/. Embryo/, exp. Morph. Vol. 52, pp. 193-202, 1979
193
Printed in Great Britain <£ Company of Biologists Limited 1979
Time-dependent effects of a-amanitin on
blastocyst formation in the mouse
By P. R.BRAUDE 1
From the Department of Anatomy, University of Cambridge
SUMMARY
Early compacting mouse morulae were placed in a-amanitin at various times after HCG
and the effect of this transcriptional inhibitor during formation of the blastocyst was noted.
No cavitation was observed in those embryos transferred into a-amanitin before 77 h after
HCG but the percentage of cavitation increased dramatically in those embryos transferred
after 80 h post HCG. The large increase in amino acid incorporation which normally occurs
during formation of the blastocyst was blocked in those embryos placed in the inhibitor
before 80 h post HCG. Two-dimensional polyacrylamide gel electrophoresis of radiolabelled
embryos showed that the changes in synthesis of certain polypeptides normally associated
with blastocyst formation did not occur in those embryos placed in a-amanitin before 80 h
after HCG. Only one cleavage division appears to occur in the presence of a-amanitin.
However, groups of embryos which had completed their fifth division before exposure to the
drug subsequently were able to form blastocysts and showed the quantitative and qualitative
changes associated with blastocyst formation despite the continued presence of the inhibitor.
These results suggest that a critical transcriptional event concerned with blastocyst formation
occurs around 80 h after HCG and may be associated with the fifth cleavage division.
INTRODUCTION
The continued development of echinoderm and amphibian embryos in the
absence of a nucleus (Denny & Tyler, 1964; Brachet, Ficq & Tencer, 1963;
Smith & Ecker, 1965) or in the presence of transcriptional inhibitors (Gross &
Cousineau, 1964; Sargent & Raff, 1976) has led to the postulate that early
embryonic development may be controlled at post-transcriptional levels by
utilization of stored presynthesized mRNAs (Gross, 1968). Some degree of
transcriptional independence has also been demonstrated in early cleavage
rabbit (Manes, 1973) and mouse (Monesi & Molinaro, 1970; Epstein, 1975;
Levey, Troike & Brinster, 1977; Johnson, Handyside & Braude, 1977) embryos,
in which maternally derived post-transcriptional information appears to substantially influence development (see Johnson, 1979). Subsequent steps in the
formation of the mammalian blastocyst appear to be more transcriptionally
dependent than the processes of blastulation in echinoderm and amphibian
embryos. We have reported previously that early mouse morulae are unable to
1
Author's address: Department of Anatomy, University of Cambridge, Downing Street,
Cambridge CB2 3DY, U.K.
194
P. R. BRAUDE
form a blastocyst in the presence of a-amanitin (Braude, 1979) at a dose which
has been shown to inhibit completely the synthesis of poly-A RNA by mouse
blastocysts (Levey & Brinster, 1978). Coupled with this observation is the
finding that certain tissue-marker polypeptides (Van Blerkom, Barton &
Johnson, 1976) which normally appear during blastocyst formation (Handyside
& Johnson, 1978) do not do so in the presence of the inhibitor (Braude, 1979).
These results could be taken to suggest that the process of blastocyst formation
is transcriptionally dependent, and that the mRNAs required for the production
of essential a-amanitin sensitive proteins are transcribed only shortly before the
process of blastocyst formation.
In contrast, other workers have reported successful blastocyst formation in
the presence of a-amanitin albeit to a limited degree (Levey et al. 1977; Golbus,
Calarco & Epstein, 1973; Warner & Versteegh, 1974). In these experiments
the embryos were subjected to the inhibitor at a slightly later stage of development than the embryos in our study. This raises the possibility that prevention
of blastocyst formation is critically time dependent and related to the synthesis
of specific mRNAs, such that transcriptional blockade after their synthesis
would have no effect on blastocyst formation.
In this study, embryos have been placed in a-amanitin at various times during
the formation of the blastocyst and the effect of this transcriptional inhibition
on both morphology and the patterns of synthesis of specific polypeptides has
been assessed.
MATERIALS AND METHODS
Recovery and allocation of embryos
Early compacting morulae were flushed from the oviducts of superovulated
female CFLP mice which had been mated with CFLP males. The pooled
embryos were randomly allocated in groups of between 11 and 39 embryos into
separate 100 /^l drops of medium 16 containing 0-4% bovine serum albumin
(BSA) under oil in 5 % CO2 in air at 37 °C. The incubations were begun 69-73 h
after HCG and not more than 2 h lapsed between killing the animals and
placing the embryos in culture. At time zero and at 3-5 h intervals thereafter,
one group of embryos was transferred to a fresh microdrop of medium 16 + BSA
which contained 11 /*g/ml a-amanitin. One group of embryos which was to
serve as a control group for normal blastocyst formation was not transferred
into a-amanitin. Before each transfer the morphological stage of the embryos
was noted. After 24-30 h in culture, by which time a high percentage of the
control embryos had formed blastocysts, each treated group was assessed for
the number of embryos which showed signs of fluid accumulation or further
degrees of cavitation.
