Degradation of sperm histones in vitro by cytoplasm of the sea urchin egg

Sperm-specific histone variants in the sea urchin Paracentrotus lividus are replaced early after fertilization with a specific embryonic set of histone variants. A possible in vitro model for the involvement of a degradation mechanism in the replacement of sperm-specific histones is presented. Soluble sperm histones are shown to be degraded quickly by egg cytoplasm. The proteolytic activity is maximal at pH 3.0; H1 and H2A histones are the most sensitive while H3 and H4 are the most resistant. H2B histones have an intermediate sensitivity. Histone degradation by egg cytoplasm or by purified fractions of it can be inhibited by chymostatin and leupeptin and, to a lesser degree, by pepstatin.     

Introduction of cloned DNA into sea urchin egg cytoplasm: replication and persistence during embryogenesis

Cloned DNA sequences were introduced into the cytoplasm of unfertilized sea urchin eggs by a simple microinjection technique. Sperm was then added, and development allowed to proceed. If linearized plasmids are injected they form random concatenates, and during the early development of the embryos replicate repeatedly. Eukaryotic sequences are not required for replication of the exogenous DNA. Injected supercoiled DNAs neither ligate nor replicate. Both forms of exogenous DNA persist in the embryo through pluteus stage.     

State of actin during the cycle of cohesiveness of the cytoplasm in parthenogenetically activated sea urchin egg

By the DNase I inhibition assay it is shown that the cytoplasmic matrix isolated 60 min after procaine activation of Paracentrotus lividus eggs contains about 20% of the total egg actin, mostly in polymerized form (85%). Electron microscopy studies on this cytoplasmic structure after treatment with heavy meromyosin (HMM), reveal that the decorated actin filaments are organized in bundles which are distributed radially, with the arrowheads pointing towards the central region. In addition few microtubules and a network of non-decorated microfilaments of about 3 nm diameter are observed. From the cytoplasmic pH determination and the DNase I inhibition assay on homogenates of eggs which were taken at different times of activation, it cannot be inferred that a direct relationship between the increase in the cytoplasmic pH and the increase in the amount of polymerized actin or of cytoplasmic matrix exists. Activation experiments carried out in the presence of colchicine shows that, although the formation of the cytoplasmic matrix is inhibited, polymerization of actin still occurs. Moreover, from the inhibition effects of cytochalasin B (CB) added before the activator it is shown that polymerization of actin is a necessary step for the organization of the cytoplasmic matrix. However, the cycles of cohesiveness of the cytoplasm observed in the course of the activation process do not appear to depend on cycles of polymerization and depolymerization of actin.     

Cytoskeleton of the unfertilized sea urchin egg

Unfertilized Paracentrotus lividus egg cytoskeleton is prepared by mild, nonionic detergent extraction at 4 degrees C in buffer systems containing either 2-methyl-2,4-pentanediol (hexylene glycol) or glycerol. These extractions allow the isolation of cytomatrices that maintain the egg form and are 70-80 micron in diameter. DNase inhibition assays show that actin is in polymerized form in these cytomatrices. Ultrastructural observations reveal that the cytoskeletons are made up essentially of 2 categories of filaments, 7-8-nm and 2-4-nm in diameter, respectively. After heavy meromyosin labelling, short, radiating actin filaments are seen in the cortical region, while longer actin filaments are found in the internal region of these cytomatrices. The 2-4-nm filaments of still unknown biochemical nature are organized in a meshwork. In contrast to results found with fertilized eggs, bundles of actin filaments and microtubules are absent; 8-13-nm filaments are not detected.     

Substructure of sea urchin egg cytoplasmic dynein

The substructure of the cytoplasmic dynein molecule was studied using the quick-freeze, deep-etch technique. Cytoplasmic dynein purified as a 12 S form from the eggs of the sea urchin Hemicentrotus pulcherrimus was composed of a single high molecular weight polypeptide. Rotary shadowing images of cytoplasmic dynein either sprayed on to a mica surface or quick-frozen on mica flakes demonstrated a single-headed molecule, in contrast to the two-headed molecule of sea urchin sperm flagellar 21 S dynein. More detailed substructure was visualized by rotary shadowing after quick-freeze deep-etching. Cytoplasmic dynein consisted of a head and a stem. The head was pear-shaped (16 nm X 11 nm) and a little smaller than the pear-shaped head of 21 S dynein (18 nm X 14 nm). The form of the stem was irregular, and its apparent length varied from 0 to 32 nm. Binding of cytoplasmic dynein to brain microtubule in the solution was observed by negative staining, and that in the precipitate was examined by the quick-freeze, deep-etch method as well. Both methods revealed the presence of two kinds of microtubules, one a fully decorated microtubule and the other a non-decorated microtubule. Cytoplasmic dynein bound to microtubule also appeared as a globular particle. Neither the periodic binding nor the crossbridges that were observed with 21 S dynein were formed by cytoplasmic dynein, although cytoplasmic dynein appeared to bind to microtubules co-operatively.     

Calcium in sea urchin egg during fertilization

Calcium plays a strikingly important role in two of the major events in developmental biology: cell activation and differentiation. In this review we begin with the location and quantity of intracellular calcium in sea urchin oocytes, and then discuss the changes that occur during fertilization and egg activation, placing special emphasis on the mobilization and redistribution of intracellular calcium. We also discuss the propagation of the calcium wave and the role of the burst of calcium on the process of reorganizing the egg cortex at fertilization.     

