Calcium-induced dehiscence of cortical granules in Xenopus laevis oocytes

Microinjection of 0.1 microgram of Ca++ into Xenopus laevis oocytes induces breakdown of the cortical granules. The cortical granules disappeared in both full grown (Stage VI) and small growing (Stage IV) oocytes. Microinjection of Mg++, K+, or Na+ had no effect on cortical granules in either Stage IV or Stage VI oocytes. Small quantities (0.03 microgram) of Ca++ induced dehiscence of the cortical granules only in proximity to the injection site.     

Export of proteins from oocytes of Xenopus laevis.

When human lymphoblastoid mRNA was microinjected into X. laevis oocytes, titers of interferon rapidly reached a maximum inside the oocyte while accumulation of interferon continued in the incubation medium for at least 45 hr. If interferon protein was injected into oocytes it was rapidly inactivated. Significantly, newly synthesized interferon but not injected interferon was found to be membrane-associated. Further experiments involving the co-injection of mRNAs coding for secretory proteins (guinea pig milk proteins and human interferon) and nonsecretory proteins (rabbit globin) revealed that only the secretory proteins were exported from the oocyte. Moreover, different proteins were exported at different rates. A distinct subclass of newly synthesized oocyte proteins of unknown function also accumulated in the incubation medium. Since the information encoded in the messenger RNAs of secretory proteins is sufficient to specify synthesis, compartmentation and secretion of these proteins, the oocyte may provide a complete system for the analysis of the secretory process.     

Endogenous phosphorylated proteins during maturation of Xenopus laevis oocytes

During the course of maturation of Xenopus laevis oocyte a burst of phosphorylation occurs around germinal vesicle breakdown. At the same time a relative drop in a unique phosphoprotein (protein I; mot wt ～40,000) is observed. Enucleation of [³²P] labeled oocytes has shown the cytoplasmic localization of protein I. Methylxanthines and cholera toxin, which inhibit progesterone-induced maturation, block the burst of phosphorylation and do not change the amount or the distribution of [³²P] phosphoproteins.     

In-cell NMR spectroscopy of proteins inside Xenopus laevis oocytes

In-cell NMR is an application of solution NMR that enables the investigation of protein conformations inside living cells. We have measured in-cell NMR spectra in oocytes from the African clawed frog Xenopus laevis. ¹⁵N-labeled ubiquitin, its derivatives and calmodulin were injected into Xenopus oocytes and two-dimensional ¹H–¹⁵N correlation spectra of the proteins were obtained. While the spectrum of wild-type ubiquitin in oocytes had rather fewer cross-peaks compared to its in vitro spectrum, ubiquitin derivatives that are presumably unable to bind to ubiquitin-interacting proteins gave a markedly larger number of cross-peaks. This observation suggests that protein–protein interactions between ubiquitin and ubiquitin-interacting proteins may cause NMR signal broadening, and hence spoil the quality of the in-cell HSQC spectra. In addition, we observed the maturation of ubiquitin precursor derivative in living oocytes using the in-cell NMR technique. This process was partly inhibited by pre-addition of ubiquitin aldehyde, a specific inhibitor for ubiquitin C-terminal hydrolase (UCH). Our work demonstrates the potential usefulness of in-cell NMR with Xenopus oocytes for the investigation of protein conformations and functions under intracellular environmental conditions.Electronic Supplementary Material Supplementary material is available to authorized users in the online version of this article at .     

