Part II

Last Update: 4 November 2017 M - 50

Fertilization
Q. What are the cortical reaction? What are the role play by ZP3 and IP3 in activation of ovum during the
fertilization? Describe in brief the acrosomal reaction of spermatozoa during fertilization? Role of
Ca2+ in the fertilization. What are the fast block to polyspermy and slow block to polyspermy?
-catenin and its role in fertilization. What are the molecular changes in capacitation?

Major Events of Fertilization

1. Recognition events between sperm and egg: species specificity
2. Regulation of sperm entry into egg: block to polyspermy.
3. Fusion of sperm and egg genetic material
4. Activation of developmental process within the egg

The main function of fertilization is to combine the haploid sets of chromosomes from two individuals into
a single diploid cell, the zygote. In addition, fertilization activates the egg. Egg activation blocks entry by
additional sperm, stimulates the final meiotic division, and triggers the onset of embryonic development.

The sea urchin is one of the several model organisms that has been used to decipher the basic cellular and
molecular biology of animal fertilization. More is known about fertilization in sea urchins than is known
about fertilization in most vertebrates. In animals in general, fertilization is the direct interaction and fusion
of two germinal cells (one "egg" and one spermatozoan), resulting in the initiation of cleavage, gastrulation
and the species-specific developmental program that characterizes each organism. In sea urchins, a discrete
series of steps characterizes fertilization (Vacquier, 1998). Each of these steps is discussed below in detail
and many may be visualized in the electron microscopical study of Anderson (1968), in the video essay
provided by Mark Terasaki (1998) or the web site of Epel, 1991).

1. Attraction of spermatozoa by fully mature ova:

Peptides dissolve from the jelly coat and stimulate sperm respiration, motility and chemoattraction in a
species-specific manner (Kinoh et al., 1994). Sperm follow a decapeptide gradient in sea water towards the
region of higher concentration at jelly coat of recently spawned fully mature ova. These peptides have been
recovered from egg-conditioned medium (Garbers, 1989). In S. purpuratus and Hemicentrotus
pulcherrimus, the peptide is entitled speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) and in Arbacia
punctulata the peptide is called resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-gly-Gly-Gly-Arg-Leu)
(Ward and Kopf, 1993). Species specificity depends on the COOH end of the peptide and deletion of the Val
or Val-Gly residues results in loss of respiration-stimulating activity. A 767 amino acid protein that serves as
a voltage sensitive channel in the plasma membrane of the spermatozoan has been isolated from the testis of
S. purpuratus (Cardullo et al., 1994; Gauss et al., 1998). Such a channel might become activated by speract-
induced hyperpolarization and lead to flagellar beating.

Mechanisms of sperm-egg recognition and contact in mammals and role of ZP3

Mammalian sperm must reside in the female reproductive tract before they are able to undergo the acrosome
reaction. This maturation process is called capacitation.

-1-
The mammalian egg is surrounded by an extracellular envelope called the zona pellucida, to which sperm
must bind and penetrate before they can make contact with the surface of the egg itself. The zona pellucida
of the mouse egg contains three glycoproteins called ZP-1, ZP-2 and ZP-3 that polymerize to form a gel.
The zona of newly-ovulated eggs is also surrounded by a constellation of follicle cells in a matrix of
hyaluronic acid. Figure 1 shows a mouse ovum accompanied by polar body I surrounded by the zona
pellucida.

Figure 1. Mouse ovum and polar body I surrounded by the zona pellucida. Image courtesy of
Dr. Douglas Kline, Kent State University.

ZP3 functions as a sperm receptor. The sperm-binding activity of ZP3 is mediated by

the oligosaccharide side chains of ZP3. The role of the oligosaccharides is
demonstrated by experiments in which either removal or modification of the sugars
causes loss of sperm-binding activity. A peripheral membrane protein in the plasma
membrane overlying the acrosome of mouse sperm called sp56 binds to the
oligosaccharide moieties of ZP3. sp56 belongs to a class of carbohydrate-binding proteins called lectins.
Purified sp56 binds to the zona of unfertilized eggs, but not to that of zygotes (Bookbinder et al., 1995. This
observation suggests that the oligosaccharides on ZP3 trap incoming sperm at the zona surface of
unfertilized eggs and that this activity is lost after fertilization.

