/. Embryol. exp. Morph. Vol. 39, pp. 129-137, 1977
Printed in Great Britain
129
Developmental precocity after asynchronous
egg transfer in mice
By LARS MARSK 1
From the Laboratory of Teratology, Karolinska Institutet, Stockholm
SUMMARY
Asynchronously transferred ova were found to implant 6-8 h earlier than did nontransferred eggs, indicating that the endometrium was receptive to implantation several hours
before the native blastocysts were prepared to implant. This developmental precocity was
also preserved on day 14, according to the results of a morphological rating study on day 14.
Synchronously transferred ova showed no such developmental precocity. No statistically
significant difference in implantation time or development on day 14 was found between the
two strains, CBA and A/Jax. The importance of precocious development for planning
teratological studies is emphasized.
INTRODUCTION
Transfer of mammalian eggs, fertilized or unfertilized, has been a valuable
tool in developmental biology ever since Heape in 1890 reported success with
egg transfers in the rabbit. The method has for instance been successfully used
in separating embryonic and maternal influences on foetal development (Fekete
& Little, 1942; McLaren & Michie, 1958; Gosden, 1974).
A methodological problem has been whether the blastocyst should be transferred synchronously, i.e. the age of the transferred egg equalling the gestational
age of the recipient, or asynchronously, i.e. the age of the egg exceeding the
gestational age of the recipient. McLaren & Michie (1956) in their basic work on
egg transfer methodology achieved the best results with asynchronous transfers which was confirmed by Tarkowski (1959). No difference in foetal yield
between synchronous and asynchronous transfers could be demonstrated by
Doyle, Gates & Noyes (1963) and McLaren has reported improved results
using the synchronous technique (McLaren, 1969a). However, it seems that
asynchronous transfer has been the more commonly used technique in mice
and has been applied in my previous studies with non-surgical blastocyst
transfers (Marsk, Theorell & Larsson, 1971; Marsk & Larsson, 1974; Marsk,
Ranning & Larsson, 1974; Marsk, Larsson & Kjellberg, 1975).
The transplanted egg, which after asynchronous transfer is initially 24 h
ahead, has been assumed to retard or stop its further development for about
24 h. Thus, the alien embryo would synchronize with the gestational age of the
1
Author's address: Department of Obstetrics and Gynaecology, Karolinska Hospital
S-104 01 Stockholm, Sweden.
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L. MARSK
recipient, and with the developmental stage of the native embryos. The cause
of this retardation could be due to transfer shock or lack of implantational
response by an immature endometrium (Tarkowski, 1959; Dickmann & Noyes,
1960).
Doyle et ah (1963) showed that mouse foetuses in mothers on day 18 of
gestation were significantly heavier after asynchronous than after synchronous
transfers, indicating a sustained developmental lead. However, they did not
show any earlier implantation after asynchronous transfer, as judged from the
number of recoverable ova after uterine flushing at diflferent intervals in early
pregnancy.
Teratogenic sensitivity to a single salicylate treatment on day 9 of gestation
was less in mouse embryos after asynchronous transfer than in non-transferred
embryos. It was obvious from a time-response study that a developmental
precocity would allow the transferred embryos to pass through the period of
maximum teratogenic sensitivity before salicylate treatment was given. By treating the foster mothers 8 h earlier on day 9, the precocious development was
compensated for and the transfer protection disappeared (Marsk et ah 1975).
The aim of the present study was to investigate the magnitude of precocious
development after asynchronous egg transfer by using a morphological rating
system on day-14 embryos, and to determine if this precocious development
could be due to an earlier implantation time.
MATERIAL AND METHODS
Animal material. A/Jax strain mice, inbred at our laboratory since 1958
(Larsson, 1962), and inbred CBA strain mice obtained from the Department of
Genetics, University of Stockholm, were used. The animals were kept at constant temperature (23 °C) and automatically regulated exposure to light from
6 a.m. to 6 p.m. The animals were fed standard lab chow (Astra-Ewos, Sodertalje,
Sweden) and water ad libitum. Virgin females were mated overnight with fertile
or vasectomized males and checked for vaginal plugs the next morning, denoted
day 0 of pregnancy.
Two separate experiments were designed for (a) morphological rating of transferred and non-transferred embryos on day 14 of gestation, and (b) an implantation study where transferred or non-transferred eggs were flushed out from the
uterine horns after in vivo staining for implantation sites with Pontamine blue.
The major part of the morphological rating experiment was undertaken before
the implantation study. However, complementary morphological rating during
the implantation study did not differ significantly from the main experiment.
