Ultrastructural Study of Asters Induced by Microinjection with Sperm Centriolar Fraction in Sea Urchin Eggs

Multiple asters can be artificially induced in sea urchin fertilized eggs by the microinjection of the centriolar fraction of sperm homogenate. Investigation was continued by the electron microscopy to determine whether the multi-aster formation was due to the centrioles or the contaminants in the injected sperm fraction. Thirty three asters in 3 operated eggs were thoroughly examined, and we confirmed that the presence of centrioles in the central region of 26 asters. We considered that the rest of them might contained the centrioles in the sections lost during the preparation procedures. Fragmented axoneme, the plug of electron dense material, and the centriolar fossa, which were usually accompanied with the isolated centrioles, disappeared from the centrioles in these multiple asters. However, electron dense, amorphous materials were formed associating with the triplet blades and distributed around the centrioles. Many astral microtubules were terminated in these pericentriolar materials. Results obtained suggest that, although the pericentriolar material is acting as the microtubule organizing center, all multiple asters, except those derived from fertilization (2 asters per egg), are most likely induced by the injected centrioles and not by the contaminants.     

The Acrosome Reaction Induced by Dimethylsulfoxide in Sea Urchin Sperm

The acrosome reaction in sea urchin sperm, as judged by disappearance of the acrosomal vesicles in Nomarski optics, was induced by dimethylsulfoxide (DMSO) at concentration above 0.1% in normal artificial sea water. The number of the acrosome-reacted spermatozoa increased in proportion to DMSO concentration. The DMSO-induced acrosome reaction, as well as the jelly water- or A23187-induced one, was inhibited by nifedipine and hardly occurred in Ca²⁺-free artificial sea water. However, the DMSO-induced acrosome reaction was found in a few number of spermatozoa in the presence of Ca²⁺at above 0.5 mM, though the jelly water- or A23187-induced acrosome reaction did not occur at external Ca²⁺levels lower than 1 mM. Dependency of the acrosome reaction by DMSO on external Ca²⁺is somewhat lower than that of the reaction by jelly water. In Ca²⁺-free artificial sea water, the acrosomal regions of DMSO-treated spermatozoa attached to their own tails. In some cases, spermatozoa thus treated with DMSO in Ca²⁺free artificial sea water caused formation of fertilization membrane in a few number of eggs kept in Ca²⁺-free artificial sea water. Even in the absence of extermal Ca²⁺, preliminary step of the acrosome reaction seems to be completed probably by DMSO-induced weak Ca²⁺-mobilization in spermatozoa.     

Sperm Nuclear Transformation and Aster Formation Related to Metabolic Behaviour in Amphibian Eggs

Bufo arenarum oocyte maturation is related to important modifications in the metabolism of carbohydrates. Such changes involve a different relative participation of the main oxidative routes and are under the influence of seasonal variations of the pituitary activity.
Ovulated coelomic oocytes obtained during the winter period, were unable to initiate cleavage after injection of a sperm suspension. The extent of sperm head transformation and aster formation in the cytoplasm of oocytes with a different metabolic behaviour (obtained during the winter and summer periods) were studied.
Our results show that sperm nuclear transformation and DNA synthesis were quite similar in both types of oocytes. In contrast to summer oocytes, in which the pronucleus was sourranded by an aster, no aster structure was formed in winter ooctyes notwithstanding pronucleus formation occurred.
These results suggest that the failure to develop aster may explain the lack of cleaving in winter oocytes. It appears that the metabolic changes, aster formation and the capability to cleave are closely related and could be dissociated from oocyte nuclear maturation in this species.     

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).     

Electric Field-induced Fusion of Sea Urchin Eggs

An electrical method is described which permits the fusion of denuded eggs of the sea urchin species Paracentrotus lividus . In a nearly non-conductive medium, containing 1.2 M glucose at pH 6.0–8.4, eggs or fertilized stages are brought into close membrane contact by dielectrophoresis arising from the application of a highly inhomogeneous alternating electric field. During this process the eggs aligne parallel to the field forming "egg-chains". In well pigmented eggs pigmentcapping is observed in the areas of cell contact. After completion of the alignment, the application of an additional single high field pulse of μs duration induces fusion of two or more eggs. The mechanism underlying the fusion process is the reversible electric breakdown of membranes in the zones of cell-to-cell contact. Fusion proceeds within 1–10 min at 10–20°C. Fused eggs have intact nuclei, can be fertilized, but undergo abortive cleavage.     