Bookbinder et al. (1995) have also reported a correlation between the presence of sp56 and species
specificity of sperm-egg recognition. Mouse and hamster sperm contain sp56 and bind to the mouse egg.
Guinea pig and human sperm, however, lack sp56 and do not bind. Different lectins may be involved in
human and rabbit sperm-egg binding. In humans, the oligosaccharides on ZP3 differ from those in mice
(Aitken, 1995). The carbohydrates present on ZP3 may provide a code that facilitates species specificity of
sperm-egg binding. Investigators may be able to exploit this mechanism in developing new contraceptives or
in diagnosis and treatment of male
infertility.

2) Egg activation

Ca2+ release at fertilization results in

an increase in metabolic activity
within the egg, apparently due to an
increase in the intracellular pH of the
egg. Diacyl gycerol (DAG) causes
protein phosphorylation cascades to
be initiated, with one result being the
phosphorylation and activation of a
plasma membrane Na+:H+ ion
exchanger. Na+ is pumped into the
cell, H+ is pumped out of the cell, and
the pH inside the cell increases.
Sperm themselves are NOT required
for egg activation - injection of Ca++
can artificially induce egg activation
in many species.
Fig. Hypothetical pathway for calcium release

Role of Ca2+ in the activation of Egg

The activation of all eggs appears to depend on an increase in the concentration of free calcium ions within
the egg. Such an increase can occur in two ways: calcium ions can enter the egg from outside, or calcium
ions can be released from the endoplasmic reticulum within the egg. Both mechanisms are used to different
degrees in different species. In snails and worms, much of the calcium probably enters the egg from outside,
while in fishes, frogs, sea urchins, and mammals, most of the calcium ions probably come from the
-2-
endoplasmic reticulum. In both cases, a wave of calcium ions sweeps across the egg, beginning at the site of
sperm-egg fusion (Jaffe 1983; Terasaki and Sardet 1991).
The presence of calcium ions is essential for activating the development of the embryo. If the calcium-
chelating chemical EDTA is injected into the sea urchin egg, there is no cortical granule reaction, no change
in membrane resting potential, and no reinitiation of cell division (Kline 1988). Conversely, eggs can be
activated artificially in the absence of sperm by procedures that release free calcium into the oocyte.
Steinhardt and Epel (1974) found that injection of micromolar amounts of the calcium ionophore A23187
into a sea urchin egg elicits most of the responses characteristic of a normally fertilized egg. The elevation
of the fertilization envelope, a rise of intracellular pH, a burst of oxygen utilization, and increases in protein
and DNA synthesis are all generated in their proper order. In most of these cases, development ceases before
the first mitosis because the egg is still haploid and lacks the sperm centriole needed for division. Calcium
release activates a series of metabolic reactions. One of these is the activation of the enzyme NAD + kinase,
which converts NAD+ to NADP+ (Epel et al. 1981). This change may have important consequences for lipid
metabolism, since NADP+ (but not NAD+) can be used as a coenzyme for lipid biosynthesis. Thus, the
conversion of NAD+ to NADP+ may be important in the construction of the many new cell membranes
required during cleavage. Another effect of calcium release involves oxygen consumption. A burst of
oxygen reduction (to hydrogen peroxide) is seen during fertilization, and much of this "respiratory burst" is
used to crosslink the fertilization envelope. The enzyme responsible for this reduction of oxygen is also
NADPH-dependent (Heinecke and Shapiro 1989). Lastly, NADPH helps regenerate glutathione and
ovothiols, which may be crucial for scavenging free radicals that could otherwise damage the DNA of the
egg and early embryo (Mead and Epel 1995).
Cortical rotation
Positional information is already contained within many eggs, with the exception of mammals. Egg polarity
is due to the asymmetric distribution of cytoplasmic molecules, including mRNAs, proteins, and yolk, and is
roughly oriented along the anterior/posterior axis in most animals. A rearrangement of the egg cytoplasm
is induced by fertilization within many frog species, and this rearrangement (called cortical rotation) results
in the establishment of the dorsal/ventral axis. During cortical rotation, the plasma membrane and cortex
(cytoplasmic region just below the plasma membrane) rotate relative to the inner cytoplasm. The
pigmentation of frog eggs makes it possible to observe this process visually, and results in a gray area, called
the gray crescent.
Molecular Consequences of Cortical Rotation & -catenin
Cortical rotation helps establish
positional information by affecting
the distribution of Cytoplasmic
Determinants like -catenin.

