J. Embryol. exp. Morph. Vol. 67, pp. 101-111, 1982 \Q\Printed in Great Britain © Company uf Biologists Limited 1982

Time-lapse film analysis of cytoplasmic streamingduring late oogenesis of Drosophila

ByHERWIGO. GUTZEIT1 AND ROSWITHA KOPPAFrom the Institiit fur Biologie I der Albert-Ludwigs-Universitat, Freiburg

SUMMARYCytoplasmic 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 bymorphological criteria. Furthermore, follicles (stage 10 and younger) which were culturedin vitro for the same length of time as follicles which were filmed, developed normally invivo 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 bedetected, but at stage 10A cytoplasmic movements are initiated within the oocyte. At stage10B, when the nurse cells start degenerating, nurse cell cytoplasm can be seen to flow intothe growing oocyte. At stage 11 a central stream of nurse-cell cytoplasm reaches the oocytewithin a minute. The ooplasmic streaming is most rapid at stage 10B and stage 11 and onlyan oocyte cortex up to 7 /tm thick remains stationary. Once the bulk of the nurse-cell cyto-plasm has poured into the oocyte (stage 12) the cytoplasmic movement ceases, first in thenurse cells and later in the ooplasm. In mature oocytes no cytoplasmic streaming can bedetected.

INTRODUCTION

The analysis of maternal-effect mutants in Drosophila has shown that matureoocytes contain the information specifying the axial coordinates in the egg(Bull, 1966; Lohs-Schardin & Sander, 1976; Nusslein-Volhard, 1977). Whilethe determination of the axial coordinates may be the result of gradientsbuilding Up during oogenesis or very early embryogenesis (Nusslein-Volhard,1979), the pole plasm contains local determinants which function auto-nomously in transplantation tests (lllmensee, Mahowald &Loomis, 1976). Theinformation-carrying molecules must have been synthesized in either caseduring oogenesis and deposited at the appropriate sites in the oocyte. Little isknown about the mechanisms that would allow such specific deposition ofmolecules in the oocyte.

The analysis of oogenesis has been hampered, because of the lack of mediathat would allow oogenesis to continue in vitro. Only in case of the paedo-genetically reproducing gall midge Heteropeza conditions were found which

1 Author's address: Institut fur Biologie I (Zoologie) der Albert-Ludwigs-UniversitatFreiburg 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 bymeans of time-lapse films (Went, 1977). The oocytes were shown to pulsaterhythmically and follicles were seen to rotate in the ovary prior to their releaseinto the body cavity. The reasons for these follicular motions are unknown.

In Robb's medium (Robb, 1969) ovarian follicles of Drosophila undergoapparently normal development during late vitellogenesis in vitro (Petri,Mindrinos, Lombard & Margaritis, 1979). This observation led us to study thefollicular development in vitro by means of time-lapse films allowing directvisualization of cytoplasmic movements.

METHODS

Female wild-type flies (Oregon R) were dissected in Robb's medium andfollicles isolated with tungsten needles. The follicles were placed in a smallincubation chamber on a siliconized slide or in a shallow depression slide andcovered 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-XReversal) were prepared using a Bolex H 16 reflex camera attached to a LeitZmicroscope. The film was finally analysed using a Kodak analyst projector.Since the morphology of the follicle changes only slightly during individual filmsequences, the observed cytoplasmic streaming was plotted on photographsprepared from a single frame of the particular film sequence studied.

Cytoplasmic streaming in a total of 63 follicles of different developmentalstages 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 streamingduring development is highly reproducible, but ooplasmic streaming duringstages 10-12 may vary with respect to speed and direction of movement (seeunder 'Results').

When follicles are filmed for 30-45 min in Robb's balanced saline solutioninstead of Robb's medium the same pattern of cytoplasmic streaming isobtained.

When stage-10 follicles were left to develop in vitro misshaped chorionicfilaments and a remaining nurse-cell cap were typically observed (Petri et al.1979). Judged by morphological criteria follicles older than stage 10 developednormally in vitro after filming.

