J. Embryol. exp. Morph. Vol. 32, 3 pp. 849-855, 1974
Printed in Great Britain
g49
The effect of 5-bromodeoxyuridine on
early mouse embryos in vitro
By WENDY GARNER 1
From Department of Genetics, University of Edinburgh
SUMMARY
The effect of 5-bromodeoxyuridine (BudR) on the development of cleavage stage mouse
embryos has been examined. The analogue was found to have a deleterious effect at relatively
low doses on survival to blastocyst, and to a lesser extent on trophoblast outgrowth in supplemented medium. No tendency to suppress differentiation rather than cell division was
detected. Possible mechanisms for the BudR effect are discussed.
INTRODUCTION
The effects of 5-bromodeoxyuridine (BudR = BrdU) on a number of different
in vitro culture systems have been examined with respect to the differentiation
of specific cell types. These studies reveal that in a wide variety of cell types,
including myoblasts (Coleman, Coleman & Hartline, 1969; Bischoff & Holtzer,
1970; Coleman, Coleman, Kankel & Werner, 1970), chondrocytes (Abbott &
Holtzer, 1968; Lasher & Cahn, 1969), amnion cells (Wessells, 1964), erythrocyte
precursors (Miura & Wilt, 1971), pancreatic acinar cells (Bischoff & Holtzer,
1968), melanoma (Wrathall, Oliver, Silagi & Essner, 1973) and hepatoma cells
(Stellwagen & Tomkins, 1971a, b) BudR can inhibit differentiation both in
terms of morphology and the synthesis of cell products characteristic of the
differentiated state, without noticeably affecting cell division and viability. In
most cases suppression of differentiation occurs when BudR is introduced during
a period of cell proliferation, and is consistent with an effect mediated through
incorporation of the analogue into DNA.
BudR effects have subsequently been examined on whole organisms. Tencer
& Brachet (1973) found that morphogenesis in sea-urchins, tunicates and
amphibians was delayed and/or abnormal in the presence of BudR. In Drosophila,
Rizki & Rizki (1969) have reported the development of supernumerary structures on wings and legs, and also bristle defects, in response to BudR.
Such effects provide a potentially useful tool for studying the control mechanisms involved in differentiation. It was of interest to know whether BudR
1
Author's address: Department of Genetics, University of Edinburgh, West Mains Road,
Edinburgh EH9 3JN.
850
WENDY GARNER
produced any similar alterations in the development of mammalian embryos
in vitro, in particular in the first visible differentiation into trophoblast and
inner cell mass.
METHOD
Two-cell or 8-cell embryos were collected from pregnant females of the
randomly-bred Q strain of mice by flushing from the oviduct into phosphate
buffered saline. Morulae were flushed from the uterus. Embryos were transferred to drops of the culture medium, described by Whitten (1971), but without
phenol red and streptomycin. The drops were held in petri dishes under paraffin
oil, incubated at 37 °C and gassed with 10 % CO2 in air (Bowman & McLaren,
1970).
Bromodeoxyuridine (Sigma) was introduced by dissolving 0-0031 g in 1 ml of
medium to give a concentration of 10~2 M. A range of concentrations was
prepared from this by dilution into medium.
The development of embryos was examined by scoring the number forming
morphologically normal blastulae, and from cell counts of air-dried spreads
prepared by the method of Tarkowski (1966).
The viability of blastocysts grown in BudR was further tested in two ways;
first, by transfer to medium supplemented with 5 % foetal calf serum (Gwatkin,
1966) which allows outgrowth of trophoblast but not inner cell mass cells, and
also by transfer to the uteri of pseudopregnant females. Autopsies were performed at 2 weeks of gestation. Foetal and placental weights were recorded and
embryos were examined for any obvious morphological defects.
RESULTS
The survival to blastocyst of embryos collected at different stages and subjected to continuous culture over a range of BudR concentrations is plotted in
Fig. 1. The graph indicates a sharp drop in survival at a concentration of around
10~7 M. At concentrations above 10"7 to 10~6 M the nucleoside is lethal. Two-cell
embryos undergo at least two divisions, but die between the 8-cell stage and
late morulae; similarly, 8-cell embryos degenerate after cleaving once or twice.
