/. Embryol. exp. Morph. Vol. 39, pp. 45-57, 1977
Printed in Great Britain
45
Recent findings in oogenesis of Drosophila
melanogaster
III. Lysosornes and yolk platelets
By F. GIORGI 1 AND J. JACOB 2
From the Institute of Animal Genetics, University of Edinburgh
SUMMARY
The role played by the vitellogenic oocytes of Drosophila melanogaster in relation to the
elaboration of material taken from the haemolymph is examined by ultrastructural
cytochemistry.
As revealed by the Gomori procedure, acid phosphatase occurs widely over the forming
yolk platelets of the cortical and central ooplasm. A number of Golgi apparatuses in the
cortical ooplasm are also positively stained with lead precipitates.
With the proceeding of the ovarian development it becomes progressively more difficult
to demonstrate cytochemically the enzyme over the yolk platelets. In stage 9-10 chambers
the acid phosphatase is restricted to the so-called associated body, while the rest of the yolk
platelet appears devoid of lead deposits.
By using a osmium zinc iodide (OZ1) complex as a preferential staining method for the
Golgi apparatus, it has been shown that, apart from the apparatus itself, a number of OZI
deposits occur over the superficial layer of the forming yolk platelets. When mature yolk platelets are formed at later stages, the OZI deposits in the yolk platelets come to be restricted to
the cap-like region of the superficial layer which contains the associated body.
In vitellogenic oocytes, both the internal lining of the limiting membrane of the forming
yolk platelets and the associated body of the mature yolk platelets react positively, to cytochemical methods to demonstrate carbohydrates.
The present findings are interpreted as indicating the involvement of lysosomal enzymes
in the process of maturation of the yolk material. The suggestion is also made that such an
involvement is required to accomplish a selective hydrolysis of those blood proteins which
have been taken in by vitellogenic oocytes along with yolk precursors.
INTRODUCTION
The evidence obtained so far from oocytes of several insects (Telfer, 1961;
Roth & Porter, 1964; Anderson, 1964; Telfer, 1965; Stay, 1965; Anderson,
1969, 1971; Giorgi & Jacob, 1977) firmly establishes that the material which is
segregated within the forming yolk platelets during vitellogenesis has an extraovarian origin. The method by which this exogenous material is sequestered
into the yolk platelets has been shown to be pinocytosis (Anderson & Spielman,
1
Author's address: Istituto di Istologia ed Embriologia, Via A., Volta 4-56100 Pisa
(Italy).
2
Author's address: Institute of Animal Genetics, University of Edinburgh, Scotland.
4
EMB 39
46
F. GIORGI AND J. JACOB
1971; Giorgi & Jacob, 1976). This method involves the formation of minute
pinocytotic vesicles in the region of the oolemma and their subsequent fusion
to form large-sized yolk platelets (Mahowald, 1972).
It has been shown that by virtue of pinocytosis only one or few proteins
among those present in the yolk platelets can actually increase their concentration over that in the blood (Telfer, 1954, 1965; Bodnaryk & Morrison, 1968;
Bell, 1970; Doira & Kawaguchi, 1972). Several mechanisms have been adduced
to account for the preferential uptake of yolk proteins in vitellogenic oocytes
of insects (Ramamurty, 1964; Anderson & Telfer, 1969; Jacques, 1969; Anderson, 1971) but none of them can fully explain the fact that non-yolk proteins
do not accumulate in mature yolk platelets, in spite of the fact that a way is
open for any one of them to get into the yolk platelets. The suggestion has been
made that vitellogenic oocytes could be involved in a process of selective
hydrolysis of those non-yolk proteins which are engulfed by the forming yolk
platelets along with yolk precursors (Cone & Eschenberg, 1966; Hopkins &
King, 1966; Lockshin, 1969; Giorgi, 1975). This suggestion is based upon the
observation that in somatic tissues the uptake of exogenous material is followed
by intervention of lysosomal enzymes (Strauss, 1964, De Duve & Wattiaux,
1966; Friend & Farquhar, 1967; Strauss, 1967; Daems, Wisse & Brederoo,
1969; Jacques, 1969).
The present cytochemical study aims to throw some light on the role played
by vitellogenic oocytes in relation to the elaboration of material taken up from
the haemolymph. The question is examined by focusing attention on the structural relationship between the Golgi apparatus and the forming yolk platelets.
