Spinning response of yeast cells to rotating electric fields

The frequency-dependent rotation or spinning motion of yeast cells subjected to a fourpole rotating electric field was examined over a very wide frequency range (500 Hz to 500 MHz). In the lower frequency range (500 Hz – 700 KHz) the yeast cells were observed to spin in a direction counter to the applied field, with a small peak at about 600 Hz and a more pronounced one at 20 KHz. For frequencies above 700 KHz the spinning of the cells switched direction from counter-field to co-field, with a maximum in the rotation rate at about 70 MHz and a subpeak at 20 MHz. The rate was also observed to exhibit a square dependence on the magnitude of the applied rotating field.     

电旋转技术用于少根根霉孢囊孢子介电性的测定

利用电旋转技术研究了少根根霉（Rhizopus arrhizus）孢囊孢子的电旋转谱,在发现另外两个峰的同时,在场频500Hz附近又发现了1个明显的正旋转峰。同时还发现萌发孢囊孢子与休眠孢囊孢子的电旋转谱有明显的差别,其负旋转峰值明显地小于休眠孢囊孢子的负旋转峰值。借助经修改的椭圆细胞双壳模型（two-shell model) 模拟出了原生质膜和原生质的电常数,结果表明萌发孢子的原生质膜和原生质的电导率较休眠孢子的相应电导率有明显增大。说明电旋转技术能够反映活细胞的生理变化。     

Recovery of acetylcholinesterase activity in the axolotl embryo following inhibition with the organophosphorus inhibitor Gd-7

The restoration of acetylcholinesterase (AChE) activity in axolotl Ambystoma mexicanum embryo after treatment at 38-42 stages with irreversibly AChE-inhibiting Gd-7 phosphororganic inhibitor in concentrations, significantly decreasing AChE activity level, but not interfering with ontogenesis has been studied. The rate of AChE activity restoration in Gd-7 treated axolotl embryo depends on the level of the enzyme restraint and the stage of the embryo development. The value of maximal restoration of AChE activity differs; it is less in embryos, treated with Gd-7 at later stages of development. The ability of the embryos to swim restores parallel to the increase in AChE activity. The data obtained suggest that axolotl embryo possess compensatory mechanism for increasing AChE biosynthesis after decrease in its activity caused by Gd-7. Acetylcholine, accumulating in the organism at partial inactivation of AChE by phosphororganic inhibitor may participate in this mechanism.     

31P-NMR saturation transfer measurements of exchange between Pi and ATP in the reactions catalysed by glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase in vitro

The rotational spectrum of yeast cells changed after pre-treatment of the cells with HgCl2 or Hg(NO3)2 and became indistinguishable from that of ultrasonically produced cell walls. The spectrum of the affected cells contained a peak which could only be explained by attributing a conductivity to the cell walls that was higher than that of the medium. Theoretical models of the rotational response are fully in accord with the experimental spectra. It is shown that the rotation method is capable of measuring even the low cell wall conductivity of yeast cells (which was found to be 33 microS/cm at 10 microS/cm medium conductivity). Knowledge of the spectra allowed a field frequency to be selected at which untreated cells showed no rotation, but at which cells affected by treatment with Hg(II) identified themselves by rotating in the same direction as the field. Calculation of the percentage of cells showing this co-field rotation gave an index (termed the co-field rotation value) of the proportion of the cells that were affected. Using this technique, effects of 25 nmol/l Hg(II) could be demonstrated. In media of low conductivity (10 microS/cm) the change in the rotational spectrum was usually 'all-or-none', whereas at 200 microS/cm a graded Hg(II)-mediated change became apparent. The co-field rotation method showed that the action of small quantities of Hg(II) was still increasing after 3 h of incubation and paralleled the Hg(II)-induced K+ release. A rapid reduction of the effects of Hg(II) was seen when 3-30 mM K+ (or Na+) or when 1 mM Ca2+ were present in the incubation medium, or as the pH was increased. At high incubation cell concentrations the toxic effect of Hg(II) was reduced, apparently due to binding by the cells.     

