/ . Embryol. exp. Morph. Vol. 36, 3, pp. 623-638, 1976
Printed in Great Britain
623
Direct exposure of postimplantation
mouse embryos to 5-bromodeoxyuridine in vitro
and its effect on subsequent chondrogenesis
in the limbs
ByNARSINGH D. AGNISH1
AND DEVENDRA M. KOCHHAR1
From the Department of Anatomy, University of Virginia Medical School, U.S.A.
SUMMARY
As maternally administered 5-bromodeoxyuridine (BudR) is very quickly degraded by the
liver, a combination of whole embryo culture and organ culture techniques was adopted to
expose postimplantation mouse embryos to the analog and to study the effects of long-term
treatment on the subsequent differentiation of limb-buds. Early and mid-1.lth-day mouse
embryos were exposed to increasing concentrations of BudR for 12 or 24 h. Forelimbs of the
treated embryos were then organ-cultured in drug-free medium and the extent of cartilage
development in the explants examined. Exposure of embryos to 50-150/ig/ml of BudR for
24 h resulted in significant inhibition of chondrogenesis in the subsequent limb cultures and
the effect was related to dose. After treatment with 150 /*g/ml of the drug, the forelimbs of the
early 11-day embryos (somite stage 26-29) showed an almost complete lack of cartilage, while
the limbs of mid-1 lth-day embryos (somite stage 32-34) were not nearly as sensitive and
exhibited about 50% reduction in the amount of cartilage development. We conclude that if
embryos in which the limb development is at a very early stage of development are exposed to
BudR, the future course of limb differentiation is permanently and irreversibly damaged,
resulting in a partial or even complete suppression of chondrogenesis in the organ. As both the
dose and perhaps also the duration of treatment were critical, we suggest that the rather low
frequency of reported limb malformations after in vivo injection of teratological doses of BudR
may be due to only a small amount of the chemical reaching the embryos.
INTRODUCTION
5-Bromodeoxyuridine (BudR) - an analog of nucleoside thymidine - has
recently been described as an 'ideal teratogen' by Rutter, Pictet, Githens &
Gordon (1975). Applied to a wide variety of cells and tissues in culture, it has
been shown to inhibit differentiation both in regard to cellular morphology and
to the synthesis of specialized cell products characteristic of the differentiated
cells and yet, at least in low concentrations, it is non-toxic and appears to have
little effect on cell viability, growth or proliferation (see reviews by Wilt &
1
Authors' address: Department of Anatomy, Jefferson Medical College, 1020 Locust Street,
Philadelphia, Pennsylvania, 19107, U.S.A.
624
N. D. AGNISH AND D. M. KOCHHAR
Anderson, 1972; Rutter, Pictet & Morris, 1973; Levitt & Dorfman, 1974). While
BudR is a potent inhibitor of chondrogenesis (Abbott & Holtzer, 1968) and
myogenesis (Stockdale, Okazaki, Nameroff & Holtzer, 1964) in cell culture, the
only reported limb anomaly after maternal administration of a teratogenic dose
was polydactyly in a small proportion of the fetuses (DiPaolo, 1964; Ruffolo &
Ferm, 1965; Skalko, Packard, Schwendimann & Raggio, 1971). BudR is very
quickly and extensively degraded by the maternal liver (Kriss & Revesz, 1962) so
that the analog is available to the embryos for only a short period of time
(Packard, Menzies & Skalko, 1973). It is possible to increase the incorporation
of BudR in the proliferating cells of adult mice by employing partially hepatectomized animals (Hill, Augenlicht & Baserga, 1973) but such a procedure would
no doubt complicate the interpretations of in vivo teratological investigations.
We have instead employed whole-embryo culture - a 'mother-free' system -to
study the full potential of BudR teratogenecity.
