Egg Fragments Research Articles

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 the rapid (complete by 40 min after fertilization) and the late (occurring from ca. 60 to 120 minutes after fertilization) Na current losses are discussed.

Cations that alter surface potentials of lipid bilayers increase the calcium requirement for exocytosis in sea urchin eggs.

1. We examined the effects of cations that bind to phospholipid bilayers on the calcium requirement for exocytosis in vitro. 2. Tetracaine and trifluoperazine, two monovalent cations that bind hydrophobically to lipid bilayers, decreased the zeta-potential of lipid vesicles with a phospholipid composition similar to sea urchin plasma membranes; the decrease in the magnitude of the negative zeta-potential was consistent with the Gouy-Chapman-Stern theory. 3. Trifluoperazine and tetracaine also increased the concentration of calcium required to produce half-maximal exocytosis (Ca50) in isolated fragments of plasma membranes from sea urchin eggs. 4. The effects of trifluoperazine and tetracaine on the Ca50 of egg fragments can be explained quantitatively from the zeta-potential data obtained with phospholipid vesicles if we assume the calcium binding sites responsible for triggering exocytosis lie within a Debye length (less than 1 nm) of the surface of the plasma membranes, such that the concentration of calcium at the sites is affected by the surface potential of the membrane. 5. The divalent cation magnesium, which binds specifically to the phosphate group of lipids, affected Ca50 in a manner that can also be explained quantitatively from its effects on the zeta-potential of phospholipid vesicles. 6. The polyvalent cation neomycin, which adsorbs to the lipid phosphatidylinositol 4,5-bisphosphate with high affinity, also binds co-operatively to phosphatidylinositol; its effect on Ca50 can be explained quantitatively from its effects on the zeta-potential of vesicles containing the latter lipid. 7. A decrease in the ionic strength increases the magnitude of the surface potential of membranes; the effect of ionic strength on Ca50 can be explained qualitatively from its effect on the zeta-potential of phospholipid vesicles. 8. All our results are consistent with the hypothesis that calcium binding sites lie within a Debye length of the plasma membrane; they also indicate that drugs with specific, high-affinity sites may affect exocytosis by a non-specific mechanism.

Development of a muscle actin specified by maternal and zygotic mRNA in ascidian embryos

In this investigation, we characterize the embryonic and adult actins and describe the embryonic expression of a muscle actin in the ascidian Styela. Two-dimensional polyacrylamide gel electrophoresis showed that embryos, tadpole larvae, and adult organs contain three major and two minor isoforms of actin. Two of the major isoforms, which are present in the mantle, branchial sac, alimentary tract, and gonads of adults and in eggs, embryos, and heads and tails of tadpoles, are likely to be cytoplasmic actins. The third major isoform, which was enriched in the mantle and branchial sac of adults and localized primarily in the tails of tadpoles, is a muscle actin. The muscle actin isoform was not detected in eggs and early embryos. Radioactivity incorporation studies showed that the cytoplasmic actins were synthesized throughout early development, but muscle actin synthesis was first detected between the 16- and 64-cell stages, 2–3 hr after fertilization. Two lines of evidence indicate that embryonic muscle actin synthesis is directed in part by maternal mRNA. First, poly(A) + RNA isolated from unfertilized eggs directed the synthesis of muscle actin in an mRNA-dependent reticulocyte lysate. Second, muscle actin was synthesized in anucleate egg fragments. Arguments are also presented that muscle actin synthesis is not directed exclusively by maternal mRNA. It is concluded that embryonic and adult Styela exhibit actin heterogeneity, that one of the actin isoforms is a muscle actin, and that the muscle actin is synthesized during embryogenesis under the direction of maternal and zygotic mRNA.

Determination and morphogenesis in the sea urchin embryo.

