J. Embryol. exp. Morph. 74, 169-182 (1983)
Printed in Great Britain © The Company of Biologists Limited 1983
Quantitative aspects of RNA synthesis and
polyadenylation in 1-cell and 2-cell mouse embryos
By KERRY B. CLEGG 1 AND LAJOS PIKO 1
From the Developmental Biology Laboratory, Veterans Administration
Medical Center, Sepulveda, and the Division of Biology, California Institute of
Technology
SUMMARY
Mouse embryos at the late 1-cell and late 2-cell stages were labelled with [3H]adenosine for
periods of up to 320 min during which the specific activity of the ATP pool was constant. The
time course of the molar accumulation of adenosine was calculated for tRNA, high-molecularweight poly(A)— RNA and poly(A) tails versus internal regions of poly(A)+ RNA. Most of
the adenosine incorporation into tRNA is due to turnover of the 3'-terminal AMP but some
new synthesis of tRNA also appears to take place in both 1-cell and 2-cell embryos at a rate
of about 0-2pg/embryo/h. In the poly(A)— RNA fraction, an unstable component which is
assumed to be heterogeneous nuclear RNA is synthesized at a high rate and accumulates at
a steady-state level of about 1-5 pg/embryo in the 1-cell embryo and about 3-0pg/embryo in
the 2-cell embryo. Both 1-cell and 2-cell embryos synthesize relatively stable heterogeneous
poly(A)- RNA, assumed to be mRNA, at a rate of about 0-3 pg/embryo/h; 2-cell embryos
also synthesize mature ribosomal RNA at a rate of about 0-4 pg/embryo/h. Internally labelled
poly(A)+ RNA is synthesized at a low rate in the 1-cell embryo, about 0-045 pg/embryo/h,
but the rate increases to about 0-2 pg/embryo/h by the 2-cell stage. A striking feature of the
1-cell embryo is the high rate of synthesis of poly(A) tails, about 2-5 x 106 tails/embryo /h of
an average length of (A)43, due almost entirely to cytoplasmic polyadenylation. This and other
evidence suggests a turnover of the poly(A)+ RNA population in 1-cell embryos as a result
of polyadenylation of new RNA sequences and degradation of some of the pre-existing
poly(A)+ RNA. In the 2-cell embryo, the rate of synthesis of poly(A) tails (average length
(A)93) is estimated at about 0-8 x 106tails/embryo/h and a significant fraction of poly(A)
synthesis appears to be nuclear.
INTRODUCTION
The ovulated mouse egg inherits about 0-35 ng maternal RNA (Piko & Clegg,
1982), most of which is synthesized during the growth phase of the oocyte (Bachvarova, 1974,1981; Bachvarova & De Leon, 1980; Jahn, Baran & Bachvarova,
1976; Brower, Gizang, Boreen & Schultz, 1981). The distribution of label in
ovulated eggs that had been pulse labelled with [3H]uridine or [3H]adenosine
during oogenesis suggests that 65-70 % of the total RNA is ribosomal, about
20 % is 4S and 5S RNA and 10-15 % is heterogeneous RNA (Bachvarova, 1974;
1
Authors' address: Developmental Biology Laboratory (151B3), Veterans Administration
Medical Center, Sepulveda, California 91343, U.S.A.
170
K. B. CLEGG AND L. PIK6
Jahn et al. 1976). A striking feature of early mouse development is that 30-40 %
of the bulk maternal RNA (Bachvarova & De Leon, 1980; Olds, Stern & Biggers,
1973; Piko & Clegg, 1982) and as much as 70 % of the poly(A)+ RNA (Levey,
Stull & Brinster, 1978; Piko & Clegg, 1982) is degraded by the 2-cell stage, within
about 24 h of fertilization. Total RNA and total poly (A) content increases by
about sixfold between the 2-cell and early blastocyst stages due to a high rate of
RNA synthesis in the embryo (Clegg & Piko, 1977; Levey et al. 1978; Piko &
Clegg, 1982), resulting in a nearly complete replacement of the maternal RNA
by embryo-derived RNA components by the early blastocyst stage. A major
change in the qualitative pattern of protein synthesis during the 2- to 8-cell stage
and the early expression of paternal gene products also suggest a transition from
maternal to embryonic genetic control beginning with the 2-cell stage (see
reviews by Johnson, 1981 and Magnusson & Epstein, 1981).
Using [3H]adenosine as a labelled precursor, we have shown recently that
RNA transcription has already commenced in the pronuclei of the mouse zygote.
