On the function of the germ line chromosomes in the oogenesis of Wachtliella persicariae (Cecidomyiidae)

Chromosoma (Berl.) 30, 180--192 (1970)

On the Function of the Germ Line Chromosomes in the Oogenesis

of Wachtliella persicariae ( Cecidomyiidae) * * *

WEI~NEI~ KUNZ, I~ANS-HEYNRICI-I TI~EPTE and KARLH~INZ B I ~ (?)

Zoologisches Institut der Universitat Miinster i. Westf.

Received October 27, 1969 / Accepted February 16, 1970

Abstract. Autoradiographic and cytological investigations were performed to elucidate the role of supernumerary chromosomes (E chromosomes) in the oogenesis of WachtlieUa persicariae. In contrast to the compact S chromosomes, these extra chromosomes are despiralized during the entire oogenesis (Fig. l) and synthesize RNA (Fig. 10c). As no nucleoli could be found in the ooeyte nucleus, the E chromosomes are thought to produce messenger I~NA. - - The S chromosomes are inactive in the first period of oocyte growth and surrounded by concentric lamellate bodies (Fig. 4). After degeneration of the nurse chamber, they also despiralize and synthesize RNA. - - In certain stages of oocyte development, there is a striking correspondence in the number of bundles of the E chromosomes with the haploid number of the S chromosomes (Fig. 5). These findings could be a hint to polyploidy of the supernumerary chromosomes.

I. Introduction

The gall midges are d is t inguished b y the difference in number of chromosomes in the i r germ line as compared to somat ic tissues. Germ cells of female Wachtliella persicariae contain, in add i t i on to the 8 chromosomes p resen t in the soma (S chromosomes), more t h a n 30 chromosomes which are e l imina ted f rom the p re sumpt ive soma cells dur ing ear ly cleavage divisions and are called E chromosomes (White, 1954; Geyer-Duszyf iska , 1959). The h i ther to exis t ing publ ica t ions on E chromosomes of gall midges are ma in ly res t r i c t ed to the i r descr ipt ion, s ta te- m e n t of the i r number , and the i r w a y of ad jus t ing the chromosome complemen t a f te r meiot ic reduct ion. The hypothes i s of Pa in t e r (1966) t h a t the e x t r a chromosomes con t r ibu te to the synthes is of r ibosomes dur ing oogenesis is no t suppo r t ed b y exper iments . Inves t iga t ions on the funct ion of E chromosomes have only been made b y Ba n toe k (1961) and Geyer -Duszyf l ska (1966). These au thors have d iscovered t h a t a f te r expe r imen ta l e l imina t ion of the E chromosomes in pole cells the gonads are no longer able to produce fert i le germ cells. As oogenesis is the

* With financial support by the Deutsche Forschungsgemeinschaft. ** Herrn Professor Dr. Dr. h .e .B. Rensch zum 70. Geburtstag gewidmet.

Germ Line Chromosomes in the Oogenesis of Wachtliella 181

stage in the germ line which is metabolically most active, studies are necessary to show whether or not the E chromosomes have a function during oocyte growth.

II. Material and Methods Material. Galls of Wachtliella persicariae were collected on Polygonum amphib-

ium in the moist lowlands surrounding Mfinster during late summer. After the larvae had pupated, the cocoons were taken from the galls and placed on filter paper which was regularly moisted. In this way the pupae could be kept alive for several weeks.

Preparations. The ovaries of pupae were examined at different stages of development (1) in in vitro preparations, (2) after the Feulgen procedure and (3) under the electron microscope. The methods have been described in detail earlier (Kunz, 1969). For autoradiographicM investigation of the RNA synthesis the pupae were injected by means of very fine glass capillary pipettes with small quantities (< 1 ~zC per animal) of a H a uridine/cytidine mixture (uridine: ll,250mC/mM, eytidine: 14,000 mC/mM; concentration: 1 mC/ml).

III. Results

Already in the oogonia S and E chromosomes are morphologically distinguishable. While normally the "chromatin" in the interphase nuclei is distributed so diffusely that an identification of chromosomes is im- possible, the Cecidomyiidae make an exception herein such that only the E chromosomes are despiralized. In contrast hereto, the S chromosomes are positively heteropycnotic and ressemble in their shape metaphase chromosomes. In the oogonia the eight S chromosomes are already somat- ically paired. The four pairs are situated in the centre of the nucleus and are surrounded by the despiraiized E chromosomes (Fig. l a, b). The nuclei of the oogonia are distinctly larger than the nuclei of the neighbouring nurse cells which also differ from the oogonia by the lack of positively heteropyenotic S chromosomes.

