CSIRO PUBLISHING

www.publish.csiro.au/journals/rfd Reproduction, Fertility and Development, 2011, 23, 23–31

Recent insights into oocyte]follicle cell interactions

provide opportunities for the development of new
approaches to in vitro maturation

Robert B. Gilchrist

Robinson Institute, Research Centre for Reproductive Health, and School of Paediatrics
and Reproductive Health, Discipline of Obstetrics and Gynaecology, Medical School,
University of Adelaide, Adelaide, SA 5005, Australia.
Email: robert.gilchrist@adelaide.edu.au

Abstract. The last 5–10 years of research in ovarian and oocyte biology has delivered some major new advances in
knowledge of the molecular and cellular processes regulating oocyte maturation and oocyte developmental competence.
These new insights include, among others: (1) the knowledge that oocytes regulate granulosa and cumulus cell
differentiation, ovulation rate and fertility via the secretion of soluble paracrine growth factors; (2) new perspectives
on the participation of cyclic nucleotides, phosphodiesterases and gap junctions in the regulation of oocyte meiotic arrest
and resumption; and (3) the new appreciation of the mechanisms of LH-induced oocyte maturation and ovulation mediated
by the follicular cascade of epidermal growth factor (EGF)-like peptides, the EGF receptor and their intracellular second
messengers. These recent insights into oocyte–follicle cell interactions provide opportunities for the development of new
approaches to oocyte in vitro maturation (IVM). Laboratory IVM methodologies have changed little over the past 20–
30 years and IVM remains notably less efficient than hormone-stimulated IVF, limiting its wider application in
reproductive medicine and animal breeding. The challenge for oocyte biologists and clinicians practicing IVM is to
modernise clinical IVM systems to benefit from these new insights into oocyte–follicle cell interactions in vivo.

Additional keywords: artificial reproductive technology (ART), cAMP, embryo production, GDF9, oocyte IVM,
simulated physiological oocyte maturation (SPOM).

Background normal offspring (Schroeder and Eppig 1984; Shu-Chi et al.

Oocyte quantity and quality are fundamentally rate-limiting in
female fertility; however, the nature of the molecular and cel-
lular processes that regulate oocyte quality is not well defined.
Oocyte quality or oocyte developmental competence refers to
an oocyte’s intrinsic capacity to undergo meiosis and support
fertilisation, preimplantation embryo development and fetal
development to term to produce a healthy offspring. Oocyte
developmental competence is gradually acquired in the devel-
oping ovarian follicle. Oogenesis and folliculogenesis occur in
unison and are orchestrated by a highly complex interplay of
endocrine, paracrine and autocrine signals that are exchanged
between the oocyte and somatic compartment of the follicle.
Improving our understanding of the intricate bidirectional
communication axes between the oocyte and follicular somatic
cells is opening new opportunities in artificial reproductive
technologies (ART).
One such ART is oocyte in vitro maturation (IVM), which is a
technique where immature oocytes are collected from mid-sized
antral follicles, generally from unstimulated ovaries, and cul-
tured, matured and fertilised in vitro (Edwards 1965) to produce
embryos that, after transfer, appear to generate developmentally
2006; Eppig et al. 2009). IVM has the advantage over conven-
tional IVF as it eliminates or drastically reduces the use of
gonadotrophins for ovarian hyperstimulation, thereby reducing
the contraindications and cost to patients, and avoiding the
potential for hyperstimulation-induced abnormalities in the
developing embryo (Baart et al. 2007; Market-Velker et al.
2010). The notable disadvantage of IVM is its significantly
lower efficiency compared with hormone-driven IVF, in terms
of embryos and offspring generated per oocyte collected (Child
et al. 2002; Eppig et al. 2009). This factor alone has greatly
restricted the use of IVM in human reproductive medicine, where
it is estimated that just 1300 IVM babies have been born over
the past 30 years (Suikkari 2008). By contrast, in 2005 alone,
,133 000 viable cattle IVM offspring were produced (Thibier
2006) and currently the use of IVM and in vitro embryo produc-
tion is increasing dramatically in this sector. Hence, for human
and veterinary applications, there is great incentive to improve
the efficiency of IVM. This review will: (1) briefly examine the
substantial advances made in recent years in our understanding
of oocyte–follicle cell interactions and the mechanisms regulat-
ing oocyte maturation in vivo and (2) discuss how this new

