scholarly journals The activation wave of calcium in the ascidian egg and its role in ooplasmic segregation.

1990 ◽  
Vol 110 (5) ◽  
pp. 1589-1598 ◽  
Author(s):  
J E Speksnijder ◽  
C Sardet ◽  
L F Jaffe
Keyword(s):  
Imaging System ◽  
Sperm Nucleus ◽  
Calcium Wave ◽  
Free Calcium ◽  
S Wave ◽  
Animal Pole ◽  
Calcium Dependent ◽  
Sperm Entry ◽  
Vegetal Pole ◽  
Ooplasmic Segregation

We have studied egg activation and ooplasmic segregation in the ascidian Phallusia mammillata using an imaging system that let us simultaneously monitor egg morphology and calcium-dependent aequorin luminescence. After insemination, a wave of highly elevated free calcium crosses the egg with a peak velocity of 8-9 microns/s. A similar wave is seen in egg fertilized in the absence of external calcium. Artificial activation via incubation with WGA also results in a calcium wave, albeit with different temporal and spatial characteristics than in sperm-activated eggs. In eggs in which movement of the sperm nucleus after entry is blocked with cytochalasin D, the sperm aster is formed at the site where the calcium wave had previously started. This indicates that the calcium wave starts where the sperm enters. In 70% of the eggs, the calcium wave starts in the animal hemisphere, which confirms previous observations that there is a preference for sperm to enter this part of the egg (Speksnijder, J. E., L. F. Jaffe, and C. Sardet. 1989. Dev. Biol. 133:180-184). About 30-40 s after the calcium wave starts, a slower (1.4 microns/s) wave of cortical contraction starts near the animal pole. It carries the subcortical cytoplasm to a contraction pole, which forms away from the side of sperm entry and up to 50 degrees away from the vegetal pole. We propose that the point of sperm entry may affect the direction of ooplasmic segregation by causing it to tilt away from the vegetal pole, presumably via some action of the calcium wave.

The sperm entry point defines the orientation of the calcium-induced contraction wave that directs the first phase of cytoplasmic reorganization in the ascidian egg

Development ◽  
1995 ◽  
Vol 121 (10) ◽  
pp. 3457-3466 ◽  
Author(s):  
F. Roegiers ◽  
A. McDougall ◽  
C. Sardet
Keyword(s):  
Sperm Nucleus ◽  
Cytoplasmic Domains ◽  
Dorsoventral Axis ◽  
Rich Domain ◽  
Sperm Entry ◽  
Vegetal Pole ◽  
Ooplasmic Segregation ◽  
The Relationship ◽  
Ascidian Embryo ◽  
Contraction Wave

Ascidians eggs are spawned with their cytoskeleton and organelles organized along a preexisting animal-vegetal axis. Fertilization triggers a spectacular microfilament-dependant cortical contraction that causes the relocalization of preexisting cytoplasmic domains and the creation of new domains in the lower part of the vegetal hemisphere. We have investigated the relationship between fertilization, the cortical contraction and the localization of cytoplasmic domains in eggs of the ascidian Phallusia mammillata. We have also examined the link between this first phase of ooplasmic segregation and the site of gastrulation. The cortical contraction was found to be initiated on the side of the egg where intracellular calcium is first released either by the entering sperm or by photolysis of caged InsP3. The cortical contraction carries the sperm nucleus towards the vegetal hemisphere along with a subcortical mitochondria-rich domain (the myoplasm). If the sperm enters close to the animal or vegetal poles the cortical contraction is symmetrical, travelling along the animal-vegetal axis. If the sperm enters closer to the equator, the contraction is asymmetrical and its direction does not coincide with the animal-vegetal axis. The direction of contraction defines an axis along which preexisting (such as the myoplasm) or newly created cytoplasmic domains are relocalized. Two microfilament-rich surface constrictions, the ‘contraction pole’ and the ‘vegetal button’ (which forms 20 minutes later), appear along that axis approximately opposite the site where the contraction is initiated. The contraction pole can be situated as much as 55 degrees from the vegetal pole, and its location predicts the site of gastrulation. It thus appears that in ascidian eggs, the organization of the egg before fertilization defines a 110 degrees cone centered around the vegetal pole in which the future site of gastrulation of the embryo will lie. The calcium wave and cortical contraction triggered by the entering sperm adjust the location of cytoplasmic domains along an axis within that permissive zone. We discuss the relation between that axis and the establishment of the dorsoventral axis in the ascidian embryo.

