New approaches regarding the in vitro maturation of oocytes: Manipulating cyclic nucleotides and their partners in crime

Abstract

Several discoveries have been described recently (5-10 years) about the biology of ovarian follicles (oocyte, cumulus cells and granulosa cells), including new aspects of cellular communication, the control of oocyte maturation and the acquisition of oocyte competence for fertilization and further embryo development. These advances are nourishing assisted reproduction techniques (ART) with new possibilities, in which novel culture systems are being developed and tested to improve embryo yield and quality. This mini-review aims to describe how the recent knowledge on the physiological aspects of mammalian oocyte is reflecting as original or revisited approaches into the context of embryo production. These new insights include recent findings on the mechanisms that control oocyte maturation, especially modulating intraoocyte levels of cyclic nucleotides during in vitro maturation using endogenous or exogenous agents. In this mini-review we also discuss the positive and negative effects of these manipulations on the outcoming embryo

Full text

35
Received July 13, 2016
Accepted November 01, 2016
Review Article
New approaches regarding the in vitro maturation of oocytes:
manipulating cyclic nucleotides and their partners in crime
Ramon Cesar Botigelli 1, Eduardo Montanari Razza1, Elisa Mariano Pioltine1, Marcelo Fábio Gouveia Nogueira1,2
1Department of Pharmacology, Institute of Bioscience, University of São Paulo State, Botucatu, São Paulo,
Brazil
2Department of Biological Sciences, Faculty of Sciences and Letters, University of São Paulo State, Assis, São
Paulo, Brazil
ABSTRACT
Several discoveries have been described recently (5-
10 years) about the biology of ovarian follicles (oocyte,
cumulus cells and granulosa cells), including new aspects
of cellular communication, the control of oocyte maturation
and the acquisition of oocyte competence for fertilization
and further embryo development. These advances are
nourishing assisted reproduction techniques (ART) with
new possibilities, in which novel culture systems are
being developed and tested to improve embryo yield and
quality. This mini-review aims to describe how the recent
knowledge on the physiological aspects of mammalian
oocyte is reecting as original or revisited approaches
into the context of embryo production. These new insights
include recent ndings on the mechanisms that control
oocyte maturation, especially modulating intraoocyte
levels of cyclic nucleotides during in vitro maturation using
endogenous or exogenous agents. In this mini-review we
also discuss the positive and negative eects of these
manipulations on the outcoming embryo
Keywords: Oocytes, cumulus cells, oocyte in vitro
maturation, cyclic nucleotides, in vitro oocyte maturation
techniques
JBRA Assisted Reproduction 2017;21(1):35-44
doi: 10.5935/1518-0557.20170010
BACKGROUND
The literature concerning oocyte competence and
embryo quality has become abundant in the last few
years. Several factors are involved in oocyte metabolism,
cyto-skeletal remodeling, accumulation of molecules
(RNAs), meiosis arrest/resumption and fertilization, all
of which are key events for initiating and sustaining early
embryogenesis. This large amount of published data has
allowed researchers to pursue new strategies in assisted
reproduction techniques (ART). This mini-review focuses
on the recent progress in understanding the events
controlling the acquisition of competence in oocytes and
the new mechanisms involved in the maintenance of oocyte
meiotic arrest; and thus, it may yield future approaches
regarding the development of novel systems of in vitro
culture, and hopefully bring the status of in vitro production
of embryos (IVP) to a whole new level. For the purpose
of this mini-review the literature search was performed in
the PubMed database, to nd all relevant papers focusing
in “mammalian oocyte maturation”, “in vitro oocyte
maturation techniques” and crossing data with “cyclic
nucleotides” (“cyclic adenosine monophosphate”, “cyclic
guanosine monophosphate”), “meiosis arrest/resumption”
and “embryo yield”, from which we have selected 112
papers, among original and review papers, to discuss the
most interesting ndings and also included some of our
own.
INTRODUCTION
Mammalian oocytes pass through a long and complex
process to acquire the competence necessary for
fertilization and embryogenesis. Oocytes are formed in
fetal life, when the primordial germ cells (PGC), rst seen
in the epiblast, move outside the embryo to the yolk sac,
and then migrate from the yolk sac to the early gonad
(genital ridges). After genital ridges are colonized by PGC
they are denominated primordial gonads (review by van
den Hurk & Zhao, 2005).
