Effect of dibutyryl cAMP on the cAMP content, meiotic progression, and developmental potential of in vitro matured pre-pubertal and adult pig oocytes.

Abstract

Pre-pubertal pig oocytes display reduced developmental competence compared with adult oocytes following in vitro maturation (IVM). Exposure to dibutyryl cyclic adenosine monophosphate (dbcAMP) for the first 20 hr IVM improves development of pre-pubertal oocytes, suggesting that their cAMP content may be inadequate. This study examined the effect of 1 mM dbcAMP treatment for the first 22 hr of IVM on the cAMP content, meiotic progression, and embryo development of pre-pubertal and adult oocytes. In control groups, a two-fold increase in cAMP was observed in adult oocytes after 22 hr IVM, with no change in pre-pubertal oocyte cAMP content. At 22 hr IVM, dbcAMP treatment resulted in two- and five-fold increases in pre-pubertal and adult oocyte cAMP, respectively. After 22 hr control IVM, a greater proportion of pre-pubertal oocytes occupied metaphase I (MI) compared with adult oocytes (69% vs. 49%). dbcAMP treatment reduced the proportion of pre-pubertal and adult oocytes in MI stage at 22 hr. Despite dbcAMP treatment, the proportion of pre-pubertal oocytes in the MI stage at 22 hr remained higher than that of adult oocytes. In control groups, adult oocytes displayed a greater ability to form blastocysts compared with pre-pubertal oocytes following either parthenogenetic activation (59% vs. 25%) or in vitro fertilization (IVF) (47% vs. 19%). dbcAMP treatment increased subsequent blastocyst formation rates of pre-pubertal oocytes, whereas blastocyst formation rates of adult oocytes remained unchanged. Our results suggest that the reduced developmental capacity of pre-pubertal oocytes may be a consequence of their reduced ability to accumulate cAMP during IVM.

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MOLECULAR REPRODUCTION AND DEVELOPMENT 73:1326–1332 (2006)
Effect of Dibutyryl cAMP on the cAMP Content,
Meiotic Progression, and Developmental
Potential of In Vitro Matured Pre-Pubertal
and Adult Pig Oocytes
MELANIE A. BAGG,* MARK B. NOTTLE, CHRISTOPHER G. GRUPEN, AND DAVID T. ARMSTRONG
Department of Obstetrics and Gynaecology, Research Centre for Reproductive Health,
University of Adelaide, The Queen Elizabeth Hospital, Woodville, South Australia, Australia
ABSTRACT
reduced developmental competence compared with
adult oocytes following in vitro maturation (IVM).
Exposure to dibutyryl cyclic adenosine monophosphate
(dbcAMP) for the first 20 hr IVM improves develop-
mentofpre-pubertaloocytes,
their cAMP content may be inadequate. This study
examined the effect of 1 mM dbcAMP treatment for the
first 22 hr of IVM on the cAMP content, meiotic
progression, and embryo development of pre-pubertal
and adult oocytes. In control groups, a two-fold increase
in cAMP was observed in adult oocytes after 22 hr
IVM, with no change in pre-pubertal oocyte cAMP
content. At 22 hr IVM, dbcAMP treatment resulted in
two- and five-fold increases in pre-pubertal and adult
oocyte cAMP, respectively. After 22 hr control IVM, a
greater proportion of pre-pubertal oocytes occupied
metaphase I (MI) compared with adult oocytes (69%
vs. 49%). dbcAMP treatment reduced the proportion of
pre-pubertal and adult oocytes in MI stage at 22 hr.
Despite dbcAMP treatment, the proportion of pre-
pubertal oocytes in the MI stage at 22 hr remained
higher than that of adult oocytes. In control groups,
adult oocytes displayed a greater ability to form
blastocysts compared with pre-pubertal oocytes fol-
lowing either parthenogenetic activation (59% vs.
25%) or in vitro fertilization (IVF) (47% vs. 19%).
dbcAMP treatment increased subsequent blastocyst
formation rates of pre-pubertal oocytes, whereas
blastocyst formation rates of adult oocytes remained
unchanged. Our results suggest that the reduced
developmentalcapacity
may be a consequence of their reduced ability to
accumulatecAMPduringIVM. Mol. Reprod. Dev. 73:
1326–1332, 2006. ? 2006 Wiley-Liss, Inc.
