Richard A. Fluck

Ooplasmic Segregation in the Medaka (Oryzias latipes) Egg

Using time-lapse video microscopy, we found that ooplasmic inclusions in the fertilized medaka egg displayed two types of movement during ooplasmic segregation. The first manifested itself as the movement of many inclusions (diameter = 1.5-11 μm) toward the animal pole at about 2.2 μm min-1; this type of movement appeared to be streaming. The second type of movement was faster (about 44 μm min-1) and saltatory; inclusions displaying this type of movement were smaller (diameter ≤1.0 μm) and moved toward the vegetal pole. The movement of oil droplets toward the vegetal pole of the egg may represent a third type of motion. All these movements began only after a strong contraction of the ooplasm toward the animal pole, which at 25°C began 10-12 min after fertilization and <3 min after formation of the second polar body. In eggs treated with microtubule poisons--colchicine, colcemid, or nocodazole--oil droplets did not move toward the vegetal pole, saltatory motion toward the vegetal pole was absent, and the growth of the blastodisc was slowed. Eggs treated with β-lumicolchicine, an inactive derivative of colchicine, showed normal movements. Colchicine, while not inhibiting formation of the second polar body, did inhibit pronuclear migration. These results suggest that microtubules are involved in the movement of some ooplasmic inclusions, including oil droplets, toward the vegetal pole; the movement of ooplasmic inclusions toward the animal pole; and pronuclear migration.

ACETYLCHOLINE AND ACETYLCHOLINESTERASE ACTIVITY IN EARLY EMBRYOS OF THE MEDAKA ORYZIAS LATIPES, A TELEOST

Acetylcholinesterase (AChE) activity is present in homogenates of medaka embryos during cleavage and epiboly. The levels of AChE activity change little during this period of development and are similar in embryos grown at either 15°C or 25°C. The specific activity of AChE in cells isolated from blastulae is 0.06 mmoles substrate hydrolyzed/min/g protein, a value comparable to that of chicken myoblasts and myotubes in vitro. Acetylcholinesterase activity was detected cytochemically in all cells dissociated from blastulae and gastrulae. In deep blastomeres AChE activity is present nearly throughout the cytoplasm; it is absent from peripheral regions of the cytoplasm which are involved in circus, or limnicolor, movements. Acetylcholine‐like activity in extracts of the embryos was assayed using the clam heart ventricle bioassay. The active principle, which caused a decrease in both the frequency and magnitude of ventricular contractions, was inactivated by heating at pH 10 and by incubation at pH 7.0 with commercial AChE. The effect of the active principle on the clam heart was blocked by mytolon chloride, a drug which specifically blocks the effect of acetylcholine on the clam heart. The active principle migrated on Whatman #1 chromatography paper with the same Rf as authentic acetylcholine in three solvent systems. The amount of acetylcholine in blastulae is about 4 picomoles/embryo.

11 Cytoskeleton in Teleost Eggs and Early Embryos: Contributions to Cytoarchitecture and Motile Events

Publisher Summary Successful interaction between gametes initiates a cascade of pathways that lead to cleavage, cellular differentiation, morphogenesis, and eventual development of an adult organism. Shortly after fertilization of the teleost egg, the egg cortex undergoes a predictable sequence of structural changes in response to intracellular signals. Early transformation of the cortex includes formation of the fertilization cone and second polar body, sperm entry into the egg, cortical granule exocytosis, and surface membrane retrieval by endocytosis. Subsequently, cytoplasm moves toward the animal pole where it accumulates in preparation for cell division and cellular rearrangement. By analogy to other cell systems that show secretion, mitosis, intracellular cytoplasmic flow, and cell migration, it is predicted that similar events in eggs are mediated by action of the cytoskeleton. Most of the knowledge about the egg cytoskeleton has been obtained from analyses of sea urchin, amphibian, and mammalian eggs. By comparison, the composition, organization, and function of the egg cytoskeleton in teleost fishes have received considerably less attention. The eggs of teleost fish have long provided favorable material for the study of vertebrate development. Unlike mammalian and amphibian eggs, teleost eggs can be obtained in large numbers, are optically transparent, and are easily accessible for experimental manipulation. Furthermore, teleosts now also offer the possibility of employing genetic and molecular approaches to investigate the role of the cytoskeleton in early development. This chapter surveys the teleost egg cytoskeleton and evaluates its proposed role(s) in events of fertilization, ooplasmic segregation, and gastrulation.

