On the Mechanics of the First Cleavage Division of the Sea Urchin Egg

We describe a continuum model of the sea urchin egg during the first cleavage division. Using estimated values of the relevant mechanical parameters we then carry out numerical simulations of cytokinesis and conduct a systematic comparison of these computations with a variety of published experimental data.     

Trifluoperazine-Sensitive Step during Sea Urchin, Echiuroid and Pelecypod Egg Activation

Trifluoperazine, a calmodulin-antagonist, is shown to inhibit egg activation by ionophore A 23187 in sea urchin (I₅₀: 43 μM), by trypsin in echiufoids (I₅₀: 22 μM) and by KCl in bivalves (I₅₀: 34 μM). In each case the inhibition could be reversed by washing the eggs and the trifluoperazine-sensitive period was clearly limited. In Barnea and Urechis , trifluoperazine inhibits calcium uptake. A common trifluoperazine-sensitive step, possibly involving calmodulin, may thus be shared by a variety of animal groups during egg activation.     

Acceleration of the Cleavage in Sea Urchin Eggs by Treatmentswith Local Anesthetics (I) Procaine Treatment*

Unfertilized sea urchin eggs were pre-treated with 2–10 mM procaine sea water for 10 min and then fertilized 5 min later. These eggs hatched about 1–1.5 hr earlier than the control eggs. This acceleration of hatching seems to be the result of a reduction in the individual cleavage cycles. In the second series of experiments, eggs were inseminated at 5, 30 and 60 min after the procaine treatments. In this case, the rate of accelerated cleavage decreased gradually, as the intervals between the treatments and insemination were extended. Next, the minimum time-length of procaine treatment to accelerate cleavage was determined. When eggs were immersed in procaine for 1 min, cleavage was accelerated as in the case of 10 min-treatment, although acceleration to the same degree required higher concentrations of procaine solution. When fertilized eggs were post-treated with procaine, cleavage was also accelerated, but both the rate of acceleration and the effective concentrations decreased with time after fertilization.     

N-Linked Oligosaccharides of Sea Urchin Egg Jelly Induce the Sperm Acrosome Reaction

The sea urchin egg jelly coat (EJ) induces the acrosome reaction (AR) of sperm. We previously demonstrated that a fraction of EJ containing two glycoproteins of 82- and 138-kDa possess the AR inducing activity (8). Here we show that Peptide-N-Glycosidase-F treatment of EJ followed by precipitation and washing in 70% ethanol results in a substantial loss of AR inducing activity in the ethanol insoluble material. When a PNGase-F digest of EJ is chromatographed on a Sepacryl-200 gel filtration column, an AR inducing fraction elutes within the partitioning volume. Acrosome reaction inducing activity of undigested EJ does not elute within the partitioning volume. The chromatographed AR inducing fraction of the PNGase-F digest reacts strongly in the phenol-sulfuric assay demonstrating carbohydrate is present; silver stained gels do not detect the presence of protein. Harsh alkaline hydrolysis of EJ in an excess of NaBH₄, preserves a substantial amount of AR inducing activity. These data show that N-linked oligosaccharides released from EJ by PNGase-F digestion are capable of inducing the sperm acrosome reaction.     

Accumulation of WGA Receptors in the Cleavage Furrow during Cytokinesis of Sea Urchin Eggs

Sea urchin eggs stained with fluorescein-conjugated wheat germ agglutinin (F-WGA) before or after fixation showed a marked accumulation of fluorescence at the cleavage furrow in the first and the second cell divisions. WGA receptors (WGA-binding membrane glycoproteins) were redistributed to the equatorial region through several steps in compressed eggs. Accumulated WGA receptors showed a distribution similar to that of contractile-ring microfilaments throughout most of the steps. Therefore, the former is probably associated with the latter directly or indirectly. Labeling with F-WGA provides a simple method to detect contractile-ring microfilaments in living eggs. Treatment of eggs with colcemid shortly before cytokinesis dispersed the ring-like accumulation of WGA receptors together with contractile-ring microfilaments. This result suggests that microtubule structures, probably asters, are involved in the redistribution of WGA receptors. Cytochalasin B prevented furrowing when it was applied shortly before cytokinesis. While contractile-ring microfilaments showed a spotty distribution in the expected furrow region, WGA receptors were normally redistributed. Furthermore, a higher concentration of the drug allowed the appearance of accumulated WGA receptors in compressed eggs although the development into a ring-like configuration was inhibited. These observations suggest the possibility that the redistribution of WGA receptors is involved in the formation of contractile ring.     