Pre-pubertal pig oocytes display
suggestingthat
of pre-pubertal oocytes
Key Words: donor
oocyte developmental competence; dbcAMP; meiotic
maturation
age;intra-oocytecAMP;
INTRODUCTION
Itiswellestablishedindomesticlivestockspeciesthat
oocyte developmental competence increases from birth
with increasing maternal age (Armstrong, 2001). In
the pig, oocytes of pre-pubertal donors possess lower
developmental competence than oocytes of adult donors
followinginvitromaturation(IVM)(Grupenetal.,1997;
O’Brien et al., 2000; Marchal et al., 2001; Ikeda and
Takahashi, 2003; Sherrer et al., 2004). Low blastocyst
formation of pre-pubertal pig oocytes has been asso-
ciated with a high incidence of polyspermy following
in vitro fertilization (IVF) (O’Brien et al., 2000; Marchal
et al., 2001), which is thought to be due to incomplete
cytoplasmic maturation and abnormal fertilization
(Niwa, 1993; Nagai, 1994).
Atthetimeofaspiration,pre-pubertalporcineoocytes
displayheterogeneityatthegerminalvesicle(GV)stage
of meiosis (McGaughey and Polge, 1972; Motlik and
Fulka, 1976). Continued asynchronous progression
results in cohorts of oocytes reaching metaphase II
(MII)andagingfortheremainderofIVM(Grupenetal.,
1997; Nagai et al., 1997; Funahashi et al., 1997b). The
majority of studies that have directly compared pre-
pubertalandadultoocytesreportthattheyreachMIIat
a similar rate by the end of IVM (Marchal et al., 2001;
Grupenetal.,2003;IkedaandTakahashi,2003;Sherrer
et al., 2004). In contrast, O’Brien et al. (2000) deter-
mined that a lower proportion of pre-pubertal oocytes
reach MII at the end of IVM.
The principal regulator of the oocyte meiotic cell cycle
is cyclic adenosine monophosphate (cAMP). In vivo,
gonadotropins trigger a transient increase in oocyte
cAMP content,which subsequentlydecreases toinitiate
germinal vesicle breakdown (GVBD) (Mattioli et al.,
1994).Incontrast,physicalremovalofoocytesfromtheir
follicularenvironmentinducesspontaneousresumption
? 2006 WILEY-LISS, INC.
Grant sponsors: National Health and Medical Research Council of
Australia and the Canadian Institute for Health Research; Grant
sponsor: The Queen Elizabeth Hospital Research Foundation.
*Correspondence to: Melanie A. Bagg, Department of Obstetrics and
Gynaecology, University of Adelaide, Level 4, Maternity Building, The
Queen Elizabeth Hospital, 28 Woodville Road, Woodville, SA 5011,
Australia. E-mail: melanie.bagg@adelaide.edu.au
Received 26 March 2006; Accepted 27 April 2006
Published online 24 July 2006 in Wiley InterScience
(www.interscience.wiley.com).
DOI 10.1002/mrd.20555

Page 2

of meiosis (Pincus and Enzmann, 1935). It is thought
that premature reduction of oocyte cAMP content
during IVM has an adverse effect on the coordination
of nuclear and cytoplasmic maturation, resulting in
decreased developmental potential.
Cyclic AMP is synthesized in cumulus cells by
adenylate cyclase activity in response to gonadotropins
and enters the oocyte via gap junctions (Schultz et al.,
1983; Racowsky, 1985). High intracellular levels of
cAMP maintain oocyte meiotic arrest at the GV stage
by activating cAMP-dependent protein kinase (PKA),
which in turn maintains maturation promoting factor
(MPF),another keyregulator ofthemeioticcell cycle,in
the inactive form (pre-MPF) (Bornslaeger et al., 1986;
Spaulding, 1993; Francis and Corbin, 1994). A number
of treatments have been used to elevate oocyte cAMP
content, including phosphodiesterase (PDE) inhibitors,
stimulators of adenylate cyclase activity, and analogs of
cAMP. Previously, treatment of pre-pubertal porcine
oocytes for 20–22 hr with dbcAMP, a membrane
permeable analog of cAMP, has been shown to increase
the synchrony of meiotic progression and the incidence
of blastocyst formation following IVF (Funahashi et al.,
1997b;Somfaietal.,2003).Inanotherstudy,exposureof
pre-pubertal porcine oocytes to dbcAMP for the first 22
hr of IVM was also found to increase the incidence of
monospermic fertilization in porcine oocytes (Mattioli
et al., 1994). The effect of dbcAMP during IVM on adult
oocyte maturation and development has not yet been
reported.
The present study examined the response of pre-
pubertal and adult oocytes to dbcAMP exposure for the
first 22 hr of IVM. The cAMP content and meiotic
progression of oocytes from pre-pubertal and adult
donors were determined at various time points during
IVM with and without dbcAMP treatment. The devel-
opmental competence of dbcAMP treated and control
oocyteswerealsodeterminedfollowingparthenogenetic
activation and IVF.
