evolution of the nucleolar apparatus during oogenesis in acipenseridae … · ('granules'...

/ . Embryo!, exp. Morph. Vol. 35, 3, pp. 667-687, 1976 6 6 7

Printed in Great Britain

Evolution of the nucleolar apparatus duringoogenesis in Acipenseridae

ByE. V. RAIKOVA1

From the Institute of Cytology of theAcademy of Sciences, Leningrad, USSR

SUMMARY

Evolution of the nucleoli has been followed during oogenesis in the Acipenserid fishes,Acipenser ruthenus (the sterlet) and A. giildenstadti (the sturgeon) using light and electronmicroscopes. In the ovaries of adults, the oogonial nuclei usually have a single nucleolus withan adjacent mass of paranucleolar fibrillar material. The cytoplasm of the oogonia containstwo dense bodies peculiar only to gonocytes, one being electron dense and containing RNAand the other being electron-lucent and lacking RNA. Neither is surrounded by membrane.The fine structure of the electron-lucent body is identical to that of the paranucleolar material,while the RNA-containing body structurally resembles the nucleolus. A nuclear origin forboth cytoplasmic bodies is likely.

Leptotene-stage oocytes usually still have a single nucleolus. During zygotene, it is adjacentto the nuclear envelope and opposite to the chromosomes contracted in synizesis. At pachy-tene, a 'cap' of extrachromosomal chromatin is formed under the nuclear envelope andaround the nucleolus. Bivalents also contact this cap. In early diplotene, the primarynucleolus still persists. The material of the cap is dispersed beneath the entire nuclear envelopein the form of granules of extra DNA; each granule then produces a peripheral (secondary)nucleolus. These become typical amphinucleoli with differentially developed granular parts,depending on the age of the nucleolus and the stage of meiosis. Their fibrillar parts alwaysface the nuclear envelope. New peripheral nucleoli continue to form as long as granules ofextra DNA persist under the nuclear envelope, i.e. approximately until vitellogenesis.

In early vitellogenesis, the peripheral nucleoli become transformed, by re-distribution oftheir fine structural components, into circular threads trailing towards the centre of thenucleus. The axis of the thread consists of fibres and is coated with granules. In late vitello-genesis, the nucleoli round up and become vacuolized; they are then peripheral again.

Proteinaceous RNA-lacking structures which are also produced in the nuclei duringoogenesis in the Acipenseridae, are the 'nuclear bodies' and 'spheres'. The former areadjacent to peripheral nucleoli, the latter form on lampbrush chromosomes. Both are ultra-structurally alike. The loops of lampbrush chromosomes produce also RNP bodies('granules' of Callan & Lloyd, 1960) which are ultrastructurally similar to nucleoli but lacksegregation of granules from fibres into spatially distinct parts.

The evolution of the nucleolar apparatus during oogenesis in the Acipenseridae closelyresembles that in amphibians.

1 Author's address: Institute of Cytology, 32 Maklin Avenue, Leningrad F-J21, USSR.

668 E. V. RAIKOVA

INTRODUCTION

The Acipenseridae, a fish family including sturgeons and sterlets, is a veryancient and primitive group among the Osteichthyes. The Acipenseridae arerather widely distributed in the rivers of the USSR and present a double interestto investigators: first, a purely economic one, being a national wealth by them-selves and as producers of caviar; second, a scientific one, as the group is bothprimitive and successful (Gerbilsky, 1962). They also resemble the Amphibiain many developmental and cytological aspects, including oogenesis. Cytologic-ally, the Acipenseridae seem to be even closer to Amphibia than to Teleostei.

Unfortunately, these fishes present problems for cytological studies: theycannot be reared in the laboratory until maturity because this would take toomany years, and also they have too many chromosomes to be favourable forcytogenetic research. According to Serebryakova (1964), the diploid chromo-some number (2ri) is about 60 in Huso huso, Acipenser ruthenus (the sterlet), andA. stellatus, and double that number (about 120) in Acipenser guldenstadti (thesturgeon). According to Fontana & Colombo (1974), the respective chromosomenumbers are even higher (approximately 2 times), due to the presence ofmicrochromosomes not taken into account by Serebryakova.

All Acipenserids except the sterlet are migrators: they ascend rivers forhundreds of kilometres for spawning, which occurs in most species in spring,and thereafter they re-descend to the sea. Their larvae remain in the river forabout a year after hatching; then, they also descend to the sea and return intorivers only after several years for their first spawning. The immaturity period is13 to 15 years in the sturgeon, and only in the non-migratory fresh-waterspecies, the sterlet {A. ruthenus), it is as short as 6 to 8 years (Berg, 1948).

