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Theriogenology 71 (2009) 836–848

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Cytoplasmic maturation of bovine oocytes: Structural and

biochemical modifications and acquisition of developmental
competence
E.M. Ferreira a,*, A.A. Vireque a, P.R. Adona b, F.V. Meirelles b,
R.A. Ferriani a, P.A.A.S. Navarro a
a
Department of Gynecology and Obstetrics, Faculty of Medicine of Ribeirão Preto, University of São Paulo,
14048-900 Ribeirão Preto, SP, Brazil
b
Department of Basic Sciences, Faculty of Zootechny and Aliments Engeneering, University of São Paulo,
13630-970 Pirassununga, SP, Brazil
Received 26 April 2008; received in revised form 14 October 2008; accepted 19 October 2008

Abstract
Oocyte maturation is a long process during which oocytes acquire their intrinsic ability to support the subsequent stages of
development in a stepwise manner, ultimately reaching activation of the embryonic genome. This process involves complex and
distinct, although linked, events of nuclear and cytoplasmic maturation. Nuclear maturation mainly involves chromosomal
segregation, whereas cytoplasmic maturation involves organelle reorganization and storage of mRNAs, proteins and transcription
factors that act in the overall maturation process, fertilization and early embryogenesis. Thus, for didactic purposes, we subdivided
cytoplasmic maturation into: (1) organelle redistribution, (2) cytoskeleton dynamics, and (3) molecular maturation. Ultrastructural
analysis has shown that mitochondria, ribosomes, endoplasmic reticulum, cortical granules and the Golgi complex assume different
positions during the transition from the germinal vesicle stage to metaphase II. The cytoskeletal microfilaments and microtubules
present in the cytoplasm promote these movements and act on chromosome segregation. Molecular maturation consists of
transcription, storage and processing of maternal mRNA, which is stored in a stable, inactive form until translational recruitment.
Polyadenylation is the main mechanism that initiates protein translation and consists of the addition of adenosine residues to the 30
terminal portion of mRNA. Cell cycle regulators, proteins, cytoplasmic maturation markers and components of the enzymatic
antioxidant system are mainly transcribed during this stage. Thus, the objective of this review is to focus on the cytoplasmic
maturation process by analyzing the modifications in this compartment during the acquisition of meiotic competence for
development.
# 2009 Elsevier Inc. All rights reserved.

Keywords: Cytoplasmic maturation; Bovine oocytes; Organelles; Cytoskeleton; Molecular maturation

1. Introduction

The complex events that occur during oocyte

* Corresponding author at: Departamento de Ginecologia e Obste- maturation depend not only on the correct dynamics of
trı́cia, Setor de Reprodução Humana, HCFMRP/USP, Av. dos Ban-
chromosome separation during nuclear maturation, but
deirantes, 3900, Monte Alegre, 14048-900 Ribeirão Preto, SP, Brazil.
Tel.: +55 16 3602 2821 fax: +55 16 3633 0946. also on the redistribution of cytoplasmic organelles and on
E-mail address: [email protected] (E.M. Ferreira). the storage of mRNA, proteins, and transcription factors

0093-691X/$ – see front matter # 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.theriogenology.2008.10.023
 E.M. Ferreira et al. / Theriogenology 71 (2009) 836–848 837

