Propranolol induces polyspermy during sea urchin fertilization

Propranolol, a β-adrenergic receptor blocker, is found to induce polyspermy in sea urchin eggs. Unfertilized sea urchin eggs treated for 10 min with 50 μM of propranolol, and then inseminated, become polyspermic and show a fertilization envelope which is barely visible to the light microscope. Examination of treated eggs by transmission and scanning electron microscopy shows that the drug does not alter the cortex of the unfertilized egg. However, after insemination an incomplete cortical reaction occurs. This might well account for both polyspermy and the defective elevation of the fertilization envelope. Since the effects of the drug are reversed by simultaneous treatment with adrenalin, perhaps propranolol interferes with the monoaminergic system that has been proposed to be active. The involvement of the monoaminergic system in the fertilization process is present in the sea urchin egg. © 1996 Wiley-Liss, Inc.     

Cross fertilization between sea urchin eggs and oyster spermatozoa

Interphylum crossing was examined between sea urchin eggs (Temnopleurus hardwicki) and oyster sperm (Crassostrea gigas). The eggs could receive the spermatozoa with or without cortical change. The fertilized eggs that elevated the fertilization envelope began their embryogenesis. Electron microscopy revealed that oyster spermatozoa underwent acrosome reaction on the sea urchin vitelline coat, and their acrosomal membrane fused with the egg plasma membrane after the appearance of an intricate membranous structure in the boundary between the acrosomal process and the egg cytoplasm. Oyster spermatozoa penetrated sometimes into sea urchin eggs without stimulating cortical granule discharge and consequently without fertilization envelope formation. The organelles derived from oyster spermatozoa seemed to be functionally inactive in the eggs whose cortex remained unchanged.     

Role of calcium influx during the latent period in sea urchin fertilization

After ∼7–40 s following gamete fusion, a steadily increasing fraction of a sea urchin's zygotes initiate an activating calcium wave. The fertilization membrane then rises, the cell cycle resumes and development begins. This study focuses on the so-called latent period that occurs between the time that gamete fusion occurs and the initiation of the activating calcium wave. We inhibited calcium influx during this period by adding lanthanum or by reducing external calcium with a buffer at various time points after insemination. Both of these treatments blocked the activation of eggs that had not yet started a wave at the time of treatment. This indicates that an influx of calcium is needed during the latent period to induce egg activation. These results support the sperm conduit model of egg activation in the sea urchin, where calcium flows from the sea through the fused sperms' acrosomal process into a cortical region of the eggs' endoplasmic reticulum.     

Characterization of hatching-associated changes in the sea urchin fertilization envelope

The embryo of the sea urchin Strongylocentrotus purpuratus hatches from the fertilization envelope (FE) via synthesis and secretion of a hatching enzyme and by ciliary activity. Although the basic characteristics of the hatching enzyme are known, little is understood about changes in the FE during hatching. We have studied the biochemical changes in FEs during hatching. Polyacrylamide gel analysis revealed an increasingly complex polypeptide spectrum of the extractable fraction of FEs isolated during development. Immunoblotting of these polypeptides (using antiserum against the soluble polypeptides extracted from FEs isolated at 30 minutes postinsemination) revealed a decrease in the soluble FE components during hatching. Immunochemical analysis of hatching medium showed a strong correlation between the soluble FE components released and the hatching interval. Immunoblotting of hatching media indicated the presence of soluble FE polypeptides of similar and lower molecular weights than those obtained for extracts of FEs. These results imply that the hatching-associated changes in the FE of S purpuratus occur via proteolysis of FE components, which are derived from the paracrystalline protein fraction, a subset of cortical granule proteins.     

Bioelectric responses at fertilization: Separation of the events associated with insemination from those due to the cortical reaction in sea urchin,Lytechinus variegatus

The bioelectric responses at fertilization of the sea urchin Lytechinus variegatus are a complex series of membrane potential and resistance changes that occur concomitant with gamete fusion, ionic fluxes, and the cortical granule discharge. This work attempts to separate the electrical effects of sperm-egg interactions from those of the cortical reactions. Two approaches were taken to discern the electrical events associated with insemination, distinct from cortical granule discharge: (1) fertilization of eggs treated with 3% urethane, 10 mM procaine, or 10 mM nicotine, to prevent the cortical reaction and (2) refertilization of fertilized eggs (denuded with 1 mM aminotriazole containing 1 mg/ml soybean trypsin inhibitor). Cortical granule discharge in the absence of sperm incorporation was investigated by artificial activation with 5 μM A23187 or by fertilization in the presence of 10 μM cytochalasin D, which prevents incorporation. These results are consistent with a model in which the sperm-egg interaction triggers both a rapid (50–400 msec), but minor (?10 mV), electrical transient that leads to an action potential and then both the Na⁺-dependent fast block to polyspermy and the late block resulting from the secretion of the cortical granules.     