Elastic Properties of the Sea Urchin Sperm Flagellum

The theory of flexural vibrations in thin rods, applied to the movement of flagella, has been extended to include an investigation of the influence of the boundary conditions on the theoretical waveforms. It was found that for flagella which are flexible enough, the flexibility can be estimated solely from the wavelength of the wave traveling in it. This can be expected to hold for those flagella which do not possess a fibrous sheath. The bending moment in flagella in which the ampitude of the wave is maintained as the wave travels distally is almost completely produced by active contractile elements. This means that the active bending moment can be estimated from the radius of curvature of the flagellum and the stiffness. The above findings were applied to the case of the sea urchin sperm flagellum. One finds that the stiffness of the flagellum is caused mainly by the nine longitudinal fibers which must have a Young's modulus of slightly less than 10⁸dyne/cm². The longitudinal fibers need to develop a tension of 1.6 × 10⁸dyne/cm² to account for the bending moment in the flagellum. These two figures are in line with those found for muscle fibers.     

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.     

Fertilization Membrane Formation in Sea Urchin Eggs Induced by Drugs Known to Cause Ca2+Release from Isolated Sarcoplasmic Reticulum

Ryanodine, miconazole, clotrimazole, doxorubicin, quercetin, halothane, caffeine and chloroform, which activate Ca²⁺-induced Ca²⁺release from Ca²⁺stores, induced Ca²⁺release from a particulate fraction isolated from sea urchin eggs, Ca²⁺influx into eggs and formation of a fertilization membrane in an appreciable number of eggs. Their minimum effective concentrations for inducing a fertilization membrane increased in the order of these drugs listed above, and this order was also the same as that of their minimum effective concentrations for inducing Ca²⁺release from the isolated particulate fraction. Their effect in inducing a fertilization membrane was blocked by ruthenium red and procaine, which inhibit Ca²⁺release from Ca²⁺stores. Thus these drugs probably induced sufficient Ca²⁺release to make the cytosolic Ca²⁺level high enough in many eggs for formation of a fertilization membrane. In the absence of external Ca²⁺, fewer eggs treated with these drugs formed a fertilization membrane and more eggs did so on further treatment with either A23187 or carbonylcyanide-p-trifluoromethoxy-phenylhydrazone (FCCP). Thus, a high level of Ca²⁺is probably derived from Ca²⁺release through Ca²⁺releasing channels (by A23187), from mitochondria (by FCCP) and its transport from the external medium.     

Spicule Formation by Isolated Micromeres of the Sea Urchin Embryo

Micromeres are isolated at the 16-cell stage from three speciesof Japanese sea urchins, Hemicentrotus pulcherrimus, Pseudocentrotusdepressus, and Anthocidaris crassispina, and are cultured insea water containing a small amount of horse serum. In all speciesused, isolated micromeres first divide unequally as they doin vivo. The pattern and number of the subsequent cleavagesare also the same as in vivo, although they are not necessarilyclear in all cases, since the border of the adjacent cells becomeinvisible at each resting stage in some batches of embryos. After cleavage, passing through the stage when the contoursof the individual cell are obscure, decendants of the isolatedmicromeres form cell aggregates similar to the group of primarymesenchyme cells in a blastula. Within such aggregates, a spicularrudiment appears which develops either into a triradiate spiculeas in normal gastrulae or into a rod. The triradiate spiculegrows into a three-dimensional skeleton which is very similarto the normal pluteus skeleton, not only in its final shapeand size including species-specific characters but in its developmentalcourse and crystallographic nature. The rod, on the other hand,develops into either a one-dimensional or two-dimensional skeleton.These skeletons probably correspond to a part of the completeskeleton.     

Vegetalization Induced by Procaine and Tetracaine in Sea Urchin Embryos

Vegetalization of sea urchin embryos was induced by the treatment with procaine and tetracaine, inhibitors of Ca²⁺mobilization, for 3 hr starting 3–5 hr after insemination at 20°C. The treatment starting 7 hr after insemination sometimes produced similar type of vegetalized embryos. The pulse treatment starting at the other stages hardly yielded vegetalized embryos. The stages at which these compounds were effective to produce vegetalized embryos were almost the same to those for Li⁺to make embryos vegetalized. On the basis of known inhibitory effects of tetracaine, procaine and Li⁺on Ca²⁺mobilization, we postulate that Ca²⁺dependent reactions participate in the process of cell determination at these stages. Inhibitory effects of procaine, tetracaine and Li⁺on Ca²⁺dependent induction of fertilization membrane formation, found in the present study, indicate that these compounds block Ca²⁺mobilization in sea urchin eggs.     

Distributional Change of Membrane-associated Calcium in Sea Urchin Eggs during Fertilization

The "source and sink" for the intracellular calcium released during fertilization were examined in sea urchin eggs, Hemicentrotus pulcherrimus , with chlortetracycline as a fluorescent chelate probe. In order to distinguish the differential distribution of membrane-associated calcium in various compartments in cytoplasm, eggs were stratified by centrifugation before or after fertilization. Only the layer containing mainly mitochondria exhibited the chlortetracycline-fluorescence in unfertilized eggs. After fertilization, a new fluorescent band emerged in the membrane-rich clear layer of stratified eggs. Chlortetracycline-fluorescence in the clear layer was gradually redistributed surrounding the prophase nucleus and then incorporated into the mitotic apparatus. From these observations, we postulate that the major source(s) of released free calcium ions at fertilization is in the mitochondira layer and membranes in the clear layer are newly activated as the calcium sequestering system after fertilization.     

