The origin and composition of nucleolus-like inclusions in the cytoplasm
of fern egg cells
P. R. BELL and R. I. PENNELL*
Department of Botany and Microbiology, University College, London WCIE 6BT, UK
•Present address: John Innes Institute, Colney Lane, Norwich NR4 7UH, UK
Summary
Investigations of oogenesis in a number of lepto-sporangiate ferns have revealed nucleolus-likebodies in the cytoplasm of the maturing egg cells.In Pteridium and Lygodium these bodies appearto be aggregates of material secreted from pro-trusions of the nucleus. They are acidic in reac-tion, but autoradiography following feeding with[3H]uridine and the use of an RNase-gold probe,has failed to demonstrate the presence of RNA. It
is concluded that the bodies consist of acidicprotein. Since their appearance coincides with anamoeboid condition of the nucleus and extremedispersal of the chroma tin, it is suggested thatthey represent a temporary storage in the cyto-plasm of non-histone structural proteins of nu-clear origin.
The presence of numerous small, nucleolus-like,bodies in the nuclei of both reproductive and somaticcells of plants at certain stages of meiosis or differen-tiation is well known (see, e.g., E. G. Williams et al.1973; L. M. Williams et al. 1983). These bodies havebeen generally regarded as containing RNA, butsimilar bodies appearing in the nucleus during matu-ration of the egg cell of the fern Pteridium lack RNAand are removed by protease digestion (Bell, 1983;Pennell & Bell, 1985). Small fibrillar 'round bodies'that arise adjacent to nucleoli in the nuclei of ratspermatocytes and early spermatids (Schultz et al.1984) have also been shown to be free of RNA, and toconsist of non-histone protein.
Bodies resembling small nucleoli in both size andtexture also occur in the cytoplasm of the reproduct-ive cells of plants. Sometimes the resemblance isstrengthened by the presence of electron-transparentregions strikingly similar to the lacunae of truenucleoli. Examples are provided by the nucleoloidspresent in the cytoplasm during micro- and mega-sporogenesis in Lilium (Dickinson & Heslop-Harrison, 1970; Williams et al. 1978; Dickinson &Potter, 1978), and the 'pseudo-nucleoloids' in thecytoplasm of the spore mother cells of the fern
Pteridium (Sheffield & Bell, 1979). In sexual repro-duction nucleolus-like inclusions are found in thecytoplasm of the egg cells of conifers (Konar &Oberoi, 1969; Konar & Moitra, 1980) and Taxus(Pennell & Bell, 1985), and of ferns (see, e.g., Bell,1975a, fig. 7). Similar bodies are sometimes seen inthe cytoplasm of meristematic cells (Sato, 1981).
The chemical composition of nucleolus-like bodiesin the cytoplasm has not been extensively investigated.In Lilium (Dickinson & Willson, 1985) and in the eggcells of Taxus (Pennell & Bell, 1985) there is evidencethat they contain RNA. In the egg cells of Pteridium(unpublished experiments) the nucleolus-like bodiesin the cytoplasm resist digestion with RNase, a featurethey share with the nuclear bodies (Bell, 1983), whichthey closely resemble.
The origin of the nucleolus-like inclusions in thecytoplasm of fern egg cells has been further investi-gated, and their chemical nature explored by means ofradioactive labelling and the use of an RNase-goldcomplex as a probe for RNA.
Materials and methods
Gametophytes of Pteridium were raised in pure cultureas described previously (Bell, 1960). Fully archegoniategametophytes were transferred for 2 days to filter paper
283
Fig. 1. Portion of a newly formed egg cell of Lygodium. p, plastid; m, mitochondrion; n, nucleus. The arrows point tosmall vesicles containing osmiophilic material, which are eliminated as the egg matures. The upper surface of the cell is tothe right, x 13 500. Bar, 1 ^m. Inset: detail of small vesicles. X55 000. Bar, 0-1 [im.Fig. 2. Pleomorphic plastid in a maturing egg cell of Lygodium. The cup-shaped extension encloses mitochondria. Thearrows indicate protrusions arising from the main body of the nucleus to the left. X30 000. Bar, 1 fim.Fig. 3. Nuclear protrusions (np) in a maturing egg cell of Lygodium connected by electron-opaque granular material in theintervening cytoplasm. The section has passed to one side of the connection of the lower protrusion with the main body ofthe nucleus (left) so that it appears closed. X60000. Bar, 05 fim.
