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On the Origin and Persistence of a Cytoplasmic State Inducing Nuclear DNA Synthesis in Frogs' Eggs

Authors:
ON
THE
ORIGIN
AND
PERSISTENCE
OF
A
CYTOPLASMIC
STATE
INDUCING
NUCLEAR
DNA
SYNTHESIS
IN
FROGS'
EGGS*
By
J.
B.
GURDON
DEPARTMENT
OF
ZOOLOGY
OF
OXFORD
UNIVERSITY,
PARKS
ROAD,
OXFORD,
ENGLAND
Communicated
by
Robert
Briggs,
June
9,
1967
Nucleocytoplasmic
interactions
are
believed
to
be
of
great
importance
in
early
animal
development,
since
theresponse
of
genetically
identical
nuclei
to
different
regions
of
egg
cytoplasm
provides
the
most
satisfactory
explanation
for
the
initial
appearance
of
cell
differences.'-'
However,
very
little
is
known
at
present
about
the
mechanism
of
nucleocytoplasmic
interactions.
With
this
in
mind,
we
have
selected
an
apparently
simple
example
of
this
kind
of
interaction
for
detailed
study
in
the
frog
Xenopus
laevis.
This
is
the
induction
of
DNA
synthesis
by
egg
cyto-
plasm,
a
phenomenon
which
has
been
observed
in
male
and
female
pronuclei
shortly
after
fertilization.4
20
The
analysis
of
this
example
of
a
nucleocytoplasmic
inter-
action
is
much
facilitated
by
the
finding
that
large
numbers
of
nuclei
isolated
from
frog
brain
and
other
adult
cell
types
are
rapidly
induced
to
synthesize
DNA
after
injection
into
unfertilized
eggs,5
though
very
few
nuclei
from
these
tissues
normally
synthesize
DNA.
In
all
experiments
to
be
reported
here,
nuclei
from
adult
frog
brain
have
been
used.
Although
these
nuclei
do
not
support
normal
development
after
their
injectioii
into
unfertilized
eggs,
they
respond
to
egg
cytoplasm
by
DNA
synthesis
as
do
egg
and
sperm
pronuclei
or
single
transplanted
embryonic
nuclei,
which
do
support
normal
development.'
For
this
reason
the
induction
of
DNA
synthesis
in
adult
brain
nuclei
can
be
justifiably
used
to
study
one
aspect
of
normal
nucleocytoplasmic
interactions.
The
experiments
reported
here
contribute
to
our
understanding
of
this
nucleo-
cytoplasmic
interaction
in
three
principal
ways.
First,
they
show
that
the
state
of
egg
cytoplasm
which
induces
DNA
synthesis
is
totally
absent
from
o6cytes,
the
cells
which
mature
into
eggs.
Second,
it
has
been
found
that
this
cytoplasmic
state
arises
as
an
effect
of
pituitary
hormone
on
mature
o6cytes.
Last,
the
experi-
ments
demonstrate
the
persistence
of
the
effective
cytoplasmic
state
which
seems
to
be
sufficiently
stable
to
permit
eventual
identification
of
its
molecular
basis.
Materials
and
Methods.-Preparation
and
incubation
of
o6cytes:
A
suspension
of
nuclei
and
label
was
inserted
into
obcytes
of
Xenopus
laevis
laevis
by
microinjection
(50-100
m,4l
for
full-sized
oocytes
and
10-15
mA
for
growing
o6cytes
of
half
the
full
size
diameter).
Injected
o6cytes,
still
surrounded
by
follicle
cells,
were
incubated
at
210C
in
modified
Barth's
medium'
(i.e.,
0.176
M
NaCl,
2.0
mM
KCl,
0.33
mM
Ca(NO3)2,
0.41
mM
CaCl2,
0.82
mM
MgSO4,
2.4
mM
NaHCO3,
10
mg
per
liter
of
both
streptomycin
sulphate
and
benzylpenicillin
sodium
salt,
and
1.5
mM
Tris-
HCl
to
bring
the
pH
of
the
whole
solution
to
7.6).
Odcytes
incubated
under
these
conditions
retained
the
same
external
appearance
as
they
had
immediately
after
removal
from
the
donor
female
for
a
period
varying
from
2
to
3
days.
Isolated
brain
nuclei:
These were
used
as
a
crude
preparation
obtained
in
the
way
described
previously.5
In
any
2-hr
labeling
period,
less
than
1%
of
adult
frog
brain
nuclei
synthesize
DNA
in
vivo,
or
as
isolated
nuclei
in
vitro.5
Labeling
methods:
H'-thymidine
(H3-TdR)
was
used
as
a
specific
label
for
DNA
synthesis.'
