/. Embryol. exp. Morph. Vol. 33, 4, pp. 907-913, 1975
907
Printed in Great Britain
Developmental precocity in
transferred mouse embryos influencing the
teratogen response to salicylate
By L. MARSK, 1 K. S. LARSSON AND M. KJELLBERG
From the Laboratory of Teratology, Karolinska Jnstitutet, Stockholm
SUMMARY
Asynchronous blastocyst transfer, supposed to equalize the developmental stage of native
and alien embryos during the organogenic period, was used as a tool in a teratological
investigation.
A spurious protection by the transfer as such was shown to depend on a persisting asynchrony between native and alien foetuses. The initial difference of 24 h was not nullified, but
decreased to 8 h. This difference allowed transferred foetuses to pass the period of maximum
sensitivity before salicylate treatment.
INTRODUCTION
The expression of teratogenic effect varies according to the type of agent
used, the dose and the time of treatment (Wilson, 1973). Protecting or sensitizing
factors in the genetic constitution of the treated animal, moreover, may modify
the teratogenic response as demonstrated in inbred mouse strains (Fraser &
Fainstat, 1951; Walker & Fraser, 1956; Goldstein, Pinsky & Fraser, 1963;
Dagg, 1963; Green, Azar & Maren, 1973).
The many possible factors involved in genetic strain differences can be studied
using blastocyst transfer between resistant and susceptible strains. Maternal
influences as observed in reciprocal crosses have been examined with this
method and found likely to be caused by cytoplasmic (Marsk, Theorell &
Larsson, 1971) or by uterine factors (Vetter, 1971; Takano, Peterson, Biddle &
Miller, 1972).
Blastocyst transfer has long been used in studies other than teratological
(Heape, 1890) and transplantation of blastocysts 24 h older than the recipients'
own was shown to give the best transfer results in mice (McLaren & Michie,
1956). The preference of such an asynchronous transfer was supposed and in
part proven to depend upon a developmental arrest affecting the transplanted
eggs and hence equalizing the development in native and alien eggs after some
time (Tarkowski, 1959).
1
Author's address: Laboratory of Teratology, Karolinska Institutet, S-104 01 Stockholm,
Sweden.
908
L. MARSK, K. S. LARSSON AND M. KJELLBERG
The present study was originally undertaken to obtain further information
about the genetic factors influencing strain differences in teratogenic susceptibility. Unlike our previous study on a 4-day cortisone treatment (Marsk et al.
1970), a single salicylate treatment earlier in the organogenic period was used
(Larsson, 1970). The study included a detailed analysis of the role of developmental precocity in the teratogenic response of transferred embryos to a single
treatment.
MATERIAL AND METHODS
Virgin mice of the A/Jax and CBA strains were mated overnight and the day
when a vaginal plug was found was denoted day zero. The A/Jax mice have
been inbred in the laboratory since 1958 (Larsson, 1962) and the CBA mice
were obtained from the Department of Genetics, University of Stockholm.
The animals were kept in macrolon cages in a room with constant temperature
(23 °C) and automatically regulated light from 6 a.m. to 6 p.m. and fed standard
lab chow (Astra-Ewos, Sodertalje, Sweden) and water ad libitum.
A total of 1565 living foetuses from 212 A/Jax and CBA litters were used in
four separate experiments after randomization. Pregnant females were treated
with a single intramuscular injection of sodium salicylate at a dose of 500 mg/kg
body weight at various times on day 9 as described below. Untreated dams
served as controls.
All animals were killed on day 16, the foetuses were removed and all living
specimens were fixed in 70 % ethanol for Alizarin staining of the skeleton
(Dawson, 1926-8). The foetuses were then checked under a dissecting microscope for fused ribs as the parameter for teratogenic effect.
In order to demonstrate a strain difference for salicylate-induced rib malformations, 10 A/Jax and 12 CBA dams were treated on day 9 at 10 a.m.,
giving 74 and 92 living foetuses respectively (see Table 1).
The width of the teratogenic zone for salicylate treatment was mapped using
a further 27 A/Jax and 30 CBA females with 186 and 218 foetuses respectively
(see Table 1). These animals were divided into four experimental subgroups
according to time of treatment on day 9 at 6 a.m., 2 p.m., 6 p.m. or 10 p.m.
Controls consisted of 9 untreated CBA females with 75 foetuses and 9 untreated
A/Jax females with 67 foetuses.