The migration and colonization of the gonads by PGC
in females give rise to the oogonia, which, associated with
somatic cells, undergo a phase of mitotic proliferation
with an incomplete cytokinesis. In the developing ovary,
the oogonia and pregranulosa somatic cells progressively
organize into epithelial structures eventually recognized as
ovarian follicles. However, before follicle formation, germ
cells change from mitotic to meiotic and become primary
oocytes, committed to follicle development (reviewed by
Guigon & Magre, 2006).
Still in fetal phase, all female germ cells reach the
prophase of the rst meiotic division, but instead of
progressing to metaphase, they are kept arrested in the
diplotene stage or germinal vesicle (GV). After birth,
oocytes undergo some important processes for their
growth and maturation, such as storage of mRNA, proteins,
metabolic substrates and organelle reorganization. The
primary oocytes, arrested in the rst meiotic division, now
become enveloped by a layer of attened pregranulosa
cells and a basal membrane to become primordial follicles
(Picton, 2001).
Followed by the activation of growth, the primordial
follicle is surrounded by a complete layer of cuboidal
granulosa cells making it a primary follicle (Picton,
2001). During follicular growth, granulosa cells continue
to proliferate and the theca layer is developed, which is
required to produce androgens and to form the network
of cells that support the vascular system of the growing
follicle (Young & McNeilly, 2010).
Primordial follicles remain ‘dormant’ in the ovaries until
recruitment into the population of growing cells. Every
day, a group of primordial follicles are recruited and start
to grow based on the order in which they are initially
formed. Consequently, certain primordial follicles are rst
transformed into primary follicles after a few days and
others only after more than a year as in rodents, or after
one to ve decades later, as in women (van den Hurk &
Zhao, 2005).
Follicles are called primary follicles when the single
layer of granulosa cells surrounding the oocyte becomes
cuboidal. The transition of primordial follicles into primary
follicles is slow and the diameter of its oocyte hardly
changes. This process is associated with commitment

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JBRA Assist. Reprod. | v.21 | no1| Jan-Feb-Mar/ 2017
and subsequent stages of follicular development, and it
is independent of direct FSH action (Méduri et al., 2002).
Furthermore, when the follicle reaches several layers
of granulosa cells, it starts forming the antrum, and the
granulosa cells dierentiate into two compartments: the
mural cells, which internally surround the basal membrane,
and the cumulus oophorus cells (CCs), that are closely
associated with the oocyte. This structure forms the so-
called cumulus-oocyte complex (COC) and its intricate
interaction confers the oocyte with the competence to
resume meiosis and be fertilized (Hyttel et al., 2010;
Picton, 2001).
Bidirectional communication between oocytes
and somatic cells
The granulosa cells have clearly established roles to
support oocyte growth and the acquisition of developmental
competence (Brower & Schultz, 1982), but also participate
in the control of meiosis progression (Eppig, 1991), and
in the modulation of global transcriptional activity and
chromatin remodeling in the oocyte (De La Fuente & Eppig,
2001).
In recent years, researchers have focused on the
understanding how the oocyte can inuence granulosa
cells through the so-called oocyte derived paracrine factors
(ODPFs). Among them, the main players are the proteins
of the transforming growth factor β (TGF-β) superfamily,
such as the growth dierentiation factor 9 (GDF9) and
the bone morphogenetic protein 15 (BMP15). Fibroblast
growth factors (FGFs) are also secreted by oocytes and
are reported to regulate granulosa cell development and
function cooperatively with TGF-β proteins (reviewed by
Emori & Sugiura, 2014).
Especially within antral follicles, ODPFs guide the
dierentiation and maintenance of granulosa and CCs
(Eppig et al., 1997). In addition, ODPFs can stimulate
growth and apoptosis (Gilchrist et al., 2001), energy
metabolism (Sugiura et al., 2005; Sutton et al., 2003;
Sutton-McDowall et al., 2010), sterol biosynthesis (Su
et al., 2008) and the CCs expansion (Elvin et al., 1999;
Varani et al., 2002). Thus, the oocyte can aect the
functions of the CCs for their own benet, since the oocyte
is not able to produce all the substrates required for its
maturation. To ensure an eective development, oocyte
and cumulus/granulosa cells must communicate through a
perfectly orchestrated signaling system.
