Vol. 9, No. 3
203
REVIEW
Role of oocyte quality in meiotic maturation
and embryonic development
Gaëlle Marteil, Laurent Richard-Parpaillon, Jacek Z. Kubiak1
CNRS-UMR 6061, University of Rennes 1, IFR 140 GFAS, Rennes, France
Received: 25 May 2009; accepted: 5 October 2009
SUMMARY
The quality of oocytes plays a key role in a proper embryo development.
In humans, oocytes of poor quality may be the cause of women infertility and an important obstacle in successful in vitro fertilization (IVF).
The competence of oocytes depends on numerous processes taking place
during the whole oogenesis, but its final steps such as oocyte maturation,
seem to be of key importance. In this paper, we overview factors involved
in the development of a fully functional female gamete with Xenopus laevis
as a major experimental model. Modern approaches, e.g. proteomic analysis,
enable the identification of novel proteins involved in oocyte development.
EP45, called also Seryp or pNiXa, which belongs to the serpin (serine
protease inhibitors) super-family is one of such recently analyzed proteins.
This protein seems to be involved in the stimulation of meiotic maturation
and embryo development. EP45 is potentially a key factor in correct oocyte
development and determining the quality of oocytes. Reproductive Biology
2009 9 3: 203-224.
Corresponding author: CNRS-UMR 6061, University of Rennes 1, Institute of Genetics & Development, “Mitosis & Meiosis” Group, IFR 140 GFAS, Faculty of Medicine, 2 Av. Prof. Léon Bernard,
CS 34317, 35043 Rennes Cedex, France; email: [email protected]
1
Copyright © 2009 by the Society for Biology of Reproduction
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Key words: cell cycle, cytoplasmic maturation, EP45, female gamete formation, MPF, meiotic maturation, oocyte, Xenopus laevis
INTRODUCTION
High quality gametes produce well-developed embryos. Genomes of both
the oocyte and the spermatozoon participate equally in creation of the embryonic genome. However, no transcription occurs at the beginning of embryo development, so the very first steps of embryogenesis are controlled
exclusively by maternal information present in the oocyte. Therefore,
the embryonic genome has almost no impact on the earliest steps of the developmental program whereas the quality of maternal information plays
a major role during this period.
After fertilization, ooplasm becomes the embryo cytoplasm, while
the spermatozoon’s participation in this process is minimal. For this reason,
the quality of oocytes is a key factor in determining the quality of the earliest
steps of embryo development. Paradoxically, the simple notion of a “good
quality oocyte” is very complex. Factors affecting the first steps of embryo
development accumulate throughout the oogenesis period but the nature
of them is unknown.
Many case reports present an inability of the human oocyte to undergo successful meiotic maturation and in vitro fertilization (e.g. [4, 38,
51, 63]). However, such cases of idiopathic infertility are very difficult to
be explained in sporadically identified patients. An understanding of their
origin and research on adequate infertility treatments would require better
characterization of regulatory mechanisms governing oocyte maturation
in animal models [69]. Similarities in regulatory pathways and mechanisms
operating in oocyte development in different vertebrate species suggest new
avenues for human research [61, 71].
In vertebrates, oocytes are arrested for several weeks, months or years
in prophase of the first meiotic division. During this long period, oocytes
accumulate molecules of mRNA, proteins, lipids and sugars as well as
gradually increase in size. Accumulation of all necessary sources of energy
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and information during oocyte growth is essential for the final step of oogenesis i.e. oocyte maturation.
Meiotic maturation consists of two interlinked and mutually dependent
processes: cytoplasmic and nuclear maturation (fig. 1). The cytoplasmic
maturation of the oocyte includes cytoplasmic changes (e.g. organelle
redistribution, micro- and macromolecular changes) that occur during
the time of oocyte maturation. These modifications contribute to the oocyte’s ability to undergo: 1/ nuclear maturation, 2/ successful fertiliza-
Figure 1. Schematic representation of oogenesis, oocyte maturation and fertilization in Xenopus laevis. During oogenesis, oocytes in all stages (IVI) are
arrested at prophase of meiosis I. During this long period, oocytes accumulate
different molecules (mostly mRNA, proteins, lipids) necessary for oocyte maturation and the beginning of embryonic development. Oocyte maturation can
be divided into two processes: cytoplasmic maturation and nuclear maturation.
