DEVELOPMENTAL

BIOLOGY

153, 302-311 (19%)

Spatial and Temporal Transcellular Current Patterns during Oogenesis WILLIAM

DIEHL-JONES

AND

ERWIN

HUEBNER’

We have used the two-dimensional vibrating probe to examine spatial and temporal patterns in the transcellular current flow around telotrophic ovarioles of the insect Rhodni?ls proli~us. We demonstrate a dynamic pattern of currents which correlates with various stages of vitellogenesis. Asymmetries exist in the radial current pattern around intact ovarioles, particularly around the terminal follicle, and may correlate with early developmental axes. The extracellular current pattern is largely reflected by a similar, though weaker pattern of currents over the germ cell membranes, indicating that both germ cell and somatic cell membranes are involved in current generation. Current enters previtellogenic oocytes and leaves oocytes entering vitellogenesis. We speculate that current reversal and loss of trophic cord contact may represent an electrophgsiological feedback control mechanism during oogenesis. ~1 1992 Academic Press. Inc.

The unique structural/functional polarization of the telotrophic ovariole lends itself well to the question as to how private or semiprivate compartments can be maintained within a common cytoplasmic milieu (Telfer et al., 1981b) and to examination of mechanisms of intraovariole regulatory feedback and cytoplasmic transport (Huebner, 1981, 1983; Telfer et al., 1981b; Sigurdson, 1984). Possible effecters of these processes include the inherent electrical polarity of Rhodnius ovarioles. A lomV electropotential difference exists between the tropharium and the terminal oocyte (Telfer et ab, 1981a), and related to this phenomenon, microinjected basic proteins show restricted movement within the tropharium (Telfer ef al., 1981b). These properties are analogous to the situation in the polytrophic Hyalophora cecropia ovariole (Woodruff and Telfer, 1980). In addition, a dynamic pattern of extracellular currents has been described around Rhodnius ovarioles, and this generally corresponds to certain stages of oogenesis (Huebner and Sigurdson, 1986; Sigurdson, 1984).

INTRODUCTION

A central problem in developmental biology concerns the mechanisms by which pattern formation occurs (Jaffe and Woodruff, 1979), and within this framework is the question as to how tissues and cells differentiate and polarize. One of the most striking examples of developmental polarity is found in the insect meroistic ovariole: in this system, two divergent cell types-nurse cells and oocytes-maintain a high degree of structural and functional polarity despite sharing a common cytoplasm (Telfer et ab, 1981b). The telotrophic ovariole is a meroistic subtype. Endopolyploid nurse cells are located in a common, multilobed tropharium, and are in cytoplasmic continuity with all oocytes via long (up to 1 mm), microtubule-filled trophic cords (Huebner, 1981; Huebner and Anderson, 1972c; Burring, 1979). This polarization leads to significant physiological dependence of the oocyte on the nurse cells. The oocyte germinal vesicle does not manufacture RNA (Davenport, 19’74; Vanderburg, 1963) and most biosynthetic activities are performed by nurse cells (Huebner and Anderson, 1970; Davenport, 1974; Hyams and Stebbings, 1977; Capco and Jeffrey, 1979). Materials manufactured by nurse cells are transported to the oocyte via the trophic cords. Furthermore, in Rhodnius only one oocyte per ovariole is in vitellogenesis at any one time, while the remaining oocytes are arrested during previtellogenesis (Huebner and Anderson, 1972b; Pratt and Davey, 1972).

