Xenopus Oocyte Extracts Research Articles

Reconstitution of Intracellular Calcium Signaling in Xenopus Egg Extracts.

Generation of calcium signal in the cytoplasm of fertilized or parthenogenetically activated eggs has been extensively studied in the intact eggs of several biological species. Calcium transient was found to elicit a plethora of biochemical and cellular events in these cells. Remarkably, intracellular calcium signaling can also be reconstituted in cell-free environment. In this chapter, we describe the methods that allow reconstitution, detection, and quantification of the calcium signal in cell-free extracts of Xenopus oocytes and eggs.

Claspin - checkpoint adaptor and DNA replication factor.

Claspin was discovered as a Chk1-interacting protein necessary for Chk1 phosphorylation and activation by the upstream kinase, ATR, in response to DNA synthesis inhibition in Xenopus oocyte extracts. Subsequent investigations have defined a molecular model in which Claspin acts as an adaptor or scaffold protein to facilitate activation of Chk1 by ATR within a multiprotein complex that forms on single-stranded DNA at stalled replication forks and sites of DNA damage. Interestingly, Claspin is an unstable protein whose degradation via the proteasome is tightly regulated via ubiquitination and controlled by multiple ubiquitin ligases and deubiquitinases. As a result, Claspin levels fluctuate during the cell cycle, contributing to the regulation of checkpoint proficiency and playing a key role in terminating checkpoint-mediated cell cycle arrest. In addition to its role in signalling genotoxic stress, Claspin is required to maintain normal rates of replication fork progression during unperturbed DNA replication and may contribute to the regulation of replication origin firing. Consistent with this, Claspin can bind directly to DNA, with particular affinity for branched or forked molecules, and it interacts with multiple protein components of the replisome. As expected for a protein with key roles in checkpoint signalling and genome duplication, aberrations of Claspin expression and structure have been observed in cancer. Claspin is furthermore targeted to facilitate viral replication and plays a role in suppressing cellular DNA synthesis in response to nongenotoxic endoplasmic reticulum stress. Here, we review the functions and regulation of Claspin with a focus on areas of active research.

Isolation of Xenopus Oocytes.

Xenopus oocytes and oocyte extracts are the starting material for a variety of experimental approaches. Oocytes are obtained by surgical removal of the ovary from anesthetized females. Although oocytes may be used while they remain within their ovarian follicle, it is more practical to work with defolliculated oocytes. Defolliculation can be performed either manually or enzymatically. Here we present a protocol for the isolation and separation of Xenopus oocytes at various developmental stages, and guidelines for maintaining oocytes in culture.

Connecting macroscopic dynamics with microscopic properties in active microtubule network contraction

The cellular cytoskeleton is an active material, driven out of equilibrium by molecular motor proteins. It is not understood how the collective behaviors of cytoskeletal networks emerge from the properties of the network’s constituent motor proteins and filaments. Here we present experimental results on networks of stabilized microtubules in Xenopus oocyte extracts, which undergo spontaneous bulk contraction driven by the motor protein dynein, and investigate the effects of varying the initial microtubule density and length distribution. We find that networks contract to a similar final density, irrespective of the length of microtubules or their initial density, but that the contraction timescale varies with the average microtubule length. To gain insight into why this microscopic property influences the macroscopic network contraction time, we developed simulations where microtubules and motors are explicitly represented. The simulations qualitatively recapitulate the variation of contraction timescale with microtubule length, and allowed stress contributions from different sources to be estimated and decoupled.

