/. Embryol. exp. Morph. Vol. 38,pp. 115-124, 1977
Printed in Great Britain
Recent findings on oogenesis of Drosophila
melanogaster
I. Ultrastructural observations on the developing ooplasm
By F. GIORGI 1 AND J. JACOB
Institute of Animal Genetics, University of Edinburgh, Scotland
SUMMARY
The ultrastructure of the developing ooplasm has been examined in 2-3-day-old flies of
Drosophila melanogaster with a view to clarifying the origin of the yolk platelets.
Previtellogenic ovarian chambers have been shown to exhibit a number of cytolysosomes;
these are first formed in the nurse cells and are later brought to the ooplasm after having passed
through the ring canals.
Just prior to the beginning of vitellogenesis some of the cytolysosomes in the ooplasm
acquire in places a yolk-like appearance. As vitellogenesis commences, in 'early stage-8
chambers, the central ooplasm comes to exhibit a number of inclusions which combine in
themselves features of both yolk platelets and cytolysosomes.
The onset of vitellogenesis at stage 8 is marked by the appearance of several structural
entities on the plasma membrane of the oocyte. At this site, there appear a number of
depressions or pits in between the microvilli. Deeper in the cortical ooplasm there are also
present a number of coated vesicles and tubules. While the former exhibit a typical threelayered structure similar to that of the pits, the tubules lack the outer spiked layer.
Mature yolk platelets appear to be made up of three major components. These are a central
homogeneous dense body, an outer superficial layer with a number of small spherules and
a rounded body therein, and an external limiting membrane.
Instances of fusion between vesicles of sizes progressively larger have been observed with
frequency. This observation is discussed in relation to the formation of yolk platelets by
means of pinocytotic uptake. A contribution made by the nurse cells to the formation of the
yolk platelets is also taken into account.
INTRODUCTION
In many insects, yolk proteins are known to be immunologically identical to
sex-specific blood proteins (Telfer, 1954, 1965; Bodnaryk & Morrison, 1968;
Pan, Bell & Telfer, 1969; Bell, 1970). A number of investigators (Anderson,
1964, 1969; Roth & Porter, 1964; Stay, 1965; Beams & Kessel, 1969; Anderson
& Spielman, 1971; Mahowald, 1972a, b) have also shown that the antigenic
identity between blood and yolk proteins can be attributed to the involvement
of pinocytosis in the formation of yolk. However, in spite of the structural
similarities that vitellogenic oocytes of Drosophila bear with those of other
1
Present address: Istituto di Istologia ed Embriologia, Via A. Volta 4, 56100 Pisa, Italy.
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F. GIORGI AND J. JACOB
insects (Mahowald, 1912a) which are known to form yolk platelets by pinocytosis, it is still held by some workers (Cummings & King, 1970; King, 1970)
that in this species yolk precursors are synthesized by the oocyte itself.
The main objective of the present series of papers is to clarify this point and
with that end in view an ultrastructural, cytochemical and autoradiographical
study has been carried out on the developing oocytes of Drosophila melanogaster.
In this paper, attention is focused especially on the structural changes which
occur in the central and cortical regions of the oocytes in stages prior to and
coincident with the initiation of vitellogenesis.
MATERIALS AND METHODS
Drosophila melanogaster (Oregon-K strain) were reared on standard Drosophila food and kept at a constant temperature of 25 °C. Ovaries from 2- to
3-day-old flies were collected in Drosophila Ringer solution and transferred to
the fixative where they were kept for 2 h at low temperature (4 °C). The fixative
was prepared by mixing equal volumes of 5 % formaldehyde and 5 % glutaraldehyde, both made in 0-1 M cacodylate-HCl buffer at pH 7-2.
After fixation, the ovaries were rinsed overnight in the buffer and then postfixed in 1 % osmium tetroxide in 0-1 M cacodylate buffer, pH 7-2, for 3 h at
4 °C. The ovaries were then briefly washed in the buffer, dehydrated in a graded
series of alcohols and then taken through propylene oxide into a freshly prepared mixture of Epon-Araldite. Polymerization of the resin mixture was carried
out at 60 °C for 72 h.
