J. Embryol. exp. Morph. Vol. 67, pp. 101-111, 1982
Printed in Great Britain © Company uf Biologists Limited 1982
\Q\
Time-lapse film analysis of cytoplasmic streaming
during late oogenesis of Drosophila
ByHERWIGO. GUTZEIT1 AND ROSWITHA KOPPA
From the Institiit fur Biologie I der Albert-Ludwigs-Universitat, Freiburg
SUMMARY
Cytoplasmic streaming in follicles of Drosophila has been analysed in vitro by means of
time-lapse films. Late vitellogenic follicles develop normally in vitro as judged by
morphological criteria. Furthermore, follicles (stage 10 and younger) which were cultured
in vitro for the same length of time as follicles which were filmed, developed normally in
vivo after injection into a host fly. The recorded cytoplasmic movements are, therefore,
unlikely to be an in vitro artefact.
At early vitellogenic stages (up to stage 9; King, 1970) no cytoplasmic streaming can be
detected, but at stage 10A cytoplasmic movements are initiated within the oocyte. At stage
10B, when the nurse cells start degenerating, nurse cell cytoplasm can be seen to flow into
the growing oocyte. At stage 11 a central stream of nurse-cell cytoplasm reaches the oocyte
within a minute. The ooplasmic streaming is most rapid at stage 10B and stage 11 and only
an oocyte cortex up to 7 /tm thick remains stationary. Once the bulk of the nurse-cell cytoplasm has poured into the oocyte (stage 12) the cytoplasmic movement ceases, first in the
nurse cells and later in the ooplasm. In mature oocytes no cytoplasmic streaming can be
detected.
INTRODUCTION
The analysis of maternal-effect mutants in Drosophila has shown that mature
oocytes contain the information specifying the axial coordinates in the egg
(Bull, 1966; Lohs-Schardin & Sander, 1976; Nusslein-Volhard, 1977). While
the determination of the axial coordinates may be the result of gradients
building Up during oogenesis or very early embryogenesis (Nusslein-Volhard,
1979), the pole plasm contains local determinants which function autonomously in transplantation tests (lllmensee, Mahowald &Loomis, 1976). The
information-carrying molecules must have been synthesized in either case
during oogenesis and deposited at the appropriate sites in the oocyte. Little is
known about the mechanisms that would allow such specific deposition of
molecules in the oocyte.
The analysis of oogenesis has been hampered, because of the lack of media
that would allow oogenesis to continue in vitro. Only in case of the paedogenetically reproducing gall midge Heteropeza conditions were found which
1
Author's address: Institut fur Biologie I (Zoologie) der Albert-Ludwigs-Universitat
Freiburg Albertstr. 21a D-7800 Freiburg i.Br. (Federal Republic of Germany).
102
H. O. GUTZEIT ANDR. KOPPA
permit normal development so that oogenesis in vitro could be analysed by
means of time-lapse films (Went, 1977). The oocytes were shown to pulsate
rhythmically and follicles were seen to rotate in the ovary prior to their release
into the body cavity. The reasons for these follicular motions are unknown.
In Robb's medium (Robb, 1969) ovarian follicles of Drosophila undergo
apparently normal development during late vitellogenesis in vitro (Petri,
Mindrinos, Lombard & Margaritis, 1979). This observation led us to study the
follicular development in vitro by means of time-lapse films allowing direct
visualization of cytoplasmic movements.
METHODS
Female wild-type flies (Oregon R) were dissected in Robb's medium and
follicles isolated with tungsten needles. The follicles were placed in a small
incubation chamber on a siliconized slide or in a shallow depression slide and
covered with a coverslip. Alternatively, a flow-through chamber (Vollmar, 1972)
which allows for sufficient oxygen supply and prevents desiccation was used.
The follicles were filmed using bright-field optics. 16 mm films (Kodak Plus-X
Reversal) were prepared using a Bolex H 16 reflex camera attached to a LeitZ
microscope. The film was finally analysed using a Kodak analyst projector.
Since the morphology of the follicle changes only slightly during individual film
sequences, the observed cytoplasmic streaming was plotted on photographs
prepared from a single frame of the particular film sequence studied.
