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.     

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     

Sea urchin spectrin in oogenesis and embryogenesis: a multifunctional integrator of membrane-cytoskeletal interactions

Using indirect immunofluorescence microscopy on semithin cryosections of maturing ovarian tissue, eggs, and developing embryos, we have mapped the cellular distribution and dynamic redistribution of spectrin in oogenesis and early embryogenesis. During oogenesis, spectrin is initially found in the cortex of oogonia and previtellogenic oocytes, and later accumulates in the cytoplasm of vitellogenic oocytes on the surfaces of cortical granules, pigment granules/acidic vesicles, and yolk platelets. Following egg activation, spectrin undergoes a rapid redistribution coincident with three major developmental events including: (1) restructuring of the cell surface, (2) translocation of pigment granules/acidic vesicles to the cortex during the first cell cycle, and (3) amplification of the embryo's surface during the rapid cleavage phase of early embryogenesis. The synthesis and storage of spectrin during oogenesis appears to prime the egg with a preestablished pool of membrane-cytoskeletal precursor for use during embryogenesis. Results from this study support the hypothesis that spectrin may function as a key integrator and modulator of multiple membrane-cytoskeletal functions during embryonic growth and cellular differentiation.     

Early Events in Anuran Amphibian Fertilization: An Ultrastructural Study of Changes Occurring in the Course of Monospermic Fertilization and Artificial Activation

In unfertilized frog eggs, the plasma membrane displays an animal vegetal polarity characterized by the presence of short microvilli in the vegetal hemisphere and long microvilli or ridge-like protrusions in the animal hemisphere. The densities of microvilli are similar in the two hemispheres.
The fertilizing sperm always fuses with the animal hemisphere of the egg and induces a wave of exocytosis of cortical granules from its site of penetration. Similar spreading of the cortical reaction is seen on activation by pricking the egg cortex. The integration of the cortical granule membrane with the plasma membrane is rapidly followed by elongation of microvilli, which is progressively realized all over the egg surface from the site of sperm entry or the site of pricking. At this time, the length and shape of the microvilli in the animal and vegetal hemispheres are similar and their densities are the same as in unfertilized eggs.
A "smoothing" wave can be seen on the living egg, 40–60 seconds after pricking, starting around the site of pricking. This wave of microvillar elongation is accompanied by changes in intensity of diffracted light spots observed at the surface of the egg. This pattern might result from rapid and progressive thickening of the cortex that would drive pigment granules into the cytoplasm. The Brownian movement of these granules is thought to be responsible for the observed diffracted light spots.
Electrical stimulus or the ionophore A23187 induced activation reactions similar to those triggered by the sperm or by pricking, except that the cortical reaction began simultaneously in several distinct sites of the cortex.     

REFRACTIVE INDEX OF THE PROTOPLASM IN SEA URCHIN EGGS*

The distribution of the refractive index (RI) of the protoplasm in sea urchin eggs was determined from the optical path differences at various regions of the cell measured by interference microscopy assuming that the cell structure is symmetrical about the line passing through the center of the cell and that of the nucleus in unfertilized eggs and about the spindle axis in fertilized eggs during mitosis and cleavage. The RI of the cytoplasm in the unfertilized egg was uniform except for the cortical region, which had the RI higher than that of the underlying endoplasm. The RI of the cortex was generally higher than that of the underlying endoplasm, which did not appreciably change during mitosis and cleavage. The RI of the nucleus was lower than that of the cytoplasm. The RI of the mitotic apparatus was lower than that of the surrounding cytoplasm. The fertilization membrane had a thickness of about 0.6 μm in hydrated state and about 25 nm in dried state (mean values). The RI of the perivitelline space was about 0.00015 higher than that of seawater, equivalent to 0.08 g/100 ml of contents.     