In the zygotene the S chromosomes of the oocytes uncoil into long threads running parallel in pairs (Fig. 1 e). In contrast, the E chromosomes stay nearly unchanged as diffuse, elondlike bodies. This state is retained during the following two stages of meiosis when the S chromosomes pass through typical pachytene and diplotene. After diplotene the S chromosomes show chiasmata in the way characteristic of diakinesis (Fig. 2). This phase of the nucleus is kept up till the nurse chamber degenerates. By then the growing oocyte has reached only one third of its final volume.

At this stage concentric lamellae become visible in the centre of the nucleus. At first they envelope each S bivalent separately. Later on all S chromosomes are surrounded by common lamellar structures (Matu- szewski, 1962) (Fig. 2). From the observation of living ooeyte nuclei,

182 W. Kunz, H.-H. Trepte and K. Biert:

Fig. 1 a--d. S chromosomes (S) in oogonia (a, b) and in oocytes (e, d) are arranged in 4 pairs in a condensed state and are situated in the centre of the nucleus and surrounded by the despirMized E chromosomes (E). e Probably zygotene;

d diakinesis, a Unfixed, phase contrast; b - -d Feulgen squash

in whose centre the g lobular l amel la te bodies are set off d i s t inc t ly f rom the diffuse, pe r iphera l ly s i tua ted E chromosomes, i t is ev iden t t h a t the pe r ichromosomal shea ths are no t ar tefacts .

The electron microscope reveals t h a t the lamel lae which consist of fine fibrils (Fig. 3) have a th ickness of 270 A and are sepa ra t ed f rom each o ther by an in te rspace of 500 A bread th , 6 to 12 lamel lae form a group a round one centre (Fig. 4). The exter ior ones bend a w a y from


Fig. 2. Diakinesis. The S chromosomes in the centre of the nucleus are enveloped by lamellae. The peripherally s i tuated E chromosomes are grouped in 4 loose

bundles

Fig. 3. The lamellae which surround the S chromosomes consist of fine fibrils

184 W. Kunz, tt.-H. Trepte and K. Bier?:

Fig. 4a--c. Ultrastructure of the lamellate bodies. Fixation in 1% O s O 4 accord- ing to the method of tCyter and Kellenberger, embedding in Epon (Luft)

the concentr ic fo rma t ion and pass cont inuous ly over in to the neighbouring l amel la te bodies (Fig. 4a). I n the nuclei of the oocytes which are a l i t t le older, the lamel lae t u rn a l i t t le fu r ther a w a y from each other. Their course which was p r e p o n d e r a n t l y closed so far is i n t e r r u p t e d b y l i t t le gaps a t i r regular in tervals . Besides tha t , compac t globules which


Fig. 5. Late diakinesis. The E chromosomes are arranged in 4 bundles, the number of which corresponds to the haploid number of the S chromosomes. Feulgen squash

in sections have an average diameter of 700 A appear in the lamellae (Fig. 4c).

The lamellate bodies have also been described in the oocyte nuclei of the dragonfly Cordu l ia aenea (HMkka, Halkka and Nyholm, 1969). The similarity in details and dimensions with Wacht l i e l la is so great that we are most probably dealing with identical structures.

During diakinesis, the E chromosomes which are at first still diffusely distributed in the nucleus (Fig. l d), contract more and more, until they finally from four loose bundles (Figs. 2, 5). These bundles are, however, present for a short time only; for, after degeneration of the nurse chamber, again a strong despirMization sets in which goes so far that the limits of the visible Feulgen staining are reached. A definition of individual bundles is no longer possible. In contrast to the stages before degeneration of the nurse chamber, this second despirMization affects the S chromosomes too. They become increasingly diffuse and finally cannot be distinguished from the E chromosomes.

Towards the end of oocyte growth the metaphase contraction of the S and E chromosomes commences. The E chromosomes again appear

185 W. Kunz, H.-H. Trepte and K. Biert:

Fig. 6. Beginning of the metaphasic contraction; above the S chromosomes, below one of the 4 E chromosome bundles. E chromosome threads are more or less

parMlely arranged. Feulgen squash

in four groups in which regularly chromosome threads run roughly parallel over long distances (Fig. 6). The S chromosomes at this stage are still lying in the centre of the nucleus, and can again be distinguished by their stronger spiralization.

