Download The Program of and Mechanisms of Fertilization in the Echinoderm

MER. ZOOL., 15:507-522 (1975).
The Program of and Mechanisms of Fertilization in the Echinoderm Egg
DAVID EPEL
Marine Biology Research Division, Scripps Institution of Oceanography, University of
California, San Diego, La folia, California 92037
SYNOPSIS. Current research on the mechanisms of sperm-egg fusion, the block to polyspermy, and metabolic activation are described. A cinemicrographic analysis of fertilization
reveals that fusion of sperm and egg occurs between non-motile gametes, indicating that the
flagellar motion of sperm is not required. The block to polyspermy is reviewed, emphasizing
recent work on the role of cortical granule protease in altering sperm receptors of the
vitelline layer.
Metabolic activation or derepression at fertilization is highly regulated and occurs in a
definite sequence. The primary event appears to be release of intracellular Ca2+. The timing
of metabolic derepression is different in starfish oocytes. Here, a part of the derepression
occurs during maturation and another part at fertilization.
The contact between sperm and egg
marks a turning point between life and
death. Life if a successful fertilization occurs, since the resultant sperm-egg interaction will initiate the programmed development of a new individual. Death in the absence of such interaction, since the unfertilized egg will soon degenerate.
For studies of fertilization, the gametes
of echinoderms, especially those of sea urchins, have long provided an ideal model
system. The major reasons for this are the
ease of in vivo fertilization and development, the large amounts of available material, and the presence of a prolonged reproductive season.
In this paper, I will describe three aspects
of fertilization as learned from studies on
sea urchin eggs. These are (i) the
mechanisms of sperm attachment and
sperm entry into the egg, (ii) the biochemical mechanisms for the block to polyspermy, and (iii) the mechanism of the programmed metabolic activation or metabolic
derepression that occurs at fertilization. Finally, we will compare these studies on sea
urchins to the gametes of other organisms.
This article reports on research done in collaboration with Dr. Edward J. Carroll, Jr., Dr. Margaret
Houk, Dr. Richard Steinhardt, Ms. Mia Tegner, and
Dr. Victor Vacquier and supported by grants from the
National Science Foundation and Population Council.
A BRIEF OVERVIEW
Ultrastructural aspects of the changes in
sperm and egg and the fusion of the
sperm-egg plasma membranes that accompany fertilization have been reviewed by a
number of authors (recent reviews by Colwin and Colwin, 1967; Anderson, 1968;
Austin, 1968; Millonig, 1969) and are also
discussed by Summers et al. (1975). Briefly,
fertilization can be viewed as a sequence of
four successive membrane fusions. TYxfirst
fusion takes place when the sperm makes
contact with overlying egg jelly. This contact can be envisaged as a receptor-effector
system which results in the fusion of sperm
membranes and resultant extrusion of the
acrosomal process (Dan, 1970). If this extrusion occurs in the immediate vicinity of
the egg, the tip of this process will attach in
a species-specific fashion to an extracellular
layer of the egg, the vitelline layer. This
attachment, seen in the scanning electron
micrograph shown in Figure 1, is apparently to a sperm receptor protein of the
vitelline layer. Aketa and his colleagues
have recently isolated this protein from sea
urchin eggs and find that (i) sperm attach to
films of this protein in a species-specific
fashion, (ii) the binding requires calcium,
and (iii) antibodies to this protein will prevent fertilization (Aketa et al., 1972).
It has long been realized that many ex-
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DAVID EPEL
FIG. 1. Scanning electron micrograph of sperm attached to vitelline layer of 5. purpuratus egg. (Courtesy
of M. Tegner.)
cess or supernumerary sperm also attach to
the egg during insemination, but the magnitude of this supernumerary binding has
not been previously appreciated. Recent
scanning electron micrographs have
dramatically revealed the extent of this
binding (Tegner and Epel, 1973), and
quantitation of the attachment has revealed
that under saturating conditions up to 1500
sperm can attach per egg (Vacquier and
Payne, 1973).
In spite of this redundancy of sperm,
only one sperm normally fuses with the
plasma membrane and enters the egg. This
fusion constitutes the second membrane/usion of fertilization. Shortly after or coincident with this fusion the cortical reactions
are initiated (Fig. 2), which represent the
third membrane fusion of fertilization. This
fusion is between the cortical granule
membrane and the overlying egg plasma
membrane with a resultant exocytosis and
extrusion of the cortical granule contents
into the space between the overlying vitelline layer and the plasma membrane (see,
e.g., Millonig, 1969). In eggs of Strongylocentrotus purpuratus, this fusion of corti-
cal granule membrane and cell plasma
membrane takes about 20 sec to propagate
over the 75/i diameter egg (Paul and Epel,
1971) and results in the formation of a
mosaic membrane with an 80% increase in
the surface area of the egg. The contents of
the granules are also involved in elevating
(Vacquier et al., 1972a) and structuralizing
(Bryan, 1970) the vitelline layer as it transforms into the fertilization membrane.
