BIOLOGY
OF
REPRODUCTION
15,
632-646
(1976)
Incorporation
of [3 H] Uridine by Mouse Embryos
Abnormalities
Induced by Parental Hyperthermia
ANTHONY
R.
with
BELLVE
Laboratory
of Human
Reproduction
and Reproductive
Biology
and Department
of Physiology,
Harvard
45
Medical
School,
Shattuck
Street,
Massachusetts
02115
Boston,
ABSTRACT
experiment
to study
the incorporation
of [3 HI uridine
by embryos
exhibiting
abnormal
development
that was induced
by a 24 h exposure
of parent mice
to an ambient
temperature
of 34.5#{176}C
and 65 percent
relative
humidity.
Embryos
recovered
from
females
killed
at 64, 72, 88 and 96 h after HCG were pulse-labeled
by incubating
in culture
medium
containing
[3 HI uridine
for either 5 h (two-cell),
90 mm (four-cell),
45 mm (eight-cell),
20
mm
(morula),
or 10 min
(blastocyst).
The incorporation
of [3I-{Iuridine
was
examined
by
Albino
ICR
mice
were
used
in a factorial
autoradiography.
Following
exposure
of the parental
female
to high temperature
approximately
40 percent
of
the embryos
are arrested
at the two-cell
stage and fail to incorporate
[3 HI uridine.
An additional
20 percent
of the two-cell
embryos
are only partially
affected
in that
one blastomere
is able to
undergo
a limited
number
of cleavage
divisions.
The ‘cleaving’
blastomeres
of these embryos
demonstrate
a distribution
of isotope
comparable
to the blastomeres
of normal
embryos
at the
same
chronological
stages
of dvelopment.
In contrast,
the
incorporation
of isotope
into
the
‘arrested’
blastomere,
if it occurs,
is confined
to the nucleus
and never assumes
the pattern
typical
of blastomeres
at later stages of development.
Paternal
hyperthermia,
an effect
conveyed
by the fertilizing
spermatozoan,
allows
normal
embryonic
development
retarded
at the
during
morula
stage
the
early
although
cleavage
stages.
However,
the
rate
of
development
is
blastulation
eventually
occurs.
A significant
proportion
of
embryos
at the 8-cell
and morula
stages
also contain
blastomeres
which
fail to incorporate
the
isotopic
precursor:
Consequently,
the retardation
in embryonic
development
is preceded
by a
partial
failure
to synthesize
RNA.
These
embryos
are considered
to be responsible
for the
embryonic
mortality
that is known
to occur at, or shortly
after, implantation.
INTRODUCTION
Limited
mice
exposure
to
a
known
of
to
high
of
et al.,
1972).
However,
embryonic
ing
the
upon
parental
the
female
after
mating
is primarily
stage
death
whether
male
to
the
a high
in
occurs
hyperthermia
or female.
ambient
differs
temperature
(Bellv#{128}, 1973).
to be only partially
of
(ElBellv,
at
depend-
is induced
Thus,
exposure
in
of
August
13,
blastomere
3,
5
is
embryos
or
embryos
des
and
eventually
blastocyst-like
to
arrested,
the
but
leads
finite
to an
described
those
which
forming
by
composed
with
Tarkowski
In contrast,
similar
male
to
temperature
prior
and
that
stage
at
with
shortly
Other
2-cell
affected
in
this
16, 1976.
instance,
to
632
mortality
implantation
the
embryonic
factor(s)
death
affected
by
either
of the
causes
mating
developblastocyst
occurring
(Bellv,
at or
1973).
responsible
must
fertilizing
spermatozoan.
The
biochemical
processes
mentally
1976.
embryonic
after
Wroblew-
exposure
an apparent
retardation
in embryonic
ment
at the
late
morula
to early
just
some
trophoblastic
vesistructures
comparable
(1967).
high
are
9 blastomeres,
ska
the
May
of
either
delayed
Accepted
Received
one
continues
through
a variable
of cleavage
divisions.
This
accumulation
development
induces
embryonic
mortality
due to an arrest
in development
the
2-cell
stage
embryos
appear
is
incidence
1970;
while
second
number
female
mortality
et al.,
Burfening
the
or
temperature
increase
embryonic
1968;
which
male
environmental
substantially
preimplantation
liott
that,
either
be
In
for
mediated
the
by
which
are
detri-
maternal
or
pater-
ABNORMAL
nal
hyperthermia
detail.
One
that
mouse
have
preliminary
embryos,
nal hyperthermia
reduction,
if
at
whether
the
2-cell
some
have
not
examined.
been
during
made
embryogenesis.
were
a
later
But,
arrested
at
the
No
RNA
comparable
to
discern
whether
effects
RNA
synthesis
However,
effect
and
mortality
at
1972).
to incorporate
hyperthermia
the paternal
spermatozoan
stage,
exhibit
to
incorporate
which
fail
in
reported
to mater-
blastomeres
also
was
precursor
studied
(Bellv,
embryos
stage
paternal
1-cell
failure,
development
those
studies
the
a
into
of
been
observation
when
subjected
not
3 H] uridine
stages
not
EMBRYONIC
the
fact
that
is conveyed
by the fertilizing
results
in delayed
embryonic
suggests
an
impairment
in gene
ex-
In
[3
consequence,
an autoradiographic
undertaken
to determine
the
H] uridine
incorporation
following
hyperthermia,
the
assessing
the
Evidence
onstrates
that
stage
following
capable
gradation
onic
of
in
synthesizing
of effects
RNA.
RNA
on
that
occurs
incorporate
by
on
which
dem-
the
2-cell
are not
Furthermore,
synthesis
are
only
treatment.
the delayed
after
of
MATERIALS
were
on
all
animals
before
and
a control
or treatment
group.
Females
designated
for
treatment
were subjected
to hyperthermic
stress
during the period
from 24 to 48 h after the injection
of
Sixty
is evi-
partially
Signif-
embryhyper-
embryos
to
Design
females,
after the
embryos.