RESULTS

Controls for normal development in vitro

When vitellogenic follicles (stage 10) are isolated and cultured in Robb'smedium for up to 11 h they develop into mature oocytes (stage 14) and, further-more, 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/ovtfemales after incubation in Robb's medium for 30-45 min

Stages

Number ofinjectedfollicles

Implant notfound after

in vivoculture

Normallydevelopedfollicles

Abnormalor

degenerated

Normaldevelopment

(%)

Ovanoles containingPrevitellogenic stages

Stages 9/10

27

37

5

19

22*

15t

— 81

3 41

* 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 cyto-plasmic streaming only for 30—45 min following the isolation of the folliclesince in this way possible artefacts as a result of longer incubations in vitromight be avoided or reduced. However, we have no evidence that this precautionwas necessary.

Stage-9 and younger follicles do not develop to maturity in vitro, presumablydue to a lack of essential growth factors in the medium. It is, therefore, importantto show that these follicles remain viable in vitro for the period of filming. Totest the viability of follicles (stage 10 and younger) after culturing in vitro for30-45 min the follicles were injected into female flies homozygous for thefemale-sterile mutation ovarian tumor (ovt). In these flies no ovarian follicles areformed (Dr E. Gateff, personal communication) and, therefore, the implant caneasily be detected in the host fly at the end of the in vivo incubation. The in-jected 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 develop-ment of the implanted follicles assessed (Table 1). The results show that a largepercentage of the injected follicles completed their development in vivo. Thesmaller percentage of successful implantations with stage-9 and -10 follicles ascompared to previtellogenic follicles (Table 1) is most probably due to technicaldifficulties since mid-vitellogenic follicles are so large that they can easily bepunctured or slit open by the sharp edges of the injection pipette. Occasionallyparts of damaged follicles were recovered; in most cases these fragments con-sisted of the posterior pole containing ooplasm surrounded by columnarfollicle cells.

In three cases the implanted follicles had not yet reached the final stage ofoogenesis when their host fly was dissected (Table I, asterisked). These folliclescompleted oogenesis in vitro and hence in these cases the follicles had gonethrough three changes of in vitro/in vivo culture and yet they developed intostage-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-9follicle in Robb's medium. Parallel arrows pointing in opposite directions: back andforth movement of cytoplasm. Intersecting double-headed arrows: strong oscillatorymovements in all directions but no cytoplasmic streaming. Triangle: Cortex areawhich appears almost motionless.

Film analysis ofoogenesis 105

Fig. 2. Posterior part of a stage-10 follicle (film frame). The arrow length is pro-portional to the speed of the cytoplasmic movements and mark the distances coveredby cytoplasmic particles within 100 sec. Bar: 20/im. Other symbols as in Fig. 1.

106 H. O. GUTZEIT AND R. KOPPA

With respect to protein synthesis no quantitative or qualitative change during1 h of culture in vitro could be detected when stage-10 follicles were labelledwith [35S]methionine immediately after their isolation or after pre-incubationfor 1 h and the radioactive polypeptides analysed on SDS-gels (not shown).

Cytoplasmic streaming prior to the centripetal migration of follicle cells. between nurse cells and oocyte

In late stage-9 follicles no cytoplasmic streaming can be observed but theooplasm shows random oscillatory motions in all directions (Fig. 1). In the nursecells characteristic back and forth movements can be observed which again donot result in any lasting displacement of cytoplasm. At this developmentalstage there is no visible indication of cytoplasmic transport from the nursecells to the oocyte.

At stage 10A the ooplasm begins to stream (Fig. 2). The central area of theoocyte could not be analysed with the methods used because of the thicknessof the follicle and strong absorption of light by the yolk platelets. However, theobserved pattern of cytoplasmic movements clearly suggests that the streamingextends into the axial area as well (Fig. 1, arrows). Since at stage 10A the nurse-cell cytoplasm does not visibly stream into the oocyte, the observed ooplasmicmovements do not seem to be the result of cytoplasmic influx from the nursecells.

