the cytology of the vitellogenic stages of oogenesis in drosophila melanogaster

Z. Zellforsch. 118, 482-492 (1971) �9 by Springer-Vcrlag 1971

The Cytology of the Vitellogenic Stages of Oogenesis in Drosophila melanogaster

I I I . F o r m a t i o n o f the Vi te l l ine M e m b r a n e *

MICHAEL R. CUMMINGS, NANCY M. BROWN and ROBERT C. KING

Department of Biological Sciences, University of Illinois at Chicago Circle, Chicago and Department of Biological Sciences, Northwestern University, Evanston, Illinois/U.S.A.

Received February 19, 1971

Summary. :Electron microscopic studies of oogenesis in Drosophila melanogaster suggest that the ovarian follicle cells alone are responsible for the secretion of the vitelline membrane and chorion. The synthesis and assembly of the vitelline membrane is a complex process involving several stages of development and different populations of follicle cells. This combined autoradiographic and ultrastructural investigation of vitelline membrane formation has led to the conclusion that the protein component of the vitelline membrane is synthesized in the follicle cells, and that these cells possess a mechanism which directs the polarized synthesis and deposition of vitelline membrane and chorion in response to contact by a specific cell, the oocyte. Under certain aberrant conditions, however, other cell types may serve to induce formation of these membranes. The concept of Drosophila egg coverings as maternal cuticle is also discussed, with regard to the embryonic origin of secreting cells, the requirement for adjacent cells as inducers, and the differences in ultrastructural mechanisms of formation.

Key-Words: Oogenesis - - Drosophila melanoffaster - - Follicle cell - - l~embrane formation - - Vitelline membrane.

Introduction

Insect oocytes are surrounded by an inner vitell ine membrane and an outer

chorion. Ea r ly entomologists having only the l ight microscope a t their disposal

in te rpre ted the vi tel l ine membrane and chorion as products of the oocyte and

follicle cells, respect ively (Imms, 1957). However , studies of the u l t ras t ruc ture

of these membranes in the frui t fly, Drosophila melanogaster, (King and Devine,

1958; King, 1960; King and Koch, 1963) have led to the conclusion tha t the

follicle cells alone are responsible for the secretion of both membranes . I t is the

purpose of this paper to present fur ther evidence to support this conclusion.

Materials and Methods

Five-day-old, mated female Drosophila melanogaster belonging to the inbred Chicago wild type strain were used in this study. The flies were cultured at 2043 ~ C in half pint milk bottles containing David's medium (David, 1962), in a normal cycle of daylight and darkness. Ovaries were dissected from females immersed in Schneider's Drosophila medium (Grand Island Biological Co.) and transferred to 0.5 ml of fresh medium to which L-leucine 4-5-H 3 (50 ~zc/ml, Schwarz BiG Research) had been added. After incubation for varying time intervals (1-15 minutes), the ovaries were rinsed three times in distilled water and

* This research was supported by U.S. Public Health Service Grants 5TIGM903-3 and 1-F 1-GM-33, 385,01, and National Science Foundation Grant GB 7457.

Vitelline Membrane Format ion in Drosophila 483

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f

9

I

5

OC

7 8

5jJ

Fig. 1. A diagram showing portions of a stage 10B egg chamber including the vitelline membrane (vm), and adjacent areas of oocyte (oc) and follicle cells (]c). An overlay, composed of adjacent sections, each having an area equivalent to 10 ~2, is shown as positioned for grain counts. The strip was placed in as many non-overlapping sites as

possible for grain counts along the entire length of the vitelline membrane

fixed in 1% OsO 4 in 0.05 M cacodylate buffer (pH 7.2) a t 0 ~ C for 60 minutes. After fixation, the ovaries were washed for 1 hour in buffer a t 0 ~ C, dehydrated in acetone, and embedded in Araldite according to the technique of King, Aggarwal and Aggarwal (1968). Two- micron thick sections were cut on a Leitz Fernandez-Moran microtome, and mounted on cleaned, gelatinized microscope slides. Sections were coated with undiluted Kodak NTB-3 liquid emulsion at 43 ~ C. After drying, the slides were placed in slide boxes and stored for various t imes at 4 ~ C. After exposure, the slides were developed in Kodak Dektol (15 ~ C, for 2 minutes) and fixed in Kodak F-5 fixer (5 min).