On the contents of the cortical granules from Xenopus laevis eggs

The extruded contents of the cortical granules in eggs of Xenopus laevis were solubilized by exposure to divalent metal ion chelators. Chelator extraction of cortical granule (CG) material from intact fertilized or artificially activated eggs was quantitated by fluorescence spectroscopy. The isolated fertilization envelope, formed upon interaction between CG material and the preexisting vitelline envelope, was also subject to extraction. An ultrastructural analysis revealed that chelator exposure resulted in the disruption of the structural integrity of the CG-derived F-component of the fertilization envelope. CG material was isolated from Xenopus ova by three procedures: (1) extrusion from artificially activated, dejellied eggs; (2) extraction of intact, fertilized eggs; and (3) extraction of isolated fertilization envelopes. Only 4–5% of the CG protein recovered by extrusion or by extraction of the intact fertilized egg could be associated with the isolated fertilization envelopes. One predominant polypeptide fraction with an identical relative mobility was demonstrated in all CG preparations upon polyacrylamide gel electrophoresis in SDS. Polymeric forms of CG protein were detected in chelator extracted preparations. The presence of an intact jelly coat during CG breakdown was a prerequisite to the transformation of the vitelline envelope to a fertilization envelope with altered physicochemical characteristics. Further, the CG-derived F-component of the fertilization envelope did not appear to play a critical role in determining the physicochemical properties of the fertilization envelope.     

Phosphorylation activity associated with plasma membrane-cortical granule complex of Xenopus laevis oocytes

Plasma membrane-cortical granule complexes from Xenopus laevis oocytes were isolated by a differential centrifugation procedure. Membrane preparations incorporated 32P from [gamma-32P]ATP predominantly into three protein bands of Mr 128,000, 54,000 and 29,000, as determined by sodium dodecyl-polyacrylamide gel electrophoresis. Progesterone and insulin, added in vitro to membranes, did not produce any change in this phosphorylation pattern, contrary to some previously published results. The availability of enriched membrane complexes should permit further biochemical dissection of signal transduction pathways that are activated during oocyte maturation and fertilization.     

Structural proteins associated with ribosomal cistrons in Xenopus laevis chromosomes

The N banding technique to define the location of nucleolus organiser in mammalian and marsupial chromosomes was applied to the Xenopus laevis chromosomes. Results obtained are: 1. The N bands coincide with the location of all the clustered ribosomal cistrons including the 18S + 28S RNA genes as well as the 5S RNA genes. 2. The N bands are consistently detected in both metabolically active (interphase) and metabolically inactive (metaphase) nuclei. 3. Cytochemical and chemical extraction tests indicate that the N bands show typical biochemical properties requested for non-histone (residual) chromosomal proteins. 4. Proteins associated with the 5S RNA genes differ, in their acid-solubility, from those for the 18S+28S RNA genes. 5. The N banding proteins comprise a small portion of a total nuclear protein. These findings strongly suggest the existence of ribosomal gene-specific non-histone proteins which probably represent the structural chromatin element rather than the primary gene product. The possible role of N banding proteins in eukaryotes is discussed.     

Purification and characterization of an N-acetyl-beta-D-glucosaminidase from cortical granules of Xenopus laevis eggs

The enzyme N-acetyl-beta-D-glucosaminidase was purified from the cortical granules of Xenopus laevis eggs using affinity chromatography, gel filtration, and density gradient centrifugation. The enzyme had a molecular weight of 37,000-40,000 as determined by polyacrylamide gel electrophoresis and density gradient centrifugation, had a Km for p-nitrophenyl-beta-D-N-acetyl-glucosaminide of 0.66 mM and a Ki for glucosamine of 4.3 mM. The kinetic properties of the cortical granule enzyme were similar to the enzyme isolated from jack bean. Treatment of unfertilized eggs with the enzyme isolated from cortical granules or jack bean rendered eggs unfertilizable. Loss of fertilizability was proportional to the product of time and enzyme concentration, consistent with an enzymatic mechanism being responsible for the loss of fertilizability. The amount of enzyme present in the perivitelline space was approximately the same as that which reduced fertilizability by 50% in one hour. We suggest that the action of cortical granule N-acetyl-beta-D-glucosaminidase on egg integuments may function as a block to polyspermy at fertilization.     