Morphological rating study. Foetal development can be estimated by scoring
the developmental stage of the foetal hind feet, fore feet, hair follicles, eyes and
ears as described by Walker & Crain (1960) and Trasler (1965). The total score
represents the developmental stage.
Developmental precocity after asynchronous egg transfer in mice
131
Control groups consisting of A/Jax and CBA foetuses were randomly divided
into three groups and killed on day 14 at 10 a.m., 4 p.m. or 10 p.m. The number
of implantations was recorded, live foetuses were fixed in Bouin's fluid and later
scored according to the morph rate schedule.
The developmental stage of transferred embryos was determined in three combinations of transplantation: (a) A/Jax to CBA, day 3-day 2, (b) A/Jax and CBA
to CBA, day 3-day 2, and (c) A/Jax and CBA to CBA, day 3-day 3. Difference
in eye pigmentation made it easy to distinguish the two strains when raised
within the same uterus. In each of the three groups the animals were killed on
day 14 at 10 a.m. and handled as described for the controls. All transfers were
performed non-surgically (Marsk et ah 1971; Marsk & Larsson, 1974).
Implantation study. The females were killed at 6 h intervals from day 3 of
gestation at 5 p.m. to day 4 at 5 p.m. Except for the 5 p.m. group on day 3 which
was examined without previous staining, the animals were anaesthesized with a
solution of 2 % tribromoethanol in amylalcohol I.P. and injected intravenously
with 0-2 ml of 0-5% Pontamine sky blue (Gurr, GBX, London) in saline 15
min before examination. The uterine horns were examined for the Pontamine
blue reaction to assess implantation (Psychoyos, 1961; Finn & McLaren, 1967).
Each uterine horn was then separately flushed with saline and the contents
were immediately examined under microscope. The eggs retrieved were counted
and classified according to: intact zona pellucida, absent zona pellucida without trophoblastic giant cell transformation or with giant cell transformation.
Where pregnant recipients were used, the number of corpora lutea was also recorded.
The times of implantation in the two strains were mapped in control series.
Pregnant A/Jax and CBA females were randomly divided into five groups
and killed at 5 p.m. or 11 p.m. on day 3 or at 5 a.m., 11 a.m. or 5 p.m. on
day 4.
Implantation time studies on transferred embryos were carried out in pseudopregnant recipients. Three-day-old A/Jax or CBA blastocysts were transferred
to pseudopregnant CBA recipients on day 2. The recipients were killed at 5 p.m.
or 11 p.m. on day 3 or at 5 a.m. or 11 a.m. on day 4. Sham transfers were performed on five pseudopregnant females on day 2 of gestation, with saline alone
placed in the uterus.
Statistical analysis was performed with Student's t test.
RESULTS
Morphological rating study. The morphological rating study of A/Jax and
CBA foetuses on day 14 indicated a good correlation between morphological and
chronological age and the increase in morphological rating score (MR) was
parallel in the two strains (Table 1). Thus the A/Jax foetuses increased their
MR from 4-0 at 10 a.m. to 12-2 at 10 p.m. The corresponding figures for the
132
L. MARSK
Table 1. Morphological rating (MR) score on day 14 at 10 a.m., 4 p.m. and
10 p.m. of non-transferred AjJax and CBA embryos {controls)
A/Jax
A
10 a.m.
4 p.m.
10 p.m.
Litter
no.
Foetuses
no.
Litter size
m±s.E.
MR
m±s.E.
12
10
10
92
71
80
7-7 + 0-58
7-1 ±0-64
80 ±0-56
40 0-33
8-4 0-30
12-2 0-31
CBA
10 a.m.
4 p.m.
10 p.m.
Litter
no.
Foetuses
no.
Litter size
m±s.E.
MR
m±s.E.
10
13
10
77
86
79
7-7 + 0-75
6-6 ±0-59
7-9 ±0-66
4-6 ±0-26
8-7±0-31
12-6 ±0-27
Table 2. Morphological rating (MR) score on day 14 at 10 a.m. of AjJax and
CBA embryos after asynchronous and synchronous blastocyst transfer to CBA
recipients
Transfer
Recipient
SynAsynTrue
Pseudochronous chronous pregnant pregnant
Embryos
No.
Strain examined
A/Jax
CBA
A/Jax
CBA
A/Jax
67 (T)
104 (N)
15 (T)
19 (T)
39 (T)
MR
m±s.E.
Litter
Size
No. m±s.E.