- Catenins
- Catenins are proteins found in
complexes with cadherin cell
adhesion molecules of animal cells.
The first two catenins that were
identified became known as alpha-
catenin and beta-catenin.
-catenin can bind to beta-catenin
and can also bind actin. Beta-catenin
binds the cytoplasmic domain of
some cadherins. Additional catenins
such as gamma-catenin and delta-catenin have been identified. The name "catenin" was originally selected
('catena' means 'chain' in Latin) because it was suspected that catenins might link cadherins to the
cytoskeleton.

-3-
Beta-catenin
When -catenin was sequenced it was found to be a member of the armadillo
family of proteins. These proteins have multiple copies of the so-called
armadillo repeat domain which is specialized for protein-protein binding. An
increase in beta-catenin production has been noted in those people who have
Basal Cell Carcinoma and leads to the increase in proliferation of related
tumors. When -catenin is not associated with cadherins and -catenin, it can
interact with other proteins such as ICAT and APC.
Interactions of beta-catenin with other proteins
Figure. -catenin can interact with several different proteins inside cells. The
interaction of -catenin with other proteins is often regulated by the
reversible attachment of phosphate (P). As mentioned above, -catenin contains armadillo repeats and is
able to bind to other proteins. Inside cells, -catenin can be found in complexes with cadherins, transcription
factors (TF in Figure) and other proteins such as axin, a component of the Wnt signalling pathway. The
ability of -catenin to bind to other proteins is regulated by tyrosine kinases and serine kinases such as GSK-
3.
When -catenin is not assembled in complexes with cadherins, it
can form a complex with axin. While bound to axin, -catenin can
be phosphorylated by GSK-3, which creates a signal for the rapid
ubiquitin-dependent degradation of -catenin by proteosomes.
Various signals such as the Wnt signalling pathway can inhibit
GSK-3-mediated phosphorylation of -catenin, allowing -catenin
to go to the cell nucleus, interact with transcription factors, and
regulate gene transcription.
-catenin can be phosphorylated by other kinases such as protein
kinase A (PKA). Phosphorylation of -catenin by PKA has been
associated with reduced degradation of -catenin, increased levels
of -catenin in the nucleus and interaction of -catenin with TCF
family transcription factors to regulate gene expression.

Fig. Principal interactions of structural proteins at cadherin-based adherens

junction. Actin filaments are linked to -actinin and to membrane through
vinculin. The head domain of vinculin associates to E-cadherin via -, - and
-catenins. The tail domain of vinculin binds to membrane lipids and to actin filaments.

3) Sperm capacitation

Unlike in lower animals, ejaculated sperm from mammals must undergo capacitation in order to fertilize an
oocyte. Sperm become "fertilization competent" as they reside in the female reproductive tract through a
series of physiological changes known as capacitation. Capacitation has been referred to as changes that
enable the sperm to undergo both the acrosome reaction and hyperactivation. Capacitation has been
correlated with changes in sperm intracellular ion concentration, plasmic membrane fluidity, metabolism
and motility. Changes in sperm cyclic nucleotide metabolism and protein phosphorylation have also been
implicated in sperm functions such as capacitation. However, the molecular basis behind these events has
been poorly understood (Visconti et al., 1995).

4) Acrosomal reaction of spermatozoa :

Activation of the many spermatozoa which triggers the acrosomal reaction results from direct their contact
with a highly sulfated fucose sulfate polymer in the jelly coat of the fully mature ovum (Mendoza et al.,
1993; Cardullo et al., 1994; Vacquier and Moy, 1997; Hughes et al., 1999).
This interaction causes adherence of many spermatozoa to the jelly coat and opening of incurrent
Ca2+ channels in their cell membranes that are similar to human polycistin-L (Chen et al., 1999).

