BIOLOGY OF REPRODUCTION (2014) 91(2):47, 1–11

Published online before print 25 June 2014.

DOI 10.1095/biolreprod.114.118471

Prematuration with Cyclic Adenosine Monophosphate Modulators Alters Cumulus

Cell and Oocyte Metabolism and Enhances Developmental Competence of In Vitro-
Matured Mouse Oocytes1
Hai-Tao Zeng,3,4 Dulama Richani,3 Melanie L. Sutton-McDowall,3 Zi Ren,3,5 Johan E.J. Smitz,6 Yvonne
Stokes,7 Robert B. Gilchrist,3 and Jeremy G. Thompson2,3
3
Research Centre for Reproductive Health, Robinson Institute, and Discipline of Obstetrics and Gynaecology, School of
Paediatrics and Reproductive Health, The University of Adelaide, Adelaide, South Australia, Australia
4
Center for Reproductive Medicine, Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, People’s Republic of
China
5
Center for Reproductive Medicine, General Hospital of Guangdong, Guangzhou, People’s Republic of China
6
Follicle Biology Laboratory, Center for Reproductive Medicine and Medical School, Free University Brussels (VUB),
Brussels, Belgium
7
School of Mathematical Science, The University of Adelaide, Adelaide, South Australia, Australia

ABSTRACT and glutathione levels (P , 0.05). Nevertheless, IVM increased

Downloaded from www.biolreprod.org.