9-7±0-31 19 90±0-61
3-9 ±0-22
8-3 ±1-03 8 4-3 ±0-48
8-8 ±0-63
4-3 ±0-37 14 2-8 ±0-24
T = transferred embryos; N = native embryos.
Synchronous: 3-day blastocyst transferred to day-3 recipient.
Asynchronous: 3-day blastocyst transferred to day-2 recipient.
CBA foetuses were 4-6 and 12-6 respectively, a little higher but not statistically
significant at the 0-05 level.
When 3-day-old A/Jax blastocysts were transferred to pregnant CBA recipients and foetuses examined at 10 a.m. on day 14 (Table 2) the MR was 9-7 for
the alien foetuses whereas the non-transferred CBA foetuses showed a MR of
3-9, not statistically different from CBA foetuses at 10 a.m. in the control
group.
When 3-day-old A/Jax and CBA blastocysts were transferred to pseudopregnant CBA recipients on day 2 and killed at 10 a.m. on day 14, the MRs for
Developmental precocity after asynchronous egg transfer in mice 133
Table 3. Non-transferred A/Jax and CBA ova recovered on
day 3 and 4 of gestation
Appearance of ova, number of corpora lutea and litters with Pontamine blue reactions (implantation sites). Percent ova with or without zona pellucida and %
transformed ova is calculated from number of recoverable ova.
Time of autopsy . ..
Day 3
Day 3
Day 4
Day 4
Day 4
5 p.m.
11 p.m.
5 a.m.
11 a.m.
5 p.m.
A/Jax CBA A/Jax CBA A/Jax CBA A/Jax CBA A/Jax
CBA
Litter no.
Recoverable ova no.
Corpora lutea no.
Ova with zona pellucida
9 10
78 21
94 95
100 100
Ova without zona pellucida (%)—
Transformed ova (%)
—
Implantation sites no
Corpora lutea no.
°
Litters with implantation
—
sites (%)
6
30_
58
40
10
55
89
60
6
48
56
98
6
38_
51
100
2
-
—
—
20 33
40 7
—
100
—
—
—
11
—
—
—
10
93 93 86 95
100 100 100 100
7
11
7
_36 _32 2A_
70 100 78
—
100
—
100
8
30
73
—
100
A/Jax and CBA foetuses were 8-3 and 8-8 respectively (Table 2). These results
did not differ significantly from MR of the A/Jax foetuses transferred to pregnant CBA dams. There was, however, a significant difference between transferred foetuses scored at 10 a.m. on day 14 and non-transferred foetuses scored
at the same time (P< 0-001) (Tables 1 and 2).
When A/Jax blastocysts were transferred synchronously to pseudo-pregnant
CBA recipients on day 3, the MR of 4-3 (Table 2) was not significantly different
from the non-transferred foetuses (Table 1).
Implantation study. As can be seen in Table 3, not all corpora lutea were
represented by blastocysts on day 3. This is due in part to developmental failure
after ovulation and in part probably to incomplete uterine flushing. The eggs
were recoverable even after they had elicited a Pontamine blue reaction (Tables
3 and 4). The yield is thus not represented by the sum of recoverable eggs and
Pontamine blue areas.
The true pregnant A/Jax females examined for implantation on day 3 and 4
of gestation all showed a Pontamine blue reaction at 11 a.m. on day 4 but none
at 5 a.m. on day 4 (Table 3). At 5 p.m. on day 3 all recoverable ova had an intact
zona pellucida. The zona remained intact at 11 p.m. on day 3 (98 %) and could
still be seen at 5 a.m. on day 4 (40%). At 11 a.m. on day 4 all recoverable ova
were transformed, partly or completely.
The CBA controls showed a similar pattern concerning time of implantation
and loss of zona pellucida. All females were Pontamine-blue positive at 11 a.m.
on day 4 (Table 3). Only one of ten showed a Pontamine blue reaction at 5 a.m.
on day 4. All ova had an intact zona pellucida at 5 p.m. and 11 p.m. on day 3,
134
L. MARSK
Table 4. Three-day-old AjJax and CBA blastocysts transferred to pseudopregnant CBA dams on day 2 of gestation and killed at different times on days 3 and 4
of gestation
Number of transferred ova, appearance of recoverable ova and litters with Pontamine blue reactions (implantation sites). Percent ova with or without zona pellucida and % transformed ova are calculated from number of recoverable ova.
Day 3
5 p.m.
A/Jax CBA
Day 3
11p.m.
A/Jax CBA
Day 4
5 a.m.
A/Jax CBA
Litter no.
Recoverable ova no.
Transferred ova no.
