J. Cell Sci. 19, 103-115 (1975) 103

Printed in Great Britain

ACCUMULATION OF GENE PRODUCTS IN THE

OOCYTES OF THE DRAGONFLY

CORDULIA AENEA L.

I. THE NEMATOSOMES

LIISA HALKKA AND O. HALKKA

Department of Genetics, University of Helsinki, P. Rautatiek. 13,SF-00100 Helsinki io, Finland

SUMMARY

The chromosomes of the active previtellogenic oocytes of the dragonfly Cordulia aenea L.extrude into the cytoplasm a substance, which is subsequently found there in the form ofgranulofibrillar masses. These nuclear extrusions evolve ultrastructurally into two components,nematosomes and dense masses. The nematosomes are later found transitorily adjacent to themitochondria and undergo transformations that are synchronized with changes in oocyteactivity. It is suggested that the nematosomes are storage structures for long-lived informationalRNA and that they may be widely distributed in various types of cells containing such RNA.

INTRODUCTION

In some types of somatic cells the cytoplasm contains structures which lack alimiting membrane and consist mainly of a tangled reticulum of diffuse electron-dense fibrils lying in a less electron-dense ground substance. The structures inquestion were first described by Grillo (1970) as 'nucleolus-like bodies' or 'nemato-somes'. She found them in the sympathetic ganglia of the rat, but nematosomesseem to be a fairly common component of nerve cells. Peach (1972) found them inthe trigeminal ganglia of the rat as well. In contrast to these, some other structuresfound in nerve cells and named 'nucleolus-resembling bodies or nematosomes' and'nucleolus-like bodies' (Le Beux, Langelier & Poirier, 1971; Kishi, 1972; and furtherliterature cited therein) differ from true nematosomes in being structurally homo-geneous and without alternation of electron-dense and electron-lucent elements.

The 'glomerular bodies' described by Hernandez-Verdun (1972) from the placentalsyncytia of mouse and rat and from the cytotrophoblasts of primates appearstructurally identical with the nematosomes of neural tissue. The same structureshave been found by Martin & Spicer (1973) in early human placenta, and describedas 'spherical structures comprised of a coiled thread'.

In the present work we report the presence of true nematosomes from the oocytesof a dragonfly, Cordulia aenea, and describe their structure from electron micro-graphs. Thanks to the extremely slow and periodic development of the oocytes ofthis insect, the structural transformations of nematosomes are easier to observein this material than in many other types of cells.

104 L. Halkka and 0. Halkka

MATERIAL AND METHODS

Female nymphs of the dragonfly Cordulia aenea L. belonging mainly to the last 2 instarswere collected from a pond in Tammisaari rural commune, southern Finland. Samples weretaken throughout the year, to the extent that this was possible in winter in spite of the icecover. The material was kept at the temperature prevailing at the time of collection, andoocytes were dissected either from the nymphs or from adults allowed to emerge in thelaboratory.

Individual ovarioles were detached from the ovaries in fresh fixing fluid containing 3 %glutaraldehyde (Taab) and 1 % OsO4 in insect Ringer (recipe 122 in Romeis, 1948) solution.One hour in this fluid was followed by several rinses in insect Ringer solution, all at roomtemperature. Dehydration was performed at 4 °C in a rising series of ethanol saturated withuranyl acetate. The material was embedded in Epon according to the method of Luft (i960),sectioned with a Porter-Blum MT-i ultramicrotome, and the sections mounted on Formvar-coated copper grids. The sections were stained by the lead citrate method of Reynolds (1963)and studied with a Philips EM 200 at the Electron Microscope Laboratory, University ofHelsinki.

RESULTS

Sorting of the nuclear extrusions into two components

Cordulia has a panoistic ovary in which the oocyte chromosomes are mainly inthe lampbrush state during previtellogenesis and concentrate some of the synthesizedRNA in the form of perichromatin granules (peg) along the chromosomes (Figs. 1,2). New pegs are formed only during the active phase of the oocyte cycle (warmseason). At the beginning of the inactive phase (cold season) no pegs were found inthe oocyte nucleus.