It
was
obtained
at
22.1
c/mM,
labeled
primarily
in
the
6C
position,
from
the
Radiochemical
Centre,
Amersham,
England,
and
was
made
up
at
2
mc/ml
in
modified
Barth's
solution.
In
most
experiments
it
was
mixed
with
brain
nuclei
(1:1
ratio
by
volume)
just
before
use.
A
utoradiography:
O6cytes
or
eggs,
fixed
in
Perenyi's
fixative
and
sectioned
at
6-7
ju,
were
545
ZOOLOGY:
J.
B.
GURDON
stained
and
mounted
for
identification
and
examination
of
nuclei.
The
sections
were
then
taken
down
to
water,
treated
with
cold
5%
trichloroacetic
acid
(TCA)
for
20
min
and
extensively
washed
out
uinder
running
water
before
being
dipped
in
K2
emulsion
(Ilford,
Essex,
England).
Exposures
were
for
3
weeks
unless
otherwise
stated.
The
great
majority
of
nuclei
scored
as
labeled
were
densely
covered
by
grains
(e.g.,
Fig.
iN,
P)
and
all
were
covered
by
several
times
the
background
number
of
grains.
Nuclei
described
as
unlabeled
had
no
measurably
greater
number
of
grains
over
them
than
an
equivalent
area
of
background
(e.g.,
Fig.
1J-L).
Hormone:
For
injection
into
frogs
as
well
as
for
addition
to
oocyte
incubation
media,
LH
obtained
as
"pregnyl"
from
Organon
Laboratories,
Surrey,
England,
was
used
to
induce
ovulation.
Numbers
of
frogs
and
eggs
used:
Each
of
the
main
conclusions
drawn
in
this
paper
is
based
on
experiments
carried
out
on
at
least
50
oecytes
or
eggs
obtained
from
at
least
two
different
females.
Normal
events
accompanying
the
maturation
of
oocytes
into
eggs:
In
Xenopus
the
growth
of
an
oocyte
lasts
many
months
or
even
years;
during
this
time
it
is
attached
to
the
ovary
by
an
en-
veloping
layer
of
follicle
cells
and
possesses
a
relatively
enormous
nucleus
called
the
germinal
vesicle
(Fig.
1G),
which
contains
multiple
nucleoli
(Fig.
1E)
and
chromosomes
in
the
diplotene
stage
of
meiotic
prophase.7
When
oocytes
have
reached
their
full
size,
they
are
able
to
respond
to
a
pituitary
hormone,
such
as
LH,
by
undergoing
a
series
of
events
called
maturation.
These
include
the
rupture
of
the
germinal
vesicle
whose
contents
mix
with
the
oocyte
cytoplasm,
the
release
of
the
oocyte
from
the
ovary
(ovulation),
and
its
passage
down
the
uterus;
the
oocyte
chromosomes
complete
their
first
meiosis,
releasing
a
polar
body,
and
proceed
as
far
as
the
meta-
phase
of
the
second
meiotic
division.
At
this
point
the
matured,
ovulated
o6cyte
is
called
an
egg.
All
these
maturation
events
can
take
place
in
Xenopus
within
12
hr
or
less
of
hormone
ad-
ministration.
Eggs,
but
not
oocytes,
respond
to
penetration
(whether
provided
artificially
by
pricking
with
a
micropipette,
or
naturally
by
sperm
during
fertilization)
by
undergoing
a
process
called
activation.
This
involves
the
completion
of
the
second
meiosis,
the
bursting
of
the
cortical
granules,
etc.
In
the
experiments
described
below,
living
eggs
were
considered
to
have
undergone
activation
if
they
showed
irregular
fragmentation
of
animal
hemisphere
cytoplasm.
Eggs,
but
not
oocytes,
always
respond
in
this
way
to
penetration
by
a
micropipette.
This
sequence
of
events
differs
in
detail
in
other
anuran
species.8
Results.-A
bsence
of
thymidine
incorporation
by
nuclei
injected
into
oocytes:
When
isolated
adult
brain
nuclei
and
H3-thymidine
are
injected
into
unfertilized
eggs,
over
90
per
cent
of
them
are
observed
by
autoradiography
to
have
incor-
porated
the
label
into
DNA
within
90
minutes5
(Fig.
10,
P).
