Fertilized Sea Urchin Eggs Research Articles

Involvement of glutathione in the inhibition of sea urchin egg mitosis by phenyl glyoxal.

Cell division in fertilized sea urchin eggs was reversibly inhibited when the ketoaldehyde phenyl glyoxal (PG) at a concentration of 0.1 mM was added to eggs for ten minutes prior to the formation of the mitotic spindle. We investigated whether inhibition of mitosis was due to PG binding to the cell surface (as previously suggested by Stein and Berestecky, '74) or to some intracellular effect. When 14C-PG was added to eggs, label was readily taken up into the egg cytoplasm; very little label was associated with the egg surface. In the cytoplasm PG combined with equimolar amounts of reduced glutathione (GSH), decreasing the levels of cellular GSH to less than 15% of normal and accounting for at least 50% of the PG taken up by eggs. The concentrations of oxidized and protein-bound glutathione were unaffected by PG treatment. We showed that glyoxalase enzymes were present in sea urchin eggs and were capable of metabolizing the PG-GSH complex, thereby restoring GSH to normal levels after PG was removed from the sea water. Though some other effect of PG cannot be ruled out, the major fate of PG in eggs was to combine with GSH, and the transient decrease in GSH which resulted could lead to inhibition of mitosis. While other reports (Nath and Rebhun, '76; Oliver et al., '76) have shown that reagents which oxidize GSH disrupt microtubule-related events, our results showed that such inhibition could be caused by decreased GSH levels alone.

Early electrical responses to fertilization in sea urchin eggs

The depolarizing component of the activation potential consists of an early phase having a constant duration at room temperature, and a late phase displaying evident overshoot. The early phase is built up by one to several depolarizing steps, each of them being due to single interactions between spermatozoa and egg. The number of steps in the early phase is related to the sperm concentration. The first step in the sequence may be regarded as the trigger of egg activation. The late phase of the activation potential is related to the cortical reaction, and therefore to the complete block of polyspermy. This phase is absent both in oocytes at the germinal vesicle stage, which are naturally susceptible to polyspermic fertilization lacking cortical granules, and in mature oocytes made polyspermic by nicotine treatment.

Metabolic similarities between fertilization and phagocytosis. Conservation of a peroxidatic mechanism.

At the time of fertilization, sea urchin eggs release a peroxidase which, together with H2O2 generated by a respiratory burst, is responsible for hardening of the fertilization membrane. We demonstrate here that the ovoperoxidase of unfertilized eggs is located in cortical granules and, after fertilization, is concentrated in the fertilization membrane. Fertilization of sea urchin eggs or their parthenogenetic activation with the ionophor A23187 also results in (a) the conversion of iodide to a trichloroacetic acid-precipitable form (iodination), (b) the deiodination of eggs exogenously labeled with myeloperoxidase and H2O2, (c) the degradation of thyroxine as measured by the recovery of the released radioiodine at the origin and in the inorganic iodide spot on paper chromatography, and (d) the conversion of estradiol to an alcohol-precipitable form (estrogen binding). The iodination reaction and the binding of estradio occurs predominantly in the fertilization membrane where the ovoperoxidase is concentrated. From the estimation of the kinetics of incorporation of iodine, we determine that the peroxidative system is active for 30 min after fertilization, long after hardening of the fertilization membrane is complete. Most of the bound iodine is lost during the hatching process. Iodination of albumin is catalyzed by the material released from the egg during fertilization, when combined with H2O2 and iodide. Iodination, thyroxine degradation, and estradiol binding are inhibited by azide, cyanide, aminotriazole, methimazole, ascorbic acid and ergothioneine, all of which can inhibit peroxidase-catalyzed reactions. These responses of the sea urchin egg to fertilization are strikingly similar to the changes induced in polymorphonuclear leukocytes by phagocytosis and, in both instances, a peroxidative mechanism may be involved.