Figs. 1-3 show the transport process of peg material from the nucleus intothe cytoplasm to form gradually enlarging nuclear extrusions (nex). These massescomprise 2 components, electron-dense structures intermingled with a fibrillarlow-contrast ground substance. These components separate from each other andaccumulate in 2 different kinds of cytoplasmic bodies, namely nematosomes (ns) anddense masses (dm) (Fig. 3). From their first appearance, the nematosomes seem tobe composed of small electron-dense granules (Figs. 4, 5). Simultaneously, thefibrillar ground substance of dense masses accumulates into large spheres, whichare less electron-dense but compact and homogeneous-looking.

The ns and dm structures are often seen in intimate contact (Figs. 3, 6, 8), pre-sumably as a consequence of their common origin from the nuclear extrusions. Thens-dm contacts are present during both the active and the inactive phases of theoocyte cycle.

Structural transformation of the nematosomes during previtellogenesis

In the present study, further previtellogenic development of the 2 derivatives ofthe nuclear extrusions is described only with respect to the nematosomes. The structuraltransformation of the dense masses and their part in the formation of the annulatelamellae will be discussed elsewhere (L. Halkka, in preparation).

In the active oocyte, the nematosomes (ns) contain granules which are a little

Gene products in dragonfly oocytes 105

smaller and with a less sharp boundary than the nuclear pegs (Figs. 1-5). The nsgranules are arranged in a 3-dimensional network with electron-dense structuresrather regularly interspersed with electron-lucent areas.

During the interval between the active and inactive phases, at the end of thesummer up to the onset of winter, the nematosomes undergo a structural trans-formation. The granules unfold and fuse into an electron-dense reticulate structurecontaining less-dense lacunae (Figs. 6, 7). Thin fibrils cross the lacunae, runningfrom one electron-dense area to another (/ in Fig. 7). At this stage the Cordulianematosomes appear identical in structure with the bodies described by Grillo(1970) in rat sympathetic ganglia.

In the middle of the inactive phase, in nymphs collected from ice-cold water, theelectron-dense component of the nematosomes appears very compact and thestructure as a whole resembles a piece of gruyere cheese with gas holes (Figs. 8, 9).The fibrils which earlier bridged the 'holes' are now irregular in form and do notpenetrate the occasional vacuoles typical of this stage.

At the end of the cold season the overwintered nematosomes lose their rigid-lookingspherical structure and collapse and later disintegrate into several units of smallersize (Fig. 10).

The late previtellogenesis and the vitellogenesis proper begin in Cordulia oocytesin the spring preceding emergence into adult female. The nuclear activity continuesover the period of emergence and produces new nuclear extrusions and freshnematosomes until late previtellogenesis. The new nuclear extrusions disperserapidly as small pieces throughout the oocyte cytoplasm and assort later into ns anddm components.

Tn addition to contacts maintained with the dense masses, the nematosomes enterinto contact with mitochondria, in particular at the activation period of oocytes(Figs. 11, 12). Not infrequently, mitochondria are seen to encircle single nemato-somes or nematosome bundles on all sides. With cell organelles other than densemasses or mitochondria, the nematosomes are found in contact only occasionally,and such contacts are probably fortuitous.

DISCUSSION

The specific carrier proteins active in the transport of RNA from the nucleusto the cytoplasm are known to be components of the nuclear informofers (peri-chromatin granules) and the cytoplasmic informosome particles (see e.g. Spirin,1969; Georgiev & Samarina, 1971; Samarina, Lukanidin & Georgiev, 1973). Themain component of these paniculate structures is mRNA, and the length of lifeof the mRNP particles depends mainly on the rate of utilization of the RNA com-ponent. Really long-lived mRNP particles are to be found in oocytes and embryos.Most of the production and accumulation of RNA in oocytes takes place duringprevitellogenesis and much of the RNA utilization after vitellogenesis, in theembryo. The storage RNA consists of material preformed for the protein-syntheticmachinery of the early embryo (rRNA and tRNA), and of mRNA destined to

106 L. Halkka and O. Halkka

participate in late egg cell development and embryogenesis. Examples of the earlyprocesses making use of storage mRNA are glycogen synthesis (Engels & Bier,1967), synthesis of microtubule protein (Raff, Colot, Selvig & Gross, 1972) andother similar basic operations (cf. the review by Davidson, 1968).

