Ascidian Eggs Research Articles

Polarity and reorganization of the endoplasmic reticulum during fertilization and ooplasmic segregation in the ascidian egg.

During the first cell cycle of the ascidian egg, two phases of ooplasmic segregation create distinct cytoplasmic domains that are crucial for later development. We recently defined a domain enriched in ER in the vegetal region of Phallusia mammillata eggs. To explore the possible physiological and developmental function of this ER domain, we here investigate its organization and fate by labeling the ER network in vivo with DiIC16(3), and observing its distribution before and after fertilization in the living egg. In unfertilized eggs, the ER-rich vegetal cortex is overlaid by the ER-poor but mitochondria-rich subcortical myoplasm. Fertilization results in striking rearrangements of the ER network. First, ER accumulates at the vegetal-contraction pole as a thick layer between the plasma membrane and the myoplasm. This accompanies the relocation of the myoplasm toward that region during the first phase of ooplasmic segregation. In other parts of the cytoplasm, ER becomes progressively redistributed into ER-rich and ER-poor microdomains. As the sperm aster grows, ER accumulates in its centrosomal area and along its astral rays. During the second phase of ooplasmic segregation, which takes place once meiosis is completed, the concentrated ER domain at the vegetal-contraction pole moves with the sperm aster and the bulk of the myoplasm toward the future posterior side of the embryo. These results show that after fertilization, ER first accumulates in the vegetal area from which repetitive calcium waves are known to originate (Speksnijder, J. E. 1992. Dev. Biol. 153:259-271). This ER domain subsequently colocalizes with the myoplasm to the presumptive primary muscle cell region.

Net calcium and acid release at fertilization in eggs of sea urchins and ascidians

Sea urchin eggs lose about 10–30% of their total calcium content upon fertilization. We have investigated the mechanism of this calcium-loss with an ion-selective vibrating probe system. Upon fertilization of Arbacia punctulata and Lytechinus pictus eggs we could measure a calcium efflux signal with an average duration of 204 ± 26 s and 146 ± 46 s, respectively. Measurements of hydrogen ion signals in normal and in low sodium media showed that the release of cortical vesicle material from these eggs lasts for about 30 and 50 s, respectively. The data indicate that most of the calcium that is lost from sea urchin eggs originates from the cytosol in which it is released during fertilization and then pumped out through the plasma membrane. Calcium loss due to cortical granule release accounts for less than 14% of the total loss measured. We also measured a substantial post-fertilization calcium efflux in eggs of Phallusia mammilata, with an average duration of 265 ± 18 s followed by smaller periodic effluxes that corresponded to oscillations in the [Ca 2+] i during contractile waves in these eggs. These data, together with the lack of cortical granules in ascidian eggs, indicate that Phallusia eggs also pump out a substantial amount of calcium through the plasma membrane after fertilization.

Introduction and Expression of Recombinant Genes in Ascidian Embryos.

In order to examine the expression of exogenous genes introduced into ascidian eggs, two recombinant plasmids pmiwZ and pHrMA4aCAT were microinjected into the cytoplasm of fertilized eggs of Ciona savignyi and Halocynthia roretzi, respectively. The plasmid pmiwZ contains the coding sequence of bacterial β-galactosidase gene (lac-Z) fused with animal gene promoters, while pHrMA4aCAT was constructed by fusing about 1.4-kb long 5' flanking region of H. roretzi muscle actin gene HrMA4a with bacterial chloramphenicol acetyltransferase gene (CAT). Injection of approximately 160 pl of 10 μg/ml pmiwZ DNA into Ciona eggs did not affect the embryogenesis, although introduction of the same volume of 30 μg/ml pmiwZ DNA resulted in abnormal development of injected eggs. When the expression of lac-Z was examined by histochemical detection of the enzyme activity, the expression was evident in the early tailbud embryos and later stage embryos, and larvae, irrespective of linear or circular form of the plasmid. The enzyme activity appeared in various cell-types including epidermis, nervous system, endoderm, mesenchyme, notochord, and muscle. In contrast, when pHrMA4aCAT was introduced into Halocynthia eggs and the appearance of CAT protein was examined later by the anti-CAT antibody, the CAT expression was restricted to muscle cells. These results indicate that the recombinant genes introduced into ascidian eggs could express during embryogenesis and that the 1.4-kb long 5' flanking region of HrMA4a contains regulatory sequences enough for the appropriate spatial and temporal expression of the gene.