-4-
 Na+ ions flow into the sperm head and the plasma membrane depolarizes, opening voltage-sensitive
Ca2+ channels.
Ca2+ ions flow into the sperm head, and the high Ca2+ causes the fusion of the acrosomal vesicle
(green) with the sperm plasma membrane, releasing acrosomal vesicle contents into the extracellular
space.
The acrosomal reaction results in fusion of the acrosomal vesicle membrane with the
spermatozoan plasma membrane and permits the release or secretion of the contents of the
acrosomal vesicle.
Hydrolytic enzymes from the acrosomal vesicle begin to digest constituents of the jelly coat and
vitelline membrane. Additionally, excurrent proton pumps in sperm membrane are also opened at
this time and their activity leads to alkalinization of the cytoplasm in the head of the spermatozoan.
This alkaline cytoplasm promotes polymerization of actin subunits into microfilament cables that
thrust acrosomal processes toward the egg plasma membrane.
Extensive polymerization of actin generates microfilaments that extend a membrane covered
acrosomal process from the bottom of the vacant acrosomal vesicle.
The acrosomal process projects through the
remains of the jelly coat towards the vitelline
membrane of the egg.
As this occurs, bindin (not a glycoprotein or
a transmembrane protein) becomes
associated in an unknown manner with the
tip of the acrosomal process.
Sea urchin bindins are not related to any
other proteins, but they do contain a central
domain of 60 amino acids that has been
conserved for over 150 MY (Biermann,
1998).
Also released from the acrosomal vesicle at
this time are syntaxin and VAMP which
become involved in secretion of the contents
of the granule granules of the fully mature ovum following fertilization (Schulz et al., 1997).

i. Binding of the membrane of spermatozoa with the vitelline membranes of fully mature ova:
Binding receptors on the vitelline membrane recognize and attach
to bindin protein on the tip of the acrosomal process (Gao et al.,
1986; Glaser et al., 1999). Sea urchin egg receptor for bindin is a
complex molecule that is still very controversial. The putative
receptor is a single protein that has several domains and is unlike any
known class of receptors. On its carboxyl terminus it has a short
cytoplasmic domain, followed by a single membrane spanning
domain. Extracellular domains on the amino terminus include a
potential vitelline membrane spanning domain (Hirohashi and
Lennarz, 1998) followed by a domain that is similar to cytoplasmic
heat shock 70 proteins in the vertebrates. Since the function of heat
shock proteins is to bind to and fold other proteins, perhaps this
HS70 domain can bind bindin (Foltz and Lennarz, 1993). Subsequent to these analyses, other researchers
have disputed the validity of this interpretation, even suggesting that the protein identified as the bindin
receptor is actually a soluble, cytoplasmic HS110 protein (Mauk et al., 1997). Resolution of this issue will
await further sequence data.

-5-
a. Fusion of the cell membrane of the spermatozoon with that of the fully mature ovum:
While thousands of spermatozoa can potentially achieve this level of interaction with the fully mature ovum,
the vitelline membrane is finally breached by the acrosomal process of a single spermatozoon. The
membrane of this spermatozoon fuses with that of the fully mature ovum. Why only one spermatozoon is
capable of this act, when many are marsheled to accomplish the same task is one of the unanswered
questions of sea urchin biology.

b. Activation of the cytoplasm, the cortical reaction of the fully mature ovum and assembly of the
fertilization envelope:
After fusion, the metabolism of the egg changes radically during activation. The membrane of the fully
mature ovum is depolarized radially away from point of fusion (Jaffee, 1976). Gamete membrane fusion
elicits a wave of Ca2+ release from intercellular stores (Henson et al., 1990) that initiates at the point of
fusion and causes exocytosis of some 15,000 cortical granules (Sommers et al., 1989; Chandler, 1991).
Included in these granules is
1. hyalin which forms a layer immediately surrounding the egg,
2. a colloid that raises the fertilization membrane by imbibing H2O,
3. a serine protease (CGSP1) that destroys receptors for sperm on the vitelline membrane (Haley and
Wessel, 1999),
4. a protein termed
vitelline delaminase
that may cleave the
connection between the
vitelline membrane and
the oolemma (Carrol and
Epel, 1975),
5. a structural protein that
in the presence of H2O2
is polymerized onto the
inner surface of the old
vitelline membrane, now
called the fertilization
membrane (Kay et al.,
1982) and
6. ovoperoxidase, a heme-
dependent peroxidase that functions to block polyspermy by interacting with the structural protein
mentioned above in the presence of H2O2 to form the new fertilization membrane (Kay et al., 1982;
Somers et al., 1989; LaFleur et al., 1998).