Structure of actin filament bundles from microvilli of sea urchin eggs

The detailed substructure of actin filament bundles in microvilli of fertilized sea urchin eggs has been studied by analysing electron microscope images of negatively stained specimens. Transverse stripes which repeat about every 130 Å along the axis of a bundle, as previously observed by Burgess & Schroeder (1977), reflect the positions of cross-bridges that connect the filaments into a bundle. Analysis of optical transforms of the micrographs reveals that there are approximately 14 actin monomers between cross-overs of the two long-pitch helical strands of the actin filaments, with three cross-bridges in this interval. The structure is basically similar to that of the hexagonally packed bundles prepared in vitro from high speed supernatants of sea urchin eggs by Kane (1975) and analyzed by DeRosier et al. (1977). One clear difference, however, is that the in vivo microvillar filament bundles are supercoiled, giving rise to long axial repeats of 1500 to 2000 Å. Computationally filtered images of regions that were only slightly supercoiled reveal the relative alignment of filaments within the bundles and show that crossbridges appear to interact with four actin monomers, apparently linking two actin monomers on one strand of one filament to the nearest two monomers on a neighbouring filament. However, the cross-bridges are not spaced at equal intervals corresponding to four actin subunits, presumably because of the lack of hexagonal symmetry in the individual filaments, which have about 14 actin monomers between cross-overs. Instead, the cross-bridges are arranged quasiequivalently along the longitudinal axis of the bundles, in steps of four or five actin subunit spacings (28 Å each).

A spiral cortical fiber system in fertilized sea urchin eggs

Fiber systems of fertilized eggs of the sea urchin Strongylocentrotus purpuratus become aggregated and thus visible in phase-contrast light microscopy, when cells are fixed in 2% glutaraldehyde in 0.45 M Na-acetate buffer at pH 6.0 and embedded in epoxy. Studies of whole mounts and of 1-μm stained sections of the first-division cycle revealed a spiral array of subcortical fibers that apparently grow inward from the cell surface shortly after sperm entry and disappear prior to streak stage. They are independent of the microtubule system associated with the sperm aster, amphiaster, and mitotic apparatus. Their chemical identity is not known, but they may very likely be actin.

Inhibitory effect of α-hydrazinoornithine on egg cleavage in sea urchin eggs

In fertilized sea urchin eggs which are kept in sea water containing α-hydrazinoornithine (αHO) at a concentration above 1 m M from the time of fertilization, cleavage is delayed markedly. The third cleavage is almost completely blocked by 3 m M αHO. Hydrazine, as well as ornithine, exerts no harmful effect on egg cleavage. αHO causes competitive inhibition of ornithine decarboxylase (ODC) in the egg homogenate. Polyamine levels decrease in fertilized eggs treated with αHO. The addition of ornithine (above 3 m M) to an egg culture containing αHO prevents the αHO-induced delay of cleavage. Putrescine (0.2–0.5 m M), which is the product of the reaction catalyzed by ODC, also relieves egg cleavage from the inhibited state. The same effect occurs in the presence of spermidine (0.2–0.5 m M) or spermine (0.1–0.8 m M). Especially, spermine (0.5 m M) completely cancels the inhibitory effect of αHO on egg cleavage. Egg cleavage is delayed only slightly in the presence of each polyamine (above 2 m M).

Prevention of the cortical reaction in fertilized sea urchin eggs by injection of calcium-chelating ligands

Eggs from the sea urchin, Lytechinus pictus, were injected with either EGTA or EDTA, and were subsequently fertilized. EGTA prevented cortical vesicle discharge and formation of the fertilization membrane. EDTA had either no effect, or sometimes retarded the elevation of the fertilization membrane, or reduced the percentage of eggs with elevated membranes. Theoretical considerations lead to estimates of the probable effects of EGTA and EDTA on the internally released calcium which triggers the cortical reaction. Whether or not cytoplasmic calcium buffers are considered, it is concluded: (1) that normally several times the threshold calcium concentration for the cortical reaction is released into a subsurface space; (2) that if a rapidly-equilibrating high-affinity buffer is present, it is locally saturated by the calcium released internally; (3) the injected EDTA reduces the subsurface free calcium concentration normally reached to approximately threshold for the cortical reaction, while injected EGTA reduces the calcium concentration to below this threshold; and (4) a rise in the internal ionic calcium concentration is a necessary step in the activation of the cortical reaction at fertilization.