The study of the sea urchin embryo has contributed importantly to our ideas about embryogenesis. This essay re-examines some issues where the concerns of classical experimental embryology and cell and molecular biology converge. The sea urchin egg has an inherent animal-vegetal polarity. An egg fragment that contains both animal and vegetal material will produce a fairly normal larva. However, it is not clear to what extent the oral-aboral axis is specified in embryos developing from meridional fragments. Newly available markers of the oral-aboral axis allow this issue to be settled. When equatorial halves, in which animal and vegetal hemispheres are separated, are allowed to develop, the animal half forms a ciliated hollow ball. The vegetal half, however, often forms a complete embryo. This result is not in accord with the double gradient model of animal and vegetal characteristics that has been used to interpret almost all defect, isolation and transplantation experiments using sea urchin embryos. The effects of agents used to animalize and vegetalize embryos are also due for re-examination. The classical animalizing agent, Zn2+, causes developmental arrest, not expression of animal characters. On the other hand, Li+, a vegetalizing agent, probably changes the determination of animal cells. The stability of these early determinative steps may be examined in dissociation-reaggregation experiments, but this technique has not been exploited extensively. The morphogenetic movements of primary mesenchyme are complex and involve a number of interactions. It is curious that primary mesenchyme is dispensable in skeleton formation since in embryos devoid of primary mesenchyme, the secondary mesenchyme cells will form skeletal elements. It is likely that during its differentiation the primary mesenchyme provides some of its own extracellular microenvironment in the form of collagen and proteoglycans. The detailed form of spicules made by primary mesenchyme is determined by cooperation between the epithelial body wall, the extracellular material and the inherent properties of primary mesenchyme cells. Gastrulation in sea urchins is a two-step process. The first invagination is a buckling, the mechanism of which is not understood. The secondary phase in which the archenteron elongates across the blastocoel is probably driven primarily by active cell repacking. The extracellular matrix is important for this repacking to occur, but the basis of the cellular-environmental interaction is not understood.(ABSTRACT TRUNCATED AT 400 WORDS)

Reversal of cellular polarity and early cell-cell interaction in the embryo of Caenorhabditis elegans

During early embryogenesis of Caenorhabditis elegans the serial stem cell-like cleavages of the germ line cells P 0-P 3 generate a number of somatic founder cells with different developmental potentials. Observations on partial embryos show that in the first two of these unequal divisions in the germ line the somatic daughter cell comes to lie anterior to the new germ line cell. In the following two, however, the somatic daughter cell comes to lie posterior to the new germ line cell, suggesting a reversal of polarity in the germ line. By the use of a laser microbeam, egg fragments can be extruded from young embryos; the fragments often cleave like partial twins. Depending on whether the fragment is derived from the posterior region of the uncleaved zygote P 0 or its daughter P 1, the mirror image duplications that are generated are joined at their larger soma-like cells or at their smaller germ line-like cells, respectively. This result is best explained as a reversal of polarity taking place in the germ line cell P 2. This notion is strengthened by the finding that partial embryos derived from the posterior region of the P 2 cell in late interphase do not undergo stem cell-like (i.e., unequal) cleavages in contrast to those derived from P 0 or P 1. Finally, an apparent early cell-cell interaction is described which is inconsistent with the classical notion of “mosaic” nematode development: removal of the germline cell P 2 results in an altered developmental pattern of its somatic sister cell EMS. A working model is presented linking reversal of polarity and cell-cell interaction and offers an explanation for the unique behavior of the EMS cell in normal development.

