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OCR for page 262
MOLECULAR EVENTS PRE- AND POST-FERTILIZATION OF MOUSE EGGS: OOCYTE
MATURATION, EGG ACTIVATION, AND POLYSPERMY BLOCK
R. M. Schultz,. S. Kurasawa, Y. Endo, and G. S. Kopf
INTRODUCTION
Prior to the fusion of the sperm with the egg, the egg undergoes a
series of complex biological processes that allow it to mature and be
fertilized. In turn, fertilization initiates a complex series of events
teemed egg activation. One aspect of egg activation results in cell
cleavage and further development. Another aspect of egg activation
prevents polyspermy, which leads to aberrant development.
This brief review will first discuss events during oogenesis and
oocyte maturation that are involved in production of a fertilizable
female gamete. Events comprising the fertilization-induced block to
polyspermy will then be addressed. The discussion will be mainly
restricted to the mouse, since this is the best characterized system for
processes involved in mammalian oocyte maturation and fertilization.
ACQUISITION OF MEIOTIC COMPETENCE
During the period of oocyte growth, which takes about 14 days,
mouse oocytes that are arrested in the first meiotic prophase grow from
about 15 Am to 80 Am in diameter. Acquisition of meiotic competence is
correlated with a specific stage of oocyte growth. In mice, oocytes
that are obtained from juvenile mice less than 15 days of age are less
than 60 Am in diameter and will not resume meiosis, i.e., undergo
meiotic maturation, when placed in a suitable culture medium (Sorensen
and Wassarman, 1976). One of the earliest morphological manifestations
of meiotic maturation is breakdown of the nuclear membrane, which is
called the germinal vesicle. Subsequent to germinal vesicle breakdown
(GVBD), a spindle forms. Separation of homologous chromosomes then
occurs, with emission of the first polar body and arrest at metaphase
II. The frequency at which growing oocytes can resume meiosis increases
with increasing diameter, which is a linear function of the age of the
donor juvenile mice (Sorensen and Wassarman, 1976).
Stage-specific differences in the spectrum of synthesized
polypeptides are correlated with acquisition of meiotic competence
(Schultz et al., 1979a). These changes are likely to underlie, in part,
the biochemical basis for the acquisition of meiotic competence.
Nucleate fragments obtained from fully-grown, meiotically competent
oocytes that are less than 60 Am in diameter undergo GVBD, emit the
first polar body and arrest at metaphase II (Balakier and Czolowska,
1 977 ; Schultz et al ., 1 97 8 ) . Thus, it is likely that the quality of the
cytoplasm, and not the amount of cytoplasm, is involved in acquisition
of meiotic competence.
Oocyte growth per se may not be involved in acquisition of meiotic
competence, since acquisition of meiotic competence can be dissociated
from oocyte growth (Canipari et al., 1984). Oocytes obtained from
juvenile mice about 15 days of age are about 6 0 em in diameter. oocytes
obtained from juvenile mice 10 days of age are less than 60 em in
— 262 —
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diameter and meiotically incompetent. When there meiotically
incompetent oocytes obtained from juvenile mice 10 days of age are
cultured for 5 days in a medium that does not support oocyte growth but
does support oocyte viability a significant fraction of the oocytes
undergo GVBD. Results from a series of similar experiments indicated
that a constant amount of combined time that totals 15 days of in Vito
growth or in vitro culture is necessary for acquisition of meiotic
con etence. To provide a tighter correlation between the changes in the
proteins synthesized during oocyte growth and the acquisition of meiotic
competence, it should be demonstrated that the changes in protein
synthesis that occur during oocyte growth also occur under the
conditions of in vi tro culture that do not sustain oocyte growth but do
foster meiotic competence.
OOCYTE MATURATION
Oocytes undergo meiotic maturation, which culminates in the
production of an egg; eggs, not oocytes, are capable of being fertilized
and giving rise to normal development. Although oocytes that have not
reached and arrested at metaphase II can be penetrated by sperm, such
oocytes do not develop normally and very quickly degenerate. The
subsequent section will discuss briefly some molecular aspects of oocyte
maturation.
1.
Role ot cAME and protein-pho~phQrylaLlQn
The follicle exerts an inhibitory influence on oocyte maturation,
since oocytes present in preovulatory ant ral follicles do not resume
meiosis, whereas liberation of these oocytes from their follicles
results in resumption and completion of meiotic maturation (Pincus and
Enzmann, 1935). A substantial body of evidence implicates cAMP in the
maintenance of meiotic arrest, although a number of molecules are likely
to participate in this process (See Schultz, 1988 and reference therein
for a more complete review of this area.).
In vitro maturation is reversibly inhibited by membrane permeable
c AMP analogs, (e.g., dibutyryl cAMP;(dbcAMP), or inhibitors of cyclic
nucleotide phosphodiesterase (PDE), (e.g., 3-isobutyl-1-methyl
xanthine;(I8MX) (Cho et al., 1974; Bornslaeger et al., 1984); the
corresponding cGMP analogs do not inhibit maturation in vitro. In
addition, treatment of oocytes with either the activator of adenylate
cyclase, forskolin, or microinjected cAMP inhibits GVBD (Schultz et
al., 1983).
