Download the extracellular electrical current pattern and its variability in

J. Cell Sci. 81, 189-206 (1986)
189
Printed in Great Britain © The Company of Biologists Limited 1986
THE EXTRACELLULAR ELECTRICAL CURRENT
PATTERN AND ITS VARIABILITY IN VITELLOGENIC
DROSOPHILA FOLLICLES
1
JOHANNES BOHRMANN
, ALFRED DORN2, KLAUS SANDER1 AND
1
HERWIG GUTZEIT *
l
Biologisches Institut I (Zoologie), Universitdt Freiburg, Albertstr. 21a, D-7800 Freiburg,
West Germany
2
Botanisches Institut I, Technische Universitdt Karlsruhe, Kaiserstr. 2, D-7500
Karlsruhe, West Germany
SUMMARY
We determined the extracellular electrical current pattern around Drosophila follicles at different
developmental stages (7-14) with a vibrating probe. At most stages a characteristic pattern can be
recognized: current leaves near the oocyte end of the follicle and enters at the nurse cells. Only at
late vitellogenic stages was an inward-directed current located at the posterior pole of many
follicles. Most striking was the observed heterogeneity both in current pattern and in current
density between follicles of the same stage. Different media (changed osmolarity or pH, addition of
cytoskeletal inhibitors or juvenile hormone) were tested for their effects on extrafollicular currents.
The current density was consistently influenced by the osmolarity of the medium but not by the
other parameters tested. Denuded nurse cells (follicular epithelium locally stripped off) show
current influx, while an accidentally denuded oocyte produced no current. Our results show that
individual follicles may be electrophysiologically different, though their uniform differentiation
during vitellogenesis does not reflect such heterogeneity.
INTRODUCTION
Extracellular electrical currents have been found in a number of different
developing systems ranging from regenerating amphibian limbs to ovarian follicles of
insects (for review, see Nuccitelli, 1983). The inferred electrical polarity in these
systems is generally believed to be an essential part of the differentiation process. In
fucoid eggs (Nuccitelli & Jaffe, 1974) and in carrot embryos (Brawley, Wetherell &
Robinson, 1984) the electrical polarity foreshadows the future embryonic axis and in
lily pollen the axis of germination (Weisenseel, Nuccitelli & Jaffe, 1975). Electrical
currents may, therefore, play a role in axial determination (Jaffe, 1981). In
polytrophic insect ovarioles (like those of Drosophila and Hyalophora) axial polarity
is already manifest in the germarium (Telfer, 1975; King, Cassidy & Rousset, 1982).
However, the maximal extracellular electrical currents in Hyalophora follicles were
found at a much later developmental stage (during vitellogenesis). A possible
developmental role of these currents was suggested by the discovery that injected
• Author to whom reprint requests should be addressed.
Key words: vibrating probe, extracellular currents, Drosophila oogenesis.
190
J. Bohrmann, A. Dorn, K. Sander and H. Gutzeit
proteins migrate in the follicle according to their charge (Woodruff & Telfer, 1980;
but see also Bohrmann, Huebner, Sander & Gutzeit, 1986).
We began to study the extrafollicular electrical current pattern in Drosophila
follicles since the large number of available mutants offers a promising approach for
analysing the functional consequences of the electrical phenomena. We report here
the changes in the current pattern of follicles of different developmental stages and
include some data on the mutant dicephalic, in which the main axis of follicle and
embryo is altered so that two anterior ends differentiate (Lohs-Schardin, 1982). We
also analysed follicles of the mutant bicaudal D (T. Schupbach, unpublished data)
in which no oocyte differentiates and, therefore, no apparent anteroposterior follicle
polarity can be recognized. Our results extend in several respects the recently
published work of Overall & Jaffe (1985) on extracellular currents in Drosophila
follicles and eggs.
MATERIALS AND METHODS
The electrical current patterns of 177 Drosophila melanogaster wild-type follicles (strain Oregon
R), 13 dicephalic (die; Lohs-Schardin, 1982) and six bicaudalD (bicD) follicles (kindly provided
by Dr T . Schupbach, Princeton, NJ) were determined at different stages of oogenesis by more than
2000 current density measurements using a vibrating probe (based on the method of Jaffe &
Nuccitelli, 1974; improved by Dorn & Weisenseel, 1982). Most of the analysed follicles were
cultured in Robb's medium (R-14) and some in Robb's balanced saline (DPBS; Robb, 1969) at
22 ± 1 deg. C. The current patterns in both solutions were not significantly different but follicles
develop from stage 10 up to stage 14 (King, 1970) only in the complete medium R-14 (Petri,
Mindrinos, Lombard & Margaritis, 1979; Bohrmann, 1981).
