/. Embryol. exp. Morph. Vol. 34, 2, pp. 29J-298, 1975
Printed in Great Britain
291
The RNA polymerase activity of the
preimplantation mouse embryo
By G. P. M. MOORE 1
From The Finsen Laboratory, The Finsen Institute, Copenhagen, Denmark
SUMMARY
The activation of the mouse embryo genome has been studied during early cleavage,
in vivo. Individual embryos, prepared as whole mounts, were assayed for endogenous RNA
polymerase activity. RNA synthesis was detected by autoradiography as the incorporation
of [3H]UMP into an acid-insoluble product.
No RNA polymerase activity could be detected in the pronuclei of one-cell embryos.
Radioactive incorporation was first evident in the nuclei of two-cell embryos. This appeared
to be confined to the nucleoplasm and could be abolished by a-amanitin but not by low
concentrations of actinomycin D. Polymerase activity which was not affected by a-amanitin
was first detected in the four-cell embryo, predominantly at the peripheries of the nucleoli.
Nucleolar labelling increased markedly between subsequent cleavages, reaching a peak in
early morulae. In one- and two-cell embryos, label incorporation could be found in the
nucleus of the persisting polar body.
INTRODUCTION
Nuclear RNA synthesis is initiated in the mouse embryo soon after fertilization. Both autoradiographic and biochemical studies have shown that RNA
precursors are incorporated into the two-cell embryo and that RNA synthesis
increases as cleavage progresses (Mintz, 1964; Monesi & Salfi, 1967; Ellem &
Gwatkin, 1968; Hillman & Tasca, 1969; Tasca & Hillman, 1970; Siracusa,
1973). The types of RNA species synthesized in early cleavage embryos have
been partially characterized. Woodland & Graham (1969) demonstrated that
small quantities of both high and low molecular weight RNA are synthesized
in two-cell embryos. A 4S component of the low molecular weight RNA was
described in four-cell embryos and further characterized as transfer RNA at
the eight-cell stage. RNA precursor incorporation into 4S and DNA-like RNA
species increases between the eight-cell and blastocyst stages (Ellem & Gwatkin,
1968; Piko, 1970). Ribosomal RNA synthesis has been reported in two-cell
(Knowland & Graham, 1972) and four-cell embryos (Mintz, 1964; Woodland &
Graham, 1969), and increases between the eight-cell and blastocyst stages
(Ellem & Gwatkin, 1968; Piko, 1970).
1
Author's address (to which all correspondence should be sent): Department of Zoology,
Australian National University, Canberra, A.C.T. 2600 Australia.
292
G. P. M. MOORE
So far, the low rate of RNA synthesis during the early phase of mouse embryo
cleavage has hindered efforts to determine whether transcriptional activity is
actually initiated in the two-cell embryo or whether it occurs earlier. Indications
of RNA precursor incorporation into one-cell embryos have been reported
(Mintz, 1964; Monesi & Salfi, 1967; Knowland & Graham, 1972) but these
require verification. The present study investigates the activation of the mouse
embryo genome during early development, in vivo, by assaying isolated embryos
for endogenous RNA polymerase activity. Earlier studies of changes in RNA
polymerase activity during cleavage in the mouse have utilized enzymes solubilized from whole embryo homogenates (Siracusa, 1973; Warner & Versteegh,
1974). The experimental method used here is based on observations which
indicate that cells briefly fixed in organic solvents retain the capacity to incorporate ribonucleoside triphosphates into trichloroacetic acid-insoluble, ribonuclease-digestible products (Moore, 1971; Moore & Ringertz, 1973). The
activity of different forms of the nuclear polymerases have been localized within
the embryo nuclei using actinomycin D and a-amanitin. Actinomycin D at low
concentrations preferentially inhibits the nucleolar, polymerase I activity which
is involved in the synthesis of ribosomal RNA. Alpha-amanitin is a specific
inhibitor of the nucleoplasmic enzyme polymerase II, which catalyses the
synthesis of heterogeneous nuclear RNA (Zylber & Penman, 1971).
MATERIALS AND METHODS
Preparation of embryos
Female mice of the Bagg albino strain were caged with males and examined
daily for the appearance of vaginal plugs. For the purposes of dating, the day
on which a plug was found was regarded as the first day of gestation. Cleavage
embryos at various stages of development were either dissected from the oviducts or recovered from the uteri by flushing with saline. With the aid of a
dissecting microscope each embryo was drawn into a fine pipette and transferred to a glass slide in a drop of saline. Excess fluid was drained and the
embryo fixed in fresh ethanol and acetone (1:1 by vol.; see Moore, LinternMoore, Peters & Faber, 1974). The preparations were then air-dried and stored
at 4 °C in a desiccator until required.
