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.     

An improved purification method for cytoplasmic dynein

An improved method has been devised for the purification of cytoplasmic dynein from sea urchin eggs (Strongylocentrotus droebachiensis and S purpuratus). This protocol introduces three changes over a previously published procedure (Hisanaga and Sakai: J Biochem 93:87, 1983)--the substitution of diethylaminoethyl (DEAE)-cellulose for hydroxylapatite chromatography, the elimination of sucrose density gradient centrifugation, and the use of phosphocellulose chromatography. These changes reduce the time and increase the efficiency of the purification procedure. The purified egg cytoplasmic dynein has enzymatic properties in common with axonemal dynein, including ionic specificity (Ca++ATPase/Mg++ ATPase = 0.8) and inhibition by sodium vanadate and erythro-9-2,3-hydroxynonyl adenine (EHNA). As assayed by silver staining of polyacrylamide gels, the cytoplasmic dynein is composed of two high molecular weight polypeptides (greater than 300 kilodaltons) that comigrate with flagellar dynein heavy chains, and lesser amounts of three lower molecular weight bands. None of these polypeptides appears to contain bound carbohydrate. The purification procedure can be modified slightly to allow the preparation of cytoplasmic dynein in only 2 days from as little as 3-5 ml of packed eggs, a 20-fold reduction over the previous method. This more rapid and efficient method will facilitate the investigation of cytoplasmic dynein in other systems where starting material is limited, including tissue culture cells and nerve axoplasm.     

Ultrastructural organization of heterochromatin within sea urchin sperm nuclei

Chromatin within swollen or lysed isolated sperm nuclei of the sea urchin, Strongylocentrotus purpuratus, was examined by electron microscopy. Spread preparations of lysed sperm nuclei demonstrated dense aggregates of nondispersed material and beaded filaments radiating from these aggregates. These beaded fibers are similar in size and appearance to the “beads-on-a-string” seen as characteristic of chromatin spreads from numerous interphase nuclei. The beads are nucleosomes that have an average diameter of 130 Å. The interconnecting string is 40 Å indiameter and corresponds to the spacer DNA. In thin sections of swollen nuclei the sperm chromatin appears to be composed of 400 Å superbeads that are closely apposed to form 400 Å fibers. As the chromatin disperses, the superbeads are seen to be attached to one another by chromatin fibers of 110 Å diameter. In thin sections, the 400 Å superbeads appear to disperse directly into the 110 Å fibers with no intervening structures. This work demonstrates that the heterochromatin in Strongylocentrotus purpuratus sperm nuclei is composed of nucleosomes that form 100 Å filaments that are compacted into 400 Å superbeads. The superbeads coalesce to give the morphological appearance of 400 Å fibers.     

Isolation and identification of novel sulfated and nonsulfated oligosialyl glycosphingolipids from sea urchin sperm