Egg yolk platelet proteins from Xenopus laevis are amyloidogenic

The association of amphibian (Xenopus laevis) egg yolk platelet proteins, represented predominantly by lipovitellin, was studied as a model of the formation of amyloid deposits. Two kinds of molecular organization formed by this protein material - native and heat-denatured - were found to exhibit amyloid properties although they differ significantly in structural organization. The first consisted in protein molecules arranged in the natural, physiological, net-like platelet organization, with a tendency to orient uni-directionally. The second was obtained by the gradual removal of Congo red from lipovitellin denatured by heating in an excess of dye. This procedure produced the twisted fibrillar organization of molecules typical for amyloids, represented predominantly by end-to-end associated major polypeptide chains of lipovitellin. Both native and denatured structural forms bind Congo red and produce a green birefringence effect, confirming the near parallel alignment of the complexed Congo red molecules. However, a dye(1,4-bis(1-amino-4-sulfonaphtyl-2-azo)phenylene) closely related to Congo red but with a very weak self-assembling tendency appeared inactive when the spectral shift was studied in a cross-polarization system, indicating in this way that dye supramolecularity is an extra factor which may determine binding to amyloid proteins and specific spectral effects.     

The metabolism of ribosomal proteins microinjected into the oocytes of Xenopus laevis

When the total proteins from Xenopus laevis 60 S ribosomal subunits (TP60) were 3H-labeled in vitro and injected back into X. laevis oocytes, most 3H-TP60 are integrated into the cytoplasmic 60 S subunits via the nucleus during 16 h of incubation. In the oocytes whose rRNA synthesis is inhibited, 3H-TP60 are rapidly degraded with a half-life of 2-3 h. This degradation ceased as soon as rRNA synthesis was resumed, suggesting that ribosomal proteins unassociated with nascent rRNA are unstable in the oocytes. The degradation of 3H-TP60 in the absence of RNA synthesis was inhibited by iodoacetamide, a cysteine protease inhibitor, resulting in the accumulation of 3H-TP60 in the nucleus reaching about a threefold concentration in the cytoplasm. Considering the results with enucleated oocytes, we suggest that the X. laevis nucleus has a limited capacity to accumulate ribosomal proteins in an active manner but that those ribosomal proteins accumulated in excess over rRNA synthesis are degraded by a cysteine protease in the nucleus. By contrast, ribosomal proteins from Escherichia coli only equilibrate between the nucleus and the cytoplasm and are degraded by serine protease(s) in the cytoplasm without being integrated in the form of ribosomes in the nucleus.     

Isolation and characterization of a lectin from the cortical granules of Xenopus laevis eggs

A cortical granule lectin was isolated from eggs of the South African clawed toad Xenopus laevis. The lectin was released from the cortical granules by activation of dejellied eggs with the Ca2+ ionophore A23187. The lectin was purified by affinity chromatography with its natural ligand, the egg jelly coat, chemically coupled to a Sepharose matrix. The purified lectin was homogeneous by the criteria of isoelectric focusing (pI = 4.6), immunodiffusion, and immunoelectrophoresis but existed in two different molecular weight isomers as determined by sedimentation velocity ultracentrifugation and disc gel electrophoresis. Molecular weights of the isomers were determined by ultracentrifugation, disc gel electrophoresis, and gel filtration and found to be 539,000 and 655,000. Chemically, the lectin was a metalloglycoprotein, composed of 84.0% protein, 15.8% carbohydrate, and 0.19% calcium. No unusual types or amounts of amino acids were present. The carbohydrate moiety was composed of fucose, mannose, galactose, glucosamine, galactosamine, and sialic acid. The monosaccharide specificity of the lectin was investigated with the sugar inhibition of the precipitin reaction in gels. The lectin was specific for D-galactosyl sugars with the configuration at carbon atoms 2-4 of primary importance.     

