vitellogenic ovarian follicles of drosophila exhibit a ...directory.umm.ac.id/data...

Journal of Insect Physiology 46 (2000) 1239–1248www.elsevier.com/locate/jinsphys

Vitellogenic ovarian follicles ofDrosophilaexhibit a charge-dependent distribution of endogenous soluble proteins

Russell W. Cole, Richard I. Woodruff*

Department of Biology, West Chester University, West Chester, PA 19383-8102, USA

Received 17 September 1999; accepted 19 January 2000

Abstract

In ovarian follicles ofDrosophila, soluble endogenous charged proteins are asymmetrically distributed dependent upon their ioniccharge. Reversal of the normal ionic difference across the intercellular bridges which connect nurse cells to their oocyte results ina redistribution of these proteins. Twelve soluble endogenous acidic proteins were identified by 2-D gel electrophoresis as beingpresent in both oocytes and nurse cells in samples run on four or more gels. Of these, following osmotically induced reversal ofthe electrical transbridge gradient the concentration of seven proteins decreased in the oocyte while nurse cell concentrations of alltwelve proteins increased. Of seven basic proteins analyzed, following reversal of the electrical gradient the concentration of allseven increased in oocytes. Four of these decreased in nurse cells, while nurse cell concentrations of the remaining three basicproteins also appeared to decrease, but yielded spots too faint for measurement. Data presented here demonstrate that, as in theSaturniidae, the ionic gradient across the nurse cell-oocyte intercellular bridges of the dipteran,Drosophila, can influence thedistribution of soluble endogenous charged molecules. 2000 Elsevier Science Ltd. All rights reserved.

Keywords:Ionic gradient; Cytosolic proteins; Membrane potential

1. Introduction

In insects, as throughout most of the animal kingdom,developing oocytes are most commonly supported bysibling cells called nurse cells, to which they are joinedby open channels of cytoplasm called intercellularbridges (Telfer, 1975). During pre-ovulation develop-ment the oocyte nucleus becomes a dormant germinalvesicle (GV) and the oocyte acquires yolk via endo-cytosis (Telfer, 1965). Meanwhile, the nurse cell nucleibecome highly endopolyploid and active, with most ofthe RNA produced destined to pass through the inter-cellular bridges and become sequestered in the oocyte(Bier, 1963a,b; Pollack and Telfer, 1969). Intercellularbridges, the products of incomplete cytokinesis, mostoften function to maintain synchrony among syncytialcells (Fawcett et al., 1959), yet in ovarian follicles thisfunction must be circumvented as oocyte and nurse cells

* Corresponding author. Tel.:+1-610-436-2417; fax:+1-610-436-2183.

E-mail address:[email protected] (R.I. Woodruff).

0022-1910/00/$ - see front matter 2000 Elsevier Science Ltd. All rights reserved.PII: S0022-1910 (00)00044-5

become both morphologically and physiologically differ-ent (Fig. 1). As the two cell types differentiate alongseparate lines some mechanism must exist which allowsthem to retain autonomy while still maintaining the openbridges needed to transport copious amounts of RNA

Fig. 1. Micrograph of a stage 10Drosophila ovarian follicle.Ooc=oocyte, NC=nurse cells. Accumulated yolk renders the largeoocyte opaque. Also visible are individual epithelial cells and thenuclei of several of the 15 nurse cells. Scale bar=100µm. SSEE optics.

1240 R.W. Cole, R.I. Woodruff / Journal of Insect Physiology 46 (2000) 1239–1248

from their sites of synthesis in the nurse cells to the siteswhere these molecules will be sequestered in the oocyte.

Particularly in Drosophila, the oocyte is preciselystructured, eventually containing organization whichinfluences post-fertilization development (Nusslein-Vol-hard et al., 1987). This organized “pre-programming” isachieved within a multicellular complex which mustitself be highly and actively regulated. Indeed, the devel-opmental sequence of follicular activities that generateegg structure implies an exacting set of cellular controls.Morphological polarity, manifested as an anterior-pos-terior orientation (with respect to both the ovariole andthe whole organism) of the oocyte-nurse cell syncytium,is apparent when the follicle is first formed. InHyalo-phora cecropia, an electrically-based physiologicalpolarity initiates close to the onset of vitellogenesis(Woodruff and Telfer 1973, 1980), and continues as asteady-state phenomenon for several days, until the nursecells disintegrate about 24 h before the end of vitellogen-esis. This physiological polarity involves a metabolicallydriven difference in [Ca2+]i between the oocyte and thenurse cells (Woodruff et al., 1991; Woodruff and Telfer,1994), which establishes an electrical gradient focusedacross the bridges connecting the two cell types.Recently, this transbridge ionic gradient has been shownto influence the distribution of charged endogenous cyto-solic proteins (Cole and Woodruff, 1997). This current,seemingly through action of the cytosolic proteins whosedistribution it regulates, also enforces changes in thetranscriptional activity of the oocyte nucleus (Woodruffet al., 1998).