Effect of Microtubular Poisons on Cleavage Furrow Formation and Induction of Furrow-like Dent in Amphibian Eggs

The effects of the microtubular poisons colchicine, vinblastine and nocodazole, on cleavage furrow formation and induction of furrow-like dents in eggs of the newt, Cynops pyrrhogaster , were examined.
Solutions of the poisons were injected beneath the cortex around the small initial furrow, or around the advancing tip of the furrow of eggs during the first cleavage. This resulted in prompt block of the progress of the furrow at the injection site, and subsequent total regression of the furrow or incomplete cleavage.
The ability of the cortex of a cleavage-arrested blastomere to form a furrow-like dent was tested by inhibiting furrow formation of one blastomere of two-cell embryos by injection of the microtubular poisons, and then transplantation of the blastomere under the cortex of the animal half with furrow-inducing cytoplasm (FIC) taken from normally cleaving eggs. No dent was formed. Moreover, FIC from eggs treated with a poison had no ability to induce a dent on the surface of normally cleaving eggs.
These results show that microtubule structures are directly involved in formation of a cleavage furrow.     

Genesis of Epidermal Action Potentials in Cytokinesis Arrested Xenopus Embryos

When Xenopus embryos were treated continuously with cytochalasin B (3–10 μg/ml) from the 8 cell stage, cleavage arrested embryos in various degrees were observed. In 3–5 μg/ml cytochalasin B, cytokinesis was inhibited at the midblastula stage and pigment granules remained at the cell cortex of the animal pole. These cells showed epidermal like action potentials when the control embryos (St. 26/28) generated epidermal action potentials. In 5–7 μg/ml cytochalasin B, furrows, following their formation at early cleavage stages, regressed and no further cleavage from the 16 cell stage to morula stage took place. The pigment granules were dispersed throughout the interior of the cytoplasm. These cells showed no epidermal action potentials. Thus, it is considered that cytokinesis per sé , following the midblastula stage, is not a prerequisite for the genesis of epidermal action potentials, and that chronological times corresponding to the tailbud larva stage and a stable structure of the cellular cortex are required to bring about these potentials.     

A CYTOLOGICAL STUDY OF THE CENTRIFUGED WHOLE, HALF, AND QUARTER EGGS OF THE SEA URCHIN ARBACIA PUNCTULATA

While the ooplasmic components of centrifuged eggs of Arbacia punctulata do not stratify in homogeneous layers, we have obtained the following strata beginning with the centripetal end: lipid droplets, pronucleus, clear zone, mitochondria, yolk, and pigment. Whereas mitochondria may be found mingled with yolk bodies, we have never observed lipid droplets nor pigment bodies among any of the other inclusions. The so-called clear zone contains a heterogeneous population of inclusions: annulate lamellae, heavy bodies, Golgi complexes, and rod-containing vacuoles. The peripheral cortical granules of immature (germinal vesicle stage) and of mature eggs are not dislodged from the cortical ooplasm with the centrifugal force utilized. When the eggs are treated with urethane, prior to centrifugation, the cortical granules of mature eggs abandon their peripheral position. Further centrifugation of the initially stratified eggs produces nucleated and nonnucleated halves and the centrifugation of the halves results in quarters. The cytology of the halves and quarters is discussed. The halves and quarters have been activated with either sperm or hypertonic sea water. With the exception of the nucleated halves, we were unable to obtain plutei larvae from the other fractions (red halves and quarters). We believe that the lack of development of the various fragments is a function of the balance of particular inclusions necessary for differentiation.     

Polarization of the Surface Membrane and Cortical Layer of Sea Urchin Blastomeres, and its Inhibition by Cytochalasin B

Sea-urchin blastomeres have two domains of the plasma membrane which can be distinguished immunocytochemically. An egg-surface antibody (anti-ES), which binds to the membrane of the entire surface region of eggs before cleavage, binds to the membrane of the outer surface region of blastomeres after cleavage, but not to that of the cleavage furrow region or interblastomeric surface region.
The anti-ES binding sites on the egg membrane were chased after cleavage by labeling the egg plasma membrane with FITC conjugated monovalent anti-ES (FITC-Fab anti-ES) before the first cleavage, and then allowing the eggs to cleave. The surface fluorescence increased in intensity in the cleavage furrow region with progress of furrowing, but after completion of the furrowing, the fluorescence became uniform and finally decreased in the interblastomeric surface region.
The distributions of pigment granules and NBD-phallacidin stainable microfilaments in the cortex after completion of furrowing were polarized in the same way as the anti-ES binding area. As cytochalasin B completely inhibited the polarization in both the surface and cortical layer but colchicine did not, polarization of the anti-ES binding area was concluded to be due to the post-cleavage polarized distribution of submembranous microfilaments in the cortical layer.     