Effects of a-amanitin on mouse blastocyst formation
195
Labelling of polypeptides for electrophoresis
After 24-27 h in culture 10 /A [35S]methionine (~ 1000 Ci/mmole, 5 mCi/ml,
The Radiochemical Centre, Amersham) was added to each 100/tl culture drop
and the embryos cultured for a further 3 h. They were then washed through
protein-free phosphate buffered medium (Whittingham, 1971) (to dilute out the
BSA) before being collected into 20 /A of lysis buffer (O'Farrell, 1975). At the
outset of each experiment for electrophoresis, one group of embryos was
transferred immediately to medium containing [35S]methionine. After culture
for 3 h it was harvested into lysis buffer as above.
Two-dimensional electrophoresis
Two-dimensional polyacrylamide gel electrophoresis was performed as described previously (Handyside & Johnson, 1978; Braude, 1979). The iso-electric
focusing (first dimension) was performed over a pH range of 4-5-7-0 and the
second dimension on SDS 7-5-15% exponential gradient polyacrylamide slab
gels. After electrophoresis, the gels were impregnated with 2,5-Diphenyloxazole
(PPO) (Bonner & Laskey, 1974) and exposed to preflashed Fuji Rx X-ray film
(Laskey & Mills, 1975) for 2-6 weeks. The fluorographs were analysed as described previously (Handyside & Johnson, 1978; Braude, 1979).
Quantitative aspects of protein synthesis
One hundred and ten morulae (73 h post HCG) were divided into groups
containing between 9 and 20 embryos. One group (20 embryos) was added
immediately to medium 16 +BSA containing 100/AM [35S]methionine (488 /tCi/
ml) and incubated for 3 h. At the end of the 3 h period these embryos were
transferred in batches of five to the wells of a microtitre plate each containing
100 /d distilled water. This group was used to measure incorporation into protein at the start of the experiment. Four of the original six groups of embryos
were transferred at 73, 76-5, 80 and 83 h post HCG, from the medium 16 +BSA
in which they had been cultured, to a fresh 100/d drop of medium containing
11/tg/ml a-amanitin. The sixth group of embryos was retained in medium
16 +BSA throughout and was designated the control group (Fig. 2). After a
total culture period of 24 h, the embryos were labelled for 3 h and the number
of embryos which had cavitated was noted. Embryos were then transferred in
batches of four or five (for the earlier groups) or individually (for the 83 h and
control group) to wells containing 100/A distilled water. After twice freezethawing the embryos, 10 /A carrier BSA (1 mg/ml) and 100 [A 25 % trichloroacetic acid (TCA) was added to each well and the plate allowed to stand overnight at 5 °C. The following morning the samples were individually filtered
through glass fibre discs (Whatman GF/C) by use of a multiple sample harvester
(Otto Hiller, Michigan, U.S.A.). After thorough washing with cold 8 % TCA,
the filter discs were dried and counted in a triton-toluene scintillant in an ICN
scintillation counter at 70 % efficiency.
196
P. R. BRAUDE
100
=i
50
70
75
80
85
Time after HCG into cc-amanitin (h)
Controls
Fig. 1. Percentage cavitation of embryos subjected to a-amanitin at various times
after HCG. Early compacting morulae were explanted between 69 and 73 h after
HCG and transferred at various times into media containing 11 /*g/ml a-amanitin.
After 27-30 h in culture the embryos were examined for signs of fluid accumulation or overt cavitation. The percentage of embryos cavitating in each group is
plotted as a function of the time (after HCG) at which the embryos were placed
into the inhibitor. The percentage cavitation in untreated embryos cultured for the
same length of time in each of the seven experiments is shown on the right, where
each open circle represents one experiment.
Counting of nuclei
The number of nuclei in individual embryos was counted using an air drying
technique (Tarkowski, 1966). Embryos were incubated in 1 % Na Citrate for
2-30 min at room temperature until the cells swelled visibly at which stage they
were transferred to a slide with minimal fluid. Drops of a 3:1 mixture of
absolute ethanol and glacial acetic acid were applied directly to fix the embryos.
The slide was air dried and the spreads stained with 5 % Giemsa before being
counted under the microscope. In two separate experiments following a similar
protocol to that outlined above, embryos were placed at various times after
HCG into medium containing ll/*g/ml a-amanitin (Table 1). Before each
transfer a number of embryos were taken for counting. Twenty-four hours after
beginning the experiment, when most of the control embryos formed blastocysts, the embryos in each group were individually fixed, stained and counted.
