J. Embryol. exp. Morph. 73, 111-133, 1983
\\\
Printed in Great Britain © The Company of Biologists Limited 1983
Control of events during early cleavage of the mouse
embryo: an analysis of the ^-cell block'
By MARTIN J. GODDARD AND HESTER P. M. PRATT 1
From the Department of Anatomy, University of Cambridge
SUMMARY
Embryos from certain strains of mice do not develop into blastocysts when cultured in vitro
from the 1- or 2-cell stages but arrest development as 2-cell embryos - a phenomenon referred
to, as the '2-cell block'. Reciprocal crosses between eggs and sperm of a 'blocking' (CFLP) and
'non-blocking' (Fj) strain show that in this combination the genotype of the egg alone determines whether the embryo 'blocks' at the 2-cell stage (or continues retarded development to
the 4- to 6-cell stage in a minority of cases). A comparison of molecular and cellular development in normal and 'blocked 2-cell' embryos was therefore undertaken to investigate the
influence of these maternal components on early mouse development.
The results show that the majority of 'blocked 2-cells' arrest development at a stage
equivalent to the late 2-cell stage in terms of cellular and nuclear division, DNA synthesis,
activation of the embryonic genome, qualitative and quantitative changes in amino acid
uptake, polypeptide synthesis and morphological maturation of organelles. These observations are compatible with the notion that maternally inherited developmental information
plays an important role in controlling early cleavage of the mouse embryo.
INTRODUCTION
In many animals the earliest stages of development occur independently of
major activity of the embryonic genome (reviewed Davidson, 1976) and are
controlled by oocyte or follicular components assembled during oogenesis
(discussed for the mouse, Johnson, 1981a). In the mouse embryo this period of
post-transcriptional, maternal control appears to extend from fertilization to the
mid 2-cell stage (approx. 40 h post hCG). During this period there is little, if any,
RNA synthesis (Moore, 1975; Young, Sweeney & Bedford, 1978; Clegg & Piko,
1977,1982) and both cleavage to the 2-cell stage and translation of stored mRNA
transcribed during oogenesis occur normally in embryos which have been physically enucleated or treated with transcriptional inhibitors (Braude, Pelham,
Flach & Lobatto, 1979; Cullen, Emigholz & Monahan, 1980; Petzoldt, Hoppe
& Illmensee, 1980). The first sign of major transcription by the embryonic
genome appears at the mid 2-cell stage (approx. 39-42 h post hCG, reviewed
Johnson 1981a) and involves the synthesis and translation of many new species
of mRNA that produce a dramatic qualitative transition in the profile of
1
Author's address: Department of Anatomy, Downing Street, Cambridge, CB23DY, U.K.
112
M. J. GODDARD AND H. P. M. PRATT
polypeptides synthesised. This qualitative change and the subsequent cleavage
to the 4-cell stage are both inhibited by a-amanitin (Braude etal. 1979; Johnson,
1981a; Flach et al. 1982). Contemporary with this expression of the embryonic
genome there appears to be a selective inactivation or destruction of much of the
pre-existing maternal mRNA. The total content of RNA decreases (Olds, Stern
& Biggers, 1973), poly-A is reduced in amount and average chain length (Levey,
Stull & Brinster, 1978; Clegg & Piko, 1978,1982; Bachvarova & de Leon, 1980)
and the translatability of both exogenous (injected) mRNA (Brinster, Chen,
Trumbauer & Avarbock, 1980) and endogenous maternal mRNA (Petzoldt etal.
1980; Flach et al. 1982) declines rapidly in normal, enucleated or a-amanitin
treated embryos.
During this transition the metabolic requirements of some embryos appear to
be difficult to satisfy in vitro, and cleavage of 1-cell to 2-cell embryos can occur
under conditions which are not compatible with subsequent development to
blastocysts (Biggers, 1971; Cross & Brinster, 1973; Whittingham, 1974). Embryos from outbred strains do not generally develop into blastocysts when cultured from the 1-cell stage in chemically denned media containing lactate,
pyruvate and bovine serum albumin, but arrest development at the 2-cell stage
(a phenomenon referred to as the '2-cell block'). On the other hand embryos
from inbred strains, or from hybrids between them, can develop into normal
blastocysts in the same culture media (Whittingham & Biggers, 1967; Whitten
& Biggers, 1968; Biggers, 1971; Whittingham, 1974). The 1-cell stage is unusually sensitive to the type and concentration of energy source and the differences
between 'blocking' and 'non-blocking' strains of mice may be related to different
lactate: pyruvate ratios necessary for balancing the oxidation-reduction potential of the embryo (Whittingham & Biggers, 1971; Biggers, 1971; Cross & Brinster, 1973; Whittingham, 1974).
For embryos from 'blocking' strains, this deficiency in vitro could operate on
the developmental programme inherited from the egg and/or processes involved
in the activation of the embryonic genome. 'Blocked 2-cells' and their normally
developing counterparts were therefore compared in an attempt to assess the
extent to which these two factors contribute to the 'block' in development at the
2-cell stage.
