Partial purification and characterization of the maturation-promoting factor from eggs of Xenopus laevis

Maturation-promoting factor (MPF) was purified 20- to 30-fold from unfertilized eggs of Xenopus laevis, by ammonium sulfate precipitation and chromatography on pentyl-agarose and arginine-agarose. The final material induces maturation in 50% of the recipient oocytes when 5 ng of protein is injected in a volume of 20 ml. The maturation response includes precocious germinal vesical breakdown, elevated protein phosphorylation, amplification of cytoplasmic MPF, and formation of an activatable egg blocked at second meiotic metaphase. These eggs are capable of cleavage and, in some cases, of gastrulation. A quantitative in vivo assay of MPF is described and a unit of MPF activity is defined as that amount causing a 50% maturation frequency when oocytes are injected each with a 20-nl test volume. Maturation frequency has a very high-order dependence on MPF concentration. The purification procedure selects simultaneously for endogenous protein phosphorylation systems containing kinases, protein substrates, and phosphatases. This fact, as well as the finding that ATP enhances MPF activity at least twofold when included in the dilution medium for assay, is discussed in terms of the possible involvement of protein phosphorylation in MPF activation and inactivation.     

Purification of a DNA-binding protein from Xenopus laevis unfertilized eggs.

A DNA-binding protein from Xenopus laevis unfertilized eggs has been purified to apparent homogeneity. It is a heat stable, lysine-rich protein and has a molecular weight corresponding to 8,200 daltons, measured by sodium dodecyl sulphate gel electrophoresis. The protein, which is active in a monomeric form, stimulates DNA polymerase alpha, and binds to single and double stranded DNA. One egg contains about 4 x 10(12) molecules (minimum estimate) of the protein; since we calculate that 4 x 10(8) molecules are sufficient to cover the entire genome (haploid complement), there is much more protein than is needed to cover chromosomal DNA.     

Massive phosphorylation distinguishes Xenopus laevis nucleoplasmin isolated from oocytes or unfertilized eggs

Nucleoplasmin isolated from unfertilized Xenopus laevis eggs possesses an in vitro chromatin assembly activity which is superior to nucleoplasmin isolated from oocytes. It is demonstrated here that the two forms of the protein differ in the amount of attached phosphate, with the egg protein possessing nearly 20 phosphate groups per protein monomer and the oocyte protein possessing less than 10 phosphate groups per monomer. A kinase preparation from unfertilized eggs is shown to be capable of modifying oocyte nucleoplasmin so that it displays the electrophoretic heterogeneity of egg nucleoplasmin. Furthermore, when the egg protein is treated with phosphatase and repurified, the chromatin assembly activity deteriorates to the level of the oocyte protein.     

Biochemical characterization of lysosomes in unfertilized eggs of Xenopus laevis.

Relations between lysosomes and yolk platelets of amphibian eggs have been suggested. This work demonstrates the presence of acid hydrolases in oocytes induced to ovulate in vitro. About 40% of the acid hydrolases are found in a sedimentable fraction, and, in accordance with the lysosomal concept, they display structural latency. Biochemical data did not indicate any association between lysosomal enzymes and yolk platelets. The mechanism of yolk resorption is discussed and it is suggested that the fusion of lysosomes and yolk platelets might be one of the mechanisms involved in yolk digestion.     

Properties of a cytostatic factor from Xenopus laevis eggs.

The cytoplasm of mature eggs of Xenopus laevis was found to contain a cytostatic factor (CSF) which induces cleavage arrest at metaphase when microinjected into one blastomere of a two-cell embryo of Xenopus laevis or Rana pipiens. The Rana CSF was found to be incapable of arresting mitosis in Xenopus embryos. Both Xenopus and Rana CSF were stabilized during the transfer procedure by Ca²⁺-chelation in the donor egg. The Xenopus CSF was not present in the germinal vesicle of immature oocytes, but arose in the cytoplasm at the time of germinal vesicle breakdown and subsequently disappeared at the time of fertilization or egg activation.     

RNA 3' cleavage and polyadenylation in oocytes and unfertilized eggs of Xenopus laevis

Xenopus laevis histone H4 and H1 genes were transcribed in vitro to generate artificial precursor mRNAs (pre-mRNAs). These pre-mRNAs were microinjected into oocytes, matured oocytes, and unfertilized eggs of Xenopus laevis and their 3' cleavage and polyadenylation were investigated. In the oocyte nucleus both H4 and H1 pre-mRNAs were 3' cleaved but were not detectably polyadenylated. In the oocyte cytoplasm there was neither 3' cleavage nor polyadenylation of these histone pre-mRNAs. When injected into either matured oocytes or unfertilized eggs, the pre-mRNAs underwent 3' cleavage but this was inefficient when compared to the oocyte nucleus. In addition approximately 50% of the remaining uncleaved pre-mRNA was subject to a polyadenylation activity which added A tails of approximately 70 A residues. In contrast, artificial mouse beta-globin pre-mRNAs were not detectably 3' cleaved or polyadenylated in either microinjected oocytes or unfertilized eggs.     

