Abstract
The acrosome reaction in sperm is an essential requirement for
fertilization. This involves several interrelated changes in
sperm plasma membrane permeability to Ca', Na', H', and K ions.
The mechanism by which the acrosome reaction occurs was studied
in the marine snail Tegula funetralis and an attempt to define
the conditions surrounding the acrosome reaction was made using
various inhibitors and inducers of the acrosome reaction. In¬
hibition of the acrosome reaction was achieved in both pH 7.6
artificial sea water and in 10 * mM EDTA in pH 8 artificial sea
water. Inhibition was effective in 85% of the sperm evaluated.
Several inducers were effective at normally inhibitory pH 7.6.
It should be noted that a 100% acrosomal reaction was never achieved
due to a mixture of mature and immature sperm. Ca in concen¬
trations ranging from 20mM to 100mM induced acrosome reactions in
48% of sperm after a period of 10 minutes. Heavy metals such as
Zn and Sn were found effective in inducing acrosome reaction in
concentrations ranging from .1 uM to 100 uM. In particular it
was found that at luM concentrations, Zn was most effective in
that 62% of sperm had undergone acrosome reactions after ten minutes. The
most dramatic results were achieved with a combination of solutions
containing 20mM to 100mM Ca and luM Zn'. Under these conditions
73% of sperm had undergone an acrosome reaction after 10 minutes.
cAMP was effective in concentrations from.luM to 10mM, the highest
percent of acrosome reactions occuring at lOuM concentrations (55%).
The results presented here and data from previous work
done on
the mechanism governing acrosome raaction indicate that Ca, Zn.
and cAMP may be involved in the mechniam which triggers and/or mediates
the acrosome reaction in T. funebralis
Introduction
In order for fertilization to occur several changes in gamete
surfaces must take place. One of these alterations, the acrosome
reaction in sperm involves an alteration in sperm plasma membrane
permeability to specific ions. The result is a transformation in
morphology and subsequent activation of the sperm necessary for
fertilization.
The conditions under which the acrosome reaction occurs have
been studied extensively in the sperm of the sea urchin, Stronglylo¬
centrotus purpuratus and is believed to be elicited by contact with
components in the jelly surrounding the outer surface of the egg.
(Shapiro, 1980). Upon contact with the egg jelly, sea urchin sperm
undergoes a morphological change characterized by membrane fusion,
exocytosis, and extension of the acrosomal filament. (Shapiro, 1980).
As in the sea urchin, fertilization in the snail, Tegula fune-
bralis takes place externally with seasonal spawning of gametes. The
eggs are surrounded by a jelly layer and the spermatozoa are rela¬
tively large with a well formed acrosome thus facilitating assessment
of the acrosome reaction under phase contrast microscopy. Although
the conditions under which the acrosome reaction in T. funebralis
occurs have not been determined, it seems reasonable to assume that
the acrosome reaction takes place near the egg and is affected by a
component or components contained in the egg or egg jelly. Studies
of an egg membrane lysin released by sperm have been made on several
species of Tegula (Haino and Kigawa) and the lysin was found to be
released after contact with the outer surface of the egg. The mechan¬
ism by which the lysin is released is still under investigation and
it remains to be seen whether this is an acrosomal enzyme. Assuming
that its release is somehow dependant on the acrosome reaction it can
be shown that there is a correlation between contact with egg jelly and
the mechanism governing the acrosome reaction.
Like many other excitable cells, sperm responds to changes in
specific ion gradients through altered permeability of the sperm
plasma membrane (Shapiro,1980). More specifically he has shown that
induction of the acrosome reaction corresponds to changes in sperm
plasma membrane permeability to Ca, H, K, and Na.
It was first shown by Dan (1954) that extracellular Ca was
an essential requirement for the acrosome reaction in sea urchin
sperm. Upon triggering of the acrosome reaction there is an influx
of Ca ions which accumulate in the mitochondria (Cantino, unpublished
data). The mechanism by which this Ca'" influx occurs is not clear.
Epel (1977) showed that an ionophore A23187 which allows for Ca
uptake triggers the acrosome reaction in sea urchin sperm thus it is
postulated that Ca influx is an initial step in the mechanism
governing the acrosome reaction. Data suggest that there is a close
relationship between the acrosome reaction and the increase in sperm
plasma membrane permeability to Ca ions. (Shapiro, et.al.).