Cytoplasmic streaming in a total of 63 follicles of different developmental
stages was analysed (stages 7-9/7 follicles; stage 10A/9; stages 10-B12/25;
stage 13/8; stage 14/14). The observed time pattern of cytoplasmic streaming
during development is highly reproducible, but ooplasmic streaming during
stages 10-12 may vary with respect to speed and direction of movement (see
under 'Results').
When follicles are filmed for 30-45 min in Robb's balanced saline solution
instead of Robb's medium the same pattern of cytoplasmic streaming is
obtained.
When stage-10 follicles were left to develop in vitro misshaped chorionic
filaments and a remaining nurse-cell cap were typically observed (Petri et al.
1979). Judged by morphological criteria follicles older than stage 10 developed
normally in vitro after filming.
RESULTS
Controls for normal development in vitro
When vitellogenic follicles (stage 10) are isolated and cultured in Robb's
medium for up to 11 h they develop into mature oocytes (stage 14) and, furthermore, the time course of this development in vitro is comparable to that in vivo
(Petri et al. 1979).
Film analysis of oogenesis
103
Table 1. Development of ovarian follicles injected into ovt/ovt
females after incubation in Robb's medium for 30-45 min
Stages
Number of
injected
follicles
Implant not
found after
in vivo
culture
Normally
developed
follicles
Abnormal
Normal
or
development
degenerated
(%)
—
81
Ovanoles containing
27
5
22*
Previtellogenic stages
3
41
Stages 9/10
37
19
15t
* Ovariole contains a terminal stage-14 follicle and other vitellogenic stages,
t Stage-14 follicles except in 3 cases: stage 10B (2), stage 12 (1): these 3 follicles thereafter
completed normal development in vitro.
We chose to isolate follicles of each developmental stage and to film cytoplasmic streaming only for 30—45 min following the isolation of the follicle
since in this way possible artefacts as a result of longer incubations in vitro
might be avoided or reduced. However, we have no evidence that this precaution
was necessary.
Stage-9 and younger follicles do not develop to maturity in vitro, presumably
due to a lack of essential growth factors in the medium. It is, therefore, important
to show that these follicles remain viable in vitro for the period of filming. To
test the viability of follicles (stage 10 and younger) after culturing in vitro for
30-45 min the follicles were injected into female flies homozygous for the
female-sterile mutation ovarian tumor (ovt). In these flies no ovarian follicles are
formed (Dr E. Gateff, personal communication) and, therefore, the implant can
easily be detected in the host fly at the end of the in vivo incubation. The injected follicles were allowed to develop in vivo for 48 h (previtellogenic stages)
or 24 h (stages 9 and 10). Finally, the host flies were dissected and the development of the implanted follicles assessed (Table 1). The results show that a large
percentage of the injected follicles completed their development in vivo. The
smaller percentage of successful implantations with stage-9 and -10 follicles as
compared to previtellogenic follicles (Table 1) is most probably due to technical
difficulties since mid-vitellogenic follicles are so large that they can easily be
punctured or slit open by the sharp edges of the injection pipette. Occasionally
parts of damaged follicles were recovered; in most cases these fragments consisted of the posterior pole containing ooplasm surrounded by columnar
follicle cells.
In three cases the implanted follicles had not yet reached the final stage of
oogenesis when their host fly was dissected (Table I, asterisked). These follicles
completed oogenesis in vitro and hence in these cases the follicles had gone
through three changes of in vitro/in vivo culture and yet they developed into
stage-14 oocytes with apparently normal morphology.
104
H. O. GUTZEIT AND R. KOPPA
Fig. 1. Single frame of a time-lapse film showing cytoplasmic motions in a stage-9
follicle in Robb's medium. Parallel arrows pointing in opposite directions: back and
forth movement of cytoplasm. Intersecting double-headed arrows: strong oscillatory
movements in all directions but no cytoplasmic streaming. Triangle: Cortex area
which appears almost motionless.
Film analysis ofoogenesis
Fig. 2. Posterior part of a stage-10 follicle (film frame). The arrow length is proportional to the speed of the cytoplasmic movements and mark the distances covered
by cytoplasmic particles within 100 sec. Bar: 20/im. Other symbols as in Fig. 1.
105
106
H. O. GUTZEIT AND R. KOPPA
With respect to protein synthesis no quantitative or qualitative change during
1 h of culture in vitro could be detected when stage-10 follicles were labelled
with [35S]methionine immediately after their isolation or after pre-incubation
for 1 h and the radioactive polypeptides analysed on SDS-gels (not shown).
