Effects of pH on the Fertilization Response of the Medaka Egg

The effects of changing the intra- and extracellular pH on the cortical reaction and sperm penetration into eggs were examined in Oryzias latipes. When eggs were inseminated in a saline adjusted to various pH values with different buffers, fertilization took place normally over a wide range of external pH (pH₀) from about 6 to 10 with a peak at 7 to 8.5. The primary causes of the reduced fertilization were impaired motility or immobility of the spermatozoa in the acidic saline and the swelling of the chorion in the alkaline saline. A rise in the intracellular pH (pH_i) shortly after commencement of the cortical reaction was found by monitoring the color of a pH indicator micro-injected into the cytoplasm. The experimental results obtained by microinjection of various pH buffers (pH 4–12) suggest that the intracellular membrane fusion of the plasma membrane with the cortical alveolar membrane (CABD) is affected by the acidic pH_i while the propagation of the CABD and the intercellular membrane fusion between the plasma membranes of the egg and the spermatozoon are affected by the acidic pH₀.     

Glucose-induced acid tolerance appearing at neutral pH in log-phase Escherichia coli and its reversal by cyclic AMP

Escherichia coli shifted from broth at external pH (pH₀) 7·0 to pH₀ 7·0 broth plus glucose rapidly induced marked acid tolerance which also appeared, albeit to a lesser extent, plus maltose, sucrose or lactose. Tolerance appeared without the medium pH becoming acidic. Tolerance was most substantial when glucose was added at pH₀ 7·0 but was also appreciable at pH₀ 7·5, 8·0 and 8·5. Induction of tolerance by glucose was markedly reduced by cyclic AMP and essentially abolished plus NaCl or sucrose ; the induction process was also reduced but not fully inhibited by chloramphenicol, tetracycline and nalidixic acid. Glucose-induced organisms showed less acid damage to DNA and β-galactosidase and it is likely that this is because glucose induces a new pH homeostatic mechanism which keeps internal pH close to neutrality at acidic pH₀. In conclusion, it is clear that glucose induces a novel acid tolerance response in log-phase E. coli at pH₀ 7·0 ; it is now known that induction of this response involves the functioning of extracellular induction components including an extracellular induction protein.     

Signalling gates in abscisic acid-mediated control of guard cell ion channels

Multiple signalling pathways and their messengers – entailing changes in cytosolic-free Ca²⁺([Ca²⁻]). pH (pH) and protein phosphorylation – underpin K⁺and anion channel control during stomatal movements. This redundancy is wholly consistent with the ability of the guard cells to integrate the wide range of environmental and hormonal stimuli that affect stomatal aperture. Signal redundancy effects a spectrum of graded responses by linking pathways to gate signal transmission, and so boosts or mutes the final 'integrated signal' that reaches each ion channel. All evidence supports a role for the AB11 protein phosphatase and protein kinase elements in gating K⁺channel sensitivity to pH and ABA. Changes in [Ca²⁺] I . in turn, are demonstrably sensitive to pH₁. Because each of these signal elements modulate and, in turn, are influenced by the activity of different sets of ion channels, the additional couplings engender a remarkably complex network, layering positive and negative controls with the ion channels that facilitate ion fluxes for stomatal movement.     

Seasonal variation in egg production and batch fecundity of European hake Merluccius merluccius (L.) in the Bay of Biscay

Sexually mature female hake Merluccius merluccius with hydrated ovaries were sampled on a monthly basis in the Bay of Biscay, from May 1996 to October 1997 and from March to April 1998. The batch fecundity was positively related to total length. The relative batch fecundity ( F _Brel) varied significantly among months and years, but not between areas, i.e. International Council for the Exploration of the Sea Divisions VIIIa and VIIIb within the Bay of Biscay. Two levels of F _Brel were found in 1997: the highest between January and April (mean ± s . e . 167 ± 5 eggs g⁻¹) and the lowest from May to October (112 ± 3 eggs g⁻¹). Population condition factor and gonado-somatic indices ( I _G) followed the expected trend in relation to the monthly changes in F _Brel during 1997. The F _Brel variation between years was 9% for 1996–1997 and 28% for 1997–1998, and the difference of the I _G was c. 14 and 36%, respectively. Population relative egg production varied from a high value in January to March (985 eggs g⁻¹) to a low egg production between April and October 1997 (445 eggs g⁻¹).     

