Localization of cortical granule lectin ligand in Xenopus laevis egg jelly

The block to polyspermy in Xenopus laevis involves an interaction between a cortical granule lectin, released at fertilization, and a ligand located in the egg extracellular matrix. The egg extracellular matrix in X. laevis consists of a vitelline envelope and three distinct jelly layers, designated J1, J2 and J3. To localize cortical granule lectin ligand in the egg extracellular matrix, we used enzyme-linked lectin assays that showed that cortical granule lectin ligands were absent in J2, J3 and the vitelline envelope. Cortical granule lectin bound to a ligand(s) in J1 in a galactose-dependent fashion. In addition, we separated egg jelly macromolecules electrophoretically and, in conjunction with western blotting, have shown that J1 contains two major, high molecular weight ligands for cortical granule ligand. Finally, using confocal microscopy, we demonstrated that the ligand(s) for cortical granule lectin occupies a 20–30 μm thick band in a region of J1 just proximal to the vitelline envelope.     

Xenopus laevis egg jelly contains small proteins that are essential to fertilization.

The eggs of Xenopus laevis are surrounded by investment layers of egg jelly that interact with the sperm immediately prior to fertilization. Components of these egg jelly layers are necessary for the fertilization of the egg by incoming sperm. Eggs which are stripped of their jelly layers are refractile to fertilization by sperm, but the addition of solubilized jelly promotes fertilization. We have shown previously that the egg jelly layers are composed of a fibrous network of glycoconjugates which loosely hold smaller diffusible components. Extracts of these diffusible components were prepared by incubation of freshly ovulated eggs in high-salt buffers for 12 h at 4 degrees C. This diffusible component extract, when incubated with sperm, promoted the sperm's ability to fertilize dejellied eggs in a dose-dependent manner. In contrast, the high-molecular-weight "structural" glycoconjugates of jelly that remain after extraction of the diffusible components did not increase fertilization efficiency of dejellied eggs nor did nonspecific proteins, carbohydrate polymers, or organic polymers. The diffusible components, analyzed by SDS-PAGE, consisted of a mixture of proteins from 4 to 180 kDa. The protein responsible for fertilization rescue appeared to be <50 kDa and appeared to self-aggregate or to bind to larger proteins. This protein component was required during sperm binding to the egg, its action required an intact egg vitelline envelope, and its action was independent of large soluble polymers such as Ficoll.     

Xenopus laevis lectin is localized at several sites in Xenopus oocytes, eggs, and embryos

The endogenous lectin of Xenopus laevis oocytes, unfertilized eggs, and blastula-stage embryos was immunohistochemically localized using a highly specific antiserum. Each tissue was examined with several techniques, including paraformaldehyde or glutaraldehyde fixation, frozen or plastic sections, and immunofluorescence or immunoperoxidase staining. In oocytes and unfertilized eggs, lectin was detected in association with yolk platelets, cortical granules, and the vitelline envelope. In embryos, cortical granules had disappeared and lectin was found in the cleavage furrows between the embryonic cells. The distribution of the lectin suggests that it plays more than one role in this developing system.     

Purification and multimer formation of allurin, a sperm chemoattractant from Xenopus laevis egg jelly

Allurin, a sperm chemoattractant isolated from Xenopus laevis egg jelly, can be purified in one step from an extract of diffusible jelly proteins (egg water) using a FPLC or HPLC anion exchange column and a multi-step NaCl gradient. Allurin homomultimers were detected by Western blotting with antibodies prepared against the purified protein or peptides within the protein. Allurin multimers were stable and resisted dissociation by SDS and beta-mercaptoethanol. Alkylation of allurin provided evidence for two free sulfhydryl groups but did not eliminate multimer formation, suggesting that intermolecular disulfide bond formation is not required for allurin aggregation. Concentration of egg water was accompanied by a reduction of chemoattractant activity that could not be fully accounted for by homomultimer formation. Rather, the presence of a multiphasic dose-activity curve upon partial purification and formation of hetero-allurin complexes during concentration suggested that egg water may contain allurin-binding proteins that reduce multimer formation and activity.     