Cytoplasmic streaming following centripetal migration of follicle cells

After the follicle cells at the nurse-cell/oocyte border have completed theircentripetal migration (stage 10B) a very different picture emerges. Nurse-cellcytoplasm can be seen streaming into the oocyte through the four ring canalswhich connect the oocyte with four neighbouring nurse cells (King, 1970). Atfirst only the cytoplasm of these four nurse cells is poured into the oocyte whilethe more anteriorly located nurse cells are not affected (not shown). Later, how-ever, when the nurse cells degenerate rather rapidly, cytoplasm flows towardsthe central area forming a fast and massive stream which reaches the oocytewithin a minute (Fig. 3). When the nurse-cell cytoplasm passes the gap left bycentripetally migrated follicle cells, it gains the fastest speed and the streamingcan even be observed directly under the microscope. Nurse-cell cytoplasmflowing towards the central cytoplasmic stream is occasionally seen to be pushedback into a nurse cell (Fig. 3, dashed lines). Minutes later when the built-uppressure is apparently equilibrated, the cytoplasm flows out the same way it wasfirst pushed back, and merges with the central stream of cytoplasm.

The flow of cytoplasm through the intercellular bridges connecting the nursecells with each other is indicated by the observed pattern of cytoplasmicstreaming (Fig. 3). However, at this stage the cell membranes start to breakdown (Cummings & King, 1970), presumably a prerequisite for the formationof the large and fast-moving central cytoplasmic stream.

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H. O. GUTZEIT AND R. KOPPA

Fig. 4. Posterior part of the follicle shown in Fig. 3. Symbols and scales as in Figs. 1and 2. The lengths of the arrows mark the distances covered by cytoplasmic particleswithin 38 sec.

During the development of a follicle from stage 10B to stage 11 the volume ofthe nurse cells was found to decrease at a rate of about 13000 /tm3/min.

The diameter of the cytoplasmic stream passing through the four intercellularbridges connecting the nurse cells with the oocyte was difficult to determine instage-1 OB follicles since the depth of focus was not small enough to measure thestreaming through each cytoplasmic bridge separately. As a result, the areasbetween the intercellular bridges, where there is no streaming, cannot bemeasured reliably since immobile regions and fast-moving cytoplasmic streamsmay alternate on different levels of focus. However, at stage 11, when the inter-cellular bridges move closer together, the cytoplasmic stream was found to beabout 12-13 /tm in diameter.

From the above data the speed of the cytoplasm passing through the cyto-plasmic bridges connecting the nurse cells with the oocyte can be predicted tobe about 1-8/mi/sec. When the speed of streaming was measured directly infilm sequences it was found to be 1 -9 ± 0-4 /tm/sec. Figure 3 shows an exampleof 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 isroughly the same it seems likely that the bulk of the nurse cell cytoplasm andnot just large-sized cytoplasmic inclusions is affected by the streaming.

The ooplasm is also in continuous motion. The cytoplasm either flows in acircular fashion (Fig. 4) or in a way described earlier (Fig. 2). In general, thegeometry and speed of ooplasmic streaming does not seem to follow any strictrules. By following the cytoplasmic streaming near the periphery of the growingoocyte in time-lapse films, we calculated that a cytoplasmic particle travellingthe 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 cyto-plasmic streaming from the nurse cells to the oocyte ceases and the ooplasmicmovements slow down. The cortical cytoplasm at the anterior end of the oocyte(facing the degenerating nurse cells) gains in thickness and can measure up to15 fim. It is not known whether the increased cortex width is due to the specificdeposition of nurse-cell products. Nurse cells of this late stage were previouslyshown to synthesize several stage-specific proteins (Gutzeit & Gehring, 1979),but it is not known whether these proteins become incorporated into the growingcortex.

