Human Reproduction Update, Vol.21, No.4 pp. 427–454, 2015
Advanced Access publication on March 4, 2015 doi:10.1093/humupd/dmv011
Oocyte maturation: gamete-somatic
cells interactions, meiotic resumption,
cytoskeletal dynamics and cytoplasmic
reorganization
Giovanni Coticchio*, Mariabeatrice Dal Canto, Mario Mignini Renzini,
Maria Cristina Guglielmo, Fausta Brambillasca, Diana Turchi,
Paola Vittoria Novara, and Rubens Fadini
Biogenesi Reproductive Medicine Centre, Istituti Clinici Zucchi, Via Zucchi 24, 20900 Monza, Italy
*Correspondence address. Tel: +39-039-83833369; E-mail: [email protected]
Submitted on September 29, 2014; resubmitted on February 4, 2015; accepted on February 11, 2015
table of contents
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Introduction
Methods
Literature search
Results
Cellular bases of oocyte –CC interactions during maturation
Oocyte–CC signalling for meiotic arrest and resumption
Global chromatin remodelling, meiotic resumption and developmental competence
DNA damage and meiotic progression
GV positioning and oocyte polarity
Cytoskeletal forces governing spindle positioning
Cortical polarization and polar body extrusion
Changes in cortical mechanics important for oocyte maturation
Cytoplasm reorganization during maturation
Clinical correlates of oocyte maturation
Conclusions
background: In a growth phase occurring during most of folliculogenesis, the oocyte produces and accumulates molecules and organelles
that are fundamental for the development of the preimplantation embryo. At ovulation, growth is followed by a phase of maturation that, although
confined within a short temporal window, encompasses modifications of the oocyte chromosome complement and rearrangements of cytoplasmic components that are crucial for the achievement of developmental competence. Cumulus cells (CCs) are central to the process of maturation,
providing the oocyte with metabolic support and regulatory cues.
methods: PubMed was used to search the MEDLINE database for peer-reviewed original articles and reviews concerning oocyte maturation
in mammals. Searches were performed adopting ‘oocyte’ and ‘maturation’ as main terms, in association with other keywords expressing concepts
relevant to the subject. The most relevant publications, i.e. those concerning major phenomena occurring during oocyte maturation in established
experimental models and the human species, were assessed and discussed critically to offer a comprehensive description of the process of oocyte
maturation.
results: By applying the above described search criteria, 6165 publications were identified, of which 543 were review articles. The number of
publications increased steadily from 1974 (n ¼ 7) to 2013 (n ¼ 293). In 2014, from January to the time of submission of this manuscript, 140
& The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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Coticchio et al.
original manuscripts and reviews were published. The studies selected for this review extend previous knowledge and shed new and astounding
knowledge on oocyte maturation. It has long been known that resumption of meiosis and progression to the metaphase II stage is intrinsic to
oocyte maturation, but novel findings have revealed that specific chromatin configurations are indicative of a propensity of the oocyte to
resume the meiotic process and acquire developmental competence. Recently, genetic integrity has also been characterized as a factor with important implications for oocyte maturation and quality. Changes occurring in the cytoplasmic compartment are equally fundamental. Microtubules, actin filaments and chromatin not only interact to finalize chromosome segregation, but also crucially co-operate to establish cell
asymmetry. This allows polar body extrusion to be accomplished with minimal loss of cytoplasm. The cytoskeleton also orchestrates the rearrangement of organelles in preparation for fertilization. For example, during maturation the distribution of the endoplasmic reticulum undergoes
major modifications guided by microtubules and microfilaments to make the oocyte more competent in the generation of intracellular Ca2+ oscillations that are pivotal for triggering egg activation. Cumulus cells are inherent to the process of oocyte maturation, emitting regulatory signals via
direct cell-to-cell contacts and paracrine factors. In addition to nurturing the oocyte with key metabolites, CCs regulate meiotic resumption and
modulate the function of the oocyte cytoskeleton.
conclusions: Although the importance of oocyte maturation for the achievement of female meiosis has long been recognized, until recently much less was known of the significance of this process in relation to other fundamental developmental events. Studies on chromatin dynamics
and integrity have extended our understanding of female meiosis. Concomitantly, cytoskeletal and organelle changes and the ancillary role of CCs
have been better appreciated. This is expected to inspire novel concepts and advances in assisted reproduction technologies, such as the development of novel in vitro maturation systems and the identification of biomarkers of oocyte quality.
Key words: oocytes / meiosis / cytoplasm / organelles / in vitro maturation of oocytes
Introduction
The unparalleled size and unique ability to develop into a totipotent
zygote place the fully grown mature oocyte at the very centre of the reproductive strategy of mammals and other metazoans. In fact while, with
the exception of mitochondrial DNA (mtDNA), oocytes and spermatozoa contribute equally to the genetic make-up of the new individual, the
cellular and molecular continuum across generations is ensured almost
exclusively by the female gamete. This justifies the phenomenal investment in regulatory processes by which oocytes are generated, nurtured
and matured for a period that can span as much as several decades in a
woman.
The natural history of the oocyte begins during fetal life when its generation as a distinct cell type coincides approximately with the initiation of
meiosis and assembly into the primordial follicle (Gougeon, 1996). Entry
into meiosis is immediately followed by arrest at the diplotene stage and
establishment of a condition of relative metabolic quiescence. In such a
suspended state, the oocyte can pause for up to 40 years until when,
under the impulse of a still not fully understood mechanism, it undertakes
a phase of growth while remaining meiotically arrested. In the human,
oocyte growth unfolds over 110– 120 days, during which cell mass
undergoes an astounding more than 100-fold increase and cell diameter
shifts from ,40 to 120 mm. Meanwhile, macromolecules and organelles are produced and stored in very large amounts (Gosden and
Lee, 2010). In such a way, the oocyte meets an essential precondition
for preimplantation embryo development, stockpiling sufficient cytoplasmic mass required for the fertilized egg to achieve multicellularity
in the absence of net growth. Notably, oocyte growth is much more
than a quantitative increase in cell mass finalized to the housekeeping
needs of the early embryo. Rather, growth is accompanied by precisely
programmed qualitative changes in the endowment of organelles and informational molecules that will regulate crucial developmental functions.
For example, maternal effect genes have been characterized as
sequences whose transcriptional and translational products are generated during oocyte growth and whose regulatory function is required
only after fertilization (Li et al., 2010). Typically, many maternal effect
gene products are transcriptional regulators whose loss of function
causes major developmental failures, such as arrest at cleavage or blastocyst stages, or inability to activate the zygote genome.
Oocyte growth occurs concomitantly and is mutually interdependent
with the development and differentiation of the follicle (Hutt and Albertini, 2007). However, oocyte growth is already finalized at the early antral
stage, i.e. significantly before follicle development is completed. In fact,
while for several days the antral follicle experiences a further expansion
in preparation for ovulation, no increase in oocyte size is observed
(Griffin et al., 2006). Finally, stimulated by the mid-cycle LH surge, the
fully grown diplotene stage-arrested oocyte resumes meiosis and progresses to the metaphase II stage (MII) in 30 h in the human, thereby
achieving meiotic maturity. Therefore, growth and maturation may be
discerned as temporally and functionally distinct phases of oogenesis.
By definition, the importance of oocyte growth for embryo development
has appeared almost self-explanatory to scientists. On the contrary, a
proper appreciation of maturation beyond the meiotic imperative has
been, and still is, less obvious to appreciate. Oocyte meiosis, whose molecular regulation is not discussed here because it is thoroughly described
elsewhere (Holt et al., 2013), is a complex process. In fact, not only does
it require substantial modification of the mitotic cell cycle, but also it
necessitates a machinery able to attain chromosome segregation with
minimal loss of cytoplasm. To this end, the oocyte develops an asymmetric cell design and a peripheral position of the meiotic spindle. This dictates that the cleavage planes of meiotic divisions lie just beneath the
oolemma. In this fashion, the extrusion of two small polar bodies is
achieved. Attainment of asymmetry and polar body extrusion demand
major rearrangements of the cytoskeleton, co-ordinated cytoplasmic
dynamics and fine control of tension forces governing the cortex, elements that only recently have emerged in their elegant complexity
(Brunet and Verlhac, 2010; Yi et al., 2011).
Oocyte maturation also involves many other intercellular, intracellular
and molecular processes whose implications for embryo development
are as important as the achievement of haploidy. Studies accomplished
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Oocyte maturation
especially in the last decade have allowed us to better understand the
complexity, choreography and significance of oocyte maturation. Examples of the intense cellular activity occurring during oocyte maturation are
major changes in chromatin configuration that accompany meiotic resumption (Luciano et al., 2012), modifications in mitochondrial distribution, clustering and activity in response to changing energy demands of
the cell machinery (Dalton and Carroll, 2013), and endoplasmic reticulum (ER) reorganization and maturation finalized to intracellular Ca2+ signalling (Mann et al., 2010). These and other transformations, illustrated in
this manuscript, are finely co-ordinated, and are often interdependent
processes. Explicative in this respect is the accumulation of mitochondria
and ER elements around the newly formed spindle, without which
spindle positioning to the cortex in preparation for the first meiotic division does not occur (Dalton and Carroll, 2013).
In addition to the extreme asymmetry of the meiotic divisions and redistribution and regulation of organelles, another major theme of oocyte
maturation, and indeed the larger processes of oogenesis and folliculogenesis, is the intense cross-talk and co-operativity between the
oocyte and the surrounding follicle cells (Fig. 1). These cells, referred
to as cumulus cells (CCs), originate as a subpopulation of granulosa
cells as soon as the antrum is formed in the developing follicle (Matzuk,
2002). Oocyte– CC interactions are implemented through different signalling modalities and are recognized as a founding element of oocyte
maturation. In fact, CCs support a wide variety of functions of the maturing oocyte, including metabolism, meiotic arrest and resumption, and
cytoskeletal rearrangements.
In providing scope for this review, meiotic progression and attendant
cytoplasmic changes are brought together in the same view to appreciate
the importance of maturation as a process having direct implications for
oocyte quality. The investigation of mammalian oocyte maturation
became an increasingly attractive object of investigation in the 1930s,
when it was first possible to culture fully grown oocytes and preimplantation embryos (Pincus and Enzmann, 1935). Most of the previous and
current knowledge has been developed in experimentally amenable
species, such as the mouse, a fact that is reflected in the contents of
this review. Nevertheless, the implications of oocyte maturation
studies in animal models for the understanding of human reproduction
and the treatment of infertility are obvious. Indeed, the first human
embryo generated extracorporeally was derived from an in vitro-matured
oocyte as a result of the pioneering efforts of John Rock and Miriam
Menkin in the 1940s (Rock and Menkin, 1944). Therefore, in the
context of this manuscript, oocyte in vitro maturation (IVM) is not illustrated as a clinical approach, but rather it is adopted as an experimental
paradigm able to shed light on many aspects of the final act of oogenesis
and inspire better criteria of oocyte quality and novel in vitro manipulation
strategies.
Methods
Literature search
PubMed was systematically searched for peer-reviewed original articles and
reviews identified by relevant keywords, such as ‘oocyte’, ‘oocyte maturation’, ‘in vitro maturation’, ‘meiosis’, ‘germinal vesicle’, ‘chromatin’, ‘DNA
damage’, ‘actin’, ‘meiotic spindle’, ‘endoplasmic reticulum’, ‘mitochondria’,
‘Golgi apparatus’, ‘cortical granules’ and ‘cumulus cells’. Keywords were
used in multiple and overlapping combinations in order to identify those publications strictly relevant to oocyte function. Additional studies were identified by thorough analysis of reference lists from relevant publications. The
‘English language’ limit was applied. The most relevant publications, i.e.
those concerning major phenomena occurring during oocyte maturation,
were assessed and discussed critically to offer a comprehensive description
of the process of oocyte maturation. Concerning animal studies, priority
was given to publications relevant to species more consistently used as experimental models for the human.
Results
By applying the above described search strategy, 6165 publications were
identified, of which 543 were reviews. The number of publications
increased steadily from 1974 (n ¼ 7) to 2013 (n ¼ 293). In 2014, from
Figure 1 Human immature GV-stage (left) and in vitro matured (right) oocytes recovered from antral follicles (6– 10 mm in diameter) of women undergoing IVM cycles. The GV-stage oocyte is enclosed in a compact vestment of CCs organized in multiple layers. Following culture for 30 – 32 h, in the in vitro
matured oocyte the CC vestment has undergone expansion as a consequence of abundant production of a viscoelastic extracellular matrix. Micrographs
were acquired at ×200 magnification.
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January to the time of submission of this manuscript, 140 original manuscripts and reviews were published. By focusing on studies concerning
mainstream phenomena occurring during maturation and described in
established experimental models and the human species, the number
of publications of interest was narrowed down to a few hundreds,
many of which are extensively described or cited in this review. The
selected studies, grouped systematically in specific topics and illustrated
in the below sections, extend previous knowledge and shed new and
astounding knowledge on oocyte maturation.
Cellular bases of oocyte– CC interactions
during maturation
The interaction between the oocyte and follicle cells is known to occur
uninterruptedly from the formation of the primordial follicle until ovulation, but the modalities by which it is implemented can differ over time in
response to the challenges imposed by the changing design of the developing follicle. For example, in the minimalist structure of the primordial
follicle, direct cell-to-cell contacts prevail as a form of interaction
(McLaren, 1988) while, as soon as the follicle becomes more complex
and physical barriers are raised between cells, additional systems are
deployed to ensure cell communication. Five types of interactions are
recognized in the germinal-somatic regulatory loop: (i) direct contactmediated signals in the absence of intercellular junctions, (ii) typical
ligand-receptor interactions, (iii) paracrine signalling pathways, (iv) gap
junctions and other junctional contacts via transzonal projections
(TZPs) and (v) receptor tyrosine kinases (RTKs) (McGinnis et al., 2013).
Among these modalities, paracrine regulation is active throughout oogenesis and plays a substantial role also during maturation. Typical in such
respect is the case of the transforming growth factor (TGF)-b family
members growth differentiation factor 9 (GDF9) and bone morphogenetic factor 15 (BMP15). These agents are profoundly involved in
oocyte –granulosa cell regulatory loops at early stages of oogenesis,
but their action extends temporally also to the maturation phase
(Edson et al., 2009). Produced by the oocyte, they diffuse through the
cumulus mass and act in a paracrine fashion on follicle cells, by interaction
with morphogenetic protein receptor type-II and activin receptor-like
kinases to ultimately stimulate Sma- and Mad-related intracellular signalling (Kaivo-oja et al., 2006). During maturation, GDF9 and BMP15 modulate a number of effects on surrounding CC, some of which are discussed
elsewhere in this review, including growth, inhibition of apoptosis and
luteinization, stimulation of energy and cholesterol metabolism, and
cumulus expansion.
During a large part of oocyte growth and maturation, the presence of a
thick zona pellucida clearly prevents extensive oocyte– CC contact.
Instead, physical interaction takes the form of thin axon-like projections,
the TZPs, that depart from the CC more closely arranged around the
oocytes, extend through the entire depth of the zona pellucida and
make contact with the oolemma (Fig. 2), where they form connexinbased gap junctions, desmosome-like structures and zonulae adherens
(Albertini et al., 2001). Interestingly, abundance and characteristics of
TZPs can vary under different phases and conditions. In fact in the
human, at the pre-antral stage, TZPs are particularly numerous and
form deep invaginations at the points of contact with the oolemma,
while during the following antral stages their number and complexity of
contact at the oocyte surface decrease (Combelles et al., 2004). Even
more intriguingly, FSH appears to directly modulate TZP stability at
Coticchio et al.
the late pre-antral stage, as shown by the fact that in FSHb knockout
mice TZP development is enhanced, while retraction and a decrease
in number occur upon treatment with FSH (Kumar et al., 1997). This
has potentially significant implications for in vivo ovarian stimulation or
IVM, in the context of which supra-physiological or inappropriate gonadotrophin dosage could affect TZP dynamics and ultimately
oocyte –CC interaction (Luciano et al., 2011). Many or most of the
TZP-mediated interactions occur via heterologous gap junctions
formed from connexins 37 and 43, contributed by the oocyte and CC,
respectively (Ackert et al., 2001; Mora et al., 2012). Through these intercellular contacts, oocyte, CC and indeed mural granulosa cells (MGCs)
form a functional syncytium by which small molecules (up to 1 kDa in
mass) are exchanged to meet the metabolic and regulatory demands
of the oocyte, as described in following sections of this manuscript.
Notably, gap junction communication can be promptly down-regulated
not only by physiological stimuli, as in the case of meiotic resumption, but
also by in vitro manipulation conditions (Lodde et al., 2007), with downstream effects that are not always compatible with the acquisition of developmental competence.
