BIOLOGY
OF
46,
REPRODUCTION
Influence
of Cell
492-500
(1992)
Cycle
Stage
of the
Donor
Transplant
PHILIPPE
Department
of Veterina?y
and
COLLAS,3
Animal
Nucleus
Rabbit
Sciences,
of Nuclear
Embryos1
J. BALISE,
JOHN
on Development
and
University
JAMES
M. ROBL3
of Massachusetts,
Amherst,
Massachusetts
01003
ABSTRACT
We
evaluated
embryos.
The
of
G I phase
in
the
the
in
G1
phase
early
nuclear
30
DNA
by
the
to arrest
synthesis
donor
donor
nuclei
the nuclear
responsible
and
synthesis,
G I phase
rate,
generates
at the
to the
late
and
INTRODUCTION
The transplantation
vated mature
oocytes
of nuclei
into enucleated,
results
in extensive
remodeling
nucleus.
The profile
of nuclear
remodeling
has
been
characterized
in rabbit
nuclear
transplant
embryos
[1]. Remodeling
nuclear
envelope
of the
donor
breakdown
and
condensation
(PCC),
followed
tion and swelling
of the nucleus
cyte [1]. Because
chromosome
limited
chromatin
necessary
for
modeling
observed
f 2-5]
plants
nonactivated
optimum
and
nuclear
in
nuclei
mouse
[2] and pig
in these
embryos.
embryos
[6] suggest
In addition,
PCC
have
to be
bit
been
shown
nuclear
sumably,
ming
sion
transplant
are
embryos
necessary
of the
donor
PCC has
studies
beneficial
for
nucleus
[7-10].
to metaphase
(M phase)
phology
of the condensed
November
Received
This
July
work
was
‘Correspondence,
‘Current
address:
GenMark,
Inc.,
421
Wakara
Was’,
No.
Suite
con-
that
only
have
cytoplasm.
a greater
In addition,
potential
of the
hybrid
pre-
201,
Salt
the
into
cleavage
of the
was
turely
UT
492
and
G2 chro-
markers
than
prematurely
[12].
This
suggests
chromatin,
cell
cycle
conthat
pre-
as opposed
to the genome.
in cell hybrids
the
of
controlling
into
zygotes
influence
mature
stage
cell
cell
stage
embryos
cycle
cell
to
Therefore,
seems
to be
of the
donor
of mice
cycle
in these
species
performed.
condensing
were
in
The
stage
of nuclei
been
investigated.
[15, 16] and
Species.
for
chromatin,
cell
fusions
In
cattle
[17],
most
cell
cycles
[19], and rabbits
also occupies
[20],
most
It is therefore
used
trans-
Short
S phase.
S phase,
since
more
detrimental
S phase
in somatic
when
[13, 14];
not
by the
of nuclei
stage
reported
cycle
has
is occupied
cycle
has been
of cell
oocytes
proportion
G2 chromatin,
84108.
chromo-
cell
in early embryros
of pigs [18], sheep
presumably
indicate
that DNA synthesis
morstage
Lake City,
upon
Gi
into
of contributing
of S phase
nuclei
however,
106 to J.M.R
88-37240-4
chromosomes
importance
transplanting
reprogram-
condenses
chro-
at fusion.
The
of rab-
studies
chromatin
dependent
cell
these
grant
are
indicate
These
largely
in reconstituted
a large
by USDA
of
which
and
G2, is more
detrimental
of the donor
genome
[1].
in pan
progression
fragments
Gi and
integrity
1991.
supported
that
embryos,
to heterogenous
[1]. Nuclear
renuclear
trans-
4, 1991.
12,
of
leads
donor
genome
of the
Accepted
effect
to blastocysts
dispersed
condensation
In particular,
the
depends
on the
beneficial
embryos
small
[7-11].
in M phase
ferred
cytoplasm.
chromatin
of
as assessed
S phase
with
regions
to the
extensively
described
in somatic
cell fuPCC is induced
in interphase
cells fused
been
in the
mature
[1] and,
The
in 0.5
JLg/mI
reversible,
transplant
is
functional
in 0.1
It is suggested
in nuclear
elements
somes
of the
development
(approximately
to cleave
embryos.
0.001).
<
late-stage
incubation
was
S phase
to blastocysts
complete
p
nonreplicating
that PCC also occurs
and nuclear
swelling
for
treatment
nucleus.
and
a 10-h
of manipulated
defects
mosomes
cytoplasm
thymocyte
This
of treated
(15%,
cellular
allowed
donor
mid-
embryos
densed
oocyte
of donor
S phase
other
were
transition.
densed
when
exposure
swelling
mouse
swelling
occurs
embryos
G 1 phase
synchronized
of the
with
first,
Duration
nuclei
cycle
obtained
transplant
in the
donor
with
of the
entailed,
nuclear
embryros
32-cell-stage
and
This
of development
matin
by chromatin
decondensaupon
activation
of the ooswelling
phase.
condensation
first,
chromosome
is prevented,
to
involves,
premature
or no nuclear
condensation
denuded
nucleus
rate
donor
stage
of
determined.
was
of the cycle
of the interphase
nucleus.
Prematurely
condensed
G 1 and G2 nuclei
display
elongated,
yet unfragmented,
chromosomes
with single
and double
chromatids,
respectively
[7-11].