To demonstrate t ha t the labeled leucine was incorporated into protein, ovaries were incubated, fixed in glutaraldehyde, and embedded in glycol methacrylate (GMA). Enzymat ic digestions of two-micron thick sections were carried out according to the methods of Klug, King and Wat t i aux (1970), and the slides were subsequently processed for autoradiography.

To facilitate the localization of radioactivity in chambers engaged in vitelline membrane formation, enlarged drawings of sections of several stage l0 egg chambers were constructed using a Wild M-20 microscope equipped with a drawing tube. The silver grains were then brought into focus, and these were added to the illustration. Each drawing was fur ther magnified with a Goodkin Image Enlarger so tha t in the final enlargement, 1 inch equalled l0 ~ in the original section. A t ransparent overlay divided into a grid of rectangles, each having an area equivalent to l0 ~2 in the original section, was used to count grains along a median longitudinal strip, 10 ~ wide extending the length of the egg chamber. Next, the distr ibution of silver grains above the vitelline membrane and the adjacent cytoplasm of the oocyte and follicle cells was studied. A similar strip was placed perpendicularly in as many non-overlapping sites as possible along the length of the vitelline membrane (Fig. l) and counts of developed silver grains were made and recorded as a function of distance from the surface of the vitelline membrane.

An LKB Ultrotome was used to cut u l t ra th in sections from blocks of single egg chambers embedded in Maraglas as previously described (Cummings and King, 1969). Sections were mounted on 100mesh Formvar-coated grids, stained with lead acetate according to the method of Bjorkman and Hellstrom (1965), and photographed with an Hitachi HU-11A electron microscope.

33*

484 M.R. Cummings, N. M. Brown and R. C. King:

Results and Conclusions

Introduction. The vitelline membrane of the Drosophila ooeyte is secreted during at least three distinct stages by different populations of follicle cells. Early in stage 9 (see Cummings and King, 1969, their Fig. 4 for orientation) vitelline membrane is secreted over the posterior and lateral surfaces of the oocyte by the adjacent columnar follicle cells. Sometime in late stage 9 or early stage 10 (Cummings and King, 1969, their Fig. 5) the border cells tha t migrate from the anterior surface of the egg chamber between the nurse cells reach the anterior surface of the oocyte. The border cells then migrate dorsally to a position opposite the oocyte nucleus and begin the secretion of the micropylar vitelline membrane. Concurrently, in stage 10, a centripetal migration of follicle cells occurs between the oocyte and the nurse cells, and those follicle cells in contact with the oocyte surface secrete vitelline membrane. By late stage 10 or early stage 11 the oocyte is surrounded by vitelline membrane, except a t the points of a t tachment of the micropyle and four ring canals. Synthesis of vitelline membrane from stages 9-11 requires 11-12 hours (Cummings and King, 1969, their Table 1). The closing of the ring canals, which completes the formation of the vitelline membrane, occurs during stage 12.

Autoradiographic Results. Incorporation of leucine-H a was evident in sections from all four incubation periods (1, 5, ]0, 15 minutes). In each incubation series, silver grains were not observed over egg chambers in stages 13 and 14. In vitellogenic egg chambers, all cell types showed an uptake of label. However, the follicle cells, nurse cells and oocyte showed consistent differences in the amounts of label taken up. The highest concentration of silver grains was seen in the cytoplasm of the follicle cells, the cytoplasm of the nurse cells was less labeled than that of the follicle cells, and the oocyte showed the lowest level of incorporation. The amount of label increased with longer periods of incubation, but the pat tern remained the same.