Live-cell fluorescence and time-lapse microscopy observations
suggest that mitochondria, endosomes and lysosomes can be moved
bi-directionally throughout the TZPs extension (Albertini et al., 2001).
Consistent with this, microtubules, which normally mediate organelle
movement, contribute to the formation of the TZPs cytoskeleton in
bovine CC (Allworth and Albertini, 1993). It therefore appears possible
that paracrine factors could be released or taken up at the TZP –
oolemma interface. Therefore, in different ways, TZPs represent a fundamental dimension of oocyte– CC interaction.
Figure 2 Three-dimensional reconstruction of a cortical portion of a
human immature COC. The COC was fixed immediately after recovery
and stained for actin before laser scanning confocal microscopy analysis.
The image was acquired in order to emphasize the presence of actin-rich
TZPs connecting the CC and the oocyte. Image acquisition was performed at ×63 magnification.
Oocyte maturation
RTKs and their respective ligands are well-known elements of intercellular regulatory networks of several phases of oogenesis, but only recently they have been described as a distinct route by which CC and the
oocyte communicate during maturation. In particular, it has been
observed in the mouse that tyrosine phosphorylation of cortical proteins
is modest in fully grown germinal vesicle (GV) oocytes obtained from preovulatory follicles, but undergoes a significant increase after maturation in
vitro (McGinnis and Albertini, 2010). This change is thought to be supported by CC because growth factors that act through CC, such as epidermal growth factor (EGF), enhance cortical phosphorylation
(McGinnis and Albertini, 2010). Fyn kinase, a member of the Src-Family
kinases, has been identified as an another element able to mediate phosphorylation of cortical proteins (Luo et al., 2009), but full elucidation of
this phenomenon, although biochemically prominent, remains elusive.
Collectively, it appears that the maturation process is supported by
diverse manifestations of the interaction between the oocyte and CC.
Among these, TZPs are of particular importance, not only for their function and regulation, but also because the preservation of their delicate
structure, involving extremely thin cellular projections extending
several microns in length, may be compromised by in vitro manipulation.
Oocyte–CC signalling for meiotic arrest and
resumption
Throughout the long period of quiescence at the primordial stage and
during follicle development, the oocyte remains arrested at the prophase
stage of meiosis. The process of chromosome segregation is resumed
only at ovulation, when the GV breaks down, the first meiotic division
is accomplished and the meiotic cycle progresses, before pausing again
at MII in preparation for fertilization. It has long been known that as
soon as a well-defined antrum is formed oocytes become progressively
competent to undergo GV breakdown (GVBD) and progress to MII.
However, during antral expansion meiotic resumption is prevented by
an inhibitory influence of the follicular environment, as shown by the
fact that in many mammalian species GVBD and extrusion of the polar
body I (PBI) occur spontaneously when oocytes are released from
their follicles (Pincus and Enzmann, 1935; Edwards, 1965). For many
years, different lines of evidence generated mainly in the mouse model
have indicated cyclic adenosine monophosphate (cAMP) as the fundamental factor by which meiotic arrest is ensured before ovulation.
First, the mid-cycle LH surge is preceded by a reduction in intraoocyte
levels of cAMP (Schultz et al., 1983). Secondly, oocyte release from
the follicle and meiotic resumption in vitro also coincide with a decrease
in intraoocyte cAMP (Vivarelli et al., 1983). Thirdly, membranepermeable cAMP analogues (Schultz et al., 1983) or inhibitors of the
oocyte-specific cAMP phosphodiesterase type 3 (PDE3) (Nogueira
et al., 2003), the enzyme by which cAMP is hydrolysed, prevent spontaneous meiosis resumption in vitro. cAMP levels dictate the arrest or reinitiation of meiosis by regulating the protein complex referred to as
M-phase promoting factor (MPF), formed from the kinase CDK1 and
cyclin B (Jones, 2004). High intraoocyte concentrations of cAMP activate
protein kinase type A (PKA, types I and II) (Viste et al., 2005). In turn, PKA
phosphorylates the phosphatase CDC25 and the kinases WEE1B and
MYT1, causing inactivation of the phosphatase and activation of the
two kinases, respectively (Jones, 2004). In this fashion, tyrosine and
threonine residues of CDK1 are phosphorylated, MPF activity is
repressed and GVBD is inhibited. A decrease in intraoocyte cAMP
431
levels overturns this state, ultimately allowing activation of MPF and, as
a direct consequence of this, meiotic resumption (Jones, 2004).
As a result of evidence mainly generated in murine oocytes, it has long
been thought that, to ensure meiotic arrest, cAMP was generated in the
granulosa cells and transferred to the oocytes through gap junctions
(Dekel, 2005). Initial evidence showed that forskolin, an adenylate
cyclase stimulant, caused meiotic arrest in cumulus-enclosed, but not
denuded, oocytes (Dekel et al., 1984; Racowsky, 1984). However, the
hypothesis that intraoocyte cAMP has a somatic origin has been questioned by more recent studies. In fact, while it is true that gap junction
communication between oocyte and somatic cells is crucial to maintain
meiotic arrest in mouse and rat oocytes (Sela-Abramovich et al., 2006;
Norris et al., 2008), gap junction permeability to cAMP is unknown.
Also, in the somatic compartment of the rabbit antral follicle cAMP
levels are rather low before LH surge (Hunzicker-Dunn, 1981), making
unlikely the possibility that granulosa cells can produce sufficient cAMP
and deliver it to the oocyte. On the contrary, the evidence has
become progressively stronger that the mouse oocyte has the potential
to generate autonomously high intracellular concentrations of cAMP
(Vaccari et al., 2008). This is suggested by the fact that mouse and
human oocytes are equipped with a complete system of cAMP production, including a G-protein (Gas) (Mehlmann et al., 2002, 2004; DiLuigi
et al., 2008) that is known to activate the cAMP signalling pathway,
GPR3, a G-protein coupled receptor (Vaccari et al., 2009), and adenylate
cyclase type 3, that converts ATP in cAMP (Tsafriri et al., 1996; Freudzon
et al., 2005; Mehlmann, 2005; Conti, 2011). Importantly, inactivation of
any of such elements in vivo or in vitro leads to spontaneous meiotic resumption (Vaccari et al., 2008). Regardless of the compelling evidence
that argues against an extraoocyte origin of cAMP, it is possible
however that in non-murine species cAMP can be generated both endogenously by the oocytes as well as exogenously by somatic cells.
This can be inferred by experiments in the bovine in which forskolin
and a PDE3 inhibitor together, but not separately, were able to maintain
denuded oocytes in meiotic arrest, while forskolin alone caused an increase in cAMP in cumulus-enclosed oocytes (Thomas et al., 2002).
Therefore, caution should be exercised in extrapolating concepts from
one species to another, considering realistically that alternative pathways
involved in intraoocyte cAMP regulation may coexist and have different
significance depending on specific conditions.
Previous and more recent findings have clarified how the follicular environment controls meiotic arrest and resumption in those species
where cAMP is endogenously generated by the oocyte. Early experiments carried out in the rat established that shortly before GVBD, in addition to cAMP, the intraoocyte levels of cyclic guanosine monophosphate
(cGMP) also fall dramatically following stimulation with LH or release
from the follicle (Törnell et al., 1990). Furthermore, it was found that
high concentrations of cGMP are inhibitory to the activity of PDE3 in
mouse oocytes (Bornslaeger et al., 1984). Further suggestion of a role
of cGMP in meiotic arrest derived from studies showing that experimental closure of gap junctions causes a drop in mouse intraoocyte cGMP
levels that shortly precedes GVBD (Norris et al., 2009). Consequently,
cGMP has become the object of many studies aimed at identifying the
substance that is produced by follicle cells and conveyed to the oocyte
to prevent meiotic resumption. Indeed, cGMP has emerged as the key
regulator of a finely orchestrated inhibitory system that involves different
factors and follicular compartments. It appears that MGCs and CCs play
complementary roles to ensure the generation of cGMP, which can then
432
be transferred to the oocyte. In particular in the mouse model, CCs preferentially express the mRNA of the membrane bound guanyl cyclase
natriuretic peptide receptor 2 (NPR2), while MGCs express much
more intensely the mRNA of the cognate ligand, natriuretic peptide precursor type C (NPPC) (Zhang et al., 2010, 2011b). Such differential expression suggests a scenario in which NPPC is primarily produced by
MGC, diffuses throughout the follicle and, by binding to its receptor
NPR2, stimulates CC to produce cGMP. Finally, the cyclic nucleotide is
transferred via gap junctions from CC to the intraoocyte environment,
where it represses the activity of PDE3 maintaining high cAMP levels.
Several in vivo and in vitro observations confirm the above hypothesis:
(i) NPPC prevents GVBD in cumulus– oocyte complexes (COCs), but
not denuded oocytes; (ii) following exposure of COC to NPPC, cGMP
increases in both CCs and oocytes; (iii) treatment of COC with NPPC
induces a rise in cAMP in oocytes but not CCs and (iv) in Nppc or
Npr2 mutant mice oocyte meiosis in resumed precociously in antral
follicles (Zhang et al., 2010, 2011b). Similar to the mouse, in the pig
(Hiradate et al., 2014) and cow (Franciosi et al., 2014) models, the
Nppc and Npr2 genes were found to be preferentially expressed in
MGC and CC, respectively. In such species, NPPC was also shown to
inhibit spontaneous meiotic resumption in COC, suggesting that the
NPPC/NPR2 system is shared among several mammalian species.
Recent observations have indicated that expression of the NPPC/
NPR2 system is subject to regulation by different factors. In vivo in the
mouse, gonadotrophin stimulation of follicular development induces expression of Nppc by MGC and Npr2 mRNA by CC (Zhang et al., 2010,
2011b). In addition, the ability of NPPC to prevent meiotic resumption in
vitro and the competence of CC to produce cGMP are transient unless
estradiol is present in the culture environment (Zhang et al., 2011b). In
fact, this steroid is essential for the expression of Npr2 in CC. It also
co-operates with FSH to enhance the expression of Nppc by MGC. Importantly, oocytes take an active and important part in this regulatory
network, as indicated by the synergistic ability of the oocyte-derived
factors GDF9 and BMP15, as homo- or heterodimers, to stimulate in
CC the expression of Npr2 and the inosine monophosphate dehydrogenase gene, whose product is the limiting element for the generation
of cGMP from guanyl metabolites (Wigglesworth et al., 2013).
Further experiments in the mouse have also shed light on the events by
which the midcycle LH surge neutralizes the inhibitory mechanisms
based on cGMP. Suppression of gap junction communication, which actively participate in the mechanism of meiotic arrest, is achieved by activation of a mitogen-activated protein kinase (MAPK)-dependent
pathway that causes phosphorylation of connexin-43 on several regulatory serines (Norris et al., 2008). However, other elements concur to the
regulation of meiotic resumption. In fact, acute LH stimulation is also
followed by a decrease in cGMP levels in the entire follicle (Norris
et al., 2009; Vaccari et al., 2009) and a suppression of NPPC synthesis
(Kawamura et al., 2011). Recent evidence obtained in the rat model
indicates that a fall in cGMP concentration in the follicle may derive
from positive regulation of the cGMP PDE5, but it is also caused by
inhibition of the guanyl cyclase activity of NPR2 in the CC compartment
(Egbert et al., 2014). Thus, reduced production of the ligand NPPC, inhibition of the guanyl cyclase activity of the cognate NPR2 receptor
and suppression of gap junction-mediated communication act in parallel
to reduce the intraoocyte levels of cGMP. In this fashion, within 1 h of LH
stimulation the intraoocyte cGMP concentration falls dramatically. In
species other than the mouse and the rat, similar mechanisms may
Coticchio et al.
mediate meiotic resumption, as suggested by the finding that in the
human exposure to ovulatory doses of hCG causes a reduction in
NPPC present in the follicular fluid (Kawamura et al., 2011).
Different lines of evidence indicate that members of the EGF family
amphiregulin (AREG) and epiregulin (EREG) play an important role as
mediators of the pathway linking LH stimulation and cGMP regulation.
It has long been known that EGF can induce meiotic resumption in
intact rat follicles (Dekel and Sherizly, 1985) or mouse COC cultured
in the presence of hypoxanthine (Downs et al., 1988), conditions that
otherwise prevent GVBD. More recent investigations have established
that, rather than EGF, AREG and to a lesser extent EREG are involved
in signalling meiotic reinitiation at ovulation. Stimulation with LH of
mouse MGC in vitro or rat follicles in vivo causes prompt expression of
AREG and EREG mRNA before GVBD (Park, 2004). Production of
such transcripts is likely to depend on the typical increase in cAMP by
which follicle cells respond to gonadotrophins. This is suggested by the
fact that forskolin, an activator of adenylate cyclase, is as efficient as LH
in promoting the synthesis of AREG and EREG in cultured equine and
bovine MGC (Sayasith et al., 2013). The two EGF-like factors are synthesized as precursors integrated in the oolemma and released as active
forms following cleavage of their extracellular domains by the transmembrane metalloprotease ADAM17/TACE (Blobel et al., 2009). The involvement of AREG and EREG in the regulation of meiotic resumption
is indicated by genetic, pharmacological and in vitro studies: (i) mice carrying inactivating mutations of the EGF receptor (EGFR), activated by
AREG and EREG, fail to support oocyte meiotic resumption following
acute LH stimulation (Hsieh et al., 2007), consistent with a delayed or
reduced oocyte maturation observed in animals deficient in AREG or
EREG synthesis; (ii) suppression of the phosphorylation-mediated activation of the EGFR, which occurs in vivo in MGC following LH treatment
(Park, 2004), by the kinase inhibitor AG1478 prevents the stimulatory
effect of LH on meiotic resumption in the mouse (Norris et al., 2010)
and (iii) in vitro, exposure to AREG or EREG induces GVBD in intact follicles or COC cultured in the presence of hypoxanthine (Park, 2004).
Other experiments have clarified further details of the involvement of
EGF-like factors in the mechanism of meiotic resumption. Adopting
the whole follicle culture as a model, it was observed in the mouse
that the ability of AREG or EREG to promote GVBD is accompanied
by a large decrease in cGMP in follicle cells (Vaccari et al., 2009; Norris
et al., 2010). However, suppression of the EGFR kinase activity with
the inhibitor AG1478 only partially prevents the large decrease in
cGMP induced by LH (Norris et al., 2010), a sign that the effect of this
hormone on cGMP levels derives from two or more redundant pathways. Also, treatment with EREG alone or in association with AREG
determines only a partial down-regulation of gap junction communication, while exposure to the inhibitor AG1478 prevents LH-induced
gap junction closure (Norris et al., 2010). Therefore, activation of the
EGFR by EGF-like factors appears to play an important role in gap junction closure and decrease in cGMP that are instrumental for GVBD, although other as yet unidentified pathway(s) co-operate to achieve the
same effect.
In the last few years, research on a possible involvement of EGF-like
factors in oocyte meiotic resumption has been extended to the human
and non-human primates. By using material donated by IVF patients,
AREG was found at detectable concentrations in the follicular fluid
only after a spontaneous LH surge or exogenous administration of
hCG (Zamah et al., 2010), suggesting an accumulation of the factor as
433
Oocyte maturation
Table I Factors involved in CC –oocyte interactions that are believed to control meiotic arrest and resumption.
Factor
Function
Species
References
.............................................................................................................................................................................................
cAMP
High intraoocyte concentrations of cAMP activate PKA
(types I and II) leading ultimately to inhibition of MPF activity and
maintenance of meiotic prophase arrest. cAMP is generated
endogenously by the oocyte, but some studies suggest it is also
produced by the somatic compartment and subsequently
transferred into the oocyte compartment.
Mouse, rat,
cow, human
Schultz et al. (1983), Dekel et al. (1984), Racowsky
(1984), Mehlmann et al. (2002), Thomas et al. (2002), Thomas
(2004), Vaccari et al. (2008), DiLuigi et al. (2008)
cGMP
cGMP is produced by CC and transferred through gap junctions
into the oocyte, where it represses the activity of PDE3
preventing cAMP degradation.
Mouse, rat
Bornslaeger et al. (1984), Törnell et al. (1990), Norris et al.
(2009), Vaccari et al. (2009)
NPPC
NPPC is primarily produced by MGC and released in the
follicular environment. By binding to its receptor NPR2, it
stimulates CC to produce cGMP that prevents meiotic
resumption after transfer into the oocyte.
Mouse, cow,
pig, human
Zhang et al. (2010, 2011a, b), Kawamura et al. (2011), Hiradate
et al. (2014), Franciosi et al. (2014)
NPR2
NPR2 is a membrane bound guanyl cyclase NPPC receptor
preferentially expressed by CC. It is positively regulated to
produce cGMP by binding with NPPC.
Mouse, rat,
cow. pig
Zhang et al. (2010, 2011b), Hiradate et al. (2014), Egbert et al.