Assay for endogenous RNA polymerase activity
Embryos were assayed for RNA polymerase activity using the method previously described for fixed preparations (Moore & Ringertz, 1973; Moore et al.
1974). The ribonucleoside triphosphates ATP, GTP and CTP were purchased
from the Sigma Chemical Company, Missouri, U.S.A., and [3H]UTP from the
Radiochemical Centre, Amersham, U.K. Polymerase I and II activities were
distinguished by including either a-amanitin (5 /*g/ml) or actinomycin D
(0-5/^g/ml) in the assay solution (Moore, 1972; Moore & Ringertz, 1973).
RNA polymerase activity of cleaving embryos
293
Each embryo was incubated with one or two drops of the assay solution for
30 min at 37 °C. The reaction was terminated by rinsing slides in distilled water
and immersion in ethanol and acetic acid fixative solution (3:1 by vol.) for
30 min. Unincorporated nucleotides were extracted with cold 5 % trichloroacetic acid (TCA) for 5 min, and the slides were washed in running water for
30 min. The autoradiographic procedures performed were similar to those
described previously for isolated mouse oocytes (Moore et ah 1974).
The results of this study are based on observations made on approximately
300 embryos prepared and assayed in this way.
RESULTS
3
Mouse embryo cells utilized [ H]UTP in the presence of ATP, GTP and
CTP, indicating that endogenous RNA polymerases were active (Figs. 1-12).
Radioactivity was localized in the nucleus and was not removed by cold TCA.
Inclusion of ribonuclease or high concentrations of actinomycin D in the assay
solution caused a reduction in label incorporation, indicating that the reaction
was dependent on DNA and that RNA was synthesized.
No polymerase activity was detected in the developing pronuclei of one-cell
embryos (Figs. 1 and 2), nor was there any associated with the chromosomes of
the first cleavage division (Fig. 3). However, one polar body was frequently
found to be labelled in these embryos (Fig. 3). Incorporation into the embryo
nuclei was first detected at the two-cell stage. Radioactivity was predominantly
located within the nucleoplasm (Fig. 4A). No incorporation into the nucleoli
was evident although polymerase activity associated with the peripheral regions
cannot be excluded. Label incorporation in two-cell embryos was not greatly
reduced by the inclusion of actinomycin D in the assay solution (Fig. 5) but was
abolished by a-amanitin (Fig. 6). Polymerase activity was also present in the
persisting polar body of two-cell embryos (Fig. 4B).
Radioactivity located over the nucleolus could be first observed in the fourcell embryo (Fig. 7). In the presence of a-amanitin, the label was largely confined to the edges of the nucleoli (Fig. 9). Nucleolar incorporation was low at
this stage and actinomycin D-resistant polymerase activity of the nucleoplasm
predominated (Figs. 8A, B). However, as the embryo progressed through later
cleavages, the amount of label associated with the nucleolus increased markedly
(Fig. 10). The levels of nucleolar and nucleoplasmic polymerase activities
attained at the morula stage (Fig. 11) appeared to be maintained during blastocyst formation (Fig. 12).
DISCUSSION
Model studies have shown that the utilization of [3H]UTP by cells after
fixation is comparable with their incorporation of [3H]uridine, in vivo (Carlsson,
Moore & Ringertz, 1973). However, the polymerase assay permits the relative
294
G. P. M. MOORE
RNA polymerase activity of cleaving embryos
295
changes in the transcriptional activity of the cell genome to be estimated during
differentiation, unhampered by fluctuations either in the rates of precursor
transport into the cell or in their concentrations in the intracellular pools (Ellem
& Gwatkin, 1968; Daentl & Epstein, 1971; Epstein & Daentl, 1971; see also
Zetterberg, Auer & Moore, 1974). The polymerase activity demonstrable is
probably involved in the elongation of RNA molecules which have already
been initiated (Zetterberg et al. 1974; Moore unpublished observations). Thus
the amount of [3H]UMP incorporated is a function of the quantity of active,
DNA-bound enzymes which are present.