Alkaline phosphatase expression in ascidian egg fragments and andromerogons

Alkaline phosphatase (AP) activity is expressed in the endodermal cell lineage of ascidian embryos beginning at gastrulation. AP expression is resistant to levels of actinomycin D which completely suppress the appearance of other tissue-specific enzyme and morphological markers including acetylcholinesterase (AchE), a larval muscle enzyme whose expression requires embryonic transcription. The resistance of AP expression to actinomycin D has led to the proposal that AP may be expressed independent of embryonic transcription by the translational activation of maternal AP mRNA. To test this hypothesis, AP expression was examined in fragments of unfertilized and fertilized Styela plicata eggs by histochemical methods. As expected, nucleate fragments from fertilized eggs developed into larvae which exhibited AP activity in their endodermal cells and AchE activity in their muscle cells. In contrast, anucleate fragments from fertilized eggs, cultured until controls reached the larval stage, did not develop AP or AchE activity. The lack of AP activity was unrelated to the absence of cleavage or to the ooplasmic composition of the anucleate fragments. Anucleate fragments from unfertilized eggs were also AP negative, unless they were inseminated, after which they often developed to the larval stage as andromerogons and exhibited AP activity in their endodermal cells. The development of endodermal AP in andromerogons suggests that the factors responsible for AP expression are not localized in or attached to the maternal nucleus. In summary, the results suggest that AP expression requires nuclear events and is not determined exclusively by maternal cytoplasmic factors such as preformed mRNA.

Endogenous photoproteins, calcium channels and calcium transients during metamorphosis in hydrozoans.

There are species of hydrozoans, Eutonina victoria, Mitrocomella polydiademata, and Phialidium gregarium whose eggs contain calcium-specific photoproteins. These cytoplasmic photoproteins are synthesized during oogenesis. During the cleavage stages of embryogenesis they are distributed to all of the cells of the developing planula larva. The amount of photoprotein slowly declines during the development of the planula larva, and markedly declines when the planula undergoes metamorphosis to become a polyp.Oocytes, unfertilized eggs, and fertilized eggs prior to the first cleavage do not produce light when treated with KCl. The ability to respond to KCl appears about the time of first cleavage, and is correlated with the appearance of active membrane responses. Both the KCl response and the action potentials will occur in sodium-free sea water, and both are inhibited by calcium channel blockers. These and other experiments suggest that voltage sensitive calcium channels first become active at about the time of first cleavage. These channels also appear on the same schedule in both unfertilized eggs and in enucleated egg fragments, which have been artificially activated with A23187.Developing planulae produce few or no spontaneous light responses before gastrulation. Later the frequency and magnitude of spontaneous light production increases presumably due to an increasing frequency and magnitude of calcium transients. Both the natural trigger of metamorphosis (bacteria) and an artificial trigger (CsCl) cause a conspicuous series of calcium transients. When these transients are inhibited by calcium channel blockers, metamorphosis is also inhibited.

Close Correlation of the Periodicities of the Cleavage Cycle and Cytoplasmic Cycle in Early Newt Embryos: (newt/cleavage/cytoplasmic cycle).

The correlation between autonomous cyclic activity of the cytoplasm (cytoplasmic cycle) and the cleavage cycle was studied by using animal and vegetal half fragments of newt eggs formed by bisecting the uncleaved eggs after those eggs had been rotated through 90° off the vertical axis so as to alter the allocations of the cytoplasmic content in the two halves. When the bisection was made shortly after the rotation (Early Bisection), the resulting vegetal fragment showed 1.5 times longer intervals in the cytoplasmic cycle than its animal partner when cleavage was prevented by injection of colchicine, while when the bisection was made 30 min after the rotation (Late Bisection), the resulting pair of halves displayed equal intervals in the cytoplasmic cycle. The intervals of cell division of vegetal halves formed by the two kinds of bisection in the absence of colchicine were then examined. In these conditions, the vegetal half formed by Early Bisection still showed 1.5 times longer intervals in the cleavage cycle than its counterpart, and the half formed by Late Bisection displayed the same intervals in the cleavage cycle as its animal partner.

Electrical characteristics of ascidian egg fragments

Fragments of ascidian eggs, but at random in any plane and ranging in size from 10 to 90% of the total egg volume, displayed the electrical characteristics of the intact egg, having a resting potential of −86 mV and giving rise to an action potential upon stimulation by electrical current injection. Following insemination, the fragments generated fertilization potentials, comparable to those of intact eggs, although the repolarization phase was shorter. Our data show that there are sufficient ion channels throughout the egg surface to generate action potentials and fertilization potentials in excised egg fragments, irrespective of their global origin. Furthermore, the fertilizing spermatozoon is capable of activating fertilization channels in areas of the egg plasma membrane not destined for sperm entry.