needed for this process to occur. The transcripts and Ultrastructural analysis of bovine oocytes subjected to
proteins stored in the cytoplasm of the oocyte are of in vitro maturation (IVM) has shown that the mitochon-
fundamental importance for the maturation process and dria move from a more peripheral position to a more
for ensuring the progression of early embryo development disperse distribution throughout the cytoplasm after 12–
to the eight-cell stage (in cattle), when the embryonic 18 h of culture [4]. This event is similar to what occurs in
genome is activated and the synthesis of new proteins vivo, which involves a more peripheral distribution
becomes necessary. This phase is denoted as embryonic before the luteinizing hormone (LH) surge, a clustered
genome activation (EGA) and the expression of certain cortical formation in the final stages of nuclear
genes during this period will determine the success of maturation, and a dispersed distribution after the
embryogenesis in the pre-implantation stage [1]. extrusion of the polar body, approximately 19 h after
Although they are distinct processes, nuclear the LH surge [5,6]. Upon reaching metaphase II (MII),
maturation and cytoplasmic maturation are interlinked the mitochondria in bovine oocytes, together with lipid
events that occur simultaneously at determined times, droplets, occupy a central position in the cell [6] (see
even though the molecular programming of the Fig. 1A). In addition, recent studies have shown that the
cytoplasm may have already started during the phase number of mitochondria present in the cytoplasm of
of oocyte growth. The cytoplasmic maturation process mammalian oocytes varies according to the stage of
can be divided into three main events: (1) redistribution development of the cell. During the pre-migratory stage
of cytoplasmic organelles, (2) dynamics of the of germ cells, the number of mitochondria is approxi-
cytoskeletal filaments, and (3) molecular maturation. mately 10 units, increasing to 200 units in the oogonium
Thus, the objective of the present review is to focus stage. Primary oocytes contain approximately 6000
on the process of cytoplasmic maturation, in light of the mitochondria, and this number increases to more than
changes that occur in this compartment during the 100,000 copies of mtDNA during oocyte maturation,
acquisition of meiotic competence for development. with one or two copies of mtDNA per organelle [7–10].
Evidence from heteroplasmic murine and human
2. Redistribution of cytoplasmic organelles oocytes, showed that the segregation of mtDNA variants
occurs as early as during the first mitotic divisions of
It is well established that several ultrastructural germinal cell precursors and that these variants are then
changes regarding morphology and redistribution can rapidly transmitted to future generations. Analysis of
be observed in cytoplasmic organelles during oocyte the distribution of pathogenic mtDNA mutations in the
maturation. Trafficking of cytoplasmic organelles offspring of carrier mothers shows that chances of
during maturation occurs through the actions of inheriting a pathogenic mutation increase with the
cytoskeletal microfilaments and microtubules and proportion in the mother, but there is no bias toward
repositioning of the organelles depends on the needs transmitting more or less of the mutant mtDNAs. This
of the cell during each stage of development. suggests that there is no strong selection against this
type of mutation. One of the possible factors responsible
2.1. Mitochondria for the variations in the amount of inherited mutant
mtDNA may be the massive reduction of these
The activation of determined metabolic pathways molecules that are transmitted by the mother. Observa-
involved in protein synthesis and phosphorylation is tions of this type gave rise to the concept of
indispensable for cytoplasmic maturation. Within this mitochondrial genetic bottleneck or developmental
context, mitochondria play an extremely important role bottleneck for the transmission of mtDNA [10,11].
since they are a key component of the metabolic Based on this concept, it was proposed that the large
machinery responsible for the supply of energy that is amount of mtDNA and, by inference, of the mitochon-
consumed during the maturation process [2,3]. The dria present in the oocyte, may represent a genetic
movement of mitochondria to areas of high energy mechanism to guarantee the distribution of these
consumption is crucial for the oocytes and the embryo organelles and molecules to the gametes and somatic
blastomere during critical periods of the cell cycle. cells of future generations. Thus, not only are the
Previous studies showed that during the maturation mitochondria important for oocyte metabolism, but also
period, mitochondria synthesizes the ATP necessary for their numbers are a predictor of functional competence,
the synthesis of proteins which, in turn, supports the since a reduced number of mitochondria may cause
completion of subsequent maturation processes and abnormal distribution of the organelle during early
embryo development [2,3]. embryogenesis [10].
838 E.M. Ferreira et al. / Theriogenology 71 (2009) 836–848

Fig. 1. Schematic overview of the distribution of cytoplasmic organelles during maturation, fertilization and bovine zygote formation. A. Nuclear
maturation progression and cytoplasmic organelle movement from the immature stage of germinal vesicle to the mature stage of metaphase II and
zygote formation. B. Organelle distribution and the mechanism of cortical granule content release, secondary to intracellular calcium (Ca2+) release
after the entry of the spermatozoon into the oocyte during fertilization.

Mouse oocytes derived from small antral follicles complement [15]. The presence of this subcortical
and matured in vitro had a reduced potential for region of highly polarized mitochondria in human and
development compared to oocytes of pre-ovulatory murine oocytes and embryos was proposed to be a
follicles and to oocytes matured in vivo. This statement microzone of differential activities related to the
is attributed to the observation of significant reductions acquisition of developmental competence [13,14],
in the number of mitochondrial DNA copies, the which may affect the normal occurrence of the
amount of ATP, and the proportion of oocytes with fertilization process [14].
peripheral mitochondrial distribution. This reduced The current discussion regarding the relation of
number of mitochondria may lead to a reduced amount mitochondria to the acquisition of developmental
of ATP, which is essential for subsequent stages of competence involves the definition of developmental
development [12]. Although these data demonstrated a potential itself, which is still controversial [9]. Zygotes
correlation between the variables analyzed (number of that initiate the first mitotic cleavages faster than others
copies of mitochondrial DNA, quantity of ATP and have a better probability of reaching the blastocyst stage
proportion of oocytes with peripheral mitochondrial and therefore have a better developmental potential. On
distribution versus developmental potential), they are this basis, the timing between the insemination and first
still insufficient to prove a causal relationship. cleavage is emerging as a determinant factor in defining
New data suggest that the mitochondria of human developmental competence [16]. Considering that
and murine oocytes and embryos may have hetero- during this process all events are controlled by the
geneous intracellular domains from the viewpoint of the molecular machinery within the oocyte [1], we can
inner mitochondrial membrane potential (deltapsim). establish a direct relationship between competent
The magnitude of deltapsim would be involved in some oocytes and zygotes with a high developmental
mitochondrial activities, including ion flow regulation potential. Among several other parameters, the relation-
and ATP release. Highly polarized mitochondria, i.e., ship between mitochondrial activity and distribution
mitochondria with high deltapsim, have a specific and (inherited from the mother, i.e., from the oocyte) and the
stable domain in the subcortical/sub-plasmalemmal amount of ATP during the early stages of development
region of the cytoplasm in these oocytes [13,14], and preceding EGA has also emerged as another important
represent a small fraction of the total mitochondrial factor to be considered in determining bovine embryo
 E.M. Ferreira et al. / Theriogenology 71 (2009) 836–848 839

