Development 101. .W43 (19S7)Primed in Grc.il Bntain © The Company of Biologists Limited 19S7

33

Distribution of yolk polypeptides in the follicle cells during the

differentiation of the follicular epithelium in Sarcophaga bullata egg

follicles

JOHAN GEYSEN. JOHAN CARDOEN and ARNOLD DE LOOF

Zoological Institute, NMimsestruiit 59. B-3000 Leuven, Belgium

Summary

In S. bullata, the ovaries contribute to the synthesis ofyolk polypeptides. A specific antiserum for yolk poly-peptides was used to visualize the presence of yolkpolypeptides in the follicle cells during their differen-tiation. After vitellogenesis has started, all folliclecells contain yolk polypeptides. The squamous folliclecells covering the nurse cells and the border cells loseyolk polypeptides before mid-vitellogenesis, whereasthe follicle cells over the oocyte contain yolk polypep-tides until after late vitellogenesis. All follicle cells areimmunonegative afterwards. In vitro translation ofpoly(A)+ RNA demonstrated that the presence of yolkpolypeptide mRNA correlates well with follicle cellimmunopositivity for yolk polypeptides. This suggests

that the follicle cells synthesize the ovarian yolkpolypeptides. Differences in cellular and nuclear mor-phology, total and poly(A)+ RNA synthesis and therate of yolk polypeptide synthesis were shown to becorrelated with the presence or absence of yolk poly-peptides in the differentiating follicular epithelium.The possible relationship between these different as-pects of follicle cell differentiation, follicle cell poly-ploidy and the extracellular current pattern aroundfollicles are discussed.

Key words: differentiation, immunocytochemistry, yolkpolypeptides, Sarcophaga bullata, Diptera, ovary, folliclecells.

Introduction

Meroistic polytrophic egg follicles of Sarcophagabullata consist of 16 sibling daughter cells, surroundedby a follicular epithelium of mesodermal origin. Fourfollicle cell types with distinct morphology and distri-bution are distinguished: squamous, columnar, bor-der and polar cells (Geysen & De Loof, 1983).Although polarized morphological differentiation ofthe follicular epithelium is a common feature ofpolytrophic follicles (King & Aggerwal, 1965; Cum-mings & King, 1969; Chia & Morrison, 1972; Khurad& Thakabe, 1980), the idea that they probably dealwith different functions has not been elaborated untilrecently. It was suggested that the follicle cellscovering the nurse cells are involved in generatingextracellular current patterns (Woodruff, Huebner &Telfer, 1986), whilst, in some species, those overlyingthe oocyte may contribute to the accumulation ofyolk precursors in the intercellular space (Kelly &Telfer, 1979; Telfer, Huebner & Smith, 1982) and/orthe formation of different egg envelopes. Brennan,

Weiner, Goralski & Mahowald (1982) demonstratedwith in situ hybridization that the follicle cells ofDrosophila are a major ovarian site of yolk proteinsynthesis. The temporal evolution of ovarian yolkpolypeptide synthesis in Drosophila has been studiedby Isaac & Bownes (1982), but no information wasprovided about the spatial pattern of this synthesis.In this paper, we focus on the distribution of yolkpolypeptides in the follicle cells during their differen-tiation. In addition to this, we present evidence for acorrelation between general morphology, the degreeof polyploidy, the rate of messenger RNA synthesisand the presence (or absence) of yolk proteins in thefollicle cells.

Materials and methods

Morphology and stage determinationS. bullata was reared in crowded conditions as described byHuybrechts & De Loof (1977). Ovaries were dissected in abalanced Ringer solution (Chan & Gehring. 1971) andimmersed in Bouin-Hollandes fixative supplemented with

34 / . Gey sen, J. Cardoen and A. De Loof

10 % of a saturated aqueous mercury chloride solution for24h, rinsed in tap water for 24h, dehydrated throughstandard series of graded ethanols, ethanol-xylol and xylol.Finally, ovaries were embedded in Paraplast and sectionedat 5 (im. Vitellogenic stages were classified according to therelative ratio of oocyte length over follicle length (modifiedclassification of Pappas & Fraenkel, 1977) and by means ofcytological features. Vitellogenic stages 4A, 4B and 4C willbe referred to as early, mid and late vitellogenesis.