Although it can be shown indirectly (by blocking synthesis of fresh RNA) thatthe stored mRNA is utilized in a given synthesis, the processes by which thendividual mass-manufactured mRNAs are stored and utilized are virtually unknown.It may be speculated that the more ubiquitous and abundant an RNA-containingstructure is in oocytes, the more probable is it a depository for material to be usedin the synthesis of basic mass components of the embryonic cells. In the oocytesof Cordulia, the abundance of nematosomes (and dense masses) suggests that theseare responsible for some dynamic or storage functions of considerable importance.That they are concerned with storage rather than with dynamic functions (at leastduring previtellogenesis) is indicated by the structural changes they undergo duringthe growth and activity cycle of the oocyte.

In autoradiographic experiments (Halkka & HaJkka, 1968) with tritiated uridine,synthesis of insoluble RNA was shown to take less than 30 min in the primarynucleolus and the chromosomes of Cordulia oocytes. The synthesis of nucleolarRNA greatly exceeded the chromosomal synthesis of RNA in 1-, 3-, 9- and 30-hexperiments. Transport of labelled RNA into the oocyte cytoplasm was seen tooccur in 3-h and longer experiments. The rapidly transported RNA was distributedthroughout the oocyte cytoplasm and not in aggregates corresponding to the nuclearextrusions, nematosomes and dense masses. In an unpublished experiment withincubation times prolonged to several days, the labelled material of the primarynucleolus was seen to be transported away more rapidly than the labelled RNAbound to the chromosomes. Thus, the rapidly labelled and rapidly transported RNAcan be interpreted to be nucleolar in origin. This provides indirect evidence thatthe nuclear extrusions, nematosomes and dense masses are not aggregates of RNA-containing material produced by the primary nucleolus.

If there is any substance in the hypothesis that the Cordulia nematosomes anddense masses are storage structures consisting of mRNP, then counterparts to thesebodies should be present in the oocytes of other organisms and in storage cellsother than oocytes. The dense masses and their structural counterparts will bediscussed in another paper (L. Halkka, in preparation).

The nematosomes of Cordulia oocytes are often found in close contact with themitochondria. With regard to electron density, but not in structural detail, the nsare similar to the RNP-containing masses reported by a number of authors fromoocytes and called 'intermitochondrial substance' (ims) (Weakley, 1971), 'cimentintermitochondrial' (Ulrich, 1969), 'ciment dense homogene' (Petit, 1973), etc.The ims has been suggested to contain RNAs produced by the oocyte nucleus andstored for use in connexion with mitochondrial functions (Weakley, 1971). But thecontacts with an ns and mitochondria, although regular in the previtellogenic stagesof oocyte development in Cordulia, are not persistent enough to imply that the onlyor main function of ns is to assist mitochondria.

Gene products in dragonfly oocytes 107

Among all the structures described in oocytes, the 'polar granules' (pg), whichhave been extensively investigated in insects with telotrophic ovaries, are closestto the nematosomes in general external appearance, and perhaps also in function.The pgs have their origin in the nurse cells, whose RNA, after combining witha protein component, moves into the oocyte cytoplasm. The pgs of the oocyte andthe mitochondria form contacts which in appearance strongly resemble those betweenns and mitochondria. After fertilization, the ^ s are known to be transported to thepole cells leading to the germ line of the embryo. During this cellular differentiationprocess, the pgs make contacts with ribosomes and probably mobilize their storedRNA (cf. review by Mahowald, 1972). How far the functions of the pgs are takenover by the nematosomes of Cordulia and other insects with panoistic ovaries is notknown.

Fawcett (1972) suggests that a counterpart of the polar granules is to be foundin the male germ cells, in which there are cytoplasmic, electron-dense 'chromatoidbodies' (cb). The cbs can be seen in contact with the nuclear membrane andmitochondria, and later on they migrate round to the base of the developingflagellum. Obviously, if homology exists between nematosomes, polar granules andchromatoid bodies, it will be revealed only by careful investigations as to thefunctions of these bodies.

