J. Cell Sci. io, 833-855 (-972) 833

Printed in Great Britain

THE EFFECT OF ACTINOMYCIN D IN VIVO

UPON PERIPHERAL NUCLEOLI AND OTHER

NUCLEAR ORGANELLES IN OOCYTES OF

TRITURUS CRISTATUS

M.H.L.SNOW

Institute of Animal Genetics,West Mains Road, Edinburgh, EHg 3JN, Scot/and

SUMMARY

Exposure of the ovaries of Triturus cristatus to actinomycin D at a concentration of 100 /tg/mlcauses characteristic changes in the peripheral nucleoli and other nuclear organelles in oocytesof o-6-i-i mm diameter. Viewed with the light microscope untreated oocytes contain nucleolithat stain uniformly with a variety of dyes. They also appear homogeneous under phase-contrast optics. After 2 or 4 h of in vivo incubation with actinomycin D, oocyte sectionsstained with Haidenhain's haematoxylin or viewed under phase-contrast optics show nucleolicomposed of 2 regions. The more heavily stained or contrasted zone is crescent-shaped anddirected away from the nuclear membrane. Neither sections stained with azure B bromide norgallocyanin chrome alum show this feature. Ribonuclease digestion does not eliminate or alter it.Autoradiography with [3H]uridine indicates that all recently synthesized RNA is lost from thenucleolus during actinomycin D treatment. The zonation is not therefore a reflexion of RNAdistribution. During recovery from actinomycin D poisoning there is a reduction in the degreeof zonation shown by nucleoli which re-establish a normal appearance some 48 h aftertreatment.

Electron microscopy of peripheral nucleoli in oocytes sampled during this treatment indicatesthat the zonation is not associated with reorganization of ultrastructural components. Duringincubation with actinomycin D the coarse granules (20 nm diameter) are completely lost fromthe nucleolus. There is associated shrinkage of the nucleolus which after treatment is foundto consist entirely of fibrils (5 nm thick) and small granules. The reappearance of the coarsegranules during recovery is completed in about 48 h. It is thought that the loss of the granularcomponent during treatment represents the movement of the 30-s precursor and the 18-sribosomal unit from the nucleolus.

Some 20-30 fim inside the nucleus of untreated oocytes is a region containing manyspheroidal bodies, less than i-o /irn diameter. They have been termed micronucleoli and consistof granules 2-5-5 n m ' n diameter and fibrils of similar thickness. Actinomycin D treatment causesthese components to segregate and eventually (within 24 h of treatment) the granular componentis extruded. This component reappears during the second day after treatment. It is postulatedthat these micronucleoli represent the sites at which the 30-s ribosomal precursor undergoes itsfinal maturation. The segregation of components induced by actinomycin D is probably themorphological manifestation of an abnormal metamorphosis of this precursor.

Treatment with actinomycin D also induces the immediate formation within the nucleus ofcrystalline bodies composed of lamellae 16 nm wide, 4 nm thick and with a centre-to-centrespacing of 8—10 nm. They are not present 24 h after treatment. They are thought to representa protein fraction normally associated with periods of intense RNA synthesis.

834 M- H- L- Snow

INTRODUCTION

There is no reason to suppose that the process of ribosome biogenesis in amphibianoocytes is fundamentally different from that in other cell types, but there is evidencewhich suggests that the nucleolus itself may be directly involved in ribosomal RNAproduction to a lesser extent than, for example, mammalian nucleoli.

Labelling experiments with cultures of mammalian cells have shown that theearliest incorporation of nucleotides into ribosomal-like RNA can be located in aprecursor molecule which has a sedimentation constant of 45 S. Labelled RNA issubsequently found in 32- and 18-s particles, and finally in 28- and 18-s particles(Girard, Penman & Darnell, 1964; Girard, Penman, Latham & Darnell, 1965;Perry, 1965). It is postulated that the 18-s rRNA molecule is formed directly fromthe 45-s precursor which consequently becomes the 32-s particle. The 32-s particlesubsequently undergoes further cleavage to yield the 28-s rRNA molecule (seePerry, 1969; Birnstiel, 1967). Since both the 28-s RNA molecule and the 45-s pre-cursor have been found in nucleolar extracts from mammalian cells (Busch et al. 1966)it would appear that the production of ribosomal RNA is completed within thenucleolus in some cases.

