Embryonic rotational behaviour in the pond snail Lymnaea stagnalis: influences of environmental oxygen and development stage

Responses of freshwater organisms to environmental oxygen tensions (PO₂) have focused on adult (i.e. late developmental) stages, yet responses of embryonic stages to changes in environmental PO₂ must also have implications for organismal biology. Here we assess how the rotational behaviour of the freshwater snail Lymnaea stagnalis changes during development in response to conditions of hypoxia and hyperoxia. As rotation rate is linked to gas mixing in the fluid surrounding the embryo, we predicted that it would increase under hypoxic conditions but decrease under hyperoxia. Contrary to predictions, early, veliger stage embryos showed no change in their rotation rate under hyperoxia, and later, hippo stage embryos showed only a marginally significant increase in rotation under these conditions. Predictions for hypoxia were broadly supported, however, with both veliger and hippo stages showing a marked hypoxia-related increase in their rotation rates. There were also subtle differences between developmental stages, with hippos responding at PO₂s (50% air saturation) greater than those required to elicit a similar response in veligers (20% air saturation). Differences between developmental stages also occurred on return to normoxic conditions following hypoxia: rotation in veligers returned to pre-exposure levels, whereas there was a virtual cessation in embryos at the hippo stage, likely the result of overstimulation of oxygen sensors driving ciliary movement in later, more developed embryos. Together, these findings suggest that the spinning activity of L. stagnalis embryos varies depending on environmental PO₂s and developmental stage, increasing during hypoxia to mix capsular contents and maintain a diffusive gradient for oxygen entry into the capsule from the external environment (“stir-bar” theory of embryonic rotational behaviour).     

Structural and physiological differences between montane and lowland avian eggs and embryos

A few avian species breed at altitudes up to 6500 m. Embryos in eggs laid at high altitudes are confronted with the problem that gases diffuse more rapidly at low barometric pressure than at sea level. Data on birds breeding up to 4500 m indicate that modifications in eggshell structure and embryonic physiology foster successful development in these groups. At moderate altitudes (up to 3600 m), shell conductance to gases (corrected to 760 torr) is decreased in approximate proportion to the reduction in barometric pressure, thus offsetting the increased tendency of gases to diffuse. At altitudes above 4000 m, the conductance is increased above levels at moderate altitudes, thus fostering improvement in oxygen availability, while increasing rates of water and CO2 losses. Above 4000 m, embryonic physiological properties become increasingly important for coping with hypoxic, hypocapnic, and dehydrated conditions inside the shell. Nothing is known about characteristics of eggshells and embryos in eggs laid between 4500 and 6500 m. Despite years of artificial selection, domestic fowl do not breed successfully much above 3000 m. Embryos of domestic fowl appear highly sensitive to the effects of hypoxia.     

The effects of hypoxia and temperature on metabolic aspects of embryonic development in the annual killifish Austrofundulus limnaeus

Embryos of Austrofundulus limnaeus are exceptional in their ability to tolerate prolonged bouts of complete anoxia. Hypoxia and anoxia are a normal part of their developmental environment. Here, we exposed embryos to a range of PO₂ levels at two different temperatures (25 and 30 °C) to study the combined effects of reduced oxygen and increased temperature on developmental rate, heart rate, and metabolic enzyme capacity. Hypoxia decreased overall developmental rate and caused a stage-specific decline in heart rate. However, the rate of early development prior to the onset of organogenesis is insensitive to PO₂. Increased incubation temperature caused an increase in the developmental rate at high PO₂s, but hindered developmental progression under severe hypoxia. Embryonic DNA content in pre-hatching embryos was positively correlated with PO₂. Citrate synthase, lactate dehydrogenase, and phosphoenolpyruvate carboxykinase capacity were all reduced in embryos developing under hypoxic conditions. Embryos of A. limnaeus are able to develop normally across a wide range of PO₂s and contrary to most other vertebrates severe hypoxia is not a teratogen. Embryos of A. limnaeus do not respond to hypoxia through an increase in the capacity for enzymatic activity of the metabolic enzymes lactate dehydrogenase, citrate synthase, or phosphoenolpyruvate carboxykinase. Instead they appear to adjust whole-embryo metabolic capacity to match oxygen availability. However, decreased DNA content in hypoxia-reared embryos suggests that cellular enzymatic capacity may remain unchanged in response to hypoxia, and the reduced capacity may rather indicate reduced cell number in hypoxic embryos.     