METHODS
in
three
replicates,
were
killed
in a
factorial
experiment
at each of the periods
64, 72, 88
and 96 h post
HCG (Fig. 1). In each replicate
of 20
females,
10 were mated to control
males and 10 mated
to treated
males.
Five females
within
each
male
treatment
group
were then designated
to be control
females
and
5 to be subjected
to hyperthermia.
This
procedure
gave
the hyperthermia
treatment
combinations; control,
female
stress, male stress and combined
stress.
In addition
to the basic experimental
design, an
additional
AND
recorded
after treatment
using procedures
outlined
previously
(Bellv#{233},1972). Males were subjected
to hyperthermia
on Day 1 and, together
with
control
males, were then
mated
with females
on Days 3 to 8, inclusive.
This
mating
period
was selected
to maximize
embryonic
mortality
and
to avoid
the aspermia
which
occurs
beyond
ten days after treatment
(Burfening
et al.,
1970).
Females,
6 to 10 weeks
old, were induced
to
superovulate
by giving an intraperitoneal
injection
of 4
to 6 iu PMSG between
4.0 and 5.0 p.m.,
followed
by 4
to 6 iu HCG 48 h later.
Immediately
after
the
injection
of HCG (0 h) two females
were placed
with
each male in the control
and
treatment
groups.
The
Experimental
Fl] uridine.
[3
temperatures
HCG.
a
paternal
a failure
2#{176}C
during
the experimental
period.
Animals
designated for hyperthermic
treatment
were exposed
to an
ambient
temperature
of 34.5
±
0.2#{176}C and
65
±5
percent
relative
humidity
for a period of 24 h. Rectal
individual
arrested
at
hyperthermia
embryos
which
by the hyperthermic
it is established
that
is preceded
placed
on
is presented
embryos
maternal
mortality
thermia
effect
of
parental
being
differential
blastomeres.
dent
affected
icantly,
emphasis
study
pattern
633
presence
of a copulatory
plug the following morning
was used
to determine
whether
mating had occurred.
Mated
females
were then allocated
at random
to either
pression.
was
DEVELOPMENT
15
females
injection
were
of HCG
killed
at both
to provide
38 and
control
43 h
2-cell
Materials
The ICR mice were obtained
from the Institute
of
Cancer
Research,
Philadelphia.
Pregnant
mares’ serum
gonadotropin
(PMSG)
was
purchased
from
Ayerst
Laboratories;
human
chorionic
gonadotropin
(HCG)
from Elanco Products;
pronase
(B grade)
nuclease
free,
from
Calbiochem;
the vital dye eosin
y, C.I. No.
45380,
from
Harleco,
Philadelphia;
ribonuclease
A
from
Sigma
Chemical
Co., and the [3 HI uridine
(sp.
act 15.1 Ci/mM)
from
New England
Nuclear.
The
radiographic
emulsion,
NTB-3,
is a product
of Eastman Kodak,
Rochester,
New York.
The embryo
culture
medium
consisted
of 94.6 mM
NaCI, 4.78 mM KCI, 1.19 mM KH2PO4,
1.19 mM
MgSO4.7H20,
1.71
mM
calcium
lactate,
21.58
The
an
Procedure
240
female
environmental
and
control
58
male
chamber
mice
were
maintained
4-?Y
Combined
Mole
stress
treS$
1J
mM
sodium
lactate,
0.33 mM sodium
pyruvate,
25.07 mM
NaHCO3,
5.56 mM glucose,
4 mg/mI
BSA, 100 i.u./ml
of pencillin
and 50 ig/ml streptomycin
as defined
by
Biggers
and Stern (1973).
Experimental
Female
Stress
Control
housed
at 21
in
±
FIG.
1.
Schematic
presentation
torial experiment
for studying
and paternal
hyperthermia
on
development.
This basic design,
treatment
groups
depicted,
was
to give 60 females
for each of
and 96 h after the administration
of the basic facthe effect of maternal
subsequent
embryonic
which
provides
the
replicated
three times
the
periods
of HCG.
64,
72,
88
634
BELLVE
Procedure
for
Incorporation
of
(3
H) uridine
Embryos
were recovered
from individual
females
by flushing
excised
oviducts
or uteri
with culture
medium
containing
0.005
percent
eosin y (w/v), and
then classified
according
to the developmental
stages;
1-cell,
2-cell,
3-cell,
4-cell
(4 to 6 blastomeres),
8-cell
(7 to 12 blastomeres),
morula
(13 or more blastomeres
but no blastocoelic
cavity) and blastocyst
(blastocoelic
cavity
present).
Embryos
of each developmental
stage
were then incubated
separately
in 0.2 ml of culture
medium
containing
20 #Ci/ml
[3 HI uridine
under light
paraffin
oil in 60 mm Petri dishes. The incubator
was
maintained
CO2
at
37.5#{176}C and
in air. Two-cell
embryos
supplied
were
with
5 percent
incubated
in the
isotopic
medium
for
5 h; 3-cell
4 h, 4-cell
90 mm,
8-cell
45 mm, morula
20 mm,
and blastocyst
10 mm.
The incubation
periods
for the respective
embryonic
stages
were selected,
after preliminary
experimentation, so as to maximize
the incorporation
of 3 Hi undine
but still allow
distinction
between
incorporation
into nucleoli,
nucleoplasm
and cytoplasm.
At the end
of the
incubation
through
two
period
the embryos
were transferred
nonradioactive
culture
medium and then incubated
in medium
containing
2 X
10
M uridine for 10 mm at 22#{176}C.
changes
of
positive
identification
of
[3 HI uridine
is incorporated
bryos at each developmental
either
ribonuclease
for
Autoradiogyaphy
Statistical
(Comings,
A naiysis
terms
for total
embryos
autoradiographed
at a given
cell stage were significant. When
certain mean squares
were
not significant,
they
were
pooled
with
the
appropriate
error
term
(Bellv#{233}, 1970).