Nuclear protein changes in the maternally and paternally derived chromatin at fertilization.

The proteins which become associated with the paternally derived chromatin during fertilization may be instrumental in its activation and in the dramatic structural metamorphosis of the sperm nucleus during pronuclear development. Proteins associated with sperm and zygote nuclei and male and female pronuclei of fertilized sea urchin eggs were analysed by polyacrylamide gel electrophoresis in order to examine nuclear protein changes in the paternally and maternally derived chromatin following insemination. Results demonstrate major changes in both the solubility characteristics and polypeptide profiles of sperm nuclei upon insemination. Evidence is presented which indicates that at fertilization the paternally derived chromatin acquires proteins of molecular weights greater than 80,000 and a nuclear protein composition similar to that of the female pronucleus. The nuclear proteins associated with zygote nuclei were compared to those of combined male and female pronuclei and showed many similarities and some differences. Several polypeptides were present in zygote nuclei which were not observed in pronuclear extracts.

Fine Structural Analysis of Animal Cell Surfaces: Membranes and Cell Surface Topography

Current research on the mechanism of transmembrane regulation of topographic modulation at the cell surface is described for the sea urchin egg and Sarcoma 180 ascites tumor cells of Swiss Webster white mice. The transmembrane system is characterized in terms of three components: glycocalyx, membrane , and cytofibrillar structures. The importance of membrane molecular architecture per se relative to the other surface components is assessed in terms of freeze fracture analysis of both cell types as well as concanavalin A (ConA)-mediated long term agglutination, cytochalasin B effects, and other drug-induced changes at Sarcoma 180 cell surfaces. A quantitative and qualitative assessment of intramembranous particle (IMP) sizes and density distributions reveals intrinsic structural changes of the fusing membranes at cortical reaction during sea urchin egg fertilization and also with the post-fertilized accumulation of surface microvilli. Comparable changes in IMP are noted for microvillus retraction and membrane smoothing in Sarcoma 180 cells under a variety of experimental conditions. On the other hand, chemical perturbation of S-180 cell surfaces reveals a rather non-ubiquitous, though identifiable, involvement of microfilaments and no microtubule involvement in these topographic changes. These observations suggest that the plasma membrane is a dynamic structure poised between “restrictive” and “less restrictive” states of fluidity or deformability and, hence, is a determinant component in topographic change.

Uptake of Ca2+ is one of the earliest responses to fertilization of sea urchin eggs.

Sea urchin eggs take up Ca for 10 min following insemination (determined as uptake of 45Ca from the SW (3)). Although 90% of this uptake occurs after the beginning of the cortical reaction and may represent external binding of Ca to the egg surface coats, there is a brief phase of uptake (o-30 sec) which precedes the cortical reaction; this may represent a Ca flux into the eggs.

Microvilli in sea urchin eggs: Differences in their formation and type

The elongation of microvilli, which normally occurs upon fertilization in sea urchin eggs, was also observed in unfertilized eggs treated with the enzyme, papain. Cortical granule exocytosis, which is thought to be the source of membrane used in microvillar elongation, does not occur in the papain-treated eggs. It appears, therefore, that there is more than one way in which the egg plasma membrane can increase very quickly and to a great extent. In addition, the kinds of microvilli formed in the two instances appear to be different. Previous work with reaggregating sea urchin cells is also cited to support the suggestion that microvilli can form in different ways and are of different types.

The problem of sea urchin egg fertilization and its implications for biological studies.

The analysis of sea urchin egg fertilization shows that several phenomena common to other biological systems are involved: cell recognition, cell fusion, exocytosis and initiation of mitotic activity. Both the role of calcium ions in cell fusion and exocytosis and the function of the cell surface in the initiation of mitotic activity appear to have general applicability. The study of fertilization can contribute to the elucidation of these processes and, reciprocally, progress in this field can help to advance our understanding of the mechanisms of fertilization in sea urchins and other organisms.