Postimplantation rat (New, 1967; Cockroft, 1973) and mouse (Kochhar,
1975a) embryos can be grown in culture for a limited period of time. Under
suitable conditions, growth and differentiation of embryos in vitro can be
maintained almost as well as in vivo (Cockroft, 1976). Whole-embryo culture has
not only proved useful in the understanding of normal differentiation processes
(Moore & Metcalf, 1970; Deuchar, 1971) and in metabolic studies (Shepard,
Tanimura & Robkin, 1970; Tanimura & Shepard, 1970) but also in teratological
investigations (Turbow, 1966; Turbow & Chamberlain, 1968; New & Brent,
1972; Morriss & Steele, 1974; Robkin, Shepard & Baum, 1974; Kochhar,
1975b). Teratological studies in whole-embryo cultures have certain advantages
in that the effect of a drug can be investigated without the chemical being
screened out, degraded or modified in some way by the maternal system. However, the system has the limitation that the embryos can be maintained for only a
fraction of their in utero life, so that no long-term effects of a teratogen can be
studied. We have tried to alleviate this limitation by combining whole-embryo
culture with limb-bud organ culture. In this 2-step culture procedure, isolated
mouse embryos were exposed to BudR in vitro for 24 h, after which their limbs
were excised and cultured for another 9 days. It was hoped that such an approach
might prove to be useful in evaluating the full teratogenic capabilities of BudR,
and might provide some information on how BudR treatment of embryos would
affect the subsequent development and differentiation of the limbs. The choice
of limb as the target organ was deliberate; the mesodermal regions which would
differentiate into the various cartilages are already present in their normal seriation in the earliest visible limb-buds (Stark & Searls, 1973) and technique of
limb-bud organ culture is well established (Aydelotte & Kochhar, 1972;
Kochhar & Aydelotte, 1974; Neubert, Merker & Tapken, 1974).
Effect of BudR on mouse embryos
625
MATERIALS AND METHODS
Animals
Random-bred ICR/DUB mice (obtained from Flow Laboratories, Dublin,
Virginia) were maintained on Purina Mouse Chow and tap water ad libitum and
kept in the light from 6 a.m. to 6 p.m. The female mice were mated either overnight or, when more precise time of mating was required, for 4 h between 8 a.m.
and noon. The presence of a vaginal plug immediately afterwards was regarded
as evidence of successful mating; this day was designated as the 1st day of
pregnancy.
Culture procedure
To study the effect of the incorporation of BudR by mouse embryos on the
subsequent development of their limbs, a 'two-step culture procedure' was
adopted. Isolated embryos were maintained in vitro for 12-24 h in the presence
of the drug. Afterwards, their limb-buds were excised and grown in organ
culture for another 9 days.
Whole-embryo culture
On the early or mid-1 lth or 12th day of pregnancy, female mice were killed by
cervical dislocation, dissected and the intact uteri transferred to a dish containing Tyrode saline solution. Using standard sterile techniques, the horns of
the uterus were cut open and the conceptuses released into the saline. An attempt
was made to select embryos that appeared similar in size and nearly identical in
their developmental stage (within 2-3 somites). Reichert's membrane and the
parietal layer of the yolk sac of each individual embryo was dissected away. This
operation lasted approximately 30-45 min. Leaving the conceptuses in the saline
at room temperature for this period of time did not seem to affect the eventual
growth of the embryos in culture, a fact also noted by New (1971). The embryos
with their visceral yolk sacs intact were then cultured for 12-24 h in Waymouth's
medium diluted with an equal amount of fetal calf serum, as described in detail
elsewhere (Kochhar, 1975a). Embryos were put singly in 10 ml screw-cap vials,
each with 2 ml of the medium. The remaining volume was filled with a gas
mixture of 95% O2 + 5% CO2. The vials were then clamped on to a motordriven rotator and rotated constantly at 36-40 rev./min. At the time of medium
renewal, all of the old medium was removed and replaced with the same amount
(2 ml) of fresh medium and the cultures regassed. After enough had been set up
in culture, the few remaining embryos were used for staging and their precise
somite number recorded. It was assumed that this somite count was representative of all the embryos and is referred to as the 'somite number at the time of
embryo explant' in the text. The culture medium used was Waymouth's medium
MB 752/1 supplemented with 50% fetal calf serum (both obtained from Flow
Laboratories, Rockville, Md.). The well-being of the embryos was monitored at
40
EMB 36
626
N. D. AGNISH AND D. M. KOCHHAR
various times during the in vitro growth by checking their heart beat and circulation in the blood vessels of the yolk sac.
Treatment with drugs
BudR (obtained from Sigma, St Louis, Missouri) at various concentrations in
Waymouth's medium was added to the culture media at the initiation of the
embryo cultures. Usually 8-12 embryos were used in each experiment; half the
embryos were grown in control medium and the other half in medium containing
the drug. Enough experiments were conducted to collect samples of 12-24 treated
and untreated limbs for comparative studies.
