A fast block to polyspermy in frogs mediated by changes in the membrane potential

The role of the egg membrane potential in the prevention of polyspermy in Rana pipiens was studied with intracellular microelectrodes and ion-substituted media. At fertilization, the egg membrane potential shifts from a resting value of ?28 to +8 mV in a single step of less than 1 sec. A second, slower shift reaches a maximum amplitude of +17 mV; the membrane potential is positive for a total of 21 min. When the membrane potential of unfertilized eggs exposed to sperm was held at +1 to +22 mV for 30 min by injecting current through a second intracellular electrode, the initiation of the first cleavage furrow was delayed about 20 min, suggesting that the eggs were not fertilized while the membrane potential was positive. Injection of a similar amount of current after fertilization did not delay cleavage. Furthermore, fertilization in ion-substituted media suggests a correlation between the maximum amplitude of the positive-going shift and the incidence of polyspermy. Up to 25% of eggs were polyspermic when inseminated in the presence of NaI, and the maximum amplitude was reduced to ?20 mV when eggs were fertilized in 40 mM NaI. In contrast, fertilization in 40 mM NaCl reduced the maximum amplitude only to +6 mV, and produced no polyspermy. In solutions of NaBr, intermediate effects on the membrane potential and polyspermy were seen. Comparable results were obtained with the toad, Bufo americanus. We conclude that the membrane potential shift prevents polyspermy.     

Maturation and fertilization of the sea urchin oocyte: An electrophysiological study

Some electrical properties of the sea urchin oocyte during germinal vesicle breakdown (GVBD) and fertilization have been studied using two intracellular electrodes. Oocytes with distinct germinal vesicles have resting potentials of ?70 to ?90 mV and the specific membrane resistance may range from 3 to 10 kΩ·cm². Around rest the I–V relationship is concave toward the axis origin and the membrane is K⁺ selective. A second electrical state, of lower potential and higher resistance, preexists in the membrane. Following GVBD, the K⁺-selective system is lost and the oocyte attains the characteristics of the second state with a resting potential of ?10 to ?50 mV and specific membrane resistance of 10–50 kΩ·cm². At rest the I–V relationship tends to be convex toward the axis origin. The majority of sea urchin eggs (which have undergone GVBD and completed meiosis) have a resting state of ?10 to ?30 mV; 10–50 kΩ·cm². The I–V relationship around rest is convex toward the axis origin and the resting potential is sensitive to changes of Na⁺, Cl^?, and K⁺ in the external medium. There is probably no major change in the electrical properties of the oocyte during the completion of meiosis. A small percentage of eggs from suboptimal animals have high resting potentials of ?70 to ?90 mV and specific membrane resistance of 5–50 kΩ·cm². Such eggs have predominantly K⁺-selective membranes and we suggest that they are either underripe or aged. The first electrical event across the egg plasma membrane during fertilization is a step-like depolarization which occurs about 2 sec after the attachment of the fertilizing spermatozoon to the vitelline layer. There is no change—at the level of the light microscope—either in the egg surface or in the behavior of the spermatozoon until the second event, the fertilization potential (FP), is initiated 11 sec later. The cortical reaction occurs simultaneously with the FP and during the rising phase of the FP the spermatozoon stops gyrating around its point of attachment. Oocytes, which do not have cortical granules, upon insemination exhibit step events but no FP; in contrast artificially activated eggs, either spontaneous or induced by the ionophore A23187, give rise to only the FP. We suggest that the FP is the electrical result of the modification of the egg plasma membrane during cortical exocytosis.     

