PATTERN OF INCORPORATION OF [»H]URIDINE INTO RNA OF AMPHIBIAN OOCYTE NUCLEOLI

Transcription

1 J. Cell Sci. a, (1967) 145 Printed in Great Britain PATTERN OF INCORPORATION OF [»H]URIDINE INTO RNA OF AMPHIBIAN OOCYTE NUCLEOLI H. C. MACGREGOR Department of Zoology, The University, St Andrews, Fife SUMMARY Amphibian oocytes were incubated in vitro in the presence of [ s H]uridine, and autoradiographs were made of nucleoli isolated from these oocytes and of sections of oocytes. After incubations of a h or less the nucleoli of oocytes larger than o-6 mm diameter are asymmetrically labelled. With longer incubations nucleoli from oocytes of o-6 to ri mm diameter become more uniformly labelled. Those of oocytes larger than i - 2 mm diameter remain asymmetrically labelled whatever the incubation time. Autoradiographs of i-/t sections through oocytes larger than o-6 mm diameter show, after short incubations, asymmetrically labelled nucleoli. In these autoradiographs silver grains are concentrated over a distinct component of each nucleolus which is eccentrically placed towards the nuclear envelope. Thin sections of oocytes show nucleoli consisting of core and cortex. The core material is always concentrated into the half of the nucleolus which, lies nearer the nuclear envelope. Autoradiographs of separated nucleolar cores and cortices from oocytes larger than o-6 mm diameter show, after short incubations, silver grains over cores but not over cortices. Similar autoradiographs prepared from oocytes of o - 6 to ri mm diameter, after longer incubations, show grains over cores and cortices. These results appear to indicate that nucleolar RNA is synthesized in the nucleolar core, in association with the nucleolar DNA, and is thence transferred to the cortex where it is built into ribonucleoprotein particles. Initial asymmetrical labelling is a consequence of the eccentric location of the nucleolar core. The nucleoli of oocytes smaller than o-6 mm diameter always label symmetrically; such nucleoli consist entirely of core material. It is suggested that the nucleoli of oocytes larger than 1-2 mm diameter always label asymmetrically because transfer of RNA from core to cortex proceeds more slowly than in smaller oocytes. INTRODUCTION The germinal vesicle nucleus of a newt oocyte has about 1000 nucleoli. Apart from one or two nucleoli which may be attached to chromosomal nucleolar organizer sites (Callan, 1966), none of these nucleoli are attached to a chromosome, and as a rule all of them are situated around the periphery of the nucleus immediately adjacent to the inner surface of the nuclear envelope. Isolated unfixed nucleoli from a yolky Triturus oocyte of about 1 mm diameter appear round, solid, and refractile when viewed in phase contrast. They are about 5-10 fi in diameter. They contain RNA and protein, and about 15 % of the nucleolar mass is attributable to RNA (Brown & Ris, 1959). The nucleolar RNA has a base composition which closely resembles that of the cytoplasmic ribosomal RNA (Edstrom & Gall, 1963). Thin sections through oocyte nucleoli from Rana clanritans show these to be structurally differentiated into two sharply defined zones (Miller, 1962). The inner of these zones, the nucleolar 'core', is tightly packed and consists of fibrils A 10 Cell Sci. i

