/ . Embryol. exp. Morph. Vol. 35, 3, pp. 521-533, 1976
Printed in Great Britain
521
Cell death in ovarian chambers of
Drosophila melanogaster
By F. GIORGI 1 AND P. DERI 1
From the Istituto di Istologia e Embriologia,
Universita diPisa
SUMMARY
An ultrastructural analysis has been made of certain ovarian chambers undergoing abnormal development. The earliest morphological change in these chambers consists of the
alteration of the nuclear material which is then followed by engulfment of portions of the
nurse cell cytoplasm, including the nuclear fragments, into the overlying follicle cells. The
continuation of this process leads to the progressive disappearance of nurse cells with the
concomitant formation of huge dense vacuoles in the follicle layer. The morphological features
described in the present investigation are similar to those found in other tissues and interpreted as leading to cell death. It is suggested that certain ovarian chambers undergo cell
death as a result of the incapability of furthering their development. The role played by cell
death in oogenesis is also discussed on the basis of the current literature.
INTRODUCTION
The study of cell death in the normal chick limb and in such mutants as
wingless (Hinchliffe & Ede, 1973) and talpicP (Hinchliffe & Thorogood, 1974)
has provided an experimental basis for understanding the morphogenetic role
played by this process in normal embryonic development. Similarly, morphological studies carried out on imaginal discs of several mutants of Drosophila
melanogaster (Fristrom, 1968, 1969) have offered a satisfactory explanation of
the abnormal appearance of various organs at the adult stage. The occurrence
of a characteristic degeneration has also been reported in ovarian cells of different organisms (Waddington & Okada, 1960; Bielanska-Osuchowska, 1973;
Gondos, 1973; El-Shersaby & Hinchliffe, 1974), but as yet too little is known
about it to establish the role played by cell death in oogenesis.
The evidence obtained so far from a number of somatic tissues indicates that
cell death is genetically controlled (Saunders & Fallon, 1966), but the mechanisms by which such a control is brought into action at the cellular level are
still unknown. A number of studies, however, suggest that hormones may act
as inducers for cell death (Kerr, Wyllie & Currie, 1972). Cells destined to death
are known to undergo a stereotyped sequence of changes which begins with the
1
Authors' address: Istituto di Istologia e Embriologia, Universita di Pisa, Via Volta 4,
56100 Pisa, Italy.
522
F. GIORGI AND P. DERI
condensation of the nuclear chromatin, is followed by the rupture of the nucleus
and ends up with the release of cell fragments, occasionally containing nuclear
portions, into the extracellular spaces (Kerr, Harmon & Searle, 1974). The cell
fragments so released are then phagocytosed by adjacent cells and are ultimately sequestered into telolysosomes (Kerr, 1973).
Present evidence (Ballard & Holt, 1968) tends also to suggest that release of
hydrolytic enzymes by the host cells, rather than being a causal factor for cell
death, may simply represent a response to the presence of engulfed ' dead' cell
fragments within the cell.
The present ultrastructural investigation was undertaken to clarify the
sequence of events leading to degeneration of some ovarian chambers in
Drosophila melanogaster. Such a sequence of morphological changes occurs
according to a typical pattern of cell fragmentation followed by phagocytosis of
cell fragments, as previously described in other somatic tissues undergoing
cell death.
MATERIAL AND METHODS
Or-K Drosophila flies were reared in glass vials containing standard Drosophila food and kept at the temperature of about 25 °C.
In order to estimate the frequency of degenerate chambers as well as their
location within the ovarioles, ovaries taken from females of different ages
(from 1 to 30 days) were stained for 1 h in a 0-01 % solution of neutral red and
photographed without prior fixation.
As routine procedure, ovaries were dissected from 2- to 30-day-old flies and
fixed for 3 h in 5 % glutaraldehyde made up in 0-1 M cacodylate buffer at pH 7-2.
After a prolonged wash in the buffer, the ovaries were post-fixed for 6 h in 1 %
osmium tetroxide in 0-1 M cacodylate buffer, pH 7-2; they were then dehydrated
in alcohol, passed through propylene oxide and embedded in Epon-Araldite
mixture.
