Benzohydroxamic acid induces polyspermic fertilization in the sea urchin Arbacia punctulata

Benzohydroxamic acid (BHA) is a competitive inhibitor of the sea urchin sperm peroxidase. We now report that addition of BHA to fertilization cultures of Arbacia punctulata promotes polyspermy. This effect is dose and sperm density dependent. The cortical reaction (elevation of the fertilization envelope) is not retarded by BHA. BHA must be added to the cultures before the eggs complete the cortical reaction at 60 sec post insemination in order to induce polyspermy. Since sea urchin eggs release H2O2 during the cortical reaction at fertilization, these findings support our hypothesis that the sperm peroxidase has a functional role in helping to prevent polyspermy.     

ELECTRICAL POLYSPERMY BLOCK IN SEA URCHINS: NICOTINE AND LOW SODIUM EXPERIMENTS1

Nicotine reduces the amplitude of the fertilization potential in sea urchin eggs, at least in part because it decreases the slope of the current voltage relation of the unfertilized egg membrane. The reduced fertilization potential amplitude provides an electrophysiological explanation for previous observations that nicotine impairs the fast block to polyspermy. The block to polyspermy is also impaired by fertilization in low sodium sea water, a medium which has been reported to reduce fertilization potential amplitude.     

Major components of a sea urchin block to polyspermy are structurally and functionally conserved

One sperm fusing with one egg is requisite for successful fertilization; additional sperm fusions are lethal to the embryo. Because sperm usually outnumber eggs, evolution has selected for mechanisms that prevent this polyspermy by immediately modifying the egg extracellular matrix. We focus here on the contribution of cortical granule contents in the sea urchin block to polyspermy to begin to understand how well this process is conserved. We identified each of the major constituents of the fertilization envelope in two species of seaurchins, Strongylocentrotus purpuratus and Lytechinus variegatus, that diverged 30 to 50 million years ago. Our results show that the five major structural components of the fertilization envelope, derived from the egg cortical granules, are semiconserved. Most of these orthologs share sequence identity and encode multiple low-density lipoprotein receptor type A repeats or CUB domains but at least two contain radically different carboxy-terminal repeats. Using a new association assay, we also show that these major structural components are functionally conserved during fertilization envelope construction. Thus, it seems that this population of female reproductive proteins has retained functional motifs while gaining significant sequence diversity-two opposing paths that may reflect cooperativity among the proteins that compose the fertilization envelope.     

Studies of the mechanism of the electrical polyspermy block using voltage clamp during cross-species fertilization

Prevention of polyspermic fertilization in sea urchins (Jaffe, 1976, Nature (Lond.). 261:68-71) and the worm Urechis (Gould-Somero, Jaffe, and Holland, 1979, J. Cell Biol. 82:426-440) involves an electrically mediated fast block. The fertilizing sperm causes a positive shift in the egg's membrane potential; this fertilization potential prevents additional sperm entries. Since in Urechis the egg membrane potential required to prevent fertilization is more positive than in the sea urchin, we tested whether in a cross-species fertilization the blocking voltage is determined by the species of the egg or by the species of the sperm. With some sea urchin (Strongylocentrotus purpuratus) females, greater than or equal to 90% of the eggs were fertilized by Urechis sperm; a fertilization potential occurred, the fertilization envelope elevated, and sometimes decondensing Urechis sperm nuclei were found in the egg cytoplasm. After insemination of sea urchin eggs with Urechis sperm during voltage clamp at +50 mV, fertilization (fertilization envelope elevation) occurred in only nine of twenty trials, whereas, at +20 mV, fertilization occurred in ten of ten trials. With the same concentration of sea urchin sperm, fertilization of sea urchin eggs occurred, in only two of ten trials at +20 mV. These results indicate that the blocking voltage for fertilization in these crosses is determined by the sperm species, consistent with the hypothesis that the fertilization potential may block the translocation within the egg membrane of a positively charged component of the sperm.     