competence [9], which is in contrast to the data reported transcripts, and by the addition of countless ribosomal
by Van Blerkom et al. [17] in a previous study on human proteins to their two subunits. The nucleolus is the site
embryos. of formation of the ribosomal subunits and during the
Recent studies on bovine and murine oocytes and phases of oocyte growth and activation of the
embryos have also correlated with the reorganization of embryonic genome the nucleolus is present in the
the mitochondria in the oocytes after IVM, the ATP fibrillogranular form, reflecting the high activity of
levels and the total number of cells in the blastocysts. ribosome synthesis and therefore protein synthesis. The
Embryos with less ATP in the cytoplasm had slower oocyte of a primordial follicle is transcriptionally
development and resulted in a smaller number of cells quiescent and the nucleolus is exclusively composed of
[3,18]. Before embryonic genome activation (approxi- the granular portion, signaling an absence of ribosome
mately 72 h of culture), mitochondria have intermediate synthesis activity [22,23]. During metaphase I of
levels of activity, a fact that may be explained by meiosis, protein synthesis in the oocyte is approxi-
adaptive protection against reactive oxygen species mately three times greater than that during the germinal
(ROS) as a result of mitochondrial metabolism [7,19]. vesicle breakdown (GVBD) stage (Fig. 1A). When the
This protection is provided by scavenger molecules cell reaches metaphase II, however, the oocyte exhibits
such as glutathione and peroxidases, which are basal levels of mRNA translation. Perhaps the absence
produced during molecular oocyte maturation or during of a functional nucleolus leads to an absence of rRNA
the 2-cell embryo stage by permissive transcription transcription or ribosome production for mRNA
[20]. Thus, if mitochondrial activity is high during the translation [24]. Previous evidence suggested that the
early stages of embryo development, the embryos production of ribosomes in the GV stage, where there is
probably would not survive, because they would be a functional nucleolus and therefore presence of rRNA
unable to eliminate the excessive production of ROS in transcription or ribosome production for mRNA
these processes [9]. After embryonic genome transcrip- translation, may favor greater storage of these
tion has begun, mitochondrial activity decreases as the organelles in the oocytes during the MI stage [17].
embryo starts to utilize other metabolic pathways such Thus, the higher levels of protein synthesis observed in
as anaerobic glycolysis to produce energy [9,21]. MI as opposed to MII in oocytes may be due to the
Perhaps mitochondria have an important role in the larger ribosomal storage observed during MI. As a
acquisition of oocyte competence during development. consequence of the high utilization of these organelles
However, none of the studies available in the literature during the maturation process until MII, the protein
was able to test this link directly, and a correlation does stores are reduced and therefore the amount of
not mean that a causal relationship is present. That the ribosomes and the levels of protein synthesis may be
real measure of competence, i.e., term development, comparatively lower. In any case, this evidence supports
was almost never the endpoint of these experiments, a the idea that the presence of ribosomes is directly linked
causal relationship is difficult to justify. Regarding ATP to protein synthesis during crucial periods of develop-
content in the oocyte, several controversial data have ment.
been published, where no correlation was found The dynamics of the Golgi membranes during
between developmental competence and ATP content maturation and fertilization in mammals requires more
in the oocytes. Thus, this gap in understanding will need study [25]. It is known that bovine oocytes in the GV
to be filled from future studies that analyze the presence stage present Golgi fragments that are transformed into
or absence of a causal relationship between oocyte vesicles during GVBD [4,25,26].
competence and mitochondrial distribution, as well as Despite the information obtained thus far, data
ATP content. regarding the role of the Golgi complex during
maturation and the subsequent events remain con-
2.2. Ribosomes, Golgi complex and endoplasmic troversial. A recent study has shown that, in contrast to
reticulum (ER) what is occurring in somatic cells, extracellular matrix
proteins (such as GM130) found in the membranes of
Protein synthesis is indispensable not only for oocyte the Golgi complex in germinal cells during MII are
maturation per se, but also for zygote formation and phosphorylated and co-localized only with sites of ER
early embryogenesis. To this end, an appropriate export and not with the meiotic spindle. This suggests
quantity of ribosomes must be present during matura- that the organizing mechanism of the Golgi complex is
tion. Ribosomes are synthesized by the transcription of independent of the centrosome. This difference can be
ribosomal RNA (rRNA) genes, by the processing of the explained by the fact that during mitosis, there is a
840 E.M. Ferreira et al. / Theriogenology 71 (2009) 836–848