An electrical block is required to prevent polyspermy in eggs fertilized by natural mating of Xenopus laevis

In 27% DeBoer's saline (DBS), which yields maximum fertility rates, Xenopus eggs fertilized in vitro are monospermic, regardless of sperm concentration. One block to polyspermy (the “slow” block), described previously, occurs at the fertilization envelope that is elevated in response to the cortical reaction. This paper describes properties of an earlier, “fast” block at the plasma membrane and evaluates the functional significance of the two blocks at physiological sperm concentrations in natural mating conditions. Unfertilized eggs have a resting membrane potential of ?19 mV in 27% DBS. Fertilization triggers a rapid depolarization to +8 mV (the fertilization potential, FP); the potential remains positive for ca. 15 min. Activation of eggs with the ionophore, A23187, produces a slower but similar depolarization (the activation potential, AP). As in other amphibian eggs, the FP appears to result from a net efflux of Cl^?, since the peak of the FP (or the AP in ionophore-activated eggs) decreases as the concentration of chloride salts in the medium is increased. In 67% DBS no FP or AP is observed; eggs fertilized in 67% DBS become polyspermic and average 2 sperm entry sites per egg. In the 5–37 mM range, I^? and Br^?, but not F^?, are more effective than Cl^? in producing polyspermy. In 20 mM NaI the plasma membrane hyperpolarizes in response to sperm or ionophore; 100% levels of polyspermy and an average of 14 sperm entry sites per egg are observed. NaI does not inhibit or retard elevation of the fertilization envelope; the cortical reaction and fertilization envelope are normal in transmission electron micrographs. In 67% DBS, which also inhibits the fast block, the slow block was estimated to become functional 6–8 min after insemination. Eggs fertilized by natural mating in 20 mM NaI exhibit polyspermy levels of 50–90% and average 5 sperm entry sites per egg. Since eggs become polyspermic when fertilized by natural mating under conditions that inhibit the fast, but not the slow, block to polyspermy, we conclude that the fast block is essential to the prevention of polyspermy at the sperm concentrations normally encountered by the egg.     

Changes in membrane potential during mouse egg development

The electrical membrane potential (E_m) was measured in the developing mouse egg with intracellular microelectrodes. The oocyte had a low negative E_m of ?8.3 ± 0.8 mV (mean ± SE) when immature, which decreased and reversed polarity to a small positive value (+1.9 ± 0.2 mV) in the mature ovulated oocyte. After fertilization E_m returned to a negative value (?9.2 ± 0.5 mV) similar in magnitude to that observed in immature oocytes and then increased significantly (P < 0.01) at both the two-cell (?10.7 ± 0.3 mV) and morula stage (?12.8 ± 0.7 mV) and leveled out at the blastocyst stage (?12.9 ± 0.7 mV). Average potential difference recorded across the blastocoele wall of not fully expanded blastocysts was ?5.0 ± 0.5 mV. These data represent the first report on membrane potentials of the mammalian egg during development. A striking similarity is seen in the relative changes in E_m throughout development of the mouse egg in comparison to those seen in other invertebrate and vertebrate eggs.     

The electrical changes accompanying fertilization and cortical vesicle secretion in the medaka egg

The electrical properties of the egg of the medaka, Oryzias latipes, were studied before, during, and after fertilization. The resting potential of the unfertilized egg averaged ?39 ± 9 mV in Yamamoto's Ringers (Y. Ringers), but 20% of the values were between ?50 and ?60 mV. Fertilization triggers a small depolarization of 4 ± 3 mV in 10% Y. Ringers with an average duration of 20 ± 10 sec. The amplitude of this depolarization is independent of [Na⁺]_o, [Ca²⁺]_o, and [Cl^?]_o, so it appears to be due to a nonspecific leak triggered by sperm-egg fusion. The depolarization is followed by a longer hyperpolarizing phase with an average amplitude of 31 ± 12 mV. Recovery from this hyperpolarization has a fast phase lasting 155 ± 18 sec, followed by a slower phase which reaches a steady average membrane potential of ?19 ± 1 mV by 9 min after fertilization. The membrane resistance falls 10-fold during the first 2 min after fertilization, from 40 (1520 kΩ-cm²) to 3 MΩ. This is largely due to an increase in the K⁺ conductance. At the peak of the hyperpolarization, the membrane potential exhibits a 28 mV/decade [K⁺]_o dependence and a 6 mV/decade [Na⁺]_o dependence. The membrane resistance slowly recovers over the next 8 min to a value about 30% larger than before fertilization. The relation of current vs voltage was linear before, during, and after fertilization and indicated a reversal potential of ?98 ± 20 mV for the hyperpolarization peak. The egg's capacitance averaged 0.04 ± 0.01 μF (0.9 μF/cm²) before fertilization and approximately doubles within 90 sec after fertilization. It then decreases over a 9-min period, reaching a value 25% smaller than before fertilization.     