The best documented mode of action of cAMP in eukaryotes is to
activate a cAMP-dependent protein kinase (protein kinase A, PK-A).
Accordingly, it was proposed that cAMP is involved in maintenance of
meiotic arrest by activating PK-A. This enzyme phosphorylates, either
directly or indirectly, a protein fs) X, which is capable of promoting
GVBD (Bornslaeger et al., 1986a). The phosphorylated form, XP, is
inactive. Resumption of meiosis in vitro is proposed to occur by a
decrease in cAMP, which would result in a decrease in PK-A activity.
Consequently, a protein phosphatase would shift the equilibrium between
XP and X to the dephosphorylated form of X and GVBD would ensue.
Consistent with this model is that microinjection of oocytes,
which are incubated in medium containing a concentration of dbcAMP that
inhibits maturation, with protein kinase inhibitor (PKI) undergo GVBD
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(Bornslaeger et al., 1986a); PKI inhibits PK-A by combining with the
free catalytic subunit (C). This result in anticipated, since
inactivation of C would result in the conversion of XP to X, and hence
maturation would resume. In addition, microinjection of oocytes,.which
are incubated in a medium that supports oocyte maturation, with the
catalytic subunit of PK-A results in inhibiting GVBD. Presumably the
excess of C..relative to regulatory subunit maker C essentially a cAMP-
independent protein kinase. C continues to phosphorylate X and to keep
it in its inactive phosphorylated form and accordingly maturation is
inhibited.
A decrease in oocyte clamp does in fact occur prior to GVBD during
maturation in vitro (Schultz et al., 1983; Vivarelli et al., 1983).
This decrease may be causally related to GVBD, since PDE inhibitors,
which inhibit GVBD, inhibit this maturation-associated decrease in
oocyte cAMP that occurs during a period of time in which the oocytes
become committed to resume meiosis (Schultz et al., 1983). Commitment
is experimentally defined an follows: After a given period of time in
culture, oocytes possessing an intact germinal vesicle are transferred
to medium containing either dbcAMP or IBMX and then scored for GVBD at
later times. If an oocyte resumes meiosis it is termed "committed".
Moreover, microinjection of oocytes, which are incubated in medium
containing a concentration of IBMX that inhibits maturation, with
purified phosphodiesterase undergo GVBD (Bornslaeger et al., 1986a) .
Presumably, even though the exogenous microinjected PDE is inhibited by
>85% by the IBMX, the excess amount of PDE activity is sufficient to
hydrolyze oocyte cAMP. This decrease in cAMP would then occur and GVBD
would ensue in the presence of IBMX.
A maturation-associated set of changes in protein phosphorylation
occurs during the commitment period (Bornslaeger et al., 1986a) and may
be causally related to GVBD. A basic phosphoprotein of Mr 60,000
undergoes an apparent dephosphorylation and a set of phosphoproteins of
Mr 24,000, 29,000, and 36,000 exhibit an apparent increase in
phosphorylation. These same changes occur in oocytes incubated in
medium containing dbcAMP and microinjected with PKI, which induces GVBD.
In addition, activators of the calcium, phospholipid- dependent protein
kinase, protein kinase C (PK-C) and antagonists of calmodulin, which
could regulate a calmodulin-modulated protein kinase, inhibit maturation
and the maturation-associated set of changes in protein phosphorylation
(Bornslaeger et al., 1986b; Bornslaeger et al., 1984). These agents do
not inhibit the maturation-a~sociated decrease in.cAMP and
microinjection of oocytes incubated in these agents with PKI does not
result in GVBD. Thus, the mode of action of there compounds may be
distal to that of cAMP.
These changes in protein phosphorylation do not occur in
meiotically incompetent oocytes, but do occur in the lO% of oocytes 60
Am in diameter that can undergo GVBD. Injection of meiotically
incompetent oocytes with an amount of PKI sufficient to induce
maturation in fully-grown oocytes does not result in GVBD in these
incompetent oocytes but does elicit the decrease in phosphorylation of
the Mr 60,000 phosphoprotein. The increase in apparent phosphorylation
of phosphoproteins of Mr 24,000, 29,000 and 36,000 is not observed. It
is likely that meiotically competent oocytes lack the ability to
phosphorylate these phosphoproteins, wince low levels of phosphorylation
of these phosphoproteins are detected. Thus, dephosphorylation of the
60,000 Mr protein is not sufficient to induce GVBD and meiotic
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incompetence may entail deficiencies in the ability of the oocytes to
execute phosphorylations that occur downstream to the dephosphorylation
of this protein (Bornslaeger et al., l9B8). Future work may provide
additional insights regarding the proteins involved in this
phonphorylation cascade.