Gamete Recognition and Egg Activation in Sea Urchins

SYNOPSIS. Free-spawning marine invertebrates face the challengeof ensuring that gametes of the same species come into contact,recognize, bind to and fuse with one another once they havebeen released by the adults. Coordinated spawning, chemoattractionand specific cell-cell recognition events help to overcome thischallenge. One marine invertebrate, the sea urchin, has servedas a model system for the study of gamete recognition and fertilizationfor over 100 years. Recent biochemical and molecular advancesin this area have begun to address the questions that have beenraised by the results of elegant physiological observations.The picture of fertilization that is emerging is characterizedby highly specific cell-cell interactions between proteins onthe surfaces of the gametes. These proteins then mediate thebinding and subsequent events that lead to activation of theegg and delivery of the male genetic material. Because of theserecent insights, the sea urchin egg is in a position to provideanswers to one of the central debates in developmental biology—themechanism of egg activation. Does the sperm deliver an activatingfactor? Does sperm binding trigger a receptor-mediated signal?Or is the mechanism a complex combination? With the tools andknowledge gained from the study of sea urchin fertilization,testing of these hypotheses should be feasible in the near future.     

Sperm Bind but Do not Unbind from the Fixed Sea Urchin Egg

In S. purpuratus sperm remained bound to aldehyde-fixed eggs and did not exhibit a detachment phase. Sperm bound in high numbers and the binding curve was similar to that for unfixed homologous gametes. Binding kinetics were analyzed using sequential still photographs of slightly flattened, fixed eggs under supported coverslips. In this way, a single egg could be observed. Alternatively, sperm remaining in the supernatant over settled eggs were counted at successive time points postmixing using spectrophotometry (340 nm) and hemocytometry. Additionally, aliquots of mixed gametes were fixed at successive time points and the numbers of bound sperm determined microscopically on egg perimeters. The maximum number of sperm bound by 2 min; this number remained at or near maximum for as long as 10 min when the experiments were terminated. When sperm were jelly-reacted before addition to eggs, fewer sperm bound, although binding curves were similar to the above experiments. It appears that the binding efficiency of sperm decreases with time after initiation of the acrosome reaction: (1) fewer prereacted sperm bound; and, (2) sperm did not continue to bind after 2 min even though the egg surface was not saturated. Whether these effects are related to motility or other factors is unclear. Furthermore, the above results indicate the importance of the cortical reaction to sperm unbinding. Such studies enable one to observe sperm behavior while precluding the effects of egg secretion during the initial stages of fertilization.     

Effect of Aminopterin and Deoxyribonucleosides on the Cleavage and Embryogenesis of the Sea Urchin, Hemicentrotus pulcherrimus

In the embryos of the sea urchin, Hemicentrotus pulcherrimus , reared with 150 μM aminopterin from the time of fertilization, cessation of the development occurred at the blastula stage, at which the dTTP level became quite low. Another addition of thymidine to the embryo culture containing aminopterin resulted in an elevation of dTTP concentration in the embryos and allowed them to develop normally. Decrease in the dTTP level, resulting from the inhibition of thymidylate synthesis by aminopterin, probably causes a failure of egg cleavage and development. 5-Bromo-2'-deoxyuridine (BUdR) also released the aminopterin-inhibition of egg cleavage and allowed the treated embryos to develop to early gastrulae. Thereafter, the degeneration of archenteron occurred and these embryos became large permanent blastulae. Other deoxyribonucleosides failed to cancel the inhibition by aminopterin of egg cleavage. In the embryos kept with both BUdR and aminopterin, BUdR incorporation into DNA occurred at a similar rate as in thymidine incorporation in the embryos kept with thymidine and aminopterin, and was inhibited by another addition of thymidine. Without aminopterin treatment, BUdR incorporation hardly occurred and the embryos developed normally. BUdR incorporation into DNA in place of thymidine probably occurs in aminopterin-treated embryos, resulting in abnormal development.     