The parental influence on salicylate-induced teratogenicity was studied in
reciprocal crosses in which the dams were treated at 10 a.m. on day 9 (Table 2).
The group consisted of 12 A/Jax females with 100 A/Jax x CBA foetuses and
23 CBA females with 160 CBA x A/Jax foetuses.
The parental influence was further investigated with blastocyst transfer (see
Tables 3 and 4). Donor blastocysts from A/Jax and CBA females on day 3
were transferred to CBA and A/Jax dams, respectively, on day 2. The recipients
were mated with fertile males of their own strain (see Table 3). The transfer was
performed by a non-surgical method as described in detail elsewhere (Marsk
909
Developmental precocity in transferred embryos
Table 1. Difference in frequency of salicylate-induced rib malformations according
to time of injection {sodium salicylate, 500 mg/kg i.m. as a single dose on day 9)
Crosses
A/Jax x A/Jax
CBA x CBA
A/Jax x A/Jax
CBA x CBA
A/Jax x A/Jax
CBA x CBA
A/Jax x A/Jax
CBA x CBA
A/Jax x A/Jax
CBA x CBA
A/Jax x A/Jax
CBA x CBA
Treatment
time
6 a.m. day 9
10 a.m. day 9
2 p.m. day 9
6 p.m. day 9
10 p.m. day 9
Untreated
Control
No. of
litters
No. of foetuses
with rib
malformations
No. of foetuses
investigated
Malformations
(%)
7
7
10
12
6
8
6
8
8
7
9
9
18/53
35/55
27/74
69/92
19/39
53/59
6/46
8/51
6/48
7/53
0/67
0/75
34
63
36
75
49
89
13
16
13
13
0
0
et al. 1971; Marsk & Larsson, 1974). Altogether 38 living CBA foetuses were
raised in 20 A/Jax mothers together with 101 non-transferred A/Jax foetuses,
and 76 living A/Jax foetuses were raised in 27 CBA mothers together with 117
non-transferred CBA foetuses. These animals were treated with sodium
salicylate at 10 a.m. on day 9 of gestation. A/Jax and CBA foetuses within the
same female were easily distinguishable by the pigmented CBA eyes.
In another group designed to allow for the precocity of development in the
transferred embryos, 14 CBA females with 60 native CBA foetuses and 43
transferred A/Jax foetuses were treated with salicylate on day 9 at 2 a.m.
(Table 4). Also 10 A/Jax females with 78 living native A/Jax foetuses were
treated on day 9 at 2 a.m. and 9 A/Jax females with 80 native A/Jax foetuses
were treated on day 9 at 10 a.m.
Statistical analysis was performed with 'Students' Mest.
RESULTS
From a frequency of 34 % malformed foetuses in the A/Jax and 63 % in the
CBA strain injected at 6 a.m. on day 9 there was a rather slow increase in malformation frequency until 2 p.m. when both strains reached their maximum
sensitivity with 49 % and 89 % respectively. A sudden decrease was then observed
in both strains and at injection time 6 p.m. a frequency of approximately 15 %
was reached, persisting at 10 p.m. (Table 1, Fig. 1).
A maternal influence for the CBA strain was seen in reciprocal crosses (see
910
L. MARSK, K. S. LARSSON AND M. KJELLBERG
100
^ 80 -
0—O
\
A/Jax
CBA
\
\
\
60
\
\
a
\
40
\
Rib
6
o
^
1
1
.
\
\
20
•n
0
6 a.m.
1
10 a.m.
2 p.m.
6 p.m.
10 p.m. gestation day 9
500 mg/kg sodium salicylate i.m. single injections
Fig. 1. Graphical illustration of sensitive period for sodium salicylate treatment in
two strains of mice. For details see Table 1.
Table 2. Salicylate-induced rib malformations after a single injection of 500 mgjkg
bodyweight on day 9 at 10 a.m.
Crosses
No. of litters
No. of foetuses
with rib
malformations
No. of foetuses
investigated
A/Jax x CBA
CBA x A/Jax
12
23
42/100
88/160
Rib malformations (%)
42
55
Table 3. Salicylate-induced rib malformations after blastocyst transfer
{sodium salicylate, 500 mgjkg i.m. as a single dose on day 9 at 10 a.m.)