Large heterodisperse poly(A)- RNA was identified as the major product. The
evidence also suggested the synthesis of small amounts of transfer RNA and
poly(A)+ RNA but no detectable synthesis of ribosomal RNA at this stage. A
significant amount of label was recovered in poly(A) sequences as a result of
cytoplasmic adenylation of stored maternal RNA. The synthesis of all the major
classes of RNA, including ribosomal RNA, was evident in the 2-cell embryo
(Clegg & Piko, 1982). In order to obtain information on the rates of synthesis and
stability of the RNA products in 1-cell and 2-cell mouse embryos, we have
studied the time course of incorporation of [3H]adenosine into different RNA
fractions under conditions of constant specific activity of the [3H]ATP pool.
MATERIALS AND METHODS
Embryo culture and radioisotope labelling
The general procedures were those described by Piko & Clegg (1982).
Prepubertal female Swiss albino mice (Charles River) were superovulated and
allowed to mate. Embryos were recovered at the following intervals after injection of human chorionic gonadotropin: 22-24 h for 1-cell fertilized eggs and
46-48 h for 2-cell embryos. In 1-cell embryos the follicle cells were carefully
removed by successive treatments with hyaluronidase and 0-25 % subtilisin. The
embryos were set up for culture in the same Petri dish in four droplets (containing
about 150 embryos each) of Brinster's pyruvate-lactate medium (PLM) under
mineral oil. After 1 h of culture, the culture medium was exchanged with prewarmed PLM containing 50/iM [2,8-3H]adenosine (37Ci/mM, Amersham) and
incubation continued for 40min. The droplets were then washed free of label
with six changes of PLM (over a period of about 5 min); at this time, from one
droplet five embryos were taken out for the measurement of the specific activity
RNA synthesis and polyadenylation in mouse embryos
111
of the ATP pool and the remaining embryos transferred into 300 [A RNA extraction buffer (see below). The other three groups of embryos continued to be
incubated in the absence of label for additional periods of 40,120 and 280 min,
respectively, at which time they were sampled and removed as above. For both
1-cell and 2-cell embryos, the labelling period involved the latter part of the cell
cycle (the G2 phase; Sawicki, Abramczuk & Blaton, 1978) and ended 2-4h
before the impending cleavage division. In our conditions, the median time of
first cleavage is about 33 h and that of second cleavage about 55 h after HCG
injection (unpublished data).
Assay of [3H]ATP pool
The nucleotides in the embryo samples were extracted with 0-5 M-HCIO4 and
the [3H]ATP in the extract fractionated by chromatography on polyethyleneimine (PEI) cellulose as described (Clegg & Piko, 1977, 1982). The specific
activity of the [3H] ATP pool was derived by adding the amount of [3H] ATP per
embryo to the endogenous ATP pool (1-lpmole per embryo; Clegg & Piko,
1977) to obtain the total ATP per embryo and expressing the [3H]ATP as a
percentage of the total ATP. In some experiments, the specific activity of the
ATP pool was measured directly in a portion of the PCA-soluble extract using
an RNA polymerase assay (Clegg & Piko, 1977). The values obtained by the two
methods were within 10 % of each other.
Fractionation of labelled RNA
The RNA extraction buffer contained 100 mM-NaCl, 50 mM-Tris-HCl, pH 7-5,
5mM-EDTA, 0-5% SDS, 100/ig/ml proteinase K, carrier RNAs (mouse
ribosomal RNA and yeast soluble RNA of about 40jUg/ml each) and about
300 c.p.m. of 14C-labelled 18S ribosomal RNA as recovery marker. The procedures for the extraction and fractionation of the three major RNA species were
essentially the same as described previously (Clegg & Piko, 1982). The embryo
lysates were digested at 37 °C for 30 min and high-molecular-weight RNA precipitated with 2 M-LiCl. The LiCl-precipitable RNA was separated into poly (A) - and
poly(A)+ fractions by binding to poly(U)-Sepharose (Pharmacia). The
poly ( A ) - RNA fraction was counted directly in PCS scintillation fluid (Amersham) in a Beckman LS-355 scintillation counter at efficiencies of 35 % for 3 H,
64 % for 14C and 40 % spillover of the 14C counts into the 3H-channel. Routinely,
over 90 % of the 14C-labelled 18S RNA marker was recovered in this fraction.
