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V D Vacquier

Publications and source records attributed to V D Vacquier.

At least 19 recordsLinked to original sources
PubMed1995

Liposome fusion induced by a M(r) 18,000 protein localized to the acrosomal region of acrosome-reacted abalone spermatozoa.

A M(r) 18,000 protein is secreted by abalone spermatozoa during the acrosome reaction. Immunofluorescence of acrosome-reacted sperm localizes the protein as a coating on the spent acrosomal granule hull and on the surface of the acrosomal process. The membrane of the acrosomal process fuses with the egg plasma membrane at fertilization. The M(r) 18,000 acrosomal protein aggregates negatively charged (but not neutral) large unilamellar liposomes and renders them permeable to internal probe. The M(r) 18,000 proteins from two abalone species are potent inducers of intervesicular lipid mixing in the resonance energy transfer assay, suggesting that they mediate the fusion of lipid bilayers. Predicted secondary structures of these proteins show the presence of strongly amphipathic alpha-helices that may be active in the perturbation of phospholipid bilayers. The M(r) 18,000 protein may mediate sperm-egg fusion during fertilization.

Acrosome↗
PubMed1995

Extraordinary divergence and positive Darwinian selection in a fusagenic protein coating the acrosomal process of abalone spermatozoa.

During fertilization in marine invertebrates, fusion between sperm and egg cell membranes occurs at the tip of the sperm acrosomal process. In abalone sperm the acrosomal process is coated with an 18-kDa protein. In situ, this protein has no effect on the egg vitelline envelope, but in vitro it is a potent fusagen of liposomes. Thus, the 18-kDa protein may mediate membrane fusion between the gametes, a step in gamete recognition known to restrict heterospecific fertilization in other species. The cDNA and deduced amino acid sequences of the 18-kDa protein were determined for five species of California abalone. The deduced amino acid sequences exhibit extraordinary divergence; the percent identity varies from 27% to 87%. Analysis of nucleotide substitution shows extremely high frequencies of amino acid-altering substitution compared to silent substitution, demonstrating that positive Darwinian selection promotes the divergence of this protein. However, amino acid replacement is conservative with respect to size and polarity of residue. The data support the developing idea that in free-spawning marine invertebrates, the proteins mediating fertilization may be subjected to intense, and as yet unknown, selective forces. The extraordinary divergence of fertilization proteins may be related to the establishment of barriers to heterospecific fertilization.

Acrosome↗
PubMed1995

Structural features of the abalone egg extracellular matrix and its role in gamete interaction during fertilization.

Abalone eggs are surrounded by a complex extracellular coat that contains three distinct elements: the jelly layer, the vitelline envelope, and the egg surface coat. In this study we used light and electron microscopy to describe these three elements in the red abalone (Haliotis rufescens) and ascribe function to each based on their interactions with sperm. The jelly coat is a spongy matrix that lies at the outermost margin of the egg and consists of variably sized fibers. Sperm pass through this layer with their acrosomes intact and then go on to bind to the vitelline envelope. The vitelline envelope is a multilamellar fibrous layer that appears to trigger the acrosome reaction after the sperm binding. Next, sperm release lysin from their acrosomal granules, a nonenzymatic protein that dissolves a hole in the vitelline envelope through which the sperm swims. Sperm then contact the egg surface coat, a network of uniformly sized filaments lying directly above the egg plasma membrane. This layer mediates attachment of sperm, via their acrosomal process, to the egg surface.

Animals↗
PubMed1995

Crystal structure and subunit dynamics of the abalone sperm lysin dimer: egg envelopes dissociate dimers, the monomer is the active species.

Lysin is a 16-kD acrosomal protein used by abalone spermatozoa to create a hole in the egg vitelline envelope (VE) by a nonenzymatic mechanism. The crystal structure of the lysin monomer is known at 1.9 A resolution. The surface of the molecule reveals two tracks of basic residues running the length of one surface of the molecule and a patch of solvent-exposed hydrophobic residues on the opposite surface. Here we report that lysin dimerizes via interaction of the hydrophobic patches of monomers. Triton X-100 dissociates the dimer. The crystal structure of the dimer is described at 2.75 A resolution. Fluorescence energy transfer experiments show that the dimer has an approximate KD of 1 microM and that monomers exchange rapidly between dimers. Addition of isolated egg VE dissociates dimers, implicating monomers as the active species in the dissolution reaction. This work represents the first step in the elucidation of the mechanism by which lysin enables abalone spermatozoa to create a hole in the egg envelope during fertilization.