MATERIALS AND METHODS
Culture Media
All chemicals and reagents were from Sigma-Aldrich
[St. Louis, MO] unless otherwise stated. Pre-IVM
washing was conducted in 25 mM HEPES-buffered
tissue culture medium-199 [H-TCM; ICN Biomedicals
Inc., Costa Mesa, CA] supplemented with 25% (v/v)
porcine FF. The basic IVM medium was TCM-199 [B-
TCM; ICN Biomedicals] supplemented with 2.2 mg/ml
sodium bicarbonate, 0.1 mg/ml sodium pyruvate, anti-
biotics (75 IU/ml penicillin-G, 75 mg/ml streptomycin
sulfate, CSL Bioscience, Victoria, Australia), 1.5 mg/ml
porcine FSH (100 IU/ml) [Follitropin-V, Vetrepharm
Canada,Inc., London, Canada],1.0 mg/ml estradiol-17b,
0.5mMcysteamine,10ng/mlEGF,and25%porcineFF.
In addition to control media, separate H-TCM and B-
TCM supplemented with 1 mM dbcAMP was also
prepared. FF was prepared from a pool of follicular
aspirates from pre-pubertal ovaries by centrifugation
(2,000gfor30min)ofthematerialaspiratedfromantral
folliclesof3–8mmindiameter,filteredthroughasterile
0.22 mm pore filter [Millipore, Bedford, MA] and stored
at ?808C. Pre-activation/IVF washing and polar body
searching was conducted in phosphate buffered North
Carolina State University-23 medium (PB-NCSU-23;
(Petters and Wells, 1993). For IVF, Sperm Pre-incuba-
tion Medium (SPM199) consisted of B-TCM supplemen-
ted with 0.91 mM Sodium Pyruvate, 2.92 mM Calcium
Lactate, 1.0 mM L-glutamine, 75 mg/L penicillin-G,
50 mg/L streptomycin sulfate, and 0.4% heat inacti-
vated-fetalcalf serum(FCS). Activation withionomycin
and fertilization procedures were carried out in tyrode-
albumin-lactate-pyruvate-poly-vinyl
PVA) medium, supplemented with 3.0 mM calcium
lactate and 2.0 mM caffeine-sodium benzoate (Bavister,
1989). Activation was performed with 6-DMAP and
subsequent in vitro culture (IVC) was conducted in
NCSU-23 medium (Petters and Wells, 1993).
alcohol(TALP-
Oocyte Maturation
Ovaries were collected from pre-pubertal and adult
LargeWhite,LandraceandLargeWhite-Landracecross
breeds of pigs and transported in saline (0.9% w/v NaCl
supplemented with antibiotics (40 IU/ml penicillin G
and 40 mg/ml streptomycin sulfate, CSL Bioscience) to
the laboratory and processed at 38.58C. Ovaries from
the two donor age groups were collected on separate
days, with time from ovary collection to start of
maturation kept constant at approximately 3 hr. Antral
follicles (3–8 mm diameter) were aspirated using an
18-Gneedlethroughwhichconstantsuctionwasapplied
(1 L/min). COC with more than three intact, compact
cumulus layers were selected for culture, sorted
and washed in H-TCM. Before transfer to treatments,
COCs were washed two times in dishes containing B-
TCM, with or without 1 mM dbcAMP as appropriate.
Groupsof15–20COCsweretransferredto50mldroplets
of B-TCM, with or without 1 mM dbcAMP as appro-
priate, and incubated at 38.58C in a humidified atmo-
sphere of 5% CO2in air. Following 22 hr IVM, COCs
werewashedandtransferredto50mldropletsofB-TCM
without 1 mM dbcAMP, and incubated for another 22–
24 hr, depending on the experiment. Embryo tested
mineral oil was used to cover all media droplets. All
bicarbonate-buffered medium was equilibrated for at
least 3 hr prior to use in a humidified atmosphere of 5%
CO2in air.
Cumulus Cell Removal
Two different methods of cumulus cell removal were
employed, depending on whether denuded oocytes were
required for embryo IVP or for the purpose of nuclear
maturation staining and cAMP analysis. For embryo
IVP,dropletscontainingoocytesweretreatedwith1mg/
ml hyaluronidase in phosphate buffered saline (PBS)
supplemented with 5% FCS for 1 min at the end of IVM
and aspirated in PB-NCSU-23 with a small-bore glass
pipette to gently remove cumulus cells. When oocytes
were required for nuclear maturation staining and
Molecular Reproduction and Development. DOI 10.1002/mrd
EFFECT OF DIBUTYRYL cAMP ON IN VITRO MATURED PIG OOCYTES1327

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cAMP analysis, cumulus cells were removed by vortex-
ing for 4 min in a 10 ml tube containing 2 ml H-TCM.