Within the follicle cells, the distribution of silver grains was denser over the cytoplasm adjacent to the developing vitelline membrane. Label was uniformly distributed throughout the cytoplasm of the nurse cells, although the posterior nurse cells showed a higher grain density than those in the anterior end of the nurse chamber. Label was detected in the yolk of the oocytes in stages 8-12, and the label was distributed in a uniform manner throughout the ooplasm. Labeling of nuclei in follicle cells and nurse cells in vitellogenic stages was apparent only after five minutes of incubation. No label was observed in the

oocyte nucleus. To determine the distribution of newly synthesized protein in the devel-

oping vitelline membrane, a count was made of the silver grains covering the vitelline membrane and adjacent cytoplasm of the oocyte and follicle cells. The results are presented graphically in Fig. 2. The largest concentration of grains was found to be over the follicle cell cytoplasm nearest the vitelline membrane and along the vitelline membrane itself. The concentration of grains over the peripheral ooplasm and its vitelline membrane inte1=[ace was only about one- fourth of tha t seen on the follicle cell side.

We assume the silver grains in the developed autoradiographs were located above protein molecules tha t had been synthesized during the time of incubation

Vitelline Membrane Formation in Drosophila 485

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70

6O %

0 50

E g 40

30

o 20

I0

3 2

Fol l ic le cells VM I Oocyte

Fig. 2. Grain distribution over vitelline membrane (vm) and adjacent cytoplasmic regions in a stage 10B egg chamber. The numbers in the histogram bars refer to the corresponding

slots in the grid in Fig. 1. Fifteen minute incubation

with the labeled leucine. The differences in grain counts presumably reflect differences in the rates of absorption of amino acid and in the rates of protein synthesis in the cytoplasmic areas by the cells in question. The grains are thought to represent protein rather than labeled precursor because the grain density in adjacent trypsin-digested sections is no higher than the background. Since an in vitro system was used, the labeled proteins were endogeneously synthesized.

Ultrastruetural Observations The Follicle Cell-Oocyte interlace. The follicle cell divisions and migrations

which occur during oogenesis have been previously described (King and Vanoucek, 1960; King and Koch, 1963; Cummings and King, 1969). Early in stage 9, when vitelline membrane formation begins at the lateral and posterior surfaces of the oocyte, the follicle cells above the oocyte are columnar, while those over the nurse cells are squamous or euboidal. In the cytoplasm of the columnar follicle cells, the rough surfaced endoplasmic reticulum is organized into concentric whorls surrounding secretion droplets of lipid (King and Koch, 1963, their Fig. 4; Quattropani and Anderson, 1969, their Fig. 2). These deposits, which are visible under the light microscope (Cummings and King, 1969, their Figs. 3-6), have been named epithelial bodies by King and Koch (1963).

The onset of vitellogenesis in the Drosophila oocyte is marked by the appearance of convolutions and interdigitating folds in the plasma membranes of the oocyte and adjacent follicle cells. In the oocyte, small membranous vesicles and tubules which have pinched off the oolemma are seen in the peripheral ooplasm (Fig. 3; see also Cummings and King, 1970). Beginning in stage 9, vitelline bodies accu- mulate in the spaces between the interdigitating plasma membranes of the ooeyte and folhele cells, and these bodies later coalesce to form the vitelline membrane. The vitelline bodies presumably arise from small, dense vesicles about 100 m~t

486 M.R. Cummings, N. M. Brown and 1%. C. King:

Fig. 3. An electron micrograph showing the border between the oocyte (oc) and follicle cells (/c) in a stage 9 egg chamber. Vitelline bodies (vb) deposited at the interface between follicle cells and oocyte will later coalesce to form the vitelline membrane. The oocyte

periphery contains tubules and vesicles which apparently have pinched off the oolemma

in d i ame te r which lie in the cy top lasm of ad j acen t follicle cells. These vesicles can somet imes be seen fusing with vi tel l ine bodies (King and Koch, 1963, the i r Fig , 5 ; Q u a t t r o p a n i and Anderson, 1969, the i r Fig. 5), and t hey occur only in the follicle cell cy top la sm ad j acen t to vi te l l ine bodies or segments of vi te l l ine membrane . The dense vesicles m a y be der ived d i rec t ly f rom blebs p inched off the endoplasmic re t iculum, or from the numerous Golgi complexes ( Q u a t t r o p a n i and Anderson, 1969, thei r Fig. 6).

