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J L Hedrick

Publications and source records attributed to J L Hedrick.

At least 19 recordsLinked to original sources
PubMed2005

Persistence of anti-zonae pellucidae antibodies following a single inoculation of porcine zonae pellucidae in the domestic equine.

In this study of equids, we investigated the antibody response and the effect on the estrous cycle following a single inoculation of porcine zonae pellucidae (pZP) employing controlled-release methodology. We also investigated the use of two different water-soluble adjuvants as an alternative to oil-based adjuvants. Twenty-seven domestic mares were inoculated with various formulations of pZP and adjuvant. We showed that the anti-pZP antibodies generated as a result of the inoculations persisted for at least 43 weeks (length of the study). Of the various formulations used in the study, pZP and QS-21 water-soluble adjuvant, administered in combination with an emulsified preparation of pZP and Freund's Complete Adjuvant generated a significantly (P < 0.05) higher titer of anti-pZP antibodies when compared with other formulations employing the water-soluble adjuvant, Carbopol. Hormone analyses for cyclicity indicated a high incidence and extended duration of persistent corpora lutea among the treated mares. The positive control group of mares receiving two standard inoculations of pZP and Freund's Complete and Incomplete Adjuvants, as well as the placebo group of mares injected with QS-21 only, also exhibited high incidences of persistent corpora lutea. However, all mares eventually returned to normal cyclicity. The basis for the high incidence and extended duration of persistent corpora lutea was unexplained. The results demonstrate for the first time the persistent generation of anti-pZP antibodies following a single inoculation of pZP incorporated into a controlled-released preparation in the horse. This study further suggests that a single inoculation of pZP sequestered in a controlled-release lactide-glycolide polymer may serve as an alternative to traditional two-inoculation protocols for contraception investigations in the equine.

Acrylic Resins↗
PubMed2003

Cross-fertilization and structural comparison of egg extracellular matrix glycoproteins from Xenopus laevis and Xenopus tropicalis.

While the anuran amphibian Xenopus laevis is a widely used vertebrate model system, it is not optimal for genetic manipulations due to its tetraploid genome and long generation time. A current alternative amphibian model system, Xenopus tropicalis, has the advantages of a diploid genome and a much shorter generation time. We undertook a comparative investigation of X. tropicalis egg extracellular matrix glycoproteins in relation to those already characterized in X. laevis. Fertilization methods and isolation of egg extracellular molecules were directly transferable from X. laevis to X. tropicalis. Cross-fertilizations were successful in both directions, indicating similar molecules involved in sperm-egg interactions. Egg envelopes analyzed by SDS-PAGE were found to have almost identical gel patterns, whereas jelly component profiles were similar only for the larger macromolecules (>90 kDa). The cDNA sequences for egg envelope glycoproteins ZPA, ZPB, ZPC, ZPD and ZPAX, and also egg cortical granule lectin involved in the block to polyspermy, were cloned for X. tropicalis and showed a consistent approximately 85% amino acid identity to the X. laevis sequences. Thus, homologous egg extracellular matrix molecules perform the same functions, and the molecular and cellular mechanisms of fertilization in these two species are probably equivalent.

Amino Acid Sequence↗
PubMed2001

Targeted use of exoglycosidase digestion for the structural elucidation of neutral O-linked oligosaccharides.

Exoglycosidase digestion in combination with the catalog-library approach (CLA) is used with matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) to obtain the complete structure of oligosaccharides. The CLA is a collision-induced dissociation (CID)-based method used to determine the structure of O-linked neutral oligosaccharides. It provides both linkage and stereochemical information. Exoglycosidases are used to confirm independently the validity of the CLA. In some cases, the CLA provides structural information on all but a single residue. Exoglycosidase is used to refine these structures. In this way, exoglycosidase use is targeted employing only a small number of enzymes. Exoglycosidase arrays, which have been used with N-linked oligosaccharides, is avoided despite the larger variations in structures of O-linked species.

Animals↗
PubMed2001

Profiling with structural elucidation of the neutral and anionic O-linked oligosaccharides in the egg jelly coat of Xenopus laevis by Fourier transform mass spectrometry.

A strategic method with high speed and sensitivity is outlined for the analysis of mucin-type oligosaccharide from the jelly coat of Xenopus laevis. The method relies primarily on mass spectrometric techniques, in this case matrix-assisted laser desorption/ionization Fourier-transform mass spectrometry (MALDI-FTMS) and collision-induced dissociation (CID). Separation with isolation of the oligosaccharides was streamlined to couple well with mass spectrometry allowing the rapid determination of all detectable components from both neutral and anionic species. Partial structures of anionic components, composed primarily of sulfate esters, were obtained with CID. For neutral species, a method that allowed the complete structural determination using mass spectrometry was used. The method builds on the structure of small number of known compounds to determine unknown structures from the same biological source. In this example, a small number of oligosaccharides, elucidated previously by NMR, were used to develop a set of substructural motifs that were characterized by CID. The presence of the motifs in the CID spectra were then used to determine the structures of unknown compounds that were in abundances too small for NMR analysis.

