Unfertilized Eggs Of Xenopus Laevis Research Articles

Inhibition of DNA replication initiation by silver nanoclusters.

Silver nanoclusters (AgNCs) have outstanding physicochemical characteristics, including the ability to interact with proteins and DNA. Given the growing number of diagnostic and therapeutic applications of AgNCs, we evaluated the impact of AgNCs on DNA replication and DNA damage response in cell-free extracts prepared from unfertilized Xenopus laevis eggs. We find that, among a number of silver nanomaterials, AgNCs uniquely inhibited genomic DNA replication and abrogated the DNA replication checkpoint in cell-free extracts. AgNCs did not affect nuclear membrane or nucleosome assembly. AgNCs-supplemented extracts showed a strong defect in the loading of the mini chromosome maintenance (MCM) protein complex, the helicase that unwinds DNA ahead of replication forks. FLAG-AgNCs immunoprecipitation and mass spectrometry analysis of AgNCs associated proteins demonstrated direct interaction between MCM and AgNCs. Our studies indicate that AgNCs directly prevent the loading of MCM, blocking pre-replication complex (pre-RC) assembly and subsequent DNA replication initiation. Collectively, our findings broaden the scope of silver nanomaterials experimental applications, establishing AgNCs as a novel tool to study chromosomal DNA replication.

Endoplasmic Reticulum Network Formation with Xenopus Egg Extracts.

The endoplasmic reticulum (ER) consists of morphologically distinct domains, including a polygonal network of tubules that is connected by three-way junctions. This network is found in all eukaryotic cells. Extracts from Xenopus laevis eggs contain stockpiles of components that allow the assembly of an ER network in vitro. Here we provide protocols for assembly of ER networks in extracts that are arrested at different stages of the cell cycle. Unfertilized Xenopus laevis eggs contain a cytostatic factor (CSF) that keeps them in the metaphase stage of the cell cycle. Disruption of the eggs by low-speed centrifugation releases calcium and the eggs cycle into interphase. This state can then be maintained by the addition of cycloheximide, which prevents the synthesis of cyclin B. CSF extracts can be also prepared in the presence of a calcium chelator, thus keeping the extract in metaphase. In this protocol, we outline procedures for the assembly of an ER network using either interphase- or metaphase-arrested Xenopus egg extracts. The network assembled is strikingly similar to the network observed in tissue culture cells. The extract allows easy biochemical manipulation, permitting the effects of purified proteins or small molecules, or the depletion of cytosolic components to be tested.

Preparation of Cellular Extracts from Xenopus Eggs and Embryos.

Cell-free cytoplasmic extracts prepared from Xenopus eggs have been used extensively to recapitulate and characterize intracellular events in vitro. Egg extracts can be induced to transit the cell cycle and reconstitute assembly of dynamic structures including the interphase nucleus and the mitotic spindle. In this protocol, methods are described for preparing crude cytoplasmic extracts from Xenopus eggs and embryos that are arrested in metaphase of the cell cycle. The basic protocol uses unfertilized Xenopus laevis eggs, which are crushed by centrifugation in the presence of EGTA to preserve the natural cytostatic factor (CSF) activity that maintains high levels of Cdk1/cyclin B kinase and metaphase arrest. In the second method, the basic procedure is adapted for Xenopus tropicalis eggs with minor modifications to accommodate differences in frog size, timing of egg laying, and temperature and salt sensitivity. The third variation takes advantage of the synchronous divisions of fertilized X. laevis eggs to generate extracts from embryos, which are arrested in metaphase by the addition of nondegradable cyclin B and an inhibitor of the anaphase-promoting complex (APC) that together stabilize Cdk1/cyclin B kinase activity. Because they are obtained in much smaller amounts and their cell cycles are less perfectly synchronized, extracts prepared from embryos are less robust than egg extracts. X. laevis egg extracts have been used to study a wide range of cellular processes. In contrast, X. tropicalis egg extracts and X. laevis embryo extracts have been used primarily to characterize molecular mechanisms regulating spindle and nuclear size.

High-quality frozen extracts of Xenopus laevis eggs reveal size-dependent control of metaphase spindle micromechanics.