Oocyte in vitro maturation (IVM) is an important assisted
reproductive technology and research tool. The adoption of IVM
into routine clinical practice has been hindered by its
significantly lower success rates compared to conventional in
vitro fertilization. Cyclic AMP (cAMP) modulation and follicle-
stimulating hormone (FSH), independently, have long been
known to improve IVM oocyte developmental competence. This
study comprehensively examined the effects of FSH and cAMP/
cGMP modulation, alone and in combination, on IVM oocyte
metabolism and developmental outcomes. Mouse cumulus-
oocyte complexes (COCs) were subjected to a 1 h prematuration
phase 6 the cAMP modulator forskolin and cAMP/cGMP
modulator 3-isobutyl-1-methylxanthine followed by IVM 6
FSH. Prematuration with these cyclic nucleotide modulators or
IVM with FSH significantly improved oocyte developmental
competence and reduced spindle abnormalities compared to
spontaneous IVM (no treatment); however, these two treatments
in combination endowed even greater developmental compe-
tence (improved subsequent blastocyst rates and quality; P ,
0.05), albeit blastocyst yield and quality remained significantly
lower than that of oocytes matured in vivo. A significant additive
effect of combined IVM treatments was evident as increased
COC lactate production and oxygen consumption and enhanced
oocyte oxidative metabolism, ATP production, ATP:ADP ratio,
1
Supported by Cook Medical with the Cook Medical Adelaide
Fellowship awarded to H.-T.Z. and Cook Medical collaborative
research grants awarded to The University of Adelaide and the Free
University of Brussels. This work was also supported by Australian
National Health and Medical Research Council (NHMRC) Project
Grants awarded to R.B.G. and J.E.J.S. (APP1007551) and J.G.T., R.B.G.,
and M.S.M. (APP1008137), NHMRC Senior Research Fellowships
(APP1023210, APP627007), the National Natural Science Foundation
of China (30901605, 81000248), and by the Belgian Institute for the
Promotion of Innovation in Science and Industry (IWT-070719).
2
Correspondence: Jeremy Thompson, School of Paediatrics and
Reproductive Health, Faculty of Health Sciences, The University of
Adelaide, Level 2, Medical School South, Frome Road, Adelaide, SA
5005, Australia. E-mail: jeremy.thompson@adelaide.edu.au
Received: 9 February 2014.
First decision: 17 March 2014.
Accepted: 13 June 2014.
Ó 2014 by the Society for the Study of Reproduction, Inc.
eISSN: 1529-7268 http://www.biolreprod.org
ISSN: 0006-3363
1 Article 47
reactive oxygen species production, particularly as a conse-
quence of FSH addition, relative to in vivo matured oocytes. In
conclusion, improvements in the embryo yield following IVM is
associated with increased COC oxygen consumption and oocyte
oxidative metabolism, but these remain metabolically and
developmentally less competent relative to in vivo derived
oocytes.
ATP, cAMP, FSH, glycolysis, IVM, mitochondria, oocyte
metabolism
INTRODUCTION
In vitro maturation (IVM) of mammalian oocytes is an
important assisted reproductive technology and research tool in
reproductive and developmental biology. IVM is widely used
to generate mature oocytes for a range of applications,
including human infertility treatment, embryo production for
livestock artificial breeding programs, and cloning, stem cell,
and transgenic technologies [1]. Despite its clinical and
financial advantages over the commonly used reproductive
technique of in vitro fertilization (IVF), a major drawback of
IVM is its significantly lower efficiency compared with
conventional IVF, including decreased preimplantation embryo
development, pregnancy, and live birth rates [2–4].
Over the past decade, new insights have emerged
surrounding the intricate bidirectional communication axes
between the oocyte, follicular somatic cells, and the endocrine
system. These new insights include: oocyte regulation of
granulosa and cumulus cell differentiation and function
through the secretion of soluble paracrine growth factors [5];
identification of the significant role epidermal growth factor
(EGF)-like peptides, the EGF receptor, and their intracellular
second messengers play in mediating the ovulatory luteinizing
hormone (LH) signal through the follicle [6]; and new
perspectives on the participation of cyclic nucleotides,
phosphodiesterases, and gap-junctions in the regulation of
oocyte meiotic maturation [7–10].
The second messenger cyclic adenosine monophosphate
(cAMP) plays an important role in the control of meiotic
progression in mammalian, and some invertebrate, oocytes
[11–13]. Cyclic AMP is synthesized within the oocyte and
additionally by the mural granulosa and cumulus cells of the
follicle and is supplied to the oocyte via gap-junctions [14–17].
 ZENG ET AL.
Within the oocyte, cAMP both inhibits and induces oocyte
meiotic maturation. A moderate concentration of cAMP is
maintained within the oocyte to maintain meiotic arrest;
however, the ovulatory LH surge induces an acute and
transient increase in oocyte cAMP that triggers meiotic
resumption [18, 19]. In contrast, oocytes that are removed
from their antral follicle and matured in vitro exhibit a rapid
drop in cAMP and cyclic guanosine monophosphate (cGMP),
which leads to activation of the oocyte phosphodiesterase 3
(PDE3), a potent cAMP hydrolyzing enzyme [7, 10, 19]. This
loss of cyclic nucleotides leads to protein kinase A inactivation
within the oocyte, which culminates in meiotic resumption [7,
10]. IVM cumulus-oocyte complexes (COCs) are known to
suffer a significant loss in cAMP and, as such, the oocytes
undergo spontaneous maturation and consequently exhibit
decreased developmental competence [19]. It is well docu-
mented that the artificial modulation of cAMP within IVM
COCs through the use of pharmacological agents enhances
oocyte developmental competence as evidenced by improved
subsequent developmental outcomes, including embryo and
fetal development [12, 19–29]. Prevention of cAMP degrada-
tion in IVM COCs increases oocyte developmental competence
by prolonging gap-junctional communication and hence the
exchange of regulatory molecules and metabolites between the
oocyte and its surrounding cumulus cells [12, 19, 25, 30, 31].
Cyclic AMP modulation during IVM has been achieved
through the use of nonspecific PDE inhibitors such as 3-
isobutyl-1-methylxanthine (IBMX), which also prevents the
hydrolysis of cGMP, or agents that increase COC cAMP such
as dibutyryl cAMP or forskolin (an adenylate cyclase activator
that stimulates cAMP synthesis) [12, 19–21, 29, 32, 33]. We
have developed a new IVM system that modulates cAMP prior
to maturation [12, 19]. This system involves prematuration of
COCs with IBMX and forskolin to generate a large (.100-
fold) and rapid increase in specifically cAMP levels (whilst
simultaneously maintaining cGMP levels) that mimics the
spike seen in vivo in response to the ovulatory LH surge [14,
18, 19, 34]. Importantly, this prevents spontaneous meiotic
resumption and slows the associated loss of oocyte-cumulus
gap-junctional communication during maturation.
Metabolism of the mature COC is a dynamic process during
oocyte maturation, with bovine COCs consuming 2-fold more
glucose, oxygen, and pyruvate than immature COCs [35, 36].
COCs preferentially use glucose as an energy substrate [37].
Oocytes have a poor capacity for glucose uptake and hence rely
on glucose metabolism by the cumulus cells, which provide the
oocyte with energy substrates such as pyruvate and lactate via
gap-junctions [38]. Hence, the premature cessation of gap-
junctional communication that occurs in standard IVM COCs
likely affects its metabolic activity; there is abundant evidence
that COC metabolism is an important contributing factor to
oocyte developmental competence. Glucose is predominantly
metabolized via glycolysis for ATP synthesis and the
production of metabolites including pyruvate and lactate [37,
39]; pyruvate is involved in ATP synthesis as it is metabolized
via the tricarboxylic acid (TCA) cycle that is coupled with
mitochondrial oxidative phosphorylation [40].
FSH is routinely used during IVM because it improves
oocyte developmental competence relative to spontaneous
maturation [41] and stimulates cumulus expansion [42]. FSH
has a major impact on metabolism of cumulus cells during
IVM by acting on several metabolic pathways. A series of
studies detailing the influence of energy substrates on oocyte
maturation have shown that FSH induction of meiotic
maturation, but not spontaneous maturation, requires the
presence of glucose [43–45]. FSH is known to stimulate both
2 Article 47
the glycolytic and hexosamine biosynthesis metabolic path-
ways [38], and conditions that trigger meiotic maturation are
associated with an increased flux through the pentose
phosphate pathway [46].
A comprehensive examination of COC and oocyte metab-
olism in mouse COCs during IVM in comparison to in vivo
matured oocytes has yet to be conducted. This study aims to
remedy this by examining extensively the metabolic profile of
COCs and oocytes in addition to the in vitro developmental
outcomes of in vitro matured oocytes. The study included IVM
conditions that combined prematuration of COCs with IBMX
and forskolin to modulate cAMP/cGMP levels as well as FSH
during IVM, and compared many of the measured parameters
within in vivo matured oocytes. Furthermore, oocyte meiotic
stability (correct pairing, recombination, and segregation of
chromosomes) and cumulus expansion were also examined.
We hypothesized that the metabolic profile of COCs and
oocytes will alter as a consequence of treatments known to
enhance developmental competence.
MATERIALS AND METHODS
Mice were maintained in accordance with the Australian Code of Practice
for the Care and Use of Animals for Scientific Purposes and with the approval
Downloaded from www.biolreprod.org.
of the University of Adelaide Animal Ethics Committee. Unless otherwise
stated, all the reagents used were purchased from Sigma Aldrich.
Oocyte Collection and Culture
Immature female 129/Sv inbred mice (21–24 days of age) were used and
housed in a temperature- and light-controlled environment. For IVM, immature
COCs were collected 46 h after administration of 5 international units of equine
chorionic gonadotrophin (eCG) (Folligon; Intervet). For in vivo oocyte
maturation, 5 international units of human chorionic gonadotrophin (hCG)
(Organon) was administered 46 h following eCG priming, and COCs were
collected 16 h later. COCs were freed from antral follicles into HEPES-buffered
alpha minimal essential medium (aMEM) (12000-022; Invitrogen) supple-
mented with 3 mg/ml bovine serum albumin (BSA) (ICP Biologica) and their
respective treatments. Treatment groups were: 1) IVM without FSH
(spontaneous maturation); 2) IVM with FSH (Puregon); 3) prematuration with
cAMP-elevating agents (defined below) þ IVM without FSH; 4) prematuration
with cAMP-elevating agents þ IVM with FSH. The 1 h prematuration phase
(termed pre-IVM treatment) involved incubation of COCs in HEPES-aMEM
6 the adenylate cyclase activator forskolin (50 lM) and the PDE inhibitor
IBMX (50 lM) at 378C for 1 h in atmospheric O2. At the end of the pre-IVM
phase, COCs were washed twice in bicarbonate-buffered aMEM containing 3
mg/ml fatty acid-free (FAF) BSA, 1 mg/ml fetuin, and their respective IVM
treatments, and transferred to drops of the same medium and cultured for 16 h
at 378C with 5% CO2 in air. Data presented represents the mean of four
replicate experiments.
Cumulus Expansion Assessment
IVM COCs were cultured for 16 h in media containing BSA. In vivo COCs
were collect 16 h post-hCG administration. Blinded scoring by an experienced
assessor of the cumulus expansion index was then performed using the reported
scoring system [47]. At least 109 COCs were assessed per treatment group over
four replicate experiments.
IVF and Embryo Culture
The effect of treatments on oocyte developmental competence was assessed
by examining the capacity of the oocyte to support preimplantation embryo
development following IVM. IVF and embryo culture were performed as
previously described [19]. All the media used were from the Vitro Research
Media series generously donated by Cook Medical (Brisbane, Australia). Data
presented represent the mean of four replicate experiments. At least 193
oocytes were used per treatment group over three replicate experiments.
Blastocyst Differential Staining
On Day 6 of embryo culture, blastocysts and hatching blastocysts were
differentially stained for inner cell mass (ICM) and trophectoderm (TE) cells as
 EFFECT OF cAMP AND FSH ON IVM COC METABOLISM
previously described by Hardy et al. [48]. Briefly, zona pellucidae were
dissolved by incubation in 0.5% pronase at 378C. Blastocysts were then
transferred to 10 mM tri-nitrobenzenesulfonic acid for 10 min at 48C, followed
by incubation in 0.1 mg/ml of anti-dinitrophenyl-BSA for 10 min at 378C, then
5 min at 378C in guinea pig serum containing 10 lg/ml of propidium iodide.
Blastocysts were then stained overnight in 6 lg/ml of bisbenzimide in ethanol
at 48C. Blastocysts were washed in 100% ethanol and mounted on siliconized
slides in glycerol drops. The number of pink (i.e., TE) and blue (i.e., ICM)
fluorescent cells was assessed, blinded to treatment group, using a fluorescent
microscope and Hg lamp, with an ultraviolet excitation wavelength dichroic
mirror at 4003 magnification (TE 2000-E; Nikon; excitation 340–380 nm,
emission 440–480 nm). At least 29 blastocysts were stained per treatment
group over three replicate experiments.
Immunofluorescent Microscopy
For staining of a-tubulin, oocytes were fixed in 4% paraformaldehyde in
PBS (pH 7.4) for at least 30 min at room temperature. Following
permeabilization with 0.5% Triton X-100 at room temperature for 20 min,
oocytes were blocked in 1% BSA-supplemented PBS for 1 h and incubated
overnight at 48C with 1:200 anti-a-tubulin antibody. Following three washes in
PBS containing 0.1% Tween 20 and 0.01% Triton X-100 for 5 min each,
oocytes were incubated with 1:100 fluorescein isothiocyanate-conjugated
immunoglobulin G for 1 h at room temperature. Oocytes were then washed in
PBS containing 0.1% Tween 20 and 0.01% Triton X-100 and were costained
with Hoechst 33258 (10 lg/ml in PBS). Oocytes were then mounted on glass
slides and examined using a FluoView FV10i Confocal Microscope
(Olympus). At least 55 oocytes were examined per treatment group over three