While Gall (1966) agrees that in principle the same process occurs in oocytes ofTriturus viridescens he failed to find significant quantities of newly synthesized 28-sand 18-s rRNA in nuclear extracts. Gall found that recently synthesized nuclearRNA showed large sedimentation peaks at 40 s and 30 s and equates these fractionswith the 45-s and 32-s precursors of mammalian cells. To explain the absence of new28-S or 18-S rRNA from the nucleus Gall concludes that the conversion of the pre-cursor molecules to the ribosome subunits occurs simultaneously with the passageof these rRNA particles to the cytoplasm.

The bulk of nuclear RNA in the amphibian oocyte is contained in the nuclear sap(Edstrom & Gall (1963) suggest a figure of 50% but this is a gross underestimate andGall's (1966) revised opinion of 85-90% is certainly nearer the true value) and it isargued that as the bulk of nuclear RNA has a sedimentation peak at 30 s much of itmust be contained in the nuclear sap rather than in the nucleoli. Gall thereforesuggests that in the oocyte of T. viridescens conversion of the 40-s to the 30-s pre-cursor results in the immediate release of the 18-s rRNA molecule to the cytoplasm,and also in release of the 30-s precursor to the nuclear sap, where it will complete itsmetamorphosis to the 28-s rRNA almost concomitantly with the migration of thisribosomal particle to the cytoplasm.

Attempts have been made to correlate the above biochemical data with ultrastruc-tural aspects of nucleoli in normal and abnormal conditions. Normal nucleoli in mostcell types studied exhibit 2 distinct regions, one characterized by a preponderance offibrous material and the other almost entirely granular. In some cell types these com-ponents are intermixed but in other, particularly amphibian oocyte nucleoli, thegranular component forms a shell or cortex around the fibrous component (see Miller& Beatty, 1969). In mammalian cells both components have been shown to containRNA and protein (Marinozzi, 1963, 1964; Bernhard & Granboulan, 1963; Granbou-

Effect of actinomycin D on newt oocyte nuclei 835

Ian & Granboulan, 1964.fi, b, 1965) but morphologically it has not been possible toequate either component with a specific stage in ribosome production nor hasthe autoradiographic study of the synthesis and movement of RNA in the nucleolusgiven conclusive results. Label, usually [3H]uridine incorporated in RNA, is firstlocated in the fibrous component and subsequently in the granular areas of thenucleolus; in terms of time this sequence of events correlates only approximatelywith the appearance of the larger RNA precursor molecule in nucleolar fractions ofmammalian cells (Granboulan & Granboulan, 1965; Simard & Bernhard, 1966;Weinberg, Loening, Willems & Penman, 1967) and amphibian cells (Macgregor,1967; Lane, 1967; Gall, 1966).

Actinomycin D is a potent inhibitor of RNA synthesis and has been shown to beparticularly effective in preventing ribosomal RNA synthesis (Perry, 1962, 1963). Inmammalian cells treated with actinomycin D the conversion of the 45-s to the 32-sribosomal precursor appears to be normal (Perry, 1962; Girard et al. 1964; Scherrer& Darnell, 1962; Scherrer, Latham & Darnell, 1963) but the conversion of the 35-sprecursor to the 28-S rRNA molecule is either inhibited or abnormal (Penman, Smith,Holtzman & Greenberg, 1966). The ultrastructural studies of nucleoli under theseconditions, in a variety of organisms, shows a rearrangement of componentsvariously described as coalescence, redistribution, segregation (Schoefl, 1964; Jacob &Sirlin, 1964; Stevens, 1964; Lane, 1969) or as nucleolar-cap formation (Reynolds,Montgomery & Hughes, 1964) whereby the granular component of the nucleolusbecomes concentrated at the periphery or to one side of the nucleolus. This is followedby a separation of granular, fibrous and an amorphous zone in the nucleolus (see alsoSuter & Salomon, 1966; Geuskens, 1966; Geuskens & Bernhard, 1966; DeMan &Noorduyn, 1967). None of these abnormal processes has been definitely correlatedwith any single aspect of the biochemical data.