RESULTS
In seven separate experiments using a total of 574 embryos, no cavitation was
observed in embryos which were placed in a-amanitin before 77 h after HCG
and less than 6 % of those placed in a-amanitin between 77 and 80 h post HCG
were able to cavitate (Fig. 1). In those groups placed in a-amanitin at, or later
than 80 h after HCG, the percentage of embryos able to cavitate increased
Effects of a-amanitin on mouse blastocyst formation
197
1500
1000
•5
500
Start
73
76
80
-S3
Time iiS'icr I ICC! into oc-unumitin ( h )
Control
Fig. 2. Acid insoluble incorporation of [35S]methionine into developing mouse
morulae placed in a-amanitin at 73, 76, 80 and 83 h after HCG. All embryos were
explanted at 73 h post HCG at which stage one group of embryos immediately was
labelled for 3 h in medium containing 100/*M [35S]methionine. After 24 h in culture
the rest of the groups were similarly labelled for 3 h. The points represent the
incorporation for individual embryos in the 83 h and control groups, and for
groups of four or five for the earlier groups of embryos. The arrows show the
means for each group. Those embryos which had cavitated are shown by the open
circles, and those which showed no signs of cavilation by the closed circles. (100
cpm ~ 19 x 10~15 mole.)
dramatically, tending toward control levels in those embryos placed in the
inhibitor from 83 h after HCG onwards.
The large increase in amino acid incorporation, which normally occurs
during blastocyst formation (Monesi & Molinaro, 1970; Johnson et ah 1977;
Braude, 1979), was blocked in embryos placed in a-amanitin before 80 h post
HCG (Fig. 2). At or after this time, protein synthesis did increase. However, in
embryos placed in the inhibitor at 83 h after HCG, levels of incorporation had
still not reached those in untreated embryos (Fig. 2). The increase in amino acid
incorporation observed appeared to be related to the extent of successful
blastocyst formation, fully expanded and expanding blastocysts tending to
incorporate more than non-cavitating embryos. However, this difference was
not statistically significant.
198
P. R. BRAUDE
The majority of polypeptides synthesized by the compacting morula are also
synthesized by the blastocyst. These, as noted previously, did not appear to
change intensity in the presence of a-amanitin (Class A, Braude, 1979). There
are also a number of polypeptides which increased in intensity during blastocyst
formation as was also reported previously (Handyside & Johnson, 1978). In
those embryos placed in a-amanitin before about 80 h post HCG, this increase
in intensity could not be demonstrated (Figs. 3, 4). These spots correspond to
the Class B polypeptides reported previously (Braude, 1979). A further group
of polypeptides decreased in intensity over the same period that the Class B
polypeptides were increasing. This reduction in intensity was less marked in
those embryos placed in a-amanitin earlier than 80 h post HCG than those
placed in the inhibitor after this time (Fig. 4). These spots correspond to the
Class C polypeptides previously reported (Braude, 1979).
The air drying technique for estimating cell number suffers from the disadvantage that only the nuclei of the embryos are preserved and stained.
Although most of these are usually easily discernible in the normal embryo, any
interference with nuclear function although not necessarily with cytokinesis may
result in an incorrect count. We have noted that many of the nuclei of the
a-amanitin-treated embryos appear smaller, do not stain clearly or indeed may
have fragmented. Thus the estimate of cell number in these embryos may be
artifactually low. Nevertheless, whereas untreated embryos are able to undergo
two or three divisions in the allocated time (from 8 cells to between 20 and 60
cells), a-amanitin-treated embryos probably only divide once (Table 1).
DISCUSSION
Blastocyst formation in the mouse under the culture conditions described
is normally first observed at about 83 to 85 h after HCG administration.
Accompanying this change, is a quantitative increase in protein synthesis and a
qualitative change in the patterns of polypeptides synthesized. All these developmental events are affected if the embryos are placed in a-amanitin by 80 h post
HCG. This brief interval of 3-5 h between the end of a-amanitin sensitivity and
overt initiation of blastocyst formation suggests that the drug affects molecular
Fig. 3. Two-dimensional SDS polyacrylamide fluorographs of [35S]methioninelabelled polypeptides from mouse embryos cultured from 72 h post HCG and
transferred into medium containing a-amanitin at 75 h post HCG (3a) and 88 h
post HCG (b). Polypeptides which normally increase in intensity during development from morula to the blastocyst are shown by (^) and those which decrease in
intensity are denoted by (./). The numbering system follows that of Van Blerkom
et al. (1976) and Handyside & Johnson (1978). The iso-electric focusing first
dimension was performed over a pH range 7-0-4-5 and the second dimension on
exponential gradient (7-15 %) polyacrylamide slab gels.