Thin sections were cut on a Porter-Blum MT-1 ultramicrotome and collected
on copper grids covered with a carbon coated Formvar membrane. Following
double staining in uranyl acetate and lead citrate, the sections were examined
in a AEI-EM6 electron microscope operating at 60 kV.
RESULTS
The ultrastructure of the developing oocyte of Drosophila melanogaster has
already been described in some detail (Cummings & King, 1970; King, 1970;
Mahowald, 1912 b), and the account which follows reviews some of the observations made by earlier workers. The present ultrastructural analysis deals only
with those aspects of oogenesis which appear to be relevant to the process of
yolk formation, and are also meant to serve as a basis for the experimental
study described in the papers which follow.
In early previtellogenic chambers (stages 1-3), the main feature of the cytoplasm of both nurse cells and the oocyte is an abundance of free ribosomes.
Other typical cytoplasmic constituents at these stages are the Golgi apparatus,
mitochondria, microtubules, centrioles and rickettsia-like bodies. In addition to
this, however, the cytoplasm of nurse cells, from stage 1 onwards, presents
a number of small membrane-limited inclusions (Fig. 1). These inclusions usually
Oogenesis o/Drosophila. /
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Fig. 1. Part of the nurse cell cytoplasm from a stage-5 ovarian chamber showing
a cytolysosome (Cy) containing a large number of membranous whorls. Dense masses
are also discernible within the cytolysosome (see arrows), x 15000.
Fig. 2. Part of the ooplasm from a stage-7 ovarian chamber. Note the cytolysosomes
(Cy) of various sizes and consistency. No cytoplasmic organelles are visible within
them, x 12000.
consist of an assembly of a number of minute vesicles and membranous bodies,
some of which are typical myelin forms. On occasions, especially in the early
stages, the inclusions are seen to contain cytoplasmic constituents obviously in
a process of degradation. These inclusions, which in all probability are cytolysosomes, have also been encountered within ring canals joining nurse cells and also
within those joining nurse cells and oocyte.
As stage 4 is reached, the ooplasm shows a larger number of cytolysosomes.
In following stages, as the ovarian chamber enlarges in size, there is a progressive
increase in the concentration of the various cytoplasmic organelles mentioned
above, including the cytolysosomes.
In stage 7 a bewildering variety of cytolysosomes is observable in the central
ooplasm (Fig. 2). They range from inclusions with a very irregular outline and
a highly heterogeneous internal structure, to those with a fairly homogeneous
internal region surrounded by a narrow periphery made up of vesicles of more
or less uniform size.
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Fig. 3. The central ooplasm from an early stage-8 ovarian chamber. Amidst what
look like yolk platelets (y) there are a number of lysosomal derivatives which range
from multivesicular bodies (mvb) to myelin figures (m/). x 25000.
Fig. 4. Yolk platelets (y) in the central ooplasm of an early stage-8 chamber, x 20000.
Fig. 5. Ooplasmic inclusions from an early stage-8 ovarian chamber. Note that the
inclusions consist of two distinct regions with a yolk-like appearance (y) and
a myelin configuration (w/). x 10000.
Stage 8 marks the beginning of vitellogenesis. The central ooplasm at this
stage presents a far greater variety of lysosomal inclusions than ever before.
A typical example of this is shown in Fig. 3. The main characteristic of the
central ooplasm at this stage is a type of inclusion which combines in itself
features of both yolk platelets and cytolysosomes (Figs. 4 and 5).
Another noteworthy feature of the central ooplasm in stage-8 chambers is the
high incidence of the Golgi apparatus: these organelles occupy a subcortical
position in the ooplasm and some of them are in contact with the yolk platelets.
This is an interesting observation and it will be dealt with extensively in the third
paper of this series.
With the onset of vitellogenesis, an interesting modification occurs on the
oocyte surface facing the vitelline membrane. This is the appearance of numerous pits or depressions of the oolemma between the microvilli (Fig. 6). The pits
show an electon-dense fuzzy material on the external side and a spiked layer on
the inner cytoplasmic side. In the region of the cortical ooplasm immediately
below the microvilli there are also present numerous small vesicles and tubules
Oogenesis o/Drosophila. /
119
*• 9,
Fig. 6. Follicle-oocyte border from a stage-10 ovarian chamber. Pits (p), coated
vesicles (cv) and tubules (/) are discernible in the cortical ooplasm. Forming yolk
platelets (y) are also visible. (Km), Vitelline membrane, x 15000.