The following report is of the in vivo effect of actinomycin D upon the nucleoli inoocytes of Triturus cristatus and describes a system where some aspects of nucleolarsegregation are spatially separated, and in which the stages of ribosome biogenesisare possibly similarly separated. The terms 'core' and 'cortex' for the fibrous andgranular components of the oocytes nucleolus will be used, in accordance with theterminology established by Miller (1966) and Macgregor (1967).

MATERIALS AND METHODS

Mature females of the crested newt, Triturus cristatus cristatus were obtained from L. Haig& Son, Newdigate, Surrey, England. Experiments were performed during winter and earlyspring when all fixed material was prepared, and detailed microscopical analysis carried outduring the summer while the newts were out of breeding condition.

Actinomycin D was kindly supplied by Dr H. J. Robertson, of the Merck Institute forTherapeutic Research. ['H]uridine, specific activity 760 mCi/mM was purchased from theRadiochemical Centre, Amersham.

Exposure of the ovaries in vivo to actinomycin D was carried out under anaesthesia in themanner established by Snow & Callan (1969). Ovaries were exposed through a small ventro-lateral incision in the body wall, the blood supply was ligated with 3/0 gut and the whole ovaryimmersed in frog Ringer solution containing 100 fig actinomycin D/ml. A small ovary sample(some 20 oocytes in the size range 0-7-1-1 mm diameter) was excised immediately before

836 M. H. L. Snow

treatment, after 2 h of treatment and a third sample removed after 4 h exposure to actinomy-cin D, when the ligature was released. When it had been seen that the blood supply to theremainder of the ovary had re-established the ovary was returned to the body cavity and theincision closed with 2 loops of 3/0 gut. Further small samples of oocytes were removed 24 and48 h after this actinomycin D treatment.

RNA metabolism was studied by autoradiography. ['HJundine was administered as a singlesubcutaneous injection of 200 /tCi given in a ventrolateral position about 1 cm in front of thehind limbs. All autoradiographs were for light microscopy and made on sectioned material.Kodak dipping emulsion, NTB-2, was used for sections less than 3 fim thick and strippingfilm, AR-10, for thicker sections. After suitable exposure they were developed in Kodak D- igband fixed in Kodak Metafix. NTB-2 coated preparations were stained through the film foriomin in 000375% toluidine blue; AR-10 coated material was stained in 0 1 5 % methylgreen in o-i M acetic acid/sodium acetate buffer at pH 4 7 . Autoradiographs were covered withNo. o cover-glasses mounted in Euparal.

Fixed material was prepared in several ways. Fixatives used were either Sanfelice's fluid or10% neutral formalin. The material was embedded either in paraffin wax or in methacrylate.Methacrylate was preferred for yolky eggs. Wax-embedded material was cut on a Leitz rotarymicrotome to give sections 8-12 fim thick; methacrylate-embedded mateiial was cut with aglass knife on a Porter-Blum Ultramicrotome to give sections 1 or 2 fim thick.

Heidenhain iron haematoxylin was used as a general nuclear stain; RNA distribution wasstudied using the azure B bromide technique of Flax & Himes (1952) or the gallocyanin/chrome alum technique described by Swift (1955). This material was examined and photo-graphed using a Zeiss Photomicroscope with automatic exposure.

Oocyte material for electron microscopy was fixed for 20 min in 10% glutaraldehyde inphosphate buffer pH 6-5, and postfixed for 60-90 min in 1 % osmium tetroxide in veronal/c i N HC1 buffer pH 72—75. The material was embedded in Vestopal-W polyester. Silver orgrey (90-nm) sections were cut with a glass knife on a Huxley-type ultramicrotome. They werestained for 5 min in 2 % uranyl acetate and 2 min in 0 3 % lead citrate, and then examined ina Siemens Elmiskop I Electron microscope.