Brain
nuclei
prepared
in
the
usual
way
were
injected
together
with
the
label
into
the
cytoplasm
of
full-
sized
o6cytes
which
were
fixed
90
minutes
later.
Exposure
to
autoradiographic
film
for
the
same
length
of
time
as
eggs
revealed
no
grains
over
the
nuclei.
The
obcytes
which
received
the
injected
nuclei
retained
a
normal
external
appearance
in
vitro
for
2-3
days,
and
the
injected
nuclei
remained
indistinguishable
from
nuclei
in
fixed
brain
tissue
for
several
hours
after
injection
into
oocytes.
Even
when
nuclei
were
allowed
to
remain
in
o6cytes
for
3
days
after
injection
at
the
same
time
as
H3-thymidine,
no
DNA
synthesis
could
be
detected.
Similar
experiments
have
been
carried
out
on
young
odcytes
that
are
growing
very
actively.
These
were
obtained
from
a
female
which
had
been
induced
by
pituitary
hormone
to
spawn
large
numbers
of
eggs
3
days
before.
After
females
have
spawned,
their
young
o6cytes
are
very
active
in
growth
and
RNA
synthesist
10
Brain
nuclei
injected
together
with
H3-thymidine
into
oocytes
of
half
the
full-size
diameter
(in
the
active
lampbrush
phase)
showed
no
DNA
synthesis,
even
if
left
in
the
odcytes
for
2-3
days
and
if
exposed
to
autoradiographic
film
for
2-3
months
(Fig.
1A,
E,
J).
It
seemed
possible
that
the
factor
responsible
for
the
induction
of
DNA
synthesis
by
egg
cytoplasm
might
accumulate
in
the
oocyte
nucleus
(germinal
vesicle)
and
be
liberated
into
the
cytoplasm
when
the
germinal
vesicle
ruptures
just
before
546
PROC.
N.
A.
S.
ZOOLOGY:
J.
B.
GUIDON
54
l
I
L
F.40
It
*
40,
hi
t.
#
....
I
mm
....i_
S"w_
ij
I-,~
FIG.
1.-(A-D)
Whole
o6cytes
or
eggs;
(E-I)
vertical
sections;
(J-P)
adult
brain
nuclei
90
min
after
injection
at,
the
same
time
as
H3-thymidine.
(A,
E,
F)
Young
odcyte;
in
(F)
germinal
vesicle
sap has
been
mixed
with
oocyte
cytoplasm;
(B,
G)
full-sized
o6cyte;
(C,
H)
nonovulated
odcyte
with
ruptured
germinal
vesicle;
(D,
I)
unfertilized
egg.
Magnification
in
(A)-(I)
is
the
same
as
in
(I).
(J,
K,
L)
Autoradiographs
of
nuclei
in
oAcyte
cytoplasm
(J),
in
an
o&cyte
germinal
vesicle
(K),
and
in
o6cyte
cytoplasm
mixed
with
germinal
vesicle
contents
(L);
(Al,
N\)
nuclei
in
an
obcyte
with
a
ruptured
germinal
vesicle
(as
in
C,
H)
before
and
after
autoradiography;
(0,
P)
nuclei
in
an
unfertilized
egg
before
and
after
autoradiography.
Magnification
in
(J-P)
and
(S)
is
the
same
as
in
(J)
and
(K).
(Q,
R)
O6cyte
follicle
cell
nuclei
all
of
which
have
incor-
porated
H3-turidine
(Q),
and
only
some
of
which
have
incorporated
H3-thymidinle
(R).
(S)
H3-uridine
incorporation
by
nucleoli
of
injected
odcytes
which
were
maintained
in
vitro
for
3
hr.
VOL.
5SI
196
7
5,47
!-4
."
V,
Ir;
.:
--.imk.
.;W.
..
"'i
.,o
#""
.,
ZOOLOGY:
J.
B.
GURDOP.-
ovulation.
This
was
tested
by
injecting
brain
nuclei
and
H3-thymidine
inside
the
germinal
vesicle.
In
the
case
of
small
o6cytes
taken
from
a
female
which
had
spawned
3
days
earlier,
it
was
found
that
10-20
per
cent
of
the
injected
o6cytes
contained
apparently
normal
brain
nuclei
in
the
germinal
vesicle.
O6cytes
of
this
kind
were
incubated
in
vitro
for
6
hours,
but
no
incorporation
of
H3-thymidine
was
observed
in
either
the
resident
germinal
vesicle
or
the
injected
nuclei
(Fig.
1K).