Genotype of
embryos
A/Jax x A/Jax (T)
CBA x CBA
CBA x CBA (T)
A/Jax x A/Jax
Genotype of
mothers
CBA x CBA
CBA x CBA
A/Jax x A/Jax
A/Jax x A/Jax
No. of
litters
} - {
No. of foetuses
with rib
malformations
No. of foetuses
investigated
10/76
89/117
13/38
25/101
Rib malformations (%)
13
76
34
25
T = Transplanted
Table 2). Foetuses raised in CBA mothers had rib malformations in 55 %
compared to 42 % in the A/Jax mothers (P<0-05).
The malformation frequency in both strains was reduced by about 50 %
(CBA 76-34 %, P< 0-001; A/Jax 25-13 %, P<0-05) after blastocyst transfer and
salicylate treatment at 10 a.m. as seen from Table 3. The non-transferred native
embryos showed a malformation rate of the same magnitude as demonstrated
for the two strains in Table 1.
Developmental precocity in transferred embryos
911
Table 4. Salicylate-induced rib malformations after blastocyst transfer and
correction for precocious foetal development relative to that of the recipients'1 own
foetuses {sodium salicylate, 500 mg/kg i.m. as a single dose at various times on
day 9 of gestation)
Genotype of
embryos
Genotype of
mothers
A/Jax x A/Jax
A/Jax x A/Jax (T)
CBA x CBA
A/Jax x A/Jax
A/Jax x A/Jax
CBA x CBA\
CBA x CBA j
A/Jax x A/Jax
Treatment
time
(day 9)
No. of
litters
2 a.m.
10
/ a.m.
14
10 a.m.
9
T = Transplanted
No. of foetuses
with rib
malformations Rib malNo. of foetuses formations
investigated
(%)
18/78
f 17/43
\ 19/60
33/80
23
40
32
41
When transferred A/Jax foetuses were treated 8 h earlier, i.e. on day 9 at
2 a.m., they showed the same frequency of malformations as did non-transferred
A/Jax foetuses treated at 10 a.m. on day 9 (Table 4).
DISCUSSION
Embryo transfer as such gave protection against salicylate-induced rib
malformations in transferred foetuses regardless of recipient strain. This
puzzling observation is most likely explained by precocious development of the
transferred embryos, which unlike the non-transferred ones had passed the
sensitive period for that particular malformation.
The asynchronous uterine transplantation of day-3 blastocysts to day-2
recipients, giving the transferred embryos an initial gain of 24 h, was proven by
McLaren & Michie (1956) to give the best yield. Tarkowski (1959) confirmed
the advantage of a 24 h asynchronous transfer. He transferred 2-cell blastomeres
to the oviduct and found a temporary developmental arrest equalizing the
developmental stage of transferred and non-transferred eggs at the moment of
transition of the eggs to the uterus. Discussing the results presented by McLaren
and Michie, Tarkowski also stated that' it is very probable that as in the case
of 2-blastomere eggs, initial check in development also takes place when more
advanced eggs are transplanted. (Several unconnected observations confirming
this viewpoint have already been made.)' In most investigations using egg
transfer, the question of timing has been of minor importance. In teratological
studies, however, the treatment time can be of the utmost importance for the
results and the need for a more exact knowledge of the developmental stage
in transferred embryos compared to non-transferred has been clearly demonstrated in the present study. Thus the persistence of the initial developmental
precocity of the transferred embryos, involving a difference of just a few hours
912
L. MARSK, K. S. LARSSON AND M. KJELLBERG
on day 9, resulted in a spuriously lower malformation rate after salicylate
treatment. Support for the existence and magnitude of precocious development
in transferred embryos has been established in a separate study of morphological development on day 14 (Marsk, unpublished observations). The transferred embryos were shown to be about 8 h ahead of non-transferred in their
morphological development, although, as also reported by Takano et al.
(1972), no gross differences were to be seen. The increase in malformation rate
after advancing the time of treatment by 8 h on day 9 in the present study on
A/Jax embryos transferred to CBA foster-mothers corroborates the hypothesis
that already on this day the initial precocity was reduced to about 16 h. This
combination, giving a higher yield, was chosen although a greater reduction
in response was seen after transferring CBA embryos to A/Jax recipients. The
developmental arrest might depend on a transfer shock or might be due to an
immature endometrium unable to respond to implantation. McLaren (1969)
claimed the importance of good timing between development of egg and
endometrium, although the blastocyst is able to wait in a resting stage.
The present experimental conditions show very clearly the importance of the
careful time control over embryonic development required in teratological
studies after blastocyst transfer. Thus, the difference in sensitivity between pure
strains was marked, the type of malformation studied was easily detectable and
the sensitivity curve showed a steep fall after a slow increase for both strains.