Distinctive subcellular alterations induced by hypertonic stress in sea urchin eggs

Sea urchin eggs continuously exposed to a hypertonic solution were ultrastructurally examined for osmotic-stress induced alterations. No fertilization membranes formed during the treatment and the surface-cortex complexes remained unaltered from the unfertilized state. However, the osmotic stress did induce a number of subcellular changes. During the first 30 minutes of the treatment the eggs formed many endoplasmic reticulum whorls and compacted Golgi body aggregations. Both of these new formations can be correlated with rapid changes in intracellular calcium, known to occur in hypertonic stressed eggs. Aggregations of mitochondria could be observed at later stages; these aggregations can also be related to subcellular stress and possible changes in internal calcium concentrations. The various morphological transitions within the cytoplasm, along with the lack of a cortical reaction in these eggs, not only supports the idea that calcium is released during parthenogenetic activation, but also suggests that this free calcium originates from stores other than the stores that are involved during fertilization or simple artificial activation.     

Localization of cortical granule constituents before and after exocytosis in the hamster egg

Electrical activation of the hamster egg was used to study cortical granule constituents before and after exocytosis. The activated hamster eggs underwent cortical granule decondensation just prior to and at the time of exocytosis. Some of the cortical granules of aged, unactivated eggs underwent similar changes. FITC- and gold-conjugated Lens culinaris agglutinin (LCA) bound intensely to the surfaces of activated but not unactivated eggs. This labelling was associated with the microvilli. Permeabilized eggs exhibited discrete cortical labelling before activation, with a subsequent decrease following the cortical reaction. Gold-conjugated LCA specifically bound to cortical granules when incubated with thin sections. FITC-soybean trypsin inhibitor (SBTI) bound in discrete foci in the cortex of unactivated eggs. Following activation, cortical labelling by SBTI decreased. Aprotinin and benzamidine hydrochloride inhibited FITC-SBTI from binding to the egg cortex. Gold-avidin localization of biotin-SBTI in the electron microscope demonstrated that condensed cortical granules did not bind SBTI but decondensed or exocytosing granules did. This suggests that a cortical granule protease is exposed just prior to exocytosis. Activated eggs exhibited dramatic decreases in the number of hamster sperm penetrating the cytoplasm, suggesting that a plasma membrane block to polyspermy is temporally related to cortical granule exocytosis.     

Evidences for direct involvement of microtubules in cleavage furrow formation in newt eggs

This paper aims at examining the effect of colchicine, a microtubular poison, on the process of furrow formation in whole eggs and egg fragments as well as the process of artificial induction of furrow-like dents, in eggs of the newt, Cynops pyrrhogaster. To apply colchicine locally to eggs, the eggs were slit across or along a furrow in a colchicine solution during first cleavage. When a slit was made across or in front of a growing furrow at the onset of its growth, the furrow quickly ceased growing and often regressed. Cortices containing an entire growing furrow were isolated along with a thin layer of subcortical cytoplasm immediately after the start of the first cleavage. Furrows in the cortices degenerated when the cortices were cultured in a colchicine solution, whereas they continued growing when they were cultured in Holtfreter's saline. Furrow-inducing cytoplasm was injected to a site beneath the cortex in the animal half of the egg during first cleavage. When a small slit was made close to the site of the injection in a colchicine solution, no furrow-like dent was induced. These results imply that microtubules are directly involved in the generation and growth of cleavage furrows.     

