/. Embryol exp. Morph. Vol. 57, pp. 79-93, 1980 79Printed in Great Britain © Company of Biologists Limited 1980

Polar granules and pole cells in the embryoof Calliphora erythrocephala: ultrastructure

and [3H]leucine labelling

By ANDERS LUNDQUIST1 AND HADAR EMANUELSSON1

From the Zoophysiological Institute, University of Lund, Sweden

SUMMARYThe polar granules in Calliphora undergo a gradual fragmentation during early cleavage,

but reaggregate after pole-cell formation. Autoradiographic analysis showed that the polecells in Calliphora acquire a higher [3H]leucine label than the rest of the embryo during theblastoderm stage. Such an increased label was not seen in the pole plasm before pole-cellformation or in the pole cells during gastrulation. Electron microscopic autoradiographyrevealed that the polar granules are substantially labelled during the blastoderm stage. Atthe same time, characteristic nuclear blebs appear in the pole cells. The observations areconsistent with the hypothesis that polar granules contain maternal messenger RNA, whichis released and translated into proteins.

INTRODUCTION

The idea that the egg contains cytoplasmic determinants has long been heldin developmental biology. It was most successfully applied to the determinationof the germ cells experimentally studied in chaetognath.es, amphibians, nema-todes, and insects (reviewed by Beams & Kessel, 1974; Eddy, 1975).

The pole plasm is the hindmost cytoplasmic region in the fly egg. It containsdeterminants required for germ-cell differentiation (reviewed by Mahowald,1977; Agrell & Lundquist, 1973; Counce, 1973). The polar granules are specificorganelles found in the pole plasm, but absent from other parts of the egg.The pole cells bud off from the posterior pole during cleavage after the nucleihave reached the surface of the egg syncytium. They enclose the pole plasm withthe polar granules. The pole cells are the sole progenitors of the adult germ cells.

If the pole plasm of the Drosophila egg is damaged or destroyed, no pole cellsare formed (e.g. Geigy, 1931; Poulson & Waterhouse, 1960; Hathaway &Selman, 1961; Graziosi & Micali, 1974) and if it is transplanted to an incapaci-tated posterior pole or to ectopic loci, potentially functional pole cells are formedthere (Illmensee & Mahowald, 1974, 1976; Okada, Kleinman& Schneiderman,1974; Warn, 1975; Illmensee, Mahowald & Loomis, 1976). Thus, it is well

1 Authors' address: Zoophysiological Institute, University of Lund, Helgonavagen 3B,S-223 62 Lund, Sweden.

6-2

"80 A. LUNDQUIST AND H. EMANUELSSON

established that some factor necessary for pole cell formation resides in the poleplasm.

The polar granules in Drosophila contain RNA (Mahowald, 1962, 1971;Counce, 1963). They begin to lose their RNA after fertilization, and the RNAis no longer cytochemically detectable by the blastoderm stage when the polecells have stopped dividing (Mahowald, 1971). During pole-cell formation, thepolar granules in Drosophila are fragmented and surrounded by polysomes(Counce, 1963; Mahowald, 1968). Therefore, the suggestion that the polargranule RNA is a long-lived maternal messenger RNA, which is translated intogerm-cell-determining proteins (Mahowald, 1968). Light microscopy has shownthat the polar granules are fragmented during pole-cell formation also inCalliphora (Noack, 1901; Alleaume, 1971) and they undergo conspicuous ultra-structural changes in Coelopa (Schwalm, Simpson & Bender, 1971). As the polargranules in higher dipterans seem to be active when the pole cells appear, it wassuggested that they are the factor causing pole-cell formation, but the directevidence is uncertain.

The available general studies on the protein synthesis in the fly egg disagreeabout the pole plasm. In Musca, an increased label was found in the pole plasmbefore pole-cell formation (Pietruschka & Bier, 1972). In Drosophila, labellingincreased only in the early pole cells (Zalokar, 1976). In this study, the aminoacid labelling of Calliphora pole cells and polar granules was examined with~both light and electron microscopic autoradiography. Moreover, some ultra-structural features of polar granules and pole cells in Calliphora are describedfor the first time.

MATERIALS AND METHODS

The culture of the flies {Calliphora erythrocephala Meig.), the collection of the•eggs, the development of the embryos at 20 °C, and the treatment of eggs forelectron microscopy were earlier described in detail (Lundquist & Emanuelsson,1979). The eggs were fixed with 2-5 % glutaraldehyde and 4 % formaldehyde incacodylate buffer, pH 7-2 (Karnovsky, 1965), and postfixed with 1 % OsO4.