MATERIALS AND METHODS
1) Collection and culture of embryos
HC-CFLP (Hacking and Churchill Ltd., Huntingdon, U.K.) and laboratory
bred Fx (C57BL/10ScSn/Ola$ x CBA/Ca/Olacf) female mice were
superovulated at 3-4 weeks of age using 5i.u. PMS (pregnant mare's serum
gonadotrophin) (Folligon, Intervet, U. K.) followed 46 to 50 h later by 5 i. u. hCG
(human chorionic gonadotrophin) (Chorulon, Intervet, U.K.). The females
were then placed with HC-CFLP or Fj males and detection of a vaginal plug the
'2-cell block' in mouse embryos
113
following morning was taken to indicate successful mating. Embryos were staged
chronologically by defining the time of hCG injection as Oh. Mice were killed by
cervical dislocation and the oviducts removed into phosphate-buffered medium 1
+ 4 mg/ml BSA (PB1 + BSA) (Whittingham & Wales, 1969). The cumulus cells
were removed by a brief treatment with hyaluronidase (1 mg/ml in phosphatebuffered saline (PBS) containing 20 mg/ml polyvinylpyrrolidone). Embryos
were then cultured in medium 16 + 4 mg/ml BSA (M16 + BSA) (Whittingham,
1971) under liquid paraffin oil in 5 % CO 2 in air at 37 °C. Later stages were
obtained by flushing oviducts (late 2-cell embryos at 42 to 46 h post hCG and
8-cell embryos at 66 to 68 h post hCG) with PB1 + BSA and then transferring
them to drops of culture medium. Blastocysts were obtained after development
of 8-cell embryos in vitro.
For fertilization in vitro male mice (HC-CFLP and Fx) were killed 12 h after
the females were injected with hCG, the vasa deferentia and the epididymides
were dissected out and one of each was placed in a 0-5 ml drop of pregassed
Whittingham's medium + 30mg/ml BSA (Fraser & Drury, 1975). The sperm
were squeezed out gently and allowed to capacitate for 2 h at 37 °C. At 13? h post
hCG the females were killed, the oviducts dissected out and the cumulus masses
released into Whittingham's medium containing 30 mg/ml BSA (eight cumulus
masses per lml drop). At 14h post-hCG, 100 jul of the sperm suspension was
added to each lml drop to give a final sperm concentration of approx.
1-2 x 106 sperm/ml. Eggs and sperm were incubated together for 4 h at 37 °C at
which time the eggs were transferred to M16 + BSA and incubated as before.
2) Microdensitometric analysis of DNA content
The DNA content per cell was analysed by microdensitometry (Barlow, Owen
& Graham, 1972) using liver cells as standards. Embryos were incubated for
15 min at 37 °C in Mg24"- and Ca2+-free Hank's medium, dried in air in a minimal
volume onto a clean glass slide and fixed in 3:1 absolute ethanol: acetic acid
(vol/vol) for 5 min followed by 85:5:10 absolute ethanol: acetic acid: formalin
(vol/vol) for l h . Embryos were then stained with Feulgen's reagent (as
described by Barlow, Owen & Graham, 1972) and analysed using a Vickers M85
microdensitometer.
3) Measurements of uptake and incorporation of methionine
Embryos were incubated at 37 °C for 1 h in 50 /il drops of M16 + BSA containing 100 iM. unlabelled methionine to which was added 5/i [35S] methionine
(specific activity HOOCi/mmol, Amersham International, U.K.). The embryos
were then washed through ten 0-1 ml drops of PB1 + BSA and groups of ten
embryos were placed in 50JU1 of PB1+BSA and frozen at — 70 °C together with
samples of the final wash fluid and diluted incubation medium. Samples were
frozen and thawed twice and then precipitated with an equal volume of 20 %
TCA for 4h at 4°C. The precipitate was pelleted by centrifugation at approx.
114
M. J. GODDARD AND H. P. M. PRATT
8000 g for 5 min, the supernatant retained and the precipitate washed by resuspension in 100 [A 5 % TCA and repelleted. The supernatants were combined and
the pellet was again suspended in 5 % TCA and filtered on to glass fibre discs.
The supernatants and pellets together with final wash fluid samples and diluted
incubation medium were counted in a scintillation counter with an efficiency (for
35
S) of approximately 70%. Total uptake was calculated as the sum of acid
soluble and acid insoluble counts and the total amount of methionine taken up
or incorporated into protein was calculated as described by Holmberg & Johnson
(1979).
Assay for Na + dependence of methionine uptake was carried out according to
Borland & Tasca (1974) as described by Pratt, Chakraborty & Surani (1981).
4) Qualitative analysis of polypeptide synthesis using 2-dimensional
poly aery lamide gel electrophoresis
Embryos were incubated in 50jul of M16 + BSA containing 5/d
35
[ S]methionine (specific activity approx. llOOCi/mmol Amersham International, U.K.) for 4 h. The BSA was then removed by a brief wash in PB1 and the
embryos were harvested into 15 jul lysis buffer (O'Farrell, 1975). Twodimensional electrophoresis was performed using the method of O'Farrell (1975)
as modified by Johnson & Rossant (1981). The isoelectric focusing was carried
out over a pH gradient of 4-5 to 7-0 and the second dimension on a SDS 10 %
polyacrylamide gel. After electrophoresis the gels were impregnated with PPO
(Bonner & Laskey, 1974) dried down on to card and exposed to preflashed Fuji
RX X-ray film (Laskey & Mills, 1975) for 1 to 6 weeks. The use of preflashed film
effectively establishes a linear relationship between c.p.m. and grain density
until the silver grains become limiting (Laskey & Mills, 1975).