Cytoplasmic maturation consists of cytoplasmic modification occurring during
oocyte maturation. These changes are necessary for nuclear maturation, fertilization and very early steps of embryonic development. Nuclear maturation consists
of chromatin changes from prophase I to metaphase II. Only fully grown stage
VI oocytes can resume meiosis under the control of progesterone and become
arrested at metaphase of meiosis II at the end of oocyte maturation. Meiotic resumption is characterized by the appearance of a “white spot” on the animal pole
of the oocyte which is the consequence of germinal vesicle breakdown (GVBD;
Mature VI).
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tion, 3/ cleavages and 4/ development at least until the activation of the
embryonic genome (midblastula transition or MBT in Xenopus laevis).
In many cases, maternal information does not disappear precisely at that
time and it may participate in embryo development beyond the full activation of the embryonic genome.
Nuclear maturation covers chromatin changes during oocyte maturation from germinal vesicle breakdown (GVBD) throughout Meiosis I (MI)
to Meiosis (MII). In vertebrates, complete nuclear maturation leads to
the formation of an oocyte arrested in metaphase II. At this stage the oocyte is physiologically prepared to complete the second meiotic division
upon fertilization. Only fully grown oocytes can resume meiosis which
implies that cytoplasmic changes occurring before oocyte maturation
are essential for the acquisition of maturational competence. However,
the completion of oocyte growth is not an absolute determinant to terminate nuclear maturation. Even fully grown oocytes can suffer from
a partial cytoplasmic maturation that may impair nuclear maturation
[11, 16, 17]. Thus, a very complex interplay between all processes taking
place during the entire oogenesis determines the quality of the fertilizable oocyte.
The understanding of mechanisms and processes involved in nuclear
and cytoplasmic maturation relies on the identification of involved mo­lecules
and comes from studies on different model organisms. Xenopus laevis
presents several advantages for investigating the role of nuclear maturation
components: 1/ cell-free extracts arrested in metaphase II can be produced
in large quantity and 2/ proteins can be easily manipulated (added or immunodepleted) in such extracts [39, 53]. Moreover, large oocytes can also
be easily injected with mRNA or proteins to observe effects of overexpression of chosen proteins. By analogy, intra-oocyte injection of morpholinooligonucleotides inhibits the translation of selected mRNA and consequently
reduces protein expression.
Here, we present processes important for the regulation of nuclear maturation and the acquisition of cytoplasmic maturation. Special interest will
be paid to proteomic analysis of oocyte maturation. Finally, preliminary data
of our proteomic screens on Xenopus laevis oocytes will be presented.
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NUCLEAR MATURATION
Oocyte maturation begins with the resumption of meiosis. In vertebrates,
oocytes are arrested at prophase of the first meiotic division (fig. 1). Only
fully grown oocytes can resume meiosis in response to hormonal stimulation (e.g. progesterone routinely used in Xenopus). Oocytes pass through
the first meiotic division and then become arrested at metaphase of the
second meiotic division until fertilization. The arrest in prophase I correlates with the insensitivity to hormone action which prevents precocious
nuclear maturation of small oocytes. Xenopus oocytes have been widely
used to determine molecular mechanisms responsible for nuclear maturation and particularly to reveal the identity of meiotic M-phase regulators
[3, 8, 9, 10, 13, 15, 18, 20-23, 25, 27, 29, 31-33, 39, 40, 42, 44, 45, 50, 52, 53,
58-60, 62, 65, 70, 78].
