Acrosome Reaction In Sea Urchin Sperm Research Articles

Identification of voltage-dependent Ca 2+ channels in sea urchin sperm

Functional evidence indicates that voltage-dependent Ca 2+ (Ca v) channels participate in sea urchin sperm motility and the acrosome reaction (AR), however, their molecular identity remains unknown. We have identified transcripts for two Ca 2+ channel α1 subunits in sea urchin testis similar in sequence to Ca v1.2 and Ca v2.3. Antibodies against rat Ca v1.2 and Ca v2.3 channels differentially label proteins in the flagella and acrosome of mature sea urchin sperm. The Ca v channel antagonists nifedipine and nimodipine, which inhibit the AR, diminish the intracellular Ca 2+ elevation induced by a K +-induced depolarization in valinomycin-treated sperm. These findings reveal that Ca v1.2 and Ca v2.3 channels could participate in motility and/or the AR in sea urchin sperm.

An ATP-binding Cassette Transporter Is a Major Glycoprotein of Sea Urchin Sperm Membranes

Sperm are terminally differentiated cells that undergo several membrane-altering events before fusion with eggs. One event, the sea urchin sperm acrosome reaction (AR), is blocked by the lectin wheat germ agglutinin (WGA). In an effort to identify proteins involved in the AR induction, the peptide sequence was obtained from a 220-kDa WGA-binding protein. Degenerate PCR and library screening resulted in the full-length deduced amino acid sequence of an ATP-binding cassette transporter, suABCA. The protein of 1,764 residues has two transmembrane regions, two nucleotide-binding domains, and is most closely related to the human ABC subfamily A member 3 transporter (ABCA3). Sequence analysis suggests a large extracellular loop between transmembrane spanning segments 7 and 8, with five N-linked glycosylation sites. An antibody made to the loop region binds to non-permeabilized cells, supporting that this region is extracellular. suABCA is found in sperm membrane vesicles, it can be solubilized with nonionic detergents, and it shifts from 220 to 200 kDa upon protein:N-glycanase F digestion. suABCA localizes to the entire surface of sperm in a punctate pattern, but is not detected in lipid rafts. Based on its relationship to subfamily A, suABCA is most likely involved in phospholipid or cholesterol transport. This is the first investigation of an ABC transporter in animal sperm.

Egg fucose sulfate polymer, sialoglycan, and speract all trigger the sea urchin sperm acrosome reaction

Macromolecules surrounding eggs induce the acrosome reaction (AR) of spermatozoa. In sea urchins, three egg jelly (EJ) molecules: a fucose sulfate polymer (FSP), a sialoglycan (SG), and speract mediate ionic fluxes triggering the AR. SG and speract are noninductive without FSP. Speract's role in AR induction is controversial. Here we show that speract potentiates the FSP-induced AR at pH 7.0, ∼1 pH unit lower than natural seawater. At pH 7.0, a mixture of FSP, SG, and speract produces the intracellular pH increase necessary for maximum AR induction. Each EJ component may mediate a distinct intracellular pH control mechanism, and all three may function synergistically to increase the intracellular pH permitting AR induction. Speract peptides are an ancient family. Although important for activating cyclic nucleotide-mediated pathways in today's seawater of pH ∼8, speract may have been more important in AR induction in the paleo-ocean of pH ∼7.

SuREJ3, a Polycystin-1 Protein, Is Cleaved at the GPS Domain and Localizes to the Acrosomal Region of Sea Urchin Sperm