During and following the cortical reaction, the "fertilizing" sperm fuses with the
egg, rotates 180° within the egg cytoplasm,
and begins a migration toward the egg pronucleus. Coincident with this movement is a
PROGRAM OF FERTILIZATION
plosmo membrane
vilellme later attachment
bj protease • sensitive
bond
FIG. 2. Representation of fertilization process of sea
urchin egg, showing sperm detachment and cortical
reactions. The vitelline layer contains at least two
protease-sensitive sites, a sperm attachment site and a
plasma membrane-vitelline layer attachment or connective. Many sperm attach to the layer but only one
sperm, the fertilizing sperm, initiates the cortical reactions. This results in the propagated fusion of cortical
granule membrane with plasma membrane and the
release of the cortical granule constituents. One cortical granule protease alters sperm receptors so that
supernumerary sperm detach; a second detaches connectives between plasma membrane and vitelline
layer. Swelling or hydration of hydrophilic macromolecules elevates the now detached vitelline layer
which is thus transformed into the fertilization membrane.
swelling and "hydration" of the nucleus,
activation of DNA synthesis and finally fusion of the two pronuclei. This fourth fusion
of fertilization, which occurs about 20 min
after insemination of the S. purpuratus egg,
can be considered the end of the fertilization period.
MECHANISMS OF SPERM FUSION AND ENTRY INTO
THE EGG
The mechanism of sperm entry into the
egg has long rivetted the curiosity of embryologists. The important ultrastructural
studies of the Colwin's (1967) reveal that
sperm and egg plasma membranes fuse
and suggest that fusion perse could provide
the motive force for incorporating the
sperm into the egg cytoplasm. Recent
studies have revealed the presence of
actin-like filaments within the acrosomal
process (Tilney et al., 1973; Jessen et al.,
1973). This suggests that contraction of an
actomyosin-like system may be involved in
carrying the sperm into the egg.
Direct observations on the behavior of
the sperm during sperm-egg fusion are
remarkably limited. This results in large :;
part from the difficulty of observing fusion i
509
and entry, since the process is so rapid and
since only one of the many attached sperm
will ultimately fuse with the egg. Cinemicrographic analysis had not proved useful,
primarily because the eggs freely move
when bombarded by sperm. J. Dan (1950),
using the then new phase contrast microscope, briefly noted that the fertilizing
sperm stops moving as soon as it makes
contact with the egg.
Collaborating with Naomi Epel, we recently completed a cinemicrographic study
of fertilization, which was made possible by the technique of Steinhardt et al.
(1971) of immobilizing eggs on a thin film
of protamine sulfate. Our movies reveal
that the fertilizing sperm becomes immobilized just before beginning its entry
into the egg cytoplasm. Analysis of these
films shows that each attached sperm (both
the fertilizing sperm and supernumerary
sperm) continuously moves around the axis
of its attached acrosomal process. About 15
to 20 sec after the initial attachment, one
sperm, which will be the fertilizing sperm,
ceases this movement and literally stands
up straight. Almost immediately thereafter
the fertilization membrane begins to rise
around this sperm and the other or supernumerary sperm are lifted away with the
elevating fertilization membrane.
Using single-frame analysis we have
measured the rate of entry of the fertilizing
sperm into the egg cytoplasm. Two types of
behavior were observed and are illustrated
in Figure 3. The upper curve depicts the
case where no detectable inward movement
was apparent for several seconds after
motility had ceased. The lower curve depicts the case where the sperm began to
move into the egg as soon as motility had
ceased. Analysis of six cases reveals that,
once initiated, the rate of entry is 5 to 11
microns per minute. Phase contrast microscope observations on living eggs indicate
that the tail of the fertilizing sperm remains
immobile during sperm entry.
Figure 4 depicts three possible hypotheses accounting for entry of an immobilized
sperm at a rate of 5 to 11 microns per minute. First, entry could be passive, the incorporation being simply a sequel to fusion as
when two contiguous oil droplets fuse. In
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DAVID EPEL
ROLE OF THE CORTICAL GRANULE COMPONENTS
IN THE BLOCK TO POLYSPERMY
SECONDS AFTER MOVEMENT CEASES
FIG. 3. T w o examples of rate of movement of the
non-motile fertilizing sperm into the L. pictus egg.
Measurements were m a d e from movies, using a
single-frame projector with a surveying instrument to
measure length of the sperm at the indicated times.
this case (Models, Fig. 4), entry is simply
the assumption of a most probable thermodynamic configuration. A second
mechanism could result from actin-like
filaments in the sperm or egg interacting
with cytoplasmic or cortical myosin of the
egg or sperm, leading to contraction and
resultant sperm entry (Model B, Fig. 4).