The ovarian follicles ofDrosophilaresemble those ofHyalophora, but possess more nurse cells (n=15) andreside in a more conventional blood ion environment.Reports from different labs on the existence of a trans-bridge gradient inDrosophilahave varied. Bohrmann etal. (1986) and Sun and Wyman (1987, 1993), found nosignificant difference, while Woodruff et al. (1988),Woodruff (1989), Verachtert et al. (1989), Verachtertand De Loof (1989) and Singleton and Woodruff (1994)all found significant differences between the steady-statepotentials of nurse cells and the oocyte to which theyare attached. An experimentally supported answerexplaining the differing results has now been put forth,and centers around the composition of the media inwhich the measurements were performed (Singleton andWoodruff, 1994). Experimental evidence revealed thatthe steady-state membrane potential (Em) of nurse cellswas more affected by osmolarity than Em of oocytes. Theosmolarity of adult female hemolymph was measured tobe 250 mOsmol, at which osmolarity nurse cells wereshown to be more electronegative than the oocyte towhich they were attached. At increasingly higher osmol-arity the difference between cell types first decreased to0 and then reversed.

Microinjection of fluorescently labeled lysozyme, in

either the positive or the negative form, has providedevidence that a charge-dependent asymmetric distri-bution of proteins can occur inDrosophila(Woodruff etal., 1988), but lysozyme is an exogenous protein in thissystem, and soluble endogenous proteins might be regu-lated by other means. Thus in the present study we haveutilized 2-D gel electrophoresis to analyze the distri-bution of charged endogenous cytosolic proteins fromthe ovarian follicles ofDrosophila. Soluble proteinscould be susceptible to iontophoretic effects, while pro-teins bound to cytoskeletal elements, membranes andother cytoplasmic structures would not be. Bound pro-teins are present in such perfusion that, if not removed,they obscure the influence of the electrical gradient uponthe soluble proteins. As in a previous study (Cole andWoodruff, 1997), a necessary step was to separate sol-uble proteins from those which were bound. To achievethis, we harvested soluble proteins from nurse cell orfrom oocyte extracts by centrifugation and ultrafiltration.We furthermore took advantage of the effect on thetransbridge electrical gradient wrought by changes inosmolarity (Singleton and Woodruff, 1994). This pro-vided a non-invasive non-pharmacological means toreverse the direction of the gradient. If the transbridgegradient actually does influence the distribution ofcharged soluble molecules, the distributions of bothacidic and basic proteins should be affected in oppositemanners. Relative to controls incubated in a 255 mOs-mol. medium, in follicles incubated at 400 mOsmol. theconcentrations of soluble acidic proteins should diminishin the oocytes, and increase in the nurse cells. Similarly,the relative concentrations of soluble basic proteinsshould decrease in the nurse cells and increase in theoocytes of follicles incubated at high osmolarity.

The experiments reported here show that the distri-butions of most of the soluble proteins responded tochanges in the osmolarity of the incubation mediumexactly as if they were responding to the transbridgeelectrical gradient.

2. Materials and methods

2.1. Animals

Drosophila melanogaster(Oregon Red) were raisedon Drosophila medium (Carolina Biological Supply,Burlington, NC). To obtain ovarioles containing themaximum number of follicles in stage 10 of develop-ment (Cummings et al., 1969), newly emerged flies wereplaced in fresh vials for four days. On the third day, 24h before dissection, we added a small dollop of yeastpaste to stimulate oogenesis (Tilney et al., 1996). By thetime of dissection females were highly active in vitellog-enesis, with multiple follicles of all stages.

