Fertilization kinetics of sea urchin eggs

Eggs and sperm of the sea urchin Paracentrotus lividus of the Mediterranean are used for an in vitro study of fertilization kinetics. The results are analyzed in terms of two models. One of these models assumes that all sperm-egg encounters lead to permanent attachment; the other (less realistically) assumes that sperm continue their random search after an unsuccessful encounter. More than 100 spermatozoa per egg are needed to achieve a fertilization ratio of more than 95%. There are two explanations for this: only 1% of the egg surface is subject to fertilization, or only 1% of spermatozoa are intrinsically able to fertilize. In the same context, chemotactic attraction and the role of the jelly are discussed. Comparison with earlier work of Rothschild and Swann and of Hultin and Hagström clarifies some discrepancies between and within these papers.     

Activation of maternal centrosomes in unfertilized sea urchin eggs.

Centrosomes are undetectable in unfertilized sea urchin eggs, and normally the sperm introduces the cell's microtubule-organizing center (MTOC) at fertilization. However, artificial activation or parthenogenesis triggers microtubule assembly in the unfertilized egg, and this study explores the reappearance and behavior of the maternal centrosome. During activation with A23187 or ammonia, microtubules appear first at the cortex; centrosomal antigen is detected diffusely throughout the entire cytoplasm. Later, the centrosome becomes more distinct and organizes a radial microtubule shell, and eventually a compact centrosome at the egg center organizes a monaster. In these activated eggs, centrosomes undergo cycles of compaction and decompaction in synchrony with the chromatin, which also undergoes cycles of condensation and decondensation. Parthenogenetic activation with heavy water (50% D2O) or the microtubule-stabilizing drug taxol (10 microM) induces numerous centrosomal foci in the unfertilized sea urchin egg. Within 15 min after incubation in D2O, numerous fine centrosomal foci are detected, and they organize a connected network of numerous asters which fill the entire egg. Taxol induces over 100 centrosomal foci by 15 min after treatment, which organize a corresponding number of asters. The centrosomal material in either D2O- or taxol-treated eggs aggregates with time to form fewer but denser foci, resulting in fewer and larger asters. Fertilization of eggs pretreated with either D2O or taxol shows that the paternal centrosome is dominant over the maternal centrosome. The centrosomal material gradually becomes associated with the enlarged sperm aster. These experiments demonstrate that maternal centrosomal material is present in the unfertilized egg, likely as dispersed undetectable material, which can be activated without paternal contributions. At fertilization, paternal centrosomes become dominant over the maternal centrosomal material.     

Fertilization envelope assembly in sea urchin eggs inseminated in chloride-deficient sea water: II. Biochemical effects

Eggs of the sea urchin Strongylocentrotus purpuratus were fertilized in normal and in several chloride-deficient sea waters ([ Cl-]: normal greater than isethionate greater than methyl sulfonate greater than bromide). The fertilization envelopes (FE) were thinner and failed to harden, and the characteristic I-T transition did not occur. The permeability of the experimental FEs, as determined by release of protein from the perivitelline space, increased in the order of decreasing [Cl-]. Release of the enzymes beta-1,3-glucanase and cortical granule protease were not significantly altered. On the other hand, release of ovoperoxidase was increased three to four times in bromide sea water. Furthermore, a dose-response was observed in varying concentrations of bromide-normal sea water. With decreasing chloride (increasing bromide) concentration, more ovoperoxidase activity was observed. Cytochemical localization of ovoperoxidase activity with diaminobenzidine revealed almost a total lack of staining of FEs from bromide-substituted sea water. The results suggest that in chloride-deficient sea waters protein incorporation into the nascent FE is impaired. At least in the case of bromide, the incorporation of ovoperoxidase into the nascent FE was also inhibited.     

Fertilization induced release of glycogen from bound state in sea urchin eggs

Glycogen in sea urchin eggs is found in both the precipitate and the supernatant fractions obtained by adding perchloric acid to the egg homogenate. Glycogen in the acid-insoluble fraction is apparently protein-bound (bound glycogen) while the acid-extractable form (free glycogen) seems to bind with less protein. The greatest amount of bound glycogen is found in the particulate fraction obtained by centrifugation of the egg homogenate at 10,000g for 30 minutes. The supernatant fraction obtained by centrifugation at 105,000g for two hours contained the largest amount of free glycogen of all the fractions obtained. The bound glycogen decreases and the free glycogen increases markedly following fertilization, while the total level of glycogen does not change. The glycogen release from the bound state occurs in vitro and the rate of release is higher in fertilized eggs than in unfertilized eggs. Polyamines (putrecine, spermidine, and spermine) cause an increase in the rate of glycogen release in the egg homogenate. cAMP, AMP, and ADP exert no effect on glycogen release in vitro, whereas ATP slightly enhances the rate of glycogen release. Na⁺ and K⁺ hardly accelerate the rate of glycogen release, and divalent cations, such as Ca²⁺ and Mg²⁺, cause an increase in the rate of glycogen release.     

Chloride permeability of sea urchin eggs.