Bistability analysis in mathematical model of M-phase control in Xenopus oocyte extracts

To gain more insight into the molecular mechanism that regulates eukaryotic cell cycle, we propose a modification to an existing Mathematical model of mitosis-promoting factor control in Xenopus oocyte extract. To achieve this, we redirect how cell exits from mitosis using ubiquitin ligase enzyme APCP:Cdc20 which is acting as a modifier in cyclin degradation. We present a modified wiring map using combination of mass action kinetics and Michaelis–Menten kinetics to represent the biochemical processes. This results in a system of non-linear ordinary differential equations. The equilibria of the system were analyzed analytically. Using MPF as a bifurcation parameter, we analytically derived conditions under which bistability can occur in the MPF activation module. We find that for bistability to occur, it is necessary that the rate of phosphorylation of Wee1 must be greater than the rate of its dephosphorylation and that the concentration of MPF must lie in certain range of parameter values. Numerical simulations conducted show that the time taken to exit the cell cycle is shorter than those published in some reports and the sensitivity analysis results suggest that the rate of synthesis of cyclin is the most sensitive parameter in the dynamics of cell cycle. Through numerical experiments, we found new parameter sets that will give rise to bistability in the MPF activation module.

Cytoskeleton and Cytoskeleton-Bound RNA Visualization in Frog and Insect Oocytes.

The majority of oocyte functions involves and depends on the cytoskeletal elements, which include microtubules and actin and cytokeratin filaments. Various structures and molecules are temporarily or permanently bound to the cytoskeletal elements and their functions rely on cytoskeleton integrity and its timely assembly. Thus the accurate visualization of cytoskeleton is often crucial for studies and analyses of oocyte structure and functions. Here we describe several reliable methods for microtubule and/or microfilaments preservation and visualization in Xenopus oocyte extracts, and in situ in live and fixed insect and frog (Xenopus) oocytes. In addition, we describe visualization of cytoskeleton-bound RNAs using molecular beacons in live Xenopus oocytes.

Reprogramming of somatic cells and nuclei by Xenopus oocyte and egg extracts.

Differentiated somatic cells and nuclei can be reprogrammed to a pluripotent undifferentiated state in the cytoplasm of oocytes and eggs. The ability of the gamete cells to induce reprogramming is not species-specific, so the extracts prepared from the oocytes and eggs of the African clawed frog Xenopus laevis can reprogram somatic mammalian cells. Thus, Xenopus egg extract-mediated reprogramming may constitute an alternative or complement other experimental reprogramming approaches, such as nuclear transfer, cell fusion, and transcription factor transduction. Here, we discuss the major reprogramming events induced by the extracts in somatic nuclei and cells, including remodeling of nuclear structure, replacement of somatic proteins with their embryonic counterparts, epigenetic modification of DNA and histones, transcriptional reprogramming, and initiation of DNA replication. We also address the advantages and limitations of the extract-based reprogramming approach.

Active contraction of microtubule networks.

Many cellular processes are driven by cytoskeletal assemblies. It remains unclear how cytoskeletal filaments and motor proteins organize into cellular scale structures and how molecular properties of cytoskeletal components affect the large-scale behaviors of these systems. Here, we investigate the self-organization of stabilized microtubules in Xenopus oocyte extracts and find that they can form macroscopic networks that spontaneously contract. We propose that these contractions are driven by the clustering of microtubule minus ends by dynein. Based on this idea, we construct an active fluid theory of network contractions, which predicts a dependence of the timescale of contraction on initial network geometry, a development of density inhomogeneities during contraction, a constant final network density, and a strong influence of dynein inhibition on the rate of contraction, all in quantitative agreement with experiments. These results demonstrate that the motor-driven clustering of filament ends is a generic mechanism leading to contraction.

Wee1 and Chk1 - crosstalk between key players in replicative stress.