As a third mechanism, entry could occur
via movement of the plasma membrane itself. Recent work has shown that the plasma
membrane of many cells is fluid and in constant motion, moving at a rate of about 5
microns per minute (Edidin, 1972). If the
site of sperm-egg fusion provides a directed
source for membrane motion, such that
membrane fluidity is directed towards the
area of fusion, then such movement would
incorporate the sperm into the egg as the
egg membrane fuses with the sperm membrane (Model C, Fig. 4).
A decision among these hypotheses cannot yet be made. However, comparative observations suggest a uniform mechanism of
sperm entry among different eggs. First,
membrane fusion always occurs. Second,
actual entry may be between immobile gametes; in mammals, for example, motility
of the fertilizing sperm also ceases during
entry (Austin, 1961).
All organisms have some mechanism to
prevent multiple sperm entry. In eggs of
most organisms this "block to polyspermy"
occurs at the level of sperm egg-fusion, although in some species the block occurs at
the level of fusion of the male and female
pronucleus (see Austin, 1968). The consequences of polyspermy are usually lethal.
In these cases, the chromosomes are segregated randomly and development aborts in
early embryogenesis.
Previous work on sea urchins has suggested the existence of two types of blocks.
One is fast and incomplete, presumably occurring within the first few seconds of
sperm-egg contact (Rothschild and Swann,
1952). The other is slow and complete, and is
temporally and experimentally associated
with the cortical reactions (reviewed by Allen, 1958).
Isolation of cortical granules and cortical
granule products
Studies on the late block have been considerably advanced by the development of
methods for obtaining and studying the
cortical granule components and their relationship to blocking polyspermy. Two basic
procedures are available: (i) direct isolation
of the cortical granules as cell particles or
(ii) collection of the "fertilization product"
or "cortical exudate" following fertilization
or artificial parthenogenesis. The first approach has been especially developed by
passive
filaments
from spurn
filaments
plasma membrane
fluidity - movsment
e
FIG. 4. Three models accounting for incorporation
of the non-motile sperm into the egg. Further details
in text.
PROGRAM OF FERTILIZATION
511
Schuel and his collaborators, using the
zonal centrifuge (Schuel et al., 1972). This
method yields granules of high purity, but
of poor structural integrity. To overcome
this latter problem, I have recently developed a simple discontinuous gradient
method which yields intact cortical
granules, (Fig. 5) but in only 80% purity in
terms of organelle content (described in
Vacquier et al, 1973).
The experimental approach used most in
our studies involves characterization of the
fertilization product, i.e., the components
released by eggs upon fertilization or artificial activation. As noted (Figs. 2, 6), the
cortical granule components are normally
released into the space between the plasma
membrane and the overlying vitelline layer.
If the vitelline layer is removed before insemination or activation, the soluble cortical granule components will instead be released into the supernatant sea water (Fig.
FIG. 5. Electron micrograph of cortical granules isolated by differential centrifugation procedure.
Method described in Vacquier et al. (1973).
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DAVID EPEL
FP IN NORMAL EGG
\t molecular
weight FPentrapped in
FP IN 0EMEMBRAN6TE0 EGG
soluble FP molecules
in seawoter
fertilization space
FIG. 6. Diagram depicting principle of obtaining the
fertilization product (FP) from sea urchin eggs. In the
normal egg, the FP is extruded into the space between
the fertilization membrane and egg (perivitelline
space or fertilization space). If the precursor of the
fertilization membrane is removed beforehand, the FP
is extruded directly into the surrounding sea water.
6) and can be collected after centrifugal
removal of the eggs. A non-enzymatic procedure for removing the vitelline layer has
proved especially useful (Epel et al., 1970).
A third procedure is to extract and solubilize the cortical granule components
from intact eggs by suspension of the eggs
in an isoosmotic non-electrolyte media. In
such media, the eggs are parthenogenetically activated and the cortical granule contents are released. Since there are no divalent cations, the components are released in
a soluble form. This procedure, first
suggested by Moore (1949) has been
further developed by Kane and Stephens
(1969).
Analysis of the fertilization product has
led to the description and characterization
of two enzymes, a /3, 1-3 glucanase (Epel et
al., 1969; Muchmore et al., 1969) and a
trypsin-like protease (Vacquier et al.,
1972a). Both of these enzymes have also
been localized in the cortical granules
(Schuel et al., 1972; Vacquier et al., 1973).
Bryan (1970) has also described and
characterized a structural protein of the
granules, probably involved in the "hardening" or structuralization of the fertilization
membrane.
/3, 1-3 glucanase
The biological role of this enzyme is unclear, but it does not appear to be involved
in the block to polyspermy. Indeed, the activity of this enzyme is not concerned solely
with fertilization, since only 40% of the enzyme is released following fertilization of 5.
purpuratus eggs (Epel et al., 1969). An even
smaller proportion of the glucanase is released from L. pictus eggs. The remaining
glucanase is contained within small cytoplasmic granules (Epel et al., 1969) and is
secreted slowly during cleavage such that
approximately 90% of the remaining enzyme is released into the supernatant sea
water by the time of hatching (Epel and
Vacquier, unpublished).
