J. Embryol. exp. Morph. 74, 15-28 (1983)
Printed in Great Britain © The Company of Biologists Limited 1983
Evidence for translation of HPRT enzyme on
maternal mRNA in early mouse embryos
By MARY I. HARPER 1 AND MARILYN MONK 2
From the MRC Mammalian Development Unit, Wolf son House, London
SUMMARY
This paper presents evidence that maternal mRNA is responsible for the early increase in
HPRT activity in preimplantation mouse embryos. Increase of HPRT activity is demonstrable
from as early as 6 h postfertilization when there is barely detectable synthesis of embryonic
RNA. The increase is sensitive to cycloheximide and thus requires protein synthesis, whereas
it is insensitive to a-amanitin and therefore independent of mRNA synthesis. These results
suggest that translation of HPRT occurs on pre-existing maternal mRNA.
Embryo-coded HPRT activity is detectable by the 4- to 8-cell stage when the increase in
HPRT activity becomes sensitive to a-amanitin. The transition from maternal- to embryocoded enzyme activity is completed by the time of compaction. At this stage there is an
unexplained yet reproducible loss of HPRT activity. Other maternally-inherited enzymes
show a marked degradation occurring at a similar time. It is possible that the enzyme degradation observed reflects some common mechanism directing the changeover from maternallyderived to embryonically-derived enzymes.
INTRODUCTION
Studies on non-mammalian species have shown that messenger RNA
synthesized during oogenesis (maternal mRNA) is utilized for protein synthesis
after fertilization (see review by Davidson, 1976). In the sea urchin, translation
on maternal mRNA continues through a number of cell divisions when transcription is inhibited by antinomycin D (Gross & Cousineau, 1964). In contrast to this
situation the early development of mouse embryos is very sensitive to inhibitors
of RNA synthesis (Monesi, Molinaro, Spalletta & Davoli, 1970; Golbus, Calarco & Epstein, 1973) suggesting that embryonic message is required. Indeed
expression of the embryonic mouse genome within the first few cleavages may
be observed at the biochemical and cellular levels (see reviews by McLaren,
1976; Johnson, 1981; Magnuson & Epstein, 1981). Clegg & Piko (1982) have
shown that large heterodisperse RNA is already synthesized at the 1-cell stage,
and additionally some newly synthesized polyadenylated RNA is present in very
1
Author's present address: Cold Spring Harbor Laboratory, P.O. Box 100, Cold Spring
Harbor, New York 11724, U.S.A.
1
Author's address: MRC Mammalian Development Unit, Wolfson House, 4 Stephenson
Way, London, NW1 2HE, U.K.
16
M. I. HARPER AND M. MONK
low amounts and may therefore be mRNA (see also Levey, Stull & Brinster,
1978; Piko & Clegg, 1982).
However, evidence has also been presented for stored maternal mRNA in
mammalian eggs. Schultz (1975) showed that the amount of the poly A + RNA
fraction in rabbit eggs 10 h after fertilization is equal to that in non-fertilized
eggs, and protein synthesis during this time is insensitive to almost total inhibition of RNA synthesis by a-amanitin. The RNA of unfertilized mouse ova
also contains poly A tracts (Levey et al. 1978; Piko & Clegg, 1982) and maternal
RNA, including a poly A + fraction, persists throughout mouse preimplantation
stages (Bachvarova & DeLeon, 1980).
Braude, Pelham, Flach & Lobatto (1979) have demonstrated that certain
proteins are translated on maternal message in the early mouse embryo. These
workers see a group of proteins appearing at the early 2-cell stage after twodimensional electrophoresis. When mRNA synthesis is inhibited by a-amanitin
the proteins still appear, even if eggs are fertilized in the presence of inhibitor
(Flach et al. 1982). From in vitro translates it has been found that the mRNA
coding for the proteins appearing at the 2-cell stage is already present in the
unfertilized mouse egg, which may suggest that the message is 'masked' in the
egg (Flach et al. 1982; Cascio & Wassarman, 1982).