Effects of ot-amanitin on mouse blastocyst formation
(a).
75 h post HCG
u—
39
i
34
17
37
pH7
pH4-5
IEF
88 h post HCG
'2i3uT
14
39
16
17
37
33
199
200
P. R. BRAUDE
4
Untreated morulae
(72 hours after HCG)
9
II
14
]6
17
33
34
37
30
•I!::::-:::
72
•75
Time of transfer into
oc-anianitin
(hours after HCG)
77
;••!!:••;•
iiiii
1I
1
•
•I
Iiiiiiiii
mm
Untreated bJastocysts
(96 hours after J ICG)
B
lil^
Strong synthesis detectable
|H Weaker synthesis detectable
H | Trace synthesis detectable
Q
Synthesis not detectable
§!§ Not scoreable
Fig. 4. Changes in relative intensity of [35S]methionine-labelled polypeptides synthesized by embryos transferred into a-amanitin at various times after HCG.
Embryos were cultured from 72 h after HCG and transferred into medium containing 11 /<g/ml a-amanitin at the various times after HCG indicated. After a total of
24 h in culture the embryos were labelled for a further 3 h in medium containing
[35S]methionine. Each horizontal line of blocks represents the data from one good
two-dimensional gel analysed and exposed as in Braude (1979). The relative intensity of the polypeptides shown in Fig. 3 were scored as shown in the key. (Asterisks
indicate the two gels shown in Fig. 3). The data from two gels of untreated morulae
and two gels of untreated blastocysts are also shown.
Table 1. Mean nucleus counts of early morulae placed in 11 /*g/ml a-amanitin at various
times after HCG
Time of transfer
into a-amanitin
(hr after HCG)
Untreated embryos
74*
74-5|
78f
79*
81f
83-5*
Expt. 1*
Expt. 2f
Nucleuscount
8-1 ±2-7 7-4±3-6 ll-2±4-l 17-6±4-3 12-7±4-7 24-2 + 10
8-1 ±2-7 7-4±3-6
before treatment (1)%
(8)
(11)
(8)
(9)
(5)
(7)
(8)
±S.D.
Nucleuscount
14-1 ±5-5 12-4±4-3 16-7±73 204±8-8 23±5-5 42-8± 10-8 403±14-6 42-7± 11-6
24 h after ex(7)
(16)
(13)
(9)
(13)
(6)
(13)
(7)
plantation +S.D.
* Cultured from 74 h after HCG.
t Cultured from 74-5 h after HCG.
% Figures in parentheses are the number of embryos counted in each group.
Effects of QL-amanitin on mouse blastocyst formation
201
events that are tightly coupled to developmental consequences. Since a-amanitin
is an effective inhibitor of RNA polymerase II, transcription of key mRNA
species may be required to initiate blastocyst formation. Although secondary
effects of a-amanitin after prolonged exposure have been described, the fact
that embryos exposed to the inhibitor from 83 h after HCG for a further 10-15 h
are still able to form blastocysts and to synthesize protein at near normal rates
argues against the block to blastocyst formation arising from a general deterioration of cellular function.
The effectiveness of a-amanitin as an inhibitor of blastocyst formation
appears critically dependent on cell or nuclear divisions. When at least some
embryonic cells have completed their fifth division (16-32 cells) before exposure
to the inhibitor then both blastocyst formation and qualitative and quantitative
changes in protein synthesis occur despite the presence of the drug for a further
15 h. Since cell number per se does not appear to be the critical factor in blastocyst formation (Smith & McLaren, 1977), the fifth nuclear division itself or a
critical nuclear: cytoplasmic ratio may be associated with some form of genetic
^programming and the transcription of key messages.
The transcriptional event associated with blastocyst formation could occur
at either or both of two levels. Expression of sets of specific genes coding for
'blastocyst' polypeptides could be transcribed around 80 h post HCG and, by
immediate utilization, lead to the expression of new proteins and the formation
of a blastocyst. Alternatively, much of the mRNA information required for
blastocyst formation could, by analogy with echinoderm and amphibian
embryos, be presynthesized but require activation by a transcriptionally dependent mechanism. One method of resolving these alternatives would be to
determine whether extraction of mRNA from 16-cell embryos and its translation in vitro yields blastocyst marker polypeptides. A system of sufficient
fidelity and sensitivity has been developed for the extraction and in vitro translation of mRNA from mouse ova (Braude & Pelham, 1979) and we are currently
applying this method to morulae and blastocysts.
1 wish to thank Drs Martin Johnson, Gilbert Schultz and Hester Pratt for their helpful
suggestions, and Gin Flach and Jo Close for their meticulous technical assistance. This work
was supported by grants from the Medical Research Council and the Ford Foundation to Dr
M. H. Johnson.
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{Received 9 January 1979, revised 12 February 1979)