1 To whom

0012-1606/92 Copyright

correspondence

should

be addressed. 302

$5.00

:im1992 by Academic

All rights of reproduction

Before any definitive links between the extracellular current pattern and ovariole physiology can be made, several properties of these currents must still be delineated. First, Kunkel (1986) and Bowdan and Kunkel (1990) show an obvious dorsal-ventral current axis around the panoistic ovariole Blattella germanica which correlates with the future site of embryo formation. Although there are no clear markers of a dorsal-ventral axis in Rhodnius ovarioles, it is nonetheless worthwhile to determine if there are any lateral extracellular current axes. Second, panoistic and meroistic insect

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303 ovarioles are covered by a layer of follicular epithelial cells, and it has not previously been shown whether the extracellular current pattern is a property of germ cells, follicle cells, or both. The actual site(s) of current generation and the current pathways are of obvious interest, particularly in terms of resolving the apparent anomaly between the current influx over the tropharium and its relative electronegativity. Since Woodruff et al. (1986) have demonstrated that follicle cells around the trophic region of Hyalophora polytrophic follicles mask current efflux from naked nurse cells, it is important to determine if a similar situation exists in Rhod?lius telotrophic ovarioles. The purpose of this work is to analyze the current pattern around intact Rhodnius ovarioles resolvable with the two-dimensional vibrating probe and to evaluate extracellular current pattern around ovarioles in which the follicular epithelium has been removed (denuded). This study serves as a basis for further dissection of the biological and developmental relevance of transcellular ion currents in the telotrophic system. MATERIALS

AND

METHODS

Tissue. Rhodnius prolixus (Stahl) were reared as previously described (Huebner and Anderson, 1972a); ovarioles were carefully dissected into modified Rhodnius Ringer’s (O’Donnell, 1985) and the ovariole sheath was removed immediately prior to each experiment. Ovarioles may be classified according to stage of vitellogenesis on the basis of terminal (T) follicle size (Pratt and Davey, 1972). For the purposes of comparing extracellular current patterns, ovarioles from nine different size classes were examined with the vibrating probe, starting with previtellogenic ovarioles in size class 1 (T-follicle length = 300-400 pm) and increasing in lOO-pm increments until size classes 8 and 9 (late vitellogenic, T-follicle length = 1000-1500 and 1500-2000 pm, respectively). T-Follicle length was measured on a video screen that had been calibrated to account for screen curvature and geometric distortion (Inoue, 1986). Twenty positions along the length of the ovariole were chosen for current measurements on the basis of easily recognizable morphological landmarks. Positions l-10 denote equidistant positions starting at the base of the terminal (T) oocyte and ending at the T connective. Positions 11, 12, and 13 are located over the prefollicular region next to successive subterminal oocytes, and positions 14-20 denote locations starting at the base and ending at the apex of the tropharium. Measurements along the longitudinal axis were made at equidistant intervals over the circumference of each ovariole by attaching a suction pipet to the pedicel of each terminal

FIG. 1. Cutaway diagram of Rhotl~i~s ovariole illustrating the four radial quadrants based on the location of the trophic cord to the terminal oocyte. When the vibrating probe is positioned in the plane labeled 0” the trophic cord lies in the plane labeled 270”. The curved arrow around the suction pipet indicates the direction of rotation. The numbers (l-20) identify the axial recording positions along the ovariole.

follicle and rotating ovarioles 90” on the longitudinal axis (Fig. 1). A consistent reference point was the trophic cord to the T oocyte; the 0” quadrant refers to the trophic cord facing up in a recording chamber, with the vibrating probe positioned on the left side of the ovariole from a posterior-anterior perspective. Subsequent positions refer to the sides of the ovariole as it is rotated through 90,180, and 270” (see Fig. l), the 270” quadrant being the plane containing the trophic cord. When the trophic cord to the T oocyte had detached and retracted in mid- to late vitellogenic ovarioles, the trophic cord to the T-l oocyte served as a reference point. Fresh Ringer’s solution was perfused through the recording chamber each time an ovariole was rotated, or approximately every 5 min. Demded warioles. In an effort to determine the origin of the extracellular currents, the basal lamina and surrounding epithelium were removed in the following manner. Ovarioles were washed in Ca2+-free Rhod~ius Ringer’s with 2 mM ethylene glycol bis(/3-aminoethyl ether)tetraacetic acid (EGTA) for 15 min, then briefly exposed to 0.5% trypsin (type III, Sigma Chemical Co.). When the basal lamina began to separate, the ovariole was removed from the trypsin-containing Ringer’s, gently washed in Ca’+-free Ringer’s, and then adhered to an acid-washed piece of glass slide which had been coated with 1% high-molecular-weight poly-L-lysine (Sigma Chemical Co.). The basal lamina was torn with sharpened tungsten needles, and patches of the ovariole were gently “defolliculated” by rolling it over the coated surface. Recording locations over denuded patches of the T follicles were classified as being either basal, mid, or apical areas of the oocyte. Likewise, locations measured