Bufo japonicus japonicus and Xenopus laevis laevis egg jellies contain structurally related antigens and cortical granule lectin ligands

The antigenic relationship of the egg jelly coat glycoproteins from Bufo japonicus japonicus and Xenopus laevis laevis was investigated using agar double diffusion methods. The presence of ligands in the jelly coats for the cortical granule lectin from X.l. laevis eggs was also investigated. Anti-jelly serum for both anuran species crossreacted with the jelly coat from the other species with precipitin patterns of identity. Each egg jelly coat of both species contained two ligands for the cortical granule lectin. Although the ligands in the two different jelly coats appeared to react with the lectin in a pattern of identity, the species ligands were antigenically distinguishable using anti-Xenopus jelly serum. The observations that the two anuran egg jelly coats were antigenically related and that they both contained ligands for the X.l. laevis cortical granule lectin was interpreted in terms of fertilization mechanisms in the two different species. In addition, these observations bring into question the currently accepted phylogenetic relationship of B.j. japonicus and X.l. laevis.     

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.     

Swimming of Xenopus laevis sperm exhibits multiple gears and its duration is extended by egg jelly constituents

The motility of Xenopus sperm is initiated by the osmotic shock experienced when these cells are ejaculated into low-salinity pond water. Motility is brief and is required for the sperm to penetrate the jelly layers and fertilize the egg. In this study we demonstrate that extracts of egg jelly contain factors that extend the period of sperm motility as well as providing a chemoattractant activity as previously reported. Both activities are partially dependent on extracellular calcium. Time-lapse and video microscopy show that after activation of motility the number of motile sperm decreases rapidly, with a half-time of about 2 min. Addition of 10% v/v egg jelly extract ("egg water") increased the number of motile sperm 2-fold over controls at 20 s and about 4- to 10-fold over controls at 10 min after initiation of motility. Extension of motility lifetime was not mediated by a nonspecific protein or by allurin, the egg-water protein that has chemoattractant activity. The helical path of Xenopus sperm exhibited tight coupling between rotational and forward velocities in egg jelly, but coupling changed rapidly from moment to moment in low-salinity buffer. Our observations suggest that jelly-derived factors regulate both the longevity and directionality of sperm propulsion.     

Identification of Xenopus laevis sperm and egg envelope binding components on nitrocellulose membranes

Interacting egg envelope and sperm surface components were identified for Xenopus laevis using blotting methods. Sperm were extracted with sodium dodecyl sulfate (SDS), the extracted proteins separated by gel electrophoresis and blotted, and the blots treated with 125I-labeled heat solubilized envelopes. The converse experiment was also performed where envelope components were separated by gel electrophoresis, blotted, and the blots treated with 125I-labeled sperm components. Blotted sperm components with apparent molecular weights of 14K, 19K, 25K, and 35K selectively bound the solubilized envelopes. All of the envelope binding components were found to be localized on the sperm surface by radioiodinating intact sperm using Iodo-Gen. The blotted egg envelope component with an apparent molecular weight of 37K selectively bound to solubilized sperm components, and this binding was due to the protein moiety of the glycoprotein. 125I-labeled heat solubilized envelopes from unfertilized and fertilized eggs showed the same pattern of binding to blotted sperm components. Selected sulfated carbohydrates (fucoidan, dextran sulfate, and heparin, but not chondroitin sulfate) inhibited fertilization and binding of 125I-labeled heat solubilized envelopes to blotted sperm extract. Thus, the binding of heat solubilized envelopes to electrophoretically separated and blotted sperm proteins may reflect cellular interactions.     

Egg jelly proteins stimulate directed motility in Xenopus laevis sperm

Previously we have shown that extracts from Xenopus egg jelly (egg water) increase the passage of sperm through a porous membrane in a dose‐dependent manner. Although this assay has shown that sperm accumulation occurs only in the presence of an egg water gradient, it has not revealed the dynamic features of how Xenopus sperm swim in such gradients. Here, we use video microscopic observations to trace sperm trajectories in a Zigmond chamber. Our results show that Xenopus sperm swim in linear and gently curving paths and only infrequently perform turns. In the presence of an egg water gradient, however, the percent of sperm swimming up the gradient axis and the net distance traveled by each sperm along this axis was increased significantly. There was no change in curvilinear velocity. Rather, the orientation of sperm travel was shifted to more closely match that of the gradient axis. In addition, using a porous filter assay, we demonstrate that the egg water protein allurin, in both purified and recombinant forms, stimulates directed motility of sperm. Finally, we use Oregon Green 488‐conjugated allurin to show that this protein binds primarily to the sperm midpiece; binding of allurin to the entire head was observed in a minor subpopulation of sperm. Dose dependence of allurin binding occurred over the 0–1 µg/ml range and correlated well with previously published dose‐dependent sperm attraction data. Binding was rapid with a half‐time of about 10 sec. These data suggest that egg water proteins bind to sperm and modify sperm‐orienting behavior. Mol. Reprod. Dev. 78:450–462, 2011. © 2011 Wiley‐Liss, Inc.     

