Forced calcium entry and polarized growth of Funaria spores

We have used both steady electric fields, and gradients of the divalent ionophore, A23187, to control the point at which rhizoids emerge from spores of the common moss Funaria hygrometrica. The spores were grown in a medium containing calcium nitrate as the only major salt. Spores tend to form rhizoids towards the positive electrode, with a half maximal response to a difference of 4–8 mV across each cell. They also tend to form rhizoids towards the end of higher ionophore concentration in response to A23187 gradients. Both of these responses are the same at pH 5.5 and 8.0. Our tentative explanation is that Funaria spores tend to form rhizoids where most calcium enters. However, the point of chloronema emergence is scarcely affected by steady fields of up to 45 mV/cell. Moreover, when steady fields are applied across already developed rhizoids or chloronemata, their subsequent growth is directed towards the negative electrode in both cases, with rhizoids giving a 50% response at only 3—5 mV/cell, and chloronemata being less responsive.From Tsung-Hsien Chen's Ph.D. thesis, Purdue University     

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.     

The responses of pollen to applied electrical fields

The growing pollen tubes of tomato and tobacco were exposed to electrical fields and the effect on the direction of growth was measured. They responded by turning toward the positive electrode (anode) and gave a detectable response in fields as small as 0.1 mV/tube diameter. The sites of germination of tobacco pollen grains were also affected by applied fields; the pollen tubes tended to emerge from the anodal side. This effect was detectable at a field of 0.4 mV/grain diameter.     

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.     

Regeneration of sialic acid on the surface of Chinese hamster cells in culture. I. General characteristics of the replacement process

Electropotential differences between the cell interior and the external medium have been studied with intracellular microelectrodes in ovarian oocytes, ovulated unfertilized eggs and fertilized eggs of R. pipiens. In ovarian oocytes the cytoplasm was 50 to 80 mV negative, relative to isotonic Ringer's solution. In contrast, electrode penetration of the oocyte nucleus in situ indicated that the nucleoplasm was about 25 mV positive, relative to the cytoplasm. After ovulation, the cortical cytoplasm became 20 to 50 mV positive with regard to an external solution of 0.1 strength Ringer's solution (ca. pond water). Penetration of the cytoplasm at levels from 0.3 to 0.6 mm below the egg surface revealed an inner zone with a potential which was about 15 mV negative, relative to the cortical cytoplasm. A slow hyperpolarization of the cortical membrane occurred at activation, with the potential returning to that of the ovulated unfertilized egg within ten minutes. After fertilization, the egg cytoplasm remained positive until the first cleavage. As division proceeded, the cytoplasm slowly depolarized and became 50 to 60 mV negative, relative to 0.1 strength Ringer's solution.     

Protons and calcium alter gating of the hyperpolarization-activated cation current (Ih) in rod photoreceptors

We investigated the effects of protons and calcium ions on the voltage-dependent gating of the hyperpolarization-activated, nonselective cation channel current, I_h, in rod photoreceptors. I_h is a cesium-sensitive current responsible for the peak-plateau sag during the rod response to bright light. The voltage dependence of I_h activation shifted about 5 mV per pH unit, with external acidification producing positive shifts and alkalinization producing negative shifts. Increasing external [Ca²⁺] from 3 to 20 mM resulted in a large (∼17 mV) positive shift in I_h activation. External [Ca²⁺] (20 mM) blocked pH-induced shifts in activation. Cytoplasmic acidification produced by 25 mM sodium acetate led to a negative shift in inactivation (−9 mV) and internal alkalinization produced with 20 mM ammonium chloride resulted in a positive shift (+6 mV). Surface charge binding and screening theory (Gouy-Chapman-Stern) accounted for the observed shifts in I_h activation, with the best fit achieved when protons and calcium ions were assumed to bind to distinct sites on the membrane. Since light induces changes in the retinal ionic environment, these results permit us to gauge the degree to which rod light responses could be modified via alterations in I_h activation.     

Lymphocyte transformation in vitro to RII mouse milk antigen among woman with breast disease.

The cell-mediated immune responses of 110 women with benign or malignant breast disease were tested in in vitro lymphocyte transformation assay with an antigen preparation made from RIII mouse milk containing mammary tumor virus. About 50% of patients responded positively to the milk preparation. In contrast, 25% of normal women or women with other gynecological malignancies responded positively to the antigen (P = 0.015). The data demonstrate a similar response pattern among women with malignant or benign breast disease. In addition, a subpopulation of normal women with positive response to this antigen is clearly defined.     