Glycolipid linkage of a polyspermy blocking glycosidase to the ascidian egg surface

Ascidian eggs release N-acetylglucosaminidase rapidly into the seawater following fertilization. This glycosidase is detected seconds after fertilization, and histochemical tests suggest the cell surface as the prefertilization storage site (Lambert, C. C. (1989). Development 105, 415–420). Living eggs of Ascidia ceratodes, A. callosa, and A. paratropa all cleave a fluorogenic substrate in seawater. Following cell surface biotinylation and activation of the eggs, enzyme activity binds to streptavidin further substantiating the cell surface localization. The released glycosidase has a molecular weight of 180 kDa by size exclusion chromatography and exhibits bands at 62 and 70 kDa by SDS-PAGE, suggesting a possibly multimeric enzyme. The enzyme is released by a glycophosphatidylinositol-specific phospholipase C and HNO 2 deamination, both of which are specific indicators of linkage to the cell surface via phosphatidylinositol. The enzyme from unfertilized eggs is quite hydrophobic in Triton X-114 phase partition experiments but becomes hydrophyllic after release by activation or deamination. All of these observations are consistent with the glycosidase being anchored to the cell surface via a GPI anchor that is cleaved at fertilization to yield the soluble form of the enzyme which helps protect the egg against polyspermy. We discuss the possible role of a cell surface PLC in this release.

The repetitive calcium waves in the fertilized ascidian egg are initiated near the vegetal pole by a cortical pacemaker

Ascidian eggs respond to fertilization with a series of repetitive calcium waves that originate mostly from the vegetal/contraction pole region ( J. E. Speksnijder, C. Sardet, and L. F. Jaffe, 1990, Dev. Biol. 142, 246–249 ), where the myoplasm is concentrated during the first phase of ooplasmic segregation. This suggests that the myoplasm may be involved in initiating these calcium waves. To test this possibility, the starting position of the calcium waves was determined in eggs that had the subcortical, mitochondria-rich part of the myoplasm displaced by centrifugation. Such centrifuged eggs display four cytoplasmic layers: a large centrifugal yolk zone, a narrow clear zone, a mitochondria-rich layer, and a small clear zone at the centripetal pole. Imaging of the cytosolic calcium in centrifuged eggs that were injected with the calcium-specific photoprotein aequorin reveals a series of repetitive calcium waves after fertilization. About 70% of these waves start in the vegetal/contraction pole area, which is similar to the number of waves previously found to start in this area in uncentrifuged eggs. In contrast, only about 25% of the waves start close to the displaced mitochondria-rich layer. From this result it is concluded that the main wave initiation site is not displaced by the centrifugal forces that displace the subcortical, mitochondria-rich part of the myoplasm. Moreover, the observation that the animal-vegetal polarity of cortical components such as actin filaments and the endoplasmic reticulum has been retained after centrifugation further suggests that a cortical component located in the vegetal hemisphere—most likely the endoplasmic reticulum network in the cortical region of the myoplasm—is involved in initiating the repetitive calcium waves in the fertilized ascidian egg.

Polarity of the ascidian egg cortex before fertilization.

The unfertilized ascidian egg displays a visible polar organization along its animal-vegetal axis. In particular, the myoplasm, a mitochondria-rich subcortical domain inherited by the blastomeres that differentiate into muscle cells, is mainly situated in the vegetal hemisphere. We show that, in the unfertilized egg, this vegetal domain is enriched in actin and microfilaments and excludes microtubules. This polar distribution of microfilaments and microtubules persists in isolated cortices prepared by shearing eggs attached to a polylysine-coated surface. The isolated cortex is further characterized by an elaborate network of tubules and sheets of endoplasmic reticulum (ER). This cortical ER network is tethered to the plasma membrane at discrete sites, is covered with ribosomes and contains a calsequestrin-like protein. Interestingly, this ER network is distributed in a polar fashion along the animal-vegetal axis of the egg: regions with a dense network consisting mainly of sheets or tightly knit tubes are present in the vegetal hemisphere only, whereas areas characterized by a sparse tubular ER network are uniquely found in the animal hemisphere region. The stability of the polar organization of the cortex was studied by perturbing the distribution of organelles in the egg and depolymerizing microfilaments and microtubules. The polar organization of the cortical ER network persists after treatment of eggs with nocodazole, but is disrupted by treatment with cytochalasin B. In addition, we show that centrifugal forces that displace the cytoplasmic organelles do not alter the appearance and polar organization of the isolated egg cortex. These findings taken together with our previous work suggest that the intrinsic polar distribution of cortical membranous and cytoskeletal components along the animal-vegetal axis of the egg are important for the spatial organization of calcium-dependent events and their developmental consequences.