One mechanisms of bidirectional communication
between the CCs and oocyte is through the gap junctional
communication (GJC). Gap junctions (GJ) are specialized
membrane proteins occurring in points of very close
contact between both cells. They consist of arrays of
intercellular channels that allow direct sharing of small
(less than 1 kD) molecules between the cells (Harris,
2001). Indeed, many of the molecules are known to be
transferred from granulosa cells to the growing oocyte via
GJC, e.g., amino acids, glucose, and ribonucleotides (Eppig
et al., 2005; Sugiura et al., 2005). GJ are comprised of
connexins, a homologous family of more than 20 proteins.
The connexin 43 (Cx43) is predominantly expressed by
cumulus/granulosa cells whereas Cx37 seems to be the
only connexin connecting oocyte to the granulosa cells
(Juneja et al., 1999), and the loss of Cx37 expression
is detrimental to the oocyte-granulosa communication
(Simon et al., 1997).
Macaulay et al. (2014, 2016) demonstrated by confocal
and transmission electron microscopy, in combination with
transcript detection, that somatic cells contribute to the
maternal reserves of oocytes, including mRNA and long
noncoding RNA. This communication is performed by
transzonal projections (TZPs). These recent discoveries
rened our understanding of the small molecule transport
mechanism (GJC/TZP) synthesized by cumulus cells, which
are transferred into the ooplasm.
Recently, a new mechanism of cell communication within
the ovarian follicle was demonstrated; this mechanism is
performed by extracellular vesicles (EVs). Initially, EVs
were described in ovarian follicular uid of mares using
ow cytometer and transmission electron microscopy
techniques (da Silveira et al., 2012). These EVs are lipid
bilayer structures secreted by many cell types into the
extracellular uid, serving as a vehicle for membrane and
cytosolic proteins, lipids, and RNA (Raposo & Stoorvogel,
2013). Several articles identied miRNAs in bovine (Miles
et al., 2012), equine (da Silveira et al., 2012) and human
(Santonocito et al., 2014) follicular uid, suggesting EVs
as a potential mediator of cell-to-cell communication,
impacting oocyte and follicle growth (reviewed by da
Silveira et al., 2015).
Cyclic nucleotides and maturation control
Other important molecules that also use the GJC/TZP
system to move around between CCs and oocyte are the
cyclic nucleotides. Among those, we should highlight the
adenosine 3’,5’-cyclic monophosphate (cAMP). This second
messenger acts mostly in the phosphorylation of the cAMP-
dependent protein kinase A (PKA), leading to the activation
of various cellular pathways. The cAMP is synthesized from
adenosine triphosphate (ATP) by adenylate cyclase (AC),
following the dissociation of the stimulatory-G (Gs) protein
from specic classes of G-protein-coupled receptors (Wright
et al., 2015). Variation in the intraoocyte concentration of
cAMP can modulate the resumption of meiosis. Optimum
concentration of cAMP maintains PKA active, which inhibits
the maturation-promoting factor (MPF) and keeps the
oocyte arrested at the GV stage (Sirard et al., 1998).
Another cyclic nucleotide, the cyclic guanosine
monophosphate (cGMP), also plays its role in controlling
meiotic arrest/resumption. cGMP is synthesized via
dierent pathways, such as through nitric oxide (NO),
bicarbonate, natriuretic peptides (NPPA, NPPB and NPPC),
guanylins, uroguanylins and guanylyl cyclase activating
proteins (GCAPs); those guanylin molecules can activate
various enzymes, e.g., guanyl, adenylyl cyclases and
guanylate, which act in the catalytic conversion of guanosine
triphosphate (GTP) into cGMP and pyrophosphate (Potter,
2011).
Like cAMP, the cGMP molecules participate in protein
kinase phosphorylation (cGMP-dependent protein kinase,
PKG) and inuence the activity of several phosphodiesterases
(PDEs). The PDEs are intracellular enzymes that catalyze
the hydrolysis of the cyclic phosphate bond into cAMP and
cGMP to generate the inactive products 5’-AMP and 5’-
GMP (Francis et al., 2011). The PDEs are classied into 11
families according to their anity, although each family can
have multiple isoforms (Francis et al., 2011). PDE activities
can be of short or long term, and are modulated by signals
including hormones, neurotransmitters, cytokines, light,
and oxidative inuences. The concentration of nucleotides
(cAMP and cGMP) is controlled by the balance between
their synthesis and degradation, which is carried out by
the PDEs themselves (reviewed by Francis et al., 2011).
PDEs decrease cAMP concentration in immature
oocytes to allow for meiosis resumption and, consequently,
the onset of oocyte maturation (Sadler & Maller, 1989).