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.     

Anti-inflammatory drugs promote polyspermic fertilization in sea urchins

Sea urchin eggs are known to release H₂O₂ during the cortical reaction at fertilization to help prevent polyspermy by inactivating excess sperm in the vicinity. This process resembles the peroxidatic killing of bacteria by phagocytic leukocytes during inflammation. Associated with these reactions in leukocytes, arachidonic acid is released from phospholipids and can be oxidized via the cyclooxygenase pathway to produce prostaglandins. Nonsteroidal anti-inflammatory drugs (NSAID) that are cyclooxygenase inhibitors in somatic cells were used to determine whether Arbacia punctulata and Strongylocentrotus purpuratus eggs use these processes to help prevent polyspermy. The potent cyclooxygenase inhibitor indomethacin causes a dose (10–100 μM) and sperm density dependent induction of polyspermy if added before the egg completes the cortical reaction. It does not retard elevation of the fertilization envelope and does not promote polyspermy by protecting sperm from peroxidatic inactivation by egg-derived H₂O₂. Other potent cyclooxygenase inhibitors, flufenamate and meclofenamate, also induce polyspermy at 10–60 μM. Aspirin, a weak cyclooxygenase inhibitor in somatic cells, does not cause polyspermy at 5 mM. These findings provide evidence that prostaglandins or other cyclooxygenase-derived metabolites may help assure monospermic fertilization in sea urchins.     

Proteolytic cleavage of the cell surface protein p160 is required for detachment of the fertilization envelope in the sea urchin

Sea urchin eggs secrete a serine protease activity, CGSP1, at fertilization that is essential for the block to polyspermy. Several targets of this proteolytic activity on the plasma membrane were identified here using a cell surface biotinylation approach. Amino acid microsequencing of one of these proteins led to the identification of a 4.75-kb cDNA clone from a Strongylocentrotus purpuratus ovary cDNA library that encodes a 160-kDa protein called p160. This protein contains five CUB domains and a putative transmembrane domain suggesting that p160 is an integral membrane protein with protein-protein interaction motifs facing the extracellular matrix of the egg. Whole-mount immunolocalization studies demonstrate that p160 is on the surface of the egg, enriched at the tips of microvilli. The protein is removed at fertilization in a protease-dependent manner, and functional assays suggest that p160 serves to link the plasma membrane to the vitelline layer until fertilization. Thus, p160 is a key candidate for a vitelline-layer linker protein, the selective proteolysis of which functions in the block to polyspermy in the sea urchin egg.     

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.     

Deprenyl, an Inhibitor of Monoamine Oxidase B, Delays the First Two Mitotic Cycles of Sea Urchin eggs

Sea urchin eggs are known to display a significant level of monoamine oxidase B activity. Deprenyl, a highly selective inhibitor of monoamine oxidase B, was found to prolong the length of the first two mitotic cycles of the sea urchin egg. The prophase seems to be specifically prolonged by the drug, while the length of the other phases of the cycle is unaffected. These results suggest that the monoamine oxidase B may be a component of the monoaminergic system acting during the divisions of the sea urchin eggs.     

Cold shock induces actin reorganization and polyspermy in sea urchin eggs

The effect of low temperature on the cortical actin system and polyspermy in sea urchin eggs was studied. Eggs cold-shocked at 3 degrees C for 1 hour formed a cortical system of polymerized actin and were polyspermic when fertilized. These effects were completely reversible following a 7-20 minute recovery period at room temperature. The present data raises the possibility that actin, or components of the egg cytoskeleton, regulate sperm entry in sea urchin eggs.     