In contrast,
induction
of premature
nonactiof the
exogenous
recently
in 16-
G1
Gi /S
cycle
3% were
and
to blastocysts
enhanced
chromosome
phase
colcemid,
development
by the
as opposed
(71%),
Gi
in vitro
cell
embryos
the
by
32%
in the
blocked
and
confirmed
transplant
whereas
from
development
of the
synchronizing
affected
of the
release
on
stages
reversibly
greatly
blastomeres
remained
cleavage
was
donor
in the cell cycle
for impaired
development.
duration
After
late
of nuclear
was
blastocysts,
short
in metaphase.
blastomeres
in the
The
nucleus
and
for
in vitro
synchronizing
donor
mid-,
and a procedure
transplant
0.001).
for
of the
in early,
to blastocysts
nuclear
<
cycle
of development
aphidicoliti,
of DNA
early-stage
with
to 59%
embryos
inhibitor,
resumption
using
extent
(p
cell
of nuclei
Development
a procedure
colcemid
the
the
led
of the
determined,
was
respectively
necessitated
g/ml
embryos
nuclei
donors,
mm)
stage
potential
evaluated.
was
Use of early-stage
of the
developmental
In addition,
developed.
was
influence
the
rabbit
likely
nuclear
no
in
synchronization
effect
of prema-
as opposed
raises
that
transfer
the
to Gi
possibility
or
DONOR
that
the
ical
factor
cell
cycle
in
Stage
the
of the
donor
development
CELL
CYCLE
nucleus
of
may
nuclear
STAGE
be a crit-
transplant
em-
bryos.
The present
study
therefore
investigated
the effect
of cell cycle stage of the donor
nucleus
on development
of
reconstituted
rabbit
embryos.
The developmental
potential
in vitro
of nuclei
in early,
was evaluated.
cycle
bit embryos
was
nizing
blastomeres
determined
reconstituted
The results
was
Duration
determined,
in Gi
as donor
of donor
improved
bryros
and
late
of the
stages
G 1 phase
of the
cell
in early
rab-
and a procedure
for synchrowas developed.
In addition,
we
the extent
of development
in vitro of embryos
with nuclei
in the Gi phase
(Gi transplants).
indicate
that the extent
of development
in vitro
reduced
The use
matically
mid-,
nuclei
progressed
blastomeres
development
in the
in the Gi
of nuclear
cell
cycle.
phase
thus
transplant
draem-
in vitro.
MATERIALS
Recipient
Oocyte
Mature
female
AND
METHODS
were
superovulated
with
jections
of 0.3 mg FSH (Burnes
Biotech,
followed
by a single
dose
of 75 IU hCG
MO).
Mature
oocytes
were
flushed
from
15 h after hCG administration
by means
of
37#{176}C
Earle’s
balanced
(EBSS/FCS;
pH 7.2),
mg/ml
hyaluronidase
six
in-
Omaha,
NE) [21]
(Sigma,
St. Louis,
the oviducts
14previously
gassed
salt solution
and 10% fetal calf serum
and the cumulus
was removed
with 1
(Sigma)
as previously
described
[22, 23].
TRANSPLANTATION
jection
mm
of the
Embryos
Mature
mixed-breed
mated
to an intact
male
16-cell
stage
females
were
superovulated
at the time of hCG injection.
embryos
were
oocyte
chamber
taining
current
recovered
and
Eight-
the
from
ovi-
493
donors,
Embryos
at 30#{176}C
overlaid
were
with
100 pM CaCI2 and
pulses
of 2.2 ky/cm
0.30
placed
in a 0.5-
M mannitol
con-
100 p.M MgCl2 [23]. Six direct
for 60 p.sec, each pulse
30 mm
apart,
starting
18-19
h post-hCG,
were
given
using
a BTX
Electro
Cell Manipulator
200 (BTX,
Inc., San Diego,
CA).
Embryos
were placed
in culture
(see below)
between
pulses.
After
g/ml
the
last stimulation,
embryos
were
incubated
cytochalasin
B for 1 h prior
to culture.
Blastomere
and
Embryo
Isolated
blastomeres,
manipulated
embryos
Culture
and
nuclear
were
cultured
(VH) of the eyes of female
([1); P. Collas,
RI. Duby,J.M.
sults).
Briefly,
VH
was
filtered
(0.2
paraffin
oil.
p.m)
air for 12-24
plant embryos
was monitored
DNA
rabbits
RobI,
were
non-
vitreous
as reported
unpublished
from
elserethe
eyes
centrifuged
at 15 000
The supernatant
was
overlaid
incubated
with
equilibrated
at 39#{176}C
in 5%
h prior
to use. Development
to 2-cell, 8-cell, morula,
as described
previously
Synthesis
7.5
and
in the
aseptically
and immediately
cellular
debris.
in microdrops
Dishes
embryos,
in vitro
collected
with
Development
transplant
humor
where
CO2
of nuclear
and blastocyst
[23].
in
transstages
Assay
DNA synthesis
meres
was assessed
radiography.
30 mm
midine
Donor
and
NUCLEAR
of killed female
rabbits
x g for 2 mm to pellet
Collection
rabbits
IN
in
intact
embryos
and
isolated
blastoby uptake
of 3H-thymidine
and auto-
Blastomeres
and
at 39#{176}C
in EBSS/FCS
5’-triphosphate
(ICN,
embryos
were
incubated
they were
washed
three
times
in PBS and fixed
in
acetic
alcohol
(95%
ethanol:
H20:
acetic
acid, 4:2:1)
approximately
an acid-washed
30 sec.
glass
Fixed
slide
for
containing
40 p.Ci/ml
3H-thyIrvine,
CA). After incubation,
-
specimens
were
dropped
and air-dried.
The slides
10#{176}C
for
onto
were
ducts with 37#{176}C
PBS containing
3 mg/mI
BSA, 44 and 54 h
after
mating,
respectively.
Embryos
were
then
washed
in
PBS and cultured
prior
to use or blastomeres
were
isolated
by pipetting
zona-free
embryos
in 0.25%
trypsin
as de-
dipped
Kodak,
4#{176}C.
The slides
were
D-19 (7 mm),
rinsed
then
developed
in dH2O (10 sec),
in Kodak
developer
fixed in Kodak
fixer
scribed
elsewhere
micromanipulation.
