The Effects of Cytoskeleton Inhibitors on Cytoplasmic Localization in Chaetopterus pergamentaceus

We have treated fertilized and KCl-activated eggs of Chaetopterus pergamentaceus with microfilament and microtubule inhibitors to test the relationship of these cytoskeletal components to cytoplasmic localization. Low doses of cytochalasin B inhibited cleavage in fertilized eggs. Such embryos underwent differentiation without cleavage, a process characterized by relocalization of the yolky endoplassm to the center of the uncleaved egg and by the formation of cilia. Similar treatment of KCl-activated eggs inhibited ciliation, but not endoplasmic relocalization. Reversible inhibition of the first cleavage resulted in equal cleavage and differentiation of a larva lacking an apical organ. Inhibition of the first two cleavages resulted in differentiation without cleavage. At all concentrations high enough to block mitosis, colchicine prevented ciliation and endoplasmic relocalization. Thus microtubule organization, but not microfilament organization, is required for ooplasmic reorganization and differentiation without cleavage.     

Protein synthesis in mouse embryos with experimentally produced asynchrony between chromosome replication and cell division

Summary The cleavage of fertilized mouse eggs was prevented during cytochalasin B incubation and consequently these eggs became tetraploid the following day during in vitro culture. When the eggs were cultured further in normal medium, they cleaved and gave rise to tetraploid blastocysts. Protein synthesis was analysed in these embryos at different developmental stages using two-dimensional polyacrylamide gel electrophoresis. The protein synthesis pattern of one-cell tetraploid eggs was intermediate between those of normal one- and two-cell embryos. Tetraploid two-cell embryos expressed protein sets equivalent to those of untreated four-cell embryos, and tetraploid four-cell embryos synthesized proteins similar to those of four- to eight-cell controls. At subsequent pre-implantation stages the asynchrony was no longer detectable. When fertilized eggs were cultured continuously in the presence of cytochalasin B, they became tetraploid, octoploid and more and more polyploid without cleavage occurring. The protein synthesis patterns expressed by these one-cell polyploid eggs did not resemble that of normal fertilized eggs, but were similar to those of cleaving control embryos and blastocysts of equivalent age and nuclear division. These results strongly suggest that in early mouse embryos stage-specific translation is temporally correlated with chromosome replication (karyokinesis) and independent of cell division (cytokinesis) or cell interaction.Some of these results were presented at the IX Congress of the International Society of Developmental Biologists in Basle, Switzerland, August 28–September 1, 1981     

Absence of furrowing activity following regional cortical tension reduction in sand dollar blastomere and fertilized egg fragment surfaces

The purpose of the present investigation was to test experimentally the possibility that division mechanism establishment at the equator of sand dollar eggs may be a consequence of cortical tension gradients between the equator and the poles. Cytochalasin has been shown to decrease tension at the sea urchin egg surface. The concave ends of cytochalasin D-containing agarose cylinders were held against regions of the surface of Echinarachnius parma blastomeres and enucleated fertilized egg fragments. The ability to interfere with normal furrowing activity was used as a biological indicator of the effectiveness of cytochalasin. When agarose containing 2 microg/mL cytochalasin contacted the equatorial region of the blastomeres resulting from the first cleavage, or the equatorial surfaces of nucleated fertilized egg halves, furrowing was blocked, stalled or delayed, indicating that the concentration of cytochalasin was effective. When the same concentration of cytochalasin was applied to the poles, the cells and nucleated fertilized egg fragments divided in the same way as the controls, indicating that the effectiveness of the cytochalasin did not spread from the poles to the equator and that bisection did not interfere with the division of nucleated fertilized egg fragments. When the same concentration of cytochalasin was applied to diametrically opposed surfaces of enucleated, spherical egg fragments, there was no evidence of furrowing activity between the areas that contacted the cytochalasin or in any other part of the surface. Because of the tension-reducing effect of cytochalasin, a tension gradient existed between the regions affected and unaffected by cytochalasin. The results strongly suggest that establishment of the division mechanism by simple gradients of tension at the surface is unlikely.     