RESULTS

Light microscopy

Sections through untreated oocytes, stained with iron haematoxylin, azure B bro-mide or gallocyanin/chrome alum show nucleoli in which no marked zonation canbe observed (Figs. 1-3). Nor is zonation apparent under phase-contrast optics (Fig. 4).After 2 or 4 h actinomycin D treatment in vivo oocytes above 0-7 mm diameter containmedium and large nucleoli most of which, cut perpendicular to the nuclear membrane,exhibit a characteristic zonation when stained with haematoxylin, or when viewedunder phase-contrast optics (Figs. 5, 6). The nucleolus appears to have segregatedinto 2 zones which stain with different intensities. The darker, or more contrasted zoneis crescent-shaped and is always directed towards the centre of the nucleus. Thereoccasionally appears to be a clear region separating these zones which may representa type of vacuolation (Fig. 5). In azure B bromide-stained sections it is extremelyunusual to find any evidence of nucleolar zonation. Where zonation has been seen itdoes not conform to the description above. Zonation in these cases is irregular andill-defined (Fig. 7) and most probably reflects surface irregularities of the sectionedmaterial. Gallocyanin staining on no occasion revealed zonation of nucleoli. This isan interesting finding since H. C. Macgregor (personal communication) has foundgallocyanin to be a very sensitive stain for demonstrating RNA distribution in nucleoli.For comparison Fig. 8 shows a section through an untreated oocyte, of comparable

Effect of actinomycin D on newt oocyte nuclei 837

size, from Bufo bufo stained with gallocyanin. The differentiation into core and cortexis very well marked.

After ribonuclease treatment nucleoli do not show staining with azure B or gallocya-nin but the zonation of the nucleolus is still apparent in haematoxylin-stained sections(Fig. 9)-

Although I have never observed a marked zonation in nucleoli of untreated oocytesin T. cristatus, Macgregor (1967) in his figs. 2-4 shows such a zonation in sections ofmethacrylate-embedded material prepared for autoradiography and stained withmethylene blue.

In oocytes sampled 24 h after actinomycin D treatment the nucleolar zonation isconsiderably reduced (Fig. 10) and cannot be observed 48 h after treatment, when thenucleoli are indistinguishable from those in untreated oocytes.

Autoradiographs of sections through oocytes that have incorporated pHJuridineindicate that during actinomycin D treatment almost all recently synthesized RNAleaves the nucleolus. Fig. 11 shows a preparation made immediately before actinomy-cin D treatment in which the nucleoli are heavily labelled with [3H]uridine. Fig. 12shows a comparable preparation from the same animal immediately after 4 h ofactinomycin D treatment.

Electron microscopy

The peripheral nucleoli of T. cristatus do not show a very well defined ultrastructuralzonation into core and cortex. Untreated nucleoli are composed of granules 15-20 nmin diameter among which scattered groups of fibrils, about 10 nm thick, can be located.Both these components embedded in an amorphous matrix can be seen in Figs. 13 and14. In nucleoli in which a zonation is apparent the core is usually eccentrically situatednearer to the nuclear membrane.

After the 4 h actinomycin D treatment the peripheral nucleoli are reduced in sizeand consist of fibrils 5 nm thick embedded in a finely granular matrix (Figs. 15, 16).The 20-nm diameter granules have been lost. The nucleolus has become difficult tostain and in order to obtain sufficient contrast within the section the staining times wereextended to 10 min in uranyl acetate and 5 min in lead citrate. In oocytes fixed after2 h of actinomycin D treatment these structural changes are already completed.There is no indication of the granular component in the nucleolus nor are there anytraces of it in the nuclear sap in the immediate vicinity of the nucleolus. At this stagei-^m thick sections of this treated material, stained with methylene blue in borax, donot show nucleolar zonation when viewed with the light microscope.

Twenty-four hours after actinomycin D treatment peripheral nucleoli appear tobe regaining their granular component. Throughout the nucleolus particles some10-15 nm diameter can be seen (Fig. 17). The nucleoli have increased in size but arenot yet as large as those of untreated oocytes, and furthermore they show considerablevacuolation. One day later this vacuolation is the only indication that the peripheralnucleoli have suffered damage at all. They have regained their full size and thegranular component of the nucleolus once again consists of 15-20 nm diameterparticles (Fig. 18).