ElectrophoresisRinger and haemolymph were drained from dissectedovaries, which were then pooled according to vitellogenicstage in Eppendorf tubes and weighed. Haemolymph wascollected from a wound near the mesothoracic leg inmicrocapillaries, which were blown out on an aluminiumblock cooled to liquid nitrogen temperature. This methodallows collection of haemolymph without antioxidant addi-tives. 9jtl of SDS sample buffer were added per mg ofsample (wet weight for ovaries). Samples were simul-taneously sonicated and heated to boiling temperature in aSoniprep 150 sonicator. Solid materials were sedimented inan Eppendorf centrifuge and supernatants stored at -20°C.SDS-PAGE was performed using 5-15 % gradient gelswith the Laemmli (1970) buffer system. Gels were stainedby means of Coomassie Brilliant Blue R250.

Antiserum preparationYolk polypeptides from an equivalent of 5 mg of egghomogenate (Huybrechts & De Loof, 1982), purified bymeans of SDS-PAGE, were injected into rabbits. Threesimilar booster injections were given biweekly, startingthree weeks after the first injection. Animals were ter-minally bled from the dorsal aorta one week after the fourthinjection.

Immunoblotting, 'Aurodye' staining andimmunocytochemistryProtein bands were transferred from SDS gels to nitro-cellulose by means of isotachophoresis (Kyhse-Andersen,1985) using a self-made semidry blotting apparatus andvisualized with Aurodye staining (Janssen Life Scienceproducts, Beerse, Belgium) (Moeremans, Daneels, deRaeymaeker & De Mey, 1987) or Amido Black 10B. Yolkproteins were visualized by means of peroxidase anti-peroxidase (PAP) immunostaining for blotted antigens(Geysen, Vandesande & De Loof, 1984; Geysen, 1987) andfor tissue section antigens (Vandesande, 1983; Verhaert,Geysen, De Loof & Vandesande, 1984). For both tech-niques, the optimal dilution of anti-yolk protein antiserumproved to be 1/10000.

Solid-phase immunoadsorption and antiserumspecificityHaemolymph samples of 5-day-old females and males werelinked to cyanogen-bromide-activated Sepharose (Pharma-cia) as specified by the manufacturers. 2 ml of antiserumdiluted 1/10000 was incubated three times for 2h with 5fi\equivalents of either male or female immobilized haemo-lymph. Immunoadsorbed sera were then applied to tissuesections in the PAP technique. Immunostaining could be

abolished completely with female haemolymph whereasserum treated with male haemolymph reacted as the nativeantiserum (Fig. 1A,B,C). Because yolk proteins arefemale-specific, this assay indirectly proves serum speci-ficity. Immunoblotting revealed that in ovarian prep-arations no protein other than yolk proteins are recognizedby the antiserum (Fig. ID).

Poly(A)+ RNA extractionPrevitellogenic, vitellogenic and maturing ovaries weredissected and frozen immediately in liquid nitrogen. TotalRNA was extracted as described by Cardoen, Huybrechts& De Loof (1986a). Poly(A)+ RNA fractions were isolatedby oligo(dT) chromatography (Aviv & Leder, 1972), 2-7 %to 3-0 % of total RNA proved to be poly(A)+ RNA. Afterprecipitation with two volumes of ethanol and 0-3 M-sodiumacetate, fractions were stored at —20°C.

Cell-free translation, fluorography andimmunoprecipitationThe poly(A)+ RNA was washed twice with 70% ethanoland finally dissolved in sterile deionized water at ljUg^l"1.3^g of poly(A)+ RNA was translated in 40^1 of rabbitreticulocyte lysate mixture (Amersham, N90) in the pres-ence of 60jiCi [35S]methionine (Amersham) for 1 h at 30°C.Subsequently, the samples were counted and the trans-lation products were analysed by SDS-PAGE using8-12% gradient gels (Laemmli, 1970). AboutlOOOOOctsmin"1 per lane were applied. After fixation, thegel was treated with 1 M-sodium salicylate according toChamberlain (1979), dried and finally exposed to a KodakX-omat film at —70°C. For immunoprecipitation, 5/A ofreticulocyte lysate was incubated with 5(i\ of undilutedantiserum for 30 min at 37CC and overnight at 4°C. Second-ary antibody was added and a similar incubation schedulefollowed. Immunoprecipitates were then sedimented usingan Eppendorf centrifuge, washed and resuspended in Tris-buffered saline and precipitated (five cycles).