The present research deals with only two species of Acipenseridae of theVolga: A. guldenstadti Brandt, the sturgeon, and A. ruthenus L., the sterlet.Both spawn in late April-early May. The interval between two successivespawnings is about 2-3 years in the sterlet and about 5 years or more in thesturgeon (Shilov, 1964).

During spawning, it is the senior generation of oocytes which mature and aredischarged. The junior generation cells remain in a resting state (in previtello-genesis) until preparation for the next spawning begins, about a year beforeactual spawning. The oocytes then start vitellogenesis. Among resting-stateoocytes, the larger ones are in diplotene of meiosis, the smaller ones, at earlierstages of the meiotic prophase.

The aim of this study is to make a general survey of the evolution of thenucleolar apparatus during oogenesis of the Acipenseridae. Some materialincluded in this report has previously been published in Russian (Raikova,1968, 1970, 1972, 1974a).

Nucleolar apparatus in Acipenseridae 669

MATERIAL AND METHODS

Oocytes of the sterlet {A. ruthenus) and of the sturgeon {A. giildenstadti) wereobtained from females caught during the last several years in the Kama (nearRybnaya Sloboda) and in the Volga (below the Kuybyshev dam, near Saratov,near Volgograd and above Astrakhan).

For light microscopy, pieces of the ovary were fixed with Bouin's, Champy's,Serra's, Carnoy's, Navaschin's, Sanfelice's, and Zenker's (with formalin)mixtures as well as with sublimate - acetic acid (19:1) and with 10% formalin.The material, dehydrated in ethyl alcohol, was embedded in paraffin. Sections(7 to 10 f.im thick) were stained with iron haematoxylin, Heidenhain's 'azan',Mayer's haemalum, or azur-eosine after Nocht-MaximofT. For cytochemistryof the nucleic acids, Feulgen's reaction (with deoxyribonuclease-treated control),methyl green-pyronin staining and toluidine blue staining (both after Brachet,1953), and gallocyanin staining (after Einarsson) were used. Ribonuclease-treated controls were made with the last three methods. Polysaccharides weredetected by the PAS reaction, with salivary amylase-treated control. Lipids wereosmicated according to Champy and subsequently treated with Na2S. Proteinswere stained with a sublimate solution of bromphenol blue (after Mazia,Brewer & Alfert, 1953). Danielli's tetrazonium coupling reaction and Serra'sstaining for arginin were also used, as well as staining with Fast green atpH 8-2 for basic proteins.

For electron microscopy, the material was fixed with 6 % glutaraldehyde inphosphate buffer (pH 7-4) for 2 h at 4 °C, and postfixed with Caulfield's 1 %osmium tetroxide in acetate-veronal buffer with 4-5 % saccharose (also 2 hat 4 °C). The fixed pieces were dehydrated in acetone and embedded in Araldite.Sections were collected on uncoated grids and stained with saturated aqueousuranyl acetate (4 h) and then with Reynolds' lead citrate (10 min). Observationswere made with a JEM-7A electron microscope at 50 kV.

OBSERVATIONS

Oogonia

In adult acipenserids, oogonia are the youngest stages of oogenesis available.Oogonia (Figs. 1-3) are small rounded cells with cytoplasm clearer than that ofsurrounding cells. Each oogonium is accompanied by another small cell whichadheres to it (the future follicle cell, Fig. 3). Mitoses of oogonia are rare in adultfish (Fig. 1). It is not known how many generations of oogonia follow eachother before oocytes are differentiated.

Oogonia have a large nucleus with a single nucleolus which is generallysurrounded by a clear, apparently structureless space (Fig. 2). Their cytoplasmconsistently contains two different bodies (Fig. 2). One of these stains lightlywith haematoxylin, appears oval or spherical, contains no cytochemically


detectable RNA (Raikova, 1968, 1970), but stains for proteins. The other bodyis dark, nearly always irregular in shape, and contains RNA and protein.

Electron microscopy shows that a vacuole, filled with finely fibrillar materialof low electron density, exists in the oogonial nucleus where it is adjacent to thenucleolus (Fig. 3). The content of this intranuclear vacuole is morphologicallyidentical with that of the clear cytoplasmic (proteinaceous) body (Figs. 3, 6).Both have no surrounding membrane. This suggests that the clear cytoplasmicbody has a nuclear origin and actually represents the paranucleolar vacuolematerial extruded into the cytoplasm. Inclusions of cytoplasmic origin are, incontrast to this, usually surrounded by a membrane.