In several experiments the medium was modified (see Table 1 for details). It was either diluted
1:1 with distilled water, or sucrose was added in order to test for osmotic effects on the current
pattern. The osmolarity was determined with a Knauer Halbmikro-Osmometer. We changed the
usual pH of the medium from 6-8 to pH 9 or 5 to see whether H + participated in the currents. Also
cytochalasin B or colchicine (Sigma, St Louis) were added to inhibit cytoplasmic streaming within
the follicle (Gutzeit, 1986) and to look for related changes in the current pattern. Furthermore,
various amounts of juvenile hormone (JH) or two JH analogues were added to test for hormonal
effects on extracellular currents (JH C-16 was from Serva, Heidelberg; Altosid ZR-515 and ZR2008 were a gift from Dr Staal, Zoecon Corp., Palo Alto, CA). The solutions were vigorously
mixed before use. Table 1 summarizes the different experimental conditions studied.
The follicles were placed in an open chamber (3-5 cm diameter) filled with culture medium. In
order to prevent displacement of the examined follicle by the motion of the medium caused by the
vibrating probe, we held the follicle in place by a suction pipette (30—70 /an diameter, see Fig. 1A) .
This method proved superior to fixing the follicles at the bottom of the measuring chamber, since
the extrafollicular electrical field is only minimally distorted in the vicinity of the suction pipette. A
further advantage of our technique is that we were able to turn the follicle around its longitudinal or
transverse axis so that we could measure the currents in more than one plane. The measuring
electrode had a tip diameter of 15-20/an and an amplitude of 16/an. The current density
measurements were usually made perpendicular to the follicle's surface at a distance of 25 fxm from
the probe's centre point of vibration, or at a distance of 10 [an when probe vibration was parallel to
the surface.
The measurements were carried out with the lock-in amplifier set at a sensitivity of 2-5 or 10 fiV
(including a preamplification of 100 times) and a time constant of 0-3 s (except in a few cases 0-01 or
0-1 s). A reference current density at a distance of about 600 fan from the follicle was determined
before and after every measurement.
In most cases the first measurements were made within 10 min after isolation of the follicle to be
studied. Single determinations lasted usually for 30—90s. Changes in the current over time were
studied at single positions up to 3 h and all the measurements on one follicle lasted in some cases for
Current pattern in Drosophila/o//icfes
191
4-5 h. Owing to the relatively large open surface of the chamber, which is necessary to permit
positioning of the probe around the vertical axis by means of a turntable (see Dorn & Weisenseel,
1982), the evaporation of the medium was considerable during the measurements. The rate of
evaporation was determined and compensated for by periodical addition of distilled water to the
medium (about every IS min) or by exchanging the medium (after 1 h at the latest).
RESULTS
Electrical current pattern: constant features
The extracellular electrical current pattern of vjtellogenic Drosophila follicles
shows some stage-typical features, which can be recognized despite the great
variation between individual follicles (see below). Fig. 1B shows a typical recording
of a stage 10 follicle. During stages 7 (last previtellogenic stage) to 10B a positive
charge current leaves the posterior part of the follicle (oocyte and surrounding
epithelial follicle cells) and enters at the anterior end (nurse cells and surrounding
very thin follicle cells), thus forming an extrafollicular current loop. The maximal
density of the current parallel to the long axis of the follicle was measured near the
oocyte/nurse cell border. The point of current reversal (no current entering or
leaving the follicle) was generally located in this region. The maxima of the inward
and outward-directed currents were usually found not at the follicle's poles but rather
more laterally on either side of the follicle (e.g., see Figs 1,3). Especially at stages
9—11 these maxima are relatively sharp. There is often hardly any current near the
poles and in this respect the current pattern differs from that of Hyalophora follicles
(Jaffe & Woodruff, 1979). Also a second maximum efflux at the oocyte/nurse cell
border, as found in Hyalophora, was not normally observed in Drosophila. During
stages 11 and 12 the points of current reversal move towards the anterior pole while
nurse cell regression proceeds.