The Follicle Cell-Nurse Cell Interlace. Secret ion of vi tel l ine m e m b r a n e in the in terface be tween follicle cells and nurse cells has been repor ted in s tudies of


Fig. 4. An electron micrograph showing vitelline bodies (vb) present in a stage 9 egg chamber at the border between follicle cells ([c) and a nurse cell (nc)

developing egg chambers from Drosophila females homozygous for various female- sterile genes (King and Koch, 1963; Falk and King, 1964; Smith, 1966; Dapples, 1969), and this phenomenon was therefore thought to be an aberrant condition. However, during stage 9 and 10A, vitelline bodies are often seen between the plasma membranes of follicle cells and adjacent nurse cells in chambers from wild type ovaries (Fig. 4). The vitelline bodies are interposed between the two cell types without any interdigitation or folding of the adjacent plasma membranes. Such vitelline bodies are found only a t the interfaces between follicle cells and those posterior nurse cells that are also adjacent to the oocyte.

The Oocyte-Nurse Cell Interlace. The initial stages of vitellogenesis are marked by changes in the plasma membranes of the oocyte and adjacent nurse cells (Cummings and King, 1970). Small folds appear at intervals along the plasma- lemmal interface between nurse cells and oocyte (Cummings and King, 1970, their Fig. 4). During succeeding stages, these folds develop into large, complex, overlapping structures. 0kada and Waddington (1959) originally proposed tha t such membrane activities reflected syntheses leading to the formation of vitelline membrane between nurse cells and oocyte. However, we have not seen any


Fig. 5. An electron micrograph showing portions of adjacent follicle cells (]c), nurse cells (nc), and an oocyte (oc) at stage ll , after the centripetal migration of follicle cells between the oocyte and nurse cells has taken place. Vitelline membrane (vm) forms only at the follicle

cell-oocyte interface. Endoplasmic reticulum (er). Lipid droplets (1). Mitoehondria (m)

evidence of vitelline membrane formation until after the centripetal migrat ion of follicle cells between the oocyte and nurse cells occurs in stage 10B. Following this migration, those follicle cells in contact with the oocyte surface (Fig. 5) secrete vitelhne membrane. No secretion of vitelline bodies or membrane infolding is observed at the border between migrant follicle cells and nurse cells. Similarly, vitellinc bodies are secreted by the border cells only at the interface between the ooeyte and border cells (Fig. 6).

Discussion

Evidence /or Follicle Cells as the Source o/ Vitelline Membrane. The distri- but ion of silver grains over the region of the developing vitelline membrane (Fig. 2) indicates tha t the protein component of the membrane is synthesized in the follicle cells. I n this process amino acids are removed from the surrounding


Fig. 6. An electron micrograph showing the border cells (bc) at the oocyte (oc) surface during stage ll. The border cells in contact with the oocyte have begun the synthesis

of the micropylar vitelline membrane (vm). Ring canal rim (r)

medium, incorporated into protein molecules in the follicle cell cytoplasm adjacent to the vitelline membrane, and transported to the follicle cell-oocyte interface where they are added to the developing vitelline membrane. The side of the vitelline membrane facing the follicle cells shows a denser label than the side of the vitelline membrane facing the oocyte, demonstrating tha t i t contains more newly formed protein molecules (Fig. 2).