The other cytoplasmic body, which appears dark and contains RNA (Figs.1-3, 6), is ultrastructurally an accumulation of small granules which veryfrequently are associated with several mitochondria. The granular materiallooks very much like the nucleolar cortex material and thus may also havenuclear origin, representing nucleolar granules extruded into the cytoplasm.This supposition is almost beyond doubt in the case of amphibians, as shownby Clerot (1968), Kessel (1969), and others. This makes it likely that bothspecific cytoplasmic bodies, which are markers of the sexual cells in Acipen-seridae, have a nuclear origin.

In Acipenseridae, oogonia of various generations are morphologicallydifficult to distinguish from very young oocytes. Whereas in amphibians oogoniausually have irregular (lobulated) nuclei (Al-Mukhtar & Webb, 1971; Coggins,1973), in acipenserids both oogonia and young oocytes have spherical nucleiwith a single central nucleolus and sometimes with two or three additionalsmall nucleoli.

FIGURES 1-5

Fig. 1. Three sturgeon oogonia. The top one is in metaphase of mitosis (pole view);the other two oogonia contain resting nuclei with single central nucleoli and densecytoplasmic bodies (db); nuclei of future follicle cells (fen) are also seen. Bouin,iron haematoxylin. x 1800.Fig. 2. Sturgeon oogonium showing both lucent (Ib) and dense (db) cytoplasmicbodies. A lucent zone (Iz) is seen inside the nucleus, adjacent to the nucleolus. Nava-schin, iron haematoxylin. x 1800.Fig. 3. Electron micrograph of a sturgeon oogonium showing nucleus with nucleolus(n) and paranucleolar material (pm). The cytoplasm contains both dense (db) andlucent (Ib) cytoplasmic bodies, lipid droplets (/), mitochondria (m), a Golgi body (g),and desmosomes (d). A follicular cell with lobulated nucleus (fen) is seen at top.OsO4 fixation, x 12300.Fig. 4. Young sturgeon oocyte with nucleus in leptotene, containing the nucleolus(n). fen, nucleus of a future follicle cell. Bouin, iron haematoxylin. x 1800.Fig. 5. Three sturgeon oocytes in zygotene (synizesis). The nucleolus (n) is seen inthe top cell opposite to the chromosome threadball. Bouin, iron haematoxylin.xl800.

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Oocytes in previtellogenesis

During the leptotene stage of meiosis, the nucleus of a young oocyte some-times contains one nucleolus but sometimes shows no nucleoli at all (Fig. 4).On the other hand, a nucleolus always exists during the next (zygotene) stage.It generally lies at the pole of the nucleus opposite to the threadball of chromo-somes (Fig. 5), the bouquet stage taking in Acipenserids the form of synizesis.

With the electron microscope, the nucleolus appears adjacent, during zygo-tene, to the inner side of the nuclear envelope, though separated from it by alayer of loose fibrillar material of unknown nature (Fig. 7).

During pachytene, chromosome bivalents are apparent, but there is also acap of chromatin which appears inside the nucleus at one side of it and is verycharacteristic of this stage (Figs. 8, 9). The nucleolus is always enclosed in thecap material (Fig. 8). Direct connexions between the chromatin cap and somebivalents are sometimes seen. The oocyte is already surrounded by small cellsforming a kind of follicle (Figs. 8-11).

During diplotene, chiasmata are seen on the bivalents. The cap materialbecomes more discrete and spreads beneath the nuclear envelope; this materialis now distinctly granular (Figs. 10-13). Beginning with this stage, new nucleoliare formed at the inner side of the nuclear envelope and remain attached to it(Figs. 10, 11). These are peripheral nucleoli, forming in close contact with thechromatin granules which earlier made up the cap. The cap chromatin certainlyis extrachromosomal DNA synthesized by amplification of ribosomal genes,exactly as in amphibians. Schmantzar (1976) showed cytophotometrically thatthe DNA content of oocyte nuclei is at this stage 50 times higher than that of thespermatids in the sturgeon, and 30 times higher, in the sterlet. We also observedintense incorporation of [3H]thymidine into the chromatin cap (Chmilevsky &Raikova, 1976).