Synthesis of RNA by male pronuclei of fertilized sea urchin eggs.

Experiments have been conducted in an effort to determine when the paternally derived genome of sea urchin embryos becomes active in RNA synthesis. Participation of the paternal genome in RNA transcription as early as fertilization has been indicated by the following: (1) polyspermic zygotes consistently demonstrate greater levels of incorporation of labelled precursor (3H-uridine) into RNA when compared to monospermic eggs. (2) Monospermic and polyspermic eggs treated with actinomycin D show the same levels of incorporation of label into RNA. (3) Analysis of autoradiographs demonstrates the incorporation of 3H-uridine into male and female pronuclei.

Polarized bundles of actin filaments within microvilli of fertilized sea urchin eggs.

We report on the internal ultrastructure of long, finger-like microvilli which cover the surface of the fertilized sea urchin egg. Eggs were attached to polylysine-coated surfaces; their upper portions were sheared away with a stream of buffer which left behind only their plasma membranes and adjacent cytoplasmic structures. Scanning electron microscopy (EM) of such fragments revealed intact thin protoplasmic projections radiating away from the body of the cortex. By transmission EM of cortices similarly prepared on grids, small bundles of microfilaments appear as cores within the thin cytoplasmic projections. These microfilaments are shown to be composed of actin by their ability to interact with muscle heavy meromyosin (HMM). HMM-decorated microfilaments possess repeating arrowheads which uniformly point toward the cell interior. Actin bundles in the microvilli of sea urchin eggs may mediate microvillus support and elongation.

The dynamics of maternal poly(A)-containing mRNA in fertilized sea urchin eggs

Cytoplasmic polyadenylated RNA with the characteristics of sequestered mRNA exists in the unfertilized sea urchin egg. Following egg activation, the amount of poly(A) doubles, but total RNA content stays constant. Chromatography of the RNA on poly(U)-Sepharose shows that the amount of RNA that bears a poly(A) tract increases slightly (approximately 20–30%) during the 2 hr after fertilization. When a cDNA transcript of the poly(A) + mRNA from 2 hr zygotes is reacted against poly(A) + RNA from either eggs or zygotes, the kinetics of reassociation of the two preparations seem identical; hence the RNA sequences bearing poly(A) are the same in eggs and zygotes. Measurement of the length of the poly(A) tract in eggs and zygotes shows an increase in number average length from about 45 bases to 60 bases. Measurement of tract length of poly(A) in two cell zygotes by adenosine/AMP ratios of radioactive RNA shows that the poly(A) tract of the zygote is solely accounted for by radioactive RNA, indicating extensive turnover of the poly(A). It in concluded that the poly(A) tract in these cells is subject to both lengthening and shortening, with the former predominating in this instance. The increase in poly(A) does not involve polyadenylation of different sequences, but is due to an increase in the number of polyadenylated sequences and the length of the poly(A) tracts that they bear.

Complete turnover of poly(A) on maternal mRNA of sea urchin embryos

Measurement of the incorporation of radioactive adenosine into precursor pools and into poly(A) of fertilized sea urchin eggs showed that the amount of adenosine incoporated into poly(A) after a 2 hr incubation approximated the total poly(A) content of the embryos. This was observed whether the incubation was begun at fertilization when the poly(A) content is tripling or at 2.5 hr after fertilization when the poly(A) levels are not changing, and thus indicates that poly(A) turns over continually and completely. The turnover appears to take place on polysomal mRNA, since after either 10 or 120 min of incubation, 75% of the 3H-adenosine incorporated into poly(A) is on polysomes. Poly(A) lengths before and after fertilization are not significantly different, indicating that the increase in poly(A) content reflects the addition of poly(A) sequences onto mRNA molecules which previously contained no poly(A) sequences or only short poly(A) sequences. Both the new as well as the preexisting poly(A) tracts must turn over to produce the incorporation we observe. The radioactive poly(A) tracts measured by alkaline release of adenosine begin as short sequences and gradually extend their lengths until they have reached a size consistent with the idea that the poly(A) sequences have become fully radioactive. This labeling pattern shows that the poly(A) is turning over from the 3′ end terminal probably by a shortening and lengthening mechanism.

