Abstract: Spermatozoa from the sea urchin, Strongykentrotus
Purpuratus are shown to lose the ability to fertilize soon after
dilution. Acrosomal reaction studies, using both nascent and
senescent sperm, demonstrate that sperm are becoming
insencitive to egg jelly (the natural inducer of the acrosomal
reaction 141) with time. Although unable to respond to the jelly
stimulus, aged sperm suspensions yield a high percentage of
acrosomal reactions when treated with Ca+ionophore A23187.
Hence, impaired potency arises from the inability to receive
the egg jelly ligand and initiate a Ca+ influx. Possible causes of
this acquired inability are discussed.
Introduction
Sea urchins' lives begin with the haphazard meeting of eggs and
sperm spawned into the vast ocean. The time window in which this meeting
can successfully occur is not without boundaries. Laboratory experiments
show that the sperm lose their ability to fertilize shortly after dilution into
sea water 16, 7, 8, 11, & results herein). Interestingly, sperm collected 'dry
(in concentrate form) and kept at ocean temperature (142 C) retain their
ability to fertilize for several days.
Upon dilution, sperm encounter a sea water environment (as opposed
to seminal fluid). This suggests that there is some toxic agent in sea water
leg. heavy metals) may decay the sperm 17, 9, & 141,; or that senescence
results from the loss of some component in the seminal fluid 18). Another
effect of dilution is to lead the spermatozoa from a state of quiescence to one
of great activity. In 1928, J. Gray noted that a diluted sperm suspension
respired more actively than a concentrated one, and indeed had a higher
respiratory level than an actively beating vertebrate heart (in terms of
volume oxygen consumed per gram of tissue). His explanation for the low
respiratory level of concentrated sperm was that each spermatozoon exerted
some form of inhibition on the movement of its neighbors I7l. Subsequent
studies revealed that the inhibiting factor in semen was carbon dioxide. This
product of respiration creates a low pH environment in which the sperm are
essentially immotile 110l. Diluting the sperm raises the internal pH, resulting
in heightened activity. It has been proposed that the ensuing fall in ATP
levels, or a byproduct of this process, may be the source of sperm senescence
11, 3, & 71.
The experiments described in this paper are aimed at unveiling the
physical changes which render sperm incapable of fertilizing an egg. It is
my hope that the results may help identify the instigator of these changes
Methods
Collection of Strongylocentrotus Purpuratus gametes was
accomplished by injecting 1-2mis of SM KCl into the coelomic cavity of adult
urchins. After injection, the urchins were shaken, and monitored for
subsequent spawning. If the substance emitted from their gonopores was
white (sperm), it was collected dry and stored at 42 C. Eggs (distinguishable
from sperm by their orange color) were collected in a beaker of filtered sea
water. They were later passed though a 10OuM millipore filter to remove
unwanted debris, and mechanically stirred in a 142 C water bath.
Determination of egg concentration was performed by centrifuging a sample
of the suspension in a Bauer Schenk tube for one minute. Concentration is
expressed in terms of percent (vol-vol) packed eggs in sea water. All
experiments were performed using gametes obtained the same day.
Percent Fertilization
The fertilizing capacity of sperm was determined by adding imi of the
sperm concentration indicated to 2mls of 18 egg suspension. The sperm
were given 3 min to find and fertilize an egg At the end of 3 minutes, the
interaction among gametes was halted by the addition of 2ml of 5M KCI (an
effective spermicide). A sample of the gamete suspension was then analyzed
using dark-field microscopy. Fertilized eggs were easily identifiable by the
presence of their fertilization membranes. Percent fertilization refers to the
number of eggs bearing this membrane out of the total number observed
(100 or more).
Changes in the Ability to Fertilize with Time
A series of 10-fold dilutions was made of dry sperm into sea water.
Each suspension was assessed for its ability to fertilize immediately after
dilution, and again after 2 hours. Since a 018 suspension went from close to
100% fertilization to 03 fertilization during this time period, it was chosen as
à convenient concentration for observing the decline in a suspensions
fertilizing-capacity with time lnote: this procedure is not trivial since the
fitness of sperm vary from urchin to urchin. Therefore a concentration
deemed suitable on one day may be undesirably potent the nextl. The
change in the suspension's ability to fertilize was determined by taking a
samples at successive times after dilution and performing the "percent
fertilization" procedure (described above).