After elution from the poly(U)-Sepharose, the poly(A)+ RNA was ethanol
precipitated, redissolved in 200[A of 10/ig/ml RNase A and 5i.u./ml RNase
Ti in 0-5M-NaCl, 0-05M-Tris-HCl, 0-005M-EDTA, pH7-5, and incubated at
37 °C for 1 h. The sample was chilled and 100 fig of carrier RNA and an equal
volume of ice-cold 20 % TCA were added. The precipitate was washed twice
with cold 10 % TCA, dissolved in 90 % NCS and counted in OCS scintillant
(Amersham) to measure the radioactivity incorporated into the poly (A) tails.
172
K. B. CLEGG AND L. PIK6
The TCA-soluble fraction was counted directly in PCS to measure [3H]adenosine
residues released from the non-poly(A) portion of poly(A)+ RNA. The LiClsoluble fraction was ethanol precipitated (after the addition of about 300 cpm of
14
C-labelled 4S RNA marker), redissolved in 0-lM-NaOAc, pH5,
reprecipitated with 0-5 % cetyltrimethylammonium bromide (CTAB), washed
several times in 0-1% CTAB until the radioactivity in the washfluid was at
background levels, dissolved in 0-5 ml 90 % NCS and counted. The recovery of
marker RNA was better than 90 % in each case. The incorporation data given
are uncorrected for recovery.
RESULTS
Specific activity of the [3H]A TP pool
When 1-cell embryos are incubated in the presence of [3H]adenosine for longer periods of time, the specific activity of the total cellular ATP pool increases
approximately linearly during the first 80min and continues to increase at a
slower rate for up to 5-6 h of labelling (Clegg & Piko, 1982; Fig. 1). The strategy
25 -
20
2 15
o
Z-~a-
40
80
8-
160
Time of incubation (min)
8
320
Fig. 1. Specific activity of the [3H]ATP pool in 1-cell (O) and 2-cell (•) embryos.
After an initial incubation of 40 min in the presence of 50/<tM-[2,8-3H]adenosine
(37Ci/mM), the label was removed and incubation continued in PLM only. At the
indicated times, the amount of [3H]ATP contained in a sample offiveembryos was
determined and the specific activity of the ATP pool calculated as described in
Materials and Methods. Each dot represents the average of two determinations; the
broken line gives the bestfittingregression line to the data. For comparison, the solid
line shows the changes in the specific activity of the ATP pool when 1-cell embryos
are cultured in the continued presence of 50jUM-[3H]adenosine (from Clegg & Pik6,
1982).
RNA synthesis and polyadenylation in mouse embryos
173
in the present series of experiments was to pulse label the embryos with
[3H]adenosine for 40min and then remove the label from the culture medium,
in order to keep the specific activity of the cellular ATP pool at a constant level.
This has turned out to be the case as the [3H]ATP concentration remained at a
level of about 7-8 % for at least up to 5 h following the initial [3H]adenosine pulse
(Fig. 1). It has been assumed in these experiments that the [3H]ATP equilibrates
relatively readily with the endogenous ATP and that, therefore, the specific
activity of the ATP pool available for RNA synthesis is the same as that derived
for total cellular ATP. We have explored the possibility that the mitochondria
store a substantial amount of ATP which does not exchange readily with the
cytoplasm. A mitochondrial supernatant fraction (Piko & Chase, 1973) was
prepared from 1-cell embryos 2 h after a 40-min pulse with [3H]adenosine and the
specific activity of this supernatant was compared with that of a total embryo
extract using an RNA polymerase assay (Clegg & Pik6,1977). The two specific
activities were about the same suggesting that no significant compartmentalization of ATP exists in the mitochondria. However, some evidence has been
obtained that after short incubation periods (40min) in the presence of
[3H]adenosine the nuclear ATP pool becomes preferentially labelled (see
below). For the calculation of the rates of adenosine incorporation into different
RNA species, the specific activity of the total cellular ATP pool was used in every
case without any correction.
Characterization of the RNA fractions
The fractionation procedures used in this study yielded three RNA fractions
on the basis of precipitability with LiCl and binding to poly(U)-Sepharose. RNA
fractions isolated by the same procedures from 1-cell and 2-cell mouse embryos
have been characterized previously (Clegg & Piko, 1982) as to electrophoretic
migration and sensitivity to RNase, and are briefly as follows: (1) LiCl-soluble
RNA migrates as a sharp 4S band which is fully sensitive to RNase and is
assumed to be tRNA; (2) LiCl-precipitable, poly ( A ) - fraction is also fully
RNase sensitive and consists of large heterodisperse (about 10-60S) RNA
species in 1-cell embryos; in 2-cell embryos, this fraction also contains 18S and
28S ribosomal RNA as well as ribosomal RNA precursor species; (3) poly(A)+
fraction consists of high-molecular-weight RNA of about 12-35S with a numberaverage size of about 2 kilobases in both 1-cell and 2-cell embryos. In the present
study, the poly(A)+ RNA fraction has been subdivided further into two fractions on the basis of sensitivity to RNase A + T i , in order to distinguish between
adenosine incorporation into the poly(A) tail versus internal locations in the
molecule.