It
was
not
found
possible
to
introduce
nuclei
into
the
germinal
vesicle
of
mature
oocytes
by
the
same
means,
since
in
Xenopus
laevis
the
germinal
vesicle
sap
of
full-sized
obcytes
has
a
stiff
jelly-like
consistency,
and
the
nuclei
which
can
be
as-
sumed
to
have
been
deposited
in
the
germinal
vesicle
are
believed
to
be
squeezed
out
into
the
o6cyte
cytoplasm
soon
after
withdrawal
of
the
microinjection
pipette.
For
this
reason,
germinal
vesicles
were
dissected
out
of
full-sized
o6cytes,
injected
with
nuclei
mixed
with
H3-thymidine,
and
incubated
as
isolated
germinal
vesicles.
Although
in
most
cases
nuclei
were
observed
to
be
quickly
squeezed
out
through
the
injection
pore,
some
germinal
vesicles
retained
the
injected
nuclei
and
were
incubated
in
vitro
for
11/2
hours
with
H3-thymidine
added
to
the
medium.
How-
ever,
no
incorporation
of
H3-thymidine
was
observed
in
any
of
the
nuclei.
There
remained
the
possibility
that
the
factor
promoting
DNA
synthesis
is
formed
or
activated
by
the
interaction
of
a
component
inside
the
germinal
vesicle
with
another
in
the
o6cyte
cytoplasm,
and
would
therefore
not
normally
appear
until
germinal
vesicle
breakdown.
This
possibility
was
excluded
by
reference
to
certain
injected
young
o6cytes
in
which
a
substantial
leakage
of
germinal
vesicle
contents
had
taken
place.
Injected
nuclei
present
in
the
region
where
the
germinal
vesicle
contents
had
mixed
with
the
o6cyte
cytoplasm
showed
no
H3-thymidine
incorporation
(Fig.
iF,
L).
Appearance,
during
o6cyte
maturation,
of
the
cytoplasmic
state
inducing
thyrnidine
incorporation:
It
has
been
possible
to
determine
the
extent
to
which
the
different
processes
constituting
o6cyte
maturation
(see
Methods)
contribute
to
the
appearance
of
the
cytoplasmic
state
which
induces
DNA
synthesis.
The
germinal
vesicle
ruptures
and
disperses
its
contents
before
the
egg
is
released
from
the
ovary
and
before
the
meiotic
divisions
of
the
nucleus
have
commenced.
Eggs
in
which
the
germinal
vesicle
has
burst
but
which
have
not
been
ovulated
can
be
recognized
by
their
external
appearance
(Fig.
IC),
and
brain
nuclei
injected
into
such
eggs
show
extensive
incorporation
of
H3-thymidine
within
1'/2
hours.
Evidently,
the
factor
promoting
DNA
synthesis
appears
at
the
same
time
as
rupture
of
the
germinal
vesicle.
That
this
temporal
association
is
not
fortuitous
is
indicated
by
finding
injected
nuclei
in
occasional
eggs
which
had
been
ovulated,
passed
through
the
oviduct,
and
laid
as
usual,
but
in
which
the
germinal
vesicle
had
failed
to
break
down;
nuclei
injected
into
such
eggs
always
fail
to
incorporate
the
label.
Reasons
for
the
absence
of
thymidine
incorporation
by
nuclei
injected
into
oocytes:
The
results
reported
so
far
indicate
that
at
least
some
of
the
conditions
which
pro-
mote
DNA
synthesis
in
eggs
are
absent
from
oocytes.
However,
the
inability
to
demonstrate
DNA
synthesis
by
nuclei
injected
into
obcytes
could
be
attributed
to
any
of
the
following
causes:
(1)
the
injected
H3-thymidine
might
not
be
used
for
any
DNA
synthesis
which
is
in
fact
taking
place,
(2)
the
recipient
o6cytes
might
not
remain
synthetically
active
after
removal
from
the
ovary
and
during
incubation
in
vitro,
or
(3)
the
isolated
brain
nuclei
might
be
damaged
or
permanently
inactivated
548
PROC.
N.
A.
S.
ZOOLOGY:
J.
B.
GURDON
by
contact
with
oocyte
cytoplasm.
These
possibilities
have
been
excluded
by
the
following
experiments.
Using
Dowex-1-formate
chromatography,
Woodland
(unpublished)
has
found
that
about
70
per
cent
of
the
H'-thymidine
injected
into
oocytes
is
converted
to
thymidine
triphosphate
(TTP)
when
the
o6cytes
are
maintained
in
vitro
for
3
hours.
