/. Embryol. exp. Morph. Vol. 30, 2, pp. 491-498, 1973
Printed in Great Britain
491
Genetic effects on the timing of early
development in the mouse
By ANNE McLAREN 1 AND PATRICIA BOWMAN
From the Agricultural Research Council
Unit of Animal Genetics and the Department of Genetics,
University of Edinburgh
SUMMARY
1. On the 4th day of gestation, embryos were recovered from mice of the C57BL, RIII,
JU, C3H and Q strains, and cell counts were carried out. Significant differences between
strains were seen, both in the percentage of embryos which had reached the blastocyst stage,
and in the mean number of cells per embryo. C57BL embryos had most cells, and C3H
embryos fewest.
2. Examination of earlier stages of C57BL and C3H development showed that the proportionate difference in cell number remained constant, so that the difference involved the
time at which cleavage began, and not the rate of cleavage. Activation of the eggs and the
formation of pronuclei also occurred earlier in C57BL than in C3H females.
3. The difference in cell number between C57BL and C3H embryos did not depend on
a difference in time of mating, nor on the genotype of the male, since reciprocal crosses were
similar to the maternal strain. The difference was maintained in culture from the two-cell
stage.
INTRODUCTION
Implantation is a critical stage of gestation, which the embryo often fails to
survive. In several species (mouse, rat, rabbit, sheep, cow) egg transfer experiments have established the necessity for a close degree of synchronization
between the development of the embryo and of the uterus if implantation is
to succeed. If the embryo is ahead of the uterus in development, it can remain
at the blastocyst stage (at least in the mouse and rat) until the uterus catches
up; but if the embryo develops too slowly, it misses the short period of uterine
receptivity during which implantation is possible.
The tempo of development during the pre-implantation period may therefore be an important factor in prenatal mortality. Little is known of its genetic
control. Gates, Doyle & Noyes (1961) found that (BALB/Cx 129) Fx mouse
blastocysts contained more cells than did those of the maternal BALB/C strain,
but with 129 females the Fx combination showed no superiority over the
inbred. Whitten & Dagg (1962) also found a difference in stage of development
1
Author's address: Agricultural Research Council Unit of Animal Genetics, University of
Edinburgh, West Mains Road, Edinburgh EH9 3JN, U.K.
32
EMB 30
492
A. MCLAREN AND P. BOWMAN
between (BALB/C x 129) Fx embryos and BALB/C embryos, as well as between
BALB/C and 129 embryos, at both the blastocyst and the 8-cell stage, which
they interpreted as a difference in cleavage rate. Bowman & McLaren (1970a)
found no difference in embryonic cell number 3-5 days post coitum between
strains of mice differing fourfold in adult size.
The present work is concerned with cell number in embryos of one randomly
bred and four inbred strains of mice, at various times after ovulation.
MATERIAL AND METHODS
The mice belonged to the randomly bred Q strain and to the inbred strains
C57BL/McL, C3H/BiMcL, JU/Fa and RIII/Fa. Females were paired with
males, and examined for vaginal plugs daily. For purposes of dating, coitus
was assumed to occur at 1 a.m. on the morning that the plug was found; this
day is termed the first day of gestation. In some experiments, females were
paired with males at 9 a.m. and examined for plugs at 10 a.m.
Embryos were recovered from the oviducts or uterus in phosphate-buffered
saline, and examined with a dissecting microscope. Up to the 8-cell stage, cell
number could be determined by inspection; at later stages, embryos were
classified into morulae or blastocysts according to whether cavitation had
occurred, and were then placed in 0-25 % sodium citrate for 5 min at room
temperature, before being transferred to aceto-carmine for 24 h. Squash preparations were made, stained with basic fuchsin, and examined by phase
microscopy.
Embryos were cultured as described by Bowman & McLaren (1970/?).
RESULTS
In the first series, embryos of the five strains were recovered 3-5 days post
coitum (p.c.) and cell counts carried out. The results are given in Table 1 and
Fig. 1. A preliminary account of the data on inbred embryos was given in
McLaren (1968).
Although the distributions of cell number were somewhat skew, the departures from normality were not enough to affect an analysis of variance, and
accordingly transformation of the data was not undertaken.