The data obtained on the overall incorporation of adenosine into the different
RNA fractions in two series of experiments are summarized in Table 1 and
illustrated diagrammatically in Figs 2-5. The quantitative aspects of adenosine
incorporation into the individual fractions are examined below.
EMB74
174
K. B. CLEGG AND L. PIK6
Table 1. Adenosine incorporation into different classes ofRNA in 1-cell and 2-cell
mouse embryos*
Length of incubation
femtomoles adenosine incorporated per embryo into
High m.w.
Poly(A)+ RNA
tRNA
poly(A)- RNA Poly(A) tail
Internal
1-cell embryos
40min
80min
160 min
320 min
0-85 ± 0-46
1-11 ±0-46
2-24 ±0-21
3-47 ± 0-43
2-0710-73
1-14 ±0-43
1-72 ±0-62
3-3210-93
0-1110-01
0-2010-06
0-4610-03
0-9610-04
0-1210-03
0-1310-01
0-201 0-03
0-2810-07
2-cell embryos
40 min
80 min
160 min
320 min
0-61 ±0-14
0-92 ± 0-20
1-24 ±0-19
1-77 ±0-30
1-9610-28
2-5510-05
3-3310-07
4-55 ±0-35
0-1210-02
0-25 10-05
0-4010-07
0-66 ±0-03
0-23 10-03
0-3610-01
0-5610-12
0-93 ±0-15
* The data show the average and the standard error of the mean of two separate experiments. For fractionation of RNA see Materials and Methods. The molar accumulation of
adenosine was calculated from the amount of radioactivity recovered in the individual fractions and the specific activity of the total cellular ATP pool monitored at the end of each
incubation period.
Adenosine incorporation into tRNA
A major fraction of the total adenosine incorporation is found in tRNA in both
1-cell and 2-cell embryos (Table 1). The time course of incorporation (Fig. 2)
indicates that both incorporation and turnover of adenosine take place over a 5 h
period. The 4S RNA obtained after 320 min of labelling was isolated by
electrophoresis in 10% poly aery lamide gel, eluted from the gel slice and
analysed for the distribution of label in the nucleotides by PEI cellulose
chromatography after alkaline hydrolysis. In 1-cell embryos, 83 % of the label
was in adenosine and 17 % was in AMP; in 2-cell embryos, 70 % was in adenosine
and 30 % in AMP. These results indicate that the bulk of adenosine is incorporated into the - C C A terminus of tRNA, presumably because of turnover of
the 3'-terminal adenosine residue (Deutscher, 1973). However, a small portion
of adenosine is incorporated into internal locations, suggesting new synthesis of
tRNA. From the amount of adenosine incorporated internally during 320 min
and using an average content of 14 adenosine residues per tRNA (Randerath,
Chia, Morris & Randerath, 1974), the rate of synthesis of tRNA can be estimated
at about 0-2pg/embryo/h in both 1-cell and 2-cell embryos.
Adenosine incorporation into high-molecular-weight poly (A)- RNA
High-molecular-weight poly (A)— RNA is the major product of RNA
synthesis in the 1-cell embryo and is by far the most abundant RNA fraction
RNA synthesis and polyadenylation in mouse embryos
175
synthesized in the 2-cell embryo (Table 1; Fig. 3). The time course of adenosine
incorporation in the 1-cell embryo reveals a complex pattern inasmuch as the
apparent rate of adenosine incorporation is very high during the first 40min
(during the period of pulse labelling) but it drops substantially during the 40 min
following the removal of [3H]adenosine from the medium. As the initial
adenosine incorporation rates appear to be consistently elevated for nuclear
RNA species (large poly ( A ) - RNA and the non-poly (A) portion of poly(A) +
RNA) in both 1-cell and 2-cell embryos, the simplest explanation seems to be
that the specific activity of the nuclear ATP pool during the pulse labelling is
higher (about two times) than the specific activity derived for the total cellular
pool. Upon removal of the [3H]adenosine from the medium, the cellular ATP
pool would equilibrate relatively rapidly, resulting in a drop in the specific activity of the nuclear ATP pool. Under these conditions, the decrease in the amount
of label recovered in poly(A)- RNA in 1-cell embryos at the 80-min time point
would indicate that a substantial portion of this RNA is unstable and is turning
over rapidly. A rapid turnover would be consistent with the presence of
heterogeneous nuclear RNA in this fraction as suggested by the electrophoretic
pattern (Clegg & Piko, 1982). Nevertheless, the time course of incorporation
during 80-320 min of incubation suggests that relatively stable poly (A)— RNA
species are also synthesized in the 1-cell embryo. From the amount of adenosine
incorporated during the last 160 min of incubation, the rate of synthesis of this
stable RNA fraction can be estimated at about 0-3 pg/embryo/h (assuming a
25 % content in adenosine). From this rate of synthesis, of the total poly(A)RNA accumulated in the 1-cell embryo over 320 min of incubation, or about
tRNA
1-cell
2 2
2-cell
o
I
40
80
160
Time of incubation (min)
320
Fig. 2. Time course of adenosine incorporation into tRNA in 1-cell (O) and 2-cell
(•) embryos (see also Table 1).