Comparisons
of
treatment
means
were made
range test (Duncan,
1955).
using
Duncan’s
multiple
RESULTS
of Parental
Hyperthermia
on Embryonic
Development
zona pellucida
was digested
by incubating
the
in culture
medium
containing
0.5 percent
(w/v)
at 22#{176}C.When
the zona
pellucida
ture
became
of
the
indistinct
blastomeres,
both
male
(P<0.01).
(4 to 6 mm),
but
the
digestion
prior
was
carefully
transferring
the embryos
to
ethanol
(1:3)
for 2 mm.
Following
embryos
were then pipetted
on to clean
slides in approximately
10 .rl of solution
complete
drying,
an additional
20 rI of
to separation
stopped
by
acetic
acid:
fixation
the
microscope
and, prior to
fresh acetic
to
increases
ated
and
This
with
it
tempera-
temperature
parents
by 2.0
of hyperthermia
increase
in
the
apparent
1973).
stage
the
of
similar
(Bellv,
that
of
± 0.01#{176}C
is associ-
incidence
development
previously
is
ambient
rectal
embryonic
obtained
over,
elevated
mean
female
degree
an
abnormal
that
the
the
to
More-
in
develop-
ment
at which
the abnormalities
occur
differs
depending
upon
whether
the parental
male
or
female
is subjected
to hyperthermia.
This
differential
response
is exemplified
by two obser-
warmed
the dark
to 45#{176}C.
The preparations
were exposed
in
for 12 to 14 days at 5#{176}C
and subsequently
using
recommended
procedures
(Kodak,
vations.
1966).
On examination,
embryos
from
individual
females
were classified
according
to the proportion
of blastomeres
with labeled
nucleoli
as follows:
a.) nonlabeled,
none of the blastomeres
incorporated
[3 HI uridine;
b.)
partially
labeled,
some but less than 50% of blastomeres labeled;
c.) incompletely
labeled,
50 percent
or
more,
but not all, blastomeres
were
labeled;
d.)
completely
labeled,
all blastomeres
were labeled.
The
partially
labeled
category
was omitted
for 2-cefl
embryos
due to the limited
number
of blastomeres.
Owing to the long incubation
period in 3 HI uridine,
the presence
of silver grains over functional
nucleoli
was generally
in marked
contrast
to both background
grains
and to those
nucleoli
of blastomeres
which had
embryos
not incorporated
incorporation
ten
silver
was
placed
Exposure
directly
on to the embryos
to
area and ensure
fixation
of blastomeres
to
the slide. Prepared
slides
were transferred
to a dark
room
and
coated
with
autoradiographic
emulsion
which
had been
diluted
1:1 with
deionized
water
and
developed
was
em-
with
The data were analyzed
within
cell stages
using
a
modified
general
least
squares
procedure
(Harvey,
1960).
The analyses
for comparison
of the treatment
combinations,
control,
female stress,
male stress and
combined
stress,
were restricted
to those periods
in
which an adequate
number
of embryos
were
present
at
the required
developmental
stage.
All
females
not
having embryos
at the required
cell stage were deleted
from the appropriate
analyses.
Adjustment
by covaniance
techniques
were used where linear and quadratic
The
embryos
pronase
acid:ethanol
wash
the
A or deoxyribonuclease
that
1966).
Effect
Procedure
nucleoli.
To verify
into RNA
control
stage were digested
the isotope.
marginal,
grains
over
the
slides
were
stained
In those
blastomeres
nucleoli
instances
with
less
where
than
were
classified
Selected
stages
with
azure
B to enable
maternal
normal
marked
at
2),
cleavage
of
Following
mately
40
the
the
88
embryos
that
can
h after
80
contain
dye
HCG.
blastomeres
eosin
y
cleavage
do not
be seen
percent
and
there
in the
3).
are
approxiarrested
25
percent
(Fig.
that
many
2).
The
show
any gross
by light
micros-
However,
of
is a
of morula
(Fig.
a further
of
during
h,
hyperthermia
embryos
and
rate
approximately
4-cell
decrease
maternal
percent
of
stage
and
number
a commensurate
(P<0.05)
of
hyperthermia
88
at
a
number
development
but,
blastocysts
arrested
2-cell
abnormalities
the
3-cell
in the
a retarded
at
2-cell,
embryonic
2-cell
exhibit
causes
in
2.) paternal
stages
and
(P<0.05)
vital
the
and,
accumulation
number
copy
(P<0.01)
arrested
(Fig.
early
at
hyperthermia
increase
allows
bryos
negative.
1.)
substantial
the
by
arrested
incorporate
are
necrotic
96
h
em-
the
in
ABNORMAL
TWO
-
EMBRYONIC
CELL
DEVELOPMENT
Nucleoli
635
Labelling:
j:E::
None
=
Partial
Incomplete
=
Complete
40
20
FOUR-
60
-
40
-
20
EP
-
0
HOURS
CELL
I
1-i--’
I
I
43
64
72 88
96
64
72
88
64
96
I
I
2.
appearance.
