ELECTRIC IMPEDANCE OF ASTERIAS EGGS

The alternating current resistance and capacity of suspensions of unfertilized eggs of Asterias forbesi have been measured at frequencies from one thousand to sixteen million cycles per second. The plasma membrane of the egg has a static capacity of 1.10µf/cm.² which is practically independent of frequency. The suspensions show a capacity dependent on frequency at low frequencies which may be attributable to surface conductance. The specific resistance of the cytoplasm is between 136 and 225 ohm cm. (4 to 7 times sea water), indicating a relatively high concentration of non-electrolytes. At frequencies above one million cycles there is definite evidence of another element of which the nucleus is presumably a part.     

ELECTRIC IMPEDANCE OF SINGLE MARINE EGGS

Alternating current impedance measurements have been made on several single marine eggs over the frequency range from 1 to 2500 kilocycles per second. The eggs were placed in the center of a short capillary made by heating the end of a 2 mm. thin walled glass tube until it nearly closed, and electrodes were placed in the sea water on each side of the egg. When it is assumed that the membrane conductance is negligible, the membrane capacity and internal resistances of unfertilized and fertilized Arbacia eggs agree with the values obtained from suspensions. Preliminary data on centrifugally separated half Arbacia eggs, and whole Cumingia and Chaetopterus eggs are given.     

ELECTRIC IMPEDANCE OF FERTILIZED ARBACIA EGG SUSPENSIONS

From the low frequency alternating current impedance and the volume concentrations of suspensions of Arbacia eggs, it is shown that the high resistance membrane is either at or very near the plasma membrane for both unfertilized and fertilized eggs, and that the specific resistances of the perivitelline space and fertilization membrane are not greatly different from that of sea water. The effect of the capacity element which appears after fertilization at intermediate frequencies is considerably less than in the earlier experiments on Arbacia and Hipponoë eggs. These findings indicate that the fertilization membrane does not have the high capacity previously attributed to it and that the increase in membrane capacity takes place at or near the plasma membrane.     

ELECTRIC IMPEDANCE OF ARBACIA EGGS

The alternating current resistance and capacity of suspensions of unfertilized and fertilized eggs of Arbacia punctulata have been measured at frequencies from 10³ to 1.64 x 10⁷ cycles per second. The unfertilized egg has a static plasma membrane capacity of 0.73 µf./cm.² which is practically independent of frequency. The fertilized egg has a static membrane capacity of 3.1 µf./cm.² at low frequencies which decreases to a value of 0.55 µf./cm.² at high frequencies. The decrease follows closely the relaxation dispersion of the dielectric constant if the dissipation of such a system is ignored. It is considered more probable that the effect is due to a fertilization membrane of 3.1 µf./cm.² capacity lifted 1.5 µ. from the plasma membrane, the interspace having the conductivity of sea water. The suspensions show a frequency-dependent capacity at low frequencies which may be attributable to surface conductance. The equivalent low frequency internal specific resistance of both the unfertilized and fertilized egg is about 186 ohm cm. or about 6 times that of sea water, while the high frequency data extrapolate to a value of about 4 times sea water. There is evidence at the highest frequencies that the current is penetrating the nucleus and other materials in the cytoplasm. If this effect were entirely due to the nucleus it would lead to a very approximate value of 0.1 µf./cm.² for the capacity of the nuclear membrane. The measurements do not indicate any change in this effect on fertilization.     

ELECTRIC IMPEDANCE OF THE FROG EGG

Electrical impedance measurements were made upon unfertilized and fertilized eggs of the leopard frog, Rana pipiens, over a frequency range of 0.05 to 10 kc. Average values of 170 ohm cm.² were obtained for the plasma membrane resistance of the egg, 2.0 µf/cm.² for the plasma membrane capacity, 86° for the phase angle of the membrane, and 570 ohm cm. for the specific resistance of the interior. These values did not change upon fertilization. No spontaneous rhythmical impedance changes such as have been found by Hubbard and Rothschild in the trout egg were found in frog eggs.     