Effect of Added Protein
Stock concentrations of superoxide dismutase and BSA solutions were
prepared by adding img of the respective proteins to 10 mis of filtered sea
water (FSW). Two five-fold dilutions were made for each solution, yielding
solutions with 02, and 004mgs of protein per mi of FSW. To each of these
media, as well as to a FSW control, was added enough dry sperm to
constitute a O1 suspension. The resulting sperm suspensions were
assessed for changes in ability to fertilize with time.
Percent Acrosomal Reactions
Imi samples of the sperm suspension were fixed by adding 1OOul of
378 formaldehyde solution. These mixtures were then spun in an Eppendorf
microfuge for 1 min. to concentrate the sperm. Sul was then transfered from
the bottom of each tube to colloidion-coated grids. 10 minutes were given
for the sperm to settle and adhere. The grids were then rinsed in water and
set to dry on filter paper. Observation and scoring of the acrosomal reactions
were performed with a transmission electron microscope (see 14, 5, & 9l and
figure 6).
Percentage Spontaneous Acrosomal Reactions
A 0058 suspension was made and assessed for changes in fertilizing
capability with time (described above). At the same time points the
suspension was analyzed for percent acrosomal reactions (also described
above).
Egg lelly and Ca+ lonophore Induced Acrosomal Reactions
A 0058 sperm suspension was allowed to reach a fertilizing capacity
of 08 (130 min.). It was then concentrated 100-fold using a technique
described by Vaquier (15 min at 1500g in a swinging-bucked
centrifuge) l151. A 'fresh“ 0052 suspension was made by resuspension and
subjected to the same re-concentration process immediately after dilution.
Rechecking the fertilization percentage confirmed that centrifugation
procedure did not diminish the sperm's potency (958 fertilization before
and after centrifuging). Vaquier's re-concentrating technique made it
possible to get à more dense distribution of sperm on each colloidion-coated
grid, thus economizing the time spent using the TEM.
The scoring procedure was the same as describe under "percent
acrosomal reactions" heading, however, egg jelly or Ca-ionophore A23187
was added before fixing. Initially, set amounts of these acrosomal reaction-
inducing agents were added. Either 500ul jelly or 5ul of lOmM ionophore
(in DMSO) was added to Iml of sperm. Five minutes were given for
triggering of the reaction before fixing. Additions of 500ul FSW and 5ul
DMSO served as controls.
To determine the concentration dependence of these agents in
initiating the acrosomal reaction, 0058 suspensions of fresh" and aged
(180 min.) sperm were prepared and re-concentrated when the fresh sperm
had lost potency. Two hundred microliter aliquots of sperm were added to
800ul, 400ul, 200ul, and 10Oul of egg jelly. To study the effect of the
ionophore, additions of 1, 2.5, 5, and 1Oul of 1OmM ionophore (in DMSO)
were made to iml of sperm (yielding final concentrations of 10, 25, 50, and
100uM A23137). Fixing and scoring procedures were identical to those
described above.
The egg jelly was obtained by dropping the pH of a concentrated egg
suspension to pH 5 by the addition of IM HCl. After 1 min., NaOH was added
to bring the pH back to pH 8 (that of sea water). The suspension was then
hand-centrifuged and the supernatent (jelly-containing) fraction frozen for
future use. The A23187 Ca+ ionophore was obtained from Sigma Chemical
Company
Resuits
Figure 1 shows that upon dilution of the sperm to 018, there was a
loss in the ability to fertilize with time. One min after the dilution was
made, the sperm yielded 938 fertilization. Seventy-five minutes later, the
same suspension was incapable of fertilizing eggs (08 fert.). This confirms
the results of Pennington I1 11 and Hayashi 181. Higher concentration
suspensions retained their ability to fertilize longer. 12 suspensions kept at
14° Cwere observed to vield »958 fertilization even after 2 days (data not
shown).
In accordance with previous studies (13l, 1141, and 1161), the addition
of protein into the dilution medium preserved the fertilizing capacity of the
spermatozoa. Figure 2 illustrates that the sperm maintained higher
fertilization percentages with the addition of 02 mg/mi of superoxide
dismutase or bovine serum albumin (as compared to the FSW control).