2 146 H. C. Macgregor thick embedded in a matrix. The outer zone, the nucleolar 'cortex', consists of closely packed granules each about 150 A in diameter and fibrils of about the same thickness. In oocytes from T. cristatus nucleolar core and cortex are distinguishable but, when seen in thin sections, nucleoli of newt oocytes appear less sharply zoned than those of frog oocytes. In Triturus, nucleoli from oocytes of less than 300 //, diameter consist entirely of core material. As the oocyte grows the nucleoli grow by accumulation of cortex material around the nucleolar cores. Nucleolar core and cortex can easily be separated from one another by isolating the nuclear contents into neutral buffered sodium veronal (Miller, 1961) or 0-03 M ammonium acetate (Macgregor, 1965). In ammonium acetate slight dissolution of both components probably occurs, but core and cortex remain as compact objects and each has a characteristic appearance in phase contrast. If nucleoli are isolated into ammonium acetate more dilute than 0-02 M then the cortex disintegrates completely in a matter of seconds and the core material disperses until all that remains is a small granular ring. Such rings contain the DNA component of the nucleolus, and the demonstration by Miller (1964) that these rings can be broken by DNase provided the first convincing evidence of DNA in amphibian oocyte nucleoli. If amphibian oocytes are incubated in a medium containing ["CJadenine (Gall, 1958), [ 3 H]cytidine(Ficq, 1961), or phjuridine, then the RNA of the nucleoli becomes labelled. Some investigators (Gall, personal communication) have observed in autoradiographs that nucleoli from oocytes which had been supplied with a labelled nucleotide were occasionally asymmetrically labelled, with the greatest concentration of silver grains over the half of the nucleolus adjacent to the nuclear envelope. In the present study I have confirmed this observation and have tried to relate the asymmetrical labelling of oocyte nucleoli with phjuridine to nucleolar structure and function. MATERIALS AND METHODS The animals used in this study were: Triturus cristatus carmfex (Laurenti) collected near Naples, Italy, and supplied by Dr P. Dohrn; and T. c. cristatus (Laurenti) collected in the south of England and supplied by the dealer L. Haig of Newdigate,. Surrey. Pieces of ovary were removed from living anaesthetized animals, rinsed quickly in incubation medium, and transferred immediately to fresh sterile incubation medium. The incubation medium consisted of 10 ml tissue culture medium TC 199 (Difco- Laboratories Inc., Detroit, U.S.A.), 6 ml water, 0-03 g/ml crystalline bovine plasma albumin (Armour Pharmaceutical Co., Ltd., Eastbourne, England) and 50 ji\ (50 me)/ ml of a solution of uridine generally labelled with tritium (1-22 C/ITIM) and supplied by The Radiochemical Centre, Amersham, England. Oocytes were incubated in this medium for periods ranging from 30 min to 48 h at 18 C. From each batch of incubated oocytes some were fixed prior to embedding and sectioning and some were used at once for preparations of isolated nucleoli and chromosomes.

3 l?h]uridine incorporation in oocyte nucleoli 147 The oocytes selected for these experiments were from three size ranges. Those hereafter referred to as large oocytes were over 1-2 mm in diameter, as medium-sized were between o-6 and I-I mm diameter, and as small were 0-3 to 0-5 mm diameter. The techniques for isolating oocyte nuclei and removing nuclear membranes were as described by Callan & Lloyd (i960). Oocytes were transferred from incubation medium to a saline solution (o-i M KC1, 5 parts; o-i M NaCl, 1 part) in a watchglass and their nuclei were isolated freehand. Cleaned nuclei were then pipetted into a drop of saline in a flat-bottomed well slide and their membranes were dissected off. The well slides were constructed as described by Gall & Callan (1962). For preparations with whole nucleoli well chambers were filled with the above 5:1 KCl/NaCl mixture with CaCl 2 added to a concentration of 0-5 x io" 4 M. For preparations in which nucleolar cores and cortices were to be separated from one another the chambers were filled with 0-03 M ammonium acetate. Immediately after removal of nuclear membranes the preparations were placed in a sealed chamber containing vapour from 50 % neutral buffered formalin (20 % formaldehyde). After 1 h of formaldehyde vapour treatment the preparations were exposed for 2-5 min to vapour from 10 % acetic acid. This had the effect of sticking the lampbrush chromosomes and nucleoli to the slide which formed the base of the well chamber. Preparations were then placed in 10 % neutral formalin for 2 h. After fixation the well slides were dismantled and the slides bearing the chromosomes and nucleoli were immersed in 5 % (w/v) trichloroacetic acid (TCA) at 5 C for 5 min. They were then passed through 70 %, 95 %, and absolute ethanol, xylene, and acetone, and air dried from acetone. The methods of preparation of oocyte sections were as follows. Bunches of oocytes were removed from the incubation medium and fixed for 12 h in Sanfelice's fixative (Darlington & La Cour, 1942). They were subsequently washed for 24 h in running water, dehydrated in an alcohol series, and embedded in methacrylate. One-micron sections of oocytes were cut with a Servall Porter-Blum ultramicrotome and mounted on slides. Methacrylate was removed from the mounted sections with amyl acetate. Sections were then rehydrated, treated with 5 % TCA at 5 C for 15 min, dehydrated and air dried from acetone. Dry preparations of isolated chromosomes and nucleoli and oocyte sections were coated at 45 C with Kodak NTB 2 liquid emulsion diluted 1:1 with water. They were then placed in light-proof boxes and left to expose for 21 days at 18 C. Preparations were developed in Kodak D 19 b for 3 min, washed in water, fixed for 5 min in Kodak acid-fixer, washed in running water and air dried. Preparations of isolated nucleoli and I-/J sections were then stained with 0-2 % fast green at ph 2 or with 1 % methylene blue in 1 % (w/v) sodium tetraborate (borax). All preparations were dehydrated in alcohol, mounted in balsam and examined with a Carl Zeiss Photomicroscope fitted with planapochromatic objectives. For electron microscopy of oocyte nucleoli oocytes mm in diameter were fixed for 1 h in 1 % (w/v) osmium tetroxide buffered with veronal acetate to ph 7-4 (Palade, 1952). They were then dehydrated in acetone and embedded in Vestopal W. Silver to grey sections were cut with glass knives on a Cambridge Ultra-Microtome