Striking is the correspondence in number of the bundles of E chromosomes with the haploid number of S chromosomes. Kraczkiewicz (1966) discovered the same relations in Rhabdophaga saliciperda and con- cluded that these findings, if regularly appearing, could be hint to polyploidy of the E chromosome set. The observations on Wachtliella support this assumption.

Chorion production now follows, and during its course all differences between the S and the E chromosomes disappear. The bundles fall apar t into single E chromosomes which arrange themselves on the equatorial plate of metaphase I. The metaphase plate contains 20 bivalents, a number which equals the haploid number of the S and E chromosomes (Fig. 7).

At all stages of their growth the nurse cell nuclei of Wachtliella are smaller than the ooeyte nucleus (Fig. 8b). Their nucleolus is very big and in some cases divided into two arm-like parts (Fig. 8a). The nurse


Fig. 7. l\'[etaphase I, 20 bivalents. Unfixed preparation, phase contrast

cell nuclei are r ich in he te rochromat ic bodies which are pa r t i cu l a r ly concen t ra ted a t the pe r iphe ry of the nueleolus (Fig. 8b). The ul t ra- s t ruc ture of the nucleolus is charac te r ized b y a l ight, ma in ly f ibr i l lar

188 W. Kunz, H.-H. Trepte and K. Bier?:

Fig. 8a and b. Nurse cell nuclei. They contain a big, in some cases branched nueleolus (a) which is surrounded by heterochromatie bodies (b, below). In b, above, the oocyte nucleus, below, one nurse cell nucleus, and on the left two follicle cell nuclei, a Living nurse cell nuclei, phase contrast; b Feulgen squash

preparation

interior and more compact, granular outer regions. The heterochromatin is situated in the outer regions, clearly distinguishable from nucleolar structures (Fig. 9a). The nurse chamber and the oocyte are separated from each other by a cell membrane. There exists, however, a cyto- plasmic connection of at least one fusome (Fig. 9b). Inside the nurse chamber there are no cell boundaries (Fig. 9c).

In insects which have a meroistic ovary, the RNA necessary for the growing oocyte is regularly supplied by the nurse chamber, while the nucleus of the oocyte is inactive (Bier, 1963). Among the group so far examined, only the Dytiscidae make an exception. The oocyte nucleus contributes to RNA synthesis, although the chromosomes are no doubt closely packed into a karyospherc. However, a great amount of ribosomal RNA is produced by multiple nucleolus organizers forming the well known ring of Giardina in the oocyte stem line of oogonia (Bier, Kunz and Ribbert, 1967; Gall, Macgregor and Kidston, 1969).

The despiralization of the E chromosomes in Wachtliella furnishes a reason for the assumption that, here too, the nucleus of the oocyte has a share in RNA synthesis. As expected for a meroistic insect, the nurse chamber is the main supply centre of RNA for the growing egg.

Germ Line C h r o m o s o m e s in t he Oogenesis of Wachtliellc~ 189

Fig. 9. a H e t e r o c h r o m a t i c bodies a t t he border of t he nu r se cell nuc leotus (arrows). b F u s o m e be tw een nur se c h a m b e r (above) a n d ooeyte, e In s ide t he nu r se c h a m b e r

the re are no cell bounda r i e s

Fig. 10a--d. Radioautographs showing labeled RNA. a Cross-section through the nurse chamber; prominent labeling of the nueleoli after 15 rain 3H uridine in- corporation, b, e Longitudinal sections through the follicle, b After 1 h incubation radioactive RNA from the nurse chamber enters the oocyte (arrow). c The peripheral region of the oocyte nucleus with distinct RNA labeling (arrow), 1 h incubation, d After the degeneration of the nurse chamber, the central region of the oocyte nucleus takes part in RNA synthesis (arrow, oocyte nucleus), 1 h

incubation

W. Kunz et aI. : Germ Line Chromosomes in the Oogenesis of Waehtliella 19I

After application of H a uridine, the nurse cell nuclei show in the radioautographs a prominent labeling which is particularly intense over the nucleoli (]Pig. 10a). This RNA enters the ooplasm via the fusome(s) (Fig. 10b). In the ooeyte nucleus, radioactive RNA is also traceable, however, its quantity is less than in the nuclei of the nurse cells. At the beginning labeling is restricted to the peripheral region of the nucleus, where the E chromosomes are situated, while the contracted S chromosomes do not yet show any activity (Fig. 10c). After degeneration of the nurse chamber, RNA labeling is also found in the central region of the ooeyte nucleus. This labeling is found over the now also despiralized S chromosomes (Fig. 10d). Under the electron microscope no nucleoli could be found in the ooeyte nuclei at these stages, neither at the regions of RNA synthesis nor elsewhere.