The increase in respiration within the cytoplasm of the newly fertilized egg produces H 2O2 which serves as
the oxidant for the catalysis of the structural proteins to the inside of the old vitelline membrane by
dityrosine crosslinkages (Shapiro, 1991). The considerable oxidative damage that could result from the
presence of H2O2 evolved in this process is countered by an intracellular amino acid called ovothiol.
Ovothiol is oxidized to the disulfide thus consuming H2O2 (Shipiro, 1991). There results a highly protective
shield, the fertilization membrane, that protects the fertilized fully mature ovum or zygote.

-6-
After making its way through the jelly coat, the sperm makes contact with the vitelline envelope. Species-
specific bindin receptors on
the vitelline envelope are only
able to recognize bindin
molecules from the same
species. This "lock and key"
mechanism ensures that eggs
are fertilized only by sperm of
the same species. After
making its way through the
vitelline envelope, the sperm
and egg plasma membranes
fuse, and the sperm nucleus
enters the cytoplasm of the
egg.

Preventing polyspermy OR
Blocks to Polyspermy
Although many sperm attach to the coats surrounding the egg, it is important that only one sperm fuses with
the egg plasma membrane and delivers its nucleus into the egg. In gist,
Only one sperm is allowed to penetrate the oocyte
Two mechanisms ensure monospermy
Fast block to polyspermy
membrane depolarization
prevents sperm from fusing with
the oocyte membrane
Slow block to polyspermy
zonal inhibiting proteins (ZIPs):
Destroy sperm receptors
Cause sperm already
bound to receptors to
detach
Fast block to polyspermy
In marine invertebrates, including the sea urchin,
a fast block to polyspermy occurs within a tenth
of a second of fusion. The fast block to
polyspermy involves the opening of Na+
channels in the egg plasma membrane. Na+
flows into the egg cell, depolarizing the
membrane. This depolarization prevents
additional sperm from fusing to the egg plasma
membrane. The egg plasma membrane is
restored to its normal -70mV potential within
minutes of fusion as the Na+ channels close,
other + ions flow out of the cell, and Na+ is
pumped out. If depolarization is prevented,
polyspermy occurs - but how depolarization
blocks polyspermy is not yet understood.
Slow block to polyspermy:
The slow block to polyspermy begins within 10
seconds of fusion of the sperm and egg plasma
-7-
membranes. A compound called inositol triphosphate (IP3) causes the release of Ca2+ from intracellular
stores in the egg endoplasmic reticulum. Ca2+ is first released at the site of sperm entry, and during the next
minute, a wave of free Ca2+ passes through the egg. This Ca2+ results in the fusion of cortical vesicles with
the egg plasma membrane, releasing their contents into the space surrounding the egg, called the
perivitelline space. This raises the vitelline membrane, and inactivates bindin receptors on the vitelline
membrane. Thus, any additional sperm are released from the vitelline membrane and no more bind.
The image below showing the events involved in the slow block to polyspermy! This pictures shows
simultaneous double imaging of phase contrast (left) and intracellular Ca2+ concentration (right) during sea
urchin fertilization. The left image shows the approach of the sperm at about 2 o'clock and the rising of the