Eggs for autoradiography were permeabilized according to a method modifiedafter Limbourg & Zalokar (1973). Dechorionized eggs (Lundquist & Emanuel-sson, 1979) were placed on a piece of moist tissue paper (Kleenex) inside a smallcup of stainless-steel screen. The cup was immersed in n-octane for 15 sec. Theoctane was thoroughly blotted off, and any remaining octane was evaporatedfor 15 sec in a humid air stream. The cup was immersed in incubation medium,.and the eggs were covered with another piece of tissue paper. The cup was thentaken up, partly drained, and incubated suspended in a moist chamber at 20 °Cfor 30 or 60 min. In this way, the eggs and a thin layer of medium were sand-wiched between two layers of tissue paper, thus ensuring adequate oxygen.supply and least mechanical damage to the eggs. Shaw's medium (Shaw, 1956),-which is suitable for Calliphora embryo culture (Davis, Krause & Krause, 1968),

Polar granules in Calliphora 81

Table 1. The condition of permeabilized Calliphora eggsimmediately after incubation in Shaw's medium

Incubationtime (min)

0

30

60

Age of the eggsat fixation (h)

H2131411234

H21314-V

Normal

20191223

54623054

24482442

Number

Possibly*abnormal

3000

12730

6660

of eggs

Clearly!abnormal

0100

7591

121243

Total %

23201223

73744255

42663445

Immediately after incubation, the eggs were fixed with Bradley-Carnoy solution (3 partsabsolute ethanol, 1 part glacial acetic acid, 4 parts chloroform). Whole-mounts were prepared,essentially according to Agrell (1962).

* Delayed development of the nuclear sphere or an unusually large number of vitello-phages.

t Generally abnormalities of the nuclear sphere or the blastoderm.% Some eggs either did not develop or aborted after a few cleavage divisions. These eggs

are probably unfertilized and were not included.

was used. The medium contained 7-4 MBq/ml (= 200/*Ci/ml) L-[4,5-3H]-

leucine (2-1 TBq/mmol ( = 5 7 Ci/mmol); Radiochemical Centre, Amersham).The leucine content of the medium was about 300 times lower than the naturalleucine content in fly eggs (Chen, Hanimann & Briegel, 1967). This is probablyan important reason why a comparatively high radioactivity in the medium anda long incubation are necessary to ensure adequate labelling. On the other hand,the retention of free labelled leucine (Peters & Ashley, 1967) would be reducedbecause of the resulting low specific activity in the endogenous leucine pool andthe prolonged incubation. But under the present conditions, acid-insoluble labelof amino acids in the Drosophila egg is nearly half the total label (Limbourg &Zalokar, 1973; Zalokar, 1976). Therefore, such retention should present noproblem. Eggs of Calliphora incubated for 60 min with [3H]leucine (1-85 MBq/ml) during oxygen shortage did not show autoradiographically-detectable label.

Tables 1 and 2 record the development of the permeabilized eggs. Eggs of allstages develop normally in the medium for 1 h, but eggs permeabilized andincubated during cleavage do not hatch. Possibly, cleavage eggs receive mech-anical injuries, which manifest only at a later stage. The eggs lose some turgorduring permeabilization, and this makes them more sensitive to mechanical

82 A. LUNDQUIST AND H. EMANUELSSON

Table 2. The development of permeabilized Calliphora eggsafter incubation in Shaw's medium

Treatment

Permeabilized,30 min inmedium,paraffin oil

Dechorionized,paraffin oil

Dechorionized

Age of theeggs afterincubationin medium

(h)

2±33±3i3±4

——————

Hatchedlarvae

0192861642069617488

32487

359

Un-hatchedlarvae*

023528

13361

321

Number of eggs

Seg-mented

embryosf

213643151944121031

2434

A

Pig-mented

embryosj

5518

732

113752

1112

Eggswithout

pigmenta-tion§

2525191613173

2012

8

387

29

Total

101100100100100100100101100100

400100395

Permeabilized eggs were incubated in Shaw's medium for 30 min and transferred to paraffin oil in awater-saturated oxygen atmosphere. The results were scored a few hours after the end of the normalhatching period. The values were similar when the eggs were incubated in medium for 60 min except fora decrease in the number of 'hatched larvae' and a corresponding increase in the number of 'segmentedembryos'. The unpermeabilized control eggs were only decborionized. They were transferred to paraffinoil during the blastoderm stage and treated as above or allowed to develop on moist filter paper in air.The hatching percentage was always higher after the latter treatment.