(5 mm),
in dH2O
Incorporation
[22, 23] for cell
cycle
synchronization
and
Micromanipulation
Nuclear
was carried
transplantation
scribed
[1, 23]. Briefly,
oocytes
were
of gassed
EBSS/FCS
with 7.5 g/ml
erlaid
with
equilibrated
paraffin
and the membrane-bounded
drawn
into an enucleation
aspirated
space
in the
against
Blastomere
Fusion
cyte was
the
Fusion
of the
induced
same
as previously
manipulated
cytochalasin
oil.
The
enucleated
and
placed
first
in the
de-
in drops
B and ovpolar
M phase
chromosomes
pipette.
A blastomere
pipette
and
out
was
and
Track
emulsion
NY), air-dried,
washed
was
thymidine
cording
tomeres
to the
were
ground
(FL.
Barnes,
personal
of grains),
were
then
Experimental
Design
Activation
donor
blastomere
with the recipient
ooelectrically
[1, 22, 23] 18 h after hCG in-
(Kodak
NTB2,
exposed
for
(5 mm).
communication).
Eastman
6 days at
of 3H-
Also,
necessary,
incorporation
was scored
on a scale
light development
of grains above
background)
development
perivitelline
and
assessed
under
bright-field
microscopy
development
of silver
grains.
Positive
those
showing
50 grains
above
body
oocyte.
Oocyte
in Nuclear
Rochester,
based
on
staining
acbIasbackwhen
of one (very
to six (heavy
intensity.
Cell cycle stage
of the donor
nucleus
and development
of reconstituted
emtnyos.
The
first experiment
determined
the extent
of development
in vitro of nuclear
transplant
stages
lected
embryos
of the
and
cell
using
donor
cycle. Late
blastomeres
were
nuclei
8-cell
isolated
in early,
mid-,
and
stage embryos
were
prior
to culture.
late
colStart-
494
COLLAS
ing
50 h after
sessed
mating,
every
off (t
in the
=
cleavage
15 mm,
and
0 h post-pick-off,
early stage of the
opment
by
cooling
after pick-off.
metabolism
thymidmne
A previous
of isolated
was
hppo).
cell cycle
and
(data
perivitelline
space.
of blastomeres
40-50
mm.
of the
cell
cycle
(up
inhib-
developed
This
to trophoblastic
indicates
that
cooling
in a reversible
20-30
min before
from
PBS
maximum
and
manner.
Oofusion,
cooled
inserted
time
when
into
between
fusion
the
removal
the 4#{176}C
PBS and complete
blastomeres
were
still in the
to 1 hppo)
yesis effec-
the
fusion
early
for
with
each
type of donor
extent
of development
of DNA synthesis
were
dissociated
after
and
autoradiography.
Late
for
inherent
observation)
stage
(fifth
used
within
and
cycle).
intact
8-cell
cleav-
cycle
are
comparable
and
intact
embryos.
precise
timing
in vitro.
Four
cubation
of onset
observation)
of DNA synthesis
in isolated
Thus,
of onset
replicates
of blastomeres
time the
available
results
([25];
indicates
in the
stage
were
expected
that
cell
cell
fifth
blastomeres
to reflect
in whole
embryos
The proportion
incorporating
3H-thymidine
during
time was compared
of DNA synthesis
by chi-square
was determined.
analysis,
each
in-
and time
Snchronization
of donor
blastomeres
in M phase
and
phase.
A method
for synchronizing
donor
blastomeres
in the Gi phase
of the cell cycle was developed.
The
procedure
entailed,
first, synchronizing
blastomeres
in M
in PBS,
aliquoted,
inhibitor
Colcemid
and
stored
of microtubule
was reconstituted
at
-
polymerization,
at 2 mg/ml
10#{176}C.
Blastomeres
54
h after
h after
[23],
mating,
and em-
in VH containing
0.1, 0.5, 1.0, 2.0,
for 10 h. Nuclei
were
then stained
33342
(Sigma),
and the proportion
each
colcemid
was
concentration
or
in
of
deter-
mined
under
epifluorescence
at 200 X. Furthermore,
reversibility
of colcemid
treatment
was evaluated
by
cleavage
rate of isolated
blastomeres
and development
embryos
the
the
of
to blastocysts.
of embryos
blocking
colcemid
at the
G 1/S
transition
en-
to the
32-cell
16-cell
stage
embryos
in M phase
with 0.5
in VH for 10 h (concentration
previously
stage.
A period
of 6 h was
corresponded
for oocyte
to the
collection,
blastomere
three
times
fusion
(see below).
in VH and cultured
tocysts.
In addition,
uated
tration.
maximum
amount
micromanipulation,
containing
treatment.
inhibition
cessed
aphidicolin
Zona-free
for
of DNA
for the
32-cell-stage
autoradiography.
chosen
because
of time
and
necessary
complete
Embryos
were then
for development
in a group
of embryos
for
3H-Thymidine
(40 p.Ci/ml)
it
washed
to blas-
was
synthesis
eval-
each aphidicolmn
concenwas added
to the drops
last
2 h of the aphidicolmn
embryos
were
then pro-
The
number
of positive
em-
bryos
was compared
by chi-square
analysis,
and the proportion
of blastomeres
showing
grains
in these
embryos
was determined.
Rate of incorporation
of 3H-thymidine
in
these
was also scored
blastomeres
Assay.”
Development
S phase
donor
chronized
constituted
chronize
previous
of nuclear
nuclei.
as described
were
The
transplant
potential
under
“DNA
embryos
with Gi and
of donor
nuclei
syn-
in the GI phase
to direct
embryos
was determined.
donor
nuclei
in Gi were
experiments.
Sixteen-cell-stage
tured
in 0.5 p.g/ml
washed
three
times
Gi
phase
using
the
colcemid
(Sigma).
for
recovered
4 h. At 58
were
removed
Synthesis
personal
start of the 16-cell
evidence
in mice
16-cell
of DNA synthesis
were
performed.
of bIas-
mouse
[16]. Isocell cycle asyn-
embryos
to accurately
In addition,
[16] and rabbits
(personal
cycle
length
and timing
method
used
in the
to overcome
metaphases
were
for
determined;
see below).