In addition to Ca' influx there are other ionic movements
associated with the acrosome reaction. In sea urchin sperm it has
been shown that when the acrosome reaction is triggered there is an
uptake of Na and concurrent efflux of H' ions into the extra¬
cellular solution. It is suggested that the Na :H exchange is in
some way essential to the acrosome reaction. (Schackmann, unpublished
data). Of particular interest he has shown that a Ca  requiring step
preceeds the Na :H exchange thus indicating that a Ca " dependant
step is an initial step in the triggering of the acrosome reaction.
Increasing K levels from 10 mM to 20mM has been shown to
prevent acrosome reaction in sea urchin sperm (Shapiro, 1980). He
suggests then that K movement is also involved in the acrosome reaction.
Later studies by Shapiro indicate that K' efflux occurs after the
acrosome reaction is completed and thus is not directly involved in
the triggering mechanism.
The effect of cyclic nucleotides on acrosome reaction has been
studied in sea urchins using a factor isolated from the jelly layer
surrounding the egg. (Kopf and Garbers, 1980). They have suggested
that activation of the sperm adenylate cyclase is mediated by Ca
influx. This activation of adenylate cyclase then causes an elevation
in intracellular cAMP concentrations which in turn leads to activation
of a protein kinase. The subsequent phosphorylation of membrane
proteins may have an effect on the acrosome reaction. (Kopf and Garbers.
1980).
The present paper investigates the conditions surrounding the
acrosome reaction including alterations in ion permeability and
cyclic nucleotide effects. Particular emphasis is placed on the
mechanism governing the initial Ca influx and its' relationship
with Zn and cAMP.
Materials and Methods
Tegula funebralis were collected in the intertidal zone at
Mussel Point, California during the months of April and May. Snails
were kept in running sea water at 13°C and used within 24 hours of
collection. To obtain sperm, the shells were cracked in a vise,
exposing the gonads. The testis were then dissected away from ad¬
jacent hepatopancreas. Sperm was isolated from the testis using a
drawn out Pasteur pipette and then transferred to a small plastic
vial. Concentrated sperm was kept on ice for up to four hours after
which fresh sperm was obtained.
Experiments were carried out by diluting 4-5 ul of sperm in
test tubes containing 1 ml of solution. Diluted sperm was thoroughly
mixed and 5 ul drops were then placed on slides at given time inter¬
vals and covered with cover slips. Acrosomal activity was assessed
by viewing sperm under phase contrast oil immersion at 1000X. Scoring
of the acrosome reaction was done using an ocular grid divided into
25 squares. Sperm were scored as being either reacted, partially
reacted, or non-reacted. "Blunt" sperm described by Johnson (1981),
were counted as reacted. (See Fig. 1, 2, and 3.). In initial ex¬
periments 500 sperm were counted in order to assess accuracy. Five
counts of 100 sperm revealed an accuracy within 5%. Therefore results
form experiments reported here were all based on counts of 100 sperm.
All solutions were prepared using five part artificial sea water:
120 mM NaCl, 10mM KCl, 10mM Cacl,, 60mM MgCl,, and 2mM NaHCO,. Tris
buffer was used to bring solutions to proper pH. Experiments were
carried out at pH 7.6 and at 25°C unless otherwise noted.
Results and Discussion
Morphology of the Acrosome Reaction.
Sperm of T. funebralis follows a similar sequence of events as
that described for sea urchin sperm. Figures 1, 2, and 3, show
electron micrographs of T. funebralis sperm during the acrosome
reaction. In the sea urchin sperm, triggering of the acrosome
reaction results in fusion of the sperm plasma membrane with acro¬
somal membrane, exocytosis of the acrosomal granule, polymerization
of globular actin in the periacrosomal region and the extension of the
acrosomal filament (Shapiro, et al, 1980). At the point of filament
extension the sperm is considered to be acrosome reacted.
Spontaneous Acrosome Reaction.
Sperm suspended in artificial sea water at pH 8 will undergo
a spontaneous acrosome reaction over time. (Fig 4). In order to
measure accurately the effects of acrosome reaction inducers the
spontaneous triggering must be eliminated. Inhibition of spontaneous
activity was achieved in solutions at pH 7.6 as well as in 10 - mM
EDTA at pH 8.0. Inhibitory effects were found to be effective in 85%
of sperm scored over a period of 30 minutes (Fig. 4).