Evidence Against the Oocyte as the Source o/ Vitelline Membrane. In an early paper dealing with the ultrastructure of the developing Drosophila oocyte, Okada and Waddington (1959) concluded tha t the oocyte was primarily responsible for


the formation of the vitelline membrane. This conclusion was based, in part , on the observation tha t at the time the vitelline bodies appear between the interdigitating plasma membranes of the oocyte and follicle cells, the peripheral ooplasm is filled with membranous tubules and vesicles, which these authors interpreted as evidence for the secretion of vitelline membrane precursors by the oocyte. Subsequent studies (Cohn and Brown, 1968; Cummings and King, 1970) have demonstrated that the tubules and vesicles in the oocyte periphery result from the internalization of segments of the oolemma and are not secretory precursors. In addition, such ooplasmic tubules and vesicles occur at the nurse cell-oocyte interface without the production of vitelline membrane.

Cytological studies of wild type ovaries and ovaries from flies homozygous for certain recessive female-sterile genes have shown tha t vitelline membrane can form in the absence of the oocyte. Fig. 3 shows vitelline membrane forming between follicle cells and adjacent nurse cells in wild type Drosophila. This same condition has been reported in ovaries of flies homozygous for the mutants tiny (King and Koch, 1963, their Fig. 6) and singed ~a (Dapples, 1969, Fig. 13v). Vitelline membrane can also form at the interface between follicle cells and tumor cells, as reported in [emale-sterile (King and Koch, 1963, their Fig. 7) and /used (Smith, 1966, Fig. 15). Formation of vitelline membrane between adjacent follicle cells has been observed in the mutant singed ~a by Dapples (1969).

The Polarized Secretory Activity o/ Follicle Cells. The observation tha t follicle cells normally deposit vitelline membrane against an oocyte, demonstrates tha t the follicle cells normally secrete in a polarized fashion. Follicle cells over the oocyte have their outer surfaces against the tunica propria, lateral surfaces against adjacent follicle cells, and the inner surface against the ooeyte (King and Koch, 1963, their Fig. 4), yet the vitelline membrane is laid down only at the latter interface. In the case of the centripetally migrating follicle cells (Cummings and King, 1969, their Fig. 6), the follicle cells have one surface against a nurse cell, and the other against the oocyte. Vitelline membrane is laid down only against the oocyte (Fig. 5). The same holds true for the border cells (Fig. 6).

I t seems probable, therefore, tha t the follicle cells possess a secretory mechanism for the initiation of synthesis of vitelline membrane components, and a second directing mechanism which causes movement of vitelline membrane precursors away from some surfaces and towards other surfaces. This directing mechanism normally leads to viteliine membrane secretion against an oocyte, but in the absence of an oocyte, a tumorous oocyte-like cell or a nurse cell, or in rare cases, an adjacent follicle cell will serve. Other follicle cells, which do not form vitelline membrane can, however, secrete chorion, as for example, follicle cells forming the dorsal appendages. Thus, a given follicle cell can undergo a sequence of polarized secretory activity leading to the formation of 1) vitelline membrane, 2) endochorion, and 3) exochorion. Whether a cell secretes 1), 2) and 3) or 2) and 3) or nothing, depends on cues, which presumably come from adjacent cells.

Egg Coverings as Maternal Cuticle. From studies of egg coverings in the cricket, McFarlane (1962) postulated that the investing membranes of the mature oocyte correspond more or less to the fundamental layers of insect cuticle, and


that egg coverings should be regarded as a maternal cuticle. Recently, Quattropani and Anderson (1969) have proposed that the formation of exo- choi'ion in the Drosophila oocyte may be equivalent to the synthesis of insect epicuticle, and that the membranes covering the eggs of Drosophila can be regarded as maternal cuticle.