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VOLUME 153,1992

rested on a vibration-free enclosed in a wire-mesh

FIG. 2. Schematic diagram of recording chamber with ovariole and suction pipet (SP) in place. IF, inflow port; OF, outflow port; REF, reference electrode; GND, ground electrode. Straight arrows indicate direction of medium exchange

over the tropharium were basal, extending halfway from the most basal nurse cell to the most apical nurse cell, or apical, extending halfway from the most apical nurse cell to the most basal nurse cell. Damage to the denuded germ cells was evidenced by either a large current influx (25-35 pA/cm2) at the site of injury or by a current efflux of similar magnitude over adjacent areas. Ovarioles exhibiting extracellular currents in this range were therefore discarded. Injury could best be avoided by removing only portions of the follicular epithelium, so measurements reflect currents over isolated patches of cell membrane. Current measurements were made 30 min postdissection and one representative measurement was taken from each location. If both current efflux and influx were observed within the same area, neither reading was used. Current. measurements were also rejected if the current decayed within 1 hr postdissection. Recording chamber. A custom-built recording chamber based partly on a design by A. Shipley and E. Bowdan (Marine Biological Laboratories, Woods Hole, MA) was used for all experiments. The chamber consisted of a lower specimen chamber and an upper fluid reservoir (Fig. 2). A fluid intake port in the lower compartment and a suction port in the upper compartment permitted either consistent fluid exchange via gravity feed or rapid fluid exchange via a push-pull syringe system. Platinum-blacked reference and ground electrodes were permanently incorporated into the recording chamber, thereby eliminating the need for a second, bimorph-mounted reference electrode and enabling calibration of the vibrating probe in the recording chamber. An auxiliary opening in the specimen chamber permitted introduction of a suction pipet which was used for holding ovarioles by the pedicel. Fluid temperature was monitored with a Sensortek thermocouple probe, and the entire assembly held in a jig on a plexiglass gliding stage on a Zeiss IM35 inverted phase-contrast microscope. The microscope and probe manipulator assembly

table (Micro-G, Inc.) and was Faraday cage. Current measurements. Extracellular currents were measured with a two-dimensional vibrating probe purchased from the Vibrating Probe Company (Davis, CA). Briefly, the probe tip consisted of a gold and platinum 30-pm-diameter sphere; the probe was usually vibrated at a frequency of 650-700 Hz and an amplitude of 25-30 pm. The probe was routinely calibrated and checked for artifact before each experiment and was periodically checked for capacitance and recalibrated. Probe data were plotted with the Axum Graphics program (Trimetrix, Inc., Seattle, WA) and analyzed with Instat statistical software (Graphpad Inc., San Diego, CA). Statistically significant differences between different circumferential locations at each position were determined with a one-way analysis of variance. Means were considered statistically significantly different when P < 0.05. Since a limited number of comparisons were made, P values were corrected with the Bonferroni post-test (Kleinbaum et al., 1988) to avoid type 1 errors. Each current measurement consisted of X (in-phase) and Y (quadrature) components. Bowdan and Kunkel (1990) reported that, for two-dimensional current measurements around Blattella germanica oocytes, the Y current component (ly) is a more accurate measurement of current normal to the oocyte surface than the Xcurrent component (1,) or the integrated total current (1,) value. In the present study, it was appropriate to accept I, as a measure of current, except at positions 4-7, for two reasons. First, the external geometry of Rhodnius ovarioles is markedly more complex than that of Blattella, and probe vibration along the Y axis, and hence the quadrature current (I,), could not be assumed to be most normal to the surface of the follicle, except at positions 4-7. Second, it was not technically feasible to accurately reposition the probe or ovariole to ensure normality between Xor Y axis of vibration and the ovariole surface. The use of It as a measure of current at other positions can suffer the disadvantage of skewing pure I, or I, current vectors with thereby possible tangential current components, broadening any spatial current peaks. However, this effect was in part minimized by accepting I, or I, when there were obvious tangential current components (i.e., current vectors less than 15” tangent to the surface). This was particularly relevant at the base of the T follicle (positions 1 and 2) and at the T-l follicle (position 11) of mid- to late vitellogenic ovarioles, and represents a reasonable compromise given the spatial limitations of this