Dropping the concentration of protein to 004 mg /mi diminishes the sperm's
fertilizing longevity to that of the control. Although the original design of
this experiment was to see if superoxide dismutase could prolong the
lifetime of sperm above the BSA control, the negative result is confirmation
that proteins in general preserve sperm.
Attempts to associate the decline in the sperm suspension s ability to
fertilize with a rise in the number of spontaneous acrosomal reactions were
unsuccessful (contrary to studies on Lytichinus Bictus by 191). Figure 3
shows that as fertilization percentages declined, the percentage of sperm
which had undergone the acrosomal reaction remained essentially zero
A good percentage (592) of fresh sperm were observed to react when
treated with egg jelly (Table 4). However, sperm that had been allowed to
age (180 min.) until they were incapable of fertilizing eggs (08 fert.) did not
extend their acrosomal process in the presence of jelly (08 acrosomal rxn.)
No response was observed from sperm treated with FSW instead of jelly (in
agreement with the " 03 spontaneous acrosomal reactions noted earlier).
In contrast to egg jelly, the Ca+ionophore A23187 was able to initiate
a high percentage of acrosomal reactions in both fresh and aged sperm. The
percentage was actually higher (903) in the case of aged sperm than fresh
sperm (772). Experiments varying the concentration of the ionophore
suggest that the difference in response of fresh and aged sperm to A23187 is
not significant. The DMSO control was ineffective in initiating a reaction (02
acrosomal rxn.).
Varying the concentration of egg jelly did not greatly affect the
percentage of reacted sperm observed (see Table 5). There was no
observable difference between a 2:1 (jelly to sperm) and a 1:2 suspension.
The data suggest the relationship is asymptotic (limit at 458 in this case),
but further experiments using lower jelly concentrations would have to be
performed to confirm this. Aged sperm showed a slight increase percent
acrosomal reactions when the concentration of jelly was increased, but
always remained far below the percentages achieved by their freshly-
diluted counterparts.
Figure 5 shows the striking similarity of fresh and aged sperm in
response to ionophore. As the percent of acrosomal reactions observed in
fresh sperm grew from 238 to 638 (with increasing ionophore
concentrations), the values for the aged sperm emulated this rise
(progressing from 308 to 588)
Discussion
Attempts at inducing the acrosomal reaction in fresh and aged
populations of dilute sperm have suggested a new reason for the decline in
fertilizing potential in X purpuratus. The sperm of this sea urchin are not
spontaneously reacting, nor are they losing the ability to undergo a reaction
entirely; they are simply becoming unresponsive to the egg jelly stimulus
over time. It is known that extension of the acrosomal process is triggered
by a rise in both the internal pH and the Ca-level 15,10, & 121. Since 'aged'
sperm (ie sperm which have lost the ability to fertilize an egg) are
insensitive to egg jelly, but do undergo an acrosomal reaction when treated
with the Ca+ ionophore, the acquired inadequacy must be somewhere
between the binding of egg jelly and the subsequent influx of Ca- Below I
consider the two prevailing views of sperm senescence, and how these could
relate to the loss of ability of sperm to respond to egg jelly
The Toxic Heavy Metal Model
Spermatozoa are exposed to the constituents of sea water upon
dilution. If certain constituents were harmful to the sperm, this might
explain why the ability to fertilize is lost soon after dilution. The sperm
would accumulate damage with time. Studies have shown that such
damaging entities exist in the form of heavy metals (h.m.s) 19, 13, & 141. In
1953, Albert Tyler demonstrated that the addition of hm. chelating agents to
sea water greatly improved the longevity of spermatozoa vis-a-vis their
fertilizabilty. He also noted that artificial sea water of lowhm content
extended fertilizable life and motility. By decreasing the amount of exposure
to these toxic ions, sperm potency can be prolonged.