Novel sulfated and nonsulfated oligosialyglycosphingolipids were isolated from sperm of the sea urchin,Hemicentrotus pulcherrimus, and their structures were established as follows: ±HSO₃Neu5Ac2(8Neu5Ac2)_n6G1c11´Cer, wheren=0, 1, 2, 3. This provides the first evidence for the natural occurrence of a tetrasialic acid structure in glycosphingolipids. The finding of sulfated oligosialyl chains is especially noteworthy in that the sulfate group exclusively resides on the C-8 of the nonreducing terminal residues of oligo/polysialyl chains and that sulfation appears to be a termination signal for elongation of oligosialyl chains. Sulfation at the nonreducing terminal Neu5Ac residues of oligosialyl chains was also found to facilitate the formation of an inter-residue lactone between the carboxyl group at the nonreducing terminal sulfated Neu5Ac and the hydroxyl group at C-9 of the penultimate Neu5Ac residue. The long chain base was 4-hydroxysphinganine (t18:0) and the major fatty acid species were identified as C20:1, C21:1, and C22:1.Abbreviations C:M chloroform:methanol - FAB-MS fast atom bombardment mass spectrometry - GLC gas-liquid chromatography - GSL glycosphingolipid - HPTLC high performance thin-layer chromatography - Neu5Ac8HSO₃ or HSO₃ Neu5Ac 5-N-acetyl-8-O-sulforylneuraminic acid - Neu5Gc9HSO₃ 5-N-glycolyl-9-O-sulforylneuraminic acid - NMR nuclear magnetic resonance - PB sodium phosphate buffer, pH 7.2 - Sia sialic acid - HSO₃ Sia sulfated sialic acid - polySia polysialic acid - TFA trifluoroacetic acid Since the dividing line between oligo- and polysialic acids is not rigidly defined, we propose here that those containing more than penta-sialic acid chains can be referred to as the polysialic acid group. The rationale for this is that 2 8-linked pentasialic acid is the minimum chain length to exhibit the conformational property of polysialic acid [of Michon, F, Brisson, J-R, Jennings, H (1987)Biochemistry 26: 8399–405] and to act as a substrate for the endosialidase, Endo-N [Hallenbeck, PC, Vimr, ER, Yu, F, Bassler, B, Troy, FA (1987)J Biol Chem 262: 3553–61].     

Properties of an antiserum against native dynein 1 from sea urchin sperm flagella

Effects of an antiserum against native dynein 1 from sperm flagella of the sea urchin Strongylocentrotus purpuratus were compared with effects of an antiserum previously obtained against an ATPase-active tryptic fragment (fragment 1A) of dynein 1 from sperm flagella of the sea urchin, Anthocidaris crassispina. Both antisera precipitate dynein 1 and do not precipitate dynein 2. Only the fragment 1A antiserum precipitates fragment 1A and produces a measurable inhibition of dynein 1 ATPase activity. Both antisera inhibit the movement and the movement-coupled ATP dephosphorylation of reactivated spermatozoa. The inhibition of movement by the antiserum against dynein 1 is much less than by the antiserum against fragment 1A, suggesting that a specific interference with the active ATPase site may be required for effective inhibition of movement. Both antisera reduce the bend angle as well as the beat frequency of reactivated S. purpuratus spermatozoa, suggesting that the bend angle may depend on the activity of the dynein arms which generate active sliding.     

The reactivation of demembranated human spermatozoa lacking outer dynein arms is independent of pH

The effect of pH, Mg-ATP, and free calcium on activity of the inner dynein arm was investigated using demembranated human spermatozoa lacking the outer dynein arms (LODA). The results were compared with those obtained for demembranated-reactivated normal spermatozoa to evaluate the functional properties of the inner and outer dynein arms in axonemal motility. The reactivation of Triton X-100–demembranated LODA spermatozoa was analysed at various pHs and concentrations of Mg-ATP and calcium using video recordings. The percentage of reactivated LODA spermatozoa as a function of Mg-ATP concentration was not dependent on pH, whereas reactivation of normal human spermatozoa is pH dependent. This suggests that there may be a pH-dependent regulatory mechanism associated with the outer dynein arms. A delay in the principal bend propagation of normal and LODA reactivated cells was found at pH 7.1. This disappeared at pH 7.8 in normal but not in LODA populations. This suggests a role for outer dynein arms in the initiation of the propagation of flagellar bends at alkaline pH. The level of LODA and normal sperm reactivation both depended on the calcium concentration in the medium. At lower free calcium concentrations, the reactivation level and beat frequency of reactivated cells were higher. Our results suggest a functional difference between outer and inner dynein arms of human spermatozoa based on a differential pH sensitivity. Moreover, calcium seems to exert its regulatory action elsewhere than on the outer dynein arms. Mol. Reprod. Dev. 49:416–425, 1998. © 1998 Wiley-Liss, Inc.     

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.     