The ultimate aim of this analysis was to see whether 'blocked 2-cell' embryos
synthesise the same species of polypeptides as normal embryos of the same
chronological age i.e. the comparison is an essentially qualitative one. However
a quantitative element enters into the analysis since 'blocked 2-cells' incorporate
less [35S]methionine into protein than their normal counterparts from 66 h posthCG onwards (see Fig. 2) therefore the possibility of overlooking polypeptides
due to fainter gels has to be confronted. One possible way of generating gels of
equal intensity is to apply the same amount of total radioactivity to each first
dimension gel. However this approach does not work well in practice probably
due to variations in the proportion of polypeptides having isoelectric points
outside the pH range used, as well as the effects of radioactive decay. A more
satisfactory solution has been to use a combination of increased exposure time
and increased loading (70-90 'blocked 2-cells' as compared with 50 normal embryos) to generate more intense gels coupled with preliminary visual comparison
prior to final re-exposure to ensure comparability (discussed Handyside & Johnson, 1978). Comparability was achieved by the use of internal standards (Pratt
et al. 1981). Polypeptides that were synthesised throughout development were
'2-cell block' in mouse embryos
115
selected as standards (Class 4 Fig. 4) and the films exposed for a sufficient length
of time for some or all of these polypeptides to produce maximal conversion of
the film. The intensity of the remaining spots was then assessed in relation to
these marker polypeptides. A four-point scale was used: not detectable, trace
detectable, clearly detectable and maximum intensity (i.e. maximal conversion
of the X-ray film). The scores for each spot were averaged between two to three
good films for each group of embryos. Only the two latter categories were
interpreted as clear indications of the synthesis of a particular polypeptide.
5) Preparation of embryos for scanning electron microscopy
The procedure of Reeve & Ziomek (1981) was followed. 8-cell embryos were
decompacted by incubation with Ca2+-depleted medium but 'blocked 2-cell'
embryos were processed without any prior treatment. Groups of 30-40 embryos
were fixed in 6 % glutaraldehyde in 0-1 M-Na cacodylate pH 7-4 for 24 h and then
washed in 0-1 M-Na cacodylate pH 7-4. The embryos were stuck down to poly-L
-lysine-coated coverslips, stained with 1% osmium tetroxide in 0-1 M-Na
cacodylate pH7-4, dehydrated through a series of graded alcohols, washed in
acetone and critical-point dried. Samples were then coated with gold and observed with a Phillips Stereoscan S-600 scanning electron microscope.
Preparation of embryos for transmission electron microscopy
Groups of 15-20 embryos were fixed in 6% glutaraldehyde in 0-1 M-Na
cacodylate pH7-4 for 1-2 h, washed in 0-1 M-Na cacodylate pH7-4, stained with
1 % osmium tetroxide, dehydrated through a graded series of alcohols and embedded in Spurr. Sections (30-40 nm) were stained with uranyl acetate and lead
citrate and viewed with a Phillips EM 300.
RESULTS
1) Reciprocal crosses
The development of embryos derived from reciprocal crosses between F1 and
CFLP mice was studied. Fertilized 1-cell embryos were either removed from
superovulated females at 16 h post hCG or generated by fertilization in vitro, and
subsequent development was monitored visually until approx. 120 h post hCG
(Table 1). The results demonstrate that when cultured in a conventional lactateand pyruvate-containing medium (Whittingham, 1971) the majority (>90 %) of
embryos derived from CFLP eggs do not cleave beyond the 2-cell stage but
remain intact ('2-cell block'), whereas the majority of embryos from Fi eggs form
blastocysts. This occurred irrespective of the constitution of the paternal or
embryonic genomes. The possibility that the differences observed were due to
any asynchrony between the two types of egg and/or their maternal environments can be eliminated since the same result was obtained when development
116
M. J. GODDARD AND H. P. M. PRATT
Table 1. Conditions for the development of the '2-cell block'
% of embryos 'blocked' at 2-cell stage
CFLP
Fi
Fi
CFLP
Egg
Sperm
CFLP
Fertilization in
vivo
h post-hCG
38
46
70
94
(No. = 49)
100
97
93
90a
(No. =40)
100
93
93
93a
(No. = 62)
100
100
12
Fertilization in
vitro
h post-hCG
(No. = 24)
(No. = 69)
(No. = 29)
(No. = 64)
35
100
100
100
100
46
70
94
100
95
92a
99
95
91a
100
4
100
14
T
9b
4d
Fi
(No. = 135)
100
100
11
lc
a
The remainder arrested at the 4-6 cell stage.
80 % had developed into blastocysts by 114 h post-hCG.