Results in this paper show that the addition of non-specific protein can
enhance the fertilizing capacity of spermatozoa. This confirms previous
observation lby 9, 13, & 151. Tyler asserts that the prolonging effect of
amino acids and proteins is due to their ability to bind hms present in the
dilution medium. Their action is thus akin to that of chelators. The fact that
exogenously added protein can bind h.ms (primarily due to the presence of
sulfhydryl groups on amino acids such as cysteine 13 & 91) suggests that the
site of hm.-toxicity is on sperm proteins. Cell membrane-imbedded proteins
would be particularsy vulnerable due to their intimate contact with sea
water. This model, then, suggests that a prime suspect for the loss of
sensitivity to egg- jelly would be damage to the jelly receptor itself. Upon
dilution (and the entailing increase in exposure to hm.s), this receptor would
quickly accumulate damage and soon lose the ability to bind or respond to
its ligand. This would explain the inability of aged sperm to acrosomally
react in the presence of egg jelly
Energy Depletion Model
The energy depletion model argues that this inability resusts from a
lower ATP level rather than physical damage. It is known that the internal
pH of sperm rises upon dilution I10l, and in their 1985 paper, Christen,
Schackmann, and Shapiro (the seminal figures" in advocating this theory)
cite a correlation between the internal pH and the endogenous ATP levels.
They propose that as pH(i) increases, so does internal ATPase activity (there
is a fall in ATP levels). Respiration is shown to increase to meet these
energy demands 121, but only up to a point. As the pH(i) rises above 7.4, the
oxygen consumption (and hence ATP production) remains constant 13l. This
creates an energy imbalance, and with time, the ATP levels drop. This model
could be interpreted to show that the drop in ATP prevents the egg jelly
stimulus from inducing the acrosomal reaction. Although this assumes that
the translating process requires energy, such an assumption is not
unwarranted. It is reasonable to believe that some step in the signal-
transduction system is ATP-consuming (eg the phosphorylation of Ca-
channel proteins may be necessary for their opening).
How does this model account for the fact that removal of hmns
prolongs fertilization potential? It has been shown that the addition of
chelators lowers the pH(i) l3 & 9l. This implies that their preservational
action is not directly through the sequestration of odious toxins, but rather
indirectly through the maintenance of high ATP levels. From this standpoint,
we can equate the effect of hm. chelators to that of a pH buffer. Indeed, it
nas been shown that lowering the pH of sea water enhances sperm viability
131. This does not disprove the heavy metal model since the affinity ofhms
for sulfhydryl groups is known to decrease with decreasing pH: Granted the
sperm activity is decreased, but so is h.m. binding.
One experiment performed by Christen et al. potentially disproving
the heavy metal hypothesis involves the addition of potassium ions (in the
form of KCl) to the sea water medium. Their results indicate that Sea Water
containing 50 mM in K- preserves sperm longevity far longer that their SW
control (1OmM K*). The proposed action of this added ion involves
decreasing the membrane potential set up by high intracellular, and low
extracellular IK-l. This inhibits a H- efflux, and, hence, maintains a low pH(i)
(effectively curbing energy usage). The added K-should not effect the
binding of membrane proteins by hms. Therefore, the ability of K-
additions to prolong sperm potency seemingly invalidates the heavy metal
model. My own attempts at preserving fertilizing potential with K-- both by
direct addition of KC to filtered sea water, and by emulation of the ASW
conditions of Christen et al. -were entirely unsuccessful. Since the mempers
of the Shapiro lab are far more experienced in working with sea urchin
gametes than myself, I regard my failure to reproduce their results as a
reflection of my own competency rather than a discredit to theirs. Still,
confirmation of their resuîts must be achieved. Until that time, we cannot
rule out the possibility of h.m. damage to the receptor as an explanation for
the acquired insensitivity to egg jelly
Other Possibilities
Although there is good evidence for the correspondence of heightened
activity and subsequent inability to fertilize, this does not necessarily mean
that the ATP levels are directly responsible. Perhaps some indirect
conformational change occurs as a result of declining ATP levels. For
instance, this change may trigger a programmed cels death sequence.
Another possibility is that deleterious byproducts of respiration (e g. reactive
oxygen species such as the superoxide anion) may accumulate with time,
damaging the signal transduction mechanism of the egg jelly receptor. R.I
Aitkin has shown evidence for the accumulation of ROS with time in human
spermatozoa 11).
It is my hope that the question of sperm senescence will continue to
be addressed until its mechanism is confirmed. Such confirmation should
provide insight on how to better preserve sperm. If the mechanism proves
similar to that seen in human spermatozoa, future research may suggest
ways of improving artificial insemination procedures. Furthermore, certain
types of impotency (those stemming from senescence) may prove treatable
Although these practical applications strongly merit further investigation of
the matter, the most compelling argument in favor of future research is
simply this: the subject is intriguing
Acknowedgements
Foremost, I would like to thank Dr. David Epel-a veritable cornucopia
of insights, suggestions, and knowledge - for spending countless hours
guiding a stray lamb this quarter. I would also like to thank Chris Patton
and Rob Swezey for acquainting me with all of the technical equipment I
needed to conduct my goose-chases.