Ó IETS 2011 10.1071/RD10225 1031-3613/11/010023

24 Reproduction, Fertility and Development R. B. Gilchrist

knowledge might be used to modernise oocyte IVM methodol-
ogies specifically to improve embryo yield from IVM.
New insights into oocyte]follicle cell interactions
Oocyte control of granulosa and cumulus cell function
An important new paradigm in ovarian and oocyte biology to
emerge over the past decade is the concept of oocyte-secreted
factor (OSF)-regulation of folliculogenesis. This topic has
already been the subject of several reviews and so will not be
dealt with extensively here (some reviews on this topic: Eppig
2001; Gilchrist et al. 2004, 2008; McNatty et al. 2006;
Scaramuzzi et al. 2010). Oocytes regulate granulosa cell growth
and differentiation through the production of soluble paracrine
growth factors. The two most important OSFs are growth dif-
ferentiation factor 9 (GDF9) and bone morphogenetic protein 15
(BMP15), as both are essential for folliculogenesis and female
fertility in a species-specific manner (Dong et al. 1996;
Galloway et al. 2000). Oocyte-secreted GDF9 and BMP15 act in
a paracrine mode on granulosa and cumulus cells (CCs) through
their receptors, bone morphogenetic protein receptor type-II
(BMPRII) and activin receptor-like kinases (ALKs), to activate
Sma- and Mad-related (SMAD) intracellular signal transducers
(Kaivo-oja et al. 2006). Aberrant expression of any of these
ligands, receptors or second messengers causes sterility,
enhanced fecundity or ovarian disease in a highly species-
specific manner (McNatty et al. 2004).
Pertinent to the subject of this review is the knowledge
that a principal function of OSFs during the antral phase of
folliculogenesis is to direct the differentiation of granulosa
cells into cumulus cells and to maintain this distinct phenotype
in the follicle (Eppig et al. 1997; Li et al. 2000). This appears
to be achieved by OSFs establishing a localised concentration
or morphogenic gradient emanating from the oocyte (Hussein
et al. 2005). The consequences of OSF signalling to cumulus
cell function are numerous (Gilchrist et al. 2008) and this
remains an active area of research. Some of the actions of
OSFs on CCs include stimulation of growth and prevention of
death (Vanderhyden et al. 1992; Gilchrist et al. 2001, 2006;
Hussein et al. 2005), inhibition of luteinisation (Vanderhyden
et al. 1993; Eppig et al. 1997; Li et al. 2000), regulation of
energy metabolism (Sutton et al. 2003a; Sugiura et al. 2005)
and sterol biosynthesis (Su et al. 2008) and the regulation of
CC expansion (Buccione et al. 1990; Salustri et al. 1990;
Fig. 1). By means of OSFs, the oocyte appears to direct the
CCs to perform functions for which the oocyte itself has a poor
capacity (e.g. glycolysis; Sutton-McDowall et al. 2010).
Hence, the capacity of an oocyte to control such CC functions
is likely to be a crucial function required for acquisition of its
own developmental competence.