Relocalization of the calcium gradient and a dihydropyridine receptor is involved in upward bending by bulging of Chara protonemata, but not in downward bending by bowing of Chara rhizoids

The localization of cytoplasmic free calcium and a dihydropyridine (DHP) receptor, a putative calcium channel, was recorded during the opposite graviresponses of tip-growing Chara rhizoids and Chara protonemata by using the calcium indicator Calcium Crimson and a fluorescently labeled dihydropyridine (FL-DHP). In upward (negatively gravitropically) growing protonemata and downward (positively gravitropically) growing rhizoids, a steep Ca²⁺ gradient and DHP receptors were found to be symmetrically localized in the tip. However, the localization of the Ca²⁺ gradient and DHP receptors differed considerably during the gravitropic responses upon horizontal positioning of the two cell types. During the graviresponse of rhizoids, a continuous bowing downward by differential flank growth, the Ca²⁺ gradient and DHP receptors remained symmetrically localized in the tip at the centre of growth. However, after tilting protonemata into a horizontal position, there was a drastic displacement of the Ca²⁺ gradient and FL-DHP to the upper flank of the apical dome. This displacement occurred after the apical intrusion and sedimentation of the statoliths but clearly before the change in the growth direction became evident. In protonemata, the reorientation of the growth direction started with the appearence of a bulge on that site of the upper flank which was predicted by the asymmetrically displaced Ca²⁺ gradient. With the upward shift of the cell tip, which is suggested to result from a statolith-induced displacement of the growth centre, the Ca²⁺ gradient and DHP receptors became symmetrically relocalized in the apical dome. No major asymmetrical rearrangement was observed during the following phase of gravitropic curvature which is characterized by slower rates of bending. Labeling with FL-DHP was completely inhibited by a non-fluorescently labeled dihydropyridine. From these results it is suggested that FL-DHP labels calcium channels in rhizoids and protonemata. In rhizoids, positive gravitropic curvature is caused by differential growth limited to the opposite subapical flanks of the apical dome, a process which does not involve displacement of the growth centre, the calcium gradient or calcium channels. In protonemata, however, it is proposed that a statolith-induced asymmetrical relocalization of calcium channels and the Ca²⁺ gradient precedes, and might mediate, the rearrangement of the centre of growth, most likely by the displacement of the Spitzenk?rper, to the upper flank, which results in the negative gravitropic reorientation of the growth direction. Received: 13 February 1999 / Accepted: 25 June 1999     

Weak electric fields serve as guidance cues that direct retinal ganglion cell axons in vitro

Growing axons are directed by an extracellular electric field in a process known as galvanotropism. The electric field is a predominant guidance cue directing retinal ganglion cell (RGC) axons to the future optic disc during embryonic development. Specifically, the axons of newborn RGCs grow along the extracellular voltage gradient that exists endogenously in the embryonic retina (Yamashita, 2013 [8]). To investigate the molecular mechanisms underlying galvanotropic behaviour, the quantification of the electric effect on axon orientation must be examined. In the present study, a culture system was built to apply a constant, uniform direct current (DC) electric field by supplying an electrical current to the culture medium, and this system also continuously recorded the voltage difference between the two points in the medium. A negative feedback circuit was designed to regulate the supplied current to maintain the voltage difference at the desired value. A chick embryo retinal strip was placed between the two points and cultured for 24 h in an electric field in the opposite direction to the endogenous field, and growing axons were fluorescently labelled for live cell imaging (calcein-AM). The strength of the exogenous field varied from 0.0005 mV/mm to 10.0 mV/mm. The results showed that RGC axons grew in the reverse direction towards the cathode at voltage gradients of ≥0.0005 mV/mm, and straightforward extensions were found in fields of ≥0.2–0.5 mV/mm, which were far weaker than the endogenous voltage gradient (15 mV/mm). These findings suggest that the endogenous electric field is sufficient to guide RGC axons in vivo.     

A long-lasting electrically mediated block, due to the egg membrane hyperpolarization at fertilization, ensures physiological monospermy in eggs of the crab Maia squinado

In response to fertilization, the membrane potential (Em) of the crab egg hyperpolarizes from about -50 mV to about -80 mV in 400 msec. To establish whether this fast hyperpolarization is correlated with physiological polyspermy or conversely mediates an electrical block to polyspermy, we examined the morphological and electrophysiological characteristics of eggs from the crab Maia squinado. Fertilized naturally spawned eggs were found to be physiologically monospermic and their average Em was constant at -77 +/- 0.5 mV. To examine a possible electrical block ensuring this monospermy, unfertilized eggs were voltage clamped at various Em values ranging from +20 to -90 mV, inseminated, and examined morphologically. All eggs clamped at +20 to -65 mV responded by developing a fertilization current, If. It consisted of an outwardly directed K+ current in one or several steps, each caused by a single spermatozoon interacting with the egg membrane. The percentage of eggs clamped at values more negative than -65 mV, which responded at insemination by developing an If, decreased and dropped to 0 at -80 mV. This indicated that the membrane processes occurring during the contact between gametes and eliciting an electrical response by the egg membrane are voltage dependent. Further, the spermatozoon never penetrated into eggs voltage clamped at a Em between +20 and -60 mV and at voltages more negative than -75 mV. Em values between -65 and -75 mV were required for spermatozoon incorporation into the egg, indicating that sperm entry is also voltage dependent. It is proposed that the hyperpolarization of the egg membrane in response to fertilization constitutes a long-lasting electrical block to polyspermy in crab eggs.     