replicate experiments. Spindle morphology was classified as normal when there
was a barrel-shaped structure with slightly elongated poles, organized
microtubules, and the chromosomes were arranged in a compact metaphase
plate at the equator of the spindle. Spindle structure was recorded as abnormal
when there was a reduction in the longitudinal dimension of the spindle,
complete absence or remnant of dispersing spindle, or chromosomes displaced
from the plane of the metaphase plate.
Glucose and Lactate Quantification
Following 16 h of IVM, spent COC culture media were collected, snap-
frozen in liquid nitrogen, and stored at 808C until analysis. Glucose and
lactate concentrations in 50 ll of spent media samples (derived from 15 COCs
cultured in 100 ll media drops) were measured using a Hitachi 912 chemical
analyzer (F. Hoffmann-La Roche Ltd.). A minimum of 15 media samples were
measured per treatment group over three replicate experiments. Glucose
consumption and lactate production are expressed as pmol/COC/h.
Oxygen Consumption Assay
COC oxygen consumption was assayed from 0–4 h of IVM as previously
described [49]. This method is based on the fluorescent properties of pyrene, an
oil-soluble compound that is excited at 340 nm and whose fluorescence
dissipates in a linear fashion in the presence of increasing O2 concentration.
Five microliter PCR-grade micropipettes (Drummond Scientific) and stainless
steel plungers were used. One microliter of 1 mM pyrene, dissolved in mineral
oil, was drawn into the micropipette, followed by 2 ll of HEPES-buffered
maturation media containing five COCs. An airtight seal was made at the open
end of the chamber, and the plunger was fixed using sealing wax. Negative
control (0% O2) chambers were constructed in a similar manner, but with 0.1 M
sodium L-ascorbate and sodium hydroxide in HEPES-buffered maturation
media (equilibrated overnight to remove O2) replacing the media; positive
controls (20% O2) were constructed with media alone. The fluorescence
emission of pyrene at the pyrene-media and the pyrene-plunger interfaces were
measured using a fluorophotometric-inverted microscope (Leica). Fluorescence
measurements were taken at intervals of 30 min over a 4 h period. Chambers
were maintained at 378C between measurements. The amount of oxygen
consumed by each COC was determined assuming a linear relation between
fluorescence and oxygen concentration and then using a mathematical model
that describes the diffusion gradient of O2 in mineral oil as a function of O2
depletion in the adjacent medium containing the COCs [50]. Three replicate
experiments were conducted representing five to eight samples per treatment
group per replicate experiment.
ATP/ADP Assay
To determine ATP and ADP levels within MII oocytes, COCs were
denuded of cumulus cells following 16 h IVM. For each sample preparation, 10
3 Article 47
oocytes from each treatment were collected in 10 ll of ice-cold distilled water
and snap-frozen and stored at 808C until analysis. ATP and ADP content in
oocytes was measured using the bioluminescence ApoSENSOR ATP Assay Kit
(BioVision) according to the manufacturer’s instructions. A duplicated five-
point ATP (Roche Diagnostics) standard curve (0, 0.5, 1, 2, and 3 lM) was
used to calculate sample concentrations. ATP and ADP were measured in
COCs from three replicates with at least five samples per treatment group per
replicate experiment.
Autofluorescence Detection of Reduced Nicotinamide
Adenine Dinucleotide (Phosphate) and Oxidized Flavin
Adenine Dinucleotide
After 16 h of culture, live intact COCs were denuded and oocytes were
transferred on to glass-bottom confocal dishes (Cell E&G) in 5 ll of
VitroWash medium containing 3 mg/ml FAF-BSA and their respective
treatments, and overlaid with mineral oil. The fluorescent intensities of
oxidized flavin adenine dinucleotide (FAD) and reduced nicotinamide
adenine dinucleotide (phosphate) (NAD[P]H) were determined using green
(excitation, 473 nm; emission, 490–590 nm) and blue (excitation, 405 nm;
emission, 420–520 nm) filters, respectively [51]. Images were captured at 903
magnification using a FluoView FV10i confocal microscope (Olympus);
microscope and image settings were kept constant across replicate
experiments. Mean fluorescence intensities within the images of the oocytes
were quantified using Image J software (National Institutes of Health).
Fluorescence measurements were performed on at least five oocytes per
Downloaded from www.biolreprod.org.
treatment group over three replicate experiments.
JC-1 Staining
The mitochondrial membrane potential sensitive fluorescent dye, JC-1
(Molecular Probes), was used to measure the activity of oocyte mitochondria
[52]. Following 16 h IVM, oocytes were denuded and incubated for 25 min in
HEPES-buffered aMEM containing their respective treatments and 2 lM JC-1
dye. JC-1 exhibits potential-dependent accumulation in mitochondria where it
aggregates as J-aggregates in high-polarized mitochondria (dWm  140 mV)
that fluoresce red. JC-1 accumulates as multimers in low-polarized mitochon-
dria (dWm  100 mV) that fluoresce green. The red:green fluorescence
intensity ratio is used as an indicator of relative mitochondrial polarization, and
thus mitochondrial activity, whereby an increased ratio indicates increased
mitochondrial activity [53]. Oocyte fluorescence was observed using a narrow
green filter (490–540 nm) and a narrow red filter (570–620nm), using a
FluoView FV10i Confocal Microscope. Laser power and photomultiplier
settings were kept constant for all the experiments. A single optical scan
through the center of the oocyte was used for the analysis. The images were
processed, and fluorescence intensities in oocytes were measured and analyzed
using the accompanying software of the FluoView FV10i Confocal
Microscope. Three replicate experiments were performed using 10–20 oocytes
per treatment group per replicate.
Reduced Glutathione Measurement
A membrane-permeable fluorescence probe, monochlorobimane (MCB)
(Molecular Probes), is used as a sensitive and specific probe to analyze
intracellular reduced glutathione (GSH) content in living cells [54, 55].
Following 16 h IVM, oocytes were denuded and exposed to 100 lM MCB in
VitroWash medium containing 3 mg/ml FAF-BSA and their respective
treatments for 30 min. Excitation and emission wavelengths of 458 and 475
nm, respectively, were used to detect the fluorescent GSH-bimane adduct.
Laser power and photomultiplier settings were kept constant for all the
experiments. A single optical scan through the center of the oocyte was used for
the analysis. Images were processed, and oocyte fluorescence intensities were
measured and analyzed using the accompanying software of the FluoView
FV10i Confocal Microscope. Three replicate experiments were performed
using 10–20 oocytes per treatment group per replicate.
Measurement of Intracellular Reactive Oxygen Species
Intracellular reactive oxygen species (ROS) production was measured using
2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), a nonfluorescent probe
for intracellular ROS detection, which is converted to the fluorescent
compound 2,7-dichlorofluorescein when oxidized by intracellular ROS [56].
This probe is highly reactive with hydrogen peroxide and has been used in
evaluating ROS generation in mammalian cells [57]. Following 16 h IVM,
oocytes were denuded and exposed to 10 lM DCFH-DA in VitroWash media
containing 3 mg/ml of FAF-BSA and their respective treatments for 30 min.
 ZENG ET AL.
Oocyte fluorescence intensity was measured at excitation and emission
wavelengths of 500 and 529 nm, respectively. The arbitrary units, relative
fluorescence units, were based directly on fluorescence intensity. Three
replicate experiments were performed where five oocytes were examined per
treatment group per replicate.
Statistical Analyses
Statistical analyses were conducted using the Statistical Package for Social
Sciences 18.0 (SPSS). Treatment effects on spindle morphology were assessed
by chi-square test. For all the other data, one-way ANOVA followed by either
Dunnett or Bonferroni multiple-comparison post hoc tests were used to identify
individual differences between means. Developmental data (including
proportion of MII oocytes) was transformed by arcsine prior to ANOVA. All
the values are presented as means with their corresponding standard error of the
mean (SEM). P , 0.05 was considered statistically significant.
RESULTS
Combined Pre-IVM Treatment and IVM with FSH Promoted
the Greatest Cumulus Expansion
As expected, cumulus expansion did not occur in the
absence of pre-IVM treatment or FSH during IVM (Fig. 1, A
and B). Pre-IVM treatment with forskolin and IBMX
significantly increased cumulus expansion compared to control
(1.07 6 0.02 vs. 2.06 6 0.07, respectively; P , 0.05),
however cumulus was significantly less expanded than with
standard IVM with FSH (3.58 6 0.06, P , 0.05). Interestingly,
pre-IVM treatment and FSH during IVM had a significant
interactive impact on cumulus expansion whereby expansion
was significantly greater than with either treatment alone and
was statistically similar to in vivo (3.81 6 0.04, P , 0.05).
As well as deficient cumulus expansion, a significant
reduction in first polar body extrusion (Fig. 1C) and an increase
in abnormal spindle structure (Fig, 1, D and E) was observed in
the absence of pre-IVM treatment or FSH during IVM
following 16 h IVM (P , 0.05). No significant differences
in the levels of polar body extrusion and abnormal spindle rate
were observed between the pre-IVM treatment and/or IVM
with FSH (P . 0.05).
Combined Pre-IVM Treatment and FSH During IVM Yielded
the Greatest Subsequent Blastocyst Yield and Quality
The effects of either pre-IVM treatment and/or IVM with
FSH on subsequent embryo development were determined.
Fertilization, cleavage, blastocyst, and hatching blastocyst rates
yielded from in vivo matured COCs were significantly higher
than from any IVM treatment (Fig. 2; P , 0.05). Unsurpris-
ingly, COCs matured spontaneously (no FSH or pre-IVM
treatment), yielded the lowest fertilization, cleavage, blastocyst,
and hatching blastocyst rates of all the treatments (P , 0.05).
IVM COCs matured with FSH IVM 6 pre-IVM treatment
yielded the second highest fertilization and cleavage rates,
which were significantly higher than those derived from COCs
matured in the absence of FSH (P , 0.05). Pre-IVM treatment
significantly increased fertilization and cleavage rates com-
pared to control (spontaneous IVM) regardless of the presence
of FSH during IVM (P ,0.05). Most notably, combined pre-
IVM treatment þ FSH IVM yielded significantly higher
blastocyst and hatching blastocyst rates compared to IVM with
FSH (no pre-IVM) (P , 0.05; Fig. 2, C and D), although the
rates were not as high as those from in vivo matured COCs.
Blastocysts derived from COCs subjected to combined pre-
IVM treatment þ FSH IVM were also of greater quality than
those from COCs subjected to only one treatment or no
treatment. This is evidenced by significantly higher numbers of
ICM cells and a higher proportion of ICM (Fig. 3; P , 0.05).
4 Article 47
However, despite this, blastocyst cell numbers from combined
pre-IVM treatment þ FSH IVM COCs remained significantly
lower than those from blastocysts from in vivo matured COCs
(P , 0.05).
FSH Exhibited a More Profound Effect on Glucose
Metabolism than Pre-IVM Treatment
Glucose is the preferred energy substrate of COCs during
maturation with a large proportion of consumed glucose
metabolized via glycolysis, producing lactate. Hence, glucose
consumption and lactate production of IVM COCs subjected to
pre-IVM treatment and/or IVM with FSH were measured
following 16 h of IVM. As expected, COC glucose
consumption and lactate production were significantly affected
by FSH during IVM with levels significantly higher than in all
the treatments devoid of FSH. Intriguingly, pre-IVM treatment
did not affect glucose consumption but significantly (P , 0.05)
increased lactate production compared to no treatment
(spontaneous maturation) (Fig. 4B), with the highest lactate
production occurring within the combined prematuration
treatment and maturation with FSH. One possible source of
this additional lactate is the cytoplasmic conversion of
Downloaded from www.biolreprod.org.
glycolysis-derived pyruvate to lactate to regenerate oxidized
NAD þ and maintain the cytoplasmic redox state. Alternatively,
aMEM medium contains 1 mM pyruvate, which may also
participate in this reaction.
Combined Pre-IVM Treatment and IVM with FSH Yielded
Greater Oocyte Energy Production and Oxidative
Metabolism than Individual Treatment
The ATP and ADP levels were measured in denuded
oocytes that were either matured in vivo or cultured as COCs
for 16 h 6 FSH 6 pre-IVM treatment (Fig. 5, A and B).
Oocyte ATP levels were similar across all the treatment groups
with the exception of the combined pre-IVM treatment þ FSH
IVM group, which exhibited significantly higher ATP level
than the those without a pre-IVM treatment (P , 0.05; Fig.
5A). Corresponding ADP levels yielded by this combined
treatment group were lowest amongst all the groups and
significantly lower than both FSH treatment alone and in vivo
derived COCs (P , 0.05; Fig. 5B). A significant increase (P ,
0.05) in the ratio of ATP:ADP, a measure of energy demand,
was higher in the combined pre-IVM treatment þ FSH IVM
group than all the other IVM treatment groups (P , 0.05)
except for the pre-IVM treatment alone (Fig. 5C).
Oxygen consumption by intact IVM COCs was measured
over 0–4 h. FSH, but not pre-IVM treatment, enhanced oxygen
consumption (Fig. 5D). A significant interaction between pre-
IVM treatment and IVM with FSH was found in regards to
oxygen consumption (P , 0.05) with the combined pre-IVM
treatment and FSH IVM stimulating more consumption than
any other treatment.
Oocyte mitochondrial activity, as measured by red:green
fluorescence JC-1 staining, was significantly higher in oocytes
matured in vivo compared with all the IVM treatments (Fig. 5,
E and F). Amongst IVM groups, FSH significantly up-
regulated mitochondrial activity compared with control both
in the presence or absence of pre-IVM treatment.
Intraoocyte Redox State Was Altered During IVM
In order to evaluate the reduction-oxidation of IVM oocytes,
the antioxidant GSH was measured using MCB, which forms a
fluorescent adduct following an enzyme-catalyzed reaction
 EFFECT OF cAMP AND FSH ON IVM COC METABOLISM