* Otherwise apparently normal.t Usually with minor segmental defects or abnormal mouth hooks.% Usually with grossly abnormal segmentation or no visible segments. The cuticular pigmentation

appears shortly before hatching.§ Unfertilized eggs included.

FIGURE 1

Light-microscopic autoradiographs of pole plasm and pole cells in Calliphora eggslabelled with [3H]leucine for 30 min. (A) Posterior pole with pole plasm duringintravitelline cleavage (60 min). (B) Pole-cell buds (120 min). (C) Pole cells duringthe cellularization of the blastoderm (180 min). (D) Pole cell with polar granules(arrows) in the yolk region near the posterior midgut invagination. This is phase IIof pole-cell migration. Early germ-band-elongation stage (270 min). PC, Pole cell;PMI, posterior midgut invagination; Y, yolk. Notice the heavy label of the polecells in the blastoderm. See also Table 3. Section thickness: 1 //,m. Exposure time:27 days.

Polar granules in Calliphora 83

84 A. LUNDQUIST AND H. EMANUELSSON

damage before they have acquired a firmer structure by the cellularization ofthe blastoderm. The Calliphora egg should be more susceptible to such damagethan the smaller Drosophila egg.

After incubation, eggs for autoradiography were fixed for 2 h at roomtemperature with Karnovsky fixative (six changes). The vitelline membrane wasremoved in the fixative after 1-1-5 h. The embryos were washed with caco-dylate buffer at + 4 °C overnight (at least five changes), postfixed with 1 %OsO4 in the same buffer, bulk-stained with 0-5 % uranyl acetate and 1 % phos-photungstic acid, and embedded in Vestopal W.

Thin sections for light microscopic autoradiography (1 pm) were covered withliquid nuclear emulsion (Ilford K2) according to the dipping method and ex-posed for 6-27 days. The auto radiograms were developed in Kodak D 19(5 min, 20 °C), rinsed in distilled water (10 sec), fixed in Kodak F24 (6 min,20 °C), and stained with Richardson's azure II.

Ultrathin sections for electron microscopic autoradiography were vacuum-coated with carbon, covered with a monolayer of nuclear emulsion (Ilford L4)according to the loop method, and exposed for 4-24 weeks. The preparationswere developed in Kodak D 19 (2 min, 20 °C), rinsed in distilled water (30 sec),fixed in 15% Na2S2O3 (3 min, 20 °C), and finally washed in distilled water(2 min). Examination was made with a Philips EM 300 electron microscope atthe Zoological Institute, University of Lund.

RESULTS

At 20 °C the Calliphora egg has completed nine intravitelline synchronousnuclear divisions 90 min after egg-laying, when the dividing nuclei reach theperiphery of the egg. The remaining four nuclear divisions are partially syn-chronous and occur in the syncytial blastoderm (Lundquist & Emanuelsson,1979). The pole cells bud off during the second or third blastodermal division(Fig. IB). They proliferate at the posterior end during the ensuing cellulariza-tion of the blastoderm (from 150 min; Fig. 1C) and most of them are broughtinto the egg interior with the posterior midgut invagination during gastrulation(between 240 and 270 min; Fig. ID). Pole cells migrate into the yolk region in

FIGURES 2 AND 3

Electron microscopic autoradiographs of polar granules in Calliphora eggs labelledwith [3H]leucine for 30 min. The stages are: (2A) Intravitelline cleavage (60 min).(2B) Pole-cell formation (120 min). (3 A) Cellularization of the blastoderm (180 min).(3B) Early germ-band elongation (270 min). dMVB, Dark multivesicular body;ER, endoplasmic reticulum; N, nucleus; M, mitochondrion; PG, polar granule.Notice that the polar granules are fragmented during pole-cell formation andheavily labelled during the cellularization of the blastoderm. Exposure time: 20weeks (2A), 9 weeks (2B, 3 A), and 24 weeks (3B).

Polar granules in Calliphora 85

FIGURE 2

86 A. LUNDQUIST AND H. EMANUELSSON

FlGURE 3

Polar granules in Calliphora 87

Table 3. Autoradiographic [sH]leucine labelling of the pole plasmand the pole cells in Calliphora eggs

Egg no.Age of theeggs (min)* Stage 1

60 Pole plasm during early 0 0 0 0cleavage

120 Pole-cell buds 0 0 0 -150 Pole cells in the +° 0 0 -

syncytial blastoderm180 Pole cells during the cellular- + + + + + + ° + +

ization of the blastoderm210 Pole cells during the cellular- + + ++° ++° + + °

ization of the blastoderm240 Pole cells at the onset of 0 +° +° + °

gastrulationf270 Pole cells during the early 0 0 - -

germ-band-elongation stageThe grain density over the pole cells was compared with that of the adjacent blastoderm

cells: 0, similar grain density; +, higher grain density; + + , much higher grain density;°, some pole cells (*S 50%) with no increased grain density.