Upon
release
from colcemid,
embryos
were
then
incubated
for 6 h in VH containing
0.05,
0.1, 0.5, 1.0, and 2.0 p.g/ml
aphidicolin
and allowed
to cleave
[24].
The length
evaluated
by
mitosis.
assessed
incubated
colcemid
Hoechst
(DMSO)
at 2 mg/mI,
synchronization
in M
BIas-
nuclei
in each
of manipulated
A similar
was
tomere
synchronization
lated blastomeres
were
chrony
in VH
pellucida
DNA synwas re-
tailed
p.g/ml
age (elongation,
t =
0). At t = 15, 30, 45, and 60 mm, a
number
of pairs
of 16-cell
stage
blastomeres
(2/16
pairs)
were picked
off, incubated
in 3H-thymidmne
for 30 mm, and
processed
embryos
with the
Aphidicolin
was
stage
occurred.
embryos
was compared
by chi-square
analysis
Length
of Gi phase in early rabbit embryos.
of the Gi phase
in early rabbit
embryos
was
timing
the onset
stage blastomeres
stage
and cultured
coat and zona
Synchronization
tomeres
used
in the mid- and late stages
of the cell cycle
were
assessed
for cleavage,
picked
off, and cultured
for 23 h and 4-7 h, respectively,
before
nuclear
transfer
and
fusion
(3-4
and 5-8
hppo,
respectively).
Four
replicates
were
performed,
replicate,
and
16-cell
bryos
were
5.0 p.g/ml
10 p.g/ml
or even
at the G 1/S boundary
aphidicolmn
(Sigma).
in pure dimethylsulfoxide
and stored
at -10#{176}C.For
phase,
at 4#{176}C.
Upon
release
from
1, 2, or
however,
DNA synthesis
resumed
The
Thus,
h immediately
constituted
aliquoted,
slowed,
removed
from
to be used
in devel-
then synchronized
thesis
inhibitor,
considerably
tive in slowing
development
cytes were enucleated,
and
were
2-3
as-
picked
had indicated
that the
assessed
by rate of 3H-
shown).
blastomeres
Blastomeres
were
arrested
for
was
were
experiment
blastomeres,
blastomeres
not
stage
mating
mucin
ited, during
incubation
3 h of 4#{176}C
treatment,
ides
16-cell
blastomeres
at 4#{176}C
in PBS
uptake,
promptly,
to the
cleaving
ET AL.
aphidicolin
to allow
colcemid
in VH
cleavage
development
Procedures
determined
embryos
for 10 h. Embryos
and cultured
with
to the
32-cell
of
to
in
were
resynthe
cul-
were
then
0.1 p.g/ml
stage
and
arrest
embryos
in GI. Meanwhile,
recipient
oocytes
were
enucleated.
Donor
blastomeres
were
then isolated,
and micromanipulation
was carried
out in 0.1 p.g/ml
aphidicolmn.
Before fusion,
in mannitol
embryos
dicolin
embryos
containing
were
allowed
0.1 p.g/ml
were
cultured
to prevent
DNA
densation
[1]. The
subsequent
pulses
to equilibrate
aphidicolin.
for 2 mm
After fusion,
for 30 mm in the presence
synthesis
before
chromosome
aphidicolin
was
were
as described
given
then
washed
above.
of aphiconout,
Late
and
S,
DONOR
TABLE
1.
embryos.
Donor
Influence
bhppo
hours
‘‘Within
49
96
142
replicates;
square
mine
analysis.
whether
licates
was
to the
of activated
with
Activated
different
obtained
to those
inhibitors.
Nuclear
on development
of e mbryos
45 (92)’
65
(68)d
32
(23)0
by culturing
synchronized
16in
was
was
in vitro of embryos
compared
h’ chi-
were
performed.
To
in development
between
recipient
oocvte,
oocytes
was
differ
deterrep-
parthenogenetic
also
de-
evaluated
in each
Ip
The
of the Donor
Embryos
developmental
(3-4
was
Nucleus
GI
and
phase
Morula
42 (86)’
52 (54)d
13 1 9)0
42 (86)’
fore,
were
the end
incubated
(0-30
mi,
joritv
With
only
however,
98%
synthesis
(p
of nuclei
in earls’
(1 hppo),
and,
mm
early
late (5-8
hppo)
stages
of the cell
by monitoring
the extent
of devel-
mm
after
rabbit
tomere
bryos.
nor
nuclei
Length
was
to evaluate
dure could
reduced
with
0.01).
Similarly,
development
with
nuclei
in mid-cycle
(32%)
of
mid-
nuclei
in
to blastocvsts
and dramati-
with nuclei
in late cycle (3%),
compared
to
cycle (59%;p
< 0.01).
The results
indicate,
the stage of the cell cycle of the donor
bIasdevelopment
of development
progressed
of the
Because
bryos
further
proportions
lower
with
<
influenced
The extent
GI
in the
Phase
development
improved
of nuclear
became
cell
in Earl)’
in vitro
with
donor
grains,
was
there-
blastomeres
after mitosis
whereas
showed
during
under
transplant
reduced
emas do-
our
The
need
together
sitated
nor
the
evidence
ma-
of DNA
successfully
was
subsequent
Rabbit
Embryos
of nuclear
nuclei
transplant
in the
early
whether
a blastomere
synchronization
he established,
we determined
the length
em-
short
for donor
in-
incubation
in the
ml (100%;p
2. Initiation
in vitro.
Incubation
0-30
mm
15-45
mm
30-60
mm
45-75
mm
60-90
mm
‘Expressed
5Embryos
cDifferent
not
phase
in
exceed
45
in M Phase
in the early
stage
of the
cycle,
necesdo-
Gi
of 32-
phase.