C
98 % had developed into blastocysts by 120 h post-hCG.
d
89 % had developed into blastocysts by 116 h post-hCG.
e
72 % had developed into blastocysts by 116 h post-hCG.
b
(including fertilization) was carried out entirely in vitro (Table 1). By removing
embryos from the oviducts at different times after hCG injection it was possible
to show that CFLP eggs require at least 36 h exposure to the CFLP oviducal
environment to ensure that they develop beyond the '2-cell block' in our chemically defined medium (Table 2). Furthermore, cleavage to the 2-cell stage in vivo
(this occurs at approx. 30-35 h post-hCG) does not protect the embryos against
subsequent arrest in vitro since both 1-cell and 2-cell embryos recovered at 31 h
post hCG showed the same tendency to 'block' (Table 2). In subsequent experiments 'blocked 2-cell' embryos were obtained by removing fertilized CFLP eggs
from the oviduct at approx. 16 h post-hCG. When aged embryos were studied the
minority (<10%) of 'blocked 2-cells' dividing beyond the 2-cell stage were
discarded. Normal control embryos were obtained either from Ft females
(removed at 16 h post-hCG) or from CFLP females at times later than 36 h posthCG.
2) Nuclei and DNA content
The number of nuclei, their staining characteristics and DNA content were
assessed in normal embryos and 'blocked 2-cells' by Feulgen staining, light
microscopy and microdensitometry. The nuclei of normal early 2-cell embryos
'2-cell block' in mouse embryos
111
Table 2. % of CFLP embryos 'blocking' at the 2-cell stage in relation to the time
of removal from the oviduct
Time of removal
(h post-hCG)
% 'blocked 2-cells'*
(at 65 h post-hCG)
25
28
31
34
37
40
98
83
61t
15
7
It
* 25-30 embryos in each group.
t Both 1-cell and 2-cell embryos when assessed separately produced similar percentages of
'blocked 2-cells'.
$ 92 % developed into blastocysts.
(32 h post-hCG) were small, densely stained and contained the diploid (2C)
amount of DNA (Fig. 1). By 46 h post-hCG the nuclei of both normal and
'blocked 2-cell' embryos had enlarged, were diffusely stained and contained 4C
Normal 2-cell
32 h
•y
2
Blocked 2-ceil
46 h
89 h
500
2C
1000
4C
Absorption at 550 nm
Fig. 1. DNA content per nucleus of normal 2-cell and 'blocked 2-cell' embryos. For
methodology see Materials and Methods. Times indicated are h post hCG. Absorption at 550 nm was measured in arbitrary units with reference to liver cells which
provided the 2C and 4C values.
118
M. J. GODDARD AND H. P. M. PRATT
Table 3. Na + dependence ofmethionine uptake*
Methionine uptake
Normal late 2-cells (46 h post-hCG)
'Blocked 2-cells' (118 h post-hCG)
Blastocysts (118 h post-hCG)
+Na+t
-Na+t
243 ± 27
147 ± 18
11819 ±367
334 ± 20
236 ± 55
4092 ± 357
* Assayed at lOOjUM-methionine, details in Materials & Methods,
ffmol methionine/embryo/hour (mean ± S.D. (n = 5)).
amounts of DNA indicating that they had undergone one round of DNA replication. Nuclei of control embryos condensed and replicated subsequently whereas
the majority from 'blocked 2-cells' remained enlarged without undergoing any
further DNA replication (Fig. 1). However, a small proportion had either
entered metaphase or undergone nuclear division (6 % in both cases) by 90 h
post-hCG. When DNA measurements were extended beyond HOh post-hCG a
spread of values for blocked 2-cells was obtained suggesting that the chromatin
was becoming unstable.
3) Methionine uptake and incorporation
Uptake of methionine and its incorporation into protein were analysed at a
methionine concentration (100 JUM) sufficient to saturate the endogenous pool
(Braude, 1979a). During normal development uptake of methionine and its
incorporation into protein is maintained at a relatively low and constant level
until approximately 60-70 h post-hCG (compacted 8- to 16-cell morula) when
both processes increase during the formation of a blastocyst (100 h post-hCG)
(Braude, 1979a,b; Kaye etal. 1982 and Fig. 2). 'Blocked 2-cells' and embryos in
which transcription had been blocked with a-amanitin showed levels of
methionine uptake and incorporation similar to normal embryos over the period
20 to 60 h post-hCG, but neither exhibited the normal increases after this time
(Fig. 2). Uptake of methionine is Na + independent in the late 2-cell embryo (46 h
post-hCG) (Table 3), develops a requirement for Na + at the late compacted
morula stage (approx. 80 h post-hCG, Kaye et al. 1982) and is predominantly
Na + dependent in the blastocyst (Table 3 and Borland & Tasca, 1974).
Methionine uptake by 'blocked 2-cells' of equivalent age (118 h post-hCG)
remained independent of Na + (Table 3).
Fig. 2. Uptake and incorporation of methionine during development of 'blocked
2-cell' embryos, and normal embryos in the presence and absence of a-amanitin. The
procedures used are described in Materials and Methods. O
O Control embryos. •
• Control embryos cultured in the presence of 11/zg/ml a-amanitin.
