/ . Embryol. exp. Morph. Vol. 37, pp. 59-64, 1977
Printed in Great Britain
59
The effect of synchronous and
asynchronous egg transfer on foetal weight in mice
selected for large and small body size
By R. J. AITKEN, 1 P. BOWMAN2 AND I. GAULD 2
From the Department of Genetics and Agricultural Research
Council Unit of Animal Genetics, University of Edinburgh
SUMMARY
The weight difference between large, small and unselected strains of mice were maintained
when blastocysts of each genotype were transferred to unselected control recipients, suggesting
that foetal genotype was an important factor in determining prenatal body size. The asynchronous transfer of large, small and unselected embryos resulted in a highly significant
(P < 0001) elevation of foetal weight compared with synchronously transferred groups,
genetically small mice attaining a foetal weight greater than the normal large strain. This
result suggested that foetal genotype did not limit the capacity of each strain for prenatal
growth. It is postulated that foetal genotype exercises its control over embryonic growth
by affecting the state of embryonic development at implantation.
INTRODUCTION
Although it has been more than a decade since D. S. Falconer established
lines of large and small mice selected from a common base population of the
random-bred Q strain (Falconer, 1973) the physiological mechanisms underlying
the genetic determination of body size are still unclear.
At 6 weeks post partum the mean body weight of large and small mice differs
by 80-84% and as early as 11 days post coitum 15-20% deviation in foetal
weight can be detected (Murrani, 1973; I. Gauld, unpublished). In contrast, an
analysis of cell number in large and small blastocysts harvested at 3 | days
post coitum failed to reveal any significant difference between the strains
(Bowman & McLaren, 1970). Since cell size does not seem to be an important
factor in determining body weight (I. Gauld, unpublished), the point of deviation for large and small embryos appears to coincide approximately with the
time of implantation.
Noyes, Doyle & Bentley (1961) proposed that the state of embryonic development at the time of implantation may influence foetal weight, on the basis of
1
Authors' address for reprints: Department of Genetics, Kings Buildings, West Mains
Road, Edinburgh, Scotland.
2
Authors' address: Agricultural Research Council Unit of Animal Genetics, University
of Edinburgh, Scotland.
60
R. J. AITKEN, P. BOWMAN AND I. GAULD
a study which revealed that asynchronously (day 4 ova-day 3 uterus) transferred
rat blastocysts developed into significantly heavier foetuses than their synchronously (day 3 ova-day 3 uterus) transferred counterparts. The authors suggested
that the prolonged preimplantation phase experienced by the asynchronously
transferred ova resulted in a more advanced state of embryonic development at
the time of implantation and that this, in turn, influenced foetal weight. We
postulated that if such a difference in development existed between large and
small blastocysts at the time of implantation it could account for the early
deviation in embryonic weight. The embryo transfer technique was therefore
used to determine the effect of manipulating the time of implantation on the
foetal weight of large and small mice.
METHODS
Preimplantation embryos were harvested on day 2 or 3 of pregnancy (day of
vaginal plug = day 0) from either large or small mice selected at random from
the six replicate lines described by Falconer (1973), or from unselected controls.
The embryos were transferred to random-bred Q recipients of 20-30 g body
weight on either day 2 or 3 of pseudopregnancy. The transfers were carried out in
a random order using the technique of McLaren & Michie (1956), with the
exception that 1 % foetal bovine serum (Flow Laboratories) in phosphatebuffered saline was used as the transfer medium. The recipients were subsequently killed by cervical dislocation on day 16 of pregnancy and the foetuses
dissected out, cleared of extra-embryonic membranes and weighed to the nearest
100 fig.
The experiment was designed so that for each of the donor genotypes (large,
small, unselected) the transfers would be either synchronous (day 2-day 2; day
3-day 3) or asynchronous (day 3-day 2), giving nine groups in all. In addition
to the transfer groups, control data were obtained by weighing day-16 foetuses
from females mated to males of like genotype.
RESULTS
The results from the transfer experiments and normal matings are summarized
in Tables 1, 2 and 3.
Regression analysis of foetal weight on litter size failed to reveal a significant
correlation in a majority of groups so it was not possible to compensate for this
variable in presenting the data.