Physiological and Ecological Aspects of Gas Exchange by Sea Turtle Eggs

The sea turtle clutch of about 100 eggs is buried deeply inthe nesting beach.The eggs exchange respiratory gases with thesurrounding beach as their metabolic activity increases throughoutthe 60 day incubation. The O₂ consumption of individual eggsthroughout incubation is less than that of avian eggs of similarmass; however, this difference may be attributed to the differencein incubation temperature and growth rate. The O₂ consumptionof the sea turtle embryo is sufficiently low and the gas conductanceof the shell sufficiently large that only small gas partialpressure gradients occur across the shell. However, the metabolicintensity of the entire clutch is quite large, and since gasmovement through the beach is restricted, increasing gas partialpressure gradients are established between the center and peripheryof the clutch and between the clutch and surrounding beach.The rate of growth and mortality of the embryos is related torespiratory gas exchange, since maximum growth and hatchlingsuccess appear to occur in respiratory environments similarto those observed in natural nests. Embryonic growth slows andmortality increases in environments in which gas exchange isreduced below naturally occurring levels. Gas exchange considerationsmay influence nest construction, clutch size and incubationtime among sea turtles.     

Quantifying the sensitivity of barley seed germination to oxygen, abscisic acid, and gibberellin using a population-based threshold model

Barley (Hordeum vulgare L.) seeds (grains) exhibit dormancyat maturity that is largely due to the presence of the glumellae(hulls) that reduce the availability of oxygen (O₂) to the embryo.In addition, abscisic acid (ABA) and gibberellins (GAS) interactwith O₂ to regulate barley seed dormancy. A population-basedthreshold model was applied to quantify the sensitivities ofseeds and excised embryos to O₂, ABA, and GA, and to their interactiveeffects. The median O₂ requirement for germination of dormantintact barley seeds was 400-fold greater than for excised embryos,indicating that the tissues enclosing the embryo markedly limitO₂ penetration. However, embryo O₂ thresholds decreased by anotherorder of magnitude following after-ripening. Thus, increasesin both permeability of the hull to O₂ and embryo sensitivityto O₂ contribute to the improvement in germination capacityduring after-ripening. Both ABA and GA had relatively smalleffects on the sensitivity of germination to O₂, but ABA andGA thresholds varied over several orders of magnitude in responseto O₂ availability, with sensitivity to ABA increasing and sensitivityto GA decreasing with hypoxia. Simple additive models of O₂–ABAand O₂–GA interactions required consideration of theseO₂ effects on hormone sensitivity to account for actual germinationpatterns. These quantitative and interactive relationships amongO₂, ABA, and GA sensitivities provide insight into how dormancyand germination are regulated by a combination of physical (O₂diffusion through the hull) and physiological (ABA and GA sensitivities)factors. Key words: Abscisic acid, barley, germination, gibberellin, Hordeum vulgare L., model, oxygen, sensitivity Received 2 August 2007; Revised 14 November 2007 Accepted 19 November 2007     

Reactive oxygen species formation in the transition to hypoxia in skeletal muscle

Many tissues produce reactive oxygen species (ROS) during reoxygenation after hypoxia or ischemia; however, whether ROS are formed during hypoxia is controversial. We tested the hypothesis that ROS are generated in skeletal muscle during exposure to acute hypoxia before reoxygenation. Isolated rat diaphragm strips were loaded with dihydrofluorescein-DA (Hfluor-DA), a probe that is oxidized to fluorescein (Fluor) by intracellular ROS. Changes in fluorescence due to Fluor, NADH, and FAD were measured using a tissue fluorometer. The system had a detection limit of 1 µM H₂O₂ applied to the muscle superfusate. When the superfusion buffer was changed rapidly from 95% O₂ to 0%, 5%, 21%, or 40% O₂, transient elevations in Fluor were observed that were proportional to the rise in NADH fluorescence and inversely proportional to the level of O₂ exposure. This signal could be inhibited completely with 40 µM ebselen, a glutathione peroxidase mimic. After brief hypoxia exposure (10 min) or exposure to brief periods of H₂O₂, the fluorescence signal returned to baseline. Furthermore, tissues loaded with the oxidized form of the probe (Fluor-DA) showed a similar pattern of response that could be inhibited with ebselen. These results suggest that Fluor exists in a partially reversible redox state within the tissue. When Hfluor-loaded tissues were contracted with low-frequency twitches, Fluor emission and NADH emission were significantly elevated in a way that resembled the hypoxia-induced signal. We conclude that in the transition to low intracellular PO₂, a burst of intracellular ROS is formed that may have functional implications regarding skeletal muscle O₂-sensing systems and responses to acute metabolic stress. dihydrofluorescein; tissue fluorometer; ebselen; N-acetylcysteine; rat     

