/ . Embryol. exp. Morph. Vol. 34, 2, pp. 29J-298, 1975 2 9 1

Printed in Great Britain

The RNA polymerase activity of thepreimplantation mouse embryo

By G. P. M. MOORE1

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 RNApolymerase activity. RNA synthesis was detected by autoradiography as the incorporationof [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 appearedto be confined to the nucleoplasm and could be abolished by a-amanitin but not by lowconcentrations of actinomycin D. Polymerase activity which was not affected by a-amanitinwas first detected in the four-cell embryo, predominantly at the peripheries of the nucleoli.Nucleolar labelling increased markedly between subsequent cleavages, reaching a peak inearly morulae. In one- and two-cell embryos, label incorporation could be found in thenucleus of the persisting polar body.

INTRODUCTION

Nuclear RNA synthesis is initiated in the mouse embryo soon after fertiliza-tion. Both autoradiographic and biochemical studies have shown that RNAprecursors are incorporated into the two-cell embryo and that RNA synthesisincreases 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 havebeen partially characterized. Woodland & Graham (1969) demonstrated thatsmall quantities of both high and low molecular weight RNA are synthesizedin two-cell embryos. A 4S component of the low molecular weight RNA wasdescribed in four-cell embryos and further characterized as transfer RNA atthe eight-cell stage. RNA precursor incorporation into 4S and DNA-like RNAspecies 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 embryocleavage has hindered efforts to determine whether transcriptional activity isactually initiated in the two-cell embryo or whether it occurs earlier. Indicationsof RNA precursor incorporation into one-cell embryos have been reported(Mintz, 1964; Monesi & Salfi, 1967; Knowland & Graham, 1972) but theserequire verification. The present study investigates the activation of the mouseembryo genome during early development, in vivo, by assaying isolated embryosfor endogenous RNA polymerase activity. Earlier studies of changes in RNApolymerase activity during cleavage in the mouse have utilized enzymes solu-bilized from whole embryo homogenates (Siracusa, 1973; Warner & Versteegh,1974). The experimental method used here is based on observations whichindicate that cells briefly fixed in organic solvents retain the capacity to in-corporate ribonucleoside triphosphates into trichloroacetic acid-insoluble, ribo-nuclease-digestible products (Moore, 1971; Moore & Ringertz, 1973). Theactivity of different forms of the nuclear polymerases have been localized withinthe embryo nuclei using actinomycin D and a-amanitin. Actinomycin D at lowconcentrations preferentially inhibits the nucleolar, polymerase I activity whichis involved in the synthesis of ribosomal RNA. Alpha-amanitin is a specificinhibitor of the nucleoplasmic enzyme polymerase II, which catalyses thesynthesis of heterogeneous nuclear RNA (Zylber & Penman, 1971).

MATERIALS AND METHODSPreparation of embryos

Female mice of the Bagg albino strain were caged with males and examineddaily for the appearance of vaginal plugs. For the purposes of dating, the dayon which a plug was found was regarded as the first day of gestation. Cleavageembryos at various stages of development were either dissected from the ovi-ducts or recovered from the uteri by flushing with saline. With the aid of adissecting microscope each embryo was drawn into a fine pipette and trans-ferred to a glass slide in a drop of saline. Excess fluid was drained and theembryo fixed in fresh ethanol and acetone (1:1 by vol.; see Moore, Lintern-Moore, Peters & Faber, 1974). The preparations were then air-dried and storedat 4 °C in a desiccator until required.

Assay for endogenous RNA polymerase activity

Embryos were assayed for RNA polymerase activity using the method pre-viously described for fixed preparations (Moore & Ringertz, 1973; Moore et al.1974). The ribonucleoside triphosphates ATP, GTP and CTP were purchasedfrom the Sigma Chemical Company, Missouri, U.S.A., and [3H]UTP from theRadiochemical Centre, Amersham, U.K. Polymerase I and II activities weredistinguished 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 293Each embryo was incubated with one or two drops of the assay solution for30 min at 37 °C. The reaction was terminated by rinsing slides in distilled waterand immersion in ethanol and acetic acid fixative solution (3:1 by vol.) for30 min. Unincorporated nucleotides were extracted with cold 5 % trichloro-acetic acid (TCA) for 5 min, and the slides were washed in running water for30 min. The autoradiographic procedures performed were similar to thosedescribed previously for isolated mouse oocytes (Moore et ah 1974).

The results of this study are based on observations made on approximately300 embryos prepared and assayed in this way.

RESULTS

Mouse embryo cells utilized [3H]UTP in the presence of ATP, GTP andCTP, 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 assaysolution caused a reduction in label incorporation, indicating that the reactionwas dependent on DNA and that RNA was synthesized.