We have examined the content and permeability of chloride in sea urchin eggs. After fertilization there is a large increase in the permeability to chloride. We discuss the mechanism underlying this permeability change and the generalized increase in ion permeability observed after fertilization.     

Fertilization of sea urchin eggs is accompanied by 40 S ribosomal subunit phosphorylation

After fertilization of sea urchin (Arbacia punctulata) eggs, there is a single prominent alteration in the pattern of protein phosphorylation. In eggs preloaded with ³²PO₄, a 31,000 M_r protein (rp31) becomes labeled within 4 min of sperm addition. A new steady-state level of rp31 labeling is achieved by 11 min. The rate of protein synthesis in sea urchin zygotes also increases at 8–10 min after fertilization. Protein rp31 corresponds to mammalian ribosomal S6 because it cosediments with 40 S subunits on high salt-sucrose gradients, it is similar to the mammalian protein in M_r and charge, and it becomes phosphorylated during an increase in protein synthesis. The specific activity of phosphorylated rp31 (relative to rRNA) is similar between free 80 S monosomes and polysomes, indicating that rp31 phosphorylation is not sufficient for ribosomal activity. A phosphatase, highly specific for rp31, is present in extracts of eggs and very early embryos. This phosphatase becomes inactive at about the same time that the degree of labeling of rp31 increases in embryos. Evidently a control system that maintains a low level of rp31 phosphorylation is active in sea urchin eggs. Inactivation of this system shortly after fertilization leads to the accumulation of phosphorylated ribosomes.     

Dynein isoforms in sea urchin eggs

Biochemical and immunological analysis of unfertilized sea urchin eggs has revealed the presence of at least two distinct isoforms of cytoplasmic dyneins, one soluble and the other microtubule-associated. The soluble enzyme is a 20 S particle with a MgATPase activity that can be activated 5-fold by nonionic detergents. It contains heavy chain polypeptides that 1) comigrate with the dynein heavy chains of embryonic cilia; 2) cross-react with antibodies against flagellar dynein; and 3) are cleaved by UV irradiation in the presence of MgATP and sodium vanadate into specific peptide fragments. The soluble egg dynein is, therefore, closely related to axonemal dynein and may be a ciliary precursor. Egg microtubule preparations contain a distinct dynein-like polypeptide, previously designated HMr-3 (Scholey, J.M., Neighbors, B., McIntosh, J.R., and Salmon, E.D. (1984) J. Biol Chem. 259, 6516-6525). HMr-3 binds microtubules in an ATP-sensitive fashion; it sediments at 20 S on sucrose density gradients, and it is susceptible to vanadate-sensitized UV cleavage. However, HMr-3 can be distinguished from the soluble cytoplasmic dynein on the basis of its weak cross-reactivity with antiflagellar dynein antibodies, its heavy chain composition on high resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis, its low specific ATPase activity, and the molecular weight of its vanadate-induced UV cleavage fragments. HMr-3 may represent a dynein-like polypeptide that is distinct from the pool of ciliary dynein precursors.     

Fertilization envelope assembly in sea urchin eggs inseminated in Cl− deficient sea water: I. Morphological effects

Elevation and hardening of the fertilization envelope (FE) occur within 15 min following insemination of the sea urchin egg. When chloride ions were replaced in the media with various anion substitutes, including methyl sulfonate, nitrates, bromide, and isethionate, the fertilization envelope failed to harden and collapsed back to the surface of the egg of Lytechinus variegatus, L. pictus, and Strongylocentrotus purpuratus. At the light microscopy level, the collapse of the envelope was accompanied by a decrease in birefringence, compared with controls. When examined with electron microscopy, the FEs of eggs inseminated in reduced Cl^? solutions failed to transform from an amorphous layer into the more robust laminar structure observed around eggs incubated in normal sea water. Furthermore, in the case of S. purpuratus, the I-T transformation of the FE did not occur. When transfer of the inseminated eggs from the Cl^?-deficient sea water to normal sea water was carried out before 10 min elapsed, the envelope did not collapse, and the birefringence of the envelope was similar to that of controls. Partial envelope collapse was also observed in a dose-dependent manner, varying with the concentration of the Cl^? in the sea water solution. The results suggest that lack of Cl^? in the media may interfere with proper fertilization envelope assembly. Possible mechanisms, including proper incorporation of the cortical granule exudate into the nascent envelope structure, are discussed.     

Action of colcemid in sea urchin eggs

The effects of Colcemid, the deacetyl-N-methyl derivative of colchicine, on the eggs of Arbacia punctulata were investigated. Colcemid in concentrations of 2.7 x 10^-5 M or greater blocks syngamy (the fusion of the pronuclei) in these eggs. Although a tenfold decrease in concentration of Colcemid usually permits the pronuclei to fuse, the subsequent division is blocked. In the sea urchin egg, the duration of presyngamy is about 15 min during which time there is no DNA synthesis. However, DNA synthesis is recorded in Colcemid-blocked cells prior to syngamy. Radioautographs of Colcemid-blocked cells which were immersed into thymidine-³H exhibited silver grains above each of the pronuclei. The action of Colcemid on Arbacia eggs is reversible. Nevertheless, exposures to 2.7 x 10^-5 M Colcemid for only 3 min, initiated 5 min after insemination, caused delays of 70 min in subsequent division. In general, cells are more sensitive to Colcemid prior to the time when the mitotic spindle is being assembled than at presyngamy stages. The results are discussed in terms of Colcemid action on pronuclear fusion and cell division.     

