3
The Regulation of Oocyte Maturation
Ekaterina Voronina and Gary M. Wessel*
Department of Molecular and Cell Biology and Biochemistry,
Brown University, 69 Brown St, Providence, RI 02912, USA
I. Concept of Oocyte Maturation
II. Life and Death of an Oocyte
A. Oocyte Origins
B. Oocyte Development
C. Transformations of the Oocyte During Meiotic Maturation
D. Death
III. Maturation Promoting Factor
IV. Initiation of Oocyte Maturation
A. Maturation-inducing Substance (MIS)
B. Receptors for Maturation-inducing Substance
C. Follicle Cells and Gap Junctions
D. A Whole New Can of Worms
V. Cytoplasmic Control of Maturation
A. Signal Transduction through G-proteins: Ignition or Parking Brake?
B. Calcium Ions
C. cAMP
D. PI3K
E. MAPK
VI. Concluding Remarks
Acknowledgments
References
Abbreviations
1-MA
AC
cADPr
CaMKII
DHP
ER
ES cells
FF-MAS
FSH
GPCR
1-methyladenine
adenylate cyclase
cyclic ADP-ribose
calcium/calmodulin-dependent kinase
17,20b-dihydro-4-pregnen-3-one
endoplasmic reticulum
embryonic stem cells
follicular fluid meiosis-activating sterol
follicle-stimulating hormone
G-protein coupled receptor
*To whom correspondence should be addressed. Tel.: (401) 863-1051; Fax: (401) 863-1182;
E-mail: [email protected]
Current Topics in Developmental Biology, Vol. 58
Copyright ß 2003, Elsevier, Inc. All rights reserved.
0070-2153/03 $35.00
53
54
GV
GVBD
hCG
IBMX
IP3
IP3R
LH
MI
MII
MAPK
MIS
MPF
mPR
MSP
nAR
nPR
PDE
PDK1
PGC
PI3K
PKA
PKB
PKC
RPTK
RyR
SDF1
THP
Voronina and Wessel
germinal vesicle (nucleus of the oocyte)
germinal vesicle breakdown
human chorionic gonadotropin (LH activity)
3-isobutyl-1-methylxantine
inositol 1,4,5-triphosphate
inositol 1,4,5-triphosphate receptor
luteinizing hormone
meiotic metaphase I
meiotic metaphase II
mitogen-activated protein kinase
maturation-inducing substance
maturation promoting factor
membrane progesterone/progestin receptor
major sperm protein of C. elegans
nuclear androgen receptor
nuclear progesterone receptor
phosphodiesterase
phosphoinositide-dependent kinase 1
primordial germ cell
phosphatidylinositol 3-kinase
protein kinase A
protein kinase B
protein kinase C
receptor protein tyrosine kinase
ryanodine receptor
stromal cell-derived factor 1
17,20b,21-trihydro-4-pregnen-3-one
I. Concept of Oocyte Maturation
Fertilization is required for sexual reproduction, and the cells participating
in this process are highly specialized. Both types of metazoan gametes—
oocytes and sperm—arise from germ cells, undergo extensive differentiation,
and eventually unite. Growth and maturation of both male and female
gametes culminates in production of fertilization-competent eggs and sperm
(Masui and Clarke, 1979). The acquisition of fertilization competence of the
oocyte, the focus of this review, encompasses a number of critical events.
For example, the oocyte must construct and store excessive numbers of
Golgi, mitochondria, and ribosomes, make specific sperm receptors, and in
oviparous animals the cell must accumulate nutritional reserves sufficient
for embryonic development. The oocyte must also prepare mechanisms
ensuring productive fertilization since aberrations in this process result in
early death of the embryo. For instance, supernumerary sperm entering
the egg bring along extra genetic material and centrosomes resulting
in abnormal mitosis and death of the zygote. To avoid these harmful
3. The Regulation of Oocyte Maturation
55
consequences, the eggs have evolved several strategies, the prevailing theme
of which is modification of their extracellular matrix upon fertilization to
make themselves nonreceptive to additional sperm. Establishment of the
permanent block to polyspermy employs exocytosis of specialized dense
core secretory vesicles called cortical granules upon fertilization. Other
strategies include a fast but transient membrane potential change in some
marine animals that blocks additional sperm–egg fusion, and degeneration
of supernumerary sperm in physiologically polyspermic yolky eggs, such as
in birds.
The final development of fertilization competence of an oocyte—
responsible for making it a fertilizable egg—usually occurs during the
process of meiosis, and is referred to as oocyte maturation1. Maturation is
often segregated into nuclear and cytoplasmic events to delineate specific
mechanisms and functions. Cytoplasmic maturation involves the cytoplasmic changes required to prepare the cell for fertilization, activation, and
embryo development. In various model organisms, it includes acquisition by
the female germ cell of competence to fuse with sperm, decondense sperm
chromatin, form pronuclei, and prevent polyspermy. Nuclear maturation
refers to the meiotic process of chromosomal reduction to a haploid content,
so as to produce a diploid organism upon fusion with sperm. Although
oocytes may be fertilized, such gamete fusions are usually nonproductive
(Longo and Schuetz, 1982 and references therein).
While sperm have completed their meiotic divisions prior to fertilization
in every species studied, eggs are diverse in this respect. The competence to
be fertilized and start zygotic development, which defines the egg, develops
at different stages of meiotic division in different animal species.
Fertilization can occur at the prophase stage of meiosis (clams, marine
worms), at metaphase I (MI; some insects, starfish), or at metaphase II
(MII; most mammals) of meiosis. The changes induced by insemination are
commonly referred to as egg activation. The conversion of G2-arrested
oocytes into actively dividing zygotes involves two major processes:
maturation and activation. Notably, sea urchins belong to a limited group
of organisms, wherein an oocyte completes meiotic maturation forming a
haploid cell before it becomes fertilizable. Therefore, the processes of
maturation and activation are fully separate in this animal in contrast to
many others (such as starfish, frog, or mouse).
Understanding how an immature oocyte transforms into an egg during
oocyte maturation is critical for our knowledge of fertility and reproduction.
The topic of oocyte maturation has been reviewed recently from multiple
1
In this review, the term ‘‘maturation’’ is used to define the completion of meiosis by the
oocytes, and production of a fertilizable egg. This is in contrast to cases where the word
‘‘maturation’’ refers to the entire process of oocyte development, or oogenesis.
56
Voronina and Wessel
perspectives (Kishimoto, 1999; Matova and Cooley, 2001). This review
integrates these perspectives and emphasizes the regulation of oocyte
maturation by cell signaling networks.
II. Life and Death of an Oocyte
A. Oocyte Origins
Gametes develop from primordial germ cells (PGC) that are set aside during
early embryogenesis. The development of the egg beginning from the
formation of germ cell precursors is termed oogenesis. The driving factor for
specification of cells as PGCs in a variety of animals appears to be
inheritance of a defined area of egg cytoplasm, called germ plasm,
containing a set of localized determinants. However, it is clear that this
strategy is not universal, as in certain groups of organisms including
mammals and sea urchins PGCs appear to be induced de novo from other
cells in the gastrulating embryo (reviewed in Matova and Cooley, 2001;
McLaren, 2003; Raz, 2002; Wylie, 1999). Furthermore, ascidian embryos
appear to have an ability to regenerate germ cells following removal of
PGCs (Takamura et al., 2002). In animals that utilize localized cytoplasmic
determinants, germ plasm can be identified morphologically by the presence
of specialized organelles, which are collectively called germ granules, or
specifically known as P granules in C. elegans, polar granules in Drosophila,
and germinal granules in Xenopus. Determination of primordial germ cells
depends on localized RNAs and proteins, especially those comprising the
germ granules. Oskar, vasa, and tudor proteins were first identified in
Drosophila as components of polar granules and regulators of their
formation. At least one of them, vasa, appears to have a universal role and is
found consistently in the germ cells of a wide variety of animals (reviewed in
Matova and Cooley, 2001; Raz, 2002; Takamura et al., 2002).
In animals that rely on inductive events for primordial germ cell
determination, such as mouse, key regulators of this process appear to
include the newly-identified interferon-inducible transmembrane protein
known as fragilis, and a nuclear protein of unknown function termed stella
(Saitou et al., 2002). Coordinate with the upregulation of stella, fragilis,
DAZ-like, Oct-4, and (later) vasa in primordial germ cells is a
downregulation of certain homeobox genes (HoxB1, HoxA1, Lim1, and
Evx1) and signaling molecules like Smad1 (McLaren, 2003). Investigators
are currently attempting to determine the pathways and targets for each of
these candidates by using targeted gene manipulation, microarray analysis,
and other techniques.
3. The Regulation of Oocyte Maturation
57
The difference between the major mechanisms for establishing the germ
line in animals (i.e., cytoplasmic localized determinants vs induction) is
extreme even in closely related animals (e.g., frogs and newts). Johnson et al.
(2003) have proposed that the regulative mode of germ cell specification is
the primitive form, from which predetermined development by localized
determinants has evolved. With this interpretation it is also easier to
understand how multiple independent mechanisms may have evolved in
various animal lineages (McLaren, 2003).
A hallmark of germ cells is an extensive migration from the place of their
formation to the developing gonad, combined with elimination of
mistargeted cells elsewhere. This migration is regulated by somatic-germ
cell interactions, and several molecular participants of this process have
been recently identified, which appear to be distinct between different
groups of organisms. For example, in Drosophila, enzymes implicated in
lipid metabolism, wunen and columbus, affect PGC migration by generating
repulsive and attractive signals, respectively (reviewed in Matova
and Cooley, 2001). Two wunen genes are homologues of mammalian
phospholipid phosphatase type 2; they encode transmembrane proteins,
whose catalytic activity is required for generation of the repellant effect
(Starz-Gaiano et al., 2001). Columbus (or Hmgcr) is a homologue of
3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase that
generates the attractant for germ cells by an unidentified mechanism.
Additional genes required for germ cell migration in Drosophila act by
uncharacterized mechanisms and include slam (Stein et al., 2002) and
scattershot (Coffman et al., 2002). In mouse, the signaling mediated by c-kit/
Steel Factor is particularly important for germ cell migration, with
additional input from integrin b2 and Fgf8 (reviewed in Matova and
Cooley, 2001; McLaren, 2003). Finally, in zebrafish PGC guidance is
regulated through seven transmembrane G-protein coupled receptor
CXCR4 [- (CXC-) chemokine receptor type 4] expressed in the PGCs
and its ligand SDF-1 chemokine (stromal cell-derived factor 1) expressed
along the path of PGC migration. These molecules establish a system for
the guidance of fish PGCs throughout their entire migration (Doitsidou
et al., 2002; Knaut et al., 2003; Kunwar and Lehmann, 2003). Once formed,
the identity and developmental potential of the germline is maintained
throughout the animal’s lifetime.
B. Oocyte Development
Once the gonad is assembled from somatic and germ cells, populating germ
cells differentiate and proliferate. Initially, PGCs have the potential to begin
either spermatogenesis or oogenesis and this decision is directed by the
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Voronina and Wessel
gonadal environment. In a female genital ridge, or in a nongonadal
environment, PGCs differentiate as oocytes, while male gonadal somatic
cells direct PGCs to a spermatogenic fate (reviewed in Adams and McLaren,
2002). In many organisms, oogonia divide several times in the gonad to
form clusters of interconnected cells, often referred to as cysts. The
cytoplasmic bridges left after each division form the connections between
cells in such clusters allowing for continuous communication between cells
and coordinated development. Alternatively, when the interconnected cells
form ‘‘oocyte-nurse cell’’ complexes they are able to more effectively utilize
biosynthetic capabilities of multiple cells to produce maternal stores of
important macromolecules in the resulting egg. The best documented
example of the syncytial development of oogonia is in Drosophila. In this
animal, a single cyst consists of 16 cells, one of which differentiates into an
oocyte, while the remaining 15 assume the role of nurse cells that support
the oocyte’s growth and differentiation throughout the rest of the oocyte’s
growth period. Interconnected cells are also found in C. elegans, Xenopus,
zebrafish, and different mammals (reviewed in Matova and Cooley, 2001).
In contrast to Drosophila, these cysts eventually break down, such that every
cell in an individual cyst of vertebrate animals has the potential to become
an oocyte.
Proliferating germ cells are referred to as oogonia or spermatogonia, and
are pluripotent stem cells normally restricted to germ line differentiation
(Matova and Cooley, 2001). Oogonia differentiate into primary oocytes
when they begin meiosis by replicating their DNA, and arresting in
prophase of first meiosis. In the ovary, transition from a mitotic to a meiotic
program is regulated by the signals from somatic cells (Chuma and
Nakatsuji, 2001; Seydoux and Schedl, 2001). The prophase oocyte then may
spend various periods of time in this state. In the human female for example,
primary oocytes are arrested from 12 to 50 years, while in frog the arrest
lasts for 3 years, and most echinoderm primary oocytes are arrested for up
to a year depending on the species. Somatic cells of the gonad then stimulate
the primary oocyte to continue development. In most animals, the growing
oocyte contains an enlarged nucleus, called a germinal vesicle (GV), and is
active in transcribing an extensive array of genes whose products are
necessary for oocyte development and for sustaining early embryonic
development. During periods of very active RNA synthesis, especially in
animals with large oocytes, the GV contains lampbrush chromosomes, with
extended DNA loops at the sites of RNA synthesis (Smith and Richter,
1985). During oogenesis, the oocyte accumulates an extensive collection of
RNAs, proteins, and organelles, such as cortical granules, yolk vesicles,
ribosomes, and mitochondria (Wessel et al., 2001).
Female germ cells interact with the somatic gonadal cells throughout their
life. In some animals, such as mammals, somatic cells of the gonad closely
3. The Regulation of Oocyte Maturation
59
Figure 1 Schematic representation of a late mammalian follicle (after Wright et al., 2001). The
cell types comprising the follicle are shown (the oocyte and somatic granulosa cells). Also
pointed out are the oocyte’s nucleus (germinal vesicle), extracellular matrix produced by the
oocyte (zona pellucida), antrum, and the granulosar membrane of the follicle.
associate with growing oocytes to form follicles (Fig. 1), while in other
animals, such as sea cucumber, individual somatic cells establish junctions
with the oocyte surface without forming complete follicles. Follicles provide
a specialized compartment for the growth of the oocyte, accumulate and
supply nutrients for oocyte’s growth, and generate signals governing oocyte
development. Formation of the follicle in many species is essential for
oocyte growth and development of a mature egg, suggesting that somatic
cells provide both nutritional support and developmental information for
the growing gamete.