Animals↗
PubMed2001

A hatching enzyme substrate in the Xenopus laevis egg envelope is a high molecular weight ZPA homolog.

The Xenopus laevis egg envelope is composed of six or more glycoproteins, three of which have been cloned and identified as the mammalian homologs ZPA (ZP2), ZPB (ZP1) and ZPC (ZP3). The remaining glycoproteins are a triplet of high molecular weight components that are selectively hydrolyzed by the hatching enzyme. We have isolated one of these proteins and cloned its cDNA. The mRNA for the protein was found to be expressed only in early stage oocytes, as are other envelope components. From the deduced amino acid sequence, it was indicated to be a secreted glycoprotein with a characteristic ZP domain in the C-terminal half of the molecule. The N-terminal half was unrelated to any known glycoprotein. Comparative sequence analysis of the ZP domain indicated that it was derived from an ancestor of ZPA and ZPB, with the greatest identity to ZPA. This envelope component has been designated ZPAX.

Amino Acid Sequence↗
PubMed2000

In situ pH measurements of the Syrian hamster uterus during early pregnancy to determine the role of pH in zona pellucida loss in vivo.

The mechanisms of zona pellucida (ZP) loss in peri-implantation hamster embryos in vivo versus in vitro are distinctly different. To investigate if ZP loss in vivo is the result of transient uterine pH changes, the luminal pH of the pregnant uterus was measured during the ZP loss period. Prior to ZP loss, pH was 7.30 +/- 0.05 (mean +/- SE; left uterine horn) and 7.35 +/- 0.03 (right horn). During ZP loss, pH was 7.26 +/- 0.07 (left) and 7.35 +/- 0.03 (right), and after embryo attachment, 7.25 +/- 0.02 (left) and 7.27 +/- 0.02 (right). None of these values are statistically different. The pseudopregnant uterine pH was 7.30 +/- 0.04 (left) and 7.31 +/- 0.04 (right), not statistically different from each other or from pregnant uteri. Blastocyst ZP loss in vitro (pH 3.0-8.5) occurred only at pH 3.0. Loss of ZP occurred in uterine flushings from pregnant or pseudopregnant hamsters, evidence that ZP loss is related to uterine factors. Complete ZP loss occurred at pH 6.8, but was incomplete at pH 6.6, 7.0 and 7.2. No ZP loss occurred in uterine flushings from non-mated females. In summary: (i) a change in uterine pH does not cause ZP loss in vivo in the Syrian hamster; (ii) a pH-sensitive factor in pregnant and pseudopregnant uterine fluid is responsible for ZP loss.

Animals↗
PubMed2000

Independent and hetero-oligomeric-dependent sperm binding to egg envelope glycoprotein ZPC in Xenopus laevis.

Vitelline envelopes are composed of glycoproteins that participate in sperm-egg interactions during the initial stages of fertilization. In Xenopus laevis, the vitelline envelope is composed of at least 4 glycoproteins (ZPA, ZPB, ZPC, and ZPX). A sperm binding assay involving the covalent coupling of envelope glycoproteins to silanized glass slides was developed. In our assay, sperm bound to the egg envelopes derived from oviposited eggs but not activated eggs. The majority of the egg envelope ligand activity for sperm binding was derived from the complex N-linked oligosaccharides of ZPC. This sperm binding involved N-acetylglucosamine and fucose residues, as binding was abolished after treatment with cortical granule beta-N-acetylglucosaminidase and commercial beta-N-acetylglucosaminidases and was reduced by 44% after treatment with alpha-fucosidase. Although both the envelope glycoproteins ZPA and ZPC possessed independent ligand activity, ZPC was the major ligand for sperm binding (75%). Mixing of isolated ZPA, ZPB, and ZPC in a ratio of 1:4:4 (equal to that in the egg envelope) resulted in sperm binding that was greater than that of the sum of the separate components. The egg glycoproteins acted in synergy to increase sperm binding. Thus, ZPC possessed both independent and hetero-oligomeric-dependent ligand activities for sperm binding.

Acetylglucosaminidase↗
PubMed1999

Ovochymase, a Xenopus laevis egg extracellular protease, is translated as part of an unusual polyprotease.