Vitelline layer lytic activity in sperm extracts of sea urchin,Hemicentrotus pulcherrimus

A factor which dissolves the vitelline layer was extracted from sperm of the sea urchin, Hemicentrotus pulcherrimus. Turbidity of the suspension was reduced when isolated vitelline layers were mixed with this sperm factor. When the mixture was subjected to SDS polyacrylamide gel electrophoresis, some of the protein bands of the vitelline layer were seen to be missing. The lytic activity of the factor was heat labile, completely inhibited by L-1-tosyl-amide-2-phenyl-ethylchloromethyl ketone and partially inhibited by soybean trypsin inhibitor. Chymotrypsin activity was detected, but not trypsin, arylsulfatase, or glycosidase. These results suggest that a chymotrypsin-like enzyme participates in lysis of the vitelline layer by the fertilizing spermatozoon.     

A latent adenosine triphosphatase form of dynein 1 from sea urchin sperm flagella.

Treatment of demembranated sea urchin sperm axonemes with an extraction solution containing 0.6 M NaCl, pH 7.0 for 10 min at 4 degrees C yields a solution of dynein 1 having a low, latent specific ATPase activity of about 0.25 mumol of Pi mg(-1) min(-1). Exposure of this dynein solution to 0.1% Triton-X-100 for 10 min at 25 degrees C causes an increase in its ATPase activity to about 3 mumol of Pi mg(-1) min(-1). A similar activation can be obtained by treating at 42 degrees C or by reacting with 60 mol of p-chloromercuribenzene sulfonate/10(6) g of protein. The effects of these activating procedures are not additive, suggesting that they lead to a common activated state. Purification of the latent activity dynein 1 by sucrose density gradient centrifugation yields a monodisperse preparation sedimenting at 21 S, and having a molecular weight of 1,250,000 as determined by sedimentation diffusion and sedimentation equilibrium. Activation of the latent dynein 1 with Triton X-100 converts it to a form sedimenting at 10 to 14 S. The 21 S dynein is also converted to a 10 S form by dialysis against 5 mM imidazole/NaOH buffer, 0.1 mM EDTA, 5 mM 2-mercaptoethanol, pH 7, although in this case, the ATPase activity is increased only about 3-fold, with another 3-fold activation being obtainable upon subsequent treatment with Triton X-100. The 21 S latent form of dynein 1 may represent the intact dynein arms that form moving cross-bridges and generate active sliding between adjacent doublet tubules of the flagellar axoneme. Electrophoretic analysis on polyacrylamide gels in the presence of sodium dodecyl sulfate suggests a model in which the 21 S dynein 1 particle is composed of three subunits of about 330,000 daltons and one of each of three medium weight subunits of 126,000, 95,000, and 77,000 daltons. When latent dynein 1 is added back to NaCl-extracted axonemes in the presence of 0.15 M NaCl, it recombines stoichiometrically and restores the arms on the doublet tubules with a 6-fold activation of its ATPase activity measured in the absence of KCl.     

Some properties of bound and soluble dynein from sea urchin sperm flagella

Axonemes were isolated from sperm of Colobocentrotus by a procedure involving two extractions with 1% Triton X-100 and washing The isolated axonemes contained 7 x 10¹⁵ g protein per µm of their length. Treatment of the axonemes with 0 5 M KCl for 30 min extracted 50–70% of the flagellar ATPase protein, dynein, and removed preferentially the outer arms from the doublet tubules. Almost all of the dynein (85–95%) could be extracted from the axonemes by dialysis at low ionic strength. In both cases the extracted dynein sedimented through sucrose gradients at 12–14S, and no 30S form was observed The enzymic properties of dynein changed when it was extracted from the axonemes into solution. Solubilization had a particularly marked effect on the KCl- and pH-dependence of the ATPase activity. The pH-dependence of soluble dynein was fairly simple with a single peak extending from about pH 6 to pH 10. The pH-dependence of bound dynein was more complex. In 0.1 M KCl, the bound activity appeared to peak at about pH 9, and dropped off rapidly with decreasing pH, reaching almost zero at pH 7; an additional peak at pH 10 0 resulted from the breakdown of the axonemal structure and solubilization of dynein that occurred at about this pH. A similar curve was obtained in the absence of KCl, except for the presence of a further large peak at pH 8 Measurement of the kinetic parameters of soluble dynein showed that both K_m and V_max increased with increasing concentrations of KCl up to 0.5 M When bound dynein was assayed under conditions that would induce motility in reactivated sperm (0 15 M KCl with Mg⁺⁺ activation), it did not obey Michaelis-Menten kinetics, although it did when assayed under other conditions. The complex enzyme-kinetic behavior of bound dynein, and the differences between its enzymic properties and those of soluble dynein, may result from its interactions with tubulin and other axonemal proteins     