Follicle cells are intimately associated with the oocyte through numerous
interdigitating processes during the growth phase and the entire follicle is
coupled with gap junctions (reviewed in Matova and Cooley, 2001). In the
mouse ovary, gap junctions form in advanced (secondary) follicles, but are
not detected in early (primordial) follicles (Wright et al., 2001). In some
reptile species (e.g., the lizard Anolis carolinensis) cytoplasmic connections
may arise between an oocyte and follicle cells and form intercellular bridges
(Neaves, 1971). Connections between the oocyte and somatic cells allow
nutrient transfer to the developing oocyte and are essential for oocyte
growth in mammals (reviewed in Carabatsos et al., 2000; Eppig et al., 1996).
The oocyte loses direct physical contact with associated follicle cells just
prior to the onset of maturation.
Bidirectional cell–cell communications coordinate the development of
oocyte and follicle cells; these interactions take the form of autocrine,
paracrine, and endocrine regulation in addition to cytoplasmic gap
junctional contact mentioned above. The oocyte appears to be the dominant
component of the follicle, determining the overall rate of follicular
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Voronina and Wessel
development (Eppig et al., 2002; Matzuk et al., 2002). A critical role of the
oocyte was established in experiments involving the exchange of
somatic and germ cell components of the follicles at different stages of
development. Mouse oocytes dissociated from the midgrowth secondary
follicles (12 days old) were reaggregated with the somatic cells of
newborn mice and surgically implanted beneath the renal capsules of host
females. Such reimplanted oocytes dramatically accelerated follicular
development, resulting in formation of antral follicles with differentiated
follicle cells nine days after reimplantation. This was significantly faster
than normal development of antral follicles in the newborn mice, which
takes 18–24 days. Thus, the oocytes appear to provide instructive signal
that is able to direct the development of the somatic component of the
follicle. This experimental setup provides a valuable tool for dissecting the
signaling mechanisms involved in the crosstalk between the oocyte and
somatic cells of the follicle during oogenesis in mammals, and perhaps other
animals as well.
Embryonic stems cells are now known to differentiate into oocytes and
this technology may greatly enhance our ability to experiment with the germ
cell differentiation pathway. Recent work from Hubner et al. (2003) showed
that ES cells can acquire the morphology and gene expression profile
consistent with differentiation into oocytes. While the pluripotential nature
of ES cells has been appreciated for some time, the possibility of germ line
differentiation in vitro was unexpected. This result has massive technical and
ethical consequences.
Key for this observation was use of a GFP reporter system driven by a
promoter specifically active in oocytes that drives the transcription factor
Oct4. ES cells in culture could then be examined noninvasively and followed
over the culture period. The investigators found that Oct4-GFP was
expressed in a subpopulation of the ES cells after a few days in culture. In
fact, several different types of markers accumulated in the oocyte-like ES
cells. These included the zona pellucida proteins ZP2 and ZP3, the
transcription factor of germ cells Fig, a TGF-b growth factor GDF-9, and
some markers of meiosis (DCM1, SCP3). Each of the markers is consistent
with the ES cell becoming an oocyte, and in concert, the population of
markers tested give strong support to an oocyte differentiation pathway.
Coincident with this marker expression was the morphological transitions
the oocyte-like cells underwent. This includes recruiting other ES cells into a
follicle-like structure (able to express several markers of estrogen
biosynthesis), displacement of the oocyte from the recruited cluster that
resembled oocyte ovulation, and eventually even formation of structures
similar to embryonic blastocysts. These findings will have great potential in
dissecting mechanistic processes in oogenesis in particular, and gametogenesis in general.
3. The Regulation of Oocyte Maturation
61
C. Transformations of the Oocyte During Meiotic Maturation
1. Nuclear Changes and Chronology of Maturation
The process of oocyte maturation traditionally has been described by
changes in chromosome morphology during meiosis. Before maturation
starts, the oocyte contains a large germinal vesicle (GV) with a large
nucleolus (Fig. 2A–C). The chromosomes in the GV are mostly
decondensed, dispersed, and transcriptionally active (reviewed in Smith
and Richter, 1985; Wassarman, 1983). With the initiation of maturation,
transcription ceases, the chromosomes begin to condense, the GV breaks
down, and nucleoli disperse (Masui and Clarke, 1979). As maturation
progresses, the paired homologous chromosomes align in the middle of the
Figure 2 Events of nuclear maturation, exemplified by sea urchin oocytes. A–C: Oocytes spend
their growth period arrested in G2 phase of meiotic cell cycle, with replicated DNA (4N). After
resumption of meiosis, oocytes of different species arrest for the second time at different stages
of meiotic progression, such as MI (D–F), or MII (G), and are relieved from this second arrest
by fertilization. Then they finish their meiotic divisions and progress into mitosis. Sea urchin
oocytes are fertilized as haploid eggs after completing meiotic divisions (H–J). A, D, G, H:
schematic of nuclear events of meiosis, ploidies of the nuclei are noted; B, E, I: brightfield
images of sea urchin oocytes undergoing meiosis; C, F, J: corresponding fluorescent images of
DNA stained with Hoechst. PB1—the first polar body; PB2—the second polar body; pn—egg
pronucleus; MI—metaphase I stage of meiosis; MII—metaphase II stage of meiosis.
62
Voronina and Wessel
forming meiotic spindle during metaphase I (Fig. 2D–F). Separation of the
paired homologous chromosomes is followed by the first polar body
formation. Then the chromosomes remaining in the oocyte are again
arranged on a meiotic spindle at metaphase II. With the second meiotic
division, chromatids separate and the second polar body is formed. Finally,
the chromatids remaining in the oocyte decondense and a pronucleus forms
(Fig. 2H–J). The meiotic cells of different organisms become fertilizable at
different stages along the process of meiosis. For example, the eggs of most
vertebrate species arrest at the second metaphase, and await fertilization to
release them into embryonic development, while sea urchin eggs proceed
through meiosis to completion, and are stored as cells with haploid
pronuclei.
How long does oocyte maturation take? The dynamics of oocyte
maturation is difficult to assess for a number of reasons. First, exact
observation of cellular features during in vitro oocyte maturation may not
faithfully reproduce in vivo processes. Second, the progression of maturation
is often not synchronous in individual members of an oocyte population,
especially when the oocytes are obtained from females in different phases
of their seasonal reproductive cycle (Masui and Clarke, 1979). Therefore,
the timing can only be defined statistically. Finally, inaccuracy can be
introduced when determining the exact time of initiation of oocyte
maturation. While in certain species, the time of oocyte exposure to a
hormone is known precisely (as in Xenopus or starfish for example), the
oocytes of other species are able to mature in vitro without any external
stimuli. Mammals and sea urchins belong to the latter group of animals, and
therefore at this time one can only rely on obvious structural changes
indicating the onset of maturation.
In various species, oocyte maturation proceeds at different rates, even in
members of the same phylogenetic class (Masui and Clarke, 1979). For
instance, oocyte maturation in starfish takes only 1 h 40 min from GVBD to
completion (Kishimoto, 1999), whereas in the sea urchin, oocyte maturation
from the first detected migration of GV to the cell surface and GVBD to
formation of egg pronucleus takes approximately 8 h in vitro (Berg and
Wessel, 1997). The extended time it takes for the sea urchin oocyte to fully
mature may be explained in part by the fact that this cell needs to complete
meiosis and reform a haploid pronucleus, whereas other oocytes abbreviate
their maturation by arresting at MI or MII (it takes sea urchin 4 h for the
first polar body to form, so MI probably occurs at 3 h after GVBD).
Other representative durations of oocyte maturation are, approximately, on
average 5–6 h for fish (but up to 18 h in certain species, Thomas et al., 2002),
3 h for Xenopus (Terasaki et al., 2001), 6–7 h for mouse, rat, and hamster,
and 15–20 h for human (both in vivo and in vitro, Bomsel-Helmreich et al.,
1987). However, it is possible that spontaneous maturation in vitro takes
3. The Regulation of Oocyte Maturation
63
longer to proceed than the hormone-induced maturation in vivo. In a study
of brushtail possum oocytes (Glazier et al., 2002), the spontaneous in vitro
maturation of the oocytes took more than 24 h. However, when the oocytes
were allowed to mature in vivo and recovered at various times post
gonadotropin treatment for observation, intermediate stages of meiotic
maturation were not observed at all, suggesting that it is an extremely rapid
process.
2. Changes of the Organelles
The process of oocyte maturation is accompanied by fundamental changes
in cellular organelles. Oocytes possess a variety of organelles typical of most
cells (such as Golgi apparatus, mitochondria, endoplasmic reticulum) as
well as oocyte-specific (yolk granules, cortical granules, annulate lamellae,
acidic granules, and pigment granules). Some of these organelles do not
undergo any transformations during maturation (mitochondria, yolk), while
others (such as cytoskeletal elements, cortical granules, and endoplasmic
reticulum) exhibit dramatic changes—and are hallmarks of meiotic
progression.
The orchestration and execution of the nuclear events of meiosis
described above depends on the cell’s cytoskeleton. The growing oocyte is
characterized by elongated, labile cortical microtubules that undergo
dramatic changes during oocyte maturation (Boyle and Ernst, 1989; Smiley,
1990). Microtubules progressively disassemble in the cortex, and the meiotic
microtubule spindle then forms in the maturing oocyte to mediate
chromosome segregation during both divisions of meiosis (a separate
spindle forms in each division). Meiotic microtubule asters contributing to
the spindle formation are morphologically distinct from the mitotic ones,
being smaller, and not reaching distal aspects of the oocyte. In the mature
sea urchin egg, microtubules can still be found at the cortex, although they
are shorter and less numerous than in oocytes (Boyle and Ernst, 1989).
During meiosis, eggs of most species (the most notable exception being
mice) lose their microtubule organizing center, or centrosome, so that they
rely on fertilization and the parental centrosome to be able to execute
subsequent cytokinesis (see below). This process is particularly well documented in starfish oocytes.
The starfish oocyte begins meiosis with two centrosomes, as do somatic
cells in a G2 phase of the cell cycle. During the first meiotic division the cell
loses one of the centrosomes to the first polar body, which exactly mirrors
the processes occurring during a mitotic division. The remaining centrosome
does not replicate between two meiotic divisions (as it would have in
mitosis). Instead, in an event unique to meiosis, the two individual centrioles
of the remaining centrosome separate and organize the second meiotic
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Voronina and Wessel
spindle. After the second meiotic division, one centriole is segregated into
the second polar body, and the egg is left with a single centriole (Schatten,
1994 and references therein). It appears that these centrioles are not equal,
as the second polar body centriole retains the ability to replicate during the
S-phase and can support mitotic divisions, while the egg centriole is not
functional (Tamura and Nemoto, 2001). This single centriole is targeted for
degradation during the anaphase of the second meiotic division, as detected
by the loss of focal -tubulin localization shortly after completion of meiosis
(Uetake et al., 2002). In contrast, the mouse oocyte has a number of
centrosomal foci (instead of well-defined centrosomes), some of which
aggregate at the poles of the meiotic spindles, and others remain spread
throughout the cytoplasm, and evade elimination during meiotic divisions
(reviewed in Schatten and Schatten, 1987).
The cortical granules accumulated in the oocytes acquire the capacity for
exocytosis during maturation. Their function is to secrete their contents at
fertilization and modify the extracellular matrix of the egg, such that it does
not support binding and fusion of extra sperm. It is critical for the cell to
rapidly exocytose its cortical granules at fertilization to block polyspermy,
but also to prevent precocious exocytosis of these cortical granules,
otherwise the extracellular matrix would be irreversibly modified and the cell
would not be receptive to sperm. The oocyte synthesizes and accumulates
large numbers of cortical granules throughout oogenesis. In oocytes where
cortical granules have been quantified, they reach 8,000 in mice and 15,000
in sea urchin (reviewed in Wessel et al., 2001). During oocyte maturation,
cortical granules move to the periphery in nearly all species examined. This
change in distribution is linked to the acquisition of the capacity of these
granules to exocytose upon fertilization. In the sea urchin, these granules
are dispersed throughout the oocytes’ cytoplasm, and translocate to
the periphery of the oocyte during maturation (reviewed in Wessel et al.,
2001). In other animals, such as starfish and frog, the cortical granules are
already in the general vicinity of the cell cortex, and are brought to the
plasma membrane and rearranged at the cell surface during oocyte
maturation. In rodents, oocytes form a cortical granule-free domain over
the region of meiotic spindle mainly by rearrangement, with some
contribution of precocious exocytosis (Deng et al., 2003). Cortical granule
rearrangements each depend on de novo formation of microfilament
networks at maturation.
Microfilaments form the scaffold for organelle movement during oocyte
maturation. In the growing echinoderm oocyte, microfilaments
are predominantly found at the cortex of the cell (Boyle and Ernst,
1989; Heil-Chapdelaine and Otto, 1996). In starfish and sea urchin
oocytes, dramatic polymerization of actin takes place at the onset of
maturation (Heil-Chapdelaine and Otto, 1996; Wessel et al., 2002, and
3. The Regulation of Oocyte Maturation
65
references therein). Actin polymerization is detected at the cell cortex (where
actin spikes appear transiently in starfish); however, most of the
polymerized actin is detected in the nucleus. This reorganization of the
actin cytoskeleton is not critical for germinal vesicle breakdown, as
cytochalasins suppressing all actin polymerization do not inhibit GVBD
(e.g., Connors et al., 1998).
Actin filaments in the germinal vesicle have been seen in many different
oocytes, including frog, starfish, and sea urchin (Heil-Chapdelaine and Otto,
1996; Parfenov et al., 1995; Wessel et al., 2002). Although the function of the
filaments is not clear, it is intriguing for several reasons: (i) they are
transient, present just before GVBD and then disappear; (ii) their presence is
coincident with initiation of cortical granule translocation; (iii) they are
found at a time when the germinal vesicle is moving to the animal
pole; (iv) and they are correlated with a change in the germinal vesicle
shape and pending GVBD (Stricker and Schatten, 1989). One hypothesis
of function is that they contribute to vesiculation of the nuclear
envelope (Parfenov et al., 1995). In addition, several regulators of actin
polymerization are also present in the nucleus or translocate to the nucleus
(Rando et al., 2000), suggesting that there is some function of actin in the
nucleus that is subject to regulation. Finally, Zhao et al. (1998) find actin
and actin-related proteins in transcriptional complexes of mammalian cells.