284 P. R. Bell and R. I. Pennell
moistened with Moore's (1903) medium containing40/iCiml"1 5-tritiated uridine (Amersham, UK, sp. act.5 Cimmol"1). They were subsequently replaced and grownfor 4 days on unlabelled medium. Thin slices containingarchegonia were then excised and fixed in 4 '5% glutaral-dehyde (TAAB Laboratories, Reading, UK) in 0-05 M-phosphate buffer (pH6-9) for 6h at room temperature.Subsequent post-fixation in osmium tetroxide (when per-formed), dehydration and embedding in Durcupan ACM(Fluka AG, Buchs, Switzerland) were as described by Bell(1980). Two gametophytes of the same batch were fixed inacetic ethanol for 1 h at 4°C, and subsequently dehydratedand embedded in wax.
Gametophytes of Lygodium japonicum were raised onsterile soil from spores obtained from the University ofLondon Botanic Garden, Egham. The cultures were main-tained at room temperature, and illuminated (12 h light/12 hdark) by a battery of fluorescent lights supplemented withlow-power incandescent lamps, giving an irradiance at benchsurface of 23Wm~2. Individual gametophytes about 2 mmlong were transferred to moist filter paper on sterile soil innew dishes. For radioactive labelling, gametophytes about5 mm long (just beginning to produce archegonia) wereremoved and fed with tritiated uridine in the same manner asW\th Pteridium, but at a concentration of lO^Ciml"1 and for6h. After the feeding some slices were fixed immediately,other gametophytes were allowed to grow for a further 18 hin the absence of label before fixation. Subsequent pro-cedures were the same as for Pteridium. For the examinationof the cytology of the egg cells, and the cytochemistry ofunlabelled material, thin slices were taken from fully arche-goniate gametophytes about 7 mm long.
The embedded material was cut at 4 fim with a glass knife,and the sections scanned with phase-contrast optics formedian sections of egg cells. The selected sections wereremounted for fine-sectioning by the method of Woodcock &Bell (1967).
Autoradiographs of fine sections were prepared by a loopmethod (Sigee & Bell, 1971), using Ilford L4 emulsion(Ilford, UK). Exposure times were 5-6 weeks, but somepreparations were left for 19 weeks before developing. Auto-radiographs of the wax-embedded material of Pteridiumwere also prepared by a loop method, but using Ilford G5emulsion. Before application of the film half the slideswere incubated in a solution of ribonuclease (type 1A frombovine pancreas; Sigma, Poole, UK) at a concentration oflmgrnl"1 in 0-05 M-phosphate buffer (pH7-0) for 2-5 h at35°C. The controls were incubated in buffer alone. Ex-posure of the autoradiographs was for 1 week.
Tests for RNA in fine sections of unosmicated material bymeans of an RNase-gold probe followed the proceduredescribed by Pennell & Bell (1985). The affinity of thecytoplasmic inclusions for the uranyl ion was investigated bystaining fine sections of similar unosmicated material for5min in cold aqueous uranyl acetate (70gl~'). Stainingl-5/im resin sections with Azure B followed the method ofJensen (1962), allowing at least 2h in the stain.
Results
Oogenesis in Lygodium proved to be in all essentialrespects similar to that in Pteridium (Bell, 1979).Newly formed egg cells contained an irregularlyshaped nucleus (Fig. 1). Although the nucleolus wasprominent, nuclear bodies were absent. Also lackingwere distinct protrusions of the nucleus into thecytoplasm. The plastids retained a few grana andoccasional small starch grains. Numerous small ves-icles, almost all containing strongly osmiophilic ma-terial, were present in the upper part of the cytoplasm(Fig. 1 and inset).
Subsequently, as the egg cell matured markedchanges occurred. The plastids became dense andpleomorphic, and their cup-shaped extensions oftenenclosed mitochondria (Fig. 2). Simultaneously, thenucleus began to produce protrusions in a manneralready well known in Pteridium and some otherleptosporangiate ferns (Bell, 1979, 1980). Electron-opaque material, texturally similar to that lining theinner membrane of the nuclear envelope of the nuclearextension, was detected in the cytoplasm adjacent to
Fig. 4. Granular electron-opaque aggregates in thecytoplasm of a mature egg cell of Lygodium connected to anuclear protrusion (np) by less-opaque material of similartexture. X45 000. Bar, 0-5/im.
Nucleolus-like inclusions in cytoplasm 285
Fig. 5. Two large nucleolus-like bodies in the cytoplasm of an egg cell of Lygodium, each associated with a smaller less-opaque mass. X30000. Bar, l^m.