EGFR
AREG
EGFR
EREG
BTC
PGE2 PTGER2

ERK1/2 p38 ERK1/2

AREG GPR3 PKA
EREG
BTC X ? MPF
Cx43 ↓ cAMP
?
cAMP
p38 ↓ cGMP ↑ PDE3
GVBD
HAS2 Sterol biosynthesis ? 5-AMP
cAMP ↓ cGMP TSG6
PTX3 Metabolism
GDF9
GC Luteinisation BMP15
? GC
BMPRII
SMAD2/3
LHR NO FSHR ALK5/6
ANP
ERK1/2
?
SRC
LH FSH
EGFR

Granulosa cell Cumulus cell Oocyte

Fig. 1. Model of new perspectives on oocyte–follicle cell interactions and how these may interact to regulate
oocyte maturation in vivo. The resumption of oocyte meiosis in vivo is a highly orchestrated, complex process that
integrates oocyte paracrine signals with an array of maternal endocrine, granulosa/cumulus paracrine and gap-
junctional signals. This highly integrated interaction between oocyte and follicular somatic cells is unlikely
to occur during spontaneous IVM, as it is clinically practiced at present. This hypothetical model of meiotic
induction in vivo is not intended to be complete but rather to highlight the new appreciation of three processes:
(1) interaction of GDF9/BMP15–SMAD2/3 signalling and EGFR; (2) LH-induction of the EGF-like peptide
cascade in granulosa and CCs; and (3) the complex interplay between gap junctions, cGMP, PDE and cAMP.
Dotted arrows indicate signals of diminishing intensity. ‘X’ represents a hypothetical meiotic-inducing factor. See
text for other abbreviations.
Recent insights provide new opportunities for IVM
Two very recent studies demonstrated an important relation-
ship between OSF and epidermal growth factor receptor (EGFR)
signalling in CCs (Sasseville et al. 2010; Su et al. 2010; Fig. 1).
It has been known for some time that there is an important
interaction between signals from the oocyte and signals from the
follicle that intersect in CCs. This was initially demonstrated
by the dual requirement for OSFs and FSH for mouse cumulus–
oocyte complex (COC) expansion (Buccione et al. 1990;
Vanderhyden et al. 1990). Subsequently it was demonstrated
that the follicular signal is mediated by ERK1/2 in CCs, which
could be activated by FSH or epidermal growth factor (EGF;
Su et al. 2002, 2003) and that the oocyte paracrine signal is
mediated by GDF9/BMP15 activating SMAD2/3 in CCs
(Dragovic et al. 2005, 2007; Yoshino et al. 2006; Diaz et al.
2007). It is now known, in the mouse at least, that participation
of these two signalling pathways is a prerequisite not only for
CC expansion but for many of the OSF-regulated CC functions
listed above. Recently we have shown that cross-talk between
these two pathways is mediated by the EGFR–ERK1/2 pathway
enabling GDF9–SMAD3 signalling in granulosa and cumulus
cells (Sasseville et al. 2010). This appears to be mediated by
activation of ERK1/2 through EGFR, which, in turn, phosphor-
ylates SMAD3 in its linker region, enabling SMAD2/3-
mediated gene transcription. Another recent study has shown
that oocytes, via GDF9/BMP15 signalling, promote EGFR
expression in CCs via a mechanism requiring SMAD2/3 (Su
et al. 2010). Hence, it appears that there are complex, mutually
dependent interactions between GDF9/BMP15–SMAD2/3 and
EGFR–ERK1/2 signalling in CCs and that these are at the nexus
of oocyte–somatic communication (Fig. 1). This autoregulatory
loop between the oocyte and its supporting somatic cells is likely
to be crucial to oocyte development.
Follicular control of oocyte maturation
In terms of follicle cell control of oocyte maturation, major
advances in understanding have been achieved in just the past
few years. While it has long been known that the preovulatory
LH surge induces oocyte maturation, the exact molecular
mechanisms regulating this process have been the subject of