Cell-free extracts from unfertilized Xenopus laevis eggs offer the opportunity for a variety of biochemical and biophysical assays for analyzing essential cell cycle events such as metaphase spindle assembly. However, the extracts often exhibit substantial variation in quality and have low storage stability, factors that hamper their experimental utility. Here we report a simple two-step method for preparing frozen egg extracts that retain spindle assembly activity levels similar to those of freshly prepared extracts. Extract degradation associated with the freeze-thaw process can be substantially reduced by using centrifugal filter-based dehydration and slow sample cooling. Large amounts of frozen extract stocks from single-batch preparations allowed us to collect extensive data in micromanipulation experiments, which are often low-throughput, and thus enabled the clarification of correlations between metaphase spindle size and stiffness. Our method provides an assay platform with minimized biological variability and improves the accessibility of egg extracts for research.

Transient activation of calcineurin is essential to initiate embryonic development in Xenopus laevis

At fertilization, an increase of cytosolic calcium ions (Ca2+) triggers various activation responses in animal eggs. In vertebrates, these responses include exit from metaphase arrest in meiosis II (MII exit) and cortical remodelling initiated by cortical granule exocytosis. Although the essential requirement of Ca2+/calmodulin-dependent protein kinase II for inducing MII exit has been documented, a role of the Ca2+/calmodulin-dependent protein phosphatase calcineurin in egg activation has not been investigated. Here we show, using cell-free extracts from unfertilized eggs of Xenopus laevis, that calcineurin is transiently activated immediately after Ca2+ addition to a concentration that induces MII exit. When calcineurin activation is inhibited, cyclin-dependent kinase 1 (Cdk1) inactivation by means of cyclin B degradation is prevented and sperm chromatin incubated in the extracts remains condensed. Similarly, if calcineurin is inhibited in intact eggs, MII exit on egg activation is prevented. In addition, the activation contraction in the cortex is suppressed whereas cortical granule exocytosis occurs. We further demonstrate that, when a high level of calcineurin activity is maintained after activation, growth of sperm asters is prevented in egg extracts and, consistently, migration of male and female pronuclei towards each other is hindered in fertilized eggs. Thus, both activation and the subsequent inactivation of calcineurin in fertilized eggs are crucial for the commencement of vertebrate embryonic development.

Mechanistic Analysis of a DNA End Processing Pathway Mediated by the Xenopus Werner Syndrome Protein

The first step of homology-dependent repair of DNA double-strand breaks is the strand-specific processing of DNA ends to generate 3' single-strand tails. Despite its importance, the molecular mechanism underlying end processing is poorly understood in eukaryotic cells. We have taken a biochemical approach to investigate DNA end processing in nucleoplasmic extracts derived from the unfertilized eggs of Xenopus laevis. We found that double-strand DNA ends are specifically degraded in the 5' --> 3' direction in this system. The reaction consists of two steps: an ATP-dependent unwinding of double-strand ends and an ATP-independent 5' --> 3' degradation of single-strand tails. We also found that the Xenopus Werner syndrome protein, a member of the RecQ helicase family, plays an important role in DNA end processing. Mechanistically, Xenopus Werner syndrome protein (xWRN) is required for the unwinding of DNA ends but not for the degradation of single-strand tails. The xWRN-mediated end processing is remarkably similar to the end processing that has been proposed for the Escherichia coli RecQ helicase and RecJ single-strand nuclease, suggesting that this mechanism might be conserved in prokaryotes and eukaryotes.

Nuclear assembly of purified Crythecodinium cohnii chromosomes in cell-free extracts of Xenopus laevis eggs.

Incubation of dinoflagellate Crythecodinium cohnii chromosomes in cytoplasmic extracts of unfertilized Xenopus laevis eggs resulted in chromosomes decondensation and recondensation, nuclear envelope assembly, and nuclear reconstitution. Dinoflagellate Crythecodinium cohnii is a kind of primitive eukaryote which possesses numerous permanently condensed chromosomes and discontinuous double-layered nuclear membrane throughout the cell cycle. The assembled nuclei, being surrounded by a continuous double membrane containing nuclear pores and the uniformly dispersed chromatin fibers are morphologically distinguishable from that of Dinoflagellate Crythecodinium cohnii. However, incubation of dinoflagellate Crythecodinium cohnii chromosomes in the extracts from dinoflagellate Crythecodinium cohnii cells does not induce nuclear reconstitution.