The sea urchin sperm acrosome reaction (AR) is a prerequisite for sperm-egg fusion. This report identifies sea urchin sperm receptor for egg jelly-3 (suREJ3) as a new member of the polycystin-1 family (the protein mutated in autosomal dominant polycystic kidney disease). suREJ3 is a multidomain, 2,681-amino acid, heavily glycosylated orphan receptor with 11 putative transmembrane segments (TMS) that localize to the plasma membrane covering the sperm acrosomal vesicle. Like the latrophilins and other members of the secretin family of G-protein-coupled receptors, suREJ3 is cleaved at the consensus GPS (G-protein-coupled receptor proteolytic site) domain. Antibodies to the extracellular 1,455-residue NH(2)-terminal portion identify a band at 250 kDa that shifts in electrophoretic mobility to 180 kDa upon glycosidase digestion. Antibodies to the 1,226-residue COOH-terminal portion identify a band at 150 kDa that shifts to 140 kDa after glycosidase treatment. Antibodies to both portions of suREJ3 localize exclusively to the plasma membrane over the acrosomal vesicle. Immunoprecipitation shows that both portions of suREJ3 are associated in detergent extracts. This is the first report showing that a polycystin family member is cleaved at the GPS domain. Localization of suREJ3 to the acrosomal region provides the first suggestion for the role of a polycystin-1 protein (components of nonselective cation channels) in a specific cellular process.

High Molecular Mass Egg Fucose Sulfate Polymer Is Required for Opening Both Ca2+ Channels Involved in Triggering the Sea Urchin Sperm Acrosome Reaction

A linear fucose sulfate polymer (FSP), >10(6) daltons, is a major component of sea urchin egg jelly. FSP induces the sperm acrosome reaction (AR), an exocytotic process required for animal fertilization. Two Ca(2+) channels activate during AR induction, the first opens 1 s after FSP addition, and the second opens 5 s after the first. Mild acid hydrolysis of FSP results in a linear decrease in polymer size. The ability of FSP to induce the AR and activate sperm Ca(2+) channels decreases with increasing time of hydrolysis. Hydrolyzed FSP of approximately 60 kDa blocks intact FSP from inducing the AR. At 44 microg/ml hydrolyzed FSP, Ca(2+) entry into sperm is almost equal to that occurring in 3.8 microg/ml intact FSP; however the AR is not induced. The shape of the [Ca(2+)](i) increase curve and use of the Ca(2+) channel blockers nifidipine and Ni(2+) indicate that hydrolyzed FSP opens the second Ca(2+) channel, but not the first, and thus does not induce the AR. The giant size of intact FSP is required to open both Ca(2+) channels involved in triggering the AR.

The Fucose Sulfate Polymer of Egg Jelly Binds to Sperm REJ and Is the Inducer of the Sea Urchin Sperm Acrosome Reaction

The sea urchin sperm cell is an advantageous model for studying ligand-mediated exocytosis. Sperm can be obtained in vast quantities and induced to undergo exocytosis of the acrosomal vesicle with great synchrony. During sea urchin fertilization, egg jelly (EJ) triggers the sperm acrosome reaction (AR) which is required for sperm binding and fusion with the egg. Uncertainty exists as to the exact biochemical nature of the AR inducer. The following study was performed in an attempt to clarify the nature of the inducer. EJ from individual females (Strongylocentrotus purpuratus) was analyzed on SDS–PAGE gels. Each female had a unique composition of EJ macromolecules, but all females possessed the previously described fucose sulfate polymer (FSP). Two electrophoretic isotypes of FSP were discovered; 87% of females had only one isotype and 13% had both. Both FSP isotypes bound to the REJ protein (receptor for egg jelly) purified from sperm. The two FSP isotypes had almost equal potency in inducing the AR. EJ was fractionated by DEAE chromatography in 6 M urea/4% β-mercaptoethanol. All AR-inducing activity coeluted with FSP. FSP, purified by trypsin digestion followed by dialysis, was twice as active as the non-trypsin-digested control at inducing the AR. EJ was digested with proteinase K, boiled in detergent and β-mercaptoethanol, and subjected to sucrose density gradient sedimentation. The FSP and AR activity had superimposable sedimentation patterns. Purified FSP had no associated peptide component. Sperm from individual males differed in the concentration dependency of purified FSP to induce the AR. The data indicate that the 138/82 kDa EJ glycoproteins, previously thought to act as AR inducers, do not appear to be involved in triggering the AR. The data are consistent with the hypothesis that FSP is the only inducer of the AR of this sea urchin species.