1241R.W. Cole, R.I. Woodruff / Journal of Insect Physiology 46 (2000) 1239–1248

2.2. Drosophila physiological salt solution (PSS)

The physiological salt solution used during dissectionsand incubations was designed to have the same majorion composition and osmolarity as theDrosophilahemo-lymph which normally bathes developing follicles(Singleton and Woodruff, 1994). This PSS was made upof: 100 mM Na-glutamate, 25 mM KCl, 15 mM MgCl2,5 mM CaSO4, 2 mM sodium phosphate buffer (pH 6.9).Sucrose was added as needed to bring the osmolarity to250–255 mOsmol (PSS255). For reversal of the trans-bridge ionic gradient, sucrose was added to bring theosmolarity to 400 mOsmol (PSS400). Osmolarity of sol-utions was checked using commercial osmometers;either an Osmette A (Precision Systems Inc, Natick,MA) or a Vapro 5520 (Wescor, Logan, UT).

2.3. Follicle collection and preparation

Vitellogenic follicles were dissected from femaleDro-sophila in PSS255 and transferred to either freshPSS255, or to PSS400. For this report we selected onlystage 10A or 10B follicles. Following one hour incu-bation at room temperature, control or high osmolaritytreated follicles were transferred to a homogenizationmedium consisting of 20% w/v sucrose, 2.5% w/v poly-vinylpolypyrrolidone, 10 mM CaCl2, 5 mM HEPES and5 mM AEBSF protease inhibitor (Calbiochem, La Jolla,CA). A fresh 26-gauge hypodermic needle was attachedto the needle holder of a Narishige MN-151 Emerson-type micromanipulator (Narishige Instruments, Japan),and adjusted so that the sharp edge of the tip bevel waspositioned as a guillotine above the follicles. Themicromanipulator was used first to lower the sharp edgeonto the oocyte-nurse cell junction until the extreme tiptouched the glass bottom of the working chamber. Thenthe tip was drawn back in a slicing motion, severing thefollicle so that the nurse cell cap was precisely separatedfrom the oocyte. Following separation neither the nursecell cap nor the oocyte showed overt signs of leakage,suggesting that a seal was achieved as the opposing sidesof the intercellular bridges were pressed together as thecut was made. Nor did Lucifer Yellow CH iontophoret-ically microinjected into oocyte or nurse cell cap revealany sign of leakage. Since no detectable leakage wasobserved from either fraction, several follicles weresequentially “decapitated”, the oocytes being concen-trated in one area of the chamber and the nurse cell capsin another. Oocytes (OOC) or nurse cell caps (NCC)were drawn into a microtransfer pipette and transportedto a drop of homogenization medium, volume of whichwas adjusted to obtain a ratio of 20µl for every 80 OOCor 80 NCC, and thence to a chilled microcentrifuge tube.The cells were pressed with a chilled glass pestle toexpress their cytoplasm with only minimal rupture ofepithelial cells, of yolk spheres in the case of oocytes,

and of nuclei in the case of nurse cells. The sampleswere centrifuged at 12,000g for 20 min at 4°C toremove intact epithelial cells, yolk spheres, nuclei, andother cellular debris. When resuspended pellets fromcentrifuged ooplasm were examined microscopicallyusing SSEE optics (Ellis, 1978), yolk spheres and col-umnar follicle epithelial cells were present in abundance,while resuspended nurse plasm pellets contained manysquamoid epithelial cells but few if any yolk spheres.Nor had nurse cell nuclei been lysed, but insteadremained intact within the cytoplasm of nurse cells,plasma membranes of which had been ruptured (Fig. 2).No yolk spheres, epithelial cells, nuclei nor other debriswere found in the supernatant. Samples were pooleduntil each pool contained the protein extracted from 240follicles in each 60µl. In some cases, to more stringentlyinsure the sample contained only the soluble proteins,the supernatant was centrifuged through a 300 kDa cut-off filter (Millipore, Bedford, MA)(6,000g, 20–40 min,4°C). However, because the amounts of sample were sosmall, and to include a small number of bound proteinswhich served as references as described below, this wasnot routinely done.