Two heavy chains of 21S dynein from sea urchin sperm flagella

The biochemical properties of 21S dynein derived from sea urchin sperm flagella and of its components dissociated by low salt treatment were studied. SDS-urea gel electrophoresis and two-dimensional gel electrophoresis showed that the 21S dynein preparation contains two distinct heavy chains. These two heavy chains, termed A alpha and A beta, had apparently the same molecular weight of 500,000 but showed different mobilities on SDS-urea gels. The isoelectric points of A alpha and A beta heavy chains were 5.7 and 5.2, respectively, in the presence of urea. Proteolytic digestion patterns of these two heavy chains were clearly different, but the amino acid compositions were similar. Low salt treatment and sucrose density gradient centrifugation could partially separate the components of 21S dynein into two fractions: the one with larger sedimentation coefficient contained the A alpha heavy chain, and the other with smaller sedimentation coefficient contained the A beta heavy chain and three intermediate chains. These two fractions showed distinctly different kinetic properties, and thus may play different roles in dynein-microtubule interaction.     

Multiple GTP-binding proteins in sea urchin sperm: Evidence for Gs and small G-proteins

Sea urchin sperm plasma membranes isolated from heads and flagella were used to examine the presence of Gs (stimulatory guanine nucleotide-binding regulatory protein) and small G-proteins. Flagellar plasma membranes incubated with [³²P]NAD and cholera toxin (CTX) displayed radiolabeling in a protein of 48 kDa, which was reactive by immunoblotting with a specific antibody against mammalian Gs. CTX-catalyzed [³²P]ADP-ribosylation in conjunction with immunoprecipitation with anti-Gs, followed by electrophoresis and autoradiography, revealed one band of 48 kDa. Head plasma membranes, in contrast, did not show substrates for ADP-ribosylation by CTX. In flagellar and head plasma membranes pertussis toxin (PTX) ADP-ribosylated the same protein described previously in membranes from whole sperm; the extent of ADP-ribosylation by PTX was higher in flagellar than in head membranes. Small G-proteins were investigated by [³²P]GTP-blotting. Both head and flagellar plasma membranes showed three radiolabeled bands of 28, 25 and 24 kDa. Unlabeled GTP and GDP, but not other nucleotides, interfered with the [α-³²P]GTP-binding in a concentration-dependent manner. A monoclonal antibody against human Ras p21 recognized a single protein of 21 kDa only in flagellar membranes. Thus, sea urchin sperm contain a membrane protein that shares characteristics with mammalian Gs and four small G-proteins, including Ras . Gs, Gi and Ras are enriched in flagellar membranes while the other small G-proteins do not display a preferential distribution along the sea urchin sperm plasma membrane. The role of these G-proteins in sea urchin sperm is presently under investigation.     

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.     