Downloaded from www.biolreprod.org.

FIG. 1. Effect of pre-IVM with forskolin and IBMX, and follicle stimulation hormone (FSH) exposure during IVM, on COCs maturation. Following 16 h
IVM or in vivo maturation, cumulus expansion (A and B) was assessed. IVM oocytes were then denuded and fixed for detection of oocyte maturation rate
(C) and spindle morphology of mature oocyte (D and E). Data represent the mean 6 SEM of three independent experiments: cumulus expansion rate (A)
and percentage of MII oocytes (C). Differences in percentage of abnormal spindles (D) was determined by chi-square analysis. Bars not sharing a common
letter are significantly different (P , 0.05). Original magnification 310 (B) and 390 (E).

with GSH [58]. Individually, pre-IVM treatment and FSH IVM Mitochondrial and cytoplasmic oocyte redox state was
significantly increased oocyte MCB fluorescence compared to determined by measurement of the autofluorescence of two
control (P , 0.05), however, a significant interaction between endogenous fluorophores, FAD (mitochondrial localization)
pre-IVM treatment and FSH IVM was observed (P , 0.05), and NAD(P)H (mitochondrial and cytoplasmic localization)
which yielded significantly higher MCB fluorescence than following 16 h post-hCG administration or 16 h IVM [59, 60].
either treatment alone and was statistically similar to that of FAD fluorescence was highest in the FSH IVM group and
oocytes matured in vivo (Fig. 6A). lowest in vivo (Fig. 6, B and F; P , 0.05). NAD(P)H
5 Article 47
 ZENG ET AL.

Downloaded from www.biolreprod.org.

FIG. 2. Effect of pre-IVM with forskolin and IBMX, and follicle stimulation hormone (FSH) exposure during IVM, on embryo development. In vitro
fertilization (A), Day 2 cleavage rate (B), Day 6 blastocyst rates (blastocysts per cleaved embryos) (C), and Day 6 hatching blastocyst rates (hatching
blastocysts per cleaved embryos) (D) were measured following 16 h of IVM or 16 h following hCG. Data represent the mean 6 SEM of three independent
experiments representing at least 40 oocytes per replicate. Bars not sharing a common letter are significantly different (P , 0.05).

