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Maintenance
of
bovine
oocytes
in
prophase
of
meiosis
I
by
high
[cAMP]i
H.
Aktas,
M.
B.
Wheeler,
C.
F.
Rosenkrans,
Jr,
N.
L.
First
and
M.
L.
Leibfried-Rutledge
1
Department
of
Meat
and
Animal
Science,
University
of
Wisconsin,
Madison,
WI
53706
USA;
and
Department
of
Animal
Sciences,
University
of
Illinois,
Urbana,
IL
61801
USA
The
effects
of
high
intracellular
cAMP
concentrations
([cAMP]i)
on
germinal
vesicle
maintenance
of
bovine
cumulus\p=n-\oocyte
complexes
were
investigated,
using
8-bromo-3\m='\,5\m='\\x=req-\
cAMP
(8-Br-cAMP)
or
an
invasive
adenylate
cyclase
from
Bordetella
pertussis
to
increase
the
[cAMP]i.
The
effects
of
interactions
of
these
agents
with
macromolecular
supplements
in
culture
medium
(fetal
calf
serum,
FCS;
polyvinylpyrrolidone,
PVP;
BSA),
and
different
methods
of
processing
complexes
before
culture,
on
subsequent
germinal
vesicle
mainten-
ance
by
invasive
adenylate
cyclase
were
studied.
While
8-Br-cAMP
was
unable
to
maintain
germinal
vesicle
arrest
in
the
majority
of
oocytes
for
20
h
(36%
with
FCS,
24%
with
BSA,
18%
with
PVP),
it
maintained
germinal
vesicle
arrest
in
a
high
proportion
of
cumulus\x=req-\
enclosed
oocytes
when
BSA
or
PVP
was
used
(37%
with
FCS,
52%
with
BSA,
53%
with
PVP).
The
difference
in
frequency
of
germinal
vesicle
maintenance
between
macromolecular
supplements
was
not
related
to
[cAMP]i
when
assayed
after
culture
for
2
h
with
invasive
adenylate
cyclase.
Complexes
processed
in
whole
follicular
fluid
were
not
maintained
in
meiotic
arrest
(26%)
when
cultured
with
invasive
adenylate
cyclase
and
PVP.
Complexes
processed
in
follicular
fluid
with
3-isobutyl
1-methylxanthine
(IBMX)
plus
invasive
adenylate
cyclase
were
arrested
at
the
germinal
vesicle
stage
at
high
frequencies
(65%),
while
those
processed
in
IBMX
or
IBMX
plus
8-Br-cAMP-supplemented
follicular
fluid
had
intermediate
(43%
and
49%,
respectively)
frequencies
of
intact
germinal
vesicles.
Oocyte
complexes
processed
in
follicular
fluid
supplemented
with
IBMX
and
invasive
adenylate
cyclase
formed
morulae
and
blastocysts
(27.2%),
as
did
oocytes
processed
in
follicular
fluid
alone
(26%).
Phosphoprotein
profiles
showed
that
control
oocytes
and
8-Br-cAMP-treated
oocytes
share
a
profile
that
is
different
from
that
of
oocytes
treated
with
invasive
adenylate
cyclase.
These
results
show
that
increased
[cAMP]i
reversibly
maintains
bovine
oocytes
in
meiotic
arrest
for
an
extended
period
without
the
occurrence
of
the
post-translational
protein
modifications
observed
during
meiotic
resumption
or
transient
arrest.
Introduction
Oocytes
of
most
mammals
begin
meiosis
during
fetal
develop¬
ment
and
arrest
at
prophase
I
(germinal
vesicle,
GV,
stage).
Oocytes
overcome
this
arrest
after
either
hormonal
induction
in
situ
or
spontaneously
after
removal
from
antral
follicles.
The
mechanism
by
which
oocytes
are
kept
in
meiotic
arrest
in
antral
follicles
has
been
the
subject
of
many
investigations.
Cho
et
al
(1974)
first
implicated
intracellular
cAMP
([cAMP]¡)
in
main¬
taining
meiotic
arrest
in
mammals.
In
both
mice
(Schultz
et
al,
1983;
Vivarelli
et
al,
1983)
and
rats
(Aberdam
et
al,
1987),
agents
that
maintain
high
intracellular
concentrations
of
cAMP
prevent
spontaneous
oocyte
maturation.
In
mice
(Schultz
et
al,
1983;
Vivarelli
et
al,
1983)
and
rats
(Aberdam
et
al,
1987),
spontaneous
maturation
of
oocytes
is
correlated
with
decreased
[cAMP]¡
in
both
cumulus—oocyte
complexes
and
oocytes.
In
amphibians
(Mailer
and
Krebs,
1977),
in
which
hormonal
induction
of
meiotic
maturation
is
required,
agents
that
main¬
tain
high
[cAMP]¡
also
interfere
with
the
induction
of
meiotic
maturation.
Studies
of
domestic
species
with
long
oestrous
cycles,
com¬
pared
with
laboratory
species
with
short
cycles,
have
failed
to
produce
such
clear-cut
results
and
increases
in
[cAMP];
elicit
only
a
transient
delay
in
meiotic
resumption,
as
evaluated
by
germinal
vesicle
breakdown.
Whereas
testosterone
and
a
cAMP
analogue
maintain
meiotic
arrest
cooperatively
in
pig
oocytes,
cAMP
alone
gives
only
a
small
decrease
in
maturation
fre¬
quency
(Rice
and
McGaughey,
1981).
Bovine
oocytes
respond
*Present
address:
Dana-Farber
Cancer
Institute,
Harvard
Medical
School,
Boston,
MA
02115,
USA.
Present
address:
Department
of
Animal
Sciences,
University
of
Arkansas,
Fayetteville,
AR
72701,
USA.
'Reprint
requests.
Received
28
December
1994.
to
agents
with
only
a
transient
maintenance
of
meiotic
arrest
(Homa,
1988;
Sirard
and
First,
1988),
with
the
exception
of
NaF
(Sirard,
1990)
or
pharmacological
doses
of
3-isobutyl
1-methyl
xanthine
(IBMX;
Homa,
1988).
The
effects
of
NaF
are
irrevers¬
ible,
while
the
reversibility
of
IBMX
has
not
been
reported.
Agents
such
as
cholera
toxin
and
forskolin
that
directly
stimulate
adenylate
cyclase
by
affecting
regulatory
G-proteins
do
cause
an
increase
in
[cAMP]¡
in
pig
(Racowsky,
1985
a)
and
sheep
(Crosby
et
al,
1985)
oocytes,
but
either
do
not
maintain
(Crosby
et
al,
1985)
or
only
transiently
maintain
(Racowsky,
1985a;
Sirard,
1990)
meiotic
arrest.
Adenylate
cyclase
is
found
in
cattle
oocytes
(Kuyt
et
al,
1988),
but
it
is
unclear
whether
it
contributes
significantly
to
[cAMP]¡
in
domestic
species
or
whether
cumulus
cells
are
the
main
mediators
for
cAMP
concentrations
in
oocytes,
as
has
been
proposed
for
laboratory
species
(Racowsky,
1984;
Bornslaeger
and
Schultz,
1985).
It
is
possible
that,
although
meiotic
arrest
is
dependent
on
high
[cAMPjj,
resumption
of
meiosis
I
in
oocyte
complexes
is
not
dependent
on
decreasing
[cAMP]¡
in
the
face
of
gonadotrophin
stimulation.