Ribosomal proteins of sea urchin eggs

Summary Ribosomal proteins from unfertilized eggs of three sea urchin species, Pseudocentrotus depressus, Hemicentrotus pulcherrimus, and Anthocidaris crassispina, were analyzed. Species-specific differences were observed in the profiles of large subunit proteins on two-dimensional slab gels, though the number of ribosomal proteins and the molecular weights of their counterparts were the same. The small subunit proteins revealed similarities in the electrophoretic profiles and in the phosphorylation patterns among these three species.     

Localization of tropomyosin in sea urchin eggs

Localization of tropomyosin in sea urchin eggs was investigated immunohistochemically. A rabbit antiserum against tropomyosin prepared from lantern muscle of the sea urchin was used for the indirect immunofluorescence staining of unfertilized and fertilized eggs. The tropomyosin-specific fluorescence was observed at the peripheral region beneath the plasma membrane, mitotic apparatus and contractile ring. The mitotic apparatus isolated from sea urchin eggs was also stained with the anti-tropomyosin serum.     

NAADP-induced calcium release in sea urchin eggs

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent activator of Ca2+ release from intracellular stores described. It acts on a mechanism distinct from inositol trisphosphate and ryanodine receptors, the two major Ca2+ release channels characterised. NAADP-gated Ca2+ release channels do not appear to be regulated by Ca2+ and may be better suited for triggering Ca2+ signals rather than propagating them. They exhibit a remarkable pharmacology for a putative intracellular Ca2+ release channel in that they are selectively blocked by potassium and L-type Ca2+ channel antagonists. Furthermore, in contrast to microsomal Ca2+ stores expressing IP3Rs and RyRs, those sensitive to NAADP are thapsigargin-insensitive, suggesting that they may be expressed on a different part of the endoplasmic reticulum. Perhaps the most unusual feature of the NAADP-gated Ca2+ release mechanisms is its inactivation properties. Unlike the mechanisms regulated by IP3 and cADPR in sea urchin eggs which after induction of Ca2+ release appear to become refractory to subsequent activation, very low concentrations of NAADP are able to inactivate NAADP-induced Ca2+ release fully at concentrations well below those required to activate Ca2+ release. The mechanism and physiological significance of this most unusual desensitisation phenomenon are unclear. More recently, NAADP has been shown to mobilise Ca2+ in ascidian oocytes, brain microsomes and pancreatic acinar cells suggesting a more widespread role in Ca2+ signalling. A possible role for this novel Ca2+ release mechanism in sea urchin egg fertilisation is discussed.     

Giant pools of DNA precursors in sea urchin eggs.

Tritiated cytochalasin D (³H-CD) is rapidly taken up by monolayers of HEp-2 HeLa and rhabdomyosarcoma cells, reaching a maximum incorporation within 5 min at 37 °C. Upon rinsing and refeeding, 80% of the bound drug rapidly dissociates from the cells; the remaining 20 % is lost more slowly. Binding is dose-dependent in a non-linear fashion; Scatchard plots are biphasic, suggesting binding of higher and lower affinity. Inhibitors of energy metabolism do not diminish binding of ³H-CD. The plasma membrane fraction of HEp-2 exhibits the highest specific binding activity (146 dpm/μg protein) and contains both high and low affinity binding sites. Endomembranes (microsomes) have moderate specific binding activity (35 dpm/μg protein) and appear to contain only low affinity binding sites. Nuclear, mitochondrial, and cytosol fractions exhibit low, probably negligible, binding. These results are consistent with the evidence afforded by radioautography. Selective enzyme digestions of whole cells and the plasma membrane fraction indicate that binding of CD requires proteins not exposed on the outer surface of the cell. Because electron micrographs of the plasma membrane fraction demonstrate microfilaments attached to the membrane, the binding data may be interpreted as evidence for an interaction of CD either with the subplasmalemmal microfilaments or directly with the plasma membrane.     

Metal-binding proteins in eggs of various sea urchin species.

Metallothionein presence and amount were determined in the unfertilized eggs of six sea urchin species by silver saturation assay and gel-chromatography of cell extracts. The results showed high levels of metallothionein in the egg cytoplasm of the two Mediterranean species Paracentrotus lividus and Sphaerechinus granularis. No metallothionein was found either in the eggs of Arbacia lixula, or in those of the three Eastern species Strongylocentrotus intermedius, Temnopleurus hardwickii and Clypeaster japonicus. However, the extracts of the latter three species revealed the presence of zinc bound in a macromolecular form, thus suggesting the existence of metal-binding proteins distinct from metallothioneins.