The effect of mercuric salts on the electro-rotation of yeast cells and comparison with a theoretical model

The rotational spectrum of yeast cells changed after pre-treatment of the cells with HgCl₂ or Hg(NO₃)₂ and became indistinguishable from that of ultrasonically produced cell walls. The spectrum of the affected cells contained a peak which could only be explained by attributing a conductivity to the cell walls that was higher than that of the medium. Theoretical models of the rotational response are fully in accord with the experimental spectra. It is shown that the rotation method is capable of measuring even the low cell wall conductivity of yeast cells (which was found to be 33 μS/cm at 10 μS/cm medium conductivity). Knowledge of the spectra allowed a field frequency to be selected at which untreated cells showed no rotation, but at which cells affected by treatment with Hg(II) identified themselves by rotating in the same direction as the field. Calculation of the percentage of cells showing this co-field rotation gave an index (termed the co-field rotation value) of the proportion of the cells that were affected. Using this technique, effects of 25 nmol/l Hg(II) could be demonstrated. In media of low conductivity (10 μS/cm) the change in the rotational spectrum was usually ‘all-or-none’, whereas at 200 μS/cm a graded Hg(II)-mediated change became apparent. The co-field rotation method showed that the action of small quantities of Hg(II) was still increasing after 3 h of incubation and paralleled the Hg(II)-induced K⁺ release. A rapid reduction of the effects of Hg(II) was seen when 3–30 mM K⁺ (or Na⁺) or when 1 mM Ca²⁺ were present in the incubation medium, or as the pH was increased. At high incubation cell concentrations the toxic effect of Hg(II) was reduced, apparently due to binding by the cells.     

Characterization and development of rotational behavior in Helisoma embryos: role of endogenous serotonin.

Cilia-driven rotational behavior displayed by embryos of the pond snail Helisoma trivolvis was characterized in terms of its behavioral subcomponents, developmental changes, and response to exogenous serotonin. Rotation was found to be a complex behavior characterized by four parameters; rotational direction, rotation rate, rotational surges, and periods of inactivity. These parameters all exhibited characteristic developmental changes from embryonic stage E15 through stage E30. Notably, both rotation rate and frequency of rotational surges increased from stage E15 to E25 and declined to an intermediate level by stage E30. It appeared that the developmental increase in overall rotation rate was caused primarily by an increase in surge frequency, rather than an increase in the rate of nonsurge rotation. Immersion of embryos inserotonin-containing pond water resulted in a dose-dependent, reversible increase in rotation rate as well as a dose-dependent, reversible decrease in surge frequency. The serotonin antagonist, mianserin, abolished the excitatory effect of exogenous serotonin. Furthermore, application of mianserin alone reduced rotation rate and virtually abolished rotational surges. Taken together, these pharmacological results suggest that endogenous serotonin is responsible for generating rotational surges. Given that early embryos contain only a single pair of serotonergic neurons (Goldberg and Kater, 1989) during the stages when rotational surges are expressed, these results also prompt the hypothesis that these neurons, embryonic neurons C1, act as cilioexcitatory motor neurons during embryonic development.     

Characterization and development of rotational behavior in Helisoma embryos: Role of endogenous serotonin

Cilia-driven rotational behavior displayed by embryos of the pond snail Helisoma trivolvis was characterized in terms of its behavioral subcomponents, developmental changes, and response to exogenous serotonin. Rotation was found to be a complex behavior characterized by four parameters; rotational direction, rotation rate, rotational surges, and periods of inactivity. These parameters all exhibited characteristic developmental changes from embryonic stage E15 through stage E30. Notably, both rotation rate and frequency of rotational surges increased from stage E15 to E25 and declined to an intermediate level by stage E30. It appeared that the developmental increase in overall rotation rate was caused primarily by an increase in surge frequency, rather than an increase in the rate of nonsurge rotation. Immersion of embryos inserotonin-containing pond water resulted in a dose-dependent, reversible increase in rotation rate as well as a dose-dependent, reversible decrease in surge frequency. The serotonin antagonist, mianserin, abolished the excitatory effect of exogenous serotonin. Furthermore, application of mianserin alone reduced rotation rate and virtually abolished rotational surges. Taken together, these pharmacological results suggest that endogenous serotonin is responsible for generating rotational surges. Given that early embryos contain only a single pair of serotonergic neurons (Goldberg and Kater, 1989) during the stages when rotational surges are expressed, these results also prompt the hypothesis that these neurons, embryonic neurons C1, act as cilioexcitatory motor neurons during embryonic development.     

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).     