As
has
been
suggested
in
mice
(Downs
et
al,
1988)
and
cows
(Aktas
et
al,
1991a;
Aktas,
1994),
a
positive
signal
originating
from
the
follicle
cells
surrounding
the
oocyte
may
cause
meiotic
resumption
despite
unaltered
[cAMP]¡.
A
series
of
studies
to
determine
the
effects
of
agents
that
increase
[cAMP]¡
on
the
maintenance
of
meiotic
arrest
in
vitro
was
initiated
to
clarify
the
role
of
[cAMP]¡
in
mediating
meiotic
arrest
in
one
species
with
long
cycles,
i.e.
cattle.
We
report
here
that
an
invasive
adenylate
cyclase
(iAC)
increases
[cAMP]¡
both
in
bovine
cumulus
cells
and
the
oocyte,
and
keeps
the
oocyte
arrested
at
the
germinal
vesicle
stage
for
an
extended
period.
The
arrest
is
reversible
and
success
of
arrest
is
affected
by
both
macromolecular
supplement
in
the
medium
and
procedures
for
recovering
the
oocyte—cumulus
complexes
from
the
ovary.
Parts
of
this
study
have
been
reported
as
an
abstract
(Aktas
et
al,
1990).
Materials
and
Methods
Oocyte
recovery
and
culture
Bovine
ovaries
were
obtained
at
an
abattoir
(Pecks,
Milwaukee,
WI)
and
transported
to
the
laboratory
in
thermos
bottles
containing
saline
(0.9%
w/v
NaCl)
at
35-36°C
(4-6
h
for
collection
and
transport).
Temperature
on
arrival
at
the
laboratory
averaged
31°C.
Contents
were
aspirated
from
antral
follicles
2—4
mm
in
diameter.
When
5
ml
of
pooled
follicular
fluid
(FF)
from
the
experimental
groups
had
been
collected,
it
was
supplemented
with
one
of
the
agents
that
increase
intracellular
[cAMP]¡,
depending
on
the
individual
experiments.
Follicular
fluid
from
control
groups
was
not
supplemented
unless
stated
otherwise.
Complete
aspiration,
washing
and
selection
of
the
oocytes
took
an
average
of
2-3
h
until
all
treatment
groups
were
filled
for
a
replicate.
Oocytes
with
an
intact,
compact
cumulus
investment
were
selected
and
washed
twice
in
C02-equilibrated
TC-199
with
Earle's
salts
(Gibco,
Grand
Island,
NY),
using
a
stereomicroscope
and
were
then
transferred
to
culture
(Leibfried
and
First,
1979).
In
Expts
1
and
2,
the
interaction
between
agents
increasing
oocyte
[cAMP]¡
and
different
macromolecular
supplements
in
the
culture
medium
was
investigated.
All
reagents
were
pur¬
chased
from
Sigma
(St
Louis,
MO),
unless
otherwise
stated.
Either
10
mmol
8-bromo-3'5'
cAMP
l"1
(8-Br-cAMP),
shown
by
Homa
(1988)
to
be
the
most
effective
analogue
and
the
most
effective
concentration
for
bovine
oocytes,
or
20
TJ
¡AC
ml
~
from
Bordetella
pertussis
(kindly
provided
by
E.
Hewlett,
University
of
Virginia
Medical
School;
activity
of
the
enzyme
that
maintained
50%
bovine
denuded
oocytes
in
meiotic
arrest
was
considered
10
U,
arbitrarily)
were
used
to
increase
[cAMP]¡.
Heat-treated
fetal
calf
serum
(FCS;
10%
v/v,
Gibco),
BSA
(3
mg
ml
~~
\
Fraction
V)
or
polyvinylpyrrolidone
(PVP;
40
kDa
molecular
mass,
3
mg
ml
~
)
were
used
as
macro¬
molecular
supplements.
Oocytes
were
cultured
in
TC-199
medium
(with
Earle's
salts)
supplemented
with
pyruvate
(0.2
mmol
1
~
'),
gentamicin
(25
µg
ml
~
:)
and
meiotic
inhibi¬
tors,
as
required
for
a
particular
experiment.
Treatment
groups
were
supplemented
with
10
mmol
8-Br-cAMP
I_
for
Expt
1,
and
20
U
invasive
adenylate
cyclase
ml
~
for
Expt
2.
Oocytes
were
cultured
in
96-well
culture
dishes,
with
25
complexes
in
125
µ
medium
in
5%
C02
in
air
with
high
humidity
at
39°C
for
20
h.
Follicular
fluid
aspirated
from
antral
follicles
was
supplemented
with
0.5
mmol
IBMX
I
~
1
+
0.5
mmol
8-Br
cAMP
1
~
:
for
Expt
1
and
0.5
mmol
IBMX
1
"
1
+
2
U
invasive
adenylate
cyclase
ml
~1
for
Expt
2.
In
these
and
all
exper¬
iments,
all
treatments
were
represented
in
each
replicate.
Experiment
3
was
designed
to
compare
different
preculture
treatments
that
might
prevent
commitment
before
culture
(i.e.,
during
aspiration,
collection
and
washing).
Follicular
fluid
was
supplemented
with
either
IBMX
(0.5
mmol
1
"
'),
IBMX
+
8-Br-
cAMP
(0.5
mmol
1
~
l),
IBMX
+
invasive
adenylate
cyclase
(2
U
ml
~
)
or
there
was
no
supplement.
Again,
each
treatment
was
represented
in
every
replicate.
Oocytes
collected
from
each
preculture
treatment
were
randomly
assigned
for
subsequent
culture
in
TC-199
with
PVP,
with
or
without
invasive
ade¬
nylate
cyclase,
for
20
h.
Intracellular
cAMP
in
whole
complexes
and
cumulus-free
oocytes
was
measured
to
determine
whether
differences
in
maintenance
of
germinal
vesicle
arrest
among
different
treatments
in
Expts
2
and
3
were
related
to
differences
in
initial
cAMP
concentrations
when
invasive
adenylate
cyclase
was
present.
In
Expt
2,
cAMP
was
measured
after
oocytes
had
been
in
culture
for
2
h.
In
Expt
3,
[cAMP]¡
was
measured
after
processing,
at
the
time
when
oocytes
would
normally
be
put
in
to
culture.
Experiment
4
was
designed
to
study
the
reversibility
of
inhibition
by
invasive
adenylate
cyclase.
Cumulus-oocyte
complexes
derived
from
FF
treated
with
IBMX
(0.5
mmol
1
~
:)
+
invasive
adenylate
cyclase
(2
U
ml
~
')
and
subsequently
cultured
in
culture
medium
supplemented
with
PVP
and
invasive
adenylate
cyclase
(20
U
ml
~
)
for
20
h
were
washed
twice
in
pre-equilibrated
TC-199,
transferred
to
TC-199
+
PVP
without
invasive
adenylate
cyclase
and
incubated
for
an
additional
20
h.
A
group
of
cumulus-oocyte
complexes
recovered
from
non-supplemented
FF
and
cultured
without
invasive
adenylate
cyclase
served
as
the
control.
Preparation
of
oocytes
for
fertilization
and
embryo
culture
In
Expt
5,
oocytes
(ten
in
a
drop)
recovered
from
FF
with¬
out
supplements
or
FF
supplemented
with
IBMX
+
invasive
adenylate
cyclase
were
subsequently
cultured
in
TC-199
with
LH
(5
µg
ml-1),
FSH
(0.5
µg
ml-1)
(both
gonadotrophins
were
ovine
derivatives
provided
by
NIH),
oestradiol
(1
µg
ml
~
:),
gentamicin
(25
µg
ml
~
)
and
pyruvate
(0.2
mmol
1
~
:)
in
50
µ
droplets
under
paraffin
oil
in
5%
C02
with
high
humidity
at
39°C
(Lenz
et
al,
1983)
for
22
h
(Sirard
et
al,
1988).