Incorporation of mouse zona pellucida proteins into the envelope of Xenopus laevis oocytes

 All vertebrate eggs have extracellular matrices, referred to as the zona pellucida in Mus musculus and the vitelline envelope in Xenopus laevis. The mouse zona, composed of three sulfated glycoproteins (ZP1, ZP2, ZP3), is critical for fertilization and early development, and mice lacking a zona pellucida produce no live offspring. The primary structures of mouse ZP1 (623 amino acids), ZP2 (713 amino acids) and ZP3 (424 amino acids) have been deduced from full-length cDNAs, but posttranslational modifications result in mature zona proteins with molecular masses of 200–180 kDa, 140–120 kDa, and 83 kDa, respectively. The vitelline envelope forms a similar structure around Xenopus eggs and contains three glycoproteins that are structurally related (39–48% amino acid similarity) to the three mouse zona proteins. To investigate whether the structural semblances are sufficient to allow incorporation of the mouse zona proteins into the Xenopus vitelline envelope, capped synthetic mRNAs encoding ZP1, ZP2, and ZP3 proteins were injected into the cytoplasm of stage VI Xenopus oocytes. After 20 h of incubation the oocytes were harvested, and posttranslationally modified zona proteins were detected with monoclonal antibodies specific to mouse ZP1, ZP2, and ZP3. The oocytes were imaged with confocal microscopy to detect individual zona proteins in the extracellular matrix of the oocytes, and this localization was confirmed biochemically. Thus the mouse zona proteins appear to have been sufficiently conserved through 350 million years of evolution to be incorporated into the extracellular envelope surrounding Xenopus eggs. Received: 5 January 1999 / Accepted: 12 February 1999     

Polyadenylic acid-containing RNA in Xenopus laevis oocytes

The quantity of poly(A)-containing RNA is measured in Xenopus laevis oocytes as a function of developmental stage. The amount of poly(A)-containing RNA per oocyte, 0.7 to 1.0% of the total RNA, remains relatively constant from early vitellogenesis until ovulation. It is largely present in the cytoplasm of the oocyte in the form of a ribonucleoprotein complex. The poly(A) sequence is approximately 100 bases in length and is attached to molecules of heterogeneous sedimentation coefficients.     

Induction of maturation in small Xenopus laevis oocytes

The competence of Xenopus laevis oocytes in various stages of growth to respond to progesterone treatment was investigated. Full-grown (stage 6) oocytes undergo nuclear membrane dissolution and resume meiosis in response to progesterone exposure, while smaller oocytes (stages 3-5; less than 1100 micron in diameter) do not. The defect which prevents 750- to 1050-micron oocytes from responding to progesterone can be overcome by microinjecting cytoplasm withdrawn from a stage 6 oocyte. Germinal vesicle breakdown in these small oocytes occurs on a timetable similar to that of stage 6 oocytes exposed to progesterone and is accompanied by a twofold increase in protein synthesis as well as the activation of MPF. The results argue that a cytoplasmic factor(s) which probably first appears at late stage 5 is required for progesterone responsiveness. The identity and role of the factor(s) in the development of maturation competence and the regulation of maternal mRNA translation are discussed.     

Mitochondria DNA synthesis in oocytes of Xenopus laevis

The process of attachment was studied in primary mouse kidney epithelial cell cultures by means of reflexion contrast microscopy, a method developed for studying the cell membrane-substrate relationship. The first in a series of events is simple adherence to the substrate, called close contact. This phenomenon is associated with the greatest extension of lamellar cytoplasm and the fewest number of cell nuclei/unit area. The nuclei of such cells are in close contact with the bottom portion of the cell membrane. Approx. 24 h after planting, as the cultures become more crowded, cells develop a different kind of attachment to the substrate—focal contacts—that are correlated with a decrease in lamellar cytoplasm. Cells detached from the substrate after close contact formation readily reattach, while cells detached after formation of focal contacts do not reattach. After incubation for periods greater than 5 days, the dense cultures degenerate and cells lose their attachment to the glass surface.