2. .~.ti ~ ~ - Li ILL -ear
An activity central to oocyte maturation and subsequent cell
cycles is that of Maturation Promoting Factor (MPF)(Masui and Clarke,
1979 and references therein; ~ishimoto, 1988 and references therein).
MPF in more aptly described an an M phase promoting factor that is
involved in the G2 to M transition of the cell cycle (Gerhart et al.,
1984). It is directly implicated in chromosome condensation and
breakdown of the nuclear membrane and its activity oscillates during the
cell cycle. MPF activity shown no evidence for species specificity,
since MPF obtained from one species will induce nuclear membrane
breakdown when injected into cells of distantly related species (Masui
and Clarke, 1979, Kishimoto et al., 1984.;;Sorensen et al., 1985) .
Until recently, MPF has defied purification, due to its instability and
cumbersome biological assay (Gerhart et al., 1984). The development of
methods for stabilizing the activity and in vitro assays that support
nuclear membrane breakdown have led to MPF's recent purification from
Xenopus oocytes (Lohka et al., 1988). The purified protein possesses
protein kinase activity, and is itself phosphorylated; it is not known
whether phosphorylation regulates its activity. Results of experiments
using extracts that contain MPF activity suggest that phosphorylation of
MPE, which may be capable of autophosphorylation, may generate the
active form of MPF (Cyert and and Kirschner; 1988). Such a mechanism
also explains the self-amplification properties of MPF. Interestingly,
the Xenopus MPF is a CDC2 homologue (Dunphy et al., 1988; Gautier et
al., 1988). The CDC2 protein is directly involved in the yeast cell
cycle. The protein has an associated protein kinase activity and is
itself likely to be regulated by phosphorylation; the level of
phosphorylation increases as cells undergo a G2 to M transition
(Simanis, V. and Nurse, 19861. Thus, MPF may be an intrinsic component
of the cell cycle.
Mouse oocytes appear to possess an MPF-like activity. Fusion of a
meiotically competent oocyte that has undergone GVBD with a meiotically
incompetent oocyte results in breakdown of the germinal vesicle of the
incompetent oocyte (Balakier, 1978). Injection of cytoplasm from mouse
oocytes that have undergone GVBD into Xenopus (Sorensen et al., 1985) or
Asterina Pectinifera oocytes (Kish~moto et al., 1984) induces
maturation; cytoplasm of mouse oocytes inhibited from maturing does
possess MPF activity as deduced by its inability to induce GRAD in the
recipient oocytes.
Mouse oocyte MPF activity oscillates during maturation (Hash~moto
and Kish~moto, 1988), as is the care in other systems (Gerhart et al.,
19 8 4 ) . In a series of elegant experiments, cytoplasm f ram oocytes at
dif ferent stages of maturation was in jected into Asterina PectiniŁera
oocytes, which were subsequently scored for GUBD . In this manner, the
amount of MPF activity was quantified and shown to increase Subsequent
to GVBD . It reaches a peak at metapha~e I, decreases during polar body
emission, and then increases again as the oocyte arrests at metaphase
II. Inhibiting protein synthesis does not affect the initial appearance
of MPF after GVBD, which occurs in the absence of protein synthesis.
265 ~
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MPF generation does not require nuclear contents, since fusion of
anucleate fragments of fully-grown oocytes with interphase blastomeres
derived from 2-cell mouse embryo n results in almost ~ ediate chromosome
condensation in the interphase nuclei (Balakier and Czolowska, 1977) ;
the anucleate fragments were used at a time when the nucleate fragments
had undergone GVBD.
Meiotically incompetent mouse oocytes also possess an "anti-MPF"
activity. Fusion of a meiotically competent oocyte with an intact GV
with a meiotically incompetent oocyte preserves the integrity of each
nucleus, which resides in the common cytoplasm (Fulka et al., 1985).
The loss of this anti-MPF activity may be required for acquisition of
meiotic competence, and further characterization of this activity is
required.
3.
The nucleus of the fertilizing sperm is highly condensed due to
chromatin-~ssociated basic proteins called protamines, which contain
high amounts of cysteine (Yanagimachi, 19 88 and references therein).
These cysteine residues are oxidized during sperm maturation and their
reduction during fertilization is necessary for the sperm nucleus to
decondense in the egg cytoplasm and thus be transformed into the male
pronucleus.
A pivotal role for glutathione in the process of sperm nuclear
Recondensation is likely, since glutathione is the major biological
thiol reducing agent and treatment of eggs with diamide, which is an
antioxidant of glutathione, reversibly inhibits the Recondensation of
mucroinjected hamster sperm nuclei (Perreault et al., 1984). In
addition, treatment of mice in viva with an inhibitor of glutathione
synthesis, L-buthionine-S,R-sulfoximine (BSO) inhibits sperm
Recondensation of mouse sperm nuclei following in vitro fertilization -
(Calvin et al., 1986). The inability of the sperm nucleus to Recondense
is apparently due to inadequate amounts of reducing power present in the
egg, since disulfide-poor sperm nuclei Recondense when microinjected
into either GV intact oocytes or eggs (Perreault et al., 1984; Zirkin et
al., 1985).