This has also been seen in yeast, with genetic evidence supporting actin
function both in regulating transcription and chromatin remodeling (Shen
et al., 2000).
In vertebrate oocytes, actin polymerization appears to be required for the
translocation of the meiotic spindle from the center of the cell to an
asymmetric cortical location, and as a consequence for the first polar body
extrusion (Connors et al., 1998; Kim et al., 2000; Maro and Verlhac, 2002;
Ryabova et al., 1986). Recent work identifies the microfilament-binding
protein formin as a necessary molecular regulator of this process, as the
spindle does not migrate and polar bodies are not extruded in oocytes from
formin knockout mice (Leader et al., 2002). Formins, or formin-homology
(FH) proteins, comprise a recently recognized protein family, which
functions in regulating cell polarity by acting as effectors of Rho family
small GTP-ases and remodeling actin cytoskeleton (reviewed in Alberts,
2002; Lew, 2002; Zeller et al., 1999). The other transport event that
actin microfilaments are required for is translocation of cortical granules in
multiple organisms, such as sea urchin, starfish, and mouse (reviewed in
Wessel et al., 2001, 2002).
The scaffold formed by an extensive network of intermediate filaments
containing cytokeratin is present within 1 m of the plasma membrane in
immature oocytes of the hamster, Xenopus, ascidians, sea urchin, and starfish
(Boyle and Ernst, 1989; Schroeder and Otto, 1991, and references therein).
66
Voronina and Wessel
Upon commencement of meiotic maturation (at GVBD) in these animals,
the intermediate fibers disassemble, and never reform in the mature
eggs (Boyle and Ernst, 1989; Schroeder and Otto, 1991). Interestingly,
an association of signaling proteins (beta-gamma subunits of heterotrimeric
G-proteins) with these cytokeratin networks has been detected in starfish
(Chiba et al., 1995). The subcortical network of intermediate filaments might
serve as a scaffold for the assembly of the signaling protein complexes,
and thus be important for signal transduction in oogenesis. The release of
G-protein b subunits is necessary for starfish oocyte maturation to occur
(see Section VA). The timing of this release is correlated with the dispersal of
intermediate filaments, so the detected structural reorganization of
intermediate filaments can be indicative of the release of the cell signaling
mediators at maturation.
The endoplasmic reticulum (ER) of many species such as sea urchin,
starfish, frog, and mouse undergoes significant changes during oocyte
maturation. The changes in the ER structure are of particular interest as
this organelle releases calcium at fertilization mediating egg activation,
and this ability to release calcium develops during oocyte maturation.
Immature oocytes of all species studied possess relatively uniform threedimensional network of ER tubules with some individual cisternae and
annulate lamellae (accumulations of cisternae) deep in the cytoplasm
(Bobinnec et al., 2003; Jaffe and Terasaki, 1994; Mehlmann et al., 1995;
Terasaki et al., 2001). The detected changes of the ER in the course of
oocyte maturation include reorganization, or formation of circular
structures around the yolk platelets in starfish (Henson et al., 1990; Jaffe
and Terasaki, 1994; Mehlmann et al., 1995; Terasaki et al., 2001) and
accumulation of ER clusters in an organized array immediately next to the
plasma membrane (Henson et al., 1990; Mehlmann et al., 1995; Shiraishi
et al., 1995; Terasaki et al., 2001).
Changes in the structure of the ER, especially dispersion of the nuclear
envelope, and fragmentation of ER tubules, are due to the cell cycle
progression, and have been documented for mitosis as well (reviewed in
Jaffe and Terasaki, 1994; Lippincott-Schwartz, 2002). However, several
features of ER behavior are unique for meiosis. In maturing oocytes, the ER
is not associated with the meiotic spindle, while such association is detected
in mitotic cells (Bobinnec et al., 2003; Terasaki, 2000). Furthermore, the
formation of cortical clusters is specific for oocyte maturation and is
ultimately required for formation of calcium release mechanisms in the egg
at fertilization. These clusters disappear some time after fertilization and are
not detected in mitotic embryonic cells (FitzHarris et al., 2003). In mouse
and probably other animals too, the disappearance of cortical ER clusters
depends on the decrease of MPF activity (FitzHarris et al., 2003 and
references therein).
3. The Regulation of Oocyte Maturation
67
3. Changes in mRNA and Protein Patterns
Oocytes are actively engaged in transcription and through their growth
accumulate extensive amounts of mRNA (Masui and Clarke, 1979; Smith
and Richter, 1985). However, the mRNA content of the oocyte changes
upon maturation. This switch in mRNA populations is brought about by
both general termination of transcription at GVBD, and degradation of the
select subset of transcripts (Smith and Richter, 1985). In sea urchin oocytes,
the mRNAs coding for yolk and cortical granule constituents each abruptly
disappear with the beginning of oocyte maturation (LaFleur et al., 1998;
Laidlaw and Wessel, 1994; Wessel et al., 1998, 2000a,b). Generally, oocyte
maturation does not require mRNA synthesis; the only exception found so
far is sheep, where oocyte maturation is inhibited by the transcription
inhibitor -amanitin (Moor and Crosby, 1986). In most cases, mature eggs
are not transcriptionally active, as they are arrested in the metaphase of the
second meiotic division. The sea urchin egg, however, is an exception, in
that it is arrested before fertilization with a haploid pronucleus, which is
transcriptionally active and selectively accumulates histone mRNA before
fertilization (Venezky et al., 1981).
Protein synthesis patterns change significantly during the transition
from an oocyte to a mature egg. Certain mRNAs are translationally
activated while others become repressed (reviewed in Hake and Richter,
1997; Smith and Richter, 1985). Translational repression results in part from
the degradation of mRNAs, but also from selective mRNA deadenylation.
In sea urchins for example, production of yolk (yp30, Wessel et al., 2000b)
and cortical granule content (Laidlaw and Wessel, 1994) proteins is
extremely active in the primary oocyte, but ceases at the beginning of oocyte
maturation, due to mRNA degradation. In Xenopus and mouse, a specific
class of maternal mRNAs is deadenylated and translationally repressed
during oocyte maturation (Paynton and Bachvarova, 1994; Varnum and
Wormington, 1990). Translational activation of select mRNAs during
oocyte maturation is achieved by regulated elongation of their poly(A) tail
(Hake and Richter, 1997), and normally leads to a several-fold increase
in the rate of overall protein synthesis (Smith and Richter, 1985; Wasserman
et al., 1986). Qualitative changes in the patterns of protein synthesis
have been observed in many animals such as starfish, frog, and mouse.
The proteins being made during oocyte maturation are mostly cell cycle
regulators needed to advance the oocyte through meiosis. The prime
example of such proteins is cyclin B, the regulatory component of
maturation promoting factor; another one is c-mos required for two
consecutive divisions of meiosis. Consistently, protein synthesis inhibitors
block oocyte maturation in many (but not all) species. Some animals appear
to have all the proteins necessary for maturation already produced and
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Voronina and Wessel
stored in the oocyte (like the clam, Spisula, Hunt et al., 1992). Others need to
synthesize cyclin proteins (mouse, fish); still others need to make different
molecular cell cycle regulators, such as ringo/speedy (Xenopus, Ferby et al.,
1999; Hochegger et al., 2001; Lenormand et al., 1999). The mature egg of the
sea urchin is translationally quiescent, while in other species such as Xenopus
or mouse the mature egg is translationally active. This may be due to the
fact that these eggs are at different phases of their meiotic cycle: frog and
mouse eggs are arrested at MII and are constantly replenishing their mitotic
cyclin proteins, while the sea urchin egg is arrested following MII, at a
‘‘G1-like’’ state.
4. Physiological Changes
Mature eggs produce the fast block to polyspermy by changes in plasma
membrane potential upon fertilization (collectively termed as ‘‘fertilization
potentials’’), which transiently depolarizes in ascidians and sea urchins,
and hyperpolarizes in certain mammalian species (reviewed in Dale,
1994). The egg’s ability to generate this fertilization potential develops
during oocyte maturation. The mature eggs of starfish as well as
bovine produce fertilization currents with greater peak amplitudes
and faster rise times than do immature oocytes (Miyazaki, 1979; Tosti
et al., 2002). These differences reflect the changes in ionic conductance
that occur during oocyte maturation, and ultimately contribute to the
establishment of the fast block to polyspermy. Thus, a decrease in potassium
conductance by downregulation of Na þ /K þ pumps was proposed to
occur in both starfish and Xenopus oocytes upon exposure to maturationinducing hormones (reviewed in Miyazaki, 1979; Smiley, 1990; Wasserman
et al., 1986).
Changes in sodium currents in Xenopus oocyte ultimately lead to an
increase in the intracellular pH after progesterone stimulation from 7.2
to 7.7. The pH change occurs due to the activity of Na þ /H þ pump in
the plasma membrane of the cell (Wasserman et al., 1986). This increase
in pH appears to be important for the progress of maturation, as stabilizing the pH below 7.0 completely blocks GVBD. This increase in pH
in the frog oocyte is dependent upon downregulation of Na þ /K þ pumps,
as a high cytoplasmic concentration of potassium may directly inhibit the
Na þ /H þ pump.
5. Competence for Calcium Release
The capacity of the mature egg to undergo activation depends on the
competence to generate a calcium transient (or transients) upon fusion with
3. The Regulation of Oocyte Maturation
69
sperm, which develops during oocyte maturation. Prophase-arrested mouse
oocytes do not generate the series of calcium transients at fertilization
characteristic of mature eggs (Cheung et al., 2000 and references therein).
In mouse and starfish oocytes, inositoltriphosphate (IP3) receptor
amounts increase during maturation (Chiba et al., 1990; Iwasaki et al.,
2002), the IP3 receptor is rearranged at the cortex of the oocytes (Mehlmann
et al., 1996), the endoplasmic reticulum is reorganized (reviewed in Section
IIC), and intracellular calcium stores increase (Tombes et al., 1992).
The maturation-associated increase in IP3 receptor protein amount results
in increased sensitivity to IP3. The increase in sensitivity in turn confers
to the cell the ability to generate a normal pattern of calcium oscillations
and undergo normal amounts of cortical granule exocytosis (Xu et al.,
2003).
D. Death
The oocyte is a terminally differentiated cell. If the oocyte does not
transform into a mature egg, or if the egg is not fertilized, it dies. The demise
of the germ cells throughout the animal kingdom occurs through
apoptosis, or programmed cell death. Apoptosis is a conserved process of
cell elimination, characterized by distinct morphological hallmarks,
and requiring activation of specific intracellular programs. Examples of
physiological cell death required for oogenesis include C. elegans oocytes
and Drosophila nurse cells that die donating their cytoplasm to the surviving
gametes (it is still unclear what prevents the spreading of death signal in
these syncytial cells to the surviving oocytes), or oocyte attrition
and follicular atresia in mammals. The environmental conditions such
as starvation or toxic substance exposure are known to cause germ
cell apoptosis as well (Matova and Cooley, 2001; Voronina and Wessel,
2001). Apoptosis induction during starvation may be a nutritional strategy
in some animals with significant resources invested into egg production
enabling the animals to make many eggs during the time of plenty, but
allowing the option to reuse these nutrients in the time of pity. This is
especially true for yolk-laden oocytes where the nutritional stockpile is great
and reusable. In many species, mature eggs that have not been fertilized age
rapidly, and apoptose (Morita and Tilly, 1999; Sasaki and Chiba, 2001;
Yuce and Sadler, 2001). This could represent the ‘‘quality control’’
mechanism in the animals that ovulate their eggs arrested at the second
metaphase. Such eggs could potentially damage meiotic spindles and
missegregate their chromosomes upon fertilization or even activate
parthenogenetically if allowed to remain in such state for a long time, and
exhaust their energy supplies.
70
Voronina and Wessel
III. Maturation Promoting Factor
The cytoplasmic signaling pathways initiating the G2/M transition during
meiotic resumption of the oocytes converge on activation of the maturationpromoting factor, or MPF (Taieb et al., 1997). MPF was identified in and
subsequently purified from mature eggs of Xenopus (Lohka et al., 1988;
Masui and Markert, 1971). It consists of cyclin B (or B1 and B2 in
vertebrates) and cdk1 kinase, also referred to as cdc2 (Fig. 3; Labbe et al.,
1989; Kobayashi, 1991, reviewed in: Maller, 1990). This complex is a
universal cell cycle regulator advancing G2/M transition in both meiotic
and mitotic cell cycles. Cyclins and their catalytic kinase partners (cdks,
or cyclin-dependent kinases) are well recognized as regulators of general cell
cycle progression (for example, reviewed in: Pines, 1999). Cyclin B is the
regulatory subunit of the complex: cdk1 kinase is structurally prevented
from activation as a monomer, and needs to complex with cyclin for activity
(Morgan, 1997). Cyclin-dependent kinases preferentially complex with
particular cyclins, and are required only at specific times during mitotic
progression. Although the levels of cdks remain constant, the levels of
cyclins usually fluctuate during the cell cycle due to periodic synthesis and
degradation, resulting in transient kinase activities. Cyclins are classified as
G1, S, or M-phase regulators based on when their activities are required in
the cell cycle (Pines, 1999).