No polymerase activity was detected in the developing pronuclei of one-cellembryos (Figs. 1 and 2), nor was there any associated with the chromosomes ofthe first cleavage division (Fig. 3). However, one polar body was frequentlyfound to be labelled in these embryos (Fig. 3). Incorporation into the embryonuclei was first detected at the two-cell stage. Radioactivity was predominantlylocated within the nucleoplasm (Fig. 4A). No incorporation into the nucleoliwas evident although polymerase activity associated with the peripheral regionscannot be excluded. Label incorporation in two-cell embryos was not greatlyreduced by the inclusion of actinomycin D in the assay solution (Fig. 5) but wasabolished by a-amanitin (Fig. 6). Polymerase activity was also present in thepersisting polar body of two-cell embryos (Fig. 4B).

Radioactivity located over the nucleolus could be first observed in the four-cell embryo (Fig. 7). In the presence of a-amanitin, the label was largely con-fined to the edges of the nucleoli (Fig. 9). Nucleolar incorporation was low atthis stage and actinomycin D-resistant polymerase activity of the nucleoplasmpredominated (Figs. 8A, B). However, as the embryo progressed through latercleavages, the amount of label associated with the nucleolus increased markedly(Fig. 10). The levels of nucleolar and nucleoplasmic polymerase activitiesattained at the morula stage (Fig. 11) appeared to be maintained during blasto-cyst formation (Fig. 12).

DISCUSSION

Model studies have shown that the utilization of [3H]UTP by cells afterfixation 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 duringdifferentiation, unhampered by fluctuations either in the rates of precursortransport into the cell or in their concentrations in the intracellular pools (Ellem& Gwatkin, 1968; Daentl & Epstein, 1971; Epstein & Daentl, 1971; see alsoZetterberg, Auer & Moore, 1974). The polymerase activity demonstrable isprobably involved in the elongation of RNA molecules which have alreadybeen initiated (Zetterberg et al. 1974; Moore unpublished observations). Thusthe 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-cellembryos. This appears to conflict with the evidence of earlier studies whichsuggest that RNA precursors may be incorporated at this stage (Mintz, 1964;Monesi & Salfi, 1967; Knowland & Graham, 1972). However, Mintz statedthat her autoradiographic observations were not consistently repeatable(Mintz, 1964), whereas in the biochemical work of Monesi & Salfi. (1967) andKnowland & Graham (1972) the possibility of RNA synthesis within the polarbody was not considered. The utilization of RNA precursors by the polar bodyof the mouse embryo has not previously been observed although [3H]leucineincorporation has been reported (Mintz 1964).

The RNA polymerase activity of the nuclei of two-cell embryos was mainlylocated 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), althoughtheir observation that small amounts of ribosomal RNA may also be synthesizedat this stage was not confirmed. Alpha-amanitin-resistant, polymerase Iactivity was first detected in the nucleoli of the four-cell embryo (see also Mintz,1964). However, the particular stage of cleavage development at which ribosomalgenes 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 andfemale 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 polarbody alone is labelled, x 700.Fig. 4. A, Two-cell embryo. Label is localized predominantly in the nucleoplasm ofthe nuclei and in the polar body. The nucleoli appear unlabelled. x 350. B, Labelincorporation 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 actino-mycinD. xllOO.Fig. 6. Two-cell embryo. Assay performed in the presence of a-amanitin. Labelincorporation is abolished. xllOO.19 E M 13 34

296 G. P. M. MOORE

11

RNA polymerase activity of cleaving embryos 297

observed that [3H]uridine was actively incorporated into the nucleoli of two-cellembryos in which the second cleavage division had been temporarily delayed.

The dependence of mouse embryo development on the early activation of thegenome during cleavage has been studied, in vitro, using actinomycin D anda-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 RNAsynthesis, actinomycin D is known to affect cell metabolism adversely. Ifa-amanitin is used to block total RNA synthesis (Jacob, 1973), limited cleavagemay occur. Manes (1973) showed, for example, that rabbit embryos couldundergo the first and a few subsequent cleavage divisions in the presence of10~4M a-amanitin, a dose found sufficient to block over 95% [3H]uridineincorporation. 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-amanitincompared with untreated controls. It is concluded from these observations andthose of the present study that the one-cell mouse embryo may undergo the firstcleavage division in the absence of pronuclear RNA synthesis.

This work was supported by the Danish Medical Research Council and the NordiskInsulinfond. I gratefully acknowledge the help of Prof. M. Faber and Dr H. Peters. Thea-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 incorporationis evident, x 350.Fig. 8. A, Four-cell embryo. Polymerase assay performed in the presence of actino-mycin D. Label incorporation in the nuclei is not markedly affected, x 350. B, Labelincorporation in one of the nuclei of the four-cell embryo of figure A. x 600.Fig. 9. Four-cell embryo. Assay performed in the presence of a-amanitin. Label islargely confined to the peripheries of the nucleoli. x 400.Fig. 10. Twelve-cell embryo. Polymerase assay performed in the presence of a-amanitin. The nucleoli are heavily labelled, x 1100.Fig. 11. Morula. Both nucleolar and nucleoplasmic regions of the nuclei arelabelled, 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)