A possible role of the glucanase is in the
formation of the hyaline layer as it forms
after fertilization. The major protein of this
layer, hyalin, is contained in the cortical
granules and released at fertilization (Kane
and Stephens, 1969). Recent studies
suggest that hyalin is continuously secreted
during early development (Kane, 1974).
Since the glucanase is also released at fertilization and continuously secreted, it may
be involved in the processing or attachment
of hyalin to cell surface.
Comparative studies on the glucanase
indicate its presence in at least six genera of
sea urchins including Allocentrotus fragilis,
Arbacia punctulata, S. purpuratus, andL. pictus (Epel, unpublished), Toxopneustes roseus,
Tripneustes depressus, and Arbacia incisa
(Vacquier, unpublished). Surprisingly, the
glucanase is not present in eggs of the sea
urchin Echinometra vanbrunti (Vacquier,
unpublished) and the sand dollar, Dendraster excentricus (Vacquier, 1971). It is also not
present in eggs of the starfish Patiria miniata (Houk and Epel, unpublished). These
data suggest that there may be considerable
diversity centering around the fertilization
reactions. Although the end result is the
same, the exact molecular mechanisms may
vary.
Protease
Lundblad (1954) first described the presence of several proteases in sea urchin eggs
and noted marked changes in activity following insemination. He postulated that
one of these protease activities was involved
with the cortical reactions and elevation of
the fertilization membrane. Several years
ago we showed that a trypsin-like protease
activity was contained in the fertilization
product (Vacquier et al., 1972a) and subsequent experiments revealed that this ac-
PROGRAM OF FERTILIZATION
tivity could be localized in the cortical
granules (Schuel et al., 1973; Vacquier et
al., 1973). The protease is trypsin-like on
the basis of its specificity towards synthetic
substrates, pH optimum, and inhibition by
natural and synthetic trypsin inhibitors
(Vacquier et al., 1972a).
The biological role of this enzyme has
been approached in two different ways.
The first determined the effects on fertilization of prior incubation of the eggs in the
crude fertilization product or in the purified protease. The second determined the
effects on fertilization when eggs were fertilized in the presence of protease inhibitors.
Using these approaches, we can ascribe
two actions to the protease. These are (i)
elevation of the vitelline layer as it transforms into the fertilization membrane and
(ii) detachment of supernumerary sperm.
Our results indicate that both of these actions are a part of the block to polyspermy.
513
bonds are hydrolyzed during the prior incubation in protease but that this hydrolysis, by itself, is not adequate to elevate
the vitelline layer. We postulate that other
components of the cortical granules are required for elevation. These may be the sulfated mucopolysaccharides of the cortical
granules. If hydrophilic, these macromolecules would hydrate when the cortical
granule contents are exposed to sea water.
This swelling would then elevate the detached vitelline layer.
These experiments also reveal that the
fertilization product protease is highly
specific, certainly much more specific than
bovine pancreatic trypsin. Treatment of
eggs with pancreatic trypsin will remove the
vitelline layer (e.g., Berg, 1967; Epel,
1970), whereas treatment with the fertilization product protease only alters the vitelline layer-plasma membrane connectives.
Alteration of sperm receptors. As noted ear-
lier, the vitelline layer contains proteinaElevation of thefertilization membrane: Two ceous sperm receptor sites. During normal
different types of evidence indicate that the fertilization, the fertilizing sperm must first
protease action is involved in fertilization attach to one of these receptors, pass
membrane elevation. First, if eggs are in- through the vitelline layer, fuse with the
seminated or artificially activated in the plasma membrane and thence enter the
presence of trypsin inhibitors, the fertiliza- egg. The vitelline layer receptors, however,
tion membrane does not elevate properly. are not essential since the vitelline layer can
Instead the vitelline layer remains attached be removed and fertilization can still occur
to the plasma membrane at many points (e.g., Runnstrom, 1966). Also, one can reand the surface of the egg looks like a bub- move the fertilization membrane after ferbly rosette (Fig. 7) (Vacquier et al., 19726). tilization and "refertilize" such eggs
This suggests that the vitelline layer is at- (Sugiyama, 1951; Tyler etal., 1956). These
tached to the plasma membrane by results, therefore, indicate that a sperm reprotease-sensitive bonds and that the pro- ceptor system also is present on the plasma
membrane of the egg.
tease detaches these linkages.