Assessment of Meiotic Stage
Denudedoocyteswerecollected,mountedundercover
slips on slides and fixed for 48 hr with ethanol: acetic
acid (3:1 v/v). After staining with 1% Orcein in 45%
acetic acid and destaining with glycerol: acetic acid:
water (1:1:3 v/v), oocytes were examined under a phase
contrast microscope at 400? magnification. Meiotic
stages were classified based on previously described
criteria (Motlik and Fulka, 1976; Homa, 1988). The
stagesofmeioticprogressionwereassessedas GV(GVI-
IV), MI (diakinesis I and MI), and MII (anaphase I,
telophase I and MII ).
Embryo Production
Only matured oocytes, displaying a polar body, were
selected for parthenogenetic activation and IVF.
Parthenogenetic Activation
Oocytes were parthenogenetically activated by incu-
bation in TALP-PVA medium containing 5 mM ionomy-
cin for 5 min. Oocytes were then washed twice and
incubated for 3 hr in NCSU-23 medium containing
2.0 mM 6-DMAP.
Fertilization
Sperm was prepared from fresh pig semen (extended
in Androhep) from Large White boars (stored for up to
2 days at 218C; Sabor Ltd, Clare, Australia) and
washed twice by centrifugation (5 min, 300g) in
SPM199. The sperm pellet was then resuspended in
10 ml pre-equilibrated SPM199 and incubated for
45 min in a humidified atmosphere of 5% CO2in air.
Following incubation of the sperm suspension, super-
natantwasremovedandthepelletdilutedinTALP-PVA
to 5?106cells/ml. Oocytes were washed once and
transferred to TALP-PVA insemination drops (90 ml)
overlaid with mineral oil (25–30 oocytes per drop). A
10 ml aliquot of the sperm containing media was added
to each insemination drop to give a final dilution of
5?105sperm/ml. Oocytes and spermatozoa were co-
culturedfor8hrat38.58Cinahumidifiedatmosphereof
6% CO2in air. Following insemination, zona-attached
sperm were removed by gentle aspirating with a small-
bore glass pipette.
Embryo Culture
Following either parthenogenetic activation or IVF,
presumptivezygoteswerewashedtwiceandtransferred
to50mldroplets(15–20presumptivezygotesperdrop)of
NCSU-23 culture medium, containing 4.0 mg/ml BSA,
and incubated at 38.58C in a humidified atmosphere of
5% O2, 5% CO2, and 90% N2. After 5 days of IVC,
cleavage was assessed and 10% FCS was added to the
culture drops. After 7 days of IVC, embryo development
was assessed.
Intra-oocyte cAMP Assay
The cAMP content of zona-free oocytes was measur-
ed using a radioimmunoassay method described and
validated previously (Reddoch et al., 1986). At 0 and
22 hr IVM oocytes were denuded by vortex as described,
and washed twice in PBS containing 10% FCS. Oocytes
weretreatedwith0.5%pronaseinH-TCMfor10–20sec
to remove the zonae pellucida and then washed five
times in PBS containing 10% FCS. Upon comparing
denuded and zona-free oocytes (data not shown), we
support previous findings that zonae pellucida removal
is important in the detection of intra-oocyte cAMP,
eliminating any possible influence of adenylate cyclase
present in the remains of the corona cell foot processes,
many of which remain embedded in the zonae pellucida
after denuding (Mattioli et al., 1994). Twenty to 30
oocytes were stored in 500 ml ethanol at ?208C in
preparation for assaying. A sample of each final wash
wasalsocollectedtoensurecompletewashingofoocytes
that were matured in dbcAMP. Samples were vortexed
for30secandpulsecentrifugedbeforebeingevaporated
at 378C using N2gas. The particulate remaining in the
tube was then resuspended in 220 ml assay buffer
(50mMsodiumacetate, pH 5.5).Thesampleswerethen
acetylated with 6.67 ml of triethylamine [AJAX Chemi-
cals,Sydney,Australia]:aceticanhydride[BDHLabora-
tory supplies, Poole, England] (2:1 v/v) and 100 ml
duplicates were aliquoted for the assay.
cAMP with specific activity of 2010 Ci/mM (prepared
by iodinating 20-0-monosuccinyladenosine-30:50-cyclic
monophosphate tyrosyl methyl ester using the chlor-
amine T method; (Hunter, 1970) and cAMP antibody
(Reddoch et al., 1986) were added to duplicate samples
and incubated for 24 hr at 48C. One milliliter of 100%
ethanol at 48C was then added and samples were
centrifuged at 3000g for 10 min. The supernatant was
decanted and the pellet was dried in air and counted
with a gamma counter. Triplicate standards were used
to produce a standard curve (0–250 fmol cAMP).