A
9-cell
cleavage
original
high
embryos
2-cell
blastomere
which
embryo
continues
cleavage
development
recovered
divisions.
is
after
of
to a type
one
blastomere
is arrested
through
This
not
observed
hyperthermia
according
STRESS
type
variable
of
among
of the
the
proportion
male,
in
to
the
both
parents
other
stress
num-
predominates
abnormal
embryos
parental
in
the
are
evident
of
the
effect
at
later
‘retarded’
to
morulae
the
of
stages
When
hyperthermic
maternal
most
that
at
recovered
from
72, 88 and 96 h
the categories
of
contained
labeled
morphologically.
subjected
effect
in
that
the
normal
development
quence,
less
even
be
88 96
-
of blastomeres
fact,
appear
of
and
a
to the
3-cell,
5-cell
of asynchro-
incidence
is due
in
Complete
I
Combined
The
and
I
6472
of the
after the
None,
Partial,
Incomplete
nucleoli
(see text for details).
of
I
96
incorporation
of (3 HI urmdine
by 2-cell,
3-cell
and 4-cell
embryos
that were
control,
female
stress,
male stress and combined
stress
treatment
groups
at 64,
administration
of HCG. Following
autoradiography
the embryos
were classified
into
FIG.
females
ber
I
88
Male
-
nous
72
Female
CONTROL
and
I
,
:
hyperthermia
embryos
2-cell
paternal
of
stage;
are
arrested
in
conse-
hyperthermia
development
is
(Figs.
BELLVE
636
Nucleol;
EIGHT-
Labelling
CELL
None
Partial
Incomplete
Complete
w
r
uJ
>
0
U
w
U-)
-
_______
MORULA
80
-
BLASTOCYST
20
-
0
>-
80
60-
40-
20-
ri
I
In
U-
0
z
0
I0
aa:
0
U
z
I
rflflAE
0
I
HOURS
64
72
88
64
96
I
72
I
I
88
96
I
64
I
72
Female
CONTROL
flfl/fl.
I
I
88
96
I
I
64
Male
72
I
88
96
Combined
STRESS
FIG. 3. The incorporation
from females
of the control,
96 h after the administration
of None,
Partial,
Incomplete
nucleoli
(see text for details).
2 and
of
female
(3
of HCG.
and
HI uridine
by 8-cell embryos,
morulae
stress, male stress and combined
stress
Following
Complete
autoradiography
according
to
3).
the
the
Embryonic
The
trol
2-cell
of
[3H]
Uridine
During
Normal
Development
incorporation
nucleus
(Fig.
overlaying
embryos
( H] uridine
is primarily
confined
by
conto
the
dent
43
were
4)
in
to 48
classified
the
are
h after
marked
labeled
clustering
nucleoli.
cytoplasmic
HCG
categories
of
When
38 h post-HCG,
few,
over
the
cytoplasm.
at
embryos
the
contained
multiple
observed
distinct
2-cell
a
were recovered
at 64, 72, 88 and
into
that
with
is initiated
grains
However,
of
groups
of blastomeres
grains
any,
that
blastocyls
treatment
embryos
proportion
incubation
Incorporation
and
labeling
that
(Fig.
are
5).
Both
cultured
nuclear
if
is evifrom
and
ABNORMAL
cytoplasmic
clease
vations
labeling
but
into
the
G2,
of
not
suggest
cytoplasm
the
a
increase
in the
nuclear
RNA.
of
synthesis
to
a
lesser
RNA
(Woodland
velopment
tion
the
increases
observation
embryos
rate
embryo
the
cell
the
10).
embryo
the
much
more
plasm
than
original
four
are
4-cell
sive
cleavages
and
1971;
Effect
of Maternal
[3H]
-Uridine
The
2-cell
from
labeling
blasto-
the
blastomeres
over
are
the
11).
The
is partly
to
partly
due
synthesis,
to
succes-
enhanced
an
increase
particularly
1969;
and
Hyperthermia
and
1971).
5. Three
embryos.
FIG.
isotope
particularly
FIG.
2-cell
embryos
of grains
15
(Figs.
are
arrested
recovered
over each
nucleus
at 43
at
is invariably
be noted,
labeling
embryo.
the
the
in
in
each
17).
to
the
this
a
are able
to
incorporation
of
[3 H] uridine
to
control
nucleus.
amount
less
comparable
increase
even
is
that
continue
but the
to
label
It should
of isotope
time
than
degree
cleavage
is
of
2-cell
blasto-
their
rate
of
though
they
another
undergoing
em-
of develop-
of a normal
the arrested
that
of
cleaving
blastomere
in considerably
achieve
is
of
stage
may
precursor
that
In
the
age
The
2-cell
isotopic
10).
which
hyperthernumber
case
chronological
confined
however,
to
Fig.
is similar
and
over
the
although
comparable
blastomere
maternal
a pattern
which
re-
is intriguing.
a variable
In
same
in
the
incorporation
compare
the
by
in a blastomere
This
suggests
incapable
which
of mouse embryos
of HCG and cultured
FIG.
4. Control
2-cell
embryo
recovered
at 38
are localized
over the nucleus
and are particularly
can be seen over the cytoplasm.
X 300.
FIG.
ment
meres
cursor
on
FIGS. 4-9. Autoradiographs
96 h after the administration
the concentration
exhibit
required
Incorporation
embryos
blastomeres
incorporation
bryos
at the
and
that
undergoing
than
14,
through
divisions.
88
blastomere
of a 3-cell
detectable
incor-
lower
continue
arrested
incorporate
uridine
can
be seen
blastomeres,
(Fig.
is incorporated
rRNA
Daentl
Daentl,
the
increased
over
due
cytoof
may
at
it appears
precludes
division
grains
cleaving
levels
particularly
embryos
of embryos
unaffected
cleavage
6-cell
in
embryos
a number
apparently
mia
nucleus
blastomeres
Graham,
a
premitotic
at
control
[3H]
the arrested
to demonstrate
while
the
a
embryos
recovered
‘asynchronous’
of
usually
but
asynchronous
(Fig.
Epstein
those
extensive
to
poration
nuclei
This
3-cell
of RNA.
of
observed
during
Similarly,
isotope,
are
of all species
pattern
12).
the
the
which
Occasionally,
embryo
fails
de-
in
incorporation
and
of RNA
and
by
labeled
stages
(Woodland
Epstein,
an
embryo
uptake
rate
the
two
3 HI uridine
of
tarded
derived
daughter
the
Knowland
instance,
confined
in
extensively
rate
precursor
in
is
is
normal
For
exhibit
but
Likewise,
which
incorpora-
blastomeres
division
The
9).
of
that
synthesis
6 to
2 and
96 h (Figs.