Total RNA synthesisFor monitoring RNA synthesis and transport, females wereinjected with 6/iCi [5,63H]uridine (Amersham, 45Cimmor1). Ovaries were dissected in Carnoy fixative, dehy-drated, embedded in Paraplast and sectioned at 4j/m.Control flies were injected with 6y\ of a 0-1 M solution ofcold undine (Serva); the specificity of the [3H]uridineincorporation was checked by including a RNAse digestionbefore application of the stripping film (Ray & Ramamurty,1979).

In situ hybridization with fH]poly(V)In order to investigate the distribution of poly(A)+ RNAovarian tissue sections were hybridized using a [3H]poly(U)probe (Amersham, 41-147 residues; 20-72 CimmoP1) as ahomopolymer probe. Hybridization and autoradiographywere performed as described by Cardoen et al. (19866).

Yolk polypeptides in Sarcophaga follicle cells 35

1A

II

Fig. 1. Specificity of the antibody in PAP immunostaining using immunoadsorption (A,BiQ and immunoblotting (D).Mid-vitellogenic ovary stained with untreated antiserum (A), after immunoadsorption with immobilized male (B) andfemale (C) haemolymph. Immunostaining is abolished after adsorption with female haemolymph only.(D) Immunoblotting. Female and male haemolymph in lanes 1 and 2, respectively; ovarium homogenate in lane 3.Lanes 4 and 5: strips of a gel overloaded with sample to check for minor contaminants to ovarial products. Aurodye-stained strip (4) PAP-immunostained strip (5). The antibody is specific for the yolk polypeptides.

36 J. Gey sen, J. Cardoen and A. De Loof

Results and discussion

Previtellogenic stagesIn all previtellogenic stages, the follicle cells areimmunonegative for yolk polypeptides. At the onsetof vitellogenesis, the fat body, haemolymph andperipheral ooplasm are immunopositive, due to thesynthesis, transport and endocytotic uptake of ex-ogenous yolk, but immunostaining is absent in folliclecells (Fig. 2A-C). At the anterior pole, the pro-liferation of the border cell complex indicates thefirst morphological differentiation of follicle cells(Fig. 2B).

The genes coding for yolk polypeptides in the fatbody and the follicle cells seem to respond to thesame hormonal environment in a different way. Thetime lag may either be due to different susceptibilitiesof the two tissues or to the action of different triggersas has been suggested for Drosophila by Isaac &Bownes (1982). Regulation of follicle cell contri-bution to vitellogenesis seems to be a complexmechanism. An explanation for tissue specificity ofhormonal action has been described previously (DeLoof, 1986a).

Vitellogenic stagesIn early vitellogenic egg follicles, the follicular epi-thelium differentiates into distinct zones (Fig. 2D);(1) a border cell complex at the anterior end of thefollicle, (2) an adjacent squamous epithelium, (3) atransition zone with follicle cells of intermediatecytological appearance (from squamous to columnar)and (4) columnar cells overlying the oocyte surface,which are highest at the nurse cell-oocyte border,and somewhat more cuboidal near the posterior end(Fig. 2D,E). At this stage, all follicle cells areimmunopositive for yolk polypeptides, but thestained material is not equally distributed. The cyto-plasm of both the border cells and the squamousepithelium reacts weakly, which contrasts with thecomplete absence of staining in the nurse cells.Posteriorwards from the transition zone, the immu-nopositivity increases to a maximum at the nursecell-oocyte border and in the epithelium covering theoocyte. Basally and apically from the follicle cellnuclei, very dense deposits of immunostaining occur,indicating that yolk polypeptides are more concen-trated at certain sites in the follicle cell cytoplasm(Fig. 2E). These deposits most probably correspondto large stacks of rough endoplasmic reticulum(RER) located basally and to RER or the Golgisystem present in the apical cytoplasm of these folliclecells (Geysen, Cardoen, Huybrechts & De Loof,1986).