particular

system.

Due to displacement of the probe along the 2 axis, errors in current direction and magnitude could result if

the probe was positioned above or below a current source (A. Shipley, National Vibrating Probe Laboratory, Woods Hole, MA); consequently, care was taken to ensure correct vertical positioning of the probe. Roth positive and negative ions can carry current, and to avoid confusion the foIlowing con~~e~tions were adopted: inward current‘ refers to either cation influx or anion effhx; outward current refers to either cation e&x or anion influx (see Nuccitelli and Jafftz, 1933.

At least two current circuits are evident in all size classes: current, exits from the base and apex of the T

follicle and returns via current influx over the middle of the T follicle, and current efflux from the T connective returns via influx of the tropharium. Overviews of the current patterns during the nine ovariole stages along the 110axiai recording positions for each radial quadrant are presented in three-dim~ns~o~~al spline plots in Fig. 3. Extra points between the actual data points aiong the size class axis are in~er~~ol~tions of the splining method for graphical purposes. Mean current densities are compiled from raw data from five to nine ovarioles per size class, and each position axial measurement per ovariol is an average of 50 vectors. With respect to qua&it&iv values that put the spline plots in perspective, the higf est total current efflux is consistentIg seen at the T eo

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nective (position lo), with a peak mean current density of 20.73 t 2.97 PA/cm’ at axial position 10, quadrant 270, size class 7 (Fig. 3d). Highest mean current influx is -8.76 + 1.19 bA/cm’, measured at axial position 6, quadrant 180, size class 5 (Fig. 3~). An advantage of the spline graphical technique is that it allows comparisons of changes in total current density and magnitude along the axial positions within any one size class as well as at any one axial position between size classes. The overall pattern of current influx and efflux is strikingly similar among radial quadrants, although there are obvious and statistically significant differences at some positions in specific radial quadrants. Current efflux over size classes at position 10 (T connective) increases gradually and linearly within quadrant 270 (Fig. 3d), while in quadrants 90 and 180, there are clear, steplike increases in mean current density between size classes (Figs. 3b and c, respectively). Moreover, there is a wavelike increase in current efllux converging on the T connective (axial position 10) over the prefollicular regions and the base of the tropharium which is evident only in quadrants 180 and 270. Since the specific values for these differences are not readily discernable, we highlight the locations and total current densities where they are significantly different (P < 0.05). In ail cases, the number of replicate measurement at each point is 5 or higher. At. axial position 2 (base of the T follicle) signi~cant decreases occur in size classes 2, 3, and 4. In size class 2, radial quadrant 0, current influx (-1.40 +- 1.04 PA/cm’) is significantly different from the current efflux (1.60 -+ 0.70 PA/cm’) in radial quadrant 180 (P = 0.034, N = 7). In class 3 ovarioles, current influx (-2.40 t 0.83 PA/cm’) in radial quadrant 180 is significantly different from the efflux (1.26 +- 1.45 @A/cm*) in radial quadrant 270 (P = 0.006, N = 9). In size class 4 current ef%x (2.54 & 1.50 ~A/crn’) at axial position 2 in radial quadrant 0 is signi~cantly different from the current influx (-1.63 rt 1.14 ~A/crn’) at radial quadrant 180 (P = 0.042, N = 7). Size class 4 (early vitellogenesis) also shows radial variations in current flux at axial position 4 (midregion of the T oocyte). Current eiRux in radial quadrant 90 (0.81 + 0.10 PA/ cm”) is significantly different from the influx (-1.25 t 0.10 PA/cm’) in radial quadrant 0 (P < 0.001, N = 7). Axial position 7 (apical end of the T oocyte) also shows significant differences between radial quadrants. In size class 4, current efilux (0.92 & 2.01 PA/em’) in quadrant 0 differs significantly from influx (-7.61 ? 1.07 pA/cm2) in quadrant 180 (P = 0.003, N = 7). In the same size class and position, efflux in quadrant 270 (3.80 + 2.21 PA/cm’) differs significantly from influx (-5.70 +2.80 PA/em”) in quadrant 180 (P = 0.004, N = 7). Finally, the only location over the rest of the ovariole that shows