debate for decades. It is evident that the follicular environment
is responsible for both meiotic arrest of the oocyte at prophase I
(GV stage) and for meiotic resumption. A focal point of oocyte
meiotic regulation and resumption is cyclic AMP (cAMP),
which is synthesised in the oocyte by constitutively active
G-protein-coupled receptors (GPR3; Mehlmann et al. 2002)
and is also supplied to the oocyte by mural granulosa cells
(MGCs) and adjacent CCs through gap junctions (Anderson and
Albertini 1976; Fig. 1). High levels of intra-oocyte cAMP keep
the oocyte meiotically arrested by suppressing maturation pro-
moting factor (MPF) activity by stimulating cAMP-dependent
protein kinase A (PKA). The oocyte possesses a potent type 3
phosphodiesterase (PDE3), the enzyme that degrades cAMP
(Tsafriri et al. 1996). Importantly, the somatic cells of the fol-
licle also supply cyclic GMP (cGMP) to the oocyte (Törnell
et al. 1991), which inhibits PDE activity. The paradox of cAMP
in the oocyte is that high levels maintain meiotic arrest
throughout folliculogenesis yet it also mediates the meiotic-
inducing effects of gonadotrophins. This may be explained by
Reproduction, Fertility and Development 25
the idea that a transient pulse in cAMP produces a meiosis-
inducing signal (Dekel 1988; Downs and Chen 2008). Two
important recent studies have revealed that the ovulatory
gonadotrophin surge causes a drop in follicular and oocyte
cGMP levels leading to upregulated oocyte PDE activity, which
causes a fall in intraoocyte cAMP and meiotic resumption
(Norris et al. 2009; Vaccari et al. 2009; Fig. 1). The above is a
substantial oversimplification of the participation of cAMP,
cGMP and PDEs in the process of oocyte meiotic resumption
and the detailed complexity of these signalling pathways
remains a topical area of investigation.
The second major recent breakthrough is that the preovula-
tory LH surge induces a rapid secondary cascade of the EGF-
like peptides – amphiregulin (AREG), epiregulin (EREG) and
b-cellulin (BTC) – in the somatic cells of the follicle (Park et al.
2004). These peptides are initially produced by the MGCs in
response to LH and have autocrine effects on MGCs and
paracrine actions on CCs via their EGF receptors (Fig. 1). An
important emerging concept is that the EGF receptor is a central
nexus in propagating the ovulatory LH signal from the granulosa
cells, through the cumulus cells to the oocyte (Hsieh et al. 2007;
Reizel et al. 2010). Central to MGC/CC signal transduction
are the EGF-like peptides that activate the EGF receptor and
ERK1/2 that transmits the intracellular signals (Park et al.
2004; Fan et al. 2009). Activation of ERK1/2 induces a cascade
of prostaglandin E2 (PGE2) and p38MAPK induction, which,
in turn, amplifies EGF-like peptide production by MGCs and
CCs (Park et al. 2004; Ashkenazi et al. 2005; Shimada et al.
2006; Downs and Chen 2008). The EGF-like peptides are potent
inducers of oocyte meiotic resumption via a mechanism requir-
ing ERK1/2 (Su et al. 2002; Hsieh et al. 2007; Fan et al. 2009).
Details of cumulus cell ERK1/2-induced meiotic resumption are
not yet fully characterised, but may involve phosphorylation of
connexin 43 (Cx43), leading to closure of granulosa–cumulus
and cumulus–cumulus gap junctions (Sela-Abramovich et al.
2005; Norris et al. 2008) and subsequent loss of the inhibitory
effects of follicular cAMP and cGMP (Sela-Abramovich et al.
2006; Norris et al. 2009), or alternatively, production of a
stimulatory meiosis-inducing factor by the CCs, or some com-
bination of the two (reviewed by Downs 2010; Fig. 1).