MATERIAL AND METHODS
For the cytochemical detection of acid phosphatase, ovaries from 2- to
3-day-old flies were fixed for 2 h at 0 °C in 5% glutaraldehyde in 0-1 M cacodylate buffer at pH 7-2. The ovaries were thoroughly washed in the buffer and
then incubated in the Gomori medium at 37 °C for 30-45 min. As controls,
some glutaraldehyde-fixed ovaries were incubated in the substrate-free Gomori
medium at 37 °C for the same length of time as the experimental tissues. Both
experimental and control ovaries were then post-fixed in 1 % osmium tetroxide
made up in 0-1 M cacodylate buffer, pH 7-2, for 3 h at 4 °C. A few ovaries were
fixed in glutaraldehyde as before, and then incubated in the Novikoff & Goldfischer's (1961) medium for 60 min at 37 °C for the cytochemical detection of
thiamine pyrophosphatase.
In another series of cytochemical experiments for identifying Golgi apparatus, 2- to 3-day-old ovaries were fixed in the osmium zinc iodide (OZ1) complex prepared according to the formula of Niebauer, Krawczyk, Kidd &
Wilgram (1969). The final pH of the OZI solution was, however, adjusted to
7-0 with 0-1 N-NaOH as suggested by Elias et al. (1972). After fixation in the
Oogenesis of Drosophila. / / /
47
OZI complex, the ovaries were washed several times in distilled water, dehydrated in alcohols and then embedded in Epon-Axaldite mixture.
Ultrathin sections were examined in the electron microscope with or without
double staining in uranyl acetate and lead citrate.
For the cytochemical detection of carbohydrates in vitellogenic oocytes of
Drosophila, the method of Thiery (1967) was employed on thin sections of
Epon-Araldite embedded material. A few ovaries were also fixed in glutaraldehyde and embedded in glycol methacrylate (GMA). Thin sections of GMA
embedded ovaries were then treated with 1 % solution of phosphotungstic acid
in 10% chromic acid as suggested by Rambourg (1967). Thin sections derived
from both these experiments were observed in an AEI-EM6 electron microscope
without further staining.
The staging criteria used in this paper for Drosophila ovarian chambers are
those described by Cummings & King (1969).
RESULTS
When ovarian chambers of previtellogenic stages were incubated in the complete Gomori medium, lead deposits could be observed over a number of
organelles in every ovarian cell. The Golgi apparatus was acid phosphatase
positive, although with varying degrees in nurse and follicle cells and also in
the oocyte. Other organelles which came to contain deposits of lead after incubation in the Gomori medium were the cytolysosomes of both nurse cells and
oocyte (Fig. 1), as well as a number of dense bodies (lysosomes) in the cytoplasm of the follicle cells (Fig. 2).
With the onset of vitellogenesis a higher number of Golgi apparatuses could
be observed in the ooplasm. Presumably this increase in number is brought
about by a transfer of Golgi apparatuses from the nurse cells to the ooplasm
through the ring canals. Besides the Golgi apparatus, the other organelles which
were positive for acid phosphatase in the ooplasm of vitellogenic oocytes were
the yolk platelets. An association between the Golgi apparatus and the yolk
platelets was frequently met with in the ooplasm of these stages. In these instances, lead deposits filled most of the Golgi cisternae, and also covered much
of the periphery of the forming yolk platelets (Fig. 3V
A study of a large number of preparations gave the impression that the
amount of lead deposited over the yolk platelets was somehow variable depending perhaps on the dimensions of the platelets themselves. The smaller the yolk
platelets, the relatively more intense the acid phosphatase reaction (Fig. 5). As
yolk platelets increased in size, the lead deposits seemed to get displaced towards the periphery (Fig. 5). In a fully grown yolk platelet the main body was
completely devoid of lead deposits, while the acid phosphatase reaction was
restricted to the associated body (Fig. 4).
In ovarian chambers of stages later than ten, the yolk platelets were no
4-2
48
F. GIORGI AND J.JACOB
Oogenesis oj Drosophila. / / /
49
longer positive for acid phosphatase. On the other hand, other ovarian cells at
these stages still exhibited deposits of lead in the Golgi apparatus and the free
lysosomes.
The nuclei of all the cells forming the ovarian chambers presented deposits
of lead to varying degrees, but a similar nuclear labelling could also be observed
in nuclei of ovarian chambers incubated in the substrate-free Gomori medium.
This observation indicates that the nuclear lead deposits are most probably an
artifact. Nurse nuclei of late ovarian chambers, however, presented a far greater
amount of lead than that normally detectable in nuclei of other stages or of
control preparations. It is conceivable that this difference may be related to the
degenerating condition of these nuclei at these stages (stages 13 and 14).