Tsafriri et al. (1996) reported that the location of PDE3A
is restricted to the oocyte and they showed an eectively
inhibition of spontaneous meiosis resumption in vitro
using specic inhibitors. Additionally, Norris et al. (2009)
demonstrated that cGMP synthesized by CCs moves across
GJ/TZP to the oocyte and inhibit cAMP degradation by
PDE3A. This process assures that the cAMP concentration,

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JBRA Assist. Reprod. | v.21 | no1| Jan-Feb-Mar/ 2017
demanded by the GV-arrest, be maintained at optimum
levels.
During the normal reproductive cycle, a surge of LH
induces oocyte maturation and ovulation (Richards et al.,
2002). Triggered by LH, a receptor coupled to G protein
is activated in the theca and granulosa cells (Breen et al.,
2013; Gudermann et al., 1992; Rajagopalan-Gupta et al.,
1998), inducing a rapid reduction in follicle cGMP, which
is diused out of the oocyte through GJ/TZP (Shuhaibar
et al., 2015). Simultaneously, LH-induced phosphorylation
and activation of PDE5 leads to decreasing levels of cGMP
and relieves the inhibition of PDE3A in the oocyte, lowering
cAMP content and allowing meiosis to resume (Egbert
et al., 2016).
Knowledge of the physiology involved in oocyte
meiotic arrest/resumption and maturation has enabled
the development and improvement of techniques for
the in vitro maturation (IVM) of oocytes. The IVM of
mammalian oocytes is an essential tool for the basic or
applied aspects of assisted reproductive technology (ART)
such as developmental biology, in vitro production (IVP)
of embryos, cloning, stem cells and embryology (Smitz
et al., 2011). However, the eciency of IVM is still low
when compared to in vivo maturation, which limits its
application in ART (Gilchrist, 2011). Drawbacks of IVM
include decreased preimplantation embryo development,
low pregnancy rates and poor live birth index (Child et al.,
2002; Eppig et al., 2009). This is probably caused by the
ineciency of the oocyte to avoid the drastic decrease
in cAMP concentration when removed from the follicular
environment during ART procedures (Luciano et al., 2004;
Mattioli et al., 1994). This spontaneous resumption of
oocyte meiosis causes incomplete cytoplasmic maturation,
and the asynchrony between cytoplasmic and nuclear
maturation, aects oocyte development and embryo
quality (Blondin et al., 1997; Gilchrist & Thompson, 2007;
Lonergan et al., 2003).
Several authors have reported reversible inhibition
of spontaneous meiotic resumption by pharmacological
methods and most of these strategies are described in the
following section of this review.
Pharmacological approaches to modulate cyclic
nucleotides during in vitro maturation
Reversible inhibition of meiotic resumption by
pharmacological methods have been long tried by many
researchers, but results on the subsequent developmental
competence is variable and often lower than in COCs
cultured without inhibition (Fulka et al., 1991; Lonergan
et al., 1997; Avery et al., 1998; Kubelka et al., 2000;
Mermillod et al., 2000). In addition, pharmacological
manipulations may, occasionally, aect oocytes and
embryos at the ultrastructural level as well (Faerge et al.,
2001; Lonergan et al., 2003; Nogueira et al., 2003, 2005;
Vanhoutte et al., 2007).
In mammalian oocytes, among the pharmacological
approaches used for in vitro maturation to maintain
meiotic arrest - or at least to retard meiotic spontaneous
resumption - we have the cAMP modulators: dbcAMP
(Sirard & First, 1988) and 8-bromo-cAMP (Chen et al.,
2009), phosphodiesterase inhibitors: specic inhibitors
of the PDE3, such as, cilostamide (Gharibi et al., 2013;
Mayes & Sirard, 2002; Shu et al., 2008; Vanhoutte et al.,
2008) and milrinone (Mayes & Sirard, 2002; Thomas et al.,
2002, 2004b), PDE4: rolipram (Mayes & Sirard, 2002;
Thomas et al., 2002, 2004b) and PDE8: dipyridamole
(Sasseville et al., 2009), nonspecic inhibitor: IBMX (Albuz
et al., 2010; Rose et al., 2013; Thomas et al., 2002) and
stimulators of adenylate cyclase: forskolin (Albuz et al.,
2010; Richani et al., 2014; Shu et al., 2008; Zeng et al.,
2014) and iAC (Aktas et al., 1995; Guixue et al., 2001;
Luciano et al., 2004).