For light microscope observations 1 /tm thick sections were cut from polymerized blocks of Epon-Araldite and stained with 1 % toluidine blue - 1 %
methylene blue.
For ultrastructural observations, sections of silver to pale gold were mounted
on formvar-coated copper grids, stained with uranyl acetate and lead citrate,
and examined in a Siemens Elmiskop 101 electron microscope.
The staging of the ovarian chambers examined in the present study was done
according to the criteria worked out by Cummings & King (1969).
RESULTS
In whole mounts of ovaries stained with neutral red, degenerate chambers
could easily be distinguished from fully developed ones, by their intense colour.
Such chambers appeared more or less randomly distributed in ovaries taken
Cell death in ovarian chambers
523
Fig. 1. Diagram to show the varying location assumed by ovarian chambers undergoing cell death (in black) in ovaries of different ages, (a) a 2- to 3-day-old ovary;
(b) a 30-day-old ovary.
from 2- to 3-day-old flies, while they tended to become more numerous and
preferentially located at the bottom, as the ovary was approaching 30 days of
age (Fig. 1).
When degenerate chambers were examined at the light microscope level,
they revealed several atypical features which allowed a clear distinction from
the majority of the ovarian chambers undergoing normal development (for a
thorough description of the normal ovarian development in Drosophila melanogaster, see King, 1970). Depending on the stage of degeneration, however, a
distinction could also be made among degenerate chambers themselves: they
will therefore be described as ranging from the least affected chambers to the
fully degenerate ones. The latter did not exhibit nurse cells or oocyte, but presented a number of densely stained bodies amidst seemingly unaffected follicle
cells (Figs. 2, 3). On the other hand, in ovarian chambers not so deeply affected
(Fig. 4) a partial morphological characterization of the various cell types could
still be made. In these instances the nurse cells could be recognized by their
location within the chamber; with respect to normal, however, the nurse nuclei
exhibited a very atypical morphology due to the condensed state of the chromatin material (compare Figs. 4 and 5). The oocyte could also be detected at the
opposite pole of these chambers by the presence of minute yolk platelets
(Fig. 4).
An examination of a large number of light microscope preparations such as
those shown in Figs. 2-4, gave the impression that the greater the number of
524
F. GIORGI AND P. DERI
Fig. 2. An ovariole taken from a 10-day-old ovary. Note the presence of a
degenerating ovarian chamber at the bottom of the ovariole following a sequence
of normal looking chambers, x 120.
Fig. 3. An ovarian chamber undergoing cell death. Amidst seemingly unaffected
follicle cells (FC), numerous dense bodies (arrows) are visible in this chamber.
x480.
dense bodies present in the degenerate chambers, the more dissimilar their
overall morphology was from that of normal chambers. When the number of
such bodies was as high as that of the chamber shown in Fig. 3, no signs of
nurse cells and oocyte could be witnessed.
Another noteworthy light microscope observation relates to the staging of
ovarian chambers. While normal chambers could be staged from stage 1 to
stage 14, although with varying frequency (David & Merle, 1968), the degenerate
chambers could never be staged beyond stage 8. On the other hand, in the
majority of instances, the degenerate chambers did not exhibit sizes smaller
than those of stage 7.
When the least affected chambers (see Fig. 4) were examined ultrastructurally,
several features of interest were noted. The chromatin material of the nurse
Cell death in ovarian chambers
525
Fig. 4. A stage-8 ovarian chamber partially affected by the process of cell death.
At the anterior end of the chamber, nurse cells (NC) can be recognized, but the
nuclear content appears highly condensed (arrows). At the posterior end minute
yolk platelets (y) can be seen in the ooplasm (00). Follicle cells (FC). x 480.
Fig. 5. A normal looking ovarian chamber at stage 10. Numerous nurse nuclei are
visible at the anterior end exhibiting a typical organization of the nucleolar masses.
