Movements of Echinochrome Granules during the Early Development of Sea Urchin Eggs

ECHINOCHROME pigment granules in unfertilized eggs of the sea urchin Arbacia punctulata undergo randomly-directed saltatory movements^1,2. After fertilization, nearly all these granules migrate to the egg cortex and become embedded. Subsequent pigment granule movements may represent mass cortical changes rather than independent granule movements^2,3. At the fourth cleavage, a quartet of micromeres containing little or no pigment forms at the vegetal pole. By the two or four-cell stage, pigment granules have begun to move out of this region, leaving a “clear area” on each blastomere (Fig. 1 and refs. 4, 5). To investigate possible mechanisms for these movements and their relation to cortical events     

Changes in the potassium content of sea urchin eggs on fertilization

In the eggs of Arbacia lixula and Paracentrotus lividus an uptake of K occurs during the first 10 minutes following fertilization. Between 10 and 40 minutes K is then released. Both in Arbacia and in Paracentrotus the minimum point of the curve coincides with the nuclear streak stage. A maximum loss of 25 per cent in Arbacia and 20 per cent in Paracentrotus with respect to the amount present in the unfertilized eggs has been found. From 40 minutes up to 1 hour K undergoes a further increase and when the first cleavage sets in the same amount of K is present as in the unfertilized eggs. By treating the eggs with K-free artificial sea water it has been established that about 60 per cent of the K content of the eggs is in a non-diffusible condition. Also under such conditions the eggs when fertilized are able to take up even the very small amount of K present in the medium that was released by them prior to fertilization.     

Fertilization alters the spatial distribution and the density of voltage-dependent sodium current in the egg of the ascidian Boltenia villosa

The spatial distribution of voltage-dependent ionic currents was characterized in Boltenia villosa eggs before and after fertilization using two-microelectrode voltage clamp of paired animal-vegetal halves of eggs (merogones) made surgically. Major voltage-dependent conductances in the Boltenia egg are a transient inward Na current, a transient inward Ca current, and an inwardly rectifying K current. These currents were randomly distributed along the animal-vegetal axis in the unfertilized egg. When paired merogones (surgically prepared egg fragments) were made at the vegetal cap stage, 15-30 min after fertilization, Ca and K currents remained randomly distributed along the animal-vegetal axis. In contrast, the relative Na current density was found to be twofold lower in the vegetal vs the animal merogones made at the vegetal cap stage. By making pairs of merogones from unfertilized eggs and subsequently fertilizing one merogone of a pair, we showed that this change in current density ratio was due to a loss of absolute Na current density in the vegetal hemisphere shortly after fertilization. These results also show that this loss was intrinsic to the vegetal hemisphere, rather than being determined solely by the point of sperm entry. A second decrease in Na current was observed during the hour before first cleavage, 60-120 min after fertilization (M.L. Block and W.J. Moody, 1987, J. Physiol. 393, 619-634), both in fertilized eggs and in animal merogones fertilized after isolation. This second loss of Na current was not observed in vegetal merogones fertilized after isolation or in either animal or vegetal merogones made from fertilized eggs at the vegetal cap stage. Possible mechanisms for te rapid (complete by 40 min after fertilization) and the late (occurring from ca. 60 to 120 minutes after fertilization) Na current losses are discussed.     

The jelly canal marker of polarity for sea urchin oocytes, eggs, and embryos

The jelly coat of a sea urchin egg possesses a narrow conical channel which identifies the animal pole. This rarely seen structure was first reported by Boveri in 1901 and is easily demonstrated by immersing freshly ovulated eggs into an ink suspension. The jelly canal, as this feature is called, fills with ink particles as the jelly swells. The jelly canal occurs on full-sized primary oocytes and unfertilized eggs. When filled with ink it serves as a useful marker of the animal pole during fertilization and early development when it is not otherwise visible. A common synonym for the jelly canal is ‘micropyle’, but this is a misnomer because sperm do not selectively pass through it for fertilization. The presence of the jelly canal on oocytes suggests how it might form and does not prove that the animal-vegetal polar axis in sea urchin eggs is defined before meiotic maturation.     

THE EXTRACELLULAR RELEASE OF ECHINOCHROME

A study was made of the diffusion of the red pigment echinochrome from the eggs of the sea urchin, Arbacia punctulata, into sea water. Unfertilized eggs retained their pigment, over periods of hours. Outward diffusion of pigment from unfertilized eggs normally is entirely negligible, or does not occur at all. Enchancing the calcium or potassium content of the artificial sea water (while retaining isosmotic conditions) did not induce pigment release. Under anaerobic conditions, unfertilized eggs release pigment in small quantities. Fertilization alone brings about echinochrome release. Fertilized eggs invariably released pigment, whether in normal sea water, or sea water with increased calcium or potassium. This diffusion of the pigment began during the first cleavage, possibly soon after fertilization. The pigment release is not a consequence solely of the cell''s permeability to echinochrome (or chromoprotein, or other pigment combination) but is preceded by events leading to a release of echinochrome from the granules in which it is concentrated within the cell. These events may be initiated by activation or by anaerobiosis. The phenomenon was not due to cytolysis.     