Germline transmission of exogenous genes in chickens using helper-free ecotropic avian leukosis virus-based vectors

We have used vectors derived from avian leukosis viruses to transduce exogenous genes into early somatic stem cells of chicken embryos. The ecotropic helper cell line, Isolde, was used to generate stocks of NL-B vector carrying theNeo ^r selectable marker and theEscherichia coli lacZ gene. Microinjection of the NL-B vector directly beneath unincubated chicken embryo blastoderms resulted in infection of germline stem cells. One of the 16 male birds hatched (6.25%) from the injected embryos contained vector DNA sequences in its semen. Vector sequences were transmitted to G₁ progeny at a frequency of 2.7%.Neo ^r andlacZ genes were transcribedin vitro in chicken embryo fibroblast cultures from transgenic embryos of the G₂ progeny.     

Factors Affecting Formation of Somatic Embryos and Embryogenic Callus from Unemerged Inflorescences of a Graminaceous Crop Pennisetum

Differentiation of somatic embryos was dependent on the concentrationof auxin and the mineral medium. Low levels of auxin 2,4-D inN₆ medium, a low ammonium nutrient, favoured the formation ofsomatic embryos, while on MS medium containing high ammoniumcompact tissues appeared. At higher levels of auxin, irrespectiveof nutrient medium, compact tissues were formed. The originof compact tissue on N₄ medium could be traced to somatic embryo-likestructures. This tissue regenerated into somatic embryos onhormone-free N₆ medium whereas on MS medium thalloid structuresappeared. Pennisetum, unemerged inflorescence, somatic embryo, embryogenic callus     

Lactate efflux from exercising human skeletal muscle: role of intracellular PO2

It remainscontroversial whether lactate formation during progressive dynamicexercise from submaximal to maximal effort is due to muscle hypoxia. Tostudy this question, we used direct measures of arterial and femoralvenous lactate concentration, a thermodilution blood flow technique,phosphorus magnetic resonance spectroscopy (MRS), and myoglobin (Mb)saturation measured by ¹H nuclearMRS in six trained subjects performing single-leg quadriceps exercise.We calculated net lactate efflux from the muscle and intracellularPO₂ with subjects breathing room airand 12% O₂. Data were obtained at50, 75, 90, and 100% of quadriceps maximalO₂ consumption at each fraction ofinspired O₂. Mb saturation wassignificantly lower in hypoxia than in normoxia [40 ± 3 vs. 49 ± 3% (SE)] throughout incremental exercise to maximalwork rate. With the assumption of aPO₂ at which 50% of Mb-binding sitesare bound with O₂ of 3.2 Torr,Mb-associated PO₂ averaged 3.1 ± 0.3 and 2.3 ± 0.2 Torr in normoxia and hypoxia, respectively. Netblood lactate efflux was unrelated to intracellular PO₂ across the range of incrementalexercise to maximum (r = 0.03 and 0.07 in normoxia and hypoxia, respectively) but linearly related toO₂ consumption(r = 0.97 and 0.99 in normoxia andhypoxia, respectively) with a greater slope in 12%O₂. Net lactate efflux was alsolinearly related to intracellular pH(r = 0.94 and 0.98 in normoxia andhypoxia, respectively). These data suggest that with increasing workrate, at a given fraction of inspiredO₂, lactate efflux is unrelated tomuscle cytoplasmic PO₂, yet theefflux is higher in hypoxia. Catecholamine values from comparablestudies are included and indicate that lactate efflux in hypoxia may bedue to systemic rather than intracellular hypoxia.