The ability of sped nuclei to Recondense in egg cytoplasm is
dependent on the maturational state of the oocyte (Perreault et al.,
1988). Sperm mucroinjection experiments indicate that Recondensing
II arrested eggs but in barely
Correlated with these dif ferences
activity is maximal in metaphase
detectable in GV intact oocytes. ~orre~acea worn enese adherences in
these decondensing potentials is a maturation-associated increase in the
amount of glutathione. Inhibiting this maturation-associated increase
in glutathione content with BSO inhibits the ability of the matured
oocyte to Recondense microinjected sperm nuclei.
Sperm nuclei in a GV-intact oocyte will not deconden~e, whereas
sperm nuclei in an oocyte that has undergone GVBD will Recondense.
Moreover, since dithiothreitol-treated hamster sperm nuclei will
Recondense in the cytoplasm of GV-intact oocytes (Perreault, et al.,
1984), the contents of the GV may be necessary for the development of
this Recondensing activity. The interaction of the nucleoplasm with the
cytoplasm may result in generating the sperm recondensing activity by
providing the cytoplasm with (1) the activity (2) a "co-factor''
necessary for activity, (3) an activity that '"activates" the sperm-
266 -
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deconden~ing activity, or ( 4 ) an activity that inhibits a sperm-
decondensing inhibitory activity . Last, it should be noted that GVBD
apparently required for development of cytoplasmic activities that
control male and female pronuclei formation (Balakier and Tarkowski,
1980; Yanag~machi, 1988 and references therein).
FERTILIZATION AND EGG ACTIVATION
IS
Although fertilization of mouse eggs induces a small increase in
the absolute rate of protein synthesis (Schultz et al., 1979b), it
initiates a dramatic series of changes in the pattern of protein
synthesis (Schultz et al., 1979b; Cullen et al., 1980). Although the
one-cell embryo supports a low level of transcription (Clegg and Piko,
1982), transcription is not necessary for these changes in protein
synthesis, since they occur in either physically enucleated zygotes or
zygotes incubated in the presence of a-amanitin (Petzoldt et al., 1980;
Braude et al., 1979). Most of these changes are due to po~t-
tran~lational modifications of existing proteins (Van Blerkom, 1981) or
recruitment of maternal ~ A (Cascio and Wassarman, 1982); In
addition, there is a small subset of changes that appear autonomous of
fertilization and are apparently initiated by oocyte maturation (Howlett
and Bolton, 1985). Both the fertilization-induced and fertilization-
independent changes in protein synthesis are likely involved, at least
in part, in the onset of DNA synthesis in the pronuclei, cleavage to the
2-cell stage, and transition from a nonproliferative to a proliferative
state with the concomitant conversion of a meiotic cell cycle to a
mutotic one. What controls these events is not known, but perturbing
protein phosphorylation during the first cell cycle can inhibit both
cleavage to the two-cell embryo, as well as activation of transcription
of the zygotic genome, which also occurs in the two-cell embryo
(Poueymurou and Schultz, 1987). This result is consistent with protein
phosphorylation being a major type of protein modification theta occurs
during the first cell cycle (Van Blerkom, 1981).
1. Block to Polyspermy
A major response of the egg to the fertilizing sperm is the block
to polyspermy. All species apparently have mechanisms to block
polyspermy. For example, sea urchins possess a fast electrical block
that operates at the level of the plasma membrane (Jaffe and Gould, 1983
and references therein).
Although mouse eggs generate a plasma membrane block to polyspermy
(Wolf, 1978; Stewart-Savage, and Baiter, 1988 for hamster), there is
no evidence to support the existence of a fast electrical block to
polyspermy at the level of the plasma membrane (Jaffe et al., 1983).
The major mechanism for the polyspermy block in mice appears to be an
egg-induced modification of the zone pellucids (ZP). The ZP is an
extracellular coat that surrounds the oocyte and is responsible for
~pecie^-specific binding of sperm and induction of the acrosome reaction
(Wasserman, 1987, 1988 and references therein). It Should be noted,
however, that rabbits do not possess a zone block to polyspermy; their
primary block apparently resides at the plasma membrane (Yanag~machi,
1988). Moreover, dispermic mouse eggs can be restored to the
monospermic condition by spend loss due to cytoplasmic blebbing (Yu, and
Wolf, 1981); this would constitute a third line of defense against
polyspermy. The mechanism of this sperm loss is poorly understood,
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although mucrofilaments are likely involved, since the process is
inhibited by cytochalasin B (Yu, and Wolf, 1981).
The mouse egg's zone pellucida is composed of three sulfated
glycoproteins called ZP1,. ZP2,. and ZP3 (Wasserman, 1988 and references
therein that pertain to structural and functional aspects; Bleil and
Wassarman, 1980a; Shimizu et al., 1983). ZP1, which has an apparent
molecular weight of about 200,000 daltons (Bleil and Wassarman, 1980a),
is a clime r connected by intermolecular disulfide bonds and is likely to
serve as a cross-linker responsible for maintaining the three
dimensional structure of the. zone pellucida (Greve and Wa~sa~man, 1985).