Ova with zona pellucida
8
7
27 25
37 36
37 28
6
6
17 19
29 30
12 5
7
7
9 18
35 40
— —
Ova without zona
pellucida (%)
Transformed ova (%)
Implantation sites no.
Transferred ova no.
63
59 53
Litters with implantation sites (%)
—
Time of autopsy...
72
—
Day 4
11a.m.
A/Jax CBA
9
20
51
6
14
32
—
—
29 42
100 100
57 63
100 100
73 59
— —
100 100
100 100
decreasing to 60 % at 5 a.m. on day 4. The first transformed ova appeared in the
5 a.m. group on day 4 and like the A/Jax animals all recoverable ova showed
signs of transformation at 11 a.m. and 5 p.m. on day 4.
When 3-day-old A/Jax blastocysts were transferred to pseudopregnant CBA
recipients on day 2 of gestation, all females had a Pontamine blue reaction at
5 a.m. on day 4 (Table 4). The zona pellucida was intact in 37 % of the recoverable ova at 5 p.m. on day 3 and in 12 % at 11 p.m. on day 3 when the first transformed ova also appeared. The same was true for the transferred CBA blastocysts (Table 4), with all females Pontamine blue positive at 5 a.m. on day 4. Of
the recoverable ova, 28 % retained their zona pellucida at 5 p.m. on day 3, decreasing to 5 % at 11 p.m. on day 3 when transformed ova were also seen.
In the sham transfer group only unfertilized eggs were recovered and no
Pontamine blue reaction was present.
DISCUSSION
The morphological scoring clearly indicated a precocious development of
6-8 h in the asynchronously transferred embryos. As seen from the delay in the
Pontamine blue reaction between the asynchronous transfer and non-transfer
groups, this precocity was apparent already at the time of implantation. Similar
differences were present when the fate of the zona pellucida was followed and
Developmental precocity after asynchronous egg transfer in mice
135
••• Zona pellucida loss
— Pontamine Blue reaction
— Giant cell transformation
5 p.m.
Fig. 1. Pre-implantational changes and appearance of Pontamine Blue reactions
in CBA mothers with native or asynchronously transferred CBA blastocysts.
also when studying the onset of giant cell transformation (Fig. 1). In the present
study no precocity was shown for synchronously transferred ova in the morphological rating study and hence was not further investigated.
Such a 6-8 h difference in implantation time between transferred and nontransferred ova emphasizes the importance of blastocyst maturity as well as
endometrial maturity in the control of nidation (McLaren, 19696). Thus the
alien 3-day-old blastocyst seemed to be waiting from recipient day 2 at 5 p.m.
to early on day 4, capable of eliciting an implantation response at least 6 h
earlier than the native blastocyst. The probable existence of a precocious implantation after asynchronous transfer has been put forward earlier by McLaren
& Michie (1956) with a discussion of the competition between alien and native
eggs, in favour of the transferred ova.
In the present investigation pre-implantational and implantational time
events were found to occur later than that described for the Q-strain by Orsini &
McLaren (1967), where 50% of the zonas were lost around midnight on day 3
post coitum and Pontamine blue reactions were seen late on day 3. An even
more accelerated course was reported for the QS-strain by Restall & Bindon
(1971). McLaren & Bowman (1973) reported genetic effects on the timing of
early development in the mouse, which together with differences in husbandry
could explain the divergent results.
The MR difference between asynchronously and synchronously transferred
embryos on day 14 might well reflect the difference in weight on day 18 as
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L. MARSK
reported by Doyle et ah (1963). Adjustments were made for correlation between
foetal weight and litter size (Healy, McLaren & Michie, 1961; Noyes, Doyle,
Gates & Bentley, 1961). In the present investigation transfers were made also to
true pregnant recipients, in order to avoid errors that might have occurred due
to the smaller litter size obtained with pseudopregnant recipients. There was no
statistically significant difference between litter size in transferred and nontransferred groups except for the groups with pseudopregnant females, but
the foetuses in these groups showed no advantage in morphological development (Tables 1 and 2).
The hazard of estimating implantation time from 50 % loss of recoverable
ova was also stressed by Doyle et ah (1963), pointing to the fact that the ova
still had an intact zona pellucida at the time of estimated implantation. They
found a 5 h difference in 50 % loss of recoverable ova between synchronously
and asynchronously transferred eggs, though this was not statistically
significant.
As has been stated earlier (Marsk et ah 1975), knowledge of the developmental precocity of asynchronously transferred ova is of great importance in teratological experiments where it must be considered in the planning of treatment
schedules.
This work was supported by grants from the Swedish Medical Research Council (No.