It is true, of course, that the homology assumed between the 'nematosomes' or'nucleolus-like bodies' described by Grillo (1970) in the sympathetic ganglia of therat and the structures called nematosomes by us rests solely on structural resemblances.The Cordulia oocyte nematosomes are similar to the ns investigated by Grillo downto the finest structural details observable in electron micrographs (compare Fig. 7to fig. 14 by Grillo). For this reason, we consider it inappropriate to add to thealready bewildering abundance of nomenclature, but instead venture to call thebodies in the oocytes 'nematosomes'. By adopting this name, we also wish to drawattention to the fact that the cells of the nervous system and cells of the germ lineperhaps build similar depositories for informational RNA. Hernandez-Verdun (1972)has already reported nematosomes from tissue other than nervous tissue, namelyfrom placental syncytia and cytotrophoblasts. The somatic nematosome structuresare often considered to be cytoplasmic depositories of nucleolar material. Thenematosomes of Cordulia oocytes, however, are not of nucleolar origin, and wewould like to suggest that the somatic nematosomes may be chromosomal ratherthan nucleolar derivatives.

We are grateful to V. Varis, M.A., for helpful cooperation in collecting the dragonflies andfor valuable information on the biology of Cordulia. The technical assistance of Mrs RiittaHovinen, M.A., during various phases of the work is also gratefully acknowledged. We areindebted to Dr J. Wartiovaara, Head of the Electron Microscope Laboratory, for criticalreading of the manuscript and for valuable suggestions. The English of the manuscript hasbeen checked by Mrs Jean Margaret Perttunen, B.Sc. (Hons.), to whom we offer our warmthanks. The study has been supported by grants from the University of Helsinki and fromthe National Research Council of Science, Academy of Finland.

io8 L. Halkka and O. Halkka

REFERENCES

DAVIDSON, E. H. (1968). Gene Activity in Early Development. New York and London:Academic Press.

ENGELS, W. & BIER, K.-H. (1967). Zur Glykogenspeicherung wahrend Oogenese und ihrervorzeitiger AuslOsung durch Blockierung der RNS-Vereorgung (Untersuchungen anMusca domestica L.). Wilhelm Roux Arch. EntwMech. Org. 158, 64-88.

FAWCETT, D. W. (1972). Observations on cell differentiation and organelle continuity inspermatogenesis. In Edinburgh Symposium on the Genetics of the Spermatozoon (ed. R. A.Beatty & S. Gluecksohn-Waelsch), pp. 37-68. Copenhagen: Bogtrykkeriet Forum.

GEORGIEV, G. P. & SAMARINA, O. P. (1971). D-RNA-containing ribonucleoprotein particles.In Advances in Cell Biology, vol. 2 (ed. D. M. Prescott, L. Goldstein & E. H. McConkey),pp. 47-110. New York: Appleton, Century, Crofts.

GRILLO, M. A. (1970). Cytoplasmic inclusion bodies resembling nucleoli in sympatheticneurons of adult rats. J. Cell Biol. 45, 100-117.

HALKKA, L. & HALKKA, O. (1968). RNA and protein in nucleolar structures of dragonflyoocytes. Science, N.Y. 162, 803-805.

HERNANDEZ-VERDUN, D. (1972). Etude cytochimique du corps glomdrulaire dans le tropho-blaste de Soleris. J. Ultrastruct. Res. 40, 68-86.

KISHI, K. (1972). Fine structural and cytocheniical observations on cytoplasmic nucleolus-like bodies in nerve cells of rat medulla oblongata. Z. ZeUforscli. mikrosk. Anat. 132, 523-532.

LE BEUX, Y. J., LANGELIER, P. & POIRIER, L. J. (1971). Further ultrastructural data on thecytoplasmic nucleolus resembling bodies or nematosomes. Their relationship with thesynaptic web and a cytoplasmic filamentous network. Z. ZeUforscli. mikrosk. Anat. 118,147-155-

LUFT, J. H. (i960). Improvements in epoxy resin embedding methods. J. biopliys. biochem.Cytol. 9, 409-414.