Fertilization and ooplasmic movements in the ascidian egg

Development ◽  
1989 ◽  
Vol 105 (2) ◽  
pp. 237-249 ◽  
Author(s):  
C. Sardet ◽  
J. Speksnijder ◽  
S. Inoue ◽  
L. Jaffe
Keyword(s):  
Polar Body ◽  
Surface Velocity ◽  
Second Phase ◽  
Animal Pole ◽  
Male Pronucleus ◽  
Vegetal Pole ◽  
Female Pronucleus ◽  
Ooplasmic Segregation ◽  
Second Polar Body ◽  
Sperm Aster

Using light microscopy techniques, we have studied the movements that follow fertilization in the denuded egg of the ascidian Phallusia mammillata. In particular, our observations show that, as a result of a series of movements described below, the mitochondria-rich subcortical myoplasm is split in two parts during the second phase of ooplasmic segregation. This offers a potential explanation for the origin of larval muscle cells from both posterior and anterior blastomeres. The first visible event at fertilization is a bulging at the animal pole of the egg, which is immediately followed by a wave of contraction, travelling towards the vegetal pole with a surface velocity of 1.4 microns s-1. This wave accompanies the first phase of ooplasmic segregation of the mitochondria-rich subcortical myoplasm. After this contraction wave has reached the vegetal pole after about 2 min, a transient cytoplasmic lobe remains there until 6 min after fertilization. Several new features of the morphogenetic movements were then observed: between the extrusion of the first and second polar body (at 5 and 24–29 min, respectively), a series of transient animal protrusions form at regular intervals. Each animal protrusion involves a flow of the centrally located cytoplasm in the animal direction. Shortly before the second polar body is extruded, a second transient vegetal lobe (‘the vegetal button’) forms, which, like the first, resembles a protostome polar lobe. Immediately after the second polar body is extruded, three events occur almost simultaneously: first, the sperm aster moves from the vegetal hemisphere to the equator. Second, the bulk of the vegetally located myoplasm moves with the sperm aster towards the future posterior pole, but interestingly about 20% remains behind at the anterior side of the embryo. This second phase of myoplasmic movement shows two distinct subphases: a first, oscillatory subphase with an average velocity of about 6 microns min-1, and a second steady subphase with a velocity of about 26 microns min-1. The myoplasm reaches its final position as the male pronucleus with its surrounding aster moves towards the centre of the egg. Third, the female pronucleus moves towards the centre of the egg to meet with the male pronucleus. Like the myoplasm, the migrations of both the sperm aster and the female pronucleus shows two subphases with distinctly different velocities. Finally, the pronuclear membranes dissolve, a small mitotic spindle is formed with very large asters, and at about 60–65 min after fertilization, the egg cleaves.

Activation currents, sperm entry and surface contractions in ascidian eggs

Zygote ◽  
1993 ◽  
Vol 1 (2) ◽  
pp. 113-119 ◽  
Author(s):  
C. Pecorella ◽  
E. Tosti ◽  
K. Kyozuka ◽  
B. Dale
Keyword(s):  
Sperm Nucleus ◽  
Initial Slope ◽  
Ostrea Edulis ◽  
Sperm Entry ◽  
Large Conductance Channel ◽  
Vegetal Pole ◽  
Inward Currents ◽  
The Mean ◽  
Contraction Wave ◽  
Surface Generate