Protein synthesis in microsurgically produced androgenetic and gynogenetic mouse embryos

Summary In order to compare paternal and maternal gene activity at the protein synthesis level during early development, androgenetic and gynogenetic mouse embryos were experimentally produced by microsurgically removing either the female or the male pronucleus from fertilized mouse eggs. The resulting haploid eggs were diploidized in a medium containing cytochalasin B and then cultured under normal conditions to the blastocyst stage. Protein synthesis was analyzed at different stages of preimplantation development using 2-dimensional polyacrylamide gel electrophoresis. Both types of uniparental embryos synthesized a similar set of proteins independent of whether the paternal or the maternal genome was present. The isodiploid embryos expressed a protein pattern that corresponded remarkably to normal embryos at the subsequent cleavage stage. This temporal change is probably due to the fact that the operated haploid eggs were kept overnight in cytochalasin B in order to allow chromosomal replication to occur without cell division, and the resulting eggs therefore corresponded to normal 2-cell embryos with respect to karyokinesis but differed as far as cytokinesis was concerned. Several 2-cell specific proteins appeared in these isodiploid eggs and, similarly, following their first cleavage some 4-cell specific proteins were detected in 2-cell androgenetic and gynogenetic embryos. The discordance between nuclear and cellular division, which was retained through the 4-cell stage, however disappeared during subsequent cleavage divisions. At the blastocyst stage, both kinds of uniparental embryos showed a similar protein pattern compared to normal embryos. Our data suggest that some stage-specific proteins are synthesized during preimplantation development and correspond to nuclear rather than cellular divisions.Some of these results were presented at the 13th Annual Meeting of the Union of Swiss Societies of Experimental Biology in Lausanne, March 1981 (Petzoldt et al. 1981)     

Regulation of stage-specific gene expression during early mouse development: effect of cytochalasin B and aphidicolin on stage-specific protein synthesis in mouse eggs

Mechanisms regulating stage-specific translation in mouse embryos were studied by inhibitor experiments. When fertilized eggs were continuously treated with cytochalasin B, cleavage was prevented, whereas karyokinesis proceeded, resulting in protein synthesis patterns changing stage-specifically as in control embryos through preimplantation development. When fertilized eggs were continuously exposed to aphidicolin, cleavage and DNA synthesis were inhibited, thus keeping their protein synthesis at the level of fertilized eggs with few new polypeptides appearing after one day. The next day these eggs stopped translation almost completely. Stage-specific translation therefore might be controlled by nuclear replications rather than by cytoplasmic clock.     

Cytoskeletal mechanisms of ooplasmic segregation in annelid eggs

Annelid embryos are comprised of yolk-deficient animal and yolk-filled vegetal blastomeres. This "unipolar" organization along the animal-vegetal axis (in terms of ooplasmic distribution) is generated via selective segregation of yolk-free, clear cytoplasm to the animal blastomeres. The pathway that leads to the unipolar organization is different between polychaetes and clitellates (i.e., oligochaetes and hirudinidans). In polychaetes, the clear cytoplasm domain, which is established through ooplasmic segregation at the animal side of the egg, is simply cut up by unequal equatorial cleavage. In clitellates, localization of clear cytoplasm to animal blastomeres is preceded by unification of the initially separated polar domains of clear cytoplasm, which result from bipolar ooplasmic segregation. In this article, I have reviewed recent studies on cytoskeletal mechanisms for ooplasmic localization during early annelid development. Annelid eggs accomplish ooplasmic rearrangements through various combinations of three cytoskeletal mechanisms, which are mediated by actin microfilaments, microtubules and mitotic asters, respectively. One of the unique features of annelid eggs isthat a homologous process is driven by distinct cytoskeletal elements. Annelid eggs may provide an intriguing system to investigate not only mechanical aspects of ooplasmic segregation but also evolutionary divergence of cytoskeletal mechanisms that operate in a homologous process.     