The Limulus sperm motility-initiating peptide initiates acrosome reactions in sea water lacking potassium.

During fertilization in Limulus, the spermatozoa first attach to the egg and then undergo an acrosomal reaction. In this reaction, the acrosomal vesicle exocytoses, and a long, preformed acrosomal filament is extruded (and subsequently penetrates the egg chorion). The egg surface component that triggers the acrosome reaction has not yet been solubilized; therefore, previous studies have examined either spontaneous acrosome reactions or acrosome reactions that were triggered by eggs (or insoluble egg fragments), elevated extracellular Ca2+, or Ca2+ ionophores. In this study, we report a new method for initiating acrosome reactions in Limulus sperm. When the Limulus sperm motility-initiating peptide (SMI) is added to sperm in K+-free sea water, greater than 90% acrosome reactions are initiated within 5 min. However, less than 5% acrosome reactions occur either in K+-free sea water lacking SMI or when SMI is added to sperm in either normal sea water or K+- and Ca2+-free sea water. Experiments with K+ ionophores (nigericin and valinomycin), a K+ channel blocking agent (tetraethyl ammonium), an Na+ ionophore (monensin), and reagents that increase the intracellular pH (monensin, nigericin, and NH4Cl) indicate that changes in intracellular K+, Na+, or H+ do not mediate SMI-initiated acrosome reactions. The K+/Ca2+ ratio determines whether or not SMI will initiate acrosome reactions, with greater than 50% acrosome reactions being initiated when this ratio is below 0.3. In that K+ movement does not appear to be the critical event, possibly the K+/Ca2+ ratio either determines the rate of Ca2+ entry or controls the conformation of sperm surface molecules to allow SMI to initiate acrosome reactions in low K+.

Protein tyrosine kinase activity of eggs of the sea urchin Strongylocentrotus purpuratus: The regulation of its increase after fertilization

Protein tyrosine kinase activity in eggs of the sea urchin, Strongylocentrotus purpuratus, increased two- to fourfold as early as several min after fertilization at 8–10°C. Artificial activation of eggs with the divalent cation ionophore, A23187, or with butyric acid induced the increase in enzyme activity. The transfer of eggs to seawater containing either no Na + or 50 m M Na + and 10 −4 M amiloride immediately after fertilization did not block the increases in enzyme activity. When eggs were activated with seawater containing NH 4OH, enzyme activity did not increase at 1 hr after activation, although the increased activity was detected at 3 hr after activation. Increased enzyme activity also was observed in enucleated egg fragments activated with butyric acid. Puromycin and emetine, inhibitors of protein synthesis, also did not inhibit the initial increases of enzyme activity after fertilization. These results demonstrated that the increased protein tyrosine kinase activity observed after fertilization of S. purpuratus eggs can be initiated independent of various other known events such as fusion with sperm cells and protein and DNA synthesis.

Developmental significance of a cortical cytoskeletal domain in Chaetopterus eggs

The cortex of Chaetopterus eggs contains a cytoskeletal domain (CD) which includes a specific class of dense granular organelles and a large proportion of the maternal mRNA. This CD, along with its constituent dense granular organelles and mRNA, can be displaced to atypical locations in the egg by centrifugation. To investigate the developmental significance of the CD, we have examined the early development of egg and zygote fragments, prepared by centrifugation, which contained the CD, the nucleus, or both. Specifically, we prepared nucleate egg and zygote fragments depleted in the CD, and two-cell embryos in which the CD was present in only one cell. Nucleate centripetal egg fragments were both unable to develop after fertilization and were depleted in the CD, as shown by electron microscopy, acridine orange staining of cortical organelles, and hybridization with poly(U) and cloned DNA probes. In contrast, about 20–35% of the nucleate centripetal fragments derived from one-cell zygotes developed into swimming larva. Correlated with this improved success of development, we found that these zygotic centripetal fragments contained significant levels of the CD, using the same methods listed above. More effective removal of the CD from zygotic centripetal fragments by stratification prior to fragmentation virtually eliminated their ability to develop. The CD and associated components could be displaced into only one of the first two blastomeres by centrifugation of zygotes immediately prior to the first cleavage. Embryos containing the CD in only one blastomere continued to cleave, but formed defective larva. The results suggest that the cortical CD is necessary for normal embryonic development.