Replicative stress is a tumor cell-associated feature that includes the accumulation of stalled or collapsed replication forks. With the DNA polymerases lagging behind the helicases, these structures contain extended regions of single stranded DNA, leading to the activation of a damage signaling pathway that includes the kinases Ataxia Telangiectasia Mutated-Related (ATR) and Chk1. The specific increase of replicative stress in tumor cells raises the perspective of further enhancing this stress condition for therapeutic purposes, ultimately resulting in cancer cell death [1]. Targeting the replicative machinery for DNA can be achieved by established chemotherapeutic drugs, such as nucleoside analogues, platinum compounds, or topoisomerase inhibitors. This can be viewed as one example of the more general concept of exploiting tumor-supportive molecular machineries as drug targets [2]. On top of interfering with DNA replication as such, targeting signaling pathways to enhance replicative stress has turned out as a viable option with clinical perspectives. In particular, interfering with ATR-Chk1 signaling promotes the death of proliferating cancer cells [3], and inhibitors of both kinases are currently evaluated in clinical trials. In addition, however, the kinase Wee1 turned out as an at least similarly promising target. Wee1 inhibition, especially when combined with conventional chemotherapy, strongly enhances cell death, at least in part by promoting premature mitosis before DNA replication can be completed during S phase [4]. The identification of Chk1 and Wee1 as targets to enhance replicative stress raises the question whether these kinases communicate with each other, and whether they affect each other's levels and activity. Current knowledge regarding this question is limited. At least in yeast cells [5] and in Xenopus oocyte extracts [6], it has long been known that Chk1 phosphorylates and activates Wee1. But what mechanisms, if any, would allow Wee1 to provide feedback on Chk1 activity? Saini et al. from the Dobbelstein lab elucidated this by dissecting a signaling chain reaching from Wee1 to Chk1 [7, in press]. We firstly induced replicative stress in our experimental systems using the nucleoside analogue gemcitabine. Under these conditions, we eliminated the activity of Wee1 and its downstream effectors by pharmacological inhibition or by siRNA. As a result, we found that Wee1 inhibition indirectly reduces Chk1 activity as well, by activating cyclin dependent kinases and subsequently suppressing the functions of ATR and the Chk1-coactivator Claspin. Thus, Wee1 inhibition not only enables premature and thus catastrophic mitosis but also enhances replicative stress by lowering the amount of active Chk1. This may, at least in part, explain the particularly effective synergism between Wee1 inhibitors and chemotherapeutics [4] and encourage the clinical evaluation of Wee1 inhibitors in combination with conventional cancer treatment. Indeed, the Wee1 inhibitor MK-1775 is currently in clinical testing (17 entries in http://clinicaltrials.gov at present). Figure Molecular communication between Wee1 and Chk1 (adapted and expanded from [7]) Despite the simultaneous impairment of Chk1 activity by Wee1 inhibitors, combining inhibitors of Wee1 and Chk1 may nonetheless prove useful to eliminate cancer cells. This is anticipated since the negative regulation of Chk1 in response to Wee1 inhibition occurs only at a delay of up to 24 hours [7]. Multiple additional drug combinations are conceivable in order to enhance replicative stress [1]. Also, inhibitors of DNA replication, such as nucleoside or base analogues, can be employed to enhance different regimens of cancer treatment. For instance, the Dobbelstein lab has recently observed that the base analogue 5-fluorouracil interferes with homologous recombination repair; it thus augments the damage induced by double strand DNA breaks, as they occur through ionizing irradiation [8]. Exploiting replicative stress for cancer treatment is a strategy that can strongly benefit from the combination of conventional DNA-damaging cancer drugs with targeted signaling inhibitors. Knowing about the cross-talks between these signaling components has the potential of further improving this approach and to identify tumor-cell associated markers for optimizing therapeutic combinations.

Pioneering the Xenopus Oocyte and Egg Extract System

Pioneering the Xenopus Oocyte and Egg Extract System

XenopusEgg Extract Treatment Reduced Global DNA Methylation of Donor Cells and Enhanced Somatic Cell Nuclear Transfer Embryo Development in Pigs