2.4. Electrophoresis

2.4.1. Sample preparationThe procedures used for two dimensional gel electro-

phoresis were essentially the same as those described inan earlier study of the soluble endogenous proteins ofthe luna moth,Actias luna(Cole and Woodruff, 1997).Because of their importance to the results, they arerepeated here. Either oocyte or nurse cell cap extracts

Fig. 2. Resuspended nurse cell cap. Nurse cells were disrupted bygentle pressure with a pistil, then centrifuged. In the resuspended pelletfraction were numerous masses of nurse cell cytoplasm with disruptedcell membranes, but containing intact nuclei. Scale bar=100 µm.SSEE optics.


were mixed 1:1 with acidic or basic overlay buffers(Basic—0.2% Bio-Lyte 3/10 (Bio-Rad, Laboratories,Richmond, CA), 1.8% ampholine 8/10.5 (Sigma), 9.5 Murea, 5.0% ß-mercaptoethanol, 0.4% Nonidet P-40(Sigma); (Acidic—0.6% Bio-Lyte 3/5, 0.7% Bio-Lyte3/10, 9.5 M urea, 5.0% ß-mercaptoethanol, 0.4% Non-idet P-40). Buffered samples were then incubated atroom temperature for 10 min.

2.4.2. First dimension: isoelectric focusing (IEF)Different acrylamide monomer solutions were pre-

pared for acidic and basic proteins. For basic gels, 600µl of 9/11 ampholine and 50µl of 3/10 Bio-Lyte wereadded to a monomer solution consisting of 4.3% acryla-mide and 0.2% piperazine diacrylamide (PDA), 9.5 Murea, 3% Nonidet P-40. For acidic gels, 450µl of 3/5Bio-Lyte and 50µl of 3/10 Bio-Lyte were added to themonomer solution. Twenty microliters of TEMED(Sigma) and 60µl 10% ammonium persulfide (APS)were added and the gels allowed to polymerize. Tenmicroliters of an overlay solution consisting of 1:1 mix-ture of the appropriate overlay buffer and distilled waterplus enough Bromophenol Blue for visibility were addedto the sample well and 30µl of prepared sample wasthen introduced between the gel and the overlay solution.(Thus each gel contained the soluble proteins from 60follicles.) Acidic protein gels were run at 5°C on a MiniPROTEAN II 2-D electrophoresis unit (Bio-Rad) at 500V for 10 min, then 750 V for 3 h 20 min. Due to theinherent instability of gradients for high pH isoelectricpoints (O’Farrell et al., 1977), basic gels were run at 500V for 10 min, then 800 V for only 1 h 30 min. Somegels from each run were extruded onto parafilm and cutinto eight pieces of equal length; each piece was elutedovernight in a separate container with 0.50 ml of 50 mMKCl, and the elutate measured with a pH meter. AfterIEF, the pH gradient of acidic gels went from pH3.85±0.06 (S.E.M.) to 5.87±0.12 (S.E.M.), while that ofbasic gels ranged from 8.0±0.1 (S.E.M.) in the first seg-ment to 9.6±0.1 (S.E.M.) in the seventh segment. Seg-ment eight, in contact with the lower tank buffer, aver-aged 11.1±0.02 (S.E.M.).

For acidic proteins, the cathode chamber was filledwith 20 mM NaOH and the anode chamber with 10 mMH3PO4. For basic proteins, the anode chamber was filledwith 250 mM HEPES (Sigma) solution and the cathodechamber with 1.0 N NaOH.

2.4.3. Second dimension: separation by molecularweight

Second dimension separation was in SDS mini slabgels (8.5×7 mm, 1 mm thick) containing 12.0% acrylam-ide and 0.26% PDA dissolved in a degassed stock sol-ution of 0.6 M Tris(hydroxymethyl)aminomethane(Aldrich Chemical Company, Inc., Milwaukee, WS),0.27 M Trizma hydrochloride (Sigma), and 1% lauryl

sulfate (Sigma). To polymerize the gel solutions, 50µlof TEMED and 250µl of 10% ammonium persulfatesolution were added. Some second dimension separ-ations were done using pre-cast 12% acrylamide “ready-gels” (Bio-Rad).

An IEF gel was placed on the top of each slab gel,and a 1µl aliquont of SDS-PAGE low molecular weightprotein standards (Bio-Rad) diluted with SDS runningbuffer was placed in the left hand sample well. Runningbuffer contained 25 mM Tris(hydroxymethyl)aminomethane, 192 mM glycine (Sigma), 0.1% laurylsulfate), 5µl glycerol, and Bromophenol Blue. Seconddimension gels were run at 100 V for 10 min then 150V for 1 h.

