Oocyte Extracts Research Articles

Phosphorylation of the proline-rich domain of Xp95 modulates Xp95 interaction with partner proteins

The mammalian adaptor protein Alix [ALG-2 (apoptosis-linked-gene-2 product)-interacting protein X] belongs to a conserved family of proteins that have in common an N-terminal Bro1 domain and a C-terminal PRD (proline-rich domain), both of which mediate partner protein interactions. Following our previous finding that Xp95, the Xenopus orthologue of Alix, undergoes a phosphorylation-dependent gel mobility shift during progesteroneinduced oocyte meiotic maturation, we explored potential regulation of Xp95/Alix by protein phosphorylation in hormone-induced cell cycle re-entry or M-phase induction. By MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS analyses and gel mobility-shift assays, Xp95 is phosphorylated at multiple sites within the N-terminal half of the PRD during Xenopus oocyte maturation, and a similar region in Alix is phosphorylated in mitotically arrested but not serum-stimulated mammalian cells. By tandem MS, Thr745 within this region, which localizes in a conserved binding site to the adaptor protein SETA [SH3 (Src homology 3) domain-containing, expressed in tumorigenic astrocytes] CIN85 (a-cyano-4-hydroxycinnamate)/SH3KBP1 (SH3-domain kinase-binding protein 1), is one of the phosphorylation sites in Xp95. Results from GST (glutathione S-transferase)-pull down and peptide binding/competition assays further demonstrate that the Thr745 phosphorylation inhibits Xp95 interaction with the second SH3 domain of SETA. However, immunoprecipitates of Xp95 from extracts of M-phase-arrested mature oocytes contained additional partner proteins as compared with immunoprecipitates from extracts of G2-arrested immature oocytes. The deubiquitinase AMSH (associated molecule with the SH3 domain of signal transducing adaptor molecule) specifically interacts with phosphorylated Xp95 in M-phase cell lysates. These findings establish that Xp95/Alix is phosphorylated within the PRD during M-phase induction, and indicate that the phosphorylation may both positively and negatively modulate their interaction with partner proteins.

Protein kinase B/Akt phosphorylation of PDE3A and its role in mammalian oocyte maturation

cGMP-inhibited cAMP phosphodiesterase 3A (PDE3A) is expressed in mouse oocytes, and its function is indispensable for meiotic maturation as demonstrated by genetic ablation. Moreover, PDE3 activity is required for insulin/insulin-like growth factor-1 stimulation of Xenopus oocyte meiotic resumption. Here, we investigated the cAMP-dependent protein kinase B (PKB)/Akt regulation of PDE3A and its impact on oocyte maturation. Cell-free incubation of recombinant mouse PDE3A with PKB/Akt or cAMP-dependent protein kinase A catalytic subunits leads to phosphorylation of the PDE3A protein. Coexpression of PDE3A with constitutively activated PKB/Akt (Myr-Akt) increases PDE activity as well as its phosphorylation state. Injection of pde3a mRNA potentiates insulin-dependent maturation of Xenopus oocytes and rescues the phenotype of pde3(-/-) mouse oocytes. This effect is greatly decreased by mutation of any of the PDE3A serines 290-292 to alanine in both Xenopus and mouse. Microinjection of myr-Akt in mouse oocytes causes in vitro meiotic maturation and this effect requires PDE3A. Collectively, these data indicate that activation of PDE3A by PKB/Akt-mediated phosphorylation plays a role in the control of PDE3A activity in mammalian oocytes.

Cytoplasmic CstF-77 Protein Belongs to a Masking Complex with Cytoplasmic Polyadenylation Element-binding Protein in Xenopus Oocytes