New opportunities to modernise oocyte IVM
Given the substantial advances made in recent years in our
understanding of oocyte–CC communication and the molecular
cascades regulating the reinitiation of oocyte maturation in vivo,
an important challenge for oocyte biologists and clinicians
practicing IVM is to capitalise on these new insights to develop
new IVM systems. Ideally, a new-generation IVM system
should recapitulate the key events that occur during oocyte
maturation in vivo; among others these should include:
(1) appropriate OSF regulation of cumulus cell SMAD2/3 sig-
nalling and associated EGFR function; (2) careful management
of fluxes of cAMP and cGMP; (3) appropriate induction of the
EGF-like peptide/ERK1/2 signalling cascade; and (4) controlled
loss of oocyte–cumulus cell gap-junctional communication.
Furthermore, designing new approaches to IVM needs to be
undertaken with the explicit purpose in mind of maximising
26 Reproduction, Fertility and Development
New insights New opportunities
Oocyte–follicle
IVM
interactions
1. Oocyte control of follicle cell 1. Appropriate cumulus cell
differentiation function during IVM
• Oocyte-secreted factors • Exogenous GDF9 and/or
BMP15 in IVM
2. Follicular control of oocyte meiotic 2. Simulated physiological oocyte
arrest and resumption maturation (SPOM)
• cGMP, cAMP, PDEs • cAMP/PDE-regulated
• EGFR, EGF-like peptides meiotic resumption
• Cumulus peptide
induction of maturation
Fig. 2. New insights into oocyte–follicle cell interactions provide oppor-
tunities for the development of new approaches to IVM. A challenge for
oocyte biologists and IVM clinicians is to modernise clinical IVM systems to
benefit from this new knowledge of oocyte–follicle cell interactions in vivo.
IVM efficiency in terms of the production of developmentally
competent healthy embryos (Fig. 2).
Problems with current IVM methodologies
The central limiting problem with current IVM practices is the
lower efficiency of IVM, in terms of embryo development,
implantation and live birth rates, compared with conventional
IVF where oocytes mature in vivo (Gilchrist et al. 2010). For
example, the live birth rate in mice from IVM was 21% com-
pared with 52% using IVF (Eppig et al. 2009). Notably, this
discrepancy in reproductive efficiency between IVM and IVF
has existed for decades and is common across mammalian
species. For example, embryo development or implantation
rates are notably lower using IVM than IVF in mice, sheep,
cattle and women (Leibfried-Rutledge et al. 1987; Thompson
et al. 1995; Child et al. 2002; Rizos et al. 2002; Nogueira et al.
2003). Moreover, the miscarriage rate in women using IVM
is significantly higher than in gonadotrophin-stimulated IVF
(Buckett et al. 2008). This lower efficiency of IVM compared
with hormone-stimulated IVF is the principal factor limiting
widespread use of IVM in human reproductive medicine. In
contrast, IVM and associated in vitro production of embryos is
in widespread use in cattle artificial breeding and its use is rising
dramatically in South America (Thibier 2006). Hence both of
these ART sectors would benefit significantly from a notable
increase in IVM efficiency.
Essentially all human and veterinary ART clinics offering
commercial IVM use a simple, standard system of IVM called
spontaneous oocyte maturation. Using this approach, COCs
are aspirated from unstimulated or mildly stimulated ovaries
and cultured for 24–48 h in vitro, typically in complex medium
supplemented with serum or albumin (Sutton et al. 2003b). By
rapidly removing COCs from the meiotic-inhibiting influence of