I
Fig. 6. Part of an autoradiograph of Pteridium showing thetextural similarity between the nucleolus-like body free inthe cytoplasm (above) and a nuclear body enclosed in anuclear protrusion (below). Labelled with tritiated uridine.X 100 000. Bar, 0-1 /im.
some of the extensions (Fig. 3). Similar material wasalso found in association with even denser masses inthe cytoplasm (Fig. 4), some of which reached 2/xm indiameter and bore a striking resemblance to nucleoli(Fig. 5). Examination of the radioactively labelledPtendtum confirmed that in this species also, asdescribed previously (Bell, 1975a), the cytoplasm ofthe egg cell contained numerous nucleolus-like bodies,although here their diameter rarely exceeded 0-5 fm\.In both Pteridium (Fig. 6) and Lygodium there was ageneral textural resemblance between the materiallying freely in the cytoplasm and that of the nuclearbodies within the nucleus.
The light-microscopic autoradiographs preparedfrom Pteridium confirmed that the unextracted ma-terial was heavily labelled. Ribonuclease removedmost of the radioactivity, the grain frequency fallingclose to background.
The high-resolution autoradiographs, particularlythose of Pteridium that had been exposed for 19 weeks,showed dense labelling of the nucleolus (Fig. 7). Inline with the previous investigation (Bell, 1983) therewas no labelling of the nuclear bodies (Fig. 7). Therewas also no firm evidence of labelling of the nucleolus-like bodies in the cytoplasm. Although silver grainswere often clustered close to these bodies, and partsof grains sometimes overlapped them, grain centreswere regularly absent from above them (Figs 6, 8).Although the frequencies of the silver grains in theautoradiographs of the Lygodium egg cell were lower,the results were exactly comparable. The presence ofradioactivity in the nucleolus-like bodies could not beconvincingly demonstrated.
286 P. R. Bell and R. I. Pennell
Fig. 7. Portion of a representative autoradiograph of a maturing egg cell of Ptendium following feeding with [3H]uridineand a 19-week exposure. Despite the high concentration of radioactivity in the nucleolus (nu) and elsewhere, the nuclearbodies both in the main body of the nucleus and in nuclear protrusions (arrows) lack silver grains. X17 000. Bar, 1 jUm.
The tests with RNase-gold gave equally consistentresults. In neither Ptendium nor Lygodium (Figs 9,10) were gold particles present above the cytoplasmicinclusions, but they were numerous above the groundcytoplasm. Even those inclusions with the closestresemblance to small nucleoli (Fig. 7) showed noaffinity for the labelled probe. As in previous tests ofthe technique (Pennell & Bell, 1985) the nucleolusitself bound the RNase-gold complex without excep-tion.
Staining unosmicated material with uranyl acetateshowed that the cytoplasmic inclusions, together withthe nucleoli and nuclear bodies, had a marked affinityfor the stain. In 1-5 (Urn resin sections stained withAzure B, the larger cytoplasmic inclusions and thenucleoli could be seen to have retained the stain.
Discussion
The similarity in oogenesis between the two fernsPteridium and Lygodium is evidently close, extendingfrom the elimination of a mass of small vesiclescontaining osmiophilic material from the cytoplasm ofthe young egg cell to the production of well-definednuclear protrusions as the egg matures. These featureshave been interpreted as indicating an initial phase ofautophagy, a characteristic of cells undergoing cyto-plasmic reorganization (Matile, 1975), followed byintense nucleocytoplasmic interaction (Bell, 1979).The formation of nucleolus-like bodies in the cyto-plasm of the maturing egg cell appears to be a furtheressential element in oogenesis.
The relatively small size of the cytoplasmic in-clusions in Pteridium makes it difficult to be certain ofthe stage at which they first appear. The inclusions ofthe Lygodium egg cell, however, cannot be over-looked. There is no doubt that they were absent fromthe cytoplasm of the cell at its formation. Theirappearance coincided with the beginning of the pro-duction of nuclear protrusions, and the micrographsstrongly suggest that the material of which they arecomposed is secreted by the nucleus. This is alsoplausible for the similar bodies in Pteridium, but theindications have not been unambiguous as they are inLygodium. The secretion appears to take place onlyfrom those parts of the nucleus that protrude into thecytoplasm, and there is a close visual similarity be-tween the finely granular material that lines the innersurface of the inner membrane of the protrusions(which is also unaffected by ribonuclease; Bell, 1983)and that which accumulates outside. In both Pteri-dium (Bell, 1979) and Lygodium the envelope of theprotrusions lacks pores, although pores are frequentelsewhere. Any outward movement of material musttherefore either take place through the two membranesof the envelope or be facilitated by their localizedcollapse. Tourte (1975) has claimed that this occurs inPteridium following separation of the protrusions fromthe main body of the nucleus. Careful examination ofthe micrographs does not, however, support thisconclusion (Bell, 1983). In Lygodium the micrographs(e.g. Fig. 3) conform with the view that materialpasses from the protrusion to the cytoplasm while the
Nucleolus-like inclusions in cytoplasm 287
8Fig. 8. Part of an autoradiograph of the same series as thatyielding Fig. 7 showing cytoplasm adjacent to the nucleus(n). Despite the numerous silver grains, grain centres donot lie unambiguously above the nucleolus-like bodies(arrows). X35 000. Bar, 0-5 fim.
protrusion is in full continuity with the main body ofthe nucleus.