Regulated mRNA translation is a hallmark of oocytes and early embryos, of which cytoplasmic polyadenylation is a major mechanism. This process involves multiple protein components, including the CPSF (cleavage and polyadenylation specificity factor), which is also required for nuclear polyadenylation. The CstF (cleavage stimulatory factor), with CPSF, is required for the pre-mRNA cleavage before nuclear polyadenylation. However, some evidence suggests that the CstF-77 subunit might have a function independent of nuclear polyadenylation, which could be related to the cell cycle. As such, we addressed the question whether CstF-77 might have a role in cytoplasmic polyadenylation. We investigated the function of the CstF-77 protein in Xenopus oocytes, and show that CstF-77 has indeed a role in the cytoplasm. The Xenopus CstF-77 protein (X77K) localizes mainly to the nucleus, but also in punctuate cytoplasmic foci. We show that X77K resides in a cytoplasmic complex with eIF4E, CPEB (cytoplasmic polyadenylation element-binding protein), CPSF-100 and XGLD2, but is not required for cytoplasmic polyadenylation per se. Impairment of X77K function in ovo leads to an acceleration of the G(2)/M transition, with a premature synthesis of Mos and AuroraA proteins. However, the kinetic of Mos mRNA polyadenylation is not modified. Furthermore, X77K represses mRNA translation in vitro. These results suggest that X77K could be involved in masking of mRNA prior to polyadenylation.

Polypeptide binding proteins: what remains to be discovered?

Many proteins have developed the potential to sequester a client polypeptide chain in its various folding states as a specific intermolecular ligand and, thus, exhibit the properties of a holding chaperone. The resulting complexes can be of a diverse nature in terms of structure and reaction dynamics and are characterized on the basis of various microscopic properties including formation and decay of encounter and Michaelis complexes as well as reactant and product stability. Interpretation of the functional consequences of complex formation in the cell generally tends to be rather complicated, with notable exceptions including complexes formed during the reaction pathways of proteases, protein kinases and protein phosphatases. Peptide bond cis/trans isomerases take up an intermediate position among the poly(oligo)peptide binding proteins because, although the relationship between chain sequestration and catalysis of isomerization can easily be delineated in vitro, it is sometimes difficult to resolve in the cell. Time-resolved studies on interactions involving peptide bond cis/trans isomerases have led to the establishment of generally applicable methods for studying protein-poly(oligo)peptide interactions that are capable of identifying new types of biocatalysis.

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.

The characterization of amphibian nucleoplasmins yields new insight into their role in sperm chromatin remodeling

BackgroundNucleoplasmin is a nuclear chaperone protein that has been shown to participate in the remodeling of sperm chromatin immediately after fertilization by displacing highly specialized sperm nuclear basic proteins (SNBPs), such as protamine (P type) and protamine-like (PL type) proteins, from the sperm chromatin and by the transfer of histone H2A-H2B. The presence of SNBPs of the histone type (H type) in some organisms (very similar to the histones found in somatic tissues) raises uncertainty about the need for a nucleoplasmin-mediated removal process in such cases and poses a very interesting question regarding the appearance and further differentiation of the sperm chromatin remodeling function of nucleoplasmin and the implicit relationship with SNBP diversity The amphibians represent an unique opportunity to address this issue as they contain genera with SNBPs representative of each of the three main types: Rana (H type); Xenopus (PL type) and Bufo (P type).ResultsIn this work, the presence of nucleoplasmin in oocyte extracts from these three organisms has been assessed using Western Blotting. We have used mass spectrometry and cloning techniques to characterize the full-length cDNA sequences of Rana catesbeiana and Bufo marinus nucleoplasmin. Northern dot blot analysis shows that nucleoplasmin is mainly transcribed in the egg of the former species. Phylogenetic analysis of nucleoplasmin family members from various metazoans suggests that amphibian nucleoplasmins group closely with mammalian NPM2 proteins.ConclusionWe have shown that these organisms, in striking contrast to their SNBPs, all contain nucleoplasmins with very similar primary structures. This result has important implications as it suggests that nucleoplasmin's role in chromatin assembly during early zygote development could have been complemented by the acquisition of a new function of non-specifically removing SNBPs in sperm chromatin remodeling. This acquired function would have been strongly determined by the constraints imposed by the appearance and differentiation of SNBPs in the sperm.