the follicle and the follicular fluid, oocytes irrevocably reinitiate
and complete meiosis in vitro (Pincus and Enzmann 1935;
Edwards 1965); hence the term ‘spontaneous IVM’. The
R. B. Gilchrist
resumption of meiosis in spontaneous IVM is hormone-
independent, even though hormones such as FSH are typically
added. Critically, oocytes that undergo meiosis spontaneously
in vitro are likely to do so in the absence of the elaborate cascade
of endocrine and paracrine molecular signals that usually induce
maturation in vivo (Park et al. 2004; Ashkenazi et al. 2005;
Shimada et al. 2006; Norris et al. 2009; Vaccari et al. 2009;
Fig. 1). The spontaneous resumption of meiosis during standard
IVM techniques is unphysiological and we propose that this
compromises subsequent oocyte developmental competence
(Gilchrist and Thompson 2007).
Application of OSFs to improve IVM
Based on the premises that OSFs are fundamental regulators
of CC differentiation and function, and that CC processes that
facilitate oocyte developmental competence are likely to be
perturbed under conditions of standard spontaneous IVM, we
hypothesised that treatment of COCs during IVM with exo-
genous OSFs would enhance oocyte developmental competence
(Fig. 3). We tested this by treating COCs during IVM with either
native OSFs (COC co-cultured with denuded oocytes) or par-
tially purified recombinant GDF9 or BMP15, or both. Both
approaches improved the capacity of bovine oocytes to proceed
to the blastocyst stage of embryo development; from 40%
(control) up to as high as ,60% (Hussein et al. 2006). Exposure
of oocytes to GDF9 or BMP15 also improved subsequent
embryo quality as evidenced by increased total and trophecto-
derm cell numbers. Treating mouse COCs during IVM with
recombinant GDF9 significantly increased the number of sur-
viving fetuses after blastocyst transfer to foster mothers without
adverse effects on fetal or placental morphology (Yeo et al.
2008). Furthermore, Romaguera et al. (2010) have recently
shown that native OSFs are able to improve the competence
of a cohort of oocytes that otherwise would have poor develop-
mental potential.
The precise molecular mechanisms underlying the improved
oocyte developmental competence with OSF-supplemented
IVM are currently unclear. Attempts to elucidate this mechan-
ism have been hampered by (1) the technical complexities of
conducting large-scale COC plus denuded oocyte co-culture
experiments and (2) the relatively poor quality of recombinant
GDF9 and BMP15 preparations available for research. Until
very recently, the only GDF9/BMP15 preparations available
were in-house generated proteins that varied widely between
research laboratories in terms of construction and purity.
Recently, commercially available purified GDF9 and BMP15
have become available from R&D Systems, although these
preparations are notable in that they lack their respective
proregions. Current and future efforts to generate recombinant
GDF9 and BMP15 proteins that are highly purified with appro-
priate proregion–mature region interactions (Mottershead et al.
2008; Li et al. 2009), are important if clinical IVM is to benefit
from the applications of these proteins.
Although the molecular mechanisms require further investi-
gation, it seems clear that OSFs participate in the regulation of
oocyte developmental competence by directing fundamental
aspects of CC differentiation and function during folliculogen-
esis and oocyte maturation (Gilchrist et al. 2008; Fig. 3). Across
Recent insights provide new opportunities for IVM Reproduction, Fertility and Development 27