Ovochymase, an extracellular Xenopus laevis egg serine active-site protease with chymotrypsin-like (Phe-X) substrate specificity, is released during egg activation. Molecular cloning results revealed that ovochymase is translated as part of an unusual polyprotein proenzyme. In addition to the ovochymase protease domain at the C terminus of the deduced amino acid sequence, two unrelated serine protease domains were present, each with apparent trypsin-like (Arg/Lys-X) substrate specificity, and thus, they were designated ovotryptase1 (at the N terminus) and ovotryptase2 (a mid domain). Also, a total of five CUB domains were interspersed between the protease domains. The presence of a hydrophobic signal sequence indicated that the polyprotein was secreted. Immunolocalization and Western blot studies of all three proteases showed that they are all present in the perivitelline space of unactivated eggs, apparently as proenzymes processed away from the original polyprotein. Western blot analysis also showed that the vast majority of the proteases in ovary, eggs, and embryos were present as the proenzyme forms, suggesting that the functions of these proteases depend on very limited levels of activation.

Amino Acid Sequence↗
PubMed1999

Catalog-library approach for the rapid and sensitive structural elucidation of oligosaccharides.

We obtained the nearly complete structural elucidation of oligosaccharide components, including sequence, linkage, and even stereochemistry in the picomolar levels. The "catalog-library" approach is used for elucidating the structures of minor components in a mixture of oligosaccharides. Oligosaccharides released from a family of glycoproteins are often composed of a small finite set of monosaccharides. In this regard, the numerous oligosaccharide species are analogous to the products found in syntheses involving combinatorial libraries. The great structural diversity in the library is the result of the nearly infinite combinations in which even a small number of monosaccharides can be arranged. Fortunately, structural similarities exist between different oligosaccharides, as specific substructural motifs are preserved among different compounds. We propose that a catalog of substructural motifs can be identified and characterized by collision-induced dissociation mass spectrometry. The catalog is constructed from a set of known compounds that have been fully structurally elucidated by, for example, nuclear magnetic resonance. The catalog consists of the characteristic fragmentation patterns belonging to a set of specific substructural motifs. Collision-induced dissociation is used to determine the presence of these motifs and reconstruct the structures of less abundant components.

Carbohydrate Conformation↗
PubMed1999

Distribution of lectin binding sites in Xenopus laevis egg jelly.

Eggs from the anuran Xenopus laevis are surrounded by a thick jelly coat that is required during fertilization. The jelly coat contains three morphologically distinct layers, designated J1, J2, and J3. We examined the lectin binding properties of the individual jelly coat layers as a step in identifying jelly glycoproteins that may be essential in fertilization. The reactivity of 31 lectins with isolated jelly coat layers was examined with enzyme-linked lectin-assays (ELLAs). Using ELLA we found that most of the lectins tested showed some reactivity to all three jelly layers; however, two lectins showed jelly layer selectivity. The lectin Maackia amurensis (MAA) reacted only with J1 and J2, while the lectin Trichosanthes kirilowii (TKA) reacted only with J2 and J3. Some lectins were localized in the jelly coat using confocal microscopy, which revealed substantial heterogeneity in lectin binding site distribution among and within jelly coat layers. Wheat germ agglutinin (WGA) bound only to the outermost region of J3 and produced a thin, but very intense, band of fluorescence at the J1/J2 interface while the remainder of J2 stained lightly. The lectin MAA produced an intense fluorescence-staining pattern only at the J1/J2 interface. Several lectins were also tested for the ability to inhibit fertilization. WGA, MAA, and concanavalin A significantly inhibited fertilization and WGA was found to block fertilization by preventing sperm from penetrating the jelly. Using Western blotting, we identified high-molecular-weight components in J1 and J2 that may be important in fertilization.

Animals↗
PubMed1999

Oviductin, the Xenopus laevis oviductal protease that processes egg envelope glycoprotein gp43, increases sperm binding to envelopes, and is translated as part of an unusual mosaic protein composed of two protease and several CUB domains.

The glycoprotein envelope surrounding the Xenopus laevis egg is converted from an unfertilizable to a fertilizable form during transit through the pars recta portion of the oviduct. Envelope conversion involves the pars recta protease oviductin, which selectively hydrolyzes envelope glycoprotein gp43 to gp41. Oviductin cDNA was cloned, and sequence analysis revealed that the protease is translated as the N terminus of an unusual mosaic protein. In addition to the oviductin protease domain, a protease domain with low identity to oviductin was present, possessing an apparent nonfunctional catalytic site. Three CUB domains were also present, which are related to the mammalian spermadhesin molecules implicated in mediating sperm-envelope interactions. We propose that during post-translational proteolytic processing of the mosaic oviductin glycoprotein, the processed N-terminal protease domain is released coupled to two C-terminal CUB domains and constitutes the enzymatically active protease molecule. In functional studies, isolated coelomic egg envelopes treated with oviductin purified from the oviduct showed a dramatic increase in sperm binding. This observation established that oviductin alone was the oviductal factor responsible for converting the egg envelope to a sperm-penetrable form, via an increase in sperm binding. Trypsin mimicked oviductin's effect on envelope hydrolysis and sperm binding, demonstrating that gp43 processing is the only requirement for envelope conversion.