DNase I activity in pig MII oocytes: implications in transgenesis

Several reliable methods to produce transgenic animals utilize the male genome. After penetration into oocyte, sperm DNA undergoes dramatic conformational changes that could represent a great opportunity for exogenous DNA to be integrated in the zygote genome. Among the enzymes responsible for sperm remodeling, a nuclease could be involved. The presence of a DNase I in oocytes has not been much investigated. To date, an immunolocalization of DNase I has been reported only in rat immature oocytes and the presence of nuclease activities has been shown in avian oocytes. The present study was conducted to verify whether a DNase-I like activity is present in MII mature pig oocytes. To do this, oocyte extracts were assessed for nuclease activity by a plasmid degradation assay and by zymography; these analyses evidenced a 33 kDa, Ca2+/Mg2+ dependent DNase I-like activity that was inhibited by Zn2+. A further identification of DNase I was achieved by Western blot, immunofluorescence and RT-PCR experiments. Moreover, the presence of the enzyme activity was confirmed by the rapid degradation of exogenous DNA microinjected into the ooplasm. Finally, the exogenous DNA transferred to oocyte by spermatozoa during sperm mediated gene transfer in vitro fertilisation protocol seemed to be protected from DNase I degradation and to persist in the ooplasm till 6 h. These results, together with the high efficiency of sperm based transgenesis methods, suggest that the association with spermatozoa protects exogenous DNA from nuclease activities.

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.

27 CYTOPLASM OF A GERMINAL VESICLE OOCYTE CAN PROMOTE SOMATIC NUCLEAR REPROGRAMMING IN MICE

A number of studies have shown that epigenetic reprogramming is severely deficient in cloned embryos, the majority of which exhibit histone hypermethylation. Xenopus geminal vesicle (GV) oocytes have been reported to have a DNA demethylating activity (Simonsson and Gurdon 2004 Nat. Cell Biol. 6, 984–990). In an attempt to develop a new method for erasing or reprogramming the epigenetic status of the donor cell prior to nuclear transfer, we examined whether the mammalian GV oocyte cytoplasm can demethylate H3-K9 of somatic cell nuclei and improve the quality of reconstructed embryos. In the first series of experiments, cumulus nuclei were injected into enucleated GV oocytes and cultured for various times, 0 h, 3 h, 7 h, 9 h, 11 h, and 16 h, before examining the chromosome morphology of somatic nuclei together with Me-H3-K9. In the second series of experiments, permeabilized cumulus cells were immersed with GV oocyte extract and injected into enucleated MII oocytes. These reconstructed oocytes were activated and cultured until the blastocyst stage. Preparation of oocyte extracts was as follows: 200 GV oocytes were collected and zonae pellucidae removed using Tyrode's solution. These zona-free oocytes were broken down in 5 �L of HEPES medium containing the ATP-generating system (1 mM ATP, 10 mM creatine phosphate, 25 �g mL-1 creatine kinase, 100 �M GTP). Permeabilized cumulus was incubated in oocyte extract for 45 min at 37�C. The demethylation of H3-K9 was analyzed in nuclear transfer embryos at the pronucleus, 2-cell, 8-cell, morula, and blastocyst stages. In 2 experiments, we examined a total of 234 enucleated GV oocytes and 358 enucleated MII oocytes. The results of first experiment showed that histone H3-K9 begins demethylation in the somatic chromosome 1 h after injection into GV oocytes (90%) and, importantly, this state was maintained until the MII-like stage. The donor somatic chromosomes in the enucleated GV oocyte can condense and undergo stages GVI (0–3 h), MI (7 h), and AI-TI (9–11 h), and the MII (16 h)-like stage. These results clearly show that enucleated GV oocytes can carry out spindle assembly and extrude the first polar body. However, some of these chromosomes are located not only on the metaphase plate of the spindle but also on spindle poles or dispersed on the spindle. In the second experiment, reconstructed embryos with GV extract-treated somatic cells showed a strong H3-K9 demethylation that was significantly different to nontreated somatic cells at the blastocyst stage. The demethylation was expressed at a rate comparable to that observed in ICSI embryos. Although there was no increase in the frequency of development at the blastocyst stage, we found an increase in cell number at the blastocyst stage. Our findings show that the GV oocyte cytoplasm has the ability to erase methylation of H3-K9 in somatic nuclei, which suggests that the incorporation of GV mammalian oocyte components may contribute to the reprogramming of somatic cell nuclei.