The overall objective of these pharmacological
manipulations is to avoid premature nuclear maturation
in vitro by means of maintaining higher concentration of
cAMP within the ooplasm. This should provide enough
time for the COC to synchronize nuclear and cytoplasmic
maturation, as similar as possible to the in vivo event
that would allegedly result in more competent oocytes
and embryo (Thomas et al., 2004a). Unsurprisingly, the
mechanism of synthesis and hydrolysis of cGMP is one of
the main targets of pharmacological strategies to control
oocyte maturation.
Nakamura et al. (2002) described an important role of
inducible nitric oxide (NO) synthase (iNOS)/NO/cGMP in the
control of oocyte maturation in rats. This technique uses
a NO donor (S-nitroso-L-acetyl penicillamine - SNAP) for 5
hours to reversibly prevent GV breakdown. This discovery
paved the way for many other studies that also reported
the activation of this pathway in several mammalian
species, including rats (Sela-Abramovich et al., 2008),
mice (Norris et al., 2009), pigs (Chmelíková et al., 2010;
Tichovská et al., 2011) and bovines (Pires et al., 2009;
Sasseville et al., 2008; Schwarz et al., 2008, 2010).
New approaches for modifying IVM and improve
developmental competence take into consideration the
knowledge from cGMP/cAMP and the use of dynamic
systems. Furthermore, non-pharmacological strategies
are trending since the 2010 paper from Dr. John Eppig,
describing the role of the granulosa cell ligand natriuretic
peptide precursor type C (NPPC) and its receptor NPR2 in
maintaining meiotic arrest in mice oocytes.
Novel systems of in vitro maturation and their
impacts in the resulting embryo
Based on the signicant advances of the mechanisms
that control oocyte maturation and their interaction with
the CCs, new paths were opened to improve the IVM
technique. One of which is the use of dynamic in vitro
systems to improve embryo quality and quantity, the so-
called prematuration or pre-IVM systems.
Interesting approaches for modifying IVM to improve
developmental competence is the use of a two-step culture
or pre-maturation systems, where during the initial step a
medium that does not promote nuclear maturation is used
(Albuz et al., 2010; Franciosi et al., 2014; Luciano et al.,
2004; Oliveira e Silva et al., 2011; Ponderato et al., 2002).
Among the systems that have been developed, the most
promising are those that pharmacologically inhibit or retard
meiotic resumption by elevating cAMP concentration in the
oocyte while sustaining GJ communication functionality
(Albuz et al., 2010; Luciano et al., 2004). It was previously
reported that modulation of cAMP levels within mammalian
COCs during IVM could substantially improve oocyte
developmental competence in several species (Albuz et al.,
2010; Funahashi et al., 1997; Luciano et al., 1999, 2004;
Nogueira et al., 2003, 2006; Shu et al., 2008; Thomas
et al., 2004b; Vanhoutte et al., 2009; Zeng et al., 2013;
Figure 1).
In 2003, Shimada et al. (2003) investigated the
formation of LH receptor in cumulus cells of swine COCs
and, after its detection, a new two-step culture system
was developed. In the rst step, the COCs were cultured
in medium supplemented with FSH and IBMX for 20h,
followed by culture in medium supplemented with LH
(second-step). This two-step system with FSH and 0.5
mM IBMX induced the expression of LH receptors in CCs,
improved the rate of blastocyst formation and increased
the number of cells in IVF blastocysts (Figure 1).

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JBRA Assist. Reprod. | v.21 | no1| Jan-Feb-Mar/ 2017
Figure 1. Summarized representation of dierent strategies used during in vitro maturation to modulate the intraoocyte
concentration of cyclic nucleotides and improve embryo yield in several mammalian species (pigs, cattle, mice, sheep or
goat). After each approach, there is a brief schematic description of the methods the authors used. The gure shows the
rates of blastocyst in each treatment in comparison to their respective control group. Statistical signicance is indicated by
the p value. Note that some authors calculate the in vitro performance by dividing the number of blastocyst by the number
of oocytes and others by the number of cleaved embryos. IVF: in vitro fertilization; IVC: in vitro culture; PA: parthenogenetic
activation.