THE EFFECT OF HYPERCAPNIA UPON INTRACELLULAR pH IN THE BRAIN, EVALUATED BY THE BICARBONATE- CARBONIC ACID METHOD AND FROM THE CREATINE PHOSPHOKINASE EQUILIBRIUM

Abstract— Abstract-Intracellular pH in the brain was evaluated by the bicarbonate-carbonic acid method and from the creatine phosphokinase equilibrium, in rats exposed to 6–40 % CO₂ for 45 min. There was a very good agreement between the two methods, indicating that the creatine phosphokinase equilibrium in vivo shows the pH dependence predicted from previous in vitro studies. The stepwise increase in the tissue CO₂ tension from 45 to 265 mm Hg resulted in a lowering of the intracellular pH from 7.04 to 6.68. The regulation of intracellular pH in hypercapnia was far better than that which can be predicted from physicochemical buffering alone, and calculations indicate that the intracellular buffer base concentration increased by more than 10 mequiv./kg at the maximal P_co2 values encountered.     

Fertilization Reaction without Changes in Intracellular Ca2+ in Medaka Eggs—An Experiment with Acetone-Treated Eggs

To investigate whether or not causal relationship exists between the increase in intracellular Ca²⁺ and other cortical reactions at fertilization in the medaka, Oryzias latipes , intracellular Ca²⁺ was determined from luminescence of aequorin previously microinjected into cortical cytoplasm in acetone-treated eggs, when they were inseminated or activated by microinjection of Ca²⁺. Neither an increase in cytoplasmic calcium nor exocytosis of cortical alveoli occurred in eggs treated with acetone, though other events of fertilization i.e. completion of meiosis, fusion of pronuclei, and accumulation of cortical cytoplasm with intact cortical alveoli in the animal pole region were observed in normal time sequence in these eggs. When denuded eggs were treated with acetone, contraction of the egg and slow resumption of meiosis (extrusion of polar body) were observed without insemination. When denuded eggs were inseminated immediately after acetone-treatment, the number of spermatozoa that penetrated into the egg was greater in the animal hemisphere than in the vegetal hemisphere. These results may indicate that acetone inactivates the egg plasma membrane or its adjacent cortical cytoplasm so that it cannot participate in a propagative increase in intracellular Ca²⁺ and exocytosis, while it also induces cytoplasmic activation leading to egg contraction, resumption of meiosis and formation of pronuclei. The present results suggest that sperm penetration, resumption of meiosis and ooplasmic segregation are regulated separately from the release of intracellular Ca²⁺ and exocytosis.     

Effects of graded hypoxia on Atlantic salmon infected with amoebic gill disease

Atlantic salmon Salmo salar with amoebic gill disease (AGD) were exposed to a graded hypoxia (135–40 mmHg water P O₂) and blood samples analysed for respiratory gases and pH at 119, 79·5 and 40 mmHg water P O₂. There were no differences in the rate of oxygen uptake between infected and control fish. However, arterial P O₂, and pH were significantly lower in the infected fish whereas P CO₂ was significantly higher in infected fish compared with controls prior to hypoxia and at 119 mmHg water P O₂. At 79·5 and 40 mmHg water P O₂ saturation, there were no significant differences in blood P O₂ or pH although blood P CO₂ was elevated in AGD affected fish at 50% hypoxia (79·5 mmHg water P O₂). The elevated levels of P CO₂ in fish affected by AGD resulted in a persistent respiratory acidosis even during hypoxic challenge. These data suggest that even though the fish were severely affected by AGD, the presence of AGD while impairing gas transfer under normoxic conditions, did not contribute to respiratory failure during hypoxia.     