125I-labeled
Statistical Analysis
Each experiment was replicated three to five times.
Maturation and developmental data were subjected to
three-way ANOVA following arcsine transformation,
andwhereappropriate
between groups were tested using the Tukey method
post hoc comparisons or t tests. For the cAMP measure-
ments, raw data was tested using three-way ANOVA,
and differences between means were detected using
the Holm-Sidak method post hoc comparison or
t tests. P<0.05 was considered to be statistically
significant.
proportionaldifferences
Experimental Design
Experiment 1. ComparisonofcAMPcontentduring
IVM and subsequent development of parthenote
embryos of pre-pubertal and adult oocytes. COCs from
each donor group were matured in control conditions or
with dbcAMP for the first 22 hr IVM. A proportion of
Molecular Reproduction and Development. DOI 10.1002/mrd
1328M.A. BAGG ET AL.

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oocytesfromeachgroupwasremovedat0and22hrIVM
forcAMPmeasurementandtheremainingoocyteswere
artificially activated after 46 hr IVM. Three replicates
eachofpre-pubertalandadultoocyteswereassessedfor
cAMP content (n¼305 and 276, respectively) and
parthenotedevelopment(n¼335and239,respectively).
Experiment 2. Comparison of meiotic progression
during IVM of pre-pubertal and adult oocytes. COCs
from each donor age group were matured in control
conditionsorwithdbcAMPforthefirst22hrIVM.Eight
replicates of pre-pubertal oocytes (n¼960) and six
replicates of adult oocytes (n¼520) were assessed at 0,
16, 22, 40, and 44 hr IVM.
Experiment 3. Comparison of embryo development
following IVF of pre-pubertal and adult oocytes follow-
ingIVM.Pre-pubertaland adult COCs were maturedin
control conditions or with dbcAMP for the first 22 hr
and then fertilized after 46 hr IVM. Three replicates
of pre-pubertal (n¼299) and adult (n¼325) IVM
oocytes were assessed for blastocyst development
following IVF. When possible, pre-pubertal and adult
IVF experiments were conducted on the same or
consecutive days using the same sperm to reduce
variation in fertilizability.
RESULTS
Experiment 1: Comparison of cAMP
Content During IVM and Subsequent
Development of Parthenote Embryos
of Pre-pubertal and Adult Oocytes
The cAMP content of pre-pubertal and adult oocytes
at 0 and 22 hr is shown in Figure 1. Zona-free oocytes
isolated from pre-pubertal and adult COC contained
similar levels of cAMP at 0 hr. Following 22 hr
incubation as an intact COC in the absence of dbcAMP,
cAMP content of adult oocytes increased 2.5-fold over
that at 0 hr, whereas no increase occurred in cAMP
contentsofpre-pubertaloocytes.Culturefor22hrinthe
presence of dbcAMP resulted in approximately twofold
greater cAMP contents of both pre-pubertal and adult
oocytes than those observed with culture in the absence
of dbcAMP. By completion of IVM after 44 hr culture,
cAMP levels had decreased to barely detectable levels
for all four groups (data not shown).
The effect of donor age and dbcAMP on the develop-
mental competence of IVM oocytes following partheno-
genetic activation is presented in Table 1. Pre-pubertal
and adult oocytes underwent similar high rates of
maturation in vitro and cleavage after activation.
However, significantly more pre-pubertal than adult
parthenotes blocked at <8-cell stage (57.4?11.9% vs.
21.1?6.7%, respectively) and this was not altered by
culturewith dbcAMP. Treatment
increased the rate of blastocyst formation of pre-
pubertal oocytes compared with the control group
cultured without dbcAMP (24.6?5.2% vs. 34.9?6.3%,
respectively), whereas rate of blastocyst formation from
adult oocytes was not affected by culture with dbcAMP.