320 MPF ACTIVITY AND DEVELOPMENTAL COMPETENCE IN DIFFERENT SIZES OF PREPUBERTAL GOAT OOCYTES

Developmental competence of prepubertal goat oocytes recovered from a slaughterhouse is low, probably due to an incomplete cytoplasmic maturation. Regulation of cytoplasmic maturation is still unknown, although maturation-promoting factor (MPF) is suggested to play an important role in this process. To better understand the role of MPF in cytoplasmic maturation, we have studied MPF kinase activity in oocytes with different developmental competence. Ovaries were obtained from a slaughterhouse, and oocytes were recovered by slicing and were selected according to morphological criteria. Some oocytes were denuded and classified in diameter groups (<110 μm, 110–125 μm, 125–135 μm, and >135 μm), placed in lysis buffer (50 mM Tris-HCl (pH 7.5), 0.5 M NaCl, 5 mM EDTA, 0.01% Brij35, 1 mM phenyl methyl sultonyl fluoride (PMSF), 0.05 mg/mL leupeptin, 50 mM 2-mercaptoethanol, 25 mM α-glycerophosphate, 1 mM Na-orthovanadate) and frozen in liquid N2. Cell extracts were stored at −80°C until use. The rest of oocytes were matured in vitro in medium TCM199 supplemented with hormones, 10% (DBS), and 400 μM cysteamine, for 27 h in 5% CO2 in air and 38.5°C. After IVM, a sample of oocytes were also denuded, classified by diameters, and frozen as described above. The rest of oocytes were used for IVF in mDM with spermatozoa capacitated with heparin and ionomicin. After 24 h, presumptive zygotes were cultured for 7 days in medium SOF in 5% CO2, 5% O2, and 90% N2 at 38.5°C. At 48 h post-insemination, we added 0.1 μL FBS per embryo. Embryonic development was evaluated with Hoechst staining after IVC. MPF kinase activity was detected using the MESACUP cdc2 kinase assay kit (MBL Woburn, MA, USA). Briefly, the oocyte extract corresponding to 10 oocytes was mixed with 10× reaction buffer (25 mM HEPES buffer (pH 7.5), 10 mM MgCl2) and 10% biotinylated MV peptide (SLYSSPGGAYC). We added 0.1 mM ATP to start the reaction. The mixture was incubated at 30°C for 30 min. The reaction was finished by adding 200 μL of PBS containing 50 mM EGTA. The phosphorylated MV peptide was detected by specific antibody using an ELISA procedure, and the OD was measured at 492 nm. Fisher's exact test was used to analyze IVC results, and ANOVA to analyze cdc2 kinase activity results. We considered differences statistically significant when P < 0.05. Results are shown in Table 1. We observed that embryonic cleavage and blastocyst rate increased with oocyte diameter. The MPF activity detected was higher in the largest oocytes after IVM. As a consequence, we could establish that oocytes with a higher MPF activity are more capable of maintaining embryonic development until the blastocyst stage, which may indicate the important role that MPF plays in cytoplasmic maturation. Table 1. Cleavage, blastocyst rate, and MPF kinase activity in different sizes of prepubertal goat oocytes

DNA polymerases η and κ are responsible for error-free translesion DNA synthesis activity over a cis–syn thymine dimer in Xenopus laevis oocyte extracts

In translesion synthesis (TLS), specialized DNA polymerases (pols) facilitate progression of replication forks stalled by DNA damage. Although multiple TLS pols have been identified in eukaryotes, little is known about endogenous TLS pols and their relative contributions to TLS in vivo because of their low cellular abundance. Taking advantage of Xenopus laevis oocyte cells, with their extraordinary size and abundant enzymes involved in DNA metabolism, we have identified and characterized endogenous TLS pols for DNA damage induced by ultraviolet (UV) irradiation. We designed a TLS assay which monitors primer elongation on a synthetic oligomer template over a single UV-induced lesion, either a cys–syn cyclobutane pyrimidine dimer (CPD) or a pyrimidine (6-4) pyrimidone photoproduct. Four distinct TLS activities (TLS1–TLS4) were identified in X. laevis oocyte extracts, using three template/primer (T/P) DNA substrates having various sites at which primer extension is initiated relative to the lesion. TLS1 and TLS2 activities appear to be sequence-dependent. TLS3 and TLS4 extended the primers over the CPD in an error-free manner irrespective of sequence context. Base insertion opposite the CPD of the T/P substrate in which the 3′-end of the primer is placed one base upstream of the lesion was observed only with TLS3. TLS3 and TLS4 showed primer extension with similar efficiencies on the T/P substrate whose 3′-primer terminal dinucleotide (AA) was complementary to the CPD lesion. Investigations with antibodies and recombinant pols revealed that TLS3 and TLS4 were most likely attributable to pol η and pol κ, respectively. These results indicate that error-free insertion in CPD bypass is due mainly to pol η (TLS3) in the extracts, and suggest that pol κ (TLS4) may assist pol η (TLS3) in error-free extension during CPD bypass.