Cyclin is a component of maturation-promoting factor from Xenopus

Highly purified maturation-promoting factor (MPF) from Xenopus eggs contains both cyclin B1 and cyclin B2 as shown by Western blotting and immunoprecipitation using Xenopus anti-B-type cyclin antibodies. Immunoprecipitates with these antibodies display the histone H1 kinase activity characteristic of MPF, for which exogenously added B1 and B2 cyclins are both substrates. Protein kinase activity against cyclin oscillates in maturing oocytes and activated eggs with the same kinetics as p34cdc2 kinase activity. These data indicate that B-type cyclin is the other component of MPF besides p34cdc2.     

Radioiodination studies of the envelopes from Xenopus laevis eggs

To investigate the molecular basis of the observed morphological and biological characteristics of coelomic egg envelopes (CE), vitelline envelopes (VE), and fertilization envelopes (FE) of Xenopus laevis eggs, envelopes were radioiodinated under a variety of conditions: in situ, isolated and intact, or solubilized. The distribution of 125I in envelope components was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Each envelope type displayed unique profiles when iodinated in the intact state. A major constituent of VE, the 41,500 molecular weight component, was not labeled in the intact state, although the corresponding component of CE was heavily labeled. After dissociation of the envelope by guanidine-HCl or sodium dodecyl sulfate, all of the components could be radioiodinated. However, when the envelopes (VE and FE) were dissolved by heating and subsequently radioiodinated by lactoperoxidase, the resulting radioactivity profile was similar to that of the intact envelopes, suggesting that in the heat-dissolved envelope, the individual components retain similar structural relations as in the intact envelope. Quantitative but not qualitative differences were found between the inner and outer aspects of VE and FE. The significance of these findings is discussed in relation to what is known about the morphological, biological, and molecular properties of the envelopes.     

Cytostatic factor (CSF) in the eggs of Xenopus laevis

Cytostatic factor (CSF) in unfertilized egg cytoplasm causes metaphase arrest when microinjected into zygotes. This was originally described in Rana pipiens eggs In Xenopus laevis, CSF has also been demonstrated. but only when the calcium-chelating agent, EGTA, was injected into the egg cytoplasm. In the present study, however, CSF was demonstrated in Xenopus eggs when donor egg activation was prevented by treatment with CO2 and Mg2+ instead of by EGTA, and recipient blastomere degeneration was prevented by increasing the KCl in the surrounding medium.     

Biochemical characterization of the p34cdc2 protein kinase component of purified maturation-promoting factor from Xenopus eggs

Genetic studies in the fission yeast Schizosaccharomyces pombe and biochemical data in oocytes and eggs of Xenopus laevis have implicated the product of the cdc2+ gene as critical for the G2 to M transition in the cell cycle. The product of the cdc2+ gene is a 34-kDa serine/threonine protein kinase, designated p34cdc2, that is a component of purified maturation-promoting factor (MPF) and also of purified mammalian growth-associated histone H1 kinase. The biochemical properties of p34cdc2 H1 kinase activity in the MPF complex were studied. Phosphorylation of the p45cyclin component in the MPF complex by p34cdc2 exhibited kinetics consistent with an intramolecular reaction. On glycerol gradient centrifugation, MPF kinase against several substrates sedimented with an apparent Mr = 45,000-55,000. p34cdc2 was found to utilize ATP, GTP, and adenosine 5'-O-(3-thiotriphosphate) with apparent Km values of 75, 700, and 250 microM, respectively. The kinase activity was inhibited by beta-glycerophosphate, NaF, and zinc, whereas p-nitrophenyl phosphate was slightly stimulatory. The relative rates of phosphorylation of various substrates by MPF and growth-associated H1 kinase were similar. These findings should prove useful in further work on the regulation of MPF kinase activity and characterization of its substrates.     

Stabilization of the maturation promoting factor (MPF) from Xenopus laevis oocytes. Protection against calcium ions

Maturation promoting factor (MPF) activity was recovered from progesterone-matured Xenopus oocytes cytosol, fractionated by polyethylene glycol-20,000 or ethanolic precipitation. An improved stabilization of the biological activity was obtained by adding adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S) (50 microM) to the preparation buffers. Neither Ca2+ ions nor calmodulin inhibit the partially purified MPF.     

DNA ligase I from Xenopus laevis eggs.