Fig. 7. A high magnification picture of the region of the pits at the surface of the
oocyte. x 45000.
Fig. 8. A coated vesicle situated close to the region of a pit. Note that the coated
vesicle exhibits features similar to those of the pit. x 45000.
Fig. 9. Part of the cortical ooplasm showing several tangential sections of the
peripheral spiked layer of the coated vesicles (cv). x 56000.
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Fig. 10. Part of the cortical ooplasm of a stage-10 ovarian chamber. Tubules (t) can be
seen contacting the forming yolk platelets (y). x 40000.
Fig. 11. Part of the central ooplasm from a stage-9 ovarian chamber taken from an
ovary fixed in glutaraldehyde and embedded in glycol methacrylate. The yolk
platelets consist of a homogeneous dense main body (mb) surrounded by a caplike region of the superficial layer (SI) within which a prominent empty-looking
associated body (Ab) can be made out. x4000.
Fig. 12. A high power micrograph of part of a yolk platelet from the central ooplasm
of a stage-9 ovarian chamber fixed in glutaraldehyde-osmium and embedded in
Epon-Araldite mixture, showing the cap-like region of the superficial layer with the
associated body (Ab) therein, x 32000.
Oogenesis of Drosophila. /
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(Figs. 6, 8 and 10). In higher power pictures the vesicles exhibit a three-layered
structure which bears a close resemblance to that of the pits (Fig. 7). In favourable
sections (Fig. 9), the outermost layer of these vesicles is seen to be composed of
a regular array of rounded granules of (15 nm) in average diameter. However, the
vesicles of the cortical ooplasm which lie further away from the microvilli are
devoid of the outer layer but still possess the inner fuzzy layer. The tubules are
ultrastructurally similar to the coated vesicles, but irrespective of their location
within the cortical ooplasm they always lack the outer spiked layer.
Deeper in the cortical ooplasm there are observable vesicles of progressively
larger size. From the disposition of the smaller vesicles around the larger ones,
one gets the impression that the larger vesicles are formed by extensive fusion of
the smaller ones. It would also appear that the tubules mentioned above are also
involved in the phenomenon of fusion (Fig. 10). While the small vesicles have
a rather empty appearance, apart from the fuzzy layer on the inner side, the
larger vesicles contain a somewhat denser material which fills them to varying
degrees (Figs. 6 and 10). On the basis of morphological criteria, some of these
vesicles may well be referred to as early yolk platelets.
In the central ooplasm of stages 9-10, many of the yolk platelets seem to
acquire a characteristic morphology. In addition to the limiting membrane and
the main body, as reported by earlier workers (King, Bentley & Aggarwal,
1966; Cohn & Brown, 1968; Cummings & King, 1970; Mahowald, 19726) there
is present an asymmetrically located superficial layer of low density within
which a number of small spherules and a rounded body - hereafter referred
to as the associated body-can be made out. The structural appearance of the
associated body varies according to the fixative and embedding medium used.
Following fixation in glutaraldehyde and embedment in glycol methacrylate
the associated body appears empty (Fig. 11), while in preparations made after
osmium fixation and embedment in Epon-Araldite mixture, the associated
body exhibits a dense content (Fig. 12).
DISCUSSION
The present electron microscope study has shown that a number of lysosomal
derivatives come to be present in the ooplasm of previtellogenic ovarian
chambers and that they increase in number progressively. The observation that
some of them contain cytoplasmic organelles in an apparent state of degradation
suggests that they are likely to originate through a process of focal degradation:
accordingly they were referred to as cytolysosomes. Our observations suggest
that these cytolysosomes are first formed in the cytoplasm of nurse cells and
then transported to the oocyte by way of the ring canals.