SummarySpermatozoa from the mollusc Ostrea edulis are capable of fusing to and entering de-chorionated ascidian eggs. During interaction they generate activation currents, comparable to the fertilisation currents induced by homologous spermatozoa. Activation currents are inward at − 80 mV, with a mean initial slope of 111 ± 124 pA/s for Ciona intestinalis eggs and 47 ± 25 pA/s for Phallusia mammillata eggs, while the mean peak currents are 2782 ± 1132 pA and 1523 ± 1668 pA, respectively. The fertilisation and activation currents reverse at a holding potential of 0 mV to + 20 mV, suggesting that oyster sperm and ascidian sperm gate the same channel precursor, a non-specific, large conductance channel described previously (Dale & DeFelice, 1984). In contrast to homologous fertilisation, the activation current is not followed by a polarised contraction of the egg surface, nor other signs of egg activation. Staining eggs with Hoechst 33342 after insemination shows the female nucleus and a single oyster sperm nucleus at the antipode. This suggests a specialised predetermined site at the vegetal pole for sperm entry. Homologous and heterologous spermatozoa delivered, in a large pipette, to localised areas of the egg surface generate fast inward currents of 200–2000 pA, but do not induce contraction of the egg surface. This shows that although channel precursors are located globally over the egg surface, channel activation does not necessarily trigger the contraction wave. Subsequent induction of both a fertilisation current and a contraction by homologous sperm added to the bath, implies a regionalised activation site with an accumulation of channel precursors and a ‘pacemaker’ for the initiation of the contraction wave.

scholarly journals Patterns of free calcium in zebrafish embryos

1998 ◽  
Vol 111 (12) ◽  
pp. 1613-1622 ◽  
Author(s):  
R. Creton ◽  
J.E. Speksnijder ◽  
L.F. Jaffe
Keyword(s):  
Cellular Differentiation ◽  
Neural Induction ◽  
Free Calcium ◽  
Vertebrate Development ◽  
Animal Pole ◽  
Direct Knowledge ◽  
Low Concentrations ◽  
Ooplasmic Segregation ◽  
Otic Placode ◽  
Placode Formation

Direct knowledge of Ca2+ patterns in vertebrate development is largely restricted to early stages, in which they control fertilization, ooplasmic segregation and cleavage. To explore new roles of Ca2+ in vertebrate development, we injected the Ca2+ indicator aequorin into zebrafish eggs and imaged Ca2+ throughout the first day of development. During early cleavages, a high Ca2+ zone is seen in the cleavage furrows. The high Ca2+ zone during first cleavage spreads as a slow wave (0.5 microm/second) and is preceded by three Ca2+ pulses within the animal pole region of the egg. When Ca2+ concentrations are clamped at the resting level by BAPTA buffer injection into the zygote, all signs of development are blocked. In later development, Ca2+ patterns are associated with cell movements during gastrulation, with neural induction, with brain regionalization, with formation of the somites and neural keel, with otic placode formation, with muscle movements and with formation of the heart. Particularly remarkable is a sharp boundary between high Ca2+ in the presumptive forebrain and midbrain versus low Ca2+ in the presumptive hindbrain starting at 10 hours of development. When Ca2+ changes are damped by injection of low concentrations of BAPTA, fish form with greatly reduced eyes and hearts. The present study provides a first overview of Ca2+ patterns during prolonged periods of vertebrate development and points to new roles of Ca2+ in cellular differentiation and pattern formation.

Vegetal egg cytoplasm promotes gastrulation and is responsible for specification of vegetal blastomeres in embryos of the ascidian Halocynthia roretzi

Development ◽  
1996 ◽  
Vol 122 (4) ◽  
pp. 1271-1279 ◽  
Author(s):  
H. Nishida
Keyword(s):  
Radial Symmetry ◽  
Second Phase ◽  
Equatorial Position ◽  
Animal Cells ◽  
Halocynthia Roretzi ◽  
Animal Pole ◽  
Vegetal Pole ◽  
Ooplasmic Segregation ◽  
Derivatives Of ◽  
Egg Cortex