Normal matings
Comparison of the foetal weights attained by large, small and unselected mice
on day 16 of pregnancy revealed a highly significant difference (P < 0-001)
between each pair of groups, 40 mg consistently separating unselected from
both large and small.
61
Large and small mice
Table 1. Mean embryonic weights on day 16 of pregnancy in
transfer groups
Genotype
Type of transfer
N
Mean embryonic weight
(g)±s.E.
Mean litter
size±s.E.
2D-2D
3D-3D
3D-2D
2D-2D
3D-3D
3D-2D
2D-2D
3D-3D
3D-2D
45
22
62
42
28
24
23
31
36
0-5286 ±01051
0-5135 ±00220
0-6682 ±0-0088
0-5599 ±00137
0-5809 ±00175
0-6543 ±00191
0-6036 ±00208
0-6387 ±00142
0-8623 ±00219
409 ±0-59
3-14 ±0-51
5-60 ±0-94
5-25 ±0-67
3-5O±H6
4-80 ±110
3-28 ±0-91
3-50 ±0-50
6-0±l-30
Small
Unselected
Large
Table 2. Mean embryonic weight for normal matings in each genotype
Genotype
N
Mean embryonic weight
(g)±S.E.
Mean litter
size ± S.E.
Small
Unselected
Large
63
65
57
0-5539 ±00081
0-5915 ±00063
0-6386 ±00102
6-40 ±0-90
710±102
11-40 ±1-69
Table 3. Details of the number of embryos transferred and the
number of foetuses recovered on day 16 of pregnancy
Transfer
anu
genotype
3D-3D
Large
Control
Small
3D-2D
Large
Control
Small
2D-2D
Large
Control
Small
Total no. embryos
transferred
1M"/"\ mi/***1*
i (jidi no. 1NU. JIUCC
Live
Mean no. embryos
Dead
transferred
embryos embryos
at
at
16 days 16 days
R
L
R
5-71
600
6-21
5-71
5-96
31
28
22
14
28
500
36
24
62
3
5
4
mice
pregnant
L
14
21
16
11
8
7
80
126
86
87
120
91
10
6
19
6
5
11
50
31
93
50
33
93
4-89
5-50
4-89
13
10
20
7
8
11
80
52
96
80
54
91
615
5-20
4-80
615
5-40
4-55
5-37
500
500
23
42
45
19
8
15
5
Synchronous transfers
The relative weight differences between large, small and unselected mice were
maintained when embryos of each genotype were synchronously transferred to
pseudopregnant control recipients. Synchronous transfers performed on day 2
EMB 37
62
R. J. AITKEN, P. BOWMAN AND I. GAULD
and day 3 of pregnancy led to highly significant differences (P < 0-001) between
the mean foetal weights of each genotype. The magnitude of this difference was
increased from 40 to 50mg by the transfer technique. However, a 'transfer
effect' was also observed, in that the absolute foetal weights attained in each
group were depressed (P < 0-001) by 20-30 mg compared with normal 16-day
values; this effect was even more apparent when differences in mean litter size
between the transfer and normal groups were taken into account. In contrast
to the large differences observed between the three genotypes, no significant
difference was observed in the foetal weights obtained from day 2-day 2 and
day 3-day 3 transfers within each group.
Asynchronous transfers
Within each genotype the asynchronous transfer of day-3 embryos to day-2
recipients resulted in a highly significant (P < 0-001) increase in foetal weight
compared with their synchronously transferred counter parts. More important,
the asynchronous transfer of genetically small blastocysts resulted in a mean
foetal weight significantly greater than that of 16-day unselected or large
foetuses obtained by either synchronous embryo transfer or normal pregnancy.
Similarly, the asynchronous transfer of unselected control blastocysts resulted in
the acquisition of a mean foetal weight significantly (P < 0-001) greater than
16-day large embryos obtained by synchronous embryo transfer or normal
pregnancy. The asynchronous transfer of genetically large blastocysts to pseudopregnant recipients resulted in the acquisition of a mean foetal weight significantly greater (P < 0-001) than all the other transfer combinations, despite the
fact that this group possessed the highest mean litter size. The mean foetal
weight for asynchronously transferred large embryos was also greater (P < 0-001)
than that observed on day 16 of normal pregnancy in large mice; in this case,
however, a proportion of the difference may have been attributable to variations
in litter size.