Effect of various ionic compositions upon the membrane potentials during activation of sea urchin eggs

The membrane potentials of sea urchin (Hemicentrotus pulcherrimus) eggs before and after fertilization and their changes during the membrane elevation induced by intracellular electrical stimulation were recorded in solutions of various ionic compositions. Upon fertilization, the membrane potential (?10 mV) depolarized and reversed polarity by a few mV, then gradually returned to a new steady level ranging between ?50 and ?60 mV. The activation potential is closely associated with a transient increase in the membrane permeability. The potential of the unfertilized egg is hyperpolarized by monovalent anions (Br^?, Cl^? and NO₃^?) and depolarized slightly by K⁺. In contrast, the membrane of the fertilized egg is markedly depolarized by K⁺. Suppression of depolarization associated with an increase of the membrane permeability was recorded in Na-free medium (Tris-HCl). The selective increase in permeability to monovalent anions is thought to alternate with the selective increase in permeability to K⁺through the mediation of a transient increase of Na⁺-permeability at the time of fertilization. No causal relationship between the membrane elevation and the depolarization was established because the breakdown of the cortical granules occurs without depolarization or an increase in membrane permeability.     

Fertilization in crabs: IV. The fertilization potential consists of a sustained egg membrane hyperpolarization

The electrophysiological properties of immature and mature oocytes of two crabs were analyzed. Growing immature oocytes of Carcinus maenas and fully grown immature oocytes of Maia squinado had essentially K⁺ dependent resting potentials, Em, of ?61 ? 1 mV, n=19, and ?67.3 ± 0.5 mV, n=68, respectively. Fully grown immature oocytes of Carcinus maenas showed an Em of ?40 ± 1.5 mV, n=19, that was k⁺ and Cl^? dependent. In mature oocytes of both species, the plasma membrane became exclusively permeable to cl^? and the Em attained–41 ± 1 mV, n=49 and ?34 ± 1.5 mV, n=27 for Carcinus maenas and Maia squinado, respectively. After in vitro insemination, a dramatic increase in egg membrane permeability to K⁺ was observed. This instantaneously caused a sustained hyperpolarization constituting, for these crabs, the fertilization potential. We observed that concurrently with this electrical response to fertilization, sperm reinitiated the oocyte meiotic maturation previously arrested at the first metaphase. The triggering mechanism of the fertilization potential as well as the possible occurrence of a physiological polyspermy are discussed.     

Spontaneous action potentials and resting potential shifts in fertilized eggs of the tunicate Clavelina

Electrical activity in the fertilized egg of the tunicate Clavelina was studied with microelectrode recording and voltage clamp techniques. The resting potential could assume either of two stable values (approximately ?70 or ?30 mV) and could be shifted between these values by direct current stimulation. Spontaneous shifts between two stable resting potentials were also seen. Egg cells produced action potentials spontaneously and in response to depolarizing stimuli. Inward currents were carried by both Na and Ca ions and a prominent outward potassium current was seen with depolarization to voltages above ?15 mV. The steady-state current-voltage relationship (I–V curve) of the membrane showed two voltages where the net membrane current equaled zero: approximately ?35 and ?70 mV. Between these two voltages, membrane current was inward and carried by noninactivating Na and Ca currents. Inward rectification, which was blocked by external Rb, occurred at voltages below ?70 mV. The voltage dependence of inward rectification is thought by the authors to be important for establishing the more negative resting potential; it is also thought the presence of inward current which does not inactivate completely at voltages more negative than about ?20 mV is an important determinant of the more depolarized resting potential.     