Effects of rapid changes in oxygen concentration on the respiration of carrot roots

Rates of CO₂ production and O₂ consumption from aged disks of carrot ( Daucus carota L.) root tissues were measured for 4 h after they were transferred from 21% to 0, 1, 2, 4 or 8% O₂ in gas mixtures. A transient peak in the rate of CO₂ production started 5 to 7 min after transfer to 2% or lower O₂ mixtures and peaked at 50 min. After the peaks in CO₂ production from the 0, 1 and 2% O₂ treatments and after the stable production from the 4 and 8% O₂ treatments, the rate of CO₂ production from all low O₂ treatments started to decline at 50 min, reaching stable rates by 160 to 240 min. Concentrations of lactate and ethanol that were significantly higher than the 21% O₂ controls had started to accumulate in disks between 10 and 50 min after exposure to atmospheres containing 2% or less O₂. Production of CO₂ started to increase 5 to 7 min after transfer to 0, 1 and 2% O₂, while the initial decline and then rise in pH and the accumulation of ethanol did not occur until 30 min after the change in atmosphere. Ethanol accumulation paralleled the increase in pH; first at 0.4 μmol g⁻¹ h⁻¹ from 30 to 60 min as the pH shifted from 5.97 to 6.11, and then at 0.08 μmol g⁻¹ h⁻¹ from 60 to 100 min as the pH stablized around 6.12. The peak at 50 min in CO₂ production roughly coincided with the shift from the rapid to the slow change in pH and ethanol accumulation.     

Nisin, temperature and pH effects on growth and viability of Pectinatus frisingensis, a Gram-negative, strictly anaerobic beer-spoilage bacterium

Pectinatus frisingensis , a Gram-negative and strictly anaerobic beer spoilage bacterium is sensitive to nisin. An increase in nisin concentration (0 to 1100 IU ml⁻¹) added to the culture medium prolonged the lag phase, and decreased the growth rate of the bacterium. In addition, late exponential cells of P. frisingensis exposed to low concentrations of nisin lost immediately a part of their intracellular K⁺. Presence of Mg²⁺ up to 15 mmol l⁻¹ did not protect P. frisingensis from nisin-induced loss of viability and K⁺ efflux. Potassium leaks were also measured in P. frisingensis late exponential phase cells exposed to combined effects of nisin addition (100–500 IU ml⁻¹), 10 min mild heat-treatment (50 °C) or rapid cooling (2 °C), and pH (4·0 and 6·2). Net K⁺ efflux from both starving and glucose-metabolizing cells, was more important at pH 6·2, whatever the temperature treatment and nisin addition. Reincubation at 30 °C of P. frisingensis glucose-metabolizing cells exposed to a preliminary combination of nisin addition and mild heat or cooling down treatment, showed that cells exposed to rapid cooling reaccumulated more K₊ than heat-treated cells, whatever the pH conditions. A combination of nisin and mild heat-treatment could thus be of interest to prevent P. frisingensis growth in beers.     

Sporulation of Clostridium putrefaciens and the Resistance of the Spores to Heat, γ-Radiation and Curing Salts

Clostridium putrefaciens grew well in most media used routinely for culturing anaerobes, but produced spores only on lactose-egg yolk agar. The D_80° was 8–14 min, z value was 4°–6° and D _γ, 0.16 Mrad. The inhibitory interactions of pH, NaCl, NaNO₂ and incubation temperature are described.     

Adrenergic responses and the Root effect in erythrocytes of freshwater fish

The effects of adrenergic-stimulation upon the oxygen-binding capacity of fish erythrocytes have been investigated. The oxygen capacity of rainbow trout, Oncorhynchus mykiss (Walbaum), erythrocytes was lowered by 44% on extracellular acidification (the so-called 'Root effect'). Addition of isoproterenol at 20° Ccaused an acid shift of the curve relating oxygen capacity to pH₀ by approximately 0.2 pH units, a value which was similar to the change in intracellular pH caused by adrenergic stimulation (Cossins & Kilbey Journal of Experimental Biology , 148 , 303–312, 1990). Moreover, when plotted as a function of pH_i, the curves for control and adrenalinstimulated erythrocytes were superimposable suggesting that the adrenergic shift in the Root curve was a result of the change in pH_i caused by activation of the adrenergic Na⁺/H⁺ exchanger.
A similarly large adrenergic shift in the Root curve was observed for pike, Esox lucius L., erythrocytes, though not for erythrocytes of carp, Cyprinus carpio L., and tench, Tinea tinea (L.). The pH for the mid-point of the Root effect in pike erythrocytes was distinctly more acid than for trout, but in both cases corresponded closely with the optimal pH for the adrenergic Na⁺/H⁺ exchange mechanism. This suggests a link between the functional characteristics of the exchanger and the oxygen-binding properties of haemoglobin.     