     

Antioxidants protect rat diaphragmatic muscle function under hypoxic conditions

In hypoxia, mitochondrial respiration isdecreased, thereby leading to a buildup of reducing equivalents thatcannot be transferred to O₂ at thecytochrome oxidase. This condition, called reductive stress, canparadoxically lead to enhanced formation of reactive O₂ species, or a decrease in theability of the cell to defend against an oxidative stress. Wehypothesized that antioxidants would protect tissues under conditionsof hypoxia. Rat diaphragm strips were incubated in tissue bathscontaining one of four antioxidants: N-acetyl-L-cysteine,dimethyl sulfoxide, superoxide dismutase, or Tiron. The strips weredirectly stimulated in an electrical field. Force-frequencyrelationships were studied under baseline oxygenation (95%O₂-5%CO₂), after 30 min of hypoxia(95% N₂-5% CO₂), and 30 min afterreoxygenation. In all tissues, antioxidants markedly attenuated theloss of contractile function during hypoxia (P < 0.01) and alsosignificantly improved recovery on reoxygenation (P < 0.05). We conclude that bothintracellular and extracellular antioxidants improve skeletal musclecontractile function in hypoxia and facilitate recovery duringreoxygenation in an in vitro system. The strong influence ofantioxidants during hypoxic exposure suggests that they can be aseffective in protecting cell function in a reducing environment as theyhave been in oxidizing environments.

     

Role of carotid body activity responsible for hypoxic ventilatory decline in awake humans

Long, W. Q., G. G. Giesbrecht, and N. R. Anthonisen. Ventilatory response to moderate hypoxia in awakechemodenervated cats. J. Appl. Physiol. 74(2): 805-810, 1993.In humans and cats the ventilatory response to 30 min ofmoderate hypoxia (arterial PO₂ 40-55Torr) is biphasic: ventilation increases sharply for the first 5 minand then declines. In humans there is evidence that the decline isdependent on the initial increase. We therefore examined ventilatoryresponses to moderate isocapnic hypoxia in awake cats with and withoutcarotid body denervation. Cats underwent denervation or a shamoperation. Then they were studied in a Drorbaugh-Fenn plethysmographwhile ventilation, arterial PO₂, and end-tidal PO₂ and PCO₂ weremeasured. Three sham-operated and four denervated cats were studiedwith room air as the control. Sham animals demonstrated a biphasicresponse: ventilation rose to 211% of control at 5 min and fell to114% of control at 25 min. Denervated animals showed neither theinitial increase nor the subsequent decrease in ventilation. Threesham-operated and three denervated cats were studied with 2%CO₂ added to the inspirate. Results were similar: intactcats showed a biphasic response to hypoxia, whereas denervated catsshowed neither an increase nor a decrease in ventilation. Preliminaryexperiments showed that hypoxia was not associated with changes inCO₂ output or systemic blood pressure in either denervatedor intact animals. We conclude that depression of ventilation does notoccur in awake denervated cats in response to moderate hypoxiaand that the decline in ventilation that occurs in intact cats is insome way dependent on peripheral chemoreceptor output.

     

Normalizing Development of Cultured Triticum aestivum L. Embryos: I. LOW OXYGEN TENSIONS AND EXOGENOUS ABA