Overexpression of truncated γ-tubulins disrupts mitotic aster formation in Xenopus oocyte extracts

Mechanisms of spindle pole formation rely on minus-end-directed motor proteins. gamma-Tubulin is present at the centre of poles, but its function during pole formation is completely unknown. To address the role of gamma-tubulin in spindle pole formation, we overexpressed GFP (green fluorescent protein)-fused gamma-tubulin (gamma-Tu-GFP) in Xenopus oocytes and produced self-assembled mitotic asters in the oocyte extracts. gamma-Tu-GFP associated with endogenous alpha-, beta- and gamma-tubulin, suggesting that it acts in the same manner as that of endogenous gamma-tubulin. During the process of aster formation, gamma-Tu-GFP aggregated as dots on microtubules, and then the dots were translocated to the centre of the aster along microtubules in a manner dependent on cytoplasmic dynein activity. Inhibition of the function of gamma-tubulin by an anti-gamma-tubulin antibody resulted in failure of microtubule organization into asters. This defect was restored by overexpression of gamma-Tu-GFP, confirming the necessity of gamma-tubulin in microtubule recruitment for aster formation. We also examined the effects of truncated gamma-tubulin mutants, which are difficult to solubly express in other systems, on aster formation. The middle part of gamma-tubulin caused abnormal organization of microtubules in which minus ends of microtubules were not tethered, but dispersed. An N-terminus-deleted mutant prevented recruitment of microtubules into asters, similar to the effect of the anti-gamma-tubulin antibody. The results indicate possible roles of gamma-tubulin in spindle pole formation and show that the system developed in the present study could be useful for analysing roles of many proteins that are difficult to solubly express.

Regulation of the G2/M Transition in Xenopus Oocytes by the cAMP-dependent Protein Kinase

Vertebrate oocytes are arrested in G(2) phase of the cell cycle at the prophase border of meiosis I. Progesterone treatment of Xenopus oocytes releases the G(2) block and promotes entry into the M phases of meiosis I and II. Substantial evidence indicates that the release of the G(2) arrest requires a decrease in cAMP and reduced activity of the cAMP-dependent protein kinase (PKAc). It has been reported and we confirm here that microinjection of either wild type or kinase-dead K72R PKAc inhibits progesterone-dependent release of the G(2) arrest with equal potency and that inhibition can be reversed by a second injection of the heat-stable inhibitor of PKAc, PKI. However, a mutant enzyme predicted to be completely kinase-dead from the crystal structure of PKAc, K72H PKAc, was much less inhibitory when carrying additional mutations that block interaction with either type I or type II regulatory subunit. Moreover, inhibition by K72H PKAc was reversed by PKI at a 30-fold lower concentration and with more rapid kinetics compared with wild type PKAc. K72R PKAc was found to have low but detectable activity after incubation in an oocyte extract. These results indicate that inhibition of the progesterone-dependent G(2)/M transition in oocytes after microinjection of dead PKAc reflects either low residual activity or binding to regulatory subunits with a resulting net increase in the level of endogenous wild type PKAc. Consistent with this hypothesis, the induction of mitosis in Xenopus egg extracts by the addition of cyclin B was blocked by wild type PKAc but not by K72H PKAc. The identification of substrates for PKAc that maintain cell cycle arrest in G(2) remains an important goal for future work.