Morulae cultured in 10~6 M or 10~7 M BudR may reach the early stage of cavitation, but fail to develop further.
At concentrations below 10~ 7 M, which are not lethal to the embryos, no
effect on differentiation is observed; blastocysts appear morphologically normal,
and, as shown in Table 1 (a, b) cell numbers are approximately equal for all
experimental groups after 2 days or 3£ days in culture. For each set of data,
Student's t test was applied to compare experimental with control results.
Results for 3^ days show no significant difference. However, the mean cell
number for one group, namely those embryos grown in 10~8 x 5 M BudR for
2\ days, is significantly higher than the control mean.
Mouse embryos cultured in 5-bromodeoxyuridine
-11
JQ-10
jQ-9
,0-8
1 Q -7
1 Q -6
851
J0
Concentration of BudR (M)
Fig. 1 Survival to blastocyst stage of embryos cultured from the 2-cell
(O),* -cell (A) or morula ( • ) stage in various concentrations of BudR.
Table 1. Cell numbers ofblastocysts grown from 2-cell in various concentrations of
BudR for (a) 2 days and (b) 3% days in culture
(a)
(b)
Molar concn
of BudR
0
io- 8
IO-8 x 5
io- 7
10~7x5
0
IO-8 x 5
io- 7
No. of
eggs
20
8
14
10
7
10
6
9
No. of cells
±S.E.M.
41-8 ±2-29
41-4 + 6-82
52-4 ±2-45**
48-1 ±3-57
41-6±3-56
75-3 ±4-95
74-8 ±14-96
83-8 ±4-78
** Significantly different from control (P < 0-01).
Transfer of blastocysts to medium supplemented with 5 % foetal calf serum
permitted trophoblast outgrowth in a high percentage of embryos in the absence
of BudR. Those concentrations of BudR allowing development to blastocyst
also allowed trophoblast outgrowth and giant cell transformation, on transfer
to supplemented medium of corresponding BudR concentration. Trophoblast
cells were counted (Table 2). BudR at doses of 10~8 x 5 M and 10~7 M resulted
in a consistent depression of cell number, although not significant in all cases,
while 10~9 M and to a lesser extent 10~8 M appeared to increase cell number.
852
WENDY GARNER
Table 2. Trophoblast cell numbers achieved after transfer of blastocysts grown
from the 2-cell or 8-cell stage in medium 1 into medium supplemented with 5 %
foetal calf serum {medium 2) using various concentrations of BudR
Molar concn of BudR
Stage into
culture
c
Medium 1
2-cell
0
10-8
10-8x5
io- 7
io- 7
0
8-cell
0
io- 9
io- 9
io- 8
10-8x5
8-cellt
8-cellf
io- 7
0
10-8
Medium 2
No. of eggs
transferred
Outgrowth
(%)
21
11
6
13
10
12
11
86
91
50
46
36
92
91
88
100
100
0
IO-8
10-8x5
io- 7
0
0
io- 5
io- 9
0
io- 8
10-8 x 5
io- 7
0
0
8
12
12
12
13
10
15
58
85
90
93
Trophoblast
cell no.
±S.E.M.
48-1 ±3-2
48-8 ±6-1
22-0 ±8-0**
34.O+3.4**
250 ±9-5*
54-1 + 6-6
32-0 ±4-3*
81-9±7-8*
91-3 ±8-3**
75-5 ±5-4*
29.9 + 3.4**
41-5±7-3
490 ±3-3
58-2 + 7-5
Comparison with non-Bud R-treated controls: * P < 005, ** P < 001.
t Outgrowths fixed after 2 days in supplemented medium; the others were outgrown for
4 days.
Table 3. The effect on post-implantation development at 2 weeks of gestation of
BudR treatment during culture from 2-cell for 48 h
Molar concn of
BudR
No. of embryos
transferred to
pseudopregnant
recipient
No. of viable
embryos
No. of
moles*
IO-8
6
0
0
6
3
3
6
0
10~7x5
2
0
5
0
io- 6
0
6
0
10-Gx5
29
3
5
Total
71
27
10
0
* Refers to number of sites in the uterus where implantation and decidua formation have
been achieved but where there has been only limited further development of the embryo.
io- 7
The same counts were obtained for embryos grown to blastocyst in 10~9 M
BudR whether they were transferred to the corresponding supplemented medium
or to control medium, suggesting that the effect, although manifested during
outgrowth, is initiated in the blastocyst before transfer.