Effects of cytochalasin B on the cortex of the unfertilized sea urchin egg

The peripheral cytoplasm of the unfertilized sea urchin egg contains approximately 18,000 cortical granules. These granules remain monolayered within the normal boundaries of the cortex when the egg is centrifuged at forces sufficient to stratify other intracellular inclusions. Exposure of unfertilized eggs to the microfilament disrupting agent, cytochalasin B (CB) causes the granules to rearrange into several layers and occasionally to undergo exocytosis or break down in situ. When these eggs are centrifuged, the cortical granules are dislodged from the cortex and migrate centrifugally among the densest intracellular components. In addition, cytoplasmic inclusions, which normally are excluded from the cortex, impinge directly upon the egg plasma membrane in CB-treated, centrifuged eggs. These results are consistent with the existence of a microfilamentous network which confines the cortical granules within and excludes other intracellular inclusions from the cortex of the unfertilized egg.     

EFFECTS OF THE SURFACTANTS ON THE CLEAVAGE AND FURTHER DEVELOPMENT OF THE SEA URCHIN EMBRYOS II. DISTURBANCE IN THE ARRANGEMENT OF CORTICAL VESICLES AND CHANGE IN CORTICAL APPEARANCE

After fertilization, two types of cortical vesicles ware examined under the electron microscope (the cortical vesicle I and II) and the light microscope (pigment granules and another kind of vesicles). The cortical vesicle I corresponds to the pigment granule and the cortical vesicle II does to the other vesicle.
The unequal division of the sea urchin embryo which occurs at the fourth cleavage was modified to an equal cleavage pattern by the treatment with sodium lauryl sulfate (SLS) or cetyl trimethyl ammonium bromide (CTAB). But other surfactants such as sodium deoxycholate, Tween 80, Lubrol PX did not have such an effect. The cell surface of the embryo which had been treated either SLS or CTAB became rough or smooth. Cortical vesicles and pigment granules disappeared and/or were dislocated from the cortex. However, cell organelles were as normal as the control. On the other hand, the cortical appearance of other surfactant-treated embryos showed no disturbance and cell organelles were also more or less normal. Therefore, the equalization of unequal cleavage is caused by the disturbance in the cortex and thus the cortex plays a major role on the micromere formation at the 16-cell stage and on the further sea urchin development.     

A marker of animal-vegetal polarity in the egg of the sea urchin Paracentrotus lividus. The pigment band

We have examined the subequatorial accumulation of pigment granules (the so-called 'pigment band') in the egg of the sea urchin Paracentrotus lividus, which constitutes an unambiguous marker of animal-vegetal polarity. Most of the reddish pigment granules are situated at the periphery of the egg. They exhibit occasional saltatory movements and can aggregate into large patches. Pigment granules are retained as a band in the isolated cortex when the egg surface complex is isolated by shearing eggs attached to polylysine-coated surfaces with calcium-free isotonic solutions. Pigment granules remain as the main vesicular component of fertilized egg cortices or of unfertilized egg cortices perfused with calcium to provoke cortical granule exocytosis. They may be anchored to the isolated cortex through associations with the plasma membrane and with an extensive subsurface network of rough endoplasmic reticulum (rough ER). Pigment granules contain antimonate-precipitable calcium and, in this respect and many others, resemble acidic vesicles recently identified in the cortex of unpigmented sea urchin eggs. We discuss the similarities observed between granules and acidic vesicles in various urchin egg species and their possible functions.     

EFFECTS OF CHEMICAL REAGENTS ON THE CYCLIC CHANGES OF CORTEX AND CYTOPLASM IN THE ACTIVATED ENUCLEATED EGG-FRAGMENTS OF THE SEA URCHIN*