The levels of newly synthesized mitotic cyclins peak in M-phase, and
mitotic cyclin/cdk1 complexes govern the transition to and progression
through M-phase (reviewed in Pines, 1999). Cyclin B-cdk1 phosphorylates
and regulates the function of numerous substrates, including nuclear
lamins, histone H1, transcription factors, kinesin-related motors, and
cytoskeletal regulators, causing structural changes in the nuclear envelope,
chromatin, and cytoskeleton (reviewed in Nigg, 1995, 2001). The cdk1 itself
is a subject of tight posttranslational regulation (reviewed in Nigg, 2001). In
addition to the required association with a cyclin, cdk1 can be
phosphorylated on multiple sites. These phosphorylation events include
both stimulatory (on Thr 161) and inhibitory (on Thr 14 and Tyr 15,
localized in the ATP-binding site) regulation. The kinases responsible for
inhibitory phosphorylations, Wee1 and Myt1, are active during the G2
phase, and their enzymatic activity is in turn regulated by cell signaling
pathways as well as by checkpoint controls (although only Myt1 is present
in oocytes). The enzyme removing inhibitory phosphates from cdk1 is a
dual-specificity phosphatase cdc25. The balance of cdc25 vs Wee1/Myt1
activities integrates the checkpoint and cell signaling inputs to determine
the activation state on cyclin B-cdk1 complex and allow progression to the
M-phase (Nigg, 2001).
3. The Regulation of Oocyte Maturation
71
Figure 3 Regulation of cdk1 activation. Activation of cdk1 depends on association with cyclin
B and the phosphorylation of cdk1 protein within the cdk1-cyclin B complex by cdk activating
kinase, CAK. Protein phosphatase 2C (PP2C) can reverse the CAK-mediated phosphorylation.
Active cdk1-cyclin B can be inactivated by the Myt1 protein kinase present in the oocyte, whose
effects are reversed by the activating phosphatase cdc25. Plx1—polo-like kinase 1, which
phosphorylates and activates cdc25. Positive feedback loops are shown as dashed lines. The
figure is adapted from Ferrell 2002 (see color plate).
The presence of multiple feedback mechanisms able to regulate cyclin Bcdk1 activity results in a switch-like, or as proposed by Ferrell, a ‘‘bistable’’
system (Ferrell, 2002). Such a system is characterized by the presence of two
stable states, and the ability of an ‘‘all-or-none’’ switch from one state to the
other in response to graded stimuli. In a bistable system, small stimuli have
little effect on cyclin B-cdk1 activity, whereas stimuli above a certain
threshold have a significant effect (Fig. 3).
The mechanisms of mitosis and meiosis regulation are quite different,
despite the same cyclins being major molecular regulators of both processes.
Oocytes spend prolonged periods of time arrested in the prophase of
first meiotic division (see Section IIB); consistently, the control of entry into
M-phase is different between mitosis and meiosis. To proceed through
meiotic divisions, the oocyte accumulates and then activates MPF in
response to the mitogenic signal (Fig. 4). Meiotic division itself is a
modification of a mitotic one, where the cell does not undergo DNA
replication between two successive rounds of division thereby decreasing the
amount of DNA to a haploid state. This is a consequence of the fact that
regulation of MPF during meiosis is different than that in the mitotic cell
cycle, namely, MPF activity increases rapidly after the first meiotic division,
and the second M-phase starts without a preceding S-phase. To gain
72
Voronina and Wessel
Figure 4 Schematic representation of MPF activity changes during growth and maturation of
oocytes compared with levels of the regulatory subunit of MPF, cyclin B (see color plate).
insight in the molecular mechanism of meiotic M-phase initiation and
progression, cell cycle dynamics of cyclin A, cyclin B, and cdk1 kinase
during oocyte maturation have been studied in many organisms (reviewed in
Kishimoto, 1999; Yamashita, 1998), and significant differences are found
between species arguing against a unified mechanism regulating MPF
activity in oocytes (Taieb et al., 1997).
One mechanism regulating MPF activity in oocytes is accumulation of
cyclin B protein, the best example being fish (reviewed in Yamashita, 1998,
Fig. 4). In this case, the newly synthesized cyclin B associates with the
catalytic subunit (cdk1), which is not subjected to inhibitory phosphorylations, immediately forming active complexes. Other layers of regulation
known for MPF include posttranslational modification of the catalytic
subunit, as exemplified in Xenopus, mouse, and starfish (Kishimoto, 1999).
In these oocytes, cyclin B is made during the growth phase of the oocytes,
and newly formed cyclin B-cdk1 complexes are immediately inactivated by
inhibitory phosphorylations. These are in turn removed in response to
signals inducing oocyte maturation, ensuring rapid activation of the
stockpile of inactive MPF. Recent data from sea urchin oocytes suggests a
similar MPF activation strategy (Voronina et al., 2003). Transport of MPF
3. The Regulation of Oocyte Maturation
73
between the cytoplasm and the nucleus is important for its activation and
function, and the dynamic changes in MPF localization may contribute to
its switch-like activation (Takizawa and Morgan, 2000). Recently, existence
of additional MPF components has been proposed based on the observation
that components of the germinal vesicle of oocyte are needed in addition to
the activated cyclin B-cdc2 to induce GVBD in the nonstimulated oocytes in
starfish (Kishimoto, 1999).
IV. Initiation of Oocyte Maturation
Hormones are the best documented stimulators of oocyte maturation.
Nevertheless, stimuli that induce oocyte meiosis in various species are quite
diverse, and include fertilization (in clam, Spisula solidissima) and even a
secreted sperm protein factor (Caenorhabditis elegans, described further in
Section IVD, Miller et al., 2001).
The somatic cells of the ovary relay the inductive signal for oocyte
maturation from the hormones2 released into the circulation. A number
of mechanisms for triggering oocyte maturation have been proposed so far
that do not necessarily exclude each other. Among those are: production
of a maturation-inducing substance by follicle cells that directs the
oocyte to mature (starfish, fish, frog, maybe mammals); inactivation
of the follicle-produced inhibitor (mammals); and inhibition of gap
junction-mediated transport to prevent transfer of a follicle-produced
inhibitor (fish, mammals).
A. Maturation-inducing Substance (MIS)
Steroid hormones have been found to induce oocyte maturation in a number
of vertebrate species. Although traditionally such hormones are known to
mediate their effects via nuclear receptors inducing transcription, it appears
that signaling in the oocyte involves activation of membrane receptors, and
is independent from new transcription. At this time, candidate MISs are
proposed for starfish, fish, amphibians, and mammals.
Mammals. The meiosis-inducing activity of human follicular fluid was
identified by an ability to promote maturation in mouse oocytes dissected
from the follicles. Gas chromatography mass spectrometry identified the
2
These hormones could generally be referred to as ‘‘gonadotropins’’ (as in Masui and Clarke,
1979), but the term appears confusing, since presently it is used only for vertebrate hormones (in
which capacity it will be used herein). However, the ‘‘gonad-stimulating substance’’ of starfish is
functionally a ‘‘gonadotropin’’ (acting on ovary) as well.
74
Table I
Voronina and Wessel
Structure of the Proposed Maturation Inducing Substances (MIS) of Various Species
Species
Name of MIS
Mammals
4,4,-Dimethyl-5-cholesta-8,
14,24-trien-3b-ol (FF-MAS)
Frog
Progesterone
Fish
17,20b-Dihydro-4-pregnen-3-one
(DHP) is shown in black;
the extra hydroxyl of 17,20b,
21-trihydro-4-pregnen-3-one (THP)
is shown in parentheses
Starfish
1-Methyladenine
Structure of MIS
See also Fig. 5 for the structure of C. elegans MIS.
active compound as 4,4,-dimethyl-5-cholesta-8,14,24-trien-3b-ol, an intermediate in cholesterol biosynthesis (Table I). It was proposed to be the
ovarian stimulatory factor inducing meiotic resumption of the oocytes, and
it was named FF-MAS for follicular fluid meiosis-activating sterol (Table I,
3. The Regulation of Oocyte Maturation
75
Byskov et al., 1995). This sterol was able to induce maturation in denuded
or cumulus-enclosed oocytes, as well as in rat ex-vivo perfused ovaries
(Grondahl et al., 2000; Hegele-Hartung et al., 2001; Ruan et al., 1998). The
origin of FF-MAS has not yet been established, but evidence suggests that
cumulus cells or follicle cells are responsible for production of this factor
(Byskov et al., 1995, 2002). However, other experimental evidence argues
against FF-MAS being the physiological MIS, as specific inhibitors of FFMAS-synthesizing enzymes fail to suppress gonadotropin-induced meiosis
in vivo in rat (Tsafriri et al., 2002), so its relevance is still a matter of debate.
It would be useful to assess whether such inhibitors are indeed effective
before completely ruling FF-MAS out.
For FF-MAS to be a physiological signal for meiosis its levels might
increase before oocyte maturation; indeed, such increase is detected. In vivo,
mammalian oocytes resume meiosis after a rise in gonadotropin levels in
blood: follicle-stimulating hormone (FSH) followed by luteinizing hormone
(LH). The amounts of FF-MAS in rabbit ovaries rapidly and significantly
increase after animals were injected with FSH followed by hCG (human
chorionic gonadotropin; LH activity) injection, whereas FSH on its own
does not significantly increase follicular FF-MAS levels (Grondahl et al.,
2003).
In amphibians, experimental evidence points to androgens being
the physiological MIS. Progesterone (Table I) had been long suggested to
be the MIS causing oocyte maturation, although other steroids such as
aldosterone and testosterone are reported to be as efficient as progesterone
(Masui and Clarke, 1979). Frog oocytes effectively mature in response to
progesterone, and preventing conversion of pregnenolone to progesterone in
the ovary by drugs (cyanoketone and eliptin) inhibits gonadotropin-induced
oocyte maturation. However, measurements of steroid content of female
frogs stimulated by hCG reveal that progesterone concentrations in the
serum and ovaries are lower than expected to be effective (Fortune and
Tsang, 1981; Lutz et al., 2001). The major constituents of steroids produced
by the ovary are the androgens testosterone and androstenedione, that
are very potent inducers of oocyte maturation, suggesting that androgens
may be the natural MIS. These steroids are metabolites of progesterone,
so inhibiting progesterone production (as in earlier experiments) would
inhibit synthesis of androgens as well. Progesterone is quickly metabolized
to androstenedione by isolated frog oocytes; however, this conversion is not
necessary for progesterone-induced maturation, thereby resolving disparate
conclusions of the earlier experiments (Lutz et al., 2001).
In fish, two progestin derivatives, 17,20b-dihydro-4-pregnen-3-one
(DHP) and 17,20b,21-trihydro-4-pregnen-3-one (THP), were identified
as major active substances produced by the follicles upon stimulation with
pituitary extract in multiple species (Table I, Petrino et al., 1993; Thomas
76
Voronina and Wessel
et al., 2002, and references therein). DHP is quickly and efficiently
metabolized to an array of steroid compounds by the oocytes, however,
none of the DHP metabolites is as active a trigger of GVBD as DHP itself,
indicating that the induction of maturation is not due to metabolic
conversion into a more active form. In a number of fish species, synthesis of
DHP takes place in the follicle cells next to the oocyte in response to
gonadotropins, and steroid accumulation occurs in the follicular layers as
well as inside the oocyte such that DHP acts locally and transiently
(Nagahama et al., 1994).
In starfish, the adenine derivative, 1-methyladenine (1-MA), is the natural
MIS (Table I, Kanatani, 1969; Schuetz, 1971). 1-MA is produced in the
follicle cells surrounding the oocyte by the transfer of a methyl group from
S-adenosylmethionine onto an ATP molecule, making 1-methylATP, which
is then converted into 1-methyladenine (Mita et al., 1999a,b). 1-MA is
quickly metabolized in the recipient oocytes and its metabolic product is not
biologically active (Toole and Schuetz, 1974). 1-MA is synthesized and
secreted into the extracellular space between the follicle and the oocyte in
response to a peptide released by the radial nerve, which is analogous in
action to vertebrate gonadotropin (reviewed in Smiley, 1990). The gonadstimulating substance of the starfish Asterias amurensis has been purified
and identified as a polypeptide, with a molecular weight of 2.1 kDa,
although its exact amino acid sequence has not been determined (Kanatani,
1985).
1-MA is effective in inducing maturation in all starfish species;
however, it seems selective for this particular group of echinoderms.
Sea urchins, sand dollars, sea lilies, and sea cucumbers do not respond
to this compound (reviewed in Smiley, 1990; and Wessel, personal
communication). Furthermore, sea cucumbers produce an unidentified
MIS, preliminary characterized as 2,8 disubstituted adenine (Smiley, 1990).
Perhaps closely related adenine derivatives fulfill the role of 1-MA for
echinoderms other than starfish.
B. Receptors for Maturation-inducing Substance
Work on characterizing the mammalian receptors for FF-MAS has only
recently begun. The hydrophobic nature of the sterol would allow it to
accumulate in the plasma membrane, or potentially permit its diffusion
through the membrane, such that it could function intracellularly. However,
intracellular injection of FF-MAS is ineffective in the induction of
maturation (Grondahl et al., 2003). Furthermore, a potent antagonist of
FF-MAS, 22-R-cholesterol, can efficiently inhibit meiotic resumption in
response to extracellular FF-MAS only when it is applied from the outside,
3. The Regulation of Oocyte Maturation
77
and not injected into the oocyte, suggesting that it competes for binding to a
surface receptor.
In amphibian oocytes, the controversy over the nature of the MIS receptor
is still not resolved, and new developments in this area are expected
considering that androgens and not progesterone are the actual MIS.
Experiments attempting to distinguish whether progesterone acts on the
outside or on the inside of the cells are still inconclusive, in large part due to
the fact that progesterone is a hydrophobic molecule, which is able to cross
biological membranes by diffusion. What appears clear, however, is that
progesterone-induced as well as androgen-induced maturation does not
depend on new transcription, as it is unaffected by the transcriptional
inhibitor, actinomycin D (Lutz et al., 2003; Smith and Ecker, 1969).
Although extracellular application of progesterone is efficient in inducing
oocyte maturation, the injection of this sterol does not always lead to
resumption of meiosis. Such injections were found effective by some
groups (Bayaa et al., 2000; Tso et al., 1982), but not others (Godeau
et al., 1978; Masui and Clarke, 1979; Smith and Ecker, 1971). Conceptually,
it is possible even for the injected steroid to diffuse out of the cell, and
still act on its surface. Attempts to prevent transmembrane diffusion
of progesterone included covalent crosslinking to a 20 kDa polymer
(polyethylene oxide), bovine serum albumin, or to polystyrene beads. These
modified progesterone compounds were still active when applied to the
surface of oocyte under conditions minimizing endocytic uptake (Godeau
et al., 1978; Masui and Clarke, 1979), but of inconsistent activity
when microinjected into the cell (Bayaa et al., 2000; Masui and Clarke,
1979). An additional confounding factor is that the preparations of
crosslinked progesterone could still contain sufficient free sterol to account
for the observed biological activity, which is difficult to control (Bayaa
et al., 2000).