(2014), Franciosi et al. (2014)
GDF9,
BMP15
Secreted as homo- or heterodimers, GDF9 and BMP15 act
synergistically and induce in CC the expression of Npr2 and the
inosine monophosphate dehydrogenase gene. The protein
product of the latter gene is the limiting element for the
generation of cGMP from guanyl metabolites.
Mouse
Wigglesworth et al. (2013)
AREG,
EREG
Produced by granulosa cells in response to acute LH stimulation,
AREG and EREG activate the EGF receptor(s) expressed by
CCs. This causes a decrease in intraoocyte cGMP and meiotic
resumption as an effect of down-regulation of gap junction
communication and inhibition of cGMP production by follicles
cells.
Mouse,
monkey, human
Park (2004), Vaccari et al. (2009), Norris et al. (2010), Peluffo
et al. (2012), Zamah et al. (2010), Ben-ami et al. (2011),
Humaidan et al. (2011)
The table includes a schematic description, divided by species, of current understanding of the function of such factors in relation to oocyte meiosis.
cAMP, cyclic adenosine monophosphate; PKA, protein kinase type A; MPF, M-phase promoting factor; cGMP, cyclic guanosine monophosphate; CCs, cumulus cells; PDE3,
phosphodiesterase type 3; NPPC, natriuretic peptide precursor type C; MGCs, mural granulosa cells; NPR2, natriuretic peptide receptor 2; GDF9, growth differentiation factor 9; BMP15,
bone morphogenetic protein 15; AREG, amphiregulin; EREG, epiregulin.
an effect of acute gonadotrophin stimulation. In addition, in follicle cells of
the same patients, AREG mRNA was highly expressed in both MGC and
CC, while the gene of the cognate receptor hErbb1 was preferentially
expressed by CC (Zamah et al., 2010), a sign that the CC compartment
is the target of AREG. Finally, it was observed that in individual follicles
low levels of AREG were associated with the recovery of immature
(GV-stage) oocytes (Zamah et al., 2010). Another investigation explored
the possibility that different modalities of triggering ovulation, i.e. hCG
administration or induction of endogenous LH surge with GnRH
agonist (GnRH-a), influence intrafollicular AREG levels and oocyte
quality (Humaidan et al., 2011). It was found that concentrations of the
EGF-like factor were significantly lower in the GnRh-a groups. This was
associated with a higher rate of mature oocytes and, following fertilization, embryos suitable for transfer, suggesting that in ovulation induction
LH and hCG may induce non-equivalent follicular responses and that the
effect of AREG on oocyte maturation and quality may be dosedependent. In the rhesus monkey, experiments carried out in ovarian
cycles involving naturally selected follicles confirmed that AREG is not detectable until several hours after the administration of an ovulatory bolus
of hCG (Peluffo et al., 2012). In the same study, immature COC retrieved
from small antral follicles were cultured for 48 h to achieve maturation in
vitro. In the presence of FSH and LH, AREG used at concentrations of 10
or 100 ng/ml had little impact on the rate of GVBD, but there was a trend
towards a higher rate of MII oocytes in the group treated with the lower
concentration of the growth factor. AREG and EREG were also tested in
IVM experiments involving a human experimental model (Ben-Ami et al.,
2011). Left-over immature GV-stage oocytes recovered from stimulated
cycles and left partially enclosed in their vestment of corona radiata cells
were exposed to AREG and EREG together. Similar to the case of rhesus
monkey oocytes, the growth factors did not appear to improve the rate
of GVBD but generated an increase in the overall availability of MII
oocytes.
In conclusion, much information has been gained on the mechanism of
meiotic arrest and resumption in the mouse and rat models, especially
with reference to the mode by which cGMP produced in the somatic
compartment generates intraoocyte conditions that are not permissive
for GVBD. Also, in the same context EGF-like factors have emerged as
important, although perhaps not exclusive, mediators of LH in the
process of meiotic resumption (Table I). Less clear is whether the
same paradigm is applicable to primates and in particular the human,
where details of the meiotic arrest mechanism and the role of EGF-like
factors have been much less investigated. This is reflected in the fact
that the use of such factors is far from reaching clinical application,
remaining entirely experimental.
434
Global chromatin remodelling, meiotic
resumption and developmental competence
During the protracted phase of quiescence in the primordial follicle, the
oocyte chromosomes are only moderately dispersed and transcriptionally active to assure a sufficient level of housekeeping functions. Subsequent follicle development is accompanied by a parallel phase of
oocyte growth, by which the female gamete reaches full size in preparation for fertilization and preimplantation development. Growth can
only be achieved through intense and sustained RNA transcription. To
this end, the oocyte chromatin acquires a condition of maximal dispersion to allow accessibility to multiple transcription sites by the transcription machinery. Once growth is achieved and the oocyte develops the
ability to resume meiosis and progress to the mature stage in the later
stages of folliculogenesis, the chromatin is subject to an extensive
process of condensation. In several species, chromatin condensation is
accompanied by intense transcription down-regulation. Such concomitance has generated the hypothesis that chromatin condensation is the
modality by which transcription silencing is achieved in preparation for
meiotic resumption. This seems to emerge from experiments showing
that in mouse oocytes knock-down of the gene encoding the developmental pluripotency-associated protein 3 causes failure of chromatin
condensation and incomplete transcriptional repression (Liu et al.,
2012). However, this matter remains controversial. In fact, in mouse
oocytes obtained from nucleoplasmin 2 (Npm22/2 ) mutants, chromatin condensation is not observed, while transcription silencing occurs
apparently undisturbed (De La Fuente et al., 2004). Progressive heterochromatinization occurs with modalities that are believed to be variations on the same theme in different species and appear to obey
precise spatio-temporal cues. In the mouse model, chromatin condensation reveals itself as a sequential transition between different conformational patterns (Mattson and Albertini, 1990). In small grown oocytes,
chromatin is dispersed and decondensed throughout the GV, with
only a few domains showing a higher degree of condensation. As cell
growth proceeds, the foci of heterochromatin increase in size and
number, tending subsequently to coalesce in larger clusters that eventually envelop the nucleolus. Following staining with fluorescent dyes, such
aggregation of chromatin appears as a rim surrounding the nucleolus and
is the origin of the classification of oocytes into two broad categories,
‘surrounded nucleolus’ (SN) and non-surrounded nucleolus (NSN)
(Zuccotti et al., 1995). It has been established that acquisition of the
SN configuration normally occurs in oocytes that have almost reached
the apex of their growth curve, at the time when follicles have already
formed a small antrum and massive transcription repression has
started (Chouinard, 1975; Parfenov et al., 1989; Mattson and Albertini,
1990). Development of the SN type appears to be a highly regulated
process because in mouse and human oocytes it occurs in close coordination with the oocyte ability to support the G2 to M phase transition
(Mirre et al., 1980; Bouniol-Baly et al., 1999). However, sometimes temporal co-ordination of chromatin condensation fails, with the consequence that both the SN and NSN types are represented among
oocytes collected from mouse antral follicles (Zuccotti et al., 1998).
Several studies indicate that timely evolution of the chromatin from an
NSN to an SN status supports the acquisition of meiotic and developmental competences. In fact, in comparison to the NSN population,
SN oocytes released from antral follicles and exposed to in vitro conditions display an increased ability to undergo GVBD and progression to
Coticchio et al.
MII. Also, while after IVM and fertilization NSN oocytes are unable to
cleave beyond the 2-cell stage, SN oocytes have the potential to
develop to the blastocyst stage (Zuccotti et al., 1998). How chromatin
condensation is achieved in oocytes approaching the stage of meiotic resumption is not fully understood. However, studies in the mouse are suggestive of a role for histone deacetylation. In this species, in fact,
pharmacological inhibition in vitro of histone deacetylase interferes with
chromatin transition from NSN to SN and meiotic progression (De La
Fuente et al., 2004). Granulosa cells are believed to be involved in the
process of chromatin condensation in the GV, as indicated by the
finding that in vitro the transition from NSN to SN is observed in
mouse oocytes cultured in association with companion granulosa cells,
but not in denuded oocytes (De La Fuente and Eppig, 2001).
Evidence derived from species other than the mouse suggests that the
association between progressive chromatin condensation and acquisition of oocyte meiotic and developmental competence is a general phenomenon, although specific details may vary. For example in the cow,
oocytes recovered from early and mid-antral follicles are characterized
by four alternative and sequential stages of chromatin condensation
(Lodde et al., 2007). In the G0 stage, chromatin presents a fibrillar constitution throughout the GV. In the G1 and G2 stages, chromatin is found
in a progressively more condensed state, displaying few foci of heterochromatin in the former and well-defined larger clumps of condensed
material in the latter. The G3 stage represents the highest condition of
condensation, with a single large clump of heterochromatin. Such
stages are indicative of oocyte meiotic and developmental potential.
G0 oocytes are in most cases unable to resume meiosis and mature in
vitro, while G1, G2 and G3 oocytes are competent to undergo GVBD
and progress to MII. However, G1, G2 and G3 oocytes are not equally
developmentally competent, because the G3 type displays a significantly
higher potential to develop to the blastocyst stage (Lodde et al., 2007).
The somatic compartment of the follicle is known to regulate in the
oocyte the process of progressive chromatin condensation. In the
same cow IVM model described above, the role of CC has been thoroughly investigated. In particular, it has been shown that conditions
that support maintenance of oocyte –CC gap junction communication
(i.e. exposure to low FSH levels), not only delay meiotic resumption
and prevent abrupt non-physiological transcriptional silencing of the
oocyte chromatin, but also induce a progressive orderly conversion of
the oocyte chromatin from a dispersed (G0) to a more condensed
form (Luciano et al., 2011). Importantly, the same conditions that
preserve oocyte –CC gap junction communications also increase the
proportion of GV oocytes that is able mature in vitro and, following
fertilization, support development to the blastocyst stage (Luciano
et al., 2011).
Data on human oocytes are limited but are nevertheless reminiscent
of those generated in animal models. An accumulation of heterochromatin around the oocyte nucleolus, described as a ‘karyosphere’, was initially observed in electron microscopy studies on the ultrastructure of
the antral follicle (Parfenov et al., 1989), but more recently chromatin
conformation was studied in left-over GV-stage oocytes derived from
stimulated assisted reproductive technology (ART) cycles. In such
oocytes, chromatin is found arranged in four alternative configurations:
(i) chromatin partially surrounding the nucleolus and dispersed in fibrillar
formations in the rest of the nucleus; (ii) all the chromatin surrounding
the nucleolus; (iii) chromatin completely surrounding the nucleolus and
partially organized in condensed masses throughout the nucleus and
Oocyte maturation
435
Figure 3 Optical sections of GVs of human fully grown immature oocytes analysed by confocal laser scanning microscopy. Micrographs represent specific
areas (A and B) or an entire section of the GV. Chromatin, stained with Hoechst 33258, appears (A) exclusively surrounding the nucleolus; (B) surrounding
the nucleolus, but also partially organized in condensed masses in adjacent domains of the nucleus; (C) only partially condensed, but mostly dispersed in
fibrillar formations in the rest of the nucleus. Scale bars represent 10 mm.
(iv) chromatin surrounding the nucleolus and organized in threads of
decondensed material in the rest of the nucleus (Combelles et al.,
2002). Interestingly, only type ‘c’ was observed to be able to mature
following culture, confirming the concept that at the GV-stage the
chromatin pattern reflects the oocyte ability to achieve meiotic maturation and attain developmental competence. Examples of chromatin
conformations visible in human fully grown GV-stage oocytes are
illustrated in Fig. 3.
Overall, these studies describe a consistent picture in which the
oocyte chromatin, largely decondensed and transcriptionally active
during follicle development, undergoes a timely and orderly process of
heterochromatinization at the end of the growth phase coinciding with
formation of the follicle antrum. This change, which may appear with different conformational manifestations in diverse species, represents an
important marker by which the relationship between oocyte growth
stage and quality can be ascertained. Importantly, these observations
suggest that GV oocytes obtained from antral follicles are apparently
morphologically similar, but in fact can be developmentally profoundly
different. This has important implications for human IVM because it
underpins the concept that meiotic failure can reflect an intrinsic
oocyte characteristic, irrespective of the ability of current IVM systems
to fully support maturation.
DNA damage and meiotic progression
DNA damage is a very common insult to which all cells are exposed
during their lifetime (Jackson and Bartek, 2009). Cells interpret DNA
single strand breaks (SSBs) or double strand breaks (DSBs) as a serious
threat to their integrity and react to that with a DNA damage response
(DDR) involving an arrest of the cell cycle as a downstream effect of the
activation of DNA damage checkpoints (DDCs), in order to give time for
repair mechanisms to intervene (Bartek and Lukas, 2007). If the scale of
damage is overwhelmingly superior to the capacity of the repair mechanism, the response takes the form of programmed cell death by which
affected cells are removed by apoptosis (Ciccia and Elledge, 2010). Mammalian oocytes are particularly exposed to DNA damage. In fact, before
undertaking growth and maturation they remain dormant in the ovary for
long periods of time (up to more than 40 years in the human). As a consequence, DNA damage can accumulate, jeopardizing oocyte function
and the genetic integrity of the ensuing embryo. DDR may be activated
principally at both the G1/S- and G2/M-phase transitions of the cell
cycle. Clearly, only the latter option can be adopted by oocytes, being
the arrest at the meiotic prophase and meiotic resumption at ovulation
reminiscent of the somatic G2/M-phase transition. At both cell phase
transitions, SSBs and DSBs trigger a DDC response by activating two
major kinases, i.e. ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (Reinhardt and Yaffe, 2009; Smith et al., 2010).
At G2, in the presence of DNA injury these regulators inhibit entry into
the M-phase by activation of the checkpoint kinases Chk1 and ChK2 that,
by destroying CDC25A or phosphorylating CD25B or CDC25C,
prevent the activation of CDK1 and, with it, the cyclin B-CDK1
complex that acts as the major regulator of the cell cycle (see also
‘Oocyte– CC signalling for meiotic arrest and resumption’ section for
the involvement of these regulators in meiotic resumption) (Bartek
and Lukas, 2007; Smith et al., 2010; Carroll and Marangos, 2013).
Taking advantage of the DDC arrest, the cell can repair various types
of DNA damage. At the DNA lesion site, ATM gives rise to the phosphorylated form of the H2AX histone (gH2AX), which acts as a catalyst
for the recruitment of the necessary checkpoint and repair factors (Burgoyne et al., 2007). Base excision repair, nucleotide excision repair and
mismatch repair can intervene in case of single strand damage, while
DSBs can be resolved by either homologous recombination or nonhomologous end joining (Jackson and Bartek, 2009). In oocytes, DSBs
have physiological significance in the context of meiotic recombination,
by allowing the exchange of portions of DNA between homologues
non-sister chromatids (McDougall et al., 2005; Burgoyne et al., 2009).
However even in such a case, if not occurring in a properly regulated
fashion, DSBs trigger a cell response that culminates in an apoptotic
event at the time of formation of the primordial follicle (Pittman et al.,
1998; Baudat et al., 2000).
At the antral and pre-ovulatory stages of follicle development, DNA
damage is anticipated to induce some form of response in fully grown
oocytes, but in such respect the DDR has remained a poorly described
phenomenon until recently. Fully grown oocytes are naturally arrested at
the meiotic prophase and a meiotic block in response to DNA damage
may not appear as a strict necessity. However, ability to resume
meiosis in response to an ovulatory stimulus is already developed at
the antral stage. Therefore, in addition to DNA repair mechanisms, it
436
is plausible that antral oocytes deploy cell cycle arrest strategies in response to DNA damage. The similarity of the mechanisms regulating
the G2/M transition in somatic cells and meiotic resumption in
oocytes would predict that fully grown oocytes can activate a DDC
cascade. If necessary, this would prevent the resumption of the
meiotic cycle for a time required for the intervention of DNA repair
mechanisms. Initial studies involving the mouse model were indicative
of a limited DDR in fully grown oocytes (Bradshaw et al., 1995), consistent with the finding that injection of females with etoposide, a topoisomerase II inhibitor and DSBs inducer, does not cause significant delay of
meiotic progression to metaphase I (MI). More recent experiments
have expanded the knowledge of oocyte response to DSBs. Mouse
fully grown oocytes released from antral follicles were initially exposed
to increasing concentrations of etoposide and their ability to resume
meiosis and progress into M phase of meiosis I was subsequently
assessed (Marangos and Carroll, 2012). At the highest concentrations
of etoposide, oocytes were unable to undergo GVBD and progress to
MI. However, at lower concentrations of the topoisomerase inhibitor,
the oocytes were capable of undergoing GVBD and entering M phase
with unaltered kinetics. Importantly, the intensity of gH2AX staining
shown by the DNA of the oocytes that underwent GVBD following etoposide treatment was much higher to that of control oocytes. This suggests that the absence of a cell cycle arrest in response to DSBs is not the
result of the intervention of an efficient DNA repair mechanism, but
rather the consequence of an inadequate DDC response. Further
experiments have demonstrated that prophase arrest after exposure
to high concentrations of etoposide, which causes extensive of DNA
damage, is ensured by sequential activation of ATM and Chk1 (Marangos
and Carroll, 2012). On the contrary, ChK1 remains inactive at low concentrations of etoposide (which produce only limited DNA damage),
explaining why GVBD occurs under such conditions. ATM also
remains inactive at low concentrations of etoposide. Therefore,
mouse oocytes are equipped with an ATM/ChK1 mechanism to
respond to DNA DSBs. However, this system is insensitive to low
levels of injury, becoming activated only above a certain threshold. It is
interesting to note how gH2AX, which is usually generated by the
active form of ATM, is detectable also at low levels of DNA damage,
an indication that phosphorylation of H2AX can also occur via an
ATM-independent regulation. Finally, phosphorylation of CDC25B
rather than degradation of CDC25A phosphatase seems to be the
downstream step promoted by the active form of Chk1 that ultimately
negatively regulates CDK1, inhibiting meiotic resumption (Marangos
and Carroll, 2012).