Expression of alkaline phosphatase in nucleate and anucleate fragments of ascidian eggs: [formula omitted] and [formula omitted] Department of Zoology, University of Texas, Austin, Tx. U.S.A. 78712

Expression of alkaline phosphatase in nucleate and anucleate fragments of ascidian eggs: [formula omitted] and [formula omitted] Department of Zoology, University of Texas, Austin, Tx. U.S.A. 78712

Localization of the factors producing the periodic activities responsible for synchronous cleavage in Xenopus embryos

This paper investigates the localization within the Xenopus egg of the factors responsible for the periodic activities such as the cyclic rounding-up and flattening, related to the cleavage cycle. Denuded eggs were bisected along the boundary line between the animal and the vegetal hemispheres immediately after being rotated through 90 degrees off the vertical axis (Early Bisection). The resulting animal halves, though prevented from cell division by colchicine, showed typical periodic rounding-up as previously observed in enucleated egg fragments, whereas the vegetal halves did not. This result indicates that the factors inducing the periodic rounding-up are not distributed uniformly throughout the egg but localized mostly in the animal hemisphere. Furthermore, the distribution of these factors between the cortex and endoplasm of the animal hemisphere was investigated. Eggs were separated into animal and vegetal halves following incubation for 30 min after the 90 degrees-off axis rotation (Late Bisection). During this incubation the endoplasmic components become relocated in the rotated egg under the force of gravity. After the rotation, the Late-Bisected vegetal halves showed typical cyclic rounding-up in contrast to those formed by Early Bisection. These results suggest that the factors inducing the periodic rounding-up (and probably also many other cyclic activities, closely linked with the rounding-up movement) are localized in endoplasmic components which can be displaced by gravity from the animal to the vegetal hemisphere of the Xenopus egg.

Periodicity of Cytoplasmic Cycle in Nonnucleate Fragments of Sea Urchin and Starfish Eggs*: (cortical tension/sea urchin/starfish/cytoplasmic cycle).

Fertilized eggs of the sea urchin, Hemicentrotus pulcherrimus and the starfish, Asterina pectinifera were separated by manual bisection to obtain pairs of nucleate and non-nucleate fragments. Simultaneous observation on the pair revealed that the cyclic change in the cortical tension in the non-nucleate fragment was definitely prolonged than the cleavage interval of nucleate partner by about 30%. Activated non-nucleate cytoplasmic fragments derived from the unfertilized eggs and colchicine treated whole eggs of H. pulcherrimus still showed the same degree of prolongation.

The specification of metameric order in the insectCallosobruchus maculatus Fabr. (Coleoptera) : II. The effects of temporary constriction on segment number.

Mechanically dividing an insect egg into anterior and posterior fragments results in a segment gap (Sander 1976), a loss of non-terminal segments in the constricted region. By altering the stage and duration of constriction, we produced different types of egg fragments in the pea beetleCallosobruchus. The patterns formed by these fragments suggest the existence of interactions between anterior and posterior egg regions that influence segment patterning and placement. Segments in excess of the numbers expected on the basis of permanent constrictions were produced in fragments when: (1) the constriction was released before cellularization occurred and (2) in addition the complementary fragment degenerated. Apparently the degenerating fragment induced the formation of excess segments in the developing fragment. Differences in the time and extent of excess segment formation in anterior versus posterior fragments suggest an asymmetric distribution of prerequisites for segment formation. This conclusion is consistent with our finding that a partial reversal of segment sequence (double abdomen formation) can be induced only in posterior fragments by a degenerating fragment, but not in anterior fragments (see companion paper).The formation of excess segments shows that the segment gap observed after permanent separation cannot be due to non-specific damage, caused by the process of constriction as such, to the egg or to localized putative segment precursors.