Respiratory and acid-base disturbances in rainbow trout blood during exposure to chloramine-T under hypoxia and hyperoxia

Rainbow trout Oncorhynchus mykiss were exposed acutely to chloramine-T at a therapeutic concentration (9mg l⁻¹) under moderately hypoxic (water P o₂: l00 mmHg) or hyperoxic (water P o₂: 430mmHg) conditions and arterial blood gas tensions ( P ao₂ and P aco₂) and pH were monitored using an extracorporeal circulation. Hypoxia, alone, resulted in an increased ventilation frequency, a decrease in both arterial P co₂ and P o₂ and an increase in arterial pH. There was no effect of chloramine-T exposure on the measured variables as compared with pre-exposure (hypoxia baseline) values. Hyperoxia, alone, resulted in a decrease in ventilation frequency, an increase in arterial P co₂ and P o₂, and a decrease in arterial pH. Chloramine-T exposure under these conditions caused a significant increase in ventilation frequency, but no significant effect on arterial blood gases or pH as compared with the hyperoxia baseline values. Despite the increase in ventilation caused by chloramine-T during hyperoxia, there was no reduction in P co₂ or increase in P o₂. Although these results were of little pathological significance, this study suggests that chloramine-T, although stimulating ventilation, was impairing the diffusion of co₂ across the gill probably by the secretion of branchial mucus and enhancing the gill boundary layer.     

Activation of protein kinase C induces cortical granule exocytosis in a Ca2+-independent manner, but not the resumption of cell cycle in porcine eggs

The effects of protein kinase C (PKC) activation on meiotic resumption and cortical granule (CG) exocytosis as well as its dependence on Ca²⁺ in porcine eggs matured in vitro were studied. Cortical granule release was judged by both confocal laser microscopy after the eggs were labeled with fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) and electron microscopy. Meiotic resumption and pronuclear formation were observed after eggs were stained with acetic orcein. When eggs were treated with PKC activators, 1-oleyl-2-acetyl-glycerol (OAG) or phorbol 12-myristate 13-acetate (PMA), the pronuclear formation percentage was significantly lower than that of Ca²⁺ ionophore A23187-treated group, but not statistically different from that in negative control group (P > 0.05), and most of the eggs were still arrested at metaphase II stage, suggesting that PKC activation does not induce the resumption of meiosis and pronuclear formation. In contrast, PKC activation induced 89.1% to 100% of the eggs completely or partially released their CG in different groups, not statistically different from A23187-treated group, and this effect could be overcome by PKC inhibition. When the intracellular free Ca²⁺ was chelated with acetoxymethal ester form of 1,2-bis(0-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM), and then treated with PMA or OAG in Ca²⁺-free medium, the proportions of eggs with CG release were 90.9% and 78.1%, respectively, not statistically different from the above-treated groups, suggesting that CG exocytosis induced by PKC activation is independent of Ca²⁺ rise. The results indicate that different events of porcine egg activation may be uncoupled from one another.     

Ca2+ release from intracellular stores by thapsigargin in sea urchin eggs: Relationship to larval development and relevance in egg activation

Thapsigargin (Tg), an inhibitor of microsomal Ca²⁺ ATPase, is used as a tool to study the changes in Ca²⁺ sequestration in sea urchin eggs and their relationship to embryonic development. Micromolar amounts of Tg inhibit ATP-dependent Ca²⁺ sequestration in a dose-dependent and non-reversible manner, depending on the bulk of biological material used. IC_5O values are 1 nmol/L and 1–10μmol/L, respectively, in the cortical Ca²⁺ stores (isolated cortices preparation) and in digitonin-permeabilized eggs, a preparation giving access to the deeper reticulum compartment. Micromolar Tg does not induce Ca²⁺ release from ⁴⁵Ca pre-loaded cortices but leads to a loss of 25% of the total Ca²⁺ content from the cortical area. Using microspectrofluorimetry of fura-2-loaded eggs, we found that 10 μmol/L Tg induced a moderate rise in cytosolic Ca²⁺ activity as compared with the fertilization-induced Ca²⁺ transient whether eggs were fertilized or not. Early events related to fertilization as, for example, elevation of the fertilization envelope, proton excretion and sustained increase of amino acid uptake, are triggered by 10μmol/L Tg but with a delayed onset relative to sperm-induced effects. The present findings indicate that although it triggers most fertilization-related events, Tg cannot be considered as a true mitotic agent in sea urchin eggs. When added after fertilization, Tg affects cleavage and the further embryonic development giving rise to abnormalities comparable to the animalized larvae obtained with other compounds responsible for the inhibition of reticular Ca²⁺ sequestration.     