4 148 H. C. Macgregor (A. F. Huxley pattern), mounted on Athene 483 grids, doubled stained with 2 % uranyl acetate for 5 min and lead citrate (Reynolds, 1963) for 2 min, and examined with a Siemens Elmiskop I (80 kv) at negative magnifications of between and OBSERVATIONS Nucleoli isolated from medium-sized carnifex oocytes are asymmetrically labelled after incubations of 2 h or less (Fig. 1). With longer incubations such nucleoli become progressively more uniformly labelled, and they are uniformly labelled after incubations lasting more than 4 h. Nucleoli from large oocytes always appear asymmetrically labelled no matter what the incubation time. Nucleoli from small oocytes are always uniformly labelled. In autoradiographs of equatorial sections through the nuclei of large oocytes the nucleoli appear asymmetrically labelled with most of the silver grains over the part of the nucleolus which lies nearest to the nuclear envelope (Fig. 2). In sections through some nucleoli stained with methylene blue a part of the nucleolus stains lightly, is eccentrically placed towards the nuclear envelope, and is flanked and backed by a crescent of more darkly staining material (Fig. 3). When these two regions are discernible in autoradiographs prepared from medium-sized oocytes after short incubations, most of the silver grains lie over the lightly stained regions of the nucleoli (Fig. 3). After longer incubations grains appear over both the light- and dark-staining regions (Fig-4)- Thin equatorial sections through the nuclei of medium-sized carnifex oocytes show nucleoli consisting of core and cortex. Invariably, the nucleolar core material is concentrated towards the nuclear envelope and the cortex forms a crescent around the sides and back of the core (Fig. 5). Autoradiographs of separated nucleolar cores and cortices prepared from mediumsized oocytes after incubations of 2 h or less, or from large oocytes after longer incubations, show silver grains over the nucleolar cores but few or no grains over the cortices (Fig. 6). After incubations lasting more than 4 h both cores and cortices of medium-sized oocytes are labelled (Fig. 7). In oocytes from T. c. cristatus the nucleoli vary in size. The smallest nucleoli in an oocyte separate in ammonium acetate into one core and one cortex each, whereas the larger nucleoli break up to give several cores and a single irregular mass of cortex material (Fig. 8). Such multi-cored nucleoli appear in autoradiographs prepared after incubations of less than 2 h with patches of silver grains marking the positions of the individual cores (Fig. 9). DISCUSSION The RNA of oocyte nucleoli becomes labelled when oocytes are supplied with phjuridine. In the presence of actinomycin D the nucleolar RNA does not become labelled (Izawa, Allfrey & Mirsky, 1963). Oocyte nucleoli contain a small amount of

