/ . Embryol. exp. Morph. Vol. 44, pp. .133-148, 1978
Printed in Great Britain © Company of Biologists Limited 1978
\ 33
Uridine and guanosine incorporation by
the mouse one-cell embryo
By R. J. YOUNG, 1 K. SWEENEY 1 AND J. M. BEDFORD 1
From the Cornell University Medical College, New York
SUMMARY
The activity of the embryonic genome prior to the first cleavage has been assessed by
studying the uptake of [3H]uridine, its phosphorylation and incorporation into RNA by
mouse one-cell embryos. One-cell embryos incorporated [3H]uridine linearly into cold
trichloracetic acid (TCA) insoluble material at a low level 1-9 h post fertilization. The
incorporation of [3H]guanosine was also low but followed a biphasic curve which had a
steeper slope at 1-3 h than during the period 4-9 h post fertilization. Unfertilized mouse
ova incorporated very little [3H]uridine or [3H]guanosine into TCA insoluble material, and
much of this was RNase insensitive. Dimethyl sulfoxide (DMSO) enhanced the uptake of
[3H]thymidine and its incorporation into pronuclear DNA by one-cell embryos, but had no
effect on the incorporation of [3H]uridine by them, or of [3H]uridine and [3H]guanosine by
unfertilized ova. The uptake and incorporation of [3H]guanosine by one-cell embryos were
enhanced by DSMO, but only during the period 1-3 h post fertilization.
Sugar derivatives of UDP, and UMP, UDP, UTP, CMP, CDP and CTP have been
identified in the soluble fraction obtained from mouse one-cell embryos incubated with
[3H]uridine 1-3 h post fertilization. Very little of the [3H]uridine taken up by the embryos is
present as [3H]UTP, or [3H]CTP; most is found as [3H]UMP or [3H]UDP or as the sugar
derivatives. Alkaline or ribonuclease (A, Tx and T2) hydrolysis of the 3H-labeled ethanol
insoluble material precipitated from the lysate of one-cell embryos incubated with [3H]uridine
1-3 h post fertilization liberated radioactive cytidine and uridine-3'-phosphates. This demonstrates that [3H]uridine is incorporated into an internal position in RNA and suggests that
RNA synthesis does occur in the one-cell embryo 1-3 h post fertilization. Since pronuclei of
one-cell embryos incubated with [3H]uridine were not labeled it appears, however, that the
RNA synthesized at the one-cell stage is not a product of the embryonic genome.
INTRODUCTION
It is not yet clear whether activity of the embryonic genome is required at all
stages of mammalian preimplantation development, and if not, when embryonic
genes are first activated. The mouse preimplantation embryo does synthesize
RNA by the two-cell stage of development, although the RNA has not been
characterized (Knowland & Graham, 1972), but whether the embryonic genome
is transcribed earlier, i.e. prior to the first cleavage, is not known. A low level of
[3H]uridine is incorporated into TCA insoluble material by one-cell embryos
1
Authors' address: Reproductive Biology Unit, Departments of Obstetrics and Gynecology
and Anatomy, Cornell University Medical College, 1300 York Avenue, New York, N.Y.
10021, U.S.A.
134
R. J. YOUNG, K. SWEENEY AND J. M. BEDFORD
but attempts to isolate [3H]uridine-labeled RNA from them have been unsuccessful (Monesi & Salfi, 1967; Woodland & Graham, 1969; Monesi &
Molinaro, 1971; Daentl & Epstein, 1971; Knowland & Graham, 1972; Graham,
1973).
The one-cell embryo has also been reported to incorporate very little [3H]guanosine, [32P]phosphate or [14C]carbonate into RNA (Woodland & Graham,
1969), and with the exception of one early autoradiographic study which
reported occasional labeling of the pronuclei of one-cell embryos (Mintz, 1964)
attempts to demonstrate RNA polymerase activity in the embryo have also
been unsuccessful (Warner & Hearn, 1977; Moore, 1975). Thus, to date there
has been no direct demonstration that RNA is synthesized by the one-cell
embryo and this possibility rests only on the observation that inhibitors of
RNA synthesis, actinomycin D and a-amanitin, inhibits cleavage of these
embryos (Golbus, Calarco & Epstein, 1973).
However, such inhibitors can affect metabolism in ways unrelated to the
inhibition of RNA synthesis, and thus the effect of these on the first cleavage
may not be due specifically to inhibition of RNA synthesis. While it is possible
that the one-cell embryo is transcriptionally inactive, it is also possible that
there may be difficulty in detecting a newly synthesized labeled RNA because
of (a) non-entry of the labeled precursor (Woodland & Graham, 1969; Knowland & Graham, 1972; Graham, 1973), (b) the non-conversion of the labeled
nucleoside to the triphosphate, (c) a level of RNA synthesis insufficient for its
isolation and characterization by the technique of polyacrylamide gel electrophoresis (Knowland & Graham, 1972; Graham, 1973), or (d) discontinuous
synthesis from the time of fertilization until the first cleavage. These possibilities
have been explored in this communication which shows that uridine can enter
the mouse one-cell embryo, is phosphorylated, and is incorporated at a low
level into newly synthesized RNA.