2 and 13). Thus,
these
embryos
the
treatment
embryonic
[3 HI uridine
two
isotope
the
(Fig.
of
division.
cytoplasm,
mere
in the
of
cell
a recent
over
stages
(Figs.
incorporate
embryos
into
precursor
1969;
do not
those
hyperthermia
[3 H] uridine
proportion
4S RNA
ribosomal
primarily
substantially
(Figs.
is strikingly
emphasized
which
are
undergoing
asynchronous
3-cell
suggest
(hnRNA),
Graham,
Graham,
1972).
During
subsequent
an
maternal
to incorporate
5 h incubation
also
may
or
fail
significant
processing
results
extent,
the
637
following
generally
late
it
and
RNA
and
phase,
pools
incorporated
nuclear
and,
and
precursor
Previous
is
heterogeneous
final
DEVELOPMENT
development
ribonu-
Alternatively,
in
rate
precursor
the
the
cycle.
change
by
These
obserRNA
is released
during
cell
reflect
of
is digestable
deoxyribonuclease.
that
nuclear
EMBRYONIC
preare
divi-
sion.
which were recovered
from control
females at 64, 72, 88 and
in [3 HI unidine
for the designated
period of time.
h after HCG and cultured
in [3 HI unidine for 5 h. The grains
dense
in areas coincident
with
multiple
nucleoli.
Few grains
h post
is similar.
HCG
Grains
and
incubated
are present
in
[3
HI uridine
over the cytoplasm
for 5 h. Note
that
of the two outside
X 160.
6. Autoradiograph
of a 4-cell
embryo
recovered
at 64 h and cultured
in [3 HI uridine
for 90 mm. The
is primarily
concentrated
in the
nuclei.
Many
grains
occur
over
the cytoplasm
of each blastomere,
in the lower
two blastomeres.
X 300.
7. Eight-cell
embryo
recovered
at 72 h and cultured
in [3 HI unidine
for 45 mm. The grains are primarily
concentrated
over the nuclei.
More
grains
occur
over the cytoplasm
of the four blastomeres
at lower left. X 300.
FIG.
8. Early
morula
recovered
at 88 h and cultured
in [3 HI uridine
for 20 mm. Numerous
grains
are located
over the nuclei.
Preferential
labeling
over the nucleoli
can be seen in four nuclei (left). The cytoplasm
has been
partially
removed
by pronase
to enhance
dispersion
of the nuclei.
X 300.
FIG.
9. Blastocyst
recovered
at 96 h and cultured
in [3 HI uridine
for 10 mm. The nuclei
are heavily labeled
and numerous
grains occur over the cytoplasm.
X 300.
638
BELLVE
ABNORMAL
EMBRYONIC
DEVELOPMENT
639
#{149}
-
i.
,.,
.
-
#{149}., ,,
-.
S
10
11
3-cell
embryo
recovered
at 64 h and cultured
in (3 HI uridine
for 4 h. The uncleaved
original
2-cell embryo
(upper)
exhibits
labeling
over the nucleoplasm;
few grains occur over
the cytoplasm.
In contrast,
the lower
two blastomeres
have grains
extending
throughout
the cytoplasm.
X 300.
FIG.
11. ‘Control’
6-cell embryo
recovered
at 64 h and cultured
in (3 HI uridine
for 90 mm. The uncleaved
blastomeres
(upper)
of the original
4-cell
embryo
exhibit
prominent
labeling
over the nuclei
with scattered
grains
over the cytoplasm.
In contrast,
the 4 blastomeres
resulting
from recent mitotic
divisions
have numerous
grains
over the cytoplasm.
X 300.
FIG.
10.
blastomere
A
‘Control’
of the
limited
number
of
4-cell
and
8-cell
em-
blastomere
arrested
bryos
are
merely
retarded
in
development
following
maternal
hyperthermia.
The
pattern
of [3 HI uridine
incorporation
observed
in these
embryos
is variable
with
many
exhibiting
a
other
sion
having
to yield
one
(Fig.
successfully
16). Usually,
reduction
differential
cursor.
chronous’
show
in
the
number
demonstrable
ration
4-cell
FIGS.
(female
FIG.
(Figs.
levels
2
embryos
12-17.
and
are
of
of
blastomeres
that
precursor
3). Furthermore,
frequently
seen
Autoradiographs
incorpo-
retarded
with
one
of embryos
recovered
blastocoelic
from
female
at
the
2-cell
undergone
another
two
blastomeres,
completed
all four
stage
and
cleavage
of which,
a further
blastomeres
division
exhibit
incorporation
of the isotopic
Embryos
which
are undergoing
or retarded
divisions
frequently
cavities
mice
that
at
were
the
same
subjected
the
divionly
a
pre‘asynform
chronological
to hyperthermia
stress).
12. Two-cell
embryo
which
was recovered
at 64 h and incubated
in (3 HI uridine
for 5 h. The effect
of
maternal
hyperthermia
is evident
by the absence
of grains over both nuclei
and cytoplasm.
X 300.
FIG. 13. Three-cell
embryo
recovered
at 88 h and incubated
in [3 HI uridine
for 4 h. No grains
are evident
over any of the 3 blastomeres
even though
the embryo
appeared
normal
and continued
to exclude
eosin y during
culture.
X 300.
FIG.
14. Partially
affected
3-cell
embryo
recovered
undeaved
blastomere
(upper)
has not incorporated
the
over the nuclei
and a few scattered
grains
in the cytoplasm.
FIG.
15. Partially
affected
5-cell embryo
recovered
arrested
Fig. 4).
blastomere
In contrast,
(upper)
exhibits
the ‘cleaving’
prominent
blastomeres
labeling
(lower)
at 64 h and incubated
isotopic
precursor.