We have presented evidence previously for a maternally-derived increase in
the activity of a specific enzyme, the X-coded HPRT (hypoxanthine phosphoribosyl transferase, E.C.2.4.2.8). The first suggestion that HPRT was maternally
derived in early cleavage stages came from investigations into X-inactivation. At
the morula stage enzyme activity is embryo coded, and since two X chromosomes
are active in female (XX) embryos they have twice the HPRT activities of males
(XY) (Monk & Harper, 1978; Epstein, Smith, Travis & Tucker, 1978; Kratzer
& Gartler, 1978). Prior to this, from the 1-cell zygote to the early 8-cell stage,
male and female embryos have equivalent HPRT activities (Monk & Kathuria,
1977) thus suggesting that the rise in enzyme activity that occurs over this period
is maternally regulated (Monk & Harper, 1978; Monk, 1978). Confirmation of
the maternal origin of HPRT came from the following findings. Monk & Harper
(1978) showed that 8-cell embryos from XX mothers contain twice the activity
of HPRT of those from XO mothers, reflecting a maternal effect of X-chromosome dosage during oogenesis (Epstein, 1972; Monk & Kathuria, 1977); and
Burgoyne, Harper & Monk (unpublished) showed that HPRT activity increases
between the 1-cell and the 4- to 8-cell stage even in YO embryos, i.e. in the
absence of an X chromosome and thus an HPRT gene. The maternal regulation
of this early increase in HPRT activity could occur either by the translation of
maternally-derived mRNA coding for HPRT or by a change in the activity of
presynthesized HPRT, possibly via post-translational modification of the
enzyme.
This paper presents evidence that the maternally regulated increase in HPRT
activity is due to translation of enzyme on maternal mRNA. The relationship
HPRT translation on maternal mRNA
17
between the HPRT-activity increase and both translation and transcription was
studied at different times throughout preimplantation development. Protein
synthesis was inhibited by culturing embryos in cycloheximide (Epstein & Smith,
1973); mRNA synthesis was inhibited by culture in a concentration of 10/ig/ml
of a-amanitin, which specifically blocks RNA polymerase II (Lindell etal. 1970;
Levey & Brinster, 1978), and which is known to block the activation of the
embryonic genome at the 2-cell stage (Flach et al. 1982).
Our results reveal some interesting points concerning the stability of HPRT
enzyme and message. Although inhibitor studies suggest both are stable in early
preimplantation stages, there is a significant and reproducible loss of HPRT
activity at the time of transition from maternal- to embryo-coded enzyme.
Degradation of several other maternally-inherited enzymes occurs at this time
suggesting some common mechanism may be involved in the changeover from
maternal to embryonic enzymes.
MATERIALS AND METHODS
Embryo collection
Mouse embryos were obtained from random-bred MF1 females (Olac 1976
Ltd.). Female mice were superovulated by intraperitoneal injection of 5i.u.
PMS (pregnant mare serum) followed 45-48 h later by 5i.u. HCG (human
chorionic gonadotrophin), and were then caged with MF1 males. Ovulation was
assumed to occur 12h after the HCG injection (Gates & Beatty, 1954).
1-cell eggs were separated from cumulus cells by 10 to 15 min digestion at room
temperature in hyaluronidase (300 i.u./ml) after which eggs were washed free of
enzyme in PB1 medium (Whittingham & Wales, 1969). 2- to 8-cell embryos were
flushed from the oviduct and morulae and blastocysts from the uterus with PB1.
Eggs and embryos were either cultured (see below) or harvested directly. To
harvest, zygotes were washed twice in PB1/PVP medium (PB1 with polyvinylpyrrolidone, 4 mg/ml, in place of albumin) and collected, singly or in groups, in
5 [A PB1/PVP in a 10 [A microcap, the ends of which were sealed by melting in
a flame. Samples were stored at - 7 0 °C. Supernatant extracts were prepared by
freeze thawing three times followed by centrifugation at 4°C.
Embryo culture
Droplets of medium 16 (Whittingham, 1971), plus or minus inhibitors of
protein or RNA synthesis, were prepared under paraffin oil in plastic culture
dishes and equilibrated in a humidified 5 % CO2 in air atmosphere at 37 °C for
at least an hour. Embryos from a number of litters were usually pooled and then
added to culture droplets, via a wash droplet, in groups of between 5 and 20.