Frozen—thawed
semen
(American
Breeders
Service,
DeForest,
WI)
was
used
for
fertilization
using
a
swim-up
procedure
(Parrish
et
al,
1986).
Fertilization
medium
was
a
modified
Tyrode's
solution
supplemented
with
fatty-acid-free
BSA
(6
mg
ml
~
),
pyruvate,
gentamicin,
hypotaurine,
penicillamine,
adrenaline
and
heparin
(Leibfried
and
Bavister,
1982;
Parrish
el
al,
1986).
Glucose
was
eliminated
from
the
formulation.
The
final
concentration
of
spermatozoa
was
106
ml-1.
Embryos
were
stripped
of
cumulus
cells
and
transferred
onto
oviductal
cell
monolayers
(Eyestone
and
First,
1989)
48
h
after
insemi¬
nation,
cultured
for
an
additional
4
days
and
then
scored
for
morulae
and
blastocyst
formation.
Radiolabelling
and
two-dimensional
gel
electrophoresis
of
oocyte
phosphoproleins
Experiment
6
compared
phosphoprotein
profiles
of
oocytes
recovered
and
cultured
with
8-Br-cAMP,
or
invasive
adenylate
cyclase,
or
in
control
media
for
a
total
of
9
h.
Cumulus-oocyte
complexes
from
each
treatment
were
cultured
in
phosphate-
free
TC-199
medium
supplemented
with
0.5
µ
[
Pjortho-
phosphate
ml
~
:
(Amersham,
Arlington
Heights,
IL)
during
the
last
2
h
15
min.
At
the
end
of
the
labelling
period,
the
cumulus
cells
were
removed
in
phosphate-free
TC-199
and
washed
in
phosphate-free
TALP-Hepes
solution
(Bavister
et
al,
1983).
Seventy-five
oocytes
were
pooled
in
4
µ ,
flash-frozen
in
liquid
nitrogen
and
stored
at
—
20°C
until
electrophoresis.
Before
electrophoresis,
the
samples
were
frozen
and
thawed
several
times
and
1
µ
RNAse-DNAse
(20
mmol
Tris-HCl
1
~
\
10
mmol
MgCl2
1
"
\
60
mmol
NaF
1
"
\
2.2
mmol
PMSF
1
"
\
0.5
mg
RNAse
ml-
and
0.1
mg
DNAse
ml"
)
was
added.
Samples
were
incubated
on
ice
for
30
min.
A
lysis
buffer
(9.5
mol
urea
\~\
10%
w/v
CHAPS,
1.5%
pH
5-7,
1.5%
pH
6-8
and
1%
pH
3-10
ampholytes;
Bio-Rad,
Herculus
CA)
with
a
trace
of
fast
green
was
added.
The
samples
were
centrifuged
at
16
000
g
for
10
min,
and
the
supernatant
was
loaded
onto
tube
gels
(7
cm
in
length).
The
gel
mixture
was
9.15
mol
urea
1"\
10%
(w/v)
CHAPS,
4%
acrylamide
and
ampholines
(1.5%
pH
5-7,
1.5%
pH
6-8,
1%
pH
3-10).
The
upper
chamber
buffer
was
20
mmol
NaOH
1
~
1
and
the
lower
chamber
buffer
was
10
mmol
H3P04
1
~
.
The
gels
were
initially
run
for
10
min
at
200
V,
15
min
at
300
V
and
15
min
at
400
V
before
the
samples
were
loaded.
Loaded
gels
were
run
at
750
V
for
3.5
h.
The
first
dimension
gels
were
extruded,
incubated
in
solubilization
buffer
and
loaded
onto
8—15%
(w/v)
SDS
slab
gels
with
molecular
weight
markers
(Laemmli,
1970).
Silver-stained
gels
were
dried
between
two
pieces
of
cellophane
paper
under
a
vacuum.
Preflashed
X-ray
film
(X-AR-OMAT)
was
exposed
for
3
days
with
intensifying
screens,
developed
and
the
pixel
intensities
normalized
using
Collage
(Photodyne,
Hartland,
WI)
software.
The
data
were
analysed
by
completely
randomized
design
to
eliminate
errors
associated
with
replicates.
Measurements
of
cAMP
Oocytes
were
either
stripped
of
cumulus
cells
by
repeatedly
aspirating
complexes
through
a
glass
pipette
(200
µ
id.),
or
left
intact
and
washed
three
times
at
4°C
in
the
presence
of
0.5
mmol
IBMX
1
~
'
in
a
modified
Tyrode's
solution
without
glucose
(TL-Hepes;
Bavister
et
al,
1983).
A
fourth
wash
was
carried
out
under
the
same
conditions
in
the
absence
of
IBMX.
Each
experiment
was
performed
three
times
and
duplicate
samples
were
obtained
for
each
replicate.
Samples
of
five
oocyte—cumulus
complexes
or
50
denuded
oocytes
were
transferred
to
150
µ
extraction
medium
(TC-199)
in
10
µ
of
the
last
wash.
Similar
amounts
of
the
last
wash
were
used
as
blanks
for
each
group
and
the
cAMP
concentration
(back¬
ground
noise)
in
this
blank
was
subtracted
from
the
cAMP
concentration
measured
from
corresponding
samples.
Tri-
chloroacetic
acid
(10
µ
of
100%
w/v,
TCA)
was
added
to
the
samples,
and
they
were
then
vortexed
for
30
s
and
centri¬
fuged
at
16
000
g
for
8
min
at
4°C.
Twice
the
volume
of
a
4:6
mixture
of
tri-n-octylamine
(Sigma)
and
1,1,2-
trichlorotrifluoroethane
(Aldrich,
Milwaukee,
WI)
were
added
to
extract
the
TCA
(Chen
et
al,
1977;
Schoff
et
al,
1989).
Samples
were
frozen
in
liquid
nitrogen
and
stored
at
—
70°C
until
assayed.
A
radioimmunoassay
kit
from
Biomedicai
Tech¬
nologies,
Inc.
(Stoughton,
MA)
was
used
to
measure
cAMP;
the
sensitivity
of
the
assay
was
5
fmol
and
highly
specific
antibodies
raised
against
succinyl
cAMP-tyrosine
methyl
ester
were
used.
[I25I]succinyl
cAMP-tyrosine
methyl
ester
was
used
as
a
tracer.
The
assay
was
validated
by
adding
increasing
numbers
(20,
40,
60,
80)
of
oocytes
to
a
constant
volume
of
extraction
medium,
as
well
as
by
adding
standard
amounts
of
cAMP
to
samples
and
recovering
proportional
amounts
of
cAMP.
No
allowance
was
made
for
extraction
losses
which
were
presumed
to
be
5-10%
(Chen
et
al,
1977).
Data
evaluation
and
statistical
analysis
For
evaluation
of
meiotic
stage
after
culture,
oocytes
were
mounted
on
slides
beneath
a
coverslip
supported
by
3:1
petroleum
jelly:paraffin,
fixed
and
cleared
in
acid
alcohol
(3:1
ethanokacetic
acid)
and
examined
at
400
magnification
using
Nomarski
optics.