Membrane potential of the unfertilized sea urchin egg

The membrane potential, specific resistance, and potassium selectivity of the unfertilized Strongylocentrotus purpuratus egg were determined by two independent methods: tracer flux and microelectrode. The potassium influx was 0.50 ± 0.2 pmole/cm²· sec, which was greater than the sodium, chloride, and calcium influxes by factors of 4, 7, and 75, respectively. By means of the constant-field equations, the flux data were used to calculate membrane potential (?70 mV) and specific resistance (420 kΩ · cm²). The effect of the external potassium concentration on the sodium influx was determined and the results closely fit the result expected if the membrane behaved as a potassium electrode. Microelectrode measurements of the potential and resistance were ?75 ± 3 mV and 380 ± kΩ · cm².     

Some electrophysiological and permeability properties of the mouse egg

Certain electrophysiological and ionic properties of the mouse egg (CF-1 and BDF 12–18 hr post ovulation) have been investigated. Membrane potential (?14 ± 0.4 mV, ± SE, inside negative), membrane resistance (2610 ± 38 ohm·cm²), and membrane capacitance (1.6 ± 0.03 μF cm^?2) have been determined by means of intracellular microelectrode recording techniques. Membrane potential and related parameters are stable for extended periods of time upon impalement and the magnitude of the cell membrane potential has been demonstrated to be sensitive to alteration in external sodium. The electrophysiological studies in conjunction with measurements of unidirectional potassium fluxes using isotope tracer-techniques have allowed determination of membrane permeability to potassium (8 × 10^?8 cm sec^?1) and membrane potassium conductance (25 μmho cm^?2). Furthermore, the use of tracer flux techniques has indicated that the exchangeable fraction of intracellular potassium is 204 ± 14 mM. This represents the bulk of egg potassium (222 ± 19 mM as determined from flame photometry). Studies of unidirectional potassium efflux have indicated that its movement out of the egg is made up of at least two components; an external potassium-independent potassium efflux and external potassium-dependent efflux, the latter possibly representing a potassium exchange mechanism. The combined electrophysiological and tracer-flux data indicate that only a small portion of the total membrane conductance is composed of potassium conductance at this stage of development. This and the fact that the membrane potential is far from the potassium equilibrium potential are similar to observations made on mature eggs of several other species.     

External Mg2+ is required for hyperpolarization to occur in ovulated oocytes of the prawn Palaemon serratus

Immediately after dissection, the ovulated oocyte of the prawn Palaemon serratus had a resting potential E_m of −42 ± 2 mV and a membrane resistance R_m of 15 ± 5 MΩ; the membrane was more permeable to Cl⁻ than to K⁺. The oocyte spontaneously hyperpolarized and E_m gradually reached −70 mV 20–30 min after removal of the oocyte from the female, due to increased membrane permeability to K⁺. However, the hyperpolarization occured only if Mg²⁺ was present in the seawater; external Ca²⁺ was not required. Long-term incubation without external Mg²⁺ depolarized the membrane and increased membrane resistance. After preincubation in Mg²⁺-free ASW, oocytes transferred to standard artificial seawater (ASW) transiently hyperpolarized and then repolarized, before gradually hyperpolarizing to a sustained value of −62 ± mV. The respective roles of external Mg²⁺ and fertilization in eliciting the electrical response of the prawn egg at natural spawning are discussed.     