The interval of the cytoplasmic cycle observed in non-nucleate egg fragments is longer than that of the cleavage cycle in normal eggs of Xenopus laevis.

Non-nucleate fragments of animal eggs are known to show a cyclic change in rigidity similar to the change observed in the cleavage of normal eggs. The interval of cyclic change was compared with that of the cleavage cycle in Xenopus eggs. Procedures employed by both Hara and co-workers and Sakai & Kubota to obtain non-nucleate fragments were carefully repeated. Non-nucleate fragments produced by either of those procedures had a longer duration of cycle than the normal cleavage cycle (about 30%). By injection of colchicine or vinblastine the cycle of the nucleate fragment was lengthened to coincide with that of the non-nucleate partner. It is suggested that the basic cycle length intrinsic to egg cytoplasm is modulated by assembly-disassembly of the mitotic apparatus, as demonstrated by Sluder in the chromosome cycle of sea urchin eggs.

Cyclic cytoplasmic activity of non-nucleate egg fragments of Xenopus controls the morphology of injected sperms.

Triton X-100-treated sperms were injected into non-nucleate egg fragments of Xenopus laevis to determine whether the structure of the injected sperm nucleus is affected by the cyclic activity of the cytoplasm. Swollen vesicular nuclei were very frequently observed when the sperms were injected and incubated during the 'rounding-up' phase of the recipient fragment, whereas no such structures were found when they were incubated during the 'relaxing' phase.

Cytoplasmic cycle in meiotic division of starfish oocytes

Nonnucleate fragments of sea urchin and amphibian eggs are known to show an autonomous cyclic activity whose interval is comparable to the cleavage interval of normal eggs. In order to examine for the presence of such a cytoplasmic cycle in meiotic division, maturing oocytes of the starfish, Asterina pectinifera, were bisected into halves about 10 min after germinal vesicle breakdown, and tension at the surface of the nonnucleate fragments was continuously measured by a compression method. The nonnucleate fragments were found to show cyclic changes in the tension, with a temporal pattern very similar to that of the changes accompanying the two successive meiotic divisions of normal oocytes; i.e., the sharp peak in the tension was found to occur always twice, and only twice. However, the nonnucleate fragment of immature oocytes showed only a gradual rise in the tension without any cyclic change even when it was induced to mature by 1-methyladenine. When the maturing oocyte was bisected before germinal vesicle breakdown, the nonnucleate fragment still did not cycle. These results indicate that cytoplasm of maturing oocytes which underwent germinal vesicle breakdown is endowed not only with cyclic activity, but also with terminating it after two cycles. It is suggested that germinal vesicle material may trigger such an autonomous cyclic activity.

Hydrozoan eggs can only be fertilized at the site of polar body formation

Hydrozoan eggs are normally fertilized at the site of polar body formation. The female pronucleus is just under the cell membrane at this site. Sperm are attracted to the eggs and aggregate at this site. This paper demonstrates that this site is the only region on the egg surface where the sperm can fuse with the egg. This has been done by cutting unfertilized eggs into fragments containing the site of polar body formation and fragments without this region. Sperm were added to the fragments and their ability to be fertilized was assayed by noting whether or not they cleaved. Only fragments containing the site of polar body formation cleaved. The absence of cleavage in fragments lacking the site of polar body formation cannot be attributed to the inability of these fragments to attract sperm. Such fragments attract sperm for several hours while fragments which contain the site of polar body formation stop attracting sperm a few minutes after fertilization. Cytological studies of egg fragments which do not contain the site of polar body formation show that they do not contain sperm nuclei. The lack of cleavage in these fragments cannot be attributed to the lack of a female pronucleus. By using centrifugation it is possible to move the female pronucleus away from the site of polar body formation. By cutting these centrifuged eggs in an appropriate way it is possible to create egg fragments with the site of polar body formation that lack the female pronucleus and egg fragments that lack the site of polar body formation but contain a female pronucleus. Only fragments which contain the site of polar body formation can be fertilized.