Several lines of evidence indicate that the
The second type of evidence comes from
recent experiments of Carroll on the effects fertilization product protease alters the
of incubating unfertilized eggs in the pur- sperm receptor sites on the vitelline layer as
ified fertilization product protease. If such a part of the block to polyspermy. First, if
eggs are artificially activated in the pres- eggs are incubated in either the crude ference of trypsin inhibitors, normal fertiliza- tilization product or a purified protease
tion membranes are elevated. Conversely, preparation, they are rendered unfertilizaif the eggs are not exposed to the purified ble. The vitelline layer is structurally alprotease but are activated in the presence tered, and sperm do not attach to this layer
of trypsin inhibitors, the fertilization mem- and do not activate or fertilize the eggs
brane does not elevate properly and is (Vacquier et al., 19726, 1973). If, however,
blebbed and bubbly (Carroll and Epel, these non-fertilizable eggs are now incubated in pronase so that the altered vitelline
1975).
Our interpretation of these experiments layer is removed, the eggs can then be feris that the vitelline layer-plasma membrane tilized by sperm (Vacquier et al., 1973).
514
DAVID EPEL
FIG. 7. Light micrograph illustrating incomplete eggs are inseminated in presence of trypsin inhibitors
elevation of fertilization membrane when S. purpuratus (lmgm/ml soybean trypsin inhibitor).
Similarly, if the eggs are first treated with
dithiothreitol or trypsin so as to remove the
vitelline layer, and then incubated in the
fertilization product protease, there is no
effect on fertilizability (Vacquier et al.,
1973).
The simplest interpretation of these experiments is that during normal fertilization, the sperm must first attach to the vitelline layer receptor sites. This attachment
PROGRAM OF FERTILIZATION
permits penetration of the layer and subsequent fusion of the sperm membrane
with the underlying cell membrane. When
eggs are incubated in the highly specific
fertilization product protease, this protease
alters only the sperm receptors of the vitelline layer; it does not remove the vitelline
layer to expose the underlying plasma
membrane. Following incubation in fertilization product protease, therefore, the
modified but still intact vitelline layer acts as
a physical barrier between sperm and
plasma membrane receptor. Recent results
indicate the presence of two proteases in
the fertilization product. One is specific for
the sperm receptors and the other is involved in vitelline layer elevation through
alteration of plasma membrane-vitelline
layer connectives (Carroll and Epel, 1975).
This proteolytic alteration of sperm receptor sites has a functional role in the prevention of polyspermy. Normally, the
supernumerary sperm attached to the vitelline layer become detached as the membrane elevates (Tegner and Epel, 1973;
Vacquier and Payne, 1973). This detachment does not occur in the presence of protease inhibitors. Rather, sperm remain attached to both elevated and non-elevated
areas of the vitelline layer. Associated with
insemination in the presence of trypsin inhibitors is an increased incidence of polyspermy (Vacquier et al., 19726, 1973).
This suggests that the supernumerary
sperm remaining attached to the vitelline
layer can still fuse with the egg. Even under
these conditions, however, only a small percentage of the attached sperm are successful in fusing with the egg (Tegner, 1973,
and unpublished). This may be because not
all sperm binding sites are equivalent or because not all sperm are functional (Tegner
and Epel, 1973) or because sperm can enter
eggs only in those areas where the vitelline
layer and plasma membrane remain in
tight apposition (Longo and Schuel, 1973).
Observations on hamster eggs reveal that
a sperm attachment-detachment sequence
also occurs in this organism (Hartmann and
Gwatkin, 1971) and that a protease released
from the egg is involved in sperm detachment and the block to polyspermy. Barros
and Yanagamachi (1971) have shown that a
515
cortical granule product results in loss of
the sperm binding sites on the zona pellucida. Furthermore, the action of this cortical
granule product on sperm detachment is
prevented if the exudate is incubated in
trypsin inhibitors (Gwatkin et al., 1973).
In making comparative observations, it
should be emphasized that the sequence of
sperm attachment and detachment is not
always apparent by direct microscopic observation of fertilization. With sea urchin
eggs, for example, attachment is quite apparent, but the detachment phase is less
dramatic and takes place over a period of
several minutes. However, detachment is
quite obvious when the eggs are fixed at
various times after insemination in aldehyde fixatives. When such eggs are
examined, sperm are found attached only
to areas of the vitelline layer which have not
yet elevated. Since detachment does not
occur when eggs are fertilized in the presence of protease inhibitors, the protease
action must alter the sperm-vitelline layer
bond such that the attachment is no longer
retained in the aldehyde fixatives.
METABOLIC ACTIVATION OR METABOLIC DEREPRESSION
During oogenesis, the developing oocyte
is synthetically active, utilizing exogenous
substances provided by its mother for
growth. At some point, probably around
maturation or ovulation, the egg becomes
independent of these exogenous substrates
and becomes metabolically quiescent or
repressed. The exact extent of this repression is unclear. In the sea urchin oocyte as
compared with the fertilized egg, the activities of respiration, transport, protein
and RNA synthesis are considerably reduced and DNA synthesis is completely
turned off.