MATERIALS AND METHODS
Collection of ova
Virgin random-bred Swiss female mice 7-12 weeks old were superovulated
by intraperitoneal injection with 7-5-10 units each of pregnant mare's serum
(Equinex, Ayerst) and 46-48 h later with human chorionic gonadotropin
(HCG) (Pregnyl, Organon). Unfertilized eggs were collected from females
sacrificed 13-16 h post HCG. To obtain fertilized eggs, a female was placed
with a male (Balb CJ x C57 BL/6J) immediately after injection with HCG, and
checked for the presence of a vaginal plug early next morning. Mated females
were sacrificed commencing 16-16-5 h post HCG. Cumulus cells were removed
from ova by incubation with hyaluronidase (Sigma, type VI, 150 units/ml) in
Whitten's medium (Whitten, 1971) containing 10 mg/ml bovine serum albumin
(BSA) (Miles) for 10 min followed by three washes with medium. The collection
Undine incorporation in mouse embryo
135
of ova was carried out at 37 °C using medium equilibrated with a gas mixture
of 5 % O2, 5 % CO2J 90 % N 2 and overlayered with silicon oil (Dow Corning
200 Dielectric Fluid).
Culture and labeling of ova
Eggs collected from females at intervals of 1 h (unmated) and 2 h (mated)
were distributed into two groups of 200-400 each, and incubated in 100/tl
drops of Whitten's medium under oil at 37 °C in an atmosphere of 5 % O2,
5 % CO2 and 90 % N 2 . The medium for one group of eggs contained 1 %
DMSO. For labeling of eggs, tritiated nucleosides were present at 100-500 /*Ci/
ml of medium. After incubation with label for 2 h the eggs were washed with
three to five changes of medium containing 0-1 mg/ml of unlabeled nucleoside,
and the eggs in each group transferred in batches of 25-60 to 25 /i\ of lysing
buffer (0-1 M Tris-HCl, pH 7-5, 1 % sodium dodecyl sulfate (SDS), 10 jug yeast
RNA (Sigma, type VI)) and frozen.
Approximately 30 % of the eggs collected from mated females had pronuclei
16-17 h post HCG, increasing to about 90 % at 23-24 h post HCG (Luthardt
6 Donahue, 1973). The percentage of pronuclear eggs was determined at the
first time point and after a further 4-6 h to provide a guide to the extent of
fertilization and development of the fertilized egg.
The maturation of mouse follicular oocytes in vitro was carried out as described by Cross & Brinster (1970) except that the prior injection of PMS was
omitted. Oocytes were cultured in the absence of cumulus cells in simple serum
medium with or without 1 % DMSO as required for the experiment. For
labeling experiments, [5, 6-3H]uridine at 500 jLtCi/mi was present. At intervals
after being placed in culture, groups of eggs were removed and washed as above.
Epididymal spermatozoa were used for in vitro fertilization experiments
(Hoppe & Pitts, 1973). Fertilization rates of 70-80 % were achieved when eggs
were collected at 13 h post HCG, with not more than 20-30 eggs per dish.
When egg numbers were increased (up to 80/dish), the fertilization rate fell to
5-20 %.
DMSO is soluble in water, but is not miscible with silicon dielectric fluid
(2 /i\ does not dissolve in 5 ml of the fluid), and is therefore not lost from the
incubation medium.
Tritiated nucleosides, [5-3H]uridine (specific activity 25-29 Ci/mmole),
[5, 6-3H]uridine (specific activity 45-50 Ci/mmole), [8-3H]guanosine (specific
activity 9-11 Ci/mmole), [methyl-3H]thymidine (specific activity 40-60 Ci/
mmole) and [5, 6-3H]uridine-5'-triphosphate (specific activity 35-50 Ci/mmole)
were obtained from New England Nuclear or Amersham/Searle.
Measurement of nucleoside incorporation
Radioactivity was measured after lysis of ova by repeated freeze-thawing by
one of two methods. For the first (a) cold 50 % TCA was added to the lysate
136
R. J. YOUNG, K. SWEENEY AND J. M. BEDFORD
to a final concentration of 10 %, and after 1 h at 0 °C the mixture was centrifuged at 10000 g in the cold. The pellet was washed three times with cold 5 %
TCA, the supernatants combined, and the TCA soluble radioactivity determined by counting the supernatant in a liquid scintillation spectrometer after
adding a toluene-based scintillation fluid (4/g Omnifluor/liter toluene) (New
England Nuclear) containing BBS3 (Beckman). The TCA insoluble pellet was
dissolved in 0-1 M ammonium hydroxide before measurement of the radioactivity.