* The eggs were labelled for 30 min in medium containing [3H]leucine (74 MBq/ml =200/tCi/ml) and fixed at (he indicated age.

t The eggs were often weakly labelled at this stage.

two phases, before and after gastrulation (reviewed by Counce, 1973; forCalliphora, see Alleaume, 1971).

The polar granules in Calliphora consist of a network of dense material.During early cleavage, most of them are irregular in shape and relatively large(up to 1 /<m), sometimes with annular profiles (Fig. 2A). Sometimes, they arechained together. Subsequently, they become smaller. During pole-cell forma-tion, small profiles (less than c. 0-5 /im) are gathered in a perinuclear zone(Fig. 2B). Towards the end of the nuclear cleavage period, large granulesreappear. Later, very large circular or annular profiles become predominant(up to 2/MTO, although small profiles are still seen at gastrulation, especiallynear the nucleus (Fig. 3).

Pieces of endoplasmic reticulum are often very close to the polar granules ofthe pole cells (Figs 2, 3). Extranuclear annulate lamellae (Lundquist & Emanuel-sson, 1979) are present in the pole-cells (Fig. 4B), but almost absent from theblastoderm. Dark multivesicular bodies were regularly found in the pole plasmand the pole cells (Fig. 2B).

Characteristic blebs are seen on the nuclear envelope in the pole cells of theblastoderm (Fig. 4). Such a bleb consists of an out-pocketing of the outernuclear envelope membrane. It contains a circular membrane-enclosed profilefilled with electron-dense material and is often associated with a nuclear pore.The blebs are first seen in the early pole cells, where they are very frequent

88 A. LUNDQUIST AND H. EMANUELSSON

during the cellularization of the blastoderm. They are only occasionally observedat the late blastoderm stage and in the early gastrula. They are never seen outsidethe pole cells.

The light-microscopic autoradiographs display an even distribution of silvergrains over the pole plasm and the pole cells after [3H]leucine labelling. Thisapplies to all stages. Both the nucleus and the cytoplasm are labelled. The graindensity over the pole plasm or the pole cells compared with the adjacent part ofthe egg was judged semiquantitatively (Table 3; Fig. 1). No difference wasobserved before pole-cell formation, but thereafter, the pole cells acquire a moreintense label. This difference disappears at the late blastoderm stage and in theearly gastrula. The highest grain density recorded in the egg was that over thepole cells during the early cellularization of the blastoderm.

For electron-microscopic autoradiography, a 30 min pulse with [3H]leucinewas generally used (Figs 2, 3). Both 30 min and 60 min pulses were made duringthe cellularization of the blastoderm, but the grain distributions were notdetectably different. Silver grains over the polar granules are present at allstages, but the polar-granule label follows the general pole-cell label. There isan approximately even distribution of label between the polar granules andother parts of the cytoplasm and between cytoplasm and nuclei. The polargranules are thus heavily labelled during the cellularization of the blastoderm.The silver grains are often seen over the periphery of the granules. The labelof the pole cell nuclei is often found close to the nuclear envelope, and silvergrains are sometimes seen near nuclear blebs.

DISCUSSION

In the CalHphora egg the time course of fragmentation and reaggregation ofthe polar granules parallels the rise and fall in amino-acid labelling of the polecells. The peak in polar-granule fragmentation during pole-cell formation pre-cedes the peak in pole-cell labelling by about one hour. That applies also toDrosophila, except that no clear-cut decrease in pole-cell label compared to othercells was detected in ovo (Mahowald, 1968; Zalokar, 1976). The results of Allis,Underwood, Caulton & Mahowald (1979) do not contradict the interpretationthat at least a slight decrease occurs in cultured Drosophila pole cells. BecauseRNA synthesis in the pole cells is very low or absent in both species (Zalokar,1976; Lamb & Laird, 1976; Lundquist & Emanuelsson, preliminary results),

FIGURE 4

Nuclear blebs in the pole cells of CalHphora eggs during the cellularization of theblastoderm (180 min). Each bleb consists of an evagination of the outer nuclearmembrane and contains a membranous vesicle. AL, Annulate lamella; B, bleb; N,nucleus; NE, nuclear envelope; M, mitochondrion; PG, polar granule. (A)Blebbingin an egg labelled with [3H]leucine. Exposure time: 9 weeks. (B) Bleb in an unlabelledegg. The black dot is an artifact.