In support of the plasma membrane being the site of progesterone
action, progesterone appears to bind specifically and with high affinity
to frog oocyte membranes (Lutz et al., 2000). These specific high affinity
binding sites can be detected on Stage V–VI (maturation-competent) oocyte
membranes, but not on Stage I–III (maturation incompetent) oocyte
membranes (Lutz et al., 2000). What is the nature of this membrane
sterol receptor? Experimental results of Lutz et al. argue against
progesterone serving as a ligand for a member of the heterotrimeric Gprotein coupled receptors (GPCR). The affinity of a GPCR agonist for its
receptor usually decreases upon activation of the associated G subunits.
When Xenopus oocyte membranes are incubated with the nonhydrolyzable
GTP analog GTPS locking all G subunits in the activated state,
progesterone binding stays at the same level as in untreated membranes,
suggesting that progesterone does not bind to its receptor in a manner
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Voronina and Wessel
typical for a GPCR agonist (Lutz et al., 2000). Thus, we are left with the
following alternatives: the membrane progesterone receptor (mPR) is not
a GPCR or, the mPR is a GPCR, but progesterone does not bind to it
as a typical agonist (and may instead function as an antagonist, see
Section VA).
Nuclear progesterone receptor (hormone-dependent transcription factor,
nPR) may be essential for the oocyte maturation in response to progesterone
(reviewed in Maller, 2001). Overexpression of nPR increased sensitivity to
progesterone and accelerated the rate of progesterone-induced meiosis
(Bayaa et al., 2000; Tian et al., 2000), while injection of nPR antisense
oligonucleotides into the oocytes inhibited the progesterone-induced oocyte
maturation after 8 days of treatment (Tian et al., 2000). The observed effects
were specific for antisense but not control oligonucleotides, and could be
rescued by subsequent injection of the oocyte with mRNA coding for
Xenopus or human nPR. However, additional experiments are required to
firmly establish the role of nPR in maturation. The antisense treatment
described (Tian et al., 2000) involves culturing oocytes in vitro for 8 days
without nutritional supply, which may very well change the cell’s
physiology. Furthermore, even the rescue experiments could be questioned
as the overexpressed nPR could initiate maturation by a nonphysiological
pathway (via direct activation of a downstream kinase Rsk; Maller, 2001).
Of particular concern is the fact that sterol binding specificity characteristic
of conventional nuclear progesterone receptors does not match the GVBDinducing potency profile of these sterols (reviewed in Maller, 2001).
Therefore, it is important to test whether the cloned Xenopus nPR exhibits
an unusual sterol-binding profile.
Following the suggestion that androgens may be the in vivo signal
inducing meiotic maturation in frog, the role of nuclear androgen receptor
(nAR) in Xenopus oocytes maturation was tested (Lutz et al., 2003).
Treatment of the oocytes with dsRNA for 2.5 days was able to significantly
inhibit oocyte maturation in response to androgen without altering the
responsiveness to progesterone. nAR protein was found expressed
throughout the Xenopus oocyte, and a fraction of the protein was associated
with the plasma membrane. A number of sterol ligands demonstrated the
ability to induce nAR-dependent transcription in a heterologous system.
Unexpectedly, when assayed for the ability to induce oocyte maturation,
two of these sterols (dihydrotestosterone and R1881) appeared to be potent
antagonists of testosterone-induced, but not progesterone-induced oocyte
maturation. This may cast a doubt on the nAR function as a MIS receptor;
however, at the same time it afforded an opportunity to test the importance
of androgen-mediated signaling in an ovarian explant. Both R1881 and
dihydrotestosterone were able to inhibit maturation of oocytes in hCGtreated Xenopus ovaries, despite efficient production of testosterone by the
3. The Regulation of Oocyte Maturation
79
ovaries under these conditions, arguing that the testosterone-induced
signaling is indeed the predominant in vivo pathway for induction of oocyte
maturation in frog (Lutz et al., 2003).
Since progesterone- and androgen-dependent maturation does not require
new transcription, the mechanism of transcription-independent nuclear
receptor effect may be through activation of Src family tyrosine kinases.
Recently, a proline-rich motif unique to nPR was uncovered in the
N-terminal domain of the protein, which appeared to mediate direct
hormone-dependent binding of nPR to SH3 domains of various cytoplasmic
proteins. This motif appeared necessary and sufficient for Src activation by a
SH3 domain-displacement mechanism, and presence of this domain in
protein overexpressed in Xenopus oocytes was required for the enhancement
of the response to progesterone (Boonyaratanakornkit et al., 2001).
However, overexpression of a fusion of nPR N-terminal DNA binding
domain with estrogen-binding domain in Xenopus oocytes did not make
these oocytes responsive to estrogen (Bayaa et al., 2000). Nevertheless, this
result could be explained by a requirement for the presence of the nPR
ligand-binding domain for the N-terminal part to bind to and activate Src
efficiently (Boonyaratanakornkit et al., 2001).
In fish the plasma membrane of the oocyte possesses specific high affinity
binding sites for MIS. Maturation-inducing substance (DHP, Table I) acts
at the cell surface and is ineffective in inducing maturation when injected
into oocytes. Investigation of the binding affinity of plasma membrane and
nuclear MIS binding sites for a range of sterols demonstrates that they are
characterized by markedly different binding affinities for a number of
steroid compounds. Thus, it is unlikely that a nuclear hormone receptor
participates in the MIS signaling at the plasma membrane. Furthermore, the
affinity of plasma membrane sites for MIS decreases with incubation of the
membranes with GTPS, consistent with the hypothesis of a GPCR being
an MIS receptor, since activation of the associated G subunits leads to a
decrease in the affinity of a typical GPCR for its ligand (reviewed in Thomas
et al., 2002).
Recently, the fish MIS candidate receptor was cloned by an innovative
approach. Monoclonal antibodies were produced against a partially
purified, solubilized oocyte plasma membrane fraction. They were then
screened for the ability to bind to the solubilized membrane receptor using a
hormone receptor capture protocol, where the efficiency of immunoprecipitation of a receptor selectively binding the radiolabeled ligand is assessed.
This assay was optimized in a way to maintain specific ligand binding in the
solubilized preparations of the membranes, which is not always possible
for GPCRs. Three monoclonal antibodies were selected, and expression
screening of the cDNA library identified a protein with no similarity to
known receptors (Thomas et al., 2002; Zhu et al., 2003b). Computer analysis
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Voronina and Wessel
of the amino acid sequence of this protein predicts seven transmembrane
domains, consistent with a GPCR structure, but suggestive of representing
a separate subclass of these receptors. In line with this, a number of proteins
homologous to the suggested fish membrane progestin receptor (mPR)
have been isolated from several vertebrate species (Zhu et al., 2003a).
Immunolocalizations, as well as subcellular fractionations, consistently
detect mPR localized on the plasma membrane of the oocyte.
Heterologously expressed mPR confers sensitivity to DHP in transfected
mammalian tissue culture cells. In these cells, DHP application reduces
cAMP production and activates MAP kinase; these effects are consistent
with proposed signaling through this receptor in the oocyte at resumption of
meiosis. The protein profile shows that mPR is low in previtellogenic
oocytes, increases with oocyte growth, and disappears upon oocyte
maturation, consistent with what would be expected for a receptor, whose
function is specific for oocyte maturation. Depletion of the mPR in
the zebrafish oocytes by morpholino antisense oligonucleotides blocks
maturation in vitro in response to DHP, consistent with this protein being
the MIS receptor (Zhu et al., 2003b). However, further experiments would
be helpful to definitely establish the proposed role for mPR in oocyte
maturation. For example, bacterially overexpressed protein displays an
unexpected sterol-binding profile, which does not match the steroid
specificity of maturation stimulation. Furthermore, a rescue of the antisense
treatment experiments in zebrafish would be useful, accompanied by
immunological detection methods of the mPR protein.
Thus, it is suggested that fish oocytes utilize a GPCR-mediated induction
of oocyte maturation, in general contradiction to signaling described for
frog. Are these differences to be reconciled in the future? It is possible that
nuclear and plasma membrane sterol receptors participate at different stages
of oocyte maturation in the MIS signaling. The homologues of the fish mPR
have been recently isolated in a number of vertebrates including frogs (Zhu
et al., 2003a). Future experiments will test the involvement of this GPCR
family in the signaling at oocyte maturation in different species, and define
the contribution of nPRs, if any.
In starfish, a variety of evidence indicates that 1-MA acts at the oocyte
surface. For example, intracellulary injected 1-MA does not cause
reinitiation of meiosis (reviewed in Kanatani, 1985). Furthermore, 1-MA
specifically binds to the oocyte surface: binding of a radiolabeled 1-MA is
inhibited in a dose-dependent manner by unlabeled 1-MA and biologically
active analogs (1-benzyladenine and 1-ethyladenine), but not by biologically
inactive analogs (1,9-dimethyladenine or 1-methylhypoxanthine) (Mita et al.,
2001; Yoshikuni et al., 1988).
It is hypothesized that a seven-transmembrane GPCR in the plasma
membrane binds 1-MA (Kishimoto, 1999). G-protein coupled receptors
3. The Regulation of Oocyte Maturation
81
(GPCRs) are known to have different affinities for receptor agonists
depending on their association with G-proteins. The binding affinity of the
starfish 1-MA receptor changes consistent with it being a GPCR: the affinity
of starfish oocyte cortices for 1-MA decreased after in vitro treatment with
GTPS that dissociates G-proteins from their receptors (Chiba and Hoshi,
1995). Induction of oocyte maturation by treatment with reducing agents
(reviewed in Kanatani, 1985) most likely does not act on the receptor,
since pertussis toxin blocks the induction of maturation by 1-MA (see
Section IVA), but not by dithiothreitol (Chiba et al., 1992; Chiba and Hoshi,
1995). The exact nature of the 1-MA receptor has not been determined yet,
despite many efforts to biochemically characterize or clone it (e.g.,
Kalinowski et al., 2003).
C. Follicle Cells and Gap Junctions
Oocytes of many species develop in close contact with the somatic cells of
the ovary. For example, in mammals, a multilayered complex of somatic
cells surrounds the oocyte in a specialized structure called a follicle (Fig. 1).
The follicle cells adjacent to the oocyte form numerous specialized contacts
(gap junctions) with the oocyte (reviewed in Matova and Cooley, 2001).
These structures are important for supplying the oocyte with nutrients, and
for communication between somatic cells and the oocyte.
Gap junctional communication plays an important regulatory role for
oocyte maturation. In mammals, hormones targeting oocyte development
exert their action on somatic cells of the follicle, which then relay the
signal to the oocyte. The pituitary gland releases the gonadotropins FSH
and LH. FSH causes the follicle cells to proliferate and differentiate, and
LH initiates the oocyte’s progression through meiosis and ovulation. The
follicle cells mediate this initiation of oocyte maturation in response to
hormones.
The oocyte is usually incapable of maturation during its growth
period (Masui and Clarke, 1979) but once meiotic competence is achieved
in a full grown oocyte, the granulosa cells become the major suppressor
of meiotic progression. Mammalian and fish defolliculated oocytes
undergo spontaneous maturation in culture, while they do not when
they are cultured enclosed in follicles (reviewed in Conti et al., 2002;
Patino and Purkiss, 1993). As follicle cells significantly influence the
state of the oocyte it was hypothesized that the oocyte is maintained in the
immature state by a follicular ‘‘arrester’’ produced by granulosa cells.
How do the follicle cells communicate with the oocyte to keep the cell
cycle blocked?
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Numerous gap junctions exist between the cells of the ovarian follicle,
providing connections among somatic cells, and between follicle cells and
the oocyte. These intercellular membrane channels can be detected by
transfer of metabolites or small fluorescent dyes. Gap junctions are essential
for oocyte growth and development and were proposed to provide the way
for a small molecule ‘‘arrester’’ to diffuse into the oocyte. The abundance of
gap junctions is regulated by the amounts of their constituent proteins,
connexins, in the cell. ‘‘Coupling efficiency’’ of existing gap junctions is
further regulated by their subunit composition and posttranslational
modifications of connexins by intracellular signaling mediators (reviewed in
Kidder and Mhawi, 2002). Regulation of follicle gap junctions within the
follicle is hypothesized to be involved in the induction of oocyte maturation.
In certain animals, like the Syrian hamster, it was determined that
downregulation (decrease in number and density) of follicle cells’ gap
junctions is correlated with an irreversible commitment of the oocytes to
maturation (Racowsky et al., 1989). On the other hand, in the mouse, the
oocyte–cumulus cells coupling index remained constant throughout the
GVBD period in hCG-stimulated animals (Eppig and Downs, 1988) and in
the explanted cumulus cell–oocyte complexes induced to mature with
gonadotropin (Downs, 2001). The molecular mechanism for downregulation of gap junction coupling efficiency is suggested to act at several levels
including phosphorylation of the protein and decrease in net levels of
protein and mRNA. In rats, the gap junction protein connexin-43 is rapidly
phosphorylated in response to exposure to LH (Granot and Dekel, 1994).
Furthermore, prolonged exposure to LH causes complete elimination of
connexin-43 protein correlated with a decrease in its mRNA level. The
experimental manipulation of gap junctions can also influence the meiotic
state of the oocyte. For example, application of chemicals such as 1-octanol
that suppresses gap junction-mediated cell–cell coupling appears to initiate
oocyte maturation in follicles of the fish Fundulus heteroclitus as well as
in mouse cumulus-enclosed oocytes (Cerda et al., 1993; Downs, 1995).
However, Xenopus follicle-enclosed oocytes cannot be stimulated to mature
in this way (Patino and Purkiss, 1993), suggesting that the input of a gap
junction-delivered arrester in mediating prophase arrest of the oocyte varies
in different animals.
Gap junctions appear to transmit both inhibitory and stimulatory signals
between follicle cells and the oocyte. If prophase I arrested, follicle-enclosed
fish or mouse oocytes are treated with stimulatory ligands, GVBD is
triggered at greater frequencies than those achieved by just removing the
follicle cells (Cerda et al., 1993; Downs, 1995, and references therein).