IV. Discussion

Bantock (1961) and Geyer-Duszyfiska (1966) have proved that the oocytes of the gall midges cannot be developed when E chromosomes are absent from the germ line. From our results follows that the E chromosomes in the oocyte nucleus of Wachtliella synthesize ~NA during the entire growth of the egg. Evidently the RNA, which is produced by the E chromosomes, is the necessary supplement for a complete growth of the oocytes.

With the synthesis of ribosomal RNA regularly the occurrence of nucleoli is correlated. As no nueleoli were found in the oocyte nuclei of Wachtliella even under the electron microscope, the RNA demon- strated in the oocyte nucleus seems to be messenger RNA. An activity in synthesis of the nucleus of the oocyte, which continues during the growth of the egg, is up to now known among the meroists only in the Dytiscidae. There, however, only the rDNA gene region is activated, while all the other genes are blocked (Bier, Kunz and Ribbert, 1967; Gall, Macgregor and Kidston, 1969). A synthesis of messenger I%NA in the nucleus of the growing ooeyte would be a novelty for meroists.

Acknowledgements. We thank Miss G. Bohr and Miss ~. Nordmann for their careful technical assistance and It. G. l%uerborn for the translation. Dr. H. G. Fromme has placed the Siemens Elmiskop I at our disposal. Prof. Dr. E. MShn from the Staatl. Museum f. Naturk. Stuttgart has confirmed our determination of Wachtliella persieariae.

References

Bantock, C. : Chromosome elimination in Cecidomyiidae. Nature (Loud.) 190, 466-- 467 (1961).

Bier, K. : Synthese, interzelluli~rer Transport und Abbau yon Ribonukleins~ure im Ovar der Stubenfliege Musca domestica. J. Cell. Biol. 16, 436440 (1963).

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Bier, K., Kunz, W., Ribbert, D. : Struktur und Funktion der Ooeytenchromosomen und Nukleolen sowie der Extra-DNS w~Lhrend der Oogenese panoistischer und meroistiseher Insekten. Chromosoma (Berl.) 211, 214--254 (1967).

Gall, J. G., Macgregor, H. C., Kidston, M. E. : Gene amplification in the oocytes of Dytiseid water beetles. Chromosoma (Berl.) ~6, 169--187 (1969).

Geyer-Duszyfiska, I. : Experimental research on chromosome elimination in Ceei- domyiidae (Diptera). J. exp. Zool. 141, 391--448 (1959).

- - Genetic factors in oogenesis and spermatogenesis in Cecidomyiidae. Chromo- somes Today 1, 174--178 (1966).

Halkka, 0., Halkka, L., Nyholm, M. : Lamellate bodies in the nuclei of dragonfly (Odonata) ooeytes. Z. Zellforseh. 94, 534--541 (1969).

Kraczkiewiez, Z. : Premiers stades de l'oogen~se de Rhabdo~)haga saliciperda (Ceei- domyiidae, Diptera). Chromosoma (Berl.) 18, 208--229 (1966).

Kunz, W. : Die Entstehung multipler Oocytenukleolen aus akzessorisehen DNS- KSrpern bei Gryllus domestieus. Chromosoma (Berl.) 26, 41--75 (1969).

Matuszewski, B. : Oogenesis in Mikiola fagi Hart. (Cecidomyiidae; Diptera). Chromo- soma (Berl.) 12, 741--811 (1962).

Painter, T. S. : The role of the E-chromosomes in Cecidomyiidae. Proc. nat. Aead. Sci. (Wash.) 66, 853--855 (1966).

White, M. J. D.: Animal cytology and evolution, 2nd ed. Cambridge: University Press 1954.

Dr. W. Kunz, H.-H. Trepte Zoologisches Insti tut der Universit/it 4400 Miinster i. Westf., Badestr. 9 W. Germany

Documents

On the function of the germ line chromosomes in the oogenesis of Wachtliella persicariae (Cecidomyiidae)