A fluorescence dequenching method for monitoring exocytotic membrane fusion in fertilization of single sea urchin eggs

A method for monitoring exocytotic membrane fusion by using a fluorescent membrane probe is presented. The method is based on the relief from concentration-dependent self-quenching (dequenching) of fluorescence of 5-N-(octadecanoyl)aminofluorescein (AF18), an amphiphilic derivative of fluorescein. The validity and usefulness of this method were shown by the following results: 1) self-quenching of AF18 fluorescence occurred in the plasma membrane of unfertilized eggs of a sea urchin, Pseudocentrotus depressus, which were heavily stained with the fluorescent dye; 2) dequenching of AF18 fluorescence occurred upon fertilization in normal eggs but not in EGTA-injected eggs; 3) Ca²⁺ induced both AF18 fluorescence dequenching and cortical granule disappearance in the isolated sea urchin egg cortex; and 4) simultaneous measurements of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and dequenching of AF18 fluorescence by using a simple one-excitation and two-emission wavelength system.     

Effect of maitotoxin on sea urchin egg fertilization and on Ca2+ permeabilities of eggs and intracellular stores.

Maitotoxin (MTX), a potent marine toxin involved in ciguatera poisoning, inhibited sea urchin egg fertilization in a dose-dependent manner with an IC50 of 7.5 x 10(-3) MU (mouse-unit)/ml. It did not affect male gametes fertilizing capabilities but provoked exocytosis in female gametes. It induced a K+ loss simultaneously with a Na+ entry into unfertilized eggs and increased the Ca2+ influx at higher concentrations. On isolated cortex preparations, high concentrations of MTX reduced the rate of ATP-dependent Ca2+ accumulation into reticulum compartments and caused a leakage of Ca2+ from a preparation pre-loaded with 45Ca2+. Verapamil (10(-4) M) similarly blocked the increase of egg permeability to Ca2+ and the effect on Ca2+ sequestering into intracellular compartment, induced by MTX. Ion transport perturbations which evolved relatively slowly are probably not the direct cause of fertilization inhibition which could be related to a modification of the plasma membrane of the female gametes by this hydrophilic toxin.     

Characterization of the vitelline envelope of the sea urchin Strongylocentrotus purpuratus

The vitelline envelope (VE) is an extremely thin, acellular, proteinaceous coat that surrounds the extracellular surface of sea urchin eggs. Despite previous studies on VE composition, structure and function, our understanding of the envelope is still incomplete at the molecular level. We have isolated VE components from intact, unfertilized Strongylocentrotus purpuratus eggs by reduction with alkaline dithiothreitol-sea water solutions and have characterized the macromolecules by SDS-PAGE. There were eight major glycoprotein bands, including two high molecular weight components at 265 and 300 kDa, and several minor components. We have revealed, by lectin blot analysis, that most components contain mannose, while a subset of glycoproteins contain fucose and N -acetylglucosamine; galactose and sialic acid were also detected. The components in the VE preparations were compared with cell surface complex preparations by immunoblot analysis, using antisera against a VE preparation, a 305 kDa electrophoretically purified VE glycoprotein and an extracellular portion of the sea urchin egg recombinant 350 kDa sperm receptor. Serum against the recombinant sperm receptor reacted with a component of ∼350 kDa on blots, but did not react with the 300 kDa component found in VE preparations. Therefore, we suggest these two glycoproteins are not the same.     