Theblastocystrateofadultoocyteswashigherthanthat
of pre-pubertal oocytes following IVM both in the
absence (58.7?1.9% vs. 24.6?5.2%) and presence
(56.9%?10.6 vs. 34.9?6.3%, respectively).
with dbcAMP
Experiment 2: Comparison of Pre-pubertal and
Adult Oocyte Meiotic Progression During IVM
Theprogressionofmeioticmaturationinpre-pubertal
and adult oocytes matured for 0, 16, 22, 40, and 44 hr
IVM is presented in Figure 2. About half of the pre-
pubertal and adult oocytes were in stages of GVBD
(GVII-IV) at the start of IVM, while the remainder were
attheintactGVIstage(datanotshown).At16and22hr
IVM in the absence of dbcAMP, a greater proportion of
pre-pubertal oocytes had progressed to the MI stage of
meiosis compared with adult oocytes (45.6?1.4 vs.
Molecular Reproduction and Development. DOI 10.1002/mrd
TABLE 1. Comparison of Developmental Competence Between Pre-Pubertal and Adult IVM Oocytes Following
Parthenogenetic Activation and the Effect of dbcAMP Treatment
GroupsN MII (%) Cleaved (%)
<8 cell (%) Morula (%)Blastocyst (%)
Pre-pubertal control
Pre-pubertalþdbcAMP
Adult control
AdultþdbcAMP
Values expressed as mean?SEM percentage <8 cell, morula, and blastocyst data are expressed as mean percentage of cleaved.
Different superscripts within columns indicate significant differences between donor age and/or treatment groups (P<0.05).
188
125
185
136
90.8?3.1a
89.1?3.3a
92.8?2.5a
90.4?1.6a
86.8?4.5a
76.2?10.7a
80.2?6.4a
78.9?4.7a
57.4?11.9a
52.5?9.7a,b
21.1?6.7b
28.3?5.2b,a
15.4?7.0a
8.2?3.9a
15.5?5.7a,b
12.2?7.5b
24.6?5.2a
34.9?6.3b
58.7?1.9c
56.9?10.6c
Fig. 1. Cyclic AMP (cAMP) concentrations of pre-pubertal (&) and
adult (&) COCs before and after 22 hr IVM in control conditions or
dbcAMP treatment. Different superscripts indicate significant differ-
ences in cAMP concentrations (P<0.05).
EFFECT OF DIBUTYRYL cAMP ON IN VITRO MATURED PIG OOCYTES1329

Page 5

26.2?2% and 68.8?2.3 vs. 48.6?1.9%, respectively).
IntheabsenceofdbcAMP,therewasnodifferenceinthe
percentages of pre-pubertal and adult oocytes reaching
MII at the time points examined.
After 16 hr IVM in the presence of dbcAMP, a similar
number of pre-pubertal and adult oocytes had reached
MI. Treatment with dbcAMP significantly reduced the
proportion of pre-pubertal oocytes in MI at 16 hr
compared with the control group (45.56?1.4% vs.
29.49?1.1%). After 22 hr the percentage of oocytes at
MI was lower in the dbcAMP group compared with the
control group for both pre-pubertal (68.75?2.3% vs.
59.65?2.3%) and adult (48.57?1.9% vs. 25.64?2.1%)
oocytes. In the presence of dbcAMP, a higher proportion
of pre-pubertal oocytes reached MI at 22 hr compared
with adult oocytes. After 40 hr IVM with dbcAMP, the
percentage of adult oocytes at MII was lower than when
matured in the absence of dbcAMP (78.26?3.1% vs.
91.8?3.13%). At the end of IVM in the presence of
dbcAMP, pre-pubertal and adult oocytes reached MII at
similar rates.
Experiment 3: Comparison of Embryo
Development Following IVF of Pre-Pubertal
and Adult Oocytes Following IVM
The effect of donor age and dbcAMP on the develop-
mental competence of IVM oocytes following IVF is
presented in Table 2. In the control group, adult oocytes
had a higher blastocyst formation rate compared
with pre-pubertal oocytes (46.8?7.1% vs. 18.6?5.4%).
Pre-pubertal oocytes displayed a significantly higher
percentage of <8-cell embryos compared with adult
oocytes (42?8.3% vs. 32.3?0.6%). The addition of
dbcAMP for the first 22 hr IVM significantly increased
the blastocyst formation rate of pre-pubertal oocytes
(18.6?5.4% vs. 35.9?9.6%), but not adult oocytes.
DISCUSSION
Thepresentstudyisthefirsttoreportadultpigoocyte
cAMP concentrations during IVM and compare these
directly with pre-pubertal oocytes matured in the
absence or presence of 1 mM dbcAMP for the first
22 hr. The results of the first experiment demonstrated
nochangeincAMPcontentofpre-pubertaloocytesanda
doubling in cAMP concentration of adult oocytes after
22 hr. This indicates that pre-pubertal oocytes have a
reduced ability to accumulate intra-oocyte cAMP level
comparedwithadultoocyteswhenstimulatedwithFSH
under IVM conditions.