Galactoside-binding serum lectin of Xenopus laevis. Estrogen-dependent hepatocyte synthesis and relationship to oocyte lectin

Xenopus laevis serum contains a lectin which binds alpha- and beta-galactosides. It was purified to homogeneity by affinity chromatography and consists of a single subunit with Mr approximately 69,000, associated in a multimer. The lectin is synthesized and secreted by hepatic parenchymal cells, and its synthesis is increased about 2-fold by estrogen treatment, both in vivo and in primary cell cultures. The serum lectin has the same carbohydrate binding properties as an oocyte lectin from X. laevis described previously, is immunologically cross-reactive, and shows similarities in its peptide map. However, marked differences in amino acid composition preclude the possibility that the serum lectin is a precursor of the oocyte lectin.     

Secretion of a cytoplasmic lectin from Xenopus laevis skin

The skin of Xenopus laevis contains a soluble beta-galactoside-binding lectin with a approximately 16,000-mol-wt subunit. It resembles similar lectins purified from a variety of tissues from other vertebrates, and differs from two other soluble X. laevis lectins from oocytes and serum that bind alpha-galactosides. The skin lectin is concentrated in the cytoplasm of granular gland and mucous gland cells, as demonstrated by immunohistochemistry with the electron microscope. Upon injection with epinephrine, there is massive secretion of the cytoplasmic lectin from the granular gland cells.     

Identification of fucosylated glycoconjugates in Xenopus laevis testis by lectin histochemistry

Glycoconjugates play roles in many physiological and pathological processes. Previous works have shown important functions mediated by glycans in spermatogenesis, and the carbohydrate composition of testis has been studied by several approaches, including lectin-histochemical methods. However, the testis of Xenopus laevis, an animal model extensively employed in biochemical, cell and developmental research, has not yet been analysed. The aim of this work was to carry out a histochemical study of the fucose (Fuc)-containing glycoconjugates of Xenopus testis by means of lectins, combined with deglycosylation pretreatments. Four Fuc-binding lectins were used: orange peel (Aleuria aurantia) lectin (AAL), gorse seed (Ulex europaeus) agglutinin-I (UEA-I), fresh water eel (Anguilla anguilla) agglutinin (AAA), and asparagus pea (Lotus tetragonolobus) agglutinin (LTA), each recognizing different forms of fucosylated glycans. Labelling with UEA-I, which preferably binds Fucα(1,2) containing oligosaccharides, did not show any appreciable staining. LTA, specific for Fucα(1,3), and AAA, which binds Fucα(1,2), labelled spermatocytes and spermatids, but no labelling was seen when the histochemical procedure was carried out after either β-elimination (which removes O-linked oligosaccharides) or incubation with PNGase F (which removes N-linked oligosaccharides), suggesting that fucosylated glycans are of both N- and O-linked types. AAL, which has its highest affinity to Fucα(1,6), but also recognizes Fucα(1,2) and Fucα(1,3), labelled the whole testis, and the staining remained when the histochemical method was performed after either β-elimination or incubation with PNGase F. Labelling with AAL could be explained by the fact that this lectin could be binding to diverse fucosylated glycans in N- and O-glycans, and even in glycolipids. The importance of these glycans is discussed.     

Sequence and specificity of a soluble lactose-binding lectin from Xenopus laevis skin.