Two
X 300.
in [3 H) uridine
for 4 h. The
blastomeres
(lower)
have grains
at 72 h and incubated
in [3 HI uridine
for 90 mm. The
over the nucleoli
but not over the cytoplasm
(compare
exhibit
a pattern
of grains typical
of an 8-cell embryo.
X 300.
FIG.
16. ‘Four-cell’
embryo
recovered
at 88 h and
incubated
in [3 HI uridine
for 90 mm.
The prominvnt
nucleus
of a blastomere
arrested
since
the 2-cell
stage
(upper)
has grains
localized
over the nucleoli
but none
over
the cytoplasm.
The remaining
blastomeres,
which
may
be retarded
in development,
exhibit
differences
in both
nuclear
and cytoplasmic
labeling.
X 300.
FIG.
17. ‘Morula’
recovered
at 88 h and incubated
in [3Hjuridine
for 20 mm. One blastomere
(uppermost),
apparently
arrested
since the 2-cell stage,
has grains
only over the contracted
nucleus.
The remaining
blastomeres
exhibit
a distribution
of isotope
comparable
to normal
blastomeres
of a late morula.
X 300.
640
BELLV’E
ABNORMAL
EMBRYONIC
DEVELOPMENT
641
#{149}
.
19
18
FIG.
18. Nine-cell
embryo
recovered,
after
maternal
hyperthermia,
at 96 h and incubated
in [3 HI uridine
45 mm.
Although
arrested
or retarded
in development
the embryo
has formed
a trophoblastic
vesicle
and
nuclei
and cytoplasm
of each blastomere
is heavily
labeled.
X450.
FIG.
19. ‘Blastocyst’
recovered
at 96 h, following
maternal
hyperthermia,
and incubated
in (3 HI uridine
10 mm. This blastocyst-like
embryo
exhibits
a pattern
of labeling
comparable
to a normal
blastocyst.
X 300.
age
as
normal
contain
vesicles
(Fig.
are
these
terns
same
embryos
fewer
blastomeres.
composed
18)
and
of three
blastocyst-like
frequently
of
Male
trophoblastic
or more
they
tal
blastomeres
19)
of
embryos
synthesis
variable
The
blastomeres
During
the
to
incorporate
that
FIGS.
were
FIG.
early
cleavage
on
[3H1
Uridine
stages
the
pattern
incorporation
appears
to be
(Fig.
2). However,
at the 8-cell
stages
increasing
numbers
of
blastomeres
which
either
fail
the
precursor
of
at
or exhibit
reduced
(Fig.
inner
these
of
male
localized
cytoplasm
affected
has been
partially
morula
recovered
removed
at 88
normal
to
a preferential
cell
mass
pole
blastomeres
effect
cannot
to improve
h (classified
are
still
normal
H] uridine
the spreading.
incompletely
for
23).
fre-
embryo
or
presumptive
determined
from
The
partially
20
after
mm.
X 300.
labeled).
a
to
to
are
by
the
pre-
mating
All
The
nuclei
effected
incorporate
females
RNA
localization
phenomenon
on the
be
which
[3
of the
preparations.
hyperthermia
from
21
regional
localized
autoradiographic
of
contain
which
fail
(Figs.
blastomeres
one
stages.
of
the
pattern
the
remainder
blastomeres
show
a definite
22).
Whether
this
20-24.
Autoradiographs
of embryos
which
were recovered
subjected
to hyperthermia
(male
stress).
20. ‘Normal’
morula
recovered
at 88 h and incubated
in
intense
labeling.
The
FIG.
21. Partially
a
20)
of
for
This detrimenis particularly
and blastocyst
60
percent
the
precursor
the
affected
reflects
3).
hyperthermia
the morula
approximately
(Fig.
number
quently
least
(Fig.
male
exhibit
incorporate
Moreover,
age.
Hyperthermia
incorporation
Although
at
of
[3 H] uridine
entirely
normal
and
subsequent
embryos
contain
of
effect
apparent
(Fig.
Incorporation
blastomeres
FIG.
incorporate
FIG.
incorporation,
incorporation.
FIG.
‘normal’
levels
generally
exhibit
labeling
patto normal
embryos
at the
chronological
Effect
though
structures
observed.
embryos
comparable
even
Thus,
for
the
nuclei
nuclei
to males
exhibit
of three
have failed
to incorporate
[3 HI uridine.
X 300.
22. Morula
recovered
at 88 h. Most
blastomeres
in one
pole
of the embryo
(upper)
have
failed
to
(3 HI uridine.
X 300.
23. Morula
recovered
at 88 h. Only
eight
of the nuclei
demonstrate
‘normal’
levels
of [3 HI uridine
the remaining
nuclei
have either
failed
to incorporate
the precursor
or exhibit
reduced
levels
of
X 300.
24.
Two
blastocysts
recovered
from
the same
female
at 96 h. While
one blastocyst
(left)
exhibits
incorporation,
the other
blastocyst
has completely
failed
to incorporate
[3 H] uridine.
X 320.
BELLVE
642
-
.2;
--
.
.
ABNORMAL
cursor
into
their
overlaying
nucleoli
the
with
nucleoplasm.
progressively
nucleoplasmic
poration
of
persists
ceased.
until
scattered
As
deteriorates
labeling
synthesis
cytoplasmic
Nucleolar
although
after
labeling
grains
RNA
both
is lost.
precursor,
EMBRYONIC
and
incor-
other
areas
RNA
synthesis
due
to activation
during
has
the restricted
immediately
mental
apparent
a particular
effect
of
that
not
female
all spermatozoa
carry
the detri-
hyperthermia.
