The sensitivity of whole,half and quarter sea urchin embryos to cytotoxic neuropharmacological drugs

1. Embryos of sea urchins Echinocardium cordatum, Echinus melo and E. esculentus are supersensitive to the same cytotoxic neurochemicals as Arbacia lixula embryos studied earlier.2. White half and clear quarter embryos of A. lixula have no obvious supersensitivity to neurochemicals.3. Supersensitivity is restored in granular quarter embryos up to the initial level, and it does not disappear in the red halves and in corresponding quarter embryos.4. There is a non-obligatory coupling between the supersensitivity receptors and cell division processes.     

Sensitivity of fragmented and stratified sea urchin embryos to cytotoxic neuropharmacological preparations

The red half embryos and related quarter embryos (yolk and pigment) of Arbacia lixula, obtained by means of centrifugation of the eggs in sucrose gradient, retain the normal level of sensitivity and supersensitivity to cytotoxic neuropharmaca, antagonists of biogenic monoamines. The white half embryos and clear quarter embryos practically lack supersensitivity whereas the granular quarter embryos restore it to the initial level. The non-pigmented blastomers of stratified embryos are characterized by somewhat weakened supersensitivity. A suggestion is put forward that the supersensitive embryos of A. lixula possess a sensibilizing factor which couples the supersensitivity receptors with the processes of cell division and moves together with the yolk granules upon centrifugation. This factor is not observed in the Strongylocentrotus granularis embryos lacking evident supersensitivity.     

Activation of respiration in sea urchin spermatozoa by calcium ionophore A23187

The respiratory rate in spermatozoa of the sea urchin, Hemicentrotus pulcherrimus, in Na+-free seawater, where sperm are immotile and their respiration remains inactive, was stimulated by calcium ionophore A23187. Addition of ionophore A23187 to Na+-free seawater induced swimming as well as activating energy metabolism in sea urchin sperm. The increase of respiratory rate and the initiation of motility in sperm were independent of external Ca2+.     

Physiological responses of sea urchin eggs to stimulation by calcium ionophore A23187 analysed with protease inhibitors

Protease inhibitors were used to study certain physiological responses (secretion of the cortical granule protease, altered resceptively to sperm penetration, initiation of cell division and embryogenesis) of sea urchin eggs to stimulation by calcium ionophore A23187. Protease activity in the secretory product released from the eggs 5 min after insemination or parthenogenetic activation with ionophore was completely inhibited by soybean trypsin inhibitor (SBTI), antipain (Ap), and leupeptin (Lp). A barrier was established to prevent subsequently added sperm from penetrating (fertilizing) ionophore-activated eggs, co-incident with the elevation of the fertilization membrane. These processes were retarded by inhibitors of the cortical granule protease in ionophore-activated eggs, just as they are when eggs are initially stimulated by sperm at fertilization. A23187-activated eggs did not divide unless they had been secondarily fertilized by sperm, even if the ionophore was subsequently removed by extensive washing. However, ionophore-activated eggs that were penetrated by a single spermatozoan in SBTI developed into normal larvae under similar conditions. These results suggest that A23187 may be an incomplete parthenogenetic agent because it cannot stimulate eggs to assemble centrioles required to organize the mitotic apparatus. The centrioles are normally provided by the sperm during fertilization. A23187 may also be toxic to the eggs. Furthermore, since cortical granules are secretory organelles, the data suggest a possible functional relationship between calcium ions and protease activation in stimulus-secretion coupling in sea urchin eggs at fertilization.     

Oral—aboral ectoderm differentiation of sea urchin embryos is disrupted in response to calcium ionophore

Intracellular signaling mediated by calcium ions has been implicated as important in controlling cell activity. The ability of calcium ionophore (A23187), which causes an increase in calcium ion concentration in the cytoplasm, to alter the pattern of differentiation of cells during sea urchin development was examined. The addition of A23187 to embryos for 3h during early cleavage causes dramatic changes in their development during gastrulation. Using tissue-specific cDNA probes and antibodies, it was shown that A23187 causes the disruption of oral–aboral ectoderm differentiation of sea urchin embryos. The critical period for A23187 to disturb the oral–aboral ectoderm differentiation is during the cleavage stage, and treatment of embryos with A23187 after that time has little effect. The A23187 does not affect the formation of the three germ layers. These results indicate that intracellular signals mediated by calcium ions may play a key role in establishment of the oralaboral axis during sea urchin development.     