To study the incorporation of labeled BudR, llth-day mouse embryos were
cultured in the presence of 2 /iCi/ml of [3H]BudR ([6-3H]5-bromodeoxyuridine,
specific activity 15-30 Ci/mmole, obtained from New England Nuclear, Boston,
Mass.). At suitable intervals, as indicated in Fig. 2, embryos were removed
from the culture medium and transfered to chilled saline containing excess
'cold' thymidine (500/*g/ml) to stop further incorporation of the isotope. The
placentas were trimmed away and yolk sacs separated from the embryos. Both
the yolk sac and embryos were rinsed briefly in fresh saline, homogenized
separately, and suspended in 10% trichloracetic acid overnight at 4 °C. DNA
was extracted by TCA-PCA precipitation method of Ruddick & Runner (1974).
The supernate after the hot PCA treatment was assumed to be the DNA fraction. An aliquot of this fraction was removed for isotope counting on a liquid
scintillation counter (ISOCAP/300, Nuclear Chicago, 111.) and another aliquot
was used to quantify DNA employing the diphenylamine reaction (Burton,
1956).
Organ culture of limbs
After exposure of embryos to BudR in vitro, their forelimbs were excised and
cultured. Only embryos with good heart beat and blood circulation were used.
The cultured whole embryos were briefly rinsed in saline, their remaining
membranes dissected away and the forelimbs explanted employing techniques
described elsewhere (Aydelotte & Kochhar, 1972; Kochhar & Aydelotte, 1974).
The limbs were maintained in vitro for 9 days. The medium (BGJ medium (Difco,
Detroit, Michigan) supplemented with 25% fetal calf serum, 12-5 /*g/ml
streptomycin, 7-5 /tg/ml penicillin G and 150/tg/ml ascorbic acid) was changed
every third day. Finally the limbs were fixed in Bouin's fluid, stained in 0-1 %
toluidine blue in 70 % ethanol, dehydrated, cleared and stored in cedar wood oil.
' Quantitative'' measurement of the cartilage
As one aspect of the present investigations was to study the dose effect of
BudR, the following method was adopted to quantify the amount of differentiated cartilage. Toluidine-blue-stained and cleared limbs were photographed
under a low magnification on a Kodachrome color film. After processing, the
Effect of BudR on mouse embryos
627
photographic slides were projected in a Tri-Simplex Micro Projector (Bausch
and Lomb, Rochester, N.Y.) onto a graph paper of uniform squares. The outline
of the cartilage was drawn on the graph paper and the various cartilage zones
(scapula, humerus, radio-ulna and digits) delimited. The number of graph paper
squares occupied by each cartilage was counted and this number is refered to as
area units for that particular cartilage. Such an approach of quantifying the
amount of cartilage has certain built-in errors. In organ culture, the various
cartilages of the limb grow in a three-dimensional aspect, rather than two as a
photograph would record it. Also sometimes two cartilages would overlap or
some parts (especially scapula and humerus) would bend downwards in the
growing cultures. The latter sources of error were easier to rectify by comparing
the enlarged outline against the original limb and applying proper corrections
wherever cartilages overlapped. The amount of cartilage area units in the limb
explants of similarly staged embryos was found to be quite similar and variation
in most cases was rather small. In cases where variation was large, the experiment was repeated one or more times to collect a larger sample so as to obtain a
statistically more valid mean value. The average cartilage area units in the limbs
of similarly staged and treated embryos is the value used in all the tables.
Although the measurements were not absolute, such a semi-quantitative approach gave us a basis to compare the effect of various concentrations of BudR
and made it easy to visualize if a certain treatment was affecting one particular
cartilage segment more than any other.
All measurements were made on toluidine-blue stained preparations. When
no metachromasia could be detected, complete suppression of chondrogenesis
was assumed to have occurred (Aydelotte & Kochhar, 1975).
RESULTS
A. Factors influencing the differentiation of cartilage in control cultures
In the limbs grown by the 2-step culture procedure, various cartilage segments
were recognizable by their relative position in the explant and by their shape
(Fig. 1). The scapula developed as a triangular or rhomboidal cartilage at the
proximal end of the explant. The humerus and radio-ulna presented themselves
as bars of cartilages with the humerus being the longest of the three. The radioulna, when developed, appeared as two rather short and parallel bars, distal to
the humerus. Development of digital cartilages was generally poor.