The transition zone is remarkable because itimplies that different types of follicle cells do not

necessarily originate from predetermined groups offollicle cells. Regulative mechanisms may determinethe differentiational pathway of a follicle cell up tomid-vitellogenesis. The pairs of polar cells at eachpole of the follicle (Geysen & De Loof, 1983) mayplay a role as organizing centres.

At mid-vitellogenesis (Fig. 3B-D; Fig. 3A showsan intermediary stage), the nurse cells are covered bysquamous follicle cells, whereas the columnar epi-thelium overlies the oocyte chamber and occupies theoocyte-nurse cell border. The border cells reach aposition between oocyte and nurse chamber. Thetransition zone is no longer present, but a sharpdelineation between the two types occurs at the nursecell-oocyte border. This differentiation is also re-flected in the immunostaining pattern: only the col-umnar follicular epithelium is immunopositivewhereas the squamous follicle cells and the bordercells have lost immunoreactive material and contrastsignificantly with the immunopositive haemolymphand the ooplasm respectively (Fig. 3B,C,D). Appar-ently, these follicle cells diminish or stop synthesizingyolk polypeptides rapidly in the course of differen-tiation. We have indications that the squamous fol-licle cells contribute to cytoplasmic transport from thenurse cells to the oocyte (for review see Gutzeit.1986) as they contain considerable amounts of micro-filaments (Geysen & De Loof, 1986).

From mid to late vitellogenesis, the immunoposi-tive follicle cells spread and change from columnar tocuboidal and finally squamoid shape. Immunoposi-tive granules in the cytoplasm predominantly appearlaterally from the oocyte. The immunonegative vitel-line membrane separates oocyte and follicle cells(Fig. 4B,C). At the oocyte-nurse cell border, thefollicle cells move centripetally and incompletelyseparate both compartments. The immunopositivefollicle cells and the squamous epithelium performthis centripetal movement in different ways: cellbodies and nuclei of the latter can be observed incentripetal position, whereas from the immunoposi-tive follicle cells only a thin layer of cytoplasm ispresent centripetally, but no cell bodies or nuclei(Fig. 4A). After the squamous cells have moved indeeply centripetally, yolk-polypeptide-positive cellbodies and nuclei can be observed in a more centri-petal position.

At late vitellogenesis, the intensely stained gran-ules have disappeared from the follicle cells coveringthe oocyte surface, but a weak cytoplasmic immuno-staining is still present.

Postvitellogenic stagesThe follicle cells become completely immunonegativefor yolk polypeptides only at the end of nurse cell

Yolk polypeptides in Sarcophaga follicle cells 37

-•"4

' .if1'

Fig. 2. PAP immunostaining for yolk protein; Normarski differential interference contrast. (A) First uptake ofexogenous yolk in the cortical ooplasm (white asterisks); all follicle cells are negative, haemolymph positive (blackasterisk). (B) Detail of border cell proliferation. (C) Follicle cells covering the oocyte. (D) Early vitellogenesis (stage4A); ooplasm and all follicle cells positive for yolk proteins. (E) Detail of the transition zone from squamous tocolumnar follicle cells. More squamous cells are poorly labelled (top), columnar cells display an immunopositivegranulation apically and basally from the nuclei (bottom). Bars in all photographs, 20jim.

31 J. Gey sen, J. Cardoen and A. De Loof

3A

Fig. 3. PAP immunostaining for yolk protein; Nomarski differential interference contrast. (A) Intermediate stagebetween 4A and 4B. Start of border cell migration (arrowhead), the transition zone shifts towards the oocyte-nurse cellborder; border cells and squamous follicle cells lose yolk protein immunoreactivity. (B) Mid-vitellogenesis (stage 4B).border cells (arrowhead) reach the oocyte and squamous follicle cells cover the nurse cell chamber. The transition zonehas turned into a sharp delineation between positive and negative cells (double arrowhead). (C) Detail of twoimmunonegative squamous follicle cells (arrowheads), contrasting with immunopositive haemolymph (asterisk).(D) Detail of oocyte-nurse cell border. Immunopositive follicle cells display granulation apically and basally from thenuclei; squamous cells (arrowhead).