Initiation of GVBD
The first studies on nuclear maturation showed that germinal vesicle
breakdown (GVBD) was due to the activation of a cytoplasmic maturation promoting factor (MPF; [44]). Further studies have shown that MPF
was a universal regulator of M-phase entry, and consequently MPF was
designated as an M-phase promoting factor. Almost twenty years later,
molecular studies on frog oocytes and yeast have enabled the purification
of MPF from Xenopus oocytes. The studies revealed that MPF is composed
of a kinase subunit, CDK1 (cyclin-dependent kinase 1; initially called p34cdc2
as a product of cdc2 gene for “cell division control 2” identified in yeasts)
associated with a regulatory subunit - cyclin B [21, 40]. During maturation
of the Xenopus laevis oocyte, MPF activity appears just before GVBD (fig. 2)
and declines at the end of the first meiotic division [23]. Then, it reappears
at the beginning of the second meiotic division and remains high until fertilization due to the MPF-stabilizing action of the cystostatic factor (CSF;
[44]). The study of MPF regulation enabled us to understand the molecular
basis of the progression of not only oocyte maturation but also of the division cycle of other cells.
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Figure 2. Profile of Maturation Promoting Factor (MPF) activity during oocyte
maturation in Xenopus laevis. MPF activity appears just before germinal vesicle
breakdown (GVBD). It falls down at the end of Meiosis I and reappears at the beginning of Meiosis II (MII). It remains high during MII-arrest of oocytes and decreases following fertilization.
In immature oocytes, MPF is already present but in an inactive form
(pre-MPF) with CDK1 phosphorylated by Myt1 on Tyr-15 and Thr-14 [15].
The removal of inhibitory phosphorylations by phosphatase Cdc25 leads to
MPF activation [22]. The Cdc25 activation also depends on its phosphorylation state: the activating phosphorylation is achieved through the CDK1and polo-like kinase-dependent phosphorylation. More recent studies have
demonstrated that MAPK is also directly involved in the activation of Cdc25
during G2/M transition [76]. On the other hand, inhibitory dephosphorylation
of Cdc25 is carried out by phosphatase 2A [32]. The pre-MPF is, therefore,
kept inactive in prophase I-arrested oocytes by a network of multiple kinases
and phosphatases whose equilibrium is controlled by a series of feedbacks
between them.
Signalling pathways leading to MPF activation
during nuclear maturation
Experimental studies on Xenopus oocytes allowed better characterization
of the signalling pathways leading to MPF activation after progesterone
stimulation.
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Inhibition of PKA. The binding of progesterone to its receptor in oocytes
induces rapid inhibition of adenylate cyclase which provokes a drop in the cytoplasmic cAMP level and consequent inhibition of PKA activity (fig. 3). This
phenomenon is essential for initiation of the meiotic maturation [41,42,75].
Indeed, progesterone-induced maturation can be inhibited by an injection
of PKA catalytic subunit. Molecular mechanisms linking the inhibition
of PKA and MPF activation remain, however, unclear. It has been shown
that PKA has numerous targets. In Xenopus laevis [13] and in the mouse
[57], the inhibitory phosphorylation of Cdc25 by PKA leads to the oocyte
arrest in prophase I. Moreover, PKA blocks de novo synthesis not only of
Figure 3. Maturation Promoting Factor (MPF) activating pathways. The binding
of progesterone to its receptor triggers numerous events leading to MPF activation:
activation of Mos/MAPK pathway, de novo synthesis of Cdc2 (CDK1) partners
as cyclin B and Ringo as well as inhibition of PKA. The Mos/MAPK pathway
contributes to the activation of MPF via activation of Cdc25 phosphatase and Myt1
kinase inhibition. De novo synthesis of CDK1 partners generates a small amount
of MPF which activates Plk and Cdc25 leading to MPF autoamplification. PKA
inhibition plays a central role via participation in activation of the Mos/MAPK
pathway, Cdc25 phosphorylation and stimulation of de novo synthesis of Cdc2
partners (dotted arrows); PP2A: protein phosphatase2A, PKA: protein kinase A,
MAPK: mitogen activated protein kinase, Cdc2: cell division control 2 or CDK1,
Plk: polo-like kinase; for details see text of the article.