Numerous studies on sea urchin eggs
have shown that fertilization "derepresses"
metabolism and activates new metabolic
pathways (for reviews see Monroy, 1965,
1973). These studies reveal a definite temporal sequence or program of metabolic
derepression (Epel et al., 1969).
The timetable or program for the S. pur-
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DAVID EPEL
puratus embryo is indicated in Figure 8.
Changes for which definite times are
known are indicated on the right; those for
which times are tentative are indicated on
the left. It is apparent that there are two
distinct clusters of changes, a large number
of "early reactions," including all those
changes occurring in the first 60 sec after
insemination and a number of "late reactions," beginning about 300 sec after insemination.
The "early changes" begin at 3 sec, with
alterations in the egg plasma membrane
resulting in an influx of Na + ion and a consequent "fertilization action potential"
(Steinhardt et al., 1971). Sperm entry and
cortical reactions begin at about 25 to 30 sec
(Paul and Epel, 1971). About 3 sec later, the
cytoplasmic activation of NAD kinase begins (Epel, 1964a), followed about 15 sec
later by a large and transient burst in respiration rate (Epel, 19646). It is postulated
that during this early period there also occurs the rapid block to polyspermy (Swann
and Rothschild, 1952), a release of intraTENTATIVE
PLACEMENT
DEFINITE
IN PROGRAM
PLACEMENT
IN PROGRAM
C o " release-ropid polyspermy block--;
Glucose-6-P0, Dehydrog
Shift
No' influx
—
Cortical reaction
—
NAD kinase
extracellular
protease .gluconase
fertilization membrane
block to
polyspermy
Respiration
Two- P- Dependent
alterations of plasma
membrane
K'- conductance
protein synthesis
omino ocid, PO^
transport
Qdenyl cyclase
nucleoside
transport
cellular calcium (Mazia, 1937; Steinhardt
and Epel, 1974), and a translocation of glucoses-phosphate dehydrogenase from a
particulate to a soluble form (Isono and Yasumasu, 1968).
The "late changes" begin about 5 min
after fertilization and include plasma
membrane changes resulting in development of potassium conductance (Steinhardt et al., 1971) and transport systems for amino acids (Epel, 1972), phosphate (Whiteley and Chambers, 1966), and
nucleosides (Piatigorsky and Whiteley,
1965). There is also initiated a large increase in protein synthesis (Epel, 1967) and
a change in the intracellular location of
adenyl cyclase (Castaneda and Tyler,
1968). However, there are no fertilizationassociated changes in cyclic AMP (Yasumasu et al., 1973).
Thus far, there are no definite changes
known to take place during the "dark"
period between 1 and 5 min. Energy requiring steps are needed for establishing potassium conductance (Steinhardt et al., 1972)
and amino acid transport (Epel, 1972), but
the nature and exact timing of these steps is
unclear.
In the following section, I will first describe some recent research, done in collaboration with Richard Steinhardt of the
University of California, Berkeley, that indicates that an increase in free calcium may
be close to the primary event of fertilization
(Steinhardt and Epel, 1974). I will then
analyze this derepression by examining the
interrelationships between three of the socalled late events, K+-conductance, protein
synthesis, and amino acid transport (Epel et
al., 1974).
Increases in free Ca2+ as a primary event
In 1937, Mazia showed that intracellular
free calcium increased after fertilization of
Arbacia punctulata eggs. Was this change
simply a result of activation? Or was it a
Pronucltor fusion
20
polyodenylation j DNA synthesis
primary
cause of activation? If primary,
ol m RNA
I
one should be able to derepress metabolism
90 *• I Division
by increasing the intracellular content of
FIG. 8. Timetable or program of post-fertilization free Ca2+. One means of accomplishing this
changes in eggs of 5. purpuratus at 17°C. Events for
which times are known are on the right; events for is with ionophorous antibiotics, which
abolish the selective permeability of memwhich times are not exactly known are on the left.
s1
517
PROGRAM OF FERTILIZATION
branes to ions. These can be specific for
monovalent cations, such as valinomycin,
or specific for divalent cations, such as
A23187 (see Pressman, 1973, for review).
Steinhardt and I observed that when
A23187 was applied in micromolar
amounts to unfertilized sea urchin eggs, the
membrane conductance changes and cortical reactions were activated just as in normal fertilization. Was A23187 acting as a
full parthenogenetic agent? We examined a
large number of the metabolic and structural changes that normally accompany fertilization and found that all of these are
activated similar to the normal activation by
sperm. These include respiration, synthesis
of protein, DNA, and chromosome condensation. Indeed, the rate of activation
was faster than other more classical, parthenogenetic agents. For example, activation by thymol or KC1 results in a delay in
DNA synthesis (von Ledebur-Villiger,
1972) which is not seen with A23187. However, we have noted that developmental abnormalities begin around the time of the
first mitotic division. Monasters develop,
but the eggs do not divide. This may reflect toxic effects of the ionophore resulting from aberrations in Ca2+ metabolism,
nonspecific side effects, or the need for
secondary parthenogenetic treatments,
such as incubation in hypertonic media.