14X-993-09,10) and from Stiftelsen Sigurd och Elsa Goljes Minne and National Foundation,
March of Dimes.
REFERENCES
Z. & NOYES, R. W. (1960). The fate of ova transferred into the uterus of the rat.
/. Reprod. Fertil. 1, 197-212.
DOYLE, L. L., GATES, A. H. & NOYES, R. W. (1963). Asynchronous transfer of mouse ova.
Fert. Steril. 14, 215-225.
FEKETE, E. & LITTLE, C. C. (1942). Observations on the mammary tumor incidence of mice
born from transferred ova. Cancer Res. 2, 525-530.
FINN, C. & MCLAREN, A. (1967). A study of the early stages of implantation in mice.
/. Reprod. Fert. 13, 259-267.
GOSDEN, R. G. (1974). Survival of transferred C57B1 mouse embryos: effects of age of donor
and recipient. Fert. Steril. 25, 348-351.
HEALY, M. J., MCLAREN, A. & MICHIE, D. (1961). Foetal growth in the mouse. Proc. R. Soc.
B 153, 367-379.
HEAPE, W. (1890). Preliminary note on the transplantation and growth of mammalian ova
within a uterine foster-mother. Proc. R. Soc. B 48, 457-458.
LARSSON, K. S. (1962). Studies on the closure of the secondary palate. 111. Autoradiographic
and histochemical studies in the normal mouse embryo. Ada morph. neerl.-scand. 4,
349-367.
MCLAREN, A. (1969 a). Transfer of zona-free mouse eggs to uterine foster mothers. J. Reprod.
Fert. 19, 341-346.
MCLAREN, A. (19696). Stimulus and response during early pregnancy in the mouse. Nature,
Lond. 221, 739-741.
MCLAREN, A. & BOWMAN, P. (1973). Genetic effects on the timing of early development in the
mouse. / . Embryol. exp. Morph. 30, 491-498.
MCLAREN, A. & MICHIE, D. (1956). Studies on the transfer of fertilized mouse eggs to uterine
foster-mothers. 1. Factors affecting the implantation and survival of native and transferred eggs. /. exp. Biol. 33, 394-416.
DICKMANN,
Developmental precocity after asynchronous egg transfer in mice
137
A. & MICHIE, D. (1958). Factors affecting vertebral variation in mice. 4. Experimental proof of the uterine basis of a maternal effect. /. Embryol. exp. Morph. 6, 645-659.
MARSK, L. & LARSSON, K. S. (1974). A simple method for non-surgical blastocyst transfer in
mice. /. Reprod. Fert. 37, 393-398.
MARSK, L., LARSSON, K. S. & KJELLBERG, M. (1975). Developmental precocity in transferred
embryos influencing the teratogen response to salicylate. /. Embryol. exp. Morph. 33,
907-913.
3
MARSK, L., RANNING, K. & LARSSON, K. S. (1974). H-Corticosterone incorporation in CBA
and transferred A/Jax embryos in CBA mothers. Biol. Neonate 1A, 49-56.
MARSK, L., THEORELL, M. & LARSSON, K. S. (1971). Transfer of blastocysts as applied in
experimental teratology. Nature, Lond. 234, 358-359.
NOYES, R. W., DOYLE, L. L., GATES, A. H. & BENTLEY, D. (1961). Ovular maturation and
fetal development. Fert. Steril. 12, 405-416.
ORSIW, M. W. & MCLAREN, A. (1967). Loss of the zona pellucida in mice, and the effect of
tubal ligation and ovariectomy. J. Reprod. Fert. 13, 485-499.
PSYCHOYOS, A. (1961). Permeabilite capillaire et decidualisation uterine. C. r. hebd. Seanc.
Acad. Sci., Paris 252, 1515-1517.
RESTALL, B. J. & BINDON, B. M. (1971). The timing and variation of preimplantation events
in the mouse. J. Reprod. Fert. 24, 423-426.
TARKOWSKI, A. K. (1959). Experiments on the transplantation of ova in mice. Ada theriol.
2, 251-266.
TRASLER, D. G. (1965). Strain differences in susceptibility to teratogenesis: survey of spontaneously occurring malformations in mice. In Teratology, Principles and Techniques (ed.
J. G. Wilson & J. Warkany), pp. 38-55. Chicago and London: University of Chicago Press.
WALKER, B. E. & CRAIN, B. (1960). Effects of hypervitaminosis A on palate development in
two strains of mice. Amer. J. Anat. 107, 49-58.
MCLAREN,
{Received 27 August 1975; revised 24 November 1976)