ZP2 isolated from oocyte~ or unfertilized eggs has an apparent molecular
weight of 120,000 daltons, which in observed under either non-reducing
or reducing conditions of gel electrophoresis (Bleil and Wassanman,
1980a). Fertilization results in the modification of ZP2 to a form
called ZP2f. Under nonreducing conditions, ZP2f has an apparent
molecular weight of 120,000, whereas under reducing conditions it has an
apparent molecular weight of 90,000 daltons (Bleil et al., 1981). This
modification is a proteolytic cleavage that results in two fragments
held together by disulfide bonds. Since ZP2 can bind to acrosome
reacted sperm, whereas ZP2f cannot, ZP2 may mediate sperm binding
subsequent to the acrosome reaction (Bleil and Wassarman, 1986). ZP3
has an apparent molecular weight of 83,000 daltons and the O-linked
carbohydrate portion accounts for the sperm receptor activity of ZP3,
which is lost after fertilization (Bleil and Wassanman, 1980b; Florman
et al., 1984). ZP3 also possesses all of the sperm acrosome reaction-
inducing activity of the ZP, and can do so to the same extent as that
induced by the ionophore A23187 (Bleil and Wassarman, 1983).
Based on the aforementioned properties of the mouse zone and that
only acrosome-intact sperm bind to the zone -acrosome-reacted sperm do
not bind- the following sequence of events has been proposed for sperm-
zona interactions and fertilization of mouse eggs. Acrosome-intact
sperm bind to the zone pelf ucida (Saling et al., 1979a; Florman and
Storey, 1982); binding is species-specific and mediated by-ZP3. Sperm
bound to ZP3 then undergo the acrosome reaction, which is also mediated
by ZP3. ZP2 mediates the binding of acrosome-reacted sperm (Bleil and
Wassarman, 1986), which then penetrate the zone pellucida and gain
access to the perivitelline space. The acrosome-reacted sperm then bind
to and fuse with the egg's plasma membrane.
In response to fertilization, the egg undergoes the "cortical
granule reaction" (Szollsi, 1967; Barros and Yanag~machi, 1971, 1972;
Wolf and Hamada, 1977). Cortical granules subjacent to the plasma
membrane are thought to fuse with the plasma membrane and release into
the perivitelline space enzymes that convert ZP2 to ZP2f and modify ZP3
such that it no longer possesses either sperm receptor activity or the
ability to induce an acrosome reaction. Thus, acrosome-intact sperm can
no longer bind to the zone and acrosome-reacted sperm bound to the zone
can no longer interact with and penetrate the zone, since these sperm do
not interact with ZP2f (Bleil and Wassarman, 1986). This series of
events is proposed to comprise the zone reaction or the zone block to
polyspermy. A membrane block to polyspermy, however, also develops with
time.
Although much is known about the cortical granule reaction in
lower species and how the contents of the granules modify the
extracellular coats surrounding eggs in these species, little is known
258 -
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about the mammalian cortical granule reaction (Gulyas, 1980). This is
in large part due to the difficulty in obtaining large amounts of
biological material from mammalian species. In turn, this has prevented
generation of molecular markers for mammalian cortical granules.
Moreover, until recently, the only way to assess accurately the status
and distribution of cortical granules was by electron microscopy, which
is a tome consuming process and difficult to quantify easily.
Polyphosphatidylinositol turnover, which has been implicated as a
response of the egg to fertilization in lower species (Turner et al.,
1984, 1986; Whitaker and I ovine, 1984; Swann and Whitaker, 1986),
generates two second messengers, i.e., an 1, 2-diacylglycerol, which
activates the calcium- and phospholipid-dependent protein kinase,
protein kinase C (PK-C), and inositol-1,4, 5-tri~phosphate (IP3) , which
releases calcium from intracellular stores (Berridge, 1984; Berridge and
Irvine, 1984) . Protein pho~phorylation(~) catalyzed by PK-C is
implicated in regulating exocytotic processes (Nishizuka, 1984; Takai et
al., 1984). Since the cortical granule reaction in mammalian eggs
involves an exocytotic process, the role of mouse egg PK-C in the early
events of the fertilization profess was examined (Endo et al., 1987
b, c ) .
Treatment of eggs with biologically active phorbol diesters or a
diacylglycerol, compounds that activate PK-C, inhibits both sperm
penetration and fertilization (Endo et al., 1987c). Biologically
inactive phorbol diesters, which do not activate PK-C, do not inhibit
either sperm penetration or fertilization. This inhibition is due to an
egg-induced modification of the zone pelf ucida, such that ZP2 is
converted to ZP2f, while ZP3 retains its sperm receptor activity. This
latter observation accounts for the finding that sperm binding is not
reduced in eggs treated with PK-C activators. The inhibition of
fertilization is due to the inability of ZP3 to induce a complete
acrosome reaction, which was determined by using an assay that monitors
of the progression of the acrosome reaction.