This
shows
that
the
enzymes
required
for
thymidine
phosphorylation
(thymidine
kinase
and
thymidine
monophosphate
(TMIP)
+
thymidine
diphosphate
(TDP)
kinase)
are
present
and
active
in
oocytes.
Evidence
that
the
labeled
TTP
is
in
fact
available
for
incorporation
into
DNA
is
conveniently
provided
by
the
follicle
cells
which
surround
each
o6cyte.
About
10-20
per
cent
of
these
follicle
cell
nuclei
are
labeled
intensely
with
H'-thymidine
after
incubation
of
o6cytes
in
vitro
for
1
'/2
hours
(Fig.
iR).
Since
the
medium
in
which
the
o6cytes
are
incubated
contains
no
added
H3-thymidine
and
only
a
very
low
concentration
of
label
by
leakage
from
injected
o6cytes,
the
follicle
cells
evidently
obtain
most
of
their
label
by
diffusion
from
the
o6cyte
cytoplasm.
This
shows
that
the
injected
H'-thvmidine
is
not
sequestered
by
or
adsorbed
onto
o6cyte
components,
but
is
freely
available
for
incorporation
into
DNA.
The
isolated
injected
oocytes
are
known
to
stay
alive
and
synthetically
active
in
vitro
from
the
fact
that
all
the
ger-
nminal
vesicle
nucleoli
of
growing
o6cytes
incorporate
H'-uridine
into
RNA
(Fig.
iS),
just
as
they
do
in
vivo.
That
the
inability
to
demonstrate
DNA
synthesis
by
nuclei
in
o6cytes
is
not
due
to
a
dilution
of
the
injected
H3-thymidine
in
a
large
DNA
precursor
pool
or
to
the
inviability
of
injected
nuclei
has
been
demonstrated
by
an
experiment
of
the
follow-
ing
design.
Brain
nuclei
mixed
with
H'-thymidine
were
injected
into
full-sized
oocytes
of
a
female
which
was
about
to
commence
ovulation
following
hormone
administration.
The
o6cytes
selected
for
injection
had
intact
germinal
vesicles
as
was
ensured
by
the
absence
of
the
white
animal
pole
region
(seen
in
Fig.
1C)
and
by
the
fact
that
intact
germinal
vesicles
could
be
dissected
out
of
o6cytes
with
an
ex-
ternal
appearance
similar
to
those
in
Figure
1B.
The
injected
odcytes
were
then
incubated
in
medium
containing
20
IU
of
LH
hormone
per
milliliter.
Many
of
the
incubated
o6cytes
matured
in
vitro
as
has
been
found
before
for
other
species
of
Amphibia,"
and
their
germinal
vesicles
ruptured.
When
this
was
observed
by
external
appearance
(Fig.
1C)
to
have
taken
place,
the
eggs
were
incubated
for
a
further
11/2
hours
and
then
fixed.
Many
of
the
brain
nuclei
in
these
in
vitro
matured
eggs
did
not
synthesize
DNA
but
contained
highly
condensed
chromo-
somes
and
entered
an
abortive
meiosis
or
mitosis
to
be
described
elsewhere.
How-
ever,
an
appreciable
number
of
nuclei
in
most
eggs
were
vesicular
in
appearance
and
had
swollen
to
some
extent
(Fig.
1111).
These
incorporated
H'-thymidine
into
DNA
(Fig.
iN).
Some
of
the
injected
o6cytes
which
were
treated
identically
to
those
above
failed
to
mature
in
vitro,
and
retained
an
intact
germinal
vesicle;
in
these,
no
incorporation
was
observed
in
any
of
the
injected
nuclei.
Apart
from
confirming
that
the
appearance
of
the
condition
inducing
DNA
synthesis
is
precisely
associated
in
time
with
germinal
vesicle
breakdown,
these
experiments
demonstrate
that
the
absence
of
DNA
synthesis
in
o6cytes
cannot
be
explained
either
as
damage
to
the
injected
nuclei
or
as
a
dilution
of
the
injected
H'-thymidine
in
a
large
precursor
pool.
Thus,
brain
nuclei
injected
into
o6cytes
remain
viable
for
at
least
several
hours,
and
are
capable
of
synthesizing
DNA
as
soon
as
the
germinal
VOL.
58,
1967
549
ZOOLOGY:
J.
1.
GURDONoN..
vesicle
ruptures.