There was significant heterogeneity in cell number among strains. C57BL
embryos had the highest mean cell number and the highest percentage of
blastocysts; they also showed the greatest variability with respect to cell number. The difference in variability was entirely accounted for by the greater variation between females in the C57BL strain: within-female variance was similar
in all strains. Variation between females exceeded variation within females for
all five strains, significantly so except for the JU strain. JU morulae and blastocysts had more cells than RIII morulae and blastocysts; but since the per-
Cleavage in the mouse
493
Table 1. Cell number and stage of development 3-5 days post coitum
in one random-bred (Q) and four inbred (C57BL, RIII, JU, C3H) strains
12-14 females of each strain were used. For each strain, about 70 % of the
embryos recovered were scored for cell number
Strain
Q
C57BL
RIII
JU
C3H
Mean no.
of embryos
per female
Blastocysts
(%)
Mean cell number
Overall
Morulae
Blastocysts
119
61
4-9
93
7-2
81-8
94-2
84-7
640
45-2
38-8*
44-5
38-3
361
28-4
23-3
300
23-2
25-3
23-6
42-6
44-7
40-9
42-8
35-7
* These cell counts of Q embryos were included in Table 2 and Fig. 1 of Bowman &
McLaren (19706).
centage of blastocysts was significantly higher in RIII than in JU females, the
overall cell number averaged slightly less in JU than in RIII embryos. C3H
embryos had the lowest mean cell number and the lowest percentage of blastocysts of any of the strains.
The two extreme strains, C57BL and C3H, were chosen for further study.
The differences between them, both for cell number and blastocyst percentage,
were significant at the 0-001 level. To determine whether the difference in cell
number 3-5 days p.c. reflected a difference in cleavage rate or in time of
commencement of cleavage, females were killed at intervals during the
first 4 days of pregnancy. To determine whether the effect depended on the
embryonic genome or on the maternal environment or cytoplasm, some females
of each strain were mated to males of the other strain, and the Fx hybrid
embryos examined. Unpublished data of McLaren & Michie, cited by McLaren
(1968), had already established that the percentage of blastocysts and
the location of the embryos (oviduct or uterus) at 3-5 days p.c. depended on
the genotype of the mother. The results of the present series of cell counts are
shown in Table 2.
From the morning of the 2nd day onwards, cell number in all four groups
showed a linear increase on a logarithmic scale (Fig. 2), as reported by Bowman
& McLaren (1970c) for embryos of the Q strain. The data for this period were
subjected to an analysis of variance, using the logarithms of litter means, rather
than cell counts on individual embryos, in order to allow for the within-female
'clumping' effect mentioned above. The four groups did not differ from one
another with respect to the slope of the regression line, showing that the differences in cell number by the 4th day of development were not due to any
differences in the rate of cleavage. In position, the (C57BL $ x C57BL $) line
did not differ significantly from the (C57BL $ x C3H <£) line, nor the
32-2
494
A. MCLAREN AND P. BOWMAN
I
C57BL
62 embryos
21
31
41
51
61
81
Number of cells
Fig. 1. The distribution of cell numbers in embryos of five strains of mice 3-5 days
post coitum. (Black areas represent morulae, hatched areas blastocysts; the arrows
indicate mean cell number for each strain.)
Cleavage in the mouse
495
Table 2. Cell number of C57BL and C3H inbred and Fx hybrid
embryos during the first four days of gestation
2nd day
C57BL
C57BL
C57BL
C3H
C3H
C57BL
C3H
C3H
3rd day
4th day
1st day
p.m.
a.m.
p.m.
a.m.
p.m.
a.m.
p.m.
10
(26)*
1-7
(14)
10
(10)
10
(15)
20
(15)
20
(17)
1-5
(11)
1-2
(15)
2-2
(28)
2-2
(16)
20
(35)
20
(8)
80
(12)
8-5
(22)
4-5
(19)
4-7
(14)
8-6
(23)
11-3
30-5
(18)
31-2
(11)
24-8
(8)
20-6
(12)
42-5
(6)
38-5
(31)
33-2
(18)
34-6
(5)
(ID
81
(ID
7-4
(16)
* Mean cell number, followed in parentheses by the number of embryos on which the mean
is based.