autofluorescence of in vivo matured oocytes was significantly
lower than that of all IVM treatments (Fig. 6, C and F; P ,
0.05). IVM treatments recorded similar NAD(P)H autofluores-
cence except for combined pre-IVM treatment þ FSH IVM,
which stimulated significantly lower fluorescence than control
and FSH IVM groups (P , 0.05). FSH stimulated the greatest
FAD:NAD(P)H ratio, which was significantly higher than
control and in vivo groups (Fig. 6, D and F; P , 0.05). DCFH-
DA fluorescence, indicative of ROS production, in vivo and
during IVM in the absence of FSH was similar; FSH 6 pre-
IVM treatment significantly increased DCFH-DA fluorescence
compared to in vivo maturation (Fig, 6, E and F; P , 0.05).
DISCUSSION
We and others have previously reported that modulation of
cAMP levels within mammalian COCs during IVM can
substantially improve oocyte developmental competence in
several species, including human [12, 19–29]. Our laboratory
recently described a combinational IVM system, termed
simulated physiological oocyte maturation (SPOM) [19], that
involves COC prematuration with the general PDE inhibitor
6 Article 47
IBMX and the adenylate cyclase activator forskolin followed
by a prolonged IVM phase in the presence of FSH and the
PDE3-specific inhibitor cilostamide. SPOM generated in-
creased embryo yield from cattle and mouse oocytes and
improved fetal survival following mouse embryo transfer [19].
The individual contributions of components of the SPOM
system on developmental competence and other aspects of
COC biochemistry, however, are yet to be described, although
in a follow-up study we demonstrated that the pre-IVM phase
alone improves oocyte developmental competence via mech-
anisms related to oocyte energy metabolism [20]. This study
further examines the impact of the IBMX/forskolin prematura-
tion and its relationship with FSH during maturation, on oocyte
developmental competence, meiotic stability, and metabolic
impact, and compares most measures to in vivo matured
oocytes. As glucose uptake, lactate production, and O2
consumption measurements are dependent on differences in
concentration over a specific incubation period, it was not
feasible to conduct these with in vivo matured oocytes.
The LH surge in vivo and FSH in vitro activate the Gs
proteins on granulosa and cumulus cells, which then stimulate
 EFFECT OF cAMP AND FSH ON IVM COC METABOLISM

Downloaded from www.biolreprod.org.

FIG. 3. Effect of pre-IVM with forskolin and IBMX, and follicle stimulation hormone (FSH) exposure during IVM, on embryo quality. Embryo quality at
Day 6 was examined by evaluation of the number of inner cell mass (ICM) cells (A), trophectoderm (TE) cells (B), total cells (C), and the ratio of cell
number of ICM to total blastocyst cells (D). Data represent the mean 6 SEM of three independent experiments with at least 193 embryos examined per
treatment group. Bars not sharing a common letter are significantly different (P , 0.05).

membrane-associated adenylate cyclase to elevate intracellular
cAMP levels [61, 62]. Cyclic AMP further activates protein
kinases to induce cumulus expansion. Both the pre-IVM
treatment and FSH stimulated cumulus expansion; however,
the pre-IVM treatment alone was significantly poorer at
eliciting expansion. Combined pre-IVM treatment with FSH
in IVM led to the greatest IVM cumulus expansion, which was
comparable to that seen in vivo. Although both treatments elicit
an increase in cyclic nucleotides, the differential levels of
cumulus expansion suggest that there are differences in
cumulus expansion stimulation that warrant further investiga-
tion. Fertilization rates across treatments paralleled the degree
of cumulus expansion, which is unsurprising given that
expansion facilitates fertilization [63]. Both pre-IVM treatment
and FSH improved meiotic competence and reduced spindle
abnormalities relative to the control (spontaneous IVM), and
meiotic competence was comparable to that of in vivo matured
oocytes, suggesting that a large pulse of cAMP (and
maintaining cGMP levels) prior to IVM is sufficient to achieve
full meiotic competence.
Our results confirm the benefit of combining the pre-IVM
treatment in conjunction with FSH during maturation [19–21,
7 Article 47
64]. Individually, both pre-IVM treatment and FSH were
equally able to enhance blastocyst development relative to
control. However, in combination, they were able to further
enhance development, albeit not to the capacity of in vivo
matured oocytes; this effect was also evident in blastocyst
quality (as evidenced by ICM and total cell numbers). These
data demonstrate that combined treatment significantly in-
creases oocyte developmental competence over either treat-
ment alone. It is well established that elevated cAMP levels
during IVM enhance and prolongs gap-junctional communi-
cation between the oocyte and cumulus cells [12, 14, 15, 19,
31], and because both these treatments elevate and maintain
cAMP, prolonged gap-junctional communication is a likely
mechanism by which these treatments increase oocyte
developmental competence and alter oocyte metabolism.
Glucose is a pivotal substrate for COC metabolism and can
be metabolized via glycolysis, the pentose phosphate pathway,
and the hexosamine biosynthesis pathway [38]. The glycolytic
pathway accounts for the majority of glucose metabolized by
the COC and facilitates energy production in the form of ATP
as well as the production of metabolites readily utilized by the
oocyte, most notably pyruvate and lactate [65, 66]. In the
 ZENG ET AL.
FIG. 4. Effect of pre-IVM with forskolin and IBMX, and follicle
stimulation hormone (FSH) exposure during IVM, on glucose metabolism.
COC glucose consumption (A) and lactate production (B) were measured
following the first 4 h of IVM. Data represent the mean 6 SEM of three
independent experiments representing at least 15 media samples per
treatment group; different letters at the top of the bars indicate significant
difference (P , 0.05).
bovine COC, glucose uptake and lactate production is reported
to occur approximately in a 1:2 relationship. Furthermore, FSH
stimulates glucose uptake in COCs, but this increase is
associated with up-regulation of the hexosamine biosynthesis
pathway, the pathway that provides the substrate for hyaluronic
acid production [63]. In mouse COCs, this relationship appears
to be markedly different. Glucose consumption and lactate
production does not occur in a 1:2 ratio [67], and prematuration
treatment-stimulated cumulus expansion was not associated
with an increase in glucose consumption (current study). This
suggests that the pre-IVM treatment of COCs with IBMX and
forskolin improved embryo yields by altering the flux of
glucose through glycolysis. Moreover, oxygen consumption by
COCs was increased by both treatments in combination,
demonstrating that this model induces increased demand for
oxidative metabolism and ATP production. Together, these
data point to a much greater dependency of mouse cumulus
cells for oxidative metabolism compared with the bovine COC
and, together with hexosamine biosynthesis metabolic activity,
a significant amount of glucose provides cumulus cells and the
oocyte with metabolic intermediates, including ATP. An
increase in ATP generation would then meet the large demand
8 Article 47
for cAMP synthesis stimulated by the combined prematuration
and FSH treatments.
This study examined several indicators of energy produc-
tion, mitochondrial activity, and redox potential within the
oocyte because the oocyte is highly dependent on oxidative
metabolism for developmental competence [68, 69]. We
demonstrated that ATP levels in oocytes did not vary between
control and treatments with the exception that more ATP was
found in oocytes subjected to combined pre-IVM and FSH
treatment, as previously demonstrated [20]. This pattern was
also observed in ADP levels, leading to a significant shift in the
ATP:ADP ratio by this treatment. An increased ATP:ADP ratio
is indicative of increased energy demand, met through
increased oxidative metabolic activity. Higher ATP levels are
associated with increased developmental potential in oocytes
and embryos [70–73]; our data agrees with this principle, yet it
is noteworthy that the in vivo matured oocyte ATP level was
not different from most of the IVM treatments. The functional
capacity of mitochondria for ATP production was examined by
measuring mitochondrial activity; FSH stimulation was
associated with increased activity, regardless of prematuration.
Unsurprisingly, this result was mirrored by FAD/NAD(P)H
and ROS levels within the oocyte. Nevertheless, in vivo
Downloaded from www.biolreprod.org.
matured oocytes exhibited significantly higher mitochondrial
activity than all the other treatments and lower ROS levels,
suggesting that the efficiency of ATP production is greatest in
these oocytes. These results reveal that the energy production
efficiency and activity of metabolic pathways utilized during
IVM to produce highly competent oocytes remain dissimilar to
their in vivo counterparts. Increasing b-oxidation within IVM
oocytes by the addition of fatty acids and L-carnitine, an
essential cofactor for fatty acid transport into the mitochondria,
may further increase mitochondrial efficiency for ATP
production [74]. An approximation of the redox state of the
mitochondrial matrix space can be determined from the redox
ratio, which refers to the ratio of oxidized FAD to reduced
NAD(P)H [75]. This fluorescence measurement indicates
relative changes in the oocyte’s redox without the use of
exogenous stains or dyes. The redox ratio is sensitive to
changes in the cellular metabolic rate and oxygen supply [75].
An increase in the redox ratio is indicative of an increased
oxidized state [75, 76]. The current study demonstrated that
cellular metabolic activity of the oocyte was greatly altered by
FSH exposure in IVM with an increased oocyte redox ratio and
by a larger ATP:ADP ratio, while oocytes matured in vivo had
a relatively quiet oxidation state, but contained more high
membrane potential mitochondria, as determined by JC-1
fluorescence. This adds to the evidence that treatment strategies
that improve oocyte competence during IVM remain subop-
timal compared with in vivo maturation.
Autofluorescence cannot distinguish NADH and NADPH
because their emission spectra are nearly identical [75]. NADH
is found in both the cytoplasm and the mitochondria and
functions as a reducing agent in numerous metabolic pathways,
such as glycolysis, b-oxidation, the TCA cycle, and oxidative
phosphorylation. NADPH is found in smaller quantities within
the cytoplasm and is reduced within the oxidative phase of the
pentose phosphate pathway and by isocitrate dehydrogenase
within the TCA cycle. An important role for NADPH is the
reduction of oxidized glutathione to GSH. We measured the
intensity of MCB within oocytes after 16 h IVM and observed
significantly higher MCB intensity following either pre-IVM
treatment or FSH. GSH is synthesized largely by cumulus cells
because denuding oocytes greatly reduces intraoocyte GSH
levels [77] and represents an important defense molecule
against oxidative stress [77]. Several studies have demonstrated
 EFFECT OF cAMP AND FSH ON IVM COC METABOLISM

