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.     

Isolation of intact sperm asters from fertilized sea urchin eggs

We have developed a procedure for isolating intact sperm asters in quantity from fertilized sea urchin eggs. This procedure is based on detergent-extraction methods developed previously for the bulk isolation of mitotic apparatuses. Using this protocol it is possible to isolate sperm asters as soon as they appear in the fertilized egg or at any subsequent point in their brief existence.     

Fertilization triggers unmasking of maternal mRNAs in sea urchin eggs.

Fertilization of sea urchin eggs results in a large increase in the rate of protein synthesis which is mediated by the translation of stored maternal mRNA. The masked message hypothesis suggests that messenger ribonucleoprotein particles (mRNPs) from unfertilized eggs are translationally inactive and that fertilization results in alterations of the mRNPs such that they become translationally active. Previous workers have isolated egg mRNPs by sucrose gradient centrifugation and have assayed their translational activity in heterologous cell-free systems. The conflicting results they obtained are probably due to the sensitivity of mRNPs to artifactual activation and inactivation. Previously, we demonstrated that unfractionated mRNPs in a sea urchin cell-free translation system were translationally inactive. Now, using large-pore gel filtration chromatography, we partially purified egg mRNPs while retaining their translationally repressed state. Polysomal mRNPs from fertilized eggs isolated under the same conditions were translationally active. The changes in the pattern of proteins synthesized by fractionated unfertilized and fertilized mRNPs in vitro were similar to those changes observed in vivo. Treatment of egg mRNPs with buffers containing high salt and EDTA, followed by rechromatography, resulted in the activation of the mRNPs and the release of an inhibitor of translation from the mRNPs. Analysis of the inhibitory fraction on one-dimensional sodium dodecyl sulfate gels indicated that this fraction contains a complex set of proteins, several of which were released from high-salt-EDTA-activated mRNPs and not from inactive low-salt control mRNPs. One of the released proteins may be responsible for the repression of egg mRNPs in vitro and be involved in the unmasking of mRNPs at fertilization.     

Immunolocalization of the sea urchin sperm receptor in eggs and maturing ovaries

The sperm receptor from Strongylocentrotus purpuratus eggs is a high molecular weight proteoglycan-like molecule that inhibits fertilization species-specifically in a competition bioassay. A preparation of highly active sperm receptor that contained one major N-terminal sequence was used to generate polyclonal antibody in rabbits. This antibody species-specifically inhibited fertilization at low concentrations without interfering with fertilization envelope elevation. On immunofluorescence microscopy, the antibody recognized determinants on the mature egg cell surface and in the cortical granules just beneath the surface. Preabsorption of the antibody with the calcium-soluble fraction of the exudate from cortical granules rendered the antibody specific for the cell surface in mature eggs and still able to inhibit fertilization at the same concentrations as before treatment with cortical granule exudate. With antibody preabsorbed with cortical granule and by counting antibody-gold particles viewed by electron microscopy, sperm receptor was almost undetectable on the cell surface of immature oocytes in preseason ovaries, present on the cell surface and intracellularly in immature oocytes of ovaries collected at the beginning or at the height of the spawning season, and present only on the cell surface of mature oocytes in the lumen of the ovaries. Our results indicate that receptor is synthesized early in oogenesis and is rapidly moved to the egg cell surface.     

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.     

Calcium influx mediates the voltage-dependence of sperm entry into sea urchin eggs

Sperm entry was monitored in voltage-clamped sea urchin eggs following insemination in a variety of artificial seawaters. In regular seawater, maintaining the membrane potential at increasingly negative values progressively inhibits sperm entry. Reducing [Ca(2+)](o) relieves the inhibition, shifting the sperm entry vs voltage relationship toward more negative potentials. Raising [Ca(2+)](o) shifts the relationship in the other direction. Large changes in [Na(+)](o) or [Mg(2+)](o) do not affect sperm entry although changing [Na(+)](o) dramatically changes the currents following sperm attachment. Applying one of seven different calcium channel blockers or replacing Ca(2+) with Ba(2+) or Sr(2+) or microinjecting calcium chelators into the cytoplasm relieves the block to sperm entry at negative potentials. We conclude that the block to sperm entry at negative potentials is mediated by calcium which crosses the membrane and acts at an intracellular site.     

A low molecular weight factor from sea urchin eggs elevates sperm cyclic nucleotide concentrations and respiration rates.

A factor associated with sea urchin eggs that increases sperm cyclic nucleotide concentrations and respiration rates was identified as having a low molecular weight. The factor was more potent at elevating cyclic GMP concentrations than cyclic AMP concentrations, and represents the first demonstration of a factor associated with eggs that is capable of causing elevations of sperm cyclic GMP. Concentration-response curves of the crude mixture of egg factors to increase sperm cyclic AMP and cyclic GMP concentrations and respiratory rates were very similar, and comparable losses of these three activities were observed after extensive dialysis and heat treatment of the crude egg factors. The factor was partly purified by ethanol precipitation of a large molecular weight egg jelly component, and by charcoal adsorption and LH-20 chromatography of the resultant ethanol-soluble material. The factor was not extracted into a variety of organic solvents and had an apparent molecular weight of between 1000 and 2000, as estimated by gel filtration.     