Measurement of the intracellular pH threshold for sperm aster formation in sea urchin eggs

In the fertilization of sea urchin eggs, intracellular [Ca2+] (Cai) increases transiently and intracellular pH (pHi) elevates accordingly. Unlinking these two activating factors experimentally, the requirement of the increase in pHi for sperm aster formation in the sea urchin, Clypeaster japonicus, was investigated. When the eggs were injected with an EGTA or BAPTA solution, they incorporated sperm but did not organize the sperm aster. Using these sperm-incorporated eggs under the condition that an increase in Cai was blocked, pHi was regulated by two methods: (i) perfusing ammonium acetate-containing seawater; and (ii) injecting pH buffer solutions of various pH values. By either of the two methods, the sperm aster formed at pHi 7.0 or more and functioned in female pronuclear migration when the sperm aster reached the female pronucleus. Hence, the step of the transient increase in Cai at fertilization can be bypassed. In contrast, a pHi increase is indispensably required for sperm aster formation in sea urchin eggs. Moreover, under the condition that there was the transient increase in Cai, the threshold pHi value for sperm aster formation was pHi 7.0 or more. Consequently, whether a Cai increase on fertilization occurs or not, the threshold pHi value for sperm aster formation is constant in sea urchin eggs.     

A new hyperpolarization-activated, cyclic nucleotide-gated channel from sea urchin sperm flagella

A sea urchin sperm flagellar hyperpolarization-activated, cyclic nucleotide-gated (HCN) channel is known (SpHCN1) that is modulated by cAMP. Here, we describe a second flagellar HCN channel (SpHCN2) cloned from the same sea urchin species. SpHCN2 is 638 amino acids compared to 767 for SpHCN1. SpHCN2 has all the domains of an HCN channel, including six transmembrane segments (S1-S6), the ion pore, and the cyclic nucleotide-binding domain. The two full-length proteins are 33% identical and 51% similar. The six transmembrane segments vary from 46-79% identity. S4, which is the voltage sensor, is 79% identical between the two proteins. The ion selectivity filter sequence is GYG in the ion pore of SpHCN1 and GFG in SpHCN2. By sequence, SpHCN2 is 73.5kDa, but it migrates on SDS-PAGE at 64kDa. Western immunoblots show localization to flagella, which is confirmed by immunofluorescence. A neighbor-joining tree shows that SpHCN2 is basal to all known HCN channels. SpHCN2 might be the simplest pacemaker channel yet discovered.     

A species-specific sperm factor dispersing the jelly coat of the egg of the sea urchin,Anthocidaris crassispina

A species-specific factor capable of disersing the jelly coat surrounding eggs has been purified from sperm of the sea urchin, Anthocidaris crassisina. It does not exert its effect on the vitelline layer. The purification has been accomlished by a four-step procedure involving ammonium sulfate fractionation, gel filtration on Sepharose CL-4B, ion-exchange column chromatography on DEAE-cellulose, and affinity column chromatograhy on heparin-Seharose CL-6B. The isolated factor is homogenous in sodium dodecyl sulfate polyacrylamide gel electrohoresis in the presence or absence of β-mercatoethanol, estimated molecular weight being about 140,000. The jelly dispersion by the present factor is activated by CaCl₂, and inhibited by KCl, MnCl₂, EDTA, and EGTA, and by sulfated saccharides such as chondroitin sulfate A and C, heparin, and glucose-6-sulfate, Inorganic sulfated such as (NH₄)₂SO₄ and Na₂SO₄ have no effect on jelly dispersion. This factor is heat-labile, its activity in 30 min at 50°C. The present factor is found also in the seminal Plasma, and released from sperm themselves by treatment with Triton X-100 .These results suggest that this factor is loosely bound to the serm surface. Although glycosidase and arylsulfatase activities are detectable in the seminal plasma, these enzyme activities are not detectable in the purified jelly disersing factor. Only trypsin and α chymotrysin among commercial enzymes tested dispersing activity is inhibited neither by trypsin inhibitors such as N-α-p-tosyl-L-lysine-chloromethyl ketone, soybean trypsin inhibitor, ovomucoid trypsin inhibitor, nor by chymotrypsin inhibitors such as L-1-tosylamide-2 pheny-ethylcholoromethyl ketone and chymostatin Participation of trysin-like and chymotrypsin-like enzymes in jelly dispersion seems unlikely.     

