Recent Advances in Insect Embryology in Japan and Poland
Edited by H. Ando and Cz. JuraArthropod. EmbryoL. Soc. Jpn.
(ISEBU Co. Ltd., Tsukuba) 1987

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Intermediate Nurse Cells in Campodea spp. (Diplura).
Differentiation and Possible Role during Oogenesis*

Szczepan BILINSKI
andWladyslawa TYLEK
Synopsis
During previtellogenesis in Campodea spp. the nurse cells differentiate into two categories.
Two cells adjoining the oocyte and connected with it, via intercellular bridges, develop into "intermediate cells" whereas other cells become typical nurse cells. The intermediate cells are morphologically and functionally transitional between the oocyte and nurse cells. The occurrence ofsuch transitional cells confirms, as previously suggested, a gradient character of determinant controlling differentiation of the oocyte and nurse cells from the uniform group of sibling cystocytes.

Introduction
Previous studies have shown that the ovaries of Campodeidae are of the polytrophic meroistic type (Bareth, 1972; Asaba and Ando, 1978; Bilinski, 1979). In Campodea spp. (Bilinski, 1979) and Lepidocampa (Asaba and Ando, 1978) each egg chamberis composed of an oocyte and 20 - 30 nurse cells, which is an exception to the N = 2nrule (for review see Telfer, 1975). During previtellogenesis in Campodea spp. the nursecells undergo additional differentiation: two cells connected with the oocyte, via intercellular bridges, develop into intermediate cells while other cells become typical nursecells (Bilinski, 1983b).
The aim of the present study was to describe the differentiation and developmentof the intermediate cells in the course of oogenesis. The results are discussed with reference to the possible function of these cells.

* Supported by Grant MR 11, I. 6. 4.

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S. Bilinski and W. Tylek

Material and Methods
Specimens of Campodea spp. were collected between May and October in the Bieszczady National Park. After dissection, ovaries were fixed in 2 % OS04 in O. 1 M phosphate buffer (pH 7.3). Sucrose was added to the fixative to produce a final osmolarityof 300 mosmol. After dehydration the material was embedded in Epon 812. Ultrathin
sections were contrasted with uranyl acetate and lead citrate and examined in a Tesla
BS 500 electron microscope at 60 kV. For routine microscopy, semi thin epoxy sections
were stained with 1 % methylene blue in I % borax. For the demonstration of RNAsemi thin sections, after removal of Epon (Maxwell, 1978), were stained with toluidine
blue at pH 5 (Stockert, 1975).

Results
Previtellogenesis
The oocyte at this stage is a flattened cell ca. 40 t-t m in diameter. Its large nucleus
is centrally located and translucent in light microscopic preparations (Fig. I). Elementsof rough endoplasmic reticulum (RER), mitochondria and numerous dense granules
are present in the ooplasm. These latest structures occur predominantly near the nucleus (Fig. 1). The apical region of the egg chamber is occupied by the typical nursecells (Figs. I, 2). In the cytoplasm of these cells mitochondria, free ribosomes andaccumulations of nuage material are observed (Figs. I, 2, arrows). Spherical nuclei ofthe nurse cells contain densely stained nucleoli and numerous chromatin aggregations(Figs. I, 2). In the central part of the egg chamber, between the oocyte and nurse cells,
two "intermediate cells" occur. These cells most frequently adhere to the central partof the oocyte containing the nucleus (Fig. 1). The intermediate cells are, in some respects, transitional between the oocyte and nurse cells. In light microscopic preparations their nuclei, while "lighter" than the nuclei of typical nurse cells, are substantially"darker" than that of the oocyte (Figs. I, 2). The accumulations of the nuage material,
typical of nurse cells, are not observed in the intermediate cells.
Staining with toluidine blue at pH 5 for RNA demonstration gives following results: the nucleoli and the accumulations of nuage material are positive (Fig. 3) while
the difference in the karyoplasm density between the intermediate and nurse cells is
less conspicuous than in routine staining (compare Figs. 1-3).