Inhibition of the sea urchin sperm acrosome reaction by a lignin-derived macromolecule

The major organic components of effluents from commercial pulping processes are lignin-derived macromolecules (LDMs), which have recently been shown to inhibit fertilization and embryonic development in a variety of marine organisms, as well as to exhibit immunostimulating activity in mammalian cells. We conducted studies on the effects of an isolated LDM from bleached kraft mill effluent (BKME), and its sub-components, at the cellular level utilizing the purple sea urchin ( Strongylocentrotus purpuratus) sperm acrosome reaction (AR) as an experimental system. The AR is an event that is induced by the egg's jelly coat and is prerequisite for successful fertilization. Sperm were preincubated with increasing concentrations of isolated LDM or electrophoretically purified LDM sub-components, followed by addition of isolated egg jelly to induce the AR in vitro. These LDM preparations significantly inhibited the AR as assessed by fluorescence (utilizing the rhodamine-conjugated phallicidin) and transmission electron microscopy. Preincubation of sperm with LDM did not have any effect on sperm motility. The level of AR inhibition was comparable to that observed in experiments assessing successful fertilization. The ability of LDM to inhibit jelly induced AR was overcome by the calcium ionophores A23187 and ionomycin. In addition, LDM was shown to inhibit the normal increase in intracellular calcium (Ca ++) associated with induction of the AR. When eggs were preincubated with LDM prior to addition of unexposed sperm, no effect on fertilization was observed, indicating that LDM specifically affects sperm function during fertilization. Fine structural studies, utilizing biotinylated LDM, confirmed LDM's specificity and revealed that its binding was restricted to the plasma membrane domain of the sperm head. The present observations on the inhibition of the AR by LDM is consistent with our hypothesis that this macromolecule inhibits the AR by blocking egg jelly interaction with the sperm surface, thus inhibiting ionic events such as increases in intracellular calcium. Our present approach also demonstrates that echinoderm sperm functions can be used as a model system for the investigation of the mode of action of toxicants at the sub-cellular level.

The sea urchin sperm receptor for egg jelly is a modular protein with extensive homology to the human polycystic kidney disease protein, PKD1.

During fertilization, the sea urchin sperm acrosome reaction (AR), an ion channel-regulated event, is triggered by glycoproteins in egg jelly (EJ). A 210-kD sperm membrane glycoprotein is the receptor for EJ (REJ). This conclusion is based on the following data: purified REJ binds species specifically to EJ dotted onto nitrocellulose, an mAb to REJ induces the sperm AR, antibody induction is blocked by purified REJ, and purified REJ absorbs the AR-inducing activity of EJ. Overlapping fragments of REJ cDNA were cloned (total length, 5,596 bp). The sequence was confirmed by microsequencing six peptides of mature REJ and by Western blotting with antibody to a synthetic peptide designed from the sequence. Complete deglycosylation of REJ followed by Western blotting yielded a size estimate in agreement with that of the mature amino acid sequence. REJ is modular in design; it contains one EGF module and two C-type lectin carbohydrate-recognition modules. Most importantly, it contains a novel module, herein named the REJ module (700 residues), which shares extensive homology with the human polycystic kidney disease protein (PKD1). Mutations in PKD1 cause autosomal dominant polycystic kidney disease, one of the most frequent genetic disease of humans. The lesion in cellular physiology resulting from mutations in the PKD1 protein remains unknown. The homology between REJ modules of the sea urchin REJ and human PKD1 suggests that PKD1 could be involved in ionic regulation.

Evidence suggesting a role for sperm metalloendoprotease activity in penetration of zona-free hamster eggs by human sperm.