• C57BL? xC57BLcf
20
x C57BLO. xC3Hcf
OC3H9
+ C3H$
go 10
0-8
0-6
0-4
0-2
Hours after ovulation
Fig. 2. Cell number in embryos of two inbred strains and their reciprocal crosses,
during the first 4 days of pregnancy. The solid line represents the calculated regression from the 2nd to the 4th day for the two groups of embryos from C57BL females,
which are homogeneous and have been combined. The broken line represents the
equivalent regression for C3H females. The two lines differ significantly in position
(P < 0-001) but not in slope.
496
A. MCLAREN AND P. BOWMAN
(C3H$xC3Hc?) from the (C3H? x C57BL<?) line; however, the two inbred
strains differed significantly from one another (P < 0-001), as also did the two
reciprocal hybrids (P < 0-01). Embryos from C57BL females, whatever the
paternal strain, were consistently about 4-5 h ahead of embryos from C3H
females in their development.
To see whether the rate of cleavage of the two strains was also similar in vitro,
C57BL and C3H embryos were removed on the afternoon of the 2nd day of
pregnancy, at the 2-cell stage, and cultured for 68 h. A higher proportion of
C57BL than C3H embryos survived in culture (41/46 v. 25/48; P < 0-001):
this probably reflects the fact that the C57BL 2-cell embryos were a few hours
further on in development when they were put into culture. Within a strain, the
later in the afternoon of the 2nd day that 2-cell embryos are removed from the
oviduct, the higher the proportion that develops in vitro (McLaren & Nay smith,
unpublished). At the end of the culture period, 95 % of the surviving C57BL
embryos were blastocysts, with a mean cell number of 49-6 ± 3-80 (S.E.), while
80 % of the C3H embryos had cavitated, with a mean cell number of
29-1 ± 1-91 (S.E.). Thus cell number attained in vitro was similar to that of the
corresponding strain in vivo on the 4th day of pregnancy (Tables 1, 2) and
differed significantly between strains (P < 0-001).
The difference in cell number thus reflects a difference in the time of commencement of cleavage, and is maternally determined. To test the possibility
that C57BL and C3H females differed in the time at which they mated, mice of
the two strains were paired with males for one hour only. Cell counts carried
out on the morning of the 3rd day of pregnancy gave means of 7-0 (50 embryos from 8 mice) and 3-9 (26 embryos from 4 mice) cells for C57BL and
C3H embryos respectively. As expected, these values are both slightly lower
than those observed after overnight mating (see Table 2), but still differed highly
significantly from one another (P < 0-001). The difference between the
strains therefore lies not in the time of mating, but in the interval between
mating and first cleavage.
In order to determine whether the retardation in C3H eggs begins at first
cleavage, or affects all post-fertilization stages of development, groups of
C57BL and C3H females were mated from 9 to 10 a.m. and killed between
12.30 and 5 p.m. The eggs were squashed, stained, and examined for the
presence of maternal chromosomes in the second meiotic division, and for pronuclei. In order to exclude infertile matings, females in which all the eggs still
contained chromosomes in the second meiotic metaphase were rejected. The
rest of the data are summarized in Table 3. Using individual females as the unit
of analysis, regression analysis following angular transformation was applied
both to the proportion of eggs in which chromosomes were no longer visible,
and to the proportion showing pronuclei. The rate of change with time (i.e. the
slopes of the regression lines) did not differ significantly between the two strains,
but C3H eggs were consistently retarded in their development, relative to
Cleavage in the mouse
497
Table 3. The tempo of development of C3H and C57BL fertilized eggs
on the first day of pregnancy (each group contained 5-7 females)
Strain
C3H
C57BL
No. of eggs in staget
Time
interval*
I
12.30-1.30
2.00-2.30
3.00-5.00
12.30-1.30
2.00-2.30
3.00-5.00
18
18
12
14
6
0
II
9
4
1
8
0
4
III
4
5
12
11
15
20
* Females were paired with males at 9.00 and examined for plugs at 10.00 a.m.
t In stage I, the egg chromosomes (in metaphase or telophase of the 2nd meiotic division)
could be seen; in stage II the chromosomes were no longer visible; in stage III pronuclei had
formed.