The ooplasm (00) contains numerous yolk platelets. The vitelline membrane
(Vm) separates the ooplasm from the overlying follicle cells (FC). Nurse cells (NC).
x750.
526
F. GIORGI AND P. DERI
Cell death in ovarian chambers
527
nuclei exhibited a rather clumped organization and this was accompanied by
a re-shaping of the nucleolar material into huge rounded masses (Fig. 6). These
masses still showed a centrally located fibrillar part and a more peripheral
granular part of fenestrated appearance (Fig. 6).
With further degeneration, the nucleolar material was no longer discernible
as such, but only material of fibrillar texture, perhaps resulting from a loosening
of the nucleolar structure, could be seen dispersed throughout the nucleoplasm.
The chromatin material, instead, tended to form masses of size even larger than
before (Fig. 7). Since the time of the early structural alteration in the nurse
nuclei, the surface of the nurse cells along the border with the overlying follicle
cells was already thrown into a series of ample infoldings (Fig. 9). Some of these
folds had acquired a pseudopodium-like shape which projected for up to about
10 fim in length into the overlying follicle cells.
Subsequently, the nuclear envelopes of nurse nuclei broke down and, as a
result, the chromatin masses together with the nucleolar remnants, were
released into the cytoplasm. In addition, numerous fragments of nuclear membranes came to lie free in the nurse cytoplasm with a characteristic parallel
orientation in tracts (Fig. 8). On occasions, the portion of the nurse cell body
forming the pseudopodium-like folds was seen to contain the chromatin masses
previously released from the nurse nuclei (Figs. 10, 11). In some micrographs,
these folds were seen to be constricted at the basis and this gave the impression
that they were about to be pinched off the nurse cell body. The nurse cell cytoplasm at the level of the folds had a more compact appearance than in the rest
of the cell and similar cytoplasmic organelles were present in both these
regions.
Chambers at a more advanced stage of degeneration did not possess identifiable nurse cells or oocyte, but exhibited normal looking follicle cells. When
such chambers were examined ultrastructurally, it was found that the bodies
which appeared densely stained at the light microscope level consisted of large
vacuoles of varying content and electron density embedded within the cytoplasm
of follicle cells. Some of these vacuoles appeared as portions of segregated cytoplasm bound by a membrane and with recognizable cytoplasmic organelles
therein (Fig. 12). In ovarian chambers undergoing cell death at the beginning
Fig. 6. Part of a nurse nucleus from an ovarian chamber assumed to be at the
beginning of a process of cell death. Both chromatin (chr) and nucleolar material
(no) are gathered into rounded masses. The nuclear envelope (ne) is still uninterrupted, x 5000.
Fig. 7. A huge mass of chromatin material surrounded by smaller masses of
similar consistency. The nucleolar material has a more dispersed appearance and
consists mainly of fibrils. Fragments of the nuclear envelope are also discernible.
Abbreviations are as in the previous figure, x 5000.
Fig. 8. Fragments of nuclear envelope exhibiting a parallel orientation in tracts,
x 12000.
528
F. GIORGI AND P. DERI
FIGURES 9-11
Fig. 9. The follicle-nurse border from a stage-7 ovarian chamber undergoing
degeneration. Note that the nurse cell cytoplasm (NC) has a higher electron density
than the rest and is partly protruding into the overlying follicle layer (FC). x 9000.
Fig. 10. Portion of the nurse cell body protruding into the overlying follicle cells.
Note the presence of a chromatin mass (chr) inside that portion, x 9000.
Fig. 11. The follicle-nurse border from a stage-7 ovarian chamber. The vacuole
present in the follicle layer - on the left of the micrograph - contains a chromatin
mass (chr) of similar consistency to that contained in the adjoining nurse cell cytoplasm. x9000.
Cell death in ovarian chambers
FIGURES
529
12-15
Fig. 12. A vacuole within the follicle layer containing a segregated portion of the
nurse cell cytoplasm with numerous organelles including myelin figures (mf) and
mitochondria (m). Note that two membranes are involved in separating the nurse
cell cytoplasm from that of the follicle cell, x 12000.