Synchronization
Culture
of blastomeres
10 h indicated
that
cells
in M phase
Reversibility
of 3H-thymidine
the
even
uptake
Total
106
82
70
58
26
in minutes
after mitosis.
with
50 grains
above
background.
from other groups
within
columns
p
at
embryos
blastomers
Positiveb
15
80
68
58
26
(p
0.001);
0.5 p.g/
<
was
de-
after
1%)
Negative
1 14)’
I 981
1 97)
(100)
(100)
<
was
p.g/ml
inhibition
in rabbit
of
in 0.1-5.0
inhibitor
at 0.1
inhibitor;
maximum
of the
Number
time
3H-thymidine’
within
Gi
duration
of the Gi phase,
of a procedure
to synchronize
vs. 8% without
of mitosis
was
<0.05).
does
Blastoneres
3).
for
(89%
metaphases
however,
inhibition
and
the
in the Gi phase
implied,
first, rein M phase
between
the 16- and
(Table
at blocking
is initiated
that
conditions.
nuclei
blastomeres
synchronization
stage,
proceof the
is very
with the short
the development
efficient
synthesis,
suggests
of Donor
blastomeres
TABLE
mitosis
DNA
This
experimental
Synchronization
in
cycle.
therefore,
embryos
colcemid
callv impaired
nuclei
in early
therefore,
that
of ti-
of thymidine
incorporation.
15-45
mm after
mitosis,
3H-Thymidmne
mitosis.
stages
Consequently,
embryos
were
were
(Table
2). When
immediately
blastomeres
0.001).
Initiation
in 16-cell-stage
embryos
of the S phase
and,
developed
in blastomeres
p.g/ml
and
emhrvos.
mitosis
initiation
14%
of the
32-cell
nuclei
7)0
not show signs
in 3H-thvmidine
<
bryos
that did not cleave,
cleavageand morula-stage
stage
29 (59)’
31 (32)d
4 ( 3)0
32-cell-stage
after
for
(86%)
did
incubation
cell-stage
versible
cycle (p
reduced
Blastocyst
of the Gi phase
in 3H-thvmidine
activation
rate was reduced
(77%
and 73%, respectively;
p
< 0.01).
In addition,
the more
advanced
the donor
nuclei
in the cell cycle, the higher
the proportion
of activated
em-
late
was
10 (
in 16- and
thvmidine
Dci ‘elopment
opment
in vitro of nuclear
transplant
embryos
(Table
1). A
very high proportion
(92%)
of embryos
activated
with donor nuclei
in the early stage
of the cycle and all activated
embryos
cleaved
to the 2-4-cell
stage.
With donor
nuclei
in the mid- and late stages
of the cell cycle,
however,
the
cycle
l%l
8-cell
thvmidmne
uptake
used
as a marker
45
potential
hppo),
and
determined
transplant
periods.
Results
were
similar
with 32-cell-stage
bla.stomeres
(not
shown).
The data
indicate
that incorporation
of 3H-
RESULTS
midcycle
nuclear
0-Oil.
<
corporated
cell (‘ycle Stage
of Reconstituted
495
of rabbit
2-4-cell
superscripts
transplantation
of development
S donor
nuclei
Five replicates
any variability
due
velopment
replicate.
blastomere
49 (100)
45 (92)’
96 (100)
74 (77)d
104 (73)d
142 (100)
5-8 hppo:
6 replicates.
values
nuclei
were
(contemporary
and extent
with late
and
donor
TRANSPLANTATION
post-pick-off.
columns,
for 16 h without
performed,
with Gi
of the
Fused
n’
3-4 hppo
5-8 hppo
‘Total
of four
donor
embryos
stage
IN NUCLEAR
Number
stage
1 hppob
Gi)
of cell cycle
STAGE
CYCLE
cell
cycle
32-cell-stage
cell-stage
CELL
0.001).
91(86)’
2 1 2)
2 1 31
0 1 0)
0 1 0)
COLLAS
496
TABLE
with
3.
Synchronization
of 16-cell-stage
rabbit
embryos
ET AL.
in metaphase
Synchronization
of Donor
The
Cleavage
after
colcemid
releaseb
No. of blastomeres
Colcemid’
(g/ml)
210
189
151
151
63
23
second
tomeres
Developmentc
0 h
1.5 h
163
122
157
169
47
16 (8)d
169
(89)’
151 (100V
151 llOO)
63 (1o0(f
23 (100)t
Total
ii
174
(6)d
230 (89)’
292 (86)0
298 (76)
90 (91(0
19
19
12
12
12
6
11
12
11
5
(92)d
(83)d
immediately
of M phase
pleted
within
aphidicolin,
(0)d
‘Length
of colcemid
incubation
was 10 h.
bData expressed
in number
of blastomeres
counted
(% increase).
cDevelopment
of intact embryos
after release from colcemid
1%).
dfValues
with different
superscripts
differ (p < 0.05).
embryos
sis (Fig.
by the
cleavage
rate
of isolated
1.5 h after release
from
ploidy
was not assessed.
16-cell-stage
bryos
ing
rate was low for nonsynchronized
blastomeres
(6%).
With
0.1-2.0
p.g/ml
colcemid,
cleavage
occurred
synchronously
within
1.5 h after
release
from
the block,
and 76-91%
of
This cleavage
colcemid-treated
velopment
rate
not different
In addition,
was
cells.
to blastocysts
2.0 p.g/ml
colcemid
controls
(83-100%;
of intact
was
p
>
that 16-cell-stage
in M phase
with
therefore
used
ments.