~~0~~~~153,1992

significant differences between quadrants is axial position 14 (prefollicular area): current efflux (1.76 + 0.79 pA/cm2) in quadrant 0 differs from influx (-0.68 k 0.63 PA/cm’) in quadrant 180 (P = 0.028, N = 9). Besides the above indications of differences in current direction, there are also significant differences in current magnitude. In size class 4, axial position 10, current efllux (5.71 i 1.14 ~A/cm2) in radial quadrant 0 is lower than in quadrants 180 (11.00 * 1.64 ~A~crn2~ (P = 0.021, N = ‘7) and 270 (11.54 t 2.03 PA/cm’) (P = 0.028, N = 7). Most strikingly, in size class 7, position 9, current efflux in quadrant 90 (8.21 rf: 1.85 PA/cm*) is significantly lower than at quadrant 270 (15.76 + 0.68 @A/cm’) (P = 0.008, N = 5). A noteworthy feature on all four spline plots is that current efllux over the apex of the T follicle and t.he T connective is significantly elevated over vitellogenic oocytes, remains significantly elevated during VitelIogenesis, then decreases significantly in late vitellogenic-chorionatin~ oocgtes. Current efflux decreases significantly in size class 9 (completion of vitellogenesis and chorionation) at axial positions 8, 9, and 10 (apex of the T follicle and T connective) (P < 0.05). At this stage the T-l oocyte (axial position 11) is beginning vitellogenesis and exhibits current efflux in all radial quadrants.

Extracellular current direction and magnitude around denuded patches of ovarioles are reported in Fig. 4. Regions of weak current efflux are evident over the base of early, mid-, and late vitellogenic ovarioles. Current at the base of only one late vitellogenic ovariole was measured due to the fragility of large terminal oocytes at this stage. Current efflux at the base of early vitellogenic ovarioles did not differ significantly from current efYux at the base of midvitellogenic ovarioles. Weak current efllux is evident over the middle of early vitellogenic oocytes (0.27 I 0.85 pA/cm2); by contrast, current influx occurs over the middle of mid- and late vitellogenic oocytes and is strongest (12.40 +- 5.25 WA/ cm’) over the latter. Differences are significant only between early and late vitellogenic ovarioles (P = 0.003). The apices of early, mid-, and late vitellogenic oocytes all have patches of current efl?ux (Fig. 4). Current ef%x is strongest over the apex of late vitellogenic oocytes (4.85 + 1.67 pA/cm’), but no significant differences exist between early, middle, and late stages of vitellogenesis. Regions of current efflux are also detected over the trophic cord to the terminal ooeytes in early and midvitellogenesis, although these were quite variable. Current vectors are usually directed toward the T oocyte; however, angular errors may be induced by positioning the

307

a

Currents

Around (Early 5

Currents

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(Mid la

Denuded vitellogenic)

Gvorioies

Denuded

Ovorioles

kiteiiogenic)

-

f

I-

2

1

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1

FIG.

diagrams i.V

are

Currents

4. Extracellular current density next to each plot summarize indicated

above

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and direction the direction

out b

*

Denuded (Late Viteliogenic)