Ionic strength-dependent isoforms of sea urchin egg dynein

Unfertilized sea urchin eggs provide a reservoir of molecules which later are involved in microtubule-mediated movements during embryonic development. Among these molecules is egg dynein, which has been isolated in two forms, 20 S and 12 S. Evidence obtained previously from our laboratory indicates that 20 S dynein is a latent activity precursor of ciliary dynein. In contrast, others have suggested that 12 S egg dynein functions in the mitotic apparatus. It is therefore important to determine the relationship between these egg dyneins. Here we demonstrate that the sedimentation velocity of the egg dynein is dependent on the ionic strength of the extraction conditions. The 20 S dynein is obtained with low ionic strength extraction, and the 12 S form is obtained in high salt (0.6 M KCl). The 20 S dynein, after collection from a sucrose gradient, can be converted quantitatively to the 12 S form by exposure to salt, and this conversion can be followed over time. Further, the 20 S dynein can be converted entirely to 12 S dynein and then partially reconstituted to a faster sedimenting species. During these conversions, the dynein high Mr heavy chains are always coincident with the MgATPase activity, and antibodies show that the dynein heavy chains of the 20 S, 12 S, and converted species are indistinguishable immunologically. These data suggest that 12 S dynein is an ionic strength-dependent isoform of 20 S dynein that results from a partial dissociation of the 20 S polypeptide complex, similar to the relationship between 12 and 21 S sperm flagellar dynein. If the 20 and 12 S enzymes are isoforms of the same dynein, then there is compelling evidence for only a single dynein in the unfertilized egg, and that dynein is probably a ciliary precursor.     

Membrane potential of the unfertilized sea urchin egg

The membrane potential, specific resistance, and potassium selectivity of the unfertilized Strongylocentrotus purpuratus egg were determined by two independent methods: tracer flux and microelectrode. The potassium influx was 0.50 ± 0.2 pmole/cm²· sec, which was greater than the sodium, chloride, and calcium influxes by factors of 4, 7, and 75, respectively. By means of the constant-field equations, the flux data were used to calculate membrane potential (?70 mV) and specific resistance (420 kΩ · cm²). The effect of the external potassium concentration on the sodium influx was determined and the results closely fit the result expected if the membrane behaved as a potassium electrode. Microelectrode measurements of the potential and resistance were ?75 ± 3 mV and 380 ± kΩ · cm².     

Unusual properties of sea urchin unfertilized egg chromatin

A typical nucleosomal pattern is not detected by electrophoretic analysis of sea urchin mature egg chromatin, following digestion with micrococcal nuclease. Moreover, at least 80 % of the egg nuclear DNA is resistant to nuclease attack. These unusual features of unfertilized egg chromatin, not shared by oocytes or sperms, are discussed in view of the unique properties and fate of mature female germ cells.     

Proteins of the sea urchin egg vitelline layer

The vitelline layers (VL) of unfertilized sea urchin eggs were isolated, and the diversity of their polypeptide constitutents estimated by two-dimensional polyacrylamide gel electrophoresis. At least 25 components are reproducibly observed. While VL polypeptides are almost certainly synthesized in the growing oocyte, they are not among the more prevalent newly synthesized proteins detected in oocytes that were isolated and labeled in vitro for 4 hr. A set of monoclonal antibodies was raised against VL components and partially characterized. The 31 monoclonals analyzed fell into 11 classes with respect to their avidity for VL proteins solubilized under mild and under strongly denaturing conditions, and to their reactions with surface components of the VLs of living eggs. Fluorescence microscopy showed diverse patterns of surface reactivity when different monoclonal antibodies were compared. Two of the monoclonal antibodies reacted with specific sets of three proteins each on VL protein blots. It is concluded that the VL is a complex structure containing a large number of different polypeptide components, the genes for several of which should now be experimentally accessible.     

Immunological purification of sea urchin egg tropomyosin.

The antiserum against lantern muscle tropomyosin of the sea urchin was prepared, and the presence of tropomyosin in the sea urchin egg was shown by immunodiffusion test between the antiserum and the egg tropomyosin fraction which was prepared according to the purification method for muscle tropomyosin. The sea urchin egg tropomyosin was isolated from the immuno-precipitate formed between the antiserum and the egg tropomyosin fraction. The subunit molecular weight of the egg tropomyosin was calculated to be 29,000.     

Characterization of the microtubule movement produced by sea urchin egg kinesin

We have used an in vitro assay to characterize some of the motile properties of sea urchin egg kinesin. Egg kinesin is purified via 5'-adenylyl imidodiphosphate-induced binding to taxol-assembled microtubules, extraction from the microtubules in ATP, and gel filtration chromatography (Scholey, J. M., Porter, M. E., Grissom, P. M., and McIntosh, J. R. (1985) Nature 318, 483-486). This partially purified kinesin is then adsorbed to a glass coverslip, mixed with microtubules and ATP, and viewed by video-enhanced differential interference contrast microscopy. The microtubule translocating activity of the purified egg kinesin is qualitatively similar to the analogous activity observed in crude extracts of sea urchin eggs and resembles the activity of neuronal kinesin with respect to both the maximal rate (greater than 0.5 micron/s) and the direction of movement. Axonemes glide on a kinesin-coated coverslip toward their minus ends, and kinesin-coated beads translocate toward the plus ends of centrosome microtubules. Sea urchin egg kinesin is inhibited by high concentrations of SH reagents ([N-ethylmaleimide] greater than 3-5 mM), vanadate greater than 50 microM, and [nonhydrolyzable nucleotides] greater than or equal to [MgATP]. The nucleotide requirement of sea urchin egg kinesin is fairly broad (ATP greater than GTP greater than ITP), and the rate of microtubule movement increases in a saturable fashion with the [ATP]. We conclude that the motile activity of egg kinesin is indistinguishable from that of neuronal kinesin. We propose that egg kinesin may be associated with microtubule-based motility in vivo.