2.5. Fixing and staining

Slab gels were fixed for 20 min with 50% methanolfor 12 h. Gels were than rehydrated and stained with ahighly sensitive silver stain (Morrissey, 1981) reportedto be 200 times more sensitive than Coomassie Blue(Deely, 1989). Stained gels were dried between twosheets of transparent cellulose (Promega, Madison, WI).Simultaneously run and stained gels of a control and ofa treated sample were dried side-by-side to aid in thecomputer analysis of protein content.

2.6. Criteria for acceptability of protein spots foranalysis

For analysis we chose proteins which gave evidenceof being soluble by being detectable in ultrafiltrated sam-ple, and for acidic proteins, appearing in at least controloocyte sample and nurse cell sample from follicles incu-bated in PSS400, and in four or more gels of each type.Basic proteins were chosen if they met the ultrafiltratecriterion, and were detectable in control nurse cells andoocytes from follicles incubated in PSS400.

2.7. Computer analysis and calculations fordetermination of protein amounts

Dried gels were scanned with either a Kodak digitalsciences DC40 camera for electrophoresis gels (Kokak,Rochester, NY) or an Epson ES-1000c flat-bed scanner(Epson Accessories, Torrance, CA) to digitize the image.No differences were found between data derived from asingle gel by either device. The image was stored usinga Power Macintosh computer (Apple Computer, Inc.,Cupertino, CA) and retrieved using IP Lab Gel LC Ver-sion 1.1.2f (Signal Analytics Corporation, Vienna, VA).Using the IP Lab Gel LC analysis program, images ofprotein spots were evaluated to determine the density ofeach protein relative to a reference standard (Cole andWoodruff, 1997) and below.

Although only stage 10 follicles were used, there was


considerable difference from fly to fly in the size ofdevelopmentally equivalent follicles. Fortunately, as inour earlier study on soluble proteins of Luna moth fol-licles, in centrifuged samples there was a small popu-lation of proteins that showed little or no change thatcould be correlated with changes in the transbridge ionicgradient resulting from experimental treatment, andwhich were removed by ultrafiltration through a 300 kDafilter. We assumed these to be proteins which in situwere bound to cytoplasmic organelles remaining in the12,000g supernatant, and which were solublized by ureaduring first dimension electrophoresis. The density ofthese spots thus reflected only the amount of sampleloaded. Absorbancy of a bound protein spot in eithercontrol or experimental gel could thus be used as a “stan-dard” against which the absorbancy of truly soluble pro-teins could be compared. For acidic proteins, the averageof several bound proteins (shown as “B” in Fig. 3) pro-vided a final normalization factor for each gel, and theabsorbancy of each soluble protein was corrected by thatfactor. Thus if the average density of the bound proteinsin an experimental gel was found to be 1.5 times greaterthan the average density of the same bound proteins ina control gel, the absorbancy of each soluble protein spotin the experimental gel was reduced by the appropriateamount. In basic gels there were far fewer proteins, andonly one could be identified as a suitable reference pro-tein. Thus for basic proteins, concentration is simplyreported as a percentage of the density of that referenceprotein. By the strategies outlined above, we determinedthe relative absorbancy of proteins from control folliclesor from follicles incubated at high osmolarity.

3. Results

3.1. Acidic proteins

As shown in Fig. 3, by centrifugation and ultrafiltr-ation we were able to identify four distinct proteinsmeeting the criteria for bound reference proteins(designated as “B”). In addition we identified 12 accept-able proteins (designated by numbers 1–12), each ofwhich met the criteria above for analysis as a cytosolicprotein. Of these, in oocyte samples from 400 mOsmolincubations, there was an overall 2±1% decrease fromcontrol levels. Seven of the proteins decreased in con-centration, three increased slightly, and two increasedmarkedly relative to their concentrations in controls (Fig.4). Among the seven proteins which did decrease, theaverage change was 3.7±1%, the largest change being9% and the smallest being 1%. By pairedt-test compar-ing the 255 mOsmol vs 400 mOsmol concentration ofeach protein on four or more gel pairs,P=0.28. For theseven which did decrease,P=0.01. Fig. 5 shows anenlarged portion of a gel on which was run sample from

control oocytes, and a similar section of a gel on whichwas run oocyte sample from follicles incubated inPSS400. One of the bound acidic proteins (marked B)can be seen, as well as several soluble acidic proteins.For convenience, the proteins of interest have beenmarked by arrowheads. Note that these proteins do notin all cases correspond to those proteins analyzed in Fig.3 and Fig. 4, since not all of the proteins so clearly seenin this pair of gels were sufficiently resolved on four ormore gel pairs to allow them to be used in statisticalanalysis. Fig. 6 shows the absorbancy profiles from aPSS255 gel and a PSS 400 gel for the bound referenceprotein and a cytosolic protein, spots of which appearalong the cursor line marked in Fig. 5.