Amino Acid Sequence↗
PubMed1998

Occurrence of reducing terminal N-acetylglucosamine 3-sulfate and fucosylated outer chains in acidic N-glycans of porcine zona pellucida glycoproteins.

Structures of acidic N-glycans released from porcine zona pellucida glycoproteins by hydrazinolysis were studied. The results indicated that the acidic glycans are of mono- to tetraantennary complex-type with and without N-acetyllactosamine repeating units. Sulfated residues are not only located at the C-6 position of GlcNAc included in the N-acetyllactosamine repeating units, but also at the C-6 position of GlcNAc in the non-repeated antennae and at the C-3 position of reducing terminal GlcNAc residue. Analysis of the oligosaccharide fragments released by endo-beta-galactosidase digestion and by hydrazine/nitrous acid treatment also revealed that various sulfated and non-sulfated forms of fucosylated structures such as Fucalpha1 --> 2Galbeta1 --> 4(+/-SO3- --> 6)GlcNAc (type 2H), Galbeta1 --> 4(Fucalpha1 --> 3)(+/-SO3- --> 6)GlcNAc(Lex) and Fucalpha1 --> 3 or 4(+/-SO3- --> 6)GlcNAc, are expressed in the repeated outer chain moieties.

Acetylglucosamine↗
PubMed1997

Characterization of neutral oligosaccharide-alditols from Xenopus laevis egg jelly coats by matrix-assisted laser desorption Fourier transform mass spectrometry.

Neutral oligosaccharides were released by alkaline sodium borohydride reduction of the jelly coating from the South African clawed toad, Xenopus laevis. The oligosaccharides were isolated by HPLC and analyzed by matrix-assisted laser desorption ionization (MALDI)-Fourier transform mass spectrometry (FTMS). The mass spectrometry analysis allowed confirmation of 12 structures first proposed by Strecker et al. using nuclear magnetic resonance. In addition, seven new oligosaccharides with weak abundances were found and characterized by mass spectrometry. A method for discriminating metastable fragments from quasimolecular ions is described. It involves doping the sample with cesium chloride. Cesium-coordinated oligosaccharides do not fragment as readily as those coordinated to sodium. Tandem MS experiments are performed on an unknown oligosaccharide illustrating the potential of MALDI-collision-induced dissociation-FTMS.

Animals↗
PubMed1997

cDNA cloning and sequence analysis of the Xenopus laevis egg envelope glycoprotein gp43.

The glycoproteins of the Xenopus laevis egg envelope function in fertilization and development. As the unfertilizable coelomic egg transits the pars recta region of the oviduct, it is converted to a fertilizable egg by limited proteolysis of the envelope glycoprotein gp43 to gp41. This conversion is caused by an oviductally secreted serine active site protease, oviductin. We cloned a cDNA for gp43 from an oocyte cDNA library. The cDNA encoded a 454 amino acid protein homologous to the ZPC family of glycoproteins previously shown to be present in mammalian and fish egg envelopes. Conserved ZPC domains and motifs present in the Xenopus sequence included a signal peptide sequence, an N-linked glycosylation site, and 12 aligned Cys residues. In mammalian and Xenopus sequences, a furin-like (convertase) site and a C-terminal transmembrane domain were present reflecting the biosynthesis of ZPC in these species via the secretory glycoprotein pathway. However, fish envelope glycoproteins lack these sequences since they are synthesized via a different route (in the liver, transported to the ovary, and assembled into the egg envelope surrounding the oocyte). Consensus amino acid residues were identified by sequence comparisons of seven ZPC family members; 19% of the amino acid residues were invariant and 48% of the residues were identical in at least four of the seven sequences. The consensus sequence was used to make structure-fertilization function predictions for this phylogenetically conserved family of glycoproteins.

Amino Acid Sequence↗
PubMed1996

The fertilization layer mediated block to polyspermy in Xenopus laevis: isolation of the cortical granule lectin ligand.

The fertilization layer of Xenopus laevis eggs is formed by the cortical granule lectin binding to its ligand. The binding requires Ca2+, is specific for galactose, and functionally establishes a block to polyspermy at fertilization. We have designed a new enzyme-linked lectin assay for the cortical granule lectin (CGL) ligand which can detect the presence of the CGL ligand at a sensitivity of 1-2 ng/ml. This assay is specifically inhibited with galactose, 50% inhibition at 9.9 mM, and produces a linear response between 2 and 20 ng of jelly adsorbed to the microtiter plate. Using this assay, the CGL ligand was purified through gel filtration, anion-exchange, and CGL affinity chromatography. Hydrolysis of the purified CGL ligand with a series of exoglycosidases showed that a terminal alpha-galactose is the ligand structure required for recognition by CGL.

Amino Acids↗