To better understand the inuence of hormones and
growth factor production on the mechanisms controlling
the in vivo maturation in pigs, Kawashima et al. (2008)
updated the two-step system into a new one called “novel
culture system - NCS”. In the NCS design, COCs are
recovered from small antral follicles (3-5mm in diameter);
rst pre-IVM uses FSH, E2 and IBMX for 10h to induce
cell proliferation; second pre-IVM takes place with FSH,
E2, IBMX and P4 for 10h to suppress cell proliferation and
induce LH receptor mRNA expression and nally, an IVM
with LH, EGF and P4 for additional 24h (Figure 1). Using
NCS system, Kawashima et al. (2008) reported the full
expansion of porcine COCs, decreased number of cumulus
cells in apoptotic process and, when oocytes obtained from
NCS were used for IVF, the developmental competence
to blastocyst was signicantly improved when compared
with the conventional culture system (FSH+LH for 48h), or
with the two-step culture system (Funahashi et al., 1997;
Shimada et al., 2003; Figure 1).
Early in the decade, Albuz et al. (2010) proposed a new
IVM system. Their methodology was seeking to mimic the
processes that occurred in the in vivo maturation; hence
their system was called simulated physiological oocyte
maturation (SPOM; Figure 1). This system consisted of
a small pre-IVM (1-2h) where the adenylate cyclase was
stimulated with forskolin, increasing cAMP levels and IBMX,
a PDE inhibitor, to prevent hydrolysis of cyclic nucleotides
(cAMP and cGMP), and after the pre-IVM, the COCs were
subjected to an extended IVM for 24 hours, where the
culture medium was supplemented with cilostamide (PDE3
inhibitor) and recombinant human (rh)-FSH. Results of the
SPOM system were quite exciting, with rates of 69% of
blastocysts per cleaved embryo. Later, the SPOM protocol
was adapted to sheep oocytes and, even though no
signicant eect on blastocyst rates were achieved, there
was an improvement in blastocyst quality observed by an
increase in total cell number (Rose et al., 2013; Figure 1).
The promising results of the rst version of the SPOM
system (SPOMv1) greatly impacted ART research; still, the
SPOM system was updated by their creators in subsequent
studies. Zeng et al. (2013) used heparin during pre-MIV
and removed the cilostamide from the extended IVM. This
approach positively aected oocyte energy metabolism,
oocyte meiotic maturation and embryo development
(SPOMv2; Figure 1).
One year later, Zeng et al. (2014) tested the presence
of rh-FSH during extended IVM (Figure 1). By now it
seems that cilostamide in extended IVM phase is gone

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for good, and now the system is no longer called SPOM,
but Prematuration System (or Pre-IVM system) instead.
They reported successful results in IVP of mice embryos
(± 70% of blastocysts per cleaved embryos), better
quality in the expansion of CCs, reduction of abnormal
spindles and a positive inuence of Pre-IVM system in the
glycolic metabolism of COCs (suggesting an eect of cAMP
production predominantly on glycolytic activity).
Several laboratories and research groups around the
world sought to repeat the success obtained by the SPOM or
Pre-IVM system; however, most failed in doing so (Bernal-
Ulloa et al., 2016; Buell et al., 2015; Guimarães et al.,
2015; Razza et al., 2015; Figure 1). Ulloa et al. (2015),
using bovine oocytes cultured in SPOM system, produced a
smaller number of blastocyst compared with the standard
IVM. However, the pattern of DNA methylation of embryos
produced in SPOM system was more similar to embryos
produced in vivo. Also, Santiquet et al. (2014a) tested a
pre-IVM treatment and could improve the developmental
competence of oocytes, as demonstrated by increased
embryo development. Additionally, pre-IVM performed
with IBMX and forskolin in the Pre-IVM system can change
ultrastructural characteristics of oocytes and blastocysts
(Razza et al., 2015; unpublished data from our group).
With the strategy used in the SPOM system of performing
a two-step culture with dierent drugs to induce dierent
eects in CCs and oocytes during IVM, new drugs and
signaling pathways have emerged as potential targets for
research seeking to improve IVM and embryo production.
Earlier in this decade, a new model discovered that the
binding of NPPC to its receptor (NPR2) in granulosa and CCs
are the main cause for the modulation of cGMP levels (Zhang
et al., 2010). Until now, studies relating the new mechanism
of NPPC/NPR2/cGMP in maturation control have been reported
in mice (Tsuji et al., 2012; Zhang et al., 2011), bovines
(Franciosi et al., 2014), swine (Santiquet et al., 2014b; Zhang
et al., 2014) and sheep (Zhang et al., 2015; Figure 1).