Cytoplasmic streaming prior to the centripetal migration offollicle cells
. between nurse cells and oocyte
In late stage-9 follicles no cytoplasmic streaming can be observed but the
ooplasm shows random oscillatory motions in all directions (Fig. 1). In the nurse
cells characteristic back and forth movements can be observed which again do
not result in any lasting displacement of cytoplasm. At this developmental
stage there is no visible indication of cytoplasmic transport from the nurse
cells to the oocyte.
At stage 10A the ooplasm begins to stream (Fig. 2). The central area of the
oocyte could not be analysed with the methods used because of the thickness
of the follicle and strong absorption of light by the yolk platelets. However, the
observed pattern of cytoplasmic movements clearly suggests that the streaming
extends into the axial area as well (Fig. 1, arrows). Since at stage 10A the nursecell cytoplasm does not visibly stream into the oocyte, the observed ooplasmic
movements do not seem to be the result of cytoplasmic influx from the nurse
cells.
Cytoplasmic streaming following centripetal migration offollicle cells
After the follicle cells at the nurse-cell/oocyte border have completed their
centripetal migration (stage 10B) a very different picture emerges. Nurse-cell
cytoplasm can be seen streaming into the oocyte through the four ring canals
which connect the oocyte with four neighbouring nurse cells (King, 1970). At
first only the cytoplasm of these four nurse cells is poured into the oocyte while
the more anteriorly located nurse cells are not affected (not shown). Later, however, when the nurse cells degenerate rather rapidly, cytoplasm flows towards
the central area forming a fast and massive stream which reaches the oocyte
within a minute (Fig. 3). When the nurse-cell cytoplasm passes the gap left by
centripetally migrated follicle cells, it gains the fastest speed and the streaming
can even be observed directly under the microscope. Nurse-cell cytoplasm
flowing towards the central cytoplasmic stream is occasionally seen to be pushed
back into a nurse cell (Fig. 3, dashed lines). Minutes later when the built-up
pressure is apparently equilibrated, the cytoplasm flows out the same way it was
first pushed back, and merges with the central stream of cytoplasm.
The flow of cytoplasm through the intercellular bridges connecting the nurse
cells with each other is indicated by the observed pattern of cytoplasmic
streaming (Fig. 3). However, at this stage the cell membranes start to break
down (Cummings & King, 1970), presumably a prerequisite for the formation
of the large and fast-moving central cytoplasmic stream.
Fig. 3(a). Degenerating nurse cells of a stage-11 follicle (film frame), (b) Cytoplasmic streaming observed in the same follicle. Symbols
and scale as in Figs. 1 and 2. The lengths of the arrows mark the distances covered by cytoplasmic particles within 23 sec.
1
H. O. GUTZEIT AND R. K O P P A
Fig. 4. Posterior part of the follicle shown in Fig. 3. Symbols and scales as in Figs. 1
and 2. The lengths of the arrows mark the distances covered by cytoplasmic particles
within 38 sec.
During the development of a follicle from stage 10B to stage 11 the volume of
the nurse cells was found to decrease at a rate of about 13000 /tm 3 /min.
The diameter of the cytoplasmic stream passing through the four intercellular
bridges connecting the nurse cells with the oocyte was difficult to determine in
stage-1 OB follicles since the depth of focus was not small enough to measure the
streaming through each cytoplasmic bridge separately. As a result, the areas
between the intercellular bridges, where there is no streaming, cannot be
measured reliably since immobile regions and fast-moving cytoplasmic streams
may alternate on different levels of focus. However, at stage 11, when the intercellular bridges move closer together, the cytoplasmic stream was found to be
about 12-13 /tm in diameter.
From the above data the speed of the cytoplasm passing through the cytoplasmic bridges connecting the nurse cells with the oocyte can be predicted to
be about 1-8/mi/sec. When the speed of streaming was measured directly in
film sequences it was found to be 1 -9 ± 0-4 /tm/sec. Figure 3 shows an example
of a particularly fast-moving cytoplasmic stream (about 2-3 /tm/sec).
Film analysis of oogenesis
109
Since the calculated and the measured speed of cytoplasmic streaming is
roughly the same it seems likely that the bulk of the nurse cell cytoplasm and
not just large-sized cytoplasmic inclusions is affected by the streaming.