176
K. B. CLEGG AND L. PIK6
Large poly(A)- RNA
2-cell
I
3
40
80
160
Time of incubation (min)
320
Fig. 3. Time course of adenosine incorporation into large heterodisperse poly(A)RNA in 1-cell (O) and 2-cell (•) embryos (see also Table 1).
3 pg, about one-half is stable RNA and the other half represents the equilibrium
level of unstable RNA.
In the 2-cell embryo, the time course of incorporation of adenosine into
poly ( A ) - RNA indicates that both unstable as well as relatively stable RNA
species are synthesized (Fig. 3). As judged from the electrophoretic pattern of
this RNA (Clegg & Piko, 1982), about 25 % of the adenosine incorporated over
a period of 320 min is localized in mature ribosomal RNA which corresponds to
a rate of ribosomal RNA synthesis of about 0-4pg/embryo/h (using an 18 %
adenosine content for ribosomal RNA). This rate of ribosomal RNA synthesis
accounts for only about one half of the increment of adenosine incorporated
during the period of 160-320 min of incubation, suggesting the synthesis of some
stable RNA species other than ribosomal RNA which would proceed at a rate
of about 0-3 pg/embryo/h (assuming a 25 % adenosine content). At the above
rates of adenosine incorporation, the synthesis of stable RNA accounts for about
half of the total adenosine incorporated over 320min of incubation, and the
other half of incorporation can be taken to represent the steady-state level of
unstable heterodisperse RNA.
Adenosine incorporation into poly (A) + RNA
Figure 4 illustrates the time course of incorporation of adenosine into the
poly (A) tail of poly(A)+ RNA. The rates of incorporation are about the same
at the 1-cell and 2-cell stages. In the 1-cell embryo, the incorporation of
RNA synthesis and polyadenylation in mouse embryos
177
10
poly(A)+ RN A/poly (A) tail
X)
^u 6
c
'35
o
c
<u
U
"o
Q.
40
80
160
Time of incubation (min)
320
Fig. 4. Time course of adenosine incorporation into the poly(A) tail of poly(A) +
RNA in 1-cell (O) and 2-cell (•) embryos (see also Table 1).
adenosine proceeds approximately linearly for 320 min, with an average rate of
about 0-18 x 10~3 pmoles/embryo/h; this corresponds to a rate of synthesis per
hour of about 2-5 x 106 poly (A) tails of an average length of (A)43 (Clegg & Piko,
1982). In the 2-cell embryo, the rate of incorporation appears to level off
somewhat after 80 min of incubation, suggesting some turnover of the label in the
poly (A) tails. The amount of adenosine accumulated in poly (A) during 320 min
of incubation would correspond to the synthesis of about 4-2 xlO 6 poly (A) tails
of an average length of (A)93 (Clegg & Piko, 1983) during this period.
The time course of incorporation into the non-poly(A) portion of poly(A) +
RNA is shown in Fig. 5. In the 1-cell embryo, incorporation of adenosine into
this RNA fraction proceeds at a low rate: for the period of incubation from
40-320 min, when the specific activity of the nuclear ATP pool is assumed to be
constant, the rate of adenosine incorporation is about 3-4 x 10~5 pmoles/embryo/h which corresponds to a rate of RNA synthesis of about 0-045 pg/h (assuming an adenosine content of 25 %). Considering the same period of incubation, the rate of adenosine incorporation in 2-cell embryos is about four times
higher, 1-5 x 10~4 pmoles/embryo/h, corresponding to a rate of RNA synthesis
of about 0-2pg/embryo/h.