Further experiments have clarified some other aspects of oocyte sensitivity and response to DNA damage. Of particular interest is the fact
that, while mouse oocytes are to some extent responsive to the presence of DSBs in their DNA as discussed above, they appear rather insensitive to interstrand cross-links (ICLs) caused by alkylating agents,
such as mitomycin C (Yuen et al., 2012). Immature mouse oocytes
treated with mitomycin C are able to resume meiosis in vitro and progress
to MII but, following parthenogenetic activation, develop into severely
compromised embryos. Unlike the case of DSBs in which the lack of sensitivity to relatively low levels of damage is not caused by the absence of
gH2AX at the injury sites, failure to respond to ICLs probably resides in
the inability to activate a specific signalling system at the damaged sites. In
fact, in mouse oocytes treated with mitomycin C, the Fanconi anaemia
protein FANCD2, which is part of the Fanconi anaemia pathway and
Coticchio et al.
as such participates in mechanism of detection and repair of ICLs,
does not appear to form associations with lesion sites, while in cleaving
embryos this protein is found where ICLs occur (Yuen et al., 2012). Additional data indicate that DNA damage not detected or not repaired
during oocyte maturation can be passed on to the embryo. DSBs can
be experimentally induced also by bleomycin or laser microbeam dissection. Immature mouse oocytes exposed to these agents show some
delay in the GVBD kinetics, but nevertheless they are able to extrude
the PBI, progress to MII and support parthenogenetic development
(Ma et al., 2013). Notably, however, also in this case the resulting
embryos display different anomalies, including multiple pronuclei and
multiple micronuclei.
In conclusion, mouse oocytes appear to have limited ability to detect
and repair DNA lesions. Such limitation may have different origins. In fact,
in the case of ICLs a suitable mechanism of detection of the injury sites
seems to be missing. On the contrary, low or moderate levels of DSBs
appear to escape the DNA repair mechanism because oocytes are apparently unable to activate ATM, the master regulator of DDR, despite
the sites of lesions are signalled by the presence of gH2AX. The fact
that oocytes do not always arrest the meiotic process in the presence
of significant levels of DNA damage has potentially important implications, because genetic instability caused by DNA lesions could be
passed on from oocytes to embryos, possibly affecting fetal and postnatal life. In the human, nothing is known of the possibility that DNA
damage can be transmitted from oocytes to embryos, although recent
observations indicate that human GV stage oocytes (Fig. 4) are affected
by DSBs (Guglielmo et al., 2012).
GV positioning and oocyte polarity
At the MI and MII stages the meiotic spindle is located cortically, separated just a few microns from the cell surface. This assures that the
two meiotic divisions occur with minimal loss of cytoplasm through
the emission of two small polar bodies. In this fashion, the endowment
of organelles and molecules stockpiled during oogenesis is maintained
almost intact, assuring maximum support in terms of cell mass to
embryo development, in the absence of net growth during cleavage
and blastocyst formation. The eccentric position of the MII spindle in
the mature oocyte and the lack of overt asymmetry that characterizes
the primordial oocyte pose the question as to when and how asymmetry
is created during oogenesis. In this scenario, the GV has attracted much
attention for its involvement in the establishment of polarity in different
systems and for the fact that its breakdown is preliminary to the formation of an asymmetrically positioned MI spindle. Examples of the involvement of the cell nucleus in the establishment of cell polarity are
numerous. For example, in somatic cells, cortical localization of the
nucleus is associated with precise cellular and functional attributes,
such as axon projection in neurons and release of secretory organelles
in epithelial cells (Drubin and Nelson, 1996). In Drosophila, repositioning
of the GV from a central position to the dorsal cortex contributes to the
design of oocyte polarity and, with it, the body plan of the future organism
(Doerflinger et al., 2003). In mammals, there are clues that at some point
in time during the growth phase the GV, initially localized centrally in the
primordial oocyte, acquires an asymmetric position. In fact, when
observed in situ, fully grown oocytes of most mammalian species
display an eccentric GV (Hertig and Adams, 1967; Allworth and Albertini, 1993; Albertini and Barrett, 2004). On the contrary, mouse and
Oocyte maturation
437
Figure 4 Confocal microscopy micrographs of a human denuded GV-stage oocyte stained for detection of actin (red), chromatin (blue) and gH2AX
(green). In the merged micrograph as revealed by staining for gH2AX, DNA damage appears at specific sites of the oocyte chromatin, but not residual
CC. Scale bar represents 15 mm.
rat oocytes released from pre-ovulatory follicles and cultured under conditions that prevent meiotic resumption exhibit a central GV (Messinger
and Albertini, 1991; Chaigne et al., 2012). This inconsistency is probably
more apparent than effective, because observation of fully grown mouse
oocytes while still enclosed in their follicles indicates that the GV is positioned eccentrically (Albertini and Barrett, 2004). Therefore, in one or
more species a pre-existing asymmetry is lost when oocytes are placed
in extracorporeal conditions. This is not a mere academic matter
because it bears implications for the morphodynamics of oocyte maturation and above all the fate of the ensuing embryo, as discussed below.
Studies of the last few years lend credit to the hypothesis that oocyte eccentricity is not entirely dictated by intrinsic regulatory mechanisms, but
depends at least to some extent on external influences (Barrett and
Albertini, 2010). Mouse oocytes were recovered and fixed for confocal
microscopy analysis to assess GV/spindle position and oocyte –CC contacts at different hours after the administration of hCG to promote
meiotic resumption in vivo. Over a period of 6 h after hCG injection,
TZPs were initially uniformly distributed around the oocyte, except
the portion of the oocyte cortex coincident with an eccentric position
of the GV, where TZPs density (number) was higher. At 4–6 h
post-hCG, the newly formed MI spindle was localized cortically in a position where TZPs were highly enriched. Therefore in vivo, while surrounding the entire oocyte, TZPs become progressively more
abundant in the region of the cortex where the GV and MI spindle are
eccentrically localized. Analysis of COC cultured in vitro in the absence
of FSH revealed a different dynamics. In fact, after 2 h of culture the
GV was repositioned centrally and the MI spindle appeared in the
same central position shortly afterwards, before being repositioned cortically at 6 h. The TZPs, initially present throughout the cortex, progressively disappeared during the same time frame. Live cell differential
interference contrast time-lapse microscopy confirmed that in vitro
the GV, initially localized cortically, is repositioned in the centre, while
the MI spindle forms centrally and only subsequently migrates to the
cortex. Interestingly, culture of immature COC in the presence of
FSH, whose receptors are expressed by CC but not the oocyte, prevents
the disappearance of TZPs and the disanchoring of the GV from the
cortex. Therefore, also in vitro the MI spindle forms eccentrically and
does not require repositioning, provided that COCs are exposed to
FSH. A direct TZP involvement in the maintenance of GV eccentricity
and oocyte asymmetry is suggested by the fact that maturation in vitro
in the presence of latrunculin, an agent that depolymerizes actin filaments
that form the TZP cytoskeleton, determines TZPs disappearance and
migration of the GV from the cortex to the centre, irrespective of the exposure of COC to FSH (Barrett and Albertini, 2010). GV disanchoring
from the cortex and repositioning to the centre involves significant developmental consequences. In fact, centrally positioned GVs become larger
438
before undergoing GVBD. In addition, they give rise to MI spindles that
are larger in comparison to in vivo matured oocytes and display dispersed
chromosomes, conditions that are associated with an increase in the rate
of oocyte aneuploidy (Barrett and Albertini, 2010). Generation of a large
MI spindle involves also another important developmental phenomenon
consisting of the emission of a large polar body (Plancha et al., 2005), a
condition that reduces the cytoplasmic mass of the MII oocyte to
levels that may not be sufficient to ensure successful development of
the ensuing embryo (Fig. 5). Collectively, the above evidence leads to
the conclusion that in mammals oocyte asymmetry, signified by cortical
localization of the GV, occurs before the oocyte achieves full size. In some
species like the mouse, such asymmetry may be lost as an effect of
removal of regulatory cues generated by the follicular environment.
Loss of asymmetry at the GV stage can be re-established after GVBD
by migration of the MI spindle to the cortex, whose formation occurs
centrally under non-physiological conditions. Therefore in the mouse
oocyte, although the forces that govern GV eccentricity display limited
stringency, polarity can be regained—if lost—by back up mechanisms
acting downstream of GVBD. However, reacquisition of polarity
comes with a cost, represented by abnormal spindle morphogenesis
and increased developmental risks. Crucial for the maintenance of the
original asymmetry is the presence of the TZPs that connect the
oocyte with surrounding CC. The pattern of distribution of such intercellular contacts mirrors the positioning of the GV and the MI, suggesting the
concept, novel for mammals but not for other model species such as
Drosophila, that elements of oocyte design are dictated by polarized
signals of somatic origin.
In the human, oocyte asymmetry has been poorly explored, although
preliminary observations seem to suggest that polarity is established
before GVBD. In left-over immature oocytes recovered from stimulated
ART cycles, in most cases the GV is found cortically although it can also be
localized centrally. Time-lapse microscopy has revealed that initial positioning may be subject to changes, with subsequent effects on meiotic resumption. In particular, cortically positioned GVs may undergo significant
and continued displacement for several hours, in the vast majority of
cases displaying the ability to undergo GVBD as long as contact with
the cortex is not lost (Coticchio et al., 2011). Centrally localized GVs
can also be subject to intense translational movements, but GVBD is unlikely to occur unless they are repositioned to the cortex. Overall, such
cellular behaviours are consistent with the hypothesis that, although
oocyte polarity may or may not be established in fully grown oocytes,
the GV has an affinity for the cortex as an elective domain where to
undergo breakdown. If confirmed, this would indicate a previously unrecognized role for the oocyte cortex. Also, while the reason why immature oocytes from stimulated cycles can exhibit the GV at different
positions remains obscure, it is tempting to speculate that the forces
that control spindle relocation in mouse oocytes may be also involved
in GV repositioning in human oocytes. Regardless, these observations
have significant clinical implications, in consideration of the use of immature oocytes for various fertility treatment approaches.
Cytoskeletal forces governing spindle
positioning
The above discussion illustrates the importance of repositioning the MI
spindle to the cortex when its assembly occurs in the vicinity of the
oocyte centre. Irrespective of whether spindle migration may be a
Coticchio et al.
Figure 5 Schematic representation of cellular and cytoskeletal
changes occurring during oocyte maturation, under normal (A) or
altered (B) conditions. In most mammals, under physiological conditions the GV (blue) is positioned eccentrically in fully grown immature
oocytes contained in antral and pre-ovulatory follicles. Following
GVBD in vivo, the MI spindle (green) forms in the same position and
the vicinity of chromosomes (blue) to the sub-oolemmal actin induces
in the adjacent cortex the organization of an actomyosin domain (red)
that is required for PBI emission. Finally, the PBI is extruded and the
oocyte pauses at MII until fertilization. In the in vitro mouse model,
under inappropriate culture conditions or in the absence of CC, maturation is subject to major perturbations. The GV disanchors from the
cortex and, while drifting towards the oocyte centre, increases in size
(broken arrow). As a consequence, the MI spindle also forms centrally.
Under such conditions, the spindle is larger in comparison to in vivo
matured oocytes, a factor that is associated with an increase in the
rate of oocyte aneuploidy. Loss of cell asymmetry during meiosis
implies that formation of the cortical actomyosin domain and extrusion
of the PBI must be preceded by repositioning of the spindle to the
cortex. Once spindle position is readjusted, the first meiotic division
can occur, but the resulting PBI is abnormally large. As a result, the cytoplasmic mass of the MII oocyte is reduced and embryo development
may be affected.
Oocyte maturation
safeguard mechanism to recover a previously lost asymmetry, it represents a major change in the design of the oocyte that exemplifies the
fundamental role of the cytoskeleton in the choreography of oocyte
maturation.
In somatic cells undergoing symmetric or asymmetric division,
spindle positioning relies on the action of astral microtubules and
microtubule-associated motor proteins (Verlhac et al., 2000). A similar
solution does not seem to be adopted by oocytes, as indicated by the
fact that the meiotic spindle is devoid of astral microtubules and chromosome migration still occurs after treatment of MI oocytes with microtubule depolymerizing agents (Verlhac et al., 2000). Instead, recent
findings highlighted a fundamental role for actin in the repositioning of
the MI spindle to the cortex of the mouse oocyte, confirming and detailing classical studies performed in the 1980s (Maro et al., 1986). In fact,
mice mutant for Fmn2, a gene encoding the actin-nucleating protein
formin-2 (FMN2), are defective for spindle migration at MI (Leader
et al., 2002). In GV-stage oocytes, FMN2 is localized at the cortex,
while following GVBD sophisticated staining techniques allow the visualization of actin filaments bridging the spindle and the cortex (Schuh and
Ellenberg, 2008). Consistent with the observation that myosin II is found
at the spindle poles and myosin II inhibitors prevent spindle migration,
this has induced some authors to hypothesize that the spindle is pulled
towards the cortex by an actomyosin-based contractile mechanism
(Chaigne et al., 2012). An alternative model has been developed from
observations that have (i) indicated additional regions where FMN2 localizes and (ii) precisely described speed and direction of spindle movement during the process of symmetry breaking and repositioning in the
mouse oocyte. Shortly after GVBD, FMN2 clusters around the forming
spindle where it generates a cloud of actin filaments (Yi et al., 2013).
FMN2 and elements of the ER colocalize in the immediate surroundings
of the spindle, suggesting also that precise positioning of FMN2 depends
on this organelle. Such colocalization was crucial in experiments in which
FMN2 distribution was experimentally disrupted to test the hypothesis
that actin polymerization is essential for spindle migration. Indeed,
agents that cause dispersion of the ER also disperse FMN2 distribution
around the spindle, but not at the cortex, and inhibit spindle migration
(Yi et al., 2013). However, how an initially symmetric distribution of
FMN2 and actin filaments around the spindle could assist polarized
spindle displacement towards the cortex remained an unsolved
439
problem until chromosome movement, as a measure of spindle displacement, was tracked and precisely analysed by time-lapse live cell imaging
techniques. During spindle migration, chromosome trajectory indicates
two types of movement. Initially, chromosomes move with low instantaneous speed and straightness within a range of 5–10 mm from the
oocyte centre. Such kinetic behaviour has been described as ‘confined
random walk’. In a second phase that occurs abruptly, chromosomes
move towards the cortex with much higher speed and much straighter
trajectory (Yi et al., 2013). This is suggestive of the existence of two distinct mechanisms by which chromosomes and the spindle are relocated
to the cortex. Interestingly, the same pattern of movement is observed if
the spindle is experimentally depolymerized shortly after GVBD.
The involvement of FMN2 in the first phase of random walk emerges
not only from the inability of Fmn22/2 mutants to support spindle movement, but also from the observation that, if the spindle is disrupted,
FMN2 accumulates opposite to the direction taken by chromosomes
as they move closer to the cortex (Yi et al., 2013). It seems plausible,
therefore, that the initial phase of slow movement of the spindle is generated by clustering of FMN2 around that spindle that, by inducing local
nucleation of f-actin, promotes an undirected pushing force that moves
the spindle randomly within a short range from the oocyte centre.
However, it is possible that as some point FMN2 accumulation and
f-actin polymerization in the vicinity of the spindle can occur stochastically in a polarized fashion, making the spindle drift more decisively to the
cortex and creating the premises for a symmetry breaking event (Li,
2013).