# - - - # 'blocked 2-cell' embryos.
'2-cell block' in mouse embryos
119
lOOi
50
V
7
|
6-
1
I"
x
0-31
20
30
40
50
60
70
80
Hrs post HCG
90
100
110
120
20
30
40
50
60
70
80
Hrs post HCG
90
100
110
120
£
jO-25
I
§ 0-20
c
c
"I 015
01
005
120
M. J. GODDARD AND H. P. M. PRATT
4) Qualitative analysis of polypeptide synthesis
Patterns of protein synthesis during normal development were analysed using
two-dimensional gel electrophoresis (O'Farrell, 1975, as modified by Johnson &
Rossant, 1981) and individual polypeptides were scored for their presence (and
intensity) or absence (details in Materials and Methods). Polypeptides falling
into the following classes were selected for the study of 'blocked 2-cell' development. Class 1 consisted of 13 polypeptides synthesised at or prior to the mid 2-cell
stage (30-35 h post-hCG). Class 2 consisted of 15 polypeptides first appearing at
the late 2-cell stage (44-48 h post-hCG) (these polypeptides are not synthesised
by embryos treated with a-amanitin at any stage up to 39 h post-hCG, (Flach et
al. 1982)). Class 3 consisted of 15 polypeptides that are first synthesised at the
morula to blastocyst stage (66h post-hCG or later), and class 4 consisted of 22
polypeptides that are synthesised throughout development from the late 2-cell
3A
Fig. 3. Two-dimensional electrophoretic profiles of [35S]methionine-labelled
polypeptides (for experimental details see Materials and Methods). Polypeptides
synthesised by:- (A) Normal early 2-cell embryos (32-35 h post-hCG); (B) Normal
late 2-cell embryos (44-48h post-hCG); (C) Normal blastocysts (96h post-hCG). A
few polypeptides representative of each of 4 classes (discussed in 'Results') are
indicated. a,b,c,d,e,f = class 1; A,B,C,D,E,F = class2; 1,2,3,4,5,6,7 = class 3; t,u,
v,w,x,y,z = class 4.
'2-cell block' in mouse embryos
•^
121
•
E
F
6
t w
122
M. J. GODDARD AND H. P. M. PRATT
stage (44h post-hCG or earlier) to the blastocyst (96h post-hCG). Figure 3
shows the profiles of polypeptide synthesis during normal development at the
early 2-cell stage (32-35h post-hCG), late 2-cell stage (44-48h post-hCG) and
blastocyst stage (96h post-hCG), and illustrates the mobility of six or seven
representative polypeptides from each of the four classes described above. The
time of appearance during normal development, and relative intensities of these
representative polypeptides are indicated diagrammatically in Fig. 4 (left-hand
column).
Polypeptides whose synthesis began at or prior to the 2-cell stage (class 1) were
synthesised by 'blocked 2-cells' at 44 h post-hCG but their synthesis declined
thereafter as it did in controls (Fig. 4). Class-2 polypeptides whose appearance
is dependent upon transcription at the 2-cell stage were synthesised strongly in
both normal and 'blocked 2-cell' embryos. However approximately half of these
polypeptides (7 out of 15) were late in appearing, being absent at 44 h post-hCG
but present by 66 h post-hCG and remaining clearly detectable during later
development (Fig. 4). These observations were made on groups of embryos
which obscured any possible difference in polypeptide synthesis between individual embryos. By using ultrathin one-dimensional gels (Van Blerkom, 1978)
and analysing 15 individual 'blocked 2-cell' embryos (50 h post-hCG) in comparison with 15 individual fertilized eggs (approx. 24 h post-hCG) it was possible
to demonstrate that there was no heterogeneity amongst 'blocked 2-cells' which
all showed the transition from synthesis of class-1 to class-2 polypeptides (not
shown). Other polypeptides which are synthesised throughout development
from the late 2-cell stage (or earlier) to the blastocyst (class 4) were synthesised
strongly in the majority of cases (19 out of 22) at 96h post-hCG. The two
exceptions (e.g. 'w' Fig. 4) declined in intensity from 66 h post-hCG. In contrast
to these observations there was no clear evidence that polypeptides appearing for
the first time during the morula to blastocyst transition (class 3) (and probably
dependent upon concomitant transcription (Braude, 1979a,b)) were synthesised
Fig. 4. Block diagram illustrating representative polypeptide synthetic profiles
during development of normal and 'blocked 2-cell' embryos. Polypeptides
synthesised during normal development were assigned to one of four classes (for
details see Results). For normal embryos (left-hand column) the number in
parenthesis beside the class designation represents the total number of polypeptides
in that class. For 'blocked 2-cells' (right-hand column) the number in parenthesis
represents the number of polypeptides in that class whose synthesis was detectable
during the development of 'blocked 2-cell' embryos. Within each class-6 or -7
polypeptides were chosen to demonstrate the main features observed (see Results
and legend to Fig. 3).
Each polypeptide is represented by a letter or number and the mobility of each
polypeptide within the two-dimensional gel is illustrated in Fig. 3.