C57BL eggs (P < 0-05 for each criterion). Indeed, Table 3 shows that the
proportion of C3H eggs (12/25) still showing meiotic chromosomes between
3 and 5 p.m. was similar to the proportion of C57BL eggs in this condition
(14/33) between 12.30 and 1.30.
DISCUSSION
By the fourth day of gestation, embryos of the five strains included in the
present survey contained significantly different numbers of cells. A similar
difference has been reported for the comparison between BALB/C embryos and
either strain 129 embryos or (BALB/C? x 129$) Fx embryos (Gates et al. 1961;
Whitten & Dagg, 1962). It therefore appears certain that genetic factors may
affect early development in the mouse. On the other hand there is no evidence,
either in published reports or in the present work, that genetic factors affect
the rate of cleavage. On the contrary, our detailed analysis of the early development of one strain combination has shown that the 50 % superiority in cell
number shown by C57BL over C3H embryos results not from any superiority
in cleavage rate, but from an advantage of some four hours in the time at which
cleavage begins.
We do not know whether the genetic effect is exerted through the cytoplasm
of the egg, or through the environment in which pre-cleavage development
takes place, the ampullary region of the oviduct. For later cleavage stages,
Wintenberger-Torres (1964) reported that rate of development of sheep blastocysts was significantly increased either after superovulation, when there is an
abnormally high number of corpora lutea, or during treatment with progesterone. For the pre-cleavage period, Krzanowska (1964) noted that fertilization
was completed earlier after mating with Fx than with inbred males. In our
498
A. MCLAREN AND P. BOWMAN
C3H/C57BL combination, not only was there no difference in cleavage rate, but
the difference in cell number was maintained in the standard environment of
in vitro culture, and was unaffected by the genotype of the male, in that
(C57BL?xC3Hc?) and (C3H ? x C57BL <J) embryos showed as great a difference from one another as did inbred C57BL and C3H embryos. Dickson
(1967) claimed that some strain differences in tempo of development were due
merely to variations in the time of copulation; in our C3H/C57BL combination
this factor can be excluded, since the difference was maintained even when
time of copulation was controlled to within an hour.
Since activation of the egg and the formation of pronuclei also appear to take
place earlier in C57BL than in C3H eggs, the difference in timing must trace back
to some event between copulation and activation. C57BL females must surpass
C3H females in the rate at which spermatozoa reach the site of fertilization, or
penetrate the eggs or their membranes, or induce activation.
We are grateful to the Ford Foundation and the Lalor Foundation for financial support.
We thank Dr Mia Buehr for her assistance with some of the C3H and C57BL one-celled
stages.
REFERENCES
P. & MCLAREN, A. (1970a). Cell number in early embryos from strains of mice
selected for large and small body size. Genet. Res. 15, 261-263.
BOWMAN, P. & MCLAREN, A. (19706). Viability and growth of mouse embryos after in vitro
culture and fusion. /. Embryol. exp. Morph. 23, 693-704.
BOWMAN, P. & MCLAREN, A. (1970 c). Cleavage rate of mouse embryos in vivo and in vitro.
J. Embryol. exp. Morph. 1A, 203-207.
DICKSON, A. D. (1967). Variations in development of mouse blastocysts. /. Anat. 101, 263267.
GATES, A. H., DOYLE, L. L. & NOYES, R. W. (1961). A physiological basis for heterosis in
hybrid mouse fetuses. Am. Zool. 1, 449. (Abst.)
KRZANOWSKA, H. (1964). Time interval between copulation and fertilization in inbred lines
of mice and their crosses. Folia Biologica, Praha 12, 231-244.
MCLAREN, A. (1968). Mechanisms affecting embryo development. In The Mammalian Oviduct
(ed. E. S. E. Hafez & R. J. Blandau), pp. 477-490. University of Chicago.
WHITTEN, W. K. & DAGG, C. P. (1962). Influence of spermatozoa on the cleavage rate of
mouse eggs. /. exp. Zool. 148, 173-183.
WINTENBERGER-TORRES, S. (1964). Influence de l'equilibre hormonal sur la vitesse de segmentation des ceufs de Brebis. C. r. hebd. Seanc. Acad. Sci., Paris 259, 1660-1662.
BOWMAN,
(Received 28 March 1973, revised 5 May 1973)