Inhibition
concentrations
2
synchronously
within
development
at
very
0.1).
from
the
that obtained
proportion
embryos
high
The
treated
with
rabbit
embryos
were
0.5 jig/mI
colcemid.
efficiently
Colcemid
ately
1.5 h of removal
TABLE
4.
inhibition.
of the
proceeded
Inhibition
inhibitor,
synthesis
32-cell-stage
0
ODMSOd
0.05
0.1
0.5
1.0
2.0
n
22
27
18
22
24
24
6
Blastocyst
22
25
17
211
22
211
Embryos
(100)
I 93)
I 94)
95)
I 92)
87)
embryos
by aphidicolin
embryos
‘Development
to blastocysts
lower
than
(+)
(-1
14
14
11
5
5
0
0
1
10
8
16
38
39
2
11
10
2
0
other
treated
em-
were
synthesizTherefore,
ap-
lb).
(2/13,
15%)
In addition,
were
the
moder-
reversibility
was determined
after release
from
and
1
reversibility
(p
of the
2
of labeling’
3
4
5
6
100
100
18
4
9
14
25
10
2
to 6 (heavy
groups
of in-
inhibition
(10/15)
of embryos
la). In the remaining
treatment
to blastocysts
Intensity
14
14
12
15
13
13
of grains)
levels
of DNA synthesis
was achieved
Similar
results
were
obtained
However,
inhibition
was stronger
and only
10% of the blasto-
synthesis.
‘Incubation
time in aphidicolmn
was 6 h following
10 h in 0.5 g/ml
colcemid
(see text).
bDevelopment
after release of 32-cell-stage
embryos
from aphidicolin
block.
‘Incorporation
of 3H-thymidine
was assessed
by the development
of grains after autoradiography
a scale of 1 (very light development
of blastomeres
showing
grains.
dDMSO
concentration
was 0.25 g/mI.
absence
of
high in all
Complete
of 3H-thymidine
of embryos
Total
0(0(0
was
by
the
incubated
in 0.05-1.0
p.g/ml,
but not 2.0
to blastocysts
at a rate similar
to that of
developed
Uptake
No.
positive
in DNA
In the
tiated
within
45 mm after
release
from
the inhibitor
(not
shown).
Therefore,
inhibition
of DNA synthesis
was
reversible
at these
concentrations.
Above
1 p.g/ml,
however,
inhibition
of DNA synthesis
was complete
but irreversible.
The results
indicate,
therefore,
that 0.1 p.g/ml
aphidicolmn
Development’
Aphidicolin’
(g/ml)
active
synG1/S
controls
(87-95%;
p > 0.1). Although
no cell count
was
done,
the morphology
of these
blastocysts
was
similar
to
that of controls.
In these
embryos,
DNA
synthesis
was ini-
arwas
and
in rabbit
inhibition
aphidicolmn.
aphidicolmn.
aphidicolmn,
p.g/ml
in the
at the
however,
variable.
in 67%
(Fig.
85%
1.0
blocks.
p.g/ml,
in vitro.
of DNA
occurred
aphidicolmn
of the colcemid/aphidicolin
assessing
development
0.1-
and
(5/15),
only 52% of the blastomeres
at a low rate (score
1-3; Fig.
meres
0.5 jig/mI
in the subsequent
experiof mitosis
was apparently
reversible
for
jig/mI.
Resumption
of mitosis
occurred
to blastocysts
lower
DNA
with
and similar
to that of
results
indicate,
there-
nuclei
blas-
DNA
no inhibition
of DNA synthep.g/ml
aphidicolmn
weakly
in-
incorporation;
were
proximately
with
of de-
arrest
of the
following
M phase
arrest
(Table
4).
16-cell-stage
embryos
in 0.05-1.0
3H-thymidmne
with 0.1 jig/mI
with 0.5 p.g/ml
the blastomeres
completed
mitosis.
In a separate
experiment, the cleavage
rate of non-colcemid-treated
late 16-cellstage
blastomeres
was
determined.
Culture
of 123 blastomeres
for 6 h resulted
in 95% cleavage
(240 blastomeres).
to
of donor
use
immediately
upon
removal
of colcemid
resumption
of mitosis.
As shown
in the precleavage
to the 32-cell
stage
was
com-
of DNA synthesis
with 0.1 p.g/ml
blas-
colcemid;
however,
chroAs expected,
the cleavage
the
1.5 h of colcemid
removal.
incorporation
of 3H-thymidmne
corporation
tomeres
mosome
synchronization
entailed
(score
6), indicating
Ic). Similarly,
0.05
hibited
termined
phase
aphidicolmn,
p.g/ml
aphidicolin
did not prevent
vious
experiment,
(92)d
(100)d
in the
Gi
inhibitor,
transition,
Incubation
Blastocyst
step
in the
thesis
in M phase
1%)
Total
0
0.1
0.5
1.0
2.0
5.0
fore,
rested
in the Gi Phase
Blastomeres
colcemid.
<
development
0.01).
of grains);
data
and was
are expressed
scored
on
as percent
DONOR
FIG. 1. 3H-Thymidine
uptake
(B) limited
uptake of 3H-thymidine
field, x 400.
efficiently
prevented
bryos
previously
initiated
upon
curred
synchronously
Development
and S Phase
synthesis
IN
Donor
was
Transplant
transplant
opment
progressed
to blastocysts
in vitro
in the cell cycle.
with
Gi
nuclei
as early
as time
of ac-
These
donors
(Table
opment
to cleavage
and morula
G1 transplants,
whereas
a gradual
stages
remained
high
in
decrease
occurred
with
the
in the
Gi
blastocyst
phase
stage
Additionally,
the
nuclear
transplant
nogenetic
Improved
with Gi
early
(15%
vs. 26% morulae;p
to S phase
nuclei,
efficiently
and
directed
71%,
extent
of development
embryos
was similar
nuclei
<
nuclear
0.001).
do-
development
respectively;p
of the
cell
cycle
have
to
0.001).