The effect of embryo age (in terms of somite stage at explantation) and culture
conditions (which determined the extent of embryonic growth in vitro) on the
development of toluidine blue-stainable cartilage in the subsequent limb cultures
is shown in Table 1. The early 11th day (somite stage 26-29) embryos could be
maintained in the same culture medium for at least 24 h during which time they
maintained an excellent heart beat and blood circulation. They gained an average
of 4-5 somites. When their forelimbs were subsequently cultured for 9 days, the
40-2
628
N. D. AGNISH AND D. M. KOCHHAR
Effect of BudR on mouse embryos
629
Table 1. The extent of cartilage development in limb cultures as related
to the ' age' of the donor embryos and their growth in culture
Somites at the
time of
embryo explants
26-29
26-29
32-34
32-34
Embryos
cultured
for
(h)
Medium
changed
after 12 h
Number of
somites at the
time of
limb explants
24
24
12
24
No
Yes
—
Yes
30-33
36-38
36-40
40-46
Average
gain in
somite
pairs
4-5
9-10
5
10-11
Amount of
cartilage development in limb
cultures
(area units ± S.D.)
171 ±16
285 ± 34
331 ±43
490 ±57
Early or mid-1 Ith-day mouse embryos were maintained in whole-embryo culture for
12 or 24 h. Their forelimbs were then excised and organ cultured for 9 days, fixed and stained
in toluidine blue. The mean cartilage area units in the last column are based on 36-64 limbs.
average amount of cartilage development was 171 area units (Table 1). If the
medium was changed after 12 h, embryonic growth was markedly improved and
they added an average of 9-10 somite pairs and also showed a much better
cartilage development in the subsequent limb cultures (Table 1). The mid-1 Ithday embryos (somite stage 32-34) could be maintained in the same culture
medium for only 12 h after which time a rapid deterioration in their heart beat
and blood circulation was noted. If these embryos were transferred to fresh
medium at this time, many of them survived for another 12 h and altogether
gained an average of 10-11 somites pairs (Table 1). In utero, the 11th day mouse
embryos add about 12 somite pairs in 24 h (Kochhar & Aydelotte, 1974) so that
under suitable culture conditions, the growth and differentiation of 11-day
mouse embryos in vitro was very nearly equal to that in utero, at least in terms of
gain in the somite pairs.
FIGURE 1
Cultured limbs from control and BudR-treated embryos. Embryos were maintained
in whole embryo culture for 12 or 24 h in control or drug-supplemented medium.
Forelimbs of these embryos were then excised and organ-cultured for 9 days in
drug-free medium, fixed and stained in toluidine blue.
(A). Early 11-day embryo, control medium 24 h.
(B). Early 11-day embryo, 150 /ig/ml of BudR for 24 h.
(C). Mid-11-day embryo, control medium 12 h.
(D). Mid-11-day embryo, 100/tg/ml of BudR for 12 h.
(E). Mid-11-day embryo, control medium 24 h.
(F). Mid-11-day embryo, 150/tg/ml of BudR for 24 h.
In all photographs, the distal tip of the limb is towards the bottom. All photographs are at the same magnification and the bar in the lower right-hand corner
represents 1 mm.
630
N. D. AGNISH AND D. M. KOCHHAR
6 i-
Yolk sac
x
P
c.
4
8
12
16
20
Exposure to isotope (h)
Fig. 2. Incorporation of [3H]-BudR by the DNA of embryonic and yolk sac tissues
after exposure of the 11-day mouse embryos, in vitro, to the isotope for varying
length of time.
B. Incorporation of BudR by embryos in culture
Before attempting detailed studies on the effect of BudR on cultured embryos,
we investigated if the embryos, in vitro, were able to incorporate the analog into
their DNA. For this purpose, 12 embryos from one litter were cultured and after
1 h - t o allow the embryos to 'adapt' to the culture environment - [3H]BudR
was added to the medium. At various time intervals after addition of the isotope,
the incorporation of the label was analyzed from a group of embryos (three for
each time interval). The results are shown in Fig. 2. As is evident, continuous
uptake of the label into embryonic and yolk sac DNA was found throughout the
20 h culture period. At increasing intervals of time during exposure to the isotope, an increase in the incorporation of the label was observed. Towards the end
of in vitro growth, there was a slight decrease in the rate of [3H]BudR incorporation in the embryonic tissue. This may be due to slight diminution in the metabolic activities of the embryos as indicated by a somewhat slower heart beat and
blood circulation at this time.