What cation is being affected by the
ionophore and is thus activating the eggs?
The two major divalent cations of sea water
are calcium and magnesium. To determine
whether the ionophore was affecting the
transport of either of these ions into the
egg, we measured cortical activations, respiration, and protein synthesis in ionically
substituted media. To our surprise, we
found that the composition of the external
environment made no difference. Eggs
could be activated in Ca2+-free or Mg2+free sea water, even in 0.55 M KC1 containing 2 mM EGTA. These results suggest that
the ionophore might be acting through release of divalent cations within the cell.
Calcium and magnesium are also the
major divalent cations within the cell. When
we estimated the content of free and bound
Ca2+ and Mg2+, we found that the bulk of
Ca2+ was bound but available for release,
whereas Mg 2+ was always "free." We estimated the content of free and bound cations by preparing homogenates in various
media and analyzing Mg2+ and Ca2+ in the
37,000 g pellet and supernatant. We found
that most of the Mg2+ was in the supernatant, and hence "free," whereas most of the
calcium was sedimentable and hence
"bound." In some media, however, the
"free" Ca 2 + content was high. These
findings indicate that although Ca2+ is
normally sequestered, it is available for release. We postulate that the ionophore is
most likely affecting calcium distribution
within the egg and that the release of this
calcium from some intracellular store results in the activation of the egg. The store
of Ca2+ may be a Ca2+-binding protein
(Nakamura and Yasumasu, 1974).
DEPENDENT AND INDEPENDENT DEREPRESS1ON
Irrespective of whether or not Ca2+ release is the primary trigger leading to derepression, we can experimentally determine
whether metabolic activations of fertilization are connected in series in a cause-effect
(dependent) relationship or whether they
are unconnected (independent) (Fig. 9).
We have examined this question, looking at
three "late" responses: K+-conductance,
protein synthesis, and Na + -dependent
amino acid transport. As noted, all of these
changes begin at about 5 min after insemination. Our analysis, however, indicates no
causal interconnections between any of
Dependent pathway model
-A — B— C— D
Independent pathway model
A
—B
-C
-E
FIG. 9. Two models accounting for the metabolic derepression occurring upon fertilization.
518
DAVID EPEL
them (Epel et al., 1974).
To examine the relationship between
potassium conductance and protein synthesis, we abolished conductance by acidifying the sea water around the eggs. This
alters the plasma membrane such that K+conductance does not take place
(Steinhardt and Mazia, 1972). Even so, protein synthesis was activated under these
conditions. Tupper (1974) has come to
similar conclusions, using barium ion to
prevent K+-conductance.
There is also no apparent relationship
between protein synthesis and amino acid
transport. This is best seen in experiments
where the metabolism of the egg is partially
activated by incubation in sea water containing 1 mM NH4C1 or NH4OH, pH 9.
This treatment has been shown to activate a
number of metabolic changes, such as K+conductance, DNA synthesis and chromosome condensation (Steinhardt and Mazia,
1972; Mazia and Ruby, 1974; Mazia, 1974)
and polyadenylation of RNA (Wilt and
Mazia, 1974). Not initiated are the cortical
reactions and Na + -conductance (Steinhardt and Mazia, 1972). We have found
that although protein synthesis and K+conductance are initiated, Na + -dependent
amino acid transport is not (Epel et al.,
OUTSIDE
1974). This indicates that although these
three events are normally initiated coincidentally, they are not causally interconnected. Assuming release of intracellular
Ca2+ is the primary activator, it may act as a
master switch initiating a series of independent events. Indeed, the "early" events may
all be dependently linked, whereas the
"late" events are independent of each other
(Epel et al., 1974).
Some reactions are, of course, causally
interconnected and I have indicated some
of these as a flow diagram in Figure 10. In
this diagram, the fertilization changes are
depicted with reference to changes within
the plasma membrane of the egg and the
resultant intracellular and extracellular
consequences.
We hypothesize that the primary effect
resulting from the interaction of the fertilizing sperm with the plasma membrane is
the release of a store of intracellular Ca2+.
This Ca2+ release affects both cytoplasm
and plasma membrane. Possibly, the increased free Ca2+ induces No?-conductance.
This Na+-conductance could be related to
the hypothetical early block to polyspermy.
Ca2+ is required for exocytosis and membrane fusion (e.g., Poste and Allison, 1973),
and the release of intracellular Ca2+ could
[late polyspermy I
block
I
|spwm receptors I
hydrophilic
substances
protease]
PLASMA
MEMBRANE
INSIDE
|dewtoproent"|
EARLY CHANGES
FIG. 10. A hypothetical flow diagram of fertilization,
showing changes and possible causal relationships oc-
LATE CHANGES
curring in plasma membrane, cytoplasm and vitelline
layer.