The comparative influence of substituted phenols (especially chlorophenols) on yeast cells assayed by electro-rotation and other methods

The toxicity of 31 phenols was studied by electro-rotation of yeast cells. Control yeast cells show both anti-field and co-field rotation, depending upon the field frequency applied. After treatment with supra-threshold amounts of phenols the anti-field rotation is weakened or abolished and a stronger co-field rotation can be seen. The proportion of cells showing the co-field rotation was found to be a sensitive measure of toxicity. Doses of 2.2 mumol/l of pentachlorophenol, or of 0.3 mumol/l of pentabromophenol were detectable after 3 h incubation at pH 4.0. At a given pH, the toxicity of the chlorophenols correlated extremely well with their octanol:water partition coefficients (Pow). The complete set of phenols showed fair overall correlation with Pow, but less good correlation with their acidity constants (pKa). In particular the toxicity of a given phenol was less than predicted from its pKa if the incubation pH was higher than the pKa. Biochemical assays on 23 of the phenols showed that the rotational sensitivity runs closely parallel to the sensitivities of cell growth rate and of the plasmamembrane ATPase, but less closely to the inhibition of purine incorporation. It appears that the electro-rotation method provides a useful and rapid test for the presence of organic ecotoxins. The test enables us to distinguish differences between single cells, and is comparable in sensitivity to biochemical tests that use vesicles or homogenates derived from a cell population.     

Melanotropin as a Potential Regulator of Pigment Pattern Formation in Embryonic Skin

Frozen tissue sections of developing axolotl embryos were labeled by indirect immunofluorescence with anti-alpha-MSH. Anti-MSH immunoreactivity is first detectable in embryos when neural crest cells are migrating from the neural tube. Antibody labeling is visible around the lateral and ventral edges of the neural tube and in the embryonic ectoderm. As development progresses, the amount of labeling increases greatly, particularly in developing ectoderm. Western blots of soluble proteins extracted from various developmental stages of axolotl embryo ectoderm reveal that MSH activity is associated directly with several high molecular weight components that may be part of the embryonic extracellular matrix. Thus, we suggest that melanotropin activity is present in embryonic axolotl skin, is associated with the extracellular matrix, and is thereby in a position to play a supportive and/or directive role in the establishment of embryonic pigment patterns.     

Myocardial cell relationships during morphogenesis in normal and cardiac lethal mutant axolotls, Ambystoma mexicanum

Sarcomere formation has been shown to be deficient in the myocardium of axolotl embryos homozygous for the recessive cardiac lethal gene c. We examined the developing hearts of normal and cardiac mutant embryos from tailbud stage 33 to posthatching stage 43 by scanning electron microscopy in order to determine whether that deficiency has any effect on heart morphogenesis. Specifically, we investigated the relationships of myocardial cells during the formation of the heart tube (stage 33), the initiation of dextral looping (stages 34-36), and the subsequent flexure of the elongating heart (stages 38-43). In addition, we compared the morphogenetic events in the axolotl to the published accounts of comparable stages in the chick embryo. In the axolotl (stage 33), changes in cell shape and orientation accompany the closure of the myocardial trough to form the tubular heart. The ventral mesocardium persists longer in the axolotl embryo than in the chick and appears to contribute to the asymmetry of dextral looping (stages 34-36) in two ways. First, as a persisting structure it places constraints on the simple elongation of the heart tube and the ability of the heart to bend. Second, after it is resorbed, the ventral myocardial cells that contributed to it are identifiable by their orientation, which is orthogonal to adjacent cells: a potential source of shearing effects. Cardiac lethal mutant embryos behave identically during these events, indicating that functional sarcomeres are not necessary to these processes. The absence of dynamic apical myocardial membrane changes, characteristic of the chick embryo (Hamburger and Hamilton stages 9-11), suggests that sudden hydration of the cardiac jelly is less likely to be a major factor in axolotl cardiac morphogenesis. Subsequent flexure (stages 38-43) of the axolotl heart is the same in normal and cardiac lethal mutant embryos as the myocardial tube lengthens within the confines of a pericardial cavity of fixed length. However, the cardiac mutant begins to exhibit abnormalities at this time. The lack of trabeculation (normally beginning at stage 37) in the mutant ventricle is evident at the same time as an increase in myocardial surface area, manifest in extra bends of the heart tube at stage 39. Nonbeating mutant hearts (stage 41) have an abnormally large diameter in the atrioventricular region, possibly the result of the accumulation of ascites fluid. In addition, mutant myocardial cells have a larger apical surface area compared to normals.     