Table 1. Modifications of culture medium
Medium
R-14 (pH 6-8)
DPBS(pH6-8)
R-14 diluted 1:1 with distilled water
DPBS diluted 1:1 with distilled water
R-14 dil. 1:3 with dist. water + 285 mgml" 1 sucrose
R-14 + 28'5 mgml" 1 sucrose
R-14 of pH 9
R-14 of pH 5
R-14 + cytochalasin B (10-20 fig ml"1)
R-14 + colchicine (20-40 ^g ml"1)
R-14 + JH C-16 (2xl0" 6 to3xl0" 5 M)
R-14 + Altosid ZR-515 (10" 4 M)
R-14 +Altosid ZR-2008 (10" 4 to 5 X 1 0 " 2 M )
n.d., not determined.
Number
of follicles
measured
Specific
resistance
at22°C
(kficm)
Osmolarity
(mosM)
103
19
15
20
1
4
8
7
4
6
12
21
5
0-13
0-11
0-24
0-20
0-66
0-15
0-14
0-13
n.d.
n.d.
n.d.
n.d.
n.d.
240
390
120
160
370
320
240
240
n.d.
n.d.
n.d.
n.d.
n.d.
192
J. Bohrmann, A. Dorn, K. Sander and H. Gutzeit
4 min
Fig. 1. A. Photograph of a stage 10B follicle held in position by a suction pipette (SP).
The tip of the vibrating probe (VP) is positioned 10/fln from the surface of the follicle
cells (FC) covering oocyte (Ooc) and nurse cells (NC). Arrows show direction of current
flow (positive charges). The numerals indicate the measuring positions around the
follicle. 0, no current (position 7). Bar, 100/im. B. Typical recordings at these positions
(stage 10 follicle). Measurements at the reference position (R) were made at a distance of
600 fim and at the other positions at a distance of 25 jttn (centre point of vibration) from
the follicle's surface except for position 1 (10 /an). Such recordinga only give information
about the current density, not about the current direction (whether positive or negative
chart recordings are obtained depends on the direction of probe vibration). In measuring
positions 1—3 the direction of vibration was as shown in A, but for measurements in
positions 4—7 the probe was rotated counterclockwise approximately 90° around its
vertical axis.
At the end of vitellogenesis (late stage 12 to stage 14) we typically measured a
rather strong inward-directed current at the posterior pole where a specialized
chorion with aeropyles differentiates (Klug, Campbell & Cummings, 1974). This
local current was also observed by Overall & Jaffe (1985) and, similarly, in late
vitellogenic Hyalophora follicles at a comparable developmental stage (Jaffe &
Woodruff, 1979).
The currents, whether inward or outward, or parallel to the follicle axis, were
always steady and even when the lock-in amplifier was set at a time constant of 0-01 or
0-1 s (instead of the usual 0-3 s) during periods up to 10 min no pulses were recorded.
Maximal current densities were of the order of 10-20 ^iAcm~2 (stage 10A-11) but
were normally in the range of only 0-l-5/iAcm~ 2 . Stages 7, 8 and 14 in particular
showed very low current densities (see Fig. 2A,c).
Current pattern in Drosophila follicles
193
Variations in current pattern
Wild-type follicles. The patterns of extrafollicular electrical current during stages
7—14 are shown in Fig. 2A—c with regard to the direction of current and the average
current density at five selected measuring positions. At a given measuring position
the current density may vary considerably between follicles of the same stage (note
large standard deviation). Unusually strong currents (larger than 10/iAcm~2) were
measured in some follicles at stages 10A-13.
The position of current reversal is always in the middle region of young
vitellogenic follicles (up to stage 9). For this reason we observed zero current or an
inward or outward current at measuring position 3 (in the latter cases the point of
current reversal was slightly posterior or anterior to this position; see Fig. 2A), while
the other measuring positions showed current flux only in one direction. This no
longer holds true for later vitellogenic stages. At stage 10B, for example, we found
inward current or outward current, or no current, at the five selected measuring
positions (though with very different frequencies; see Fig. 2B). While the overall
pattern with current leaving the oocyte and entering the nurse cell end (see above) is
maintained, neither the magnitude nor the direction of the current is strictly fixed at
any given measuring position.
Interestingly, the inward-directed current at the posterior pole of late vitellogenic
follicles can already be detected at stages 9—11 in a small fraction of analysed follicles.
At stage 12 we observed inward or outward current, or no current, with the same
probability (Figs 2c, 3m,n). At the final stage of oogenesis (stage 14) current efflux at
the posterior pole was never observed.