Dynamics of the actin microfilament system in the Tubifex egg during ooplasmic segregation

Following the second polar body formation (PBF), the Tubifex egg undergoes ooplasmic segregation consisting of two steps, i.e., centrifugal migration of membranous organelles forming a subcortical ooplasmic layer and then movements of these organelles along the egg surface. The present investigation was undertaken to examine the microfilament organization in eggs during these ooplasmic rearrangements. Microfilaments throughout the egg are identified as actin by their reversible heavy meromyosin binding. Before the second PBF, a distinct network of actin filaments is present in the endoplasmic region. It is disorganized during the second PBF; short actin filaments are caused to aggregate with membranous organelles. Following the second PBF, similar short filaments become localized in the subcortical layer but not in the underlying yolky region. However, it is not until 50-60 min after the second PBF that an elaborate actin network is established in the subcortical layer. The cortex contains a sheet-like lattice of actin filaments. It is thickest around the animal pole, and tapes toward the equator of the egg. At about 90 min after the second PBF, this polarized distribution of cortical filaments becomes more pronounced as the result of their movements. Chronologically, subcortical actin network formation and cortical reorganization correspond to the later portion of the first step and the earlier portion of the second step of ooplasmic segregation, respectively. These findings are discussed in terms of ooplasmic movements and rearrangements.     

Effect of cell-surface binding on development of Ascidian egg

Summary The early development ofPhallusia mammillata eggs, dechorionated with trypsin and treated with Concanavalin A, was studied. Vital staining with a very dilute solution of acridine orange (0.01 g/ml) helped to visualize the mitochondrial crescent by fluorescence. At high concentrations of Concanavalin A (20–200 g/ml) fertilized eggs did not cleave, but went through early ooplasmic segregation movements (formation of the crescent) and multinuclear syncytia were formed. At lower concentrations of Concanavalin A (less than 10 g/ml), cleavage occurred, but the blastomeres remained rounded, leading to a grapelike embryo. Eggs attached to Concanavalin A treated nylon surfaces either did not cleave or produced grapelike embryos. Attachment of the eggs did not affect ooplasmic segregation. Considering modern theories of membrane structure it was concluded that Concanavalin A prevented cleavage either by immobilizing surface structures connected with microfilaments or by indirectly modifying other membrane structures. These structures could not have been involved in ooplasmic segregation, but their mobility was necessary for further morphogenesis.This work was performed at the Station Zoologique, Villefranche-sur-Mer and at the Station de Biologie Marine et Lagunaire. Sète     

Ultraviolet irradiation of eggs and blastomere isolation experiments suggest that gastrulation in the direct developing ascidian, Molgula pacifica, requires localized cytoplasmic determinants in the egg and cell signaling beginning at the two-cell stage

Gastrulation in the maximum direct developing ascidian Molgula pacifica is highly modified compared with commonly studied "model" ascidians in that endoderm cells situated in the vegetal pole region do not undergo typical invagination and due to the absence of a typical blastopore the involution of mesoderm cells is highly modified. At the gastrula stage, embryos are comprised of a central cluster of large yolky cells that are surrounded by a single layer of ectoderm cells in which there is only a slight indication of an inward movement of cells at the vegetal pole. As a consequence, these embryos do not form an archenteron. In the present study, ultraviolet (UV) irradiation of fertilized eggs tested the possibility that cortical cytoplasmic factors are required for gastrulation, and blastomere isolation experiments tested the possibility that cell signaling beginning at the two-cell stage may be required for the development of the gastrula. Irradiation of unoriented fertilized eggs with UV light resulted in late cleavage stage embryos that failed to undergo gastrulation. When blastomeres were isolated from two-cell embryos, they developed into late cleavage stage embryos; however, they did not undergo gastrulation and subsequently develop into juveniles. These results suggest that cytoplasmic factors required for gastrulation are localized in the egg cortex, but in contrast to previously studied indirect developers, these factors are not exclusively localized in the vegetal pole region at the first stage of ooplasmic segregation. Furthermore, the inability of embryos derived from blastomeres isolated at the two-cell stage to undergo gastrulation and develop into juveniles suggests that important cell signaling begins as early as the two-cell stage in M. pacifica. These results are discussed in terms of the evolution of maximum direct development in ascidians.     