The efficiency to produce offspring by somatic cell nuclear transfer (SCNT) is low. It has been showed that treatment of donor cells with Xenopus oocyte extract increased live births in ovine and handmade cloned embryo development in pigs. Scriptaid treatment after oocyte activation is another approach to improve SCNT efficiency. The present study was carried out to investigate (a) the effects of treatment of donor cells with Xenopus egg extract on donor cell DNA methylation at days 0 and 4 with two digitonin permeabilization concentrations (10 and 15 μg/mL), (b) the effects of treatment of donor cells with Xenopus egg extract on early development of cloned embryos, and (c) the effects of combined treatments, treating donor cells with extract before nuclear transfer and treatment of cloned embryos with scriptaid after oocyte activation, on embryo development. Compared to the control, a decrease of DNA methylation in donor cells was observed at 2.5 h after extract treatment. However, this effect was not observed after the cells were cultured for four more days. More embryos developed into blastocysts in the Xenopus egg extract-treated group than in the control (13.4±1.9% vs. 9.1±1.9%, p=0.01). Furthermore, scriptaid treatment of cloned embryos further increased the frequency of development to blastocyst, compared to the control reconstructed with the same extract-treated cells (22.5±0.9% vs. 15.3±0.9%, p<0.01). In addition, egg extract treatments increased the cell number in the blastocysts. This study demonstrated that Xenopus egg extract treatment reduced donor cell DNA methylation and enhanced the SCNT embryo development. Moreover, the combined treatments of donor cells with egg extract before nuclear transfer and of cloned embryos with scriptaid could improve cloned embryo development additively.

Affinity-based enrichment strategies to assay methyl-CpG binding activity and DNA methylation in early Xenopus embryos

BackgroundDNA methylation is a widespread epigenetic modification in vertebrate genomes. Genomic sites of DNA methylation can be bound by methyl-CpG-binding domain proteins (MBDs) and specific zinc finger proteins, which can recruit co-repressor complexes to silence transcription on targeted loci. The binding to methylated DNA may be regulated by post-translational MBD modifications.FindingsA methylated DNA affinity precipitation method was implemented to assay binding of proteins to methylated DNA. Endogenous MeCP2 and MBD3 were precipitated from Xenopus oocyte extracts and conditions for methylation-specific binding were optimized. For a reverse experiment, DNA methylation in early Xenopus embryos was assessed by MBD affinity capture.ConclusionsA methylated DNA affinity resin can be applied to probe for MBD activity in extracts. This assay has a broad application potential as it can be coupled to downstream procedures such as western blotting, fluorimetric HDAC assays and quantitative mass spectrometry. Methylated DNA affinity capture by methyl-CpG binding proteins produces fractions highly enriched for methylated DNA, suitable for coupling to next generation sequencing technologies. The two enrichment strategies allow probing of methyl-CpG protein interactions in early vertebrate oocytes and embryos.

Histone H3 lysine 4 methylation is associated with the transcriptional reprogramming efficiency of somatic nuclei by oocytes

BackgroundWhen the nuclei of mammalian somatic cells are transplanted to amphibian oocytes in the first meiotic prophase, they are rapidly induced to begin transcribing several pluripotency genes, including Sox2 and Oct4. The more differentiated the donor cells of the nuclei, the longer it takes for the pluripotency genes to be activated after the nuclear transfer to oocytes. We have used this effect in order to investigate the role of histone modifications in this example of nuclear reprogramming.ResultsReverse transcription polymerase chain reaction analysis shows that the transcriptional reprogramming of pluripotency genes, such as Sox2 and Oct4, takes place in transplanted nuclei from C3H10T1/2 cells and from newly differentiated mouse embryonic stem cells. We find that the reprogramming of 10T1/2 nuclei is accompanied by an increased phosphorylation, an increased methylation and a rapidly reduced acetylation of several amino acids in H3 and other histones. These results are obtained by the immunofluorescent staining of transplanted nuclei and by Western blot analysis. We have also used chromatin immunoprecipitation analysis to define histone modifications associated with the regulatory or coding regions of pluripotency genes in transplanted nuclei. Histone phosphorylation is increased and histone acetylation is decreased in several regulatory and gene coding regions. An increase of histone H3 lysine 4 dimethylation (H3K4 me2) is seen in the regulatory regions and gene coding region of pluripotency genes in reprogrammed nuclei. Furthermore, histone H3 lysine 4 trimethylation (H3K4 me3) is observed more strongly in the regulatory regions of pluripotency genes in transplanted nuclei that are rapidly reprogrammed than in nuclei that are reprogrammed slowly and are not seen in β-globin, a gene that is not reprogrammed. When 10T1/2 nuclei are incubated in Xenopus oocyte extracts, histone H3 serine 10 (H3S10) is strongly phosphorylated within a few hours. Immunodepletion of Aurora B prevents this phosphorylation.ConclusionWe conclude that H3K4 me2 and me3 are likely to be important for the efficient reprogramming of pluripotency genes in somatic nuclei by amphibian oocytes and that Aurora B kinase is required for H3S10 phosphorylation which is induced in transplanted somatic cell nuclei.