Fig. 13. A vacuole formed in the follicle layer overlying the ooplasm. Note the
presence of several yolk platelets (y). x 12000.
Fig. 14. Another vacuole in the follicle layer with recognizable nuclear fragments
(nu) therein. In this instance also, the segregated portion of cytoplasm is separated
from the follicle cell cytoplasm by two membranes, x 12000.
Fig. 15. A vacuole within the follicle layer. Note the highly condensed state of the
content of this vacuole. Altered mitochondria (m), cisternae of the endoplasmic
reticulum (er) and a nuclear fragment (nu) are visible, x 12000.
530
F. GIORGI AND P. DERI
of vitellogenesis (stage 8), a few vacuoles within the follicle cells overlying the
ooplasm were seen to contain several yolk platelets (Fig. 13). In other places
the vacuoles exhibited parts of higher electron density that, at all effects,
resembled nuclear fragments (Figs. 14, 15). Other vacuoles consisted of homogeneously dense material amidst extremely altered cisternae of endoplasmic
reticulum and clusters of mitochondria (Fig. 15). Among the various inclusions
which could be detected within these vacuoles, myelin figures were of frequent
occurrence (Fig. 12). Some of the vacuoles, perhaps those at the initial stage of
their formation, were seen to be separated from the follicle cell body by means
of another membrane besides being enclosed by their own membrane. That two
membranes are actually involved in enclosing the vacuoles, could be better
appreciated in preparations where a partial dislocation, due perhaps to faulty
fixation procedure, occurred between the follicle cell cytoplasm and the vacuole
(Figs. 12-14). This observation supports the view that the vacuoles which come
to lie within the cytoplasm of the follicle cells represent a later stage in the
evolution of the pseudopodium-like folds previously described as pinching off
the nurse cell body.
DISCUSSION
The account given in the present investigation on the abnormal morphology
of certain ovarian chambers is suggestive of the occurrence of a process of cell
death. Images similar to those observed during this investigation have, in fact,
been reported by earlier workers (Gliicksmann, 1951; Bellairs, 1961; Goldsmith,
1966; Kerr et al. 1972) in a variety of animal tissues and interpreted by them as
leading to cell death.
The earliest morphological change in ovarian chambers undergoing degeneration is the alteration of nuclear material in nurse cells.
While the chromatin condenses into large masses which, upon interruption
of the nuclear envelope, are released into the cytoplasm, the nucleolar material
undergoes structural dissolution somehow resembling that observable in normal
ovarian development during stages 11-13 (Giorgi, 1975). Degeneration continues
with the formation of pseudopodium-like folds which are ultimately engulfed
into the overlying follicle cells. By means of this budding process, nurse cells
are subjected to a progressive disappearance which is concomitant with the
formation of large vacuoles in the follicle layer. That the vacuoles in question
are actually derived from portions of the nurse cytoplasm is also suggested by
the presence, at least at the initial stage of their formation, of two membranes
around them. Most probably, of these membranes, the external one is the follicle
cell plasma membrane, whereas the internal one represents the plasma membrane of the nurse cells. These findings indicate that the vacuoles are heterophagic in origin. However, the possibility has also to be considered that not all
vacuoles which are present in the follicle layer may be formed through a heterophagic process. Autophagy could also occur, but the observations reported in
Cell death in ovarian chambers
531
the present study do not suggest that it plays a major role in the formation of
dense vacuoles in the follicle cells.
Another point which deserves mention concerns the role played by follicle
cells in accomplishing the degeneration of the ovarian chamber. It is remarkable
that cells, which in normal conditions would be involved in providing the egg
chamber with its own coverings (King & Koch, 1963; Cummings, Brown &
King, 1971), may, following disruption of nurse nuclei, become capable of
phagocytosing portions of nurse cytoplasm. Previous evidence (Kerr et al.