The myoplasm of ascidian eggs: a localized cytoskeletal domain with multiple roles in embryonic development

The myoplasm of ascidian eggs is a localized cytoplasmic region containing a unique cytoskeletal domain. During ooplasmic segregation, the myoplasm moves first to the vegetal pole and then to the future posterior region of the fertilized egg, where it subsequently enters the muscle cell lineage during cleavage. In the vegetal pole region, the myoplasm defines a developmental center which later controls gastrulation and embryonic axis formation. In the posterior region, the myoplasm defines another developmental center, which specifies muscle cell development. Evidence is described suggesting that the integrity of the myoplasmic cytoskeletal domain is required for normal embryonic functions of the myoplasm.     

Artificial insemination of the American lobster (Homarus)

A technique has been developed for artificially inseminating freshly molted female lobsters (Homarus). Using the criterion that eggs were fertilized if they showed development of normal cleavage patterns, at least 49.5% of the inseminated females extruded fertilized eggs. Sixty-two percent of the fertilized eggs developed to the eyespot stage, and 26.6% of these subsequently hatched. Eggs were not fertilized in 7.7% of the extrusions. In 42% of the samples, it was not possible to establish whether fertilization had occurred because either samples were collected prior to initiation of cleavage or eggs were too poorly preserved to assess cleavage with confidence. It is probable that many of the eggs that were evaluated as equivocal were also fertilized. This technique should be valuable in the experimental and commercial aquaculture of lobsters and might be directly applicable to other crustaceans.     

Incidence of chromosome anomalies in first-cleavage mouse embryos obtained from frozen-thawed oocytes fertilized in vitro

To assess the effect of low temperature storage on mouse oocytes we (1) examined the capacity for normal development of embryos derived from frozen oocytes fertilized in vitro after transfer to pseudopregnant foster mothers and (2) analyzed the chromosome complement at the first cleavage division. Fewer frozen than control oocytes were fertilized (36% vs 66%), but after embryo transfer the proportion of fertilized eggs that implanted (67–68%) and formed normal foetuses (50–53%) was similar in the two groups. Freezing did not affect the observed incidence of aneuploidy (1.5–3.3%). The frequency of polyploid embryos derived from frozen oocytes was almost double that of controls (15.8% vs 8.5%), but it is unclear whether this is a real effect of freezing or is an artifact produced by the chromosome preparation technique.     

Germinal vesicle components are not required for the cell-cycle oscillator of the early starfish embryo

We show that certain events of the cell cycle can still occur in starfish oocytes or fertilized eggs from which the germinal vesicle (the prominent nucleus of prophase-arrested oocytes) has been removed before the induction of meiotic maturation. Two meiotic asters develop following hormonal induction of meiotic maturation in these enucleated oocytes. The asters then divide to form a transient tetrapolar figure. When enucleated oocytes are fertilized, the sperm centrosome duplicates at the times corresponding to each cleavage in control nucleated embryos. Periodic changes in the organization of the asters and in the morphology of the cell surface also occur in synchrony with controls. Decondensation of the sperm nucleus, spindle formation, and cleavage do not occur when enucleated oocytes are fertilized. Ultimately the number of asters increases to approximately 520 (about 2(9] before the pseudo-embryo arrests and cytolyzes. Fertilized eggs from which both pronuclei but not the sperm aster have been removed undergo nine cleavages and then cease cell division. The cessation of division may be related to the events that cause the midblastula transition after seven cleavages in normal nucleated embryos.     

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.     