Unfertilized eggs of sea urchins. Anthocidaris crassispina and Hemicentrotus pulcherrimus, were separated by centrifugation into two fractions (nucleated light and enucleated heavy fragments). The enucleated egg-fragments were activated by treatment with 1 M urea and then put into sea water solutions of the following three reagents; colcemid, cytochalasin B and Monogen at a concentration by which cleavage was suppressed. It was then examined whether the egg-fragments can exhibit cyclic changes of cytoplasm and cortex in correlation with the cleavage cycle in normally fertilized eggs without any influence of nuclear activity. The results obtained clearly showed that colcemid can suppress the cyclic appearance of cytoplasmic changes, but not that of cortical changes; on the contrary, in cytochalasin B- and Monogen-treated fragments, the periodicity in cortical activities is suppressed, while the periodic changes in the cytoplasm appear according to a timeschedule of the cleavage cycle. Therefore, it may be said that: 1) cyclic changes can occur in both the cytoplasm and the cortex independently, without the direct influence of nuclear activity; 2) if either of them is arrested, the cleavage does not take place; 3) the normal cleavage requires the simultaneous occurrence of periodic activities both in the cortex and in the cytoplasm after fertilization.     

A Cytochemical Study of the Sulfhydryl Groups of Sea Urchin Eggs during the First Cleavage

In the eggs of four species of echinoderms, Mespilia globulus, Pseudocentrotus depressus, Hemicentrotus pulcherrimus and Clypeaster japonicus, changes in the distribution of protein-bound SH groups from fertilization to the 2 cell stage have been studied cytochemically by use of a mercaptide-forming azo dye. In the eggs of these species, the color intensity in the cytoplasm increased upon fertilization. The astral centers and spindle during mitosis were stained deeply. When the aster formation was suppressed by ether, hyaline spots appeared in the egg cytoplasm instead of well formed astral centers and these spots were stained by the SH-specific dye. Upon recovery of such eggs in pure sea water, and when cleavage ensued, such spots disappeared and two new astral centers were reorganized. The SH-protein occurring in the centrosphere is considered to be the precursor material for the asters and spindle, and this material is apparently derived from the cytoplasm.     

Cytological changes and DNA and protein synthesis in parthenogenetically activated sea urchin eggs

Summary The present study deals with cytological observations, DNA and protein synthesis in artificially activated sea urchin eggs. The eggs were activated by means of Loeb's double treatment with butyric acid and hypertonic sea water. Most of the eggs ofHemicentrotus pulcherrimus divided when the chromosomes duplicated after formation of the first monaster and other eggs divided at a later cell cycle. In the eggs ofTemnopleurus toreumaticus, however, haploid division at the first cell cycle was observed predominantly.Activated eggs that were treated for 25 min with hypertonic sea water showed a marked uptake of³H-thymidine during the two periods of 30–40 min and 90–100 min after the double treatment. These periodic changes in the³H-thymidine uptake paralleled morphological changes within the nucleus. However, these periods of increased uptake were not observed in the eggs treated with hypertonic sea water for 60 min. During exposure to hypertonic sea water, the³H-thymidine-uptake by eggs activated with butyric acid decreased gradually. When the uptake of¹⁴C-valine by eggs was measured, a very low level was seen in unfertilized eggs. The level of uptake increased strikingly when the eggs were activated with butyric acid but was suppressed by the hypertonic treatment. However, removal of the eggs to sea water allowed the uptake to return to the former high level. This pattern suggests that the hypertonic treatment has an inhibitory effect on the synthesis of protein (or enzymes) which obstruct cleavage induction.     

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.     

Endoplasmic reticulum whorls as a source of membranes for early cytaster formation in parthenogenetically stimulated sea urchin eggs

Summary Sea urchin eggs exposed to a continuous hypertonic treatment rapidly form many concentric whorls of endoplasmic reticulum (ER) during the pre-activation period of the parthenogenetic development. These whorls, however, are only a temporary configurational alteration of ER which begin to break up just prior to egg activation. The conversion back to normal vesicles and lamellae occurs not only concurrently with the appearance of early cytastral areas, but also frequently in close association with the formation of these membranous areas. It is revealed here that membrane elements from disrupting whorls may become incorporated into adjacent, developing clear areas, early cytastral areas, and that this ER constitutes an initial major source of membranes for these early astral areas. Having previously suggested that the actual formation of ER whorls occurs in direct response to released intracellular calcium in hypertonic stressed eggs, the new findings, along with other related data and correlations, further suggest that whorl disruption and the formation of associated astral areas can be correlated with a corresponding decrease in the concentration of this released calcium in the cytoplasm.