The second method (b) is similar to that previously described (Johnson &
Young, 1969). The lysate was quantitatively transferred onto 25 mm discs of
Whatman No. 1 filter paper, dried, and the radioactivity on the discs measured.
The paper discs were then washed in toluene, and then in cold 5 % TCA, cold
95 % ethanol, and cold ether, dried and counted. The first measurement gives
an estimate of the total nucleoside uptake, and the second, the amount of TCA
insoluble radioactivity. Backgrounds were determined by adding an aliquant
of the medium used for the final wash to the lysing buffer, and carrying this
through the precipitation and washing procedure.
The second method (b) was more convenient for the assay of many batches
of embryos but the efficiency of counting was lower because of self absorption.
Larger numbers of embryos or a higher level of incorporation was required
when the time course of incorporation was assayed by this method. Samples
were counted for a time sufficient to accumulate a minimum of 1000 background
counts. Background varied between 15 and 20cpm. Each experimental point
represents the average from two or three batches of 25-60 eggs, and most
kinetic experiments were carried out three times. Labeling times given are the
hours after HCG at which the eggs were first placed into culture.
Autoradiography (Luthardt & Donahue, 1973) was carried out using Kodak
NTB2 liquid emulsion. Sample slides were checked at 10 days and then at
3-day intervals.
Isolation of labeled material
Incubation of up to 700 embryos was carried out with gentle shaking on a
reciprocal shaker for 2 h (17-19 h post HCG) in 100 jil drops of Whitten's
medium containing [5, 6-3H]uridine at 500/6Ci/ml. Embryos were then washed
4-6 times in medium containing 0-1 mg/ml of uridine and then transferred to
lysing buffer. The embryos were lysed by repeated freeze-thawing and insoluble
3
H-labeled material isolated as described (Young, 1977). This method gave
good recovery of insoluble material, but some soluble nucleotides were also
present as a contaminant. Three cycles of precipitation usually reduced contamination of the insoluble 3H-labeled material by soluble nucleotides, mainly
UTP and UDP, to 0-1-1 % of the soluble fraction. The 3H-labeled precipitate
was suspended in 0-1 M Tris-HCl, pH 7-5, incubated at 37 °C with pronase
(self-digested, nuclease free (Calbiochem)) for 2 h, and RNA isolated as
Uridine incorporation in mouse embryo
137
described by Perry, La Torre, Kelley & Greenberg (1972). Controls in which
E. coli rRNA was present in the lysate showed that this procedure did not
result in degradation of RNA.
For isolation of soluble 3H-labeled material embryos were suspended in
buffer containing 10-15 /.ig UTP but no SDS. After repeated freeze-thawing the
insoluble material was precipitated as described above, collected by centrifugation and washed with 70 % aqueous ethanol. The precipitate was dispersed,
resuspended in water and re-precipitated with 2 vol of ethanol. This procedure
was repeated three to four times. The supernatant and 70 % ethanol washes
from the precipitations were combined, concentrated and the composition of
the soluble 3H-labeled material analyzed by chromatography on polyethyleneimine impregnated (PEI) cellulose thin layer sheets. Hydrolysis by KOH of the
insoluble 3H-labeled material obtained by this procedure from three batches of
embryos (total 2500), followed by paper electrophoresis of the hydrolysate at
pH 3-5, showed that little or no UDP or UTP was present in the insoluble
fraction. Thus this procedure does not result in a loss of 3H-labeled material
from the soluble to the insoluble fraction.
Alkaline and enzymic digestion
Alkaline hydrolysis was carried out in 0-3 M - K O H at 37 °C for 16-20 h. The
hydrolysate was desalted with perchloric acid before chromatography. Embryo
lysate not extracted with phenol-chloroform and carrier yeast tRNA (7 /tg) was
digested with ribonucleases Tx (15 units), T2 (10 units) (Sigma) and ribonuclease
A (5 jug) (Worthington) in 005 M sodium acetate, pH 5 and 0-003 M - E D T A for
8 h at 37 °C. The pH was adjusted to 7-5 and incubation continued for 2 h
with pronase (200 /tg) before chromatography.
Thin-layer chromatography and paper electrophoresis
Thin-layer chromatography was carried out on PEI-cellulose sheets (Baker)
as described (Young, 1977). Solvent systems were: step formate and step LiCl
systems (Randerath & Randerath, 1964); solvent A, 0-15 M sodium borate0-5 M boric acid in 25 % ethylene glycol; B, 0-9 M acetic acid - 0-1 M LiCl;
c, 1 M LiCl; and D 1 M acetic acid. The PEI-cellulose sheets were washed with
anhydrous methanol after spotting the sample and markers and also after chromatography in the first dimension. A spot of equal area adjacent to the markers
was used as background.