The ooplasm is also in continuous motion. The cytoplasm either flows in a
circular fashion (Fig. 4) or in a way described earlier (Fig. 2). In general, the
geometry and speed of ooplasmic streaming does not seem to follow any strict
rules. By following the cytoplasmic streaming near the periphery of the growing
oocyte in time-lapse films, we calculated that a cytoplasmic particle travelling
the circular way may complete one round in about 20-40 min (Fig. 4).
Cessation of cytoplasmic streaming at late vitellogenic stages
When the nurse cell breakdown is almost completed (stage 12), the cytoplasmic streaming from the nurse cells to the oocyte ceases and the ooplasmic
movements slow down. The cortical cytoplasm at the anterior end of the oocyte
(facing the degenerating nurse cells) gains in thickness and can measure up to
15 fim. It is not known whether the increased cortex width is due to the specific
deposition of nurse-cell products. Nurse cells of this late stage were previously
shown to synthesize several stage-specific proteins (Gutzeit & Gehring, 1979),
but it is not known whether these proteins become incorporated into the growing
cortex.
At stage 13 even the cytoplasmic movements within the oocyte come to a halt
and no streaming can be detected anywhere in the ooplasm during the final
stage of oogenesis.
DISCUSSION
An inherent problem of in vitro research is the difficulty in assessing the relevance for the in vivo situation. However, we feel confident that our observations
reflect by and large the in vivo situation since (1) stage-10 follicles develop
normally by morphological criteria in vitro; (2) the developmental time required
to complete oogenesis is comparable after in vitro and in vivo culture (Petri et al.
1979); (3) young follicles (stage 10 and earlier) are not irreversibly damaged
by the in vitro treatment since they continue developing in vivo and, finally,
(4) protein synthesis in stage-10 follicles is quantitatively and qualitatively stable
for at least 60 min in vitro.
Once the follicle cells at the nurse cell/oocyte border have completed their
centripetal migration, the nurse-cell cytoplasm pours into the rapidly growing
oocyte where strong cytoplasmic streaming leads to thorough mixing within
minutes. Therefore, the movements do not appear to be involved in the transport of molecules to specific sites in the oocyte. During late stage 12 and stage 13
the cytoplasmic movements cease, first in the nurse cells and later in the oocyte.
The ooplasmic streaming can be observed not only before the nurse cytoplasm
pours into the oocyte but also after this process is completed. Therefore, it
appears that the ooplasmic movements and the cytoplasmic streaming in nurse
110
H. O. GUTZEITANDR. KOPPA
cells at the time of their rapid degeneration are independently controlled
processes.
The reported observations have interesting implications with respect to the
site-specific localization of molecules during oogenesis. Because of the rapid
ooplasmic streaming during stages 10 to 12, this period appears to be ill-suited
for the specific localization of molecules. Consistent with this notion is the
finding that polar granules appear already in stage-9 oocytes at the posterior
pole of the follicle (Mahowald, 1962). In the wasp Pimpla the oosome is already
localized at the posterior pole at the beginning of vitellogenesis long before
the nurse-cell cytoplasm streams into the oocyte (Meng, 1968). This also holds
true for the germ plasm in follicles of the ants Camponotus and Formica (Bier,
1952). Prelocalized receptors might, of course, bind specific molecules carried
around in the stream of cytoplasm and thereby acquire the necessary factors
for their function. The pole plasm may be a case in point, since transplantation
tests show that it becomes competent during late vitellogenesis to induce pole
cells in young embryos (Illmensee et ah 1976).
In the Drosophila oocyte only those molecules which are localized in the
approximately 5-7 /im wide cortex at stage 11 may be unaffected by the cytoplasmic streaming.
The polar granules at the posterior pole of late vitellogenic follicles are presumably included in the immobile cortical cytoplasm. In mature eggs of
Drosophila hydei polar granules were found to be located in the peripheral
5-10 ju,m of the egg (Mahowald, 1973) which approximates the cortex thickness
(up to 7/*m) of the smaller sized stage-11 follicles of Drosophila melanogaster
(Fig. 4). The large oocyte nucleus, however, must be anchored in some unknown
fashion to keep its place.