Polar granules in Calliphora 89

90 A. LUNDQUIST AND H. EMANUELSSON

the amino-acid label should be attributed to translation of stored messengerRNA. In Calliphora, the polar granules are substantially labelled with [3H]-leucine, especially peripherally, when the label of the pole cells is high. In thisrespect, by showing a label comparable to the ground plasm, polar granulesdiffer from the somatic inclusions: the yolk region shows less label than theperiplasm in Calliphora (unpublished) and Drosophila (Zalokar, 1976). Thecorrelation between polar-granule fragmentation and amino-acid labelling iscompatible with the idea (Mahowald, 1968, 1977) that stored mRNA is releasedfrom the fragmented polar granules and later translated into proteins.

The changes in amino-acid label are interpreted as local changes in proteinsynthetic activity. Alternatively, there is an exceedingly large, transient changein the leucine pool size or the permeability of the pole cells only. This is notlikely. Moreover, pole cells acquire increased label only some time after theirformation. As pointed out by Zalokar (1976), this indicates that the newly formedpole cells continue to share their amino-acid pools with the blastoderm. Sometranslocation of label to the polar granules may occur. But since the totalamount of polar-granule material apparently does not increase before gastrula-tion (Rabinowitz, 1941; Counce, 1963), such translocation is unlikely to be ofmajor importance.

The vesicle-containing nuclear blebs of the pole cells have not previously beenrecorded. Interestingly, the blebbing coincides with the increased amino-acidlabelling in the pole cells and silver grains were sometimes seen near the blebs.But more evidence is difficult to obtain, as it is not yet possible to shorten thelabelling pulse.

The polar granules are thought to be responsible for pole-cell formation.When the polar granules in the Drosophila egg are removed from the pole plasmby centrifugation, no pole cells are formed at the posterior pole. However, thedislodged polar granules do not give rise to pole cells in other parts of the egg(Imaizumi, 1958; Jazdowska-Zagrodziriska, 1966). Obviously, direct evidence is,as yet, lacking.

In some lower dipterans, certain chromosomes are eliminated from all somaticnuclei during cleavage, whereas the full chromosome set is retained in the polecells, i.e. in the germ line. In gall midges, the polar granule material seems toprotect the pole cells from chromosome elimination, but it is not needed forpole cells to form (Geyer-Duszyiiska, 1959; Nicklas, 1959; Bantock, 1970).Chromosome elimination is not known in higher dipterans, but somatic loss ofchromatin does occur. In Calliphora and Drosophila, a specific terminal chromo-some fragment is lost in the early embryo, presumably from all somatic nuclei.In Calliphora, such fragments appear during the last three nuclear divisions ofthe syncytial blastoderm and in the gastrula (Melander, 1963). Pseudochiasmatasuch as those believed to precede this chromosome diminution were previouslyobserved in Drosophila. They probably affect the X chromosome (Rabinowitz,1941). As a second working hypothesis, we tentatively suggest that the polar

Polar granules in Calliphora 91granules prevent chromosome diminution in the pole cells of Calliphora andDrosophila. Chromosome diminution occurs after polar-granule fragmentationand coincides with the increased [3H]leucine label and the nuclear blebbing inthe pole cells. Moreover, a unified hypothesis explaining the function of thepolar granules in all dipterans should be appealing. The hypothesis is testable,because it offers cytological markers. The tools needed to determine the function(or functions) of the polar granules might soon become available. As yet, cellfractions enriched in polar granules have been isolated (Allis, Waring &Mahowald, 1977; Waring, Allis & Mahowald 1978) and culture methods for polecells have recently been designed (Allis, Underwood, Caulton & Mahowald, 1979).

Grants from the Magnus Bergvall Foundation and the Swedish Natural Science ResearchCouncil supported this work. We express our gratitude to Mrs Annagreta Petersen for in-valuable technical aid, to Miss Inger Norling for printing the electron micrographs, and toMrs Marianne Andersson for typing the manuscript.

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Polar granules in Calliphora 93WARING, G. L., ALLIS, C. D. & MAHOWALD, A. P. (1978). Isolation of polar granules and

the identification of polar granule-specific protein. Devi Biol. 66, 197-206.WARN, R. (1975). Restoration of the capacity to form pole cells in UV-irradiated Drosophila

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(Received 6 November 1979, revised 16 January 1980)

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