Animals↗
PubMed1995

Positive selection is a general phenomenon in the evolution of abalone sperm lysin.

Lysin is a 16kDa acrosomal protein used by abalone sperm to create a hole in the egg vitelline envelope (VE). The interaction of lysin with the VE is species-selective and is one step in the multistep fertilization process that restricts heterospecific (cross-species) fertilization. For this reason, the evolution of lysin could play a role in establishing prezygotic reproductive isolation between species. Previously, we sequenced sperm lysin cDNAs from seven California abalone species and showed that positive Darwinian selection promotes their divergence. In this paper an additional 13 lysin sequences are presented representing species from Japan, Taiwan, Australia, New Zealand, South Africa, and Europe. The total of 20 sequences represents the most extensive analysis of a fertilization protein to date. The phylogenetic analysis divides the sequences into two major clades, one composed of species from the northern Pacific (California and Japan) and the other composed of species from other parts of the world. Analysis of nucleotide substitution demonstrates that positive selection is a general process in the evolution of this fertilization protein. Analysis of nucleotide and codon usage bias shows that neither parameter can account for the robust data supporting positive selection. The selection pressure responsible for the positive selection on lysin remains unknown.

Amino Acid Sequence↗
PubMed1994

A unique expression pattern for a sperm membrane protein during sea urchin spermatogenesis.

Specific mRNAs coding for a 63 kDa sperm membrane protein (63-SMP) were localised in Strongylocentrotus purpuratus testis sections using in situ hybridisation techniques. 35S-labelled antisense RNA probes transcribed from a 766 base pair fragment of the gene coding for the 63-SMP hybridised to all spermatogenic cells in the basal germinal epithelia of testicular acini, except the most peripherally located (least differentiated) spermatogonia. No hybridisation to the luminally located mature spermatozoa or somatic cells of the testis was observed. Using monoclonal antibody J17/30 and indirect immunofluorescence techniques, the 63-SMP was localised to the same subset of spermatogenic cells that contain the 63-SMP mRNA, suggesting that expression of this gene is transcriptionally controlled. In combination with previous studies on the expression of sperm histones and sperm binding, these results show that multiple, perhaps sequential, classes of gene activity contribute to the differentiation of sea urchin sperm.

Animals↗
PubMed1993

Anion channels in the sea urchin sperm plasma membrane.

Ionic fluxes in sea urchin sperm plasma membrane regulate cell motility and the acrosome reaction (AR). Although cationic channels mediate some of the ionic movements, little is known about anion channels in these cells. The fusion of sperm plasma membranes into lipid bilayers allowed identification of a 150 pS anion channel. This anion channel was enriched from detergent-solubilized sperm plasma membranes using a wheat germ agglutinin Sepharose column. Vesicles formed from this preparation were fused into black lipid membranes (BLM), yielding single channel anion-selective activity with the properties of those found in the sperm membranes. The following anion selectivity sequence was found: NO3- > CNS- > Br- > Cl-. This anion channel has a high open probability at the holding potentials tested, it is partially blocked by 4,4'-diisothiocyano-2,2'-stilbendisulfonic acid (DIDS), and it often displays substates. The sperm AR was also inhibited by DIDS.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1991

In vitro phosphorylation of sea urchin sperm adenylate cyclase by cyclic adenosine monophosphate-dependent protein kinase.

Addition of [gamma -32P]ATP to a 2% Brij-78 40,000g supernatant of sea urchin sperm results in the cAMP-dependent phosphorylation of eight to ten proteins. One phosphoprotein of Mr 190 kD is sperm adenylate cyclase (AC). An antiserum to the AC immunoprecipitates the Mr 190 kD protein. Peptide maps of immunoprecipitates show that the AC is the only phosphoprotein present in the Mr 200 kD range. With respect to the in vitro phosphorylation of AC, the endogenous kinase has a Km for ATP of 5.2 microM and is maximally stimulated by 4-8 microM cAMP. The protein kinase inhibitors H8 (9 microM) and PKI (30 U/ml) inhibit the phosphorylation of the AC. The catalytic subunit of bovine cAMP-dependent protein kinase phosphorylates the AC on the same peptides as the endogenous protein kinase. Cyanogen bromide generated peptide maps of the phosphorylated AC show a minimum of five sites of phosphorylation. No change in the Km or Vmax of the sperm AC resulted from the additional phosphorylation by bovine kinase. Calcium ions at submicromolar concentrations completely block the in vitro phosphorylation of the AC, suggesting the presence in the preparation of a Ca2(+) -activated protein phosphatase. To our knowledge, this is the first report of the phosphorylation of an AC by cAMP-dependent protein kinase.