FIGURES 6-11

Fig. 6. Electron micrograph of two sturgeon oogonia interconnected with a cyto-plasmic bridge (at arrow). Note the paranucleolar material (pm) and its similarityto that of the lucent cytoplasmic bodies (Ib) in both cells, db, dense cytoplasmic body.OsO4 fixation, x 9200.Fig. 7. Electron micrograph of the nucleolus of a sterlet oocyte in synizesis. Note thefibrillar material attaching the nucleolus to the nuclear membrane (at arrow).Glutaraldehyde - OsO4 fixation, x 45000.Figs. 8 and 9. Two focal planes of a sturgeon oocyte nucleus in pachytene. Note thedense chromatin cap with the primary nucleolus in central position inside it (inFig. 8), and edges of the cap and the bivalents (in Fig. 9). Navaschin, iron haema-toxylin. x 1800.

Figs. 10 and 11. Sturgeon oocyte nuclei in early diplotene. The chromatin cap withseveral peripheral nucleoli forming inside it is shown in side view (in Fig. 10) and inpole view (in Fig. 11). The cap is now loose and granular; its granules begin to spreadbeneath the nuclear envelope. Navaschin, iron haematoxylin. x 1800.

674 E. V. RAIKOVA

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Granules of extrachromosomal DNA, regularly interspaced at the inner sideof the nuclear envelope, can be shown very well (Figs. 12, 13) by a Feulgen-typefluorescent reaction specific for DNA, the auramine-SO2 staining after HC1hydrolysis (Rosanov & Kudriavtsev, 1967). Some chromosomal bivalents areseen to contact certain peripheral chromatin granules (Fig. 12).

Diplotene-stage oocytes enter the period of rapid growth, the bivalentstransforming into lampbrush form. Many peripheral nucleoli are alreadypresent, as are several central intranuclear bodies which stain very intenselywith iron haematoxylin but not with pyronine or toluidine blue (Fig. 14).These are 'spheres', resembling those described by Callan & Lloyd (1960) inTriturus. They seem to form on the loops of the lampbrush chromosomes.

Electron microscopy reveals both the young peripheral nucleoli and thegranules of extrachromosomal chromatin adhering to the nuclear envelope(Fig. 15). At first, the nucleoli contain only fibres, but later (with the start ofrapid growth) each nucleolus develops a granular part in the form of a knob,always turned towards the centre of the nucleus (Fig. 16). Each nucleolus thusbecomes an amphinucleolus (Figs. 16, 17). The granular part of such anamphinucleolus is more basophilic than its basal (fibrillar) part, but, inversely,the latter stains more strongly for proteins. The fibrillar part thus seems tocontain more protein, and the granular part, more RNA. The granular knobcan completely detach from the base of the nucleolus. This has also beenobserved in Xenopus by Van Gansen & Schram (1968). Two neighbour nucleolisometimes have a common granular knob (Fig. 18).

The spheres are less numerous than the nucleoli. In previtellogenesis theformer are always attached to lampbrush chromosomes (Fig. 14). Sometimesa sphere is connected with two or three bivalents at once, which seems to indi-cate that several spheres can fuse. Large spheres may be as large as nucleoli.

FIGURES 12-16

Fig. 12. Fluorescence photograph of a sterlet oocyte nucleus in diplotene, stainedAuramine 00 for DNA (after Carnoy fixation). Note the chromatin granules dis-persed all around the nuclear periphery, and many bivalents and three spheres in theinterior of the nucleus, x 1800.Fig. 13. The same, showing granules of extra DNA regularly spaced under thenuclear membrane, as seen in a tangential section of a diplotene nucleus, x 2900.Fig. 14. Sterlet oocyte nucleus at the beginning of rapid growth, showing peripheralnucleoli (pn) at different stages of formation, and a sphere (s) attached to severalbivalents. Sanfelice, iron haematoxylin. x 630.Fig. 15. Electron micrograph of a young peripheral nucleolus (pn), of mainly fibrillarstructure, in a sturgeon oocyte nucleus. Chromatin granules (eg) under the nuclearenvelope (ne) are also seen. OsO4 fixation, x 51000.Fig. 16. Electron micrograph of a typical peripheral amphinucleolus in a sterletoocyte nucleus, showing differentiation into a fibrillar part (fp) and a granularknob (gk). ne, nuclear envelope, m, mitochondria. OsO4. x 19500.

676 E. V. RAIKOVA


Their fine structure is fibrillar; the fibres are loosely packed and of low electrondensity (Fig. 19).