A 16-kDa lactose-binding lectin comprises 5% or more of the soluble protein in Xenopus laevis skin. This lectin is mainly localized in the cytoplasm of granular gland cells. In response to stress, the lectin along with a variety of toxic and antibiotic peptides are released onto the skin surface by holocrine secretion. We have purified the lectin, sequenced tryptic peptides using tandem mass spectrometry and Edman degradation, and isolated full-length cDNA using a deduced oligonucleotide. Comparison of the cDNA and peptide sequences revealed expression of at least two isolectins, which differ in sequence at only two or three amino acids. Comparison of cDNA with complementary message by ribonuclease protection confirmed expression in approximately equal abundance of two nearly identical messages. The major soluble lactose-binding lectin expressed in Xenopus muscle is composed of these same isolectins, but at 100-fold lower levels. Similarities and distinctions in sequence and carbohydrate-binding specificity indicate that this lectin is a novel member of a family of soluble lactose-binding lectins expressed in a wide range of vertebrate tissues.     

Egg jelly layers of Xenopus laevis are unique in ultrastructure and sugar distribution

Jelly coats surrounding the eggs of the South African clawed toad, Xenopus laevis, consist of three transparent, gelatinous layers: the innermost layer (J1), the middle layer (J2), and the outer layer (J3). The distribution of N-acetylglucosamine within these jelly coats, as probed with FITC-conjugated wheat germ agglutinin (WGA-FITC), and the matrix ultrastructure of each layer, as visualized in platinum replicas produced by the quick-freeze, deep-etch, and rotary-shadowing technique, suggests that each layer has a unique fiber and glycoprotein composition. J1 extends nearly 200 μm from the egg surface and exhibits no WGA-FITC staining. Stereo images of platinum replicas indicates that J1 consists of a tightly knit network of 5–10 nm fibers decorated with 10–20 nm particulate components. In contrast, J2 is a relatively thin layer, extending only 25–40 μm from the outer aspect of J1. When visualized by confocal microscopy, J2 displays a multilayered WGA-FITC staining pattern. The ultrastructure of J2 consists of sheets of fine fibers that run parallel to one another and that can be identified by their ability to bind WGA-colloidal gold. The fibers of each sheet run at an oblique angle to fibers in neighboring layers. J3 extends 100 μm or more from J2. The WGA-FITC staining pattern shows high intensity in its outer region and less intensity in regions closer to J2. Like J1, the J3 ultrastructure consists of a network of 5–10 nm fibers, decorated with 10–20 nm particulate components. The results of these studies add to a growing body of information that suggests the jelly coats surrounding the eggs of many animals consist of a fibrous glycoprotein superstructure that acts as a scaffold to which globular glycoproteins are bound. © 1996 Wiley-Liss, Inc.     

Micromolar free calcium exposes ouabain-binding sites in digitonin-permeabilized Xenopus laevis oocytes.

As demonstrated previously, digitonin-permeabilized Xenopus oocytes have a large internal pool of sodium pumps which are inaccessible to cytosolic ouabain [Schmalzing, Kröner & Passow (1989) Biochem. J. 260, 395-399]. Access to internal ouabain-binding sites required permeabilization of inner membranes with SDS. In the present study, micromolar free Ca2+ was found to stimulate ouabain binding in the digitonin-permeabilized cells (K0.5 0.5 microM-Ca2+, h 1.9, average of seven experiments) without disrupting intracellular membranes. Sustained incubation at 9 microM-Ca2+ was as effective as SDS in inducing access to the ouabain-binding sites of the internal sodium pumps. Omission of either Mg2+ or ATP completely abolished the Ca2+ effect. Half-maximal stimulation by Ca2+ required approx. 0.4 mM-MgATP. Of a variety of nucleotides tested, none was as effective as ATP (rank order ATP greater than ADP greater than ATP[S] (adenosine 5''-[gamma-thio]triphosphate) greater than CTP greater than UTP greater than ITP = XTP greater than GTP). Pi, AMP, cyclic AMP, cyclic GMP, GTP[S] (guanosine 5''-[gamma-thio]triphosphate) and a stable ATP analogue p[NH]ppA (adenosine 5''-[beta gamma-imido]triphosphate), were ineffective. The metalloendoproteinase inhibitor carbobenzoxy-Gly-Phe-amide reduced the Ca2+ effect by some 50%. Inhibitors of chymotrypsin and the Ca2+ proteinase calpain had no effect. Ca2+ ionophores (A23187 and ionomycin) and the polycations neomycin and polymixin B blocked the Ca2+ response entirely. Neomycin also abolished a Ca2(+)-independent stimulation of ouabain binding by the wasp venom mastoparan. The requirements for increasing the accessibility of ouabain-binding sites are remarkably similar to those for exocytosis in secretory cells, suggesting that oocytes and eggs possess a Ca2(+)-regulated pathway for the plasma membrane insertion of sodium pumps.     

