Vol. 9, No. 3 203

Copyright © 2009 by the Society for Biology of Reproduction

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 inferti-lity 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.

1Corresponding author: CNRS-UMR 6061, University of Rennes 1, Institute of Genetics & Develop-ment, “Mitosis & Meiosis” Group, IFR 140 GFAS, Faculty of Medicine, 2 Av. Prof. Léon Bernard, CS 34317, 35043 Rennes Cedex, France; email: jacek.kubiak@univ-rennes1.fr

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Key words: cell cycle, cytoplasmic maturation, EP45, female gamete forma-tion, 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 em-bryonic genome. However, no transcription occurs at the beginning of em-bryo 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 de-velopmental 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 un-dergo 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 oo-genesis 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 oo-cyte’s ability to undergo: 1/ nuclear maturation, 2/ successful fertiliza-

Figure 1. Schematic representation of oogenesis, oocyte maturation and fertil-ization in Xenopus laevis. During oogenesis, oocytes in all stages (IVI) are arrested at prophase of meiosis I. During this long period, oocytes accumulate different molecules (mostly mRNA, proteins, lipids) necessary for oocyte matu-ration 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, fertiliza-tion 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 re-sumption 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 activa-tion of the embryonic genome.

Nuclear maturation covers chromatin changes during oocyte matura-tion 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 oo-cyte 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 ter-minate 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 fertiliz-able oocyte.

The understanding of mechanisms and processes involved in nuclear and cytoplasmic maturation relies on the identification of involved mo lecules 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 im-munodepleted) in such extracts [39, 53]. Moreover, large oocytes can also be easily injected with mRNA or proteins to observe effects of overexpres-sion of chosen proteins. By analogy, intra-oocyte injection of morpholino-oligonucleotides inhibits the translation of selected mRNA and consequently reduces protein expression.

Here, we present processes important for the regulation of nuclear matu-ration 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 stimula-tion (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 cor-relates 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 matura-tion 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 matura-tion 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 fer-tilization 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 divi-sion 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 be-ginning of Meiosis II (MII). It remains high during MII-arrest of oocytes and de-creases 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 phosphory-lation state: the activating phosphorylation is achieved through the CDK1- and 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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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.

Inhibition of PKA. The binding of progesterone to its receptor in oocytes induces rapid inhibition of adenylate cyclase which provokes a drop in the cy-toplasmic 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

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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 proges-terone 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 cyclohexim-ide) prevents MPF activation. It was shown that the translation of specific maternal mRNAs stored in oocytes is necessary for the initiation of me iotic 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 appear-ing 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 sub-strates of MAPK/ERK2 is p90rsk. At the end of the kinase cascade, p90rsk directly phosphorylates, and thus inhibits, Myt1, which leads to MPF acti-vation (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 in-hibits 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 inhibi-tion of both pathways prevents M-phase entry [26]. Thus, inhibition of PKA after progesterone stimulation leads to multiple events (Cdc25 phosphory-lation, de novo synthesis of Mos, cyclin B and Ringo) which collectively trigger the initiation of MPF activation by producing a small amount of ac-tive 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 pro-gesterone 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 regu-lators 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 homo-logue 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 phos-phatase 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 overex-pression 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 in-competent 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 sug-gest 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 oo-cyte 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 mi-tochondria 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 com-petences [54, 56]. The relocalization of mitochondria may be related to increased production of energy required for the maturation process (pro-tein 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 pro-phase 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 clus-ters 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 dur-ing 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 phe-nomenon 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 spe-cies. 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 nec-essary 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 dis-tribution 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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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, microfila-ments of F-actin and intermediate filaments. Microtubules (MTs) and actin filaments are polymers of globular subunits, respectively of α-β tubulin and G-actin. 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 redis-tribution (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 forma-tion of the meiotic spindle formed of microtubules linked to chromosomes, 2/ the transient presence of cytoplasmic MTs and 3/ the relocalization of micro-filaments from cytoplasm to oocyte cortex and around the chromatin.

In porcine and bovine oocytes, MTs are not detected in prophase I-arrested (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 progres-sion 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 matura-tion 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 chro-matin. 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 posi-tioning [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 matura-tion 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 ac-tivation 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 poly-adenylation 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 in-teracts 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 matura-tion 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 de-velopmental competences. In accordance with this idea, Brevini and colleagues [5] showed in cattle that an abnormal level of mRNA polyadenylation is cor-related 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 com-petent 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 ex-pressed 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 cer-tain 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 com-pared by 2D-electrophoresis (screen 1). After silver staining, protein spots of dif-ferent 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.

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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 oo-cytes (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 regu-lation of cytoplasmic and nuclear maturation in Xenopus. By analogy, mam-malian 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 pos-sible 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 As-sociation 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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