The
labeled
thymidine
will
be
diluted
in
the
oocyte
pools
of
TdR,
TiMP,
TDP,
and
TTP
soon
after
injection,
and
yet
the
precursor
pool
is
still
suffi-
ciently
labeled
to
clearly
reveal
DNA
synthesis
when
the
same
o6cytes
have
under-
gone
maturation
and
germinal
vesicle
breakdown.
In
eonclusion,
the
inability
to
demonstrate
H3-thymidine
incorporation
by
nuclei
in
oocytes
evidently
reflects
a
true
lack
of
conditions
necessary
for
DNA
synthesis.
Persistence
of
the
cytoplasinic
state
responsible
for
the
induction
of
DNA
synthesis:
In
order
to
identify
the
cytoplasmic
component
which
induces
DNA
synthesis,
it
is
important
to
know
whether
it
has
a
transient
existence
and
disappears
soon
after
it
arises,
or
whether
it
remains
in
an
effective
state
for
a
considerable
time
after
fer-
tilization.
Two
kinds
of
experiments
have
been
used
to
answer
this
question.
In
the
first
experiment,
brain
nuclei
were
injected
into
eggs
which
thein
received
a
20-minute
pulse
of
H'-TdR
by
a
second
injection
at
various
times
after
this
(Fig.
2E-I).
The
conclusion
from
these
experiments
is
that
egg
cytoplasm
is
able
to
MINS.
AFTER
ACTIVATION
(unfertilised
*egg)-21
C
20
o
do
do
'
100
'
190
INCORPORATION
EXPERIMENT
OF
3-TdR
A
r-
4
~~~~~86%
A
Label
and
Fi
X
nuclei
IC
_
_
_
_
_
s
66
%
B
Activat
Lobel
and
nuclei
r
A
£
31%
Activate
Nuclei
and
label
Activate
Lobel
and
nuclei
A
It
Label
and
Fix
nuclei
Nc
Nuclei
L
Lbel
Nuclei
Label
Nuclei
Laeli
92%.
2%
81%
61%
24%
83%
FIG.
2.-I)esign
of
experiments
to
test
persistence
of
DNA
synthesis-inducing
factor.
H3-TdR,
I3-thymidine.
The
low
percentage
of
labeled
nuclei
inl
experiment
H
(24%7c)
may
be
due
to
the
fact
that
many
nuclei
were
not
properly
mixed
with
cytoplasm
in
these
eggs.
Two
hundred
nuclei
were
scored
for
each
per
cent
valle.
support
DNA
synthesis
from
20
to
at
least
100
nmiinutes
after
nuclear
injection
or
egg
activation.
Furthermore,
many
of
the
samie
nuclei
synthesize
DNA
during
most
or
all
of
this
period
(compare
Fig.
2F,
G,
I).
The
absence
of
DNA
synthesis
for
the
first
20
minutes
after
fertilization
is
characteristic
of
male
and
female
pronuclei.4
It
seems
that
these,
as
well
as
adult
somatic
cell
nuclei,
require
a
certain
time
to
respond
to
their
new
cytoplasmic
environment.
It
is
during
this
time
that
nuclei
undergo
a
pronounced
swelling'
and
that
their
DNA
is
thought
to
undergo
a
change
in
state
or
in
degree
of
dispersion.12
In
the
second
type
of
experiment,
egg
cytoplasm
was
shown
to
have
a
persistent
capacity
not
only
to
support
but
also
to
initiate
DNA
synthesis.
This
was
demon-
strated
by
activating
unfertilized
eggs
(with
a
prick),
and
then
injecting
nuclei
at
various
times
after
this.
As
shown
in
Figure
2A-D,
egg
cytoplasm
retains
its
ability
to
initiate
DNA
synthesis
for
at
least
65
minutes
after
egg
activation,
whether
label
was
introduced
at
activation
or
with
the
nuclei.
D
E
F
G
H
I
Nuclei
Label
*)5)(
PROCt.
N.
A.
S.
ZOOLOGY:
J.
B.
GEUI-?DO5\-
1)iscussion.
A
cytoplasmic
factor
responsible
for
the
iiiduction
of
DNA
synthesis
can
now
be
added
to
the
known
examples
of
substances
which
are
formed
during
oogenesis
or
egg
maturation
but
which
do
not
normally
become
effective
until
after
fertilization.
In
Xenopus
laevis,
ribosomes
synthesized
during
o6genesis
are
suffi-
cient
for
the
needs
of
cell
differentiation
up
to
the
swimming
tadpole
stage.3
In1
the
axolotl,
Briggs
and
Cassens'4
have
provided
a
very
clear
demonstration
that
normal
postgastrular
development
requires
a
gene
product
synthesized
during
oogenesis.