uniform partition of proteins and Golgi membranes progresses to the metaphase II stage [31,32] (Fig. 1A).
between the two daughter cells, an event that does not The sensitivity of the system to calcium release is
occur in oocytes due to the formation of the first and increased after maturation. During fertilization, the
second polar bodies. In addition, the study showed that, entry of the spermatozoon into the oocyte leads to a
in contrast to what was observed in mouse and C. marked release of calcium from the reticulum, followed
elegans embryos, neither fertilization nor cell division by the beginning of embryonic development [31]
up to the eight-cell stage required trafficking through (Fig. 1B). At the time of second polar body formation,
the secretory pathway, since the bovine zygotes cultured the ER clusters begin to disaggregate approximately 3–
in the presence of Brefeldin A (BFA), a fungal 4 h after insemination and 2 h before calcium signaling
metabolite that inhibits protein secretion by interrupting ceases in the pronuclear stage of development [32,33].
the transport of ER-Golgi vesicles, was able to complete
cytokinesis and divide up to the eight-cell stage. One 2.3. Cortical granules
explanation for this observation was that the BFA dose
used was low and therefore insufficient to inhibit Cortical granules (CG) are derived from the Golgi
secretion [25]. Thus, further studies are needed in order complex [34]. Exocytosis of CGs involves cytoskeleton
to fill the gaps regarding the dynamics of proteins and of filaments [35] and proteins homologous to members of
Golgi membrane and their direct importance for oocyte the SNARE hypothesis, a molecular model of traffick-
maturation, fertilization and early embryogenesis [26]. ing, docking and exocytosis of vesicles in the secretory
The membranes of the ER are physiologically active, compartment of somatic cells, also observed in sea
interact with the cytoskeleton and contain different urchin eggs [36]. For oocytes in the GV stage, cortical
domains specialized in different functions. Among the granules are distributed in clusters throughout the
known ER functions are protein folding and degrada- cytoplasm [37]. At the end of the maturation period,
tion, lipid metabolism, compartmentalization of the when these oocytes reach the MII stage, the granules are
nucleus, regulation of the Ca2+ ion gradient, and distributed throughout the inner surface close to the
membrane synthesis [27]. By storing and releasing plasma membrane [36,38], a pattern strategically
calcium, this system plays an extremely important role arranged to await for spermatozoon entry and egg
in intracellular signaling. Complex mechanisms activation (Fig. 1A).
involved in remodeling and in the activity of calcium The cortical granules are organelles exclusively
signaling pathways have been suggested, which points found in oocytes and their composition includes a
out the importance of calcium ion in various develop- diverse population of proteins, structural molecules,
mental events [28]. The Ca2+ signaling pathways enzymes, and glycosaminoglycans. The exocytosis of
depend on differences between intra- and extracellular cortical granules (cortical reaction) is one of the most
calcium levels, which generate different concentration common mechanisms used by the oocyte to prevent
gradients between the two compartments. This gradient polyspermy [37]. If fertilization with more than one
is regulated by the membrane potentials of the spermatozoon occurs, the resulting zygote will undergo
oolemma. In rodent and human oocytes, Ca2+ release abnormal cleavage and will become non-viable,
from cytoplasmic stores is mediated by ligand-gated ion eventually degenerating at the beginning of mitotic
channels such as the inositol 1,4,5 triphosphate receptor divisions. The mechanism of blockade is highly
(IP3R) and the ryanodine receptor, both located on the conserved among animal groups and is based on rapid
ER membrane. It was established that Ca2+ release via modification of the oocyte extracellular matrix. This
IP3 and its receptor, IP3R, is essential for oocyte modification involves releasing CG contents to the outer
activation during fertilization [29,30,28]. surface after oocyte activation is triggered by calcium
Biochemical and structural changes in the ER during release from the ER in response to spermatozoon entry
maturation are critical for proper functioning of into the oocyte oolemma [39] (Fig. 1B). Evidence
intracellular calcium regulation. In vivo analyses of indicates that in mammalian oocytes the intracellular
mouse oocytes in the germinal vesicle (GV) stage signals leading to cortical reaction involves the
showed that the ER is uniformly distributed in the activation of inositol phosphate (PIP (2)) cascade.
ooplasm. As is the case for mouse oocytes as well as Furthermore, the spermatozoon-oocyte fusion as
oocytes from other mammalian species, the ER is found mediated by the GTP-binding protein (protein G) leads
in cortical regions and accumulates in small 1–2 mm to the generation of two secondary messengers, i.e.,
wide clusters throughout the cytoplasm (except in the inositol 1,4,5 triphosphate (IP(3)) and diacylglycerol
vicinity of the meiotic apparatus) as development (DAG). Activity of IP(3) is directly involved in calcium
 E.M. Ferreira et al. / Theriogenology 71 (2009) 836–848 841