Varying Ca Concentrations
The effects of varying Ca concentrations at pH 7.6 (Fig 5)
reveal that the acrosome reaction may be induced with high extra¬
cellular Ca levels. Inhibition at the sea water Ca - level (10mM)
is overcome by concentrations of 20mM and greater. These results
further the hypothesis that Ca influx is correlated with triggering
of the acrosome reaction.
Effects of Heavy Metals.
Work done by Tyler (1953) with sea urchin sperm showed that
heavy metal chelators would prolong the life of sperm as much as 100-fold.
Iyler concluded that these chelating agents worked by chelating the
heavy metals present in sea water. Later work done by Johnson (1981)
using EDTA and cysteine gave similar results in prolonging motility
and fertilization potential in sea urchin sperm. Johnson has pos¬
tulated that the inhibitory action of chelators is likely to take
effect on the sperm surface rather than intracellularly. This
theory suggests that the addition of heavy metals should induce and ac¬
rosome reaction.
The effects of adding Zn and Sn' in concentrations ranging
from 0.1 uM to lOuM are shown in Figure 6. The most dramatic effect
Sn showed a similar effect yet not
was achieved with luM Zn.
as dramatic as the Zn effect. Addition of 1 uM Zn' proved to
be particularly interesting and warranted further experimentation.
Additionally, .luM amounts of Zn' acted to increase the number of
sperm acrosome reacted by a margin of 26 % after 10 minutes. Sea water
concentrations of Zn' were found to be between .1 uM and .3 uM along
the Pacific Coast (Potts and Todd, 1965). As seen in Figure 6,
a luM concentration of Zn' effected a 43% increase in the number of
acrosome reactions.
Triggering of the acrosome reaction in sea urchins appears to be
especially sensitive to Zn concentrations; an increase in concen¬
tration to 10"Mor greater has an inhibitory effect (Johnson, 1981).
Similarly in T. funebralis Zn concentrations above 10 M are
not nearly as effective in inducing an acrosome reaction as are lower
concentrations.
These results indicate a correlation between heavy metals and
triggering of the acrosome reaction. Further experiments tested the
effects of luM Zn at various Ca levels from .lmM to 100 mM. (Fig.7)
As shown in Figure 8, addition on Zn even at very low levels of
Ca was somewhat effective in inducing acrosome reactions. The data
presented suggests that there may be a heavy metal factor, perhaps a
Zn factor, involved in the mechanism governing the acrosome reaction.
A possible mode of action would be for Zn' to alter the sperm plasma
membrane permeability to Ca+2. The following explanation suggests
a mechanism by which Zn may act to initiate an acrosome reaction.
It is conceivable that there are certain Zn'- specific enzymes such
as histidine or another amino acid residue bound in the sperm plasma
membrane. When these enzymes or metalloproteins bind to Zn or less
strongly to other heavy metals, the structure along the entire protein
is altered creating a "hole" through which Ca' influx may occur.
Selectivity for Zn or other heavy metals over the more prevalent
Ca' ions can be explained by their respective electron configurations.
Zn' has a filled "d" shell and an empty "s'" shell making it
readily availabel to form covalent bonds with uncharged molecules such
as the amino acid residues of proteins. Ca takes on a noble
gas configuration and binds mainly to charged ligands (Gilly, in press
1982). The result is that Zn" is selected over Ca' and binds to
histidine or another amino acid residue in the sperm plasma membrane
thus forming an imidazole bond which exhibits great flexiblilty.
(Freeman, 1973). The structure along the entire protein is altered
in such a way that the membrane becomes permeable to Ca. Through
such a mechanism the presence of Zn' or perhaps other heavy metals
may be correlated triggering of the acrosome reaction.
Effects of cAMP.
The mechanism by which increased levels of extracellular cAMP
induces an acrosome reaction is not clear. As shown in Figure 9,
addition of cAMP, especially at 10 uM concentrations proved effective
in inducing an acrosome reaction in 53% of sperm. The means by which
cAMp is transperted across the sperm plasma membrane, if indeed it is,
remains a puzzle. The mechanism by which an intracellular increase in
cAMP acts in relationship to the acrosome reaction has been studied in
sea urchin sperm. (Kopf and Garbers, 1980). These results are dis¬
cussed in the following proposed mechanism for governing of the
acrosome reaction in T. funebralis.
Based on data presented here and on previous findings in the
study of acrosome reactions, the following hypothesis is suggested
for a mechanism by which the acrosome reaction might occur.