If FMN2 is responsible for the initial slow and random displacement of
the spindle, what causes the subsequent much faster and polarized
movement towards the cortex? Time-lapse video microscopy has
revealed that in the mouse oocyte the second phase of chromosome
and spindle movement is accompanied by an intense activity of cytoplasmic streaming (Yi et al., 2013). An uninterrupted flow of cytoplasmic particles may be observed departing from the area of the cortex approached
by the chromosomes and the spindle. Drawing two symmetrical paths,
these currents of cytoplasmic particles drift away cortically, reach the opposite oocyte pole and return centrally towards the spindle (Fig. 6). This
pattern is rotationally symmetric around the axis connecting the spindle
and the oocyte centre, while the speed of cytoplasmic particles mirrors
that of the spindle, increasing gradually as spindle movement progresses.
Figure 6 Schematic representation of forces governing spindle repositioning to the cortex prior to PBI emission. Initially, the spindle moves with low
instantaneous speed and straightness in the vicinity of the oocyte centre as an effect of symmetric distribution of FMN2 (pink circles) and actin filaments
(not shown). Afterwards, FMN2 accumulation and f-actin polymerization in the vicinity of the spindle acquires a polarized distribution, causing the spindle to
drift towards to the cortex. As the spindle moves towards the cell periphery, Arp2/3 (red circles) becomes progressively localized in the adjacent cortical
domain. Dynamic actin polymerization generated by Arp2/3 promotes an uninterrupted flow of cytoplasmic material (blue broken arrows) that drifts away
cortically, reach the opposite oocyte pole and return centrally towards the spindle. This is believed to produce a pushing force that ultimately determines
spindle positioning at the cortex.
440
Overall, this is strongly indicative that streaming of cytoplasmic material
generates a pushing force that accounts for the fast phase of spindle
movement. Interestingly, cytoplasmic streaming does not stop with the
repositioning of the MI spindle to the cortex. It continues even after
PBI extrusion, maintaining the MII spindle at the cortex. Therefore, cytoplasmic streaming appears to emerge as a major phenomenon by which
oocyte polarity is achieved during maturation (Yi et al., 2011).
Experiments involving mouse MI and MII oocytes indicate that cytoplasmic streaming is a downstream effect of an interplay between the
chromatin and the cortex. Central to this interaction is the actin-related
protein 2/3 (Arp2/3) complex, a factor that promotes actin nucleation
in cell motility and membrane trafficking (Goley and Welch, 2006).
Following GVBD when chromosomes are still positioned centrally,
Arp2/3 is not detectable by immunofluorescence. However, as chromosomes move towards the cell periphery, Arp2/3 becomes progressively localized in the cortical domain where the MI spindle will finally
become repositioned (Yi et al., 2013). This suggests the concept that
at some point of the journey away from the oocyte centre, the chromosomes (but not the spindle, as explained below) trigger a cortical polarized localization and activation of Arp2/3. In its turn, by inducing actin
nucleation, Arp2/3 is believed to orchestrate flows of cytoplasmic material that push decisively the MI spindle towards the cortex. The role
of Arp2/3 clearly emerges from experiments in which the specific
inhibitor CK-666 was found to prevent the generation of cytoplasmic
flows and spindle repositioning to the cortex (Yi et al., 2013). The
spindle microtubular apparatus is not essential for this mechanism,
because its depolymerization does not affect cytoplasmic streaming
and the second phase of fast chromosome movement towards the
cortex (Yi et al., 2013).
Little is known on the signal that induces Arp2/3 localization in the
cortical domain adjacent to the MI spindle. However, fast spindle movement does not occur in Fmn22/2 oocytes in which chromosomes
are positioned naturally towards the cortex, indicating that FMN2 may
play a role in assisting the communication between the chromosomes
and the adjacent cortex to achieve Arp2/3 localization or activation
(Yi et al., 2013).
Information has also been gained on the involvement of cytoplasmic
streaming in maintaining the meiotic spindle in a cortical position in MII
oocytes. As mentioned above, in such oocytes cytoplasmic streaming
occurs according to trajectories entirely comparable to those observed
in MI oocytes (Yi et al., 2011). Similarities in cytoplasmic streaming
between MI and MII oocytes also involve Arp2/3, which at the mature
stage is detectable by immunofluorescence microscopy in the cortex
overlying the spindle (Yi et al., 2011) and whose inhibition by CK-666 disrupts cytoplasmic streaming and causes spindle detachment from the
cortex (Yi et al., 2011). Neural Wiskott-Aldrich syndrome protein
(N-WASP), an upstream activator of Arp2/3 (Campellone and Welch,
2010), can also be detected in the cortex near the spindle. Its knockdown in mouse oocytes by morpholino injection determines disruption
of Arp2/3 localization and spindle detachment (Yi et al., 2011), indicating
an active role for this regulator in spindle positioning. Therefore, it is
believed that also at the MII stage cortical localization of Arp2/3 and
activation of its actin-nucleating ability generates a flow of cytoplasmic
material that pushes and maintains the spindle at the cortex. Spatiotemporal image correlation spectroscopy analysis confirmed the importance of the acting-nucleating activity of Arp2/3, showing that actin
filaments move along trajectories equivalent to those of cytoplasmic
Coticchio et al.
streaming and that Arp2/3 inhibition with CK-666 drastically attenuates
actin flow in addition to disrupting cytoplasmic streaming (Yi et al., 2011).
In mouse MII oocytes, further details are known on how chromosomes
induce Arp2/3 localization in the adjacent cortex. Previous studies had
shown that MII chromatin is source of a Ras-related nuclear protein
(Ran) GTPase signal that organizes an actin-rich cortical cap required
for polar body extrusion, as illustrated in the following section (Deng
et al., 2007). To test the hypothesis that the same signal was responsible
for Arp2/3 localization and consequent spindle positioning, mouse MII
oocytes were injected with a dominant-negative Ran protein. The treatment disrupted localization of N-WASP and Arp2/3 and caused spindle
detachment from the cortex (Yi et al., 2011). Overall, the above evidence
demonstrate that, through Ran signalling, MII chromosomes activate a
cascade of events that sequentially involve N-WASP and Arp2/3 localization, activation of Arp2/3, generation of actin flows and finally cytoplasmic streaming, by which the cortical position of the MII spindle is
maintained.
Cortical polarization and polar body extrusion
At both meiotic divisions, the peripheral positioning of the spindle not
only orientates the cleavage plane in a fashion that results in the extrusion
of a very small polar body, but also induces structural changes in the overlaying cortex. In mouse MI and MII oocytes, it has been known for long
time that chromosomes cause significant modifications of the adjacent
cortex, including disappearance of microvilli from the oocyte surface
and above all a thickening of the cortical cytoskeleton involving a prominent actin cap surrounded by a ring of myosin II (Longo and Chen, 1984,
1985; Maro et al., 1986). Such a specialized contractile actomyosin structure, referred to as cap, is crucial for the mechanics of PBII extrusion
(Brunet and Verlhac, 2010). In mouse MII oocytes, more recent findings
have shown that chromosomes (not the spindle microtubules) are the
source of a cue that, in addition to triggering cytoplasmic streaming
(see above), is responsible for the assembly of the actomyosin cap.
Chromosome action is believed to be a function of mass and distance
from the cortex. This can be inferred from experiments in which
oocytes were injected with beads coated with plasmid DNA (Deng
et al., 2007). The beads were able to induce formation of cortical actomyosin structures as long as their distance from the cortex was
shorter than 20 mm. In addition, the size and strength of the actomyosin
cap was inversely proportional to the distance of the beads from the
cortex and directly proportional to the quantity of beads. Therefore,
MII chromosomes generate a signal progressively decreasing with distance that is used as a molecular ruler to trigger changes in the nearest
region of the cortex. Such a solution ensures that the actomyosin cap
forms only when and where it will be needed to assist PBII extrusion.
The biochemical nature of this signal is believed to be similar to the
system that promotes microtubule polymerization and spindle organization in the vicinity of the chromosomes, involving a Ran-GTPase gradient.
The signal is centred at the chromosomes and depends on two
chromatin-bound factors—chromatin-associated RCC1 (regulator of
chromosome condensation 1) and RAN GEF (guanine nucleotide exchange factor)—while another factor (cytoplasmic GTPase activating
protein for RAN) is dispersed throughout the cytoplasm (Dumont
et al., 2007; Yi et al., 2011; Azoury et al., 2012). An important clue of
the involvement of a Ran gradient in the chromosome ability to influence
the cortex derives from experiments showing that in mouse dominant-
Oocyte maturation
441
Tyr kinase (Luo et al., 2009) and Ser/Thr kinase MOS (Deng et al.,
2005), a major cell cycle regulator, making it more difficult to decipher
the overall scenario by which the actomyosin cap and the mechanics of
PB extrusion are regulated.
Human mature oocytes also display a network of sub-oolemmal actin,
but the presence of an actin-rich domain in the vicinity of the spindle has
remained elusive until recently. In fact, conventional fluorescence microscopy does not show the existence of an actin cap. However, highresolution confocal microscopy has recently demonstrated that in MII,
but not GV-stage, oocytes cortical actin is indeed measurably more
abundant in the vicinity of the spindle (Fig. 7) (Coticchio et al., 2014).
In the context of human assisted reproduction, further research on the
polarity of the cortical cytoskeleton and the mechanism of PBII extrusion
is warranted. Interest in this matter is exemplified by the fact that in some
cases, often in a cycle- or patient-specific fashion, oocytes fertilized by a
single sperm develop abnormally into tripronucleate eggs as a consequence of PBII retention (Porter et al., 2003). Interestingly, the degree
of actin polarization and abundance was found to diminish with increasing
maternal age (Coticchio et al., 2014), an observation that sheds new light
on the phenomenon of oocyte ageing. In fact, while it is widely recognized
that a progressive increase in oocyte aneuploidy is the major cause of a
decrease in female fertility with age (Hassold and Hunt, 2009), it
cannot be rule out a priori that other factors, such as a weakening of
the actin cytoskeleton, contribute to oocyte ageing.
Changes in cortical mechanics important for
oocyte maturation
Figure 7 Confocal microscopy micrograph of a human MII oocyte.
Microtubule tubulin is represented in green, chromosomes in blue
and actin in red. Optical intensity profile for the actin channel allows
semi-quantitative detection of sub-oolemmal and cytoplasmic actin.
Two peaks of different height are visible, corresponding to domains of
the cortex proximal (a) and distal (b) to the spindle, suggesting cortical
polarization of actin. Bar equals 30 mm.
negative Ran mutants the formation of the actomyosin cap is inhibited. In
MI oocytes, chromosomes are also a source of a Ran gradient. However,
in dominant-negative Ran mutants disruption of the actomyosin cap is
not observed at the MI stage and PBI extrusion occurs undisturbed,
raising the question of whether cortical polarization is regulated by two
different mechanisms at MI and MII (Deng et al., 2007).
How the chromatin-borne Ran gradient is translated into a regulatory
signal that induces the formation of an actomyosin structure is only partially understood. It is known that the actin nucleator Arp2/3 is localized
in the cortex overlaying the spindle and that inhibition of Arp2/3, or its
upstream activator N-WASP, disrupts formation of the actin cap (Halet
and Carroll, 2007; Yi et al., 2011). However, evidence for other possible
upstream regulators is sketchy. CDC42 and RAC (two Rho-type
GTPases that regulate actin polymerization in other cell systems) are suspected to control N-WASP activity and therefore actin nucleation in the
vicinity of the spindle (Na and Zernicka-Goetz, 2006). Recent work has
also revealed that several other factors intervene in the process of actin
polarization, such as myosin light chain kinase (Deng et al., 2005), FYN
In mitotic cells that give rise to daughter cells of equivalent size, it is known
that cytokinesis requires a timed stiffening of the cortex (Théry and
Bornens, 2006). This prompts the question of whether and how cortical
tension changes during oocyte maturation, especially considering the
highly asymmetric divisions that characterize the meiotic process and
the polarization of the cortex described in the previous section. Two
recent studies carried out in mouse oocytes have shed light on the
mechanics of the oocyte cortex, highlighting fundamental and previously
unrecognized aspects of oocyte cytokinesis. An initial investigation
revealed the rather surprising notion that, unlike mitotic cells, the
overall stiffness of the oocyte cortex progressively decreases 6-fold as
meiosis progresses from the GV to the MII stage (Larson et al., 2010).
However, this decrease does not occur homogeneously but reflects
the polarity that is established in the cortex during maturation. In fact,
in MII oocytes the cortical tension of the domain overlying the cortex
is almost 3-fold less than that of the remaining cortex, despite sphericity
of the cell being preserved. Therefore, the very basic mechanics of the
oocyte cortex differ from what is normally observed in somatic cells,
most likely in order to assist the extreme asymmetry of polar body extrusion. Further studies have identified some of the factors that regulate cortical tension. In the first place, it has been important to rule out that an
overall decrease in stiffness is not merely caused by a progressive reduction in actin content, whose polymeric and soluble fractions do not vary
during maturation. Paradoxically, a thickening of cortical actin is progressively observed during maturation, reaching its maximum at the sites
where polar bodies are emitted (Chaigne et al., 2013). By exposing
mouse oocytes to specific inhibitors, thickening of cortical actin has
been shown to depend on at least two regulatory elements, i.e. the
actin nucleator Arp2/3 and the Mos-MAPK pathway. It is hypothesized
442
Coticchio et al.
Table II Schematic description, divided by species, of changes occurring in organelles and the cortical domain in oocytes
during the transition from the prophase (GV) stage to the MII stage.
Cytoplasmic
component or
domain
Prophase
Metaphase II
References
.............................................................................................................................................................................................
Endoplasmic
reticulum
Mouse: poorly defined organelle clusters are
visible throughout the ooplasm, while larger less
well-defined accumulations are localized only in
the cell interior and without obvious polarization.
Cow and hamster: elements of the organelle are
found in a cortical position.
Human: organelles elements are organized in a
fine network extending throughout the cortex and
the cell interior.
Mouse: organelle elements form a reticular
network extending over the entire cell, with large
accumulations in the oocyte cortex. Such clusters
are polarized, being absent from the microvilli-free
cortical domain overlaying the MII spindle.
Cow and hamster: small clusters are more
homogeneously dispersed.
Human: clusters are found throughout the oocyte,
also in the vicinity of the MII spindle.
Mehlmann et al. (1995), Shiraishi
et al. (1995), Payne and Schatten
(2003), Mann et al. (2010)
Mitochondria
Mouse: aggregates are finely dispersed
throughout the cell, showing only moderate
concentration around the GV
Mouse: aggregates are found throughout the
oocyte, with partial accumulation in the vicinity of
the meiotic spindle.
Human: mitochondria with higher potential
difference across the inner mitochondrial
membrane, and therefore more functionally
competent, are found beneath the oolemma.
Dalton and Carroll (2013), Van
Blerkom (2003, 2011)
Golgi apparatus
Mouse, cow, rhesus monkey: the organelle is not
organized in a typical single ribbon-shaped array,
but is formed from many ‘mini-Golgis’ dispersed in
the cytoplasm. These aggregates appear
moderately more concentrated in the interior
than in the cortex.
Mouse, cow, rhesus monkey: Mini-Golgis fragment
in even smaller clusters and diffuse evenly
throughout the ooplasm.
Hyttel et al. (1989), Moreno
et al. (2002)
Cortical granules
Mouse, cow, human: CGs are centrally localized a
finely dispersed in the oocyte
Mouse, cow, human: CGs are found beneath the
oolemma but in the mouse are excluded by the
cortex adjacent the MII spindle
Szollosi (1967), Nicosia et al.
(1977), Liu et al. (2003)
Cortex
Mouse: cortex characterized by high stiffness
Mouse: 6-fold reduction in cortex stiffness in
comparison to the GV prophase stage. Stiffness of
domain overlying the spindle almost 3-fold less than
that of the remaining cortex.
Larson et al. (2010)
GV, germinal vesicle; MII, metaphase II; CGs, cortical granules.
that the function of these elements is organized sequentially, according to
a model in which Mos-MAPK acts as an upstream signal that promotes
Arp2/3 activation by phosphorylation of the Arp2/3 regulator
Wave2. On another hand, softening of the cortex that takes place
during maturation seems to depend on another factor, myosin II,
whose role is well established in contractile activities that shape the
cortex of mitotic cells. Indeed, at the GV stage myosin II is predominantly
localized throughout the cortex, but as soon as maturation begins and
cortical tension decreases it is progressively displaced in the cytoplasmic
compartment by a mechanism apparently driven by Mos-MAPK
(Chaigne et al., 2013). The cortical area adjacent to the spindle is predictably spared from this action of displacement, to allow the maintenance of
an actomyosin contractile ring that will drive ingression of the cleavage
furrow during PB extrusion. Why the oocyte regulates the mechanical
properties of its cortex by reducing the overall tension is only partially
understood, but it is significant that treatment with concanavalin A that
causes stiffening of the cortex also prevents MI spindle migration from
the oocyte centre to the periphery (Chaigne et al., 2013). Therefore,
spindle –cortex interactions occur with modalities that go beyond the
creation of an actomyosin cap and cytoplasmic streaming.