The times indicated are h post-hCG at the end of the 3h labelling period. The
density of hatching represents the average intensity of individual spots on a fourpoint scale:- White: not detectable; grey: trace detectable; dark grey: clearly detectable; black: maximum intensity (for further details see Materials and Methods).
e
2-cell block' in mouse embryos
Normal embryos
Class 1
Y12>
Blocked 2-cells
(13)
(13)
a
b
c
d
e
f
a
b
c
d
e
f
D
E
F
4
5
6
7
w
x
y
z
1-cell
2-cell
2-cell
8-cell
'><> h
Blastocyst
Class 2
96 h
96 h
(15, 7 appear late)
A B
C
(15)
A
B
C
D
E
F
1-cell
29 h
2-cell
35 h
2-cell
44 h
44 h
8-cell
66 h
66 h
Blastocyst
96 h
96 h
Class 3
(15)
(7)
1 2
3
4
5
6
7
1 2
1-cell
29 h
2-cell
35 h
2-cell
44 h
44 h
8-cell
66 h
66 h
Blastocyst
Class 4
96 h
29 h
2-cell
35 h
2-cell
44 h
8-cell
66 h
Blastocyst
96 h
111
(22)
t
1-cell
111
96 h
(22)
3
u
v
w
x
y
z
t
u
v
124
M. J. GODDARD AND H. P. M. PRATT
Table 4. Degree of cell flattening in 'blocked 2-cells'
Time
(h post-hCG)
% flattened
(Total No. of embryos)
66
72
88
96
112
120
18
21
49
48
53
63
(137)
(191)
(125)
(50)
(107)
(32)
* Includes all embryos where the degree of intercellularflatteningwas greater than that seen
in control 2-cells (46 h post-hCG) (see Fig. 5A & B).
to any great extent in 'blocked 2-cells'. Only 7 out of the 15 polypeptides appeared and only two of these (3 & 5, Fig. 4) were clearly detectable.
5) Morphology
The majority (>63 %) of 'blocked 2-cell' embryos flattened in a manner
analogous to normal compaction (Figs 5B, 5E, 6C) although the process was slow
and maximal flattening was not achieved until 96-120 h post hCG (Table 4) as
compared with 66-72 h post-hCG for normal embryos. The majority of flattened
and unflattened embryos had a homogeneous distribution of surface microvilli
however a small proportion concentrated their surface microvilli into a pole at
one end of the cell (Table 5, Fig. 5F, G). This process of surface polarisation was
slow compared to the normal sequence. Only 2 % of 'blocked 2-cells' were
polarised at 72 h post hCG as compared with 95 % of normal 1/8 blastomeres
(Fig. 5G, H, Table 5 and Reeve & Ziomek, 1981), and this value only increased
to 13 % by 113-120 h post hCG (Table 5).
Aspects of morphological differentiation were also abnormal in 'blocked
2-cell' embryos. The majority of mitochondria retained the immature,
vacuolated form characteristic of late 2-cell embryos (Figs 6A, 6C, 6D) and only
a minority of embryos developed the concentric cristae characteristic of 8- to
Fig. 5. Morphology of normal and 'blocked 2-cell' embryos. (Bar represents 50 |Um.)
Phase micrographs of:- (A) normal late 2-cell embryos (46 h post hCG); (B) 'blocked
2-cell' embryos (120 h post hCG) selected to demonstrate degree of intercellular
flattening that develops in some of these embryos.
Scanning electron micrographs of 'blocked 2-cells':- (Bar represents 10 jum) (C) &
(D) non-flattened, both blastomeres non-polar (i.e. with a homogeneous
distribution of microvilli). (E) flattened, both blastomeres non-polar. (F) flattened,
one blastomere partially polarised (note microvilli-enriched (*) and -depleted (**)
areas). (G) partially flattened, one blastomere fully polarised (microvillous pole
indicated (*). (H) 8-cell embryo (decompacted with Ca2+-depleted medium) showing microvillous poles (*).
'2-cell block' in mouse embryos
125
"•v....-^
v •
5A
;P
B
H
EMB73
Non-flattened
Flattened§
72
113-120 Non-flattened
Flattened§
72
(13 %)
(94 %)
75
(2%)
3(2/2)
12(2/2)
2(1/2)
Polarizedt
4(2/2)
45(2/2)
12(2/2)
5
9(2/2)
27(2/2)
3(1/2)
(6%)
(80%)
(85 %)
Non-polarized
—
5(2/2)
5(2/2)
1(1/2)
(7%)
(13 %)
80
138
88
Total number of
blastomeres
assayed
* Assessed by scanning electron microscopy, for experimental details see Materials and Methods. Both types of embryo were processed as intact
*0
embryos. 8-cell embryos were decompacted with Ca2+-depleted medium prior to fixation, 'blocked 2-cells' were processed without any prior
treatment.
3
t Number of blastomeres (percentages in parentheses) showing a heterogeneous distribution of microvilli. For 'blocked 2-cell' embryos 2/2
indicates both blastomeres and 1/2 indicates only one blastomere was polarised. All visible blastomeres of 8-cell embryos were assessed for polarity.
tNot scoreable.