While Fig. 2 gives quantitative data on the observed variations in different follicles
of the same stage (average current densities at five selected measuring positions),
further information can be obtained by studying the pattern of individual follicles in
greater detail (e.g., see Fig. 3). In this way the differences in current densities
and direction of current flow between different measuring positions become more
obvious (compare Figs 2B,C and 3). The position of current reversal is usually near
the oocyte/nurse cell border (where follicle cells begin to migrate centripetally at
stage 10B), but variations, particularly at older vitellogenic stages, were frequently
observed (Fig. 3). Even in a single follicle the positions of maximal current densities
and of current reversal are not necessarily symmetrical on opposite sides, which is
most obvious at stages 11-14 (for examples of stages 11 and 12, see Fig. 3i-n).
The dorsal and the ventral sides can be distinguished at early vitellogenic stages by
the dorsal location of the oocyte nucleus and from stage 10 on also by the thicker
follicular epithelium at the dorsal side. The current density on the dorsal side may
differ dramatically from that of the ventral side (for examples of stages 11 and 12, see
Fig. 3i—n), but there is no ventral- or dorsal-specific direction of the current: above
the thickened dorsal epithelium we have detected influx or efflux. Such differences
were not described in Hyalophora follicles (Jaffe & Woodruff, 1979).
Some follicles (n = 6) of different stages showed no measurable current directly
after explantation (within 10—15min). Also, some stage 11 follicles (n = 3) had an
194
J. Bohrmann, A. Dorn, K. Sander and H. Gutzeit
Measuring positions
1
2
cnr
2"
Stage 7
rw^-e;^
o"M)
50
HX)
I
2
Stage 8
T
c
1
l
4
1
(I%0
50
It K)
1 1
I
1
4
5
n=4
2
4'
X
6
4
Stage 9
2
>-
3-
1
1
2-
I '
)
50
T
100
1 1
1
2
3
1
n =
13
8
7
T
4
Fig. 2A. For legend see p. 197
i
8
15
195
Current pattern in Drosophila follicles
Measuring positions
1
2
cnv
41
Stage IDA
3•
2-
I.
1 1"
d
%0
50
100
= 27
20
19
21
28
Stage 10B
3I (Measuring positions as in stage 10A)
2\
1
0%0
§
50 KK)
'•
2•
n = 57
42
43
53
3
7i
6
Stage 11
54
c 1
0%()
50 100
n = 19
15
Fig. 2B. For legend see p. 197
18
19
J. Bohrmann, A. Dorn, K. Sander and H. Gutzeit
196
Measuring positions
1
2
71
5.
Stage 12
4 •
I 1
r
.H)
50
UK)
n = U)
10
81\
6-
Stage 13
5•
4 •
.V
2.
J
i 1
T
()•
r/
I 1'
r0
50
UK)
11
(Measuring positions as in stage 13)
Stage 14
%()
§
50
HXI
11
L
[±3.
!•
n=7
Fig. 2c
Current pattern in Drosophila follicles
197
unusually low current density from the beginning, or their current decreased during
the course of the measurements down to the detection limits of the vibrating probe.
However, cytoplasmic streaming (which is most rapid at stage 11) from nurse cells
into the oocyte (Gutzeit & Koppa, 1982) continued and these follicles also continued
developing to stage 12, as judged by the nurse cell morphology. From these
observations we conclude that extracellular currents are not necessary for cytoplasmic streaming to occur from nurse cells to oocyte, nor does the streaming appear
to generate extracellular currents. It is conceivable, however, that the cytoplasmic
streaming might indirectly disturb electrical current loops in the follicle, since
charged particles from the regressing nurse cells are rapidly transported into the
oocyte. This might explain the observed large variations in the current pattern,
particularly during stage 11.
Dicephalic follicles. We analysed 13 dicephalic (die) follicles (Lohs-Schardin,
1982) in which the altered axial polarity is already manifest during oogenesis, in that
groups of nurse cells are located at both poles of the oocyte (in wild-type follicles they
are present only at the anterior end). In such mutant follicles we found even greater
variability in the extracellular current pattern than in wild-type follicles. In fact, it is
impossible to list constant features in the current pattern as has been done for wildtype follicles (see above).
In two cases we measured different current directions at opposite sides of the
follicle (a stage 10B and a stage 8 follicle, see Fig. 4a). In four cases both nurse cell
clusters differed in the direction of currents: while at one cluster an inward directed
current was measured the other cluster showed an outward current or no current at
all (Fig. 4b,c). Outward flowing current from the nurse cells (or the surrounding
epithelium) has only rarely been observed in wild-type follicles (see Fig. 2 and
below).