Tables and Figures
2 Fertilization
Time after dilution (min)
82
66
46
29
21
1#0s 1: The percentage of eggs fertilized when 1mi of 0017 sperm is added to 2ml of a
17 egg suspension. The 'time after dilution refers to the time elapsed after the dry
sperm is diluted 100.000-fold to form the 0017 suspension.
Data from Fertilization vs. Time
100
* Bfert.
40


20
20
40
60 80
Time (min)
Egure 1: Plot of data from Table 1.
Added Protein (mg/mi
Time (min) FSW 1/50 SD
1/250 SD. 1/50BSA
1/250 BSA
30
26
77
108
18
24
144
* Superoxide Dismutase
Bovine Serum Albumin
#2: The effect of adding protein to the medium (Filtered Sea Vater) that sperm are
diluted into. The values listed are fertilization percentages vielded by adding 1ml of
each sperm suspension (all 017) to 2ml of 17 eggs. Time“ refers to time after dilution.
Data from Protein Added“
100
80
B fert control
60
1750 mg/mI SD
E 17250 mg/ml S0
40
E 1/50 mg/mI BSA
D 17250 mg/mI BSA
HEAA I
5 30 47 77
108 144
Time (min)
Eime 2: Data from Table 2.
Time (min) fert  8 Acrosomal Rxn
64
24
10
11 3: The percentage of sperm vhich have spontaneously undergone an acrosomal
reaction as time elapses. At the same time points, the fertilizing capacity of the sperm
(a .0057 suspension) vas assessed. Time“ indicated the time after the sperm vere
diluted.
Data from Spontaneous Acrosomal rxn.
100
80 -
+ P fert.
+ % acrosomalrxn
40
20 -

o+
20 40 60 80 100 120
Time (min)
Eigune 9: Data from Table 3.
2 Acrosomal Rxn.
egg jelly IFSW controlA23187 in DMSODMSO control
Time 2 fer
1 min95
59
180
18199: The effect of aging on the ability of sperm (a 0057 suspension) to respond to
egg jelly and to Ca+ ionophore A23187. 1ml of sperm was added to each of the
folloving: 500ul egg jelly. 500ul Filtered Sea Vater (ESV-as a control), Sul A23187 in
DMSO (final concentration of 50uM), or 5ul DMSO (as a control). Acrosomal reaction
percentages vere obtained from scoring 60 or more sperm using a transmission
electron microscope.
2 Acrosomal Rxn.
Egg Jelly (vol: vol spern
fresh sperm
aged sper
4:1
(dilution error made)
2:1
1:1
42
3: The effectiveness of various egg concentrations (ratio of jelly volume to a
fixed 200ul of sperm) on inducing the acrosomal reaction. Scoring vas performed
visually using a transmission electron microscope. Aged sperm refers to a 0057
suspension that vas given sufficient time (120 min.) for fertilizing capacity to reach
0%. The sperm vere then concentrated by centrifugation for more efficient scoring
The fresh sperm'refers to a suspension that vas subject to the same reconcentrating
procedure immediately after disution.
Data from Acrosomal Rxn./ Jellu
50
P
40
% 50
+ fresh sperm
+ aged sperm
20
10 -

0+
jelly/sperm
Bigu d: Data from Table 5.
2 Acrosomal Rxn.
IA23187 ionophore (uM)
fresh sperm
aged sperm
100
63
60
23
119 G: The effectiveness of various Ca-ionophore concentrations on inducing the
acrosomal reaction. Scoring vas performed visually using a transmission electron
microscope. Aged sperm refers to a 005% suspension that vas given sufficient time
(120 min.) for fertilizing capacity to reach 02. The sperm were then concentrated by
centrifugation for more efficient scoring. The 'fresh sperm'refers to a suspension
that vas subject to the same reconcentrating procedure immediately after dilution
Data from  Acrosomal Rxn./ionophore
80 -

%
+ fresh sperm
40
+ aged sperm
20
0 20 40 60 80 100 120
IA231871
Eigue 3: Data from table 6.
Figure6: Example of a reacted (bottom) and unreacted (top) sperm
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