This
is
2-cell
stages
by
phase
of the cell cycle
the cleavage
division.
causes
related
an
three
forms
arrest
which
is
cytoplasmic
Mukherjee,
1973)
then
are operative
early
in
hyperthermia
stage
cleavage
genome
and
involved
but
apparently
mortality,
1)
point
early
of the
G1
after
Maternal
It is also
inseminating
643
factors
(Bernstein
the mechanisms
diminished,
in
DEVELOPMENT
in
of
discrete
embryonic
at the
development
is associated
if not
with
a failure
to
clearly
demonstrated
and blastocysts
which
hyperthermic
patterns
by the fact that
morulae
exhibit
either
normal
or
of RNA
synthesis
can be
incorporate
[3 H] uridine
into
effect
in which
one blastomere
RNA,
2) a partial
is arrested
and
the
undergo
recovered
same
cleavage
tarded
from
the
Furthermore,
a substantial
embryos,
patterns
into
female
(Fig.
proportion
of
presumably
those
that
show
of [3 HI uridine
incorporation,
viable
fetuses
24).
these
normal
develop
(Bellv#{233},1973).
other
blastomere
ent lack
embryos
of RNA
is not
mortality
since
the
eosin
y for
exclude
the
termination
Furthermore,
DISCUSSION
During
the
normal
mouse
detectable
embryonic
the
incorporation
by
autoradiography
development
of
at
cleavage
precursor
(Mintz,
1964a).
At
is almost
exclusively
nucleus
being
localized,
fibrillo-granular
which
late
1969).
of the
in
the
In
be
cell
grains
period
are
presumably
This
reflects
the
(hnRNA)
from
(Woodland
and
transfer
labeling
of 4S
nucleus
to
Graham,
RNA
The
1969;
(3 H] uridine
incor-
into
RNA
increases
substantially
stages
of development
(Monesi
consequence
RNA
of
synthesis
enhanced
and
1969;
but
uptake
intracellular
an
increase
and
kherjee,
1973).
synthesis
is
increased
incorporation
part,
precursor
into
(Daentl
Epstein
in
in
Daentl,
The
and
1971;
over
and
the
of
inhibits
for continued
also relevant
diminishing
1971;
and
thermia
Mu-
in RNA
of
emphasized
by
the
period
precursor
that
oc-
fined
increase
of
curs
in the
new
generation
normal
embryos
undergoing
sion
(Figs.
10 and
11).
Clearly,
of
blastomeres
asynchronous
if the
in
species
in
until
al.,
divi-
increase
of
first
et
to
ova
in
RNA
of
1-
al., 1973),
actinomycin
3)
D
arrest
does
cycle
(Moli-
4) exposure
of Tetraelevated
temperature
the
1-cell
arrest
a later
stage
1965;
Criblez
the
that
division
(Fig.
25).
temperature
stage
in
6 to
final
mitotic
stage
prime
the
1) RNA
rabbit
development
of hnRNA
with
2-cell
to
that,
1-cell
is essential
cell division
(Moner,
1967).
It is
that
the period
of maternal
hyperthe
of
not
prevents
hnRNA.
In
developmental
following
cell
synthesis
stage,
the
do
DNA-primed
(Golbus
exposed
and,
to
coincides
1-cell
to, or
(Criblez
to hyperthermia
1974),
2) actinomy-
further
G2 period
until
the
an
note
in
inhibitor
prevents
al., 1972),
pyriformis
of CO2
embryos
to
D,
naro
et
hymena
rate
hyperthermia
RNA
and
4S
cm
to
Epstein,
rabbit
vitro
during
the
not occur
the
levels
of rRNA
until
the
stages,
respectively
(Wood1969;
Manes,
1971),
it is
have
been
subjected
(Criblez
and Alliston,
rate
due
Bernstein
dramatic
particularly
the
of
cell mouse
embryos
when
embryos
are
Gwatkin,
1968;
WoodPik#{244},1970)
primarily
also,
of
pools
of
and
1972).
as a
rate
Knowland
incorporation
yet
is equivalent
normal
embryos
maternal
of both
an
continue
to
48 h after
pyruvate
this regard
it is pertinent
synthesis
is also diminished
synthesis,
cytoplasm
poration
succeeding
1967;
Ellem
and Graham,
and
and
detectable
morula
Graham,
evident
that
the synthesis
arrested
2-cell
embryonic
[3 HI uridine
Alliston,
1974).
Since
mouse
and
that
RNA
the
Graham,
Salfi,
land
can
background
heterogeneous
the
and
the
cytoplasmic
transcribed
newly
and
observa-
incubation
and
in the
immediate
treatment
(Bellv#{233}, 1973).
rabbit
embryos
also exhibit
of
production
from
greater
than,
that
synthesize
4-cell
and
land
and
nucleoli
earlier
when
minimal.
hyperthermia
proportion
of the
in the cytoplasm
of
discerned
levels
following
the
(Hillman
to
of
reduced
stage the
to the
further
but
reThe appar-
blastomeres
approximately
2-cell
is
of
over
cycle
contrast
a prolonged
readily
2-cell
confined
definitive
tions
(Mintz,
1964a)
a minor
isotopic
precursor
is located
after
stages
primarily,
cortex
form
Tasca,
the
all
synthesis
due
to
in
[3 HI uridine
may
divisions,
and,
3) continued
cleavage
of both
blastomeres.
7 h of
and
However,
the
sensitivity
even
cleavage
in development
and
Alliston,
is con-
though
may
the
most
in
not
some
occur
(Alliston
et
1974).
These
BELLVE
644
PMS
F.rI
!2!#{176}’
-
-48
1
0
___
Ic 14
31
-
49
lo 52
1
-
I
of
7
Iwo-cell
stages
the
the
8-cell
FIG.
25. Schematic
presentation
of the temporal
relationships
between
maternal
hyperthermia,
the
stage
of embryonic
development
and cycles
of DNA
synthesis.
Time
is referenced
to the injection
of HCG
at 4.0 pm on a daylight
regime
(open
areas)
of 7 am to
7 pm. The time of ovulation,
DNA synthesis
(hatched)
and
cleavage
divisions
were
taken
from
the data
of
Edward
and
Gates
(1959),
Whittingham
(1969),
Gamow
and
Prescott
(1970),
Barlow
et al. (1972),
Luthardt
and
Donahue
(1973)
and
Siracusa
et al.
