Using the vibrating probe technique, we have measured transcellular ionic currents ranging from 0.05 to 3.00 μA cm-2 around polar-lobe-forming embryos of Bithynia tentaculata.
During maturation and the first two mitotic division cycles, we have detected recurrent changes in the current pattern, which correlated temporally and spatially with cytokinesis and polar lobe formation. The maximum inward current at the animal pole coincides with the onset of cleavages at that pole, and the maximum inward current at the vegetal pole slightly precedes the formation of the two meiotic polar lobes and the first mitotic polar lobe at that pole. This correlation was less clear with regard to the second mitotic polar lobe. The resorption of the polar lobe correlates with a reversal of the current from inward to outward at the vegetal pole. After the resorption of the first mitotic polar lobe, the transmembrane ion flow in the membrane area, which formerly covered the lobe, is different from the surrounding membrane.
The animal-vegetal axis of the molluscan zygote originates from the apicobasal polarity of the oocyte and gives rise to the future anterior-posterior axis of the embryo. The dorsal-ventral axis, which is established later in development than the animal-vegetal axis, determines the bilateral symmetry of the embryo. Together, these two axes of polarity provide, early in development, the spatial coordinates for the molluscan body plan (van den Biggelaar and Guerrier, 1983).
During maturation and early cleavages, in embryos of some molluscan and annelid species, the animal-vegetal polarity is accentuated by the formation of a polar lobe at the vegetal pole of the zygote. The polar lobe is a cytoplasmic protuberance, which is constricted off through a process similar to cytokinesis, and temporarily set apart from the remainder of the embryo. It contains morphogenetic determinants which are indispensable for the specification of the dorsal quadrant and for mesoderm formation in the embryo (Verdonk and Gather, 1983). These determinants are progressively directed to defined cells of the embryo by fusion of the polar lobe with these cells.
The animal-vegetal polarity in polar-lobe-forming eggs is also expressed at the level of the plasma membrane. In Dentalium eggs, for instance, the plasma membrane of the microsurgically separated polar lobe appears to be electrically more excitable than that of the two remaining blastomeres without the polar lobe (Jaffe and Guerrier, 1983); in Nassarius eggs, the diffusion coefficient of membrane lipids is greater in the polar lobe area than at the animal pole (Speksnijder et al. 1985a); in the eggs of the same species, the density of intramembrane particles is considerably higher at the vegetal pole than in the animal hemisphere (Speksnijder et al. 1985b); in Bithynia, the activity of membrane-bound Ca2+/Mg2+ ATPase is localized to the membrane region around the polar lobe (Zivkovic et al. 1990). In several species, the polar lobe region also shows a distinct surface architecture such as special microvilli or surface ridges (Dohmen and van der Mey, 1977; Dohmen, 1983α). The significance of these regional surface differentiations in relation to the animal-vegetal polarity of the egg, or to the localization of morphogens at the vegetal pole, is unknown. It has been speculated that these phenomena might be instrumental in producing polar ionic currents such as have been observed in oocytes and eggs of a number of species using the vibrating probe technique (Dohmen, 1983b).
An increasing interest has recently been focussed on the possible role of extracellular electric fields and intracellular voltage gradients in polarity and localization phenomena (Nuccitelli, 1988). Intracellular gradients of specific ions, such as free cytosolic calcium, may play a role in the determination of the site of cytoplasmic localization, as shown in Fucus (Robinson and Jaffe, 1975; Robinson and Cone, 1980) and ascidian eggs (Jeffery, 1982; Bates and Jeffery, 1988). These intracellular ion gradients may be reflected in the transcellular current pattern. Especially in the case of the eggs of the brown algae, Pelvetia and Fucus, there is evidence that the transcellular ionic currents, partly carried by calcium ions, are causally related to the establishment of embryonic polarity (see Brawley and Robinson, 1985), since in these eggs the site of inward current predicts the position of the future rhizoid outgrowth (Nuccitelli, 1978).
Previous studies have shown that in Bithynia eggs, during maturation, there is a polar localization of membrane-bound Ca2+/Mg2+ ATPase that correlates with a polar pattern of ionic currents (Zivkovic et al. 1990). This Ca2+/Mg2+ ATPase may constitute an important factor in the regulation of polar transcellular ionic currents. In the present study, we provide evidence that the pattern of currents in Bithynia tentaculata embryos correlates in time and space with the formation of the polar lobe and the cleavage furrow.