<
to blastocysts
of Gi
to that of parthe-
a greater
potential
than those
in later stages
of the
also suggest
that the colcemid/aphidicolmn
procedure
proportions
conin early and mid1). Rate of devel-
embryos
(71%
and 61%, respectively,
p > 0.1).
development
in vitro
of embryos
reconstituted
nuclei
reinforces
the conclusion
that nuclei
in the
stages
did
not affect
of the
the
developmental
developmental
cycle. The results
synchronization
potential
of these
in vitro.
the influence
of the
nucleus
on development
phase
The
in 16-
in the
of nuclear
tro.
G1 phase
data
indicate
was reduced
To overcome
and
Duration
Synchronization
embryos
Gi
in several
stage
of
blastomeres
a procedure
The use
development
blastocyst
Blastomeres
A two-step
in vi-
in-
30-45
mm. The Gi phase
in 16-cellhas also been
shown
to be short
(<
studies
(reviewed
by Chisholm
[16]).
How-
of the Gi phase
(2/16)
[16],
approximately
onset
of cleavage
in our study.
Although
mias opposed
to autoradiography,
was used
study
[16], this discrepancy
may reveal
a speShort
duration
of the Gi phase,
associated
asynchrony
inherent
in the donor
embryos,
therefore,
necessitated
meres
in the Gi phase
rest donor
jig/mi
for
stage
and
ever, considerable
variability
in the length
has been
observed
in mouse
blastomeres
whereas
DNA synthesis
consistently
started
30 mm after
crodensitometry,
in the mouse
dies difference.
with cleavage
to synof donor
of 16- and 32-cell-stage
blastomeres
phase
is very short
in these blastomeres
and does
not exceed
stage mouse
embryos
2 h)
develnuclei
dura-
improved
to the
of Gi Phase in Donor
in Gi Phase
Autoradiographs
dicated
that the
that
as donor
the short
32-cell-stage
dramatically
transplant
synchronization
for nuclear
synchronization
embryos
10 h was
phase
in a reversible
microtubule
(tubulin)
DISCUSSION
The present
study
evaluated
of the cell cycle of the donor
G1
embryos.
(A) no uptake of 3H-thymidine
or
was the highest
(score 6). Bright-
(approximately
30 mm),
we developed
chronize
blastomeres
in the G1 phase.
in the G 1 phase,
as opposed
improved
development
of re(Table
5). The beneficial
effect
nuclei
(89%
in contrast
showed
uptake
nuclear
Embryos
manifested
aphidicolin
3H-thymidine
was
oc-
(93% vs. 74%;p
< 0.001).
firmed those obtained
with nuclear
/late
cell cycle stage,
respectively
nors
with 0.1 g/mI
of aphidicolin,
tion
tivation
S phase
donor
Most importantly,
treated
absence
497
TRANSPLANTATION
em-
Nuclei
nuclei
NUCLEAR
32-cell-stage
in embryos.
of Nuclear
donor
STAGE
DNA synthesis
and cleavage
in
in M phase.
of the inhibitor,
Transplantation
of nuclei
to the late S phase,
greatly
constituted
embryos
in vitro
of G1
CYCLE
in 32-cell-stage
blastomeres.
Blastomeres
(score 2 on a scale of 1 to 6). (CI In the
DNA
arrested
removal
CELL
of
method
was
at the Gus
transition.
effective
in arresting
manner.
Colcemid
polymerization.
rectly
on
assembled
microtubules;
diated
by its interaction
with
blasto-
pooi
developed
to ar-
Coicemid
blastomeres
at 0.5
in M
is an inhibitor
It does
not act
rather,
the
donor
transfer.
its
of free
effect
tubulmn
of
di-
is mesub-
498
El
COLLAS
TABLE
5.
Development
nuclear
o f rabbit
in vitro
trans plant
AL.
embryos
using
Number
Cell cycle
stage
of donor
nuclei
G1
Late S
M-II oocyte5
‘Total
of five
bMetaphase
< 0.001.
Fused
102
66
46
102 (100)’
64 (97)C
95 (93)’
95 (93)’
49
38 (58)d
(74)d
39 (85)’
S p hase
donor
nuclei.
(%)
2-4-cell
39 (85)”
-
late
of emb ryos
Activated
n’
G1 and
8-cell
Morula
Blastocyst
92 (89)’
21 (32)d
37 (80)’
91(89)’
17 (26)d
37 (80)’
72 (71)’
10 (,5)1
28 (61)’
replicates.
II oocyte.
c.dp
units
in the
cytoplasm
constituting
mitotic
cells
cells
used
[261. Therefore,
the spindle
are
causes
disruption
in M phase.
for M phase
Consequently,
synchronization
ture. Although
colcemid
inhibition
of nucleoside
its action
on
removal
versibility
of
illustrated
in this
spindle
action
colcemid
is not
at the
binds
thesis
to DNA polymerase
in eucaryotic
cells
G1/S
transition
The DNA synthesis
used
in the rabbit
0.1
inhibitor
[34].
that
aphidicolmn
and
DNA
synthesis
aphidicolmn.
in
aphidicolin.
by Spindle
were
more
were
0.5
Aphidicolin
inhibits
embryos
DNA syn[31-33].
C, however,
has been
the
mouse
indicate
jig/mI
aphidicolmn
[31]. In contrast,
aphidicolmn
and
for
did
treatment
allowed
a high
that
for 16 h
our results
up to 6 h did
not
alter
not
blastocyst
of nuclear
donors
with
rate of development
in vitro.
Although
embryos
was
this suggests
that, in our hands,
aphididetrimental
to development
of nuclear
transin vitro.