RNA polymerase activity was not detected in the pronuclei of one-cell
embryos. This appears to conflict with the evidence of earlier studies which
suggest that RNA precursors may be incorporated at this stage (Mintz, 1964;
Monesi & Salfi, 1967; Knowland & Graham, 1972). However, Mintz stated
that her autoradiographic observations were not consistently repeatable
(Mintz, 1964), whereas in the biochemical work of Monesi & Salfi. (1967) and
Knowland & Graham (1972) the possibility of RNA synthesis within the polar
body was not considered. The utilization of RNA precursors by the polar body
of the mouse embryo has not previously been observed although [3H]leucine
incorporation has been reported (Mintz 1964).
The RNA polymerase activity of the nuclei of two-cell embryos was mainly
located in the nucleoplasm. Label incorporation was prevented by a-amanitin,
indicating that the extranucleolar, type II polymerases were active (Jacob, 1973).
This is consistent with the findings of Knowland & Graham (1972), although
their observation that small amounts of ribosomal RNA may also be synthesized
at this stage was not confirmed. Alpha-amanitin-resistant, polymerase I
activity was first detected in the nucleoli of the four-cell embryo (see also Mintz,
1964). However, the particular stage of cleavage development at which ribosomal
genes are first utilized may not be defined in the mouse embryo. Mintz (1964)
FIGURES
1-6
RNA polymerase activity in one and two-cell embryos.
Fig. 1. Newly fertilized ovum. No label incorporation can be detected in the male and
female pronuclei nor in the second polar body (pb 2). x 350.
Fig. 2. Two one-cell embryos. Pronuclei (arrows) are not labelled, x 375.
Fig. 3. One-cell embryo. Chromosomes (ch) of the first cleavage division. The polar
body alone is labelled, x 700.
Fig. 4. A, Two-cell embryo. Label is localized predominantly in the nucleoplasm of
the nuclei and in the polar body. The nucleoli appear unlabelled. x 350. B, Label
incorporation in the polar body of the two-cell embryo of figure A. x 825.
Fig. 5. Two-cell embryo. Polymerase assay performed in the presence of actinomycinD. xllOO.
Fig. 6. Two-cell embryo. Assay performed in the presence of a-amanitin. Label
incorporation is abolished. xllOO.
19
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296
11
G. P. M. MOORE
RNA polymerase
activity of cleaving embryos
297
3
observed that [ H]uridine was actively incorporated into the nucleoli of two-cell
embryos in which the second cleavage division had been temporarily delayed.
The dependence of mouse embryo development on the early activation of the
genome during cleavage has been studied, in vitro, using actinomycin D and
a-amanitin to inhibit RNA synthesis (Mintz, 1964; Monesi, Molinaro, Spalletta
& Davoli, 1970; Tasca & Hillman, 1970; Golbus, Calarco & Epstein, 1973).
The results support the general conclusion that, in the presence of actinomycin D,
there is a rapid arrest of cleavage. However, in addition to its effect on RNA
synthesis, actinomycin D is known to affect cell metabolism adversely. If
a-amanitin is used to block total RNA synthesis (Jacob, 1973), limited cleavage
may occur. Manes (1973) showed, for example, that rabbit embryos could
undergo the first and a few subsequent cleavage divisions in the presence of
10~ 4 M a-amanitin, a dose found sufficient to block over 95% [3H]uridine
incorporation. In the mouse, Golbus etal. (1973) found that a maximum of 70 %
of one-cell embryos were able to cleave in the presence of 10~5-10~7 M a-amanitin
compared with untreated controls. It is concluded from these observations and
those of the present study that the one-cell mouse embryo may undergo the first
cleavage division in the absence of pronuclear RNA synthesis.
This work was supported by the Danish Medical Research Council and the Nordisk
Insulinfond. I gratefully acknowledge the help of Prof. M. Faber and Dr H. Peters. The
a-amanitin was a generous gift from Prof. T. Wieland, Max-Planck-lnstitut, Heidelberg.
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FIGURES
7-12
RNA polymerase activity in four-cell to blastocyst-stage embryos.
Fig. 7. Four-cell embryo. Both nucleolar and nucleoplasmic label incorporation
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Fig. 8. A, Four-cell embryo. Polymerase assay performed in the presence of actinomycin D. Label incorporation in the nuclei is not markedly affected, x 350. B, Label
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Fig. 11. Morula. Both nucleolar and nucleoplasmic regions of the nuclei are
labelled, x 675.
Fig. 12. Blastocyst. The distribution of label is similar to that found in the morula.
x500.
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{Received 11 December 191 A, revised 6 May 1975)