In vitro decondensation of the sperm chromatin in Holothuria tubulosa (sea cucumber) not affecting proteolysis of basic nuclear proteins

Sea urchin and sea star oocyte extracts contain proteolytic activities that are active against sperm basic nuclear proteins (SNBP). This SNBP degradation has been related to the decondensation of sperm chromatin as a possible model to male pronuclei formation. We have studied the presence of this proteolytic activity in Holothuria tubulosa (sea cucumber) and its possible relationship with sperm nuclei decondensation. The mature oocyte extracts from H. tubulosa contain a proteolytic activity to SNBP located in the macromolecular fraction of the egg-jelly layer. SNBP degradation occurred both on sperm nuclei and on purified SNBP, histones being more easily degraded than protein Ø(o) (sperm-specific protein). SNBP degradation was found to be dependent on concentration, incubation time, presence of Ca(2+), pH, and this activity could be a serine-proteinase. Thermal denaturalization of the oocyte extracts (80 degrees C, 10-15 min) inactivates its proteolytic activity on SNBP but does not affect sperm nuclei decondensation. These results would suggest that sperm nuclei decondensation occurs by a mechanism different from SNBP degradation. Thus, the sperm nuclei decondensation occurs by a thermostable factor(s) and the removal of linker SNBP (H1 and protein Ø(o)) will be a first condition in the process of sperm chromatin remodeling.

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.

Phosphorylation of Maskin by Aurora-A Participates in the Control of Sequential Protein Synthesis during Xenopus laevis Oocyte Maturation

At the end of oogenesis, Xenopus laevis stage VI oocytes are arrested at the G2/M transition (prophase) waiting for progesterone to release the block and begin maturation. Progesterone triggers a cascade of phosphorylation events such as a decrease of pK(a) and an increase of maturating-promoting factor activity. Progression through meiosis was controlled by the sequential synthesis of several proteins. For instance, the MAPK kinase kinase c-Mos is the very first protein to be produced, whereas cyclin B1 appears only after meiosis I. After the meiotic cycles, the oocyte arrests at metaphase of meiosis II with an elevated c-Mos kinase activity (cytostatic factor). By using a two-hybrid screen, we have identified maskin, a protein involved in the control of mRNA sequential translation, as a binding partner of Aurora-A, a protein kinase necessary for oocyte maturation. Here we showed that, in vitro, Aurora-A directly binds to maskin and that both proteins can be co-immunoprecipitated from oocyte extracts, suggesting that they do associate in vivo. We also demonstrated that Aurora-A phosphorylates maskin on a Ser residue conserved in transforming acidic coiled coil proteins from Drosophila to human. When the phosphorylation of this Ser was inhibited in vivo by microinjection of synthetic peptides that mimic the maskin-phosphorylated sequence, we observed a premature maturation. Under these conditions, proteins such as cyclin B1 and Cdc6, which are normally detected only in meiosis II, were massively produced in meiosis I before the occurrence of the nuclear envelope breakdown. This result strongly suggests that phosphorylation of maskin by Aurora-A prevents meiosis II proteins from being produced during meiosis I.

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.

A Tribute to the Xenopus laevis Oocyte and Egg

When I was asked to reflect as part of the celebration of the 100-year anniversary of the Journal of Biological Chemistry I recalled a talk by Seymour Cohen at the Federation meetings in Atlantic City about 1960. He began by thanking bacteriophage for providing him with so many wonderful research problems. I decided in the same spirit to pay homage to the Xenopus laevis egg and oocyte, two states of a cell that has played and continues to play a central role in most of the disciplines of modern biology including biochemistry and molecular biology. Many of us owe a debt of gratitude to this cell. The egg is the most important and interesting cell in the repertoire of any organism. In X. laevis this single cell has a diameter of about 1.3 mm. The oocyte is permeable to small molecules, but after it undergoes meiosis and becomes an unfertilized egg it is impermeable to these same molecules. The oocyte is an active site of RNA and protein synthesis but not DNA replication, whereas the unfertilized egg is poised to replicate every 10 min after it is fertilized. During the cleavage period the embryo is transcriptionally silent. An oocyte can be cultured for days, but once ovulated the egg degenerates rapidly if it is not fertilized. The oocyte is the largest single cell in the body yet it has many of the same structures as somatic cells. These compartments are so exaggerated in size that they are accessible to cell biologists for manipulation and visualization. The huge oocyte nucleus is called a germinal vesicle (GV). The premeiotic tetraploid chromosomes are expanded into a “lampbrush” configuration so that they can be studied with a light microscope. The X. laevis egg and its possibilities for experimental manipulation were introduced to modern biology in the late 1950s by John Gurdon (Fig. 1), then a graduate student in the laboratory of Michail Fischberg in the Department of Zoology at Oxford University. Gurdon’s thesis was to reproduce with the South African “clawed toad” X. laevis the famous nuclear transplantation experiments that Briggs and King had perfected with the American leopard frog, Rana pipiens (1). Embryonic nuclei were injected into eggs whose own nuclei had been removed or destroyed. These early cloning experiments addressed the question of whether a differentiated and specialized somatic cell nucleus was still capable of expressing its full genetic repertoire. Briggs and King found that nuclei from R. pipiens embryos older than gastrula never promoted normal development, suggesting that the genetic material might change in cells as they specialize during embryonic development. This result was challenged by John Gurdon’s demonstration that nuclei from X. laevis embryos are more permissive. Even a small fraction of nuclei derived from differentiated intestinal epithelial cells supported normal development (2). This crucial scientific question of whether irreversible changes occur in the genome of highly specialized cells was addressed more precisely by experimental manipulation than by biochemistry or genetics. The concepts behind these experiments have influenced the stem cell field. The topic of whether the genome is altered in specialized somatic cells was revisited just this year by the same strategy of nuclear transplantation. Postmitotic nuclei from olfactory neurons support the development of normal fertile mice when transplanted into enucleated mouse eggs (3). THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 44, Issue of October 29, pp. 45291–45299, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Modulation of Kv4.2 Channel Expression and Gating by Dipeptidyl Peptidase 10 (DPP10)