Furthermore, induction of meiotic maturation in cumulus-enclosed oocytes
depends significantly on the presence of functional gap junctions between
the oocyte and cumulus cells, as assessed by either inhibiting gap junctions
3. The Regulation of Oocyte Maturation
83
in intact follicles chemically, or by dissociating cumulus cells from the
oocyte, and subsequently coculturing them together (Downs, 1995, 2001).
Experiments in Xenopus oocytes produce comparable results: while oocyte
maturation of the follicle-enclosed oocytes can be induced by gonadotropin
treatment, gap junction-inhibiting reagents suppress this induction (Patino
and Purkiss, 1993). Thus, even though in Xenopus no inhibitory signal is
transmitted through gap junctions, there appears to be an activating input
instead. This effect appears to result specifically from disruption of gap
junctional communication, and not from detrimental effects of the 1-octanol
on the follicle cells or the oocyte itself. This conclusion is based on the result
that treatment with 1-octanol does not decrease the amounts of steroids
produced by the follicle cells, and does not inhibit maturation of follicle-free
oocytes in response to external progesterone. It is hypothesized that gap
junctional coupling of follicle cells and oocyte in Xenopus either facilitates
delivery of the hydrophobic sterol to the oocyte surface, or provides a
cytoplasmic route of progesterone delivery to the oocyte.
D. A Whole New Can of Worms
A strategy of oocyte maturation regulation unique to the worm C. elegans
has been recently described. Maturation of the oocytes in this animal is
dependent on the presence of sperm in the spermatheca (but not
fertilization). Major sperm protein, MSP (Fig. 5), secreted by sperm was
found to be the active component inducing oocyte maturation (Miller et al.,
2001). This mechanism is quite unusual in that no other known meiosisinducing hormone is a peptide (Table I). As the MSP gene family in C.
elegans consists of 40 genes whose protein products differ by one to four
amino acids, knockout experiments do not appear feasible. However, the
evidence for MSP being the maturation-inducing activity is strong, since
recombinant MSP is able to induce oocyte maturation in the absence of
sperm, while injection of antibodies to MSP into the uterus of the adult
nematode leads to a reduction in the oocyte maturation rate (Miller et al.,
2001). A screen for the receptors of MSP was carried out by a combination
of microarray-based identification of oocyte-enriched genes, and RNAimediated ablation of the candidates followed by assay for MSP binding.
These efforts identified a Vab-1 Eph (ephrin) receptor protein tyrosine
kinase (RPTK) as the only definite candidate (Kuwabara, 2003; Miller et al.,
2003). However, the phenotype of vab-1 mutants is inconsistent with this
protein positively regulating oocyte maturation. Instead, Vab-1 is a
suppressor of oocyte meiosis, and MSP induces maturation by antagonizing
Vab-1 activity. It appears that Vab-1 expressed on the oocyte surface
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Voronina and Wessel
Figure 5 Crystal structure of the major sperm protein (MSP) of a nematode C. elegans (from
Baker et al., 2002). Note that MSP is significantly larger than any other maturation-inducing
substance molecule (Table I) (see color plate).
receives a constitutive signal from the Efn-2 ephrin also expressed in
the germ line to keep up the inhibitory activity. This system thus functions
as a sperm-sensing checkpoint mechanism that inhibits oocyte meiotic
maturation until sperm is available for fertilization. Potentially, this model
of ‘‘inhibition relief’’ may be defined by the hermaphrodite reproductive
strategy of C. elegans, and appears unique to this animal. However, other
organisms could utilize hormone-mediated relief of constitutive inhibition
of cell cycle progression as a means to induce oocyte maturation as well
(see Section VA).
V. Cytoplasmic Control of Maturation
The ultimate target of signal transduction pathways initiated at oocyte
maturation is activation of MPF kinase and release of the oocyte from the
block in meiotic progression. Among candidate second messengers linking
the MIS receptor to MPF activation are cAMP and calcium; these and
more upstream components of signaling machinery will be examined in this
section.
3. The Regulation of Oocyte Maturation
85
A. Signal Transduction through G-proteins: Ignition or Parking Brake?
How is the presence of hormones interpreted by an oocyte to achieve release
of the cell from the prophase block? The most detailed analysis of an
intracellular signaling pathway activated during oocyte maturation comes
from the studies of starfish. Heterotrimeric G-proteins are universal
signaling molecules present in most of the tissues. As their name suggests,
the complex consists of three subunit types: alpha (GTPase), beta, and
gamma. The inactive state is a heterotrimeric complex with the subunit in
a GDP-bound form. Upon activation, the GDP of the subunit is
exchanged for GTP, and dissociates from the b complex. Free GTP-
and free b diffuse in the plane of membrane and regulate the activity of
their respective effectors. Upon hydrolysis of GTP by the subunit, it
returns to the inactive GDP-conformation and reassociates with b, thus
concluding the activity cycle. Different G subunits have distinct sets of
intracellular targets and some G proteins have opposing effects on the
same targets. For example, Gs and Gi both target adenylate cyclase,
however Gs activates this enzyme while Gi inhibits it.
Mouse oocytes can be maintained in the prophase arrest in culture
indefinitely when they are enclosed in their follicles. However, when the
follicle cells are removed, mouse oocytes enter maturation spontaneously. It
was documented that such cumulus-free mouse oocytes could be maintained
in meiotic arrest in a transient and dose-dependent manner when
microinjected with GTPS, an activator of G-protein signaling
(Downs et al., 1992). This data led to a model whereby spontaneous
maturation of the oocyte is induced by removal of inhibitory signal
normally present in the ovary and transduced via the heterotrimeric Gproteins (summarized in Fig. 6A). This model was recently supported by
work of Mehlmann and colleagues (Mehlmann et al., 2002) suggesting that
the inhibitory ovarian signal is mediated by the activity of Gs. Injection of
anti-Gs inhibitory antibody induced oocyte maturation in follicle-enclosed
oocytes, while injection of anti-Gi inhibitory antibody did not have any
effect on spontaneous oocyte maturation. Furthermore, injection of cholera
toxin, which hyperactivates Gs-mediated signaling, into mouse oocytes was
able to significantly inhibit both spontaneous and FF-MAS-induced
maturation suggesting that activation of Gs signaling is able to
sustain meiotic arrest of the oocyte (Downs et al., 1992; Grondahl et al.,
2000). The most likely target activated by Gs in this cell is adenylate cyclase.
It produces cAMP, a second messenger that can sustain meiotic arrest of
the oocyte (see Section VC). Consistently, the effect of cholera toxin
on spontaneous maturation is more pronounced when the oocyte is
cultured in the presence of hypoxanthine, inhibiting cAMP degradation
(Downs et al., 1992).
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Voronina and Wessel
Figure 6 G-protein signaling and oocyte maturation. A. Maintaining vertebrate oocyte
prophase arrest by G-protein signaling. Depicted is a summary of experimental data in mouse
and Xenopus oocytes (see text for details and references). 7TM GPCR—seven-transmembrane
G-protein-coupled receptor; AdC—adenylate cyclase; GRK—G-protein coupled receptor
kinase. Blue color: reagents decreasing cAMP levels in oocytes and promoting oocyte
maturation, red color: reagents found or expected to increase cAMP levels in oocytes. B.
Induction of starfish (and possibly fish) oocyte maturation by G-protein signaling (see text for
details and references) (see color plate).
This model of G-protein function in mouse oocytes is supported in
Xenopus oocytes, which can be stimulated to mature by injection of an
antibody inhibiting Gs activity. Such injected oocytes proceed through
GVBD with the same time course as progesterone-stimulated ones, and the
resulting eggs are fertilizable (Gallo et al., 1995). Conversely, GTPS and
cholera toxin antagonize the action of progesterone on Xenopus oocytes,
while GDPbS has no effect (Cork et al., 1990; Schorderet-Slatkine et al.,
1982). Increasing the amounts of Gs in Xenopus oocytes blocks
progesterone-induced oocyte maturation as well. This effect is even more
pronounced if the mutant constitutively activated form of the protein is
expressed (Romo et al., 2002). Furthermore, depletion of endogenous Gs
by injection of antisense oligonucleotides induced early events of oocyte
maturation independent of progesterone addition (Romo et al., 2002). The
activation of adenylate cyclase in response to Gs signaling can be mediated
by both Gs and Gb complex interaction with the enzyme (Simonds,
1999). In the Xenopus oocyte, b subunits released upon Gs activation
are also thought to play a significant role in the signaling pathway
further activating adenylate cyclase. Experiments by multiple research
groups demonstrate that sequestration of endogenous b enhances, while
overexpression of exogenous b inhibits progesterone-induced as well as
androgen-induced maturation in a number of studies (Lutz et al., 2000,
2003; Sheng et al., 2001; but not Gallo et al., 1995). The observation that the
amount of b subunits in mouse oocytes decreases with oocyte maturation is
consistent with the model that a relative decline in b is involved in the
commitment of the oocyte to meiotic maturation (Allworth et al., 1990).
3. The Regulation of Oocyte Maturation
87
The evidence in support of a constitutively active G-protein-coupled
receptor upstream of this inhibitory Gs signaling was recently reported
(Wang and Liu, 2003). Reagents known to promote classic GPCR
desensitization, such as G-protein receptor kinase (GRK) and b-arrestin,
were found to induce Xenopus oocyte maturation independent of progesterone, presumably by downregulating the Gs signaling originated from
an upstream GPCR (Wang and Liu, 2003).
These results are summarized in a common model of sustaining prophase
arrest in a vertebrate oocyte by a combination of signaling by Gs itself and
b subunits generated through Gs activation (Fig. 6A). The adenylate
cyclase enzymes requiring both Gs and b subunits for activation have
been previously described in mammals (Simonds, 1999).
However, it is still unclear how the effects of Gs activation are reversed in
vivo by hormone action on a mouse follicle or a Xenopus oocyte. Hormonedependent resumption of maturation in both mouse and Xenopus oocytes
includes a rapid decrease of cellular cAMP. Could this effect be mediated
through activation of Gi family of heterotrimeric G-proteins (which is
known to antagonize Gs by inhibiting adenylate cyclase)?
It is possible to experimentally detect Gi activation during mouse
oocyte maturation. The amounts of Gi proteins somewhat decrease during
mouse oocyte maturation as determined by western blotting (Jones and
Schultz, 1990). However, dissociation of the Gi from the b subunits is
detected by a substantial decrease in the amounts of pertussis toxin
ribosylation substrate in the mature egg in comparison to the oocyte,
that cannot be accounted for by the decrease in Gi protein levels
(Allworth et al., 1990; Jones and Schultz, 1990). The reduction in the
PTX substrate amount occurs as well in vitro, and can be detected after 3 h
of in vitro culture, coinciding with GVBD when mouse oocytes
mature spontaneously (Jones and Schultz, 1990). This suggests that
activation of Gi may take place upon removal of the mouse oocyte from
the follicle.
Involvement of Gi-mediated signaling in the response of mouse and frog
oocytes to maturation-inducing hormones was tested by using its specific
inhibitors (Faerge et al., 2001; Goodhardt et al., 1984; Grondahl et al., 2000;
Lutz et al., 2000). Pertussis toxin inhibits certain members of the Gi family
of heterotrimeric G-proteins by ADP-ribosylation and blocks their
respective signaling. Gi inhibition by pertussis toxin does not consistently
affect resumption of meiosis in response to MIS in both vertebrate species
suggesting that suppression of Gs signaling might not occur through
induction of ‘‘opposing’’ Gi signaling. The injected pertussis toxin Asubunit was found effective (Pellaz and Schorderet-Slatkine, 1989), while
incubation of oocytes with the whole pertussis toxin is generally not effective
(Downs et al., 1992; Faerge et al., 2001; Goodhardt et al., 1984; Kline
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Voronina and Wessel
et al., 1991; Sadler et al., 1984). It appears that even though the Gi signaling
might be activated during the oocyte maturation, it does not contribute
directly to eventual resumption of meiosis.
Activation of all G-proteins in the mouse oocyte by GTPS injection
does not significantly interfere with FF-MAS-induced maturation, nor does
it have a stimulatory effect (Grondahl et al., 2003). In contrast, when
GDPbS is injected, FF-MAS-induced meiosis resumption is significantly
decreased. These effects are markedly different from the spontaneous
maturation, which is inhibited by GTPS injection (Downs et al., 1992). It
appears, that for FF-MAS to exert its action, inhibition of G-protein
signaling is not required; instead, a (pertussis toxin-insensitive) G-protein
must be activated.
Overexpression or injection of constitutively activated Gi in frog
has minimal effects on either spontaneous or progesterone-induced
maturation, which indicates that Gi activity is not important for the
Xenopus maturation response (Kroll et al., 1991; Lutz et al., 2000).
Participation of another type of G-protein was suggested, as injection of
constitutively active Go is able to stimulate Xenopus GVBD (Kroll et al.,
1991). However, the Go-mediated maturation depends on PKC activation
while progesterone-mediated maturation does not. Finally, expression and
activation of heterologous Gi-coupled receptors does not cause Xenopus
oocyte maturation (Kalinowski et al., 2003). It is conceivable then that the
function of G-proteins is to maintain the oocyte’s cell cycle arrest rather
than to induce oocyte maturation. The action of progesterone thus might be
in relieving the Gs/b mediated inhibition. Possible mechanisms include
functioning as an antagonist of a constitutively active GPCR; in this light,
the fact that progesterone does not bind to the oocyte membranes in
the manner typical of GPCR ligands becomes less puzzling (see
Section IVB). An alternative mechanism could be hypothesized to include
a novel effect of cytoplasmic progesterone receptor on Gs, b, adenylate
cyclase, or phosphodiesterase potentially through activation of Src (see
Section IVB).
Probing the signal transduction pathway leading to oocyte maturation in
fish uncovered that—consistent with other vertebrates—hyperactivation of
Gs by cholera toxin inhibits the oocyte’s response to MIS. The involvement
of Gi signaling in MIS-induced maturation is uncertain as microinjection
of activated pertussis toxin into oocytes blocks the MIS-induced maturation
only in select species (Thomas et al., 2002). Recent experiments identified a
candidate fish MIS receptor, which is capable to signal through Gi when
expressed in mammalian tissue culture cells (Zhu et al., 2003b). In the
context of other oocytes, these contradictory results need to be further
evaluated, as they suggest that there may be a diversity in oocyte maturation
signal transduction pathways within vertebrates.