The second component of the fertilization potential in sea urchin (Pseudocentrotus depressus) eggs involves both Na+ and K+ permeability

The fertilization potential of the Pseudocentrotus depressus egg involved three transiently depolarizing components which had a different time course and a peak value. Three peaks were at less than 10 sec, 43 ± 4 sec (mean ± SD), and 182 ± 22 sec after the onset of the fertilization potential. Their peak values (mean ± SD) were 37 ± 4, 17 ± 3, and −31 ± 5 mV in standard artificial sea water. The effect of external ions on the membrane potential at the peak of the second component was measured with a conventional voltage-recording microelectrode. The peak value changed 51 mV with a 10-fold change in external Na⁺ concentration. However, it was about 65 mV more negative than the equilibrium potential of Na⁺, assuming that the internal Na⁺ concentration was 13.5 mM. H⁺, Ca²⁺, Mg²⁺, and Cl⁻ did not contribute to the peak value. The peak value was sensitive to the external K⁺ concentration. These data fitted a theoretical line obtained from the Goldman-Hodgkin-Katz equation, using a ratio of P_Na:P_K:P_Cl = 1.1:1.0:0. This means that the permeability to both Na⁺ and K⁺ is responsible for the second component of the fertilization potential. The fertilization potential was also measured in the artificial sea water containing Li⁺ or Cs⁺. The egg at the second component of the fertilization potential was almost equally permeable to Li⁺ as well as Na⁺ or K⁺ and somewhat permeable to Cs⁺. By contrast, the resting membrane potential before fertilization depended to a large extent upon K⁺ permeability.     

The electrical block to polyspermy induced by an intracellular Ca2+ increase at fertilization of the clawed frogs,Xenopus laevis and Xenopus tropicalis

Polyspermy blocking, to ensure monospermic fertilization, is necessary for normal diploid development in most animals. We have demonstrated here that monospermy in the clawed frog, Xenopus tropicalis, as well as in X. laevis, is ensured by a fast, electrical block to polyspermy on the egg plasma membrane after the entry of the first sperm, which is mediated by the positive‐going fertilization potential. An intracellular Ca²⁺ concentration ([Ca²⁺]_i) at the sperm entry site was propagated as a Ca²⁺ wave over the whole egg cytoplasm. In the X. tropicalis eggs fertilized in 10% Steinberg's solution, the positive‐going fertilization potential of +27 mV was generated by opening of Ca²⁺‐activated Cl^?‐channels (CaCCs). The fertilization was completely inhibited when the egg's membrane potential was clamped at +10 mV and 0 mV in X. tropicalis and X. laevis, respectively. In X. tropicalis, a small number of eggs were fertilized at 0 mV. In the eggs whose membrane potential was clamped below ?10 mV, a large increase in inward current, the fertilization current, was recorded and allowed polyspermy to occur. A small initial step‐like current (IS current) was observed at the beginning of the increase in the fertilization current. As the IS current was elicited soon after a small increase in [Ca²⁺]_i, this is probably mediated by the opening of CaCCs. This study not only characterized the fast and electrical polyspermy in X. tropicalis, but also explained that the initial phase of [Ca²⁺]_i increase causes IS current during the early phase of egg activation of Xenopus fertilization.     

Abundance and age of viable resting eggs of the calanoid copepod Boeckella poppei Mrázek in sediments: evidence of egg banks in two Antarctic maritime lakes

The existence of egg banks not only ensures the survival of zooplankton through harsh periods, but could also affect microevolutionary dynamics. Whether zooplankton at high latitudes can build up an egg bank in sediments, as occurs at lower latitudes, is still unknown. The distribution and age of viable resting eggs of the calanoid copepod Boeckella poppei Mrázek in sediments of two small freshwater lakes on King George Island were determined by slicing sediment cores at 1-cm intervals. Most viable resting eggs were found near the sediment surface, with abundance sharply declining to very low values at the depth of 5?cm, although eggs were present as deep as 9?cm in the sediments of Yanouhu Lake. The egg abundances in Xihu Lake and Yanouhu Lake were estimated to be 9.2?×?10⁴ and 7.2?×?10⁴?eggs?m^?2, respectively. ²¹⁰Pb dating indicated a relatively constant sedimentation rate (0.023?cm?year^?1) in Xihu Lake, which was used to estimate the mean age (46.8?year), the maximum age (195.7?year), and the mortality rate (1.64?% year^?1) of resting eggs of B. poppei in Xihu Lake. The accumulation of resting eggs with long-term viability in sediments provided the first evidence for the existence of egg banks in two Antarctic maritime lakes. An egg bank may serve as an overwintering strategy for B. poppei in Antarctica, enhance their ability to cope with random extreme changes, and contribute to their broad distribution.     