In nurse cell samples from follicles incubated inPSS400, all 12 of the analyzed proteins increased in con-centration. The overall average change for these 12 pro-teins was a 6.5±0.8% increase above control levels. Bypaired t-test, P=0.0001. The largest change was a 10%increase and the smallest change was 2%. Fig. 4 showsthe changes in all 12 acidic proteins analyzed.

3.2. Basic proteins

Generally, the number and concentration of cytosolicproteins is small compared to the great number andabundance of proteins which are bound in some waywithin the cytoplasm and its organelles. Of cytosolicproteins, the vast majority are acidic or neutral, thus itwas not surprising that there were few basic proteinswhich met our criteria of being present in both cell typesand in four or more gels of one type. We were able toidentify one bound protein which served as a reference,and seven cytosolic proteins (numbered 1+–7+) with pI’sof pH 8 or greater. Of these, three were in such lowconcentration in nurse cells that we could not meaning-fully analyze their concentrations as they decreased infollicles incubated in PSS400. Thus we provide data forseven proteins in oocytes incubated in PSS255 or inPSS400, but only for four of these in nurse cells.Because there was only one formerly bound basic pro-tein to serve as a reference, concentrations are given sim-ply as a percentage of that reference protein (Fig. 7).

Offsetting the small number of basic proteins accept-able for analysis was the clarity of their response. Inoocytes incubated in PSS400, all seven basic cytosolicproteins increased their concentration. The averageincrease was 290±20% (2.9 times) above the levelsfound in control oocytes from PSS255. The maximumchange was an increase to 385% of controls, while theminimum change was an increase to 222% of controlconcentration. By pairedt-test,P=0.008.

Of these seven proteins, in nurse cells we were ableto analyze the changes of four, all of which decreasedin concentration following incubation in PSS400. Therewas an average 79±5% decrease to a level 21% of con-


Fig. 3. Examples of second dimension gels silver stained to show soluble endogenous proteins. (A) Oocyte acidic proteins. (B) Nurse cell acidicproteins. (C) Oocyte basic proteins. (D) Nurse cell basic proteins. Numbered spots are proteins which met the criteria described in the text foranalysis of soluble proteins. The designation “+” after the identification numbers for basic proteins indicates only that they are positively charged,not their charge density. Spots marked “B” are proteins which were removed from samples passed through a 300 kDa filter, and thus are assumedto have been bound in situ. Solubilized during sample preparation, they varied only dependent upon the total amount of protein loaded in the firstdimension, and thus serve as reference points. (Note: the nurse cell basic protein gel shown as “D” had greater separation in the second dimensionthan did its oocyte counterpart shown as “C”. In “D” protein no. 7+, which would otherwise not have shown in this composite figure, appears inthe box at lower left. For A and B the pH gradient runs in each gel from pH 6 at left to pH 4 at right. For C and D the pH gradient runs in eachgel from pH 8 at left to pH 9.6 at right.

trol concentrations (P=0.003). The maximum changewas to only 6% of control values, while the minimumchange was to 30% of control values.

4. Discussion

The data reported here reveal that: (1) oocytes andnurse cells ofDrosophilaovarian follicles share several

endogenous proteins which are charged and soluble; (2)the concentrations of these proteins in each cell typechanged on average in a manner consistent with theirdistributions being influenced by an electrical gradientin the intercellular bridges and (3) this transbridge elec-trical gradient operates as a “leaky” selective gate.

Bohrmann and Gutzeit (1987) attempted to determineif a transbridge ionic gradient influenced the distributionof endogenous, rather than microinjected proteins. They


Fig. 4. Changes in the relative absorbancy (concentration) of 12endogenous soluble acidic proteins caused in 1 h by the osmoticallyinduced reversal of the transbridge electrical gradient between nursecells and their attached oocyte.

concluded that “...only a small fraction of acidic andbasic proteins may be subject to intrafollicular electro-phoresis, but the bulk of the proteins clearly does notmigrate by way of electrophoresis”. Their methods, how-ever, included treatments which would have solubilizedall proteins within the various cells of the follicle,obscuring in their number and concentrations those pro-teins which were naturally dissolved in the cytosol.Indeed, the small fraction of the proteins which theynoted as perhaps being subject to the electrical gradientmay well have constituted nearly all of the solublecharged proteins. It was for this reason that we took greatcare in obtaining only the cytosolic fraction beforeemploying any treatments containing chemicals such asurea which would solubilize previously bound proteins.

