption rate.
Discussion
These results show that ceramide disrupts fertilization in S. purpuratus in a concentration¬
dependent manner. This inhibitory action appears to affect the sperm since the effect on eggs was
reversible whereas ceramide-treated sperm were unable to regain their fertilization ability despite
the 50-fold dilution of ceramide that accompanied the addition of these sperm to the egg
suspension. Since such low concentrations of ceramide (on the order of 200 nM after dilution)
were well below the threshold above which ceramide reduced, it seems that the sperm were
unable to recover from ceramide treatment. This implies that without ceramide present in the
suspension around washed eggs, fertilization is not inhibited because sperm are not suppressed.
While it is not possible to rule out the additional inhibition of the egg, it is clear that the effect of
ceramide on sperm is large enough to account for the reduced fertilization observed. The lack of a
pronounced impact on eggs would suggest that ceramide does not regulate PKC activity in these
cells, but does not rule out the operation of a sphingolipid signaling pathway later in the
development of sea urchins.
The observed inhibition of fertilization by ceramide appears to ensue from an inability of
the sperm to bind eggs. Microscopic examination of the sperm indicated that ceramide-treated
sperm were significantly less motile and ceased moving many minutes before sperm treated with
any of the other reagents. An attempt to quantify sperm motility using the Nelson
spectrophotometer assay provided mixed results. Ceramide suppressed sperm motility and slowed
the sperm sedimentation rate in the centrifuge. Dihydroceramide had no effect on sperm
swimming ability, but the intermediate swimming rate for DMSO was puzzling since the
dihydroceramide sample also contained DMSO. It is possible that DMSO reduced sperm motility
as indicated, and that dihydroceramide reverses this effect while ceramide amplifies it. This would
make doubtful the use of dihydroceramide as a benign negative control for the specific effects of
ceramide. Nevertheless, this qualitative analysis seems to support the motility characteristics
clearly visible under the microscope
The respiration studies similarly cloud the use of dihydroceramide as a control. While
respiration and motility are not perfectly linked, a reduced rate of motility might indicate, or be
the reason for, a decline in respiratory rate. Such a correlation was found for ceramide, which
completely halted respiration in sperm. Similarly, seawater and DMSO did not cause any
appreciable difference in the respiration rate of sperm. Oddly, the addition of dihydroceramide led
to a large reduction in respiration rate exceeded only by ceramide itself. Sperm that exhibited this
drop were examined under the microscope and found to be swimming normally. It is possible that
the compound, though benign when assessing motility and fertilization, is toxic as far as
respiration is concerned. On the other hand, DMSO appeared to affect motility, but had little
impact on respiration. These results suggest that while motility seems to explain differences in
fertilization between the different treatments, respiration does not, possibly because the two need
not be directly linked
One final factor was examined as an explanation for the ceramide-mediated inhibition of
fertilization: the acrosome reaction. It is known that the premature induction of the acrosome
reaction will lead to the rapid death of sperm as calcium from the seawater floods the cytosol and
uncouples mitochondria (10). In addition, suppression of the reaction would hinder binding and
fertilization. However, neither effect is likely since no ceramide-specific inhibition or induction of
the acrosome reaction was observed. DMSO, however, did seem to diminish the incidence of the
reaction since all DMSO-containing treatments showed reduced levels of acrosome reaction in
comparison to seawater. A problem with these experiments, however, is the low percentage of
acrosomal reaction seen in the jelly-treated eggs. This suggests that the sperm or jelly may have
been defective and not suitable for experimentation.
Taken together, results from the fertilization, binding, and motility experiments suggest that
ceramide inhibits fertilization by reducing sperm motility and thereby diminishing sperm-egg
binding efficiency. There are two possible explanations for these results Ceramide may simply be
exhibiting a toxic effect on sperm and consequently diminishing their fertilization performance.