§ Includes all 'blocked 2-cell' embryos where the degree of intercellular flattening was greater than that seen in normal 2-cell embryos at 46 h H
H
post-hCG (Fig. 5A,B).
Control 8-cells
'Blocked 2-cells'
Embryos
Time
(h post-hCG)
Table 5. Surface polarization in 'blocked 2-cells' and normal 8-cell embryos*
'2-cell block' in mouse embryos
127
16-cell-stage embryos (Fig. 6B insert) when analysed beyond 100 h post hCG.
Similarly, most nucleoli retained the condensed form found in late 2-cell-stage
embryos (Fig. 6C) and few developed the type of peripheral reticulations which
normally appear at the 2- to 4-cell stage (Fig. 6B).
DISCUSSION
The results of reciprocal crosses between CFLP and Fx eggs and sperm (Table
1) show that in this combination the genotype of the egg alone determines
whether the embryo 'blocks' at the 2-cell stage or continues to develop normally
in our conventional culture medium. A maternal influence on cleavage rate and
a maternal and possible paternal influence on the tendency to block in vitro
(Shire & Whitten, 1980a,6) have been described. However in these studies
fertilization occurred in vivo and factors other than the genotype of egg and
sperm have to be taken into account (discussed Shire & Whitten, 1980<2,6).
Embryos derived from CFLP eggs are susceptible to this culture-induced effect
for a period of approx. 36 h after injection of hCG (this includes the process of
cleavage to the 2-cell stage (Table 2)), whereas embryos derived from Fj eggs do
not show such sensitivity. It is not clear whether the differences between the two
types of egg arise from characteristics induced by their respective genital tract
environments or from differences in their capacities to adapt to in vitro conditions. The ability to overcome the block by injecting cytoplasm from embryos
of a non-blocking strain (Muggleton-Harris, Whittingham & Wilson, 1982) confirms the cytoplasmic nature of the defect and provides a potentially useful
approach to identifying the factor(s) involved. Relatively small volumes of
cytoplasm (approx. 8 pi) are effective suggesting that the rescuing factor(s) is
likely to be a modifying enzyme, cofactor, or self-replicating molecule or organelle that is defective in the CFLP embryo after exposure to our culture
medium. This component(s) is unlikely to be of mitochondrial origin since these
organelles do not replicate until the blastocyst stage (Piko & Matsumoto, 1976).
The molecular and morphological studies reported here suggest that all
'blocked 2-cell' embryos reach a state equivalent to a late 2-cell embryo in terms
of numbers of cycles of cell and nuclear division, the extent and nature of
methionine transport and the qualitative profile of synthesised polypeptides.
However a small proportion show some retarded development beyond this
stage. The majority of embryos (>90 %) arrest cleavage at the 2-cell stage and
undergo one round of DNA synthesis with subsequent inhibition of nuclear
division and DNA synthesis (Fig. 1). This lack of DNA synthesis is not a direct
consequence of arrested cell division since 2-cell embryos in which cleavage is
blocked by treatment with cytochalasin D continue to replicate their DNA with
the same timing as normal embryos (Pratt et al. 1981). Activation of the embryonic genome as assessed by the synthesis of novel or-amanitin-sensitive
polypeptides (class 2, Figs 3 & 4, (Johnson, 1981a; Flach et al. 1982)) can be
128
M. J. GODDARD AND H. P. M. PRATT
•i
v
'\
Fig. 6
'2-cell block' in mouse embryos
129
detected in 'blocked 2-cell' embryos although the process may be slower than
normal as judged by their rate of appearance. Since there was complete correspondence between the polypeptide profiles of 'blocked 2-cells' analysed individually and those analysed as a population it is very likely that this transition
occurs in all 'blocked 2-cell' embryos. In normal embryos activation of the embryonic genome is accompanied by a decline in synthesis of the majority of the
polypeptides that are characteristic of the early 2-cell stage (class 1, Figs 3 & 4,
Johnson, 1981a; Flach et al. 1982) and this was also a feature of 'blocked 2-cell'
embryos (Fig. 4). Normal embryos do not cleave from the 2-cell to the 4-cell
stage if activation of the embryonic genome is inhibited with a-amanitin (Braude
et al. 1979; Flach et al. 1982) suggesting that a transcriptional event is involved
in this process. However, as discussed above, transcription of the embryonic
genome probably occurs in all 'blocked 2-cell' embryos and therefore any abnormalities of cleavage, DNA synthesis or nuclear replication that occur between
the 2-cell and 4-cell stage in these embryos are likely to be caused by cytoplasmic
and/or cytocortical anomalies induced by culture in vitro.