The spatial and temporal distribution of polarizing activity in the flank of the pre-limb-bud stages in the chick embryo

The presence of polarizing activity in the limb buds of developing avian embryos determines the pattern of the anteroposterior axis of the limbs in the adult. Maps of the spatial distribution and the strength of the signal within limb buds of different stages are well documented. Polarizing activity can also be found in Hensen's node in the early embryo. We have mapped the distribution of polarizing activity as it emerges from Hensen's node and spreads into the flank tissue of the embryo. There is a clear change in the local pattern of expression of polarizing activity between stage 8 and 18. Almost no activity is measured for stages 8 and 9. More or less uniform levels of around 10% are spread along the flank lateral to the unsegmented somitic mesoderm from somite position 12 to 22 in stage 10 embryos. Some 6 to 8 h later at stage 12, there is a distinct peak of activity at somite position 18, the middle of the wing field. This peak increases at stages 13 to 15 and its position traverses to the posterior edge of the wing field. Full strength of activity is reached shortly before the onset of limb bud formation at stage 16 to 17. Stages 16 to 18 were investigated for polarizing activity in the wing and the leg field. Low levels of polarizing activity are present in the anterior leg field at stages 16 and 17 but have disappeared by stage 18 and all activity is confined to the posterior part of the leg bud.     

Nucleolar and mitochondrial morphology in bovine embryos reconstructed by nuclear transfer

The nucleolar and mitochondrial morphology of developing reconstructed bovine nuclear transfer (NT) embryos and stage-matched in vivo-produced control embryos were examined under the electron microscope. Each reconstructed embryo at the one-cell (n = 12), two-cell (n = 5), three-cell (n = 3), four-cell (n = 5), 5–8 cell (n = 5) and blastocyst (n = 3) stages was produced by fusion of a 16–32-cell-stage blatomere with an aged enucleated bovine oocyte. The normal and reconstructed embryos showed similar mitochondrial morphology. However, NT embryos produced several pleiomorphic forms not seen in controls, and were more heterogenous at early stages of development. Control embryos exhibited nucleolar features considered indicative of rRNA synthesis from the eight-cell stage onwards. In contrast, the NT embryos presented nucleoli with morphology consistent with rRNA synthesis in all embryos examined, except in the three-cell and in two of the five four-cell embryos. From this nucleolar morphology, it was concluded that nuclear reprogramming does not occur immediately following nuclear transfer, but occurs gradually over the first two or three cell cycles. © 1996 Wiley-Liss, Inc.     

Low frequency electrorotation of fixed red blood cells.

Electrorotation of fixed red blood cells has been investigated in the frequency range between 16 Hz and 30 MHz. The rotation was studied as a function of electrolyte conductivity and surface charge density. Between 16 Hz and 1 kHz, fixed red blood cells undergo cofield rotation. The maximum of cofield rotation occurs between 30 and 70 Hz. The position of the maximum depends weakly on the bulk electrolyte conductivity and surface charge density. Below 3.5 mS/m, the cofield rotation peak is broadened and shifted to higher frequencies accompanied by a decrease of the rotation speed. Surface charge reduction leads to a decrease of the rotation speed in the low frequency range. These observations are consistent with the recently developed electroosmotic theory of low frequency electrorotation.     

Recovery of acetylcholinesterase activity after irreversible inhibition by organophosphorous compounds in embryonic development

1. Recovery of acetylcholinesterase (AChE) activity was studied using the embryos of sea urchins Strongylocentrotus intermedius and S. nudus, embryos of axolotl Ambystoma mexicanum and in the chick embryo muscle culture treated by "irreversible" organophosphorous inhibitors (OPI). 2. AChE activity was assayed by a modified Ellman's procedure. 3. It follows from the data obtained that, unlike the plutei of sea urchins and the monolayer culture of chick embryo muscle cells, the embryos of axolotl show a compensatory increase in AChE biosynthesis after inhibition by OPI. 4. This mechanism is assumed to be related to the presence of a well developed neuromuscular system in the A. mexicanum embryos. 5. It is possible that acetylcholine accumulated as a result of partial AChE inhibition is responsible for the compensatory increase in AChE biosynthesis.     

Role of enzymes and identification of stage-specific proteins in developing somatic embryos of chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.)