Microtubule-dependent assembly of the nuclear envelope in Xenopus laevis egg extract

Microtubules take part in several mechanisms of intracellular motility, including organelle transport and mitosis. We have studied the ability of Xenopus egg extract to support nuclear membrane and pore complex formation when microtubule dynamics are manipulated. In this report we show that the formation of a nuclear envelope surrounding sperm chromatin requires polymerized microtubules. We have observed that microtubule-depolymerizing reagents, and AS-2, a known inhibitor of the microtubule motor protein kinesin, do not inhibit the formation of a double nuclear membrane. However these double membranes contain no morphologically identifiable nuclear pore complexes and do not support the accumulation of karyophilic proteins. In contrast, the assembly of annulate lamellae, cytoplasmic structures containing a subset of pore complex proteins, was not affected. Our data show that not only polymerized microtubules, but also the microtubule motor protein kinesin, are involved in the formation of the nuclear envelope. These results support the conclusion that multiple nuclear envelope-forming mitotic vesicle populations exist, that microtubules play an essential and selective role in the transport of nuclear envelope-forming vesicle population(s), and that separate mechanisms are involved in nuclear envelope and annulate lamellae formation.     

Fertilization potential and electrical properties of the Xenopus laevis egg

The membrane potential of Xenopus eggs was monitored continuously from prior to fertilization until early cleavage. A rapid decay of the initial potential of -33.1 +/- 8.1 (SD) mV (N = 14) upon impalement to a value of -19.3 +/- 4.2 (SD) mV (N = 68) suggested that insertion of the first electrode caused depolarization. Outward and inward rectification were observed when the resting potential was made more positive than about 5 mV or more negative than about -30 mV. Eggs were not activated by this level of current injection. Fertilization and activation evoked a membrane depolarization which was influenced by the external Cl- concentration, the nature of the halide species, and 4,4-diisothiocyanostilbene-2,2-disulfonic acid. Smaller transient depolarizations were associated with the initial stages of the fertilization potential but not with activation. Only when the fertilization potential was significantly diminished, as in high external Cl- or in the presence of Br- or I- solutions did polyspermy ensue. The input resistance of the unfertilized egg was 13.2 +/- 9.8 M omega (N = 26) and decreased about 200-fold at the peak of the fertilization potential to 0.077 +/- 0.020 M omega (N = 9). Ninety minutes after the onset of the fertilization potential and about 6 min after the start of furrow formation the membrane began a series of cleavage cycle-associated hyperpolarizations. These were unaffected by either the external Cl- concentration or other halide species. Reduction in amplitude of the fertilization potential had no apparent effect upon the normal elevation of the fertilization envelope or upon cleavage and later development. The fast electrical block to polyspermy appears to have a lower threshold in Xenopus compared with other species and is also effective at negative membrane potentials.     

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.     

Nonhomologous DNA end joining of synthetic hairpin substrates in Xenopus laevis egg extracts.

Processes of DNA end joining are assumed to play a major role in the elimination of DNA double-strand breaks (DSB) in higher eucaryotic cells. Linear plasmid molecules terminated by nonhomologous restriction ends are the typical substrates used in the analysis of joining mechanisms. However, due to their limited structural variability, DSB ends generated by restriction cleavage cover probably only part of the total spectrum of naturally occurring DSB termini. We therefore devised novel DNA substrates consisting of synthetic hairpin-shaped oligonucleotides which permit the construction of blunt ends and 5'- or 3'-protruding single-strands (PSS) of arbitrary sequence and length. These substrates were tested in extracts of Xenopus laevis eggs known to efficiently join linear plasmids bearing nonhomologous restriction termini (Pfeiffer and Vielmetter, 1988). Sequences of hairpin junctions indicate that the short hairpins are joined by the same mechanisms as the plasmid substrates. However, the bimolecular DNA end joining reaction was only detectable when both hairpin partners had a minimal duplex stem length of 27bp and their PSS-tails did not exceed 10nt.