In conclusion, collective evidence gathered in relation to the process of
oocyte polarization and cytoskeletal dynamics strengthens the notion
that the cortex is a distinct cellular domain where events believed to
be crucial for oocyte maturation take place. Much, however, remains
to be revealed about the role of the oocyte cortex. Suggestions in this direction concern the finding that Fyn kinase-dependent protein tyrosine
phosphorylation occurs at the cortex in a preferential and progressive
fashion during maturation and that in vitro this phenomenon is more pronounced under conditions that support more efficiently oocyte developmental potential (McGinnis and Albertini, 2010).
Cytoplasm reorganization during maturation
During the transition from the prophase (GV) stage to the MII stage virtually all major organelles undergo important changes in structure, function and/or distribution. Such modifications are described in the
following sections and schematically summarized in Table II, together
with those concerning the oocyte cortex, as discussed in the previous
section.
Endoplasmic reticulum
The ER is an organelle formed from membranous tubules and vesicles
organized in multiple continuous sub-components, namely rough ER,
smooth ER and nuclear envelope. Protein synthesis and assembly and
Oocyte maturation
lipid synthesis are major functions of the ER in all cells. In oocytes, the
ability of this organelle to store and release free Ca2+ in the cytoplasm
acquires a specific and fundamental importance. In fact, at fertilization,
the ER is central to a mechanism that generates transient rises in the
concentration of intracellular free Ca2+ [Ca2+]i, referred to as oscillations, by which MII oocytes respond to gamete fusion (Swann and Yu,
2008). Amplitude, frequency and duration of these oscillations are
then decoded by downstream regulators into signals that trigger the
events of fertilization, i.e. cortical granules (CGs) release, exit from
MII, PBII extrusion and formation of pronuclei (Ducibella and Fissore,
2008). Studies carried out over decades in different species have finally
identified in the sperm-borne phospholipase C zeta (PLCz) the origin
of the stimulus that ultimately induces the Ca2+-releasing activity
of the ER (Swann, 1990; Swann et al., 2006). Introduced into the
ooplasm as a result of oocyte-sperm fusion, PLCz is in fact believed
to activate the phosphoinositide signalling pathway by breaking down
phosphatidylinositol 4,5-bisphosphate into diacylglycerol and inositol
1,4,5-trisphosphate (IP3). Downstream from this signalling pathway,
IP3 finally binds its receptor (IP3R) located in the membranes of the ER
that, as a consequence of a conformational change caused by the
binding, acts as an IP3-gated channel allowing release of Ca2+ from the
lumen of the organelle (Swann and Lai, 2013). The molecular and biochemical aspects of the regulation of the Ca2+-releasing mechanism in
mature oocytes have been extensively described in recent publications
and will not be discusses in this context (Wakai and Fissore, 2013;
Wakai et al., 2013). On the contrary, here attention will be focused on
the changes to which the ER is subject during oocyte maturation that
are part of a larger cytoplasmic reorganization and are relevant to the
acquisition of developmental competence.
In mouse oocytes, changes in the arrangement of the ER during maturation become immediately apparent from the comparison of GV and
MII oocytes. In the GV, no well-defined organelle clusters are visible
throughout the ooplasm, while larger less well-defined accumulations
are localized only in the cell interior and without obvious polarization.
A different picture emerges from the analysis of MII oocytes, in which
the ER takes the form of a reticular network extending over the entire
cell, with large (1 –2 mm in diameter) accumulations of elements in the
oocyte cortex. Such clusters display a polarized distribution because
they are not found in the microvilli-free cortical domain overlaying the
MII spindle (Mehlmann et al., 1995). In other mammalian species, the
pattern of ER rearrangement during maturation may be different, as
observed in the cow (Payne and Schatten, 2003) and hamster (Shiraishi
et al., 1995) where elements of the ER are found in a cortical position in
GV-stage oocytes and more homogeneously dispersed in small clusters
in mature oocytes. Differences in the distribution of the ER in mouse GV
and MII oocytes are associated with changes in the ability to respond to
Ca2+-releasing stimuli. In fact, MII oocytes respond to sperm penetration
or treatment with IP3 with Ca2+ transients that are much larger and more
frequent in comparison to those generated by GV oocytes under the
same conditions (Jones et al., 1995). Therefore, it is possible that the
transition of the ER from a structure organized in non-polarized,
poorly defined accumulations localized deep in the cytoplasm into a
network of more well-defined asymmetrically distributed cortical clusters represents a cellular manifestation of an increased responsiveness
to the activating stimulus triggered by the sperm. Other studies in
mouse oocytes suggest that regulation of the IP3R may account for the
increased sensitivity of the Ca2+-releasing mechanism that accompanies
443
maturation. In fact, western blot analysis indicates that the amount of the
IP3R almost doubles during the transition from the GV to the MII stage
(Mehlmann et al., 1996). As shown by immunofluorescence microscopy,
this increase is associated with a redistribution of the IP3R, which in GV
oocytes is diffusely localized in the cortex without apparent polarization,
while in MII oocytes the same molecule appears distributed in clusters of
1–2 mm occupying the cortex, except the region around the MII spindle.
In addition, during maturation the IP3R appears to increase its sensitivity,
i.e. response to IP3, as an effect of phosphorylation by M-phase kinases,
namely cdk1 (Wakai et al., 2011). Overall, the above evidence is consistent with the hypothesis that during maturation the oocyte enhances the
sensitivity of its Ca2+-releasing mechanism by redistributing to the cortex
the elements of the ER that act as intracellular stores of this ion, and by
increasing the number and reactivity of IP3R molecules, through which
the ER responds to the generation of IP3 that follows the action of the
sperm PLCz. In this fashion, the oocyte is thought to set the stage
for the initial phases of fertilization. The apparent importance of ER
redistribution during maturation has inspired studies investigating the
role of the cytoskeleton, by which organelles are usually moved within
cells. Immediately following GVBD and before becoming organized in
clusters of 1–2 mm occupying the cortex, in mouse oocytes the ER
develops into a dense array of membranes that surrounds the newly
formed MI spindle. As discussed in the previous section, this is instrumental for the localization of FMN2 and the initial phase of spindle
movement towards the cortex (Yi et al., 2013). Such a distribution in
the vicinity of the spindle appears to depend on the breakdown of the
GV because it does not occur if GVBD is experimentally prevented
(FitzHarris et al., 2007). Nocodazol, a microtubule depolymerizing
agent, or inhibition of cytoplasmic dynein, a motor protein associated
with microtubules, also prevent formation of the ER network around
the spindle. At later stages of maturation, the cloud of ER accompanies
the spindle during its journey to the cortex and, in coincidence with
the MI –MII transition, acquires the typical organization in clusters of
1–2 mm localized at the cortex. This final phase of reorganization is
not affected by depolymerization of microtubules, but is prevented
by disruption of microfilaments. Thus, reorganization of the ER during
maturation appears as a finely regulated phenomenon whose initial
phase following GVBD depends on microtubules and the motor
protein dynein, while the subsequent and final rearrangement is supported by microfilament action. Notably, in oocytes of diabetic mice
the ER network does not surround the forming MI spindle and does
not become organized into typical cortical clusters of 1–2 mm diameter
following maturation (Zhang et al., 2013), suggesting that this organelle
may be involved in developmental anomalies, reported also in the
human, dictated by maternal pathologies.
During maturation, human oocytes display modifications in the
pattern of ER distribution and a variation in the abundance of the IP3R
that are reminiscent of the changes observed in the mouse. In fact,
human GV-stage oocytes are characterized by elements of the ER organized in a fine network extending throughout the cortex and the cell interior, while MII oocytes have large distinct clusters 2–3 mm in diameter
throughout the cortex and the cell interior (Mann et al., 2010). Interestingly, in MII oocytes ER clusters are not absent from the cortex in the
vicinity of the MII spindle, consistent with the fact that in the human
the oocyte surface overlying the spindle is not devoid of microvilli,
unlike the mouse. Also, during the GV –MII transition, western blot analysis indicates that oocytes increase their endowment of IP3R by 50%,
444
while the ability to release Ca2+ in response to IP3 almost doubles (Mann
et al., 2010). Therefore, in a fashion similar to the mouse model, during
maturation human oocytes undergo changes in the distribution and constitution of the elements of the ER that account for an increased ability to
release Ca2+. Interestingly, human MII oocytes matured in vitro from
GV-stage oocytes fail to support such increases in IP3R abundance and
Ca2+-release ability despite an apparently normal ER distribution in clusters of 2– 3 mm, suggesting that a reduced response to IP3 may explain
the reportedly lower developmental competence of in vitro matured
oocytes (Mann et al., 2010). However, these results should be interpreted with caution, because they were obtained using left-over
GV-stage oocytes derived from stimulated IVF cycles, i.e. material that
is believed to be intrinsically developmentally incompetent. In addition,
in such experiments GV-stage oocytes were cultured without CC, a condition known to profoundly affect many cellular and biochemical aspects
of the process of maturation.
In human IVF, the ER has drawn the attention of clinical embryologists.
In fact, a fraction of mature oocytes recovered from treatment cycles involving ovarian stimulation with gonadotrophins show large aggregates
(many microns in diameter) of smooth ER (Otsuki et al., 2004), whose
network is not usually detectable by conventional transmitted light microscopy. Interestingly, accumulations of ER are not visible in GV-stage
oocytes, a sign that they form during maturation in vivo, irrespective of
the underlying mechanism. In several reports, an association between
such aggregates and birth anomalies was described (Ebner et al., 2008;
Sá et al., 2011), although a recent review showed that healthy babies
can be born from oocytes displaying ER anomalies (Shaw-Jackson
et al., 2014). However, data on this matter remain limited and clearly
more systematic research is needed.
Mitochondria
The massive organelle and cytoskeletal reorganization occurring during
oocyte maturation is energetically costly, requiring adequate levels
of ATP at precise times and positions. Oxidative phosphorylation
supported by mitochondrial function is the primary source of ATP
because fully grown oocytes have a limited ability to metabolize
glucose into pyruvate (Biggers et al., 1967; Leese, 1995). Therefore,
conditions that perturb mitochondrial function are expected to cause
important developmental disturbances, such as meiotic chromosome
segregation and maturation and fertilization failure. In addition, being
that the mitochondrial legacy is entirely maternal, compromised mitochondrial function in oocytes can have direct effects on the early
embryo, including cleavage arrest, abnormal cytokinesis and blastomere
fragmentation, as a consequence of mitochondrial-driven apoptosis (Van
Blerkom, 2011). Intriguingly, during maturation mitochondria efficiently
support oocyte function despite their apparently underdeveloped,
primitive aspect characterized by a small spherical shape, few truncated
cristae and highly electron-dense matrix (Motta et al., 2000).
During maturation, mitochondria are subject to changes in abundance, function and localization that are crucial for their ability to meet
the oocyte energy demands. Estimates of mitochondrial number in maturing oocytes are difficult, considering the extremely large cell size and
the challenge posed by ultrastructural morphometric analysis. However,
mitochondrial number is thought to be around 92 000 and 740 000 in
mouse and sheep oocytes, respectively (Pikó and Matsumoto, 1976;
Cotterill et al., 2013). In the human, indirect assessments based on the
assumption that each organelle contains one copy of mtDNA have
Coticchio et al.
suggested figures ranging from 30 000 to more than 1 000 000
(Brenner et al., 2000; Van Blerkom, 2004; May-Panloup et al., 2007).
Studies carried out in pig oocytes are consistent with the hypothesis
that during the transition from the GV to the MII stage, mitochondrial
number can decrease by two-thirds (Cran, 1985). However, possible numerical differences at the different maturation stages do not necessarily
reflect changes in the mitochondrial mass because individual organelles
can interact with each other and undergo phenomena of fusion and
fission, as recently shown in mouse oocytes (Wakai et al., 2014).
Another factor that may reconcile the apparent paradox of a decrease
in mitochondrial number during maturation is mtDNA copy number.
Throughout oogenesis from the primordial to the pre-ovulatory stage,
mtDNA content per oocyte expands dramatically, growing from thousands to many hundred thousand copies. In the pig, a peak in mtDNA
synthesis has been reported to occur during maturation (Spikings et al.,
2007), a phenomenon that could give rise to the presence of more
than one copy of DNA in each individual organelle and therefore counterbalance the concomitant reduction in mitochondrial number. The
prevalent importance of mtDNA copy number over mitochondrial
number also emerges from studies in which mtDNA synthesis was inhibited at various stages of maturation, from which it is possible to conclude
that a precise mtDNA threshold is essential to ensure the normal unfolding of fertilization and early development (Spikings et al., 2007). Intriguingly, mtDNA can be subject to degradation during the early
cleavage stages (Spikings et al., 2007; Cree et al., 2008), suggesting
that a balance between synthesis and degradation may represent a
homeostatic mechanism by which mitochondrial function is regulated
during maturation and early preimplantation development in response
to a changing involvement of oxidative phosphorylation in energygenerating processes. Whether a burst of mtDNA synthesis occurs in
human oocytes during maturation is currently unknown. Preliminary
data are consistent with this possibility, although it remains uncertain
whether this phenomenon can have a clinical significance (Van
Blerkom, 2011).
During transition from the GV to the MII stage, in addition to possible
changes in overall relative organelle mass and mtDNA copy number,
mitochondrial function can be regulated through other modalities.
Specific organelle distributions at the different maturation stages can profoundly influence the quality of the maturing oocyte and the destiny of the
ensuing embryo. In fact, compromised oocyte quality is associated with
aberrant mitochondrial rearrangement and low ATP levels (Van Blerkom
et al., 1995; Van Blerkom, 2004). Changes in mitochondrial distribution
during maturation have been precisely mapped in mouse oocytes. At the
GV stage, mitochondria aggregates are finely dispersed throughout the
cell, showing only moderate concentration around the GV. Following
GVBD, mitochondria accumulate in large amounts (over 40% of the
overall population) in the immediate surroundings of the forming MI
spindle (Dalton and Carroll, 2013). Importantly, as in other maturation
phases, many mitochondria appear juxtaposed to elements of the ER,
a vicinity that at this stage is motivated by the energy demands of
the mechanics of formation and translocation of the MI spindle (see
‘Cytoskeletal forces governing spindle positioning’ section). This is consistent with the fact that a dense cloud of mitochondria accompanies the
MI spindle during its relocation from the oocyte centre to the periphery
and, following PBI extrusion, reconstitutes around the forming MII
spindle (Dalton and Carroll, 2013). However, by the time the oocyte
arrest at the MII stage, the spindle is no longer enclosed in a shell of
Oocyte maturation
mitochondria, which nevertheless remain more densely localized in
this area.
Recent findings have described mitochondrial rearrangement in even
greater detail. In most eukaryotic systems, mitochondrial are known to
be transported along microtubules. In mouse oocytes this seems to be
also the case, because inhibition of microtubule polymerization with
nocodazole prevents accumulation of mitochondria around the MI
spindle while the same effect is not observed by disrupting actin filaments
with latrunculin. Predictably, motor proteins have been found to assist
mitochondrial accumulation. In fact, oocytes react to treatment with
function-blocking antibodies specific for dynein or kinesin-1, which
move cargoes towards the minus and plus ends of microtubules, respectively (Hirokawa et al., 1998). In particular, inhibition of dynein function
reduces the clustering of mitochondria around the MI spindle, while
suppression of kinesin-1 activity enhances mitochondrial accumulation
(Dalton and Carroll, 2013). Therefore, microtubules provide the
major infrastructure for mitochondrial displacement and a regulated
balance of the activity of different motor proteins accounts for the appropriate vectorial movement towards the spindle. Live cell time-lapse
microscopy and semi-quantitative data generated by confocal microscopy have revealed an intriguing aspect of mitochondrial distribution
during PBI extrusion. As described above, a massive array of mitochondria accompany the spindle during its relocalization from the centre to
the cortex. At MI this arrangement is still present, but at around the
time of anaphase I mitochondria acquire a polarized arrangement and
appear predominantly clustered at the spindle pole oriented inward
and virtually absent from the area near the pole facing the cortex.