An animal-vegetal axis exists in the unfertilized eggs of the ascidian Halocynthia roretzi. The first phase of ooplasmic segregation brings the egg cortex to the vegetal pole very soon after fertilization. In the present study, when 5–8% of the egg cytoplasm in the vegetal pole region was removed between the first and second phase of segregation, most embryos exhibited failure of gastrulation, as reported previously in Styela by Bates and Jeffery (Dev. Biol, 124, 65–76, 1987). The embryos that were deficient in vegetal pole cytoplasm (VC-deficient embryos) developed into permanent blastulae. They consisted for the most part of epidermal cells and most lacked the derivatives of vegetal blastomeres, such as endoderm, muscle and notochord. Removal of cytoplasm from other regions did not affect embryogenesis. The cleavage of the VC-deficient embryos not only exhibited radial symmetry along the animal-vegetal axis but the pattern of the cleavage was also identical in the animal and vegetal hemispheres. Examination of the developmental fates of early blastomeres of VC-deficient embryos revealed that the vegetal blastomeres had assumed the fate of animal cells. These results suggested that the VC-deficient embryos had been totally animalized. When vegetal pole cytoplasm was transplanted to the animal pole or equatorial position of VC-deficient eggs, gastrulation occurred, starting at the site of the transplantation and tissues derived from vegetal blastomeres formed. Therefore, it appears that vegetal pole cytoplasm specifies the site of gastrulation and the cytoplasm is responsible for the specification of vegetal blastomeres. It is suggested that during the second phase of ooplasmic segregation, cytoplasmic factors responsible for gastrulation spread throughout the entire vegetal hemisphere.

scholarly journals A free calcium wave traverses the activating egg of the medaka, Oryzias latipes.

1978 ◽  
Vol 76 (2) ◽  
pp. 448-466 ◽  
Author(s):  
J C Gilkey ◽  
L F Jaffe ◽  
E B Ridgway ◽  
G T Reynolds
Keyword(s):  
Calcium Release ◽  
Wide Band ◽  
Calcium Wave ◽  
Free Calcium ◽  
Ionophore A23187 ◽  
Fresh Water Fish ◽  
Animal Pole ◽  
Vesicle Exocytosis ◽  
Cortical Vesicles ◽  
Internal Sources

Aequorin-injected eggs of the medaka (a fresh water fish) show an explosive rise in free calcium during fertilization, which is followed by a slow return to the resting level. Image intensification techniques now show a spreading wave of high free calcium during fertilization. The wave starts at the animal pole (where the sperm enters) and then traverses the egg as a shallow, roughly 20 degrees-wide band which vanishes at the antipode some minutes later. The peak free calcium concentration within this moving band is estimated to be about 30 microM (perhaps 100-1,000 times the resting level). Eggs activated by ionophore A23187 may show multiple initiation sites. The resulting multiple waves never spread through each other; rather, they fuse upon meeting so as to form spreading waves of compound origin. The fertilization wave is nearly independent of extracellular calcium because it is only slightly slowed (by perhaps 15%) in a medium containing 5 mM ethylene glycol-bis[beta-aminoethyl ether]N,N'-tetraacetic acid (EGTA) and no deliberately added calcium. It is also independent of the large cortical vesicles, which may be centrifugally displaced. Normally, however, it distinctly precedes the well-known wave of cortical vesicle exocytosis. We conclude that the fertilization wave in the medaka egg is propagated by calcium-stimulated calcium release, primarily from some internal sources other than the large cortical vesicles. A comparison of the characteristics of the exocytotic wave in the medaka with that in other eggs, particularly in echinoderm eggs, suggests that such a propagated calcium wave is a general feature of egg activation.

scholarly journals Myotubes from transgenic mdx mice expressing full-length dystrophin show normal calcium regulation.

1994 ◽  
Vol 5 (10) ◽  
pp. 1159-1167 ◽  
Author(s):  
W F Denetclaw ◽  
F W Hopf ◽  
G A Cox ◽  
J S Chamberlain ◽  
R A Steinhardt
Keyword(s):  
Single Channel ◽  
Calcium Regulation ◽  
Full Length ◽  
Mdx Mice ◽  
Free Calcium ◽  
Muscle Degeneration ◽  
Channel Activity ◽  
Mouse Muscle ◽  
Calcium Dependent ◽  
Normal Calcium

A lack of dystrophin results in muscle degeneration in Duchenne muscular dystrophy. Dystrophin-deficient human and mouse muscle cells have higher resting levels of intracellular free calcium ([Ca2+]i) and show a related increase in single-channel open probabilities of calcium leak channels. Elevated [Ca2+]i results in high levels of calcium-dependent proteolysis, which in turn increases calcium leak channel activity. This process could initiate muscle degeneration by further increasing [Ca2+]i and proteolysis in a positive feedback loop. Here, we tested the direct effect of restoration of dystrophin on [Ca2+]i and channel activity in primary myotubes from mdx mice made transgenic for full-length dystrophin. Transgenic mdx mice have been previously shown to have normal dystrophin localization and no muscle degeneration. Fura-2 calcium measurements and single-channel patch recordings showed that resting [Ca2+]i levels and open probabilities of calcium leak channels of transgenic mdx myotubes were similar to normal levels and significantly lower than mdx littermate controls (mdx) that lack dystrophin. Thus, restoration of normal calcium regulation in transgenic mdx mice may underlie the resulting absence of degeneration.