838 M. H. L. Snow

From the above description it is clear that classical nucleolar segregation, asdescribed by Schoefl (1964), has not been observed in the peripheral nucleoli ofT. cristatus oocytes. However, some 20-30 /tm inside the nucleus there is a regioncontaining large numbers of spheroidal bodies, less than 1 /tm in diameter, whichdo undergo a segregation of components under the influence of actinomycin D. Thesebodies occurred in all oocytes examined and the size of these oocytes (O-6-I-I mmdiameter) excludes the possibility that they represent sections through parts of ring-nucleoli (Lane, 1967). In the 100 or more oocytes examined, from 6 differentanimals, only once has a micronucleolus been observed outside this narrow region.On that occasion it was found adjacent to the nuclear membrane. For reasons thatwill become apparent these bodies will be termed micronucleoli.

In untreated oocytes the micronucleoli are composed of granules 2-5-5 n m m

diameter and fibrils of similar thickness. The granules show a tendency to formaggregates some 20 nm across, composed of 20-50 smaller particles. Fig. 19 showsan example of these micronucleoli and it can be seen that there is some evidencefor a continuity between the nuclear sap and these bodies.

The micronucleoli undergo a considerable change during actinomycin D treatment,exhibiting all the characteristics of classical nucleolar segregation. There has beena concentration of the granular aggregates to one side, or around the periphery of themicronucleolus. These aggregates form the electron-dense regions in Figs. 20-22,and they may now be some 30-40 nm across. In the less-dense region, granules3-5 nm in diameter are embedded in a fibrillar network. The fibrils are 3-5 nm thick.In many cases the nucleoprotein network of the nuclear sap appears to be continuouswith and to be streaming either into or away from the micronucleolus (Figs. 21, 22).

Twenty-four hours after actinomycin D treatment the granular aggregates of themicronucleoli are greatly reduced and the bodies are small and consist almostentirely of the 3-5 nm diameter granules and fibrils (Fig. 23). It is not clear whetherat this stage after antibiotic treatment the granular aggregates are still being lost fromthe micronucleoli or whether the presence of a few scattered groups of particlesrepresents the reaccumulation of the granular component. It seems more reasonable tosuppose the latter case is true as 48 h after actinomycin D treatment the micronucleoliare essentially the same as in the untreated condition (Fig. 24) although the granularaggregates tend to be less well defined.

Also present in the nucleus of oocytes treated with actinomycin D are many rod-shaped bundles of what appear to be fibrils, similar to those found by Lane (1969) andnamed fibriUar bodies. These bodies are found in the nucleus immediately after anti-biotic treatment, they are not especially associated with peripheral nucleoli, nor withmicronucleoli, and can be found throughout the nucleus although they are less abun-dant in the peripheral nuclear sap, and have not been seen close to the nuclear mem-brane. They cannot be found 24 h after actinomycin D treatment. In this study thesebodies reached a length of 3-4/tm and a diameter of about 0-4 /tm. They weregenerally too thin to be observed by light microscopy. Electron microscopy showsthese structures to be composed of lamellae (not fibrils as described by Lane) about16 nm wide and 4 nm thick with a centre-to-centre spacing of 8-10 nm. Figs. 25 and

Effect of actinomycin D on newt oocyte nuclei 839

26 show these lamellar bodies in longitudinal section and Figs. 26 and 27 in cross-section. It can be seen that these bodies also are continuous with the nucleoproteinnetwork of the nuclear sap.

DISCUSSION

Bearing in mind that actinomycin D-induced nucleolar segregation had been re-ported in T. viridescens for oocyte nucleoli (Lane, 1969) and for lung cell nucleoli(Burns, 1968) it was at first sight puzzling that it had not been observed in theperipheral nucleoli of T. cristatus. The absence of the granular component from thenucleoli after treatment suggested segregation had been completed during incubationwith actinomycin D. There is, however, reason to believe that this explanation is notvalid and that nucleolar segregation of the type described by Schoefl (1964) genuinelydoes not occur in the peripheral nucleoli of these animals. In the oocytes fixed aftera 2-h actinomycin D treatment there was no evidence of rearrangement of com-ponents prior to loss of granular material. It is extremely doubtful that the processof segregation would have been completed in less than 2 h. Burns (1968) reports thatlung cell nucleoli of T. viridescens require a minimum of 3 h to complete their segre-gation when incubated in vitro with ioo/tg actinomycin D/ml of culture medium;in vivo systems are generally less susceptible to actinomycin D poisoning (Flickinger,1963; Harel, Harel, Boer, Imbenotte & Carpari, 1964).