It has been reported that metalloendoprotease (MEP) activity is involved in somatic cell membrane fusion events and in the sea urchin sperm acrosome reaction (AR). MEP activity also has been demonstrated in human and other mammalian sperm. The present study was concerned with investigating whether a human sperm MEP is important in membrane events necessary for sperm egg fusion. Ejaculated human sperm were washed, capacitated in vitro, and preincubated with the competitive MEP inhibitors phosphoramidon (50 microM) or CBZ-L-phenylalanine (1 mM), with 100 microM diethylenetriaminepentaacetic acid (DTPA), a heavy metal chelator, or as controls, with the appropriate solvents. The AR was initiated in vitro with preovulatory human follicular fluid and the sperm washed to dilute inhibitors and then coincubated with zona-free golden hamster eggs (zonae and cumuli removed with trypsin and hyaluronidase, respectively). Eggs were washed after 0.5 h, and the number of sperm remaining bound was counted. After 2.5 h further incubation, the eggs were stained with acetolacmoid or acetoorcein and penetration was assayed by counting the number of decondensed sperm heads per egg (penetration index) and the percent of penetrated eggs. The inhibitor treatments did not decrease the percentage of penetrated eggs (range 80-90%), but a significant reduction in the penetration index was observed. Phosphoramidon reduced the penetration index by 45%, CBZ-L-phenylalanine by 57%, and DTPA by 56%. None of the inhibitors decreased the penetration index or the percentage of penetrated eggs when added directly to suspensions of acrosome-reacted sperm and zona-free eggs at the diluted levels that would have been present after washing inhibitor-treated sperm.(ABSTRACT TRUNCATED AT 250 WORDS)

A fast transient hyperpolarization occurs during the sea urchin sperm acrosome reaction induced by egg jelly

The egg jelly-induced sea urchin sperm depolarization, assayed with the membrane potential-sensitive dye diS-C 3-(5), is preceded by a fast quenching, which was initially taken as an artifact. Here we show that part of this quenching results from a K + -dependent transient hyperpolarization (∼4 s). Seawater containing 25–35 mM KCl inhibits this hyperpolarization, the depolarization and the acrosome reaction induced by egg jelly, or by the acrosome reaction inducing factor purified therefrom. These results suggest that egg jelly induces a transient hyperpolarization mediated by an increase in K + permeability, which may be involved in triggering the acrosome reaction

Evidence for the involvement of metalloendoproteases in the acrosome reaction in sea urchin sperm.

An essential initial step in fertilization in the sea urchin Strongylocentrotus purpuratus is an intracellular membrane fusion event in the sperm known as the acrosome reaction. This Ca2+-dependent, exocytotic process involves fusion of the membrane of the acrosomal vesicle and the plasma membrane. Recently, metalloendoproteases requiring divalent metals have been implicated in several Ca2+-dependent membrane fusion events in other biological systems. In view of the suggested involvement of Zn2+ in the sea urchin sperm acrosome reaction (Clapper, D.L., Davis, J.A., Lamothe, P.J., Patton, C., and Epel, D. (1985) J. Cell Biol. 100, 1817-1824) and the fact that Zn2+ is a metal cofactor for metalloendoproteases, we investigated the potential role of this protease in the acrosome reaction. A soluble metalloendoprotease was demonstrated and characterized in sperm homogenates using the fluorogenic protease substrate succinyl-alanine-alanine-phenylalanine-4-aminomethylcoumarin. The protease was inhibited by the metal chelators EDTA and 1,10-phenanthroline, and activity of the inactive apoenzyme could be reconstituted with Zn2+. The metalloendoprotease substrate and inhibitors blocked the acrosome reaction induced either by egg jelly coat or by ionophore, but had no effect on the influx of Ca2+. These observations suggest that inhibition occurs at a step independent of Ca2+ entry. Overall, the results of this study provide strong indirect evidence that the acrosome reaction requires the action of metalloendoprotease.

Inhibition of sea urchin sperm acrosome reaction by antibodies directed against two sperm membrane proteins: Characterization and mechanism of action

The egg jelly-induced acrosome reaction is inhibited by polyclonal antibodies raised against either of two S. purpuratus sperm-membrane proteins, of M r 80 and 210 kD. Although the two antigens used have dissimilar CNBr peptide maps, antisera produced against each of them cross-react with both proteins. Inhibition of the egg jelly-induced acrosome reaction by the antisera is bypassed by a combination of the ionophores monensin and A23187. This result, along with data showing that the antisera inhibit egg jelly-induced uptake of 45Ca 2+, suggests that the antisera may block both Ca 2+ uptake and Na + H + exchange in the sperm. The acrosome reaction blockage appears to be caused by the same component of the polyclonal sera responsible for cross-reaction; consequently, these antisera cannot be used to determine whether one or both of the crossreacting proteins modulate a critical step in the acrosome reaction.