However,
more
comparable
with
the
DNA
synthesis
factor
described
here
for
Xenopus,
at
least
in
its
time
of
appearance
and
activity
is
the
factor
re-
quired
for
cleavage
and
aster
formation
in
many
animal
eggs.
It
has
been
known
for
a
long
time
that
the
ability
of
various
invertebrate
eggs
to
be
fertilized
arises
only
after
rupture
of
the
germinal
vesicle."
In
Bufo,
Dettlaff
et
a7.'"
have
demon-
strated
by
a
variety
of
experiments
that
a
factor
which
is
necessary
for
egg
activa-
tion
and
cleavage,
and
which
might
include
a
DNA
synthesis
inducer,
appears
at
the
time
of
germinal
vesicle
breakdown
and
was
not
present
before
in
the
germinal
vesicle
or
oocyte
cytoplasm.
The
cleavage
and
activationi
response
is
realized
in
Bufo
only
if
the
eggs
are
activated
by
pricking
after
the
release
of
the
germinal
vesicle
contents."
This
is
not
true
of
the
DNA
synthesis
inducer
in
Xenopus,
which
as
shown
above
is
operative
in
eggs
which
matured
in
vitro
and
which
are
not
pricked
after
germimial
vesicle
breakdown.
The
induction
of
DNA
synthesis
by
egg
cytoplasm
may
be
regarded
as
a
simple
form
of
a
type
of
nucleocytoplasmic
interaction
that
is
of
great
importance
in
early
development.
It
is
a
reaction
of
which
we
know
the
product
(D-NA)
and
which
may
be
controlled
by
the
availability
of
the
essential
reacting
components.
The
following
sequence
of
events
seems
to
account
most
simply
for
the
control
of
the
reaction.
The
required
component
appears
as
an
effect
of
pituitary
hormone
on
mature
o6cytes,
and
this
is
likely
to
involve
both
protein
synthesis
and
DNA-
dependemit
RNA
synthesis,
since
Dettlaff'6
has
showmi
that
germinal
vesicle
break-
down
in
Bufo
is
both
puromycin-
and
actinomycin
D-sensitive.
Smith
et
al.'7
have
observed
a
great
increase
in
the
rate
of
protein
synthesis
at
this
time
in
Rana.
The
induction
of
DNA
svmithesis
cannot
be
explained
solely
as
a
direct
effect
of
the
hlormone
on
the
injected
nuclei,
because
it
is
not
induced
in
an
incubation
of
iso-
lated
nuclei
to
which
hormone
has
been
added,
nor
by
the
microinjection
of
hormone
as
well
as nuclei
and
H'-thymidine
into
full-sized
odeytes
with
an
intact
germinal
vesicle
(Gurdon,
unpublished
observations).
It
is
of
special
interest
that
in
Xenopus
the
DNA
synIthesis-iniducing
factor
can
first
be
demonstrated
at
just
the
time
when
the
oocyte
chromosomes
enter
an
unresponsive,
condensed
state
in
which
they
remain
during
meiosis
until
fertilization
stimulates
them
to
proceed
beyond
the
second
meiotic
metaphase.
Since
they
heave
reached
at
least
the
4C
condition
by
meiotic
prophase,'8
1"
they
do
not
need
to
sIn-ithesize
DNA
during
oocyte
matura-
tion,
and
condensed
mitotic
or
meiotic
chromosomes
have
miever
been
reported
to
do
so.
Thus,
it
seems
that
the
regulation
of
this
example
of
a
nucleocytoplasmic
inter-
action
can
at
present
be
adequately
accounted
for
by
precise
control
of
the
time
at
which
responsive
chromosomes
are
exposed
to
the
cytoplasmic
factor.
The
situa-
tion
may
well
turn
out
to
be
more
complicated
than
suggested
here,
and
we
have
in
any\
case
to
explain
what
causes
chromosome
condensation
and
the
continuation
of
meiosis
at
the
time
of
germinal
vesicle
breakdown.
\
Ali,.
5-,X,
1'96
7
55.1
ZOOLOGY:
J.
B.
GURDOP
.
A
Perhaps
the
most
interesting
aspect
of
this
reaction,
not
yet
understood,
is
the
identity
of
the
DNA
synthesis-inducing
factor.
Little
is
known
about
this
at
present
except
that
it
is
not
species-specific,5
and
that
it
appears
not
to
include
the
enzymes
necessary
for
thymidine
phosphorylation
(see
above).