3. The Regulation of Oocyte Maturation
89
The 1-MA signal inducing starfish oocyte maturation proceeds through a
G-protein pathway (Fig. 6B). The only G-protein involved in regulation of
1-MA-induced maturation in starfish appears to be Gi, since the 1-MAinitiated signal is inhibited by pertussis toxin (Shilling et al., 1989). The Gi
subunit modified by pertussis toxin was biochemically purified from starfish
oocytes and subsequently cloned (Chiba and Hoshi, 1995; Tadenuma et al.,
1991). Furthermore, expression of mammalian Gi-family-linked receptors,
such as human adenosine A1 receptor and rat ADP receptor, causes the
starfish oocyte to resume meiosis in response to corresponding agonists
(Kalinowski et al., 2003). Gs protein is present in starfish oocytes as well,
as detected by ADP-ribozylation of the protein by cholera toxin (Chiba
and Hoshi, 1995). Nevertheless, injections of the anti-Gs inhibiting
antibody in starfish oocytes neither stimulated GVBD, nor inhibited GVBD
in response to 1-MA stimulus (Gallo et al., 1995).
The b subunits of Gi appear to be the key for the initiation of oocyte
maturation. Starfish Gi activation, especially release of b subunits, is
necessary and sufficient to initiate maturation, while signaling through the
activated G subunit appears less significant (Chiba and Hoshi, 1995; Jaffe
et al., 1993). The free b complexes relay the signaling further along
the pathway, including phosphatidylinositol 3-kinase (PI3K) as a likely
downstream component (Section VD, Sadler and Ruderman, 1998).
B. Calcium Ions
Calcium is a universal signaling molecule involved in many important
processes such as muscle contraction, synaptic transmission, and
fertilization. Recent research suggests that calcium also governs certain
transitions of cell division cycle, such as nuclear envelope breakdown and
separation of chromosomes at the metaphase–anaphase transition (reviewed
in Whitaker and Larman, 2001). It is therefore possible that calcium
signaling contributes to the release of the oocyte from the meiotic prophase
arrest.
A transient increase in the intracellular calcium concentration has long
been known to be required for the induction of oocyte maturation, or
GVBD, as it is blocked by injection of calcium chelators into the cell (Masui
and Clarke, 1979; Pesty et al., 1998). Hormone addition to mammalian,
Xenopus, and starfish oocytes leads to the increase in cytoplasmic calcium
concentration, under certain conditions in the form of a pronounced spike
(Su and Eppig, 2002; Wasserman et al., 1986; Witchel and Steinhardt, 1990,
and references therein). Mouse oocytes dissected from the follicles and
spontaneously resuming meiosis in vitro display repetitive calcium transients
prior to GVBD as well (Carroll et al., 1994). These maturation-associated
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Voronina and Wessel
calcium transients are generally smaller in amplitude than fertilizationassociated calcium transients (reviewed in Schultz and Kopf, 1995). These
results are comparable to calcium signaling in mitosis documented for
embryonic cells (in a host of organisms: sea urchin, starfish, Xenopus,
mouse), which exhibit calcium rises associated with nuclear envelope
breakdown (Whitaker and Larman, 2001 and references therein).
Could simply introducing calcium ions into the cell induce oocyte
maturation? Most reports conclude that calcium on its own is insufficient
for induction of oocyte maturation (see below), although the evidence in
Xenopus is still controversial (Han and Lee, 1995; Noh and Han, 1998;
Wasserman et al., 1986 and references therein). The calcium ionophore
A23187 causes oocyte maturation in a very narrow group of species (some
of them, like Spisula, resume meiosis in response to fertilization, and the
calcium transient in this species is normally induced by egg activation). For
most species, including starfish, A23187 is ineffective, and it simply kills sea
urchin oocytes (reviewed in Kanatani, 1985). This suggests that the calcium
release is not sufficient for the initiation of oocyte maturation, although
calcium ionophore treatment is likely to cause a super-physiological increase
in cytoplasmic calcium concentration, and thus cause nonphysiological
effects. Still, cytoplasmic calcium transients are necessary for the execution
of certain aspects of oocyte meiosis.
The stages of oocyte maturation that can be regulated by calcium include
nuclear envelope breakdown (in starfish, but not in mouse), extrusion of the
first polar body, and cortical granule translocation to the cell surface
(Santella and Kyozuka, 1994; Santella et al., 1999; Su and Eppig, 2002;
Tombes et al., 1992 and references therein). Starfish oocytes injected with
heparin (which intereferes with IP3-mediated calcium release) delay the
GVBD in response to 1-MA, but more importantly, do not undergo normal
transformations of the cytoskeleton and do not redistribute cortical granules
at the plasma membrane (Santella et al., 1999).
The mechanism of calcium action at meiosis is not yet fully understood.
The strongest possibility, based on comparison with the active mitotic
players, is action through the calcium/calmodulin-dependent kinase,
CaMKII. Activation of calmodulin and CaMKII has been reported during
mitosis in a number of organisms and cultured cell types (reviewed in
Whitaker and Larman, 2001). The suggested role of CaMKII at the time of
mitosis entry is activation of cdc25 phosphatase, an activator of maturation
promoting factor (Patel et al., 1999). In support of meiotic function of this
enzyme, CaMKII was found active during the response to progesterone in
Xenopus oocytes (Stevens et al., 1999). Expression of a constitutively
activated mutant CaMKII is sufficient for induction of partial Xenopus
oocyte maturation (movement of GV, but not GVBD) independent of
hormonal signal (Waldmann et al., 1990). However, the role of CaMKII in
3. The Regulation of Oocyte Maturation
91
mouse meiotic resumption is unclear, since the CaMKII inhibitors, KN-93,
myristoylated AIP (autocamptide-2-related inhibitory peptide), and specific
calmodulin antagonist W-7, do not prevent mouse oocytes from
spontaneously resuming maturation when cultured in vitro (Su and Eppig,
2002). Instead, CaMKII appears to fulfill a later function in mouse meiosis.
All the mentioned CaMKII inhibitors inhibit extrusion of the first polar
body in a dose-dependent fashion, apparently blocking the metaphase to
anaphase transition.
Another potential downstream target of calcium is protein kinase C
(PKC). Artificial activation of PKC in Xenopus oocytes can cause
GVBD in the absence of hormone (Kroll et al., 1991; Stith and Maller,
1987). However, it is probably not a physiological mediator of progesterone
signaling, since inhibition of PKC by injection of inhibitory peptide does
not interfere with progesterone-induced maturation (Kroll et al., 1991).
Furthermore, artificial PKC activation in mouse oocytes actually prevents
GVBD (Bornslaeger et al., 1986; Downs et al., 2001 and references therein).
Thus, PKC does not normally mediate calcium signaling in the maturing
oocyte.
The pathway of calcium increase during meiosis is still under
investigation. This ion can be released from the internal stores via two
main types of calcium channel receptors: the inositol 1,4,5-triphosphate
receptor (IP3R), and the ryanodine receptor (RyR) insensitive to IP3, but
stimulated by various agents such as calcium, caffeine, nicotinic acid
adenine nucleotide phosphate, or cyclic ADP-ribose (cADPr). The
prevailing route for calcium release during mitosis is through generation
of IP3 produced by PLC (reviewed in Whitaker and Larman, 2001). IP3 is
a strong candidate for the meiotic mediator of calcium release since its levels
increase in Xenopus oocytes upon progesterone stimulation (Noh and Han,
1998 and references therein). It appears that the release of calcium is mostly
IP3-mediated, since injection of reagents preventing IP3 generation or
binding to the receptor delays (in starfish) or prevents (in Xenopus) the onset
of normal maturation, while introduction of 8NH2cADPr (inactive analog
of cADPr), ruthenium red, or procaine (specific inhibitors of RyR channel)
does not (Han and Lee, 1995; Iwasaki et al., 2002; Noh and Han, 1998;
Santella et al., 1999). However, RyR-mediated calcium release is important
(at least, in starfish) since a combination of heparin and 8NH2cADPr is
more efficient than either reagent on its own, and prevents GVBD of starfish
oocytes completely (Santella et al., 1999). Producing IP3 artificially in
Xenopus oocyte by activation of heterologously-expressed serotonin
receptor does not result in induction of oocyte maturation, which is
consistent with the conclusion that calcium release, by itself, is insufficient
for the induction of maturation (Noh and Han, 1998). However, the
amounts of IP3 produced by this artificial mechanism are less than those of
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Voronina and Wessel
the control oocytes responding to progesterone, making the conclusions
somewhat questionable.
C. cAMP
Cyclic AMP is a well-known second messenger, whose regulatory targets
include kinase and GTP exchange factor activities. In somatic cells, cAMP
functions both as a positive and a negative regulator of the mitotic cell cycle
progression (depending on cell type), exerting its effects mainly in the G1
part of the cell cycle. In contrast, progression of the oocyte through the
meiotic prophase block appears to be negatively regulated by the cAMP
levels (reviewed in Conti et al., 2002).
cAMP has been repeatedly proposed to be the mysterious inhibitor of
oocyte maturation, being made in follicle cells and transferred into the
follicle-enclosed oocytes in mammals (see Section IIIC, reviewed in Webb
et al., 2002). In support of this concept, a number of reports point to
inhibition of oocyte maturation by high cytoplasmic cAMP levels (see
below). cAMP is produced by adenylate cyclases and degraded by
phosphodiesterases. In starfish, phosphodiesterase inhibitors such as
IBMX (3-isobutyl-1-methylxanthine) or other xanthine derivatives have
an inhibitory effect on the maturation of 1-MA-treated oocytes (Doree et al.,
1976), potentially by interfering with cAMP degradation, but their effects on
cAMP levels have not been tested. Furthermore, in frog, forskolin (activator
of adenylate cyclase) either delays Xenopus oocyte maturation or blocks
maturation completely in a concentration-dependent manner (Noh and
Han, 1998). Forskolin inhibits spontaneous oocyte maturation in mouse
and fish as well (Schultz et al., 1983; Thomas et al., 2002). In the sea urchin,
artificially elevating levels of cAMP in isolated oocytes also maintains the
G2 arrest (Wessel et al., 2002).
Do cAMP levels decrease naturally upon MIS exposure? In hCGstimulated mice, oocytes exhibit a 30% decrease in cAMP, as detected by
radioimmuno assay 1.5 h after gonadotropin administration (Schultz et al.,
1983). However, the concentration of cAMP in the whole follicle actually
increases up to and throughout GVBD (Eppig and Downs, 1988; Schultz
et al., 1983). To explain these dynamics, a regionalized increase in cAMP
confined to the follicle cells is proposed resulting either from blocked gap
junctional communication between the oocyte and somatic cells, or from
selective downregulation of gap junction permeability for cAMP while
maintaining permeability for other molecules (Qu and Dahl, 2002). In
explants of oocytes enclosed in cumulus cells, stimulation of cumulus cells
by FSH leads to a transient increase in cAMP levels of the oocyte as
detected by the in situ fluorescent indicator FlCRhR (Webb et al., 2002).
3. The Regulation of Oocyte Maturation
93
Key here is that the oocyte increase is dependent on the presence of
functional gap junctions between cumulus cells and the oocyte. Consistently,
oocyte maturation in FSH-treated complexes is less than in control groups
early on after FSH treatment, at the time of detected cAMP increase, and is
significantly induced later (by 10 h post-FSH), when cAMP levels return
to baseline (Downs et al., 1988). These data suggest that the changes in
cAMP levels occurring during oocyte maturation in mouse depend on the
mechanism of maturation induction.
The evidence in starfish oocytes is less clear. Some reports indicate that
maturation-inducing 1-MA causes a 10–30% decrease in cAMP, starting
within 2 min after hormone addition (Meijer and Zarutskie, 1987), but other
evidence suggests that cAMP amounts do not change significantly during
oocyte maturation (reviewed in Kanatani, 1985). Even though starfish
oocyte maturation depends on activation of Gi protein, signaling through
the Gi (adenylate cyclase-inhibiting) subunit is not required for the
induction of maturation, while it is the release of b complex that ultimately
stimulates meiotic resumption.
The cAMP levels appear to decrease in frog and fish oocytes upon
stimulation of meiosis resumption. In Xenopus, the cAMP concentration
decreases (10–15%) upon progesterone stimulation within 15 min of
treatment (Cork et al., 1990; Schorderet-Slatkine et al., 1982). Induction of
oocyte maturation in fish is correlated with a 20–60% decline in the cAMP
concentration of the follicle upon treatment with DHP (reviewed in Haider,
2003; Thomas et al., 2002). Even though these are subtle changes in cAMP
concentration, they could still be important for the oocytes’ meiotic
transitions after being amplified via changes in the activity of the cAMPdependent kinase, and exhibit a threshold effect.
The role of cAMP in the regulation of maturation appears to be mediated
by its effects on cAMP-dependent protein kinase A (PKA). Microinjections
of PKA inhibitors, such as the regulatory subunit of PKA (rPKA), the
PKA inhibitory protein (PKI), membrane-permeable cAMP antagonist
(rp-cAMP), or small molecule inhibitor H-89 are able to induce oocyte
maturation (or at least accelerate hormone-induced maturation) in some
species, such as Xenopus or mouse (Leonardsen et al., 2000; Noh and Han,
1998), but not others, such as starfish (reviewed in Kanatani, 1985). On the
other hand, stimulating the signaling output of PKA by injection of the
catalytic subunit of PKA (cPKA) inhibits the response to hormone in both
Xenopus and starfish (reviewed in Kanatani, 1985; Masui and Clarke, 1979).
Interestingly, the total PKA activity in Xenopus oocytes does not
change after exposure to progesterone (Cicirelli et al., 1988). One proposed
mechanism of PKA action is by phosphorylation and inhibition of cdc25,
a cyclin B-cdk1 activating phosphatase (Duckworth et al., 2002).