Fertilization induced changes in sea urchin sperm: mitochondrial deformation and phosphatidylserine exposure

This study demonstrates that the single mitochondrion of the sea urchin sperm undergoes a shape change at fertilization that is linked to respiration. The mitochondrion swells and shifts to the lateral side of the sperm head on contact with the homologous egg jelly or egg surface; Mg(2+)- or Na(+)-free seawater or respiratory inhibitors also induce this change. During the mitochondrial deformation, the sperm decreases the rate of oxygen consumption and their redox-state of cytochromes is disrupted b-c(1)/c. Simultaneously, the adenine nucleotides content changes precipitously. This suggests that mitochondrial morphology is strongly associated with respiratory activities in the sea urchin sperm. These changes in mitochondrial morphology and function are similar to the mitochondrial changes in apoptotic cells such as swelling, decrease in its membrane potential, and release of cytochrome c. In apoptotic cells, the exposure of phosphatidylserine from the inner to outer leaflet of the plasma membrane is one of prominence phenomena. This change was visualized by staining the sea urchin sperm with Annexin V-Fluorescein. It is possible that mitochondrial deformation is an initial sign of sperm destruction, which like as apoptotic cells.     

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.     

Sulfhydryl groups are involved in the activation of sea urchin eggs

Unfertilized sea urchin eggs exposed to the sulfhydryl reagents Ag⁺ or N-ethylmaleimide either elevated fertilizationlike membranes, formed surface protrusions, developed a clear cortical layer devoid of organelles, or cytolysed. The relative fraction of each modification varied from batch to batch and was also dose and time dependent. With Ag⁺ and higher doses of N-EMI (10^?3 M), the most common effect was the elevation of a membrane indicating cortical exocytosis, while at lower doses of N-EMI protrusions were predominant. Glutathione (GSH) protected eggs against these reagents also in a dose-dependent manner. Eggs exposed to equimolar amounts of N-EMI and GSH, which otherwise formed membranes, produced protrusions, while increasing GSH tenfold afforded complete protection. We suggest there are two targets for the sulfhydryl reagents–the first, SH groups on proteins that regulate the release of Ca²⁺ from the intracellular sequestering mechanism which subsequently triggers cortical exocytosis; the second, SH groups on the egg surface that may regulate cortical organization.     

Induction of cross-fertilization between sea urchin eggs and starfish sperm by polyethylene glycol treatment

Cross-fertilization between sea urchin eggs (Strongylocentrotus nudus) and starfish sperm (Asterina pectinifera) was induced by treatment with polyethylene glycol (PEG). Without treatment with PEG, the denuded egg surface (jelly coat- and vitelline coat-free) engulfed the head of acrosome-reacted sperm; however, sperm penetration did not occur [Kyozuka and Osanai, 1988]. When these eggs were exposed briefly to PEG (molecular weight 3,000) in seawater, the sperm entered the egg by membrane fusion. Cortical granules were discharged, and embryogenesis began following sperm penetration. PEG did not induce parthenogenesis in Strongylocentrotus eggs. Egg activation is thus closely linked with gamete membrane fusion.     

Progression of flagellar stages during artificially delayed motility initiation in sea urchin sperm

Transition from immotile to motile flagella may involve a series of states, in which some of regulatory mechanisms underlying normal flagellar movement are working with others being still suppressed. To address ourselves to the study of starting transients of flagella, we analyzed flagellar movement of sea urchin sperm whose motility initiation had been retarded in an experimental solution, so that we could capture the instance at which individual spermatozoa began their flagellar beating. Initially straight and immotile flagella began to shiver at low amplitude, then propagated exclusively the principal bend (P bend), and finally started stable flagellar beating. The site of generation of the P bend in the P-bend propagating stage varied in position in the basal region up to 10 microm from the base, indicating that the ability of autonomous bend generation is not exclusively possessed by the very basal region but can be unmasked throughout a wider region when the reverse bend (R bend) is suppressed. The rate of change in the shear angle, the curvature of the R bend and the frequency and regularity of beating substantially increased upon transition from P-bend propagating to full-beating, while the propagation velocity of bends remained unchanged. These findings indicate that artificially delayed motility initiation may accompany sequential modification of the motile system and that mechanisms underlying flagellar motility can be analyzed separately under experimentally retarded conditions.     

Microtubules are required for completion of cytokinesis in sea urchin eggs.