Vitellogenesis
The intermediate cells at this stage of oogenesis still differ markedly from thenurse cells (Fig. 4). In the cytoplasm of the intermediate cell mitochondria, elements ofRER, lipid droplets and numerous dense granules are observed (Figs. 4, 5). Theselatest structures are enclosed in dilated vesicles of RER (Fig. 5, arrows). It has beenreported previously (Bilinski, 1979), that identical granules are formed also in theoocyte cytoplasm (there termed type I yolk spheres). The ovoid nucleus of intermediate

Intermediate nurse cells in Campodea

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Figs. 1, 2. Semiserial sections of an egg chamber during previtellogenesis. Note oocyte (00),intermediate cells (ic) and nurse cells (nc). Arrows indicate accumulations ofnuage material in nurse cell cytoplasm. Methylene blue. XI, 300.

26Fig. 3.

S. Bili6ski and W. Tylek

Photomicrograph of an egg chamber during previtellogenesis. Note nucleoli (nu)and aggregations of nuage material (arrows). ic, intermediate cell; ne, nutse cells;
00, oocyte. Toluidine blue at pH 5. X 900.
Fig. 4. Photomicrograph of an egg chamber during vitellogenesis. Note dense granules andtransparent lipid droplets in the cytoplasm of intermediate cells (ic). Arrow indicates weakly stained structure in the nucleus of intermediate cell. ne, nurse cell; nu,nucleolus; 00, oocyte; small arrows, aggregations of nuage material. Methylene
blue. X 1,000.
Fig. 5. Portion of an intermediate cell. Note electron dense granules (arrows), mitochondria, RER elements and ribosomes. X 24,000.

Intermediate nurse cells in Campodea

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cell contains only a single weakly stained structure (Fig. 4, arrow), that might be interpreted as residual, nonfunctional nucleolus.
Typical nurse cells are markedly larger than intermediate cells (Fig. 4). The nurse
cell cytoplasm contains numerous mitochondria and RER elements, while dense granules, typical for the intermediate cells, are unfrequent. Elongated accumulations ofnuage material are observed in close contact with nuclear envelope (Fig. 4, smallarrows). Within the nucleus, large densely stained nucleolus occur (Fig. 4).
During vitellogenesis the ribosomes, lipid droplets and dense granules, producedby the nurse and/or intermediate cells, are transported to the oocyte. At the end of thisstage both the nurse and intermediate cells degenerate.

Postvitellogenesis
During this developmental stage a thin granular chorion is deposited on the oocyte
surface (Bilinski, 1983a). Concurrently, segregation of reserve material takes place
within the ooplasm. Dense granules migrate to peripheral ooplasm and make up a distinct cortical layer (Figs. 6, 7). Larger spheres, originating from pinocytotic vesicles, remain in the oocyte centre (Fig. 6).

Discussion
Previous studies have shown that the principal function of typical nurse cells in
Campodea is to synthesize rRNA, that is later transported to the oocyte (Biliiiski, 1979).
This sequence agrees well with generally accepted view of the role of trophocytes inoogenesis (reviewed by Telfer, 1975). It seems likely that the participation of the intermediate cells in rRNA synthesis is limited, as may be judged by the disappearanceof their nucleoli. On the other hand, the intermediate cells show some features in common with the oocyte, i. e. they are able to synthesize dense granules and lipid droplets.
A comparison of results obtained by the routine and toluidine blue stainings showthat the difference in the karyoplasm density between the nurse and intermediate cells
results from different DNA rather than RNA content. This may suggest that thesetypes of cells differ in the degree of endopolyploidy.
All these data show that the intermediate cells are transitional between oocytes
and typical nurse cells not only morphologically but also functionally. The occurrenceof such transitional cells confirms earlier reports (Biliiiski, 1983b) on gradient characterof determinant responsible for differentiation of the oocyte and nurse cells from the originally uniform group of sibling cystocytes.
The egg chamber of Campodea shows much similarity to that of another apterygotan insect, Tetrodontophora bielanensis. In this species two nurse cells, connected directlywith the oocyte, differ from remaining nurse cells (Krzysztofowicz, 1971, 1975). Autoradiographic and ultrastructural studies have shown that these two cells play no partin RNA synthesis (Krzysztofowicz, 1971, 1975). Among pterygotan insects all nursecells, of a given chamber, are morphologically and functionally similar. It has beenshown, however, that in some species nurse cell nuclei contain various amounts of

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S. Bilinski and W. Tylek

Fig. 6.