To determine whether trophoblast itself is detrimentally affected by BudR
Mouse embryos cultured in 5-bromodeoxyuridine
853
without pretreatment up to the blastocyst stage, expanded blastocysts derived
from 8-cell embryos in control medium were transferred to either control or
BudR-containing medium for outgrowth. It was found that trophoblast would
attach and outgrow at concentrations of at least 10~ 3 M. Table 2 includes a
comparison of mean cell number after 4 days in supplemented medium, with
experimental outgrowths subjected to a comparatively high dose of BudR
(10 5 M). Values are significantly different at the 5 % level.
Data on experiments involving transfer of treated embryos to pseudopregnant females are presented in Table 3. Embryos were transferred at the
morula stage after 2 days in culture. It can be seen that 35 % of control transferred morulae developed normally, whereas only 10% of BudR-treated
embryos were viable at autopsy. The latter showed no obvious morphological
defects and foetal and placental weights of individuals derived from BudRtreated blastocysts were similar to those of controls.
DISCUSSION
The experiments reported here show that treatment of cleaving mouse embryos
with BudR at concentrations between 10~9 and 10~5 M leads to either morphologically normal development at the low doses, or death at the higher doses,
with no intermediate effects on differentiation to blastocyst, nor on differentiation of trophoblast cells during outgrowth. Thus the effect that BudR in some
systems exerts on differentiation rather than cell division is not apparent in
early mouse embryos.
In comparison with the BudR-dose effects reported by Tencer & Brachet
(1973) on sea-urchin, amphibian and tunicate embryos, and those of in vitro
cell cultures, mouse embryos seem particularly sensitive to the analogue. Possibly
mouse embryos are particularly permeable to exogenous nucleosides, since these
are known to be readily incorporated into mouse embryo DNA (Mintz, 1964;
Snow, 1973). Also, BudR may compete with natural nucleosides, as adenosine,
cytidine, guanosine, thymidine and uridine, in concentrations above 10~6 M,
are all inhibitory to the development in vitro of 2-cell mouse embryos into
blastocysts (Ten Broeck, 1968). This effect may be related to the finding (Steck,
Nakata & Bader, 1969) that these nucleosides can restrict the entry of heterologous nucleosides into the nucleotide precursor pool. Another relevant factor
may be that mouse embryos are dependent on the synthesis of new RNA much
earlier in development than are sea-urchins and amphibia. Sea-urchin embryos
can develop up to blastocyst stage in the presence of Actinomycin D, while
mouse embryos are sensitive to this drug as early as the 2-cell stage (Graham,
1973). If BudR incorporation into DNA resulted in erroneous transcription,
synthetic processes dependent on newly transcribed RNA would be affected.
Tencer & Brachet (1973) point out that the arrest in development of sea-urchin
and amphibian embryos in BudR is correlated with the timing of an increased
854
WENDY GARNER
synthesis of m'RNA, an event which in mouse embryos probably occurs at the
8- to 16-cell stage, when total RNA synthesis increases sharply.
The depression of trophoblast outgrowth after culture in BudR concentrations
of around 10~7 M is not surprising, since this is near the lethal level. The increase
in number of outgrowing trophoblast cells at lower dose levels, and in one
experiment the increased number of cells in the blastocyst, is more difficult to
explain but may nonetheless be a real effect, since Bischoff & Holtzer (1970)
also reported the occasional enhancing effect of BudR at relatively low doses
on cell number in myogenic cell culture.
This work was carried out during an M.R.C. studentship at the Institute of Animal Genetics,
Edinburgh, under the supervision of Dr Anne McLaren, to whom grateful thanks are due. I
am also grateful to Dr McLaren for doing the embryo transfers, and to the Ford Foundation
for equipment.
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(Received 20 June 1974)