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CDK 1-binding proteins belonging to the cyclin B family [20], but also of
Mos, an activator od MAPK pathway involved in nuclear maturation as well
([45]; see the following paragraph).
Protein synthesis. One of the key effects of PKA inhibition after progesterone stimulation is the synthesis of new proteins. This plays a pivotal
role in triggering the initiation of meiotic maturation. Accordingly, protein
synthesis inhibition in prophase I-arrested oocytes (e.g. with cycloheximide) prevents MPF activation. It was shown that the translation of specific
maternal mRNAs stored in oocytes is necessary for the initiation of me­iotic
maturation. One of the main regulators of M-phase entry is a proto-oncogene
c-Mos, a serine/threonine kinase, absent in immature oocytes and appearing during progesterone-induced oocyte maturation [62]. An activator
of the MAPK/ERK2 pathway – Mos activates MEK (MAPK kinase) which,
in turn, activates MAPK/ERK2 in Xenopus oocytes (in mouse oocytes,
ERK1 and ERK2 are concomitantly activated). One of the important substrates of MAPK/ERK2 is p90rsk. At the end of the kinase cascade, p90rsk
directly phosphorylates, and thus inhibits, Myt1, which leads to MPF activation (for review see [8]). In agreement with those data, injections of Mos
or the downstream kinases trigger nuclear maturation without the need
of progesterone stimulation [25, 27, 78].
Progesterone action also provokes synthesis of CDK1-partners, such
as B-type cyclins. Accordingly, an experimental injection of cyclin B into
prophase I-arrested oocytes rapidly induces GVBD. Newly synthesized
or exogenous cyclin B associates with CDK1 (present in large excess
in oocytes), and generates small amounts of active MPF. This, in turn,
initiates a positive feedback loop by phosphorylating and activating Cdc25
and polo-like kinase Plx1. Concomitant activation of this phosphatase
and this kinase allows extremely rapid and efficient MPF amplification
by removing the inhibitory phosphorylations of the stockpile of pre-MPF
and consequently inducing a burst of MPF. However, newly synthesised
cyclin B is not absolutely required for MPF activation during meiosis I, but
it is necessary for the second wave of MPF activation during meiosis II [29,
50]. Another regulatory subunit of CDK1, different from cyclin, is Ringo
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[18], which is also able to induce MPF activation. Translation of this CDK1
partner seems to be essential for MPF activation since its knock-down inhibits progesterone-induced maturation [18].
In summary, it seems that two distinct pathways can induce meiotic
maturation: the Mos/MAPK pathway and the synthesis of CDK 1 regulators.
A recent study shows that these two pathways are redundant i.e. inhibition
of either does not prevent nuclear maturation, whereas concomitant inhibition of both pathways prevents M-phase entry [26]. Thus, inhibition of PKA
after progesterone stimulation leads to multiple events (Cdc25 phosphorylation, de novo synthesis of Mos, cyclin B and Ringo) which collectively
trigger the initiation of MPF activation by producing a small amount of active MPF. The appearance of the threshold amount of active MPF triggers
a positive feedback loop characterized by the activating phosphorylation
of Cdc25 by MPF itself, and by activation of polo-like kinase leading to
auto-amplification of MPF activity.
Regulators that are missing in progesterone-insensitive oocytes. Oogenesis
in Xenopus is divided into six major stages (stage I to stage VI; [14]). Only
fully grown stage VI oocytes can resume meiosis under the control of progesterone while oocytes in the other stages are insensitive to this hormone.