1972) also suggests that cells other than macrophages as, for example, epithelial
cells, may be involved in the phagocytosis of the cell fragments. The mechanism
by which events occurring at the level of nurse nuclei may lead to phagocytosis
by follicle cells remains unclear. It may be possible, however, that changes in
the molecular architecture of the plasma membrane of the nurse cells adjoining
the follicle layer are responsible for inducing phagocytosis.
The observation that degenerate chambers vary in number and location with
ageing of the female recalls the earlier findings of David (1961) that those ovarioles which contain degenerate chambers (referred to as 'atrophic' by him)
are not deposited and so accumulate in the ovary.
It was mentioned earlier that the dimensions of the ovarian chambers undergoing degeneration are within a narrow range corresponding to that of stages
7-8. This makes it likely that the initiation of vitellogenesis represents a critical
stage in the process of ovarian development so as to determine degeneration of
those ovarian chambers which are incapable of furthering their maturation.
Available evidence indicates that vitellogenesis is a hormone-controlled process (Masner, 1968; Bell, 1969; Bell & Barth, 1971), and that the retention of
mature eggs in the ovary, which may be caused, among other factors, by a diminished mating frequency (Hinton, 1974), inhibits the development of the younger
oocytes (Meola & Lea, 1972; Caussanel, 1972). According to Adams, Hintz &
Pomonis (1968) retained eggs secrete an 'oostatic hormone' which could either
reduce the haemolymphal titre of juvenile hormone or prevent certain ovarian
chambers from responding specifically to the hormone. As a result of the
inhibition exerted by the retained eggs, some oocytes, among those destined to
initiate their vitellogenic maturation, would thus be prevented from doing so.
It could therefore be that the process of cell death takes place as a result of the
inhibition exerted by the mature eggs retained in the ovary. This, however,
would not completely rule out the possibility that some ovarian chambers
could already be genetically determined to undergo cell death so as to degenerate
prior to becoming subject to the influence of the retained eggs. The question of
the factors causing cell death thus remains to be worked out. It is, however, conceivable that, by whatever cause, cell death should ensure a recycling of those
macromolecules which would otherwise be lost as components of those chambers
which had become incapable of completing their development.
532
F. GIORGI AND P. DERI
REFERENCES
ADAMS, T.
S., HINTZ, A. M. & POMONIS, J. G. (1968). Oostatic hormone production in houseflies, Musca domestica, with developing ovaries. /. Insect Physiol. 14, 983-993.
BALLARD, K. J. & HOLT, S. J. (1968). Cytological and cytochemical studies on cell death and
digestion in foetal rat foot: the role of macrophages and hydrolytic enzymes. /. Cell Sci.
3, 245-262.
BELL, W. J. (1969). Dual role of juvenile hormone in the control of yolk formation in Periplaneta americana. J. Insect Physiol. 15, 1279-1290.
BELL, W. J. & BARTH, R. H. JR. (1971). Initiation of yolk deposition by juvenile hormone.
Nature, New Biology 230, 220-222.
BELLAIRS, R. (1961). Cell death in chick embryos as studied by electron microscopy. /. Anat.
95, 54-60.
BIELANSKA-OSUCHOWSKA, Z. (1973). Oogonia and oocytes degeneration and the nutritive
macrophages in the process of the development of the ovary in embryos of the domestic
pig (Sus scrofa dom. L.). Z. Anat. EntwGesch. 142, 37-52.
CAUSSANEL, C. (1972). Influences de la prise de nourriture et de la copulation sur le cycle
ovarien et endocrine de la Labiduria riparia (Insecte, Dermaptere). C. r. hebd. Seanc. Acad.
Sci., Paris 274, 560-563.
CUMMINGS, M. R., BROWN, N. M. & KING, R. C. (1971). The cytology of the vitellogenic
stages of oogenesis in Drosophila melanogaster. III. The formation of the vitelline membrane. Z. Zellforsch. mikrosk. Anat. 118, 482-492.
CUMMINGS, M. R. & KING, R. C. (1969). The cytology of the vitellogenic stages of oogenesis
in Drosophila melanogaster. I. General staging characteristics. J. Morph. 128, 427-442.