The dipeptidyl aminopeptidase-like protein DPPX (DPP6) associates with Kv4 potassium channels, increasing surface trafficking and reconstituting native neuronal I SA-like properties. Dipeptidyl peptidase 10 (DPP10) shares with DPP6 a high amino acid identity, lack of enzymatic activity, and expression predominantly in the brain. We used a two-electrode voltage-clamp and oocyte expression system to determine if DPP10 also interacts with Kv4 channels and modulates their expression and function. Kv4.2 coimmunoprecipitated with HA/DPP10 from extracts of oocytes heterologously expressing both proteins. Coexpression with DPP10 and HA/DPP10 enhanced Kv4.2 current by approximately fivefold without increasing protein level. DPP10 also remodeled Kv4.2 kinetic and steady-state properties by accelerating time courses of inactivation and recovery ( τ rec: WT = 200 ms, +DPP10 = 78 ms). Furthermore, DPP10 introduced hyperpolarizing shifts in the conductance-voltage relationship (∼19 mV) as well as steady-state inactivation (∼7 mV). The effects of DPP10 on Kv4.1 were similar to Kv4.2; however, distinct biophysical differences were observed. Additional experiments suggested that the cytoplasmic N-terminal domain of DPP10 determines the acceleration of inactivation. In summary, DPP10 is a potent modulator of Kv4 expression and biophysical properties and may be a critical component of somatodendritic I SA channels in the brain.

A Feedback Loop in the Polo-like Kinase Activation Pathway

The Xenopus polo-like kinase Plx1 plays important roles during entry into and exit from mitosis (M phase). Previous studies revealed that Plx1 is activated by phosphorylation on serine and threonine residues, and purification of an activating enzyme from mitotic Xenopus egg extracts led to cloning and characterization of Xenopus polo-like kinase kinase (xPlkk1), which can phosphorylate and activate Plx1 in vitro. In the present study, a positive feedback loop between Plx1 and xPlkk1 was shown to result in each kinase phosphorylating and activating the other. Sequencing of radiolabeled xPlkk1 after phosphorylation by Plx1 in vitro identified three phosphorylation sites each spaced three amino acids apart, two of which have the consensus acidic-X-pSer-hydrophobic described for other polo-like kinase substrates. In addition, endogenous xPlkk1 in oocytes was phosphorylated on these sites in M phase but not in interphase. A mutant xPlkk1 in which these three amino acids were changed to alanine (xPlkk1(SA3)) was unable to be phosphorylated or activated in vitro by Plxl. Depletion of Plx1 from oocyte extracts prior to stimulation of the G(2)/M transition blocked the activation of xPlkk1, but depletion of xPlkk1 before stimulation did not block Plx1 activation. These results indicate that xPlkk1 may function downstream as a target of Plx1 rather than as an upstream activating kinase during the G(2)/M transition.