We have purified the major DNA ligase from Xenopus laevis eggs and raised antibodies against it. Estimates from SDS PAGE indicate that this DNA ligase is a 180 kDa protein. This enzyme is similar to the mammalian type I DNA ligase which is presumed to be involved in DNA replication. We have also analysed DNA ligase activity during X. laevis early development. Unfertilized eggs contain the highest level of activity reflecting the requirement for a large amount of DNA replicative enzymes for the period of intense replication following fertilization. In contrast with previous studies on the amphibians axolotl and Pleurodeles, the major DNA ligase activity detected during X. laevis early development is catalysed by a single enzyme: DNA ligase I. And the presence of this DNA ligase I in Xenopus egg before fertilization clearly demonstrates that the exclusion process of two forms of DNA ligase does not occur during X. laevis early development.     

Cytostatic factor (CSF) activity in cytosols extracted from Xenopus laevis eggs

Cytostatic factor (CSF), found in the cytoplasm of unfertilized eggs of amphibians, causes metaphase arrest when microinjected into cleaving blastomeres. Although CSF from Rana pipiens eggs has been extracted and characterized, little is known about CSF extracted from eggs of other species. We investigated the conditions required to preserve CSF activity in cytosols extracted from Xenopus laevis eggs and found that it was necessary to expose the eggs to CO2 prior to extraction and that the extraction buffer must contain sodium beta-glycerophosphate. CSF activity disappeared after 24 h of storage at 2 degrees C. Cytological examination showed that the arrested blastomeres injected with cytosols had been arrested at metaphase and contained a spindle lacking polar asters, in which highly condensed chromosomes were embedded.     

Initiation of replication at specific origins in DNA molecules microinjected into unfertilized eggs of the frog Xenopus laevis

Initiation of DNA replication at specific origins was observed by electron microscopy after microinjection of pXlr11, pXlr14 or Col E1 plasmid DNA molecules into unfertilized eggs of the frog, Xenopus laevis. These results are in apparent contradiction with published reports (Harland and Laskey, Cell 21, 761-771, 1980; Laskey and Harland, Cell 24, 283-284, 1981) that specific origin sites were not used in Xenopus laevis eggs. We suggest that eucaryotic origins exist that both increase the probability of replication of contiguous sequences and determine the site at which replication is most likely to begin.     

Membrane potential measurements of unfertilized and fertilized Xenopus laevis eggs are affected by damage caused by the electrode

Upon penetration in an unfertilized Xenopus egg bathed in 1/10 Ringer, the voltage recorded by a microelectrode shows an abrupt jump to a negative voltage (Ep) followed by a rapid depolarization to a steady value (Er) (Ep = -39.4 +/- 1.9 mV and Er = -11.5 +/- 0.5 SE, 54 eggs from 9 females). The same is true for fertilized eggs impaled 16-35 min after insemination (Ep = -29.5 +/- 2.1 mV, Er = -11.5 +/- 0.9 mV, SE, 18 eggs from 3 females). The voltage recorded by a second microelectrode inserted into the same egg does not show the transient initial negativity. The stationary level of the membrane potential is close to the diffusion potential calculated from the Goldman equation with equal permeabilities for all the relevant ions. It is concluded that the low resting potentials measured in Xenopus eggs before and after fertilization are largely due to damage caused by the electrode. Using an upper limit of -39 mV for the true membrane potential and correlating the input resistance with the stationary membrane potential, a lower limit of 22 M omega (about 1 M omega cm2) for the membrane resistance can be obtained. Insertion of a microelectrode during the first 3 min after insemination shows a steady positive potential while, at later times (3-16 min post-insemination), a positive peak followed by a repolarization can be observed. This indicates that the measurement of the peak of the fertilization potential is not seriously affected by the electrode penetration while its time course after the first 3 min may be deformed by the presence of a large leakage conductance.     

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.     

Neural inducing factors from Xenopus laevis eggs and embryos

Large foreheads can be induced by ribonucleoprotein particles from Xenopus laevis eggs and embryos. The host embryos develop only a rudimentary primary axis. A neural inducing factor from the cytosol of gastrula-neurula stages has been partially purified. The factors are associated with other proteins in larger complexes.     

A possible maternal-effect mutant of Xenopus laevis I. Cytological and biochemical analyses of the unfertilized eggs and embryos

In an effort to define the cause of the developmental arrest of offspring from a certain Xenopus female (designated as No. 65), we have examined eggs and embryos from the female both cytologically and biochemically. Light and electron microscopic observations revealed that all of the blastomeres from embryos of female No. 65 had multiple small spherical nuclei, while wild-type counterparts had a single lobulated nucleus. Two-dimensional gel electrophoretic analyses demonstrated that a major acidic protein, whose molecular weight was 38 kDa, was always found in samples from wild-type unfertilized eggs and embryos, but was not recognizable in those from female No. 65. These facts, coupled with the death of the offspring at gastrulation, suggest the possibility that female No. 65 carries a mutation of the ova-deficient type.