Oocytes
were
classified
as:
germinal
vesicle
stage,
with
intact
nuclear
membranes;
intermediate,
if
after
germinal
vesicle
breakdown
to
metaphase
I;
or
mature,
if
at
anaphase
I
to
metaphase
II.
Percentage
data
were
analysed
by
one-way
analysis
of
variance
(ANOVA)
with
or
without
arcsin
transformation,
and
the
same
results
were
obtained
in
each
case.
Means
were
compared
using
the
Newman—Keuls
method,
if
ANOVA
revealed
significant
differences
between
treatments.
The
per¬
centage
of
oocytes
remaining
at
the
germinal
vesicle
stage
was
used
as
the
end
point
for
comparison
of
treatments
with
inhibitors.
The
percentage
of
oocytes
matured
was
the
end
point
for
comparison
of
control
groups
and
reversal
of
invasive
adenylate
cyclase
maintained
arrest.
The
percentage
of
morulae
and
blastocysts
was
used
as
the
end
point
for
embryo
development.
The
content
of
cAMP
(fmol)
per
oocyte
or
per
oocyte—cumulus
complex
was
used
as
the
end
point
for
comparison
of
cAMP
measurements.
The
cAMP
data
(obtained
Table
1.
Effect
of
8-bromo
cAMP
(8-Br-cAMP)
on
germinal
vesicle
(GV)
maintenance
in
bovine
cumulus-oocyte
com¬
plexes
cultured
with
different
macromolecular
supplements
8-Br-cAMP
GV
Intermediate
Mature
Supplement"
(mmol
1
"
l)
(%)
±
SEMb
(%)
+
SEMb
(%)
±
SEMb
FCS
10
36+
7.4
58
±
6.9
6
+
2.4
FCS
0
7±
2.1
23+
3.8
70
±3.7
PVP
10 18
+
10.7
66
+
10.2
16
+
2.4
PVP
0
6+3.0
19
±
5.3
75
+
4.0
BSA
10
26
±
5.1
55
±
4.1
19
±
4.5
BSA
0
2±
2.2
28+
2.8
70
+
2.8
FCS:
fetal
calf
serum;
PVP:
polyvinylpyrrolidone.
aCumuIus—oocyte
complexes
were
collected
in
follicular
fluid
with
3-isobutyl
1-methylxanthine
(IBMX
0.5
mmol
1
"
')
+
8
bromo-cAMP
(0.5
mmol
1
"
')
added
at
5
ml
volume.
After
three
washes
in
control
medium,
complexes
were
placed
in
TC-199
plus
indicated
additives
for
20
h.
^Total
number
of
oocytes
within
each
treatment
ranged
from
71
to
100
over
five
replications.
No
difference
between
treatments
was
observed.
in
conjunction
with
Expt
3)
were
analysed
both
by
including
and
excluding
samples
derived
from
follicular
fluid
supple¬
mented
with
IBMX
+
8-Br-cAMP.
Correlations
between
cAMP
concentration
and
the
frequency
of
oocytes
remaining
at
the
germinal
vesicle
stage
were
calculated
by
simple
linear
regres¬
sion
after
excluding
the
treatment
group
derived
from
FF
supplemented
with
IBMX
+
8-Br-cAMP.
Results
Interaction
of
macromolecular
supplements
with
8-Br-cAMP
The
results
from
Expt
1,
in
which
cumulus-oocyte
com¬
plexes
recovered
from
FF
supplemented
with
IBMX
+
8-Br-
cAMP
were
cultured
for
20
h
with
different
macromolecular
supplements
(BSA,
PVP,
FCS)
with
or
without
8-Br-cAMP
(10
mmol
I~
),
are
given
in
Table
1.
Regardless
of
macro¬
molecular
supplement,
only
a
small
proportion
of
the
oocytes
remained
at
the
germinal
vesicle
stage
after
20
h
of
culture
when
incubated
with
8-Br-cAMP.
The
frequency
of
germinal
vesicle
arrest
did
not
differ
statistically
between
treatments
containing
8-Br-cAMP.
The
majority
of
the
oocytes
were
at
metaphase
I,
indicating
a
transient
delay
in
meiotic
resumption.
No
further
attempt
was
made
to
use
lower
or
higher
doses,
since
this
has
been
done
by
others
(Homa,
1988).
Oocytes
in
control
groups
reached
metaphase
II
at
reasonably
high
fre¬
quencies
regardless
of
the
macromolecular
supplement
present.
Interaction
of
invasive
adenylate
cyclase
with
different
protein
supplements
In
Expt
2,
oocytes
were
recovered
from
FF
supplemented
with
IBMX
+
invasive
adenylate
cyclase
and
cultured
for
20
h
with
different
macromolecular
supplements
(FCS,
PVP,
BSA),
with
or
without
20
U
invasive
adenylate
cyclase
ml
~
1
(Table
2).
Oocytes
cultured
in
medium
containing
FCS
had
a
lower
Table
2.
Effect
of
adenylate
cyclase
on
germinal
vesicle
(GV)
maintenance
in
bovine
cumulus-oocyte
complexes
cultured
with
different
macromolecular
supplements
iAC
GV
Intermediate
Mature
Supplement"
(U
ml
"
l)
(%)
±
SEMb
(%)
+
SEMb
(%)
+
SEMb
FCS
20
36±3.8C
60
±
3.7
4
+
2.2
FCS
0
8
+
5.4
37
±3.9
55
±4.1
BSA
20
52±3.7d
46
±
4.3
2
±1.9
BSA
0
7
±3.0
32
±4.6
61
±
2.2
PVP
20
53±4.7d
43
±5.1
3
+
1.8
PVP
0
16
±3.4
37
±2.7
47
±
4.3
FCS:
fetal
calf
serum:
PVP:
polyvinylpyrrolidone;
iAC:
invasive
adenylate
cyclase.
^Cumulus—oocyte
complexes
were
collected
in
follicular
fluid
with
invasive
cyclase
(2
U
ml
~
')
plus
3-isobutyl
l-methylxanthine
(IBMX;
0.5
mmol
1
"
')
added
at
5
ml
volume.
After
three
washes
in
control
medium,
complexes
were
placed
in
TC-199
medium
plus
the
indicated
additives
for
20
h.
The
total
number
of
oocytes
within
each
treatment
ranged
from
82
to
120
over
five
replications.
c
Maintenance
of
germinal
vesicle
(GV)
arrest
with
iAC
present
within
treatments
with
different
superscripts
is
significantly
different
(P<
0.05).
The
percentage
intermediate
was
not
analysed.
Table
3.
[cAMP]¡
in
bovine
oocyte
complexes
and
in
oocytes
cultured
with
adenylate
cyclase
and
different
macromolecular
supplements
cAMP
cAMP
Macromolecular
(fmol
per
complex)3
(fmol
per
oocyte)3
supplement
±
sem
+
SEM
FCS
119.4
±32.7
7.7
±
0.7
PVP
152.8
±11.4
7.5
±1.6
BSA
177.0
±21.8
9.0
±1.7
FCS:
fetal
calf
serum;
PVP:
polyvinylpyrrolidone.
^Complexes
were
incubated
for
2
h
in
invasive
adenylate
cyclase
(20
U
ml
~
J),
and
then
processed
for
cAMP
assay.
Some
were
stripped
of
cumulus
(50
oocytes
in
a
sample),
while
others
were
measured
as
whole
complexes
(five
complexes
in
a
sample).