519
PROGRAM OF FERTILIZATION
initiate cortical granule fusion. (Indeed, in long after the completion of meiosis. In the
order to isolate intact cortical granules, other echinoderm classes, fertilization and
chelating agents must be included in the meiosis (or maturation) are tightly coordihomogenizing media.) Increased cytoplas- nated. Does a similar program of metabolic
mic Ca2+ could activate the transient res- derepression occur in these organisms as in
piratory burst (Epel, 1969; Nagazawa et the sea urchin? Is derepression initiated by
aL, 1970) and also activate the enzyme NAD maturation? Or by fertilization?
kinase (Epel, 1967; Blomquist, 1973) and
Margaret Houk and I have recently
glycogen phosphorylase (Shoger et al., examined these questions, using eggs of the
1973). The resultant increase in NADP and starfish, Patiria miniata. We found that the
glucose-6-phosphate would release starfish oocyte is similar to the sea urchin
glucose-6-phosphate dehydrogenase from egg in that a major derepression in rate of
the particulate to supernatant fraction respiration (Houk, 1974) and protein syn(Isono and Yasumasu, 1968). Changes in thesis (Houk and Epel, 1974) takes place. It
this enzyme, plus the increased levels of is dissimilar in that the major changes occur
NADP and glucose-6-phosphate could during maturation: fertilization results in
maintain increased respiration of the eggs, no change in protein synthesis (Houk and
which is primarily through the pentose Epel, 1974) and only a transient increase in
phosphate pathway (Krahl, 1956). It is un- respiration (Houk, 1974).
clear how protein synthesis is activated at
Figure 11 shows the per cent incorporathe translational level, but activation of a tion of amino acid into protein at various
cytoplasmic protease may be important times after initiation of maturation by
(e.g., Monroy et al., 1965; Mano and 1-methyladenine. As seen, the level inNagano, 1970), perhaps to remove an in- creases twofold by 40 min and fivefold by 2
hibitory protein from the ribosomes, such hr. This protein synthesis is required for
as described by Metafora et al. (1971). completion of meiosis; if protein synthesis
Eventually, steps in the program result in is inhibited with pactamycin, meiosis is arinitiation of DNA synthesis, cleavage and rested at metaphase or anaphase. Fertilizadevelopment.
tion has no effect on the rate of protein
synthesis. This can be seen in Figure 12,
which depicts an experiment measuring
METABOLIC DEREPRESSION IN OTHER ORGANISMS protein synthesis after insemination.
Ca2+ release (ionophore effects) in eggs of other
organisms.
Does this general picture of metabolic
derepression, as described for sea urchins,
hold for other organisms? Assuming that
activation of eggs by A23187 results from a
release of intracellular calcium, an increase
in this cation may be the primary event in
activation of all eggs. So far, we have found
ionophore activation in eggs or oocytes of
five different organisms including starfish
(Patiria miniata), tunicates (Ciona intestinalis), amphibians (Xenopus laevis), and
hamsters (Mesocricetus auratus) (Steinhardt
et al., 1974).
40
80
120
160
200
MINUTES AFTER 1-MA STIMULATION
Metabolic derepression during maturation and
fertilization of starfish oocytes
Fertilization of the sea urchin egg occurs
FIG. 11. Incorporation of amino acid increases during 1-methyladenine-induced maturation of starfish
(Patiria miniata). O, oocytes exposed to
1-methyladenine; A, unactivated oocytes. (Data of
Houk and Epel, 1974).
520
DAVID EPEL
A similar activation of protein synthesis
during maturation has previously been described in amphibians (Smith and Ecker,
1970). Indeed, the parallels between
starfish and amphibians are striking. In
both, protein synthesis increases in response to a hormonal stimulus, and in both
this protein synthesis is necessary for the
completion of meiosis. In both, there is no
increase in protein synthesis after fertilization.
Information about the metabolic
changes occurring during maturation and
fertilization is lacking for the echinoderm
classes other than the asteroids and
echinoids. If maturation is also under hormonal control in these groups, their
metabolic activation may be more similar to
the starfish than to the sea urchin.
Based on this limited comparative data, it
is probable that in organisms where maturation and fertilization are closely coordinated, as in asteroids, amphibians, and
other vertebrates, a partial derepression
occurs during maturation and this derepression is completed at fertilization. Conversely, if maturation occurs long before
fertilization as in the echinoids, the eggs are
ovulated in a metabolically repressed state
and are completely derepressed by fertilization. Since ionophore A23187 activates
60
120
MINUTES AFTER FERTILIZATION
FIG. 12. Incorporation of amino acid is not altered
by fertilization of P. miniata eggs. A, Unfertilized eggs;
O, fertilized eggs. (Data of Houk and Epel, 1974.)
both classes of eggs, the common event at
fertilization may be an increase in the intracellular content of calcium.
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