The ability to enter the first meiotic M-phase seems to rely on certain regulators appearing or being activated in stage VI and missing or present in an
inactive form in the remaining stages. The stage IV small oocytes contain
functional progesterone receptors and their stimulation by progesterone
induces a decrease in cAMP level [52]. Therefore, the inability to resume
meiosis in these oocytes is caused by factors located downstream from
the cAMP level. Small oocytes also contain an inactive pool of MPF (i.e.
pre-MPF) and MPF-activating phosphatase Cdc25 but are incompetent for
MPF auto-amplification [59, 60]. Among the known regulators of the MPF
auto-amplification loop, Plx1 is the only one absent in incompetent oocytes at
stage IV [33]. Accordingly, the microinjection of Plk1 mRNA (human homologue of Plx1) into stage IV oocytes allows the MPF auto-amplification loop
to be restored both in vivo and in vitro. To observe this effect, protein phosphatase 2 (PP2) must be inhibited by okadaic acid. Thus, Plx1 is the missing
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regulator responsible for Cdc25 activation during the auto-amplification loop
and, consequently, for the MPF auto-amplification incompetence of stage
IV oocytes. In addition, Plx1 seems to restore the MPF auto-amplification
loop via down-regulation of Myt1 [33, 55].
In stage IV oocytes, the progesterone stimulation does not lead to Mos
synthesis and, consequently, cannot activate the MAPK/ERK2 pathway,
even though both ERK2 and p90rsk are present. Moreover, the Mos overexpression in incompetent oocytes activates the MAPK pathway but does not
trigger MPF activation as it is in fully grown stage VI oocytes [33]. Thus,
the links between MAPK/ERK2 and MPF are clearly not functional in incompetent oocytes. Interestingly, a similar absence of reciprocal feedback
between these two major M-phase kinases (MPF and ERK2) was reported
in Xenopus embryos during the first embryonic mitosis [3]. This may suggest that the full dialog between ERK2 and MPF operates only in stage
VI and maturing/mature oocytes. Further studies on stage IV oocytes are
necessary to better characterize the oocyte incompetence.
CYTOPLASMIC MATURATION
Cytoplasmic maturation plays an important role in the development of oocyte
competences. Selection of good quality oocytes is particularly important for
the improvement of assisted reproduction. For this reason the cytoplasmic
maturation of oocytes is widely studied in mammals and is less documented
in Xenopus. Cytoplasmic maturation is often divided into three major
processes: 1/ organelle redistribution, 2/ cytoskeleton dynamics, and 3/
molecular maturation [19].
Organelle redistribution
Mitochondria. One of the earliest studies on mitochondria reorganization
during oocyte maturation [68] was performed by Van Blerkom and Runner
on fixed mouse oocytes [73] and confirmed later by Calarco [7] in a live oocyte study. In mouse, porcine and bovine oocytes, a homogenous distribution
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of mitochondria in the cell cortex is observed before the beginning of oocyte
maturation, whereas a translocation of mitochondria to the perinuclear region
is observed following GVBD [7, 66, 68, 73]. After nuclear maturation, large
foci of mitochondria are dispersed throughout the cytoplasm except the very
central region of oocytes. This mitochondrial reorganization appears to
be a very important event for the nuclear maturational competence. Indeed,
oocytes with a low maturation rate show no mitochondria redistribution [66].
In addition, oocytes which failed to mature show a distinct mitochondria
redistribution pattern characterized by the presence of small clusters of mitochondria in the cellular cortex and around GV [7]. More recent studies on
mice confirmed that the aggregation of mitochondria around the nucleus
is correlated with the acquisition of maturational and developmental competences [54, 56]. The relocalization of mitochondria may be related to
increased production of energy required for the maturation process (protein phosphorylation, protein synthesis etc.). Studies on bovine and human
oocytes have correlated the maturational competences with mitochondria
reorganization and ATP level [66, 74]. Actually, oocytes which undergo
a correct reorganization of mitochondria during maturation, have a higher
ATP content and better developmental capacity than the oocytes with no
mitochondria relocalization. Nevertheless, the nuclear maturation does not
seem to be directly correlated to ATP content because meiotic maturation
takes place in a wide range of ATP levels in mouse and human oocytes.