DISCUSSION
Three major factors are known to influence prenatal body weight: (a) nongenetic maternal effects such as litter size, (b) maternal genotype, and (c) foetal
genotype. The inverse relationship frequently observed between foetal weight
and litter size does not appear to contribute towards the considerable difference
in large and small foetal weights. Although several workers (Falconer, 1965;
Roberts, 1966; Murrani, 1973) have observed litter size/foetal weight correlations within each strain, the fact that genetically large mice come from significantly larger litters than small mice (Bowman & McLaren, 1970) indicates that
this parameter is unimportant in determining the difference between the lines.
This conclusion is supported by the present study in which the weight differences
between each genotype were maintained despite differences in mean litter size,
Large and small mice
63
while even within each transfer group litter size/foetal weight correlations were
rarely observed.
The impact of maternal genotype on foetal size has been studied in a wide
range of mammals using a variety of techniques (Pani, 1968; Legates, 1972). In
mice, the importance of maternal factors appears to vary with the stocks of
mice used. Separate studies by Brumby (1960), Cox, Legates & Cockerham
(1959) and El-Oksh, Sutherland & Williams (1967) suggested that the maternal
uterine environment has a profound effect on prenatal uterine environment has
a profound effect on prenatal embryonic growth. However, the opposite
conclusion was reached by Moore, Eisen & Ulberg (1969) and Murrani (1973);
the latter used the same strains of large and small mice as the present study. The
fact that the expected differences in foetal weights were maintained when large,
small and unselected embryos were synchronously transferred to unselected
control recipients confirms Murrani's conclusion that maternal effects are of
minor importance in determining embryonic weight in these mice. If such an
effect had existed one would have expected the foetal weight of small embryos in
unselected control uteri to be relatively higher than that of small embryos in
small uteri; a decrease in embryonic weight was in fact observed. The results
obtained in the synchronous transfer experiments therefore suggest that foetal
genotype is the most important single factor controlling prenatal growth in
these strains of large and small mice.
The interpretation of the asynchronous transfer data is complicated by our lack
of knowledge concerning the fate of the blastocysts after transfer. It is possible
that the implantation and development of day-3 blastocysts continues unhindered
in day-2 uteri and that the higher foetal weights observed on day 16 of pregnancy
merely reflect a more advanced stage of pregnancy. However, Murrani (1973)
did not observe a significant difference in gestation length when normal pregnancies and pregnancies derived from asynchronously transferred ova were
compared. In addition the induction of implantation is known to be controlled
by the uterus rather than the blastocyst (Psychoyos, 1973). One would therefore
expect the implantation of asynchronously transferred blastocysts to be held
in abeyance until the uterus reaches a state of maximum receptivity late on day
3 (Finn & Martin, 1974).
In the light of these considerations we tentatively propose that the elevated
foetal weights observed on day 16 of pregnancy represent differences in the rate
of growth rather than the state of development of the embryos. When the data are
interpreted in this way it is evident that neither large nor small mice are limited
in their capacity for prenatal growth; in both cases asynchronous egg transfer
resulted in highly significant increases in foetal weight compared with the
respective normal or synchronously transferred embryos. The fact that asynchronously transferred small blastocysts managed to acquire foetal weights in
excess of those normally observed for the large strain may, as Noyes et al. (1961)
suggest, have been due to the influence of the extended preimplantation phase
5-2
64
R. J. AITKEN, P. BOWMAN AND I. GAULD
on the state of embryonic development at the time of implantation. It is also
possible that under normal circumstances genetically large blastocysts are relatively more advanced at implantation than those of the small strain and that this
advantage is subsequently expressed as a higher foetal weight. The state of
development at implantation rather than cell number is suggested as the decisive
factor because chimaeric mouse blastocysts, containing twice the normal
number of cells, regulate their size by 5£ days p.c. (Buehr & McLaren, 1974;
McLaren, 1975). Critical anatomical studies are now required to test this
hypothesis.
We are very grateful to Mrs C. Cherrie for her expert technical assistance. R. J. Aitken
was supported by an MRC postdoctoral fellowship.
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{Received 14 April 1976, revised 27 July 1976)