This proposal seems unlikely for a number of reasons. First, the rite]line membrane and chorion are secreted by mesodermally-derived cells, while cuticle is secreted by cctodermally-derived cells. Secondly, in Drosophila, the production of egg coverings by follicle cells is an intercellular phenomenon, usua]]y the result of contact between two cell types as discussed earlier, while cuticle formation is predominately, if not entirely an extracellnlar event, dependent only upon the action of a single cell type, the epidermal cell. A follicle cell will not spon- taneously form vitelline membrane unless it contacts another type of cell, usually the oocyte. There is no evidence to suggest that an epidermal cell reqtfires the presence or action of another cell type to induce cuticle synthesis. Finally, one of the most common components of cuticle, chitin, is missing from egg coverings. More importantly, the various layers which have been equated with one another in cuticle and egg coverings differ in cytochemical properties and nltrastructural characteristics (Hackman, 1964; Locke, 1964, 1966; King, 1960, 1964).

I t seems more logical, therefore, to regard the synthesis of Drosophila egg coverings and insect cuticle as separate phenomena produced by embryologically dissimilar cell types with significant differences in both nltrastructure and cytochemistry.

References

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Cohn, R. H., Brown, E. H. : The formation of alpha (proteoid) yolk spheres in the ooeyte of Drosophila melanogazter. Dros. ]nfo. Serv. 43, 117.

Cummings, M.R., King, R.C.: The cytology of the vitellogenic stages of oogenesis in Drosophila melanogaster. I. General staging characteristics. J. Morph. 128, 427-442 (1969).

- - - The cytology of the vitellogenic stages of oogenesis in Drosophila melanogaster. II. Ultrastructural investigations on the origin of protein yolk spheres. J. Morph. 130, 467-478 (1970).

Dapples, C.C.: The development of the nucleoli of nurse cells of the wild type and mutant egg chambers of Drosophila melanogaster. University Microfilms 70-6456, Ann Arbor Michigan, U.S.A. (1969).

David, J. : A new medium for rearing Drosophila in axenic conditions. Dros. Info. Serv. a6, ~2S (I962).

Falk, G., King, R.C.: Studies on the developmental genetics of the mutant tiny of Drosophila melanogaster. Growth 28, 291-321 (1964).

Hackman, R. H. : Chemistry of the insect cuticle. In: Physiology of the insecta, vol. III, ed. by M. Rockenstein, p. 471-506. New York: Academic Press 1964.

Imms, A.D.: A general textbook of entomology. (Revised by O.W. Richards and R. G. Davies), 9th ed. London: Methuen 1957.

King, R. C.: Oogenesis in aduit Drosophila melanogazter. IX. Studies on the cytochemistry and ultrastructure of developing oocytes. Growth 24, 265--323 (1960).

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King, 1%. C., Aggarwal, S. K., AggarwM, U. : The development of the female Drosophila reproductive system. J. Morph. 124, 143--166 (1968).

- - D e v i n e , 1%. L.: Oogenesis in adult Drosophila melanogaster. VII. The submicroscopic morphology of the ovary. Growth 22, 299-326 (1958).

- - Koch, E . A . : Studies on ovarian follicle cells of Drosophila. Quart. J. micr. Sci. 104, 297-320 (1963).

- - Vanoucek, E.: Oogenesis in adult Drosophila melanogaster. X. Studies on the behavior of the follicle cells. Growth 24, 333-338 (1960).

Klug, W. S., King, 1%. C. Wattiaux, J. : Oogenesis in the suppressor o/Hairy-wing mutant. of Drosophila melanogaster. II. Nucleolar morphology and in vitro studies of RNA and protein synthesis. J. exp. Zool. 174, 125-140 (1970).

Locke, M. : Insect integument. In: Physiology of insecta, vol. I II , edit. by M. Rockstein, p. 380-470. New York: Acad. Press 1964.

- - T h e structure and formation of the cuticulin layer in the epicutiele of an insect, Calpodes ethlius (Lepidoptera, Hesperiidae). J. Morph. 118, 461-494 (1966).

McFarlane, J. E. : The cuticle of the egg of the house cricket. Canad. J. Zool. 40, 13-21 (1962).

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Dr. Michael R. Cummings University of Illinois at Chicago Circle Department of Biological Sciences Box 4348 Chicago, Illinois 60680/U.S.A.

the cytology of the vitellogenic stages of oogenesis in drosophila melanogaster

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