Patterns in the binding of cytotoxic neuropharmacologic preparations by early sea urchin embryos]

Early embryos of Arbacia lixula, Paracentrotus lividus and Sphaerechinus granularis intensively bind cytotoxic neuropharmacological drugs, such as antiserotonine indole derivatives, cholinolytics and tricyclic antidepressants. The binding intensity decreases markedly upon quaternization of the drugs. Quantitative analysis indicates that: a)with respect to the drugs, the suspension of living embryos may be described as a single adsorbing system following the Langmuir equation; b) at least two independent binding pools exist in embryos; c) the magnitude of cytotoxic effect of a given drug is not proportional to its binding.     

Pulse treatment of sea urchin embryos with A23187 blocks their hatching out

Summary Pulse treatment of sea urchin embryos with 3 µM A23187 for 2 h at 20° C, starting from 3 to 6 h of development, prevented the embryos from hatching. Many embryos thus treated with A23187 produced mesenchyme cells and underwent gastrulation while still enclosed within the fertilization membrane. The pulse treatment in this pre-hatching period exerts markedly stronger inhibitory effects on hatching than on other events in early development. Treatment beginning at times earlier than 2 h and later than 8 h of development caused only a slight delay of hatching. The activity of hatching enzyme, known to increase between 6 and 8 h after fertilization, was quite low, if present at all, in embryos in which hatching was blocked by A23187. Hatching enzyme synthesis is probably blocked by the preceding pulse treatment. However, overall protein synthesis, estimated with methionine S 35 incorporation, was somewhat augmented in embryos by the pulse treatment. The blockage of hatching and the augmentation of overall protein synthesis by A23187 were appreciably reversed by procaine, tetracaine, ruthenium red or verapamil. Probably, an artificial Ca²⁺ signal induced by A23187 activates protein synthesis but blocks the induction of hatching enzyme synthesis.     

Local perinuclear calcium signals associated with mitosis-entry in early sea urchin embryos

Using calcium-sensitive dyes together with their dextran conjugates and confocal microscopy, we have looked for evidence of localized calcium signaling in the region of the nucleus before entry into mitosis, using the sea urchin egg first mitotic cell cycle as a model. Global calcium transients that appear to originate from the nuclear area are often observed just before nuclear envelope breakdown (NEB). In the absence of global increases in calcium, confocal microscopy using Calcium Green- 1 dextran indicator dye revealed localized calcium transients in the perinuclear region. We have also used a photoinactivatable calcium chelator, nitrophenyl EGTA (NP-EGTA), to test whether the chelator- induced block of mitosis entry can be reversed after inactivation of the chelator. Cells arrested before NEB by injection of NP-EGTA resume the cell cycle after flash photolysis of the chelator. Photolysis of chelator triggers calcium release. TreatmenT with caFfeine to enhance calcium-induced calcium release increases the amplitude of NEB- associated calcium transients. These results indicate that calcium increases local to the nucleus are required to trigger entry into mitosis. Local calcium transients arise in the perinuclear region and can spread from this region into the cytoplasm. Thus, cell cycle calcium signals are generated by the perinuclear mitotic machinery in early sea urchin embryos.     

Effect of calcium ionophore A23187 on pregnenolone metabolism to progesterone in rat granulosa cells

The effect of calcium ionophore A23187 on the metabolism of pregnenolone to progesterone was examined in rat granulosa cells during a 24-h culture period. Granulosa cells harvested from pregnant mare's serum gonadotropin treated immature rats were incubated in the presence and absence of the divalent cation ionophore A23187. The ionophore induced progesterone synthesis from both endogenous sterol substrate and exogenous pregnenolone in a time- and concentration-dependent manner. Pregnenolone metabolism was examined in the presence of aminoglutethimide phosphate, an inhibitor of endogenous pregnenolone production. Steroid secretion resulting from metabolism of endogenous substrate was more sensitive to A23187 in that a lower concentration of the ionophore was required to induce a significant increase than that noted for exogenous pregnenolone metabolism. In addition, progesterone production from endogenous sterol occurred 6 h earlier than the observed increase in the conversion of pregnenolone to progesterone. These results indicate that A23187 and therefore possibly enhanced calcium influx may play a significant role in the regulation of pregnenolone metabolism in granulosa cells depending on the duration of incubation. The earlier steroidogenic response from endogenous substrate may be a reflection of an acute effect of A23187 on certain steroidogenic steps proximal to pregnenolone production.     