Extreme Toxicity of Ethanol During Activation of Sea Urchin Eggs

We have examined the effects of ethanol on early fertilization events and later development in the sea urchin Strongylocentrotus purpuratus . Eggs can still be fertilized in ethanol concentrations as high as 480 mM (2.0%); egg cytolysis was rapidly observed postinsemination in 50% of the cells at 220 mM ethanol. Yet, sperm motility was essentially normal in 250 mM ethanol; 940 mM ethanol was required to affect a 50% reduction. To determine the effect of ethanol on K⁺-efflux from eggs induced by fertilization, we used parthenogenetic activation induced by the Ca²⁺-ionophore A23187. Surprisingly, ethanol at only 0.2 mM caused an abnormal K⁺-efflux, but only when added between 1 and 3 min after induction of activation. The K⁺-efflux rates of unfertilized eggs were not influenced by up to 730 mM ethanol. Finally, normal embryonic development through the mesenchyme blastula stage was observed in egg suspensions which were treated for 30 min with ethanol concentrations as high as 240 mM, but washed with normal seawater prior to insemination. Normal plutei were obtained from cultures which were continuously cultured in 24 mM ethanol from 15 min postinsemination. We conclude that an extreme ethanol sensitivity of embryogenesis is apparent only during the cortical reaction.     

A Cytochemical Study of the Sulfhydryl Groups of Sea Urchin Eggs during the First Cleavage

In the eggs of four species of echinoderms, Mespilia globulus, Pseudocentrotus depressus, Hemicentrotus pulcherrimus and Clypeaster japonicus, changes in the distribution of protein-bound SH groups from fertilization to the 2 cell stage have been studied cytochemically by use of a mercaptide-forming azo dye. In the eggs of these species, the color intensity in the cytoplasm increased upon fertilization. The astral centers and spindle during mitosis were stained deeply. When the aster formation was suppressed by ether, hyaline spots appeared in the egg cytoplasm instead of well formed astral centers and these spots were stained by the SH-specific dye. Upon recovery of such eggs in pure sea water, and when cleavage ensued, such spots disappeared and two new astral centers were reorganized. The SH-protein occurring in the centrosphere is considered to be the precursor material for the asters and spindle, and this material is apparently derived from the cytoplasm.     

Anti-peptide Antibody Identifies a 57 kDa Protein Tyrosine Kinase in the Sea Urchin Egg Cortex

The egg plasma membrane and cortical structures are highly enriched in protein tyrosine kinase activity which is thought to play an important role in the fertilization process. In order to identify the tyrosine protein kinases in the egg cortex, a site directed polyclonal antibody was produced against a peptide duplicating a conserved region of the catalytic domain of the sea urchin c-abl gene product. The region chosen as an antigen had a high degree of homology (57%) to other protein tyrosine kinases. The antibody was found to bind with a high degree of specificity to a 57 kDa protein tyrosine kinase in S. purpuratus eggs. The antibody was capable of immunoprecipitating the enzyme as a 57 kDa phosphoprotein from purified egg cortex fractions solubilized in NP-40. Immunoprecipitation was completely inhibited by prior incubation of the antibody with the synthetic peptide used as an antigen. Binding of the antibody completely inhibited kinase activity. However, the immunoprecipitated kinase activity could be eluted from the Sepharose-coupled antibody and was shown to have catalytic activity towards a tyrosine containing peptide substrate. The enzyme also underwent autophosphorylation on tyrosine in vitro. Ultrastructural localization of the kinase by immuno-electron microscopy revealed that the enzyme was primarily restricted to the egg plasma membrane.     

Differential Effects of the Egg Jelly Molecules FSG and SAP-I on Elevation of Intracellular Ca2+ and pH in Sea Urchin Spermatozoa

We examined the effects of two egg jelly components, a fucose sulfate glycoconjugate (FSG) and sperm-activating peptide I (SAP-I: Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly), on the intracellular pH (pH_i) and Ca²⁺ ([Ca²⁺]_i) of spermatozoa of the sea urchin Hemicentrotus pulcherrimus . FSG and/or SAP-I induced elevations of [Ca²⁺]; and pH_i in the spermatozoa at pH 8.0. At pH 8.0, a second addition of FSG did not induced further elevation of the [Ca²⁺]_i or pH_i of spermatozoa treated with FSG, but addition or FSG after SAP-I or of SAP-I after FSG induced further increases of [Ca²⁺]_i and pH_i, At pH 6.6, FSG and/or SAP-I did not induce significant elevation of the [Ca²⁺]_i, although SAP-I elevated the pH_i, its half-maximal effective concentration being 10 to 100 pM. At pH 8.0, tetraethyl-ammonium, a voltage-sensitive K⁺-channel blocker, inhibited induction of the acrosome reaction and elevations of [Ca²⁺]_i and pH_i by FSG, but did not affect those by SAP-I. These results suggest that FSG and SAP-I activate different Ca²⁺ and H⁺ transport systems.     