C. Effect of BudR treatment on chondrogenesis
Embryos were exposed to increasing concentrations of BudR in vitro and the
effect of this treatment on the differentiation of cartilage in the limb-buds was
studied after the latter had been cultured for 9 days in drug-free medium. The
BudR treatment of the embryos interfered with the differentiation of cartilage in
the limb explants. The degree of chondrogenic inhibition was found to depend
Effect of BudR on mouse embryos
631
Table 2. Exposure of early 11-day {somite stage 26-29) mouse embryos to
BudR and its effect on the subsequent chondrogenesis in forelimbs
Cone, of BudR
in the medium
(/<g/ml)
,
Scapula
0
25
50
100
125
150
90
86 (-4)*
72 (-20)
68 (-24)
34 (-62)
8 (-98)
Extent of cartilage development (in area units)
*
^
Humerus Radio-ulna
Digits
Total (± S.D.)
75
73 (-2)
46 (-39)
22 (-71)
11 (-85)
0(-100)
6
0
0
0
0
0
0
0
0
0
0
0
171 ±16
159±13(-7)
118 ±27 ( — 31)
90±20 (-47)
45 ±11 (-74)
8 ±7 (-95)
Early 11 th-day mouse embryos were cultured for 24 h in the presence of various concentrations of BudR. Forelimbs of treated embryos were then organ cultured for 9 days in drug free
medium.
* Figures in parentheses is the percentage reduction in mean cartilage area units as compared to those in control explants. Each mean value represents 16-24 limbs.
Table 3. Exposure of early 11-day {somite stage 26-29) mouse embryos to
BudR and its effect on the subsequent chondrogenesis in forelimbs
Scapula
Humerus
Radio-ulna
0
50
100
150
112
75 (-33)*
44 (-61)
18 (-84)
164
110 (-33)
63 (-62)
15 (-91)
OS O O O
Extent of cartilage development (in area units)
Cone, of BudR
in the medium
(/ig/ml)
A
(-100)
(-100)
(-100)
Digits
Total (± S.D.)
0
0
0
0
285 ±34
185±17(-35)
107±15 (-63)
33 ±17 (-88)
Early 11-day mouse embryos were cultured for 24 h (with change of media after 12 h) in
the presence of various concentrations of BudR. Forelimbs of treated embryos were then
excised and organ cultured for 9 days in drug-free medium.
* Figures in parentheses is the percentage reduction in mean cartilage area units as compared to those in control explants. Each mean value represents 16-24 limbs.
on the drug dose as well as on the developmental stage of the donor embryos.
For this reason, great care was taken to ensure that comparisons were made on
limbs that had been excised from embryos of similar somite stage and also grown
under identical conditions. With this in mind, the embryos were divided into two
groups depending on the number of somites at the time of embryo explants: early
11th day = somite stage 26-29; mid-1 lth day = somite stage 32-34.
(i) Early 11-day embryos. These embryos were grown in the same medium
for 24 h and exposed to various concentrations (0-150/*g/ml) of BudR. The
results are presented in Table 2. The data was subjected to an analysis of variance
and this coupled with Dunnett Test (Dunnett, 1955) revealed that the treatment
of embryos with the drug had a highly significant inhibitory effect {P < 0-01)
632
N. D. AGNISH AND D. M. KOCHHAR
Table 4. Exposure ofmid-llth-day {somite stage 32-34) mouse embryos to BudR
(for 12 h) and its effect on the subsequent chondrogenesis in the forelimbs
Cone, of BudR
Extent of cartilage development (in area units)
K
in the medium
(/tg/ml)
,
Scapula
Humerus
Radio-ulna
Digits
N
Total (±S.D.)