Rearrangements of sea urchin egg cytoplasmic membrane domains at fertilization

Fertilization in the sea urchin is accompanied by rapid reorganization of the egg endoplasmic reticulum (ER). ER-derived vesicles contribute to one of three classes of membranes used in assembling the male pronuclear envelope in vitro. We provide here biochemical evidence for the rearrangement of sea urchin egg cytoplasmic membrane domains at fertilization up to the first mitosis, with respect to two nuclear envelope markers, lamin B and lamin B receptor (LBR), using purified vesicles prepared from homogenates fractionated by floatation on sucrose gradients. In unfertilized eggs, immunoprecipitation data indicate that most of lamin B and LBR are localized in the same vesicles but do not interact. By 3 min post-fertilization, both proteins are more widely distributed across the gradients and by 12 min most of lamin B and LBR are localized in vesicles of different densities. This partitioning is maintained throughout S phase. At mitosis, most lamin B and LBR remain in distinct vesicles, while a small proportion of lamin B and LBR, likely derived from the disassembled nuclear envelope, associate in a minor subset of vesicles. The results illustrate a dynamic reorganization of egg cytoplasmic membranes at fertilization, and the establishment of distinct membrane domains enriched in specific nuclear envelope markers during the first cell cycle of sea urchin development. Additionally, we demonstrate that male pro-nuclear membrane assembly occurs only when both cytosol and membranes originate from fertilized but not unfertilized eggs, suggesting that fertilization-induced membrane rearrangements contribute to the ability of the egg to assemble the male pronuclear envelope.     

Mastoparan induces Ca2+-independent cortical granule exocytosis in sea urchin eggs

In most species, cortical granule exocytosis is characteristic of egg activation by sperm. It is a Ca(2+)-mediated event which results in elevation of the vitelline coat to block permanently the polyspermy at fertilization. We examined the effect of mastoparan, an activator of G-proteins, on the sea urchin egg activation. Mastoparan was able to induce, in a concentration-dependent manner, the egg cortical granule exocytosis; mastoparan-17, an inactive analogue of mastoparan, had no effect. Mastoparan, but not sperm, induced cortical granule exocytosis in eggs preloaded with BAPTA, a Ca(2+) chelator. In isolated egg cortical lawns, which are vitelline layers and membrane fragments with endogenously docked cortical granules, mastoparan induced cortical granule fusion in a Ca(2+)-independent manner. By contrast, mastoparan-17 did not trigger fusion. We conclude that in sea urchin eggs mastoparan stimulates exocytosis at a Ca(2+)-independent late site of the signaling pathway that culminates in cortical granule discharge.     

False fertilization in sea urchin eggs induced by diabolin, a 120K kelp protein

A 120K lectin-like protein was isolated from the kelp Laminaria diabolica (Oni-kombu), with a unique activity to induce false fertilization specifically in the eggs of the sea urchin Hemicentrotus pulcherrimus. The protein designated as "diabolin" rendered the unfertilized egg forms and elevated the fertilization envelope without insemination at 18 nM half-maximally. Those eggs with elevated fertilization envelopes, however, could not enter into normal cleavage or further development, and hence the proliferation of the sea urchin was hindered. Diabolin, thus, by its unique defense mechanism protects the kelp from the predator sea urchin. It was partially sequenced and found to have the highest homology with phytoene dehydrogenase from the plant virus Erwinia uredovora. A question was left to be solved as to how the kelp on the southeast coast of Hokkaido Island could develop the defense mechanism against the sea urchin on Honshu Island separated by Tsugaru Straits.     

The Chaetopterus vitelline envelope is not necessary for the gamete interactions that lead to fertilization

It has been recently shown that, in several genera of annelids, including Chaetopterus, fertilizing sperm attach to and fuse with egg microvilli which penetrate the vitelline envelope. This suggests that the annelid vitelline envelope may have no direct or obligatory role in normal fertilization. The present study was undertaken to investigate the involvement of the vitelline envelope in fertilization in Chaetopterus experimentally, by examining the fertilization of vitelline envelope-free eggs quantitatively and qualitatively. Brief exposure of the eggs to isotonic sucrose-EDTA removed the vitelline envelope as determined by both phase-contrast and electron microscopy, rendered the eggs more sensitive to polyspermy and substantially reduced the binding of supernumerary sperm to eggs but did not decrease fertilizability as determined by sperm dilution assay and did not make the eggs more sensitive to cross-fertilization. The events of fertilization were examined by electron microscopy and found to be very similar in vitelline envelope-free eggs to those in intact eggs. We conclude that the vitelline envelope in Chaetopterus has binding sites for sperm but that it has no obligatory role in fertilization and is primarily involved in the prevention of polyspermy.     