Localized defects of blastoderm formation in maternal effect mutants of Drosophila

In the three maternal effect lethal mutant strains of D. melanogaster described in this report, the homozygous mutant females produce defective eggs that cannot support normal embryonic development. The embryos from these eggs begin to develop for the first 2 hr after fertilization in an apparently normal way, forming a blastula containing a cluster of pole cells at the posterior end and a layer of syncytial blastoderm nuclei. During the subsequent transition from a syncytial to a cellular blastoderm, cell formation in the blastoderm is either partially or totally blocked. In mutant mat(3)1 no blastoderm cells are formed, indicating that there are separate genetic controls for pole cells and blastoderm cells. The other two mutants form an incomplete cellular blastoderm in which certain regions of the blastoderm remain noncellular. The noncellular region in mutant mat(3)3 is on the posterior-dorsal surface, covering about 30% of the total blastoderm. In mutant mat(3)6 blastoderm cells are formed only at the anterior and posterior ends, separated by a noncellular region that covers about 70% of the total blastoderm. The selective effects on blastoderm cell formation in the three mutants emphasize the importance of components present in the egg before fertilization for the transition from a syncytial to a cellular blastoderm.The genes defective in the three mutants are essential only for oogenesis and not for any other period of development, as indicated by a strict dependence of the lethal phenotypes on the maternal genotypes. Heterozygous embryos from the eggs of homozygous mutant females die, whereas homozygous mutant embryos from the eggs of heterozygous females develop into viable adults.One of the mutants, mat(3)3, has a temperature-sensitive phenotype. Homozygous mat(3)3 females maintained at a restrictive temperature of 29°C show the lethal maternal effect. However, at a permissive temperature of 20°C the females produce viable adult progeny. The temperature-sensitive period in mat(3)3 females occurs during the last 12 hr of oogenesis, consistent with the maternal effect phenotype of the mutant.     

Voltage clamp studies of fertilization in sea urchin eggs: I. Effect of clamped membrane potential on sperm entry,activation, and development

To analyze the role of the activation potential (a positive shift of the membrane potential which occurs following sperm attachment) in fertilization and development of the sea urchin egg, unfertilized Lytechinus variegatus eggs were voltage clamped at membrane potentials (E_m) from +20 to ?90 mV, and then inseminated. Either a fast two electrode voltage clamp, or a single electrode switched voltage clamp was used. The clamp was maintained for 3 to 15 min after initiation of a conductance increase. At E_m more positive than +18 mV, even though many sperm may attach, the egg remains completely inert (Jaffe, Nature (London)261, 68–71, 1976). At E_m from +17 to ?90 mV, all inseminated eggs elevate normal fertilization envelopes, although substantially increased concentrations of sperm are required at E_m from +17 to +12 mV. Whether cleavage occurs depends on the clamped E_m. When clamped at E_m from +17 to ?25 mV, 100% of activated eggs cleave. However, when clamped at E_m from ?26 to ?75 mV the percentage of activated eggs which cleave progressively decreases. At clamped E_m between ?76 and ?90 mV, none of the activated eggs cleave. All monospermic voltage clamped eggs that cleave develop to normal swimming blastulae. In all eggs that fail to cleave (clamped at E_m more negative than ?30 mV), sperm penetration is blocked, the sperm is lifted off the egg surface as the fertilization envelope rises, and a sperm aster never forms. Preventing formation of the fertilization envelope by prior disruption of the vitelline layer with dithiothreitol does not promote entry of the sperm. In conclusion, preventing the depolarization normally associated with fertilization suppresses sperm entry in the sea urchin egg, yet activation proceeds. Present evidence suggests an effect of the electrical field across the plasma membrane in suppressing sperm entry.     