The progression of capacitated, acrosome-intact sperm to acrosome-
reacted sperm can be monitored by changes in staining patterns using-the
fluorescent probe chlortetracycline (Saling and Storey, 1979b). Three
major fluorescent staining patterns have been characterized with this
assay. The B-pattern of capacitated sperm is correlated with acrosome
intact sperm, as assessed by transmission electron microscopy (Flonman
and Storey, 1982). The S-pattern represents an intermediate stage and
appears prior to completion of the acrosome reaction. This pattern
correlates with loss of the ability of sperm to maintain a transmembrane
pH gradient (Lee and Storey, 1985). The AR-pattern corresponds to
acrosome-reacted sperm, as determined by transmission electron
microscopy (Saling et al., 1979b).
ZP3 isolated from untreated eggs possesses the ability to induce
the B to S to AR transitions. In contrast, ZP3 isolated from eggs
treated with PK-C activators can induce the B to S transition, but not
the S to AR transition. Accordingly, sperm treated with ZP3 isolated
from these eggs treated with PK-C activators accumulate in the S
pattern. Although previous studies indicated that ZP3 isolated from 2-
cell embryos does not induce the acrosome reaction, the methods used in
these studies assayed an end point, i.ee, the completion of the acrosome
reaction, and would not detect intermediates in this process (Bleil and
Wassarman, 1983). When tested with the chlortetracycline assay, sperm
269 -
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incubated with ZP3 isolated from 2-cell embryos do not even undergo the
B to S transition.
Sperm arrested in the S pattern by zonae isolated from PK-C
activator-treated eggs can be induced to undergo the S to AR transition
by treatment with either ionophore A 23187 or solubilized zonae from
untreated eggs; solubilized zonae from 2-cell embryos do not induce this
transition (Kligman, Storey, and Kopf, unpublished results). Thus, the
S pattern in which the sperm accumulate by treatment with ZP3 isolated
from PK-C activator-treated eggs represents an intermediate stage of the
acro~ome reaction that can be induced to undergo subsequent steps and
complete the acrosome reaction. ~
i , ~ .
These studies demonstrate that treatment of eggs- with activators
of protein kinase C results in an egg-induced modification of the zone
such that there is a dissociation of the spe,`~-receptor activity from
the acro~ome reaction-inducing activity of- ZP3. In contrast,
fertilization results in the loss of both-the sperm receptor and
acrosome reaction-inducing activities of ZP3. Presumably, differences
in ZP3 obtained from untreated and phorbol diester-treated eggs reflect
those portions of the molecules that participate in the acrosome
reaction. Studies examining biochemical differences in ZP3 obtained
from untreated and phorbol diester-treated eggs, therefore, should.
facilitate analysis of those portions of ZP3 that are involved in
inducing the acrosome reaction. The use of zonae from phorbol diester-
t rested eggs should also facilitate studies on the mechanism(s) of the
acrosome reaction, since it is now possible to use these zonae to study
independently the B to S transition from the S to AR transition. Such
an experimental system may be of great value in determining the
biochemical correlates of the S pattern, which has characteristics of an
intermediate stage prior to the completion- of the acrosome reaction.
The effects of IP3 microinjected into mouse eggs have also been
examined with respect to its effect on egg activation, zone
modifications, and sperm receptor activities of the zone (unpublished
results ) . A low percentage ( 15% ) of eggs microin jected with IP3 at a
final concentration of 4 AM become activated, as evidenced by second
polar body emission within 1.5 h. In contrast, -eggs injected with the
vehicle do not activate. Zonae from the IP3-microinjected eggs that
activate always show the loss of BP2 and its conversion to ZP2f.
Although IP3-injected eggs do not activate, 85% of these eggs reveal a
conversion of ZP2 to ZP2f. In about 70% of these cases the conversion
is total and in the other 30% about 50% of the ZE2 is modified. Of the
vehicle injected eggs, about 30% reveal a modification of ZP2, which is
usually only partially modified.
The half-maximal concentration of IP3 necessary to elicit the
change in ZP3 is about 5 no, and this corresponds well to that necessary
to induce the cortical granule-snediated elevation of the fertilization
envelope in sea urchin eggs, as well as calcium release f ram
intracellular stores in other systems (Whitaker and Irvine, 1984; Swann
and Whitaker, 198 6) . Extracellular calcium is not required for mouse
eggs injected with IP3 to display the ZP2 modification, and this is
consistent with release of calcium from intracellular stores in the egg.
Microin jection of either I(1,4)P2, I(2,4,5)P3, or I(1,3,4)P3, each of
which does not release intracellular calcium, to a final concentration
of 4 AM fails to induce a modification of ZP2. Moreover, microinjection
of inositol 1, 3, 4,5-tetrakisphosphate, which is implicated in regulating
270 -
OCR for page 271
calcium channels in the plasma membrane of eggs of lower species (Irvine
and Moor, 1986, 1987),.does.not induce a conversion of ZP2 to ZP2f.