Seasonal variation in numbers of eggs laid byCulex quinquefasciatus Say (Diptera: Culicidae) at Khartoum

Regular counts of the eggs ofCulex quinquefasciatus Say were recorded during the period April 1979 – March 1981. The results indicate that the lowest numbers of eggs laid were during summer months (May and June) and the highest ones were during winter months (November and January). The decline of numbers during summer was mainly due to small size of adults (0.57 mg, average wt of female). Populations flourished during the rainy season (July – October) and reached their peak in August. Bigger batches of eggs were then obtained. In winter prolongation of larval life gave rise to large-sized adults and hence bigger batches of eggs.     

Electrical Properties of Toad Oocytes During Maturation and Activation

The full-grown oocytes of the toad Bufo bufo japonicus , whether in follicular layer or not, had a membrane potential of about -50 mV in De Boer's solution (DB), but underwent a deep hyper-polarization of up to -90 mV when pretreated with Ca, Mg-free EDTA-solution. Regardless of the magnitude of their resting potentials, the defolliculated oocytes exposed to progesterone underwent a gradual depolarization before the germinal vesicle breakdown and retained membrane potential at a level of -10 mV until 18 hr post hormone treatment (PHT), the stage of the second meiotic metaphase. A positive-going activation potential of a magnitude of 70 mV was recorded in the oocytes when pricked at 18 hr PHT as well as in uterine eggs 3–5 min after insemination. A low magnitude of activation potential in response to pricking was recorded in 63% of the oocytes at 13 hr PHT, and premature oocytes exhibiting the activation potential always underwent cortical granule breakdown (CGBD) and perivitelline space formatión. Oocytes where the germinal vesicle had been removed before the hormone treatment exhibited an activation potential and underwent CGBD in response to pricking at 18 hr PHT, whereas those pulse-treated with cycloheximide (10 μg/ml) during the 8–11 hr PHT exhibited neither of these cortical responses. These results indicate that the syntheses of proteins independent of germinal vesicle taking place at 9–11 hr PHT enable the oocytes to undergo cortical responses.     

Phototropism in a red alga, Griffithsia pacifica

In the red alga, Griffithsia pacifica, shoot portions of a plantare positively phototropic and rhizoids are negatively phototropic.We have studied the phototropic response of rhizoids which elongateby tip growth. For 45 min after the beginning of unilateralillumination a rhizoid grows straight, then phototropic curvaturebegins and continues rapidly until the rhizoid is growing awayfrom the light. Curvature is 70–80% complete after 3 hr.If the unilateral stimulus is given for a short time (15 min),curvature again begins at 45 min. However, within an additional30–45 min the rhizoid stops growing away from the lightand wanders back towards its original direction of growth. Phototropismis elicited by light of wavelengths from 350 nm to 500 nm; inlight of wavelengths above 550 nm, little, if any, responseoccurs. ¹Present address: Division of Natural Sciences, University ofCalifornia, Santa Cruz, California 95064, U.S.A. (Received December 10, 1976; )     

Control of wave propagation in a biological excitable medium by an external electric field

We present an experimental evidence of effects of external electric fields (EFs) on the velocity of pulse waves propagating in a biological excitable medium. The excitable medium used is formed by a layer of starving cells of Dictyostelium discoideum through which the waves of increased concentration of cAMP propagate by reaction-diffusion mechanism. External dc EFs of low intensities (up to 5 V/cm) are shown to speed up the propagation of cAMP waves towards the positive electrode and slow it down towards the negative electrode. Electric fields were also found to support an emergence of new centers, emitting cAMP waves, in front of cAMP waves propagating towards the negative electrode.     

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.     