Calcium signaling during the early development of medaka and zebrafish

The ex-utero fertilization and development of the optically clear embryos of teleost fish have long been favorites of developmental biologists. They have, therefore, provided considerable insight with regards to our understanding of embryonic pattern formation and the early development of vertebrates. These attributes have also been most helpful in the visualization of Ca(2+) signaling events that have been reported to accompany many of the fundamental steps and processes that constitute early embryonic development. These include egg activation; segregation of the cytoplasm from the yolk; cytokinesis; axis determination; cellular rearrangement and germ layer establishment; as well as the formation of the first tissue domains. The developing eggs and embryos of medaka (Oryzias latipes) and zebrafish (Danio rerio) have for many decades been a favorite choice of investigators attempting to visualize Ca(2+) signaling events. In this short review, we have attempted to catalog and present a comparative study of the developmental Ca(2+) signals recorded in these most amenable of vertebrate models.

Microinjection of cyclic ADP-ribose triggers a regenerative wave of Ca2+ release and exocytosis of cortical alveoli in medaka eggs.

Medaka (Oryzias latipes) eggs microinjected with the Ca(2+)-mobilising messenger cyclic adenosine diphosphate ribose (cADPR) underwent a wave of exocytosis of cortical alveoli and were thus activated. The number of eggs activated was sharply dependent on the concentration of cADPR in the pipette, the threshold concentration was approximately 60 nM. After injection, a pronounced latency preceded the onset of cortical alveoli exocytosis; this latency was independent of the concentration of cADPR but decreased markedly with increasing temperature. Heat-treated cADPR, which yields the inert non-cyclised product ADP-ribose, was ineffective in activating eggs. When cADPR was injected into aequorin-loaded eggs, a wave of luminescence arose at the site of cADPR injection and then swept out across the egg with a mean velocity of approximately 13 microns/s; the velocity was independent of the concentration of injected cADPR. In such a large cell (diameter of around 1 mm), this is considerably faster than that possible by simple diffusion of cADPR, which unambiguously demonstrates that cADPR must activate a regenerative process. cADPR has been demonstrated to modulate Ca(2+)-induced Ca2+ release (CICR) via ryanodine receptors (RyRs) in many cell types, and consistent with this was the finding that microinjection of the pharmacological RyR modulator, ryanodine, also activated medaka eggs. These results suggest that a cADPR-sensitive Ca2+ release mechanism is present in the medaka egg, that cADPR is the most potent activator of medaka eggs described to date, and that it activates eggs by triggering a wave of CICR from internal stores that in turn stimulates a wave of exocytosis.

High Calcium Zones at the Poles of Developing Medaka Eggs

We have injected medaka fish zygotes with recombinant aequorin and visualized the resulting patterns of luminescence to reveal patterns of free calcium during early development. We have co-injected fluorescein-labeled aequorin to correct for nonuniformities in aequorin (as opposed to calcium) distributions by visualizing the resulting patterns of fluorescence as opposed to luminescence. We have also coinjected a calcium buffer to facilitate calcium diffusion, dissipate apparent calcium gradients, and thus confirm their reality. An exploratory study shows zones of elevated free calcium at the vegetal as well as the animal pole during the first day of development and thus up to the beginning of gastrulation. A closer study during the first 6 h, and thus through ooplasmic segregation and early cleavage, shows a steady zone of high calcium at the vegetal pole and a slowly oscillating one at the animal pole. The latter is particularly strong during ooplasmic segregation and cytokinesis. This report contains the first unambiguous evidence of relatively steady zones of high cytosolic calcium during the development of an animal egg.