Another region of the vitellogenic chamber which gave rise to artifactual
acid phosphatase reaction was the vitelline membrane. Lead deposits were, in
fact, observable at this site in both experimental and control preparations.
A similar observation was also reported by Kessel & Decker (1972) in oocytes
of Rana. In ovarian chambers of Drosophila, however, this artifact could be
avoided by prolonging the rinse in the buffer up to several days after the
incubation in the Gomori medium (this procedure was suggested to us by
Dr A. Jurand).
Ovarian chambers incubated in the Novikoff & Goldfischer's (1961) medium
for the cytochemical detection of thiamine pyrophosphatase revealed lead deposits only on lysosomes and the Golgi apparatus of the follicle cells. By this
method no lead deposits were ever seen on any site of the nurse cells or the
oocyte.
The lack of structural clarity in preparations of ovarian chambers treated for
the cytochemical detection of acid phosphatase makes it difficult to establish
the origin of the enzyme in the yolk platelets. It is, on the other hand, well
established that acid phosphatase and presumably other lysosomal enzymes
are released from the Golgi apparatus by means of membrane-bound vesicles
FIGURES
1-5
Fig. 1. Part of the central ooplasm from an early stage-8 ovarian chamber. The
heavy deposits of lead are most probably lying over cytolysosomes. x 6000.
Fig. 2. The follicle-oocyte border from a stage-8 ovarian chamber. The Golgi
apparatus (G) and the nucleus (N) of the follicle cell appear very well labelled with
lead. Heavy deposits occur also in the ooplasm (00). (L), lysosome. x 6000.
Fig. 3. A growing yolk platelet (y) in the cortical ooplasm of a stage-9 ovarian
chamber. Lead deposits are visible over the margin of the yolk platelet and also on
the cisternae of the Golgi apparatus (G) associated with it. x 24000.
Fig. 4. Two yolk platelets in the central ooplasm of a stage-10 ovarian chamber.
Note the heavy deposition of lead over the associated body (Ab). The main body
(mb) is devoid of deposits, x 12000.
Fig. 5. A low power micrograph of part of the ooplasm from a stage-9 ovarian
chamber. Several yolk platelets with lead deposits on are visible in the area, x 3000.
50
F. G I O R G I AND J. JACOB
Fig. 6. A yolk platelet from the central ooplasm of an early stage-8 ovarian
chamber. Note that the main body (mb) is asymmetrically located within a less
dense structure with numerous OZI deposits therein, x 30000.
Fig. 7. A high power micrograph of a stage-9 ovarian chamber showing a growing
yolk platelet. Note the presence of a number of OZI deposits in the superficial layer
(SI) of the yolk platelet, (mb), main body, x 36000.
Fig. 8. Part of a yolk platelet from the central ooplasm of a stage-10 ovarian
chamber. Note the presence of several OZI deposits in the cap-like region of the
superficial layer (SI). The associated body (Ab) is devoid of OZI deposits,
x 30000.
Fig. 9. The cortical ooplasm from a stage-9 ovarian chamber. A number of vesicles
are stained with OZI (see arrows). The coated vesicles and tubules are free of OZI
deposits. (G), Golgi apparatus, x 9000.
Oogenesis of Drosophila. / / /
51
(Novikoff, Essner & Quintana, 1964; Novikoff, Novikoff, Quintana & Hauw,
1971). It seemed, therefore, desirable to identify the Golgi apparatus and the
vesicles derived from it by means of some other cytochemical method. One
such approach is the osmium zinc iodide (OZ1) method and this was applied
to ovarian chambers of Drosophila melanogaster.
In previtellogenic chambers fixed for 4 h in the OZI complex, the Golgi
apparatus of every ovarian cell appeared to contain several electron-dense
deposits. At these stages the Golgi apparatus is mainly formed of vesicles and
tends to occupy a perinuclear position within the cytoplasm of these cells. By
using this method it was possible to confirm that the number of Golgi apparatuses decreases progressively in the cytoplasm of nurse cells as the chamber
approaches vitellogenesis. At the same time there is a concomitant increase in
the number of Golgi apparatuses present in the ooplasm.
Apart from the Golgi apparatus, the other organelles which contained OZI
deposits in vitellogenic chambers were the yolk platelets. In the central ooplasm
of early stage-8 chambers, these platelets consist in part of lysosomal inclusions, while the remaining part forms an asymmetrically located dense body.