Materials and methods
Embryos and their handling for vibrating probe measurements
Bithynia tentaculata is a freshwater snail. The eggs are fertilized internally. Spontaneously laid eggs were allowed to develop in their capsules until they had reached the desired stage. After decapsulation with fine forceps, they were rinsed several times in filtered copper-free tap water, which normally contains about 0.7 mM calcium. All measurements were performed at 25°C, in the lids of 35 mm Petri dishes (Costar, Cambridge, Massachusetts) filled with copper-free tap water supplemented with 2 mM CaCl2 .The specific resistivity of this medium is about 1400 Ωcm and the pH is 8.2. To perform vibrating probe measurements, the embryos had to be immobilized. Contrary to Lymnaea eggs (Zivkovic and Dohmen, 1989), the eggs of Bithynia do not spontaneously adhere to plastic or other surfaces, and they lyse immediately upon adhesion onto surfaces coated with poly-L-lysine or protamine sulphate. However, they adhere to glass coverslips coated with 2.5 mg poly-L-omithine hydrobromide, Mr=30 000–70 000 (Sigma) per ml distilled water, and subsequent development is normal. Coated coverslips were attached to. the bottom of the Petri dish with high-vacuum grease. The eggs were usually oriented with the animal-vegetal axis in the horizontal plane, but they could be detached and manipulated without adverse effects.
Measurement of extracellular currents
The magnitude and pattern of the ionic currents were measured using the extracellular vibrating probe (Jaffe and Nuccitelli, 1974) as described previously (Zivkovic and Dohmen, 1989). A one-dimensional probe system equipped with wire electrodes (Scheffey, 1988) was used. The electrodes had platinum-black tips of 15–25 μm, and the amplitude of vibration was 15–30μm. The electrodes were calibrated by measuring a known current which was passed through a glass microelectrode filled with 3M KC1. Current densities are indicated as detected at the actual measuring position, which was adjusted in such way that the center of vibration was about 40 μm away from the egg surface. The minimum recording time at one measuring site was 0.5 min with 3 s time constant of the lock-in amplifier.
Only currents normal to the surface of the embryo were measured by orienting the embryo in such a way that the probe would vibrate perpendicular to the surface. This was achieved by rotating the microscope stage. The direction of the current refers to the flow of positive charge.
Analysis of the measurements
Since there is some variability, both in the length of the cell cycles and in the peak levels of the current, the duration of the cell cycle for each particular embryo was expressed as a 100%. This relative cell cycle time was plotted on the abscissa. The currents were normalized by setting the maximum current density measured within one cell cycle for each embryo at 100%. These relative currents were then pooled for corresponding cell cycle times. The mean and the standard error of the mean were then calculated at 10% intervals of the cell cycle and these data were plotted on the ordinate as a percentage of peak current density. In the figures, inward current is designated as positive and outward current as negative.
Embryos of Bithynia form a polar lobe four times, at the first and second meiotic divisions and at the first and second cleavage. Meiotic polar lobes are only slightly constricted. The first mitotic polar lobe which has a diameter of about 20 μm is almost completely constricted off and subsequently resorbed into the CD blastomere. The second mitotic polar lobe which is only slightly constricted off is resorbed into the D macro-mere (Dohmen and Verdonk, 1979; van Dam, 1986).
When the eggs are left in their capsules, each of the first four cleavages lasts about 120 min at 25 °C, but the cleavages of decapsulated eggs are extended by 10–20 %. We assume that this extension of the cell cycle occurs in the same manner in all embryos, by the proportional prolongation of all, or corresponding cell cycle phases.
We measured ionic currents from oviposition through the fourth mitotic division cycle, when the embryo consists of 4 macromeres and 12 micromeres. Measurements were done mainly at the animal and at the vegetal pole, but other areas of the embryo were also measured regularly. Since we were primarily concerned with the temporal aspects of the current, in order to study its pattern in relation to the occurrence of the polar lobe and cytokinesis, we did not extensively explore the spatial characteristics of the electric field around the eggs by measuring the current from other areas besides the poles.