Paper electrophoresis was carried out in citrate buffer as previously described
(Young & Fraenkel-Conrat, 1971).
RESULTS
Nucleoside incorporation by one-cell mouse embryos and unfertilized mouse ova
There was a low level of incorporation of [3H]thymidine by one-cell embryos
into TCA insoluble material throughout the period 17-25 h post HCG
138
R. J. YOUNG, K. SWEENEY AND J. M. BEDFORD
130
A
110
+ DMSO
7-0
E.
50
+ DMSO
21
23
Time post HCG (h)
25
27
Fig. 1. Effect of DMSO on uptake and incorporation of [3H]thymidine by one-cell
mouse embryos. (A) TCA soluble radioactivity; O, 1% DMSO; • , no DMSO.
(B) TCA insoluble radioactivity; 0 , 1 % DMSO; • , no DMSO. Each time point is
the time post HCG when the embryos were placed in the culture medium for a 1 h
incubation. Radioactivity was measured by method (b). One experiment is shown
and each experimental point is the average value from 2 or 3 batches of embryos.
The nucleoside was present at a concentration of 500 /tCi/ml.
(Fig. IB). However, at about 21 h there was an increase in the level to reach
a maximum at 23-25 h before returning to the basal level at about 26 h post
HCG or about 4-6 h before the first cleavage division. The TCA insoluble
radioactivity was reduced by 70-80 % after incubation of embryo lysate with
DNase showing that DNA is synthesized during this period. Because of asynchrony of fertilization, pronuclear DNA synthesis will commence at different
times in different embryos and [3H]thymidine incorporated into TCA insoluble
material in the period 17-20 h post HCG may also represent pronuclear DNA
synthesis. The latter has also been studied by autoradiographic and cytophotometric methods (Luthardt & Donahue, 1973; Siracusa, Coletta & Monesi,
1975; Ambramczuk & Sawicki, 1975), and the results are in excellent agreement
139
Uridine incorporation m mouse embryo
1-5
-
A
50.
-
10
"
n
30
LJ
0-5
5 oo
1
1
40
-
20
-
10
1
B
_->
S" 20
-• 2 0
fi-
inios
~" 1-5
-
\
-- 15
\
10
- 10
0-5
-
1
00
16
17
18
19
1
5
1
20
Time post HCG
Fig. 2. Uptake and incorporation of tritiated nucleosides by one-cell mouse embryos
in the presence or absence of DMSO. (A) [5-3H]uridine; • , TCA soluble, no DMSO;
O, TCA soluble, 1 % DMSO; • , TCA insoluble, no DMSO; • , TCA insoluble,
1% DMSO. (B) [3H]guanosine; • , TCA soluble, no DMSO; O, TCA soluble,
1% DMSO; • , TCA insoluble, no DMSO; D, TCA insoluble, 1% DMSO.
Each time point is the time post HCG when the embryos were placed in the culture
medium for 2 h. The figure shows an experiment in which the radioactivity was
measured by method (a). Each point is the average value from two-three batches
of embryos. The concentration of the nucleosides was 500 /tCi/ml.
with those of the present study showing that the present method is sufficiently
sensitive to measure low levels of incorporation of label.
[3H]Uridine was found to be incorporated at a low level into TCA insoluble
material in agreement with the studies of other workers (Monesi & Salfi, 1967;
Monesi & Molinaro, 1971; Knowland & Graham, 1972). The incorporation of
[3H]uridine remained at this low level throughout the period 16-24 h post
HCG (Fig. 2 A), but in contrast to the behaviour of [3H]uridine, [3H]guanosine
incorporation was not linear being higher 16-18 h post HCG than during later
stages of development, i.e. the kinetic curve for guanosine is biphasic (Fig. 2B).
These results suggest that [3H]uridine is incorporated into macromolecules at a
140
R. J. YOUNG, K. SWEENEY AND J. M. BEDFORD
Table 1. Incorporation of [zH]uridine by maturing mouse follicular oocytes
Total radioactivity
(cpm/egg)
Time in
culture (h)
A
,
>
TCA insoluble radioactivity
(cpm/egg)
,
-*
>
-DMSO
-DMSO
+ 1 % DMSO
1-3
2-2
4-5
12
11
14
16
17
66
1-9
2-6
3-3
3-5
7-4
11-2
240
78
83
25-0
310
constant low rate throughout the development of the one-cell embryo, whereas
more [3H]guanosine is incorporated 1-3 h after fertilization than at later periods
during development of the one-cell embryo. The estimate of the time of fertilization (15-16 h post HCG) after natural mating as in the present experiments is
imprecise, and in vitro fertilization (Hoppe & Pitts, 1973) was attempted in
order to determine more accurately the commencement of [3H]uridine and
[3H]guanosine incorporation. However, the percentage of eggs fertilized was
found to vary inversely with the number of eggs present per drop of medium
and in vitro fertilization could not provide sufficient fertilized eggs to enable a
kinetic study of nucleoside incorporation.