Downloaded from www.biolreprod.org.

FIG. 5. Effect of pre-IVM with forskolin and IBMX, and follicle stimulation hormone (FSH) exposure during IVM, on COC energy metabolism. Oocyte
ATP (A) and ADP (B) content were measured following 16 h of IVM or 16 h following hCG. The ATP:ADP ratio was calculated as an indicator of energy
demand (C). COC oxygen consumption (D) following the first 4 h of IVM was measured. Oocyte mitochondrial activity following 16 h IVM or 16 h
following hCG was evaluated by mitochondrial membrane potential sensitive JC-1 staining (E and F), where a green fluorescence is emitted as JC-1
monomers in low-polarized mitochondria and a red fluorescence as J-aggregates in highly polarized mitochondria. Original magnification 390 (F). Data
represent the mean 6 SEM of three independent experiments representing the mean of at least five samples per replicate experiment. Bars not sharing a
common letter are significantly different (P , 0.05).

FIG. 6. Effect of pre-IVM with forskolin and IBMX, and follicle stimulation hormone (FSH) exposure during IVM, on oocyte redox state. A) The relative
level of monochlorobimane (MCB), a measure of GSH, was evaluated following 16 h IVM or 16 h following hCG. The relative autofluorescence of FAD (B)
and NAD(P)H (C) were also measured. D) The ratio of FAD:NAD(P)H was determined as a measure of redox state. E) Intracellular ROS production was
examined following 16 h IVM or 16 h following hCG using 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), a nonfluorescent probe for intracellular
ROS detection. F) Representative images of oocyte fluorescence for each parameter explored. Original magnification 390. Data represent the mean 6
SEM of three replicate experiments representing at least five oocytes per replicate experiment. Bars not sharing a common letter are significantly different
(P , 0.05).