5 ^H\uridine incorporation in oocyte nucleoli 149 DNA (Miller, 1964). It therefore seems reasonable to suppose that oocyte nucleoli are sites of DNA-dependent RNA synthesis. I suggest that this synthesis occurs in the nucleolar core, where the DNA component resides, and that the RNA product is passed from core to cortex. In the cortex it becomes built into particles of ribonucleoprotein. The exact nature of these particles and their relationship to cytoplasmic ribosomes is unknown. After short incubations in the presence of ph]uridine most of the RNA newly synthesized in association with the nucleolar DNA is likely to be found in the nucleolar core. In T. cristatus the nucleolar core is asymmetrically placed with respect to the cortex, and I think this is the reason why Triturus nucleoli which have both core and cortex components label asymmetrically after short incubations. With longer incubation times labelled RNA accumulates in the cortex and asymmetry of labelling is obscured. Nucleoli from small oocytes are always uniformly labelled. They consist mainly of core material. The situation in large oocytes is more complex. Here the nucleoli are asymmetrically labelled after incubations of 48 h or more. It is therefore necessary to suppose either that in large oocytes the transfer of newly synthesized RNA from core to cortex proceeds more slowly than in small ones, or that a substantial proportion of the cortex RNA comes from an extra-nucleolar source, possibly from the chromosomes, and that the synthesis of core RNA continues long after the supply of cortex RNA has ceased. Perhaps in this instance a significant difference between small or medium-sized oocytes and large ones is that in the former the loops of the lampbrush chromosomes are extended and actively engaged in RNA synthesis, whereas in the latter the loops of the chromosomes are being withdrawn and the synthesis of chromosomal RNA is proceeding more slowly. In Triturus the nucleolar core is situated on that side of the nucleolus which lies nearest the nuclear membrane. What is the significance of this feature? Painter & Taylor (1942) identified in oocytes of Bufo valuceps large numbers of Feulgen-positive granules scattered over the inner surface of the nuclear envelope. Each of these granules has associated with it one peripheral nucleolus, and the granule lies between the nuclear envelope and the nucleolus. A similar arrangement has recently been observed in nuclei of small oocytes from Triturus viridescens (J. G. Gall, personal communication) and from T. cristatus (H. C. Macgregor, unpublished). Accordingly, I suggest that nucleolar DNA is behaving in a manner comparable to a loop-chromomere complex on a lampbrush chromosome. The Feulgen-positive granule which appears on the nuclear membrane in young oocytes may be compared with a chromomere on a lampbrush chromosome. The DNA of this isolated chromomere then becomes spun out into the form of a loop; and in this nucleolar situation the spinning out of the DNA is complete, and the end product is an extended ring. This ring of DNA is localized within the nucleolar core and is extensively, though loosely, folded within the core. Throughout its length it actively supports the synthesis of nucleolar RNA (Lane, 1966).

6 150 H. C. Macgregor REFERENCES BROWN, C. A. & Ris, H. (1959). Amphibian oocyte nucleoli. J. Morph. 104, CALLAN, H. G. (1966). Chromosomes and nucleoli of the axolotl, Ambystoma mexicanum.j. Cell Sci. 1, CALLAN, H. G. & LLOYD, L. (i960). Lampbrush chromosomes of crested newts Triturus cristatus (Laurenti). Phil. Trans. R. Soc. B 243, DARLINGTON, C. D. & LA COUR, L. F. (1942). The Handling of Chromosomes. London: Allen and Unwin. EDSTROM, J.-E. & GALL, J. G. (1963). The base composition of ribonucleic acid in lampbrush chromosomes, nucleoli, nuclear sap, and cytoplasm of Triturus oocytes. J. Cell Biol. 19, FICQ, A. (1961). Metabolisme de l'oogenese chez les amphibiens. In Symposium on the Germ Cells and Earliest Stages of Development (ed. S. Ranzi), pp Pavia: Prem. Tip. Successori Frat. Fusi. GALL, J. G. (1958). Chromosomal differentiation. In A Symposium on the Chemical Basis of Development (ed. W. D. McElroy & B. Glass), pp Baltimore: The Johns Hopkins Press. GALL, J. G. 85 CALLAN, H. G. (1962). HMJridine incorporation in lampbrush chromosomes. Proc. natn. Acad. Sci. U.S.A. 48, IZAWA, M., AI.T.FREY, V. G. & MIRSKY, A. E. (1963). The relationship between RNA synthesis and loop structure in lampbrush chromosomes. Proc. natn. Acad. Sci. U.S.A. 49, LANE, N. J. (1966). Uridine incorporation in amphibian oocyte nucleoli. J. Cell Biol. 31, 65 A. MACGREGOR, H. C. (1965). The role of lampbrush chromosomes in the formation of nucleoli in amphibian oocytes. Q. Jl microsc. Sci. 106, MILLER, O. L. (1961). Chromosome and nucleolar structure in Triturus oocytes. In First Annual Meeting of the American Society for Cell Biology. MILLER, O. L. (1962). Studies on the ultrastructure and metabolism of nucleoli in amphibian oocytes. Proc. 5th Int. Conf. Electron Microsc. (ed. S. S. Breese), p. NN-8. New York and London: Academic Press. MILLER, O. L. (1964). Extrachromosomal nucleolar DNA in amphibian oocytes. J. Cell Biol. 23, 60 A. PAINTER, T. S. & TAYLOR, A. N. (1942). Nucleic acid storage in the toad's egg. Proc. natn. Acad. Sci. U.S.A. 38, PALADE, G. E. (1952). A study of fixation for electron microscopy. J. exp. Med. 95, REYNOLDS, E. S. (1963). The use of lead citrate at high ph as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, {Received 30 January 1967)