Cytoplasmic and sperm nuclear transformations in fertilized ammonia-activated sea urchin (Arbacia punctulata) eggs

Studies examining cytoplasmic and sperm nuclear transformations in sea urchin (Arbacia punctulata) eggs inseminated at different periods after ammonia activation have been caried out at the light- and electron-microscopic levels of observation. Arbaca eggs treated with ammonia-seawater demonstrated chromosome condensation after DNA synthesis and underwent a chromosome cycle similar to that described for Lytechinus [Mazia, 1947]. Cortical granule reaction, fertilization cone formation, and sperm aster development in eggs fertilized at 20 (interphase), 50 (prometaphase), and 180 (interphase) min after ammonia activation were structurally simialr to processes in untreated zygotes. Cyclical changes in the formation of fertilization cones and sperm asters, as reported for eggs fertilized after activation by agents that induce a cortical granule reaction, were not observed. Although sperm nuclear transformations were prolonged (14 vs 18 min), male pronuclei that developed in eggs fertilized 20 min after ammonia activation were morphologically similar to those observed in fertilized, untreated ova and incorporated ³H-thymidine. Sperm incorporated into eggs at 50 min after ammonia activation underwent nuclear envelope breakdown and chromatin despersion; however, ³H-thymidine incorporation was not observed, and male pronuclei rarely developed (less than 5% of all specimens examined). Subsequent to dispersion, the paternal chromatin condensed into chromosomes which were associated with an aster. These results demonstrate that although ammonia-activated eggs inseminated at interphase or prometaphase undergo similar cytoplasmic alterations, sperm nuclear transformations vary with the chromosome cycle of the egg.     

Single-channel activity in sea urchin sperm revealed by the patch-clamp technique

Ionic fluxes are deeply involved in the response of spermatozoa to the egg. Using the patch-clamp technique, we show for the first time single ion channel activity in sea urchin spermatozoa and spermatozoa heads. Due to their small size gigaseals were obtained in suspended cells by applying suction through the pipette. The rate of gigaseal formation was very low and improved to 6% (n = 1145) when flagella were detached from sperm. Current-voltage curves created from single-channel events showed conductances of approx. 65 and 170 pS, suggesting the presence of two types of channels. At least one appears to be a K⁺ channel.     

Activation of Ca2+channels during the acrosome reaction of sea urchin sperm is inhibited by inhibitors of chymotrypsin-like proteases

Probable participation of sperm protease in the acrosome reaction was investigated using several inhibitors and substrates. Among those examined, L-l-tosylamide-2-phenylethyl chloromethyl ketone (TPCK) and chymostatin, chymotrypsin inhibitors, p-nitrophenyl-p′-guanidinobenzoate (NPGB), a serine protease inhibitor, and N-benzoyl-L-tyrosine ethyl ester (BTEE), a chymotrypsin substrate, inhibited the egg jelly-induced acrosome reaction of Strongylocentrotus intermedius. TPCK and BTEE, however, did not inhibit the reaction caused by ionophores, A23187, or nigericin. To know the mechanism of inhibition by chymotrypsin inhibitors and substrates of the egg jelly-induced acrosome reaction, intraccllular Ca²⁺ concentration ([Ca²⁺]_i) and pH (pH_i) were measured with fura-2 and 2′,7′-bis (carboxy-ethyl)carboxyfluorescein (BCECF), respectively. Egg jelly caused increase of [Ca²⁺]_i which was depressed by BTEE. Egg jelly also caused a transient rise of pH_i, which was not depressed by BTEE. In the presence of verapamil, the acrosome reaction by egg jelly was significantly inhibited concomitant with depressed increase of [Ca²⁺]_i. The rise of pHj was not depressed by verapamil. Thus, modes of action of BTEE and of verapamil are similar to each other. Bringing these findings together, the authors present a view that a chymotrypsin-like protease of sea urchin sperm activates verapamil-sensitive Ca²⁺ channels, which take part in the acrosome reaction.