Following fixation with the OZI complex, the lysosomal part of these platelets
was seen to contain a number of OZI deposits (Fig. 6).
In vitellogenic chambers of succeeding stages (stages 8-10), the OZI deposits
were most frequently observed within the superficial layer of the yolk platelets
(Fig. 7).
In fully grown yolk platelets which are usually found in the deeper regions of
the ooplasm in stage 9-10 ovarian chambers, the OZI deposits were restricted
to the cap-like region of the superficial layer which contains the associated body
(Fig. 8). The associated body itself, however, was free of OZI deposits.
A number of small vesicles containing OZI deposits could also be seen in the
cortical ooplasm of vitellogenic oocytes. These vesicles, however, were distinguishable from the coated vesicles which are also present in this region
(Fig. 9).
Vitellogenic chambers (stages 8-10) embedded in GMA and treated with
PTA according to the procedure of Rambourg (1967) revealed several intensely
stained sites. These were the region of the follicle-oocyte border underneath the
vitelline membrane (Fig. 11), which also includes the various entities, such as
pinocytotic vesicles, tubules and small yolk platelets, previously described in
the corticaJ ooplasm (Giorgi & Jacob, 1976). In high resolution electron micrographs a very thin layer of PTA-stained material could be made out within these
structural entities. Fig. 10 shows the PTA staining of the limiting membrane of
small yolk platelets; most probably this layer corresponds to the fuzzy layer
which lines internally the coated vesicles and the tubules. When sections passing
through the ooplasm were examined after PTA staining, it could be seen that
the associated body of the mature yolk platelets was intensely stained while
the main body remained unaffected by this treatment (Fig. 13).
52
F. G I O R G I AND J . J A C O B
Fig. 10. The cortical ooplasm from a stage-10 ovarian chamber embedded in glycol
methacrylate and stained with PTA. Note that the fuzzy layer of the limiting membrane of the forming yolk platelets (y) is stained, x 25000.
Fig. 11. The follicle-oocvte border from a stage-10 ovarian chamber embedded in
glycol methacrylate and stained with PTA. Note a heavy stained layer (see arrows)
between the vitelline membrane (Vm) and the surface of the oocyte. x 9000.
Fig. 12. Yolk platelets in the central ooplasm of a stage-10 ovarian chamber treated
according to Thiery's (1967) method. Note the heavy deposits of silver on the
associated body (Ab). x 25 000.
Fig. 13. Part of the central ooplasm from a stage-10 ovarian chamber embedded in
glycol methacrylate and stained with PTA. Note the dense staining of the associated body (Ab) in several yolk platelets, x 9000.
Oogenesis of Drosophila. / / /
53
The results obtained with the PTA method were confirmed by using another
cytochemical method for detecting carbohydrates: that of Thiery (1967). To
get comparable results by these two methods, however, the exposure to thiosemicarbazide in the second method had to be prolonged up to 72 h (Fig. 12).
In fact, following an exposure of only 30-40 min or of 48 h, no silver deposits
could be observed in any site of the vitellogenic chambers. Ovarian chambers
in stages later than ten exhibited some deposits of silver even with short exposure to thiosemicarbazide (Giorgi & Deri, 1976).
The results obtained with Thiery's method show that the reaction sites in
vitellogenic chambers most probably possess glycoproteins which during
vitellogenesis come to be present in the cortical ooplasm and are eventually
conveyed to the associated body of the forming yolk platelets.
DISCUSSION
The aim of the present cytochemical study was to verify whether or not the
process of yolk formation in vitellogenic oocytes of Drosophila can be functionally related to that of endocytosis and intracellular digestion as it occurs
in somatic cells. Evidence has been provided to indicate that in vitellogenic
oocytes of Drosophila the forming yolk platelets contain acid phosphatase and
that as the yolk platelets increase in size the enzyme can no longer be demonstrated by the Gomori procedure.
Much of the evidence in the present study comes from cytochemical techniques and therefore, as in many other techniques, there is the risk of artifact
formation which could lead to misinterpretation (Holt & Hicks, 1962). However, to ensure reproducible detection of both acid phosphatase and thiamine
pyrophosphatase, care was taken to work in conditions which are known to
minimize the occurrence of artifacts (Anastasia-Sawicki & Mclntyre, 1976).