Activation of ascidian eggs with lectins

Eggs of Phallusia mammillata, dechorionated with trypsin, were activated in solutions of concanavalin A and wheat germ agglutinin. The polar bodies were extruded, and an aster appeared, but there was no cleavage. Ooplasmic segregation took place and mitochondria accumulated at the vegetal pole. Large chromatic granules formed in older eggs. The striking parallel between the migration of lectin receptors to the vegetal pole and “capping” was noted.     

Intracellular expression profiles measured by real-time PCR tomography in the Xenopus laevis oocyte

Real-time PCR tomography is a novel, quantitative method for measuring localized RNA expression profiles within single cells. We demonstrate its usefulness by dissecting an oocyte from Xenopus laevis into slices along its animal-vegetal axis, extracting its RNA and measuring the levels of 18 selected mRNAs by real-time RT-PCR. This identified two classes of mRNA, one preferentially located towards the animal, the other towards the vegetal pole. mRNAs within each group show comparable intracellular gradients, suggesting they are produced by similar mechanisms. The polarization is substantial, though not extreme, with around 5% of vegetal gene mRNA molecules detected at the animal pole, and around 50% of the molecules in the far most vegetal section. Most animal pole mRNAs were found in the second section from the animal pole and in the central section, which is where the nucleus is located. mRNA expression profiles did not change following in vitro fertilization and we conclude that the cortical rotation that follows fertilization has no detectable effect on intracellular mRNA gradients.     

Deep cytoplasmic rearrangements during early development in Xenopus laevis

The egg of the frog Xenopus is cylindrically symmetrical about its animal-vegetal axis before fertilization. Midway through the first cell cycle, the yolky subcortical cytoplasm rotates 30 degrees relative to the cortex and plasma membrane, usually toward the side of the sperm entry point. Dorsal embryonic structures always develop on the side away from which the cytoplasm moves. Details of the deep cytoplasmic movements associated with the cortical rotation were studied in eggs vitally stained during oogenesis with a yolk platelet-specific fluorescent dye. During the first cell cycle, eggs labelled in this way develop a complicated swirl of cytoplasm in the animal hemisphere. This pattern is most prominent on the side away from which the vegetal yolk moves, and thus correlates in position with the prospective dorsal side of the embryo. Although the pattern is initially most evident near the egg's equator or marginal zone, extensive rearrangements associated with cleavage furrowing (cytoplasmic ingression) relocate portions of the swirl to vegetal blastomeres on the prospective dorsal side.     

Mitochondrial regulation in sea urchins. I. Mitochondrial ultrastructure transformations and changes in the ADP:ATP ratio at fertilization.

Mitochondrial ultrastructural transformations have been examined in intact eggs and embryos from three sea urchins, Arbacia punctulata, Strongylocentrotus purpuratus, and Lytechinus pictus. Following fertilization, naturally occurring ultrastructural transformations (designated as condensed to orthodox) were observed to occur in mitochondria of all three families. The ratios of the soluble ADP and ATP pools were examined in eggs of S. purpuratus before and after fertilization in order to test whether the ultrastructural transformations reflect a decrease in relative size of the ADP pool following fertilization. Our data indicate that there is a decrease in the ADP:ATP ratio at fertilization. These findings and their implications are discussed with respect to presently accepted theories on mitochondrial regulation.     

Fertilization alters the orientation of pigment granule saltations in Arbacia eggs.

Unfertilized eggs of the sea urchin Arbacia punctulata contain pigment granules distributed throughout their cytoplasm. During the first 15 minutes after fertilization, these vesicles move out to the cortex where they become firmly anchored. We have used time-lapse video differential interference microscopy to analyze the motility of these organelles in unfertilized and fertilized Arbacia eggs. Pigment granules exhibit saltatory movement in both unfertilized and fertilized eggs. Quantitation of vesicle saltations before and after fertilization demonstrates that while there is no significant difference in the speed or path-length of vesicle movement, there is a dramatic change in the orientation of these saltations. Saltations in the unfertilized egg are very non-radial and are as likely to be directed toward the cortex as away. In contrast, saltations in the fertilized egg are more radially oriented and more likely to be cortically directed. This transition must reflect underlying changes in the cellular structures necessary for pigment granule saltations. The change in the orientation of pigment granule saltations following fertilization requires both a transient increase in the cytoplasmic concentration of Ca2+ and an elevation of cytoplasmic pH. Similarly, the ability of pigment granules to adhere to the cortex requires both the transient elevation of cytoplasmic Ca2+ and the alkalinization of the cytoplasm. As the reorganization of cortical actin at fertilization is regulated by these ionic fluxes, and both movement and adhesion are sensitive to cytochalasins, we hypothesize that the alterations in directed motility and adhesion reflect underlying changes in the actin cytoskeleton.     