Due to its lipophilic nature, ceramide might intercalate into the membrane and disrupt normal
cellular processes by distorting the membrane architecture. Its C4 double bond may have the same
effect of increasing membrane fluidity as do the double bonds of unsaturated fatty acyl chains.
This would explain why dihydroceramide, without the double bond, did not cause similar
disruptions of sperm behavior. It should be noted, however, that ceramide does not appear to
produce similar disturbances in membrane dynamics in mammalian and yeast studies, despite its
double bond. This suggests that ceramide may, in fact, have a true physiological role in the sperm
of S. purpuratus.
Perhaps a compound in the environment may signal unfavorable conditions for
development and trigger a ceramide signaling cascade that serves to prevent fertilization when
subsequent development is not likely to be successful. Since a concrete paradigm of ceramide
signaling has not been established, there is no reason to believe that CAPP must always act in
opposition to PKC and therefore affect the egg. A hypothetical sperm CAPP could serve to
dephosphorylate many of the proteins that are phosphorylated upon sperm activation in seawater,
thereby limiting motility. Alternatively, it could depress respiration by a similar mechanism.
Since little is known about sphingolipid signaling in invertebrates, and signal cascades of
sperm in general, hypotheses concerning a role for ceramide in the cellular processes of sperm
must all be viewed with caution until conclusive studies are done. Nevertheless, if ceramide is
found to inhibit sperm in a non-toxic fashion, it would not be surprising to find many of the
common elements of a sphingomyelin signal cascade inside the sperm, themselves. In addition to
providing another example of the widespread presence of a role for ceramide, such a discovery
would also help to deepen our understanding of the factors influencing and regulating fertilization
and development in sea urchins.
Though the sperm “apoptosis" presented in this discussion may seem wasteful since a sea
urchin could save much energy by not releasing its gametes in the first place, it must be
remembered that evolution does not create the perfect solution, but merely one that is better than
its predecessor. Still, one may ask how such a system could have ever evolved, since the sperm,
once it is spawned, doesn’t have much to lose by attempting fertilization despite a less than
favorable environment. Future research to clarify the method of ceramide inhibition and to
demonstrate the presence of a sphingolipid signaling cascade could help to distinguish between
the two opposing views of ceramide action in the sperm of purple sea urchins. If a true
physiological role is found, it will send us all scratching our heads at evolution's often mysterious
course.
Reference
1) Stryer, L. Biochemistry: Third Edition. New York: W. H. Freeman & Co., 1996
2) Nishizuka, Y. (1992) Intracellular signaling by hydrolysis of phospholipids and activation of
protein kinase. Science 258: 607-614
3) Okazaki, T., et al. (1989) Sphingomyelin turnover induced by vitamin D3 in HL-60 cells
Role in cell differentiation. J. Biol. Chem. 264: 19076-19080.
4) Hannun, Y. A. (1996) Functions of Ceramide in Coordinating Cellular Responses to Stress.
Science 274: 1855-1859.
5) Hannun, Y. A., et al. (1986) J. Biol. Chem. 261: 12604-12609.
6) Strum, J. C., et al. Ceramide Triggers Meiotic Cell Cycle Progression in Xenopus Oocytes.
J. Biol. Chem. 270: 13541-13547.
7) Gilbert, S. F. Developmental Biology: Third Edition. Sunderland, MA: Sinauer Associates,
Inc., 1996.
8) Vacquier, V. D. Sea Urchin Spermatozoa in Methods in Cell Biology. Orlando: Academic
Press, Inc., 1986.
9) Nelson L. (1972) Exp. Cell Research. 74: 269-274.
10) Lee, H. C., et al. (1983) Changes in Internal pH Associated with Initiation of Motility and
Acrosome Reaction of Sea Urchin Sperm. Developmental Biology 95: 31-45.