7 Journal of Cell Science, Vol. 2, No. 2 20// n 2, _ J «J»l 20// >$* n 20// j 4, * 20^ f 5 ' For legend see next page. H. C. MACGREGOR (Facing p. 150)

8 The preparations shown in Figs. 1-4 are autoradiographs and were stained with methylene blue. Cytoplasm, c; nucleus, n. Fig. 1. Isolated asymmetrically labelled nucleoli, prepared after 2 h incubation from a medium-sized carnifex oocyte. Fig. 2. Part of a i-fi section, prepared after 2 h incubation from a large carnifex oocyte. The nucleolus in the centre of the picture is asymmetrically labelled with most of the silver grains overlying the half of the nucleolus adjacent to the nuclear envelope. Fig. 3. Part of a i-fi section, prepared after 2 h incubation from a medium-sized carnifex oocyte. Two regions of the nucleolus are discernible. An inner region is more lightly stained and is eccentrically placed towards the nuclear envelope. Most of the silver grains overlie this lightly stained region. Fig. 4. Part of a i-ft section prepared from a medium-sized carnifex oocyte after 8 h incubation. In this preparation there are silver grains overlying both the light- and dark-staining regions of the nucleoli. Fig. 5. Electron micrograph of part of a section through a medium-sized carnifex oocyte showing a single peripheral nucleolus. The right half of the nucleolus lies nearer the nuclear envelope (ne) and consists mainly of compact fibrous core material (circumscribed in white). The left half of the nucleolus consists mainly of granular cortex.

9 Journal of Cell Science, Vol. 2, No p 20 n The preparations shown in Figs. 6, 7 and 9 are autoradiographs stained with fast green at ph 2. Fig. 6. A nucleolus from a medium-sized carnifex oocyte after 2 h incubation. The nucleolus was isolated into 0-03 M ammonium acetate so that core (co) and cortex partially separated from one another. Most of the silver grains overlie the core. Fig. 7. A preparation similar to that shown in Fig. 6 from a medium-sized carnifex oocyte after a 12 h incubation. Silver grains overlie both core (co) and cortex. Fig. 8. Phase-contrast micrograph of three nucleoli from an oocyte of T. c. cristatus isolated in 0-03 M ammonium acetate. Nucleolar cores and cortices have partially separated from one another. The large nucleolus on the left shows three cores and an irregular mass of cortex. The other two nucleoli show one core and one cortex each. Arrows indicate cores. Fig. 9. Nucleoli isolated from a medium-sized cristatus oocyte after 2 h incubation. Clusters of silver grains overlie the individual cores in these nucleoli. The number beside each nucleolus indicates the number of cores in the nucleolus estimated from the number of grain clusters H. C. MACGREGOR

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