IP3 in jection of mouse eggs may also modify ZP3, since IP3-
in jected eggs bind fewer sperm than vehicle in jected eggs . The lower
extent of binding could represent the inability of acrosome-reacted
bound sperm to establish a secondary binding with ZP2, for the following
reason: If ZP2 mediates the binding of acrosome-reacted sperm and ZP2f
cannot interact with acrosome-reacted sperm, then although the sperm can
bind to IP3-injected eggs and undergo the acrosome reaction, they cannot
establish the secondary binding with ZP2, which has been modif fed to
ZP2f. Thus, the bound sperm will dissociate. This explanation for the
lower level of binding is made less likely since the sperm used in these
experiments are treated with pertussis toxin, which prevents the spear`
from undergoing the acrosome reaction (Endo et al., 1987a). Thus, their
interaction with ZP2 is prevented and their interaction with the zone is
restricted to ZP3. The lower level of sperm binding to IP3-injected
eggs is therefore likely to be due to a reduced level of ZP3 sperm
receptor activity. -
Fertilization is associated with a characteristic set of changes
in the pattern of protein synthesis. Although IP3 injected eggs display
a modification in ZP2, they do not reveal the changes in the pattern of
protein synthesis associated with fertilization. This is consistent
with the very low level of egg activation, as assessed by pronuclear
formation. Thus, although IP3 can apparently bring about an egg-induced
modification of the zone, it does not elicit a full egg activation
response, but rather a subprogram of events that occurs during egg
activation.
The mechanism of IP3-induced modifications of the zone is likely
to be via an IP3-stimulated release of intracellular calcium, which
somehow is involved in cortical granule exocytosis. Periodic increases
in intracellular free calcium concentration occur following
fertilization in hamster eggs (Miyazaki et al., 1986)~. Injection of IP3
to a final concentration of 80 nM induces a transient increase in
intracellular free calcium that spreads over the entire egg within a
second (Miyazaki, 1988). In addition, IP3 also stimulates a
hyperpolarization of the membrane potential. These changes are very
similar to those that normally occur after fertilization.
G proteins are a family of guanine nucleotide binding proteins
that are activated by GTP and serve to couple various extracellular
signals to their intercellular effectors, which can be involved in
generation of second messengers (Oilman, 1987 and references therein;
Neer and Clapham, 1988, and references therein). Generation of an 1,2-
diacylglycerol and IP3 is believed to be mediated by a G protein
stimulated phospholipase C, and in sea urchins, fertilization is .
correlated with a rapid turnover of phosphatidylinositol bisphosphate
(Turner et al. , 1984). In addition, activation of G.proteins is
believed to result in cortical granule exocytosis in sea.urchins (Turner
et al., 1986).
If mammalian sperm initiate a signal transduction sequence that is
mediated by-a G protein, a cascade of fertilization-induced events
should be triggered by microinjected GTP. Consistent with this
hypothesis is the observation that microinjection of GTPgS into hamster
- 271 ~
OCR for page 272
oocytes also triggers transient increases in intracellular free calcium
and hyperpolarization of the membrane potential, and GDPbS inhibited
this GTPgS-induced response (Myasaki, 1988).
Although these data are consistent with a fertilization-induced, G
protein coupled, PK-C/IP3 mediated stimulation of a cortical granule
reaction, which in turn effects the modifications of the zone that
constitute the zone block to polyspermy, experiments have not yet
correlated the biological and biochemical changes with changes in the
number of cortical granules in response to there agents. [ens culinaris
agglutinin is a lectin that apparently stains cortical granules, and
accordingly provides a convenient marker to monitor the cortical granule
reaction (Cherr et al., 1988). Other lectins may also provide potential
markers for cortical granules (Lee et al., 1988). Results of recent
experiments indicate that fertilized or ionophore-activated eggs have
dramatically reduced numbers of there lectin staining granules
(Ducibella, personal communication). Moreover, eggs treated with PK-C
activators have a partially reduced number of these granules (Ducibella,
Kopf, and Schultz, unpublished observations).
Future studies are required to ascertain effects of IP3 and GTP on
the release of these granules, as well BS characterizing the cortical
granules with respect to their enzymatic contents. The development of
micro-assay procedures will facilitate these studies, which may reveal
the types of enzymes involved in the modification(s) of both ZP2 and
ZP3. In addition, the development of cortical granule probes may be
used to study cortical granule biogenesis and to reveal if there is a
heterogeneity in the cortical granule population. Electron microscopy
studies reveal the existence of light and dark staining populations of
cortical granules in mouse eggs (Nicosia et al., 1977). This may
reflect granules at different stages of packaging their internal
contents, or could ref. lect heterogeneity of mature granules . Such
heterogeneity exists in lower species. For example, in sea urchins,
although the cortical granules appear homogeneous in the transmission
electron microscope, only about 20% of them contain a cortical granule
antigen, as determined by immunoelectronmicroscopy (Anstrom et al.,
1988). The partial modification of ZP3 coupled with the partial
reduction in the number of LCA-staining granules in response to PK-C
activators is consistent with cortical granule heterogeneity in
mammalian eggs, but further studies are clearly required to support this
conjecture.