MICROTUBULE ARRAYS DURING OOPLASMIC SEGREGATION IN THE MEDAKA FISH EGG (ORYZIAS LATIPES)

We used indirect immunofluorescence to study microtubule arrays in the medaka egg between fertilization (normalized time, Tn, - 0) and the first cleavage (Tn = 1.0). Eggs were fixed at various times after fertilization and examined with conventional fluorescence microscopy, laser scanning confocal microscopy, and three-dimensional fluorescence microscopy. Soon after the eggs were fertilized (Tn = 0.02), we saw microtubules oriented perpendicular to the plane of the plasma membrane but none parallel to the plasma membrane. Later (Tn = 0.08), we saw an array of microtubules oriented more or less parallel to the plasma membrane but having no apparent preferred orientation with respect to the animal-vegetal axis of the egg. In the interpolar regions of the egg, this network increased in density by Tn = 0.24 and remained a constant feature of the ooplasm until the first cleavage. From Tn = 0.30 to 0.76 the polar regions of the egg contained dense arrays of organized microtubules. At the animal pole, microtubules radiated from a site near the pronuclei; while at the vegetal pole, an array of parallel microtubules was present. Injection of the weak (KD = 1.5 {mu}M) calcium buffer 5,5`-dibromo-BAPTA disrupted the radial pattern of microtubules near the animal pole but had no apparent effect on the parallel array of microtubules near the vegetal pole. Because this buffer has previously been shown to suppress a zone of elevated cytosolic calcium at the animal pole and to disrupt ooplasmic segregation in this egg, the results of the present study (1) are consistent with a model in which microtubules are required for ooplasmic segregation in the medaka egg, and (2) suggest that the normal function of a microtubule-organizing center at the animal pole of the egg requires a zone of elevated calcium.

Localization of acetylcholinesterase activity in young embryos of the medaka Oryzias latipes, a teleost

Abstract 1. Acetylcholinesterase activity is present at the surface of unfertilized eggs and of embryos throughout early development of the medaka Oryzias latipes , a teleost fish. 2. Acetylcholinesterase activity is present in yolk from blastulae and gastrulae. 3. Acetylcholinestease activity is present in cells of the enveloping layer, in deep blastomeres, and in the internal and external yolk syncytial layers.

Microtubule-Based Movements During Ooplasmic Segregation in the Medaka Fish Egg (Oryzias latipes)

We used time-lapse video microscopy to monitor the effects of cytochalasin D (CCD) and demecolcine on cytoplasmic streaming toward the animal pole of the medaka egg, the formation of the blastodisc at the animal pole, the movement of oil droplets in the cytoplasm toward the vegetal pole, and the saltatory movement of small cytoplasmic parcels toward the animal pole and vegetal pole. Cytochalasin D inhibited both cytoplasmic streaming toward the animal pole and the formation of the blastodisc, suggesting a role for microfilaments in these processes. However, CCD had no apparent effect on saltatory movement or on the movement of oil droplets toward the vegetal pole. Thus, the segregation of oil droplets toward the vegetal pole is not the result of the bulk movement of ooplasm toward the animal pole. In eggs treated with demecolcine, oil droplets did not move toward the vegetal pole but instead floated to the uppermost portion of the egg, and saltatory movement was absent, suggesting that microtubules are required for these movements. The effects of demecolcine on oil droplet movement and saltatory movement could be reversed by irradiating the eggs with UV light (360 nm). Using indirect immunofluorescence, we showed that irradiation of demecolcine-treated eggs with UV light regenerated microtubules within the irradiated region. The specificity of the mechanism responsible for the vegetal poleward movement of oil droplets was assessed by microinjecting droplets of five other fluids--mineral oil, silicone oil, vegetable oil, and two fluorinated aliphatic compounds--into the ooplasm. None of these fluids segregated with the endogenous oil droplets. These results suggest that a specific mechanism, probably involving microtubules, is responsible for the segregation of oil droplets to the vegetal pole.

Cholinergic molecules modify the in vitro behavior of cells from early embryos of the medaka Oryzias latipes, a teleost fish

Abstract 1. A method is described that enables one to collect with a micropipette cells from several hundred early medaka ( Oryzias latipes ) embryos in less than 30 min; the cells can subsequently be dissociated by ethylenediamine tetraacetic acid. 2. d -Tubocurarine (10 μM) decreased and carbamylcholine (10 μM) increased the rate of aggregation of these cells in vitro . 3. d -Tubocurarine also decreased the proportion of cells that translocated a measurable distance in vitro . 4. Extracts of the cells contain acetylcholine, and the amount they contain is about 25% of that present in whole embryos. 5. No acetylcholine was detected in the culture medium bathing aggregating cells.