After glutaraldehyde fixation, electron microscopy shows that oocyte nucleiwith amphinucleoli contain also small fibrillar masses of loose structure and oflow contrast, which lie near the peripheral nucleoli (Fig. 18). These masses aresimilar to the 'nuclear bodies' described by Kessel (1969) in amphibian oocytes.They might serve to fix nucleoli on the nuclear membrane (Lane, 1967). Thenuclear bodies still cannot be seen in the light microscope at this stage. Theirfine structure (Fig. 18 A) is comparable to that of the paranucleolar materialand to that of the clear cytoplasmic body found in oogonia.

The fate of the primary nucleolus, which persists in the previtellogeneticoocyte nucleus from the pachytene stage, is difficult to ascertain, because it israther similar to secondary (peripheral) nucleoli. However, one nucleolus issometimes larger and more spherical than others. This nucleolus is usuallyvacuolized and can be found either near the nuclear envelope or near the centreof the nucleus (Fig. 20). With the electron microscope, its vacuole is seen tocontain a finely fibrillar substance (Fig. 20) which again resembles the para-nucleolar material of the oogonia. We suppose this to be the primary nucleoluswhich has grown and acquired a fibrillar vacuole since the pachytene stage.

Oocytes in vitellogenesis

At the time of beginning of vitellogenesis, the number of peripheral granulesof extrachromosomal chromatin, which produce new nucleoli, rapidly decreases.Formation of peripheral nucleoli thus gradually ceases.

The start of vitellogenesis coincides with a spectacular transformation of thenuclear structure. This is also the time when an oocyte, which before belonged tothe junior generation, differentiates to become member of the senior generationof oocytes destined to mature for the next spawn, the junior generation remain-ing blocked in previtellogenesis. During early vitellogenesis, the lampbrush

FIGURES 17-20

Fig. 17. Peripheral amphinucleoli (pri) in a sturgeon oocyte nucleus. Two of them areapparently extruded into the cytoplasm, if not lying in deep evaginations of thenuclear envelope. Two spheres (s) are also seen. Bouin, azan. x 900.Fig. 18. Electron micrograph of two peripheral nucleoli having separate fibrillarparts (fp) but a common granular part (gp). A nuclear body (nb) is seen near thenuclear envelope (ne). Sterlet oocyte, OsO4 fixation, x 9000.Fig. 18 A. Enlargement of a part of a nuclear body. Sterlet oocyte, OsO4 fixation,x 45000.Fig. 19. Electron micrograph of a sphere in a sterlet oocyte nucleus. Glutaralde-hyde - OsO4 fixation, x 24500.Fig. 20. Electron micrograph of the primary nucleolus in a sterlet oocyte diplotenenucleus. Note the fibrillar material (/m) filling the vacuoles inside the nucleolus.Glutaraldehyde - OsO4 fixation, x 7800.

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678 E. V. RAIKOVA


chromosomes stretch to maximum; they are practically invisible in sections(Fig. 21). They are very active in synthesis, producing at their loops a largenumber of spheres and of RNP bodies ('granules' of Callan & Lloyd, 1960).The nucleus becomes filled with various small inclusions.

The peripheral nucleoli undergo a striking transformation at the time ofappearance of the first yolk bodies in the cytoplasm (Fig. 22). At first, they roundup (Figs. 21-22, 26), then they elongate into circular threads extending towardsthe centre of the nucleus (Figs. 22, 23). At that time, there is already muchyolk (Fig. 23).

Electron microscopy shows that the secondary rounding up of the peripheralnucleoli is due not to loss of their granular knobs but to uniform re-distributionof the granular material all around the fibrillar part. Also, the nucleolus becomesvacuolized, and the granular component coats not only the outer surface of thenucleolus, but also the surfaces of the vacuoles (Fig. 26 A).

The small fibrillar bodies ('nuclear bodies' of Kessel, 1969) also round up,become more compact and follow the movements of their correspondingnucleoli. They are no longer attached to the nuclear membrane (Fig. 26). Thesebodies can now be seen under the light microscope as well, especially in semi-thin Araldite sections. They do not stain with toluidine blue. At later stagesthey seem to disappear.

When nucleoli extend into circles formed by a more or less beaded thread(Fig. 23), electron microscopy shows that the axis of the circular thread isfibrillar and that granular material covers only the periphery of the thickeningsoccurring on the thread (Fig. 27).