A gastrulation center in the ascidian egg

A gastrulation center is described in ascidian eggs. Extensive cytoplasmic rearrangements occur in ascidian eggs between fertilization and first cleavage. During ooplasmic segregation, a specific cytoskeletal domain (the myoplasm) is translocated first to the vegetal pole (VP) and then to the posterior region of the zygote. A few hours later, gastrulation is initiated by invagination of endoderm cells in the VP region of the 110-cell embryo. After the completion of gastrulation, the embryonic axis is formed, which includes induction of the nervous system, morphogenesis of the larval tail and differentiation of tail muscle cells. Microsurgical deletion or ultraviolet (UV) irradiation of the VP region during the first phase of myoplasmic segregation prevents gastrulation, nervous system induction and tail formation, without affecting muscle cell differentiation. Similar manipulations of unfertilized eggs or uncleaved zygotes after the second phase of segregation have no effect on development, suggesting that a gastrulation center is established by transient localization of myoplasm in the VP region. The function of the gastrulation center was investigated by comparing protein synthesis in normal and UV-irradiated embryos. About 5% of 433 labelled polypeptides detected in 2D gels were affected by UV irradiation. The most prominent protein is a 30 kDa cytoskeletal component (p30), whose synthesis is abolished by UV irradiation. p30 synthesis peaks during gastrulation, is affected by the same UV dose and has the same UV-sensitivity period as gastrulation. However, p30 is not a UV-sensitive target because it is absent during ooplasmic segregation, the UV-sensitivity period. Moreover, the UV target has the absorption maximum of a nucleic acid rather than a protein. Cell-free translation studies indicate that p30 is encoded by a maternal mRNA. UV irradiation inhibits the ability of this transcript to direct p30 synthesis, indicating that p30 mRNA is a UV-sensitive target. The gasturlation center may function by sequestration or activation of maternal mRNAs encoding proteins that function during embryogenesis.

Lithium and phorbol ester modify the activating capacity of ascidian spermatozoa

In this paper we have shown, using the whole-cell voltage clamp technique, that two parameters of the fertilization current in ascidian eggs may be modified by exposing spermatozoa to lithium or to phorbol ester. When spermatozoa were pre-treated in 250 mM lithium sea water for up to 30 min there was a significant increase in the mean initial slope of the fertilization current, from 116±90 to 169±84 pA/s (p 0.05). Pre-treatment in 200–600 nM phorbol 12-myristate 13-acetate also increased the activating capacity of ascidian sperm, as monitored by a significant increase in the initial slope current in control eggs; however, there was no increase in peak current. Furthermore, we have shown, using NH4Cl, that an increase in intracellular pH alone is insufficient to change the activating capacity of spermatozoa. This is the first report showing that the kinetics of an egg activation event depend upon the physiological status of the spermatozoon.

Analysis of morphogenetic determinants in ascidian eggs and embryos by cytoplasmic deletion and microinjection

The determination of cell fates in ascidian embryos is thought to be mediated primarily by factors localized in the cytoplasm. Morphogenetic factors were studied in ascidian eggs and early cleavage stage embryos by the partial deletion of blastomeres and by microinjection of sperm cells into anucleate, myoplasm-deficient egg fragments. In the first series of experiments, regions of blastomeres comprised of 20–30% of the volume of one blastomere were deleted from two- and four-celled embryos. Most of the embryos exhibited regulative capabilities, in that normal tadpole larvae developed from the operated embryos, irrespective of the deletion site. In a second series of experiments, the expression of endodermal alkaline phosphatase (AP) and muscle acetylcholinesterase (AchE) were tested in myoplasm-deficient egg fragments that were injected with sperm cells. After the unoperated controls had developed into tadpoles, myoplasm-deficient fragments injected with sperm cells were scored for their ability to express AP and AchE activity. Many of the myoplasm-deficient fragments that contained injected sperm cells expressed AP activity, whereas uninjected myoplasm-deficient fragments did not exhibit AP activity. When myoplasm-deficient fragments injected with sperm cells were tested for their ability to express AchE activity, none of the fragments expressed this muscle cell marker. Similar results were obtained using two species, Halocynthia roretzi and Styelaplicata. These results suggest that nuclear events are required for the expression of AP and that the myoplasmic region of the fertilized egg contains muscle cell determinants.