Wheat (Triticum aestivum L.) embryos form in dynamically-regulatedovular environments. Our objectives were to improve developmentof cultured immature wheat embryos by simulating, in vitro,abscisic acid (ABA) levels and O₂ tensions as found in wheatovules during zygotic embryogenesis. We characterized from intactwheat kernels embryo respiration, embryo morphology and embryoand endosperm + ABA levels at 13, 19 and 25 d post-anthesis(DPA). Young (13 DPA) embryos were then excised and culturedin vitro, where they were exposed to 0·2 or 2·Ommol m^–3 ±ABA and 2.·1, 2·5 or 7·4mol m^–3 (6, 7 and 21%, respectively) gaseous O₂. At 6and 12 d in culture, + ABA levels, embryo respiration and embryomorphology were characterized by treatment. Thirteen-day-oldembryos from two different plant populations differed by 17-foldin initial ABA content. However, this difference did not affectprecocious germination in vitro, nor did it affect the amountof exogenous ABA required to reduce precocious germination by40%. In this respect, embryos from both populations were equallysensitive to exogenous ABA. Cavity sap O₂ levels (2·1to 2·5 mol m^–3) were much more effective in preventingprecocious germination of cultured embryos than were cavitysap levels of ABA (0·2 to 2·0 mmol m^–3).The combination of physiological levels of both ABA and O₂ largelynormalized DW accumulation and embryo morphology without alteringendogenous + ABA levels. Residual respiration of cultured embryoswas higher than that of embryos grown in situ, and was not influencedby the exogenous O₂ and ABA treatments Key words: Abscisic acid, embryo development, oxygen tensions, respiration, wheat     

Accelerated Germination of Maize Seeds Under Chilling Stress by Osmotic Priming and Associated Changes in Embryo Phospholipids

Osmotic priming of maize seeds (Zea mays L. cv. Partap) usingpolyethylene glycol or potassium salts (K₂HPO₄, KH₂PO₄, KNO₃and K₂HPO₄+KNO₃) resulted in accelerated germination at a chillingtemperature (10 °C). The response of seeds primed in solutionsof either 2.5% K₂HPO₄ or 2.5% K₂HPO₄+ KNO₃ was particularlymarked compared with the untreated seeds, and the effect ofpriming was largely retained after seeds had been dried back.All embryo phospholipid fractions and sterols increased duringsalt-priming and the proportion of phospholipid which was diphosphotidylglycerol(DPG) also increased. It is suggested that the marked increasein the DPG content of primed embryos may be due to enhancedinternal organization of their mitochondrial membranes, andthat the benefit of osmotic priming may be at least partly dueto an increased potential for ATP accumulation. Germination, Zea mays L., osmotic priming, phospholipid changes     

Oxygen sensitivity of mitochondrial metabolic state in isolated skeletal and cardiac myocytes

In striatedmuscle the coupling of blood flow to changes in tissue metabolism ishypothesized to be dependent in part on release of vasodilatingmetabolic by-products generated when mitochondrial metabolism becomesO₂ limited. Cytochrome oxidase,the terminal step in oxidative phosphorylation, is half-maximallysaturated at <1 mmHg PO₂ inisolated mitochondria. However, blood flow is regulated at tissuePO₂ of ~20 mmHg. If the affinity ofmitochondrial respiration for O₂were higher in vivo than in vitro,O₂ limitation of mitochondrialmetabolism near mean tissue levels could occur. In the present studythe PO₂ at which mitochondrialmetabolism becomes inhibited (criticalPO₂) was measured for cardiacmyocytes in suspension (1.1 ± 0.15 mmHg) and single cells (1.0 ± 0.22 and 1.25 ± 0.22 mmHg in cardiac myocytes and ratspinotrapezius cells, respectively). These measurements are consistentwith those from isolated mitochondria, indicating that vasodilatorsproduced when oxidative phosphorylation becomes inhibited may beimportant for regulating blood flow only in highly glycolytic musclesor under conditions of severe O₂limitation.

     

Intravascular oxygen distribution in subcutaneous 9L tumors and radiation sensitivity

Cerniglia, George J., David F. Wilson, Marek Pawlowski,Sergei Vinogradov, and John Biaglow. Intravascular oxygendistribution in subcutaneous 9L tumors and radiation sensitivity.J. Appl. Physiol. 82(6):1939-1945, 1997.Phosphorescence quenching was evaluated as atechnique for measuring PO₂ in tumors and for determining the effect of increasedPO₂ on sensitivity of the tumors toradiation. Suspensions of cultured 9L cells or small pieces of solidtumors from 9L cells were injected subcutaneously on the hindquarter ofrats, and tumors were grown to between 0.2 and 1.0 cm in diameter.Oxygen-dependent quenching of the phosphorescence of intravenouslyinjected Pd-meso-tetra-(4-carboxyphenyl) porphine was used to image thein vivo distribution of PO₂ in thevasculature of small tumors and surrounding tissue. Maps (512 × 480 pixels) of tissue oxygen distribution showed that thePO₂ within 9L tumors was low(2-12 Torr) relative to the surrounding muscle tissue (20-40Torr). When the rats were given 100% oxygen or carbogen (95%O₂-5%CO₂) to breathe, thePO₂ in the tumors increasedsignificantly. This increase was variable among tumors and was greaterwith carbogen compared with 100% oxygen. Based on irradiation andregrowth studies, carbogen breathing increased the sensitivity of thetumors to radiation. This is consistent with the measured increase inPO₂ in the tumor vasculature. It isconcluded that phosphorescence quenching can be used for noninvasivedetermination of the oxygenation of tumors. This method for oxygenmeasurements has great potential for clinical application in tumoridentification and therapy.