Figure Legends
Figure 1: Ceramide Inhibition of Fertilization. Eggs were treated as indicated and fertilization
assayed using darkfield microscope. Ceramide was found to inhibit fertilization in a concentration-
dependent manner. Lowest levels of fertilization (68%) were seen at 20 uM ceramide, though
ceramide-inhibition was significant in comparison to the three controls at all concentrations tested
(p5.05)
Figure 2: Reversible Inhibition of S. purpuratus Eggs. Eggs were treated with 20 uM ceramide
and fertilization reduced to 58% in unwashed eggs. This value was significantly different from the
controls (p#.01). Washed eggs did not differ in their ability to be fertilized.
Figure 3: Irreversible Inhibition of S. purpuratus Sperm. A1:1,000 sperm dilution was treated
with 10 uM ceramide and added to 1 ml of a 0.3% egg suspension in the volumes indicated.
Significant inhibition of fertilization was observed at I and 5 ul of dilution in comparison to
controls (ps.01). Fertilization in these conditions was reduced to 45 and 80%, respectively
Figure 4: Ceramide Inhibition of Sperm-Egg Binding. AO.3% egg suspension was fixed 20
seconds after addition of a sperm dilution and sperm-egg binding assayed using darkfield
microscopy. The number of sperm bound to each egg at its maximum diameter was recorded,
Ceramide-treated sperm bound significantly less than control sperm (ps.05) with only 0.5 sperm
bound per egg. Controls ranged between 2.5 and 3.2 sperm per egg.
Figure 5: Ceramide Effect on the Acrosome Reaction. Treated sperm were incubated with egg
jelly and the incidence of acrosomal reaction assayed under phase-contrast microscopy. Ceramide
dihydroceramide, and DMSO-treated sperm all inhibited the reaction significantly in comparison
to seawater (pS.05). 22% of seawater-treated sperm reacted while only 11-13% of sperm treated
with the other reagents showed any acrosomal process.
Figure 6: Ceramide Effect on Sperm Motility. Absorbance at 540nm was assessed in a
spectrophotometer before and after centrifugation of sperm treated as indicated. Changes in
ODsao were plotted as a percent of original readings. Ceramide-treated sperm (22%) were most
similar to formaldehyde-treated sperm (19%) in their change in absorbance after centrifugation.
Highest changes in absorption were observed in seawater and dihydroceramide controls (SI and
50%, respectively). DMSO was curiously in the middle (39%)
Figure 7: Sperm Respiration Rates. Sperm were placed in the chamber of an oxygen-sensing
electrode and basal respiration established (right). Treatments were added (middle), the system
allowed to equilibrate, and final rates of oxygen consumption (lest) compared to initial rates.
Ceramide-treated sperm appeared to cease respiring, while DMSO and seawater treatments
appeared to little affect sperm respiration. Dihydroceramide approached ceramide in its ability to
suppress respiration. Numbers refer to the slopes corresponding to each treatment and are meant
for qualitative comparisons only.
100
80
60
40
20
Figure 1: Ceramide Inhibition of Fertilization
ceramide d-ceramide DMSO seawater
Treatment
20UM
— 1OUM
5UM
Figure 2: Reversible Inhibition of S. purpuratus Eggs
100
80
60
40
20
ceramide d-ceramide DMSÖ seawater
Treatment
pre-wash
post-wash
Figure 3: Irreversible Inhibition of S. purpuratus Sperm
120
100
80
60
40
20
20
Sperm Volume (uL)
ceramide
— d-ceramide
DMSO
Seawater
Figure 4: Ceramide Inhibition of Sperm-Egg Binding
0
ceramide d-ceramide DMSÖ seawater
Treatment
Figure 5: Ceramide Effects on the Acrosomal Reaction
25
20
≈ 10
ceramide d-ceramide DMSO
seawater
Treatment
60
50
8 40
E 30
820
10
Figure 6: Ceramide Effect on Sperm Motility
CH2O ceramide DMSO FSW d-ceramide
Treatment
Figure 7: Sperm Respiration Rates
eamide
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