Meiotic cell cycles
First meiotic cell cycle
This period is defined as the interval between oviposition and the first meiotic anaphase (zero in Fig. 1A, B). The latter phase is externally distinguishable in the living eggs as the onset of bulging-out of the first polar body. This cycle lasts about 70 min. A few minutes before anaphase of meiosis I, the polar lobe constriction starts to appear. The earliest measurements were made 20 min before the onset of first polar body formation (Fig. 1A). During most of this 20 min period, ionic currents were inward at both poles, and ranged from 0.05 to 0.20 μA cm-2.
At the animal pole, the mean inward current showed two maxima: the first one occurred at 10 min prior to the onset of anaphase, and the second one occurred right at the onset of anaphase. The second maximum coincided with the onset of bulging-out of the first polar body (Fig. 1A).
At the vegetal pole, maximum inward current was measured at about 15 min prior to anaphase (zero in Fig. 1A). This peak coincided with the beginning of the elongation of the animal-vegetal axis of the egg, which is the first morphological change associated with the forthcoming formation of the first meiotic polar lobe (see drawings in Fig. 1A).
In two out of nine observed eggs, the first and second meiotic polar lobes did not form, whereas both first and second polar body were normally extruded. In these two eggs, the current at the vegetal pole was either zero or outward and not inward as measured in eggs that formed polar lobes. Both eggs, lysed at the onset of the first cleavage. These data suggest that the inward current measured at the vegetal pole during meiosis is somehow related to the appearance of the polar lobe.
Second meiotic cell cycle
The second meiotic cell cycle is defined as the interval between the anaphase of the first meiotic division and the anaphase of the second meiotic division, which is externally distinguishable as the onset of bulging-out of the second polar body (100% in Fig. 1B). It lasts about 50 min. This cell cycle includes the maximal constriction of the first polar body and that of the first meiotic polar lobe, which occur simultaneously at 10% of the cycle. At this point in time the egg is pear-shaped (see drawings in Fig. 1B). The resorption of the first polar lobe takes place between 10% and 25% of the cell cycle. At 70 % of the cell cycle, the second meiotic polar lobe starts to appear (see drawings in Fig. 1B). Comparable to meiosis I, the mean current was inward at both poles during most of meiosis II (Fig. 1B). The current density ranged from 0.00 to 0.30 μA cm-2. At the animal pole, the mean inward current showed two maxima. The first maximum occurred between 10% and 20 % of the cell cycle and its onset coincided with the maximal constriction of both the first polar lobe and the first polar body (10% in Fig. 1B). The second maximum, at 100 % of the cell cycle, was concomitant with the onset of second polar body formation (Fig. 1B).
At the vegetal pole too, the mean current showed two maxima: the first one coincided with the maximal constriction of the first meiotic polar lobe (until and including 10% in Fig. 1B), whereas the second one, at 60% of the cell cycle (Fig. 1B), coincided with the elongation of the animal-vegetal axis of the egg pending formation of the second meiotic polar lobe.
Summarizing, the above data show that the maximum inward current at the animal pole coincided with the formation of the polar bodies (0 in Fig. 1 A; 100 % in Fig. 1B). The maximum inward current at the vegetal pole coincided with the onset of the elongation of the animal-vegetal axis which preceded both first (—15 min in Fig. 1A) and second polar lobe formation (60 % in Fig. 1B). The maximum inward current at the vegetal pole also coincided with the maximal constriction of the first meiotic polar lobe (10% in Fig. 1B).
Mitotic cell cycles
First cell cycle
The first mitotic cell cycle is defined as the interval between the second meiotic anaphase (0 % in Fig. 2) and the onset of the first cleavage (100% in Fig. 2). In eggs in their capsules, it lasts about 120 min at 25°C.
This cycle includes the maximal constriction of the second meiotic polar lobe (at about 5 % of the cycle), its resorption (5-10% of the cycle), and the formation of the first mitotic polar lobe (80-100% of the cycle) (Fig. 2).
At the animal pole, the mean current was inward during the first 30 % and the last 5 % of the cell cycle (Fig. 2). The highest density of the inward current at the animal pole (0.10μ0.20 μA cm-2) was measured at the onset of the first cleavage (100% of the cycle in Fig. 2). The current was outward at the animal pole from 30 to 90% of the cell cycle with a maximum (0.20–0.50 μA cm-2) at 70% of the cell cycle. This maximum coincided with the shape changes of eggs prior to polar lobe formation and preceded by about 10 % of the cell cycle the onset of polar lobe formation (Fig. 2).