A linear vector carrying human telomeres is replicated in unfertilized eggs of Xenopus laevis.

A linear vector carrying human telomeres is replicated in unfertilized eggs of Xenopus laevis.

Decondensation of human sperm nuclei and HP1 protamine degradation from normospermia and asthenospermia in Xenopus egg extracts.

The process of human sperm decondensation has been studied in vitro in cytoplasmic extracts prepared from unfertilized Xenopus laevis eggs. The chromatin decondensation-recondensation cycle was divided into four stages according to chromatin appearance. Spermatozoa from normospermia and asthenospermia were evaluated according to their capacity to reach these stages, and their DNA integrity was assessed by acridine orange (AO) staining. We observed a significant difference between normospermia and asthenozoospermia in the ability to achieve the cycle of chromatin decondensation-recondensation. These results correlated with AO staining. The role of human protamine 1 degradation in the decondensation process was evaluated by immunostaining. It was found not to be a prerequisite for the earlier stage of chromatin decondensation and it was not implied in the latest stages of pronuclear development.

Characterization of early DNA synthesis in Xenopus eggs after injection of circular plasmid DNA.

Circular plasmid DNA is known to replicate when injected into unfertilized eggs of Xenopus laevis. We characterized early DNA synthesis which precedes the replication. Incorporation of a radioactive precursor into covalent closed circular DNA becomes detectable as early as 2 minutes after injection. Judging from the sensitivity to aphidicolin, replicative DNA polymerase alpha or delta is involved in this reaction. However, analysis of density-labeled product as well as detection of newly synthesized, unmethylated strands by restriction endonuclease DpnI digestion, both indicated that most if not all, of the synthesized DNA is present as short repair-like patches in the injected DNA molecules. They are present randomly in all the HaeIII fragments of injected DNA, and transcriptional activation of the 5S RNA gene inserted in the plasmid does not affect the distribution. Only a minor fraction of injected DNA is utilized for this reaction, and 12% of such DNA molecules are further chased into replicated progeny DNA. This efficiency of replication is nearly the same as that of bulk injected DNA which has not been subjected to the early DNA synthesis. We concluded that, despite the common use of replicative DNA polymerase, the early DNA synthesis and the subsequent DNA replication are mutually independent processes.

Involvement of Proliferating Cell Nuclear Antigen (Cyclin) in DNA Replication in living Cells

Proliferating cell nuclear antigen (PCNA) (also called cyclin) is known to stimulate the activity of DNA polymerase delta but not the other DNA polymerases in vitro. We injected a human autoimmune antibody against PCNA into unfertilized eggs of Xenopus laevis and examined the effects of this antibody on the replication of injected plasmid DNA as well as egg chromosomes. The anti-PCNA antibody inhibited plasmid replication by up to 67%, demonstrating that PCNA is involved in plasmid replication in living cells. This result further implies that DNA polymerase delta is necessary for plasmid replication in vivo. Anti-PCNA antibody alone did not block plasmid replication completely, but the residual replication was abolished by coinjection of a monoclonal antibody against DNA polymerase alpha. Anti-DNA polymerase alpha alone inhibited plasmid replication by 63%. Thus, DNA polymerase alpha is also required for plasmid replication in this system. In similar studies on the replication of egg chromosomes, the inhibition by anti-PCNA antibody was only 30%, while anti-DNA polymerase alpha antibody blocked 73% of replication. We concluded that the replication machineries of chromosomes and plasmid differ in their relative content of DNA polymerase delta. In addition, we obtained evidence through the use of phenylbutyl deoxyguanosine, an inhibitor of DNA polymerase alpha, that the structure of DNA polymerase alpha holoenzyme for chromosome replication is significantly different from that for plasmid replication.

Involvement of proliferating cell nuclear antigen (cyclin) in DNA replication in living cells.