Embryogenesis in the Presence of Blockers of Mechanosensitive Ion Channels: (embryogenesis/mechanosensitive ion channels/channel blockers/Xenopus/ascidians).

Certain developmental events are thought to be controlled by mechanical tension, but the nature of the transduction mechanism for sensing and responding to tension changes is unknown. A good candidate for such a sensing system would be stretch-activated (SA) ion channels, a type of mechanosensitive (MS) ion channel found in many preparations including the oocytes or embryos of ascidians, fish, and amphibians. To test the hypothesis that SA channel activation is important for early embryogenesis, we treated amphibian and ascidian eggs and embryos with inhibitors of MS ion channels. Xenopus laevis eggs and embryos were treated with gadolinium (Gd3+ ) concentrations up to 100 times the Kd for SA channel inhibition. Boltenia villosa eggs and embryos were exposed to three agents (Gd3+ , tubocurarine, and gallamine) which are known to block SA channels in other organisms. None of these drugs interfered with morphogenesis in a manner that would suggest SA channel activity is critical to early embryogenesis.

Identification of a cytoskeletal protein localized in the myoplasm of ascidian eggs: localization is modified during anural development

The myoplasm of ascidian eggs is a localized cytoskeletal domain that is segregated to presumptive larval tail muscle cells during embryonic development. We have identified a cytoskeletal protein recognized by a vertebrate neurofilament monoclonal antibody (NN18) which is concentrated in the myoplasm in eggs and embryos of a variety of ascidian species. The NN18 antigen is localized in the periphery of unfertilized eggs, segregates with the myoplasm after fertilization, and enters the larval tail muscle cells during embryonic development. Western blots of one-dimensional and two-dimensional gels showed that the major component recognized by NN18 antibody is a 58 x 10(3) Mr protein (p58), which exists in at least three different isoforms. The enrichment of p58 in the Triton X-100-insoluble fraction of eggs and its reticular staining pattern in eggs and embryos suggests that it is a cytoskeletal protein. In subsequent experiments, p58 was used as a marker to determine whether changes in the myoplasm occur in eggs of anural ascidian species, i.e. those exhibiting a life cycle lacking tadpole larvae with differentiated muscle cells. Although p58 was localized in the myoplasm in eggs of four urodele ascidian species that develop into swimming tadpole larvae, this protein was distributed uniformly in eggs of three anural ascidian species. The eggs of two of these anural species contained the actin lamina, another component of the myoplasm, whereas the third anural species lacked the actin lamina. There was no detectible localization of p58 after fertilization or segregation into muscle lineage cells during cleavage of anural eggs. NN18 antigen was uniformly distributed in pre-vitellogenic oocytes and then localized in the perinuclear zone during vitellogenesis of urodele and anural ascidians. Subsequently, NN18 antigen was concentrated in the peripheral cytoplasm of post-vitellogenic oocytes and mature eggs of urodele, but not anural, ascidians. It is concluded that the myoplasm of ascidian eggs contains an intermediate filament-like cytoskeletal network which is missing in anural species that have modified or eliminated the tadpole larva.

L-type Ca 2+ currents in ascidian eggs

We have studied Ca 2+ currents in ascidian eggs using the whole-cell clamp technique. T and L components, as observed in somatic cells, are present and the L-type current predominates. Since the IV relationship for these inward currents overlap at −30 mV, separation of the two components using different voltage regimes is not feasible. Increasing external Ca 2+ results in larger currents. The L-type current decreases in a dose-dependent fashion in the presence of Mn 2+ and Nifedipine, while the T-type current is inhibited in Ni 2+. When Ba 2+ was used as the carrier ion, channel kinetics and conductance were completely altered. Considering the density and kinetics of L-type channels in unfertilized eggs it is probable they play an important role in regulating cytosolic Ca 2+ during early developmental processes.