release from intracellular stores. Concurrently, DAG movement towards the minus-end in Xenopus laevis
activity activates protein kinase C (PKC) which in turn oocytes [44]. These observations were confirmed by
leads to exocytosis of cortical granules. Calmodulin- FitzHarris et al. [45] in a recent study in which the
dependent kinase II is another key molecule involved in movement of the ER was analyzed during IVM of mouse
the translation of the calcium signal. The release of GC oocytes. Their study demonstrated that the ER reorga-
content promotes changes in the sperm receptors on the nization during oocyte maturation is a complex multi-
zona pellucida, consequently leading to hardening of step process involving distinct microtubule- and micro-
the zona (zona reaction). Another factor acting on the filament-dependent phases and indicated a role for
blockade of polyspermy is the modification of the dynein in the cytoplasmic changes which prepare the
oolemma after the spermatozoon-oocyte fusion and the oocyte for fertilization. The cytoplasmic dynein is also
consequent formation of CG envelopes after the cortical directly involved in various cell functions, such as
reaction [40]. chromosome movement, organization and positioning of
the meiotic spindle, and nuclear migration [46,47]. On
3. Dynamics of cytoskeletal filaments during the basis of this information, we will describe below the
maturation events associated with chromosome movement during
oocyte maturation and the involvement of the cytoske-
The cytoskeletal filaments are dynamic and adap- letal filaments in these events.
table structures that can remain unchanged or undergo During the GV stage of oocyte growth, the spatial
modifications according to the needs of the cell. In rearrangement of the organelles is related to the modified
addition, this system is responsible for chromosome organization of the cytoskeleton that forms a network in
segregation during meiosis and mitosis, for cell division which the organelles encased by a membrane move and
during cytokinesis, and for trafficking molecules and occupy defined positions [48] (Fig. 2). When entering the
organelles inside the cells [41]. The three filament types M phase of the cell cycle (meiosis in the case of female
of the cytoskeleton are formed by subunits that are gametes) microtubule asters appear close to the
characteristic for each one. The microtubules consist of condensed chromatin in bovine oocytes after GVBD.
globular and compacted tubulin subunits, whereas actin Furthermore, during the transition from the GV stage to
filaments consist of similarly globular and compacted anaphase I, the microfilaments or actin filaments are
actin subunits. The intermediate filaments consist of distributed in the cortical area below the oolemma,
elongated and fibrous polypeptide subunits arranged in without connecting to the microtubules [49] (Fig. 2). In
a tetramer analogous to both the aß-tubulin subunits in metaphase I (MI), the microtubules are nucleated by
the microtubule and the actin monomer. Their function tubulin polymerization in the oocyte cytoplasm with the
is mainly related to mechanical resistance in response to centrosome, forming the meiotic spindle and the
stress. These three types of cytoskeletal ‘‘polymers’’ are metaphase plate in which the chromosomes are arranged
maintained by weak noncovalent interactions, and can in an equatorial manner [3,48,50] (Fig. 2A). In MI, the
rapidly associate and dissociate without the need for metaphase plate is proportionally larger than that formed
new formation or breakage of covalent bonds [41]. in MII and in this phase the actin filaments are abundantly
Among the three types of cytoskeletal filaments, distributed in the cortical region but are absent among the
microtubules are more directly involved in the microtubules (Fig. 2). The spindle is barrel-shaped and its
processes of organelle movement [42] even though poles are flattened [49]. Although the microfilaments are
the participation of microfilaments was also observed absent among the microtubules, there seems to be an
during ER redistribution in starfish oocytes [43]. The interaction between these polymers since the polarized
microtubule subunits adhere to motor proteins such as movement of the chromosomes also depends on
dynein, dynactin and kinesin, which bind to molecules processes mediated by actin filaments [42,51].
and to membranes of the organelles, and promote As the cell approaches anaphase I, the chromosomes
movement along the microtubules [44]. start to separate and therefore a large portion of
The microtubules have two distinct regions: a growth microtubules can be seen located between the two
end facing the periphery of the oocyte (plus-ends) and segregating chromosome sets. The spindle elongates
another end facing the interior of the cell or the and a large quantity of actin filaments can be observed
microtubule organizing center (minus-ends). The two around the chromosomes (Fig. 2). In telophase I (TI),
major motor protein families are kinesin, which mediates the microtubules located between the two chromosome
the movement towards the plus-end region, and dynein sets form a cone-like triangular structure having a wider
which, in association with dynactin, mediates the and a more tapered shape (Fig. 2B). The wider portion
842 E.M. Ferreira et al. / Theriogenology 71 (2009) 836–848

Fig. 2. Dynamics of cytoskeleton filaments during cytoplasmic and nuclear maturation of bovine oocytes. (A) Detail of the meiotic spindle
apparatus at metaphase I and the centriole/centrosome structure. (B) Detail of the meiotic spindle at telophase I, in which we can see the microtubules
among the chromosome sets.