Adenylyl Cyclases↗
PubMed1990

Diffraction quality crystals of lysin from spermatozoa of the red abalone (Haliotis rufescens).

Single crystals of the protein lysin (Mr = 16,070) from the spermatozoa of the red abalone (Haliotis rufescens) have been obtained by the vapor diffusion technique, using as precipitants a 32.5% saturated solution of (NH4)2SO4 (incubation at 18 degrees C) or a 5% w/v polyethyleneglycol 8000 solution (incubation at 29 degrees C), both in Bis-Tris-iminodiacetic acid buffers of pH 7.0. The addition to the droplets of EDTA, other carboxylate-containing polyanions, and/or organic solvents improved the size and quality of the crystals and, especially with (NH4)2SO4, addition of EDTA, and/or organic solvents produced a change in crystal habit which resulted in crystals more elongated in the b direction. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with a = 52.3 A, b = 46.0 A, and c = 81.5 A and one molecule per asymmetric unit. The crystals diffract to 2.3 A resolution. The molecular structure of lysin is relevant to the nonenzymatic mechanism by which the protein dissolves a hole in the egg vitelline layer during fertilization.

Animals↗
PubMed1990

Species-specific sequences of abalone lysin, the sperm protein that creates a hole in the egg envelope.

Abalone eggs are contained within a rigid, elevated vitelline envelope through which the sperm must pass before reaching the egg cell membrane. Abalone spermatozoa possess an acrosomal protein called lysin that creates a hole in the egg vitelline envelope by a nonenzymatic mechanism. Lysins from two species of abalone, termed pink and red, which share the same habitat, exhibit species specificity in the dissolution of isolated egg envelopes. Cloning and sequencing the cDNAs for pink and red abalone lysins reveal transcript lengths of approximately 660 nucleotides. The open reading frames of 465 (pink) and 462 (red) nucleotides show a 13% difference. The 3' untranslated regions before the poly(A) tails are 170 (pink) and 165 (red) nucleotides long and differ from each other by about 7%. The protein sequences show nearly identical signal sequences of 18 amino acids for both lysins. The mature protein is 137 amino acids in the pink abalone and 136 in the red abalone; the two mature lysins differ in 29 of 137 amino acids (21%). The most variable region, which may account for lysin's species specificity, is at the NH2 terminus, where 11 of the 15 amino acids differ between the two species. Predictions of secondary structure indicate that both lysins contain four homologous amphiphilic alpha-helices.

Amino Acid Sequence↗
PubMed1990

Identification of sea urchin sperm adenylate cyclase.

Calmodulin (CaM) affinity chromatography of a detergent extract of sea urchin sperm yielded approximately 20 major proteins. One of these proteins, of Mr 190,000, was purified and used to immunize rabbits. After absorption with living sperm, the serum reacted monospecifically on one- and two-dimensional Western immunoblots with the Mr 190,000 protein. The anti-190-kD serum inhibited 94% of the adenylate cyclase (AC) activity of the CaM eluate. An immunoaffinity column removed 95% of the AC activity, and the purified (but inactive) Mr 190,000 protein was eluted from the column. The antiserum also inhibited 23% of the activity of bovine brain CaM-sensitive AC and 90% of the activity of horse sperm CaM-sensitive AC. These data support the hypothesis that the Mr 190,000 protein is sea urchin sperm AC. Although this AC bound to CaM, it was not possible to demonstrate directly a Ca2+ or CaM sensitivity. However, two CaM antagonists, calmidazolium and chlorpromazine, both inhibited AC activity, and the inhibition was released by added CaM, suggesting the possibility of regulation of this AC by CaM. Indirect immunofluorescence showed the Mr 190,000 protein to be highly concentrated on only the proximal half of the sea urchin sperm flagellum. This asymmetric localization of AC may be important to its function in flagellar motility. This is the first report of the identification of an AC from animal spermatozoa.

Adenylyl Cyclases↗
PubMed1989

Egg jelly induces the phosphorylation of histone H3 in spermatozoa of the sea urchin Arbacia punctulata.