DISCUSSION
3
[ H] Adenosine was used as a precursor in these experiments because it is taken
up at a very high rate and is converted efficiently into ATP (which represents
about 90 % of the acid-soluble radioactivity) by 1-cell and 2-cell mouse embryos
178
K. B. CLEGG AND L. PIK6
10 -
poly(A) + RNA/internal
>^
<u 6 -
2-cell
s"
4 -
1-ccll
--°
2 -
40
80
160
Time of incubation (min)
320
Fig. 5. Time course of adenosine incorporation into the internal regions of
poly(A)+ RNA in 1-cell (O) and 2-cell (•) embryos (see also Table 1).
(Clegg & Piko, 1977). The specific activity of the total cellular ATP pool reached
7-8 % at the end of the initial 40-min pulse labelling and remained at this level
after removal of the label throughout the time-course studies (Fig. 1). Although
there is evidence of preferential labelling of the nuclear pool during the initial
pulse, we assume that during the succeeding labelling period the total cellular
pool has been in equilibrium, without major compartmentalization of ATP.
Evidence suggesting that in cleavage-stage mouse embryos the intracellular UTP
and ATP pools equilibrate relatively rapidly has been discussed (Clegg & Pik6,
1977).
The RNA fractions obtained in the present work have been characterized
previously (Clegg & Piko, 1982) and consist of 4S RNA (assumed to be tRNA),
high-molecular-weight poly(A)- RNA and poly(A)+ RNA. A major
qualitative difference between the 1-cell and 2-cell stages is the presence of label
in ribosomal RNA species (isolated in the poly(A)- RNA fraction) in 2-cell
embryos whereas there is no detectable synthesis of ribosomal RNA in 1-cell
embryos. Essentially all the incorporation of label could be attributed to the
synthetic activity of the embryo proper, without significant contribution by the
mitochondria or the polar bodies (Clegg & Piko, 1982).
In the tRNA fraction, the bulk of the label was localized in the - C C A terminus, but the minor fraction of label (about 17 % in 1-cell and 30 % in 2-cell
embryos) recovered from internal locations suggests some new synthesis of
tRNA as early as the 1-cell stage. Previously the earliest evidence of incorporation of [3H]uridine label into 4S RNA was obtained in the 2-cell mouse embryo
(Knowland & Graham, 1972). Our data suggest a rate of tRNA synthesis of
RNA synthesis and polyadenylation in mouse embryos
179
about 0-2 pg/embryo/h. After adjustment is made for this stable component,
the remaining incorporation into the —CCA termini appears to approach
equilibrium at the end of 320 min of incubation. This suggests a rate of turnover
comparable to that observed in rabbit reticulocytes in which the time needed for
complete turnover of the 3'-terminal AMP was estimated at 4-8 h (Holt, Joel &
Herbert, 1966). The reason for the about two times higher overall level of
incorporation of adenosine in 1-cell versus 2-cell embryos is not clear. It could
be due in part to a reduction in tRNA content at the 2-cell stage as has been found
for total RNA (Bachvarova & De Leon, 1980; Olds et al. 1973; Piko & Clegg,
1982).
The time course of incorporation into high-molecular-weight heterodisperse
poly(A)— RNA in the 1-cell embryo suggests the synthesis of an unstable component and some relatively stable RNA species (Fig. 3). The stable RNA fraction which may consist of poly ( A ) - mRNA (e.g., histone mRNA) accumulates
at a rate of about 0-3 pg/embryo/h. The steady-state level of the unstable RNA,
assumed to be heterogeneous nuclear RNA, is estimated at about l-5pg/embryo. This value is similar to the content of heterogeneous nuclear RNA in
mouse L cells, about 1-8 pg (Brandhorst & McConkey, 1974), but ten times less
than that accumulated in growing mouse oocytes, about 15pg/nucleus (Bachvarova, 1981). From the steady-state level of 1-5 pg and assuming a t\ of between
10 min (an estimate derived in mouse blastocysts; Piko, 1970) and 20 min (mouse
L cells; Brandhorst & McConkey, 1974), the instantaneous rate of synthesis of
heterogeneous nuclear RNA in the 1-cell embryo can be estimated at
0-05-0-1 pg/min (calculated from Eqs. 5.2 and 5.4 of Davidson, 1976) or
10-20x the rate of accumulation of the stable RNA fraction described above.