Around

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I

L

c

A /

12

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Ovarioles

over denuded germ cells during early (a), miti- (III, and lute cc) \Gtcllonenesis. of current flow. EITOP bars represent standard errors of the mean, Numbers

Schematic of rq)licates

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DEVELOPMENTALBIOLOGY

vibrating probe slightly above or below the plane tangent to the surface being measured. A remarkable finding concerns the current direction over denuded T-l oocytes. Extracellular current over T-l oocytes in early vitellogenic follicles is directed inward, averaging -3.62 * 1.81 PA/cm’ (Fig. 4a); current influx is also observed over denuded T-l oocytes in midvitellogenic ovarioles, averaging ~2.02 & 0.71 PA/cm2 (Fig. 4b). However, denuded T-l oocytes from late vitellogenic follicles exhibit patches of current efflux (2.99 + 1.09 pA/cm2; Fig. 4~). These currents are statistically different between early and late vitellogenic oocytes (P = 0.006) and between mid- and late vitellogenic oocytes (P < 0.001). This roughly corresponds to the current patterns observed around the same areas over intact follicles; current influx is usually measured at position 11 on ovarioles from classes 2, 6, and 7, and current efflux was consistently measured over class 9 ovarioles (Fig. 3). Extracellular current flows outward over denuded trophic cords connected to T-l oocytes (Fig. 4~). Current vectors are usually directed away from the T-l oocyte in early and midvitellogenic ovarioles and toward the T-l oocyte in late vitellogenic ovarioles, although the same restriction noted above also applied to these measurements. Current influx is obvious over patches of denuded T-2 oocytes at all stages of vitellogenesis. Three trophic cords connected to T-2 oocytes were also measured for extracellular currents. Both cords measured in midvitellogenic ovarioles exhibit tangential current efflux, whereas the single T-2 trophic cord measured in a late vitellogenic oocyte has a tangential current influx. A limitation in all of these measurements is the proximity of the T-2 cords to the base of the tropharium. The basal portions of denuded tropharia in all three stages of vitellogenic oocytes have mean positive or outward currents, and the apical portions of tropharia have mean negative or inward currents. Mean current efflux over tropharia of early vitellogenic oocytes is not significantly different from the mean current efflux over the apical portions of tropharia in the same size class. This is mainly due to the weak and variable nature of apical currents (~0.62 i 1.00 pA/cm2). However, basal current efflux and apical current influx are significantly different in mid- (P = 0.027) and late (P = 0.015) vitellogenic ovarioles. DISCUSSION

Radiul Asymmetries As stated by Bohrmann et crl. (1986), “current patterns from the whole follicle cannot be determined from mea-

VOLUME 153,1992

surements in one plane of the follicle.” A primary, novel finding of this study is that radial asymmetries exist in the extracellular current pattern around a meroistic ovariole. These asymmetries are statistically significant in previtellogenic (class l), early and midvitellogenie (classes 2, 4, and 5), and late vitellogenic (class 8) stages of development. Previtellogenic ovarioles are the only group to have statistically significant radial differences over the prefollicular region; all other significant asymmetries occur over the T follicle. The present study extends and is in general agreement with the Sigurdson (1984) study. In both the latter study and the present one, current efflux from the base and apex of the T follicle returns via current influx over the middle of the T follicle, and current efflux at the T connective completes a circuit via current influx over the tropharium. Since efflux at the T connective is sometimes directed anteriorly toward the tropharium, part of the return circuit is probably by way of the tropharium. The increase in current efflux as ovarioles enter vitellogenesis and the subsequent decline in current density in very late vitellogenic ovarioles are also very consistent between both studies. A novel finding in the present study is that the area of current influx over the T follicle is more extensive than previously believed, and demonstrates a general balance between total current influx and efflux. The morphological reference point for current measurements around the axis of Rho&Gus ovarioles is the trophic cord. The relationship between the trophic cord location and physiological-developmental axes is at present unknown. In several instances, currents enter one side of the T follicle or tropharium and exit via the opposite side of the ovariole; these phenomena cannot be consistently ascribed to any known axis and are obscured in the pooled, averaged data. It is noteworthy that Kunkel(l986) and Bowdan and Kunkel(l990) have reported a dorsal-ventral polarity in Blattella oocytes which have morphologically distinguishable axes. Rhodr~iusoocytes do not present any distinguishable morphological axes. An alternative marker is the germinal vesicle, which was used in electrophysiological measurements of Locusta, ovarioles (Verachtert, 1988). However, yolk spheres in vitellogenic Rhodnius ovarioles greatly obscure the germinal vesicle; furthermore, the germinal vesicle is usually centrally located in subterminal oocytes during early and midvitellogenesis. One possible solution to this problem may be to look for oolemmal or ooplasmic markers. There is precedence for the existence of discrete ooplasmic domains in insect eggs: RNA and protein-rich polar granules are localized in the cortical layer of the posterior pole of Drosophila