Moreover, the ATP content is not modified by the time of the first polar
body extrusion in bovine oocytes [66, 74]. Thus, the direct relationships
between mitochondria and maturational or developmental competences
remain unclear and need further studies.
Endoplasmic reticulum. Endoplasmic reticulum (ER) is the organelle where
protein and lipid synthesis occurs. ER is also implicated in Ca2+ intracellular
regulation because it is the major cellular store of Ca2+. For this reason, ER
plays an essential role in oocyte activation during fertilization. After oocyte
activation, Ca2+ release from ER is mediated by IP3 receptor located on the ER
membrane. Structural changes in ER have been observed in numerous animal
species during oocyte maturation. In mature mouse oocytes, an accumulation
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of ER in the oocyte cortex, specifically in the region with cortical granules
(CG), may be observed. This accumulation was not found in immature prophase I arrested oocytes [47]. In addition, a fine reticular network is present
throughout the MII oocyte cytoplasm, whereas homogenous large clusters
of ER are found in deeper cytoplasm of immature oocytes. Thus, during
nuclear maturation, an accumulation of ER clusters is observed in the same
region as CG exocytosis and sperm-egg fusion. In Xenopus, large ER clusters appear in the vegetal cortex in mature, stage VI oocytes [70]. In various
species, ER clusters accumulate on the side opposite to the meiotic spindle.
In addition to ER reorganization, an increase in IP3 receptor is observed during mouse oocyte maturation [48]. These events could explain why calcium
release after fertilization can be efficiently enhanced [46].
Cortical granules. In many vertebrate and invertebrate species, exocytosis
of cortical granules (CGs) after spermatozoon penetration into the oocyte
triggers a rapid modification of extracellular matrix of oocytes. This phenomenon plays an essential role in the block against polyspermy assuring
that only one spermatozoon enters the oocyte upon fertilization. During
oocyte maturation, a reorganization of CGs is observed in various species. In immature mouse oocytes, CGs are found in the whole cytoplasm
(the cortex cytoplasm and the inner cytoplasm) whereas in mature oocytes,
an asymmetric distribution of CGs in the cortex is observed with no CGs
around the MII spindle [12]. In porcine and bovine oocytes, the CGs also
migrate to the cortex. However, they form a continuous layer just underneath
the membrane during oocyte maturation [30, 77]. The relocalization of CGs
during maturation prepares oocytes for CGs extrusion upon fertilization.
Lipid droplets. In oocytes, lipid droplets (LD) constitute energy supply necessary for meiotic maturation, fertilization and early embryo development.
During meiotic maturation and fertilization, a rearrangement of LD has been
observed in horses and pigs [24, 34]. In equine oocytes, a homogenous distribution has been reported in GV-stage oocytes contrary to mature oocytes,
in which LD contribute to the oocyte polarization by surrounding the MII
spindle [24]. A recent study shows the relationship between the polar aggre-
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215
gation of liquid droplets and nuclear maturation [1]. Oocytes with uniform
distribution of LD show a lower maturation rate as compared to oocytes with
polarized distribution of LD. However, the fertilization rate and early embryo
development seem not to be influenced by LD organization [1].
Cytoskeleton dynamics
Cytoskeleton is composed of three types of filaments: microtubules, microfilaments of F-actin and intermediate filaments. Microtubules (MTs) and actin
filaments are polymers of globular subunits, respectively of α-β tubulin and Gactin. Intermediate filaments consist of tetramers of fibrous polypeptides.
Among these filaments, microtubules and actin filaments are most specifically
involved in oocyte maturation. Microtubules participate in organelle redistribution (principally mitochondria) and in chromosome segregation within
the meiotic spindle. Actin filaments also take part in dynamic events during
oocyte maturation and fertilization by controlling CG and ER redistribution
and chromosomes positioning [67, 70]. During nuclear maturation a drastic
reorganization of the cytoskeleton occurs. It is characterized by 1/ the formation of the meiotic spindle formed of microtubules linked to chromosomes, 2/
the transient presence of cytoplasmic MTs and 3/ the relocalization of microfilaments from cytoplasm to oocyte cortex and around the chromatin.