Summary.
-Adult
frog
brain
nuclei
have
been
injected
into
oocytes
at
various
stages
of
growth
and
maturation
in
order
to
determine
the
stage
at
which
egg
cyto-
plasm
acquires
its
capacity
to
induce
DNA
synthesis
in
gamete
and
other
nuclei.
DNA
synthesis
was
recognized
autoradiographically
by
incorporation
of
H3-
thymidine.
The
factor
which
induces
DNA
synthesis
is
absent
from
the
nucleus
and
cytoplasm
of
growing
oocytes
and
from
a
mixture
of
nucleus
and
cytoplasm.
The
factor
appears
a
few
hours
after
the
administration
of
pituitary
hormone,
that
is,
just
after
the
rupture
of
the
oocyte
nucleus.
It
persists
in
the
egg
cytoplasm
for
at
least
an
hour
after
fertilization
or
activation,
but
is
effective
in
the
absence
of
fertilization.
The
author
is
most
grateful
to
H.
Pt.
Woodland
for
permission
to
quote
unpublished
results
on
thymidine
phosphorylation;
also
to
Professors
R.
W.
Briggs
and
H.
G.
Callan,
F.R.S.;
Drs.
E.
R.
Creed,
C.
F.
Graham,
11.
E.
Offord;
and
to
K.
Arms
and
11.
It.
Woodland
for
comments
on
this
manuscript;
he
is
indebted
to
-Miss
J.
Rooney
for
expert
technical
assistance.
*
This
work
was
supported
by
a
research
grant
from
the
-Medical
Research
Council
of
Great
Britain.
I
Dreisch,
H.,
Analytische
Theorie
der
organischen
Entwicklung
(Leipzig,
1894).
2
Morgan,
T.
H.,
Embryology
and
Genetics
(New
York:
Columbia
University
Press,
1934).
3Briggs,
R.
W.,
and
T.
J.
King,
in
The
Cell,
ed.
J.
Brachet
and
A.
E.
Mirsky
(New
York:
Academic
Press,
1959),
vol.
1,
p.
537.
4Graham,
C.
F.,
J.
Cell
Sci.,
1,
363
(1966).
5
Graham,
C.
F.,
K.
Arms,
and
J.
B.
Gurdon,
Develop.
Biol.,
14,
349
(1966).
6
Barth,
L.
G.,
and
L.
J.
Barth,
J.
Embryol.
Exptl.
Morphol.,
7,
210
(19.59).
7
Callan,
H.
G.,
Intern.
Rev.
Cytol.,
15,
1
(1963).
8
Subtelny,
S.,
and
C.
Bradt,
Develop.
Biol.,
3,
96
(1961).
9
Brown,
D.
1).,
and
E.
Littna,
J.
Mol.
Biol.,
8,
688
(1964).
10
Davidson,
E.
H.,
G.
G.
Allfrey,
and
A.
E.
.Mirsky,
these
PROCEEDINGS,
52,
501
(1964).
11
Dettlaff,
T.
A.,
L.
A.
Nikitina,
and
0.
G.
Stroeva,
J.
Embryol.
Exptl.
Morphol.,
12,
851
(1964).
12
Gurdon,
J.
B.,
in.
Ciba
Foundation
Symposium,
Cell
Differentiation,
ed.
J.
Wolstenholme
(London:
Churchill,
1966),
irl
press.
13
Brown,
1).
D.,
and
J.
B.
Gurdon,
these
PROCEEDINGS,
51,
139
(1964).
14
Briggs,
R.,
and
G.
Cassens,
these
PROCEEDINGS,
55,
1103
(1966).
15
Delage,
Y.,
Arch.
Zool.
Exptt.
et
GMn.,
3e
Ser.,
9,
284
(1901).
16
Dettlaff,
T.
A.,
J.
Embryol.
Exptl.
Morphol.,
16,
183
(1966).
17
Smith,
L.
D.,
1t.
E.
Ecker,
and
S.
Substelny,
these
PROCEED)INGS,
56,
1724
(1966).
18
Alfert,
M.,
J.
Cell.
Comp.
Physiol.,
36,
381
(1950).
19
Izawa,
M.,
V.
G.
Allfrey,
and
A.
E.
Mirsky,
these
PROCEEDINGS,
50,
811
(1963).
20
Simmel,
E.
B.,
and
l).
A.
Kanriosky,
J.
Biophys.
Biochenr.
Cytol.,
10,
59
(1961).
PROC.
N.
A.
S.
5
5
2