At stage 13 even the cytoplasmic movements within the oocyte come to a haltand no streaming can be detected anywhere in the ooplasm during the finalstage of oogenesis.

DISCUSSION

An inherent problem of in vitro research is the difficulty in assessing the rel-evance for the in vivo situation. However, we feel confident that our observationsreflect by and large the in vivo situation since (1) stage-10 follicles developnormally by morphological criteria in vitro; (2) the developmental time requiredto 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 damagedby the in vitro treatment since they continue developing in vivo and, finally,(4) protein synthesis in stage-10 follicles is quantitatively and qualitatively stablefor at least 60 min in vitro.

Once the follicle cells at the nurse cell/oocyte border have completed theircentripetal migration, the nurse-cell cytoplasm pours into the rapidly growingoocyte where strong cytoplasmic streaming leads to thorough mixing withinminutes. Therefore, the movements do not appear to be involved in the trans-port of molecules to specific sites in the oocyte. During late stage 12 and stage 13the cytoplasmic movements cease, first in the nurse cells and later in the oocyte.The ooplasmic streaming can be observed not only before the nurse cytoplasmpours into the oocyte but also after this process is completed. Therefore, itappears 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 controlledprocesses.

The reported observations have interesting implications with respect to thesite-specific localization of molecules during oogenesis. Because of the rapidooplasmic streaming during stages 10 to 12, this period appears to be ill-suitedfor the specific localization of molecules. Consistent with this notion is thefinding that polar granules appear already in stage-9 oocytes at the posteriorpole of the follicle (Mahowald, 1962). In the wasp Pimpla the oosome is alreadylocalized at the posterior pole at the beginning of vitellogenesis long beforethe nurse-cell cytoplasm streams into the oocyte (Meng, 1968). This also holdstrue for the germ plasm in follicles of the ants Camponotus and Formica (Bier,1952). Prelocalized receptors might, of course, bind specific molecules carriedaround in the stream of cytoplasm and thereby acquire the necessary factorsfor their function. The pole plasm may be a case in point, since transplantationtests show that it becomes competent during late vitellogenesis to induce polecells in young embryos (Illmensee et ah 1976).

In the Drosophila oocyte only those molecules which are localized in theapproximately 5-7 /im wide cortex at stage 11 may be unaffected by the cyto-plasmic streaming.

The polar granules at the posterior pole of late vitellogenic follicles are pre-sumably included in the immobile cortical cytoplasm. In mature eggs ofDrosophila hydei polar granules were found to be located in the peripheral5-10 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 unknownfashion to keep its place.

Niisslein-Volhard (1979) suggested that the anteroposterior and the dorso-ventral coordinates of the embryo are defined by two gradients. If gradients areset up by concentration differences of diffusible molecules (Meinhardt, 1977) itseems reasonable to assume that such gradients can only be established in theabsence of cytoplasmic streaming. These conditions are met prior to stage 10Aand 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 forfinancial support (SFB 46).

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BULL, A. (1966). Bicaudal, a genetic factor which affects the polarity of the embryo inDrosophila melanogaster. J. exp. Zool. 200, 149-156.

CUMMINGS, M. R. & KING, R. C. (1970). Ultrastructural changes in nurse and follicle cellsduring late stages of oogenesis in Drosophila melanogaster. Z. Zellforsch. mikrosk. Anat.110, 1-8.

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ILLMENSEE, K., MAHOWALD, A. P. & LOOMIS, M. R. (1976). The ontogeny of germ plasmduring 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 Drosophilamelanogaster. Wilhelm Roux Arch, devl Biol. 179, 159-162.

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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.

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NUSSLEIN-VOLHARD, C. (1977). Genetic analysis of pattern-formation in the embryo ofDrosophila 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 coordinatesof 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 vitrodevelopment of the Drosophila chorion in a chemically defined organ culture medium.Wilhelm Roux Arch, devl Biol. 186, 351-362.

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{Received 10 June 1981, revised 22 September 1981)