Embryos were harvested after culture as above.
18
M. I. HARPER AND M. MONK
Drugs
Solutions of cycloheximide (Sigma) in medium 16 (1, 10 or lOOjUg/ml) were
prepared fresh before embryo culture. A stock solution of a-amanitin (1 mg/ml,
Boehringer Mannheim) in medium 16 was aliquoted in 10/il volumes and stored
at — 70 °C; prior to use lml medium 16 was added to the tube giving a final
concentration of
/
Enzyme assays
HPRT was measured in embryo extracts as described in Monk & Harper,
1978. The specific activity of [3H]guanine sulphate was 700Ci/M unless otherwise stated. The incubation period was either 3 or 4 h at 37 °C. Male and female
embryos around the morulae stage have different HPRT levels; however, embryos were assayed in pools of seven or more to give the mean value for both
sexes combined. LDH activity was measured by the method of Brinster (1965).
RESULTS
Evidence for translation of HPRT on maternal mRNA
Fig. 1 shows the profile for HPRT activity during preimplantation development, the enzyme increasing in activity during cleavage. The rise in HPRT
activity is statistically significant even by the 2-cell stage (Table 1).
Fig. 2 shows that culture in the drug cycloheximide inhibits the rise in HPRT
activity at all preimplantation stages from the 2-cell stage to blastocyst. The rise
Table 1. Increase in HPRT activity from the 1-cell stage
HPRT activity (pM/h/embryo ± S.E.M.)
Experiment
number
1-cell
2-cell
4-cell
Expt. I
0-05 ±0-01
(18h)|
0-15 ±0-02*
(42 h)
0-21 ±0-02*
(48 h)
0-2510-01*
(64 h)
Expt. II
0-16 ±0-01
(20 h)
0-35 ±0-02**
(44 h)
Expt. Ill
0-1 ±0-01
(20 h)
0-24 ±0-01**
(42 h)
0-25 ±0-01**
(48 h)
8-cell
0-35 ±0-07*
(64 h)
0-82 ±0-05**
(68 h)
1-22 ±0-02**
(68 h)
0-5 ±0-04**
(64 h)
0-59 ± 0-03**
(64 h)
0-99 ±0-08**
(68 h)
thpostHCG.
The increase in activity from the 1-cell stage is significant, * P<0-05, * * P< 0-001.
HPRT translation on maternal mRNA
19
2-
OH
X
Ou
1-
42
48
64
68
72
76
88
92
Hours post HCG
v
v v
rH
CN
T v v f
CN
4
oo
oo
°£
«
Ex>
Fig. 1. HPRT activity during in vivo preimplantation development. Litters from two
females were collected at each time point. Embryos were harvested in groups of
seven to ten, assayed for HPRT, and the values pooled. The specific activity of
[3H]guanine was 84Ci/M. The dashed and dotted lines respectively indicate the
postulated maternal and embryonic origin of the HPRT activity.
in enzyme activity is therefore dependent on protein synthesis, consistent with
de novo synthesis of HPRT.
To test the effect of inhibiting mRNA synthesis, embryos were cultured in the
presence of 10 /ig/ml of a-amanitin. Fig. 3 compares the effect of a-amanitin treatment with cycloheximide treatment during a very early phase of development.
Fertilized eggs (18 h post HCG) cultured for 24 h in a-amanitin show the same rise
in HPRT activity as in the control, whereas the increase is inhibited by
cycloheximide. As the significant rise in HPRT activity (tio= 19-956, P<
0-001) is insensitive to inhibition of transcription while being eradicated by inhibition of translation, the simplest conclusion is that this early increase in HPRT
activity is due to translation of new enzyme on preexisting maternal message.
20
M. I. HARPER AND M. MONK
4-
E >•>
H
2-
Q
70
Hours post HCG
90
Fig. 2. Effect of cycloheximide on HPRT activity. 2-cell embryos were cultured in
medium 16 (
• ) , samples being transferred to cycloheximide (—O) at different
stages. Cycloheximide concentration: 1,10 or 100|Ug/ml (values from different drug
concentrations are pooled). Embryos were harvested in groups of seven.