Release of acid and changes in light-scattering properties following fertilization of Urechis caupo eggs.

The release of a fertilization acid, monitored by measuring the pH of egg suspensions, begins within 10 sec of insemination of Urechis caupo eggs. This is 4 min before the vitelline layer begins to elevate and is apparently unrelated to that process. The eggs of two molluscs, Mytilus californianus and Acmaea incessa, do not form a fertilization acid. The acid of Urechis eggs is not accompanied by release of “fertilization” carbohydrate, sulfate, or a nonvolatile weak acid into the seawater. The light-scattering properties of Urechis eggs change during the first 10 min after insemination. A decrease in light scattering begins by 10 sec and is complete by 1 min (Phase I). This is followed by a further decrease (3–6 min, Phase II) and an increase (6–10 min, Phase III). In striking contrast to an overtly similar situation in sea urchin eggs (fertilization acid and coincident light-scattering decrease), the release of acid and the initial light-scattering change are not the result of cortical granule discharge, and the acid, at least, is not related to the changes in shape or surface area which the eggs undergo. The processes underlying these rapid events are not yet known.     

Site of sperm entry and a cortical contraction associated with egg activation in the frog Rana pipens

The region of the frog egg that is receptive to fertilization was determined. As an approximation to the site of sperm entry, the start of the male pronuclear penetration path within the egg was made visible externally by bleaching fixed eggs. A bleached egg had a pigment accumulation on its surface corresponding to the start of the penetration path. The accumulation characteristically changed shape with cortical movements prior to first cleavage, and most accumulations (path starts) were within 60° of the animal pole.Localized inseminations and an analysis of the distribution of failures of fertilization at the egg plasma membrane demonstrated that few if any sperm entered the vegetal region of the egg. Localized inseminations, however, demonstrated that sperm entered between 60° from the animal pole and the animal-vegetal margin.Although sperm entry occurred throughout the animal region, most penetration paths started within 60° of the animal pole. To account for this, the sperm nucleus must move towards the animal pole prior to starting the penetration path. This movement appeared to be due to a contraction of the cortex towards the animal pole that occurred 3–4 min after activation of the egg.     

Determinants of Early Amphibian Development

The animal-vegetal organization of the amphibian egg may originatefrom the axis of organelles and cytoskeletal elements establishedin the oocyte as it divides from the oogonium. Along this axis,cytoplasmic materials are localized during oogenesis: yolk platelets,for example, are translocated toward the vegetal pole, increasingtheir amount and size in that region. In the first cell cycleafter fertilization, the egg cortex rotates 30° relativeto the cytoplasmic core, modifying animal-vegetal organization.The direction of this rotation, biased by the point of spermentry, defines the site of development of anatomical structuresof the dorsal midline of the embryo. As its immediate effect,rotation activates the cytoplasm of a subregion of the vegetalhemisphere, causing cells cleaved from this subregion to bemore effective than other vegetal parts in inducing marginalzone cells to initiate gastrulation movements. The most stronglyinduced part of the marginal zone begins gastrulation first(the dorsal lip of the blastopore) and proceeds through a seriesof cell interactions leading to its determination as the anteriordorsal mesoderm of the embryo. If these cell movements are inhibitedin the gastrula stage, or if vegetal induction is inhibitedin the blastula stage, or if cortical rotation is inhibitedin the first cell cycle after fertilization, the embryo alwaysfails to develop dorsal structures of the anterior end of itsbody axis; the more inhibition, the more posterior is the levelof truncation, until a radial ventralized embryo develops, derivedfrom the animal-vegetal organization of the oocyte.     

Dynamic organization of cortical actin filaments during the ooplasmic segregation of ascidian Ciona eggs

Spatial reorganization of cytoplasm in zygotic cells is critically important for establishing the body plans of many animal species. In ascidian zygotes, maternal determinants (mRNAs) are first transported to the vegetal pole a few minutes after fertilization and then to the future posterior side of the zygotes in a later phase of cytoplasmic reorganization, before the first cell division. Here, by using a novel fluorescence polarization microscope that reports the position and the orientation of fluorescently labeled proteins in living cells, we mapped the local alignments and the time-dependent changes of cortical actin networks in Ciona eggs. The initial cytoplasmic reorganization started with the contraction of vegetal hemisphere approximately 20 s after the fertilization-induced [Ca²⁺] increase. Timing of the vegetal contraction was consistent with the emergence of highly aligned actin filaments at the cell cortex of the vegetal hemisphere, which ran perpendicular to the animal–vegetal axis. We propose that the cytoplasmic reorganization is initiated by the local contraction of laterally aligned cortical actomyosin in the vegetal hemisphere, which in turn generates the directional movement of cytoplasm within the whole egg.     