Other die follicles showed current influx at both nurse cell clusters and efflux at the
oocyte as would be expected from the normal current pattern in wild-type follicles
(see also Overall & Jaffe, 1985). In two cases we detected only an outward current
around the entire die follicle in the plane of the measurements (Fig. 4d).
Bicaudal D follicles, bicaudal D (bicD) is one of several mutants in which
homozygous females produce follicles with no recognizable oocyte but 16 nurse cells
(T. Schupbach, unpublished data). These follicles develop to the size of stage 6/7
wild-type follicles and afterwards degenerate. The measured current densities were
low (0-1-0-8 /iAcm~2) and the pattern was quite heterogeneous. Two follicles had a
normal current pattern with efflux at the posterior and influx at the anterior end
(defined with reference to the axis of the ovariole). Two follicles had influx at the
posterior end and no current at the anterior end (but efflux laterally), and two others
Fig. 2A-C. Mean current densities (bars indicate 1/2 standard deviation) and frequency
of current influx, efflux or zero current (in % of n; n, numbers of follicles or follicle halves
in positions 2—4) at five selected measuring positions of stage 7-14 follicles (see insets).
The symmetrical measuring positions in stage 7-11 follicles are indicated on one side
only. In some cases measurements were not made at all measuring positions in the
analysed follicles.
198
jf. Bohrmann, A. Dorn, K. Sander and H. Gutzett
Stage 10A
(a-d)
200 ;im
Fig. 3
Current pattern in Drosophila follicles
199
Fig. 4. Schematic drawings of dicephalic follicles. Ooc, oocyte; NC, nurse cells; FC,
follicle cells. Other symbols as in Fig. 3.
showed no current at all. Thus in most cases an electrical polarity was established
although no apparent anteroposterior polarity can be recognized in these follicles.
Changes in current density over time
In both DPBS and R-14 culture medium the current densities of most follicles
changed upon prolonged culture. The observed changes may be classified as follows,
(a) In most follicles the current density (influx, efflux or parallel current) decreased
during the measurements at a rate of 0-01-0-8/zAcm~2 min" 1 at all measuring
positions. In seven follicles no electrical current could be detected around their
circumference after culturing for about 1 h. (b) In six follicles the current at certain
measuring positions remained constant for 20min up to 100 min. (c) In many cases
(n = 36) the current density (influx, efflux or parallel current) increased for some
time at a rate of 0-01 to 0-04/iA cm min at some measuring positions. At the
same time we occasionally measured decreasing current densities at other positions.
Fig. 3. Current patterns of selected stage 10A to stage 12 follicles (or halves of different
follicles in a-h) showing the range of heterogeneity observed at a particular developmental stage. The arrows indicate the current directions; arrow length represents the
current density measured at a distance of 25 pm from the follicle's surface (centre point of
vibration). Dorsal (d) and ventral (v) are indicated in follicles in which measurements
were made in the dorsoventral plane. The points of current reversal are marked with
triangles ( A ) ; o, zero current.
200
y. Bohrmann, A. Dorn, K. Sander and H. Gutzeit
While the overall pattern of extrafollicular current (external current flowing from
oocyte to nurse cells) was generally stable for even up to 4 h in culture, we found four
follicles that were exceptions to the rule. In these cases the current pattern became
inverse (external current flowing from nurse cells to oocyte) after 1—3 h in vitro.
The observed qualitative and quantitative instability in the pattern of current
densities during culture suggests that the control of the electrical phenomena is
complex and not just the result of a single experimental variable such as, for example,
evaporation of the medium during the experiments (see Materials and Methods).
Experimentally induced variations
Effects of pH and osmolarity of the medium. Changing the pH of the culture
medium to pH 5 or 9 had no effect on the current pattern and little effect on the size
of the currents. Therefore, H + does not seem to play a major role in the extracellular
current flow. In the acidic medium we observed in a few follicles a rather slow
current pulse after 10-100min in culture (an increase of up to 3-5^Acm~ 2 min~ 1
and a decrease of up to 5-5/xAcm~ min~ ).