0
12-5
25
50
100
170
171
168
140
180
109
116
126
111
108
52
30
47
64
56
0
0
0
0
0
331 ±43
317 ±27
341 ±34
315 ±21
344 ±27
Mid-1 lth-day mouse embryos were cultured for 12 h in the presence of various concentrations of BudR. Forelimbs of treated embryos were then organ cultured for another 9 days in
drug-free medium. Each mean value represents 8—12 limbs.
on the differentiation of cartilage in limb cultures. There was no significant effect
on the total cartilage area units at the drug level of 25 /tg/ml. As the dosage was
increased, a 'thinning out' of the humerus and a reduction in the size of scapula
was observed with the effect being more severe on the long bone. Eventually, at
the highest concentration tested (150 /*g/ml), cartilage development was severely
inhibited and a small nodule of metachromasia was all that could be detected in
the toluidine-blue-stained preparations (Table 2, Fig. IB).
It was possible to 'improve' the growth of embryos in culture by changing the
medium after 12 h. As noted earlier, such a procedure resulted in an average gain
of 9-10 somites per embryo and the subsequent cartilage development in limb
organ cultures was enhanced considerably (Table 1). Exposure of these embryos,
in vitro, to BudR (0-150 /*g/ml) affected the eventual development of cartilage in
limb cultures in a manner similar to that described earlier. The degree of cartilage
inhibition was related to dose (Table 3); at the highest dose tested (150/tg/ml),
there was almost a 90 % reduction in the cartilage development.
(ii) Mid-1 lth-day embryos (somite stage 32-34). If grown in the same medium,
these embryos could be maintained well for only about 12 h, after which time
there was a very rapid deterioration in their heart beat and blood circulation.
BudR treatment in these experiments was therefore restricted to 12 h (rather
than 24 h as was the case with the early 1 lth-day embryos). The treatment of
these embryos with BudR (0-100 /*g/ml) for 12 h had no significant effect on the
subsequent differentiation of forelimb-buds as judged by the development of
cartilage in these cultures (Table 4). All limb cartilage components appeared
well developed and similar to those observed in control cultures (Fig. 1C, D).
In a few preliminary experiments, doses higher than 150 /tg/ml of BudR proved
toxic and the embryos failed to survive for longer than 5-6 h in culture.
We were interested to see if the mid-1 lth-day embryos had indeed become
refractory to the chemical or whether the treatment was not long enough. To
Effect of BudR on mouse embryos
633
Table 5. Exposure of mid-1 lth-day (somite stage 32-34) mouse embryos to BudR
(for 24 h) and its effect on the subsequent chondrogenesis in the forelimbs
Cone, of drug
in the medium
(/ig/ml)
,
Scapula
Extent of cartilage development (in area units)
Humerus
Radio-ulna
Digits
*
Total (± S.D.)
None
BudR 100
BudR 150
Thymidine 150
186
140 (-25)*
85 (-54)
313
202
212 (+5)
155 (-23)
305
78
15 (-81)
0(-100)
38
21
0(-100)
0(-100)
23
490 ±57
367±39(-25)
240±41 (-51)
678 ±74
A
Mid-1 lth-day mouse embryos were cultured for 24 h (with change of media after 12 h) in
the presence of various concentrations of BudR or thymidine. Forelimbs of the treated
embryos were then excised and organ cultured for 9 days in drug-free medium.
* Figures in parenthesis is the percentage reduction in the mean cartilage area units as
compared to those in control explants. Each mean value represents 16-24 limbs.
keep the embryos alive for a longer period of time, we transferred the embryos
to fresh, pre-warmed medium containing the same amount of BudR that was
added to the original medium. Under these culture conditions, many, but not
all, of the older embryos could be kept alive for at least another 12 h and all
surviving embryos showed a much better growth and gained an average of 10-11
somites (Table 1), almost the same as in vivo (Kochhar & Aydelotte, 1974).
Table 5 summarizes the results of experiments where the mid-1 lth-day embryos were exposed to a variety of BudR concentrations (0-150 /*g/ml) using the
modified culture technique. A significant and dose-related inhibition of chondrogenesis in limb cultures was observed. In all cases,the radio-ulna and digits exhibited the maximum sensitivity (Fig. 1F). Embryos grown in the presence of
150/tg/ml of thymidine in the medium showed an improvement in cartilage
development in the subsequent limb cultures (Table 5). High levels of thymidine
are toxic to cells in culture (Morris & Fischer, 1960, 1963).
Unfortunately, these studies could not be extended to the 12th- or the 13thday embryos, which proved very difficult to maintain in vitro for longer than
4-6 h. In a few preliminary studies, such a short treatment with BudR did not
affect the subsequent differentiation of cartilage in the limb explants.