In oocytes filled with yolk, the lampbrush chromosomes somewhat contract;their axes are now well seen in light microscopical sections (Fig. 24). Thespheres are at this stage usually rounded and rather small (Fig. 24). No more

FIGURES 21-25

Fig. 21. Sterlet oocyte in late previtellogenesis. The nucleus contains many peri-pheral nucleoli (pn) and several spheres (s); new peripheral nucleoli still form (atarrows). Bouin, mercuric bromphenol blue, x 420.Fig. 22. Nucleus of a sterlet oocyte in early vitellogenesis. Some peripheral nucleolibegin to transform into circles (at arrows). The interior of the nucleus is filled withvarious bodies formed on lampbrush chromosomes (Icb). y, yolk. Bouin, ironhaematoxylin. x400.Fig. 23. Nucleus of a sterlet oocyte in middle vitellogenesis. Circular nucleolarthreads (at arrow) trail towards the centre of the nucleus, occupied by lampbrushchromosome products (Icb). y, yolk. Bouin, iron haematoxylin. x 280.Fig. 24. Central part of the nucleus of a sterlet oocyte in vitellogenesis, showingspheres (s) and 'granules' (g) attached to lampbrush chromosomes. Zenker, ironhaematoxylin. x 900.Fig. 25. Electron micrograph of a lampbrush chromosome 'granule'. Sterlet oocyte,glutaraldehyde - OsO4 fixation, x 32500.

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680 E. V. RAIKOYA

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fusion of spheres is observed. Their attachment to chromosomes becomesdifficult to observe. They seem to be mostly detached, as in Reptilia (Agamidae)(Arronet, 1975). As before, the spheres contain no nucleic acids.

The small RNP bodies formed on the chromosome loops (the 'granules' ofCallan & Lloyd, 1960) consist of both fibrils and submicroscopic granules, astrue nucleoli do. But here the two components are mixed together (Fig. 25).These bodies can detach from the chromosomes without changing their aspect.Their RNA is generally supposed to be messenger RNA because they form onthe loops of lampbrush chromosomes and not at nucleolus-organizing loci oron their extra copies. On the other hand, peripheral nucleoli obviously containribosomal RNA. This makes us consider misleading the use of the term 'micro-nucleoli' to designate these RNP bodies (Sterba, 1961; Sterba & Schaffner,1965; Schaffner, 1968; Sanchez, 1969).

At the end of vitellogenesis, the nucleoli contract to the nuclear peripheryand round up again (Fig. 28). They are now clearly vacuolized and remain inthis condition until maturation of the oocyte.

Just before spawn, the oocyte nuclei generally show no nucleoli at all andlook optically empty. The cytoplasmic RNA concentrates at the animal pole.According to Votinov (1947, 1963) and Kazansky (1953), the nucleoli graduallydissolve while moving towards the centre of the nucleus.

DISCUSSION

The data presented above demonstrate that the Acipenserid nucleolarapparatus undergoes a definite series of transformations during oogenesis.The primary nucleolus, which can be traced from oogonia to pachytene and,with some doubt, to diplotene, shows no budding or fragmentation. Nocontinuity thus exists between the primary nucleolus and the peripheral nu-cleoli except that the former may become one among the latter. But all other

FIGURES 26-28Fig. 26. Electron micrograph of a rounded-up peripheral nucleolus (pri) before itsstretching into a circle, with its nuclear body (nb). Nucleus of a sterlet oocyte inearly vitellogenesis. ne, nuclear envelope. Glutaraldehyde - OsO4 fixation, x 15500.Fig. 26 A. Electron micrograph of a part of a rounded-up vacuolized peripheralnucleolus showing granules mainly at the surface of an intranucleolar vacuole (v);the fibrillar material is more peripheral (/m). Sterlet oocyte, glutaraldehyde - OsO4fixation, x 26000.Fig. 27. Electron micrograph of parts of nucleolar circular threads, showingfibrillar cores (/) and granular cortexes (g). Sterlet oocyte, glutaraldehyde - OsO4fixation, x 56000.Fig. 28. Periphery of sturgeon oocyte nucleus in late vitellogenesis, showing indentednuclear envelope (ne) and secondarily rounded peripheral nucleoli (pri). y, yolk.Bouin, azan. x 900.

682 E. V. RAIKOVA

peripheral nucleoli arise independently of it. In this respect, the oogenesis ofAcipenseridae fits into the general pattern of alimentary type oogeneses(Raven, 1961).