The process of maturation of the ribosomal RNA molecules from the 45-s RNAprecursor has been reported to take 60-90 min (Penman et al. 1966; Perry, 1962,1965) but Weinberg et al. (1967) report that labelled 28-s ribosomal RNA can bedetected by acrylamide gel electrophoresis 42 min after administration of [Me-14C]-methionine. As the above observations involve a 2- or 4-h incubation with actinomycin Dthe metamorphosis of the 40-s precursor would, under normal conditions, be expectedto be complete. If the production of the 30-s rRNA precursor and 18-s rRNA moleculeis not affected by actinomycin D (as reported for the mammalian system by Perry,1962; Girard et al. 1964; Scherrer & Darnell, 1962; Scherer et al. 1963), then theloss of [3H]uridine labelling from the nucleolus during treatment is strong supportfor Gall's hypothesis that the 30-s precursor leaves the nucleolus in Triturus. If theconversion of the 30-s precursor to the 28-s molecule occurred in the nucleolus itwould be expected that the prevention of this step by actinomycin D would resultin retention of label in the nucleolus as reported by Schoefl (1964). Nevertheless thepossibility of breakdown of this RNA species cannot be ruled out; Harris (1963) andSchwarz & Garofalo (1967) present evidence for the intranuclear breakdown of RNAin the presence of actinomycin D.

The maturation of the 40-s precursor and the migration of its products from thenucleolus would account for the loss of the granular component seen in electronmicrographs of nucleoli treated with actinomycin D. There is, however, only indirect•evidence to link this nucleolar component with specific RNA molecules. Woodland& Graham (1969) have demonstrated that during the cleavage stages of mouse•embryos 28-s and 16-s RNA can first be detected during the 4-cell stage. After 6 h

840 M. H. L. Snow

in 4-cell stage RNA of high molecular weight sediments in 2 peaks, one coincidentwith the 16-s RNA and one sedimenting slightly faster than marker 28-s RNA.Allowed a further 6 h of development the heavier peak becomes coincident with the28-s RNA marker. The timing of this 'maturation' of the 45-s precursor into ribo-somal RNA molecules exactly coincides with the appearance and development ofa granular cortex in the nucleoli of these embryonic cells (Calarco & Brown, 1969;Hillman & Tasca, 1969).

The large reduction in size of the peripheral nucleoli suggests a genuine loss ofmaterial rather than an in situ breakdown of the granules. It is unlikely that thismaterial has migrated to the cytoplasm; actinomycin D has been shown to inhibitthe passage of RNA from nucleus to cytoplasm, at least in mammalian cells (Girardet al. 1964; Schwarz & Garofalo, 1967). This leaves the most likely possibility thatthe granular material or at least its breakdown products are still in the nucleus.

It has been suggested by Lane (1969) that the lamellar bodies found in the nucleusafter actinomycin D treatment may represent accumulation of ribosomal proteinsince they apparently contain neither DNA nor RNA, although there is not anoticeable spatial relationship between lamellar bodies and nucleoli. The reports ofidentical bodies in a variety of cells do little to confirm or reject Lane's suggestionbut they do lead to the belief that these bodies are associated with cycles of RNAsynthesis. Mann (1894) reported intranuclear rods in dog nerve cells stained withmethylene blue. More recently ultrastructural studies on mammalian nerve cells havefound similar intranuclear rods in a small percentage of cells (Siegesmund, Dutta &Fox, 1964; Popoff & Stewart, 1968; Hirano & Zimmerman, 1967). Such structureshave also been found in normal human thymus cells (Henry & Petts, 1969) and inkidney cells of monkey infected with SV 40 virus (Granboulan, Tournier, Wicker &Bemhard, 1963). All these ultrastructural studies show the intranuclear rods to beindistinguishable from the lamellar bodies described above. A very similar lattice-like structure has been found in neurons of rats and mice (Chandler, 1966; Chandler& Willis, 1966). It is noteworthy that the cells in which these rods have been found arealso cells rich in and normally actively synthesizing RNA.

