Lionel F. Jaffe

Sources of calcium in egg activation: A review and hypothesis☆

A careful reanalysis of the literature indicates that the initial mechanism of activation in sea urchin eggs is remarkably similar to the mechanism established in medaka eggs: i.e., sea urchin eggs are activated by a qualitatively and quantitatively similar calcium explosion; one which is propagated in a wave sustained by the calcium-stimulated release of calcium from internal sources. These sources are probably in the endoplasmic reticulum. An exhaustive survey of the literature reveals that a wide variety of other activating eggs in the vertebrate line also exhibit secretory waves which are propagated at about 10 microns/sec, and can thus be assumed to reflect the same basic mechanism. Activating protostome eggs on the other hand do not exhibit such waves. This and other systematic differences from deuterostomes suggest that unlike deuterostome eggs, protostome eggs are primarily activated by calcium ions which enter the cytosol from the medium, and do so in response to depolarization of the eggs plasma membrane.

CALCIUM EXPLOSIONS AS TRIGGERS OF DEVELOPMENT

A few years ago, Gilkey et al. showed that the development of medaka fish eggs begins with a free calcium explosion within the cytoplasm. This paper summarizes those findings; provides an interim report on the effects of injecting calcium and hydrogen ion buffers into medaka eggs; reviews recent evidence of similar calcium increases in other activating eggs, as well as sperm and oocytes (Table 1); and attempts to put these explosions in a broader context (Figures 1 and 4).

The major growth current through lily pollen tubes enters as K(+) and leaves as H (+).

Growing lily (Lilium longiflorum Thunb.) pollen always drive a current into their tubes and out of their grains. The only external ions needed for growth (and the growth current) are K+, H+, and Ca2+. Increases in K+ immediately stimulate the current; while decreases in K+ immediately inhibit it. Comparable changes in H+ have the opposite effect; while those in Ca2+ have very little effect. We infer that most of the steady growth current is carried in by a potassium leak and out by a proton pump; but other considerations indicate that a minor, but controlling, component of the inward current consists of calcium ions.

LOCAL CATION ENTRY AND SELF‐ELECTROPHORESIS AS AN INTRACELLULAR LOCALIZATION MECHANISM*

We are concerned with the mechanisms of intracellular localization that contribute to development. How, for example, does an ameboid cell form a protrusion at one point and not a t another? How does a neuron initiate an outgrowth a t one point and not a t another? How is a neurite’s continued growth oriented? How does a plant egg or spore initiate an outgrowth at one point and not a t another? How are “vegetal” materials localized in one end of an animal egg so that it develops into gut, not skin? Genetic mechanisms have proven to have considerable generality; much of what is true of the genetics of bacteria is likewise true of man. Similarly, we expect morphogenetic mechanisms, in particular those of intracellular localization, to have much generality. Therefore, we have focused our study upon the early development of the fucoid egg. Unlike animal eggs, this common seaweed egg has no preformed animal-vegetal axis. The fucoid zygote is essentially apolar. Then, in the course of a day or less, it initiates growth at one pole, visibly polarizes, and divides into two quite different cells: a rhizoid, or attachment, cell a t the growth pole and a thallus cell a t its antipode (FIGURE 1). This first day of the fucoid egg’s development is a prototype of the localization process. We are further focusing our study upon an essentially electrical hypothesis of localization. According to this hypothesis, the plasma membrane in a growth region, or presumptive growth region, becomes relatively leaky to certain cations that are normally a t a much higher electrochemical potential outside of the cell than within it. These cations could include Caz+, MgZ+, Na+, and H+. The resultant movement of these cations into this region constitutes entry of an electrical current. Movement of this cation flux or current through the resistance of the cytoplasm under the leak will necessarily generate a cytoplasmicjeld that is relatively positive under the leaky portion of the membrane. This field will generate movement. It will tend to pull vesicles and other cytoplasmic constituents with a negative surface charge toward the leaky membrane region. This movement, in turn, may act to make the local membrane leakier, which thus provides the last link in apositive feedback loop. This loop would serve to establish and maintain localized growth, expansion, segregation. and other functions. Specifically, this movement could give such feedback by causing fusion of certain vesicles with the plasma membrane if these vesicles were themselves relatively leaky to particular cations or if they thus released substances that made preexisting parts of the membrane leaky. In our view, the mature nerve synapse may serve as a model of this hypothesis,

Electric fields and wound healing

Abstract There are many points of view from which one may consider the healing of wounded skin, several of which are dealt with in earlier chapters. We would like to focus on one aspect of wound healing that has received only scant attention: the possible role of electric fields in the migration of epithelial cells that must occur in order to heal wounded skin. This cell migration is one of the earliest signs of epithelial repair of epidermal wounds, both in mammals and in amphibians. 1 One important reason for the lack of attention to electrical aspects of skin healing can be attributed to the paucity of information, useful to an understanding of wound healing, about the mammalian skins ability to generate electrical currents. The bulk of the literature on the electrical properties of mammalian skin is concerned with psychophysiology. 2 4 There is some information about electrical properties of skin that may be relevant to wound healing. Some years ago, Herlitzka 5 confirmed DuBois-Reymonds 1860 observation 6 that about 1 microampere of current leaves small epidermal wounds made in human fingers when the wounds are immersed in saline. Much more recently. Illingworth and Barker 7 have found currents with densities ranging from 10 to 30 μA/cm 2 leaving the stumps of accidentally amputated childrens fingertips when the stumps are immersed in saline. We, working with Barker, have added to this information 8 by studying the current-generating capacity of the glabrous epidermis of the cavy, and, particularly, the gradients of electrical potential that exist in the vicinity of wounds made in such skin. Subsequently, we have begun study of wound healing in a simpler system, larval Xenopus skin. We here will review this information, present some previously unpublished observations, and speculate on the possible relevance of this information to wound healing.