When the osmolarity of the medium was changed the current densities were
markedly affected but the overall pattern of current flow was maintained. In
hypotonic solution (DPBS or R-14 diluted 1:1 with distilled water) most follicles of
all analysed stages produced strong currents at first (about 40 % above normal) but
by the time the follicle looked swollen (after 20—30 min) the currents had rapidly
decreased (initially at a rate of about 13 % per min compared to about 4 % per min in
normal medium). Again, in some follicles a slow current pulse was observed (after
10—15 min in culture an increase of up to 0-8/iAcm min~ and a decrease of up to
l-2^Acm~ 2 min~ 1 ).
In hypertonic media (R-14 with added sucrose) we mostly observed high currents
(above 10/zAcm~2) for up to 40 min of culture. A single stage 10B follicle was
cultured in an isotonic medium but with altered ion composition (R-14 diluted 1:3
with distilled water and osmolarity adjusted with sucrose, see Table 1). After
transfer from R-14 into the modified culture medium the currents decreased to about
1/3 of the normal value but the pattern was still normal after 2h and the follicle
looked healthy.
Effects of cytoskeletal inhibitors and juvenile hormone. When cytochalasin B was
added to R-14 (see Table 1) to inhibit cytoplasmic streaming from nurse cells to
oocyte during stages 10B—12 (Gutzeit, 1986) the currents were not affected. Also
colchicine (see Table 1), which stops ooplasmic streaming during stages 10A-12
(H. Gutzeit, unpublished data) does not affect the electrical current pattern either
quantitatively or qualitatively.
inRhodnius, juvenile hormone (JH) is known to affect the intracellular potential
of the oocyte (Telfer, Woodruff & Huebner, 1981) as well as the extracellular
currents (Sigurdson, 1984). This finding and other considerations have led several
authors to suggest that electrical phenomena may be related to vitellogenesis
(Bohrmann et al. 1984; Dittmann, Ehni & Engels, 1981; Sigurdson, 1984). We
tested the effect of JH and two JH analogues (see Table 1) on extrafollicular
Current pattern in Drosophila follicles
201
electrical currents in Drosophila follicles in vitro. The response was heterogeneous
for all three drugs tested. Most follicles showed no significant change in current
pattern or magnitude compared to the control medium (R-14 without JH). In several
follicles the decrease in current density with incubation time (see above) seemed to
slow down. On the other hand there were some cases with very fast decrease (of up to
2-0/zA cm min" 1 ). In a few follicles (n = 5, stages 10B—13) we found a strong local
current influx (of up to 15 /jAcm"2) somewhere at the lateral sides of the nurse cell
cap after 10-100 min of culture, and parallel current flowing from both poles of the
follicle to these sites. In three stage 10 follicles a small region with current influx
could be detected at the posterior pole within 30 min of incubation. However, since
such variations were also occasionally observed in JH-free media, a clear effect of JH
or its analogues (irrespective of the concentration used) on extrafollicular currents in
Drosophila follicles could not be demonstrated. Also, a stage 7 follicle of the JHdeficient mutant apterous 4 (kindly provided by Dr M. Bownes, Edinburgh) showed
currents with normal pattern and magnitude in JH-free medium. In this mutant
oogenesis stops at stage 7 but the follicles can be initiated to continue vitellogenesis
with ZR-515 in vivo (Wilson, 1982); ZR-S15 is a JH analogue, which was shown to
promote pinocytotic activity of the Drosophila oocyte in vitro (Giorgi, 1979). Our
results with JH show that Drosophila differs from Rhodnius in that the extracellular
currents are not consistently affected by the hormone. However, the extracellular
currents may still in some way be related to vitellogenesis. We found that follicles
from well-fed young flies (with high rate of oogenesis) as a rule produced stronger
current than follicles from older flies in which mature follicles accumulated in the
ovary (egg deposition inhibited in crowded culture vessels).
Currents in injured or denuded follicles. We occasionally observed in all media
used a slight shift of the points of current reversal or of maximum current density
after local current increase or decrease. Such spatial changes of the current pattern
could also be induced by local injury of a follicle. When a nurse cell, the oocyte or
follicle cells of several stage 9-12 follicles were injured with the probe or deliberately
punctured with the tip of a glass capillary a local and rather strong inward-directed
'injury current' (9—14/iAcm~2) was consistently observed, irrespective of the cell
type and site of injury. This injury current may affect the overall pattern of current
flow in the entire follicle; for example, when a strong inward current is induced by
injury of the follicular epithelium around the oocyte, where previously an exit
current had been demonstrated, the exit current in the vicinity of the induced current
reversal can increase and even more distant regions over the nurse cells can then show
an unexpected exit current.
