Short Communications: Steady State Potential in the Developing Oocytes of Locusta Migratoria: Passive and Active Components

Ovarioles of the African migratory locust (Locusta migratoria) are of the panoistic type, where each oocyte is surrounded by a layer of follicle cells, and a single ovariole forms a longitudinal chain of ‘follicles’ held together by an ovariole sheath. In an earlier publication (Wollberg, Cohen & Kalina, 1976) we demonstrated that in this species, a potential difference exists across the ‘follicle’ wall (defined hereafter as ‘oocyte potential’), ranging between 30 and 55 mV (inside oocyte negative in respect to external grounded medium), with the more mature oocytes possessing larger potential differences than the less-mature oocytes. We have also shown that all the follicles along a single ovariole were electrotonically and histologically coupled by oocyte follicle cell gap junctions. The ionic basis of oocyte and egg potentials have been investigated in several animal species, invertebrates and vertebrates (e.g. Powers & Tupper, 1975; Miyazaki & Hagiwara, 1976; Shen & Steinhardt, 1976; Ziegler & Morrill, 1977). In most of these cases, oocyte membrane potentials were determined, primarily, by the passive distribution of potassium ions and to a lesser extent by sodium ions. For some species the involvement of an undefined metabolic electrogenic pump, in maintaining the steady-state oocyte potential, was proposed (Powers & Tupper, 1975; Miyazaki & Hagiwara, 1976; Ziegler & Morrill, 1977). In the present report we show that the steady-state oocyte potential of Locusta migratoria is mainly determined by the passive distribution of potassium ions and by an ouabain-sensitive electrogenic Na/K pump.

The insect material, ovariole preparation, general experimental conditions and electrophysiological techniques used in the present study were essentially the same as those described in a previous paper (Wollberg et al. lyfo). Here we describe only some modifications. The composition of the standard physiological solution was as follows (concentrations expressed in mm): NaCl 170, KC16·4, MgClg 3·5, CaCl₂ 2·1, NaHCO₃ 2·1, NaH₂PO₄ 4·3, sucrose 10. In experiments where sodium concentrations were varied, choline ions replaced the sodium. Variations in potassium concentration were achieved by replacing potassium for or by sodium ions. Osmotic pressure, ionic strength, and pH (6·3) were kept constant in all experimental solutions.

For electrophysiological measurements an isolated ovariole was stretched in a plexiglass perfusion chamber with a Sylgard-coated bottom. The preparation was continuously perfused with the experimental solutions. The effective volume of the amber was 0·5 ml and the rate of solution exchange was controlled with the aid of a peristaltic pump. The routine rate of flow of perfusion solution was about 1ml/min Potential measurements were conducted while oocytes were continuously impaled and monitored on a Tektronix dual-beam oscilloscope (Model 502 A) with a Beckman paper recorder (model RP) connected to its vertical signal output. Potential differences are expressed hereafter in mean values ± 1 S.D.

The potential difference across the wall of 65 developing oocytes (45 females) ranging in size between 0·6 and 2·9 mm (oocytes no. 1–3, numbered according to their location along the ovariole, starting with the terminal oocyte) was — 53·7 ± 10·1 mV. This value of oocyte potential is in agreement with our previous findings (Wollberg et al. 1976).

A total of 25 oocytes (20 females) were tested for the effect of reduced external sodium concentration (from 176·4 to 10 mm) on the potential difference. Most oocytes (16) disclosed some depolarization (5·0 ±3·4 mV) as a result of this treatment. Three oocytes did not show any response, and three oocytes were slightly hyperpolarized (— 2·1 ± 0·9 mV). In some of the depolarized oocytes, depolarization was preceded by a small and short transient hyperpolarization. The possibility that these three alternative effects of reduced external sodium concentration, on oocyte potentials, are randomly scattered was statistically rejected (x² test; P < 0·01, D.F. 2). Routine potential measurements across the micropipette and the agar bridge, both immersed in the experimental solutions, ruled out the possibility that we measured junction potentials.