Control of ventilatory movements in the aquatic insect Corydalus comutus: central effect of hypoxia

ABSTRACT. The influence of hypoxia and hypercapnia on the ventilatory rhythm of the hellgrammite Corydalus cornutus L. (Megaloptera) was studied. In intact animals the frequency of rhythmic retractions and protractions of abdominal gills is increased by hypoxia (10% O₂, 90% N₂) but no ventilatory response is elicited by hypercapnia (1–5% CO₂, 20% O₂, 75–79% N₂).
The ventilatory motor pattern was examined by recording extracellularly from the gill retractor muscle or its efferent nerve. In response to hypoxia (8% 0₂, 92% N₂), there are decreases in the cycle-time, the interspike interval, and the burst length of the gill retractor motorneuron. In addition, previously quiescent motorneurons associated with gill protraction are recruited.
Individual ganglia or small groups of abdominal ganglia can be isolated both from the central ganglionic chain and from the periphery by selective cutting of roots and connectives. When exposed to hypoxia, preparations that include the first abdominal ganglion show characteristic changes in the ventilatory motor pattern similar to those in intact animals. Thus sensitivity to oxygen appears to be located centrally and not peripherally. In small animals (head width < 7 mm), abdominal ganglia 2–3 and 2–7 respond characteristically to hypoxia, but in larger animals (head width > 9 mm), chains of ganglia lacking abdominal ganglion 1 fail to respond. In larger animals oxygen sensitivity may thus be concentrated in abdominal ganglion 1, whereas in smaller animals the ability to initiate a ventilatory response to hypoxia is distributed among the abdominal ganglia.     

Improvement of the 15N dilution method for estimation of absorption of NOx by plants supplied with 15N-labelled fertilizer

To develop further the methods for estimation of NOx absorption by plants supplied with ¹⁵N-labelled fertilizer, we proposed a new calculation method, total N fixed method (TNF), and compared with the ¹⁵N dilution method and the classical mass balance method (MB).
Hydroponically grown soybean plants were supplied with ¹⁵N-labelled nitrate and exposed to 200–250 nl l⁻¹ NO₂ for 7 d. The proportions of the N derived from NO₂ to total N in exposed plants were estimated by the three methods.
The reported rates of NO₂ absorption by several plant species, estimated by the ¹⁵N dilution method, were recalculated using the TNF method. The results of the two methods were compared and showed that: (1) The ¹⁵N dilution method overestimated the content of NO₂-N in exposed plants compared with the MB method whilst the TNF method produced estimations of NO₂-N closer to those by the MB method when the plants were supplied with 5 m M nitrate. (2) The differences in estimations between the MB method and either the ¹⁵N dilution method or the TNF method increased with decreasing supply of ¹⁵N-labelled nitrate to roots.     

The Role of Intracellular pH in Fertilization of Sand Dollar Eggs Analyzed by Microinjection Method

The change in intracellular pH (pH_i) upon fertilization and the effects of changing the pH_i by microinjection of pH buffers were investigated in the eggs of the sand dollar, Clypeaster japonicus. The pH_i was determined by the tint of a pH indicator, phenol red, microinjected into eggs. The pH_i ranged from 6.5 to 6.75 in unfertilized eggs and it rose by 0.4 to 0.5 unit within 3 min upon fertilization. The elevated pH_i ranging from 7.0 to 7.25 was maintained at least until the first cleavage. As reported in eggs of other species of sea urchin (1–4), development of fertilized eggs which had been transferred to Na-free sea water immediately after insemination was arrested and the pH_i did not rise remaining at the level of unfertilized eggs. Development was initiated in eggs arrested in Na-free sea water when the pH_i was elevated up to the level of fertilized eggs, i.e. 7.0 to 7.25, by microinjecting 1 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)-KOH buffer at pH 8.0. By microinjection of pH 7.5 buffer, some eggs started development though none of them underwent cleavage. By microinjection of pH 7.0 or pH 6.5 buffer, development was not initiated. The initiation of development depended on the pH value of microinjected pH buffer, and in consequence, on the final pH_i. The elongation of microvilli which had been arrested in eggs in Na-free sea water was also induced by microinjection of pH 8.0 or 7.5 buffer.     