Duplicate
samples
were
obtained
for
every
treatment
within
each
replicate.
Values
are
means
for
three
replications.
frequency
(P
<
0.05)
of
germinal
vesicle
maintenance
(36%)
than
did
those
cultured
with
either
PVP
(53%)
or
BSA
(52%).
Approximately
half
of
the
oocytes
cultured
with
either
BSA
or
PVP
in
the
presence
of
invasive
adenylate
cyclase
maintained
an
intact
germinal
vesicle
for
20
h.
Most
of
the
remaining
oocytes
in
these
three
treatments
also
showed
a
transient
delay
in
germinal
vesicle
breakdown,
since
few
were
classified
as
meiotically
mature
at
20
h.
Again,
oocytes
matured
equally
well
in
all
three
macromolecular
supplements.
In
conjunction
with
this
experiment,
cAMP
concentrations
in
whole
complexes
and
in
oocytes
alone
were
measured
after
the
complexes
had
been
cultured
in
invasive
adenylate
cyclase
(20
U
ml
~
)
for
2
h
with
different
macromolecular
supplements
to
determine
whether
differences
in
frequency
of
germinal
vesicle
maintenance
could
be
correlated
with
differences
in
cAMP
concentrations
(Table
3).
Neither
complexes
nor
Table
4.
Effect
of
preculture
collection
and
wash
procedures
on
germinal
vesicle
(GV)
maintenance
in
bovine
oocyte
complexes
subsequently
cultured
with
invasive
adenylate
cyclase
(iAC)
Inhibitor
in
iAC
GV
Intermediate
Mature
follicular
fluid3
(U
ml
"
')
(%)
±
sem
(%)
±
sem
(%)
±
sem
IBMX
+
iAC
20
65±2.8b
31
±4.7
4+1.5
0
10
±3.1
33
±5.2
57
±5.0
IBMX+
20
49±4.9C
43
±5.1
9
±
2.7
8-Br-cAMP
0
10
±
2.9
37
+
3.7
53
±
4.4
IBMX
20
43±4.9C
52
±5.4
5
+
2.1
0
11
+
5.1
33
±4.6
56
+
6.6
None
20
26
±
5.0d
62
±
3.8
13
+
3.4
0
9
±2.7
24
±4.4
67
+
3.5
a3-Isobutyl
l-methylxanthine
(IBMX)
and
8-bromo
cAMP
(8-Br-cAMP)
were
used
at
0.5
mmol
1~
\
iAC
at
2
U
ml~
'.
The
total
number
of
oocytes
used
within
a
treatment
ranged
from
83
to
110
over
five
replications.
Values
for
treatments
having
iAC
present
with
different
superscripts
are
significantly
different
(P
<
0.05).
The
percentage
of
intermediates
was
not
analysed.
cumulus-free
oocytes
had
statistically
different
cAMP
concen¬
trations,
suggesting
that
the
lower
frequency
of
germinal
vesicle
maintenance,
when
cultured
with
FCS,
was
due
to
interaction
of
yet
unknown
factors
in
FCS
with
cAMP.
The
use
of
FCS
as
a
supplement
in
culture
medium
when
studying
meiotic
arrest
was
discontinued
from
this
point.
In
this
exper¬
iment
no
control
measurements
were
made
of
untreated
oocytes
or
complexes;
however,
such
observations
were
made
in
a
later
experiment
(Table
5).
Comparison
of
the
estimates
of
cAMP
concentrations
show
that
they
were
about
ten
times
higher
in
the
presence
of
invasive
adenylate
cyclase.
Effect
of
supplementing
follicular
fluid
during
collection
on
subsequent
maintenance
of
meiotic
anest
Sirard
and
First
(1988)
suggested
that
bovine
oocytes
commit
to
undergo
meiosis
soon
after
removal
from
the
follicle.
Our
preliminary
studies
also
suggested
that
care
should
be
taken
to
prevent
premature
meiotic
commitment
during
aspiration,
collection
and
wash
procedures.
For
that
reason,
it
was
necessary
to
develop
a
practical
system
to
prevent
commitment
while
collecting
and
washing
the
bovine
material.
This
was
done
in
Expt
3.
Follicular
fluid
was
supplemented
with
IBMX
(0.5
mmol
l"1),
IBMX
plus
8-Br-cAMP
(0.5
mmol
1),
IBMX
plus
invasive
adenylate
cyclase
(2
U
ml
~
),
or
for
the
controls
there
was
no
supplement.
Oocytes
recovered
from
these
four
different
preculture
collection
treatments
were
allocated
randomly
for
culture
with
or
without
invasive
adenylate
cyclase
(20
U
ml_
I)
for
20
h
(Table
4).
Concen¬
trations
of
cAMP
in
complexes
or
oocytes
were
measured
at
the
time
when
they
would
have
been
transferred
to
culture,
to
determine
whether
preculture
procedures
affected
[cAMP]¡
at
the
onset
of
culture
(Table
5).
Oocytes
from
FF
supplemented
with
IBMX
+
invasive
ade¬
nylate
cyclase
had
the
highest
frequency
of
germinal
vesicles
(65%),
whereas
arrested
oocytes
from
FF
supplemented
with
IBMX
alone
(43%)
or
IBMX
plus
8-Br-cAMP
(49%)
were
Table
5.
[cAMP]¡
after
preculture
procedures
for
collecting
bovine
cumulus-oocyte
complexes
cAMP
cAMP
Inhibitor
added
to
(fmol
per
complex)
(fmol
oocyte
per)
follicular
fluid
±
sem
±
sem
IBMX
+
¡AC
27.9
±
4.5a
0.97
±0.16"
IBMX
+
8
Br-cAMP
240.4
±
94.7b
13.47
±5.90b
IBMX
15.2
±
2.9C
0.54±0.05c
None
10.9
±
1.5C
0.50±0.04c
IBMX:
3-isobutyI
1-methylxanthine;
iAC:
invasive
adenylate
cyclase;
8
Br-cAMP:
8-bromo
cAMP.
a-cWithin
a
column,
values
for
treatments
with
different
superscripts
are
significantly
different
(P<0.05).
Means
are
presented
for
three
replications.
Fifty
denuded
oocytes
and
five
complexes
were
assayed
per
sample.
Duplicate
samples
of
each
treatment
were
taken
at
every
replication.
intermediate
(Table
4).
The
oocytes
treated
with
invasive
adenylate
cyclase
recovered
from
unsupplemented
FF
(26%)
were
similar
to
controls
(P
>
0.05).
These
data
suggest
that
bovine
oocytes
begin
commitment
to
undergo
meiotic
maturation
if
they
are
kept
in
FF
for
>
2
h.
None
of
the
pretreatments
affected
the
frequency
of
maturation
in
control
groups.
The
[cAMP]¡
of
both
complexes
(28
fmol)
and
oocytes
(0.97
fmol)
recovered
from
IBMX
+
iAC
FF
supplement
was
higher
than
that
of
complexes
(10.9
fmol)
or
oocytes
(0.5
fmol)
derived
from
unsupplemented
FF
(
<
0.05,
Table
5)
when
measured
after
preculture
treatments.
The
complexes
and
the
oocytes
recovered
from
FF
with
IBMX
added
had
intermediate
[cAMP]¡
(15.15
and
0.54
fmol
in
complexes
and
oocytes,
respectively),
which
correlates
with
their
degree
of
retention
of
the
germinal
vesicle.