Analysis of histones and chromatin in Xenopus laevis egg and oocyte extracts

Histones are the major protein components of chromatin, the physiological form of the genome in all eukaryotic cells. Chromatin is the substrate of information-directed biological processes, such as gene regulation and transcription, replication, and mitosis. A long-standing experimental model system to study many of these processes is the extract made from the eggs of the anuran Xenopus laevis. Since work in recent years has solidified the importance of post-translational modification of histones in directing biological processes, the study of histones in a biochemically dissectible model such as Xenopus is crucial for the understanding of their biological significance. Here we present a rationale and methods for isolating and studying histones and chromatin in different Xenopus egg and oocyte extracts. In particular, we present protocols for the preparation of: cell-free egg and oocyte extract; nucleoplasmic extract (“NPE”); biochemical purification of maternally-deposited, stored histones in the oocyte and the egg; assembly of pronuclei in egg extract and the isolation of pronuclear chromatin and histones; and an extract chromatin assembly assay. We also demonstrate aspects of the variability of the system to be mindful of when working with extract and the importance of proper laboratory temperature in preparing quality extracts. We expect that these methods will be of use in promoting further understanding of embryonic chromatin in a unique experimental system.

Ectopic expression of prelamin A in early Xenopus embryos induces apoptosis

Lamin proteins are components of metazoan cell nuclei. During evolution, two classes of lamin proteins evolved, A- and B-type lamins. B-type lamins are expressed in nearly all cell types and in all developmental stages and are thought to be indispensable for cellular survival. In contrast, A-type lamins have a more restricted expression pattern. They are expressed in differentiated cells and appear late in embryogenesis. In the earliest steps of mammalian development, A-type lamins are present in oocytes, pronuclei and during the first cleavage stages of the developing embryo. But latest after the 16-cell stage, A-type lamin proteins are not any longer detectable in embryonic cells. Amphibian oocytes and early embryos do not express lamin A. Moreover, extracts of Xenopus oocytes and eggs have the ability to selectively remove A-type lamins from somatic nuclei. This observation and the restricted expression pattern suggest that the presence of lamin A might interfere with developmental processes in the early phase of embryogenesis. To test this, we ectopically expressed lamin A during early embryonic development of Xenopus laevis by microinjection of synthetic mRNA. Here, we show that introducing mature lamin A does not interfere with normal development. However, expression of prelamin A or lamin A variants that cannot be fully processed cause severe disturbances and lead to apoptosis during gastrulation. The toxic effect is due to lack of the conversion of prenylated prelamin A to its mature form. Remarkably, even a cytoplasmic prelamin A variant that is excluded from the nucleus drives embryos into apoptosis.

Inhibition of apoptotic potency by ligand stimulated thyroid hormone receptors located in mitochondria

We recently reported that shortened thyroid hormone receptor isoforms (TRs) can target mitochondria and acutely modulate inositol 1,4,5 trisphosphate (IP3)-mediated Ca2+ signaling when activated by thyroid hormone 3,5,3'-tri-iodothyronine (T3). Stimulation occurs via an increase in mitochondrial metabolism that is independent of transcriptional activity. Here, we present evidence that T3-bound xTRbetaA1s inhibit apoptotic activity mediated by cytochrome c release. An assay for apoptotic potency was modified to measure the ability of Xenopus oocyte extracts to induce morphological changes in isolated liver nuclei. Apoptotic potency was significantly decreased when oocyte extract was prepared from xTRbetaA1 expressing oocytes and treated with T3. The ability of T3 treatment to inhibit apoptosis was dependent on the expression of xTRbetaA1s in the mitochondrial fraction, not in the cytosolic fraction. T3 treatment also increased the membrane potential of isolated mitochondria prepared from oocytes expressing xTRbetaA1s but not from wildtype controls. We conclude that T3 acutely regulates cytochrome c release in a potential dependent manner by activating TRs located within mitochondria.