(1975).
observations,
together
ment,
imply
with
that
the
incurred
during
the
first
cleavage
division.
observations
of
G2
1-cell
current
experi-
affect
7 h period
On the
is entirely
it
the
the
hyperthermic
preceding
basis
of
plausible
stage,
that,
hyperthermia
‘asynchronous’
RNA
synthesis
ceed
through
tively,
another
in
these
thermia
may
cessing
or the
be
in
available
for
hnRNA
of the
explain
under-
division.
with
of RNA
which
or
are
capable
unable
to
case
of
pro-
aberrations
pattern
be
the
cavitate
of
of
are
(Smith,
1956;
1970;
Hillman,
to
Mintz,
1972;
2-cell
1953;
that
ex-
observed
the
of
Hillman
et al.,
1975),
following
(Goldstein
1975)
embryos
to
to
al.,
and
in
(Austin
first
and
In
evident
paternal
embryos
near
genome
(Brinster,
is
1974).
days
be transported
the vas deferens
1975).
et
1967).
is
Wudl,
eight
in em1 2 allele
development
effect
and
period
(BeIlv#{233} et
the
delayed
paternal
hy-
for
Tarkowski,
lethal
Chapman
spermatozoa
epididymis
mortality
implantation
Bomsel-Helmreich,
the
an
incorporation
embryos
1973;
is
exhibit
parthenogenetic
of polyploid
It
which
1964b;
Bennett,
development
case
blas-
a decidual
precursor
(Witkowska,
each
appear
these
to form
some
respects
induced
by
during
Braden,
of
inducing
homozygous
of
not
implantation.
is similar
which
of
partial
It is possi-
for
the
embryonic
or shortly
after
at
a
does
many
embryos
(Bellv#{233}, 1973).
In
embryonic
mortality
A
would
cannot
responsible
occurs
1973;
pro-
translation.
potential
abnormal
cytoplasm
mRNA
mortality
since
undergoing
that
1975),
and
the
time
at which
the
initially
activated
in normal
hypernuclear
and
rate
[3 HI uridine.
capable
presumed
beyond
retarded
failure
in RNA
synthesis
an incipient
cell
death.
eventually
are
reaction
al.,
a
early
proportion
blastulation
may
and
that
the
developed
exhibit
embryonic
immediately
embryos
tocyts
have
apparent
reflect
merely
However,
to
ensue
apparent
during
a significant
incorporate
the
X-irradiation
Alterna-
to the
may
bryos
during
blastomeres
interferred
transfer
1975)
Other
cleavage
particular
have
(Greenberg,
not
division
but are
to
that
are
that
yet
which
stage
perthermia
the
these
directly
indirectly
represses
the
synthesis
of
that
is essential
for further
development
2-cell
embryo.
This reasoning
may also
why the arrested
blastomeres
of embryos
going
limited
is
ble
and
delayed
failure
peod]
is
occurs
embryos
cleavage,
I Ir,OIn’enl
It
development
cleavage
-
One-Cell
spermatozoan.
normal
1
I
fertilizing
Sond
Ic 33
II
-
U-
I
Since
is
required
from
in
for
the
caput
the
embryonic
mouse
lethality
cluded
at this time.
For instance,
hyperthermia
may have interferred
with DNA synthesis
or the
mitotic
process
(Zeuthen,
1972;
Edwards
et al.,
1974)
although
the absence
of developmental
occurs
only
when
males
are mated
after
exposure,
it can be concluded
thermia
has a detrimental
effect
spermatozoa
in the
epididymis
3 to 8 days
that hyperon immature
and
is not
a
arrest
consequence
that
at
the
1-cell
possibilities.
RNA
synthesis
imbalance
in
the
early
1974;
1973;
applied
period
that
parthenogenetic
Witkowska,
24
that
h
stages
et
the
argues
feasible
al.,
of
the
(Biggers
1974).
hyperthermic
these
effect
1973)
paternal
since
and
It
on
some
of
Bellv#{233},
is unlikely,
treatment
development
since
after
mating
and
is optimal
for
the
particularly
against
that
may
be secondary
to
unique
energy
metabolism
cleavage
parthenotes.
The
effect
triguing,
the
Barbehenn
however,
duces
stage
It is also
hyperthermia
it is mediated
of the
stages
inis
the
of
is inby the
of
and Steinberger,
regard
it is
cause
tation
an
when
and
that
normal
process
mechanisms
to an effect
drial
recovered
oxidative
occurs
at
(Chowdhury
1973
a,b).
immature
In this
rabbit
from
the
into
does,
corpus
also
in the incidence
of preimplanmortality
(Bedford,
1966;
1971).
paternal
Ploen,
that
inseminated
increase
embryonic
Orgebin-Crist,
degeneration
spermatogenesis
1970;
pertinent
spermatozoa,
epididymis
(Komar,
treatment
following
induction
earlier
It
hyperthermia
of
sperm
involved,
which
of hyperthermia
metabolism
is
feasible,
interferes
maturation.
therefore,
with
the
But,
the
may be secondary
on the mitochonof immature
sperma-
ABNORMAL
tozoa
(Voglmayr
speculated
questions
by
of
the
uents
of
tinued
the
ovum
the
is
to
the
It
that
there
embryonic
that
in
for
be
than
essential
to
constit-
for
its
con-
development.
ACKNOWLEDGMENTS
The
author
extends
his appreciation
to Steven
Borack
of the
Photographic
Unit
and
to Mrs.
Mary
Forte
for preparation
of the manuscript.
This
research
was supported,
in part,
by NICHD
grants
HD-06916
and HD-06645
and by the Rockefeller Foundation,
RF-6 5040.
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