These data disagree, however, with Olschwang's (1936) description, accordingto which the primary nucleolus of the oocyte of A. ruthenus would grow,become polymorphous and then fragment into many small peripheral nucleoli.Such polymorphous intranuclear bodies can be seen also in some figures ofIonescu-Varo & Grigoriu (1963) concerning the same species, though no inter-pretation of them is given in the text. Some of our preparations also show largeirregular nucleoli at one side of the oocyte nucleus, but these are atypical andlikely to be stages of oocyte degeneration or fixation artifacts. At any rate, theyappear only in diplotene, when peripheral nucleoli are already present andoocytes are surrounded by follicle cells.

A cap of extrachromosomal DNA is produced in pachytene nuclei. Its com-ponent DNA granules progressively spread beneath the nuclear membrane indiplotene, assuming a very regular distribution. Each granule is a centre ofneoformation of a peripheral nucleolus (Raikova, 1968). Nucleologenesiscontinues until the start of yolk formation. It is now beyond doubt that thechromatin cap consists of amplified copies of the ribosomal RNA gene: thecap material is strongly Feulgen-positive, incorporates [3H]thymidine (Chmilev-sky & Raikova, 1976), the total DNA content of the oocyte nucleus reachingin result 30-50 times that of a spermatid (Schmantzar, 1976). We also obtainedgood hybridization in situ of Acipenserid (sterlet and sturgeon) oocyte DNAwith iodinated rRNA isolated from oocytes of Xenopus laevis. The chromatincap in pachytene oocyte nuclei was strongly marked (unpublished resultsobtained during the author's stay in the Laboratory of Molecular Cytology andEmbryology of the Free University of Brussels, in collaboration with C. Thomas,G. Steinert, and A. Schram).

These observations are in good agreement with Arndt's (1960) and Chmilev-sky's (1970) data on oogenesis of some Teleosts. Arndt reported that, in someCyprinidae, a substance beneath the nuclear envelope remained Feulgen-positive much longer than the bivalents did, and supposed this 'heterochroma-tin', localized in the folds of the nuclear envelope, to participate in formation ofperipheral nucleoli. Chmilevsky (1970) also found a chromatin cap in oocytenuclei of Acerina cernua and demonstrated that incorporation of [3H]thymidineinto it, indicating synthesis of extra DNA, was limited to pachytene.

The mode of formation of peripheral nucleoli in the oocytes of Acipenseridaeis also very similar to that in Xenopus laevis (Gall, 1968; Macgregor, 1968),although the primary nucleolus in Acipenser oocytes, unlike that of Xenopusoocytes (Van Gansen & Schram, 1972) shows no budding. On the other handthe 'supernumerary' nucleoli were reported to arise in oocytes of Salvelinusfontinalis (Salmonidae) in succession, on a single heterochromatin segment of abivalent (Chouinard, 1963).


In spite of the fact that lampbrush chromosomes produce in Acipenseridaean enormous quantity of 'granules', most of which are of ribonucleoproteinnature and some of which are rather large and nucleolus-like, these 'granules*are unlikely to become peripheral nucleoli as has been said in Triturus (Mac-gregor, 1965). In Acipenser at least, the 'granules' differ from true peripheralnucleoli in size, fine structure, and mode of formation.

The peripheral nucleoli of the Acipenseridae undergo a definite sequence ofchanges: at first they develop into amphinucleoli carrying a granular 'knob',later, during early vitellogenesis, they round up and then pull into circles. At theend of vitellogenesis, they return to the nuclear periphery and become sphericalvacuolized bodies.

Amphinucleoli are common in oocytes of fishes and other animals. They havebeen observed in Acipenser stellatus (Votinov, 1947), in Salmonidae (Persov,1966), some Cyprinidae (Arndt, 1960), and in Amphibia (Brown & Ris, 1959).In all these cases, both cytochemical (Guyenot & Danon, 1953; Tandler, 1958)and ultrastructural differences (Miller, 1962; Raikova, 1972; Van Gansen &Schram, 1972) exist between the two parts of an amphinucleolus. Amphinucleoliare most typical of molluscan oogenesis (Hacker, 1899); many authors reportdifferential staining properties and density of their two parts (Bolognari, 1959;Romanova, 1963, 1964), as well as differential incorporation of precursors ofnucleic acids and proteins into them (Romanova & Gazarian, 1966).