At the vegetal pole, the mean current was inward throughout the cell cycle, except from 10 to 30 % of the cell cycle when it was outward (Fig. 2). The onset of this period of outward current corresponds to the end of resorption of the second meiotic polar lobe at about 10% of the cycle. The inward current at the vegetal pole was maximal from 70 % to 80 % of the cell cycle (0.15 to 0.30 μA cm-2), coinciding with the maximum outward current at the animal pole, and concurrent with the shape changes preceding the appearance of the first mitotic polar lobe (Fig. 2).
Second cell cycle
The second cell cycle is defined as the interval between the beginning of the first cleavage (0% in Fig. 3) and the beginning of the second cleavage (100 % in Fig. 3). During this period a cleavage cavity is formed between the two blastomeres. The second cell cycle includes the maximal constriction of the first mitotic polar lobe (15–20% of the cell cycle), its subsequent resorption (starting after 20% of the cell cycle), and the onset of the formation of the second mitotic polar lobe (80 % of the cycle). The current densities measured during this cycle were generally higher than those in the previous cell cycles, and showed maxima ranging from 0.50 to 1.00 μA cm-2.
At the animal pole, the current was inward from 0 to 15 % of the cell cycle, coincident with the progressive constriction of the cleavage furrow and the polar lobe contractile ring (Fig. 3). Throughout the remaining period, the current was outward at the animal pole, reaching its maximum from 40 to 60 % of the cell cycle (Fig. 3). At the onset of the second cleavage this outward current decreased and even turned into inward current in some eggs, which resulted in a mean current ranging from weakly inward to weakly outward (100 % of the cycle in Fig. 3).
At the vegetal pole, the current was outward from about 25 % of the cycle onwards (Fig. 3). This is the opposite from what we observed during the first mitotic cell cycle, in which we measured inward current at the vegetal pole most of the period (Fig. 2). At 80-100 % of the cell cycle when the second mitotic polar lobe forms (Fig. 4D,E), the vegetal current was weakly outward in most embryos or no net current was measured at all (Fig. 3). The maximum inward currents occurred simultaneously at both poles at about 10 % of the cell cycle and just preceded by 5 – 10 % of the cycle both the maximal constriction of the first mitotic polar lobe and the maximal constriction of the cleavage furrow. The maximum outward current at both poles was measured at about 40% of the cell cycle. At this time, shape changes in the embryo are prominent: the blastomeres are flattening against each other and the cleavage cavity forms (Fig. 3).
The measurements from other locations on the embryo (see Fig. 4) which were all on the outline of the cross section through the animal-vegetal plane, revealed that the pattern of the current, considering both its direction and density, was bilaterally symmetric with regard to the first cleavage plane (see Fig. 4A – E). If one considers only the direction, this pattern appears during interphase to be also bilaterally symmetric with respect to the equator (Fig. 4A – C). It is interesting to note that in the interval from 40 to 60 % of the cell cycle (Fig. 4B,C), the area at the animal pole which generated outward current corresponded to the region of hyaline plasm surrounding the nuclei. At the margin of the hyaline zone, the current was zero (x in Fig. 4) and a few microns more laterally it was inward. At the vegetal pole, the current was outward in a similarly sized region (see x for zero reversal points at the vegetal hemisphere in Fig. 4B,C), and more laterally it was inward, fully symmetrical to the spatial pattern at the animal pole. The region of outward current at the vegetal hemisphere cannot be correlated, however, with any particularity in the cytoplasm. At the onset of mitosis and concurrent increase in the size of the cleavage cavity, the correspondence between zero current points and margins of hyaline plasms was lost (see x points in Fig. 4D,E).