0-2
0-1-
18
30
Hours post HCG
42
Fig. 3. Effect of cycloheximide and a-amanitin on HPRT activity from 1-cell to
2-cell embryos. Early 1-cell zygotes were cultured in either medium 16 (
•),
lOjUg/ml cycloheximide (—O) or lOjUg/ml a-amanitin (... A). Eggs were harvested in groups offive;the data are pooled from two experiments.
In order to test later stages of development, embryos were cultured in
a-amanitin for 24 h periods at different (overlapping) stages as follows: 2-cell to
4/8-cell (40-64 or 46-70h post HCG); 4-cell to 8-cell (52-76 or 58-82h post
HPRT translation on maternal mRNA
21
2-
0-5-
0-25
+i
40
52
64
46
58
70
52
64
76
70
82
94
Q.
2-
4-
4-
2-
58
70
82
64
76
Hours post HCG
Fig. 4. Effect of a-amanitin on HPRT activity from 2-cell to blastocyst. Embryos at
different stages were cultured in either medium 16 (
• ) or in lOjUg/ml aamanitin (—O), and harvested in groups of five or seven. Graphs give results from
single experiments except: (B) - four experiments pooled; (C) - three experiments
pooled; (E) - two experiments pooled. Note different times along the X-axis, and
different scales of enzyme activity along the Y-axis, for different periods. * HPRT
activities of control and treated samples are significantly different, F<0-05.
HCG); 8-cell to morula and blastocysts (64-88 or 70-94h post HCG). HPRT
activities of control and experimental cultures were compared after 12 and 24 h.
The results are shown in Fig. 4.
Early 2-cell embryos of the MF1 strain do not culture well in vitro. Nevertheless a significant increase in HPRT activity (tt = 3-33, P < 0-05) occurs over 24 h
culture which is insensitive to a-amanitin (Fig. 4A), as it is from the 1-cell to
the 2-cell stage shown in Fig. 3. However some caution should be exercised in
interpreting this period of transition from maternal to embryonic mRNA. It is
conceivable that some embryonic mRNA for HPRT is synthesized prior to
commencement of culture of the 2-cell embryos in a-amanitin (Fig. 4A) and
22
M. I. HARPER AND M. MONK
that the rise in HPRT activity occurs on both embryonic and maternal message.
From the late 2-cell stage onwards preimplantation embryos culture well. For
these later stages of development, except the latest period (Fig. 4F), a-amanitin
reduces the HPRT activity significantly after 24 h treatment, showing that newly
synthesized embryonic mRNA is now required for the normal enzyme increase,
a-amanitin in fact inhibits a change in rate of enzyme increase between 12 and
24 h after addition of the drug, indicating that the increased rate requires transcription. The delay in the effect of a-amanitin on the increase in enzyme activity
suggests a period of at least 12 h between synthesis of new mRNA and its expression as active enzyme. In Fig. 4F, the latest period tested, there is no change
in rate of enzyme activity increase. This may indicate cessation of further mRNA
synthesis or enzyme degradation (see Fig. 2) as embryos enter the late blastocyst
stage. In all these later stages (Fig. 4B, C, D, E, F) a-amanitin allows a rise in
HPRT activity to continue throughout the 24 h of treatment, presumably by
translation on stable mRNA present at the time of addition of the drug.
HPRT activity decrease
During the period of changeover from maternally- to embryonically-derived
HPRT there is a transient loss in enzyme activity. Fig. 1 shows that after 64 h post
HCG the HPRT activity rises steeply for a short time, dips at 72 h (when the
embryonic population consists of compacted 8-cell andmorulae stages), and
then resumes the high rate of increase up to the blastocyst stage where it appears
to plateau. The decrease at 72 h post HCG is reproducible but variable (see Figs
1 and 5 (HPRT)); HPRT activity may drop significantly (Fig. 5 (HPRT), t2g =
2-574, P<0-02) or only slightly (Fig. 1) or it may plateau. A similar drop in
activity is observed at the same time for adenine phosphoribosyltransferase
(APRT, E.C.2.4.2.7; data not shown).