DIEHL-JONESANDHUEBNER

Trnnscellulnr

eggs (Mahowald, 1971; Hay et al., 1988). Thus far, no analogous structures are apparent in Rhodnius oocytes. The discovery of such elements in Rhodnius oocytes and a means by which to visualize them would yield another valuable positional marker and would strengthen the developmental implications. There are no conspicuous electrical polarities that suggest an obvious dorsoventral axis in Rhodnius ovarioles, although this does not preclude the existence of such axes around Rhodnius ovarioles. The asymmetries that are evident imply that at least subtle electrical polarities are associated with different developmental stages. The longitudinal pattern of transition from current influx to current efflux at the apex of the T follicle, for example, may be physiologically significant with respect to the oocyte cortical cytoskeleton which changes dramatically at this location (Graham and Huebner, in preparation). Along this line, it seems possible that current asymmetries may relate to cytoskeletal organization and possibly localization of mRNA. A basis for such speculation is provided by Larabell and Capco (1988); these authors implicate Ca2+ influx in the delocalization of poly(A)+ mRNA and tubulin mRNA in Xenopus oocytes. A role for ionic currents in general and current asymmetries in particular as effecters or modifiers of mRNA translocation and/or localization is appealing. Cytoplasmic Ca2+ and pH influence the cytoskeleton, and localized ion fluxes may influence the cytoskeleton and ultimately the location of message transcripts. Additionally, transmembrane currents may mediate mRNA localization by influencing factors binding mRNA to the cytoskeleton or by exerting an effect other than via the cytoskeleton. For example, the net negative charge of mRNA could enable its electrophoretic redistribution toward areas of current influx such as the sites over the tropharium or lateral aspects of the T oocyte. Finally, intracellular ionic concentration gradients produced by these currents could likely regulate other dynamic cellular processes central to oogenesis. Evidence to this effect will be presented in a subsequent paper on the effects of various ion substitutions on non-receptor-mediated endocytosis.

Currents around the T oocyte are generally in the same direction as those over the corresponding areas over intact ovarioles, although they are of lower magnitude. Currents over the trophic cord of the T oocyte are also consistent with the current efflux observed at the T