Niisslein-Volhard (1979) suggested that the anteroposterior and the dorsoventral coordinates of the embryo are defined by two gradients. If gradients are
set up by concentration differences of diffusible molecules (Meinhardt, 1977) it
seems reasonable to assume that such gradients can only be established in the
absence of cytoplasmic streaming. These conditions are met prior to stage 10A
and after stage 12.
We wish to acknowledge the invaluable advice and support of Dr H. Vollmar and Prof. K.
Sander during the course of this work. We thank the Deutsche Forschungsgemeinschaft for
financial support (SFB 46).
REFERENCES
BIER, K. (1952). Beziehung zwischen Nahrzellkerngrosse und Ausbildung ribonucleinsaurehaltiger Strukturen in den Oocyten von Formica rufa rufopratensis minor (Gossw.) Verh.
Dtsch. Zool. Ges. Freiburg, 46, 369-374.
BULL, A. (1966). Bicaudal, a genetic factor which affects the polarity of the embryo in
Drosophila melanogaster. J. exp. Zool. 200, 149-156.
CUMMINGS, M. R. & KING, R. C. (1970). Ultrastructural changes in nurse and follicle cells
during late stages of oogenesis in Drosophila melanogaster. Z. Zellforsch. mikrosk. Anat.
110, 1-8.
Film analysis of oogenesis
111
H. O. & GEHRING, W. J. (1979). Localized synthesis of specific proteins during
oogenesis and early embryogenesis in Drosophila melanogaster. Wilhelm Roux Arch, devl
Biol. 187, 151-165.
ILLMENSEE, K., MAHOWALD, A. P. & LOOMIS, M. R. (1976). The ontogeny of germ plasm
during oogenesis in Drosophila. Devl Biol. 49, 40-65.
KING, R. C. (1970). Ovarian Development in Drosophila melanogaster. New York, London:
Academic Press.
LOHS-SCHARDIN, M. & SANDER, K. (1976). A dicephalic monster embryo of Drosophila
melanogaster. Wilhelm Roux Arch, devl Biol. 179, 159-162.
MAHOWALD, A. P. (1962). Fine structure of pole cells and polar granules in Drosophila
melanogaster. J. exp. Zool. 151, 201-215.
MAHOWALD, A. P. (1973). Oogenesis. In Developmental Systems: Insects, vol. 1 (ed. S. J.
Counce & C. H. Waddington), pp. 1-47.
MEINHARDT, H. (1977). A model of pattern formation in insect embryogenesis. J. Cell Sci.
23, 117-139.
MENG, C. (1968). Strukturwandel und histochemische Befunde insbesondere am Oosom
wahrend der Oogenese und nach der Ablage des Eies von Pimpla turionellae L. (Hymenoptera, Ichneumonidae). Wilhelm Roux Arch. EntwMech. Org. 161, 162-208.
NUSSLEIN-VOLHARD, C. (1977). Genetic analysis of pattern-formation in the embryo of
Drosophila melanogaster. Characterization of the maternal-effect mutant bicaudal.
Wilhelm Roux Arch, devl Biol. 183, 249-268.
NUSSLEIN-VOLHARD, C. (1979). Maternal effect mutations that alter the spatial coordinates
of the embryo of Drosophila melanogaster. In Determinants of Spatial Organisation (ed. J.
Konigsberg & S. Subtelney), pp. 185-211. Academic Press.
PETRI, W. H., MINDRINOS, M. N., LOMBARD, M. F. & MARGARITIS, L. H. (1979). In vitro
development of the Drosophila chorion in a chemically defined organ culture medium.
Wilhelm Roux Arch, devl Biol. 186, 351-362.
ROBB, J. A. (1969). Maintenance of imaginal discs of Drosophila melanogaster in chemically
defined media. /. Cell Biol. 41, 876-885.
VOLLMAR, H. (1972). Fruhembryonale Gestaltungsbewegungen im vitalgefarbten DotterEntoplasma-System intakter und fragmentierter Eier von Acheta domesticus L. (Orthopteroidea). Wilhelm Roux Arch. EntwMech. Org. 171, 228-243.
WENT, D. F. (1977). Pulsating oocytes and rotating follicles in an insect ovary. Devl Biol.
55, 392-396.
GUTZEIT,
{Received 10 June 1981, revised 22 September 1981)