In addition,
complete
inhibition
of
was
not
achieved
clear
transfer
(0.1 jig/mi).
thymidine
incorporation
inhibition
effects
involved
releasing
M
reversibly
arresting
of G I transplants
to the blastocyst
stage
development
to term
of aphidicolmn-treated
not determined,
cohn was not
plant embryos
by
to term.
effects
of
such
mitomycin
formation
Furthermore,
jig/mI
step
with
Data
1 jig/mI
blastocyst
morphology.
upon
only
by development
of negative
a and thereby
[30] and mouse
of embryos
with
blastocyst
development
indicated
and
the data indicate
that colsynchronizing
blastomeres
synchronization
from
coicemid
them
prevent
effects,
such as
cell types
[27],
of DNA
synthesis
at the
dose
used
However,
quantification
showed
that approximately
was
achieved
with
0.1
G 1/s
of PCC
transition,
they
were
arrested
until
time
embryos.
of Donor
Cell Cycle
Transplant
Progression
duced
where
in manipulated
Influence
Nuclear
Stage
on Development
of
Embryos
of donor
development
nuclei
in the
of reconstituted
Gi donor
nuclei
directed
plant
embryos
to blastocysts
cell
cycle
embryos.
led
to re-
In particular,
development
of nuclear
transat the highest
rate. The ben-
eficial
effect of transferring
early-stage
nuclei
was
manifested
at pronuclear
formation
and persisted
throughout
development
to the biastocyst
stage. Several possibilities
may
explain
improved
development
with early-stage
nuclei.
In
this study,
in contrast
to mid- and late-stage
blastomeres,
early-stage
donors
ing at 4#{176}C
for 2-3
may
in M phase.
The second
phase
blastomeres
affects
widely
in cul-
assessed
excluded,
not detected
in our study,
and
cemid
was efficient
in reversibly
treatment
been
cells
[27, 28],
it was
and not
possibility
in biastomeres
to the
microtubules
fibers
re-form
[28, 29]. Rein rabbit
embryos
was also
however,
to blastocysts
although
the
development
Therefore,
negative
in some
is reversible
study;
the
colcemid
has
of somatic
may have
transport,
microtubules
the drug,
of coicemid
since
labile,
addition
of colcemid
to
of spindle
fibers
and blocks
have
were
arrested
in development
by coolh prior
to transfer.
Thus,
cooling
itself
enhanced
the
developmental
nuclei.
However,
in a separate
of mid- and late-stage
rabbit
potential
experiment,
blastomeres
of donor
cooling
for 2 h
prior
to nuclear
transfer
did not enhance
development
to biastocysts
of reconstituted
embryos
(20%,
3/15
and 0%, 0/18,
with midand late-stage
biastomeres,
respectively;p
> 0.1).
Also, the
fact that Gi donor
nuclei
were
not cooled
prior
to transfer
in the
latter
experiment
blastomeres
A second
transplant
indicates
per se did
alternative
embryos
not
affected
was
that
cooling
of early-stage
improve
embryro
is that
development
by the
development.
of nuclear
length
of in vitro
culture
cultured
of the donor
nucleus.
Late-stage
blastomeres
were
for 4-6
h longer
than
early-stage
and mid-cycle
nuclei.
However,
mid-cycle
cultured
embryo
nuclei,
for the
as mentioned
last experiment
development
compared
same length
previously,
were
reduced
with
late S and
contemporaneous
in the
This
the
of blastomere
lines of evidence
jig/mI
of the donor
blastomere
affected
development
of reconstituted
embryos.
In a previous
nuclear
transfer
study in rabbits, higher
rates of development
to biastocysts
were
found
with parthenogenetic
than with nuclear
transplant
embryos
This is similar
to rates reported
in the mouse
et al. 131]. Doses
of aphidicohmn
above
1 jig/mI
effective
in preventing
DNA synthesis;
however,
reversibility
tended
to be affected.
In addition,
0.1 jig/mi
aphidicolin
did not prevent
cleavage
after release
from colcemid.
Therefore,
biastomeres
proceeded
through
mitosis
culture.
indicate
that
the
5).
of 3H85%
length
Several
that
embryos
biastomeres
(Table
rules
out
of nuclear
nu-
hypothesis
transplant
GI
therefore
velopment
for
the
was
to early-stage
donors
that were
of time (Table
1). In addition,
was
the
extent
of
affected
cell
cycle
[1]. In the present
paper,
however,
similar
proportions
development
to the blastocyst
stage with parthenogenotes
and Gil transplants
are reported
for the first time. This
deby
stage
of
sug-
DONOR
gests
have
that previous
been
caused
donor
nucleus
and
that
a 32-cell-stage
G1
equivalent
to the parthenogenetic
any problems
in early development
artificial
activation
from
the
use
exist
to
some
of prematurely
transplant
sults
than
similarities
phology
cell
[35] and
fusion
the
cell
cycle
genetic
has
been
contribution
extensive
cells
fragmentation
Fragmentation
in the condensed
lication
is taking
detrimental
place
to the
of the
the
in
the
to the
for
genome
and
[10, 12].
been
of
genome
of
Fusion
of
PCC, with
[7, 8, 11].
shown
In addition,
to be
due
chromatmn
condensed
indicate
that the extent
chromatmn
in
of
into
chromosomes
within
formation
is dramatically
portantly,
morphologically
tained
in GI and early
portion
of
bility,
therefore,
that
S transplants
allow
whereas
chromosome
S transplants.
This,
bility that
velopment
In the
in
abnormalities
[35].
normal
chromosomes
whereas
late
in G 1 and
study,
we
contribute
evaluated
nor cell cycle stage
on development
embryos
in vitro. We also investigated
the cell cycle
in early rabbit
embryos.
the
for valuable
discussions.
Szollosi
influence
early
in vitro,
of late
the possi-
to reduced
Johnson
of nuclear
transplant
ways of manipulating
The results
indicate
that a high rate of development
to the blastocyst
stage was
obtained
in Gi transplants
with
the synchronization
procedure
developed.
However,
the extent
of development
in
vivo of GI transplants
remains
to be determined.
In addi-
time
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