Proliferating cell nuclear antigen (PCNA) (also called cyclin) is known to stimulate the activity of DNA polymerase delta but not the other DNA polymerases in vitro. We injected a human autoimmune antibody against PCNA into unfertilized eggs of Xenopus laevis and examined the effects of this antibody on the replication of injected plasmid DNA as well as egg chromosomes. The anti-PCNA antibody inhibited plasmid replication by up to 67%, demonstrating that PCNA is involved in plasmid replication in living cells. This result further implies that DNA polymerase delta is necessary for plasmid replication in vivo. Anti-PCNA antibody alone did not block plasmid replication completely, but the residual replication was abolished by coinjection of a monoclonal antibody against DNA polymerase alpha. Anti-DNA polymerase alpha alone inhibited plasmid replication by 63%. Thus, DNA polymerase alpha is also required for plasmid replication in this system. In similar studies on the replication of egg chromosomes, the inhibition by anti-PCNA antibody was only 30%, while anti-DNA polymerase alpha antibody blocked 73% of replication. We concluded that the replication machineries of chromosomes and plasmid differ in their relative content of DNA polymerase delta. In addition, we obtained evidence through the use of phenylbutyl deoxyguanosine, an inhibitor of DNA polymerase alpha, that the structure of DNA polymerase alpha holoenzyme for chromosome replication is significantly different from that for plasmid replication.

In vitro formation of the “S” layer, a unique component of the fertilization envelope in Xenopus laevis eggs

The extracellular matrix (ECM) of unfertilized Xenopus laevis eggs consists of an elaborate filamentous network in the perivitelline space (PS) and a thick fibrillar vitelline envelope (VE), with a thin layer of horizontal filaments (HF) separating the two. At fertilization this ECM is converted into the fertilization envelope (comprised of the fertilization (F) layer and altered VE), and a third layer, the smooth (S) layer, is formed at the upper boundary of the PS ( Larabell and Chandler, 1988). In this report, we use quick-freeze, deep-etch, rotary-shadow electron microscopy to show that an intact S layer can be formed in vitro by incubation of unfertilized eggs in an exudate obtained from cortical granules. Within 5 min numerous 36-nm-diameter particles assemble in a highly ordered array at the microvillar tips. These particles appear to “melt” and to form patches of smooth material and within 10 min one continuous sheet has formed. The presence of the VE is required for formation of the S layer, and we suggest that the HF layer is the site of assembly.

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 unfertilised 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 unfertilised 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 β-globin pre-mRNAs were not detectably 3′ cleaved or polyadenylated in either microinjected oocytes or unfertilised eggs.

Endogenous lectin secretion into the extracellular matrix of early embryos of Xenopus laevis

An endogenous galactoside-binding lectin with subunit molecular weight of 43,000–45,000, previously detected in unfertilized eggs of Xenopus laevis, persists at high levels in embryos through gastrulation. During embryonic development the lectin is found in cytoplasmic vesicles, and then is secreted into extracellular matrix which is prominent around the blastopore and on the roof of the blastocoel. The lectin is also found in the extracellular material in the developing neural fold. The presence of lectin at sites of active morphogenetic movements raises the possibility that it participates in the formation of an extracellular matrix that influences these processes.

Partial purification of the maturation-promoting factor MPF from unfertilized eggs of Xenopus laevis.

A 200-fold purification of the maturation-promoting factor or MPF from unfertilized eggs of Xenopus laevis is reported for the first time. Purification was achieved by three successive column chromatographies on hydroxyapatite, trisacryl blue and L-arginine-agarose. The presence of MPF was assessed by the usual maturation criteria after injections of test material into immature stage VI unstimulated X. laevis oocytes: the precocious appearance of the maturation spot (within 45-120 min), the germinal vesicle breakdown, the presence of the first polar body and the second metaphase spindle. Purification was monitored by the decrease of the minimal amount of protein injected in a constant volume (50 nl) required to induce 50% frequency of germinal vesicle breakdown. This amount decreased from 500 ng in the crude extract to 2.5 ng in the 200-fold purified material. Analysis by SDS-PAGE of the crude extract showed about 40 Coomassie-blue-stained polypeptides with molecular masses ranging from 300 kDa to 20 kDa, whereas in the 200-fold purified MPF only 5 stained polypeptides were revealed, with molecular masses of 62, 53, 49, 39 and 37 kDa. In vitro phosphorylations for the detection of kinase activities for endogenous and exogenous substrates were monitored by analysis of autoradiograms of SDS-PAGE, after treatment of fractions with [gamma-32P]ATP. Only inactive fractions eluted from columns ahead of MPF, and fractions containing MPF activity were tested. Phosphorylation of numerous stained polypeptides was demonstrated in the crude MPF extract and exogenous substrates such as phosvitin, casein and histone type II-AS were also strongly phosphorylated. In the MPF fraction, purified on hydroxyapatite, a polypeptide of 53 kDa was more highly and specifically phosphorylated and the presence of kinase activities was observed for the above three exogenous substrates. In the 100-fold and 200-fold purified MPF, phosphorylation of endogenous substrates could not be shown and kinase activities for the above three substrates were drastically decreased as compared with the crude and purified MPF obtained after hydroxyapatite column chromatography. However, neither endogenous phosphorylations nor kinase activities with the above exogenous substrates could be shown in inactive fractions eluted ahead of MPF at the different purification steps. Some characteristics of the purified material are also described in this paper.

