Ilyanassa Egg Research Articles

FORMATION, ORGANIZATION, AND COMPOSITION OF THE EGG CAPSULE OF THE MARINE GASTROPOD, ILYANASSA OBSOLETA.

Embryos of the marine mud snail Ilyanassa obsoleta undergo early development within an egg capsule. After about a week of encapsulation, embryos hatch by releasing a chemical substance that removes the plug found at the apex of a capsule. However, the mechanism of action of this hatching substance remains poorly understood. To study how the hatching substance functions, we examined the composition of the egg capsule, particularly the plug region, to determine what the "substrate" of the hatching substance might be. We have also examined the formation and organization of the egg capsule to determine the origin and identity of the regions of a capsule that the hatching substance must remove. The results show that the Ilyanassa egg capsule is organized into four layers, the outer three of which are composed of protein and carbohydrate. Portions of the two inner layers of the capsule wall extend into the capsule apex and form the plug, which is dissolved by the hatching substance. The isolated capsule plug region contains three major glycoproteins resolved on sodium dodecyl sulfate-polyacrylamide gels. Therefore, the hatching substance may be a protease similar in action to the enzymes released by many other embryos at hatching.

PROTEIN SYNTHESIS IN THE POLAR LOBE AND LOBELESS EGG OFILYANASSA OBSOLETA

The relative rates of protein synthesis in the lobeless egg and polar lobe of Ilyanassa obsoleta were measured and found to be the same. Two-dimensional electrophoresis of 35S-methionine in vivo labeled polypeptides extracted from the Ilyanassa egg and its isolates resolved the same set of polypeptides from the egg, lobeless egg, and isolated polar lobe. For those peptides detected it was concluded that (1) the lobeless egg and the polar lobe contain the same set of mRNAs, (2) these mRNAs are not differentially translated in either of these isolates, (3) the polar lobe does not regulate the synthesis of these proteins, either quantitatively or qualitatively, during early cleavage, and (4) the peptides synthesized in the isolated polar lobe were translated from mRNAs produced during oogenesis.It was shown by one-dimensional electrophoresis of 35S-methionine-labeled basic peptides that the egg, lobeless egg, and isolated polar lobe synthesized, at approximately the same rate, two variants of H1 histone, histones H2A, H2B, H3, and H4, and high mobility group peptides 14 and 17. Because these peptides were synthesized in the isolated polar lobe it was concluded that they were translated from maternal mRNAs.

Characterization of actin from fertilized eggs of Ilyanassa obsoleta during polar lobe formation and cytokinesis

Actin was identified in fertilized eggs of the marine snail, Ilyanassa obsoleta, by heavy meromyosin (HMM) decoration of cortical microfilaments and by tryptic peptide analysis of presumptive egg actin. Eggs beginning to undergo first cleavage after third polar lobe formation were glycerinated, incubated with or without HMM, and examined by electron microscopy. In both sets of eggs, microfilaments were observed in the cleavage furrow and the polar lobe constriction, as well as in the remaining cortical cytoplasm. Microfilaments also were found in the cortex of glycerinated spherical eggs incubated without HMM. Microfilaments in glycerinated eggs treated with HMM had a barbed or fuzzy appearance; however, this type of decoration was not observed in glycerinated eggs incubated without HMM or when ATP or sodium pyrophosphate were present with the HMM. Microfilaments in the cleavage furrow and the third polar lobe constriction of glycerinated Ilyanassa eggs were stabilized by incubation in solutions of skeletal muscle tropomyosin or of 50 m M MgCl 2. Acetone powders prepared from Ilyanassa eggs contained a major protein which comigrated with actin purified from several vertebrate tissues when subjected to gel electrophoresis under denaturing conditions. The presumptive egg actin and purified actin standards were iodinated and compared by two-dimensional tryptic peptide analysis. The resulting peptide patterns from Ilyanassa egg actin were very similar to those of chick brain actin, but were distinctly different from those prepared from mouse skeletal muscle and chick gizzard actins.