9 Article 47
 ZENG ET AL.
the benefit of increasing intraoocyte GSH levels on subsequent
development [77–80]. An increase in MCB fluorescence
possibly reflects an increased demand for GSH for protection
from increased ROS levels, especially within FSH-treated
COCs as a result of increased oxidative metabolism. In
contrast, in vivo matured oocytes had comparable levels of
GSH, yet the lowest ROS levels.
In conclusion, we found that oocyte developmental
competence was positively influenced by the combined
presence of prematuration with IBMX/forskolin and FSH
during oocyte IVM, and this was reflected in COC and oocyte
metabolism. Lactate production (hence glycolytic activity) by
COCs measured at the cessation of IVM was stimulated by pre-
IVM treatment prior to IVM, suggesting an effect of
predominantly cAMP production on glycolytic activity. More
significantly, both treatments improved meiotic stability and
enhanced oxidative metabolism and ATP production. Howev-
er, these treatments, especially FSH, increased ROS produc-
tion, revealing that a major difference between in vitro matured
oocytes and those derived in vivo is the efficiency of
mitochondrial function and requirement for ATP production,
demonstrating that further work is needed to bridge the
metabolic gap between these two modes of oocyte maturation.
REFERENCES
1. Smitz JE, Thompson JG, Gilchrist RB. The promise of in vitro maturation
in assisted reproduction and fertility preservation. Semin Reprod Med
2011; 29:24–37.
2. Child TJ, Phillips SJ, Abdul-Jalil AK, Gulekli B, Tan SL. A comparison of
in vitro maturation and in vitro fertilization for women with polycystic
ovaries. Obstet Gynecol 2002; 100:665–670.
3. Gremeau AS, Andreadis N, Fatum M, Craig J, Turner K, McVeigh E,
Child T. In vitro maturation or in vitro fertilization for women with
polycystic ovaries? A case-control study of 194 treatment cycles. Fertil
Steril 2012; 98:355–360.
4. Eppig JJ, O’Brien MJ, Wigglesworth K, Nicholson A, Zhang W, King
BA. Effect of in vitro maturation of mouse oocytes on the health and
lifespan of adult offspring. Hum Reprod 2009; 24:922–928.
5. Gilchrist RB, Lane M, Thompson JG. Oocyte-secreted factors: regulators
of cumulus cell function and oocyte quality. Hum Reprod Update 2008;
14:159–177.
6. Park JY, Su YQ, Ariga M, Law E, Jin SL, Conti M. EGF-like growth
factors as mediators of LH action in the ovulatory follicle. Science 2004;
303:682–684.
7. Vaccari S, Weeks JL II, Hsieh M, Menniti FS, Conti M. Cyclic GMP
signaling is involved in the luteinizing hormone-dependent meiotic
maturation of mouse oocytes. Biol Reprod 2009; 81:595–604.
8. Zhang M, Su YQ, Sugiura K, Xia G, Eppig JJ. Granulosa cell ligand
NPPC and its receptor NPR2 maintain meiotic arrest in mouse oocytes.
Science 2010; 330:366–369.
9. Tsuji T, Kiyosu C, Akiyama K, Kunieda T. CNP/NPR2 signaling
maintains oocyte meiotic arrest in early antral follicles and is suppressed
by EGFR-mediated signaling in preovulatory follicles. Mol Reprod Dev
2012; 79:795–802.
10. Norris RP, Ratzan WJ, Freudzon M, Mehlmann LM, Krall J, Movsesian
MA, Wang H, Ke H, Nikolaev VO, Jaffe LA. Cyclic GMP from the
surrounding somatic cells regulates cyclic AMP and meiosis in the mouse
oocyte. Development 2009; 136:1869–1878.
11. Vaccari S, Horner K, Mehlmann LM, Conti M. Generation of mouse
oocytes defective in cAMP synthesis and degradation: endogenous cyclic
AMP is essential for meiotic arrest. Dev Biol 2008; 316:124–134.
12. Shu YM, Zeng HT, Ren Z, Zhuang GL, Liang XY, Shen HW, Yao SZ, Ke
PQ, Wang NN. Effects of cilostamide and forskolin on the meiotic
resumption and embryonic development of immature human oocytes.
Hum Reprod 2008; 23:504–513.
13. Bilodeau-Goeseels S. Bovine oocyte meiotic inhibition before in vitro
maturation and its value to in vitro embryo production: does it improve
developmental competence? Reprod Domest Anim 2012; 47:687–693.
14. Bornslaeger EA, Schultz RM. Regulation of mouse oocyte maturation:
effect of elevating cumulus cell cAMP on oocyte cAMP levels. Biol
Reprod 1985; 33:698–704.
15. Webb RJ, Marshall F, Swann K, Carroll J. Follicle-stimulating hormone
10
induces a gap junction-dependent dynamic change in [cAMP] and protein
kinase a in mammalian oocytes. Dev Biol 2002; 246:441–454.
16. Dekel N, Lawrence TS, Gilula NB, Beers WH. Modulation of cell-to-cell
communication in the cumulus-oocyte complex and the regulation of
oocyte maturation by LH. Dev Biol 1981; 86:356–362.
17. Horner K, Livera G, Hinckley M, Trinh K, Storm D, Conti M. Rodent
oocytes express an active adenylyl cyclase required for meiotic arrest. Dev
Biol 2003; 258:385–396.
18. Yoshimura Y, Nakamura Y, Oda T, Ando M, Ubukata Y, Karube M,
Koyama N, Yamada H. Induction of meiotic maturation of follicle-
enclosed oocytes of rabbits by a transient increase followed by an abrupt
decrease in cyclic AMP concentration. J Reprod Fertil 1992; 95:803–812.
19. Albuz FK, Sasseville M, Lane M, Armstrong DT, Thompson JG, Gilchrist
RB. Simulated physiological oocyte maturation (SPOM): a novel in vitro
maturation system that substantially improves embryo yield and
pregnancy outcomes. Hum Reprod 2010; 25:2999–3011.
20. Zeng HT, Ren Z, Guzman L, Wang X, Sutton-McDowall ML, Ritter LJ,
De Vos M, Smitz J, Thompson JG, Gilchrist RB. Heparin and cAMP
modulators interact during pre-in vitro maturation to affect mouse and
human oocyte meiosis and developmental competence. Hum Reprod
2013; 28:1536–1545.
21. Luciano AM, Pocar P, Milanesi E, Modina S, Rieger D, Lauria A,
Gandolfi F. Effect of different levels of intracellular cAMP on the in vitro
maturation of cattle oocytes and their subsequent development following
in vitro fertilization. Mol Reprod Dev 1999; 54:86–91.
22. Nogueira D, Albano C, Adriaenssens T, Cortvrindt R, Bourgain C,
Devroey P, Smitz J. Human oocytes reversibly arrested in prophase I by
Downloaded from www.biolreprod.org.
phosphodiesterase type 3 inhibitor in vitro. Biol Reprod 2003; 69:
1042–1052.
23. Nogueira D, Cortvrindt R, De Matos DG, Vanhoutte L, Smitz J. Effect of
phosphodiesterase type 3 inhibitor on developmental competence of
immature mouse oocytes in vitro. Biol Reprod 2003; 69:2045–2052.
24. Nogueira D, Ron-El R, Friedler S, Schachter M, Raziel A, Cortvrindt R,
Smitz J. Meiotic arrest in vitro by phosphodiesterase 3-inhibitor enhances
maturation capacity of human oocytes and allows subsequent embryonic
development. Biol Reprod 2006; 74:177–184.
25. Luciano AM, Modina S, Vassena R, Milanesi E, Lauria A, Gandolfi F.
Role of intracellular cyclic adenosine 3 0 ,5 0 -monophosphate concentration
and oocyte-cumulus cells communications on the acquisition of the
developmental competence during in vitro maturation of bovine oocyte.
Biol Reprod 2004; 70:465–472.
26. Vanhoutte L, Nogueira D, De Sutter P. Prematuration of human denuded
oocytes in a three-dimensional co-culture system: effects on meiosis
progression and developmental competence. Hum Reprod 2009; 24:
658–669.
27. Vanhoutte L, Nogueira D, Dumortier F, De Sutter P. Assessment of a new
in vitro maturation system for mouse and human cumulus-enclosed
oocytes: three-dimensional prematuration culture in the presence of a
phosphodiesterase 3-inhibitor. Hum Reprod 2009; 24:1946–1959.
28. Thomas RE, Thompson JG, Armstrong DT, Gilchrist RB. Effect of
specific phosphodiesterase isoenzyme inhibitors during in vitro maturation
of bovine oocytes on meiotic and developmental capacity. Biol Reprod
2004; 71:1142–1149.
29. Funahashi H, Cantley TC, Day BN. Synchronization of meiosis in porcine
oocytes by exposure to dibutyryl cyclic adenosine monophosphate
improves developmental competence following in vitro fertilization. Biol
Reprod 1997; 57:49–53.
30. Luciano AM, Franciosi F, Modina SC, Lodde V. Gap junction-mediated
communications regulate chromatin remodeling during bovine oocyte
growth and differentiation through cAMP-dependent mechanism(s). Biol
Reprod 2011; 85:1252–1259.
31. Thomas RE, Armstrong DT, Gilchrist RB. Bovine cumulus cell-oocyte
gap junctional communication during in vitro maturation in response to
manipulation of cell-specific cyclic adenosine 3 0 ,5 0 -monophosophate
levels. Biol Reprod 2004; 70:548–556.
32. Rose RD, Gilchrist RB, Kelly JM, Thompson JG, Sutton-McDowall ML.
Regulation of sheep oocyte maturation using cAMP modulators.
Theriogenology 2013; 79:142–148.
33. Guixue Z, Luciano AM, Coenen K, Gandolfi F, Sirard MA. The influence
of cAMP before or during bovine oocyte maturation on embryonic
developmental competence. Theriogenology 2001; 55:1733–1743.
34. Schultz RM, Montgomery RR, Belanoff JR. Regulation of mouse oocyte
meiotic maturation: implication of a decrease in oocyte cAMP and protein
dephosphorylation in commitment to resume meiosis. Dev Biol 1983; 97:
264–273.
35. Sutton ML, Gilchrist RB, Thompson JG. Effects of in-vivo and in-vitro
environments on the metabolism of the cumulus-oocyte complex and its