Many of the inclusions present in the ooplasm of stage-7-8 chambers of
Drosophila exhibit a characteristic intermediate between lysosomal derivatives
and yolk platelets. In an earlier study Mahowald (19726) had observed that some
of the multivesicular bodies which are present in the ooplasm of previtellogenic
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chambers exhibit an internal structure which makes them indistinguishable
from fully grown yolk platelets. Oocytes from several other species have also
been shown to acquire yolk-like material prior to the initiation of pinocytotic
activity (Steinert & Urbani, 1969; Spornitz & Kress, 1973). In Drosophila
melanogaster this has been regarded as indicating the occurrence of an
endogenous synthesis of yolk precursors by the oocyte itself (Mahowald, 19726).
In the light of our findings, it would seem more appropriate to attribute the
appearance of yolk platelet-like bodies in previtellogenic chambers to a contribution made to the yolk by the lysosomal derivatives. The cytolysosomes
which are formed at earlier stages in the nurse cell cytoplasm may, in fact,
acquire such a state of condensation as to make them morphologically indistinguishable from the yolk once in the ooplasm. It would thus appear that yolk
platelets are formed not only through a heterophagic process which involves
pinocytosis, but also by means of an autophagic activity. The latter would
convey to the yolk platelets breakdown products derived from the intracellular
sequestration of the cytoplasmic organelles present in the nurse cytoplasm. This
suggestion is supported by a number of examples where somatic cells have been
shown to acquire nutrients by either heterophagic or autophagic activities
(Swift & Hruban, 1964; De Duve & Wattiaux, 1966; Locke & Collins, 1975;
Gordon, Miller & Beusch, 1965; Arstila, Jauregui, Change & Trump, 1971).
The onset of vitellogenesis is marked by the appearance of several structural
entities in the cortical ooplasm; these are the pits of the oolemma, the coated
vesicles and the tubules in the region immediately below it. On the basis of the
structural similarities of these entities, it seems highly probable that the coated
vesicles represent internalized pits which have lost contact with the oolemma.
There are also indications that the continued fusion of coated vesicles and
tubules results in the formation of small yolk platelets in the cortical ooplasm.
This structural analysis has led us to conclude that pinocytosis is most probably the mechanism by which yolk precursors are taken into vitellogenic oocytes
from the haemolymph and ultimately sequestered into growing yolk platelets.
Several other workers (Roth & Porter, 1964; Stay, 1965; Anderson, 1969;
Anderson & Spielman, 1971; Mahowald, 1912a) have also come to the conclusion that uptake of proteins by pinocytosis is a method common to all
insects to form yolk platelets. However, quite a different opinion has been
expressed by Cummings & King (1970) in their study of the origin of yolk
platelets in Drosophila. They maintain that only 'raw material' is taken in by
pinocytosis and that proteins forming the yolk platelets are synthesized by the
ribosomes attached to the endoplasmic reticulum of the oocyte. Their argument
is based upon the assumption that the tubules which make contact with the yolk
platelets originate from the endoplasmic reticulum. Our structural analysis
shows that this is not the case and experiments to be described in the following
paper further corroborate the view that the tubules in question originate from
the oolemma presumably by progressive fusion of coated vesicles.
Oogenesis 0/Drosophila. /
123
Cummings & King's (1970) criticism of the formation of yolk platelets in
Drosophila melanogaster lies in the observation that pinocytosis, because of the
relative surface-to-volume ratios between pinocytotic vesicles and yolk platelets,
would actually result in the transfer of more membrane than protein content.
Our findings show that there is no obvious accumulation of membranes at the
sites where pinocytotic vesicles fuse to form yolk platelets. We may predict that
this is so because the excess membrane is broken down to its molecular constituents. The existence of a mechanism involved in the breaking down of
membranous components in the yolk platelets of Drosophila oocytes is made
likely by the presence in them of lysosomal enzymes, as will be reported in the
third paper of this series. A similar conclusion was reached in their study of the
fat body of an insect by Locke & Collins (1968) who suggested that an organelle
which contains hydrolytic enzymes such as for instance multivesicular bodies,
may be concerned with the breaking down of both the content and the limiting
membrane of any exoplasmic vesicle which fuses with it.
These considerations as well as the lack of evidence for the presence of a layer
of endoplasmic reticulum interposed between the oolemma and the forming
yolk platelets make the view expressed by Cummings & King (1970) on the
origin of the yolk platelets in Drosophila melanogaster untenable.
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{Received 27 July 1976)