The potassium dependence of the oocyte potential was investigated on 15 oocytes (12 females). This was done by substituting K ions with or for sodium ions. The total concentration of these two ions was kept constant so that [K]₀+[Na]₀ = 182·8 mm. Chloride concentration was kept constant. After each test solution, and before applying a new concentration, the oocyte was equilibrated in the standard physiological solution. Increasing external [K] beyond the standard concentration (6·4 mm) caused in all oocytes a depolarization the magnitude of which was related to the potassium concentration. Reducing external [K] below 6·4 mm revealed either no clear change in oocyte potential, or some depolarization. In several cases it produced a slight hyperpolarization. The depolarization which followed the increase of potassium concentration reached a new steady state within 2–4 min. When membrane potentials were plotted against log [K]₀(Fig. 1) it was found that a tenfold change of [K]_o at concentrations beyond 20 mm caused a depolarization of 50·1 ±7·4 mV. This is somewhat smaller than a slope predicted by the Nemst equation for a potassium electrode. For potassium concentrations lower than 20 mm the deviation from the predicted potassium equilibrium potential could not be explained solely by a passive contribution of other ions, less permeable than potassium, to the potential difference. This is especially true for potassium concentrations lower than 5–6· 4 mm, where in most cases a depolarization was disclosed.

A depolarization induced by reducing external potassium concentration can be explained by either the existence of a Na/K electrogenic pump which is blocked by reducing or removal of external potassium, or alternatively by a change in the relative permeability of an ion whose equilibrium potential is more positive. The first alternative was tested by studying the effect of ouabain on the oocyte potential upon adding the drug to: (a) a standard saline solution (8 oocytes, 8 females); (b) a free potassium solution (14 oocytes, 11 females); (c) when increasing [K]_o after exposure of the same 14 oocytes to a free K_o solution; and (d) when investigating the potassium dependence of the oocyte potential (4 oocytes, 4 females). Results were as follows: application of ouabain to the standard solution (a final concentration of 5 x 10−⁴ m) caused within about 12 min a depolarization of 4·2 ±3·5 mV (Fig. 20). Complete removal of external potassium (Fig. 2b) produced, within 2 · 3 min, a similar depolarization (4·7–2·2 mV). A t test for equality of means did not show any significant difference between these two results (P > 0·6, D.f. 20). In the presence of ouabain, removal of K<, does not cause any additional depolarization (Fig. 2 a), and conversely, there is virtually no response to adding ouabain once external potassium is removed (Fig. 2 c). This suggests that both the removal of K_oand ouabain act upon the same process. Replacing the potassium-free solution by a high potassium solution caused an initial transient hyperpolarization followed by a depolarization the magnitude of which was [K]₀-dependent (Fig. 2 b). This last phenomenon is generally characteristic of cells which possess an electrogenic Na/K pump and is accounted for by the reactivation of the pump in the presence of potassium ions after a period in which intracellular sodium concentrations increased (e.g. Lovengood & Kusano, 1072). Adding ouabain to the K-free solution completely eliminated the transient hyperpolarization, and the following depolarization was slightly deeper (Fig. 2 Investigation of the potassium dependence of oocyte potential, in the absence and presence of ouabain, showed that without ouabain, potentials were at all concentrations (except of [K]_o = 0) somewhat more negative (Fig. 1). These results suggest the existence of a Na/K electrogenic pump which is abolished by both ouabain and the removal of external potassium. The validity of this pump is strongly supported by the fact that reducing external sodium concentration causes in most oocytes a slight depolarization. Considering these findings it is suggested that in Locusta migratoria, oocyte steady-state potential consists of two components: (1) the passive distribution of ions, mainly potassium, and (2) a metabolic Na/K electrotonic pump which contributes about 8 · 5 % of the total negative oocyte potential

In what follows we discuss, briefly, a possible role of a Na/K pump in the process of oocyte maturation.

Several investigators suggested that specific factors which regulate oocyte maturation might be transmitted from the follicle cells to the oocytes by means of gap junctions (Anderson & Albertini, 1976; Gilula, Epstein & Beers, 1978; Browne, Wiley & Dumont, 1979). As mentioned earlier, we have shown that oocyte follicle cell gap junctions exist also in the follicles of the migratory locust. Browne et al. (1979) had demonstrated that in Xenopus laevis, uncoupling or assembly of such junctions are regulated by gonadotropic hormones.

In some tissues the maintenance of a trans-membranal sodium electrochemical gradient by an active sodium pump is essential for preservation of cell-to-cell junctions (De Mello, 1976; Cohen, 1977). In such a system the higher the Na/K coupling ratio is, the more favourable are the conditions for preserving these junctions. It is possible, thus, that the regulation of oocyte-follicle cell gap junctions by gonadotropic hormones is mediated by an active sodium pump. That possibility is consistent with some recent findings revealing that in Rhodnius prolixus the appearance of large spaces between the follicle cells and oocyte (at the final stages of oocyte development), which is induced by the gonadotropic juvenile hormone (JH), is inhibited by ouabain, and in vitro activity of an ovarian Na/K ATPase is increased by JH (Davey & Huebner, 1974; Abu-Hakima & Davey, 1979).