Sulfated acid mucopolysaccharides in the cortical granules of eggs. Effects of quaternary ammonium salts on fertilization

Sulfated acid mucopolysaccharides have been shown to be constituents of cortical granules in sea urchin and vertebrate eggs. These observations were made possible by retaining soluble acid mucopolysaccharides in situ within the eggs by precipitation during fixation with cetyltrimethyl-ammonium bromide, a quaternary ammonium salt. The sulfated mucopolysaccharides were then identified by staining with Astrablau at pH 0.2 and also by reaction with sodium rhodizonate. Staining reaction with Alcian blue at pH 2.5 showed that carboxylated mucopolysaccharides may also be present in cortical granules. The natural ionic environment of these eggs would favor the formation of very stable complexes between sulfated mucopolysaccharides and quaternary ammonium salts. Brief exposure of unfertilized sea urchin eggs to several quaternary ammonium compounds produced a residual adverse effect on subsequent fertilization in terms of increased vulnerability to polyspermy and reduced fertilizability. These results suggest that sulfated acid mucopolysaccharides participate in the function of the cortical granules and the establishment of the block to polyspermy at fertilization, and possibly in other cellular secretory processes.     

U.V. Irradiation Inhibits The Electrical Block to Polyspermy in Echinoderms*

Oocytes of the sea urchin Sphaerechinus granularis and the startish Marthasterias glacialis have been submitted to U.V. irradiation before fertilization. This treatment significantly increased the incidence and severity of polyspermy in the sea urchin and was also found effective on starfish oocytes. These were found more resistant to damage than sea urchin eggs and U.V. irradiation did not affect either their response to the hormone l-methyladenine or the rate of elevation of the fertilization envelope, which assures the late and definitive block to polyspermy. Electrophysiological measurements performed on M. glacialis oocytes definitively demonstrate that U.V. irradiation completely inactivates voltage-dependent sodium channels, without altering the other main conductances, Cl^?, K⁺ or Ca²⁺. After such a treatment, the relative permeability of the membrane to Na⁺ as compared to K⁺ shifted from 0.019±0.003 to 0.003±0.002 and only the calcium component of the action potentials could be observed. However, a fertilization potential, preceded by small sperm induced steps, is still present in these conditions, although its peak and plateau level are greatly reduced. These new findings are discussed, which confirm the electrical nature of the fast block to polyspermy but question about the specificity of those sperm-gated channels which are supposed to trigger the fertilization potential.     

Propranolol, a beta-adrenergic receptor blocker, affects microfilament organization, but not microtubules, during the first division in sea urchin eggs

Propranolol, a beta-adrenergic receptor blocker, blocks the formation of the cleavage furrow, while karyokinesis is unaffected during first division in the sea urchins Paracentrotus lividus or Lytechinus pictus. This effect is reversed by adrenalin, indicating that it is mediated by an adrenergic mechanism. The staining of F-actin microfilaments by rhodamine phalloidin in eggs in which the cleavage is blocked by the drug has revealed that propranolol affects both the distribution and the organization of actin microfilaments. A low-voltage scanning electron microscopy (LVSEM) study of microvilli in these eggs shows an extensive rearrangement of the egg surface. Anti-tubulin immunofluorescence microscopy of eggs treated with propranolol shows that they form normal mitotic asters. This indicates that while cleavage is affected, mitotic spindle formation is not. These results suggest that neurotransmitter monoamines known to be present in the sea urchin egg might be involved in the reorganization of the actin cytoskeleton underlying the formation of the cleavage furrow.     

