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 sites where these molecules will be sequestered in the oocyte.
Particularly in Drosophila, the oocyte is precisely structured, eventually containing organization which
influences post-fertilization development Nusslein-Vol- hard et al., 1987. This organized “pre-programming” is
achieved within a multicellular complex which must itself be highly and actively regulated. Indeed, the devel-
opmental sequence of follicular activities that generate egg structure implies an exacting set of cellular controls.
Morphological polarity, manifested as an anterior-pos- terior orientation with respect to both the ovariole and
the whole organism of the oocyte-nurse cell syncytium, is apparent when the follicle is first formed. In Hyalo-
phora cecropia
, an electrically-based physiological polarity initiates close to the onset of vitellogenesis
Woodruff and Telfer 1973, 1980, and continues as a steady-state phenomenon for several days, until the nurse
cells disintegrate about 24 h before the end of vitellogen- esis. This physiological polarity involves a metabolically
driven difference in [Ca
2 +
]
i
between the oocyte and the nurse cells Woodruff et al., 1991; Woodruff and Telfer,
1994, which establishes an electrical gradient focused across the bridges connecting the two cell types.
Recently, this transbridge ionic gradient has been shown to influence the distribution of charged endogenous cyto-
solic proteins Cole and Woodruff, 1997. This current, seemingly through action of the cytosolic proteins whose
distribution it regulates, also enforces changes in the transcriptional activity of the oocyte nucleus Woodruff
et al., 1998.
The ovarian follicles of Drosophila resemble those of Hyalophora
, but possess more nurse cells n =
15 and reside in a more conventional blood ion environment.
Reports from different labs on the existence of a trans- bridge gradient in Drosophila have varied. Bohrmann et
al. 1986 and Sun and Wyman 1987, 1993, found no significant difference, while Woodruff et al. 1988,
Woodruff 1989, Verachtert et al. 1989, Verachtert and De Loof 1989 and Singleton and Woodruff 1994
all found significant differences between the steady-state potentials of nurse cells and the oocyte to which they
are attached. An experimentally supported answer explaining the differing results has now been put forth,
and centers around the composition of the media in which the measurements were performed Singleton and
Woodruff, 1994. Experimental evidence revealed that the steady-state membrane potential E
m
of nurse cells was more affected by osmolarity than E
m
of oocytes. The osmolarity of adult female hemolymph was measured to
be 250 mOsmol, at which osmolarity nurse cells were shown to be more electronegative than the oocyte to
which they were attached. At increasingly higher osmol- arity the difference between cell types first decreased to
0 and then reversed.
Microinjection of fluorescently labeled lysozyme, in either the positive or the negative form, has provided
evidence that a charge-dependent asymmetric distri- bution of proteins can occur in Drosophila Woodruff et
al., 1988, but lysozyme is an exogenous protein in this system, and soluble endogenous proteins might be regu-
lated by other means. Thus in the present study we have utilized 2-D gel electrophoresis to analyze the distri-
bution of charged endogenous cytosolic proteins from the ovarian follicles of Drosophila. Soluble proteins
could be susceptible to iontophoretic effects, while pro- teins bound to cytoskeletal elements, membranes and
other 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 upon the soluble proteins. As in a previous study Cole and
Woodruff, 1997, a necessary step was to separate sol- uble proteins from those which were bound. To achieve
this, we harvested soluble proteins from nurse cell or from oocyte extracts by centrifugation and ultrafiltration.
We furthermore took advantage of the effect on the transbridge electrical gradient wrought by changes in
osmolarity Singleton and Woodruff, 1994. This pro- vided a non-invasive non-pharmacological means to
reverse the direction of the gradient. If the transbridge gradient actually does influence the distribution of
charged soluble molecules, the distributions of both acidic and basic proteins should be affected in opposite
manners. Relative to controls incubated in a 255 mOs- mol. medium, in follicles incubated at 400 mOsmol. the
concentrations of soluble acidic proteins should diminish in the oocytes, and increase in the nurse cells. Similarly,
the relative concentrations of soluble basic proteins should decrease in the nurse cells and increase in the
oocytes of follicles incubated at high osmolarity.
The experiments reported here show that the distri- butions of most of the soluble proteins responded to
changes in the osmolarity of the incubation medium exactly as if they were responding to the transbridge
electrical gradient.
2. Materials and methods
2.1. Animals Drosophila melanogaster
Oregon Red were raised on Drosophila medium Carolina Biological Supply,
Burlington, NC. To obtain ovarioles containing the maximum number of follicles in stage 10 of develop-
ment Cummings et al., 1969, newly emerged flies were placed in fresh vials for four days. On the third day, 24
h before dissection, we added a small dollop of yeast paste to stimulate oogenesis Tilney et al., 1996. By the
time of dissection females were highly active in vitellog- enesis, with multiple follicles of all stages.