Isolation of cDNA Clones for Epidermis-Specific Genes of the Ascidian Embryo

The present investigation was conducted to isolate cDNA clones that correspond to epidermis-specific genes of the ascidian embryo. When cleavage of fertilized eggs of Halocynthia roretzi is blocked by treatment with cytochalasin B and the arrested eggs are reared as one-celled embryos for about 30 hr, they develop features of differentiation of the epidermis only. Translation in vitro of poly(A)⁺ RNA from cleavage-arrested embryos and analysis of the products by two-dimensional gel electrophoresis revealed several predominant polypeptides that were not detected in a similar analysis of fertilized eggs, suggesting the appearance of epidermis-specific mRNAs in cleavage-arrested embryos. A cDNA library was constructed from arrested one-celled embryos. Differential screening of the library with a total cDNA probe from cleavage-arrested embryos and with a similar probe from fertilized eggs yielded eight different cDNA clones specific for the cleavage-arrested embryos. Northern blot analysis revealed that the mRNAs that corresponded to these cDNAs were present in normal tailbud embryos. In addition, in situ hybridization of whole-mount specimens showed that the mRNAs were restricted to the epidermal cells of tailbud embryos.     

ELECTROPHORETIC MOBILITY OF SEA-URCHIN EGGS DURING THE DIVISION CYCLE

The electrokinetic potential of fertilized sea-urchin eggs, without the fertilization membrane and hyaline layer, was investigated by measuring the electrophoretic mobility of the eggs from fertilization to the second cleavage. A cyclic change in mobility was found to accompany the division cycle: the peak of the change was observed about 15 min before the appearance of both the first and second cleavage furrows.
A smaller peak was observed at 20–30 min after fertilization, but such a peak was not repeated between the first and the second cleavage.
Fertilized eggs with the fertilization membrane intact did not show a significant change in electrophoretic mobility throughout the division cycle.     

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

We have studied egg activation and ooplasmic segregation in the ascidian Phallusia mammillata using an imaging system that let us simultaneously monitor egg morphology and calcium-dependent aequorin luminescence. After insemination, a wave of highly elevated free calcium crosses the egg with a peak velocity of 8-9 microns/s. A similar wave is seen in egg fertilized in the absence of external calcium. Artificial activation via incubation with WGA also results in a calcium wave, albeit with different temporal and spatial characteristics than in sperm-activated eggs. In eggs in which movement of the sperm nucleus after entry is blocked with cytochalasin D, the sperm aster is formed at the site where the calcium wave had previously started. This indicates that the calcium wave starts where the sperm enters. In 70% of the eggs, the calcium wave starts in the animal hemisphere, which confirms previous observations that there is a preference for sperm to enter this part of the egg (Speksnijder, J. E., L. F. Jaffe, and C. Sardet. 1989. Dev. Biol. 133:180-184). About 30-40 s after the calcium wave starts, a slower (1.4 microns/s) wave of cortical contraction starts near the animal pole. It carries the subcortical cytoplasm to a contraction pole, which forms away from the side of sperm entry and up to 50 degrees away from the vegetal pole. We propose that the point of sperm entry may affect the direction of ooplasmic segregation by causing it to tilt away from the vegetal pole, presumably via some action of the calcium wave.     

Studies on differentiation without cleavage in Chaetopterus: effects of inhibition of DNA synthesis with aphidicolin

The effects of aphidicolin - a powerful inhibitor of DNA polymerase alpha and of DNA replication - on normal development and on differentiation without cleavage of Chaetopterus eggs have been studied with cytological, cytochemical, and biochemical methods. The experiments show that the initial period of pseudocleavage can take place in the absence of nuclear DNA synthesis, but further development (segregation, hatching, ciliation) requires DNA synthesis. However ciliated unicellular larvae can be obtained under conditions where the DNA content of the embryos in only 40% of the controls. In fertilized eggs, aphidicolin immediately stops cleavage. The significance of these results is discussed.