In the 2-cell embryo, the time course of accumulation of adenosine in
poly ( A ) - RNA also suggests the synthesis of stable and unstable RNA components (Fig. 3). Two stable RNA species appear to be synthesized: mature
ribosomal RNA at a rate of about 0-4 pg/embryo/h and stable heterogeneous
poly(A)- RNA, presumably mRNA, at a rate of about 0-3 pg/embryo/h. The
amount of the unstable component, assumed to consist almost entirely of
heterogeneous nuclear RNA, is about 3-0pg/embryo or l-5pg/cell (assuming
an adenosine content of 25 % ) .
The high rate of incorporation of adenosine into poly(A) tails versus internal
locations in the 1-cell embryo confirms the previous finding that an active
cytoplasmic polyadenylation takes place at this stage (Clegg & Piko, 1982). From
an approximately 20 % increase in poly(A) content and some shortening in the
average length of the poly (A) tracts, we have estimated that the number of
poly(A)+ RNA molecules increases by about 40 %, from 1-7 x 107 to 2-4 x 107
molecules, between fertilization and the late 1-cell stage (Clegg & Piko, 1983).
The rate of synthesis of poly(A) tails, about 2-5 x 106 tails/embryo/h, derived
in the present study would lead to a much greater increase in the number of
poly(A)+ RNA even if extensive cytoplasmic polyadenylation may be restricted
180
K. B. CLEGG AND L. PIK6
only to the latter part of the 1-cell stage (see Clegg & Piko, 1982). This
discrepancy suggests that cytoplasmic adenylation as well as degradation of
poly(A), or poly(A)+ RNA, take place concurrently. However, the
approximately linear rate of incorporation of adenosine into poly (A) (Fig. 4) and
the distinctly different electrophoretic pattern of newly labelled poly(A) versus
total poly(A) in the 1-cell mouse embryo (Clegg & Piko, 1983) argue against a
general turnover of poly (A) tails as observed in sea urchin eggs (Wilt, 1977;
Dolecki, Duncan & Humphreys, 1977). The data thus suggest a turnover in the
poly(A)+ RNA population itself, due to polyadenylation of previously nonpolyadenylated stored RNA and degradation of some of the pre-existing
poly (A)-I- RNA, resulting in an overall net increase in poly (A) and the number
of poly(A) + RNA molecules. Such a turnover could lead to changes in the
relative amounts of individual mRNAs which would be consistent with the observed quantitative changes (both increase and decrease) in the synthesis of individual proteins following fertilization (Chen, Brinster & Merz, 1980).
Cytoplasmic polyadenylation may also be involved in the reported selective
utilization of some of the stored maternal mRNA (Braude, Pelham, Flach &
Lobatto, 1979; Cullen, Emigholz & Monahan, 1980; Petzoldt etal. 1981; Van
Blerkom, 1981; Cascio & Wassarman, 1982).
The incorporation of adenosine into internal locations suggests new synthesis
of a small fraction of stable poly(A)+ RNA in the 1-cell embryo, at a rate of
about 0-045 pg/h; this corresponds to the synthesis of about 5 x 104 poly(A) +
RNA molecules of an average size of 1-7 kilobases (Clegg & Piko, 1982) or about
2 % of the number of poly(A) tails synthesized per hour. The rate of synthesis
of poly(A)+ RNA increases to about 0-2 pg RNA or about 2 x 105 molecules
synthesized per hour in the 2-cell embryo. Presumably an equal number of
poly(A) tails or about 25 % of the total poly(A) synthesized is also derived from
nuclear synthesis. That significant nuclear synthesis of poly (A) occurs in the
2-cell embryo is supported by the observation that the nuclei become heavily
labelled when embryo sections are hybridized with [3H]poly(U) in situ; the same
procedure results in very little label detectable in the pronuclei of 1-cell embryos
thus providing further evidence that most if not all of the poly (A) synthesis at this
stage is cytoplasmic (Piko & Clegg, 1982).
This work was supported by the Medical Research Service of the Veterans Administration
and by PHS research grant CA24989 from the NCI.
REFERENCES
R. (1974). Incorporation of tritiated adenosine into mouse ovum RNA. Devi
Biol. 40, 52-58.
BACHVAROVA, R. (1981). Synthesis, turnover, and stability of heterogeneous RNA in growing
mouse oocytes. Devi Biol. 86, 384-392.
BACHVAROVA, R. & DE LEON, V. (1980). Polyadenylated RNA of mouse ova and loss of
maternal RNA in early development. Devi Biol. 74, 1-8.