1241 R.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 dissections
and incubations was designed to have the same major ion composition and osmolarity as the Drosophila hemo-
lymph which normally bathes developing follicles Singleton and Woodruff, 1994. This PSS was made up
of: 100 mM Na-glutamate, 25 mM KCl, 15 mM MgCl
2
, 5 mM CaSO
4
, 2 mM sodium phosphate buffer pH 6.9. Sucrose was added as needed to bring the osmolarity to
250–255 mOsmol PSS255. For reversal of the trans- bridge ionic gradient, sucrose was added to bring the
osmolarity 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 female Dro-
sophila in PSS255 and transferred to either fresh
PSS255, or to PSS400. For this report we selected only stage 10A or 10B follicles. Following one hour incu-
bation at room temperature, control or high osmolarity treated follicles were transferred to a homogenization
medium consisting of 20 wv sucrose, 2.5 wv poly- vinylpolypyrrolidone, 10 mM CaCl
2
, 5 mM HEPES and 5 mM AEBSF protease inhibitor Calbiochem, La Jolla,
CA. A fresh 26-gauge hypodermic needle was attached to 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 was
positioned as a guillotine above the follicles. The micromanipulator was used first to lower the sharp edge
onto the oocyte-nurse cell junction until the extreme tip touched the glass bottom of the working chamber. Then
the tip was drawn back in a slicing motion, severing the follicle so that the nurse cell cap was precisely separated
from the oocyte. Following separation neither the nurse cell cap nor the oocyte showed overt signs of leakage,
suggesting that a seal was achieved as the opposing sides of the intercellular bridges were pressed together as the
cut was made. Nor did Lucifer Yellow CH iontophoret- ically microinjected into oocyte or nurse cell cap reveal
any sign of leakage. Since no detectable leakage was observed from either fraction, several follicles were
sequentially “decapitated”, the oocytes being concen- trated in one area of the chamber and the nurse cell caps
in another. Oocytes OOC or nurse cell caps NCC were drawn into a microtransfer pipette and transported
to a drop of homogenization medium, volume of which was adjusted to obtain a ratio of 20
µ l for every 80 OOC
or 80 NCC, and thence to a chilled microcentrifuge tube. The cells were pressed with a chilled glass pestle to
express their cytoplasm with only minimal rupture of epithelial cells, of yolk spheres in the case of oocytes,
and of nuclei in the case of nurse cells. The samples were centrifuged at 12,000 g for 20 min at 4
° C to
remove intact epithelial cells, yolk spheres, nuclei, and other cellular debris. When resuspended pellets from
centrifuged ooplasm were examined microscopically using SSEE optics Ellis, 1978, yolk spheres and col-
umnar follicle epithelial cells were present in abundance, while resuspended nurse plasm pellets contained many
squamoid epithelial cells but few if any yolk spheres. Nor had nurse cell nuclei been lysed, but instead
remained intact within the cytoplasm of nurse cells, plasma membranes of which had been ruptured Fig. 2.
No yolk spheres, epithelial cells, nuclei nor other debris were found in the supernatant. Samples were pooled
until each pool contained the protein extracted from 240 follicles in each 60
µ l. In some cases, to more stringently
insure the sample contained only the soluble proteins, the supernatant was centrifuged through a 300 kDa cut-
off filter Millipore, Bedford, MA6,000 g, 20–40 min, 4
° C. However, because the amounts of sample were so
small, and to include a small number of bound proteins which served as references as described below, this was
not routinely done.
2.4. Electrophoresis 2.4.1. Sample preparation
The procedures used for two dimensional gel electro- phoresis were essentially the same as those described in
an earlier study of the soluble endogenous proteins of the luna moth, Actias luna Cole and Woodruff, 1997.
Because of their importance to the results, they are repeated here. Either oocyte or nurse cell cap extracts
Fig. 2. Resuspended nurse cell cap. Nurse cells were disrupted by
gentle pressure with a pistil, then centrifuged. In the resuspended pellet fraction were numerous masses of nurse cell cytoplasm with disrupted
cell membranes, but containing intact nuclei. Scale bar =
100 µ
m. SSEE optics.