Using the NPPC during pre-IVM (8h) in bovine COCs,
Franciosi et al. (2014) could successfully arrest meiosis re-
sumption and extend the functional communication among
oocytes and CCs through GJ. After IVF and embryo culture,
the NPPC treatment in pre-IVM has also increased blasto-
cyst cell number and hatching rates.
In a more recent approach of two-step maturation with
caprine COCs, Zhang et al. (2015) used the NPPC and
estradiol during pre-IVM (8h), followed by conventional IVM
(18h). With this system, meiosis was eectively arrested in
pre-IVM and the maturation rate was also increased after
conventional IVM. They also increased embryo production
and quality, evaluated by total cell number per blastocyst
(Figure 1).
Conventionally, oocyte competence has been assessed
by embryo morphology and blastocyst rates; however,
these aspects alone do not provide sucient information to
fully endorse the IVM system eciency. Several strategies
are being used to study the quality of oocytes (before and
after IVM) and embryos. Some of the most promising and
approaches that focus on the identication of biomarkers.
New perspectives and nal considerations
A few non-invasive strategies are already being used
to predict oocyte competence to become a viable embryo,
even before the oocyte is fertilized. These approaches aim
to identify oocyte competence biomarkers mostly in cumulus
cells. In this context, the morphology of CCs can be used to
rst categorize oocyte potential (de Loos et al., 1991) and then
to compare the morphological data with CCs transcriptome
(dierentially expressed genes in CCs surrounding the good
oocytes versus poor-quality oocytes). At present, many genes
are identied as potential biomarkers (reviewed by Labrecque
& Sirard, 2014). Still, many research groups are working on
the identication of miRNAs as biomarkers as well. Proles
of miRNAs isolated from EVs present in follicular uid were
described and associated with proper cytoplasmic oocyte
maturation; hence, these miRNA proles can be used to
predict oocyte competence (Sohel et al., 2013).
The use of non-invasive strategies, such as analysis
of follicular uid and culture media (after culture) also
appears to be quite useful on the search for molecular
biomarkers for oocyte competence. The presence of
cytokines and growth factors in follicular uid is crucial for
determining oocyte quality (reviewed by Dumesic et al.,
2015). In this context, the metabolic characterization of
the culture media, in which IVP embryos are kept for many
hours, may represent an important non-invasive tool to
either indicate possible predictive biomarkers of viability
or to explain IVP outcome afterwards (Muñoz et al., 2014).
Lipid metabolism is induced in COCs during oocyte
maturation and contributes to oocyte and embryo
development (Gardner & Harvey, 2015). Specic fatty acids
have distinct eects on oocyte maturation. In general,
saturated fatty acids (palmitic acid and stearic acid) are
elevated in follicular uid and, in some metabolic contexts,
are detrimental, while the presence of unsaturated
non-esteried fatty acids (oleic acid and linoleic acid)
can counteract these detrimental eects and promote
developmental competence (reviwed by Dunning et al.,
2014). Thus, in vitro oocyte and embryo development may
be optimized through the provision of appropriate energy
substrates and essential co-factors during ART in domestic
animals and subfertility women.
Despite the large number of publications in the eld
we still have a long way to go to deeply understand and
manipulate the mechanisms controlling oocyte maturation.
Overcoming these gaps may allow us to improve ART
results. Therefore, it is necessary to design studies aiming
at nding eective biomarkers for oocyte competence. The
eld of the OMICs seems to be quite promising, especially
regarding the new ndings in transcriptomics, proteomics
and lipidomics in oocytes, CCs, embryos and in the EVs
within the follicular uid. Future studies on this subject
might enable the design of more complex, dened and
ecient culture conditions for oocytes to be fully matured
and able to generate optimum IVP embryos.
ACKNOWLEDGEMENTS
We acknowledge the São Paulo Research Foundation
(FAPESP) for funding (2012/50533-2 and 2013/05083-1) and
fellowships for RCB (2014/25072-7), EMR (12/23409-9) and
EMP (13/07730-4).
CONFLICT OF INTERESTS
No conict of interests has been declared.
Corresponding author:
Ramon Cesar Botigelli
Multi-user laboratory Phytomedicine
Pharmacology and Biotechnology (FitoFarmaTec)
Institute of Bioscience, University of São Paulo State
Botucatu - SP, Brazil
E-mail: ramonbotigelli@gmail.com
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