I P
PRI SMAD2/3
BM
L K 5 P
A
F9
GD P
II
BMPR SMAD1/5/8

BMP15 ALK6 P

? P
Ga arac
p rine
jun
cti Regulation of cumulus cell function
on
al Proliferation Metabolism
Apoptosis Expansion
Oocyte quality Luteinisation

Embryo development
Fetal viability

Fig. 3. Bidirectional communication between the oocyte and cumulus cells is required for oocyte
developmental competence. Oocytes control the differentiation of CCs through the secretion of GDF9
and BMP15. These act via CC receptors BMPRII, ALK5 and ALK6 to activate SMAD intracellular
transducer molecules, which, in concert with maternal signals such as FSH and the EGF-like peptides,
regulate a large range of CC functions. CCs in turn make substantial contributions to oocyte growth and
development via paracrine and gap-junctional-mediated processes. This oocyte–CC feedback loop is
likely to be a crucial determinant of oocyte developmental competence.

mammalian species, activation of the GDF9 signalling cascade
in CCs by oocytes, including ERK1/2-mediated SMAD2/3
activation (Sasseville et al. 2010) and subsequent EGFR regula-
tion (Su et al. 2010), is probably fundamental to the regulation of
oocyte quality (Figs 1, 3). Indeed, oocyte developmental com-
petence or embryo post-transfer fetal survival is compromised in
mutant mice with perturbed GDF9 and BMP15 expression (Su
et al. 2004) or when GDF9 signalling is inhibited during IVM
(Yeo et al. 2009). Considerable recent effort to identify pre-
dictors of oocyte quality has identified a considerable number of
CC genes that are specifically under OSF control (see review
Li et al. 2008). These include, among others, OSF-regulated
genes such as Has2, Ptgs2, Ptx3, Grem, Areg, Star and Ereg
(McKenzie et al. 2004; Feuerstein et al. 2007; Assidi et al. 2008;
Fig. 1). These studies provide further weight to the notion that
GDF9/BMP15 regulation of CC function regulates oocyte
quality (Gilchrist et al. 2008) and provide the basis for devel-
oping a rapid quantitative test of oocyte quality using a biopsy of
CCs (Smitz et al. 2010).
Simulated physiological oocyte maturation (SPOM): a new
IVM system based on induced oocyte maturation in vivo
Building on the significant new insights into mechanisms
mediating oocyte maturation in vivo – specifically the roles of
cAMP/cGMP/PDEs and the induction of maturation via EGFR-
ERK1/2 signalling (Fig. 1) – we have recently developed a new
approach to oocyte IVM called ‘simulated physiological oocyte
maturation’ (SPOM; Albuz et al. 2010). SPOM is an integrated
IVM system that includes a short pre-IVM phase (1–2 h) and an
extended IVM phase that synergise to generate high embryo and
fetal yields following embryo transfer (Fig. 4). The pre-IVM
phase is specifically designed with the human and veterinary
clinical scenario in mind, whereby at pick-up, COCs are
removed from the follicular environment and typically placed in
a simple buffered collection medium that leads to a rapid and
precipitous drop in oocyte cAMP levels (Vivarelli et al. 1983).
Accordingly, in SPOM we designed a pre-IVM phase to be used
at oocyte pick-up, containing cAMP-modulating agents (for-
skolin þ IBMX), that generate a large rapid increase (4100-fold)
in COC cAMP levels, as occurs in COCs immediately after the
preovulatory gonadotrophin surge in vivo (Albuz et al. 2010).
This approach is analogous to the use of invasive adenylate
cyclase that has previously been used in bovine oocyte collection
medium (Aktas et al. 1995; Luciano et al. 1999; Guixue et al.
2001). The pre-IVM phase was designed to cause an immediate
increase to both CC and oocyte cAMP levels. This has the effect of
preventing immediate loss of oocyte–cumulus gap-junctional
communication at pick-up (Albuz et al. 2010) and simultaneously
loads the oocyte with cAMP, thereby preventing precocious
spontaneous resumption of meiosis.
The extended IVM phase of SPOM attenuates meiotic
resumption, which is overridden or induced by FSH. An
important feature of the IVM system is that oocytes are
continuously exposed throughout maturation to a low to
moderate concentration of a type 3-specific PDE3 inhibitor.
As PDE3s are principally expressed in the oocyte and not in
CCs (Tsafriri et al. 1996; Sasseville et al. 2009), this approach
specially targets intraoocyte cAMP. The PDE3 inhibitor con-
centration is low but sufficient to notably impair meiotic
maturation in the absence of a meiotic-inducing hormone. In