1242 R.W. Cole, R.I. Woodruff Journal of Insect Physiology 46 2000 1239–1248
were mixed 1:1 with acidic or basic overlay buffers Basic—0.2 Bio-Lyte 310 Bio-Rad, Laboratories,
Richmond, CA, 1.8 ampholine 810.5 Sigma, 9.5 M urea, 5.0 ß-mercaptoethanol, 0.4 Nonidet P-40
Sigma; Acidic—0.6 Bio-Lyte 35, 0.7 Bio-Lyte 310, 9.5 M urea, 5.0 ß-mercaptoethanol, 0.4 Non-
idet P-40. Buffered samples were then incubated at room 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, 600 µ
l of 911 ampholine and 50 µ
l of 310 Bio-Lyte were added to a monomer solution consisting of 4.3 acryla-
mide and 0.2 piperazine diacrylamide PDA, 9.5 M urea, 3 Nonidet P-40. For acidic gels, 450
µ l of 35
Bio-Lyte and 50 µ
l of 310 Bio-Lyte were added to the monomer solution. Twenty microliters of TEMED
Sigma and 60 µ
l 10 ammonium persulfide APS were added and the gels allowed to polymerize. Ten
microliters of an overlay solution consisting of 1:1 mix- ture of the appropriate overlay buffer and distilled water
plus enough Bromophenol Blue for visibility were added to the sample well and 30
µ l of prepared sample was
then introduced between the gel and the overlay solution. Thus each gel contained the soluble proteins from 60
follicles. Acidic protein gels were run at 5 °
C on a Mini PROTEAN II 2-D electrophoresis unit Bio-Rad at 500
V for 10 min, then 750 V for 3 h 20 min. Due to the inherent instability of gradients for high pH isoelectric
points O’Farrell et al., 1977, basic gels were run at 500 V for 10 min, then 800 V for only 1 h 30 min. Some
gels from each run were extruded onto parafilm and cut into eight pieces of equal length; each piece was eluted
overnight in a separate container with 0.50 ml of 50 mM KCl, and the elutate measured with a pH meter. After
IEF, the pH gradient of acidic gels went from pH 3.85
± 0.06 S.E.M. to 5.87
± 0.12 S.E.M., while that of
basic 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 filled with 20 mM NaOH and the anode chamber with 10 mM
H
3
PO
4
. For basic proteins, the anode chamber was filled with 250 mM HEPES Sigma solution and the cathode
chamber with 1.0 N NaOH. 2.4.3. Second dimension: separation by molecular
weight Second dimension separation was in SDS mini slab
gels 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 Trishydroxymethylaminomethane Aldrich Chemical Company, Inc., Milwaukee, WS,
0.27 M Trizma hydrochloride Sigma, and 1 lauryl sulfate Sigma. To polymerize the gel solutions, 50
µ l
of TEMED and 250 µ
l of 10 ammonium persulfate solution 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 1
µ l aliquont of SDS-PAGE low molecular weight
protein standards Bio-Rad diluted with SDS running buffer was placed in the left hand sample well. Running
buffer contained
25 mM
Trishydroxymethyl aminomethane, 192 mM glycine Sigma, 0.1 lauryl
sulfate, 5 µ
l glycerol, and Bromophenol Blue. Second dimension gels were run at 100 V for 10 min then 150
V for 1 h. 2.5. Fixing and staining
Slab gels were fixed for 20 min with 50 methanol for 12 h. Gels were than rehydrated and stained with a
highly sensitive silver stain Morrissey, 1981 reported to be 200 times more sensitive than Coomassie Blue
Deely, 1989. Stained gels were dried between two sheets of transparent cellulose Promega, Madison, WI.
Simultaneously run and stained gels of a control and of a treated sample were dried side-by-side to aid in the
computer analysis of protein content.
2.6. Criteria for acceptability of protein spots for analysis
For analysis we chose proteins which gave evidence of being soluble by being detectable in ultrafiltrated sam-
ple, and for acidic proteins, appearing in at least control oocyte 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 ultrafiltrate
criterion, and were detectable in control nurse cells and oocytes from follicles incubated in PSS400.
2.7. Computer analysis and calculations for determination of protein amounts
Dried gels were scanned with either a Kodak digital sciences 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 a single gel by either device. The image was stored using
a 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 of
protein spots were evaluated to determine the density of each protein relative to a reference standard Cole and
Woodruff, 1997 and below.
Although only stage 10 follicles were used, there was
1243 R.W. Cole, R.I. Woodruff Journal of Insect Physiology 46 2000 1239–1248
considerable difference from fly to fly in the size of developmentally equivalent follicles. Fortunately, as in
our 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 that could be correlated with changes in the transbridge ionic
gradient resulting from experimental treatment, and which were removed by ultrafiltration through a 300 kDa
filter. We assumed these to be proteins which in situ were bound to cytoplasmic organelles remaining in the
12,000 g supernatant, and which were solublized by urea during first dimension electrophoresis. The density of
these spots thus reflected only the amount of sample loaded. Absorbancy of a bound protein spot in either
control 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 average of several bound proteins shown as “B” in Fig. 3 pro-
vided a final normalization factor for each gel, and the absorbancy of each soluble protein was corrected by that
factor. Thus if the average density of the bound proteins in an experimental gel was found to be 1.5 times greater
than the average density of the same bound proteins in a control gel, the absorbancy of each soluble protein spot
in the experimental gel was reduced by the appropriate amount. In basic gels there were far fewer proteins, and
only one could be identified as a suitable reference pro- tein. Thus for basic proteins, concentration is simply
reported as a percentage of the density of that reference protein. By the strategies outlined above, we determined
the relative absorbancy of proteins from control follicles or from follicles incubated at high osmolarity.
3. Results