DISCUSSION
The 11-day mouse embryos, in vitro, maintained an excellent heart beat and
blood circulation at least for the first 24 h. Under suitable culture conditions,
they added an average of 9-11 somite pairs after 24 h growth in vitro (Table 1)
and this compared well to the gain of 12 somites in utero during the same time
(Kochhar & Aydelotte, 1974). Cockroft (1976) also found that the growth and
differentiation of rat embryos was nearly normal for the initial 24 h in culture.
Because of the limited life span of embryos in vitro, whole-embryo culture has
634
N. D. AGNISH AND D. M. KOCHHAR
only a limited application in teratological investigations as no long-term effects
of a drug can be investigated. The 2-step culture technique used in the present
investigations provided a means to extend the BudR treatment of embryos to
24 h and study the long-term effect of the drug on developing limbs. This was a
distinct advantage because in vivo the drug is very quickly degraded by the liver
(Kriss & Revesz, 1962). Although these studies were carried out in vitro, the
experimental protocol was similar to the classical teratological approach.
Embryos-in a 'mother-free' system-were exposed to the drug for only a
fraction of their in utero life span after which the organ under study (limb-buds)
was allowed to grow and differentiate in the absence of the drug.
Although no systematic investigation was undertaken to study the effect of
the length of BudR treatment, there was an indication that this may be important.
When mid-1 lth-day embryos were exposed to up to 100 /*g/ml of the analog for
12 h, no significant effect on chondrogenesis in the subsequent limb cultures was
observed (Table 4, Fig. 1C, D). When the treatment was extended to 24 h (which
necessitated a slight modification in culture techniques), significant inhibition of
chondrogenesis at 100 and 150 /^g/ml levels of BudR became apparent (Table 5,
Fig. IF).
In all cases, a dose-related effect of the analog was observed. As the embryos
were exposed to increasing concentrations of BudR, there was a corresponding
increased inhibition of the cartilage development in the subsequent limb explants
(Tables 2, 3, 5). As our studies show fhat both the dose and possibly also the
length of treatment were critical for the manifestation of chondrogenic inhibition
in the limbs, it would appear that the rather mild teratogenic effects of BudR
in vivo on the limbs could be due to only a small amount of the drug reaching the
embryos in utero. This conclusion is supported by the observation that after
maternal administration of a teratogenic dose of BudR in mice, the half life of
the injected drug was short (1 h) and only 2 % of the thymine was replaced by
bromouracil in the embryonic DNA (Packard et al. 1973). This contrasts with
the reported 14-80% replacement in vitro (Djordjevic & Szybalski, 1960; Stellwagen & Tomkins, 1971; Fabian & Wilt, 1973) which could explain why BudR,
in culture, is such a potent inhibitor of differentiating processes. Packard,
Skalko & Menzies (1974) have argued that ' . . . t h e suppression of cellular
differentiation in vitro is (not) analogous to the production of malformations in
mammalian embryos.. .(and).. .may indicate a basic dissimilarity between the
production of fetal malformations by BrdU in vivo and its ability to inhibit
cellular differentiation in vitro...' In our studies where by employing the wholeembryo culture technique we could treat the embryos to relatively high concentrations of the drug for an extended (24 h) period of time, we were able to
reproduce the almost total inhibition of chondrogenesis (Fig. 1B) reported in
cultured chondrocytes and limb-bud cells (Abbott & Holtzer, 1968; Levitt &
Dorfman, 1972).
Excess thymidine and BudR (10~5 M or higher) have been shown to be highly
Effect of BudR on mouse embryos
635
cytotoxic to cells in culture as the conversion of cytidine nucleotide to its deoxynucleotide form was affected (Morris & Fischer, 1960, 1963; Reichard,
Canellakis & Canellakis, 1961; Morris, Reichard & Fischer, 1963; Meuth &
Green, 1974). BudR at levels of 10~5 M or higher was also found to be toxic to
preimplantation mouse embryos where cleavage was severely affected and the
embryos died soon after (Garner, 1974; Golbus & Epstein, 1974; Pollard,
Baran & Bachvarova, 1976). Garner (1974) raised the possibility that the drug
may be exerting its influence by competing with and thus restricting the entry of
heterologous nucleosides into the nucleotide precursor pool in a manner suggested by Steck, Nakata & Bader (1969). In contrast to the above studies, we did
not find BudR or thymidine to be nearly as toxic to the postimplantation mouse
embryos grown in vitro. Even in the presence of 100-150 /*g/ml of the analog almost 50-fold higher concentration than that used by the above authors - most
of the llth-day mouse embryos stayed healthy (in as far as no deleterious effect
on their heart beat or blood circulation could be detected in the first 24 h of
culture), grew and differentiated as indicated by an increase in their somite
number. The average gain in somite pairs was nearly the same whether the
embryos were grown in control or drug-supplemented medium. In a few preliminary experiments where the embryos were exposed to 200 /*g/ml of BudR, the
drug proved toxic and only a very few embryos survived in culture for longer
than 2-4 h.
