/. Embryol. exp. Morph. Vol. 24, 1, pp. 203-207, 1970
203
Printed in Great Britain
Cleavage rate of mouse embryos in vivo and
in vitro
By PATRICIA BOWMAN1 AND ANNE McLAREN 2
From the Department of Genetics and the Agricultural Research
Council Unit of Animal Genetics, Edinburgh University
SUMMARY
Mouse embryos (Q strain) developing in vivo from the 2-cell to the blastocyst stage showed
a constant cell doubling time of about 10 h. Embryos cultured in vitro over the same period
showed a rate of cleavage which was initially almost as great as in the reproductive tract, but
subsequently declined to give a doubling time of about 24 h. Addition of oestrogen to the
culture medium increased the diameter of blastocysts but did not increase cell number.
INTRODUCTION
Mouse embryos take about 3 days to develop from a one-celled zygote to a
blastocyst containing several dozen cells. Data on cleavage rate are scattered in
the literature (e.g. Lewis & Wright, 1935; Edwards, 1957; Kiihl, 1941; Whitten
& Dagg, 1961), but are mostly limited to the first two or three cleavages where
cell number can be estimated from direct inspection. Little evidence exists as yet
on cleavage rate of mouse embryos maintained in vitro, although mouse embryo
culture is now a routine procedure in many laboratories (see Mintz, 1967).
We have studied the cleavage rate of spontaneously ovulated eggs of the
randomly bred Q strain, flushed from the reproductive tract or cultured from
the 2-cell up to the blastocyst stage. Embryo diameters were measured at
x 100 magnification, using an eyepiece micrometer. Up to the 4-cell stage, cells
were counted under a stereo-microscope at x60 magnification; at later stages
embryos were placed for 5 min in 0-25% sodium citrate, then fixed in 0-5%
acetocarmine for 24 h, squashed, and stained with basic fuchsin. Culture was
in microdrops under liquid paraffin (Brinster, 1963), using the medium of
Brinster (1965), as modified by Bowman & McLaren (1970). The incubator
was gassed with 10% CO2 in air, and maintained at about 36 °C.
A large number of embryos were examined between 36 and 40 h after ovulation. (The time of ovulation is taken to be 1 a.m. on the morning that the
1
Author's address: Department of Genetics, Institute of Animal Genetics, West Mains
Road, Edinburgh EHJ 3JN, U.K.
2
Author's address: Agricultural Research Council Unit of Animal Genetics, Institute of
Animal Genetics, West Mains Road, Edinburgh EHJ 3JN, U.K.
204
P. BOWMAN AND A. McLAREN
vaginal plug was found.) At this time development is still closely synchronized,
so that although a small but significant increase in cell number was detected
during the 4 h period, over 80 % of all embryos were at the 2-cell stage (Table 1).
Over the next 2 days, embryos developing in the reproductive tract showed a
mean doubling time of about 10 h (Table 2, Fig. 1). As cleavage proceeded,
Table 1. Cell number in mouse embryosflushedfrom the
oviduct on the second day of pregnancy
Time after
ovulation
(h)
No. of
females
56
105
37
36
38
40
3 4
Cell number
(mean ± S.E.)
Females
with 2-cell
embryos only
(%)
19 625 1 2
23 1235 6 29
2 436 1 12
1-98 ±0-008
203 ±0009
205 ±0016
82
81
84
No. of embryos
with cell no.:
12
Table 2. The effect of culture on cleavage rate of mouse embryos
(Some of these observations have been briefly mentioned in McLaren, .1968.)
Time after
ovulation (h)
No. of
females
58
64
82
86
89
2
2
2
12
2
57
64
81
88
105
113
129
137
154
—
—
—
—
—
—
—
—
—
No. of
embryos
Cell number
(mean ± S.E.)
In vivo group
24
7-9 + 0-36
12
12-5 ±0-56
15
28-3 ±1-99
102
38-8 ±1-20
11
56-9 ±7-81
Diameter
(/*, mean ± S.E.)
89 ±1-5
—
90 ±1-4
—
95 ±2-1
//•/ vitro group
18
19
21
19
17
20
18
16
9
61 ±0-40
7-5 ±0-29
8-9 ±0-96
191 ±1-65
18-1 ±2-98
31-7 ±2-57
43-8 ±1-53
43-3 ±1-41
53-0 ±3-48
82 ±0-9
76 ±1-2
84+1-1
78 ±0-9
81 ±0-8
91 ±2-6
105 ±2-8
109 ±4-9
126 ±4-1
synchrony diminished, and considerable variation in cell number was encountered, as reported by Gates (1965). An analysis of variance carried out on the
86 h sample showed that the greater part of the variation occurred between
rather than within females (F 1 1 9 0 = 7-9, P < 0-001).