Completion of cytokinesis, abscission, has been studied little despite the intensive studies of the onset and contractile mechanism of the earlier phases of division. It has been well documented that microtubule (MT) disruption before furrow stimulation prevents furrowing, while MT disruption after furrow stimulation allows division to proceed. We have confirmed those findings using the MT inhibitors, nocodazole and demecolcine. In addition, we have found that MT disruption after furrow stimulation but before completion of division prevents abscission as evidenced by the observation that prospective daughter cells in MT-disrupted eggs maintain electrical continuity. Continued observation of eggs revealed that the furrow in MT-disrupted eggs did not result in abscission, but rather held steady until the time when controls underwent second cleavage, at which point the furrows regressed. These findings extend the recent reports that MTs are required for completion of division in mammalian tissue culture cells and frog eggs, to invertebrates, suggesting a common mechanism of abscission for animal cells.     

Effects of aphidicolin on premature condensation of sperm chromosomes in fertilized sea urchin eggs

Unfertilized Strongylocentrotus purpuratus eggs may be treated with ammonia to initiate maternal DNA replication and the maternal cell cycle. When these eggs are polyspermically fertilized 75 min after the beginning of ammonia treatment, the nuclei of the fertilizing spermatozoa undergo premature chromosome condensation (PCC) in an apparent attempt to conform to the advanced maternal cell cycle. PCC is inhibited if maternal DNA replication is blocked by exposing the eggs to aphidicolin but will proceed if this exposure begins after replication is complete. Additionally, PCC will proceed in ammonia-activated, polyspermically fertilized anucleate merogons in the continuous presence of aphidicolin. These results suggest that the direct inhibitory effect of aphidicolin may well be limited to the replication of DNA and that the unreplicated maternal nucleus itself exerts negative control over the development of chromosome-condensing conditions in the maternal cytoplasm.     

Cytoplasmic and sperm nuclear transformations in fertilized ammonia-activated sea urchin (Arbacia punctulata) eggs

Studies examining cytoplasmic and sperm nuclear transformations in sea urchin (Arbacia punctulata) eggs inseminated at different periods after ammonia activation have been caried out at the light- and electron-microscopic levels of observation. Arbaca eggs treated with ammonia-seawater demonstrated chromosome condensation after DNA synthesis and underwent a chromosome cycle similar to that described for Lytechinus [Mazia, 1947]. Cortical granule reaction, fertilization cone formation, and sperm aster development in eggs fertilized at 20 (interphase), 50 (prometaphase), and 180 (interphase) min after ammonia activation were structurally simialr to processes in untreated zygotes. Cyclical changes in the formation of fertilization cones and sperm asters, as reported for eggs fertilized after activation by agents that induce a cortical granule reaction, were not observed. Although sperm nuclear transformations were prolonged (14 vs 18 min), male pronuclei that developed in eggs fertilized 20 min after ammonia activation were morphologically similar to those observed in fertilized, untreated ova and incorporated ³H-thymidine. Sperm incorporated into eggs at 50 min after ammonia activation underwent nuclear envelope breakdown and chromatin despersion; however, ³H-thymidine incorporation was not observed, and male pronuclei rarely developed (less than 5% of all specimens examined). Subsequent to dispersion, the paternal chromatin condensed into chromosomes which were associated with an aster. These results demonstrate that although ammonia-activated eggs inseminated at interphase or prometaphase undergo similar cytoplasmic alterations, sperm nuclear transformations vary with the chromosome cycle of the egg.     

Dispersal of sperm surface antigens in the plasma membranes of polyspermically fertilized sea urchin eggs

Polyspermically fertilized Strongylocentrotus purpuratus eggs were fixed at varying times after insemination and exposed to a monoclonal antibody (mAb J18/29) directed against a group of sperm surface antigens. Indirect immunofluorescence microscopy reveals that the sperm surface components recognized by mAb J18/29 are quickly incorporated into the egg plasma membrane and begin to disperse as early as 1.5 min after insemination. At subsequent times after insemination, they undergo further dispersal so that by 45 min they are distributed evenly over the entire surface of the egg. These results provide evidence for the free lateral mobility of sperm membrane components in the fertilized egg.     

Heavy metal chelators prolong motility and viability of sea urchin sperm by inhibiting spontaneous acrosome reactions

A variety of heavy metal chelating agents is known to prolong the fertilizing capacity and motility of sea urchin sperm. We report here that these agents maintain fertilizing capacity by preventing acrosome reactions which occur spontaneously after dilution of sperm into seawater. These chelating agents also inhibit acrosome reactions induced by high pH or egg jelly. Since induction of the acrosome reaction leads to steps that abolish motility, specifically a massive Ca2+ uptake and concomitant acidification of the cytoplasm, motility is prolonged by these chelators. These observations also suggest that heavy metals play a role in controlling the acrosome reaction in sea urchin sperm.