of the microtubules is linked to the chromosome set opment. The dynamics of the cytoskeletal filaments
destined for extrusion out of the cell, thus forming the discussed thus far has been observed and related to the
first polar body. The tapered portion is associated with acquisition of nuclear developmental competence in
the set that will remain in the oocyte and enter meiosis studies on bovine [52] and swine [53] oocytes. In
II, again forming the metaphase plate. Thus, during murine and bovine oocytes, the events involving the
oocyte meiosis, this cytoskeletal structural system still cytoskeleton during oocyte maturation are regulated by
plays a fundamental role by ensuring that almost the molecules such as the serine/threonine kinase Aurora-A
entire cytoplasm of the dividing cell will remain in the [54,55] and the cytoplasmic polyadenylation element-
secondary oocyte, which has accumulated all the binding protein (CPEB) [55], which is synthesized and
information needed for the progression to subsequent accumulated in the oocyte cytoplasm during its
steps. Only a small cytoplasmic portion will then be molecular maturation [55]. The protein kinase Aur-
eliminated at the time of polar bodies extrusion. Based ora-A and other Auroras kinases co-localize with the
on this, the actin filaments associated with the centrosomes, chromosomes and midbody and their
chromosomes will also be eliminated at the time of activity can be observed in the regulation of chromo-
extrusion [49]. some segregation, in the maintenance of MII and in the
During the transition from TI to MII there is no formation of the first polar body. On the other hand, the
additional interphase period, but only a rapid chromatin massive destruction of the CPEB protein is related to the
condensation and the disappearance of the microfila- transition from MI to MII [55]. These activities may be
ments and microtubules of the meiotic spindle in a a consequence of these molecules acting on the other
process referred to as interkinesis [50]. The metaphase crucial molecular mechanisms that are described below.
plate reappears soon after this event. As stated earlier,
the meiotic apparatus is smaller in MII than in MI, a fact 4. Molecular maturation
possibly explained by the reduction of chromosome
number to half the initial amount [49] (Fig. 2). Molecular maturation corresponds to the phases of
Thus, in addition to what was described earlier oocyte growth and maturation and it involves the
regarding the movement of cytoplasmic organelles, the transcription, storage and processing of the mRNAs
filaments of the cytoskeleton still actively participate in expressed by the chromosomes, which will be further
the capacitation of the oocyte as a whole for the nuclear translated into proteins by the ribosomes. The proteins
acquisition of competence to progress through devel- derived from these mRNAs are involved both in
 E.M. Ferreira et al. / Theriogenology 71 (2009) 836–848 843

maturation and in subsequent cellular events such as published. In a study on the polyadenylation of various
fertilization, pronucleus formation and early embry- mRNAs involved in the beginning of oocyte meiosis
ogenesis. Thus these proteins are being stored until the resumption to the first embryo cleavages, Brevini et al.
appropriate time for their utilization [56]. [68] observed that, depending on the transcript
Since many transcripts will be consumed, until the investigated, mRNA polyadenylation may vary accord-
activation of embryonic genome, their correct storage in ing to four distinct patterns: no changes, gradual
the oocyte cytoplasm is of great importance. After reduction, gradual elongation, and reduction followed
meiosis is resumed there is no longer any gene by elongation. When evaluating oocytes from the GV
expression [56], and therefore what was produced stage to the MII stage, the authors observed that the
during the growth phase will be metabolized at the poly-(A) tail of the mRNAs was significantly shorter at
appropriate time. MII than during GV, although an elongation or no
The mRNA transcribed during the molecular change in size in the poly-(A) tail was observed in some
maturation of the oocyte is accumulated in a stable, transcripts. In subsequent stages (i.e., during fecunda-
but transiently inactive manner [24,56]. The biosyn- tion and the formation of zygote as well as the two-cell
thetic machinery of the cytoplasm processes this mRNA embryo), the polyadenylation pattern was, with few
into ribonucleoprotein particles [57]. In this ‘‘packed’’ exceptions, the same. When comparing better quality
form, the mRNA is protected from nucleolytic embryos (i.e., embryos that cleaved up to 27 h after
degradation and remains stored until the signals for insemination) to embryos with low developmental
translation are generated during maturation and early potential (cleavage after 27 h post-insemination), the
embryo development [58]. authors observed several differences in the polyadeny-
lation patterns and concluded that these specific
4.1. Polyadenylation changes in mRNA polyadenylation are associated with
modulation of gene expression in the embryos during
Several mechanisms are involved in the activation of this phase. In addition, they also concluded that
translationally inactive mRNA [24,59]. These mechan- abnormal levels of polyadenylation in some maternal
isms involve the phosphorylation of many factors that mRNAs are accompanied by a lower potential for
initiate translation (such as eIF-4F [60]), the phosphor- embryo development [68].
ylation of S6 protein on the 40S ribosomal subunit In addition to those directly related to the acquisition
[50,61], and the dephosphorylation of poly(A)-poly- of developmental competence, the main transcripts
merase [62]. According to this model, polyadenylation produced during molecular maturation of the cumulus-
(the addition of adenine) of the 30 terminal portion of the oocyte complexes (COCs) encode regulators of the cell
cytoplasmic mRNA would stimulate the release of cycle: maturation promoting factor (MPF) and its
repressor molecules linked to the 50 portion [24,63], forming subunits, cyclin B and p34cdc2; the protein of
thereby initiating translation. This polyadenylation the c-mos pro-oncogene (MOS), and mitogen-activated
process starts in the nucleus, and 250 to 300 adenine protein kinase (MAPK) in addition to countless other
(A) residues are added to mRNA for the formation of mRNAs to be described later on [69]; the proteins and
the poly-(A)-30 tail. The transport of this mRNA to the molecules that are markers of cytoplasm maturation
cytoplasm occurs through a characteristic splicing of such as glutathione [70] and ATP molecules [3]; and the
the poly-(A) tail which, after reaching the cytoplasmic components of the antioxidant enzyme system catalase,
compartment, becomes smaller and heterogeneous in superoxide dismutase, and glutathione peroxidase [71].
size [24,64]. When mRNA molecules have short
poly(A) tails they are not effectively translated [65] 4.2. Regulators of the cell cycle
and deletion of this sequence is an early step in the
process of degradation [24,66]. The cytoplasmic We know that an event preceding the resumption of
prolongation of the poly-(A) tail, however, is related meiosis in bovine oocytes is the synthesis of new
to the activation of translation [67] and this means that proteins [72,73]. Most of the proteins and factors
the addition of adenine to mRNA in the oocyte involved in this process are synthesized during the first
cytoplasm during maturation leads to the translation hours of in vitro culture, with MPF being responsible for
of proteins and deadenylation. This in turn leads to the the resumption of meiosis [73]. MPF is a heterodimer
degradation of the particular mRNA. consisting of the regulatory subunit cyclin B1 and the
Recently, supporting evidence on the patterns of catalytic subunit p34cdc2 [74]. Maternal stores of cyclin
polyadenylation in bovine oocytes and embryos was B1 mRNA are detected in bovine oocytes in the GV
844 E.M. Ferreira et al. / Theriogenology 71 (2009) 836–848