Article 47
 EFFECT OF cAMP AND FSH ON IVM COC METABOLISM
influence on oocyte developmental capacity. Hum Reprod Update 2003; 9:
35–48.
36. Gutnisky C, Morado S, Dalvit GC, Thompson JG, Cetica PD. Glycolytic
pathway activity: effect on IVM and oxidative metabolism of bovine
oocytes. Reprod Fertil Dev 2013; 25:1026–1035.
37. Donahue RP, Stern S. Follicular cell support of oocyte maturation:
production of pyruvate in vitro. J Reprod Fertil 1968; 17:395–398.
38. Sutton-McDowall ML, Gilchrist RB, Thompson JG. The pivotal role of
glucose metabolism in determining oocyte developmental competence.
Reproduction 2010; 139:685–695.
39. Eppig JJ. Analysis of mouse oogenesis in vitro. Oocyte isolation and the
utilization of exogenous energy sources by growing oocytes. J Exp Zool
1976; 198:375–382.
40. Steeves TE, Gardner DK. Metabolism of glucose, pyruvate, and glutamine
during the maturation of oocytes derived from pre-pubertal and adult
cows. Mol Reprod Dev 1999; 54:92–101.
41. Izadyar F, Zeinstra E, Bevers MM. Follicle-stimulating hormone and
growth hormone act differently on nuclear maturation while both enhance
developmental competence of in vitro matured bovine oocytes. Mol
Reprod Dev 1998; 51:339–345.
42. Eppig JJ. Gonadotropin stimulation of the expansion of cumulus oophori
isolated from mice: general conditions for expansion in vitro. J Exp Zool
1979; 208:111–120.
43. Downs SM, Humpherson PG, Martin KL, Leese HJ. Glucose utilization
during gonadotropin-induced meiotic maturation in cumulus cell-enclosed
mouse oocytes. Mol Reprod Dev 1996; 44:121–131.
44. Downs SM, Mastropolo AM. The participation of energy substrates in the
control of meiotic maturation in murine oocytes. Dev Biol 1994; 162:
154–168.
45. Fagbohun CF, Downs SM. Requirement for glucose in ligand-stimulated
meiotic maturation of cumulus cell-enclosed mouse oocytes. J Reprod
Fertil 1992; 96:681–697.
46. Downs SM, Humpherson PG, Leese HJ. Meiotic induction in cumulus
cell-enclosed mouse oocytes: involvement of the pentose phosphate
pathway. Biol Reprod 1998; 58:1084–1094.
47. Vanderhyden BC, Caron PJ, Buccione R, Eppig JJ. Developmental pattern
of the secretion of cumulus expansion-enabling factor by mouse oocytes
and the role of oocytes in promoting granulosa cell differentiation. Dev
Biol 1990; 140:307–317.
48. Hardy K, Handyside AH, Winston RM. The human blastocyst: cell
number, death and allocation during late preimplantation development in
vitro. Development 1989; 107:597–604.
49. Sutton ML, Cetica PD, Beconi MT, Kind KL, Gilchrist RB, Thompson
JG. Influence of oocyte-secreted factors and culture duration on the
metabolic activity of bovine cumulus cell complexes. Reproduction 2003;
126:27–34.
50. Stokes YM. Quantifying oxygen diffusion in paraffin oil used in oocyte
and embryo culture. Mol Reprod Dev 2009; 76:1178–1187.
51. Aubin JE. Autofluorescence of viable cultured mammalian cells. J
Histochem Cytochem 1979; 27:36–43.
52. Reers M, Smiley ST, Mottola-Hartshorn C, Chen A, Lin M, Chen LB.
Mitochondrial membrane potential monitored by JC-1 dye. Methods
Enzymol 1995; 260:406–417.
53. Smiley ST, Reers M, Mottola-Hartshorn C, Lin M, Chen A, Smith TW,
Steele GD Jr, Chen LB. Intracellular heterogeneity in mitochondrial
membrane potentials revealed by a J-aggregate-forming lipophilic cation
JC-1. Proc Natl Acad Sci U S A 1991; 88:3671–3675.
54. Kamencic H, Lyon A, Paterson PG, Juurlink BH. Monochlorobimane
fluorometric method to measure tissue glutathione. Anal Biochem 2000;
286:35–37.
55. Fernandez-Checa JC, Kaplowitz N. The use of monochlorobimane to
determine hepatic GSH levels and synthesis. Anal Biochem 1990; 190:
212–219.
56. Rosenkranz AR, Schmaldienst S, Stuhlmeier KM, Chen W, Knapp W,
Zlabinger GJ. A microplate assay for the detection of oxidative products
using 2 0 ,7 0 -dichlorofluorescin-diacetate. J Immunol Methods 1992; 156:
39–45.
57. Hagar H, Ueda N, Shah SV. Role of reactive oxygen metabolites in DNA
damage and cell death in chemical hypoxic injury to LLC-PK1 cells. Am J
Physiol 1996; 271:F209–F215.
58. Igosheva N, Abramov AY, Poston L, Eckert JJ, Fleming TP, Duchen MR,
McConnell J. Maternal diet-induced obesity alters mitochondrial activity
and redox status in mouse oocytes and zygotes. PLoS One 2010; 5:
e10074.
59. Sutton-McDowall ML, Mottershead DG, Gardner DK, Gilchrist RB,
11
Thompson JG. Metabolic differences in bovine cumulus-oocyte
complexes matured in vitro in the presence or absence of follicle-
stimulating hormone and bone morphogenetic protein 15. Biol Reprod
2012; 87:87.
60. Chance B, Schoener B, Oshino R, Itshak F, Nakase Y. Oxidation-
reduction ratio studies of mitochondria in freeze-trapped samples. NADH
and flavoprotein fluorescence signals. J Biol Chem 1979; 254:4764–4771.
61. Downs SM, Hunzicker-Dunn M. Differential regulation of oocyte
maturation and cumulus expansion in the mouse oocyte-cumulus cell
complex by site-selective analogs of cyclic adenosine-monophosphate.
Dev Biol 1995; 172:72–85.
62. Cooke BA. Signal transduction involving cyclic AMP-dependent and
cyclic AMP-independent mechanisms in the control of steroidogenesis.
Mol Cell Endocrinol 1999; 151:25–35.
63. Gutnisky C, Dalvit GC, Pintos LN, Thompson JG, Beconi MT, Cetica PD.
Influence of hyaluronic acid synthesis and cumulus mucification on
bovine oocyte in vitro maturation, fertilisation and embryo development.
Reprod Fertil Dev 2007; 19:488–497.
64. Richani D, Wang X, Zeng H, Smitz JE, Thompson JG, Gilchrist RB. Pre-
maturation with cAMP modulators in conjunction with EGF-like peptides
during in vitro maturation enhances mouse oocyte developmental
competence. Mol Reprod Dev 2014; 81:422–435.
65. Downs SM, Utecht AM. Metabolism of radiolabeled glucose by mouse
oocytes and oocyte-cumulus cell complexes. Biol Reprod 1999; 60:
1446–1452.
66. Harris SE, Adriaens I, Leese HJ, Gosden RG, Picton HM. Carbohydrate
metabolism by murine ovarian follicles and oocytes grown in vitro.
Downloaded from www.biolreprod.org.
Reproduction 2007; 134:415–424.
67. Richani D, Sutton-McDowall ML, Frank LA, Gilchrist RB, Thompson JG.
Effect of epidermal growth factor-like peptides on the metabolism of in
vitro matured mouse oocytes and cumulus cells. Biol Reprod 2014; 90:49.
68. Rushmer RA, Brinster RL. Carbon dioxide production from pyruvate and
glucose by bovine oocytes. Exp Cell Res 1973; 82:252–254.
69. Leese HJ. Metabolism of the preimplantation mammalian embryo. Oxf
Rev Reprod Biol 1991; 13:35–72.
70. Van Blerkom J, Davis PW, Lee J. ATP content of human oocytes and
developmental potential and outcome after in-vitro fertilization and
embryo transfer. Hum Reprod 1995; 10:415–424.
71. Zeng HT, Ren Z, Yeung WS, Shu YM, Xu YW, Zhuang GL, Liang XY.
Low mitochondrial DNA and ATP contents contribute to the absence of
birefringent spindle imaged with PolScope in in vitro matured human
oocytes. Hum Reprod 2007; 22:1681–1686.
72. Stojkovic M, Machado SA, Stojkovic P, Zakhartchenko V, Hutzler P,
Goncalves PB, Wolf E. Mitochondrial distribution and adenosine
triphosphate content of bovine oocytes before and after in vitro
maturation: correlation with morphological criteria and developmental
capacity after in vitro fertilization and culture. Biol Reprod 2001; 64:
904–909.
73. Tamassia M, Nuttinck F, May-Panloup P, Reynier P, Heyman Y,
Charpigny G, Stojkovic M, Hiendleder S, Renard JP, Chastant-Maillard
S. In vitro embryo production efficiency in cattle and its association with
oocyte adenosine triphosphate content, quantity of mitochondrial DNA,
and mitochondrial DNA haplogroup. Biol Reprod 2004; 71:697–704.
74. Dunning KR, Cashman K, Russell DL, Thompson JG, Norman RJ,
Robker RL. Beta-oxidation is essential for mouse oocyte developmental
competence and early embryo development. Biol Reprod 2010; 83:
909–918.
75. Skala M, Ramanujam N. Multiphoton redox ratio imaging for metabolic
monitoring in vivo. Methods Mol Biol 2010; 594:155–162.
76. Chance B. Metabolic heterogeneities in rapidly metabolizing tissues. J
Appl Cardiol 1989; 4:207–221.
77. Curnow EC, Ryan JP, Saunders DM, Hayes ES. Developmental potential
of bovine oocytes following IVM in the presence of glutathione ethyl
ester. Reprod Fertil Dev 2010; 22:597–605.
78. de Matos DG, Furnus CC, Moses DF. Glutathione synthesis during in
vitro maturation of bovine oocytes: role of cumulus cells. Biol Reprod
1997; 57:1420–1425.
79. de Matos DG, Gasparrini B, Pasqualini SR, Thompson JG. Effect of
glutathione synthesis stimulation during in vitro maturation of ovine
oocytes on embryo development and intracellular peroxide content.
Theriogenology 2002; 57:1443–1451.
80. de Matos DG, Herrera C, Cortvrindt R, Smitz J, Van Soom A, Nogueira D,
Pasqualini RS. Cysteamine supplementation during in vitro maturation and
embryo culture: a useful tool for increasing the efficiency of bovine in
vitro embryo production. Mol Reprod Dev 2002; 62:203–209.
Article 47