Furthermore, the OZI complex was prepared at neutral pH and used for a
short time interval, conditions which are known to enhance its specificity for
the Golgi elements (Elias et al. 1972). So, although relying on cytochemical
approaches, it seems reasonable to conclude that products of Golgi origin get
transferred to the forming yolk platelets during vitellogenesis. Both the Gomori
procedure and the OZI staining method point in fact to the yolk platelets as
another site where a chemical specificity similar to that of Golgi apparatus can
be detected. In the present material it could not be established whether the
acid phosphatase-rich region of the Golgi apparatus bears any functional
relationship to the nearby endoplasmic reticulum as recently suggested by
Novikoff(1976).
The present findings confirm earlier observations that oocytes in several
animal species may acquire lysosomes during the early phases of vitellogenesis
(Brachet, 1942; Yao, 1950; Cone & Eschenberg, 1966; Hopkins & King, 1966;
Bluemink, 1969). Hitherto, however, there has not been general agreement as to
the possible role of lysosomal enzymes in relation to vitellogenesis.
54
F. GIORGI AND J. JACOB
In a recent review article on lysosomes, Pasteels (1973) expressed the view
that the acid phosphatase detected in the yolk platelets of the snail oocyte
during vitellogenesis is an artifact. In a study on amphibian oocytes, Denis
(1964) suggested that no particular function could be attributed to the acid
phosphatase detected in the yolk platelets during vitellogenesis, and that
presumably the enzyme becomes active only during embryogenesis when
utilization of yolk occurs. On the other hand, a large body of evidence,
mainly from biochemical work carried out on somatic cells, has established
that lysosomal enzymes, like other enzymes and proteins, are synthesized in the
ribosomes attached to the endoplasmic reticulum, transferred to the Golgi
apparatus and then released as vesicles to form primary lysosomes in the cytoplasm (De Duve & Wattiaux, 1966). Numerous cytochemical investigations
have further corroborated this concept (Novikoff, 1963; Novikoff & Shin,
1964; Daems et al. 1969; Ericsson, Trump & Weibel, 1965), although recent
knowledge has been gained as to the actual site of primary lysosome release
(Novikoff, 1976). It is also current knowledge that fusion between lysosomes
and heterophagosomes determines the initiation of the process of digestion of
the ingested material in the resulting phagolysosomes (Strauss, 1964, 1967).
As a result of the lytic activity of the lysosomal enzymes, it has been shown that
low molecular weight degradation products will rapidly diffuse out of the
phagosomes into the surrounding area of the cytoplasm (Mego & McQueen,
1965; Gordon, Miller & Beusch, 1965; Gordon, 1973; Neely & Martinore,
1974). Based on this evidence it is conceivable that lysosomal enzymes in
vitellogenic oocytes function in a manner similar to that in somatic cells, that
is to say, they may be involved in some sort of selective hydrolysis of proteins
taken up from the haemolymph. An indication that some degradation of proteins takes place in oocytes of Drosophila may be found in our autoradiographical experiments (Giorgi & Jacob, 1976) where the density of silver
grains over the yolk platelets following long incubation periods in tracer16 or 20 h - w a s seen to be lower than that after shorter exposure. This is
probably due to a loss of the radioactivity brought about by degradation of
non-yolk proteins. This hypothesis, however, does not explain how yolk proteins escape degradation by lysosomal enzymes. This problem requires further
study. However, a brief reference to this may not be unwarranted and we therefore present the following considerations at least to provoke further thought
and research.
Brambell and co-workers (1964, 1966) put forward a hypothesis to explain
the mechanism of immunity transmission in mammals. They assumed that the
plasma proteins taken up by the placenta are transported to the foetus through
a lysosomal system. During the transport the y-globulin alone was assumed
to escape degradation by virtue of their binding to certain protecting substances. Jacques (1969) has more recently proposed that the mucopolysaccharide cell coat could function as the protecting substance referred to above.
Oogenesis of Drosophila. / / /
55
Support for this idea has come from the work of Snellman & Sylven (1974) who
have actually demonstrated that a substance may be protected from hydrolytic
enzymes by virtue of their binding to a carbohydrate moiety.
In the present study it was shown how in later stages of yolk development
there is a segregation of yolk material into a large main body while the carbohydrates get segregated to form an associated body. One could therefore
envisage that the carbohydrates earlier served to protect the specific yolk proteins from degradation by lysosomal enzymes.
It is pertinent to point out that subsequent to the segregation mentioned
above the acid phosphatase activity appears to be confined to the associated
body as though this body could serve as a device to somehow isolate the residual
lysosomal enzymes at the end of the process of yolk formation.
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{Received 27 July 1976, revised 30 November 1976)