Unfertilized ova incorporated very little [3H]uridine and [3H]guanosine into
cold TCA-insoluble material during the period 1-6 h post ovulation. More than
80 % of the [3H]guanosine labeled and 60-70 % of [3H]uridine-labeled material
was insensitive to RNase A, showing that no [3H]guanosine and little [3H]uridine was incorporated into RNA; the latter incorporation is most probably
turnover incorporation into the -CCA end of tRNA. This result is in agreement
with the expectation that RNA synthesis should not occur in the ovulated ovum
where the chromosomes are in condensed state. The identity of the cold TCAinsoluble 3H-labeled material is unknown.
Effect of DMSO on nucleoside incorporation
The low level of [3H]nucleoside incorporation by the one-cell embryo has
been postulated to be due to its restricted entry (Woodland & Graham, 1969;
Knowland & Graham, 1972; Graham, 1973). Since DMSO can increase the
permeability of cellular membranes to many solutes its effect on the incorporation of nucleosides by one-cell embryos was studied.
Preliminary experiments showed that 1 % DMSO did not inhibit the cleavage
of fertilized mouse ova. Fig. 1 shows that 1 % DMSO enhanced both the
uptake and incorporation of [3H]thymidine by one-cell embryos. This was
observed first at about 21 h, the time of commencement of pronuclear DNA
synthesis (Fig. 1B) with the maximum enhancement occurring during the time
of maximum pronuclear DNA synthesis at 23-25 h post HCG. Its enhancement
Uridine incorporation in mouse embryo
141
3
of [ H]thymidine incorporation during the pronuclear DNA synthetic period
is low (assayed by method b) but since the percentage increment in uptake
(Fig. 1 A) is approximately equal to the percentage increment in incorporation
(Fig. 1 B), the incorporation of [3H]thymidine is most probably maximal under
the conditions of incubation. Thus, although low, the enhancement by 1 %
DMSO of [3H]thymidine incorporation into pronuclear DNA is real, and was
reproducible.
The effect of 1 % DMSO on the incorporation of [3H]uridine by mouse
follicular oocytes maturing in vitro was also studied since [3H]uridine is incorporated by follicular oocytes after 2-6 h in culture (Bloom & Mukherjee,
1972). The data presented in Table 1 show that [3H]uridine uptake and incorporation by follicular oocytes is in fact enhanced by 1 % DMSO. Therefore
in the maturing follicular oocyte and the one-cell embryo, where the synthesis
of RNA and DNA respectively has been demonstrated by other methods, 1 %
DMSO has been found to enhance the uptake and incorporation of nucleoside
precursor.
By contrast, the uptake and incorporation of [3H]uridine by one-cell embryos
(Fig. 2 A) was not affected by DMSO over the period 16-24 h post HCG.
However, DMSO enhanced (Fig. 2B) the uptake and incorporation of [3H]guanosine 16-18 h post HCG, a period in which the nucleoside is incorporated
into macromolecules, but at 18-24 h post HCG when less [3H]guanosine was
incorporated, DMSO had no effect on this. Thus the curve of [3H]guanosine
incorporation by the one-cell embryo 16-24h post HCG is biphasic, whether
DMSO is present or absent.
The uptake and incorporation of [3H]uridine and [3H]guanosine by ovulated
unfertilized mouse ova were unaffected by 1 % DMSO.
These results indicate that while DMSO can enhance the uptake and incorporation of nucleosides into macromolecules by the ovum, their transport
into the ovum appears to depend on their utilization for synthesis. The low
level of [3H]uridine incorporation into macromolecules by the one-cell embryo
is probably therefore not due to the inability of the nucleoside to enter the
embryo.
Composition of the labeled soluble fraction
The failure of nucleoside precursor to be converted to the triphosphate seems
a possible reason for the low level of incorporation of [3H]uridine into a TCA
insoluble product by the one-cell embryo.