stage from ovaries recovered at abattoirs, but the protein equilibrium and coordination of the cellular events
is not yet present. The mRNA of cyclin B1 is stored involved in the acquisition of competence as a whole.
along with a short poly-(A) tail in a translationally Gonadotrophins play an early role in physiological
inactive manner and the accumulation of this transcript coordination and therefore mRNAs that code for FSH,
leads to extensive cytoplasmic polyadenylation and LH and connexin 43 receptors (FSHr, LHr and Cx 43r)
subsequent translation and synthesis of the protein must also be transcribed by the cumulus-oocyte
cyclin B, which is accumulated and detected in the complex so that the hormones will act during oocyte
oocyte cytoplasm 3 h after the beginning of IVM maturation when added in vitro, as it occurs in vivo.
[72,73]. Studies on Xenopus oocytes and on human Connexins are transmembrane proteins that form gap
somatic cells cultured in vitro have revealed that the junctions, as well as being responsible for communica-
mRNA of the protein kinase p34cdc2 or Cdk1 also tion between the oocyte and the surrounding cumulus
undergoes lengthening of the poly-(A) tail by poly- cells [69]. Connexin 43 (Cx43), in particular, is
adenylation. This process involves the phosphorylation involved in follicular growth and its mRNA and protein
of CPEB through the action of the mitotic kinase are present during the maturation of ovine COCs [81].
Aurora-A. In bovine oocytes, it has been observed that The mRNAs of FSHr, LHr and Cx43 may be considered
both CPEB and Aurora-A are synthesized and to be predictors of oocyte competence in vitro by
accumulated in the cytoplasm during oocyte maturation performing such important functions in cell signaling
[55]. CPEB is bound in a repressive manner to a specific and folliculogenesis. Recent studies have shown that
RNA sequence (UUUUUAU). The cytoplasmic poly- levels of mRNA coding these proteins depend on
adenylation element (CPE), located in the non- maturation time and oocyte quality [70,82].
translated 30 region of the mRNAs, consists of an
mRNA recognition site for the beginning of poly- 4.4. Markers of cytoplasmic maturation and the
adenylation. Thus the Aurora-A-CPEB phosphorylation antioxidant system
pathway leads to the beginning of polyadenylation and
consequently to the translation of p34cdc2 [75–77]. After In addition to cell cycle regulators, other critical
translation, the p34cdc2 protein joins cyclin B1 and this molecules for the development and progression of
complex is then phosphorylated by the Wee1/Myt1 maturation and early embryogenesis are synthesized
kinases. This leads to the formation of the so-called and accumulated inside the oocyte, with their presence
preMPF which, after undergoing dephosphorylation by considered to be a marker of having acquired
phosphatase cdc25, is activated and starts to have the cytoplasmic competence to support the subsequent
functional activity of MPF proper [74]. phases. We consider good biological markers of
The MOS protein, a product of the c-MOS proto- cytoplasmic maturation to be those that can be analyzed
oncogene, is also coded by maternal mRNA stored during by reproducible, precise and minimally invasive
oocyte growth. During the maturation period, this mRNA techniques [83]. Within this context, glutathione is
is translated and degraded together with other mRNAs in one of the markers of cytoplasm maturation that has
later phases of development according to the species been extensively investigated. Several studies have
studied. The MOS protein activates the MAPK cascade, shown that this enzyme plays a fundamental role in cell
which modulates MPF activity and leads to entry into the protection against oxidative damage [84] by eliminating
cell cycle (transition from the G2 phase to the M phase) the ROS produced during mitochondrial metabolism. Its
[78]. In bovine COCs, the polyadenylation of mRNAs intracellular concentration increases as the oocytes
that code for the proteins necessary for MPF and MAPK develop from the GV stage to MII [70,85]. After
activation occurs between 9 and 12 h of in vitro culture fertilization, its activity is related to the decondensation
[79,80]. Thus, appropriate synthesis and storage of these of sperm chromatin, with consequent oocyte activation
transcripts in the oocyte cytoplasm will also be crucial for and also the transformation of the sperm head into a
the continuity of nuclear resumption and progression of male pronucleus [84]. If the medium used for IVM is
meiosis. deficient in cysteine, a precursor of glutathione [86], the
glutathione synthesis will be affected by substrate
4.3. Other mRNAs involved in the acquisition of insufficiency and then the oocytes will be cultured
competence for development under suboptimal conditions resulting in unsatisfactory
embryo development [70,84]. In addition to glu-
Other transcripts coded during COC maturation are tathione, ATP molecules have also been used as
added to those listed here as a function of the markers of cytoplasmic maturation in bovine [3] and
 E.M. Ferreira et al. / Theriogenology 71 (2009) 836–848 845