Stretching of nucleoli into threads, especially during vitellogenesis and beforethe meiotic divisions, is a rather common phenomenon during oogenesis invarious animals, such as Hydrozoa, Mollusca, Aranei, Insecta, and Vertebrata(Jorgensen, 1913; Kedrovsky, 1959; Sokolov, 1960). This phenomenon occursalso among fishes, e.g. during vitellogenesis in sharks (Marechal, 1907) andsome deep sea fishes (Jorgensen, 1913; Nusbaum, 1913), and before meioticdivisions in some teleosts (Yamamoto, 1956; Sakun, 1961). The nucleoli ofthe newt oocyte also transform into thick strands (Guyenot & Danon, 1953).In axolotl oocyte nuclei, the peripheral nucleoli transform into circular threadswhich later condense again into spherical nucleoli (Callan, 1966; Lane, 1967).These phenomena are very similar to those in Acipenseridae. It is quite possiblethat in other animals also, where nucleoli are reported to transform into'strands' or 'threads', these structures are in reality circular.

It is now known that an axis of extrachromosomal DNA exists inside eachperipheral nucleolus (Miller, 1964) and that the transcription activity of thisDNA axis affects both the morphology and the metabolism of the nucleolus(Miller, 1964,1966,1967; Macgregor, 1967; Lane, 1967; Ebstein, 1969, Miller &Beatty, 1969; and others). According to Ebstein, the cycle of changes of thisDNA axis can be compared to that of a chromomere which transforms into aloop of a lampbrush chromosome. Transformation of the DNA axis fromchromomere state into loop state may bring about a corresponding change ofthe shape of the entire nucleolus which becomes circular and beaded. Lane

684 E. V. RAIKOVA

(1967) demonstrated that this transformation is correlated with a change in thepattern of incorporation of [3H]uridine into the nucleolar RNA: while pre-viously the label was incorporated into the fibrillar core only, it now becomesdistributed all along the circle.

No experiments with uridine incorporation have yet been made with Acipenseroocytes. However, the morphological transformations of their nucleoli beingvery similar to those of Amphibia, we have reasons to believe that the intrinsicmechanism of these transformations is the same in both groups.

Apart from both primary and peripheral nucleoli, RNA-lacking protein-aceous structures, topographically related to nucleoli, are consistently observedduring oogenesis in Acipenseridae. These structures are: (1) the paranucleolar'vacuole' in the oogonial nucleus, which probably gives rise to the less stainableof the two cytoplasmic bodies considered as markers of germ line cells; (2) the'nuclear bodies' of Kessel (1969), which are satellites of the peripheral nucleoliin oocytes; (3) the spheres, which are formed on lampbrush chromosomes inoocyte nuclei.

The first two types of structure undergo a somewhat similar evolution, duringdevelopment of the respective nucleoli: they grow, become more compact andtransform into well delimited bodies visible even with the light microscope andaccompanying their respective nucleoli. The last type of structure is the spheres;these are never in contact with nucleoli and are produced on lampbrushchromosomes. They consist of thicker fibrils (10-11 nm) than those of the'nuclear bodies' (9 nm) and of the paranucleolar 'vacuoles' in oogonia (7 nm).

The paranucleolar fibrillar material is also reported to exist in oogonialnuclei of Amphibia (Coggins, 1973). Cytoplasmic bodies similar to those inAcipenser oogonia have been found in gonocytes and oogonia of the teleost fishOryzias (Satoh, 1974). Finally, nuclear fibrillar inclusions, closely resemblingthose of Acipenseridae, are known to exist in oogonia of Lacerta (Hubert,1970a, b).

Spheres are rather common in oocytes where much yolk is produced; in thenuclei of such oocytes, spheres often occur in enormous quantities, e.g. in somefishes, amphibians, and especially reptiles (Arronet, 1975) and birds (Gagin-skaya, 1972). The function of the spheres remains, however, obscure.

In conclusion, we can state that a great cytological resemblance existsbetween oogenesis in the Acipenseridae on one hand, and in the Amphibia onthe other. This includes not only nucleolar and other nuclear structures, whichwe have discussed, but also the cytoplasmic organelles of the oocytes. Oocytesof the two groups show, in particular, the same structure of yolk plates, thesame presence of cortical granules, the same absence of ribosomes in pre-vitellogenesis, the same extrusions of nucleolar material into the cytoplasm,and the same dense material cementing mitochondria (Raikova, 1973, 19746).

Nucleolar apparatus in Acipenseridae 685The author is greatly indebted to the late Professor I. Sokoloff for his guidance and valuable

advice during the larger part of this study, and to Mrs I. Schmantzar for technical assistance.The author also wishes to thank Professor J. Brachet for most helpful discussion of theresults.

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{Received 12 January 1976)

evolution of the nucleolar apparatus during oogenesis in acipenseridae … · ('granules'...

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