The polar lobe area constitutes an anomaly in the bilaterally symmetric electric field. This area is positioned exactly at the vegetal pole before and at the beginning of the first cleavage. At the end of cleavage, the polar lobe is resorbed into the CD blastomere (the onset of the resorption process is shown in Fig. 4A) and this yolk-free area (which we call the polar lobe patch from now on) is readily visible in the living eggs (pip in Fig. 5), which allows the currents pertaining to this area to be measured. From about 20% of the cell cycle onwards, the current pattern at the polar lobe region was as follows. Concurrent with the progress of the polar lobe resorption, at about 20% of the cycle, the net current at the vegetal cleavage furrow was more inward than the current at the polar lobe region (Fig. 5A). From 25% of the cell cycle onwards, the current at the vegetal cleavage furrow was in most eggs outward, whereas at the same time, the current at the polar lobe patch was less outward (Fig. 5B). However, in some eggs, the current entered the vegetal cleavage furrow region. In these eggs, even stronger current entered the polar lobe patch (Fig. 5C, at 40 % and 70/80% of the cell cycle). The current pattern at the polar lobe patch differed also from the current pattern at the corresponding area in the AB blastomere, being more inward at the polar patch region (Fig. 5C). We infer from these data that, at least during the second mitotic cell cycle, after the resorption of the first mitotic polar lobe, the membrane area which formerly covered the lobe has distinct ion transport properties.
Third cell cycle
This period is defined as the interval between the beginning of the second (0 % in Fig. 6) and that of the third cleavage (100% in Fig. 6). At the animal pole the current was inward (0.05 – 1.20 μA cm-2) during the first 15 – 20% of the cycle, which corresponded to the progression of cytokinesis and the presence of the second polar lobe. During the remaining period, the current at the animal pole was outward (Fig. 6). During the last 20 % of the cell cycle, pulses of outward current were measured (up to 4.5μAcm-2), which coincided with the expulsion of the cleavage cavity fluid at the animal pole.
At the vegetal pole, the current was outward (up to 1.1 μAcm-2) throughout the cell cycle (Fig. 6), with no significant changes in the current pattern after resorption of the polar lobe (20% of the cycle).
Fourth cell cycle
At the animal pole, the current was outward throughout most of the cycle, except for the beginning (0 % and 10 – 20 % of the cycle) when it ranged between individual eggs from inward to outward, and at the end of the cycle (100% in Fig. 7), when it was weakly inward (up to 0.10μAcm2) (Fig. 7). These periods are associated with cytokinesis. During the first 10 % of the cell cycle, the current may vary extremely between different eggs, ranging from 0.50 μA cm-2 outward to 0.75μAcm-2 inward. During this period, outward current pulses were measured at the animal pole, associated with the expulsion of the cleavage cavity fluid. Although we did not include these particular peaks into the calculation of outward current, but always used for calculation the current density preceding such a pulse, the outward current measured in some eggs may have resulted from the outward current that probably accompanies a gradual leaking-out of the cleavage cavity fluid, which may have masked the inward current related to cytokinesis. In support of this possibility is the fact that at about 20 % of the cell cycle, when the emptying of the cleavage cavity is completed, the current becomes stably inward.
At the vegetal pole, the current was outward during most of the cell cycle, showing maxima (up to 3.00 μA cm-2) at about 10 %, 50 % and 90 % of the cell cycle. The outward current reached minima from 30 to 40% and from 70 to 80% of the cycle (Fig. 7).
The extracellular electric field around Bithynia embryos shows considerable resemblance to the field generated by embryos of Lymnaea stagnalis, which do not form a polar lobe (Dohmen et al. 1986; Zivkovic and Dohmen, 1989) (see Fig. 8). During meiosis and early cleavages, the embryos of both species generate polar fields with maximum current densities at the animal and vegetal pole. Current densities usually do not exceed 3 μA cm-2, except for some short pulses associated with the expulsion of the cleavage cavity contents. The maximum inward current at the animal pole is correlated, in both species, with the extrusion of the polar bodies and with cytokinesis. Short periods of inward current at the animal pole correlate with first, second and fourth cleavage. At third cleavage this correlation is less clear.
However, the current pattern also shows characteristic differences between the two species. The most conspicuous one is that, in Bithynia, the current pattern correlates temporally and spatially with the formation of the polar lobe. The maximum inward current at the vegetal pole precedes and correlates with the formation of the first three polar lobes. This correlation was less pronounced or not at all present with regard to the second mitotic polar lobe. This may be due to the fact that the second mitotic polar lobe never becomes entirely constricted.