When spermatozoa of Arbacia punctulata are labeled with 32P and treated with soluble egg jelly, radiolabel is incorporated into histone H3. The time course of labeling correlates with the period of chromatin decondensation of sperm pronuclei in eggs. Phosphorylation is on serine and may result from increased turnover of phosphate on H3. The macromolecular fraction of egg jelly (and not the peptide fraction) is the inducer of H3 phosphorylation. The reaction is dependent on external Ca2+ and is induced by monensin and A23187. H3 phosphorylation is not induced by the phosphodiesterase inhibitor IBMX and relatively high (250 microM) concentrations of the protein kinase inhibitor H8 are needed to block the reaction, suggesting that it is cAMP independent. A surprising finding is that merely diluting the cells into Na+ free media is the most effective method to induce the radiolabeling of H3. These results are in contrast to findings on the egg jelly induced phosphorylation of histone H1 in S. purpuratus spermatozoa. These species differences must reflect the great evolutionary divergence between these two sea urchin species in the mechanism of regulation of the phosphorylation of nuclear proteins during fertilization.

1-Methyl-3-isobutylxanthine↗
PubMed1989

The amino terminal sequence of sea urchin sperm histone H1 and its phosphorylation by egg cytosol.

1. The amino acid sequence of the first 34 residues of sperm histone H1 (SpH1) from Strongylocentrotus purpuratus shows striking similarity with sequences from three South African species. 2. Five contiguous repeats of the tetrapeptide SPBB (where B is R or K) occur between positions 10 and 29. 3. SpH1 was phosphorylated in vitro using egg cytosol as the source of protein kinase and approximately 4.2 mol phosphate were incorporated per mol H1. 4. Sequences of five phosphopeptides of SpH1 show the egg possesses protein kinase activity capable of phosphorylating multiple seryl residues including SPBB in the NH2-, and BBSP in the COOH-end of the protein.

Amino Acid Sequence↗
PubMed1988

Extraction of phosphorylated sperm specific histone H1 from sea urchin eggs: analysis of phosphopeptide maps.

Sea urchin spermatozoa contain a unique histone H1 (SpH1) with an unusual primary structure. Within 12 minutes postinsemination SpH1 is phosphorylated and lost from the chromatin. Both sperm and egg kinases phosphorylate SpH1 in vivo. Until now the analysis of the phosphorylation of SpH1 in eggs in vivo during the first 12 minutes has been impossible because of the enormous volume of the egg relative to the sperm nucleus. Here we present the first such analysis based on the direct extraction of [32P]-labeled SpH1 from eggs, fractionation by HPLC, cleavage by CNBr and isolation of the two CNBr-generated fragments of SpH1 by HPLC. Two dimensional maps of the [32-P]-peptides show that at least five sites in the amino-terminal fragment, and five sites in the carboxyl-terminal fragment, are phosphorylated by 1 minute postinsemination.

Animals↗
PubMed1988

CAMP-dependent protein kinase of sea urchin sperm phosphorylates sperm histone H1 on a single site.

The phosphorylation of sperm specific histone H1 in the sea urchin Strongylocentrotus purpuratus occurs both in vivo and in vitro on a single serine site in the sequence Arg-Lys-Gly-Ser(P)-Ser-Asn-Ala-Arg. This is a preferred sequence for cAMP-dependent protein kinase. The in vitro phosphorylation is completely dependent on cAMP and is inhibited by the peptide protein kinase inhibitor. The protein kinase inhibitor H-8 blocks the in vivo phosphorylation of H1 without damaging motility, the acrosome reaction or the ability of sperm to fuse with and activate eggs.

Amino Acid Sequence↗
PubMed1988

Monoclonal antibodies induce the translocation, patching, and shedding of surface antigens of sea urchin spermatozoa.

Monoclonal antibodies reacting with external domains of plasma membrane proteins of sea urchin spermatozoa cause the reversible aggregation of sperm. The rate of disaggregation is temperature dependent indicating the involvement of membrane fluidity. Immunofluorescence shows that disaggregation is temporally correlated with the movement of surface-bound antibody into large aggregates which are then shed from the cell surface. Electrophoretic analysis of the shed antigens shows that they resemble the total complement of proteins of isolated sperm membranes. The translocation and shedding of surface-bound ligands may be a common property of eukaryotic flagellar membranes.

Animals↗