Polar lobe formation and cytokinesis in fertilized eggs of Ilyanassa obsoleta: III. Large bleb formation caused by Sr 2+, ionophores X537A and A23187, and compound [formula omitted

Fertilized eggs of Ilyanassa obsoleta form a protuberance which resembles a normal polar lobe when injected with Sr 2+ or Ca 2+ by microiontophoresis. Eggs also form a lobe-like protuberance when exposed to any of three drugs: compound 48 80 , ionophore X537A, and ionophore A23187. Protuberances form more quickly and at lower drug concentrations if additional exogenous Ca 2+ is added, whereas higher concentrations of Mg 2+ do not have such an effect. When eggs are exposed to these drugs in Ca 2+-, Mg 2+-“free” seawater, with or without 10 m M EDTA, the eggs are still able to undergo extensive shape changes and form protuberances. Drug-induced shape changes are prevented by cytochalasin B, but will still occur in the presence of colchicine. Approximately 75% of Ilyanassa eggs are capable of forming and resorbing their third polar lobe and undergoing cytokinesis in Ca 2+-, Mg 2+-“free” artificial seawater (even containing 10 m M EDTA), solutions which by atomic absorption spectroscopy are shown to contain low concentrations of Ca 2+ (3–5 μ M) and Mg 2+ (1.0–3.5 μ M). The data suggest that if Ca 2+ is required for normal polar lobe formation and cytokinesis, it is derived from intracellular sources or is required in only very low exogenous concentrations (i.e., less than 10 −2 μ M free Ca 2+, in the presence of 10 m M EDTA).

An unusual cytoplasmic organelle in oocytes of Ilyanassa obsoleta.

An unusual organelle in the oocytes of the marine mud snail Ilyanassa obsoleta is described and called a polymerosome, because of its several components. Polymerosomes appear at the beginning of vitellogenesis and persist, in part, through postvitellogenesis. The persistent part is similar to an organelle that has been described by other workers in the vegetal region and the polar lobe of the Ilyanassa egg.

An Electrophoretic Analysis of RNA Synthesis in Normal and Lobeless Ilyanassa Embryo

RNA from pulse-labelled normal and lobeless Illyanassa embryos was analysed by electrophoresis on polyacrylamide gels. The proportional distribution of radioactivity among the RNAs made by these two types of embryos was compared statistically. It was shown that removal of the third polar lobe from the Ilyanassa egg at first cleavage did not produce any change in the proportion of several size classes of RNA transcribed during the first day od development. The post-gastrular lobeless embryo did show a significant deviation from the normal pattern of RNA synthesis, though it is not clear that this was a direct effect on transcription by the polar lobe cytoplasm.

Nucleic Acid Chemistry of theIlyanassaEmbryo

A morphological study of Ilyanassa embryogenesis was made from sections of several stages and the nucleic acid content was measured throughout the course of development. The kinetics of uptake and incorporation of radioactive precursors into RNA was determined. The RNAs synthesized at different stages of embryogenesis were fractionated by methylated albumin kieselguhr chromatography, and the polyadenylation of RNA during development was measured. From these studies it was concluded that the Ilyanassa embryo, beginning with the 4-cell stage, synthesizes dRNA, rRNA, 4S RNA, and 5S RNA throughout all stages of development. The extensive synthesis of dRNA and the higher proportion of RNAs polyadenylated by the pregastrula embryo was considered to be of particular significance. The localization of nucleic acids and nucleic acid precursors in the polar lobe and the effect of removing the polar lobe on nucleic synthesis was reviewed. Recent work on the ultrastructure of the Ilyanassa egg was reviewed in relation to the problem of ooplasmic segregation. The discovery of a new cytoplasmic organelle in the Ilyanassa oocyte was reviewed.

Cell Determination and Organogenesis in Molluscan Development: A Reappraisal Based on Deletion Experiments inIlyanassa

The vegetal region of the Ilyanassa egg, incorporated into the polar lobe and segregated to the D blastomere, appears to have an organizing influence in development. The results of deleting individual micromeres of the first three quartets, and the 4d cell, indicate that most of these cells play rather distinctive developmental roles. Both cytoplasmic segregation and embryonic induction are believed to be involved in determining the fate of cells during development in Ilyanassa . Comparisons are made with other forms.