Follicle cells (fc) during postvitellogenesis. Note chorion (arrows) and dense granules forming cortical layer of ooplasm. y, yolk spheres. Methylene blue. X 1,500.

Fig. 7.

Cortical ooplasm filled with dense granules (asterisks). fe, follicle cell; arrows, chorion. X 19,000.

Intermediate nurse cells in Campodea

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DNA, i. e. achieve various degree of endopolyploidy. In Drosophila the degree of ploidy
in the nurse cells adjacent to the oocyte is 2 or 4 times greater than those of more distally located cells (reviewed by Telfer, 1975).
The dense granules, formed in the oocyte and intermediate cells have been previously described and called "type I yolk spheres" (Bilinski, 1979). The mode of theformation of these granules, their migration to the oocyte periphery as well as theirbiochemical composition different from that of the yolk granules (Bareth, 1972; Asabaand Ando, 1978) show, however, that they represent yet another kind of reserve material. It might be suggested, that these granules contain the precursors of a substancewhich having been released from the oocyte, forms additional egg envelope or reinforces the chorion. This supposition is in line with the results of previous studies(Bilinski, 1983a) showing that the chorion in Campodea spp. is thin and composed ofloosely aggregated granules.

References
Asaba, H. and H. Ando, 1978. Ovarian structures and oogenesis in Lepidocampa weberi Oudemans(Diplura: Campodeidae). Int. j. Insect Morphol. Embryol. 7: 405-414.
Bareth, C., 1972. L'ovogenese chez un Arthropode du sol: Campodea (C. ) remyi (Diploures).
Structure et evolution de l'ovaire. C. R. Acad. Sci. Paris 275: 1887-1890.
Bilinski, S., 1979. Oogenesis in Campodea sp. (Diplura). The ultrastructure of the egg chamber
during vitellogenesis. Cell Tiss. Res. 202: 133-143.
- - - - - , 1983a. Oogenesis in Campodea sp. (Diplura): chorion formation and the ultrastructure offollicle cells. Cell Tiss. Res. 228: 165-170.
- - - - - , 1983b. Differentiation of the oocyte and nurse cells in an apterygote insect (Campodea). Tiss. Cell 15: 965-973.
Krzysztofowicz, A., 1971. Histochemical and autoradiographic analysis of RNA synthesis introphic cells of the female gonad in Tetrodontophora bielanensis (Waga) (Collembola). Acta Biol.
Crac.,.Ser. Zool. 14: 299-305.
- - - - - , 1975. Histochemical and ultrastructural analysis of blocking nurse cells in thefemale gonad of Tetrodontophora bielanensis (Waga) (Collembola). Acta Biol. Crac., Ser. Zool. 18:45-53.
Maxwell, M. H., 1978. Two rapid and simple methods used for removal of resins from 0.1 p. mthick epoxy sections. j. Microsc. 112: 253-255.
Stockert,]. C., 1975. Nucleic acid metachromasy by toluidine blue in Epon thick sections. Microsc. Acta 76: 433-436.
Telfer, W. H., 1975. Development and physiology of the oocyte-nurse cell syncytium. Adv. InsectPhysiol. 11: 223-319.

Authors' address: Dr. S. Bilinski and MSc. W. Tylek
Department of Systematic Zoology, Institute of Zoology, Jagiellonian University, ul. Karasia 6, 30-060 Krak6w,Poland