MAGNESIUM ION-REQUIRING STEP IN FERTILIZATION OF SEA URCHINS*

The magnesium ion-requiring step in fertilization of sea urchins was investigated. When eggs were inseminated in Mg-free sea water, several spermatozoa were found to bind to each egg surface with their reacted acrosomes without elevation of fertilization membrane. The number of binding jelly-treated spermatozoa to an egg did not differ regardless of the presence or virtual absence of magnesium ions. Although fertilization did not occur in Ca, Mg-deficient sea water (CM-deficient SW) even when jelly-treated spermatozoa were employed, some eggs could be fertilized by the addition of magnesium to the CM-deficient SW 60 sec after insemination, when jelly-treated spermatozoa had completely lost their fertilizing capacity in the CM-deficient SW. The acrosomal process of jelly-treated spermatozoa appeared to penetrate the vitelline layer in the CM-deficient SW. DTT- or pancreatin-treated eggs could not be fertilized in the virtual absence of magnesium. Re-fertilization using the fertilized eggs deprived of fertilization membrane did not occur under conditions of magnesium deficiency. These results suggest that external magnesium ions are indispensable at least for the fertilization process following penetration of the vitelline layer by the spermatozoa, such as fusion of the plasma membrane between an egg and a reacted spermatozoon, or the subsequent step(s) such as sperm penetration into egg interior and egg activation which precedes the cortical reaction.     

ELECTRIC IMPEDANCE OF SUSPENSIONS OF SPHERES

A general expression has been derived for the electric impedance of a suspension of spheres each having a homogeneous non-reactive interior and a thin surface layer with both resistance and reactance. The applications and limitations of impedance measurements on such suspensions are discussed.     

THE EFFECT OF HYDROGEN ION CONCENTRATION UPON THE INDUCTION OF POLARITY IN FUCUS EGGS : II. THE EFFECT OF DIFFUSION GRADIENTS BROUGHT ABOUT BY EGGS IN CAPILLARY TUBES

1. When a Fucus egg develops near one end in a close fitting capillary tube of pyrex glass or silica (quartz), diffusion of substances passing to and from the egg is more impeded on the side of the egg toward the far end of the tube. 2. The egg therefore develops in a gradient of its own diffusion products, and of oxygen tension. 3. More than 600 eggs have been reared, each near one end in a capillary, in sea water at various regulated and measured pH values. 4. When the medium, which is initially homogeneous inside and outside the capillary, is initially at pH 6.5 to 7.6, nearly all of the eggs develop rhizoid protuberances on the sides of the eggs toward the far ends of the capillaries. This is on the sides of the eggs where the concentration of substances diffusing from the eggs is greatest. 5. The polarity and developmental pattern of the egg is thus determined either by a concentration gradient of products diffusing from it, or by a gradient of oxygen tension. The former interpretation is favored. 6. This is regarded as an extension of earlier observations that rhizoid protuberances form on the sides of two neighboring eggs in the direction of the neighbor if the sea water is acidified. 7. It appears hardly possible that a mitogenetic effect could be responsible for the response of an egg to its own diffusion gradients. 8. When the medium is made more basic, the percentage of the eggs which form rhizoid protuberances toward the far end of the tube decreases to about 20 or 25 per cent between pH 8.1 and 8.6. The response of the egg to the gradients which it produces is thus statistically reversed. The determination of the polarity of the eggs by the diffusion gradients does not become as complete in alkalinized as in acidified sea water. 9. When the pH of the sea water is elevated to 9.1 or 9.2, salts precipitate out. The type of development is altered and the control of the diffusion gradients over the polarity of the eggs decreases.     

THE EFFECT OF HYDROGEN ION CONCENTRATION UPON THE INDUCTION OF POLARITY IN FUCUS EGGS : I. INCREASED HYDROGEN ION CONCENTRATION AND THE INTENSITY OF MUTUAL INDUCTIONS BY NEIGHBORING EGGS OF FUCUS FURCATUS

1. The eggs of Fucus furcatus develop perfectly in sea water acidified to pH 6.0. They are retarded at pH 5.5. At pH 5.0 they do not develop, nor do they cytolize. 2. In normal sea water in the dark at 15°C., eggs develop rhizoids on the sides in the resultant direction of a mass of neighboring eggs. The polarity and the whole developmental pattern of the embryo is thereby induced. This inductive effect does not operate, however, unless the directing mass is an appreciable aggregation of cells (10 or more), or unless there are numerous other eggs in the dish. A group of five eggs alone in a dish do not carry out mutual inductions. Two eggs alone in a dish do not develop rhizoids toward each other. 3. When the sea water is acidified to pH 6.0 all sizes of aggregations carry out mutual inductions. Two eggs alone in a dish now develop rhizoids on the sides toward each other, provided they are not more than about 4 egg diameters apart. 4. Increased hydrogen ion concentration thus augments or intensifies the mutual inductive effect. 5. This may explain why only larger masses of eggs show inductions in normal sea water, since presumably the larger masses considerably increase the hydrogen ion concentration locally. 6. The nature of the inductive action is discussed. 7. In acidified sea water at pH 6.0, compared with normal sea water at pH 7.8–8.0, the rhizoids originate and extend with a strongly increased downward component. The substrate then forces further extension or growth of the rhizoid to be in the plane of the substrate.     