It was noted from the literature that a number of other enzymes that increase
their activity during preimplantation development display a drop in activity (Fig.
5), the troughs coinciding at 72 h post HCG e.g. malate dehydrogenase, fructose
1,6-diphosphate aldolase (MDH, FDA; Epstein, Wegienka & Smith, 1969),
aspartate aminotransferase (AA; Moore & Brinster, 1973), /3-glucuronidase
(Gus h; Chapman, West & Adler, 1977). There is another class of enzymes that
are maternally inherited at relatively higher activities. These enzymes are stable
for 2\ days and then show an exponential decline in activity initiated at 72 h post
HCG (see Fig. 5) e.g. lactic dehydrogenase, glucose 6-phosphate dehydrogenase
(LDH, G6PD; Epstein et al. 1969), phosphoglycerate kinase (PGK; Kozak &
Fig. 5. Enzyme activities during preimplantation development. Except for HPRT
(at top right of Figure) the graphs are redrawn from the literature. MDH - malate
dehydrogenase, FDA - fructose 1,6-diphosphate aldolase, LDH - lactate
dehydrogenase, G6PD - glucose 6-phosphate dehydrogenase (Epstein et al. 1969);
AA - aspartate aminotransferase (Moore & Brinster, 1973); Gush - ^3-glucuronidase
(Chapman et al. 1977); PGK - phosphoglycerate kinase (Kozak & Quinn, 1975).
HPRT translation on maternal mRNA
32
72
112
32
Hours post HCG
23
72
24
M. I. HARPER AND M. MONK
Quinn, 1975). Indeed Spielmann, Erickson & Epstein (1974) have shown by
immunological identification that the lowered activity of both LDH and G6PD
is due to loss of enzyme molecules, and Epstein et al. (1969) have suggested that
specific degradative processes may be involved. However HPRT enzyme is
stable for 12 to 24 h in the presence of cycloheximide up to the late morula stage
(Figs 2 and 3). Even transfer to the drug at 66 h post HCG and 70 h post HCG
(by which time the decline is usually underway) gives no evidence for degradation (data not shown). This apparent contradiction would be resolved if
cycloheximide itself inhibits the process of enzyme degradation leading to the
loss of activity.
Lactic dehydrogenase (LDH) is inherited at high levels by the fertilized egg
and shows striking degradation after 72 h post HCG (Epstein et al. 1969), the
same time as the decrease in HPRT activity. As little or no synthesis of LDH
occurs during preimplantation development we examined this enzyme as a
model system to investigate the effect of cycloheximide on degradation. Fig. 6
shows that unlike the situation with HPRT, the drop in LDH activity is insensitive to cycloheximide, despite embryos being cultured in the drug for 20 h prior
to the decline; by 80 h post HCG both control and experimental samples have the
same lowered activity (tg = 5-9942, P<0-01).
40-
+1
o
X)
E
20-
-/A
60
80
Hours post HCG
100
120
Fig. 6. Effect of cycloheximide on LDH activity. 2- to 4-cell embryos were cultured
in either medium 16 (
• ) or in 10/zg/ml cycloheximide (—O). Embryos were
harvested in groups of five. The differences between treated and untreated samples
after several days incubation are not considered meaningful.
HPRT translation on maternal mRNA
25
DISCUSSION
Translation of HPRT on maternal mRNA
There are several lines of evidence from our laboratory that the increase in
HPRT activity during early cleavage stages of the mouse is maternally derived
(Monk & Kathuria, 1977; Monk & Harper, 1978; Monk, 1978; Burgoyne, Harper & Monk, unpublished). An activity increase is demonstrable in eggs from as
early as 6 h postfertilization when there is very little synthesis of embryonic RNA
(Clegg & Piko, 1977; Piko & Clegg, 1982; Young, Sweeney & Bedford, 1978).
In addition activity increase occurs up to 64 h post HCG in the presence of
a-amanitin but is inhibited by cycloheximide. The most likely and simplest interpretation of these results is that the number of active enzyme molecules increases
due to translation of HPRT on stable maternal message up till the late 2-cell
stage, and, as supported by X-chromosome dosage studies, probably until the
4- to 8-cell stage.