Coupled transcription-and-translation in Xenopus oocyte and egg extracts

Coinjection of T7 promoter-driven plasmids and T7 RNA polymerase (T7 RNAP) into Xenopus oocytes results in robust protein synthesis, due to simultaneous gene transcription-and-translation (TnT) in the oocyte cytoplasm [Geib, S., Sandoz, G., Carlier, E., V. Cornet, Cheynet-Sauvion, V., De Waard, M., 2001. A novel Xenopus oocyte expression system based on cytoplasmic coinjection of T7-driven plasmids and purified T7-RNA polymerase. Receptors Channels 7, 331-343; Tokmakov, A.A., Matsumoto, E., Shirouzu, M., Yokoyama, S., 2006. Coupled cytoplasmic transcription-and-translation--a method of choice for heterologous gene experession in Xenopus oocytes. J. Biotechnol. 122, 5-15]. In the present study, we demonstrate that the TnT reaction of protein synthesis can be reconstituted in cell-free extracts of Xenopus oocytes and eggs. Similar to the reaction in oocytes, the effective coupling of bacteriophage T7 RNAP-mediated transcription with the eukaryotic translation machinery takes place in the Xenopus oocyte and egg extracts. However, the kinetics of protein and RNA production in the extracts are quite different from those observed in oocytes. Potent RNA synthesis in the extracts starts immediately after the addition of T7 promoter-driven DNA and T7 RNAP and continues for about 30 min, followed by RNA degradation. The protein product is detectable in the extracts in 15 min after the initiation of the TnT reaction. Efficient protein synthesis in the extracts continues for about 1h. The productivity of this expression system can be boosted by the additions of an RNase inhibitor and an ATP-regeneration system, and by extract dilution. Kinetic analyses suggested that extending the lifetime of the extracts would further increase their productivity.

Mammalian Emi2 mediates cytostatic arrest and transduces the signal for meiotic exit via Cdc20

Fertilizable mammalian oocytes are arrested at the second meiotic metaphase (mII) by the cyclinB-Cdc2 heterodimer, maturation promoting factor (MPF). MPF is stabilized via the activity of an unidentified cytostatic factor (CSF), thereby suspending meiotic progression until fertilization. We here present evidence that a conserved 71 kDa mammalian orthologue of Xenopus XErp1/Emi2, which we term endogenous meiotic inhibitor 2 (Emi2) is an essential CSF component. Depletion in situ of Emi2 by RNA interference elicited precocious meiotic exit in maturing mouse oocytes. Reduction of Emi2 released mature mII oocytes from cytostatic arrest, frequently inducing cytodegeneration. Mos levels autonomously declined to undetectable levels in mII oocytes. Recombinant Emi2 reduced the propensity of mII oocytes to exit meiosis in response to activating stimuli. Emi2 and Cdc20 proteins mutually interact and Cdc20 ablation negated the ability of Emi2 removal to induce metaphase release. Consistent with this, Cdc20 removal prevented parthenogenetic or sperm-induced meiotic exit. These studies show in intact oocytes that the interaction of Emi2 with Cdc20 links activating stimuli to meiotic resumption at fertilization and during parthenogenesis in mammals.