In sea urchin eggs, Gontcharoff & Mazia (1967) found that the introduction
of BudR during the first 5 h of development prevented the formation of echinochrome pigment which does not appear until 24 h of development. They found
it intriguing t h a t ' . . .a known modification of DNA, such as substitution of BU
(bromouracil) for thymine, at one stage of development can affect expressions
of development that become manifest only at a much later stage.' In the present
study, we have demonstrated a similar 'delayed effect' of BudR on the chondrogenesis in the mouse limb-buds. In the 1 lth-day mouse embryos the limbs are at
a very early stage of development ('protodifferentiated', Rutter et al. 1968): all
the mesenchymal cells appear structurally alike even under the electron microscope and it is not possible to distinguish between the future muscle, cartilage or
connective tissue cells (Kochhar, 1976). First signs of chondrogenesis do not
appear till late 12th day in utero or the 3rd day of in vitro growth (Neubert et al,
1974; Kochhar, 1976). When these early embryos were treated with 150 /*g/ml of
BudR for 24 h, an almost complete inhibition of chondrogenesis in the subsequent limb cultures was observed (Tables 2, 3). In some way treatment with the
drug had altered or damaged the course of events that lead to the development
of cartilage in the limbs. Once affected, chondrogenesis in the organ remained
severely suppressed even when the limb was grown in drug-free medium for up
to 9 days. It appears that much before the onset of chondrogenesis, the determined (according to Stark & Searls, 1973) but not fully differentiated chondrogenic cells pass through a stage of cytodifferentiation in which they are extremely
636
N. D. AGNISH AND D. M. KOCHHAR
vulnerable to the teratogen. Levitt & Dorfman (1972) have reported a similar
irreversible suppression of chondrogenesis in the BudR-treated cultures of
mesenchymal cells taken from very early chick wing-bud.
Whereas the limbs of early 1 lth-day mouse embryos were very sensitive to the
BudR treatment and at appropriate concentrations of the drug exhibited an
almost complete inhibition of chondrogenesis, the slightly older mid-1 lth-day
embryos were not nearly as sensitive to the analog. At the highest dose tested
(150 mgl/A), only a 51 % reduction in the total cartilage area units was observed
(Table 5) in contrast to 95 % inhibition that was recorded in the limbs of the
younger embryos exposed to the same dose (Tables 2, 3). A stage-dependent
effect of BudR has been shown in cleaving echinoderm eggs. Up to 8-cell stage,
the sea urchin eggs were able to cleave in the presence of the drug although
blastulation was abnormal and the embryos failed to gastrulate. Applied to
older embryos (100-cell stage or older), BudR even at 1000 /*g/ml level had a
minimal effect on embryogenesis (Gontcharoff &Mazia, 1967; Tencer & Brachet,
1973). Whether developmentally advanced embryos actually become refractory
to the BudR treatment could not be ascertained in these studies. Older embryos
(12th and 13th day) proved difficult to maintain in vitro for longer than a few
hours. However, it is possible to grow the limbs from these embryos for an extended period of time in organ culture. In the accompanying communication,
we studied the effect of BudR on these limbs and found that they were indeed
more resistant to the drug.
We thank Dr P. M. Rodier for her help with the statistical analyses; Mr John Penner for
his assistance with photography and Miss Pamela Baker for typing the manuscript. This
research was supported by NIH grant no. HD-06550. Some of the results described here were
presented orally at the 15th annual meeting of the Teratology Society (May, 1975), Pocono
Manor, Pennsylvania.
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(Received 14 June 1976, revised 6 August 1976.)