Examination of the soluble [3H]uridine-labeled material present in the lysate
of embryos 17-19 h post fertilization by paper electrophoresis at pH 3-5 and
by thin layer chromatography with the step formate system showed that UMP,
UDP and UTP were present in the soluble fraction, but also that the composition of the soluble fraction was complex. However, a two-dimensional thin
layer chromatographic system (Randerath & Randerath, 1964) resolved the
10
EMB 44
142
R. J. YOUNG, K. SWEENEY AND J. M. BEDFORD
Table 2. Undine nucleotides present in one-cell embryos 1-3 h post
fertilization*
Nucleotide
UMP
UDP
UTP
CMP
CDP
CTP
UDPG
UDPGAJ
CDPG
X
Percentagef
42-7
29-9
30
1-5
0-83
0-42
16-3
0-66
0-77
3-9
* Components resolved by two-dimensional chromatography on PEI-cellulose. First
dimension: step LiCl; second dimension: step formate (Randerath & Randerath, 1964).
t Average value from four experiments.
% Uridine diphosphate N-acetylglucosamine.
3
H-labeled material present in the soluble fraction into nine radioactive components which co-migrated with added markers. A tenth radioactive component,
that migrated slightly ahead of both uridine diphosphate glucose (UDPG) and
cytidine diphosphate glucose (CDPG) in the step LiCl system and had the same
mobility as CDPG in the step formate system, was also detected in the twodimensional chromatogram.
It is clear that some UTP, the immediate precursor of RNA, is present in the
one-cell embryo but that most of the [3H]uridine is present as the UMP or
UDP or as nucleoside diphosphate sugars (Table 2). This result shows that
[3H]uridine is able to enter the mouse one-cell embryo, and upon entry is made
available for RNA synthesis by conversion to the triphosphate. The difficulty
in detecting [3H]uridine incorporation into RNA is probably not due to the
unavailability of the nucleoside or its triphosphate.
Paper electrophoresis and paper chromatography showed that cytidine was
not present as a contaminant in the [5, 6-3H]uridine precursor. The presence of
small amounts of CTP, CDP and CMP in the soluble fraction therefore demonstrates that pyrimidine interconversion can occur in the one-cell embryo,
although this interconversion apparently does not take place at the later stages
of development (Woodland & Graham, 1969).
The nature of the labeled insoluble fraction
Even when 500-2000 one-cell embryos were used in previous studies, labeled
RNA could not be resolved by gel electrophoresis although TCA insoluble
material was present (Knowland & Graham, 1972; Graham, 1973); this
material however was not tested for its stability toward RNase or alkali by
Undine incorporation in mouse embryo
143
C5
Solvent B
then
Solvent C
UDPG
c
Solvent A
Fig. 3. Two-dimensional thin layer chromatogram of an alkaline hydrolysate of 3Hlabeled RNA isolated by phenol-chloroform extraction of one-cell embryos that
had been incubated with [5, 6-3H]uridine 1-3 h post fertilization. First dimension:
solvent A (right to left). Second dimension: solvent B, then dried and washed with
anhydrous methanol and developed further in solvent C until front was 1 cm below
Up spot (bottom to top).
these or other workers (Monesi & Salfi, 1967; Daentl & Epstein, 1971). Incubation of the lysate of embryos labeled with [5, 6- 3H] uridine or [3H]guanosine
overnight with RNase A or 0-3 M - K O H reduced the amount of TCA insoluble
radioactivity present, but only by 40-60 %. Thus the labels are incorporated
into RNA as well as into material which is not RNA. An attempt to isolate
RNA by phenol-chloroform extraction of the pronase-digested lysate of
embryos which had been incubated with [3H]guanosine was unsuccessful; most
(85-95 %) of the radioactivity was present in the aqueous layer, but very little
was precipitated from solution by ethanol. Therefore very little of the nucleosides uridine or guanosine are incorporated into RNA by the one-cell embryo
and the failure of gel electrophoresis (Graham, 1973; Knowland & Graham,
1972) to detect and resolve labeled RNA was most likely due to the small
quantity of material available.
Since pyrimidine interconversion occurs in the one-cell embryo (see above)
the 3H-labeled TCA insoluble material present in embryos incubated with
[3H]uridine may represent turnover incorporation of [3H]cytidine into the
-CCA terminus of tRNA rather than [3H]uridine incorporation into newly
synthesized RNA. Chemical degradation of the labeled material was studied to
determine whether both UTP and CTP are incorporated into RNA. Alkali or
144
R. J. YOUNG, K. SWEENEY AND J. M. BEDFORD
a mixture of the enzymes ribonuclease A, Tx and T2 will degrade RNA into a
mixture of nucleoside-3'-phosphates. Since these nucleotides do not occur
naturally, their presence in such digests would prove the existence of RNA.
Using this approach, 3H-labeled material isolated by ethanol precipitation
from one-cell embryos incubated 17-19 h post HCG with [3H]uridine was
digested with pronase and the digest extracted with phenol-chloroform. The
labeled RNA in the aqueous layer was hydrolyzed with alkali and the hydrolysate examined by two-dimensional thin layer chromatography (Fig. 3).