Fig. 5. Sections of a pair of 2-D gels illustrating the change in oocyteconcentration of cytosolic acidic proteins caused by reversal of thetransbridge electrical gradient. The gel at left shows proteins(arrowheads) from oocytes incubated at control osmolarity of 255mOsmol. The gel at the right shows these same proteins harvestedfrom oocytes incubated for 1 h in medium of 400 mOsmol. Protein“B” is a normally bound protein, the concentration of which serves asa normalization factor. After incubation in medium of 400 mOsmol,while density (concentration) of protein “B” is nearly unchanged, eachof the eight acidic soluble proteins shown here decreased in concen-tration, presumably due to back-diffusion into nurse cells. The cursorsconnecting “B” and a cytosolic protein mark the lines of theabsorbancy profiles shown in Fig. 6.

By centrifugation and ultrafiltration we were able tolimit our investigations to those proteins which, beingsoluble in the cytoplasm, would be available to be influ-enced by an electrophoretic field confined to the inter-cellular bridges. Of the acidic proteins which appearedto be soluble, all 12 increased in nurse cells followingreversal of the transbridge electrical gradient. In oocytes,seven behaved in a charge-dependent manner, decreas-ing in their concentrations. Four others, each of whichincreased strikingly in nurse cells, also increased inoocytes, but only slightly. Another protein (designatedas no. 6, Fig. 4) increased markedly in concentration inboth cell types after incubation at high osmolarity withthe strongest increase in the nurse cells. Two possiblecauses, both speculative at this time, present themselves.Firstly, all five may in fact be proteins which were veryloosely bound in situ, with binding constants decreasedby incubations at high osmolarity. Alternatively, thesefive may represent a group of proteins for which ratesof synthesis are strongly affected by the osmolarity ofthe medium (Mizuno et al., 1984; Prince and Villarejo,1990; Hengge-Aronis et al., 1993; Muffler et al., 1996).Osmolarity of the incubation medium has been shownto cause changes in the rates of uptake of certain precur-sor amino acids, and precursor availability would pre-sumably have an effect on rates of protein synthesis.Methionine uptake in particular has been shown to behigher at increased osmolarity (Bohrmann, 1991).Osmolarity also has an effect on the uptake of K+, theprincipal ion setting the value of Em (Miyazaki and Hagi-wara, 1976). Changes in [K+]i would, in turn, causechanges in internal ion activities of other ions, whichmay up- or down-regulate the synthesis of given pro-


Fig. 6. Absorbancy profiles of gel spots representing a bound refer-ence protein and near-by cytosolic acidic protein no. 12. Area undereach peak is determined by the amount of protein in the spot. Theplots are from digital data of pixel intensity taken along the cursorlines shown in Fig. 5. Upper plot shows the profiles of the two proteinsextracted from follicles incubated in PS255, while the lower profilesare from follicles incubated 1 h in PSS400.

Fig. 7. Changes in the relative absorbancy (concentration) of sevenendogenous soluble basic proteins caused by the osmotically inducedreversal of the transbridge electrical gradient between nurse cells andtheir attached oocyte.

teins. If these five acidic proteins do have rates of syn-thesis which are greater at 400 than at 255 mOsmol.,one would expect increased concentration in both celltypes, with the greatest increase occurring in the celltype on the “favored” side of the intercellular bridge.This is what was observed. At present we have no hardevidence for any of these possibilities, although someproteins with similar behavior were observed in our earl-ier study of protein distributions inActias luna (Coleand Woodruff, 1997). If the five “rogue” proteins are notnaturally soluble proteins, or if their rates of synthesisincrease dramatically in PSS400, they would not be can-didates for statistical analysis. Analysis of the sevenremaining oocyte proteins shows that their concen-trations decreased at high osmolarity, withP=0.01. Inthe nurse cells, all 12 proteins increased in relative con-centration, with a very highly significantP-value of0.0001.