     

Single locus typing of MHC class I and class II B loci in a population of red jungle fowl

In species with duplicated major histocompatibility complex (MHC) genes, estimates of genetic variation often rely on multilocus measures of diversity. It is possible that such measures might not always detect more detailed patterns of selection at individual loci. Here, we describe a method that allows us to investigate classical MHC diversity in red jungle fowl (Gallus gallus), the wild ancestor of the domestic chicken, using a single locus approach. This is possible due to the well-characterised gene organisation of the ‘minimal essential’ MHC (BF/BL region) of the domestic chicken, which comprises two differentially expressed duplicated class I (BF) and two class II B (BLB) genes. Using a combination of reference strand-mediated conformation analysis, cloning and sequencing, we identify nine BF and ten BLB alleles in a captive population of jungle fowl. We show that six BF and five BLB alleles are from the more highly expressed locus of each gene, BF2 and BLB2, respectively. An excess of non-synonymous substitutions across the jungle fowl BF/BL region suggests that diversifying selection has acted on this population. Importantly, single locus screening reveals that the strength of selection is greatest on the highly expressed BF2 locus. This is the first time that a population of red jungle fowl has been typed at the MHC region, laying the basis for further research into the underlying processes acting to maintain MHC diversity in this and other species.     

Chronic in ovo hypoxia decreases pulmonary arterial contractile reactivity and induces biventricular cardiac enlargement in the chicken embryo

Although chronic prenatal hypoxia is considered a major cause of persistent pulmonary hypertension of the newborn, experimental studies have failed to consistently find pulmonary hypertensive changes after chronic intrauterine hypoxia. We hypothesized that chronic prenatal hypoxia induces changes in the pulmonary vasculature of the chicken embryo. We analyzed pulmonary arterial reactivity and structure and heart morphology of chicken embryos maintained from days 6 to 19 of the 21-day incubation period under normoxic (21% O(2)) or hypoxic (15% O(2)) conditions. Hypoxia increased mortality (0.46 vs. 0.14; P < 0.01) and reduced the body mass of the surviving 19-day embryos (22.4 +/- 0.5 vs. 26.6 +/- 0.7 g; P < 0.01). A decrease in the response of the pulmonary artery to KCl was observed in the 19-day hypoxic embryos. The contractile responses to endothelin-1, the thromboxane A(2) mimetic U-46619, norepinephrine, and electrical-field stimulation were also reduced in a proportion similar to that observed for KCl-induced contractions. In contrast, no hypoxia-induced decrease of response to vasoconstrictors was observed in externally pipped 21-day embryos (incubated under normoxia for the last 2 days). Relaxations induced by ACh, sodium nitroprusside, or forskolin were unaffected by chronic hypoxia in the pulmonary artery, but femoral artery segments of 19-day hypoxic embryos were significantly less sensitive to ACh than arteries of control embryos [pD(2) (= -log EC(50)): 6.51 +/- 0.1 vs. 7.05 +/- 0.1, P < 0.01]. Pulmonary vessel density, percent wall area, and periarterial sympathetic nerve density were not different between control and hypoxic embryos. In contrast, hypoxic hearts showed an increase in right and left ventricular wall area and thickness. We conclude that, in the chicken embryo, chronic moderate hypoxia during incubation transiently reduced pulmonary arterial contractile reactivity, impaired endothelium-dependent relaxation of femoral but not pulmonary arteries, and induced biventricular cardiac hypertrophy.