The Role of External Potassium Ion in Activation of Sea Urchin Spermatozoa

When sperm of the sea urchin, Hemicentrotus pulcherrimus, are diluted into K⁺-free seawater, the pH of the suspension gradually decreases, whereas a rapid decline in pH is observed following dilution into regular seawater. Sperm motility and respiration are also activated after dilution into K⁺-free seawater, but levels of activity are less than those observed following dilution into regular seawater. Upon addition of 10 mM K⁺ to K⁺-free seawater, rapid acid release occurs and motility and respiratory rate in sperm are reactivated. The effect of K⁺ on respiration was competitive with respect to the external Na⁺ concentrations. Harmaline, a potent inhibitor of Na⁺/K⁺-ATPase, causes a decrease in movement and respiration of the sperm. Harmaline does not inhibit the rapid decline in pH, although it depresses the release of acid from mitochondria. These results suggest that external K⁺ plays an important role in intracellular alkalinization of sea urchin sperm.     

Activation of Sea Urchin Eggs by Halothane and Its Inhibition by Dantrolene

Eggs of the sea urchin, Hemicentrotus pulcherrimus , were stimulated by halothane, known to induce Ca²⁺ release from sarcosome, to cause fertilization membrane formation in normal and Ca²⁺ free artificial sea water. In the absence of external Ca²⁺, halothane-induced formation of fertilization membrane was inhibited by dantrolene, an inhibitor of Ca²⁺ release from sarcosome, but was not blocked by nifedipine, a Ca²⁺ antagonist specific to Ca²⁺ channels in plasma membrane. Ca²⁺ release from sedimentable fraction isolated from eggs was induced by halothane and was inhibited by dantrolene, but was not blocked by nifedipine. In normal artificial sea water, halothane-caused egg activation was not inhibited either by dantrolene or by nifedipine, but was blocked in the presence of both compounds. ⁴⁵Ca²⁺ influx was substantially stimulated by halothane in eggs exposed to ⁴⁵CaCl₂. Halothane-induced ⁴⁵Ca²⁺ influx into eggs was inhibited by nifedipine but was not blocked by dantrolene. When Ca²⁺ release from intracellular organellae is blocked, Ca²⁺ transport through Ca²⁺ channels in plasma membrane probably acts as a "fail-safe" system to induce an increase in cytosolic Ca²⁺ level, resulting in egg activation.     

Activation of Proteasome in Sea Urchin Sperm by Lysophosphatidylinositol and by Sperm Lipids

Effects of phospholipids, their metabolites and endogenous sperm lipids on the chymotrypsin-like activity of proteasome purified from sperm of the sea urchin, Strongylocentrotus intermedius were examined. Some lysophospholipids remarkably enhanced the activity. The most prominent activation was found in lysophosphatidylinositol (LPI) which enhanced about 12-fold at 2.5 μg/ml. On the other hand, higher concentrations (above 250 μg/ml) were required for the enhancement of the activity by some saturated fatty acids and phospholipids. Lipids extracted from sperm also were effective in the enhancement, and those from sperm which were treated for 15 sec in egg jelly were more effective than those from untreated sperm. These results suggest that certain metabolites belonging to lysophospholipids are produced during the acrosome reaction and activate sperm proteasome. Also, they are not inconsistent with our view that the chymotrypsin-like activity of sperm proteasome participates in the acrosome reaction (23, 24).     

Mobility of Membrane Lipids and Proteins at the Animal and Vegetal Pole of the Sea Urchin Egg

We have compared the mobility of a fluorescent lipid analogue and of fluorescently labeled membrane proteins at the animal and vegetal poles of the egg of the sea urchin Paracentrotus lividus. Translational diffusion coefficients have been measured by fluorescence microphotolysis ("photo-bleaching") on the egg which was rotated on its poles. Lipid and protein diffusion coefficients averaged 0.8 μm²/sec and 0.04 μm²/sec, respectively at both animal and vegetal pole of the egg. Substances which were known to animalize (Zn⁺⁺) or vegetalize (Li⁺) the sea urchin egg had no significant effect on protein diffusion coefficients.     

Effects of Aphidicolin on Arylsulfatase Gene Expression in Sea Urchin Embryos

Expression of the arylsulfatase (Ars) gene in sea urchin embryos begins just before hatching and ceases at the pluteus stage. Initiation of the Ars gene expression is inhibited by aphidicolin, which inhibits DNA synthesis without arresting the total RNA synthesis. Based on these finding it is supposed that DNA replication is a prerequisite for initiation of the Ars gene expression in developing sea urchin embryos.