Action of insulin and cell calcium: Effect of ionophore A23187

Summary We have measured the effects of the carboxylic Ca⁺⁺ ionophore A23187 on muscle tension, resting potential and 3-O-methylglucose efflux. The ionophore produces an increase in tension that is dependent on external Ca⁺⁺ concentration since (a) the contracture was blocked by removing external Ca⁺⁺ and (b) its size was increased by raising outside Ca⁺⁺. Neither resting potential nor resting and insulin-stimulated sugar efflux were modified by the ionophore. These data imply that the action of insulin is not mediated by increasing cytoplasmic [Ca⁺⁺]. Additional support for this conclusion was obtained by testing the effects of caffeine on sugar efflux. This agent, which releases Ca⁺⁺ from the reticulum, did not increase resting sugar efflux and inhibited the insulin-stimulated efflux. Incubation in solutions containing butyrated derivatives of cyclic AMP or cyclic GMP plus theophylline did not modify the effects of insulin on sugar efflux. Evidence suggesting that our experimental conditions increased the cytoplasmic cyclic AMP activity was obtained.     

Sensitivity of starfish oocytes and whole, half and enucleated embryos to cytotoxic neuropharmacological drugs

In early embryos of eight starfish species both usual sensitivity and supersensitivity to cytotoxic neurochemicals have been found. This sensitivity is unaffected by the removal of the cell nucleus. In the starfish Aphelasterias japonica the supersensitivity in the embryos' red halves is higher and in embryos' white halves lower than in the whole embryos.     

Action of serotonin antagonists on cytoplasmic calcium levels in early embryos of sea urchin Lytechinus pictus

Possible interaction of the serotonergic system with intracellular calcium mechanisms was investigated using techniques of ratio imaging measurement of intracellular Ca2+ and confocal microscopy in cleaving embryos of sea urchin Lytechinus pictus. Some serotonin antagonists specifically increase free intracellular Ca2+ and evoke transient regression of the first cleavage furrow, suggesting possible linkage of serotonergic and calcium mechanisms in the regulation of cellular events during cleavage divisions. These effects were more pronounced in the experiments with hydrophilic 5-HT-antagonists, quarternary ammonium salts that do not penetrate the cell membrane. Thus, it appears that 5-HT-receptors which mediate these effects are localised on the cell membrane, whereas previously studied receptors mediating the cytostatic action of lipophilic 5-HT-antagonists are localised intracellularly.     

Biphasic stage sensitivity to UV suppression of gastrulation in sea urchin embryos

The effects of ultraviolet light (UV) on the gastrulation of sea urchin embryos were examined. The results suggest that gastrulation is inhibited by UV irradiation and that stage sensitivity to UV suppression of gastrulation changes biphasically: higher sensitivity at early and late blastula, and lower sensitivity at the mid-blastula stages. The UV-induced inhibition of gastrulation was completely reversible by subsequent exposure to visible light.     

Development of membrane sensitivity to the parthenogenetic agent calcium ionophore A23187 during meiotic maturation in the hamster oocyte

The response to the parthenogenetic agent Ca-ionophore A23187 was studied in hamster oocytes undergoing meiotic maturation, by using electrophysiological techniques. Following germinal vesicle breakdown, the activating agent induces a long-lasting hyperpolarization accompanied by an increased membrane conductance. The duration of the response progressively shortens during the long metaphase I stage. Terminal metaphase I oocytes respond to A23187 by a hyperpolarization that is very similar to that seen in metaphase II oocytes. The ionic mechanism of the change in the membrane sensitivity to A23187 during meiotic maturation is discussed.