Specification and differentiation processes of secondary mesenchyme-derived cells in embryos of the sea urchin Hemicentrotus pulcherrimus

Four types of mesoderm cells (pigment cells, blastocoelar cells, coelomic pouch cells and circumesophageal muscle cells) are derived from secondary mesenchyme cells (SMC) in sea urchin embryos. To gain information on the specification and differentiation processes of SMC-derived cells, we studied the exact number and division cycles of each type of cell in Hemicentrotus pulcherrimus. Numbers of blastocoelar cells, coelomic pouch cells and circumesophageal muscle fibers were 18.0 +/- 2.0 (36 h post-fertilization (h.p.f.)), 23.0 +/- 2.5 (36 h.p.f.) and 9.5 +/- 1.3 (60 h.p.f.), respectively, whereas the number of pigment cells ranged from 40 to 60. From the diameters of blastocoelar cells and coelomic pouch cells, the numbers of division cycles were elucidated; these two types of cells had undertaken 11 rounds of cell division by the prism stage, somewhat earlier than pigment cells. To determine the relationship among the four types of cells, we tried to alter the number of pigment cells with chemical treatment and found that CH3COONa increased pigment cells without affecting embryo morphology. Interestingly, the number of blastocoelar cells became smaller in CH3COONa-treated embryos. In contrast, blastocoelar cells were markedly increased with NiCl2 treatment, whereas the number of pigment cells was markedly decreased. The number of coelomic pouch cells and circumesophageal muscle fibers was not affected with these treatments, indicating that coelomic pouch and muscle cells are specified independently of, or at much later stages, than pigment and blastocoelar cells.     

Activator of G-protein signaling in asymmetric cell divisions of the sea urchin embryo

An asymmetric fourth cell division in the sea urchin embryo results in formation of daughter cells, macromeres and micromeres, with distinct sizes and fates. Several lines of functional evidence presented here, including pharmacological interference and dominant negative protein expression, indicate that heterotrimeric G protein Gi and its interaction partner, activator of G-protein signaling (AGS), are necessary for this asymmetric cell division. Inhibition of Gi signaling by pertussis toxin interferes with micromere formation and leads to defects in embryogenesis. AGS was isolated in a yeast two-hybrid screen with G alpha i as bait and was expressed in embryos localized to the cell cortex at the time of asymmetric divisions. Introduction of exogenous dominant-negative AGS protein, containing only G-protein regulatory (GPR) domains, selectively prevented the asymmetric division in normal micromere formation. These results support the growing evidence that AGS is a universal regulator of asymmetric cell divisions in embryos.     

Reaction of sperm with egg-derived hydrogen peroxide helps prevent polyspermy during fertilization in the sea urchin

Recent evidence suggests roles for egg derived hydrogen peroxide (H₂O₂) and ovoperoxidase (secreted by cortical granules) in both fertilization envelope hardening and the block to polyspermy in sea urchins. Strongylocentrotus purpuratus eggs were found to release H₂O₂ during the cortical reaction at fertilization. Treatment of sperm with equivalent concentrations of H₂O₂ resulted in a rapid loss of sperm fertilizing ability. Attempts were made to induce polyspermy by utilizing ovoperoxidase inhibitors at concentrations known to inhibit fertilization envelope hardening. Eggs fertilized in phenylhydrazine became polyspermic, while 3-amino-1,2,4-triazole-treated eggs did not. These data suggested that a sperm peroxidase might be involved in preventing polyspermy. This hypothesis was tested by the addition of phenylhydrazine or 3-amino-1,2,4-trizaole to H₂O₂-treated sperm. Phenylhydrazine acted to protect sperm fertility from H₂O₂, while 3-amino-1,2,4-triazole increased the adverse effect of H₂O₂. Simultaneous addition of both inhibitors to sperm incubated in H₂O₂ gave an intermediate value of sperm fertility. These data indicate that (1) H₂O₂ generated by sea urchin eggs during the cortical reaction at fertilization is used for two separate processes, fertilization envelope hardening and the prevention of polyspermy; (2) ovoperoxidase is probably not involved in preventing polyspermy; and (3) egg-derived H₂O₂ reacts directly with sperm enzymes to prevent polyspermy. The phenylhydrazine-sensitive enzyme in the sperm is probably a peroxidase that acts to inactivate sperm, while the 3-amino-1,2,4-triazolesensitive enzyme is probably a catalase which protects sperm from H₂O₂. This hypothesis is consistent with model experiments on horseradish peroxidase and bovine liver catalase.     