Two-cell embryos placed in culture at 38-40 h after ovulation showed a rate
of cleavage which was initially almost as great as that of embryos growing in
the uterus, but subsequently declined to give a doubling time of about 24 h
Cleavage of mouse embryos
205
(Table 2, Fig. 1). For the first 3 days in culture, embryos were consistently
smaller than those recovered from the uterus (Table 2). A day later, when
cleavage had almost ceased, the cultured blastocysts began to emerge from their
zonae pellucidae. At 129, 137 and 154 h after ovulation, 25%, 62% and 100%
respectively were zona-free.
64
32
-
/
o
//
E
I"53 8
/
o
I 16
U
o
/
0
/
4
/*
2
30
1
1
1
1
40
50
60
70
1
1
1
1
1
80 90 100 110 120 130 140 150 160
Hours after ovulation
Fig. 1. Cleavage rate of mouse embryos developing in the reproductive tract
(•
•) and in culture (o
o).
Table 3. The effect of oestrogen on mouse embryos maintained
in vitro from 81 h to 105 h after ovulation
Oestrogen in
medium f
10-4M
10 6M
10-8 M
None
No. of
embryos
cultured
No.
surviving
.12
5
9
4
18
18
20
20
Blastocyst
diameter
(/*, mean ± S.E.)
114 ±6-8*
—
126±50***
99 + 2-8
No. of cells
(mean ± S.E.)
34.3+1.7**
54-0 ±3-1
57-4 ±3-9
560 ±3-1
* P < 005 for comparison with controls.
** P < 001 for comparison with controls.
*** P < 0001 for comparison with controls.
f A10~2 solution of oestradiol-17/? in absolute ethanol was diluted in
culture medium to give the required final concentrations.
When embryos were removed from the reproductive tract at 81 h after
ovulation and cultured for 24 h, the presence of oestrogen in the culture medium
(Table 3) resulted in a significant increase in diameter. At a concentration of
10~ 4 M some of the eggs failed to develop, and the survivors contained significantly fewer cells than in the other groups; at lower concentrations mean cell
number was not affected.
The slower rate of cleavage of embryos in culture might be due to the lower
temperature at which they were maintained. The body temperature of mice
varies with strain, age and sex (McLaren, 1961), but is unlikely to be as low as
206
P. BOWMAN AND A. McLAREN
36 °C, our mean incubator temperature. No information is yet available as to
the relation of cleavage rate to temperature in mammals. An alternative explanation of the reduced rate of cleavage in culture would be that the conditions
of culture constituted a less favourable environment than did the reproductive
tract. This conclusion would agree with the findings that blastocysts cultured
from the 8-cell stage, under the same conditions as were used in the present
study, have a lower metabolic rate than blastocysts taken from the uterus
(Menke & McLaren, 1970), and develop less well when transferred to the uteri
of foster-mothers, with respect both to viability and to the growth of foetuses
derived from them (Bowman & McLaren, 1970).
The conditions of culture, as judged by cleavage rate, were not improved by
adding oestrogen to the medium: the increased diameter, even where cell
number was actually depressed, presumably reflects some effect of oestrogen on
the water-regulating mechanisms of the blastocyst.
No satisfactory explanation has yet been put forward for the strikingly slow
cleavage rate of mammalian embryos. Doubling time in the frog is about
1 h and in the goldfish about 20 min. According to Samoshkina (1968), the
period of DNA synthesis in mouse embryos cleaving in vitro lasts about 3^—4 h,
and hence is unlikely to be the factor limiting the rate of cell division.
RESUME
Taux de clivage chez les embryons de souris, in vivo et in vitro
Lors du developpement in vivo d'embryons de souris (Q strain) depuis le stage de deux
cellules jusqu'au stade de blastocystes, la duree de duplication des cellules est constante et
d'environ 10 h.
Le taux de clivage des embryons cultives in vitro pendant la meme periode, et qui etait
initialement le meme que dans le tractus genital, decroit ulterieurement jusqu'a une duree
double, d'environ 24 h.
L'addition d'oestrogene au milieu de culture accroit le diametre des blastocystes mais
n'accrit pas le nombre des cellules.
We gratefully acknowledge financial support from the Lalor Foundation and from the
Ford Foundation.
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Cleavage of mouse embryos
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(Manuscript received 10 November 1969)