There are two other arguments that favour the existence of a relationship between the inward current at the vegetal pole and lobe formation. The first one is that the embryos that did not form polar lobes during meiosis, did not show any inward current at the vegetal pole. The second one is based on preliminary deletion experiments (data not shown). When, in these experiments, the AB blastomere is deleted at the 2-cell stage, the CD blastomere cleaves as if it were an uncleaved egg, since a fully constricted polar lobe is formed, such as appears in undisturbed embryos during the first, but not during the second cleavage. During this (second) cell cycle, the half-embryo generates a current pattern at the vegetal pole which is almost identical to that produced by a zygote during the first mitotic cell cycle, and which differs considerably from the current pattern normally observed during the second mitotic cell cycle.
Not only the formation, but also the resorption, of the polar lobe can be correlated with a special current pattern. If we consider the first mitotic polar lobe, it appears that the inward current, which enters this region during the formation of the lobe (until about 15 % of the cycle), becomes less inward (Fig. 5A) and eventually changes its direction into outward during the resorption of the lobe (Fig. 5B). The current pattern in the area where the first polar lobe has been resorbed is distinct from that of the surrounding egg surface throughout the cell cycle, including the appearance of the second mitotic lobe. In most eggs, the current in this region was more inward or less outward as compared to the current at the cleavage furrow at the vegetal pole or at the symmetrically positioned region on the AB blastomere (Fig. 5C). This pattern allows one to detect the region where the lobe was resorbed and to predict the location of the second mitotic polar lobe before the actual bulging-out starts. This suggests that the plasma membrane that covers the polar lobe region has ion transport properties that differ from its surroundings.
If we consider some similarities and differences between current patterns around eggs of Lymnaea (Dohmen et al. 1986; Zivkovic and Dohmen, 1989; Zivkovic, 1990) and Bithynia during the first two mitotic cell cycles (Fig. 8), it appears that cytokinetic events such as cleavage and polar lobe formation may, at least in part, be related to establishing the observed current patterns. The relationship between inward current and polar lobe formation in Bithynia resembles the relationship between inward current and polar body formation in both Bithynia and Lymnaea. Polar lobes as well as polar bodies are constricted off by means of a contractile ring of microfilaments. In the eggs of both Bithynia and Lymnaea, the contractile ring associated with the formation of the polar bodies is located at the animal pole. In Bithynia, however, there is also a contractile ring activity associated with polar lobe formation at the vegetal pole. Since the first (Fig. 8A) and the second cleavage furrow (Fig. 8B) bisect the eggs of both species in the same fashion, unilaterally from the animal to the vegetal pole, the difference between Lymnaea and Bithynia is that, in Lymnaea, there is only one contractile ring, at the animal pole, whereas in Bithynia there are two rings, one at the animal and the other at the vegetal pole. The current pattern is correspondingly different in the two species, which suggests that this cytokinetic process is somehow involved in producing the inward current prior and during the visible contraction of the rings.
The underlying mechanism might be an activation of Ca2+ channels allowing the influx of Ca2+ that is needed for the contraction of the ring to occur. A direct relationship between a localized increase in cytoplasmic calcium concentration and polar lobe formation has been demonstrated by Conrad and Davis (1977), who injected calcium into Ilyanassa eggs and found that a polar-lobe-like protrusion developed, associated with the site of microinjection. It is interesting to note that the localized activity of the cytoskeleton, caused by microinjecting calcium, could never be induced at the time of polar lobe resorption in Ilyanassa, i.e. the time when in Bithynia the inward current at the vegetal pole decreases to zero and eventually reverses into outward. The authors postulate the existence of efficient systems to sequester and/or pump out calcium at the time of resorption of the polar lobe in Ilyanassa.
However, it is not known whether transmembrane Ca2+ fluxes are involved in formation and/or resorption of the polar lobe in Bithynia eggs. Experiments to be done to address this question would be the use of Ca2+ channel blockers to interfere with transmembrane Ca2+ fluxes and the direct measurements of extracellular calcium gradients resulting from such fluxes using a calcium selective extracellular vibrating electrode (Kühtreiber and Jaffe, 1990).
We thank Professor Dr N. H. Verdonk and Dr J. E. Speksnijder for their critical comments on the manuscript. The Department of Image Processing and Design is thanked for the services rendered. Special thanks are due to the Animal-Care Unit for collecting the snails and maintaining them in the laboratory.