A gradient of poly(A)+ RNA sequences in Xenopus laevis eggs and embryos

Xenopus laevis eggs and gastrula stage embryos were fractionated into three equal sections normal to the animal-vegetal axis, and poly(A)⁺ RNA was isolated from each section. Hybridization of these poly(A)⁺ RNAs with [³²P]cDNA synthesized using animal or vegetal poly(A)⁺ RNAs showed no detectable differences in the extents or rates of reaction. Thus, the vast majority of poly(A)⁺ RNAs are not segregated along the animal-vegetal axis. To increase the sensitivity of these experiments, [³²P]cDNAs were prepared which had reduced levels of RNA sequences from the animal region of the gastrula stage embryo or spawned unfertilized egg. Hybridization reactions with these probes showed that 3 to 5% of the input cDNA represents poly(A)⁺ RNA sequences enriched 2- to 20-fold in the vegetal region of the egg or gastrula stage embryo.     

Cortical Expressions of Polarity in the Starfish Oocyte*

The animal-vegetal (A-V) axis in oocytes of the starfish Pisaster ochraceus and Asterina pectinifera is described in terms of new cytological observations. Ordinarily, this axis of polarity is revealed by the eccentric germinal vesicle that is displaced toward the animal pole where polar bodies eventually form. In addition, however, the cortex at the animal pole is characterized by a pre-meiotic aster (centrosomes and bundles of microtubules lying within folds of the nuclear envelope), an absence of large, so-called “acidic vacuoles” that occur abundantly everywhere else, a diminished ability to form actin-filled spikes, and local mechanical weakness. These expressions of the A-V axis within the cortex are documented by micrographs. The problem of distinguishing if any feature is an “expression” or a “determinant” of axial differentiation is briefly discussed.     

Localized binding of Dolichos lectin to the early Ascidia embryo

A fluorescent conjugate of Dolichos lectin has been used to investigate the surface of eggs and early embryos of Ascidia malaca. Unfertilized eggs show a patchy distribution of fluorescence. After fertilization, this pattern is retained until about the time of the emission of the second polar body, when the fluorescence becomes localized at the vegetal pole. This localization is retained in early development and by the 64-cell stage, binding is displayed by the two micromeres and the posterior vegetal macromeres.     

Fertilization of sea urchin eggs is accompanied by 40 S ribosomal subunit phosphorylation

After fertilization of sea urchin (Arbacia punctulata) eggs, there is a single prominent alteration in the pattern of protein phosphorylation. In eggs preloaded with ³²PO₄, a 31,000 M_r protein (rp31) becomes labeled within 4 min of sperm addition. A new steady-state level of rp31 labeling is achieved by 11 min. The rate of protein synthesis in sea urchin zygotes also increases at 8–10 min after fertilization. Protein rp31 corresponds to mammalian ribosomal S6 because it cosediments with 40 S subunits on high salt-sucrose gradients, it is similar to the mammalian protein in M_r and charge, and it becomes phosphorylated during an increase in protein synthesis. The specific activity of phosphorylated rp31 (relative to rRNA) is similar between free 80 S monosomes and polysomes, indicating that rp31 phosphorylation is not sufficient for ribosomal activity. A phosphatase, highly specific for rp31, is present in extracts of eggs and very early embryos. This phosphatase becomes inactive at about the same time that the degree of labeling of rp31 increases in embryos. Evidently a control system that maintains a low level of rp31 phosphorylation is active in sea urchin eggs. Inactivation of this system shortly after fertilization leads to the accumulation of phosphorylated ribosomes.     

Autonomous mesoderm formation in blastocoelic roof explants from inverted Xenopus embryos.

Xenopus eggs, artificially fertilized, were prevented from undergoing equilibrium rotation by incubation in medium containing ficoll. Three orientations were selected: normal, with animal pole uppermost; inverted, with vegetal pole away from gravity; and an off-axis orientation, with embryos tilted approximately 90 degrees from the animal-vegetal axis. At blastula stage 8, cells forming the blastocoelic roof were cultured in isolation as explants. These cells are normally fated to from epidermis ventrally and neural derivatives dorsally. Unexpectedly, in the fragments originating from inverted or 90 degrees-off-axis embryos, axial structures were found: notochord, somites, neural cells, cement glands, and sometimes sensory organs. Inverted eggs could be exploited in studies of mesodermal specification.