Proteolysis of Xenopus laevis egg envelope ZPA triggers envelope hardening

The egg envelope of most animal eggs is modified following fertilization, resulting in the prevention of polyspermy and hardening of the egg envelope. In frogs and mammals a prominent feature of envelope modification is N-terminal proteolysis of the envelope glycoprotein ZPA. We have purified the ZPA protease from Xenopus laevis eggs and characterized it as a zinc metalloprotease. Proteolysis of isolated egg envelopes by the isolated protease resulted in envelope hardening. The N-terminal peptide fragment of ZPA remained disulfide bond linked to the ZPA glycoprotein moiety following proteolysis. We propose a mechanism for egg envelope hardening involving ZPA proteolysis by an egg metalloprotease as a triggering event followed by induction of global conformational changes in egg envelope glycoproteins.     

Protein tyrosine kinase-dependent release of intracellular calcium in the sea urchin egg

The aminoguanide, methylglyoxal bis(guanylhydrazone) (MGBG), was shown to stimulate phosphorylation of RR-SRC, a synthetic protein tyrosine kinase (PTK) substrate, and different levels of tyrosyl phosphorylation of endogenous proteins in a sea urchin egg membrane-cortex preparation. Stimulating protein tyrosine kinase activity in the sea urchin egg stimulated intracellular Ca2+ release, because microinjection of 1-5 mM of MGBG into unfertilized eggs triggered a transient rise in intracellular Ca2+ activity ([Ca2+]i) after a brief latent period. Pretreating eggs with PTK-specific inhibitors, genistein or tyrphostin B42, significantly inhibited the MGBG-induced rise in [Ca2+]i. Methylglyoxal bis(guanylhydrazone) stimulation of PTK activities in the unfertilized sea urchin egg appeared to trigger Ca2+ release through phospholipase C (PLC)-dependent inositol 1,4,5-trisphosphate (InsP3) production. The MGBG-induced Ca2+ response could be suppressed in eggs preloaded with the InsP3 receptor antagonist, heparin, and was reduced in eggs pretreated with U73122, a PLC inhibitor. However, the response was unchanged in eggs treated with nicotinamide, an inhibitor of ADP-ribosyl cyclase, or nifedipine, an inhibitor of nicotinic acid adenine dinucleotide phosphate activity. These results suggest that MGBG may be useful as a chemical agonist of PTK in sea urchin eggs and allow direct testing of the PTK requirement for the transient rise in [Ca2+]i in sea urchin eggs during fertilization. Although genistein was observed to significantly delay the onset, the sperm-induced Ca2+ response in PTK inhibitor-loaded eggs otherwise appeared normal. Therefore, it was concluded that sea urchin eggs contain a PTK-dependent pathway that can mediate intracellular Ca2+ release, but PTK activity does not appear to be required for the fertilization response.     

Free-radical crosslinking of specific proteins alters the function of the egg extracellular matrix at fertilization

All animal embryos begin development by modifying the egg extracellular matrix. This protein-rich matrix protects against polyspermy, microbes and mechanical stress via enzyme-dependent transformations that alter the organization of its constituents. Using the sea urchin fertilization envelope, a well-defined extracellular structure formed within minutes of fertilization, we examine the mechanisms whereby limited permeability is established within this matrix. We find that the fertilization envelope acquires a barrier filtration of 40,000 daltons within minutes of insemination via a peroxidase-dependent mechanism, with dynamics that parallel requisite production of hydrogen peroxide by the zygote. To identify the molecular targets of this free-radical modification, we developed an in vivo technique to label and isolate the modified matrix components for mass spectrometry. This method revealed that four of the six major extracellular matrix components are selectively crosslinked, discriminating even sibling proteins from the same gene. Thus, specific free-radical chemistry is essential for establishing the embryonic microenvironment of early development.