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.     

Movements of Echinochrome Granules during the Early Development of Sea Urchin Eggs

ECHINOCHROME pigment granules in unfertilized eggs of the sea urchin Arbacia punctulata undergo randomly-directed saltatory movements^1,2. After fertilization, nearly all these granules migrate to the egg cortex and become embedded. Subsequent pigment granule movements may represent mass cortical changes rather than independent granule movements^2,3. At the fourth cleavage, a quartet of micromeres containing little or no pigment forms at the vegetal pole. By the two or four-cell stage, pigment granules have begun to move out of this region, leaving a “clear area” on each blastomere (Fig. 1 and refs. 4, 5). To investigate possible mechanisms for these movements and their relation to cortical events     

Vegetalization of Sea Urchin Larvae Induced with Cycloheximide

The embryos, kept at 20°C for 3 hr–6 hr from the time of fertilization (at the morula stage), were cultured in sea water containing cycloheximide (10–16 mM) for successive 3 hr and then transferred to normal sea water. The embryos, thus treated, became vegetalized larvae. With the same treatment performed at a developmental stage prior to 3 hr of fertilization, most of embryos developed to small blastulae filled with mesenchyme-like cells. The treatment at a stage after 6 hr of fertilization yielded normal plutei. From the embryos exposed to both ¹⁴C-leucine and ³H-thymidine during the treatment, labelled chromatin was isolated. Only in the presumptive vegetalized embryos obtained by the cycloheximide treatment of morulae, ratio of ¹⁴C-radioactivity found in proteins of chromatin to ³H-radioactivity in DNA was markedly lower than that observed in chromatin from control embryos. The rate of ³H-radioactivity-decrease by DNase I treatment was higher in chromatin isolated from the presumptive regetalized embryos than that observed in chromatin isolated from control ones. Probable failure of chromatin structure formation, due to cycloheximide-inhibition of chromatin protein synthesis, seems to disturb the determination in the embryos at the morula stage, resulting in an induction of vegetalized embryos.     

Induction of Fertilization Membrane Formation and Cyanide-insensitive Respiration in Sea Urchin Eggs by the Treatment with Dimethylsulfoxide Followed by an Incubation in an Ice Bath

In unfertilized eggs of the sea urchin Hemicentrotus pulcherrimus , fertilization membrane formation was induced by an incubation with dimethylsulfoxide (DMSO) for several min at 20°c followed by another incubation in an ice bath. The number of eggs with fertilization membrane, thus obtained, increased in relation to the concentration of DMSO between 1 and 3% (v/v) and was higher than 75% at concentrations above 3%. Fertilization membrane formation by this treatment occurred in Ca²⁺ free- or Ca²⁺, Mg²⁺ free- artificial sea water containing EGTA (50 mM) and was inhibited by verapamil. In the presence of DMSO, the membrane formation was also induced by 2, 4-dinitrophenol or cyanide in considerable number of eggs at 20°c. Eggs remained fertilizable, even when they were kept with DMSO for 1 hr at 20°c. DMSO slightly enhanced respiratory rate in unfertilized eggs and substantially reduced it in fertilized eggs. DMSO-treated eggs exhibited cyanide-insensitive respiratory burst following chilling in an ice bath or by adding DNP or cyanide, in a similar manner to the burst induced by sperm.     

Distribution of Membrane-Associated Calcium in Fertilized Sea Urchin Eggs during Mitosis*

The distribution of membrane-associated calcium in dividing sea urchin eggs was examined with chlortetracycline as a fluorescent chelate probe. The fluorescence of bound chlortetracycline in fertilized eggs was initially evenly distributed, but began to gather around the nucleus in prophase, and formed a dumb-bell shaped condensation enclosing the mitotic apparatus by metaphase. During anaphase and telophase, the fluorescence was observed in kinetochore-to-pole regions of the spindle, with little fluorescence in the interkinetochore region. The astral regions showed intense fluorescence. The distribution of the chlortetracycline-fluorescence coincided with that of ER-like membranes seen in electron micrographs. The distribution of the fluorescence was obscure and the birefringence of spindles disappeared on perfusion on perfusion of the cells in metaphase with 1 mM tetracaine, which is known to displace membrane-bound calcium. These results suggest that intracellular free calcium ions are sequestered in the membrane system associated with the mitotic apparatus during mitosis.     

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.