Oocytes
recovered
from
FF
supplemented
with
IBMX
plus
8-Br-cAMP
had
very
high
contents
of
[cAMP]¡,
mostly
attributable
to
the
cAMP
analogue,
yet
maintenance
of
meiotic
arrest
was
only
comparable
with
oocytes
recovered
from
FF
supplemented
with
IBMX
alone.
The
role
of
cAMP
in
meiotic
arrest
was
assessed
by
estimating
the
correlation
between
the
maintenance
of
the
germinal
vesicle
and
the
concentration
of
cAMP
after
the
different
pretreatments.
The
correlation
was
0.93
for
cumulus-oocyte
complexes
and
0.78
for
denuded
oocytes,
when
oocytes
derived
from
IBMX
+
8-Br-cAMP
supplemented
FF
were
not
considered.
This
group
was
excluded
from
further
analysis
as
representing
an
outlier.
Reversibility
of
inhibition
Cumulus-oocyte
complexes
recovered
from
IBMX
+
invasive
adenylate
cyclase
supplemented
FF
and
cultured
in
the
presence
of
iAC
for
20
h
were
washed
twice
and
cultured
in
the
same
medium
without
invasive
adenylate
cyclase
for
an
addi¬
tional
20
h
to
test
whether
the
effect
of
iAC
on
oocytes
was
reversible.
The
results
(Table
6)
show
that
inhibition
of
germinal
vesicle
breakdown
by
invasive
adenylate
cyclase
was
reversible.
Although
not
statistically
different,
the
slightly
lower
frequency
for
completion
of
meiotic
maturation
may
be
Table
6.
Reversibility
of
meiotic
arrest
in
vitro
of
bovine
oocytes
maintained
by
invasive
adenylate
cyclase
(iAC)
Inhibitor
GV
Intermediate
Mature
in
culture
(%)
±
sem
(%)
±
sem
(%)
±
sem
iAC
58±4.4b
41
+
3.3
3
±
1.4b
Control
11
±5.Ie
29
±5.8
60
±
6.7e
Reversal3
15
±
3.8e
36
±
3.6
49
±
7.0e
GV:
germinal
vesicle.
""Reversal:
complexes
cultured
with
iAC
for
20
h,
and
then
cultured
for
a
further
20
h
without
iAC
Total
number
of
oocytes
within
each
treatment
ranged
from
73
to
99
over
five
replications.
^Within
a
column,
values
for
treatments
with
different
superscripts
are
significantly
different
(P
<
0.05).
The
percentage
of
intermediates
was
not
analysed.
Table
7.
Development
of
bovine
oocytes
after
suppressing
meiotic
resumption
during
collection
procedures
Inhibitor
added
to
>
2
cell
Morulae
and
follicular
fluid
(%)a
blastocysts
(%)b
IBMX
+
iAC
283
54 27
None
342
55
26
aThe
number
of
fertilized
oocytes
cleaving
at
least
once
48
h
after
adding
spermatozoa/total
number
of
oocytes
cultured
(n)
averaged
over
three
replicates.
^Number
of
morulae
and
blastocysts
observed
6
days
after
fertilization/total
oocytes
(n)
cultured
averaged
over
three
replicates.
due
to
the
time
necessary
for
neutralization
or
clearance
of
invasive
adenylate
cyclase
from
the
cells.
Developmental
capacity
of
oocytes
recovered
from
follicular
fluid
supplemented
with
IBMX
+
invasive
adenylate
cyclase
Experiment
5
was
designed
to
evaluate
possible
detrimental
effects
of
supplementing
FF
with
IBMX
plus
invasive
adenylate
cyclase
during
the
2—3
h
processing
period
on
the
subsequent
developmental
potential
of
oocytes.
Treated
oocytes
were
cultured
in
standard
maturation
medium
for
24
h,
and
were
then
fertilized
and
evaluated
for
development
with
a
control
group
recovered
from
FF
that
was
not
supplemented.
Oocytes
in
both
groups
cleaved
and
progressed
to
morula
and
blasto¬
cyst
stages
in
vitro
at
equal
frequencies
(Table
7),
indicating
that
supplementation
of
FF
with
IBMX
plus
invasive
adenylate
cyclase
during
collection
was
not
detrimental.
Phosphoprotein
profiles
of
bovine
oocytes
In
Expt
6,
phosphoprotein
profiles
of
oocytes
maintained
in
meiotic
arrest
permanently
by
treatment
with
invasive
adenylate
cyclase
or
transiently
by
8-Br-cAMP
were
analysed.
Cumulus-enclosed
oocytes
were
cultured
for
9
h
with
either
20
U
iAC
ml"1,
10
mmol
8-Br-cAMP
1
or
in
control
medium.
The
treatment
groups
were
transferred
to
similar
Fig.
1.
Phosphoprotein
profiles
of
bovine
oocytes
maintained
in
meiotic
arrest
with
(a)
(iAC)
invasive
adenylate
cyclase,
(b)
transiently
with
8-bromo-cAMP
or
(c)
maturing
spontaneously.
Complexes
were
cultured
in
20
iu
iAC
ml-1,
10
mmol
8-Br-cAMP
1~:
or
control
medium
only
for
a
total
of
9
h.
For
the
last
2
h
15
min,
the
complexes
were
transferred
to
phosphate-free
medium
supplemented
with
[32P]orthophosphate
(0.5
µ
ml
~
l)
and
the
appropriate
inhibitor.
At
the
end
of
culture,
cumulus
cells
were
removed
and
oocytes
were
washed
in
phosphate-free
TL-Hepes
several
times.
A
gel
is
shown
that
used
75
oocytes
per
lane,
selected
from
five
replications,
where
each
replicate
used
oocytes
collected
on
separate
days.
Two
29
kDa
proteins
(circled)
are
shown
which
become
minimally
phosphorylated
in
transiently
arrested
and
spontaneously
maturing
oocytes.
Arrows
indicate
24, 25,
and
2
kDa
proteins,
which
become
more
heavily
phosphorylated
in
these
two
types
of
oocyte.
culture
medium
(phosphate-free
TC-199
medium
with
added
[32P]orthophosphate)
for
the
last
2
h
15
min
of
culture.
Table
8.
Phosphoproteins
in
bovine
oocytes
maturing
sponta¬
neously,
or
maintained
in
germinal
vesicle
arrest
with
invasive
adenylate
cyclase
(iAC),
or
transiently
with
8-bromo-cAMP
(8-Br-cAMP)
Intensity
(pixels
IO3)
±
sem3
Protein
_
(kDa)
Control
8-Br-cAMP
iAC
29
1±1
5±
0.2
9±2
29
3±1
4±2
10
±2
27
115
±8
107
±10
25
±
3
25
47
±5
43
±
6
8
+
0.8
24
30
±
1
29
±
6
12
±
5
aPixel
intensities
from
gels
were
averaged
over
five
replications,
each
consist¬
ing
of
gels
run
with
oocytes
recovered
on
different
days.
Oocytes
completely
maintained
in
meiotic
arrest
(i.e.,
cultured
in
the
presence
of
invasive
adenylate
cyclase,
90%
at
germinal
vesicle
stage
at
9
h)
showed
phosphoprotein
profiles
dissimilar
to
those
of
mature
oocytes
(Fig.
1;
Table
8).
These
oocytes
contained
two
prominent
29
kDa
phosphoproteins.
Moreover,
27,
25
and
24
kDa
proteins
seen
in
mature
oocytes
were
only
minimally
phosphorylated.