In order to investigate whether 'blocked 2-cells' were capable of developing
beyond the late 2-cell stage we studied other morphological and molecular
features in aged 'blocked 2-cells'. These experiments could be subject to the
criticism that the process of ageing in 'blocked 2-cell' embryos is a degenerative
one however these embryos remain macroscopically (Biggers, 1971) and
ultrastructurally intact for more than 48 h in vitro (Fig. 6) as compared with aged
unfertilized eggs or 2-cell embryos treated with a-amanitin which lyse within 48 h
with morphologically disrupted organelles and vacuolated cytoplasm. The
overall impression gained from these studies of aged 'blocked 2-cells' is that these
embryos are in a state of developmental arrest rather than degeneration during
the 48 h following 'block' at the 2-cell stage. In contrast to evidence for the initial
activation of embryonic transcription we could find no clear indications of any
subsequent gene activity in 'blocked 2-cells'. In normal embryos later rounds of
transcription result in a general increase in uptake and incorporation of
methionine (beginning approx. 70h post-hCG, Fig. 2 & Braude, 1979a,b) the
transition from an exchange-mediated to Na+-dependent methionine transport
system (Table 3 and Borland & Tasca, 1974; Kaye et al. 1982) and the synthesis
Fig. 6. Morphology during development of normal and 'blocked 2-cell' embryos:
(A) One blastomere of normal late 2-cell embryos (46 h post-hCG) (X4300). Inset:
Typical morphology of mitochondria (x 14 200). (B) Normal 4-cell embryo (approx.
52 h post-hCG) showing localisation of mitochondria to areas adjacent to intercellular apposition (X1750). Inset: typical morphology of mitochondria from compacted 8-cell embryo (approx. 72 h post-hCG) (x 17 500). (C) 'Blocked 2-cell' embryo (90 h post-hCG) showing intercellularflatteninglocalisation of mitochondria to
areas adjacent to cell apposition and lack of reticulation in the nucleoli (x2940). (D)
'Blocked 2-cell' embryo (90 h post-hCG) showing a peripheral aggregate of
mitochondria and other vesicles (x4340). Inset: High magnification of mitochondria
(x 14 280).
130
M. J. GODDARD AND H. P. M. PRATT
of novel species of polypeptides (class 3, Fig. 4Nos. 1,4,6 &7, Braude, I979a,b)
or the continued synthesis of others (class 4, Fig. 4 and Braude, 1979a,b). None
of these features was observed in populations of 'blocked 2-cells'. Uptake and
incorporation of methionine remained low (Fig. 2), uptake stayed Na + independent (Table 3) and polypeptides that were sensitive to a-amanitin during
the transition from morula to blastocyst were absent or only synthesised at very
low levels (e.g. class 3, Nos. 1, 4, 6 & 7, class 4 'w' Fig. 4). Levels of protein
synthesis were too low to assess any potential heterogeneity within the population by analysing individual embryos.
'Blocked 2-cell' embryos also remained morphologically immature. During
normal development, activation of ribosome assembly (Bachvarova & de Leon,
1977) and changing energy metabolism (Biggers & Borland, 1976) are associated
with structural and functional maturation of nucleoli and mitochondria (Stern,
Biggers & Anderson, 1971; Hansmann, Gebauer, Bihl & Grimm, 1978; Van
Blerkom & Motta, 1979) which begin at the 2- to 4-cell stage and are regulated
to an extent by embryonic transcription and protein synthesis (Piko & Chase,
1973; Hansmann et al. 1978; discussed Johnson, 1981a). However in 'blocked
2-cell' embryos nucleoli rarely showed any signs of reticulation (Fig. 6C) and
mitochondria remained small and vacuolated (Fig. 6D). The main elements
which could contribute to this failure to mature are faulty organelles themselves,
defects in their interactions with cytoplasmic factors involved in activating DNA
transcription (Gurdon, 1974) or in modifying mitochondrial structure function
(Piko, 1971; Piko & Chase, 1973) and a requirement for transcription of the
embryonic genome beyond the 2-cell stage. Cell polarisation (involving reorganisation of both the cytoplasm and cytocortex) and cell flattening are independent features of the normal 8-cell-stage embryo which have important
consequences for development of the blastocyst (Johnson, 1981b; Ziomek, Pratt
& Johnson, 1982; Pratt, Ziomek, Reeve & Johnson, 1982). Only 13 % of the
'blocked 2-cells' showed any surface polarity although approximately 60 % flattened (Table 4). These processes were slow compared with normal development
and the retardation and heterogeneity observed are presumably related to events
other than the initial activation of the genome.
In conclusion, these experiments demonstrate that the '2-cell block' described
here is due to a culture-induced cytoplasmic defect which does not permit normal
development beyond the late 2-cell stage in the majority of cases. These observations are compatible with studies involving physical enucleation or using transcriptional inhibitors (discussed Johnson, 1981a) which demonstrate that early
cleavage of the mouse embryo is under a degree of maternal (cytoplasmic)
control in common with other vertebrate and invertebrate embryos (Davidson,
1976; McLaren, 1981).
We wish to thank Dr Hilary MacQueen for help with the analysis of single embryos, Gin
Flach and Mike Parr for invaluable technical assistance, Wally Mouel and Jeremy Skepper for
'2-cell block' in mouse embryos
131
help with SEM and TEM, Raith Overhill for help with thefigures,Ian Edgar for photographic
assistance and Dr B. Hewitt of Department of Biology, University of East Anglia, Norwich,
U.K. for the use of his Vickers M85 microdensitometer. This work was supported by a grant
from the M.R.C. to H.P.M.P. and from the C.R.C. to M. H. Johnson.
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(Accepted 30 September 1982)