It is conceivable that the protein required to be synthesized is not HPRT, but
some other protein that activates or modifies an HPRT precursor. The results
would then suggest that this 'activator' is translated on maternal message.
The conclusion that the early increase in HPRT activity is due to translation
on stable maternal mRNA is based on the assumption that a-amanitin is an
active inhibitor of mRNA synthesis at this stage. Levey & Brinster (1978) have
shown that 10/ig/ml of a-amanitin inhibits polymerase II activity in the blastocyst within an hour, but no such precise information is available for other
cleavage stages. As a labelled form of the drug is not commercially available the
uptake of a-amanitin has not been tested directly. Nevertheless Braude et al.
(1979) subjected fertilized eggs to a 6h in vitro pulse of a-amanitin treatment
which resulted in retarded development after subsequent culture in control
medium, and Flach et al. (1982) have shown that eggs fertilized in vitro in the
presence of a-amanitin did not later synthesize new polypeptides characteristic
of the late 2-cell stage.
Even in the presence of a-amanitin the activity of HPRT rises at all preimplantation stages (Figs 3 and 4), due to synthesis on stable pre-existing mRNA.
If we assume there is less than a 6 h lag in effectiveness of the drug, and possibly
less than 1 h, it appears that embryonic message is preformed a significant time
before it is translated as there is an interval between exposure to a-amanitin and
the inhibition of a new rate of increase. Our results suggest that embryonic
message for HPRT is synthesized by the late 2-cell or early 4-cell stage. However, because there is a delay between transcription and translation, active
HPRT enzyme is still synthesized on maternal mRNA until the 4- to 8-cell stage.
The delay in translation of embryonic mRNA may be due to the time normally
taken to process the message or be due to active 'masking' and 'demasking' of
the message. Other workers have evidence of delays between transcription and
translation of messages required at blastulation, the lag period ranging from a
26
M. I. HARPER AND M. MONK
few hours (Braude, 1979) to over 24 h (Schindler & Sherman, 1981). The following implications for protein studies in early development are worth noting: (1)
a gene may be actively transcribed long before translation of its product is
identified, and (2) a gene may be switched off while increasing levels of its
product are still being measured (important in studies of X-inactivation).
Transition from maternally to embryonically derived proteins
The present results, and those of Monk & Harper (1978), suggest that HPRT
is translated on maternal mRNA up to the 4- to 8-cell stage, after which the
message is embryonic. Around this proposed time of changeover there is a
decrease in HPRT activity which consistently occurs at 72 h post HCG. The
coincidence in timing of the activity loss of HPRT (and APRT), and the seven
other enzymes shown in Fig. 5, suggests some common mechanism may be
responsible for removal of maternally-inherited enzymes. The decrease in
HPRT activity was not observed in embryos incubated in cycloheximide. The
reason for this is not known. In contrast the marked LDH decline is insensitive
to cycloheximide. It is worth noting that the process of compaction does not
appear to be required for enzyme activity decrease. Culture of early 8-cell embryos in medium without calcium or magnesium to inhibit compaction (Ducibella & Anderson, 1975) did not inhibit the drop in either HPRT or LDH activity
(data not shown).
A consideration of the profile of the HPRT curve in Fig. 1 shows extrapolation
of the known maternal and embryonic contributions to produce hypothetical
curves (broken lines, Fig. 1) based on the assumption that maternal enzyme is
removed. At 64 h post HCG nearly all HPRT enzyme is maternally derived and
at 76 h post HCG all, or nearly all, HPRT enzyme is embryo coded (Monk &
Harper, 1978). Between these two times, we have shown that HPRT enzyme is
no longer translated on maternal mRNA and HPRT activity declines. The composite curve implies that maternally-derived HPRT and its message are specifically removed. With reference to the maternal message the results of Bachvarova
& DeLeon (1980) support such a model. Following an initial postfertilization
decrease, maternally-derived polyadenylated RNA (putative mRNA) remains
stable throughout the second day of gestation in the mouse. After 60 h postfertilization (or 72 h post HCG) the amount of maternal mRNA again declines,
roughly consistent with the time postulated in the composite curve. However this
degradation may not be specific to maternal message. Non-specific degradation
would also serve to 'flush out' maternal enzyme and maternal message as these
have no means of being renewed.