Preliminary experiments showed that small quantities of uridine-5'-phosphate
were present in the insoluble 3H-labeled material and UMP (pU) as well as
UTP, UDP were found to be radioactive. In addition the pyrimidine-3'phosphates, Up and Cp, products of alkaline hydrolysis, were radioactive, but
CMP (pC), which is not a product of alkaline hydrolysis and was not present
as a contaminant, was not radioactive. The same result was obtained if the
ethanol insoluble 3H-labeled material was digested with alkali or with a mixture
of ribonucleases A, Tx and T2 without prior extraction with phenol-chloroform.
The separation of Cp, pC, Up and pU from each other and from contaminating
UDP, UTP and uridine diphosphate sugars could also be effected by using
step LiCl in the first dimension followed in the second dimension by solvent D
until the front was 9 cm from the origin, then with further development without
intermediate drying in solvent B until the front reached the top of the thin layer
sheet. In this system radioactivity was also associated with Cp, Up and pU but
not with pC. In three experiments using between 3000 and 3600 embryos per
experiment, the amount of radioactivity found in RNA, measured as Cp and
Up, averaged 383 cpm. Thus both uridine and cytidine are incorporated at a
low level into internal positions in RNA by the one-cell embryo, and terminal
addition of [3H]cytidine is not the sole contributor of 3H-label to the 3 Hlabeled RNA.
Autoradiography
Autoradiographs of embryos incubated with [3H]thymidine showed distinct
labeling of both pronuclei. When [3H]uridine or [3H]UTP was used neither the
pronuclei nor the ooplasm was labeled even after an exposure period of 3-4
weeks.
DISCUSSION
Examination of nucleoside uptake by mouse preimplantation embryos has
indicated that uridine and thymidine share the same or a similar transport
system (Daentl & Epstein, 1973). Since thymidine (Fig. 1) as well as [3H]guanosine (Fig. 2B), [3H]adenosine and [32P]phosphate are taken up by the
one-cell embryo (Young, 1976) and [3H]uridine is able to enter the maturing
oocyte it is unlikely that its cell membrane is impermeable to [3H]uridine.
The present results (Fig. 2 A) and those of Warner & Hearn (1977) show that
Undine incorporation in mouse embryo
3
145
[ H]-uridine is taken up by the one-cell embryo. Difficulties in labeling its RNA
is therefore unlikely to be due to failure of [3H]uridine to enter the embryo.
Thus the low level of incorporation of [3H]guanosine after 18 h post HCG
(Fig. 2B), and the biphasic nature of the incorporation curve may explain the
failure of early workers to detect incorporation of this nucleoside by the onecell embryo (Woodland & Graham, 1969).
Since the chromosomes of unfertilized ova are at the metaphase II stage and
are condensed, RNA synthesis would be unexpected and indeed DMSO does
not enhance the uptake of [3H]uridine or [3H]guanosine. This solvent does
enhance the uptake of [3H]thymidine (Fig. 1A) and [3H]guanosine (Fig. 2B)
by the one-cell embryo and [3H]uridine by the maturing oocyte (Table 1) when
the embryo and oocyte are active in the synthesis of nucleic acids. It is possible
that the entry of nucleosides and the ability of DMSO to enhance the entry of
nucleosides into mouse embryos and ova are related to the level of utilization
of the nucleoside for nucleic acid synthesis. Therefore the inability of DMSO
to enhance the uptake or incorporation of [3H]uridine by one-cell embryos
(Fig. 2 A) may mean that the level of its RNA synthesis is low. This conclusion
is supported by the recent observation that the amount of [3H]uridine taken up
by the one-cell embryo is in the same order of magnitude as that taken up by
the two-cell stage (Warner & Hearn, 1977). The latter is active in RNA synthesis
whereas the former is not (Knowland & Graham, 1972).