Time-dependent increases in the cAMP content have
been reported for pre-pubertal pig oocytes matured
either within COCs (Shimada and Terada, 2002) or
within everted follicles (Mattioli et al., 1994). Our
Molecular Reproduction and Development. DOI 10.1002/mrd
Fig. 2. Progressionof pre-pubertal (
fromGV(A)toMI(B),andMII(C)stagesofmeioticmaturationat0,16,
22, 40, and 44 hr IVM. Open symbols indicate dbcAMP treatment (pre-
pubertal
,adult).Differentsuperscriptsindicatesignificant
differences in meiotic progression at a particular time point (P<0.05).
) and adult () oocytes
TABLE 2. Comparison of Developmental Competence Between Pre-pubertal and Adult IVM Oocytes Following
IVF and the Effect of dbcAMP Treatment
GroupsnMII (%)Cleaved (%)
<8 Cell (%) Morula (%) Blastocyst (%)
Pre-pubertal control
Pre-pubertalþdbcAMP
Adult control
AdultþdbcAMP
Values expressed as mean?SEM. Percentage <8cell, morula, andblastocyst data are expressed as mean percentage ofcleaved.
Different superscripts within columns indicate significant differences between donor age and/or treatment groups (P<0.05).
159
162
188
150
93.0?3.0a
91.4?5.7a
96.1?0.6a
95.7?0.8a
80.7?5.8a
87.6?6.9a
78.3?2.7b
79.7?1.6b
42.0?8.3a
39.3?8.7a
32.3?0.6b
34.2?7.9b,a
36.9?7.3a
24.0?6.9a
18.4?6.2a,b
12.2?6.0b
18.6?5.4a
35.9?6.9b,c
46.8?7.1c
50.1?6.0c
1330M.A. BAGG ET AL.

Page 6

findings for zona-free oocytes are consistent with
those of Mattioli et al. (1994), who observed no
significant change from 0 hr in the amount of cAMP in
pre-pubertal oocytes matured as COCs or denuded
oocytes throughout IVM (Mattioli et al., 1994). In
contrast, Shimada and Terada (2002) have reported
that the cAMP concentration of pre-pubertal oocytes
significantly increases at 4 hr IVM, reaches a peak level
at8hr,andthendramaticallydropsat12hrandagainat
28hrtoreachbasallevelsat32hrandremainslowuntil
the end of maturation (Shimada and Terada, 2002). The
reason for these differences is unknown, but might be
explained by variation in the methods used to
assay cAMP, for example, the use of zona-free versus
zona-intact oocytes.
Treatment with dbcAMP for the first 22 hr of IVM
resulted in significant increases in the intra-oocyte
cAMP concentration of both pre-pubertal and adult
oocytes at 22 hr. The dbcAMP derivative of cAMP
mimics the action of endogenous cAMP (Budavari,
1989). In comparison with cAMP, dbcAMP has a
lipophilic nature and is therefore used preferentially
with intact cells due to its greater permeability and
resistance to hydrolysis by PDEs (Henion et al., 1967;
Swislocki,1970).Toourknowledge,thisisthefirststudy
to directly measure cAMP content of dbcAMP treated
oocytes during IVM.
Intermsofmeioticprogression,wedemonstratedthat
a higher proportion of pre-pubertal oocytes had under-
gone GVBD and progressed to the MI stage of meiosis
compared with adult oocytes by 16 and 22 hr of control
IVM. Previous studies have described variation in
meiotic stage in pre-pubertal pig oocytes at the start of
IVM with continued asynchronous meiotic progression
thatresultedinacohortofagedoocytesattheendofIVM
(McGaughey and Polge, 1972; Motlik and Fulka, 1976;
Ocampo et al., 1993; Grupen et al., 1997; Funahashi
et al., 1997b). Our findings are in agreement with
thoseofpreviousstudiesinpre-pubertalpigoocytesand
extends these findings by directly comparing the
meiotic progression of pre-pubertal and adult oocytes.
Our results indicate that pre-pubertal oocytes have a
reduced ability to accumulate cAMP compared with
adult oocytes, which may account for their asynchro-
nous meiotic progression.