Polar lobe formation and cytokinesis in fertilized eggs of Ilyanassa obsoleta: II. Large bleb formation caused by high concentrations of exogenous calcium ions

Fertilized eggs of the mollusk Ilyanassa obsoleta ( Nassarius obsoletus) form large blebs resembling polar lobes within 12 min of exposure to solutions of isotonic CaCl 2, whereas control eggs in sea water remain spherical. Under identical conditions, fertilized eggs of the sea urchin, Strongylocentrotus purpuratus, an organism which normally does not form polar lobes, do not form blebs upon exposure to solutions of isotonic CaCl 2. The calcium-induced blebbing in Ilyanassa still occurs if other cations such as Na +, Mg 2+, or Mn 2+ are present in addition to Ca 2+, but not if comparable concentrations of K + are present. Cytochalasin B prevents the calcium-induced blebbing, whereas colchicine does not. Cytokinesis in both Ilyanassa and Strongylocentrotus and normal polar lobe formation in Ilyanassa appear to require exogenous K + but not exogenous Ca 2+. Preliminary electron microscopy of Ilyanassa eggs exposed to isotonic solutions of CaCl 2 has shown microfilaments in the cortical cytoplasm in the region of the bleb constriction but no microfilaments in spherical control eggs in sea water. These data suggest that high concentrations of exogenous Ca 2+ trigger the polymerization and contraction of a ring of microfilaments in the cortical cytoplasm of the Ilyanassa egg which results in the formation of a lobelike bleb of cytoplasm. The observation that K + antagonizes this Ca 2+-induced blebbing has led to the formulation of a theory which postulates that the ratio of intracellular Ca 2+ to intracellular K + is critical in the control of polar lobe formation and cytokinesis.

Distribution of ribosomes in the egg of Ilyanassa obsoleta

The cytoplasm of the Ilyanassa egg at the trefoil stage contains a large population of electrondense particles of about 700 Å and few free ribosome-like particles. The 700 Å particles are composed of granules of about 120 Å embedded in a homogeneous matrix. These particles are sparsely distributed in the perinuclear cytoplasm and are concentrated in very large patches at the periphery of the egg. In the polar lobe these particles occupy almost all the cytoplasm not filled with yolk. Amylase and RNase digestion experiments have characterized these 700 Å particles as clusters of ribosomes embedded in an amylase-sensitive matrix.

Development of the vegetal half of the Ilyanassa egg after removal of most of the yolk by centrifugal force, compared with the development of animal halves of similar visible composition

Summary By centrifuging fertilized, mature Ilyanassa eggs in raffinose solution, three cytoplasmic zones were produced, and the eggs were pulled into light and heavy fragments of approximately equal size. Light halves, which contained the lipid cap, intermediate zone, pronuclei, and only a small portion of the original yolk of the egg, could undergo further development. Two classes of light halves, animal and vegetal, were produced, and their development was compared. Animal halves were produced by centrifuging directly in raffinose solution, without prior treatment. Since Ilyanassa eggs orient under gravity with the vegetal pole downward, the light halves of unrestrained eggs represented the animal hemisphere. As a rule, these cleaved equally without formation of a polar lobe, and differentiated into partial larvae which lacked lobe-dependent structures (eyes, foot, shell, intestine, heart, etc.). A few exceptional cases are considered to have contained a portion of the vegetal hemisphere. Light halves containing all or part of the vegetal hemisphere were produced by a two-step centrifugation treatment. Eggs were first held upside down in a gelatin-seawater gel, and centrifuged until stratified internally. They were then freed from the gel and recentrifuged in raffinose solution to separate them into halves. In eggs with complete reversal of the usual stratification pattern, the nucleated light half would contain all the vegetal hemisphere; light havles of incompletely reversed eggs would contain a part of the vegetal hemisphere. Nucleated vegetal fragments, free of most of the yolk, formed a polar lobe of approximately normal proportions and underwent unequal first cleavage. Approximately half of the vegetal fragments differentiated lobe-dependent structures. It is concluded that the morphogenic influence of the vegetal polar area (polar lobe region) probably does not depend upon the presence of the yolk particles normally found there, and is able to withstand centrifugal force sufficient to displace lipid droplets, yolk particles, and nuclei.