Insulin Receptor Sites as Membrane Markers during Embryonic Development: I. Data Obtained with Unfertilized and Fertilized Sea Urchin Eggs

Binding of insulin to sea urchin egg plasma membrane has been studied by biochemical and immunocytochemical methods. Unfertilized and fertilized eggs as well as embryos during the first cell division have been used. 1. Competition experiments between 125I-insulin (1 nM) and an excess of native insulin (30 muM) indicate a specific hormone fixation to membrane crude extracts from unfertilized and fertilized eggs. The magnitude of "specific binding" is comparable to values recorded for mammalian cells. 2. Inhibition of insulin fixation by concanavalin A (100 mug/ml) suggests the glycoprotein composition of plasma membrane receptors. 3. An 30-min incubation of unfertilized and fertilized eggs in the presence of insulin leads to a significant increase in cyclic AMP content. 4. An immunocytochemical method demonstrates that insulin is selectively and specifically bound to the plasma membrane of eggs incubated in the presence of insulin before fixation. It can be concluded that insulin receptor sites are components of sea urchin eggs plasma membrane. Insulin binding which leads to cyclic AMP accumulation is not deeply modified by fertilization and does not include visible morphological changes in the eggs.

Influence of different organochlorine pesticides on the development of the sea urchin embryo

The action of the pesticides aldrin, dieldrin, endrin, heptachlor, as well as DDT and methoxychlor, on the development of fertilized sea urchin eggs was investigated. Whereas dieldrin did not affect development, endrin demonstrated slight effects while aldrin led to greatly abnormal embryos. Heptachlor proved to be more toxic than aldrin and led to abnormal development and lysis of the embryos. DDT also affected development but less than aldrin. Methoxychlor demonstrated toxic effects similar to those observed with heptachlor. Independent of these morphological observations, data on the incorporation of [ 3H]thymidine in early developmental stages were obtained. Unlike all other compounds methoxychlor significantly inhibited deoxyribonucleic acid synthesis. In contrast to the effects produced here, methoxychlor is much less toxic to mammals than DDT. The meaning of these results for practical purposes is discussed.

Uptake and incorporation of [ 3H]glycerol into lipids of unfertilized and fertilized sea urchin eggs

Various types of labeling experiments have revealed the pattern of uptake of [ 3H]glycerol and its incorporation into lipids during the early cleavage period of the sea urchin, Strongylocentrotus purpuratus. It has been found that within the first minute after fertilization there is a marked increase in the incorporation of [ 3H]glycerol into chloroform-extractable materials and evidence is presented that this post-fertilization response represents a stimulation in synthetic activity rather than simply an increase in the specific activity of the precursor pool. Thin layer chromatography of newly synthesized lipids indicates that the increased synthesis at fertilization is primarily a quantitative one, with a similar variety of lipid classes (primarily neutral lipids and phosphatidylcholine) being synthesized before and after fertilization as well as throughout the entire first cleavage period. Autoradiographic analysis has failed to demonstrate localization of newly synthesized lipids over the nuclear or plasma membrane and, therefore, the relationship between lipid synthesis and membrane formation during cleavage remains obscure.

CHANGES IN THE LEVEL OF ARGININE PHOSPHATE FOLLOWING FERTILIZATION OF ECHINODERM EGGS.

The content of arginine phosphate was measured following fertilization of sea-urchin eggs and starfish oocytes. In sea-urchin eggs, a rise in the level of arginine phosphate occurred within 2 min after insemination: this was not accompanied by any detectable alteration in the level of ATP. On the other hand, the level of arginine phosphate in starfish oocytes did not change on fertilization.