With regard to the chemical nature of the cyto-plasmic inclusions in Pteridium and Lygodium, theiraffinity for Azure B and the uranyl ion indicatesacidity, a feature they share with the nucleolus and thenuclear bodies. Nevertheless, although the presence ofa trace of RNA cannot be ruled out, there is noevidence that RNA is a principal component of theinclusions. It seems very likely therefore that, like thenuclear bodies (Bell, 1983), they consist principally orentirely of an acidic protein. Their composition doesnot support the view that they consist of aggregates ofribosomal precursors. The scheme of Tourte (1975),which envisages the transfer of ribonucleoprotein tothe cytoplasm of the egg cell of Pteridium by way ofthe nuclear protrusions, seems entirely fanciful.
The evidence currently available does not supportany similarity between the nucleolus-like inclusions inthe cytoplasm of fern egg cells and the nucleoloids ofmicro- and megasporogenesis in Ulium, despite the
•V*
*>/.:
4
10Figs 9, 10. Sections of the cytoplasm of maturing egg cellsof Lygodium treated with RNase-gold. Representativeresults showing failure of the probe to bind to thenucleolus-like bodies (arrows), despite general bindingelsewhere in the cytoplasm. Fig. 9, X60000. Bar, 0-5 jUm.Fig. 10, X37500. Bar, 0-5
striking visual similarity. Certainly in Lygodium, andmost probably in Pteridium, they do not enter thecytoplasm at times when the continuity of the nuclearenvelope is disturbed, as may be the situation inUlium (Williams et al. 1973; but see Dickinson &Willson, 1985). Nor are they formed by the engulfingof regions of cytoplasm by pleomorphic plastids, asappears to be the manner in which the nucleolus-likebodies arise in the cytoplasm of the egg cells of Taxus(Pennell & Bell, 1987) and conifers (Camefort, 1960),despite similar (but less extensive) changes in theplastids of the egg cell of Lygodium during maturation.It is significant that the bodies appear in the cytoplasmof the maturing egg cells at a time when the nucleusis dilated, highly irregular in shape, and with thechromatin finely dispersed and not responsive toFeulgen staining (Bell, 1960). The acidity of thebodies would be in line with their representing atemporary storage in the cytoplasm of non-histoneproteins from the nucleus. Proteins of this kind are
288 P. R. Bell and R. I. Pennell
known, for example, to play a crucial role in thehigher-order structure of chromosomes (Hadlaczky,1985). There is no evidence that the bodies areconcerned with the transfer of information fromnucleus to cytoplasm, or with the generation ofribosomes. The egg cells of ferns, unlike newly formedspores of ferns (Sheffield & Bell, 1979) and themicrospores of Ulium (Dickinson & Heslop-Harrison,1970), are well provided with ribosomes ab initio(Cave & Bell, 1974). If the material of the bodies iscorrectly identified as being involved with the organ-ization of chromosomes its presence in the cytoplasmmay contribute to the rapid decondensation of themale chromatin following fertilization. Deconden-sation begins as soon as the sperm nucleus enters theegg cell (Bell, 19756). The helical form of the malenucleus is already lost, and its chromatin well relaxedby the time karyogamy occurs.
The investigation of the nucleolus-like inclusions inthe cytoplasm of the egg cells of Pteridium andLygodium has made clear the danger of assumingchemical identity from resemblances of texture, mor-phology, and affinity for basic stains. Firm evidencefor the presence of RNA is provided by the incorpor-ation of [3H]uridine, or the binding of RNase-gold,with appropriate enzyme controls in each instance.The regressive staining technique of Bernhard (1969),making use of the retention of the uranyl ion by acidicmolecules, depends upon relative charge. It cannot beregarded as specific for RNA. In a number of instancesclaims for the presence of RNA in nuclear (Williams etal. 1983) and cytoplasmic (Tourte, 1975) bodies haverested solely upon the inconclusive evidence providedby the Bernhard technique.
The authors are indebted to Dr C. Cave for doingextensive uridine incorporation experiments with Pteridium,and to the Science and Engineering Research Council forfinancial support.
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