The localization of ribonucleic acid in the egg of Ilyanassa obsoleta

Using quantitative microchemical methods the RNA content of the egg, lobeless egg, polar lobe, AB and CD blastomeres was determined in the egg of Ilyanassa obsoleta.The concentration of RNA, calculated from three different reference units, was significantly greater in the AB than in the CD blastomere. The CD cell and the polar lobe had approximately the same concentration of RNA.No RNA was found in the yolk platelets of the Ilyanassa egg.

Simultaneous effects of metabolic inhibitors on the viscosity, surface rigidity and cleavage in Ilyanassa eggs.

Journal of Cellular and Comparative PhysiologyVolume 47, Issue 3 p. 341-356 Article Simultaneous effects of metabolic inhibitors on the viscosity, surface rigidity and cleavage in ilyanassa eggs Joseph M. Butros, Joseph M. Butros Marine Biological Laboratory, Woods Hole, Mass., and Emory University, Emory University, GaSearch for more papers by this author Joseph M. Butros, Joseph M. Butros Marine Biological Laboratory, Woods Hole, Mass., and Emory University, Emory University, GaSearch for more papers by this author First published: June 1956 https://doi.org/10.1002/jcp.1030470304Citations: 6AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Citing Literature Volume47, Issue3June 1956Pages 341-356 RelatedInformation

Behavior of the Cell Surface During Cleavage

1) Linear stretching and shrinkage of the surface of Ilyanassa eggs as viewed from the side (perpendicular to the egg and spindle axes) was studied by the method of attaching kaolin particles.2) During the second maturation division with its accompanying form change, the animal pole region (animal furrow region) follows a continuous course of shrinkage, and after the cell regains a spherical form, it remains shrunken. The region of the spindle pole (polar region) sometimes shrinks and sometimes stays unchanged, which is more characteristic of the vegetal area; that is, this region is transitional in nature. In both cases, a temporary stretching can be seen in connection with the lobe formation. Around the constriction between the main cell body and the polar lobe, the temporary stretching is more marked, and in the spherical stage following division, the surface returns to the initial state with no shrinkage. In the surface of the lobe, the temporary stretching is much increased, and the surface remains stretched, even after the cell acquires a spherical shape.3) During cleavage, the animal pole region (animal furrow region) shows a characteristic initial shrinkage phase which is followed by a stretching of about 250%. After the cleavage, a residual stretching of about 190% is observed. The polar region has no initial shrinkage phase and the maximum stretching is 120 to 150%, with a correspondingly slighter residual stretching. The surface of the constriction starts to stretch with no initial shrinkage phase and attains a maximum of nearly 500%, the greatest expansion in any part of the egg surface. However, the residual stretching is comparatively slight. The surface of the polar lobe exhibits an expansion of a little over 200% with no initial shrinkage. The residual stretching, when compared with the spherical stage before the first cleavage, must be very slight if any exists at all. The cleavage furrow in the vegetal side of the smaller blastomere (vegetal furrow region) behaves much like the animal furrow region, but the initial shrinkage phase is lacking. This is presumably due to the presence of the polar lobe.4) A shrinkage of the animal pole region during the second maturation division, which is maintained through the subsequent spherical stage, and a permanent stretching of the surface of the polar lobe during the same period indicate that the surface material of Ilyanassa eggs is shifted from the vegetal side to the animal side during the second maturation division.5) In cleavage, the surface of Ilyanassa eggs behaves fundamentally like the surface of sea urchin eggs.6) The possibility of the formation of a new surface at the contact area of the blastomeres is discussed.