The prerequisites for the evaluation of the maternal influence on strain difference
in teratogenic susceptibility were less favourable. It can, however, be speculated
that the uterine effect in the CBA mothers is less important, since after transfer
with correction for precocious development the frequency was only raised to
40 %. This did not exceed the frequency for non-transferred A/Jax embryos
(see Table 4). Moreover, no difference in incorporation of 14C-labelled salicylic
acid in 14-day-old CBA and A/Jax embryos was observed (Eriksson & Larsson,
1971).
Causes of retarded or accelerated foetal development induced by factors
other than egg transfer might influence the teratological results. Less drastic
environmental factors than egg transfer might influence developmental rate.
Inadequate diet or change in diurnal rhythm could for instance push the
embryos out of their normal time schedule and thus displace the maximum
sensitivity to a teratogenic treatment.
A preliminary communication was in part given at the Third Conference of European
Teratology Society, Helsinki, 3-6 June 1974.
This work was supported by grant no. 14X-993-09 from the Swedish Medical Research
Council.
Developmental precocity in transferred embryos
913
REFERENCES
DAGG, C. P. (1963). The interaction of environmental stimuli and inherited susceptibility to
congenital deformations. Am. Zool. 3, 223-233.
DAWSON, A. B. (1926-8). A note on the staining of the skeleton of cleared specimens with
Alizarin Red S. Stain Technol. 1, 123-124.
ERIKSSON, M. & LARSSON, K. S. (1971). Salicylate-induced foetal damage in two mouse
strains: Studies on the distribution of 14C-labelled salicylic acid. Acta pharmac. tox. 29,
256-264.
FRASER, F. C. & FAINSTAT, T. D. (1951). Production of congenital defects in the offspring of
pregnant mice. Pediatrics, Springfield 8, 527-533.
GOLDSTEIN, M., PINSKY, M. F. & FRASER, F. C. (1963). Genetically determined organ specific
responses to the teratogenic action of 6-aminonicotinamide in the mouse. Genet. Res.,
Camb. 4, 258-265.
GREEN, M. C, AZAR, L. A. & MAREN, T. H. (1973). Strain differences in susceptibility to the
teratogenic effect of Acetazolamide in mice. Teratology 8, 143-145.
HEAPE, W. (1890). Preliminary note on the transplantation and growth of mammalian ova
within a uterine foster-mother. Proc. R. Lond. Soc. B 48, 457-458.
LARSSON, K. S. (1962). Studies on the closure of the secondary palate. III. Autoradiographic
and histochemical studies in the normal mouse embryo. Acta morph. neerl.-scand. 4, 349367.
LARSSON, K. S. (1970). Action of salicylate on prenatal development. In Malformations
congenitales des Mammiferes (ed. H. Tuchmann-Duplessis). Paris: Masson.
MCLAREN, A. (1969). Stimulus and response during early pregnancy in the mouse. Nature,
Lond. Ill, 739-741.
MCLAREN, A. & MICHIE, D. (1956). Studies on the transfer of fertilized mouse eggs to uterine
foster-mothers. I. Factors affecting the implantation and survival of native and transferred
eggs. /. exp. Biol. 33, 394-416.
MARSK, L. & LARSSON, K. S. (1974). A simple method for non-surgical blastocyst transfer in
mice. /. Reprod. Fert. 37, 393-398.
MARSK, L., THEORELL, M. & LARSSON, K. S. (1971). Transfer of blastocysts as applied in
experimental teratology. Nature, Lond. 234, 358-359.
TAKANO, K., PETERSON, A. C, BIDDLE, F. G. & MILLER, J. R. (1972). Analysis of cleft palate
induction by a glucocorticoid in mice: An application of the egg transfer technique in
teratology. Teratology (Abstract) 6, 119-120.
TARKOWSKI, A. K. (1959). Experiments on the transplantation of ova in mice. Acta theriol. 2,
252-266.
VETTER, M. S. (1971). The development of transplanted blastocysts in cortisone-treated A/Jax
and CBA mice. Anat. Rec. (Abstract) 169, 447.
WALKER, B. E. & FRASER, F. C. (1956). Closure of the secondary palate in three strains of
mice. /. Embryol. exp. Morph. 4, 176-189.
WILSON, J. G. (1973). Environment and Birth Defects. New York and London: Academic
Press.
{Received 22 August 1974)