UNRESOLVED QUESTIONS AND FUTURE DIRECTIONS
Little is known regarding the molecular basis for the acquisition
of meiotic competence. In the future, subtraction hybridization of cDNA
libraries generated from meiotically competent and incompetent oocytes
may allow the cloning of cDNAs specific to meiotically competent
oocytes. Analysis of such clones may provide insights regarding how
oocytes develop and differentiate. This may be of extreme importance,
since an understanding of factors involved in acquisition of meiotic
competence may lead to improved systems that support oocyte growth and
acquisition of meiotic competence in vitro.
Although protein phosphorylation is implicated in regulating
Asiatic maturation, we know very little concerning the sequence of
events that comprise meiotic maturation and the proteins involved in
thin process. Meiotic maturation entails a G2 to M transition in the
279 -
OCR for page 273
cell cycle, and therefore is a problem in cell cycle regulation.
Specific cellular oncogenes, some of which are protein kineses, are
Implicated in cell cycle regulation in other systems. Interestingly,
recent studies show changes in the temporal patterns of expression of
specific oncogenes, e.g., c-mos, which is a serine/threonine protein
kinase, during oocyte growth and maturation (Propst et al., 1988).
Future studies addressing functional aspects of these gene products,
coupled with the recent purification and identification of maturation
promoting factor as a homolog of the fission yeast cell cycle control
protein encoded by the cdc2+ gene will undoubtedly shed light on the
process of meiotic maturation at the molecular level and define more
clearly the role of protein phosphorylation in regulating this process.
Again, an understanding of the molecular mechanisms of the process of
meiotic maturation may lead to Improvements in culture systems that
support maturation in vitro.
Not all oocytes that mature into eggs during maturation are
capable of being fertilized and giving rise to normal development. This
type of maturation is teemed 'tcytopla~mic" maturation. An example of
cytoplasmic maturation was discussed above, namely, the acquired ability
of the egg during maturation to Recondense the sperm nucleus. We still
do not understand at the molecular level what constitutes "cytoplasmic"
maturation and its regulation. A major problem in medically assisted
conception is to identify eggs that are capable of being fertilized and
giving rise to normal development. Although matured eggs may appear
similar on morphological grounds, they are likely to possess profound
differences with respect to their state of cytoplasmic maturation.
These differences may compromise their ability to be fertilized and
develop.
The sequence of events from sperm fusion with the egg's plasma
membrane to egg activation and the zone polyspermy block is still not
well defined. Although it is likely that this process is mediated by a
G protein(s), the biochemical nature of the sperm receptor on the egg's
plasma membrane, the molecular identity of the G protein(s) involved,
and the coupling mechanism of the putative receptor with the G
protein(s) and the consequence of this interaction are still unknown.
For example, does fertilization of mammalian eggs result in activation
of a phospholipase C with the subsequent production of diacylglycerol
and IP3? Analysis at the biochemical and molecular levels should
provide basic and essential information regarding these issues. Such an
understanding may lead to refinement of conditions that foster
fertilization.
The molecular basis for the mammalian cortical granule reaction in
response to fertilization is still unknown. Although studier in lower
species and initial studies in the mouse and hamster implicate calcium,
much more work is required to understand this exocytotic event at the
molecular level. The biochemical composition of mammalian cortical
granules is still undefined and how the contents of these granules
modify the zone to elicit the zone block to polyspe,~`y remains to be
determined. Moreover, the actual biochemical changes that occur in the
zone proteins following egg activation needs to be elucidated. Work
directed at isolating and characterizing mammalian cortical granules
will help resolve many of these issues. In addition, studies that focus
on first determining the biochemical identity of determinants on ZP3 and
ZP2 that are involved in sperm binding and the acrosome reaction will
pave the way for subsequent studies that address the nature of the
OCR for page 274
changes in these proteins that result in loss of these biological
activities associated with these zone proteins. Results of such studies
should establish the biochemical basis for the block to polyspenmy.
Such knowledge may result in in vitro fertilization culture systems in
which the incidence of polyspenmy is further reduced.
ACKNOWLEDGMENTS
Research performed by the authors was supported by grants from the-
National Institutes of Health (HD 180604 to R.M.S. and 19096 to G.S.K.
and HD 22732 to G.S.K. and R.M.S.) and grants from the Mellon Foundation
(G.S.K.) and the University Research Fund (R.M.S.). S.K. was supported
by the Rockefeller Foundation. G.S.K. and R.M.S. would like to thank
Philip Hugo for assistance with some of the experiments described above
and Jeff Bleil for stimulating discussions about the role of ZP2 in
sperm binding.
- 274
OCR for page 275
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Representative terms from entire chapter:
cortical granule