Accumulation of proline, activities of peroxidase (POX), catalase (CAT), phenylalanine ammonia lyase (PAL) and malate dehydrogenase (MDH) were studied during different developmental stages of somatic embryos in chickpea. Callus cultures that did not form somatic embryos served as control. While increased levels of proline and POX activity were noticed in globular stages of embryos, CAT activity increased during early and late heart-shaped embryo formation indicating tissue-specific activation of these enzymes. The activity of PAL reached a peak during torpedo and cotyledonary stages of embryo development. On the other hand, MDH activity enhanced during the germination of somatic embryos inferring more requirement of energy during this stage. Electrophoretic (sodium dodecyl sulfate polyacrylamide gel electrophoresis) pattern of proteins revealed that ten bands are associated with non-embryogenic tissues, whereas 11 bands with globular, heart, torpedo and cotyledonary stages of embryo development and nine bands during the germination stage of embryos. Two extra stage-specific protein bands with molecular masses of 16 and 18 kDa appeared during globular, heart, torpedo, and cotyledonary stages. But, these bands disappeared during germination of embryos and are absent in non-embryogenic cultures. This study thus may help in the identification of proteins and the role of above enzymes during different developmental stages of somatic embryo induction and their maturation in a recalcitrant leguminous crop plant chickpea.     

The distribution of fibronectin and tenascin along migratory pathways of the neural crest in the trunk of amphibian embryos

It is generally assumed that in amphibian embryos neural crest cells migrate dorsally, where they form the mesenchyme of the dorsal fin, laterally (between somites and epidermis), where they give rise to pigment cells, and ventromedially (between somites and neural tube), where they form the elements of the peripheral nervous system. While there is agreement about the crest migratory routes in the axolotl (Ambystoma mexicanum), different opinions exist about the lateral pathway in Xenopus. We investigated neural crest cell migration in Xenopus (stages 23, 32, 35/36 and 41) using the X. laevis-X. borealis nuclear marker system and could not find evidence for cells migrating laterally. We have also used immunohistochemistry to study the distribution of the extracellular matrix (ECM) glycoproteins fibronectin (FN) and tenascin (TN), which have been implicated in directing neural crest cells during their migrations in avian and mammalian embryos, in the neural crest migratory pathways of Xenopus and the axolotl. In premigratory stages of the crest, both in Xenopus (stage 22) and the axolotl (stage 25), FN was found subepidermally and in extracellular spaces around the neural tube, notochord and somites. The staining was particularly intense in the dorsal part of the embryo, but it was also present along the visceral and parietal layers of the lateral plate mesoderm. TN, in contrast, was found only in the anterior trunk mesoderm in Xenopus; in the axolotl, it was absent. During neural crest cell migration in Xenopus (stages 25-33) and the axolotl (stages 28-35), anti-FN stained the ECM throughout the embryo, whereas anti-TN staining was limited to dorsal regions. There it was particularly intense medially, i.e. in the dorsal fin, around the neural tube, notochord, dorsal aorta and at the medial surface of the somites (stage 35 in both species). During postmigratory stages in Xenopus (stage 40), anti-FN staining was less intense than anti-TN staining. In culture, axolotl neural crest cells spread differently on FN- and TN-coated substrata. On TN, the onset of cellular outgrowth was delayed for about 1 day, but after 3 days the extent of outgrowth was indistinguishable from cultures grown on FN. However, neural crest cells in 3-day-old cultures were much more flattened on FN than on TN. We conclude that both FN and TN are present in the ECM that lines the neural crest migratory pathways of amphibian embryos at the time when the neural crest cells are actively migrating. FN is present in the embryonic ECM before the onset of neural crest migration.(ABSTRACT TRUNCATED AT 400 WORDS)     

Label-free imaging of lipid-droplet intracellular motion in early Drosophila embryos using femtosecond-stimulated Raman loss microscopy

Lipid droplets are complex organelles that exhibit highly dynamic behavior in early Drosophila embryo development. Imaging lipid droplet motion provides a robust platform for the investigation of shuttling by kinesin and dynein motors, but methods for imaging are either destructive or deficient in resolution and penetration to study large populations of droplets in an individual embryo. Here we report real-time imaging and quantification of droplet motion in live embryos using a recently developed technique termed "femtosecond-stimulated Raman loss" microscopy. We captured long-duration time-lapse images of the developing embryo, tracked single droplet motion within large populations of droplets, and measured the velocity and turning frequency of each particle at different apical-to-basal depths and stages of development. To determine whether the quantities for speed and turning rate measured for individual droplets are sufficient to predict the population distributions of droplet density, we simulated droplet motion using a velocity-jump model. This model yielded droplet density distributions that agreed well with experimental observations without any model optimization or unknown parameter estimation, demonstrating the sufficiency of a velocity-jump process for droplet trafficking dynamics in blastoderm embryos.     