The conversion of [3H]uridine to [3H]uridine phosphates has been noted in
two- and eight-cell embryos, and in the blastocyst (Woodland & Graham,
1969; Daentl & Epstein, 1971). The present results show that [3H]uridine is
taken up and phosphorylated by the one-cell embryo as early as 3 h post
fertilization, but that most is found as UMP and UDP and as sugar derivatives
of UDP rather than as UTP (Table 2). Sugar derivatives of UDP have not
previously been found in mouse embryos, but can be anticipated there in view
of the increase in glycogen during development from the one-cell to the two-cell
stage (Stern & Biggers, 1968; Ozias & Stern, 1973). The large amount of UDPsugar derivatives present in the soluble uridine nucleotide pool indicates that
the low percentage of UTP in the pool is not due to low uridylate kinase
activity. The percentage of [3H]uridine found as UTP at the two-cell and later
stages of development has been reported to vary between 40 % and 70 % and
these stages are also active in RNA synthesis (Daentl & Epstein, 1971; Ellem
& Gwatkin, 1968; Woodland & Graham, 1969; Monesi & Salfi, 1967; Piko,
1970; Tasca & Hillman, 1970; Monesi & Molinaro, 1971; Knowland &
Graham, 1972). In contrast the present work shows that only 3 % of the [3H]uridine taken up by the one-cell embryo 1-3 h post fertilization is present as
[ 3 H]UTP; thus there is little UTP available for RNA synthesis at the one-cell
stage and consistent with this only a small amount of TCA-insoluble 3H-labeled
material has been isolated from such embryos (Knowland & Graham, 1972;
Graham, 1973). The difficulty in detecting newly labeled RNA in the one-cell
146
R. J. YOUNG, K. SWEENEY AND J. M. BEDFORD
embryo is therefore most probably due to the very low level of RNA synthesis
rather than to restricted entry of nucleoside precursors or to inactivity of the
kinases required to convert the nucleoside to the triphosphate. Much of the
UTP appears to be utilized for polysaccharide rather than for RNA synthesis,
since the percentage of UDP-sugar derivates is high.
The presence of radioactive Up and Cp in alkaline and enzymic digests of
one-cell embryos incubated with [3H]uridine up to 4 h after fertilization or in
their 3H-labeled material shows that the 3H-labeled material is RNA and that
both Cp and Up are present in internal positions in the RNA. The one-cell
embryo is active in protein synthesis (Epstein & Smith, 1973; Van Blerkom &
Brockway, 1975) and incorporation of CTP into the 3'-terminal-CCA segment
of tRNA would be expected. At least part of [3H]Cp found in the alkaline and
enzymic digest would be derived from this source. Uridine is not known to have
a similar turnover role, and unless [3H]uridine incorporation is simply a terminal
addition to existing RNAs, the finding of Up means that RNA is synthesized
by the one-cell embryo albeit at a low level. Furthermore, this low level of
incorporation occurs continuously soon (1-3 h) after fertilization until after
the onset of pronuclear DNA synthesis (Fig. 2 A) suggesting that the RNAs
made have similar functions and are synthesized continuously after activation
at fertilization. On the other hand the biphasic nature of the [3H]guanosine
incorporation curve (Fig. 2B) indicates that the [3H]guanosine-labeled macromolecules synthesized soon after fertilization are either different from those
appearing at a later stage, or simply that the macromolecules are the same but
the rate of synthesis is higher soon after fertilization. For example, after fertilization [3H]guanosine may be incorporated into pre-existing mRNA as the 7methylguanosine capped structure, thus converting the mRNA into an active
form (Mulhukrishnan, Both, Furuichi & Shatkin, 1975; Both, Banerjee &
Shatkin, 1975a; Both, Furuichi, Mulhukrishnan & Shatkin, 19756; Young,
1977). In an autoradiographic study of [3H]uridine incorporation by mouse
one-cell embryos, Mintz (1964) reported that pronuclei of some embryos but
not the cytoplasm were labeled. A later autoradiographic study employing a
technique which enables [3H]UTP to enter the embryo failed to confirm pronuclear labeling although one polar body was frequently found to be labeled
(Moore, 1975). We also have not observed labeling of pronuclei in one-cell
embryos incubated with [3H]uridine. Thus, the RNA synthesized soon after
fertilization may not be a transcript of the embryonic genome; it is also unlikely
that the RNA is a product of the mitochondrial genome since mitochondria
of the embryo appear not to be active in RNA synthesis before the eight-cell
stage (Piko, 1975; Piko & Chase, 1973) and the cytoplasm of the embryo was
not labeled. It is possible therefore that the [3H]uridine-labeled RNA present
in the one-cell embryo is synthesized by a polar body. If this is the case it is
unlikely that this RNA plays a role in the first cleavage of the fertilized ovum,
and development of the embryo to the two-cell stage does not require
Uridine incorporation in mouse embryo
147
pronuclear RNA synthesis. The possibility cannot be excluded that the embryonic genome is active and that the level of RNA synthesized is too low to be
detected consistently by autoradiography. This putative RNA is unlikely to be
mRN A but may have a regulatory role or may serve as a primer for DNA replication. Thus, although a low level of RNA synthesis is shown here to occur in
the one-cell embryo, this may not be a transcript of the embryonic genome and
development of the mouse embryo up to the two-cell stage may depend on a
store of maternal RNA.
Part of this work was carried out in the Laboratory of Reproductive Physiology, University
of Pennsylvania. The hospitality and encouragement extended by Dr R. L. Brinster and
discussions with Dr P. Cross and Dr G. B. Stull are gratefully acknowledged. This investigation was supported by The Rockefeller Foundation and The National Institutes of Health.
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{Received 11 August 1977, revised 26 October 1977)