Ultrastructural changes in the avian vitelline membrane during embryonic development

Development ◽  
1969 ◽  
Vol 21 (3) ◽  
pp. 467-484
Author(s):  
Cynthia Jensen
Keyword(s):  
Embryonic Development ◽  
Mechanical Strength ◽  
Dual Role ◽  
Early Embryonic Development ◽  
Ultrastructural Changes ◽  
Vitelline Membrane ◽  
Entire Surface ◽  
Animal Pole ◽  
The Third ◽  
Vegetal Pole

The vitelline (yolk) membrane of the avian egg plays a dual role during early embryonic development; it encloses the yolk and provides a substratum for expansion of the embryo (Fig. 1). Expansion appears to be dependent upon the movement of cells at the edge of the blastoderm which is intimately associated with the inner layer of the vitelline membrane (New, 1959; Bellairs, 1963). The blastoderm (embryonic plus extraembryonic cells) has almost covered the entire surface of the yolk by the third and fourth days of incubation, and when this stage has been reached the vitelline membrane ruptures over the embryo and slips toward the vegetal pole. Rupture of the membrane during development appears to be the consequence of a decrease in its mechanical strength (Moran, 1936), which changes most rapidly at the animal pole (over the embryo).

GSK3beta/shaggy mediates patterning along the animal-vegetal axis of the sea urchin embryo

Development ◽  
1998 ◽  
Vol 125 (13) ◽  
pp. 2489-2498 ◽  
Author(s):  
F. Emily-Fenouil ◽  
C. Ghiglione ◽  
G. Lhomond ◽  
T. Lepage ◽  
C. Gache
Keyword(s):  
Sea Urchin ◽  
Wnt Pathway ◽  
Dominant Negative ◽  
Early Gene ◽  
Expression Domain ◽  
Animal Pole ◽  
Axial Patterning ◽  
Sea Urchin Embryo ◽  
Vegetal Pole ◽  
Dominant Negative Activity

In the sea urchin embryo, the animal-vegetal axis is defined before fertilization and different embryonic territories are established along this axis by mechanisms which are largely unknown. Significantly, the boundaries of these territories can be shifted by treatment with various reagents including zinc and lithium. We have isolated and characterized a sea urchin homolog of GSK3beta/shaggy, a lithium-sensitive kinase which is a component of the Wnt pathway and known to be involved in axial patterning in other embryos including Xenopus. The effects of overexpressing the normal and mutant forms of GSK3beta derived either from sea urchin or Xenopus were analyzed by observation of the morphology of 48 hour embryos (pluteus stage) and by monitoring spatial expression of the hatching enzyme (HE) gene, a very early gene whose expression is restricted to an animal domain with a sharp border roughly coinciding with the future ectoderm / endoderm boundary. Inactive forms of GSK3beta predicted to have a dominant-negative activity, vegetalized the embryo and decreased the size of the HE expression domain, apparently by shifting the boundary towards the animal pole. These effects are similar to, but even stronger than, those of lithium. Conversely, overexpression of wild-type GSK3beta animalized the embryo and caused the HE domain to enlarge towards the vegetal pole. Unlike zinc treatment, GSK3beta overexpression thus appeared to provoke a true animalization, through extension of the presumptive ectoderm territory. These results indicate that in sea urchin embryos the level of GSKbeta activity controls the position of the boundary between the presumptive ectoderm and endoderm territories and thus, the relative extent of these tissue layers in late embryos. GSK3beta and probably other downstream components of the Wnt pathway thus mediate patterning both along the primary AV axis of the sea urchin embryo and along the dorsal-ventral axis in Xenopus, suggesting a conserved basis for axial patterning between invertebrate and vertebrate in deuterostomes.

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