vitelline membrane. Intracellular Ca2+ is monitored by an indicator that becomes more fluorescent when it
binds Ca2+. A transient Ca2+ rise around the entire cortex (fifth frame) is followed by the Ca2+ wave, which
begins at the sperm entry site.
It appears that the media in which the sperm are incubated play an integral role in many sperm processes.
Lee and Storey (1986); Kopf and Gertion (1991); Yanagimachii (1994) have suggested that Ca2+ and
NaHCO3 are required for the induction of the acrosome reaction by the zona pellucida.
Visconti et al. investigated the need for CaCl2, bovine serum albumin (BSA) and NaHCO3 to induce the
capacitation process in mouse caudal epididymal sperm. They also investigated the relationship between the
capacitation state of sperm and protein tyrosine phosphorylation and proposed a possible mechanism by
which this may occur via a cAMP pathway. They hypothesized that Ca2+, HCO3- and BSA are required for
the capacitation process and that capacitation is accompanied by a time dependent increase in protein
tyrosine phosphorylation.
Visconti et al. performed five different tests and assays to determine which factors were necessary in sperm
capacitation, acrosomal reaction and ability to form pronuclei. They performed an experiment to test how
much BSA, if any, was required for sperm capacitation. In the absence of BSA, they discovered that protein
tyrosine phosphorylation was restricted to p95/116 hexokinase. As BSA concentrations were increased,
degrees of protein tyrosine phosphorylation increased as well, displaying a maximum at about 3mg/ml of
BSA.
The correlation of capacitation with the increase in protein tyrosine phosphorylation could be demonstrated
if the sperm now possessed the ability to undergo ZP induced acrosomal reaction as determined by the
Chlortetracycline (CTC) assay and to form pronuclei.
In a similar way, the researchers performed experiments to test the role of calcium ion and the bicarbonate
ion. In both cases, they found that a sufficient concentration of the ions produced protein tyrosine
phosphorylation as determined by probing the fixed proteins with an anti-phosphotyrosine antibody. It
should be noted that the sodium bicarbonate was not merely a pH affect, and the researchers performed tests
to establish this.
In their accompanying report, Visconti et. al. (1995b) continued their work and showed that protein tyrosine
phosphorylation and capacitation are regulated by a cAMP-dependent pathway. Subsequent investigations
revealed that sperm incubated in media devoid of NaHCO3, CaCl2 or bovine serum albumin could be
induced to undergo capacitation by the addition of cyclic AMP analogues.
The role of bovine serum albumins may involve accelerating the loss of cholesterol from the sperm plasma
membrane. Changes in membrane composition led to an increased permeability of the membrane to calcium
and bicarbonate ions. Ca2+ and HCO3- subsequently activate the adenylyl cyclase or as in the case of
calcium ion stimulate the activity of phosphodiesterases. A rise in cAMP then results in the activation of
protein kinase A, which in turn leads to the activation of sperm tyrosine kinases and/or inactivation of
phosphoprotein phosphatases. The result is an increase in protein tyrosine phosphorylation.