Yolk polypeptides in Sarcophaga follicle cells 39

Fig. 4. PAP immunostaining for yolk proteins; Nomarski differential interference except B and E: transillumination.(A) Stage 4C. Centripetal movement. Squamous cells (arrowheads) migrate first whereas positive follicle cells seem tosend cytoplasmic projections along (double arrowhead); haemolymph (asterisk) stains weakly. (B,C) Positive folliclecells spread over the growing oocyte surface. Positive granules now are mainly located laterally from the nuclei. Theimmunonegative vitelline membrane (arrowhead) separates follicle cells and oolemma. (D) Mature follicle (stage M),nurse cell degeneration is completed. All follicle cells are negative, border cells proliferate (arrowhead); egg shells aredeposited (double arrowhead). (E,F) The stretched follicle cells are fully immunonegative (E) but still display clearnuclear morphology (F). Arrowheads, nucleoli.

40 J. Gey sen, J. Cardoen and A. De Loof

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Yolk polypeptides in Sarcophaga follicle cells 41

degeneration. However, differential interference mi-croscopy reveals that the follicle cells covering theoocyte surface have not yet disintegrated (Fig. 4E.F).They probably make a functional switch from yolkpolypeptide to chorion protein synthesis. Morpho-logically, the border cells do not change significantlythroughout vitellogenesis. although transcriptionallythey are as active as the follicle cells covering theoocyte (Cardoen et al. 19866). During and after nursecells degeneration they proliferate and form themicropyle. Also, the proliferation of the squamousepithelium continues during and after nurse cellbreakdown (Fig. 4D).

Nuclear morphology and transcriptional activity of thefollicle cells

The nuclei of the follicle cells adjacent to the oocyteare round while those of the follicle cells surroundingthe nurse cells are flattened (Fig. 5A). This morpho-logical differentiation entails differences in DNAcontent (Cardoen et al. 19866). Time-dependentlabelling experiments with [3H]uridine (Fig. 5B)suggests that the columnar follicle cells are moreactively involved in RNA synthesis than thesquamous ones. In situ hybridization with[3H]poly(U) suggests an intensive transcriptional

Fig. 5. Nuclear morphology, polyploidy andtranscriptional activity. (A) Feulgen staining of stage-4Bvitellogenic follicles; sections were hydrolysed in 4N-HC1for 50min prior to staining (Cardoen et al. 19866).Nuclear morphology (and polyploidy) of squamousfollicle cells (arrowheads) differs from that of thecolumnar follicle cells and the border cells (doublearrowhead). (B) In vivo labelling with [3H]uridine for 1 h.The columnar follicle cells (double arrowhead)incorporate more label than the squamous in nucleus andcytoplasm. There is no evidence for mRNA transportfrom follicle cells into the oocyte (C). In situhybridization with labelled poly(U)+ RNA. mRNAsynthesis is intense in the follicle cells overlaying theoocyte (double arrowhead) and low in squamous folliclecells (single arrowheads). The karyosome also displaysweak activity (triple arrowhead). (D) Fluorography of invitro translated mRNA fractions from: lane 1.previtellogenic ovaries; lane 2, vitellogenic ovaries;lane 3, fat body from vitellogenic females; lane 4, matureovaries; lane 5, female haemolymph, labelled in vivo byinjection of tritiated amino acids. Due to processing ofyolk proteins by the fat body, the yolk proteins fromhaemolymph (lane 5) have a smaller molecular weightthan unprocessed yolk polypeptides.(E) Immunoprecipitation with anti-yolk protein antiserumof translated mRNA from: lane 1, mature ovaries; lane 2,previtellogenic ovaries; lane 3, fat body of vitellogenicfemales; lane 4. vitellogenic ovaries. Labelled yolkproteins appear in. and were only immunoprecipitatedfrom, vitellogenic ovaries and fat body (weak reaction) ofvitellogenic females.

activity in the follicle cells covering the oocyte and inthe border cells (Fig. 5C) (see also Cardoen et al.19866). Although these differences may also be dueto different RNA-turnover rates, preservation ofRNA in the section or accessibility to the probe, theseeffects reflect aspects of the divergent differen-tiational pathways of the two follicle cell types.