Role of glucose in mouse preimplantation embryo development

Mouse preimplantation embryos consume pyruvate preferentially during the early developmental stages, before glucose becomes the predominant energy substrate in the blastocyst. To investigate the importance of the switch to glucose utilization at the later developmental stages, mouse embryos from F1 hybrid mice (CBA/Ca × C57BL/6) were cultured from the one-and two-cell stages (22 and 46 h post hCG, respectively) for 5 days in a modified medium, M16, containing 0.33 mM pyruvate and 5 or 23 mM D+L-lactate, in the presence and absence of 1 mM glucose (M16+G and M16-G, respectively). Nutrient uptakes were also determined over this time. Some embryos cultured in M16-G were transferred to M16+G at 94 or 118 h post hCG. Embryos cultured from the two-cell stage in M16+G exhibited the characteristic fall in pyruvate consumption between the morula and the blastocyst stage; those cultured from the two-cell stage in M16-G compensated for the lack of glucose by consuming increasing amounts of pyruvate, from 2.78 pmol/embryo/h at 58 h post hCG to 5.21 pmol/embryo/h at 154 h post hCG. However, the percentage of embryos developing to the blastocyst stage, the hatching rate, and blastocyst cell numbers (50.6 ± 2.5 [28] vs. 105 ± 3.8 [37]) were all lower in this group. When exposed to glucose at 94 or 118 h post hCG, embryos cultured from the two-cell stage in M16-G readily consumed glucose in preference to pyruvate, although the characteristic fall in pyruvate consumption was not observed. One-cell embryos cultured continuously in M16-G were only able to develop to the morula stage, after which time they degenerated. In these embryos pyruvate was readily consumed between 22 and 94 h post hCG, before falling from 2.77 pmol/embryo/h at 83 h post hCG to 0.045 pmol/embryo/h at 130 h post hCG. Transfer of these embryos to M16+G at 94 and 118 h post hCG did not support development to the hatching blastocyst stage. The results show that mouse preimplantation embryos from F1 hybrid mice (CBA/Ca × C57BL/6) need only be exposed to glucose for less than 24 h between 22 and 94 h post hCG in order to develop from the morula to the blastocyst stage in vitro. However, the exposure time needs to be increased to between 24 and 72 h in order that blastocyst cell numbers reach control levels. The importance of glucose before the morula stage may relate to the need to synthesize glycogen for later use. If the obligatory requirement for glucose is fulfilled, embryos are able to utilize pyruvate in the absence of glucose at the later stages of development. These results show that the mouse preimplantation embryo can, to some extent, adapt metabolically to changes in its external environment. © 1995 Wiley-Liss, Inc.     

Changes in protein profiles associated with somatic embryogenesis in peanut

The somatic embryogenesis potential of zygotic embryo axes of peanut (Arachis hypogaea L. cv. DRG-12) at different stages of development was evaluated by culturing on MS medium with 18.1 μM 2,4-dichlorophenoxyacetic acid (2,4-D). A 100 % frequency with 18.3 somatic embryos per explant was observed from 4 mm long immature zygotic embryo axes collected 31 – 40 d after pollination. Medium supplemented with 16.6 μM picloram resulted in slow development of somatic embryos whereas in the presence of 21.5 μM α-naphthaleneacetic acid (NAA), the explants underwent maturation with induction of roots after 30 d. The changes in protein profiles in zygotic embryo axes at different stages of development correlated with their potential to form somatic embryos. Immature zygotic embryo axes exhibited high frequency somatic embryogenesis in the stage preceding abundant accumulation of 22 and 65 kDa proteins. The content of 22 and 65 kDa proteins decreased immediately after culture on medium fortified with 18.1 μM 2,4-D and increased again after 12 d of culture coinciding with the development of somatic embryos on the explants. The content of 22 and 65 kDa proteins was low at 15 d of culture on medium supplemented with 16.6 μM picloram possibly due to slow development of the somatic embryos on the explant. On maturation medium containing 21.5 μM NAA, a marked increase in the content of 22 and 65 kDa proteins in 15 d-old cultures was observed.