Current

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iw 0oqenesi.s

309

connective (position 10). Current patterns over denuded tropharia, however, are not fully compatible with the current patterns over intact tropharia. The basal current efflux extends over positions where influx is consistently observed in intact tissue; only over the apical half of the tropharium do currents mirror those measured over intact tissues. One possible explanation of this phenomenon may be that the thick basal lamina in this region offers enough resistance to effectively shunt current efflux more basally. Since these currents are manifest 30 min postdissection and are within the same order of magnitude as currents over intact ovarioles, it is unlikely they are the result of physical insult to the germ cell membrane. Perhaps the most noteworthy finding is that current direction reverses over the T-l oocyte during the transition from mid- to late vitellogenic T-l oocytes. This compares well with the change in direction at position 11 between size classes 8 and 9, and is a potential electrophysiological link with the activation of subterminal oocytes. Two possibilities suggest themselves. The first is that the reversal in current represents a change in the internal electrophoretic fields between the T-l oocyte and the tropharium. These changes may set in motion physiological processes such as increased nurse cell oocyte transport, oocyte nuclear migration, and oolemma and cortex changes associated with growth and differentiation. Alternately, such reversals in current flux probably alter the intracellular concentrations of key ions, and it may be this change that signals a change in developmental state. Justification for this idea is strong: Woodruff and Telfer (1990) show that ooplasmic pH increases and nurse cell and oocyte membrane potentials hyperpolarize in activating Hyulophoru follicles. The discovery of an apical/basal current loop around the tropharium provides a simple model for explaining intracellular potential differences between the tropharium and terminal oocyte. Basically both the terminal oocyte and tropharium maintain complete transcellular current loops. Efflux over the base and apex of the T oocyte returns via the middle of the follicle. Current efflux from the base of the tropharium is completed by influx over the tip. By maintaining separate current loops, the T oocyte and tropharium are able to maintain different intracellular potentials, thereby reconciling the net electronegativity of the tropharium relative to the T oocyte. In fact, ion inhibitor and ion substitution experiments show that the trans-tropharium current loop can be inhibited independently of the transcellular current around the T oocyte (Diehl-Jones and Huebncr, in preparation). Undoubtedly, these circuits can interact in some manner via the trophic cord, the extent of which must decrease upon its attenuation and sever-

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DE~ELOPME~ALBIOLOGY

ante. This model fits well the suggestion of Telfer et al. (1981b) that a potential gradient radiates outward from the trophic core to the nurse cell compartments. Woodruff et al. (1986) report current reversals over denuded patches of Hyalophora nurse cells, and they provide convincing evidence that the epithelium over the trophic cap forms a tight barrier to dye movement. By extension, this epithelium could theoretically block or impede current et&x from the trophocytes. Since the transbridge potential between nurse cells and oocytes is nlaintained after enzymatic and mechanical removal of the follicular epithelium, it is unlikely that the reversal of current over the trophic region is artifactual. Differences between the current patterns over denuded Hyalophora and Rhodnius trophocytes may simply represent differences between polytrophic and telotrophic ovarioles or could be related to the different procedures used for removing the follicular epithelium. Initial attempts to denude Rhodnius ovarioles according to the method of Woodruff et al. (1986) were unsuccessful, so it is not possible to compare these methods directly. On the one hand, the convincing nature of the Woodruff et aZ. (1986) data and the fact that many epithelia are morphologically adapted to the task of ion transport argue strongly for a broader role for the follicular epithelium in producing transcellular ionic currents. On the other hand, it makes electrophysiological sense that whatever current enters the follicle cells should also enter (or leave) the germ cells (see Nuccitelli, 1990), since the two are known to be well coupled in both Hyalophora (Woodruff, 1979) and Rkodnius (Huebner, 1981) ovarioles during certain periods of vitellogenesis. One other concept. to consider is whether or not the follicular epithelium offers enough resistance to effectively block current flow. Although Overall and Jaffe (1985) dispute this possibility, there is still no firm evidence either supporting or refuting it. The role of the follicular epithelium has not been directly examined herein, although it is clearly important in ovarian physiology. Early work by Wollberg et al. (1915) and Woodruff (1979) demonstrate electrical coupling between adjacent follicles in panoistic and polytrophic ovarioles, respectively, and recently Woodruff and Telfer (1990) extended observations on ~~/a‘~o~~l~a follicles to clearly demonstrate that interfollicular coupling via follicle cells plays a pivotal role in the onset of vitellogenesis. In RhodrGus ovarioles the oolemma is involved in at least a component of the extracellular current pattern. The situation is less clear with respect to the tropharium. Now that the basic current paths are known it should be possible to further explore the roles of transcellular currents in development.

~0~~~~15~,1992 We thank Karen Yeow for the drawings in Fig. 4. This research supported by the Canadian Natural Sciences and Engineering search Council grants to E.H.

was Re-

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