Effects of development, temperature and salinity on metabolism in eggs and yolk-sac larvae of milkfish, Chanos chanos (Forsskål)

Oxygen uptake rates and yolk-inclusive dry weiGhts were measured during the egg and yolk-sac larval stages of milkfish, Chanos chanos (Forsskal). Oxygen uptake by eggs and yolk-sac larvae was measured to assess the effects of four salinities (20,25,30,35 ppt) at 28°C. The effects of three temperatures (23,28,33°C) on oxygen uptake by yolk-sac larvae were determined at a salinity of 35 ppt. Dry weights were measured throughout embryonic development at 28°C and the yolk-sac stage at 23.28 and 33°C.
Oxygen uptake rates of eggs increased more than fivefold during embryogenesis (0.07±0.03 to 0.40 ± 03 μl O₂ egg ⁻¹ h ⁻¹;blastula to prehatch stage). Larval oxygen uptake did not change with age but was affected by rearing temperature (0.33 ± 0.08, 0.44 ± 0.07 and 0.63 ± 0.13 μl O₂ larva ⁻¹ h⁻¹ at 23, 28 and 33°C, respectively; Q₁₀= 1.93). Acute temperature changes from 28 to 33°C caused significant increases in oxygen uptake by embryos (Q ₁₀= 1.69–3.58) and yolk-sac larvae (Q ₁₀=2.55). Salinity did not affect metabolic rates.
Dry weight of eggs incubated at 28°C decreased 13% from fertilization to hatching. Incubation temperatures from 23–33°C did not affect dry weights at hatching. Rearing temperatures significantly affected the rate of larval yolk absorption (Q ₁₀= 2.25).     

Physiological mechanisms of buoyancy in eggs from brackish water cod

Newly fertilized eggs of brackish water (Gotland, Baltic Sea) and marine (Lofoten, Norway) cod were investigated with regard to specific gravity, wet and dry weight, water content, chorion weight, and content of protein, free amino acids (FAA), and ions. The eggs had neutral buoyancies equivalent to a salinity of 14^.3% (range 11^.5–16^.2%) in brackish water, and 33^.0% (range 31^.8–34^.5%) in the marine environment. A buoyancy model was developed and showed that this difference was mainly caused by differences in egg water content which was 96.6 ± 0^.47% and 92.7 ± 0.45% in the brackish and marine eggs, respectively. The higher water content of the brackish eggs resulted from increased water uptake during final oocyte maturation due to higher intracellular contents of FAA, Cl ^– and NH₄⁺. SDS polyacrylamide gel electrophoresis of eggs and oocytes, and measurements of egg protein content suggested that the FAA pool of both egg types originated from hydrolysis of specific yolk proteins. The main contributor seemed to be a protein with a molecular weight of 100 kDa.     

The effect of endotoxin lipopolysaccharide from different bacterial species on the generation of intracellular inositol triphosphate and superoxide in a human phagocytic cell line

Abstract The acute effects of endotoxins and lipid A on two intracellular responses, inositol phosphate generation and superoxide production were analysed in the DMSO differentiated premyelocytic leukamic HL-60 cell line. Short-term incubation (1–10 min) with Escheria coli -type LPS, Salmonella -type LPS and Lipid A caused significant increases in cellular InsP₁ and InsP₃, compared with control cells ( P < 0.5 − P < 0.001). The Escheria coli -type B LPS released approximately twice the quantity of InsP₃ compared with Salmonella -type LPS ( P < 0.001). Lipid A-dependent stimulation of InsP₃ production was also detected. The rate of superoxide production increased 1–10 min after addition of both Escheria coli - and Salmonella -type LPS and Lipid A. Endotoxins and Lipid A caused a dose-dependent increase in intracellular oxidative activity. The superoxide response showed less species dependence and a higher response to particulate lipid A compared with the inositol phosphate response.