Questions relating to this event are whether the embryo is capable of distinguishing its own gene products from those of its mother and, if so, whether the
latter are removed specifically or not. In this respect preliminary experiments
(Harper, unpublished) have revealed no differences in the Km values for the two
substrates for HPRT made on maternal or embryonic mRNA. Irrespective of the
HPRT translation on maternal mRNA
27
mechanisms involved, it may be important for the embryo to control its differentiation exclusively from its own genome from the time of compaction onwards.
We wish to thank Peter Braude, Paul Burgoyne, Martin Johnson and Dallas Swallow for
stimulating discussion, and Paul Burgoyne and Dallas Swallow for many helpful comments on
the manuscript.
REFERENCES
BACHVAROVA, R. & DELEON, V. (1980). Polyadenylated RNA of mouse ova and the loss of
maternal RNA in early development. Devi Biol. 74, 1-8.
BRAUDE, P. R. (1979). Time dependent effects of a-amanitin on blastocyst formation in the
mouse. /. Embryoi exp. Morph. 52, 193-202.
BRAUDE, P. R., PELHAM, H., FLACH, G. & LOBATTO, R. (1979). Post-transcriptional control
in the early mouse embryo. Nature 282, 102-105.
BRINSTER, R. L. (1965). Lactate dehydrogenase activity in the preimplanted mouse embryo.
Biochim. Biophys. Acta 110, 439-441.
CASCIO, S. M. & WASSARMAN, P. M. (1982). Program of early development in the mammal:
post transcriptional control of a class of proteins synthesized by mouse oocytes and early
embryos. Devi Biol. 89, 397-408.
CHAPMAN, V. M.,WEST, J. D. & ADLER, D. A. (1977). Genetics of early mammalian development. In 'Concepts in Mammalian Embryogenesis' (ed. M. I. Sherman), pp. 95-135. Cambridge, Mass.: M.I.T. Press.
CLEGG, K. P. & PIK6, L. (1977). Size and specific activity of the UTP pool and overall rates
of RNA synthesis in early mouse embryos. Devi Biol. 58, 76-95.
CLEGG, K. B. & PIK6, L. (1982). RNA synthesis and cytoplasmic polyadenylation in the onecell mouse embryo. Nature 295, 342-345.
DAVIDSON, E. H. (1976). Gene Activity in Early Development. 2nd Ed. New York: Academic
Press.
DUCIBELLA, T. & ANDERSON, E. (1975). Cell shape and membrane changes in the eight-cell
mouse embryo: prerequisites for morphogenesis of the blastocyst. Devi Biol. 47, 45-58.
EPSTEIN, C. J. (1972). Expression of the mammalian X chromosome before and after fertilization. Science 175, 1467-1468.
EPSTEIN, C. J. & SMITH, S. A. (1973). Amino acid uptake and protein synthesis in preimplantation mouse embryos. Devi Biol. 33, 171-184.
EPSTEIN, C. J., SMITH, S., TRAVIS, B. & TUCKER, G. (1978). Both X chromosomes function
before visible X-chromosome inactivation in female mouse embryos. Nature 274, 500-503.
EPSTEIN, C. J., WEGIENKA, E. A. & SMITH, C. W. (1969). Biochemical development of preimplantation mouse embryos: In vivo activities of fructose-l,6-diphosphate aldolase, glucose
6-phosphate dehydrogenase, malate dehydrogenase, and lactate dehydrogenase. Biochem.
Genet. 3, 271-281.
FLACH, G., JOHNSON, M. H., BRAUDE, P. R., TAYLOR, R. A. S. & BOLTON, V. (1982). The
transition from maternal to embryonic control in 2-cell mouse embryo. Embo Journal, 1,
681-686.
GATES, A. H. &BEATTY, R. A. (1954). Independence of delayed fertilization and spontaneous
triploidy in mouse embryos. Nature 174, 356-357.