APOLAR EMBRYOS OF FUCUS RESULTING FROM OSMOTIC AND CHEMICAL TREATMENT

Embryos of the brown alga Fucus vesiculosas L. were grown as populations in glass petri dishes in seawater at 15 C in continuous low-intensity unilateral fluorescent illumination for periods up to 2 weeks. A quantitative estimate of increase in nuclear number was made from acetocarmine squash preparations of samples taken at 12-or-24 hr intervals. Over the period of 2-6 days embryos showed a doubling time of about 12-18 hr. Under normal seawater culture conditions each embryo formed a single rhizoid. When grown in seawater supplemented with sugar concentrations above 0.4 m , Fucus embryos developed as multicellular spherical embryos lacking rhizoids. In 0.6 m sucrose-seawater, 97% of the embryos were apolar at 2 days; only 37% were apolar at 4 days, many having recovered from the sucrose inhibition. Some embryos remained apolar after growth in 0.6 m sucrose for 2 weeks. Nuclear counts showed that sucrose-seawater markedly inhibited the rate of cell division. Other sugars including D-glucose, D-fructose, D-galactose and the sugar alcohol D-mannitol were also effective. When apolar embryos grown in sucrose-seawater were returned to seawater, embryo growth resumed at the normal seawater rate, judged from nuclear counts. Such embryos formed multiple rhizoids, varying from two to eight rhizoids per embryo, which developed on the embryo quadrant or half away from the unilateral light. Each of the multiple rhizoids originated from a single small cell in the periphery of the multicellular spherica embryo. Thus the rhizoid-forming stimulus apparently had been subdivided among a number of the cells of the apolar embryos. The implications of this finding are discussed. Attempts to produce multiple rhizoids by treatment of embryos with indoleacetic acid or 2,4-dichlorophen-oxyacetic acid failed. However, embryos treated with 10⁻⁴ M or 5 × 10⁻⁵ m 2,3,5-triiodobenzoic acid formed 40 and 30% multiple rhizoids, respectively, suggesting that some chemical, perhaps hormonal, mechanism is involved in polarization and rhizoid initiation in Fucus embryogenesis.     

ζ-Potential and surface charge of Thermoplasma acidophila

The surface charge density and the ζ-potential of Thermoplasma acidophila was estimated from microscopic electrophoresis experiments. The cells moved towards the positive electrode. The mobility remained constant from pH 2 to 5, and increased for pH values higher than 6. The mobility at pH 6 decreased dramatically with increased external Ca²⁺ concentration. At pH 2 and an ionic strength similar to that of the growth medium, the ζ-potential was about 8 mV, negative relative to the bulk medium; the surface charge density was 1360esu/cm^-2 which corresponds to one elementary charge per 3500 A².     

Membrane potential measurements of unfertilized and fertilized Xenopus laevis eggs are affected by damage caused by the electrode

Upon penetration in an unfertilized Xenopus egg bathed in 1/10 Ringer, the voltage recorded by a microelectrode shows an abrupt jump to a negative voltage (Ep) followed by a rapid depolarization to a steady value (Er) (Ep = -39.4 +/- 1.9 mV and Er = -11.5 +/- 0.5 SE, 54 eggs from 9 females). The same is true for fertilized eggs impaled 16-35 min after insemination (Ep = -29.5 +/- 2.1 mV, Er = -11.5 +/- 0.9 mV, SE, 18 eggs from 3 females). The voltage recorded by a second microelectrode inserted into the same egg does not show the transient initial negativity. The stationary level of the membrane potential is close to the diffusion potential calculated from the Goldman equation with equal permeabilities for all the relevant ions. It is concluded that the low resting potentials measured in Xenopus eggs before and after fertilization are largely due to damage caused by the electrode. Using an upper limit of -39 mV for the true membrane potential and correlating the input resistance with the stationary membrane potential, a lower limit of 22 M omega (about 1 M omega cm2) for the membrane resistance can be obtained. Insertion of a microelectrode during the first 3 min after insemination shows a steady positive potential while, at later times (3-16 min post-insemination), a positive peak followed by a repolarization can be observed. This indicates that the measurement of the peak of the fertilization potential is not seriously affected by the electrode penetration while its time course after the first 3 min may be deformed by the presence of a large leakage conductance.     

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.     

Transmission of crimean-congo haemorrhagic fever virus from experimentally infected sheep to hyalomma truncatum ticks

Crimean-Congo haemorrhagic fever (CCHF) virus was inoculated into West African sheep that were simultaneously infested with adult Hyalomma truncatum ticks. Certain sheep developed a viraemia and antibodies, indicating virus infection and replication; however, the length and magnitude of the viraemia and serological responses corresponded to the animals' immunological status. Tick attachment and feeding was not influenced by sheep infection. CCHF virus infection was acquired by 11–33 % of female and 0–60 % of male ticks. Infection in the ticks did not influence their feeding success, as judged by weight at drop-off, and the weight of eggs produced by infected and non-infected ticks was similar. Transovarial transmission of CCHF virus was demonstrated in 2 of 12 (17 %) egg batches from infected female ticks, but in none of 19 egg batches from ticks that tested negative for CCHF virus. Our results suggest that under certain ecological conditions, sheep may serve to amplify CCHF virus in nature through horizontal transmission and that the maintenance cycle also may be influenced by transovarial transmission to the next generation of ticks.