Upon contact with egg jelly components similar to those presented
by Kopf and Garbers (1980), the sperm plasma membrane permeability to
Ca ions is increased. This step may also be potentiated by presence
of Zn'. The increase in intracellular Ca activates sperm adenylate
cyclase levels which leads to elevation of sperm cAMP concentrations.
The result is an activation of a cAMP-dependant protein kinase and
subsequent phosphorylation of membrane proteins which could then allow
for further Ca influx, also potentiated by Zn. The end result is
the triggering and actual process of the acrosome reaction.
This hypothesis is in agreement with findings by Levine and Walsh
(1979) suggesting that protease activation occurs prior to any morphe¬
logical changes characterizing the acrosome reaction. It is conceivable
then that a correlation exists between the presence of Zn, Ca per¬
meability increase in sperm plasma membrane, and increased levels of
cAMP in the triggering of the acrosome reaction.
Acknowledgements
I owe a very special thanks to Dr. Don Abbott for kicking
me in the rear and encouraging me to finish this paper in time
for graduation. Many thanks to Dr. Epel for putting up with my
antics and especially for all the help and guidance given me
throughout the quarter. Without the help of Dr. Dave Clapper in
writing this paper I would have been lost and I thank him for all
the time he put in. Thanks to Chris Patton for the pictures, and
to "Joe" Gilly for all he has done in helping me. Special thanks
to Willy and Chris for taking me trout fishing so I could come back
and write this paper with a clear mind.
Literature Cited
Collins,F. and Epel,D. (1977). Exp. Cell Res. 106, 211-222.
Dan, J.C. (1954). Biol Bull. 107, 335-349.
Freeman, H.C. (1973). Inorganic Biochemistry, G.L. Eichorn (ed.),
Elsevier, New York, pp. 121-166.
Gilly, W. (in press 1982). Journal of General Physiology.
Haino,K. and Kigawa, M. (1966). Exp. Cell Res. 12, 625-633.
Johnson, C. (1981). Doctoral Thesis. Stanford University. pp151-193.
Kopf, G.S. and Garbers, D.L. (1980). Biology of Reproduction, 22;
1118-1126.
Levine, A.E. and Walsh, K.A. (1979) Develop. Biol. 72, 126-137.
Potts, W.T.W. and Todd, M. (1965) Comp. Biochem. Physiol.,16, 479-189.
Shapiro,B.M., Schackmann, B.W., Gabel, C.A., Foerder, C.A., Farance,
M.L., Eddy, E.M. Klebanoff, S.J., (1980). The Cell Surface:
Mediator of Developmental Processes. Academic Press Inc.
New York. PP128-148.
Tyler, A. (1953) Biol Bull. 104: 224-239.
Figure Legends
Figure 1. Electron micrograph of T. funebralis sperm before undergoing
acrosome reaction. Sperm measures approximately lu at the base and
7u in length excluding flagella. Sperm morphology: flagella (f);
mitochondria (M), nucleus (n), periacrosomal region containing
actin(pa) and acrosomal granule (ag).
Figure 2. Electron micrograph of T. funebralis sperm partially reacted.
Exocytosis has begun, contents of the acrosomal granule are about
to be exposed. Tail not visible due to fixation.
Figure3. Electron micrograph of a reacted or "blunt" sperm.
The acrosomal filament and tail were destroyed in fixation.
Figure 4. Sperm undergoes a spontaneous acrosome reaction in
pH 8.1 sea water over time. The effects of inhibition with .1
mM EDTA at pH 8.0 and with pH 7.6 artificial sea water are
shown.
Figure 5. Effects of increased Cay concentration are shown. Normal
sea water concentration of Ca is about lOmM.
Figure 6. Effegts of Zn and Sn' at normally inhibitory pH 7.6.
At luM Zn concentration 62% of spermghad undergone an acrosome
reaction. A control without added Zn exhibited a 14% acrosome
reaction.
Figure 7. Effects of luM Zn' concentrations at various Ca
concentrations. The highest percentage of acrogome reactions
was seen to occur at levels of 50mM to LOOmM Ca
Figure 8, Effejts of luM Zn at low levels of Ca Ljas compared
with a Ca control without Zn. As shown, Zn induces an
acrosome even at low levels of Ca
Figure 9. Effects of Addition of Cyclic AMP to pH 7.6 ASW.
Figure 1
Figure 2
Figure 3
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