Differential nuclear remodeling of mammalian somatic cells by Xenopus laevis oocyte and egg cytoplasm

The mechanisms governing nuclear reprogramming have not been fully elucidated yet; however, recent studies show a universally conserved ability of both oocyte and egg components to reprogram gene expression in somatic cells. The activation of genes associated with pluripotency by oocyte/egg components may require the remodeling of nuclear structures, such that they can acquire the features of early embryos and pluripotent cells. Here, we report on the remodeling of the nuclear lamina of mammalian cells by Xenopus oocyte and egg extracts. Lamin A/C is removed from somatic cells incubated in oocyte and egg extracts in an active process that requires permeable nuclear pores. Removal of lamin A/C is specific, since B-type lamins are not changed, and it is not dependent on the incorporation Xenopus egg specific lamin III. Moreover, transcriptional activity is differentially regulated in somatic cells incubated in the extracts. Pol I and II transcriptions are maintained in cells in oocyte extracts; however, both activities are abolished in egg extracts. Our study shows that components of oocyte and egg extracts can modify the nuclear lamina of somatic cells and that this nuclear remodeling induces a structural change in the nucleus which may have implications for transcriptional activity. These experiments suggest that modifications in the nuclear lamina structure by the removal of somatic proteins and the incorporation of oocyte/egg components may contribute to the reprogramming of somatic cell nuclei and may define a characteristic configuration of pluripotent cells.

121 DEMETHYLATION OF MAMMALIAN SOMATIC DNA BY XENOPUS EGG AND OOCYTE EXTRACTS

In mammals, the successful development of live offspring by somatic cell nuclear transfer (SCNT) has demonstrated the ability of oocyte or egg cytoplasm to reprogram the differentiated status of somatic DNA. However, the efficiency of development is low, and this has been attributed to incomplete or inappropriate reprogramming of epigenetic status. One such epigenetic marker is methylation of genomic DNA at CpG islands. In SCNT, derived embryo abnormal DNA methylation patterns have been reported by a number of groups; in particular, it has been observed that the methylation pattern of embryonic cells resembles that of the donor cell (Santos et al. 2003 Curr. Biol. 13, 1116-1121). One strategy to improve reprogramming and, hence, development is to erase or reprogram the epigenetic status of the donor cell prior to nuclear transfer. We have previously reported that Xenopus egg and oocyte extracts show a differential effect on transcription. In oocyte extracts Pol I and II transcripts are maintained in the somatic cells; in egg extracts, these are abolished (Alberio et al. 2005 Exp. Cell. Res. 307, 131-141). To extend these studies, we have investigated the ability of oocyte and egg extracts to demethylate bovine somatic DNA. Preparation of Xenopus oocyte and egg extracts, culture, permeabilization of donor cells, and incubation conditions were all as previously described (Alberio et al. 2005 Exp. Cell. Res. 307, 131-141). Cells were incubated in extracts for 1 and 3 h at 21�C, centrifuged onto glass slides fixed in 4% Para formaldehyde for 15 min, followed by 4 M HCL for 1 h at 39�C, and blocked for 1 h. Cells were stained with mouse monoclonal anti-1MeC (1:50) overnight at 4�C followed by FITC-conjugated goat anti-mouse antibody (1:20) for 1 h at room temperature and mounted in Vectashied containing 10 �g of propidium iodide/mL. Nuclei were scored as positive or negative for 5MeC staining. In control cells, 90% of nuclei stained positively for 5MeC. In both oocyte and egg extracts the number of positive nuclei decreased with time showing demethylation of the somatic DNA 68 and 58% and 38 and 42% positive, respectively, after 1 and 3 h of incubation. Addition of apyrase (2%) to hydrolyze ATP inhibited demethylation in both extracts (90% nuclei positive). High rates of DNA replication were observed in somatic cells in egg extracts in contrast to no replication in oocyte extracts. Aphidicolin (1 �g/20 �L) was added to egg extracts to inhibit DNA replication, and under these conditions, DNA demethylation was abolished, suggesting a passive DNA demethylation mechanism as a result of DNA replication. In conclusion, Xenopus laevis oocyte and egg extracts can demethylate mammalian somatic DNA in an energy-dependent manner. In oocyte extracts, demethylation is independent of DNA replication, suggesting an active mechanism. In egg extracts, DNA replication is required, suggesting a passive mechanism. These studies further demonstrate the differences in reprogramming activities between oocyte and egg cytoplasm and suggest that interspecies extracts may provide a tool for nuclear reprogramming.