Bearing in mind that only a small percentage of the cells in the above-mentionedstudies possessed intranuclear rods the question that immediately arises is whetherthese cells are normal and healthy. If they are, it is unlikely that the lamellar bodiesrepresent breakdown products from a defunct nuclear metabolic process. It is moreprobable that they represent an accumulation of a particular protein, such as RNApolymerase or a structural protein, that is required to meet the demands of periods ofintense synthetic activity, and produced in excess. Such conditions would arise bythe sudden cessation of, for example, RNA synthesis and the small delay that wouldoccur before the influx of enzymes and structural proteins into the nucleus alsostopped. These conditions are produced by the inhibitory action of actinomycin Dand changes of activity, involving RNA synthesis, are a characteristic feature of nervecells and of virus-infected cells. Thymus cells also appear to undergo cyclical changesin their activity (see Everett & Tyler, 1967).

Further insight into the nature of the lamellar bodies could be obtained by

Effect of actinomycin D on newt oocyte nuclei 841

determining whether their formation is prevented by inhibitors of protein synthesissuch as puromycin cr cycloheximide administered shortly before actinomycin D.Such an analysis would show if these bodies are formed by material already in thenucleus or whether influx of new proteins is necessary.

Turning once again to Gall's (1966) scheme for ribosome biogenesis, the site atwhich the 30-s precursor is converted to a 28-s rRNA molecule must be sought.It is possible that the precursors are randomly distributed throughout the nuclearsap but haphazard organization is not a characteristic of biological systems. Neverthe-less, if the 30-s precursor carries with it all the requisite material for production of the28-s RNA molecule then an organized site for the conversion would be unnecessary.Since the primary action of actinomycin D is inhibition of DNA-dependent RNAsynthesis and protein synthesis is affected as a second order phenomenon, theabnormal conversion of the 30-s precursor in the presence of actinomycin D suggeststhe lack of an essential component, either an RNA molecule or a protein (a structuralprotein or an enzyme). The incorporation or involvement of this extra componentwould be greatly facilitated by the existence of specific conversion sites.

In this respect the presence and behaviour of the micronucleoli is of interest.Although any site for the final maturation of ribosomal particles might be expected tobe located very close to the nuclear membrane it is perhaps significant that themicronucleoli undergo a segregation that is precisely similar to that exhibited bynucleoli of cell types in which final production of the ribosomal subunits is regardedas a nucleolar function. Could the process of nucleolar segregation be the morphologi-cal manifestation of the abnormal metamorphosis of the smaller ribosomal precursor?

The nucleolar zonation seen after actinomycin D treatment in preparations madefor light microscopy is in no way similar to that seen in the ultrastructural studies. Itwas at first thought to be due to a redistribution of RNA but the failure to demonstratethis with azure B bromide, gallocyanin or autoradiography must rule out thisexplanation. Bearing in mind that contraction of nuclear contents is a recognizedfixation artifact following actinomycin D treatment (Simard, 1966; Burns, 1968) itis most likely that this is the explanation for the light-microscope observations.Nevertheless, it is noteworthy that the nucleolus splits into 2 zones under theseconditions and stimulates speculation about the protein composition of the 2 regions.

This work was carried out during tenure of an S.R.C. Research Studentship in the ZoologyDepartment, University of St Andrews, Fife, Scotland.

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HILLMAN, N. & TASCA, R. J. (1969). Ultrastxuctural and autoradiographic studies of mousecleavage stages. Am. J. Anat. 126, 151-174.

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844 M. H. L. Snow

Fig. 1. Untreated peripheral nucleoli. Stained with Haidenhain's haematoxylin. x 1500.

Fig. 2. Untreated nucleoli. Stained with azure B bromide, x 1500.