GOLBUS, M. S., CALARCO, P. G. & EPSTEIN, C. J. (1973). The effects of inhibitors of RNA
synthesis (a-amanitin and actinomycin D) on preimplantation mouse embryogenesis. J.
exp. Zool. 186, 207-216.
GROSS, P. R. & COUSINEAU, G. H. (1964). Macromolecule synthesis and the influence of
actinomycin on early development. Expl Cell Res. 33, 368-395.
JOHNSON, M. H. (1981). The molecular and cellular basis of preimplantation mouse development. Biol. Rev. 56, 463-498.
28
M. I. HARPER AND M. MONK
L. P. & QUINN, P. J. (1975). Evidence for dosage compensation of an X-linked gene
in the 6-day embryo of the mouse. Devi Biol. 45, 65-73.
KRATZER, P. G. & GARTLER, S. M. (1978). HGPRT activity changes in preimplantation mouse
embryos. Nature 21<\, 503-504.
LEVEY, I. L. & BRINSTER, R. L. (1978). Effects of a-amanitin on RNA synthesis by mouse
embryos in culture. J. exp. Zool. 203, 351-359.
LEVEY, I. L.,STULL, G. B. & BRINSTER, R. L. (1978). Poly(A) and synthesis of polyadenylated
RNA in the preimplantation mouse embryo. Devi Biol. 64, 140-148.
LINDELL, T. J., WEINBERG, F., MORRIS, P. W., ROEDER, R. G. & RUTTER, W. J. (1970).
Specific inhibition of nuclear RNA polymerase II by a-amanitin. Science 170, 447-449.
MAGNUSON, T. & EPSTEIN, C. J. (1981). Genetic control of very early mammalian development. Biol. Rev. 56, 369-408.
MCLAREN, A. (1976). Genetics of the early mouse embryo. Ann. Rev. Genet. 10, 361-388.
MONESI, V., MOLINARO, M., SPALLETTA, E. & DAVOLI, C. (1970). Effect of metabolic inhibitors on macromolecular synthesis and early development in the mouse embryo. Expl
Cell Res. 59, 197-206.
MONK, M. (1978). Biochemical studies on mammalian X-chromosome activity. In: Development in Mammals (ed. M. H. Johnson), vol. 3, pp. 189-223. Amsterdam: North-Holland.
MONK, M. & HARPER, M. I. (1978). X-chromosome activity in preimplantation mouse embryos from XX and XO mothers. /. Embryol. exp. Morph. 46, 53-64.
MONK, M. & KATHURIA, H. (1977). Dosage compensation for an X-linked gene in preimplantation mouse embryos. Nature 270, 599-601.
MOORE, R. W. & BRINSTER, R. L. (1973). Activity of mitochondrial enzymes in mouse embryos. /. Reprod. Fert. 35, 37-43.
PIK6, L. & CLEGG, K. B. (1982). Quantitative changes in total RNA, total poly (A), and
ribosomes in early mouse embryos. Devi Biol. 89, 362-378.
SCHINDLER, J. & SHERMAN, M. I. (1981). Effects of a-amanitin on programming of blastocyst
development. Devi Biol. 84, 332-340.
SCHULTZ, G. A. (1975). Polyadenylic acid-containing RNA in unfertilized and fertilized eggs
of the rabbit. Devi Biol. 44, 270-277.
SPIELMANN, H., ERICKSON, R. P. & EPSTEIN, C. J. (1974). Immunochemical studies of lactate
dehydrogenase and glucose-6-phosphate dehydrogenase in preimplantation mouse embryos. J. Reprod. Fert. 40, 367-373.
WHITTINGHAM, D. G. (1971). Culture of mouse ova. /. Reprod. Fert., Suppl. 14, 7-21.
WHITTINGHAM, D. G. & WALES, R. G. (1969). Storage of two-cell mouse embryos in vitro.
Aust. J. biol. Sci. 22, 1065-1068.
YOUNG, R. J., SWEENEY, K. & BEDFORD, J. M. (1978). Uridine and guanosine incorporation
by the mouse one-cell embryo. /. Embryol. exp. Morph. 44, 133-148.
KOZAK,
(Accepted 26 November 1982)