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 fürthering their development. The role played by cell death in oogenesis is also discussed on the basis of the current literature.

The study of cell death in the normal chick limb and in such mutants as wingless (Hinchliffe & Ede, 1973) and talpid3 (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 imaginai 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 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.

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 μm 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).

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 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).

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.

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.

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).

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, × 120.

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, × 120.

Fig. 3.

An ovarian chamber undergoing cell death. Amidst seemingly unaffected follicle cells (FC), numerous dense bodies (arrows) are visible in this chamber. ×480.

Fig. 3.

An ovarian chamber undergoing cell death. Amidst seemingly unaffected follicle cells (FC), numerous dense bodies (arrows) are visible in this chamber. ×480.

Fig. 4.

A stage-8 ovarian chamber partially affected by the process of cell death. At the anterior end of the chamber, nurse cells (AC) 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 (OO). Follicle ceils (FC), × 480.

Fig. 4.

A stage-8 ovarian chamber partially affected by the process of cell death. At the anterior end of the chamber, nurse cells (AC) 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 (OO). Follicle ceils (FC), × 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 (OO) contains numerous yolk platelets. The vitelline membrane (Vm) separates the ooplasm from the overlying follicle cells (FC). Nurse cells (AC), ×750.

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 (OO) contains numerous yolk platelets. The vitelline membrane (Vm) separates the ooplasm from the overlying follicle cells (FC). Nurse cells (AC), ×750.

An examination of a large number of light microscope preparations such as those shown in Figs. 24, gave the impression that the greater the number of 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 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).

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. × 5000.

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. × 5000.

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 μm in length into the overlying follicle cells.

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, × 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, × 5000.

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.

Fig. 8.

Fragments of nuclear envelope exhibiting a parallel orientation in tracts, × 12000.

Fig. 8.

Fragments of nuclear envelope exhibiting a parallel orientation in tracts, × 12000.

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), × 9000.

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), × 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, ×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, ×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. ×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. ×9000.

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 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. 1214). 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.

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, ×12000.

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, ×12000.

Fig. 13.

A vacuole formed in the follicle layer overlying the ooplasm. Note the presence of several yolk platelets (y). × 12000.

Fig. 13.

A vacuole formed in the follicle layer overlying the ooplasm. Note the presence of several yolk platelets (y). × 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, ×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, ×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, × 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, × 12000.

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 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 (Meóla & 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.

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