Following incubation in PSS400, the concentrationsof all seven basic proteins assayed increased in oocytes(P=0.008). Basic proteins 4, 5 and 6 were barely visiblein nurse cell samples from control follicles, and afterincubation in PSS400 their concentrations had dimin-ished to unmeasurable levels. Thus we could not obtainreliable densitometric measurements for them, and theyhave not been included in averages for basic proteinsfrom nurse cells.

Concerning the operation of a transbridge electricalgradient, there are some misconceptions which haveentered the literature. Firstly, the gradient has been seenby some as a mechanism by which molecules and cyto-plasmic particles might be moved generally throughoutthe germ line cells of follicles. Yet there is absolutelyno evidence that the reported electrical gradients inDro-sophilaor any other organism exist from one end of anovarian follicle to the other. Instead, in every reportedcase the entire measured gradient was focused acrossintercellular bridges, specifically those between oocyteand nurse cells in the case of polytrophic ovaries ininsects (Woodruff and Telfer 1973, 1980; Verachtert,1988; Woodruff et al., 1988; Verachtert et al., 1989;Woodruff, 1989; Singleton and Woodruff, 1994) andmarine polychaete worms (Emanuelsson and Anehus,1985). In the telotrophic ovaries of insects, the electricalgradient is focused between groups of co-bridged nursecells and the core of the tropharium (Telfer et al., 1981;Woodruff and Anderson, 1984; Munz and Dittman,1987). Thus the gradient could have no part in the gen-eral movement of organelles, granules and other micro-scopically visible particles, nor of molecules, from dis-tant regions of nurse cells to locals in oocytes more thana few micrometers from an intercellular bridge.

It has been suggested that negatively charged enzymessuch as those used in glycolysis would be eliminatedfrom the nurse cell cytoplasm (and positively chargedenzymes eliminated from the ooplasm) (Sun andWyman, 1993). The data presented in this study showthat nurse cells are not swept clear of acidic soluble pro-teins. This is because the gradient can serve only as a“leaky” gate mechanism, influencing the eventual con-centrations of charged molecules. Without an interven-ing membrane or membrane-like structure, at any tem-perature above absolute zero an electrical gradient, nomatter how narrowly focused, could only oppose dif-fusion to a limited extent. The establised asymmetrymust be in the form of a Gibbs–Donnan equilibrium,mathematically described by the Nernst equation. Thusa transbridge electrical gradient could serve to influencethe concentration, but could not bring about the absoluteabsence of a charged molecule.

Nor is the isoelectric point (pI) of a protein the para-mount determinant of the degree to which it would beaffected by such a gradient. Rather it is the charge den-sity on the molecule which is most important. Across an

intercellular bridge with an electrical gradient of 5 mV,a protein with a pI of pH 10, but which has an ioniccharge of only2+ would reach an equilibrium in whichit would be 1.5 times greater in concentration on theelectronegative side of the bridge than on the electro-positive side (Woodruff, 1989). Conversely, a proteinwith a pI of only pH 8, but with 12 exposed positivelycharged sites, would also be positively charged in cyto-plasm. Having a charge density of 12, it would eventu-ally reach an equilibrium in which its concentration onthe electronegative side of the bridge would be ten timeshigher than on the electropositive side. This is the mostlikely reason that some charged proteins showed agreater response then did others to changes in the trans-bridge gradient. For instance, concentrations of acidicproteins no. 9 and no. 10 each increased in nurse cellsand declined in oocytes following osmotically inducedreversal of the transbridge gradient. However, thechanges exhibited by protein no. 9 in both cell typeswere about twice those which occurred to protein no.10, behavior which would be expected if protein no. 9has a greater charge density.

Many cytoplasmic particles and the molecules whichattach to them have been shown to be moved about thenurse cells and the oocyte via cytoskeletal transport sys-tems. However, movement within the bridges seems tobe regulated by other means (Theurkauf and Hazelrigg,1998). While we would be surprised if distribution ofbound molecules was also regulated in the same manner,data reported here strongly support the hypothesis that,within germ-line cells, the transbridge electrical gradientdoes influence the distribution of charged endogenouscytosolic proteins inDrosophila ovarian follicles.

Acknowledgements

The authors wish to thank Dr G. Cassotti and R. W.Woodruff for helpful suggestions in the perpetration ofthis manuscript. This work was supported in part by agrant from the National Science Foundation, no. IBN-9903994.

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