Periodic calcium waves cross ascidian eggs after fertilization

Ascidian eggs respond to fertilization with one to two dozen periodic calcium pulses (J. E. Speksnijder, D. W. Corson, C. Sardet, and L. F. Jaffe, 1989a, Dev. Biol. 135, 182–190). We examined the spatial pattern of these pulses and found that they are initiated in discrete regions from which they propagate as waves. The first few pulses start in the animal hemisphere, whereas the later ones are mostly initiated near the vegetal pole. Such vegetal waves are often followed by a contraction of the egg surface. Since these waves are attenuated as they spread, they repeatedly expose the vegetal pole region to more calcium. The mechanism of these repetitive calcium waves and their possible role in establishing pattern or completing meiosis is discussed.

Interactions of the Golgi apparatus with microtubules

RcaEaaxu!KsL-IN~ s73 Am EGS Chiara Campanella, Dip. di Scienze e Tecnol. Biomed. e di Bicanatria, 67100, L'Aquila, Italia. In oocytes and &ryos, the cortical cytoskeleton (CC) has variuos distribution and implication according to species and stages. In ascidian eggs, CC consists of an F-actin containing network (Sawada and Osanai,1985) linked to the plasma membrane and carrying along m-RNA as well as other cell constituents necessary for embryonic development (Jeffery and Meier, 1983). At mplasmic segregation, the jellcm crescent forms as a consequence of cooperative ccntracticn of CC and subcortical microfilaments (&alla et al, 1985). In

The activation wave of calcium in the ascidian egg and its role in 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.

Detection of extracellular calcium gradients with a calcium-specific vibrating electrode.

We have developed a vibrating calcium-specific electrode to measure minute extracellular calcium gradients and thus infer the patterns of calcium currents that cross the surface of various cells and tissues. Low-resistance calcium electrodes (routinely approximately 500 M omega) are vibrated by means of orthogonally stacked piezoelectrical pushers, driven by a damped square wave at an optimal frequency of 0.5 Hz. Phase-sensitive detection of the electrode signal is performed with either analogue or digital electronics. The resulting data are superimposed on a video image of the preparation that is being measured. Depending on the background calcium concentration, this new device can readily and reliably measure steady extracellular differences of calcium concentration which are as small as 0.01% with spatial and temporal resolutions of a few microns and a few seconds, respectively. The digital version can attain a noise level of less than 1 microV. In exploratory studies, we have used this device to map and measure the patterns of calcium currents that cross the surface of growing fucoid eggs and tobacco pollen, moving amebae and Dictyostelium slugs, recently fertilized ascidian eggs, as well as nurse cells of Sarcophaga follicles. This approach should be easily extendable to other specific ion currents.

Free calcium pulses following fertilization in the ascidian egg

Using the calcium-specific, chemiluminescent photoprotein aequorin, we have measured changes in the concentration of free cytosolic calcium at fertilization in single eggs of the ascidians Phallusia mammillata and Ciona intestinalis. Shortly after insemination, the free calcium concentration rises within a minute from a resting level of about 90 n M in the unfertilized egg to a peak level of about 7 μ M in Phallusia and about 10 μ M in Ciona. The total duration time of this fertilization transient is 2–3 min. It is immediately followed by a series of 12 to 25 briefer calcium transients with peak levels of about 1–4 μ M. These postfertilization pulses occur at regular intervals of 1–3 min during the completion of meiosis, and they stop as soon as the second polar body is formed at about 25 min. An interesting exception to this pattern was observed in eggs from Ciona that had been raised at lower temperatures during the winter months. Insemination in the absence of external calcium in Phallusia results in a pulse pattern very similar to the normal pattern. From this result we infer that the bulk (if not all) of the calcium required for both the fertilization pulse and the meiotic oscillations is released from internal sources.

Ooplasmic movements and calcium waves at fertilization in ascidian eggs

Ooplasmic movements and calcium waves at fertilization in ascidian eggs

Molecular identification and purification of the myoplasmin-C1: A polypeptide localized to the myoplasm of ascidian eggs

Molecular identification and purification of the myoplasmin-C1: A polypeptide localized to the myoplasm of ascidian eggs

PH in eggs of the ascidian Ciona intestinalis at fertilization and activation

AbstractUsing liquid ion exchanger microelectrodes, we have measured the intracellular pH of unfertilized ascidian eggs. The pH in these eggs ranges from 7.1 to 7.6 (similar to values for fertilized sea urchin eggs) and does not change during the first 7 minutes of activation, whether induced by spermatozoa or by the Ca2+ ionophore A23187. Factors regulating metabolic derepression in regulative and mosaic eggs are discussed.