ELECTRON MICROSCOPIC DEMONSTRATION OF A DITHIOTHREITOL-LABILE VITELLINE COAT SURROUNDING THE UNFERTILIZED EGG OF COMANTHUS JAPONICA (ECHINODERMATA: CRINOIDEA)*

In Comanthus, the unfertilized egg is surrounded by a vitelline coat, which is separated from the underlying plasma membrane by a space several hundred Ångstroms wide. By electron microscopy, the vitelline coat is a distinct layer 100 to 150 Å thick, which consists of finely granular material of moderate electron density. Treatment for 3 min in 0.01 M dithiothreitol in sea water buffered to pH 9.2 almost completely removes the vitelline coat and causes the irregularly shaped egg to become spherical. After such DTT-treated eggs have been washed for 2 min in sea water, they cannot be fertilized, but they can undergo a cortical reaction when treated with ionophore A23187. This cortical reaction consists of the exocytosis of cortical granule material directly into the surrounding sea water. By several hours after DTT treatment, most of the eggs, whether exposed to ionophore or not, fragment into spheres of diverse sizes.     

TRANSVERSE ELECTRIC IMPEDANCE OF THE SQUID GIANT AXON

The impedance of the excised giant axon from hindmost stellar nerve of Loligo pealii has been measured over the frequency range from 1 to 2500 kilocycles per second. The measurements have been made with the current flow perpendicular to the axis of the axon to permit a relatively simple analysis of the data. It has been found that the axon membrane has a polarization impedance with an average phase angle of 76° and an average capacity of 1.1µf./cm² at 1 kilocycle. The direct current resistance of the membrane could not be measured, but was greater than 3 ohm cm.² and the average internal specific resistance was four times that of sea water. There was no detectable change in the membrane impedance when the axon lost excitability, but some time later it decreased to zero.     

An ultrastructural immunocytochemical localization of hyalin in the sea urchin egg

The fertilized sea urchin egg is invested by the hyaline layer, a thick extracellular coat which is necessary for normal development. On the basis of ultrastructural studies and the fact that hyalin is released during the time of the cortical reaction, it has been generally accepted that hyalin is derived from the cortical granules. However, this has never been proven definitely, and recently, it has been reported that hyalin is a membrane and/or cell surface protein. To determine where hyalin is stored, we carried out an ultrastructural immunocytochemical localization of hyalin in the unfertilized egg. Hyalin purified from isolated hyaline layers was used to immunize rabbits. Antisera so obtained were shown to be hyalin specific following absorption with a combination of sea urchin proteins. Immunocytochemical localizations were carried out on sections of Epon-embedded material using protein A-coated gold particles as an antibody marker. Our results demonstrate that, prior to fertilization, hyalin is stored in the homogeneous component of the cortical granule in Strongylocentrotus droebachiensis and Strongylocentrotus purpuratus. Labeling of small cortical vesicles in both unfertilized and fertilized eggs, suggests that these vesicles may contain a secondary reservoir of hyalin.     

SPECIES-SPECIFIC ADHESION OF SPERMATOZOA TO THE SURFACE OF FIXED EGGS IN SEA URCHINS

When the spermatozoa of sea urchins are added to eggs which have been fixed with glutaraldehyde and washed thoroughly, the spermatozoa swarm around the eggs and adhere to the egg surface. The mode of sperm adhesion to the fixed egg is assumed, on the evidence of electron-microscopical studies, to be the same as that of adhesion to the intact egg at the initial stage of normal fertilization. The spermatozoa and fixed eggs of five species of sea urchins were combined and heterologous crosses were studied. Species-specific adhesion of sperm to fixed eggs was clearly demonstrated. There is a direct relationship between the cross-fertilization of living gametes and the binding capacity of spermatozoa and fixed eggs in so far as the employed five species are concerned.     

Does egg dispersal occur via the ocean in the stick insect Megacrania tsudai (Phasmida: Phasmatidae)?