In porcine and bovine oocytes, MTs are not detected in prophase Iarrested (immature) oocytes, whereas asters of MTs associate with chromatin
during GVBD. This association is maintained during meiotic maturation
allowing proper positioning and segregation of chromosomes. The important
role of MTs in nuclear maturation has been clearly demonstrated in vitro,
since the inhibition of MTs polymerization by nocodazole inhibits progression in metaphase I [6, 35, 36]. In porcine oocytes, cytoplasmic MTs also
emanate from the cortex to the inner cytoplasm during in vitro maturation
and disappear at the end of meiotic maturation. Such a cytoplasmic network
seems to be crucial for the acquisition of cytoplasmic maturation and thus
developmental competences. Indeed, a transient network of cytoplasmic
MTs is observed in well-maturing oocytes during maturation whereas
oocytes with low developmental competences show no cytoplasmic MTs.
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Interestingly, the chromatin – linked MTs organization is the same in high
and low competent oocytes [6].
Actin microfilaments also undergo reorganization during meiotic maturation in mouse, porcine, bovine and equine oocytes [35, 36, 43, 72]. During
the GV stage, microfilaments are distributed throughout the cytoplasm,
and during GVBD they migrate toward the oocyte cortex and around chromatin. During metaphase II, microfilaments surround the meiotic spindle.
Inhibition of microfilament polymerization by cytochalasin B has no effect
on GVBD and metaphase but prevents chromatin against its correct positioning [36]. Recently, it was shown that actin microfilaments participate
in formation of the first meiotic spindle and that they play a major role
in the meiotic spindle positioning, probably by the generation of pushing
and pulling forces during oocyte maturation [2, 64]. In conclusion, both MTs
and actin filaments participate actively in proper positioning and segregation
of chromosomes during meiotic maturation. Reorganization of the oocyte
cytoskeleton seems to be a crucial event not only for nuclear maturation but
also for the acquisition of developmental competences.
Molecular maturation
No gene transcription occurs between the beginning of the nuclear maturation of the oocyte and the embryonic genome activation at the MBT. Thus,
proteins (or their mRNA) involved in the regulation of nuclear maturation,
fertilization and early embryo development are stored in oocytes during
oogenesis in an inactive but stable form until needed.
mRNA translation is regulated in part by cytoplasmic polyadenylation (for
review see [58]). Briefly, dormant mRNAs have a short polyA tail and the activation of translation requires mRNA polyadenylation. Different sequences
in 3’ UTR region of mRNA are important for cytoplasmic polyadenylation:
1/ cytoplasmic polyadenylation element (CPE) rich in U, 2/ a hexanucleotide
AAUAAA, 3/ a C-rich region, and 4/ polyadenylation response element (PRE).
These sequences are recognized by RNA binding proteins which control polyadenylation and subsequently the translation of mRNA. One of these proteins
is Musashi that binds to some PREs and controls temporal order of mRNA
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translation during Xenopus oocyte maturation [10]. For instance, Musashi interacts with the PRE sequence in 3’UTR of c-Mos and consequently regulates
c-Mos early polyadenylation and translation during meiotic maturation [9].
Another well-characterized RNA binding protein essential for nuclear maturation is CPEB, CPE binding protein (for review see [49]). This protein regulates
cytoplasmic polyadenylation and translation of some M-phase regulators as
cyclins and CDK2 [65]. Inhibition of CPEB by antibody injection prevents
polyadenylation of some regulators of M-phase entry and consequently blocks
meiotic maturation [65]. c-Mos is one of those regulators, and CPEB seems
to participate in early polyadenylation and translation of this kinase.
Cytoplasmic polyadenylation seems to be an essential event for nuclear
maturation by controlling the translation of important regulators of M-phase
entry [37, 65]. This process also seems to participate in the acquisition of developmental competences. In accordance with this idea, Brevini and colleagues
[5] showed in cattle that an abnormal level of mRNA polyadenylation is correlated with a decrease in the ability of the embryo to develop properly.