MAHOWALD, A. P. (1071). Origin and continuity of polar granules. In Results and Problemsin Cell Differentiation, vol. 2, Origin and Continuity of Cell Organelles (ed. J. Reinert &H. Ursprung), pp. 158-169. Berlin and New York: Springer.

MARTIN, B. J. & SPICER, S. S. (1973). Ultrastructural features of cellular maturation andageing in human trophoblast. J. Ultrastruct. Res. 43, 133-149.

PEACH, R., (1972). Nematosomes in the trigeminal ganglion. J. Cell Biol. 55, 718-721.PETIT, J. (1973). Etude morphologique et cytochemique de deux types de groupements

mitochondriaux dans les jeunes ovocytes de Polydesmus angustus Latz. (Myriapode, Diplo-pode). J. Microscopie 17, 41-54.

RAFF, R. A., COLOT, H. V., SELVIG, S. E. & GROSS, P. R. (1972). Oogenetic origin of messengerRNA for embryonic synthesis of microtubule protein. Nature, Lond. 235, 211-214.

REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain inelectron microscopy, J. Cell Biol. 17, 208-212.

ROMEIS, B. (1948). Mikroskopische Technik. Miinchen: Leibniz Verlag.SAMARINA, O. P., LUKANIDIN, E. M. & GEORGIEV, G. P. (1973). Ribonucleoprotein particles

containing mRNA and pre-mRNA. In Karolinska Symp. No. 6: Protein Synthesis inReproductive Tissue (ed. E. Diczfalusy & A. Diczfalusy), pp. 130—160. Copenhagen:Bogtrykkeriet Forum.

SPIRIN, A. S. (1969). Informosomes. Eur.J. Biochem. 10, 20-35.ULRICH, E. (1969). Etude des ultrastructures au cours de l'ovogenese d'un poisson te'ltoste'en

le danio, Brachydanio rerio (Hamilton-Buchanan). J. Microscopie 8, 447-478.WEAKLEY, B. S. (1971). Basic protein and ribonucleic acid in the cytoplasm of the ovarian

oocyte in the golden hamster. Z. Zellforsch. mikrosk. Anat. 112, 69—84.

{Received 3 April 1975)

Gene products in dragonfly oocytes 109

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Fig. i. In a young, actively RNA-producing oocyte, perichromatin granules (peg)are seen associated with lampbrush chromosomes. Cytoplasmic nuclear extrusions(nex) are most frequent near the nuclear peg aggregates. The material of thenuclear extrusions and a young nematosome (ns) have the same electron density.x 30000.

Fig. 2. Perichromatin granule material (peg) of a lampbrush chrosomosome protrudesthrough the nuclear pore and forms cytoplasmic aggregates (nex) with the sameelectron density as in the nucleus, x 126000.

Fig. 3. Cytoplasmic nuclear extrusions consist of 2 intermingled components: morecompact and electron-dense nematosome substance (ns) and less electron-densefibrillar ground substance, dense mass (dm). In the nematosome (ns), some granularinner structures can be distinguished, x 126000.Figs. 4, 5. Newly formed nematosomes are composed of small, electron-densegranules which aggregate into spongelike structures. The surrounding ribosomesare much smaller than the granules forming nematosomes. x 78000.

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Figs. 6, 7. Nematosomes at the beginning of the cold period (autumn). Thenematosome granules have fused into electron-dense reticulate sheets of even meshwidth.

Fig. 6. Shows the intimate contact between nematosome (ws) and dense mass(dm) components, x 52000.

Fig. 7. Fine, connecting fibrils (/) can be seen between the sheets of electron-densematerial and are responsible for the evenness of the lacunae, x 84000.Figs. 8, 9. Nematosomes during the cold period of the year. The electron-denseground substance has become very compact. Connecting fibrils are lacking and theinterspaces of the nematosome network are bubble-like and irregular in size andsometimes carry vacuoles surrounded by a single membrane, x 52000.

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Fig. 10. At the end of the cold period, nematosomes in oocytes of a moulting dragonflycollapse and fragment, x 16000.Figs, i i , 12. Intimate connexions between mitochondria and nematosomes can beobserved at the beginning of the active period (spring) in previtellogenic oocytes.x 52000.

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