Fat body and ovaries of vitellogenic females, butnot pre- and postvitellogenic ovaries, contain yolkpolypeptide mRNA (Fig. 5D). In fat body extracts ofvitellogenic females, yolk polypeptides are the pre-dominant transcripts, whereas in vitellogenic ovariesother messages are also transcribed. Postvitellogenicovaries display a different transcriptional pattern. Weobserve small differences in molecular weight be-tween the translated yolk polypeptides (lanes 2, 3)and the yolk polypeptides occurring in the haemo-lymph (lane 5). This difference is due to cleavage of asignal peptide (Cardoen et al. 1986a).

Synthesis of yolk polypeptides by the follicle cellsThe follicle cells of S. bullata contain and perhapssynthesize the ovarian yolk proteins (Huybrechts.Cardoen & De Loof, 1983). The correlation betweenimmunopositivity of the follicle cells and the presenceof yolk polypeptide mRNA strongly suggests that thefollicle cells are involved in yolk polypeptide syn-thesis during vitellogenesis. It is very unlikely that theyolk polypeptides would be synthesized in otherovarian cell types. The karyosome of the germinalvesicle exhibits only very weak transcriptional activity(Cardoen et al. 19866), the nurse cells do not displayimmunoreactivity and transport of RNA from thenurse cells does not occur until late vitellogenesis. Wenever obtained evidence indicating RNA transferfrom the follicle cells to the oocyte.

It is more likely that the RNA synthesis in thefollicle cells sustains translational activity. Yolk poly-peptide messengers have a high turnover rate and aconcomitant short life time, which would make trans-port inefficient and unlikely (Cardoen, Huybrechts,Theunis & De Loof, 1984; Cardoen et al. 1986a). Thepresence of a signal peptide is compatible with thefact that the yolk proteins are synthesized in cellspossessing a well-developed RER and Golgi com-plex. Compared to the fat body of vitellogenic fe-males, however, yolk polypeptide mRNA constituteonly a minor part of the ovarian transcripts. The factthat the vitelline membrane is formed while thecylindrical follicle cells are immunopositive points tothe complex pattern of functions these follicle cellsperform.

42 J. Gey sen, J. Cardoen and A. De Loof

Polar follicle cell differentiation and extracellularcurrent patterns

Superimposed on polarized morphological differen-tiation, we observed four other features in Sarco-phaga follicles: (1) the distinct nuclear morphology,(2) the different degrees of polyploidy (squamouscells 4C; columnar cells 8 to 16C; Cardoen et al.1986b), (3) the different levels of transcriptionalactivity and (4) restriction of yolk polypeptide syn-thesis to the cuboidal follicle cells overlying theoocyte.

The extracellular current patterns around poly-trophic follicles can be interpreted as a fifth elementin polar differentiation. They do not complement theintracellular potential difference between oocyte andnurse cells but follow follicle cell differentiation inSarcophaga (Verachtert & De Loof, 1986). Thiscritically suggests that the follicle cells behave asan electrically independent system from the nursecell-oocyte syncytium in the course of their differen-tiation, the fact that yolk proteins are synthesized infollicle cells of columnar to cuboidal shape with roundnuclei and that squamification of cells is followed by aloss of the ability to synthesize yolk proteins can beinterpreted following the concept of intracellularelectrophoresis by cells (De Loof, 1983, 1985) and theconcept of epigenetic regulation of gene expressionby ions (De Loof & Geysen, 1983; De Loof, 19866).The well-known morphology and new insights intothe physiology of the polytrophic follicle make it afavourable model to elaborate epigenetic aspects ofgene expression.

The authors are grateful to Prof. Spencer Berry, DrRoger Huybrechts and Dr Barend Verachtert for criticallyreading the manuscript. We also wish to thank Prof. FransOllevier for allowing access to the Reichert Polyvar micro-scope and Julie Puttemans for photographical assistance.J.C. wishes to thank the IWONL (Belgium) for financialsupport.

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(Accepted 29 April 1987)