The Roles of Egg's Sperm Receptor (ZP3)

Sixteen years ago, ZP3, one of three glycoproteins found in the extracellular coat, zona pellucida, of
mammalian eggs was determined by Bleil and Wassarman to be a sperm receptor. Six years later, they
-8-
reported the results from binding of radioiodinated egg ZP3 from mice to acrosome-intact and acrosome-
reacted sperm. As expected of a mouse egg's sperm receptor, visual evidence of ZP3 binding to the heads of
acrosome-intact sperm was obtained. Not surprisingly, ZP3 failed to bind acrosome-reacted sperm.
Additional information was obtained with labeled ZP2, the second of the zona glycoproteins. An unexpected
ability of this glycoprotein to bind to acrosome-reacted sperm heads was revealed. It would thus appear that
both ZP3 and ZP2 play key roles in mammalian sperm binding during fertilization.
Under fertilizing conditions in vitro, sperm attach loosely and without specificity to the zona pellucida
outside mammalian eggs. Species-specific binding between the sperm plasma membrane and receptors in
the zona pellucida follows. This triggers a cascade of events in which the plasma membrane at the anterior
end of the sperm head fuses with the outer acrosomal membrane. Both are shed, resulting in the exposure of
the inner acrosomal membrane and subsequent release of acrosomal enzymes. This is what is commonly
referred to as the acrosome reaction.
The fate of the acrosome-reacted sperm is remain attached to the egg and subsequently penetrate the zona
pellucida with the aid of an acrosomal protease is extensively studied by Bleil and Wassarman. The three
glycoproteins of the zona are appropriately named ZP1, ZP2, and ZP3. While the function of ZP1 at the time
of Bleil and Wassarman's research was unknown, ZP3 had already been determined to act as a sperm
receptor.
The glycoproteins isolated from the zona pellucida were radiolabeled with radioactive iodine. The
investigators reported radioiodonated-ZP3 binding to only acrosome-intact sperm. Acrosome-reacted sperm
were devoid of the labeled glycoprotein. To further characterize this finding, a competition assay was
performed in which the binding of a fixed amount of radioiodonated-ZP3 to acrosome-intact sperm was
measured in the presence of unlabeled solubilized egg zonae pellucidae (ZP1, ZP2, and ZP3). Increasing
concentrations of the unlabeled zonae inhibited ZP3 binding. Bleil and Wassarman were thus able to
conclude that there are a limited number of binding sites on the sperm head, for which the radiolabeled
and unlabeld ZP3 compete.
The role of ZP2
Preliminary studies had suggested that ZP2 in any form lacked sperm receptor or acrosome-reaction
inducing activities. It was therefore somewhat surprising that Bleil and Wassarman achieved minimal, yet
significant, binding of radioiodinated ZP2 to acrosome-intact sperm and an impressively high level of
binding to acrosome-reacted sperm (as much as 50 times the level of ZP3). These observations suggest that
ZP2 binds very well to the inner acrosomal membrane, which is exposed following the acrosome
reaction.
The preferential binding of ZP3 to acrosome-intact sperm came as no surprise. The two investigators
showed that ZP3 prevents binding of sperm to unfertilized eggs under conditions that support fertilization in
vitro. Neither ZP1 nor ZP2 had any effect on binding, suggesting that ZP3 is the sperm receptor. Only
acrosome-intact sperm bind to eggs. It thus follows that the inhibition of binding due to ZP3 is attributable
to an interaction with the plasma membrane overlying the heads of acrosome-intact sperm. Once the
acrosome reaction has taken place resulting in the loss of the plasma membrane at the anterior portion of the
sperm head, binding of ZP3 can no longer occur as observed with the acrosome-intact sperm.
Relationship of ZP3 and ZP2
The researchers proposed that ZP2 may act as a secondary receptor, interacting with the inner acrosomal
membrane after the binding and release of ZP3. In this way its function would be to retain binding between
the sperm and egg following ZP3 dissociation. This would support the finding that once bound to the zona
pellucida, sperm that have undergone the acrosome reaction remain bound. Free-swimming, acrosome-
reacted sperm, however, can not bind to zona pellucida. If ZP2 is in fact a secondary receptor, the question
arises as to why free-swimming acrosome-reacted sperm fail in this regard. Bleil and Wassarman suggested
that the interaction between ZP2 and the acrosome-reacted sperm is too weak to bring about binding to the
egg yet sufficient in strength to maintain prior binding initiated by ZP3. However, this is subject to
experimental verification.

-9-
The big surprise is the apparent binding of ZP2 to acrosome-intact sperm at a low, but significant, level. The
prevailing view is that this binding may actually reflect binding of ZP2 to the inner acrosomal membrane of
sperm heads in the process of undergoing the acrosome reaction. The methods used by Bleil and Wassarman
were unable to detect partially reacted acrosomes.
Collaborative discovery concerning the role of ZP3 in fertilization, Wassarman later showed the need for a
ZP3 in the initiation of the acrosome reaction, thus establishing a second function for this glycoprotein.
Further research is required to identify the role of the ZP1 in the fertilization process as well as to explain
the unexpected ZP2 binding results reported by Bleil and Wassarman in 1986.
Activating the Mammalian Acrosome Reaction
The idea that ZP3 can induce the acrosome reaction after the sperm bound to it was not easily accepted. The
picture of fertilization had been that the acrosome reaction should occur prior to binding (as it does in sea
urchins) and that the mammalian sperm would use the enzymes from the acrosome to lyse its way through
the cumulus. However, Endo and colleagues
(1987a,b) and Leyton and Saling (1989a)
showed that the protein part of ZP3 was
capable of inducing the acrosome reaction
on mouse sperm. The carbohydrate region
(which is essential for binding the sperm) is
not needed to induce the acrosome reaction.
Fig. Evidence that the cross linking of sperm
receptors for ZP3 is necessary to initiate the
acrosome reaction of mouse sperm. Fragments of
ZP3 can bind to the sperm, but they will not induce
the acrosome reaction until they are cross linked.