Although insects expand their distribution by various ways, generally only the adult phase has been taken into consideration in research on dispersal. In Megacrania tsudai, it has been proposed that eggs are dispersed through seawater. To test this hypothesis, eggs were treated under normal condition (NC) on wet cotton swabs, and marine condition (MC), floating on salt water for 30, 60, 90, and 365 days. In addition, eggs in the NC and MC treatment groups were dissected every 10 days to verify the developmental stage. The hatching rates in the NC and MC treatment groups were not significantly different among the five treatment groups. However, the egg period, time from laying to hatching, in the MC treatment group was significantly longer than that in any other treatment groups. The egg period was lengthened when the floating period on seawater was longer. The time of the start of egg development was similar in the NC and MC treatment groups, but the developmental speed was slower in the MC treatment group. These results support that M. tsudai can expand its distribution by dispersing its eggs through seawater, probably thanks to specific characteristics of eggs that allow their survival when they float in the sea.     

THE MEMBRANE CAPACITANCE OF THE SEA URCHIN EGG

1. The surface of the unfertilized sea urchin egg is folded and the folds are reversibly eliminated by exposing the egg to hypotonic sea water. If the plasma membrane is outside the layer of cortical granules, unfolding may explain why the membrane capacitance per unit area decreases (and does not increase) when a sea urchin egg is put into hypotonic sea water. 2. The degree of surface folding markedly increases after fertilization, which provides an explanation for the increase in membrane capacitance per unit area observed after fertilization. 3. The percentage reduction in membrane folding in fertilized eggs after immersion in hypotonic sea water is probably sufficient to explain the decrease in membrane capacitance per unit area observed in these conditions.     

OSMOTIC PROPERTIES OF THE EGG CELLS OF THE OYSTER (OSTREA VIRGINICA)

Investigations of the osmotic properties of oyster eggs by a diffraction method for measuring volumes have led to the following conclusions: 1. The product of cell volume and osmotic pressure is approximately constant, if allowance is made for osmotically inactive cell contents (law of Boyle-van''t Hoff). The space occupied by osmotically inactive averages 44 per cent of cell volume. 2. Volume changes over a wide range of pressures are reversible, indicating that the semipermeability of the cell during such changes remains intact. 3. The kinetics of endosmosis and of exosmosis are described by the equation, See PDF for Equation, where dV is rate of volume change; S, surface area of cell, (P-P_e), the difference in osmotic pressure between cell interior and medium, and K, the permeability of the cell to water. 4. Permeability to water during endosmosis is 0.6µ³ of water per minute, per square micron of cell surface, per atmosphere of pressure. The value of permeability for exosmosis is closely the same; in this respect the egg cell of the oyster appears to be a more perfect osmometer than the other marine cells which have been studied. Permeability to water computed by the equation given above is in good agreement with computations by the entirely different method devised by Jacobs. 5. Permeability to diethylene glycol averages 27.2, and to glycerol 20.7. These values express the number of mols x 10^–15 which enter per minute through each square micron of cell surface at a concentration difference of 1 mol per liter and a temperature of 22.5°C. 6. Values for permeability to water and to the solutes tested are considerably higher for the oyster egg than for other forms of marine eggs previously examined. 7. The oyster egg because of its high degree of permeability is a natural osmometer particularly suitable for the study of the less readily penetrating solutes.     

Sea urchin egg hyaline layer: evidence for the localization of hyalin on the unfertilized egg surface

The sea urchin embryo hyaline layer is an extracellular investment which develops within 20 min postinsemination of Strongylocentrotus purpuratus eggs and contains a single calcium-precipitable subunit termed hyalin. Other ultrastructural and biochemical studies have suggested that hyalin is localized in the cortical granules. We have examined the hypothesis that hyalin is a cell surface protein of the unfertilized egg using vectorial lactoperoxidase-catalyzed radioiodination. Extracts of labeled unfertilized eggs contained several labeled proteins, one of which was electrophoretically indistinguishable from authentic hyalin isolated by each of three different procedures. Pronase digestion of labeled unfertilized eggs removed 75% of the label, but the labeled hyalin-like molecule was still present in whole cell extracts. Upon insemination, pronase-digested, labeled eggs formed an apparently normal hyaline layer and whole cell extracts contained the labeled hyalin-like molecule. Denuded, labeled eggs were inseminated and the hyaline layer was selectively solubilized in calcium- and magnesium-free artificial seawater. Labeled hyalin was purified from this crude hyalin preparation to constant specific radioactivity and apparent homogeneity as shown by gel electrophoresis. These data strongly suggest that hyalin or a precursor is a cell surface protein of the unfertilized sea urchin egg.     

Maternal messenger RNA and histone synthesis in embryos of the surf clam Spisula solidissima.