SPOM a relatively high concentration of FSH (100 mIU mL1)
is crucial to override or ‘induce’ oocyte maturation in the
presence of the PDE3 inhibitor. We have previously used a
similar approach to improve the developmental potential of
28 Reproduction, Fertility and Development
Conventional IVF Ovarian hyperstimulation
(in vivo matured)
Standard IVM
Simulated physiological
oocyte maturation
(SPOM) Pre-IVM
(forskolin  IBMX)
Fig. 4. Schematic illustration of the SPOM IVM system and how it differs from standard IVM and
conventional IVF. SPOM is characterised by a short pre-IVM phase (COC collection) where COCs are
exposed to agents that cause a spike in cAMP, and by an extended IVM phase where FSH induces oocyte
maturation in the continuous presence of an oocyte-specific PDE inhibitor. SPOM generates murine
fertilisation, blastocyst and implantation rates and fetal yield comparable to hormone-driven conventional
IVF. Figure modified from Albuz et al. (2010).
bovine oocytes (Thomas et al. 2004). In the SPOM system, the
pre-IVM and IVM phases synergise to substantially improve
oocyte developmental competence relative to standard sponta-
neous IVM, to levels that match hormone-stimulated conven-
tional IVF (Albuz et al. 2010; Fig. 4).
It is notable that a model of hormone-dependent (using FSH,
EGF, FF-MAS, etc.) induced oocyte maturation has been used
for decades by mouse oocyte biologists to study the fine detail
of meiotic regulation (Downs et al. 1988), but curiously this
mode of oocyte maturation has rarely been used as a means of
generating embryos. SPOM differs from ‘biphasic IVM’, which
also typically exploits PDE inhibitors. Biphasic IVM differs
notably in that PDE inhibitors or other meiotic inhibitors are
used at sufficient concentration to block meiotic resumption
for a defined period (usually ,24 h), after which the inhibitor is
washed out so that the oocyte then undergoes spontaneous
maturation (reviews Smitz et al. 2004; Gilchrist and Thompson
2007). SPOM is a form of hormone-dependent oocyte matura-
tion as sufficient FSH is required to drive meiotic induction in
the presence of the type 3 PDE inhibitor (Fig. 4). Furthermore,
FSH induction of meiotic resumption occurs by a mechanism
that requires the EGF receptor (Albuz et al. 2010), which is
likely to involve FSH/cAMP-induced expression of EGF-like
peptides by CCs (Downs and Chen 2008). By generating high
levels of cAMP in the oocyte and the induced nature of oocyte
maturation, SPOM mimics some of the key, newly characterised
molecular signals that occur during oocyte maturation in vivo.
Continued development of IVM methodologies that build on our
new knowledge of mechanisms regulating oocyte maturation
in vivo should lead to further increases in IVM efficiency and
embryo production in vitro.
Conclusions
Substantial progress has been made in recent years, largely
using the mouse model, into long-standing controversies over
the detailed cellular and molecular processes regulating
oocyte maturation in vivo. New signalling systems that were
largely unknown 10 years ago, in particular, the oocyte signal-
ling system using GDF9/BMP15–SMAD2/3 and the EGF-like
R. B. Gilchrist
Fetal
yield
22%
IVF/ET
8%
IVM (FSH) IVF/ET
26%
Extended IVM IVF/ET
(cilostamide  FSH)
peptide–ERK1/2 cascade, have now been characterised in
detail. It is now incumbent on oocyte IVM scientists and clin-
icians to incorporate this new knowledge into new IVM systems
for genetic improvement in domestic animals and clinical
infertility treatment that are designed to improve oocyte
developmental competence and produce a high yield of healthy
embryos and offspring.
Acknowledgements
I would like to acknowledge collaborators who have worked closely with me
over many years on these topics; Jeremy Thompson, Firas Albuz, David
Armstrong, David Mottershead, François Richard, Lesley Ritter, Maxime
Sasseville, Johan Smitz and Melanie Sutton-McDowall. Critical review
of the manuscript by Maxime Sasseville, Karine Reynaud and Jeremy
Thompson is greatly appreciated. I would like to thank Graeme Martin for
the artwork provided in Fig. 3. This research area was funded by the National
Health and Medical Research Council of Australia (Grants 519235, 453556
and Fellowship 465415), by the Australian Research Council (Grant
LP0562536) and by Cook Medical Pty Ltd.
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