Cell-wall formation in Pelvetia embryos. A freeze-fracture study

The cell-wall formation in the egg of Pelvetia fastigiata (J.G. Agardh) DeToni (Fucaceae) was studied with freeze-fracture. 1. The wall is lamellated with microfibrils approximately parallel in each lamella. The average orientation of microfibrils turns about 35° in each subsequent lamella. This slow turn gives rise to bow-shaped arcs when the wall is obliquely cross fractured. 2. The organization of the fibrils in the innermost lamellae is visualized by their imprints on the plasma membrane. These imprints are the result of both turgor pressure and adhesion of fibrils to the membrane. 3. Strings of membrane particles appear on the plasma membrane shortly after fertilization. They seem to be formed by a fertilization-induced aggregation of isolated membrane particles. Later each string comes to lie under a fibril and along its imprint. Peculiar lateral rips indicate that some strings are tightly bound to a fibril and may be involved in its orientation. 4. Wall formation in Pelvetia is marked by pronounced secretory activities. Following fertilization, the fusion of cortical vesicles and other vesicles make numerous loci in the plasma membrane. In older embryos, fibril-free patches in the plasma membrane mark the position of microfibril elongation centers in the wall matrix. Prior to germination, these elongation centers and their corresponding membrane patches reach a high density at the presumptive rhizoid end.

Polarization of fucoid eggs by steady electrical fields

Abstract Pelvetia eggs were exposed to steady electric fields from 5 hr after fertilization until their rhizoids began to grow out some 6 to 10 hr later. Eleven batches of eggs responded by initiating rhizoids towards the positive electrode; two batches responded by growing towards the negative electrode; and three grew towards the negative one in small fields and towards the positive one in higher fields. Polarization, defined as the average cosine of the outgrowth directions, was proportional to field strength up to polarization values of 50% for the positive responses and 75% for the negative ones. A voltage drop of 6 mV/cell induced 10% polarization in the positively galvanotropic batches, while 3 mV/cell did this in the negative ones. We reason that both responses are mediated by faster calcium entry at the future growth point. It is supposed to be faster there in positively galvanotropic eggs because the membrane potential, hence the driving force, is highest; in negatively galvanotropic eggs because depolarization induces an overbalancing increase in calcium permeability there.

Patterns of ionic current through Drosophila follicles and eggs

Large steady electrical currents traverse Drosophila follicles in vitro as well as permeabilized eggs. During the period of main follicle growth (stages 9-11), these currents enter the anterior or nurse cell end of the follicles. This inward current acts like a sodium ion influx with some calcium involvement. During the period of chorion formation (stages 12-14), foci of inward current also appear at the posterior, posterodorsal, and anterodorsal regions of follicles in vitro. In stage 14, the posterior in current acts like a chloride ion efflux. In preblastoderm eggs substantial currents continue to enter their anterior end; while weaker and less frequent ones enter their posterior end. We present models in which the currents during follicle growth are driven by the plasma membrane of the oocyte nurse cell syncitium; the external currents during choriogenesis are driven by the follicular epithelium; while the currents through the preblastoderm egg are driven by its plasma membrane. Measurements of pole-to-pole resistances and voltages across preblastoderm eggs indicate that the transcellular currents normally maintain a steady extracellular voltage gradient along the perivitelline space, with the anterior pole kept negative by perhaps 4 or 5 mV. The developmental significance of these currents is discussed.

Membrane potential and impedance of developing fucoid eggs.

Abstract Within 7 hr after fertilization, the fucoid eggs membrane potential rises from about −20 mV to −80 mV (Fig. 4) and within 12 hr after fertilization its conductance rises 8-fold (Fig. 7). Its changing responses to rapid individual ion concentration changes (Table 1) imply permeability changes which help explain these electrical ones: Thus the resting potential and conductance of the unfertilized egg seem to be mainly explained by pathways jointly permeable to K + , Mg 2+ , and to a lesser degree Na + ; those of the developing egg by potassium channels in parallel with less conductive chloride channels. In the unfertilized state the eggs membrane is passive; by a few hours after fertilization it responds sharply and within 1 msec to imposed currents sufficient to shift its potential by more than 20 mV; responding to outward current by reducing the resultant voltage change and to inward current by increasing it (Figs. 6 and 7). It is speculated that these responses may respectively serve to start and stop natural transcellular current surges. Finally, the scattered literature on comparable electrical changes in developing eggs, from algae to amphibians, is culled and organized (Table 7); the main features shown by the fucoid egg are remarkably general.

The ionic components of the current pulses generated by developing fucoid eggs.

Abstract Using a newly developed, extracellular vibrating electrode, we studied the ionic composition of the current pulses which traverse the developing Pelvetia embryo. External Na + , Mg 2+ , or SO 4 2− , are not needed for the first 20 min of pulsing. In fact, lowering external Na + or Mg 2+ (or K + ) actually stimulates pulsing. Since tracer studies show that Ca 2+ entry is speeded by Na + , Mg 2+ , or K + reduction, these findings suggest that Ca 2+ entry triggers pulsing. A sevenfold reduction in external Cl − raises pulse amplitudes by 60%. Moreover, Cl − is the only major ion with an equilibrium potential near the pulse reversal potential. These facts suggest that Cl − efflux carries much of the “inward” current. We propose a model for pulsing in which increased Ca 2+ within the growing tip opens Cl − channels. The resulting Cl − efflux slightly depolarizes the membrane and thus drives a balancing amount of K + out. Thus, the pulses release KCl and serve to relieve excess turgor pressure. By letting Ca 2+ into the growing tip, they should also strengthen the transcytoplasmic electrical field which is postulated to pull growth components toward this tip.