gilt oocytes [87]. In a recent study, it was reported that Considering that the real measure of competence
the number of ATP molecules in morphologically (i.e., term development) was almost never the end point
competent oocytes (category 1, homogeneous oocyte of experiments that evaluated the correlation between
cytoplasm, compact multilayered cumulus oophorus; organelle quantity and distribution, it is difficult to
category 2, cytoplasm with small inhomogeneous areas, justify a causal relationship between cytoskeleton
more than five layers of compact cumulus; category 3, function and molecular events, and oocyte competence.
heterogeneous/vacuolated cytoplasm, three to five Further studies with an adequate methodology are
layers of cumulus including small areas of denuded needed to better understand this association.
zona pellucida) increases from 1.4–1.8 pmol before Despite the great interest this topic has attracted over
IVM to 2.3–2.5 pmol after maturation, with a positive the last decades with the advent of new technologies,
reflex also on early embryo development [3]. many gaps remain. As an example, controversies
Together with glutathione, other molecules in the continue regarding the function of several cytoplasmic
antioxidant enzymatic system also play an important organelles and molecules in the gradual process of
role in attenuating the deleterious effects of ROS- oocyte capacitation. There is lack of consistent
induced oxidative stress [71]. It was demonstrated that information on ribosome, ER, and Golgi apparatus
catalase, superoxide dismutase and glutathione are involvement during bovine oocyte maturation. Much of
present in bovine oocytes and in cumulus cells after what is known about the participation of these
IVM. In addition, their levels in denuded oocytes are organelles in the process of maturation comes from
lower than those observed in the cumulus-oocyte studies conducted on species other than cattle. There is
complex, although no difference has been detected some evidence indicating an important role for
between the two groups regarding ROS production [71]. mitochondria in the acquisition of oocyte competence
for development. However, with regards to the ATP
5. Conclusions content of the oocyte, several reports found no
correlation between developmental competence and
Oocyte maturation is a long process during which ATP content of the oocytes. In addition, understanding
oocytes acquire the intrinsic ability to progress through the mechanisms related to intra- and extracellular
subsequent events of development and involves com- signaling that coordinate all the events involved in the
plex and distinct, although linked, mechanisms of acquisition of competence also represents an interesting
nuclear and cytoplasmic maturation. The structural and approach that requires further elucidation.
biochemical changes described here regarding orga-
nelle redistribution, cytoskeletal filament dynamics and Acknowledgements
molecular maturation - which start during the growth
phase and are completed with the progression of We thank the Department of Gynecology and
development as a direct consequence of oocyte Obstetrics of the Faculty of Medicine of Ribeirão
cytoplasmic maturation - are linked mechanisms Preto/USP and the University Hospital of the Faculty of
depending on the functional integrity and the coordi- Medicine of Ribeirão Preto (HC-FMRP/USP) for
nated participation of all the elements involved in the financial support, and ‘‘Fundação de Amparo à Pesquisa
process. On this basis, ultrastructural analysis shows do Estado de São Paulo’’ (FAPESP) for the fellowship
that mitochondria, ribosomes, endoplasmic reticulum, granted to Elisa Melo Ferreira (process n8 05/57852-2).
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