Role of integrins and polyphosphoinositide metabolism during fertilization in sea urchin egg and hamster oocyte

Summary

In almost all species studied to date, a transient increase in the intracellular free calcium concentration (Ca_i) occurs after fertilization and is essential for egg activation. How the sperm triggers this calcium signal remains, however, to be determined. In this brief review, we compare the mechanisms that are common to mammalian and invertebrate systems. In the light of our own data, we discuss how integrins that have recently been proposed to mediate sperm-egg binding and fusion in mammals might trigger egg activation through cytoskeletal structures. We suggest a model, leading to the calcium signal and common to all species, where phosphorylation on tyrosine and phospholipase Cγ (PLCγ) are interconnected pathways stimulated by a multimolecular complex involving integrins.     

Acid release,cytoplasmic alkalinization,and chromosome condensation during sea urchin fertilization and amine-lnduced parthenogenesis

We have studied the relationship between acid release, cytoplasmic alkalinization, and the extent of chromosome condensation during parthenogenetic activation of sea urchin eggs. The relative rate of acid release in Strongylocentrotus purpuratus eggs was determined from pH measurements of egg suspensions. Acid release in inseminated eggs began after a lag of 0.4 min and the relative rate increased 108-fold, declined, and release was essentially complete by 8-min postinsemination. An average of 3.8 ± 0.23 × 10^?12moles H⁺ cell^? was released as determined by backtitration with NaOH. Acid release characteristics of eggs parthenogenetically activated with either NH₄C1, methylamine ethylamine, n-propylamine, n-butylamine, or benzylamine were qualitatively similar. There was no detectable lag peroid and the increase in relative rate of acid release was directly proportional to the carbon number of the amine used, eg, from 8.3-fold methylamine to 470-fold with benzylamine. The total equivalents of acid released ranged from 0.50–8.2 × 10^?12 moles H⁺·cell^? in direct proportion to the concentration of amine used. The degree fo cytoplasmic alkalinization induced as a function of methylamine and benzylamine concentration was determined by pH measurements fo egg homogenates; egg cultures were also prepared for microscopic examination of chromosome condensation. None of the eggs had condensed chromosomes at 0.5-mM methylamine whereas a cytoplasmic alkalinization of 0.6 pH units was observed. Increased methylamine levels up to 10mM resulted in chromiosome condensation in only 20% of the eggs. A similar result was found with benzylamine. We conclude that acid release and cytoplasmic alkalinization during chemical parthenogenesis are insufficient to mimic sperm induction of chromiosome condensation and suggest that an additional factor(s) is required for chromosome condensation by low concentration of amines.     

Sensitivity of sea urchin fertilization to pH varies across a natural pH mosaic

In the coastal ocean, temporal fluctuations in pH vary dramatically across biogeographic ranges. How such spatial differences in pH variability regimes might shape ocean acidification resistance in marine species remains unknown. We assessed the pH sensitivity of the sea urchin Strongylocentrotus purpuratus in the context of ocean pH variability. Using unique male–female pairs, originating from three sites with similar mean pH but different variability and frequency of low pH (pH_T ≤ 7.8) exposures, fertilization was tested across a range of pH (pH_T 7.61–8.03) and sperm concentrations. High fertilization success was maintained at low pH via a slight right shift in the fertilization function across sperm concentration. This pH effect differed by site. Urchins from the site with the narrowest pH variability regime exhibited the greatest pH sensitivity. At this site, mechanistic fertilization dynamics models support a decrease in sperm–egg interaction rate with decreasing pH. The site differences in pH sensitivity build upon recent evidence of local pH adaptation in S. purpuratus and highlight the need to incorporate environmental variability in the study of global change biology.     

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.     

Unequal divisions at the third cleavage increase the number of primary mesenchyme cells in sea urchin embryos

To clarify the distribution and behavior of the maternal factors that direct the differentiation of primary mesenchyme cells (PMC) in sea urchin embryos, unequal division was induced at the third cleavage with the treatment of dinitro-phenol (DNP), and the numbers of differentiated PMC were examined. The most surprising finding was that the number of PMC was considerably increased in some of the DNP-treated embryos. This increase in the number of PMC was suggested to be closely related to the size of the precocious micromeres formed at the 8-cell stage. By measuring both the size of the precocious micromeres and the number of PMC in individual embryos, it was suggested that almost all the descendants of the precocious micromeres differentiated into PMC, if the volume was less than 26 pL (about three times the volume of normal micromeres). Cell tracing experiments ascertained that precocious micromeres with small volumes behave just like micromeres formed at the fourth cleavage in normal embryos. The obtained results indicated that the maternal factors present in sea urchin embryos can direct, at least, more than three times the number of PMC, and that the number of cell divisions of the PMC lineage is not strictly regulated.     