Exposureofpre-pubertaloocytesto1mMdbcAMPfor
22hrIVMsignificantlydelayedmeioticprogressionand
increased blastocyst formation rate. The meiotic pro-
gression of adult oocytes was also delayed by dbcAMP
treatment, but this did not result in an increase in
blastocyst formation rate. Despite exposure to dbcAMP,
ahigherproportionofpre-pubertalcomparedwithadult
oocytes had attained MI at 22 hr, indicating that
dbcAMP treatment of pre-pubertal oocytes could not
prevent GVBD as effectively as in adult oocytes. Our
findings are in agreement with previous studies in pre-
pubertal pig oocytes, which demonstrated that treat-
ment with 1 mM dbcAMP decreased GVBD by
around 50% (Funahashi et al., 1997b; Somfai et al.,
2003). Our study, although reporting the same trend,
only demonstrated a 20% reduction in GVBD following
dbcAMP treatment for 22 hr. The reasons for this
differenceareunknown,butmaybeduetodifferencesin
IVM culture conditions between these studies.
In the pig, oocytes from pre-pubertal donors display
lower developmental competence than those of adult
donors (O’Brien et al., 2000; Marchal et al., 2001;
Grupenetal.,2003;IkedaandTakahashi,2003;Sherrer
et al., 2004). The findings of the present study are in
agreement with those of previous studies, having
demonstrated that treatment with 1 mM dbcAMP for
the first 22 hr IVM improves blastocyst formation
following both parthenogenetic activation and IVF in
pre-pubertal pig oocytes. The blastocyst formation rate
ofdbcAMPtreatedpre-pubertaloocytesremainedlower
than that of adult oocytes, despite the dbcAMP treat-
ment raising cAMP levels in pre-pubertal oocytes
similar to those in adult oocytes at 22 hr. Similar
differences were observed in the developmental compe-
tence of pre-pubertal and adult oocytes following
parthenogenetic activation and IVF. This finding indi-
cates that parthenogenetic development is a useful
means of assessing developmental potential to the
blastocyst stage. Parthenogenetic activation is also
convenient in that it removes the variability in fertiliz-
ing ability between sperm doses.
The intra-oocyte cAMP concentration differences
reported here have been correlated with the matura-
tional and developmental differences observed between
pre-pubertal and adult IVM oocytes. Although the
oocyte can synthesize cAMP, the lack of luteinizing
hormone (LH) receptors on the oolemma means that it
probably does not account for the gonadotropin stimu-
lated cAMP increase (Mattioli et al., 1994). However,
cAMP is also produced in cumulus cells by adenylate
cyclase when stimulated by LH or FSH and is trans-
ported into oocytes via gap junctions that communicate
between cumulus cell projections through the zonae
pellucida and the oocyte cell membrane (Schultz et al.,
1983; Racowsky, 1985). Therefore, it seems likely that
pre-pubertal COCs possess cumulus cells that either
have a lower level of LHR/FSHR expression, a different
adenylate cyclase response or different PDE activities.
In addition, pre-pubertal oocyte gap junctional commu-
nication may not be as well formed or functional as in
adult oocytes. Alternatively, the observed differences
maybeduetodifferencesinthedistributionoffolliclesin
the 3–8 mm aspiration range on ovaries of pre-pubertal
and adult donors. Previously, the ability of granulosa
cells to stimulate intra-oocyte cAMP accumulation has
been shown to progressively decrease with follicle size
(Mattioli et al., 1994).
In conclusion, this study demonstrates that pre-
pubertal pig oocytes accumulate less cAMP during
IVM, undergo a more rapid meiotic progression and
display a lower ability to develop to the blastocyst stage
thaninvitromaturedadultpigoocytes.Treatmentwith
dbcAMP for the first 22 hr IVM increased the cAMP
content of pre-pubertal oocytes, which was correlated
with slower meiotic progression during IVM and
Molecular Reproduction and Development. DOI 10.1002/mrd
EFFECT OF DIBUTYRYL cAMP ON IN VITRO MATURED PIG OOCYTES1331

Page 7

improved developmental competence. Even though the
cAMP levels of pre-pubertal oocytes were increased to
levels similar to those of adult oocytes, the develop-
mentalpotentialofpre-pubertaloocytesremainedlower
than that of adult oocytes, suggesting that factor(s)
independent of cAMP are also deficient in pre-pubertal
oocytes. Elucidation of these deficiencies and a greater
understanding of the mechanisms involved in the
accumulation of cAMP in oocytes will facilitate future
improvements to porcine embryo in vitro production
systems.
ACKNOWLEDGMENTS
TheauthorsaregratefultothestaffofBigRiverPork,
Murray Bridge for supplying the ovaries used in this
study, and Fred Amato and Samantha Schultz for
technical assistance. This work was supported by
National Health and Medical Research Council of
Australia and the Canadian Institute for Health
Research and The Queen Elizabeth Hospital Research
Foundation (Postgraduate Research Scholarship; to
Melanie A. Bagg).
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