Messenger RNA has been isolated from the postribosomal supernatant of Spisula solidissima eggs. This mRNA directs the synthesis of several proteins when added to the ascites or wheat germ cell free system. No histone except F1 is coded for by Spisula egg mRNA, in contrast to what has been reported previously for sea urchin egg mRNA. In sea urchin eggs histone mRNA is among the abundant species of maternal mRNA.Histones have been prepared from Spisula embryos at different development stages and histone synthesis followed by incubation with (¹⁴C)lysine. The analysis by electrophoresis on acrylamide gels indicates that the pattern of synthesis of histones changes during development and that a new histone F1 fraction is actively synthesized from the 32–64 cells stage. In earlier embryos a different F1 histone is synthesized and the mRNA for this protein may be the only histone mRNA present in eggs.     

STABILITY OF SUSPENSIONS OF SOLID PARTICLES OF PROTEINS AND PROTECTIVE ACTION OF COLLOIDS

1. It is shown that the concentrations of different salts required to precipitate suspensions of gelatin-coated collodion particles in water are practically identical with the concentrations of the same salts required for the "salting out" of gelatin from aqueous solutions. Neither effect shows any relation to the electrical double layers surrounding the particles. 2. It is shown that at the isoelectric point of gelatin, suspensions of gelatin-coated collodion particles are not stable and it had been shown previously that gelatin is least soluble at the isoelectric point. The addition of salt increases both the solubility of gelatin in water as well as the stability of suspensions of gelatin-coated collodion particles in water, and both effects increase with the valency of one of the ions of the salt. 3. This latter effect is not due to any charges conferred on the gelatin particles by the salts, since the cataphoretic experiments show that salts like NaCl, Na₂SO₄, or CaCl₂, which at the isoelectric point of gelatin increase the solubility of gelatin as well as the stability of suspensions of gelatin-coated collodion particles, leave the particles practically uncharged in the concentrations in which the salts are efficient. 4. It follows from all these facts that the stability of suspensions of gelatin-coated particles in water depends on the solubility of gelatin in water; e.g., on the chemical affinity of certain groups of the gelatin molecule for water. 5. Though crystalline egg albumin is highly soluble in water, the stability of collodion particles coated with crystalline egg albumin does not depend upon the affinity of the albumin molecule for water, but depends practically alone on the electrical double layer surrounding each particle. As soon as the P.D. of this double layer falls below 13 millivolts, the suspension is no longer stable. 6. The critical potential for the stability of suspensions of collodion particles coated with genuine egg albumin is the same as that for particles of boiled (denatured) white of egg. Since through the process of heating, egg albumin loses its solubility in water, it is inferred that egg albumin undergoes the same change when it forms a film around a solid particle like collodion. 7. The influence of electrolytes on the stability of suspensions of collodion particles coated with casein or edestin was similar to that of collodion particles coated with egg albumin. The experiments are, however, complicated by the fact that near the isoelectric point CaCl₂ and even NaCl cause a suspension again at concentrations of about M/2 or 1 M, while still higher concentrations may cause a precipitation again. These latter effects have no connection with double layers, but belong probably in the category of solubility phenomena. 8. These experiments permit us to define more definitely the conditions for a general protective action of colloids. Protective colloids must be capable of forming a durable film on the surface of the suspended particles and the molecules constituting the film must have a higher attraction for the molecules of the solvent than for each other; in other words, they must possess true solubility. Only in this case can they prevent the precipitating action of low concentrations of electrolytes on particles which are kept in suspension solely by the high potentials of an electrical double layer. Thus gelatin films, in which the attraction of the molecules for water is preserved, have a general protective action, while crystalline egg albumin, casein, and edestin, which seem to lose their attraction for water when forming a film, have a protective action only under limited conditions stated in the paper.     

Intracellular pH controls the development of new potassium conductance after fertilization of the sea urchin egg

Fertilization of the sea urchin egg leads to a sequence of changes at the egg surface and the interior cytoplasm. Among these changes are the transient elevation of internal calcium levels, alkalization of the cytoplasm and development of new K⁺-conductance. In the series of experiments reported here, we separate the effects on potassium activation of the calcium release and the rise in the intracellular pH. The development of new K⁺-conductance was dependent on alkalization of the egg cytoplasm, and not on a rise of internal calcium levels. The effects of 2,4-dinitrophenol, N-ethylmalemide, antimycin A and oligomycin suggest that the maintenance of the alkaline internal pH of fertilized eggs appears to be dependent on membrane ATPase activity.