BACHVAROVA,
RNA synthesis and polyadenylation in mouse embryos
181
BRANDHORST, B. P. &MCCONKEY,E. H. (1974). Stability of nuclear RNA in mammalian cells.
J. mol. Biol. 85, 451-463.
BRAUDE, P., PELHAM, H., FLACH, G. & LOBATTO, R. (1979). Post-transcriptional control in the
early mouse embryo. Nature, Lond. 282, 102-105.
BROWER, P. T., GIZANG, E., BOREEN, S. M. & SCHULTZ, R. M. (1981). Biochemical studies
of mammalian oogenesis: synthesis and stability of various classes of RNA during growth
of the mouse oocyte in vitro. Devi Biol. 86, 373-383.
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.
CHEN, H. Y., BRINSTER, R. L. & MERZ, E. A. (1980). Changes in protein synthesis following
fertilization of the mouse ovum. /. exp. Zool. 212, 355-360.
CLEGG, K. B. & 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, Lond. 295, 342-345.
CLEGG, K. B. & PIK6, L. (1983). Poly (A) length, cytoplasmic adenylation and synthesis of
poly(A) + RNA in early mouse embryos. Devi Biol., 95, 331-341.
CULLEN, B., EMIGHOLZ, K. & MONAHAN, J. (1980). The transient appearance of specific
proteins in one-cell mouse embryos. Devi Biol. 76, 215-221.
DAVIDSON, E. H. (1976). Gene Activity in Early Development. New York: Academic Press.
DEUTSCHER, M. P. (1973). Synthesis and functions of the -C-C-A terminus of transfer RNA.
Progr. Nucleic Acid. Res. molec. Biol. 13, 51-92.
DOLECKI, G. J., DUNCAN, R. F. & HUMPHREYS, T. (1977). Complete turnover of poly(A) on
maternal mRNA of sea urchin embryos. Cell 11, 339-344.
HOLT, C. E., JOEL, P. B. & HERBERT, E. (1966). Turnover of terminal nucleotides of soluble
ribonucleic acid in intact reticulocytes. /. biol. Chem. 241, 1819-1829.
JAHN, C. L., BARAN, M. M. & BACHVAROVA, R. (1976). Stability of RNA synthesized by the
mouse oocyte during its major growth phase. /. exp. Zool. 197,161-172.
JOHNSON, M. H. (1981). The molecular and cellular basis of preimplantation mouse development. Biol. Rev. 56, 463-498.
KNOWLAND, J. & GRAHAM, C. (1972). RNA synthesis at the two-cell stage of mouse development. J. Embryol. exp. Morph. 27, 167-176.
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.
MAGNUSSON, T. & EPSTEIN. C. J. (1981). Genetic control of very early mammalian development. Biol. Rev. 56, 369-408.
OLDS, P. J., STERN, S. & BIGGERS, J. D. (1973). Chemical estimates of the RNA and DNA
contents of the early mouse embryo. J. exp. Zool. 186, 39-46.
PETZOLDT, U., ILLMENSEE, G. R., BURKI, K., HOPPE, P. C. & ILLMENSEE, K. (1981). Protein
synthesis in microsurgically produced androgenetic and gynogenetic mouse embryos. Mol.
gen. Genet. 184, 11-16.
PIK6, L. (1970). Synthesis of macromolecules in early mouse embryos cultured in vitro: RNA,
DNA, and a polysaccharide component. Devi Biol. 21, 257-279.
PIK6, L. & CHASE, D. G. (1973). Role of the mitochondrial genome during early development in mice. Effects of ethidium bromide and chloramphenicol. /. Cell Biol. 58, 357378.
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.
RANDERATH, E., CHIA, L. S. Y., MORRIS, A. P. & RANDERATH, K. (1974). Transfer RNA base
composition studies in Morris hepatomas and rat liver. Cancer Res. 34, 643-653.
SAWICKI, W., ABRAMCZUK, J. & BLATON, O. (1978). DNA synthesis in the second and third
cell cycles of mouse preimplantation development. Expl Cell Res. 112, 199-205.
VAN BLERKOM, J. (1981). Structural relationship and posttranslational modification of stagespecific proteins synthesized during early preimplantation development in the mouse. Proc.
natn. Acad. Sci., U.S.A. 78, 7629-7633.
182
K. B. CLEGG AND L. PIK6
F. H. (1977). The dynamics of maternal poly(A)-containing mRNA in fertilized sea
urchin eggs. Cell 11, 673-681.
WILT,
{Accepted 21 October 1982)