Oocytes
cultured
with
8-Br-cAMP
(maintained
in
meiotic
arrest
transiently)
showed
a
phospho¬
protein
profile
indistinguishable
from
control
oocytes
(10%
at
germinal
vesicle
stage),
although
they
still
had
distinct
germinal
vesicles
(85%
intact
germinal
vesicles).
Oocytes
cultured
with
8-Br-cAMP
and
those
cultured
in
control
medium
showed
maturation
related
phosphoproteins
of
27,
25
and
24
kDa
and
two
29
kDa
proteins
that
were
minimally
phosphorylated
compared
with
oocytes
treated
with
invasive
adenylate
cyclase.
These
differences
were
obviously
and
con¬
sistently
present
in
all
five
replications
and
indicated
that,
during
transient
delay
of
meiotic
arrest,
post-translational
modifications
in
proteins
related
to
maturation
still
occur.
Discussion
These
studies
have
demonstrated
that
bovine
oocytes
can
be
reversibly
maintained
in
meiotic
arrest
for
an
extended
period
by
increasing
[cAMP]¡;
therefore,
cAMP
may
play
a
role
in
maintaining
meiotic
arrest
in
bovine
oocytes
as
in
other
animals.
Precautions
to
prevent
premature
commitment
and
choice
of
macromolecular
media
supplements
were
important
aspects
of
this
model.
Use
of
a
preculture
treatment
to
prevent
premature
commitment
to
meiotic
resumption
did
not
impair
the
developmental
potential
of
the
oocytes.
Bovine
oocytes
could
not
be
kept
in
meiotic
arrest
with
cAMP
analogues
such
as
8-Br-cAMP
or
dibutyryl-cAMP
(db-cAMP,
data
not
shown);
moreover,
the
phosphoprotein
profiles
of
oocytes
treated
with
8-Br-cAMP
clearly
demonstrated
that
during
transient
arrest,
post-translational
modification
of
proteins,
characteristic
of
maturing
oocytes,
continue
to
occur.
These
changes
were
not
found
in
oocytes
maintained
in
meiotic
arrest
with
invasive
adenylate
cyclase.
Since
the
demonstration
by
Mailer
and
Krebs
(1977)
that
progesterone-induced
oocyte
maturation
in
Xenopus
is
inhibited
by
analogues
of
cAMP
or
phosphodiesterase
inhibitors,
numerous
studies
have
been
undertaken
to
elucidate
possible
functions
of
cAMP
in
maintaining
mammalian
oocytes
in
meiotic
arrest
(for
review,
see
Schultz,
1988).
It
is
now
considered
that
intracellular
concentrations
of
cAMP
regulate
the
meiotic
state
of
oocytes
in
many
amphibians
(Mailer
and
Krebs,
1977)
and
mammals
(Bornslaeger
et
al,
1986).
Both
progesterone-induced
Xenopus
oocyte
maturation
(Cicirelly
and
Smith,
1985)
and
spontaneous
mouse
oocyte
maturation
(Schultz
et
al,
1983)
are
accompanied
by
a
decrease
in
intra-oocyte
cAMP
concentrations.
Interference
with
this
reduction
prevents
maturation
(Schultz
et
al,
1983;
Vivarelli
et
al,
1983).
Several
studies
(Rice
and
McGaughey,
1981;
Homa,
1988;
Sirard
and
First,
1988)
carried
out
with
oocytes
of
mammals
with
long
oestrous
cycles
have
failed
to
produce
unequivocal
results.
Studies
with
oocytes
(Tornell
et
al,
1984)
and
other
cells
in
vitro
(for
review,
see
Lohmann
and
Walter,
1984)
have
shown
that
cAMP
analogues
may
differ
and
that
their
effects
on
cell
growth
may
be
different
from
those
of
cellular
cAMP.
As
reviewed
by
Lohman
and
Walter
(1984),
the
action
of
cAMP
and
its
analogues
may
be
modified
by
the
presence
of
serum,
growth
factors
and
hormones
in
culture
medium,
as
we
observed
in
the
bovine
model
for
studying
meiotic
regulation.
The
conflicting
results
reported
on
the
effect
of
cAMP
on
oocyte
maturation
in
domestic
animals
may
result
from
differ¬
ent
means
of
increasing
[cAMP]¡
and
different
culture
condi¬
tions
(i.e.,
supplementation
with
serum
or
hormones).
A
further
complication
arises
because
bovine
oocytes
(Sirard
and
First,
1988),
like
those
of
other
species
(Dekel
and
Beers,
1980;
Schultz
et
al,
1983),
may
commit
to
undergo
meiosis
during
the
collection
process.
These
experiments
were
therefore
con¬
ducted
to
describe
the
optimal
culture
conditions
and
means
to
increase
[cAMP]¡
for
the
study
of
effects
of
cAMP
on
bovine
oocyte
maturation.
The
effects
of
increased
[cAMP]¡
were
investigated
by
using
cumulus-intact
bovine
oocytes
to
compare
different
agents
that
increase
[cAMP]¡
and
determining
how
these
substances
interact
with
the
macromolecular
components
of
maturation
medium.
An
attempt
was
also
made
to
describe
an
optimal
system
for
collecting
these
oocytes
before
culture.
Homa
(1988)
demonstrated
that
8-Br-cAMP
was
more
potent
than
dibutyryl-cAMP
in
inhibiting
bovine
oocyte
maturation.
Preliminary
studies
showed
that
the
same
concentration
of
8-Br-cAMP
was
more
potent
than
db-cAMP
when
inhibition
of
progression
beyond
metaphase
I
was
taken
as
an
end
point
(data
not
shown).
Our
studies
showed
that
8-Br-cAMP,
even
when
used
at
a
concentration
of
10
mmol
I
~
:
(reported
by
Homa,
1988,
to
be
the
most
effective
concentration
in
maintaining
bovine
oocytes
in
meiotic
arrest
transiently)
was
a
weak
inhibitor
of
germinal
vesicle
breakdown
of
bovine
oocytes,
and
invasive
adenylate
cyclase
could
inhibit
germi¬
nal
vesicle
breakdown
in
bovine
oocytes
at
a
high
frequency
with
reproducible
results,
when
PVP
and
BSA
were
used
as
macromolecular
supplements
in
culture
medium.
The
correla¬
tion
between
cAMP
concentration
in
oocytes
and
complexes
and
inhibition
of
germinal
vesicle
breakdown
suggests
that
the
enzyme
acts
through
cAMP.
By
contrast,
analogues
of
cAMP
were
less
effective
at
preventing
germinal
vesicle
breakdown.
Fulka
et
al.
(1993),
by
fusing
mouse
and
bovine
oocytes,
showed
that
cAMP
analogues
fail
to
block
germinal
vesicle
breakdown
in
both
mouse
and
bovine
oocyte
nuclei
within
the
same
cytoplasm,
even
at
doses
as
high
as
5
mmol
1
~
.
However,
both
types
of
nuclei
in
the
fused
cell
remained
intact
when
cultured
with
IBMX
(1
mmol
1
~
).
These
data
suggest
that,
although
bovine
oocytes
are
insensitive
to
cAMP
analogues
and
that
maturation
promoting
factor
created
in
bovine
oocyte
cytoplasm
in
the
presence
of
these
analogues
causes
germinal
vesicle
breakdown
in
both
types
of
nucleus,
bovine
oocytes
are
sensitive
to
changes
in
concentration
of
cellular
cAMP.