Leyton and Saling (1989b) then did an ingenious experiment to show that ZP3 induced the acrosome
reaction by aggregating the sperm membrane receptors for it. They capacitated mouse sperm and incubated
them with fragments of ZP3 that could bind to the sperm. Although these fragments of ZP3 were bound, the
acrosome reaction did not take place. However, when these fragments were crosslinked with antibodies
against ZP3, the acrosome exocytosis occured (Figure 1). Therefore, it appears that ZP3 induces the
acrosome reaction by crosslinking its receptors.
The use of crosslinking to induce exocytosis is not unique to the sperm acrosome. Perhaps the most well
studied case concerns the degranulation of histamine granules in our bodies' mast cells that is responsible for
allergic reactions. Here, the antigen (allergen) cossslinks the IgE antibodies in the mast cell membrane. This
initiates the reactions by which the intracellular calcium is increased and exocytosis of the granules occurs
(Benhamou et al., 1990).
Two events appear to be critical in the acrosome reaction. First, the amount of calcium ions within the sperm
increases dramatically; second, the membrane enzyme protein kinase-C (PK-C, which can be stimulated
through the G protein-PLC-b or receptor tyrosine kinase pathways) is activated. These appear to be critical
in exocytosis reactions throughout the animal kingdom (Storey, 1991). Several studies (Endo et al., 1987a,b;
Glassner et al., 1991; Ward et al., 1992; Walensky and Snyder, 1995) have provided strong evidence
implicating IP3 in the acrosome reaction. They have localized all the components of the IP3 pathway (the G
proteins, phospholipase C, and IP3 receptors) to the acrosomal cap of the sperm in several mammalian
species (Figure). Moreover, their inhibitor studies suggest that IP3-gated
calcium release is critical in the acrosome reaction.
Figure. Localization of the IP3 receptor in the acrosomes of mammalian sperm. Sperm are
visualized by Coomassie Blue dye and are outlined by arrowheads. The presence of the IP 3
receptor is shown by fluorescent antibody labeling below each of the sperm. (After Walensky
and Snyder, 1995).

- 10 -
In a field so full of controversies as that of mammalian fertilization, there is no consensus as to which ZP3-
binding protein is aggregated to start this reaction. Shur's laboratory has shown that ZP3 binds to and
aggregates the galactosyltransferases and that this binding activates a G-protein complex on the cytoplasmic
side of the sperm cell membrane (Gong et al., 1995). Saling's laboratory (Burks et al., 1995) has shown that
ZP3 binds to the ZRK and activates the kinase activity on that glycoprotein. Perhaps the tyrosine kinase and
the G-protein are both needed to induce the acrosome reaction. This would suggest that neither pathway is
sufficient and that the combined products of both pathways are critical for generating the acrosome
response. It is also possible that both the G-protein-mediated receptors and the phosphorylated tyrosine
kinases activate the phopholipase C enzymes that generate IP3. So while the pathways within the sperm
leading to the acrosome's exocytosis are accounted for, the initial receptor reaction is still controversial.
5. Pronuclear Fusion
Within the cytoplasm of the newly formed zygote, the centriole near the female pronucleus generates an
array of microtubules. Internalization of components of the sperm is accomplished using a myosin molecular
motor and the microtubules of the acrosomal process. The haploid male pronucleus and the centriole and
some cytoplasm from the spermatozoan are drawn into the cytoplasm of the egg. The nucleus of the
spermatozoan swells and becomes the male pronucleus and its centriole generates an array of microtubules.
Female and male pronuclei fuse following their migration towards each along their respective microtubular
arrays. Either the female pronucleus migrates to the male or the male pronucleus migrates to the female and
resulting fusion of the membranes of these pronuclei produces the diploid zygote nucleus (Hinchcliffe et al.
1999). The chromosomes from these nuclei have already undergone pre-meiotic S-phase during which they
replicated their DNA. Mitotic cleavage divisions follows and generate the blastula.
Sea urchins have been a convenient system for studying fertilization and egg activation for a century. In fact,
many of the key discoveries regarding species-specific sperm-egg binding and the signals that mediate the
blocks to polypsermy and egg activation were made first in this system. The next few pages discuss some of
the major events and mechanisms of fertilization in sea urchins.

- 11 -