With the progression of the first meiotic division, this has the effect of
retaining almost all mitochondria within the oocyte, while a much
smaller fraction of organelles is segregated in the PBI (Dalton and
Carroll, 2013). Although such a mechanism has not been yet elucidated
in fine detail, it appears obvious that it represents a solution to the need of
preserving for the oocyte, and therefore the ensuing embryo, the original
endowment of organelles, especially considering that mitochondrial replication does not occur during preimplantation development. Interestingly, in mouse oocytes during PBI extrusion the ER follows a similar,
although leakier, pattern of segregation (Dalton and Carroll, 2013), suggesting that the two types of organelle may be regulated by the same
mechanism. Further experiments have established that the distance of
the spindle from the cortex is crucial for mitochondrial polarization at
anaphase I. In fact, a direct relationship exists between distance of the
spindle from the cortex and degree of mitochondrial polarization
(Dalton and Carroll, 2013). Therefore, it is plausible that asymmetric
mitochondrial segregation occurs as an effect of not yet known
spindle –cortex interactions, while it would not take place in conditions
that prevent spindle migration. Indeed, following GVBD, when spindle
repositioning to the cortex is prevented by inhibiting the actin-nucleating
factor Arp2/3, the first meiotic division generates two daughter cells that
inherit comparable proportions of mitochondria (Dalton and Carroll,
2013). Mitochondrial segregation appears to be differently regulated at
the second meiotic division. As previously discussed, as soon as the
PBI is extruded, mitochondria form a dense cloud around the forming
MII spindle, but tend to disperse shortly afterwards by the time the
oocyte pauses at MII. In mouse eggs treated with ethanol to achieve parthenogenetic activation, during anaphase II mitochondrial accumulation
near the pole oriented towards the oocyte centre and rarefaction around
the pole facing the cortex are not observed, with the consequence that
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mitochondrial segregation is not asymmetric (Dalton and Carroll, 2013).
However, the contingent of mitochondria removed from the oocytes
and eliminated with the PBII is small, because mitochondrial accumulation around the MII spindle becomes much less pronounced once the
oocyte progresses through meiosis and pauses at MII. This probably
makes unnecessary the mechanism of polarization that vice versa acts
in concomitance with PBI extrusion. Overall, the changing arrangements
of mitochondria during maturation represent a notable example of the
oocyte’s ability to regulate mitochondrial function. The mitochondrial
population is redistributed in time and space in a very specific fashion
and concentrated when and where it is most needed, i.e. around the
meiotic spindle, from its formation to the time when its migration from
the cell centre to the cortex is completed. Afterwards, mitochondrial
polarization at the innermost spindle pole at anaphase I adds another
level of regulation, by which mitochondrial loss with the extrusion of
the PBI is minimized.
Compartmentalization of mitochondria subpopulations with different
functional attributes may be another option for the regulation of ATP
supply in maturing oocytes. In fact, although morphometrically and
ultrastructurally similar, mitochondria may belong to different functional
categories in oocytes and embryos. Potentiometric dyes, such as JC-1,
have revealed that mitochondria can differ in DCm, i.e. the potential
difference across the inner mitochondrial membrane (Van Blerkom,
2003). Because the magnitude of DCm is directly correlated to important mitochondrial functions, including ATP production, there is the
awareness that mitochondria are not a homogeneous population and
that specific localization of subpopulations characterized by different
DCm may correspond to precise oocyte regulatory needs. An
example of this scenario may be represented by a small contingent
(5%) of sub-oolemmal mitochondria that shift to a higher level of
DCm when the mouse oocyte approaches the MII stage, while the
vast majority of mitochondria remain in a state of low DCm (Van
Blerkom, 2011). Current findings suggest that this change is actively
prevented by nitric oxide produced by corona cells until the ovulatory
stimulus is triggered (Van Blerkom et al., 2008). Irrespective of the
mechanism of regulation, however, it is notable that the localization of
highly polarized, and therefore more functional, mitochondria occurs
in a timely fashion in a specific oocyte domain where they are believed
to be crucially needed. Several lines of evidence in fact suggest the
notion that hyperpolarized mitochondria are essential for supporting
the very first steps of fertilization at the cortical level (Van Blerkom
et al., 2002).
The relationship between ATP production/demand and mitochondrial distribution during maturation was assessed further in recent investigations. In mouse oocytes matured in vitro, mitochondrial activity is
characterized by three waves of increase in ATP production, separated
by two shorter phases of down-regulation to basal levels (Yu et al.,
2010). These waves of enhanced activity of ATP generation are temporally coincident with GVBD, migration of the spindle to the cortex and the
transition between MI and MII. Above described studies illustrate how
an accumulation of mitochondria is found around the nucleus at the
time of GVBD, surrounds the spindle during its migration from the
centre to the cortex, and become polarized at the innermost spindle
pole shortly before body emission (Dalton and Carroll, 2013).
However, these patterns do not appear to be essential to, or associated
with, the above-mentioned peaks in ATP production because when
mitochondrial rearrangement is disrupted by inhibitors of microtubule
446
polymerization, changes in ATP production remain detectable (Yu et al.,
2010). Conversely, formation of small mitochondrial aggregates appears
to be instrumental for the occurrence of the bursts in ATP production. In
fact, constitution of these small clusters is temporally coincident with the
ATP peaks observed at GVBD and the successive maturation steps. Formation of such small mitochondrial aggregates seems to be directed by
actin microfilaments. Assembly and disarrangement of this cytoskeletal
component, in fact, parallels the formation and disappearance of small
mitochondrial clusters. Furthermore, disruption of actin microfilaments
with cytochalasin B, which causes actin depolymerization, is incompatible with the formation of mitochondrial clusters and the increases in
ATP production (Yu et al., 2010). Therefore, in mouse oocytes the
actin microfilament network appears responsible for mitochondrial redistribution on a small scale and the increase in ATP production to
support GVBD, spindle translocation and first polar body emission.
However, the reasons why mitochondrial clustering in small aggregates
are a requisite for an increase in ATP generation remains unknown.
Finally, a role of CC for establishing adequate levels of ATP during maturation should not be overlooked. In this respect, it is indicative that
mouse denuded oocytes and COC display similar patterns of ATP
changes at precise maturation events, with the crucial difference that in
the absence of CC the absolute levels of such patterns are lower in
denuded oocytes (Dalton et al., 2013).
In conclusion, during maturation mitochondria undergo major
changes involving redistribution on a large scale, formation of small aggregates on a smaller scale, compartmentalization of subpopulations characterized by different DCm and activity, fusion/fission phenomena and
mtDNA synthesis. By finely orchestrating these events, the maturing
oocyte deploys a strategy by which appropriate levels of ATP, mainly
derived by oxidative phosphorylation, are made available at precise
times and spatial domains, in concomitance with crucial maturation
steps.
The Golgi apparatus
The Golgi apparatus (GA) is the organelle by which macromolecules are
post-translationally modified, sorted, packaged and destined for release
in the extracellular environment or delivery to other intracellular compartments. In fully grown oocytes, the overall function of this organelle
is not completely understood considering that protein secretion activity
is negligible, although its role in the generation of CG is well known. In
somatic interphase cells, the GA takes the form of a single ribbon-shaped
mass of membranous structures known as ‘cisternae’ associated with
the single centrosome positioned near the nucleus (Thyberg and
Moskalewski, 1999). Indeed, GA organization depends on the microtubules that radiate from the centrosome (Thyberg and Moskalewski,
1999). During the S phase the centrosome duplicates and in the following
mitosis the couple of centrosomes are crucial for the organization of
the mitotic spindle (Green et al., 2012). Meanwhile, the structure of
the GA fragments and disperses throughout the cytoplasm to ensure
that the two daughter cells inherit comparable fractions of the organelle.
This dynamic cannot be reproduced in fully grown oocytes, as the centriole—the centrosome’s core—is lost during early prophase (Szollosi
et al., 1972). Investigations concerning mouse (Moreno et al., 2002),
cow (Hyttel et al., 1989) and rhesus monkey (Moreno et al., 2002)
oocytes converge towards the notion that at the GV stage the GA is
not organized in a single ribbon-shaped array, but is formed from a
multitude of ‘mini-Golgis’ dispersed in the cytoplasm. In the mouse,
Coticchio et al.
these small Golgi aggregates appear moderately more concentrated in
the interior than in the cortex. Following GVBD, the mini-Golgis fragment into even smaller clusters and diffuse evenly throughout the
ooplasm. This condition of fine fragmentation and homogeneous distribution persists until the MII stage. Fragmentation and dispersion of miniGolgis occurs also in cow oocytes during GVBD, with the difference
that in this species a second phase of redistribution occurring at MI
concentrates a larger contingent of these organelles in the oocyte interior
(Racedo et al., 2012). Preliminary observations are consistent with the
hypothesis that redistribution and fragmentation of the GA in cow
oocytes is regulated by microtubules and cytoplasmic dynein (Racedo
et al., 2012).
During oocyte maturation, a specific role for the GA remains elusive
but different lines of evidence suggest that this organelle is not simply
subject to passive redistribution, but may actively participate in important
regulatory mechanisms. This can be inferred by the observation that
treatments, such as brefeldin A that block membrane trafficking from
the ER to the GA, have the ability to inhibit reversibly the maturation
in vitro of mouse oocytes (Moreno et al., 2002). It is presumed, therefore,
that membrane trafficking is required for the meiotic progression of
murine oocytes, to assure post-translational modification of proteins
at the Golgi level, or assignment of these proteins to appropriate (postGolgi) sites. Recent evidence suggests new, previously unsuspected possibilities for a role of the GA during oocyte maturation. GM130 is a
protein of the cis-Golgi matrix involved in the trafficking between the
ER and the GA, glycosylation and maintenance of Golgi organization
(Nakamura, 2010). Novel information indicates that GM 130 intervenes
in completely different functions, such as cell polarization and division.
This is consistent with the fact that GM130 is able to recruit microtubule
nucleating factors, namely AKAP450 and g-tubulin (Rivero et al., 2009),
allowing the GA to act as a microtubule nucleating apparatus. In immature mouse oocytes, immunofluorescence analysis reveals that
GM130-positive granules are present throughout the cytoplasm, although more abundant around the GV. At GVBD, GM130 begins to concentrate in the central part of the oocytes and, as assembly of the spindle
progresses, localizes preferentially at the spindle poles. This distribution
persists until anaphase I when the protein is found in the midbody bridging the oocyte and the PBI. Finally at MII, GM130 concentrates again at
the spindle poles (Zhang et al., 2011a). Consequently, it is tempting to
speculate that at least a subpopulation of GA elements has the ability
to promote microtubule nucleation through the activity of GM130,
thereby assisting meiotic spindle formation. Such a hypothesis seems
to find confirmation in the fact that GM130 morpholino injection
causes multiple spindle defects, i.e. elongation and disruption of pole focusing or formation of multipolar structures, and disturbances of polar
body extrusion (Zhang et al., 2011a). In mouse oocytes, an involvement
of the GA in spindle formation is also apparent from the observation that
overexpression of a negative-dominant form of the regulator CDC42,
which is involved in spindle formation in mitotic cells, causes phenotypes
very similar to those associated with GM130 inactivation (Kodani et al.,
2009). These findings open new hypotheses on the role of the GA in
oocyte maturation, beyond the well-established function of CG generation. Nevertheless, extrapolation of these conjectures to other
species should be made with caution. For example, in mouse oocytes numerous microtubule organizing centres (MTOCs) act as nucleators for
microtubule assembly and could recruit elements of the GA carrying
GM130. On the contrary, MTOCs are not present in oocytes of other
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Oocyte maturation
species, including the human, a condition that must necessarily requires
different mechanisms of microtubule nucleation. Thus, the notion that
the GA is actively involved in the formation and regulation of the
meiotic spindle in species other than the mouse will have to be tested
in future studies.
Cortical granules
CGs are small individual membranous organelles distributed just beneath
the oolemma in the mature oocyte that intervene in a very early and
crucial step during the fertilization process. At the mature stage,
CGs appear as secretory vesicles measuring 0.2–0.6 mm in diameter
(Szollosi, 1967). They are produced exclusively in oocytes and are not
replaced after release. Functionally, they are associated with the modification occurring to the zona pellucida at fertilization. Following gamete
fusion and the start of intracellular Ca2+ signalling that drives the activation events, CGs fuse their membrane with the oolemma and shed their
content of enzymes in the perivitelline space. Such GC-derived proteolytic proteins modify by cleavage one of the key zona pellucida proteins
(ZP2) ad convert it in a version (ZP2f) that is unable to bind other spermatozoa, thereby preventing further penetration through the zona pellucida (Moller and Wassarman, 1989). In this fashion, polyspermic
fertilization is prevented. Distribution of CG beneath the sub-oolemmal
domain characterizes the mature MII oocyte, but at earlier stages of
oogenesis CG formation and localization involves other cellular
domains. Formation of CG starts at different stages of folliculogenesis
in various mammalian species. In murine oocytes, CGs are present
already at the primary follicle stage (Szollosi, 1967). Conversely, in the
monkey (Hope, 1965) and human (Baca and Zamboni, 1967), these
organelles do not appear until the secondary follicle stage. CGs have a
sub-cellular origin associated with the activity of the GA. As discussed
above, during the GV –MII transition, the GA undergoes significant rearrangement (Liu, 2011) assisted by the microtubules cytoskeleton.
However, the peripheral relocation of CG, which represents final products of the GA, is thought to be guided by microfilaments (Sun et al.,
2001). In mouse mature oocytes, the sub-oolemmal distribution of
GC that is typically observed in almost all mammalian species acquires
a particular pattern. In fact, while being localized at the extreme periphery of the oocyte, GCs are virtually absent from cortical regions overlying
MI and MII spindles (Nicosia et al., 1977; Okada et al., 1986). It is not clear
how GCs are excluded from such areas, but evidence generated in the
mouse and the hamster indicates that a peri-spindle CG-free area
(CGFA) may derive from displacement and redistribution at MI of
pre-existing GC (as suggested by an increase in CG density in the area
surrounding the CGFA) or precocious and confined GC exocytosis
occurring during the MI– MII transition (Ducibella et al., 1988, 1990;
Liu et al., 2003). Doubts also remain on the reasons that lead to the
establishment of a CGFA in these species. It cannot be ruled out that
targeted release of CG in the cortex in the vicinity of the MI/MII spindles
modifies the zona pellucida or the oolemma in a way that locally prevents
sperm –oocyte fusion. This would assure that decondensation of the
sperm head and formation of the male pronucleus occurs at a safe distance from the MII spindle and that the integrity of maternal chromatin
is not affected as it undergoes remodelling. This seems to be confirmed
by the observation that in mouse oocytes sperm penetration is less likely
to occur in regions of the cortex where the MII spindle is located (Nicosia
et al., 1977).
Clinical correlates of oocyte maturation
From the studies illustrated above, oocyte maturation emerges as an extremely complex process, although it unfolds during a minute fraction of
the time taken by the follicle to convert a primordial oocyte into a mature
egg. Understanding how oocyte maturation occurs in vivo is challenging in
animal models and almost impossible in the human. Nevertheless, IVM
experiments have allowed us to observe more closely how meiosis is
resumed and, to some extent, how the cytoplasm is reshaped in preparation for fertilization. By observing oocyte maturation in vitro, we have
also tested some hypotheses on the regulatory networks that govern,
and the conditions that make possible, oocyte maturation. However,
by its very nature, IVM remains an oversimplified system. In addition,
caution should be exercised in translating evidence gathered in animal
models, which in any case are essential to generate novel proofs of principles, into axioms adopted for developing new treatment strategies in a
human ART scenario.
Regardless, over the years our perception of oocyte maturation has
dramatically improved thanks to studies that have examined the CC–
oocyte interactions, the signalling system that guides the meiotic
process, and the internal forces by which the cytoskeleton and organelles
are reorganized. For example, in the previous section we have already
discussed how studies carried out in the mouse have prompted questions on the role of AREG in oocyte maturation in vivo following controlled ovarian stimulation. Also, increasing knowledge has inspired
approaches by which maturation can be achieved in vitro to serve clinical
needs. Even more in the future, novel findings in this field will provide the
bases for the development of more efficient IVM systems and the definition of more reliable criteria of oocyte quality.
In the context of human IVM, AREG has initially attracted considerable
interest as a potential biochemical tool by which to increase yield and
quality of in vitro matured oocytes. Progress in IVM is much needed, especially considering that current maturation rates after 30 h of culture
usually do not exceed 50–55% (De Vos et al., 2011; Coticchio et al.,
2012; Guzman et al., 2012), unlike the mouse and bovine models
where 80 –90% of GV-stage fully grown oocytes progress to the MII
stage in vitro (Albuz et al., 2010). In the human, higher maturation rates
can be achieved by extending the time of culture to 48 –52 h after
removal of CC and assessment of meiotic status at 30 h (Son et al.,
2008), although culture in the absence of CC is known to be highly detrimental to oocyte quality. Data supporting the notion that AREG can
have a decisive role in improving human IVM are lacking, stressing the perception that progress in IVM can be hardly achieved by the adoption of a
single factor or condition. Rather, it seems more reasonable to focus on
general processes that regulate oocyte maturation to obtain multiple
clues suitable for designing innovative systems. Progress in this direction
has been made, leading to a new approach by which oocyte maturation
can be pursued in vitro. These attempts derive from an appreciation of the
differences occurring between conditions in vivo and in IVM, especially
during the initial phases of the process. In vivo, resumption of meiosis
and associated cytoplasmic changes result from a delicate balance
between forces that prevent precocious initiation of the maturation
process and signals that induce the oocyte to start the meiotic
process, as described in the previous section. On the contrary, IVM is
believed to occur in a rather deregulated fashion, simply as an effect of
the absence of the inhibitory influence of the follicular environment.