Fate of the sea urchin egg receptor for sperm following fertilization

The sea urchin egg receptor for sperm is a 350 kDa glycoprotein containing a large extracellular domain that contains the sperm binding site, a transmembrane domain and a short COOH- terminal intracellular domain. During oogenesis, the receptor protein is first detected in Golgi-associated vesicles and cortical granules. Not until the egg is mature does the receptor appear on the cell surface; at this stage the intact receptor is found in approximately equal quantities on the egg cell surface and in cortical granules. As a potentially unique type of receptor, we were interested in its fate following fertilization. Several techniques have revealed that, following sperm binding, the amount of receptor markedly decreases. Using western blot analysis as well as direct measurement of the receptor protein, it was found that the membrane-bound form of the receptor rapidly disappeared following sperm binding to the egg, with only 3% of the receptor remaining after 30 s. Analysis by immupoelectron microscopy revealed that 30 s after sperm binding, 30% of the initial level of receptor was present. This remaining 30% was found mostly within the perivitelline space formed by the raised fertilization envelope. The disparity between these two sets of results (i.e. 3 vs 30%) is most likely accounted for by the exocytosis of receptor molecules from cortical granules; this fraction of the receptor would have been lost during isolation of the membrane-bound form of the receptor. Thus, unlike other cell surface receptors, the sea urchin egg receptor for sperm is not endocytosed and recycled following ligand binding. Rather, it disappears, presumably as a result of proteolysis. Transiently, the cortical granule form of the receptor is found released into the perivitelline space where it may bind to sperm and thereby prevent polyspermy. Despite the apparent secretion of this form of the receptor, experiments with antibodies to the extracellular and intracellular domains indicate that the receptors in cortical granules and in the plasmic membrane are similar, if not identical.     

Change in the adhesive properties of blastomeres during early cleavage stages in sea urchin embryo

Blastomeres of sea urchin embryo change their shape from spherical to columnar during the early cleavage stage. It is suspected that this cell shape change might be caused by the increase in the adhesiveness between blastomeres. By cell electrophoresis, it was found that the amount of negative cell surface charges decreased during the early cleavage stages, especially from the 32-cell stage. It was also found that blastomeres formed lobopodium-like protrusions if the embryos were dissociated in the presence of Ca2+. Interestingly, a decrease in negative cell surface charges and pseudopodia formation first occurred in the descendants of micromeres and then in mesomeres, and last in macromeres. By examining the morphology of cell aggregates derived from the isolated blastomeres of the 8-cell stage embryo, it was found that blastomeres derived from the animal hemisphere (mesomere lineage) increased their adhesiveness one cell cycle earlier than those of the vegetal hemisphere (macromere lineage). The timing of the initiation of close cell contact in the descendants of micro-, meso- and macromeres was estimated to be 16-, 32- and 60-cell stage, respectively. Conversely, the nucleus-to-cell-volume ratios, which are calculated from the diameters of the nucleus and cell, were about 0.1 when blastomeres became adhesive, irrespective of the lineage.     

Flagellasialin: a novel sulfated alpha2,9-linked polysialic acid glycoprotein of sea urchin sperm flagella

A novel alpha2,9-linked polysialic acid (polySia)-containing glycoprotein of sea urchin sperm flagella was identified and named "flagellasialin." Flagellasialin from Hemicentrotus pulcherrimus shows a diverse relative molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of 40-80 kDa. Flagellasialin is a 96-amino acid, threonine-rich, heavily O-glycosylated (80-90% by weight) glycoprotein with a single transmembrane segment at its C-terminus and no apparent cytosolic domain. Of 12 extracellular Thr residues, eight are O-glycosylated and three are nonglycosylated. Flagellasialin is highly expressed in the testis but cannot be detected in the ovary. The amino acid sequences of flagellasialin from three sea urchin species (H. pulcherrimus, Strongylocentrotus purpuratus, and Strongylocentrotus franciscanus) are identical, but some species differences exist in the three core glycan structures to which the sulfated alpha2,9-linked polyNeu5Ac chain is linked. Finally, the treatment of sperm with a specific antibody against the alpha2,9-linked polyNeu5Ac structure results in the elevation of intracellular Ca(2+) and inhibition of sperm motility and fertilization, implicating flagellasialin as a regulator of these critical processes.