The
demonstration
by
Homa
(1988)
that
IBMX,
a
phosphodiesterase
inhibitor
that
causes
accumulation
of
cAMP,
was
far
more
effective
than
either
dibutyryl-cAMP
or
8-Br-cAMP
in
maintaining
meiotic
arrest
is
also
in
agreement
with
our
results.
The
weak
effect
of
8-Br-cAMP
may
be
due
to
its
inability
to
cross
bovine
oocyte
cell
membranes.
However,
our
measurements
of
the
cAMP
content
of
bovine
oocytes
showed
that
8-Br-cAMP
would
accumulate
in
bovine
oocytes
very
efficiently.
Under
the
assay
conditions
used
in
the
study
reported
here,
similar
concentrations
of
8-Br-cAMP
and
stan¬
dard
cAMP
displaced
I25I-labelled
tracer
to
the
same
extent.
The
difference
between
the
ability
of
8-Br-cAMP
and
cAMP
produced
by
invasive
adenylate
cyclase
to
maintain
meiotic
arrest
may
be
due
either
to
a
difference
in
their
subcellular
distribution
or
in
their
ability
to
bind
regulatory
subunits
on
the
cAMP-dependent
protein
kinase.
Earlier
work
with
sheep
(Moor
and
Heslop,
1981;
Crosby
et
al,
1985;
Moor,
1988)
suggested
that
increased
[cAMP]¡
would
not
inhibit
oocyte
maturation.
Gonadotrophins
included
during
the
culture
of
ovine
oocytes,
rather
than
species
difference,
may
account
for
the
observed
differences
in
patterns
of
[cAMPjj.
A
positive
stimulus
from
follicle
cells
induced
by
gonadotrophins
may
cause
nuclear
maturation
without
a
con¬
comitant
fall
in
cAMP
concentration
(Downs
et
al,
1988)
as
we
have
observed
in
cattle.
An
invasive
adenylate
cyclase
has
been
used
by
Aberdam
et
al
(1987)
at
higher
concentrations
and
in
combination
with
IBMX
and
pertussis
toxin
on
rat
oocytes,
and
results
com¬
parable
to
those
reported
here
were
obtained.
Aberdam
et
al
(1987)
and
others
(Wheeler
and
Veldhuis,
1988;
Gilboa-Ron
et
al,
1989;
Gordon
et
al,
1989)
showed
that
the
invasive
adenylate
cyclase
accumulated
in
the
host
cells,
and
was
inactivated
by
the
host
cells
in
a
time-dependent
fashion
if
cells
were
transferred
to
a
medium
without
invasive
adenylate
cyclase.
The
concentration
of
crude
preparations
of
invasive
adenylate
cyclase
used
initially
was
inferred
from
a
previous
study
using
the
same
preparation
(Wheeler
and
Veldhuis,
1988).
This
preparation
and
others
were
later
characterized
using
maintenance
of
meiotic
arrest
in
denuded
bovine
oocytes
as
a
bioassay.
In
rat
cumulus-oocyte
complexes,
the
inactivation
and
resulting
recovery
from
the
effect
of
invasive
adenylate
cyclase
was
detectable
by
1
h
and
completed
by
24
h.
When
invasive
adenylate
cyclase
plus
IBMX
were
added
to
FF
during
collec¬
tion
and
processing
of
the
complexes,
no
long
lasting
effects
were
noted,
since
both
the
frequency
of
meiotic
maturation
and
the
formation
of
late
stage
preimplantation
embryos
were
similar
to
control
values.
Thus,
pretreatment
during
collection
prevents
spontaneous
commitment
of
bovine
oocytes
to
meiotic
resumption
and
allows
study
of
the
mechanisms
for
meiotic
regulation
of
the
oocyte.
Our
demonstration
that
inhibition
of
oocyte
maturation
for
20
h
in
culture
by
invasive
adenylate
cyclase
is
reversible
is
in
agreement
with
other
findings
that
inhibition
of
oocyte
maturation
by
either
cAMP
analogues
or
agents
that
increase
[cAMP]¡
is
reversible
in
mouse,
rat,
pig
and
hamster
oocytes
(Rice
and
McGaughey,
1981;
Schultz
et
al,
1983;
Aberdam
et
al,
1987;
Racowsky,
1985b).
Since
reversal
of
the
extended
arrest
was
possible
without
compromising
the
developmental
potential
of
most
of
the
oocytes
(Aktas
et
al,
1991b;
Aktas,
1994),
we
consider
that
[cAMP]¡
has
a
physiologically
relevant
role
in
maintaining
meiotic
arrest.
Meiotically
arrested
bovine
oocytes
have
different
phospho¬
protein
profiles
from
transiently
arrested
or
spontaneously
maturing
oocytes.
Differences
in
the
phosphoproteins
of
mature
and
immature
oocytes
have
been
demonstrated
in
mouse
(Bornslaeger
et
al,
1986)
and
frog
(Mailer
and
Smith,
1985)
oocytes.
That
the
oocytes
incubated
with
8-Br-cAMP
show
a
phosphoprotein
profile
similar
to
that
of
control
oocytes,
even
while
possessing
intact
germinal
vesicles,
points
to
a
possible
reason
why
invasive
adenylate
cyclase,
but
not
8-Br-cAMP,
maintains
bovine
oocytes
in
meiotic
arrest
for
an
extended
period.
Whether
these
phosphorylation
and
dephos-
phorylation
events
are
the
cause
or
the
result
of
meiotic
resumption
is
not
yet
clear.
Similarly,
it
is
not
known
whether
two
29
kDa
phosphoproteins
prominent
in
meiotically
arrested
oocytes
are
responsible
for
meiotic
arrest
or
are
the
result
of
these
events.
Several
proteins
of
40—80
kDa,
which
were
heavily
phosphorylated
in
mature
or
maturing
oocytes,
are
likely
to
be
structural
proteins
(i.e.
lamins)
the
phosphorylation
of
which
is
required
for
germinal
vesicle
breakdown
to
occur.
The
small
gels
used
in
this
study
do
not
facilitate
further
separation
of
these
proteins.
In
conclusion,
the
results
of
these
studies
suggest
that
[cAMP]¡
has
a
physiological
role
in
maintaining
meiotic
arrest
in
bovine
primary
oocytes.
They
also
present
a
useful
model
for
further
study
of
meiotic
regulation
in
mammals
with
long
oestrous
cycles.
Although
previous
work
in
domestic
species
has
indicated
that
[cAMP]¡
plays
a
role
in
the
maintenance
of
meiotic
arrest,
as
in
other
animals,
the
work
presented
here
provides
a
model
for
studying
meiotic
arrest
when
post-
translational
mechanisms
regulating
meiotic
resumption
are
held
in
abeyance.
This
is
in
strong
contrast
to
the
situation
in
which
only
a
transient
delay
in
meiotic
resumption
is
induced.
The
authors
thank
E.
Hewlett
for
generously
providing
the
adenylate
cyclase
and
expert
advice,
P.
Schoff
and
L.
Hagemann
for
helpful
comments
on
the
manuscript,
L.
Hewitson
for
editorial
assistance,
J.
Busby
and
B.
Wagner
for
typing
the
manuscript
and
B.
Haley
for
obtaining
animal
material.
Research
was
supported
by
the
College
of
Agricultural
and
Life
Sciences,
University
of
Wisconsin-
Madison
and
CSRS-NRICGP
Grant
88-37240-3740
to
ML
Leibfried-
Rutledge.
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