Fig. 3. Untreated nucleoli. Stained with gallocyanin chrome alum, x 1500.

Fig. 4. Untreated nucleoli. Viewed with phase-contrast optics, x 1500.

Fig. 5. Nucleolus immediately after 4 h incubation with actinomycin D (100 /ig/ml).Stained with Haidenhain's haematoxylin. Note the lightly staining gap (vacuole??)indicated by the arrow, x 1500.Fig. 6. As Fig. s, viewed with phase-contrast optics, x 1500.

Fig. 7. As Fig. 5, showing the irregular zonation found in nucleoli stained with azureB bromide, x 1500.

Fig. 8. Nucleoli in an untreated oocyte from Bufo bufo. Stained with gallocyaninchrome alum. The differentiation of core and cortex is clearly indicated, x 1500.

Fig. 9. As Fig. 5. Stained with Haidenhain's haematoxylin, after exhaustiveribonuclease digestion, x 1500.

Fig. 10. A medium-sized nucleolus 24 h after actinomycin D treatment. Stainedwith Haidenhain's haematoxylin. x 1500.Fig. 11. Autoradiograph of nucleoli in an untreated oocyte. PHjuridine administered24 h before fixation; 31 days exposure, x 1500.

Fig. 12. Autoradiograph of nucleoli in an oocyte fixed immediately after a 4-h incuba-tion in actinomycin D (100 /ig/ml). [3H]undine administered 24 h before treatment;31 days exposure, x 1500.

Effect of actinomydn D on newt oocyte nuclei 845

846 M. H. L. Snow

Fig. 13. Electron micrograph showing a section through the centre of a peripheralnucleolus. The differentiation of core and cortex is particularly clear in this example.The nuclear membrane is just off the bottom of the picture. The area marked isshown enlarged in Fig. 14. x 20000.Fig. 14. Area of nucleolus outlined in Fig. 13, showing the different composition ofcore and cortex. Fibrils can be seen in the core at the places indicated by arrows,x 40000.

Fig. 15. Part of a nucleolus taken immediately after 4 h incubation in actinomycin D(100/tg/ml). It is composed entirely of fibrils and fine granular matrix, x 80000.

Ejfect of actinomydn D on newt oocyte nuclei 847

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Fig. 16. A complete nucleolus after actinomycin D treatment. Note the reductionin size and also the absence of any large granular component, x 40000.Fig. 17. Nucleolus 24 h after actinomycin D treatment. Vacuolation is considerablebut the granular component appears to be reforming, x 20000.

Effect of actinomycin D on newt oocyte nuclei

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Fig. 18. Nucleolus 48 h after actinomycin D treatment. Compare with Fig. 13.The composition of the nucleolus is normal but increased vacuolation is still apparent,x 20000.

Fig. 19. Section through a micronucleolus from an untreated oocyte. Granules andfibrils can be seen throughout this structure. The concentration of granules intoconglomerates is also clear, x 90000.Fig. 20. As Fig. 19, taken immediately after 4 h incubation in actinomycin D (100 figjml). Note the peripheral concentration of the granular conglomerates. The structurebelow the micronucleolus is a transverse section through a lamellar body, x 60000.

Effect of actinomydn D on newt oocyte nuclei 851

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Fig. 21. As Fig. 20. Note the apparent streaming of the nucleoprotein network ofthe nucleus, x 60000.Fig. 22. As Figs. 20 and 21. The apparent continuity between these niicronucleoliand the nuclear sap network is clearly seen all round this body, particularly in theregions indicated by arrows, x 90000.Fig. 23. A micronucleolus 24 h after actinomycin D treatment, x 60000.Fig. 24. A micronucleolus 48 h after actinomycin D treatment, x 40000.

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Fig. 25. A longitudinal section through a lamellar body found immediately afteractinomycin D treatment. Continuity with nuclear sap material is indicated byarrows, x 120000.

Fig. 26. As Fig. 25 but also showing a cross-section through a small lamellar body(arrow), x 40000.

Fig. 27. A cross-section through a large lamellar body, x 120000.

Ejfect of actinomydn D on newt oocyte nuclei 855

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