Induction of nuclear envelope breakdown, chromosome condensation, and spindle formation in cell-free extracts.

Incubation of demembranated sperm chromatin in cytoplasmic extracts of unfertilized Xenopus laevis eggs resulted in nuclear envelope assembly, chromosome decondensation, and sperm pronuclear formation. In contrast, egg extracts made with EGTA-containing buffers induced the sperm chromatin to form chromosomes or irregularly shaped clumps of chromatin that were incorporated into bipolar or multipolar spindles. The 150,000 g supernatants of the EGTA extracts could not alone support these changes in incubated nuclei. However, these supernatants induced not only chromosome condensation and spindle formation, but also nuclear envelope breakdown when added to sperm pronuclei or isolated Xenopus liver or brain nuclei that were incubated in extracts made without EGTA. Similar changes were induced by partially purified preparations of maturation-promoting factor. The addition of calcium chloride to extracts containing condensed chromosomes and spindles caused dissolution of the spindles, decondensation of the chromosomes, and re-formation of interphase nuclei. These results indicate that nuclear envelope breakdown, chromosome condensation, and spindle assembly, as well as the regulation of these processes by Ca2+-sensitive cytoplasmic components, can be studied in vitro using extracts of amphibian eggs.

Regulated replication of DNA microinjected into eggs of Xenopus laevis

Purified circular DNA of SV40 or polyoma virus has been injected into unfertilized eggs of Xenopus laevis. Injected DNA initiates and completes multiple rounds of semiconservative replication while observing cellular regulatory signals. Thus replication initiation of double-stranded templates is induced after the oocyte is matured in vitro by progesterone. Only one round of replication of injected DNA is observed in a single cell cycle. When protein synthesis is inhibited unreplicated molecules continue to initiate replication at an undiminished rate, but reinitiation on previously replicated molecules is completely and selectively abolished. The DNA sequence requirements for the replication of injected DNA have been investigated. A variety of procaryotic DNA molecules and circularized fragments of SV40 or polyoma DNA replicate, regardless of whether they contain the viral origin of DNA replication. These results suggest that a specialized DNA sequence is not essential for the initiation of semiconservative DNA replication in the Xenopus embryo, nor is a specialized sequence essential for the mechanism which prevents reinitiation on a molecule which has already replicated within a cell cycle. The possibility is discussed that viral origins of replication are not valid models for the eucaryotic chromosome but are adaptations for uncoupling viral replication from the mechanism which prevents reinitiation within a cell cycle.

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.

Developmental and ploidy characteristics of "senescent" unfertilized eggs of Xenopus laevis.

Unfertilized eggs of Xenopus laevis have been stored in a high salt physiological saline (De Boer's solution) over a period of about 24 hours. During this time, aliquots have been subjected to lowered salinity and fertilized. The stored eggs undergo a defined series of morphological changes, which are initially reversible upon fertilization. Length of storage may have little or no effect on the capacity of the eggs to undergo cleavage, but the normal developmental capacity declines with increasing storage time. Among the characteristics of this decline is a regressive cytokinesis, early arrest, and the appearance in surviving larvae of a definable syndrome of morphological type. Increasing storage times predispose the eggs to subsequent triploid development, the triploidy arising as a consequence of failure of second polar body emission. The possible interest of these findings for a model system of mammalian gamete aging is discussed.