In five follicles it was possible to injure the very thin follicle cells covering the
nurse cells with tungsten needles, so that single intact nurse cells bulged out of the
follicle. Over these 'naked' nurse cells (Fig. 5A) we also observed rather strong
current influx (of up to 11 /zAcrn"2). In Hyalophora follicles such naked nurse cells
produce an exit current (Woodruff, Huebner & Telfer, 1983).
In one case we produced a naked oocyte by injuring the follicular epithelium
covering the oocyte, so that part of the oocyte with an intact oolemma bulged out.
202
jf. Bohrmann, A. Dorn, K. Sander and H. Gutzeit
This follicle showed a normal current pattern, but without any current over the
naked oocyte (Fig. 5B), an indication that the epithelium (rather than the oocyte)
may produce the exit current normally observed.
DISCUSSION
The general current pattern of vitellogenic Drosophila follicles was found to be
constant, particularly during early stages of vitellogenesis. Current enters a follicle at
the nurse cell end and leaves at the oocyte end. Also an inward-directed current at the
posterior pole of late vitellogenic follicles (stage 14) has been typically observed.
However, within certain limits the current pattern can change and the quantitative
differences in the current flow can be as much as two orders of magnitude between
different follicles of the same stage.
In most previous reports on extracellular currents in insect follicles (Jaffe &
Woodruff, 1979; Dittmann et al. 1981; Huebner, 1984; Sigurdson, 1984; Overall &
Jaffe, 1985) little attention has been paid to the variability that is presumably
100 ;/m
Fig. 5. A. Schematic drawing of a stage 10B follicle with naked nurse cells (not covered
by follicle cells). These cells produced relatively strong current influx at first (1), which
decreased to a normal level after 40 min culture in vitro (2). The current densities marked
at the other positions were also determined after about 40 min in vitro. B. Drawing of a
stage 10B follicle with a naked oocyte held by a suction pipette (SP). The current pattern
was normal but the naked oocyte surface produced no current at all.
Current pattern in Drosophila follicles
203
present. However, we feel that this variability is very significant and has to be
considered when discussing the biological functions of electrical currents. In vitro
artifacts may occur but they cannot fully account for the observed differences.
In follicles of the mutant dicephalic (Lohs-Schardin, 1982) the inward current at
the nurse cells is not a constant feature as it is in wild-type follicles: one or even both
nurse cell clusters sometimes show outward current. Despite these abnormalities, die
follicles normally develop into mature oocytes (distinguishable from wild-type
oocytes only by the altered egg shell pattern) and a fraction of them enter embryogenesis and produce die embryos. Also follicles of the mutant bicaudal D
(T. Schiipbach, unpublished data), which consist of nurse cells and follicle cells
only, exhibit current influx or efflux at various positions on their circumference but
in this mutant oogenesis arrests early.
The described variations in the extracellular currents, both in wild-type and in
die follicles, are not reflected by corresponding morphological characteristics or
developmental properties. For example, in vivo nearly all wild-type follicles
differentiate functional germ plasm at the posterior pole (where polar granules
appear at stage 10; see Mahowald, 1962). However, at stages 10—14 not all follicles
show the inward-directed current that is typically observed at stage 14. In view of the
observed variation it is difficult to see how this current could be involved in the
specific location (or maturation) of components in the germ plasm (Jaffe &
Woodruff, 1979). Similarly, degeneration of nurse cell nuclei, cytoplasmic streaming
and other developmental processes do not seem to depend on specific extracellular
current loops, since follicles with no or little current complete the process of nurse
cell regression.
The observed heterogeneity in the current pattern clearly shows that follicles of
the same developmental stage may differ considerably with respect to their electrophysiological properties. Other parameters may also show great variability in different follicles, e.g. protein synthesis, amino acid pools (H. Gutzeit, unpublished data)
and Ca z+ concentrations (Heinrich, Kaufmann & Gutzeit, 1983). Our observation
that the rate of oogenesis affects the magnitude of the current is also likely to be the
result of changes in physiological parameters.
If extracellular currents are directly related to intra- and intercellular transport
phenomena the transport efficiency must be widely different in different follicles.
Cytoplasmic streaming from the nurse cells to the oocyte at stages 10B—12 (Gutzeit &
Koppa, 1982) is the only form of transport that can be visualized by direct observation (time-lapse films). This process does not depend on extracellular currents nor
do the currents depend on cytoplasmic streaming from nurse cells to the oocyte or in
the oocyte, as shown by inhibition experiments with cytochalasin B or colchicine.