Biophysical properties of human astrocytic brain tumor cells in cell culture

For malignant cells cultured from a human astrocytoma, electrophysiological characteristics of the plasma membrane included specific resistivity of 446.82 ± 279.5 ohm·cm², specific capacitance of 0.758 ± 0.52 microfarads/cm², time constant 0.318± 0.10 msec. The resting membrane potential averaged-14.07 ± 7.4 mV; the mean input resistance 8.1 ± 4.0 megohms. The average cell area was 1638 ± 585 ±² for contactual and 1919 ± 989 ±² for noncontactual cells. Changes in input resistance and resting membrane potential were observed with increasing time in culture, possibly reflecting cell cycling. There did not appear to be electrical coupling in this cell line.     

Decrease in the electrogenic Contribution of Na,K-ATPase and the resting membrane potential as a possible mechanism of Ca<Superscript>2+</Superscript> accumulation in rat soleus muscle in a short-term gravity unloading

The resting membrane potential and electrogenic contribution of α1- and α2-isoforms of Na⁺/K⁺-ATPase in the rat soleus muscle at early stages of gravity unloading were analyzed. The role of L-type calcium channels in accumulation of calcium ions in the myoplasm under these conditions was estimated. After 3-day antiorthostatic suspension, the resting membrane potential of the muscle fibers decreased from ?71.0 ± 0.5 to ?66.8 ± 0.7 mV, the muscle excitability reduced, and a trend of muscle fatigue acceleration appeared. The electrogenic contribution of ouabain-sensitive α2-isoform of Na⁺/K⁺-ATPase, determined as the depolarization caused by 1μM ouabain, decreased after suspension from 6.2 ± 0.6 to 0.5 ± 0.8 mV. The contribution of ouabain-resistant α1-isoform of Na⁺/K⁺-ATPase, determined as an additional depolarization after addition of 500 μM ouabain, decreased from 4.6 ± 0.6 to 2.6 ± 0.6 mV. The intensity of Fluo-4AM fluorescence in individual muscle fibers increased after suspension more than fourfold, which suggests an elevated calcium concentration in the myoplasm. A local delivery of nifedipine, a blocker of the L-type calcium channels, to the muscle removed this effect. The existence of a selective mechanism suppressing the electrogenic contribution of Na⁺/K⁺-ATPase α2-isoform, which is the main cause of the muscle fiber membrane depolarization after 3-day suspension, is postulated. The depolarization can activate part of potential-sensitive L-type Ca²⁺ channels, causing the accumulation of calcium ions in the muscle fiber myoplasm.     

The fertilization potential is not necessary for the block to polyspermy or the activation of development in the medaka egg

The egg of the medaka, Oryzias latipes, was impaled with two microelectrodes so that its membrane potential could be clamped at a constant level during fertilization. Fertilization occurred at all membrane potentials between ?80 and +48 mV. Therefore, there is apparently no electrical block to polyspermy in this egg. In 16 of these eggs the membrane potential was also clamped at a constant level during the 6- to 14-min period after fertilization and the eggs' subsequent development was studied. All of these eggs developed normally up to at least the beating heart stage. Therefore, the fertilization potential is not necessary for further development. When the egg is clamped at levels more negative than ?25 mV, the injected clamping current is usually biphasic just after fertilization with an inward current phase preceding a longer outward phase. The inward current phase corresponds well in time with the membrane depolarization normally triggered by fertilization. The outward current phase was observed in all eggs studied and the more positive the holding potential, the longer was the outward current duration.