DAVID, J. (1961). Etude quantitative du fonctionnement ovarien chez Drosophila melanogaster Meig. Bull. biol. Fr. Belg. 95, 521-535.
DAVID, J. & MERLE, J. (1968). A revaluation of the duration of the egg chamber stages in
oogenesis of Drosophila melanogaster. Drosoph. Inf. Serv. 43, 122.
EL-SHERSHABV, A. M. & HINCHLIFFE, J. R. (1974). Cell redundancy in the zona-intact preimplantation mouse blastocyst: a light and electron microscope study of dead cells and their
fate. /. Embryol. exp. Morph. 31, 643-654.
FRISTROM, D. (1968). Cellular degeneration in wing development of the mutant vestigial of
Drosophila melanogaster. J. Cell Biol. 39, 488^91.
FRISTROM, D. (1969). Cellular degeneration in the production of some mutant phenotypes in
Drosophila melanogaster. Mol. Gen. Genet. 103, 363-379.
GIORGI, F. (1975). Ultrastructural and cytochemical studies of oogenesis in Drosophila melanogaster with special reference to the formation ofyolk. Ph.D. Thesis, University of Edinburgh.
GLUCKSMANN, A. (1951). Cell deaths in normal vertebrate ontogeny. Biol. Rev. 26, 59-86.
GOLDSMITH, M. (1966). The anatomy of cell death. /. Cell Biol. 31, 41 A.
GONDOS, B. (1973). Germ cell degeneration and intercellular bridges in the human fetal ovary.
Z. Zellforsch. mikrosk. Anat. 138, 23-30.
HINCHLIFFE. J. R. & EDE, D. A. (1973). Cell death and the development of limb form and
skeletal pattern in normal and wingless (ws) chick embryos. /. Embryol. exp. Morph. 30,
753-772.
HINCHLIFFE, J. R. & THOROGOOD, P. V. (1974). Genetic inhibition of mesenchymal cell death
and the development of form and skeletal pattern in the limbs of talpid3 (ta3) mutant chick
embryos. /. Embryol. exp. Morph. 31, 747-760.
HINTON, H. E. (1974). Accessory function of seminal fluid. /. med. Ent. 11, 19-25.
KERR, J. F. R. (1973). Some lysosome functions in liver cells reacting to sublethal injury.
In Lysosomes in Biology and Pathology, vol. 3 (ed. J. T. Dingle & H. B. Fell), pp. 365-394.
Amsterdam and London: North Holland.
KERR, J. F. R., HARMON, B. & SEARLE, J. (1974). An electron microscope study of cell
deletion in the anuran tadpole tail during spontaneous metamorphosis with special
reference to apoptosis of striated muscle fibres. /. Cell Sci. 14, 571-585.
KERR, J. F. R., WYLLIE, A. H. & CURRIE, A. R. (1972). Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239-257.
Cell death in ovarian chambers
533
R. C. (1970). Ovarian Development in Drosophila melanogaster. New York and London : Adademic Press.
KING, R. C. & KOCH, E. A. (1963). Studies on ovarian follicle cells of Drosophila. Q. Jl
Microsc. Sci. 104, 297-320.
MASNER, P. (1968). The inductors of differentiation of prefoUicular tissue and the follicular
epithelium in ovarioles of Pyrrhocoris apterus (Heteroptera). /. Embryol. exp. Morph.
20, 1-13.
MEOLA, R. & LEA, A. O. (1972). Humoral inhibition of egg development in mosquitoes.
/. med. Ent. 9, 99-103.
SAUNDERS, J. W. JR. & FALLON, J. F. (1966). Cell death in morphogenesis. In Major Problems in Developmental Biology (ed. M.Locke), pp. 289-314. New York and London:
Academic Press.
WADDINGTON, C. H. & OKADA, E. (1960). Some degenerative phenomena in Drosophila
ovaries. /. Embryol. exp. Morph. 8, 341-348.
KING,
{Received 21 October 1975)
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