Cytochalasin B also inhibits elongation and cytoplasmic streaming in pollen tubes
but has no effect on the current (Weisenseel et al. 1975).
During stage 11, when cytoplasmic streaming is strongest, large extracellular
currents could frequently be observed in the region where some specialized follicle
cells migrate centripetally between nurse cells and oocyte (see Fig. 3 I - L ) . Recently,
we reported that subsets of these cells are clearly differentiated and can be
204
jf. Bohrmann, A. Dorn, K. Sander and H. Gutzeit
distinguished from neighbouring follicle cells by increased levels of calcium
(Heinrich & Gutzeit, 1985) and by ultrastructural features (Heinrich, 1985).
Whether these cells play a role in the inferred intrafollicular current loop remains to
be shown.
Unfortunately, it is very difficult to distinguish experimentally between currents
generated by the germ-line cells and those that may be generated by the follicle cells.
Therefore, intrafollicular current loops cannot be predicted reliably from the
extracellular current pattern alone. The measurements on naked nurse cells suggest
that current enters the nurse cells. This contrasts with measurements on Hyalophora
follicles, in which inward current at the nurse cell end of the follicle appears to be due
to the presence of the follicular epithelium: naked nurse cells in Hyalophora produce
an outward current (Woodruff et al. 1983). Although we are sure that the naked
nurse cells we studied in Drosophila have not been punctured by the probe or during
preparation, we cannot completely rule out the possibility that injury currents
(perhaps from the follicle cells) contribute to the observed current pattern. However,
in a follicle in which no extracellular current could be detected, 'denuding' of a nurse
cell did not lead to the local induction of an injury current, while deliberate
puncturing of such follicles did elicit the typically observed injury current. The
measurements on a naked oocyte suggest that the extracellular currents are produced
by the columnar epithelial cells surrounding the oocyte rather than by the oocyte
itself.
Overall & Jaffe (1985) reported that sodium and chloride (and possibly calcium)
ions mainly carry the currents in Drosophila follicles. It seems clear from our results
that H + does not significantly participate in the currents of Drosophila follicles (in
contrast to some plants; see, e.g., Weisenseel, Dorn & Jaffe, 1979; Brawley et al.
1984).
Jaffe & Woodruff (1979) and Overall & Jaffe (1985) calculated from the current
pattern measured in a single plane of a follicle the total current influx and efflux over
the entire surface of the follicle. Influx and efflux rarely added up to zero and the
extreme deviations that occurred were not considered. In our opinion the current
pattern of the whole follicle cannot be determined from measurements in one plane of
the follicle. First, we found that many follicles measured in different planes (dorsoventral axis rotated) exhibited different points of maximum current and different
points of current reversal. Second, in the few cases in which the pattern around the
equator (follicle standing upright) was determined, we found varying current
densities and sometimes even current loops around the equator. Third, in several
cases we found two opposite current directions parallel to the follicle axis, but in the
same plane of measurements the pattern of inward and outward current was normal.
And fourth, patterns with only outward current (see, e.g., Fig. 4D; a defect in the
probe can be excluded) can only stand for one plane of measurements, since outward
current over the total follicular surface could not possibly occur for any length of
time.
The negative intracellular potential of Drosophila follicles (approx. — 21 mV;
Bohrmann et al. 1986) and of Hyalophora follicles (approx. —40 to — 46mV;
Current pattern in Drosophila follicles
205
Telfer et al'. 1981) suggests that extracellular currents may serve to maintain this
potential. The observed inward injury current may be a consequence of this negative
intracellular potential, since rapid equilibration of the charge differences (positive
charge influx or negative charge efflux) must occur locally where the plasma
membrane was injured.
In summary, it is conceivable that the currents play a role in the maintenance of
physiological ion balance within the follicle. On present evidence, however, we
cannot exclude the possibility that extracellular currents may also play a role in
developmental processes during oogenesis, as has been suggested by other authors
(Woodruff & Telfer, 1980; De Loof, 1983), but the observed variability in the
current pattern makes this latter concept less attractive.
We thank Dr E. Huebner for helpful discussions and for critically reading the manuscript and
the Deutsche Forschungsgemeinschaft forfinancialsupport.
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{Received 30 July 1985 -Accepted 10 September 1985)