SEARCH FOR NOVEL REGULATORS OF OOCYTE
MATURATION: THE PROTEOMIC APPROACH
In order to identify new regulators of Xenopus laevis oocyte maturation,
differential proteomic screens were performed in our laboratory. Proteomes
of high-responsive stage VI oocytes vs. low-responsive stage VI oocytes
(screen 1; fig. 4) as well as incompetent stage IV oocytes vs. stage VI competent ones (screen 2; fig. 4) were compared. It should be emphasized that
low-responsive oocytes show a rate of maturation at about 36% at 24 hours
compared to 88% for high-responsive ones upon progesterone stimulation.
These screens allow us to identify a couple of proteins differentially expressed between the listed groups of oocytes (fig. 4).
Differences in the abundance of proteins in high- and low-responsive
stage VI oocytes may explain, at least partially, the oocytes’ phenotype
in respect to the reactivity to progesterone stimulation. The absence of certain proteins in stage IV oocytes could explain why these oocytes cannot
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Figure 4. Schematic representation of the search for new regulators of Xenopus
oocyte maturation by proteomic 2D analysis. A/ Fully grown, postvitellogenic
stage VI oocytes (VI) were tested for their capacity to mature upon progesterone
treatment. Cytoplasmic proteomes of low-responsive and high-responsive oocytes
arrested in prophase I (counterparts of oocytes used for maturation test) were compared by 2D-electrophoresis (screen 1). After silver staining, protein spots of different intensity were selected and identified by mass spectrometry. B/ Cytoplasmic
proteomes of vitellogenic stage IV oocytes (IV) and fully grown, postvitellogenic
stage VI oocytes (VI) were compared by 2D-electrophoresis (screen 2). After silver
staining, four proteins more abundant in stage VI oocytes than in stage IV were
identified by mass spectrometry.
Marteil et al
219
resume meiosis. Importantly, we did not find clearly less abundant proteins
in stage VI vs. stage IV oocytes, accordingly to the notion of continuous
accumulation of proteins in growing oocytes. Proteins identified in these
screens may be involved in both nuclear and cytoplasmic maturation.
One of the proteins clearly less abundant in stage IV than in stage VI oocytes (screen 2; fig. 4) is the estrogen-regulated protein 45 (45 kDa; also called
EP45, Seryp, pNiXa). We focused our attention on EP45 because previous
data suggested that it may be involved in oocyte maturation and in embryo
development in Xenopus laevis [28]. Recently, it was shown that EP45 is an
important component of yolk platelets in X. laevis and its degradation occurs
before the proteolysis of the major yolk proteins - vitellogenin derivatives,
lipovitellins 1 and 2 (LV1 and LV2, respectively; [31]). A direct role of EP45
in the rate of yolk consumption remains to be investigated. EP45 belongs
to serpin super family (serine protease inhibitors). Even if EP45 has indeed
the property to inhibit proteases, it might also be involved in processes other
than the regulation of oocyte and embryo nutrition. Analysis of this and other
proteins issued from our proteomic screens may shed a new light on the regulation of cytoplasmic and nuclear maturation in Xenopus. By analogy, mammalian homologues of amphibian proteins successfully identified as regulators
of oocyte maturation may be studied in mouse and human oocytes.
The complexity of regulatory mechanisms of oocyte maturation does
not allow to perform an efficient analysis on human oocytes as it is possible on animal models. The frog Xenopus laevis was a very useful model
for the last twenty years. Novel methodological approaches will certainly
make possible a further understanding of these processes in frogs as well
as in other species.
ACKNOWLEDGEMENTS
The authors were supported by grants from Ligue Contre le Cancer and Association pour la Recherche contre le Cancer (ARC 4900) to JZK. GM was
a recipient of a fellowship from the French Ministère de la Recherche et de
l’Enseignement supérieur.
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Oocyte quality
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