Therefore, it was hypothesized that maturation in vitro could be attained
448
more successfully by reproducing in an IVM system at least part of the
conditions that regulate maturation in vivo. In this vision, the simulated
physiological oocyte maturation (SPOM) system was developed by
using the mouse and the cow models (Albuz et al., 2010). This approach
involves a ‘pre-IVM’ phase in which, immediately after collection, COCs
are exposed for a few hours in vitro to potent stimulators of cAMP production, such as forskolin. This allows reproducing the 100-fold transient
increase in cAMP that is observed in whole COC during maturation in vivo
(Mattioli et al., 1994). As a second step, and for the remaining period of
maturation in vitro, the SPOM system implies the use of a specific inhibitor
of the oocyte PDE3, typically cilostamide, to prevent the almost instantaneous fall in cAMP that occurs in oocytes in vitro in the absence of stimulants of cAMP synthesis. In addition, the PDE3 inhibitor is used in
association with FSH to actively promote meiotic resumption. Overall,
these conditions have been suggested to recapitulate the process of maturation in vivo by (i) mimicking the spike in cAMP levels that occurs in
COC at the time of ovulation that is thought to be somehow involved
in the meiotic resumption regulatory cascade and (ii) ensuring that
meiotic resumption is specifically induced while the oocyte is still
under the influence of an inhibitory signal. It is a fact that this treatment
extends the time in which gap junctional communication remains
active during IVM, with clear implications concerning the degree and
quality of oocyte –CC co-operativity. More importantly, following IVF
of in vitro matured oocytes, the SPOM system appears to improve significantly oocyte developmental potential, as shown by an increase in blastocyst yield and quality in the bovine system and improvements in
blastocyst rate, implantation rate and fetal yield in the mouse (Albuz
et al., 2010). It is anticipated that the SPOM system is currently being
tested with human material and future developments will clarify
whether it will bring about major progress in human IVM. Indeed,
other interesting results in the human have been obtained adopting a
pre-IVM approach. In a preliminary study, prior to IVM left-over
cumulus-enclosed GV-stage oocytes retrieved in stimulated IVF/ICSI
cycles were maintained in meiotic arrest for 24– 48 h by treatment
with the PDE3 inhibitor Org9935. In oocytes surrounded by compact
layers of CC, treatment with the inhibitor increased the rate of maturation, although it did not have effects on the rate of fertilization or proportion of good quality embryos (Nogueira et al., 2006). In a similar
study, left-over denuded GV-stage oocytes obtained from stimulated
ICSI cycles were exposed to cilostamide, another specific PDE3 inhibitor, for 24 h before culture under standard IVM conditions. Transient
cilostamide treatment maintained the large majority of oocytes in
meiotic arrest and increased the proportion of in vitro-matured
oocytes having a bipolar spindle and equatorially aligned chromosomes
(Vanhoutte et al., 2007). Although such studies are affected by the
major flaw of the use of poor quality oocytes, i.e. supernumerary GV
oocytes (with or without surrounding CC) retrieved in stimulated
cycles, nevertheless they offer the proof-of-principle that control of
meiotic resumption could represent a valuable approach for the improvement of oocyte IVM systems in the human. Another downstream
implication of the studies that have attempted to shed light on oocyte
maturation is the use of oocyte-secreted factors (OSFs) as part of a
more complex environment in which mature oocytes can be achieved
in vitro. OSFs, in particular the TGF-b family members GDF9 and
BMP15, are paracrine factors produced and released by the oocyte primarily for activating or enhancing in the surrounding CC functions that
meet specific oocyte demands. In this way, the oocyte creates an
Coticchio et al.
intrafollicular microenvironment where its growth and maturation can
be maximally supported. For example, in the mouse both in vivo and in
vitro BMP15 and GDF9 appear to increase in CC the rate of transcription
of genes encoding key enzymes of the cholesterol biosynthetic pathway
(Su et al., 2007), while the same pathway is barely used by the oocyte.
Therefore, it is clear that the oocyte influences the metabolism of adjacent CC to compensate for its own need of cholesterol supply in the
absence of autonomous production. A similar situation occurs in the
case of glycolysis, a function poorly expressed by the oocyte that
however is important for its development. This problem is resolved by
the ability of CC to provide the oocyte with the products of glycolysis
through gap junction-mediated transfer. However, in CC sustained
glycolytic activity does not occur spontaneously but is supported by
two OSFs, BMP15 and fibroblast growth factor-8 (Sugiura et al., 2008).
Similar results have been observed in cow COCs, where BMP15 contributes to maintain active gap junction communication by which the products of glycolysis, a pathway positively regulated by AREG, are
transferred from CC to the oocyte (Sugimura et al., 2014). These and
other studies exemplify the importance of OSFs for the oocyte –CC
interplay and oocyte function, a fact that could have major implications
for improving the efficiency of oocyte maturation systems. This has
already been confirmed by studies carried out in the mouse and the
cow. In the mouse, oocyte exposure to GDF9 during IVM improves
the rates of blastocyst formation and hatching, the blastocyst cell
number and the fetal yield of the resulting embryos (Gilchrist et al.,
2008). In the cow, GDF9 and BMP15 used alone or in combination
during IVM also increase blastocyst rate (Hussein et al., 2006), probably
by enhancing oocyte –CC gap junction communication. Unfortunately,
however, the proof that these factors can decisively improve the
outcome of human IVM is still lacking.
Collective knowledge gained from oocyte maturation studies has
implications also for practices that are attempted with the aim of optimizing the outcome of ART procedures. The potential use of left-over
GV-stage oocytes recovered after standard ovarian stimulation is exemplifying in this respect. Such oocytes occur with a frequency of 5–10%
but, in specific cases, at retrieval much larger fractions of oocytes may
be found meiotically arrested. GV arrest can be ascertained without ambiguity only after CC removal immediately after oocyte retrieval or a few
hours afterwards. Although GV-arrested oocytes are in most cases discarded, their use after IVM for clinical purposes may be found in many
reports (Vlaisavljević et al., 2007), especially in cases in which there is
little or no availability of mature oocytes. While the effort of IVF specialists to rescue such oocytes and therefore offer the largest chances of
success to IVF patients should be recognized, the use of left-over
GV-stage oocytes should be discouraged. In fact, despite meiotic resumption and progression to MII being observed, at the current state
of knowledge the culture of immature oocytes to achieve maturation
in vitro in the absence of CC raises more concerns than hopes. Much
of the evidence discussed in this manuscript leaves little doubt on the
central role of CC in oocyte maturation and the disturbances that can
occur to oocyte function in their absence. In addition, it should be considered that GV-arrested oocytes recovered in stimulated IVF cycles are
likely to be inherently developmentally incompetent (Nogueira et al.,
2000). This can also be inferred from different chromatin and cytoskeletal patterns shown by such oocytes, most of which are incompatible
with the ability to resume meiosis (Combelles, 2003). Preliminary, but
informative, observations also suggest that a large fraction of
449
Oocyte maturation
GV-arrested oocytes are affected by significant DNA damage as indicated by positivity to gH2AX staining (Guglielmo et al., 2012). It
should not surprise, therefore, that clinical data show very poor, if any,
benefit from the use of such qualitatively compromised oocytes, especially if used after removal of companion CC (Reichman et al., 2010).
However, although negligible from a clinical standpoint, GV-arrested
oocytes can have a role in research. In fact they represent a model, although imperfect, to study human oocyte maturation, thereby alleviating
the perennial shortage of oocytes in basic research. Such opportunities
should not be overlooked because significant knowledge can be
gained, even recognizing the limits of the model. This concept is
explained by a recent study that not only laid the foundations of MII
spindle morphometry and mechanics but also, by comparing oocytes
matured in vivo and GV oocytes matured in vitro, dispelled the myth
that IVM affects spindle morphology in human oocytes (Coticchio
et al., 2013).
The importance for oocyte maturation of functional CC –oocyte contacts is relevant to the cryopreservation of immature COCs obtained
from unstimulated cycles. These oocytes may be cryopreserved by
slow cooling or vitrification before or after IVM. Several reports suggests
that while cryopreservation of in vitro matured COCs does not significantly affect oocyte developmental competence (Huang et al., 2010),
cryostorage of immature COCs poses a major threat to CC–oocyte
communication. This may be inferred by confocal microscopy studies
showing massive disruption of TZPs as an effect of cryopreservation
(VandeVoort et al., 2008; Brambillasca et al., 2013). Therefore, immature
COCs recovered from liquid nitrogen are found in a condition of functional, if not physical, dissociation from surrounding CC, with predictably
negative implications for the acquisition of oocyte developmental competence following IVM.
Oocyte maturation research can also assist in the interpretation of
phenomena involving mature oocytes. Typical in this respect is the
case of PBI morphology. Different manifestations of this trait have
been suggested to be associated with oocyte quality and therefore clinical outcome. In particular, it has been reported that oocytes displaying a
PBI characterized by large size, irregular shape, roughness of its surface or
fragmentation are developmentally less competent, cleaving more irregularly in vitro after fertilization and giving rise to lower pregnancy
rates after embryo transfer (Ebner et al., 1999, 2002). Nevertheless,
there is no consensus around this matter, except the case of oocytes
showing large polar bodies, which indeed appear to be developmentally
compromised. Animal studies can assist in interpreting such cases. In fact,
in the mouse the generation of a large polar body is a well-documented
effect of intrinsic or extrinsic factors that can perturb oocyte maturation
and ultimately affect developmental competence. Indeed, oocytes from
c-mos2/2 females generate a large PBI and are developmentally compromised (Choi et al., 1996). Similar consequences are observed when
wild-type mouse oocytes are matured in vitro under inappropriate
culture conditions (Sanfins et al., 2003; Plancha et al., 2005). On the contrary, there is no firm evidence that in non-human species PBI shape,
roughness or fragmentation may predict oocyte quality, consistent
with a lack of consensus on the hypothesis that these characteristics
can be adopted for oocyte selection in human IVF.
From the above discussion, it emerges how basic studies on oocyte
maturation not only enlighten us on a crucial phase of oogenesis, but
also have practical implications inspiring novel applications and
approaches to human ART.
Conclusions
In mammals, oocyte growth is already completed several days before
ovulation. However, at ovulation the fully grown oocyte must undergo
an intense and laborious period of final maturation to become developmentally competent. The significance of oocyte maturation for the attainment of haploidy has been a well-established concept for several
generations of scientists. In contrast, changes occurring in the cytoplasmic compartment and the role of CC during the maturation process
have been much more challenging to observe and understand. CCs
develop as a distinct cell lineage concomitantly with the formation of
the antrum as an effect of regulatory signals, such as GDF9 and
BMP15, emitted by the oocyte (Diaz et al., 2007). By modifying the
fate of the more adjacent granulosa cells, the germ cell creates a specialized intrafollicular microenvironment more suitably equipped to meet
its own metabolic and cellular demands. Before and during maturation,
CCs are instructed to activate specific metabolic pathways and make
available molecules poorly synthesized by the oocyte, such as pyruvate,
cholesterol and various amino acids (Su et al., 2009), whose shortage
would otherwise compromise the maturation process. Notably, CC
support to the oocyte is not limited to the mere supply of nutrients
and metabolites. Perhaps even more important, the CC control vital
functions. Meiotic arrest and resumption is exemplar in this respect.
Fully grown oocytes are competent to undergo GVBD and progress to
MII. Nevertheless, they pause at the GV stage as a consequence of a
concerted inhibitory action of both CC and MGC. At ovulation,
meiotic resumption is also under the control of follicle cells, which interrupt their inhibitory influence and at the same time emit a stimulatory
signal (Conti et al., 2012; Liu et al., 2014). Some elements of this
regulatory network (e.g. NPPC and EGF family members) have been
identified in the mouse model and are under close scrutiny as candidates
for the development of more advanced IVM systems (Albuz et al.,
2010; Peluffo et al., 2012; Franciosi et al., 2014). In fact, current IVM
approaches are highly imperfect and in need of major improvements
(Smitz et al., 2011). In vitro, meiotic resumption is obtained as a result
of simple removal of the inhibitory influence of the follicular environment
in the absence of specific and genuine positive stimulation, a scenario very
different from physiological conditions (Albuz et al., 2010). This has
major detrimental influences on oocyte developmental competence.
For example in the cow model, under in vitro conditions oocyte –CC
communication is interrupted prematurely or severely affected. As a
consequence, the process of chromatin condensation occurring in preparation for meiotic resumption is deregulated and oocyte ability to
mature and undergo preimplantation development is compromised
(Luciano et al., 2011). Recent studies have offered a glimpse into
another dimension of the role of CC in the maturation process. Development of cell polarity and the ability to undergo highly asymmetric divisions are characteristics that largely rely on self-organizing processes
(Li and Albertini, 2013). However, oocyte polarity appears to depend
also on CC. In vivo, pre-ovulatory oocytes are connected to CC
through a dense array of TZPs, which are particularly abundant and
polarized in the portion of cortex arching the asymmetrically positioned
GV. In vitro, inadequate culture conditions, i.e. the absence of FSH supplementation or exposure to the actin-depolymerizing agent latrunculin,
cause disruption of TZPs, GV disanchoring from the cortex, loss of asymmetry and ultimately a series of downstream cellular and developmental
consequences, suggesting that CC, through their TZPs, are instrumental
450
for the design and maintenance of oocyte asymmetry (Barrett and
Albertini, 2010).
While these findings underpin the concept that acquisition of oocyte
developmental competence during maturation is crucially influenced by
the surrounding environment, of which CCs are integral part, other lines
of evidence indicate that specific oocyte traits are entirely intrinsic.
Typical in this respect is the oocyte’s ability to respond to DNA
damage. Oocyte are particularly exposed to DNA damage as a consequence of physiological mechanisms associated with chromosome recombination in fetal life or physical and chemical insults accumulating
over long periods of time during follicular quiescence at the primordial
stage. Fully grown GV stage oocytes appear to the able to detect DSBs
but their ability to control the meiotic cycle and deploy an efficient
repair response is limited (Marangos and Carroll, 2012), with the implication that maturation can occur in the presence of significant DNA
damage and that genetic integrity of the resulting embryo may be affected
(Yuen et al., 2012).
The interactions occurring during maturation between the chromosomes positioned in the meiotic spindle and the oocyte cortex are
another major manifestation of self-determining processes occurring
during maturation. Meiotic chromosomes are in fact sources of a
Ran-GTPase gradient that promote cytoplasmic streaming through
actin polymerization achieved by activation of the Arp 2/3 complex (Yi
et al., 2013). During spindle migration to the cortex, as chromosomes
reach sufficient proximity to the cortex, the Ran-GTPase gradient triggers an intense activity of cytoplasmic streaming by which the meiotic
spindle is pushed to the extreme cell periphery. This action completes
the cell symmetry breaking process, creating the geometrical premises
for the extrusion of a small PB. Importantly, the same Ran-GTPase gradient is responsible for the formation of a specialized actomyosin domain
in the more adjacent cortex, whose function supports the mechanics of
PB extrusion (Deng et al., 2007).
Organelle rearrangement is a further major theme of oocyte maturation. Not only are elements of the ER and the GA and other organelles
regulated in their function during maturation, but they are also redistributed in response to precise spatio-temporal cues. For example, a cloud of
mitochondria and ER elements surrounds the spindle during its migration
to the cortex (Dalton and Carroll, 2013; Yi et al., 2013), providing energy
and spatial information for the localization of the actin-nucleating factor
FMN2, which promotes spindle displacement.
These and other numerous findings described in this review offer us a
picture of oocyte maturation that extends far beyond the achievement of
meiotic maturity. Despite interspecies differences which may delay the
translation of research into practice, our increasing understanding of
oocyte –CC interactions will be key to the development of more efficient
IVM systems, highly demanded by animal biotechnology and human ART.
Acknowledgements
We would like to kindly thank Prof. David F. Albertini for inspiring many
of the concepts discussed in the manuscript.
Authors’ roles
G.C.: literature search, manuscript preparation, critique and critical discussion of manuscript. M.D.C., M.M.R., M.C.G., F.B., D.T., P.V.N. and
Coticchio et al.
R.F.: critique and critical discussion of manuscript. M.C.G.: confocal microscopy imaging.
Funding
No external funding was either sought or obtained for this study.
Conflict of interest
None declared.
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