Unexpectedly, the kinase-inactive cPKA blocks progesterone-induced
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Voronina and Wessel
maturation in Xenopus as effectively as wild-type cPKA (Schmitt and
Nebreda, 2002). Apparently, this inhibition is not a result of sequestering
known regulatory proteins that inhibit endogenous cPKA activity (such as
PKI) and represents either a new signaling mechanism or sequestration of
cdc25 by the excessive kinase dead cPKA. Interestingly, while microinjection of cPKA into mouse oocytes inhibited spontaneous oocyte maturation,
it failed to suppress FF-MAS-induced maturation, suggesting that a decrease
in cAMP levels and PKA activity is more critical for initiation of spontaneous maturation than for the response to FF-MAS (Faerge et al., 2001).
Oocytes may be able to overcome the inhibitory effects of cAMP when
they are stimulated with maturation-inducing substance. For example,
isolated mouse oocytes cultured in the presence of the phosphodiesterase
inhibitor hypoxanthine do not experience significant changes in their cAMP
levels, yet they are able to efficiently respond to FF-MAS (Grondahl et al.,
2003). Furthermore, cumulus cell-enclosed mouse oocytes cultured in the
presence of cell permeable dbcAMP and stimulated by FSH still enter
meiosis (Downs, 1995). Similarly, the inhibitory influence of xanthine
derivatives on the starfish oocytes’ response to 1-MA can also be overcome
by increasing 1-MA concentration (Doree et al., 1976). Furthermore, raising
cAMP in starfish or mouse oocytes by applying forskolin delays GVBD in
response to 1-MA or FF-MAS respectively but does not block it completely
(Hegele-Hartung et al., 1999; Meijer and Zarutskie, 1987). Finally, injection
of cholera toxin into starfish oocyte is effective in increasing the levels of
cytoplasmic cAMP; still, it does not block 1-MA-induced maturation
(reviewed in Kanatani, 1985).
Artificially decreasing the levels of cAMP in the oocyte is useful to test
whether this in itself would be sufficient to cause oocyte maturation. Culture
of mouse oocytes outside of their follicular environment leads to a decrease
in cAMP levels. For spontaneous maturation of mouse oocytes to occur, the
drop in cytoplasmic cAMP concentration needs to take place to 50–60% of
initial levels (Schultz et al., 1983). Another way to decrease the levels of
cAMP is by stimulation of heterologous Gi-coupled serotonin receptor
overexpressed in the oocyte. In Xenopus, stimulation of 5-HT1aR by
serotonin lowers cytoplasmic cAMP concentration, but never induces
GVBD (Noh and Han, 1998). However, this experiment is flawed in that the
resting values of cAMP in HT1aR-expressing oocytes appear to be twice
that of controls, and serotonin treatment only serves to bring cAMP closer
to the control levels, but never below those. Finally, expression of cAMPdegrading enzymes such as mouse phosphodiesterase 3 or rat phosphodiesterase 4 in Xenopus oocytes induces GVBD to the same extent as
progesterone (Andersen et al., 1998; Conti et al., 2002). Thus, it appears that
a physiological decrease in cAMP is sufficient by itself to induce a reentry of
oocyte into meiosis.
3. The Regulation of Oocyte Maturation
95
The physiological mechanisms regulating cAMP levels in the oocytes are
currently unknown. In different animals, evidence favors cAMP regulation
at the level of synthesis (by regulating adenylate cyclase), as well as at the
level of degradation (by regulating phosphodiesterase, PDE). In starfish,
Xenopus, and fish, a decrease in adenylate cyclase activity was proposed
to be responsible for the decrease in cAMP (Meijer and Zarutskie, 1987;
Schorderet-Slatkine et al., 1982; Thomas et al., 2002). The candidate
regulators of adenylate cyclase are G-proteins, and generally Gs subunits
activate adenylate cyclase, while Gi subunits inhibit its function. However,
this simplistic view has been altered during the last decade with the
identification of multiple different types of adenylate cyclases that are
responsive to multiple regulators in addition to G subunits, e.g., Gb
subunits, calcium levels, and protein phosphorylation (Simonds, 1999).
Thus, characterization of adenylate cyclases present in the oocyte is
necessary to gain understanding in regulation of oocyte maturation.
Recently, expression of type 3 adenylate cyclase (AC3) was detected in the
oocytes of rat and mouse (Horner et al., 2003). Furthermore, the AC3
knockout mice are characterized by a significant percentage of ovarian
oocytes escaping meiotic arrest. AC3 is activated by Gs protein and is
inhibited by an increase in intracellular calcium through phosphorylation by
CaMKII (Simonds, 1999). Indeed, AC3 activity is detected in rodent
oocytes, raising intracellular calcium can inhibit it, and this inhibition can be
rescued by in turn inhibiting CaMKII (Horner et al., 2003). These
observations suggest a possible mechanism for the contribution of calcium
signaling to the regulation of meiotic resumption through its effects on
cAMP levels.
The degradation of cAMP is also important for the induction of oocyte
maturation. Type 3 PDE appears to be active in mouse oocytes, but its
regulation has not been extensively explored (Conti et al., 2002; Webb et al.,
2002). This activity is required both in vitro and in vivo, as mice injected with
PDE3 inhibitors ovulate GV-arrested oocytes (reviewed in Conti et al.,
2002). Inhibition of oocyte maturation by phosphodiesterase inhibitors
does not distinguish between the impact of constitutive and regulated
PDE activity, making important the analysis of PDE3 activity profile
during maturation. A significant increase in the PDE3 activity is detected
in rat oocytes preceding both spontaneous and LH-induced meiosis
(Richard et al., 2001). PDE activity increases as well in fish oocytes
upon stimulation of oocyte maturation with DHP (Haider, 2003).
However, in Xenopus, the basal oocyte level of PDE activity does not
change after progesterone treatment (Sadler and Maller, 1987). It appears
that animals can utilize a decrease in cAMP production, or increase in
cAMP degradation, or a combination thereof to stimulate oocytes’ meiotic
resumption.
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Voronina and Wessel
Thus, cAMP is clearly not the only intracellular messenger involved in the
initiation of meiotic divisions, but lowering its concentration is important
for oocyte maturation to occur. The hormonal action on the oocytes might
have the effect of altering the oocytes’ sensitivity to cAMP levels in addition
to decreasing these levels. The data reviewed here are consistent with the
hypothesis that meiotic reinitiation in response to hormonal treatment is
triggered by a stimulatory signal that can be effective despite sustained
inhibitory influence of cAMP, while decreasing cAMP levels is important
for spontaneous oocyte maturation. Maturation may be influenced by a
threshold effect of cytoplasmic cAMP concentration, such that supraphysiological levels of cAMP are inhibitory for the meiotic progression. If,
however, cAMP is only slightly elevated, the hormone-induced signaling is
able to overcome the inhibition and promote maturation. In this case,
normally physiological conditions reflect a balance of cAMP and hormonal
activation. Such a threshold effect would impart on the oocyte an all-ornothing property for meiotic progression, analogous to MPF activation.
D. PI3K
Phosphatidylinositol 3-kinase (PI3K) is another proposed mediator of
hormone-induced maturation. This kinase converts the plasma membrane
lipid phosphatidylinositol-4,5-bisphosphate {PI(4,5)P2} to phosphatidylinositol-3,4,5-trisphosphate {PI(3,4,5)P3} (reviewed in Cantley, 2002). Signaling
proteins with plekstrin-homology domains accumulate at the sites of PI3K
activation by directly binding to its product PI(3,4,5)P3. Particularly
important binding partners are two serine–threonine kinases, Akt/PKB and
PDK1 (phosphoinositide-dependent kinase 1). Association with PI(3,4,5)P3
at the membrane brings these two kinases in proximity, and results in
phosphorylation and activation of Akt/PKB by PDK1. Akt/PKB activity
then leads to multiple cellular changes, including cell polarization, cell cycle
entry, cell survival, and oocyte maturation (Cantley, 2002).
In Xenopus, activated forms of PI3K can induce maturation, and PI3K
activity increases about 2-fold in progesterone-treated oocytes (reviewed in
Ferrell, 1999). Moreover, microinjection of a dominant-negative fragment
of PI3K inhibits progesterone-induced maturation. Potentially PI3K could
contribute to oocyte activation through the downstream Akt/PKB kinase,
which stimulates PDE3. Consistent with this notion, expression of
constitutively active Akt/PKB kinase is sufficient to induce Xenopus oocyte
maturation through PDE3 activation (Andersen et al., 1998). However, an
inhibitor of PI3K, wortmannin, does not inhibit progesterone-induced
maturation in frog, so the role of PI3K in this species is not altogether clear
(Ferrell, 1999). On the other hand, PI3K contribution appears more definite
3. The Regulation of Oocyte Maturation
97
in starfish oocyte maturation. It is thought to be activated by the
heterotrimeric G-protein b subunits, and PI3K inhibitors, wortmannin and
LY294002, inhibit 1-MA-induced maturation (Sadler and Ruderman, 1998).
The downstream kinase, Akt/PKB is activated in 1-MA stimulated oocytes,
and this activity is necessary for 1-MA-induced maturation (Okumura et al.,
2002). Accordingly, constitutively active Akt/PKB protein is able to induce
1-MA-independent oocyte maturation. The proposed target of Akt/PKB is
Myt1, the kinase that phosphorylates and inhibits cyclin B-associated cdk1.
Myt1 activity decreases upon phosphorylation by Akt/PKB, thereby
shifting the equilibrium towards production of active cyclin B-cdk1
complexes and resumption of meiosis (Fig. 7).
E. MAPK
Activation of the MAPK (mitogen-activated protein kinase) cascade is
associated with resumption of oocyte meiosis in many different species. Mos
codes for a serine/threonine kinase that functions upstream of MAP kinase,
and its normal expression is confined to germ cells. Oocytes accumulate mos
mRNA, which is translated into protein during meiotic maturation and
causes activation of downstream MAPK. For a number of years, it was
inferred that the signal inducing meiotic resumption is transduced through
Figure 7 Signaling pathways regulating the state of MPF activation in the maturing oocyte
(details in text). In red: protein kinase A activity is inhibitory to MPF activation. In green: PI3K
activity resulting in Akt/PKB activation leads to activation of MPF (see color plate).
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Voronina and Wessel
MAPK and requires mos protein accumulation, based on the ability of mos
and its downstream targets to induce hormone-independent maturation
(reviewed in Dupre et al., 2002). Furthermore, initial experiments in
Xenopus suggested that mos synthesis is required for GVBD. However, later
data obtained in various animals and with many techniques such as specific
MAPK inhibitors (starfish, Sadler and Ruderman, 1998), transgenic mos
RNAi (mouse, Stein et al., 2003), mos gene knockouts, mos DNA antisense
(mouse, O’Keefe et al., 1989), and morpholino antisense treatments
(Xenopus, Dupre et al., 2002) suggest that MAPK activation is not required
for the resumption of meiosis (or GVBD) in any animal. Instead, MAPK
activity plays a role in acceleration of MPF activation in the oocyte at
meiotic resumption (Dupre et al., 2002), and is needed for suppression of
interphase (DNA replication) between two divisions of meiosis and
subsequent arrest at the second metaphase of meiosis (Dupre et al., 2002;
O’Keefe et al., 1989; Stein et al., 2003).
VI. Concluding Remarks
In this review of the signaling networks bringing about oocyte maturation
of different species, we encountered significant variability among animals
at almost every level. The maturation-inducing hormones range from
a nucleotide (starfish) and complex sterols (vertebrates) to a protein
(nematode), or even to the uncertainty of whether such physiological
inducers even exist (mouse). The receptors of these hormones are mostly
unknown, but the evidence for them ranges from a receptor tyrosine kinase
(nematode), to a seven-transmembrane G-protein-coupled receptor (fish and
starfish), to a cytoplasmic transcription factor hormone receptor, or an
unidentified membrane receptor (Xenopus). Consequently, the identity of the
signaling mediators varies, and the heterotrimeric G-proteins, which are
strongly implicated in the induction of oocyte maturation in certain species
(starfish), appear inhibitory to maturation in other species (mouse,
Xenopus), or not involved in regulation of oocyte maturation in yet other
species (nematode). Significant discrepancies are encountered even in the
involvement of the particular second messengers such as levels of cAMP,
calcium, or phosphatidylinositol-3,4,5-trisphosphate (product of PI3K
activity) in the regulation of meiotic resumption. The common thread to
all these regulatory mechanisms appears to be the final downstream
requirement to activate the cyclin B-cdk1 complex (MPF).
To identify more common principles of oocyte maturation regulation, it
would be useful to compare and contrast the regulatory mechanisms present
in evolutionary close organisms, which nevertheless exhibit noticeable
differences in the progression of oocyte maturation. An example of a pair
3. The Regulation of Oocyte Maturation
99
suitable for comparative studies is starfish and sea urchin. Since they are
both members of the echinoderm phyla, we would expect to find similarities
in their oocytes.
However, several aspects are divergent. For example, the starfish
stores its gametes as oocytes, and resumption of oocyte meiosis coincides
(and is caused by the same hormone, 1-MA) with ovulation and release of
gametes by the animal. In contrast, the sea urchin stores its gametes
as mature eggs, which have already resumed and completed meiotic
division within the gonad. Accordingly, fertilization occurs at different
meiotic stages in these animals. Furthermore, meiotic divisions are relatively
quick in starfish (90 min), while in sea urchin meiosis takes considerably
longer (9 h).
Sea urchin eggs have been an extremely popular model for studies of
fertilization and early development. Nevertheless, the regulation of sea
urchin oocyte maturation has not been thoroughly scrutinized despite
providing an important advantage of ‘‘divorcing’’ oocyte maturation and
egg activation. We find it very strange that something as fundamental
as meiotic maturation and fertilization appears to have diverged in
mechanisms so greatly between animals, when other key developmental
events are far better conserved. Perhaps this divergence in germ cells reflects
the positive selection pressure on the genes involved in reproduction relative
to the rest of the genome (Swanson and Vacquier, 2002). Finding other
highly divergent genes in the future may be suggestive of their involvement
in this process. In addition, with recent advances in genomics, development
of databases comparing gene expression patterns in the oocytes of various
animals might help in identifying new important molecular regulators of
oogenesis on the basis of evolutionarily conserved pattern of expression
(Schlecht and Primig, 2003).
Acknowledgments
The authors would like to thank Laurinda Jaffe for the helpful comments on this manuscript.
Our research is supported by grants from the NSF and the NIH.
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