Amphibian Oocytes Research Articles

Characterization of somatic cell nuclear reprogramming by oocytes in which a linker histone is required for pluripotency gene reactivation

When transplanted into Xenopus oocytes, the nuclei of mammalian somatic cells are reprogrammed to express stem cell genes such as Oct4, Nanog, and Sox2. We now describe an experimental system in which the pluripotency genes Sox2 and Oct4 are repressed in retinoic acid-treated ES cells but are reprogrammed up to 100% within 24 h by injection of nuclei into the germinal vesicle (GV) of growing Xenopus oocytes. The isolation of GVs in nonaqueous medium allows the reprogramming of individual injected nuclei to be seen in real time. Analysis using fluorescence recovery after photobleaching shows that nuclear transfer is associated with an increase in linker histone mobility. A simultaneous loss of somatic H1 linker histone and incorporation of the oocyte-specific linker histone B4 precede transcriptional reprogramming. The loss of H1 is not required for gene reprogramming. We demonstrate both by antibody injection experiments and by dominant negative interference that the incorporation of B4 linker histone is required for pluripotency gene reactivation during nuclear reprogramming. We suggest that the binding of oocyte-specific B4 linker histone to chromatin is a key primary event in the reprogramming of somatic nuclei transplanted to amphibian oocytes.

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.

Tips and tricks for preparing lampbrush chromosome spreads from Xenopus tropicalis oocytes

Due to their large size and fine organization, lampbrush chromosomes (LBCs) of amphibian oocytes have been for decades one of the favorite tools of biologists for the analysis of transcriptional and post-transcriptional processes at the cytological level. The emergence of the diploid Xenopus tropicalis amphibian as a model organism for vertebrate developmental genetics and the accumulation of sequence data made available by its recent genomic sequencing, strongly revive the interest of LBCs as a powerful tool to study genes expressed during oogenesis. We describe here a detailed protocol for preparing LBCs from X. tropicalis oocyte and give practical advice to encourage a large number of researchers to become familiar with these chromosomes.

Novel domains in the hnRNP G/RBMX protein with distinct roles in RNA binding and targeting nascent transcripts

The heterogenous nuclear ribonucleoprotein G (hnRNP G) controls the alternative splicing of several pre-mRNAs. While hnRNP G displays an amino terminal RNA recognition motif (RRM), we find that this motif is paradoxically not implicated in the recruitment of hnRNP G to nascent transcripts in amphibian oocytes. In fact, a deletion analysis revealed that targeting of hnRNP G to active transcription units depends on another domain, centrally positioned, and consisting of residues 186-236. We show that this domain acts autonomously and thus is named NTD for nascent transcripts targeting domain. Furthermore, using an RNA probe previously characterized in vitro as an RNA that interacts specifically with hnRNP G, we demonstrate a new auxiliary RNA binding domain (RBD). It corresponds to a short region of 58 residues positioned at the carboxyl terminal end of the protein, which recognizes an RNA motif predicted to adopt an hairpin structure. The fact that the NTD acts independently from both the RRM and the RBD strongly suggests that the initial recruitment of hnRNP G to nascent pre-mRNAs is independent of its sequence-specific RNA binding properties. Together, these findings highlight the modular organization of hnRNP G and offer new insights into its multifunctional roles.

Molecular characterization of Xenopus lamin LIV reveals differences in the lamin composition of sperms in amphibians and mammals

Lamins are nuclear intermediate filament proteins. They are involved in most nuclear activities and are essential for retaining the mechano-elastic properties of the nucleus. Somatic cells of vertebrates express lamins A, B1, and B2 while lamin LIII, a major component of the amphibian oocyte lamina is absent in mammals. The organization of the lamina of germ cells differs significantly from that of somatic cells. Mammalian spermatogenic cells express two short lamins, C2 and B3, that are splice isoforms of lamin A and B2, respectively. Here we identify the previously described Xenopus lamin LIV as splice variant of the lamin LIII gene. LIV contains 40 extra residues in coil 2A of the rod domain, which results in altered assembly properties. Xenopus lamin LIV and mammalian B3 assemble into short structures rather than into long IF-like filaments. Expression of lamin LIV is restricted to male germ cells suggesting that it might be the functional equivalent of mammalian lamin B3. We provide evidence that lamins C2 and B3 are restricted to the mammalian lineage and describe the lamin composition of Xenopus sperm. Our results show that the evolution of germ cell-specific lamins followed separate and distinctly different paths in amphibians and mammals.

Progesterone modulation of transmembrane helix-helix interactions between the alpha-subunit of Na/K-ATPase and phospholipid N-methyltransferase in the oocyte plasma membrane.

BackgroundProgesterone binding to the surface of the amphibian oocyte initiates the meiotic divisions. Our previous studies with Rana pipiens oocytes indicate that progesterone binds to a plasma membrane site within the external loop between the M1 and M2 helices of the α-subunit of Na/K-ATPase, triggering a cascade of lipid second messengers and the release of the block at meiotic prophase. We have characterized this site, using a low affinity ouabain binding isoform of the α1-subunit.ResultsPreparations of isolated plasma membranes from Rana oocytes demonstrate that physiological levels of progesterone (or the non-metabolizable progestin R5020) successively activate phosphatidylethanolamine-N-methyltransferase (PE-NMT) and sphingomyelin synthase within seconds. Inhibition of PE-NMT blocks the progesterone induction of meiosis in intact oocytes, whereas its initial product, phosphatidylmonomethylethanolamine (PME), can itself initiate meiosis in the presence of the inhibitor. Published X-ray crystallographic data on Na/K-ATPase, computer-generated 3D projections, heptad repeat analysis and hydrophobic cluster analysis of the transmembrane helices predict that hydrophobic residues L, V, V, I, F and Y of helix M2 of the α1-subunit interact with F, L, G, L, L and F, respectively, of helix M3 of PE-NMT.ConclusionWe propose that progesterone binding to the first external loop of the α1-subunit facilitates specific helix-helix interactions between integral membrane proteins to up-regulate PE-NMT, and, that successive interactions between two or more integral plasma membrane proteins induce the signaling cascades which result in completion of the meiotic divisions.

IGF-1 Receptors in Xenopus laevis Ovarian Follicle Cells Support the Oocyte Maturation Response

Insulin-like growth factor 1 (IGF-1)-stimulated amphibian oocyte maturation has been studied extensively by a number of laboratory groups, but in previous studies possible effects of IGF-1 on ovarian follicle cells had not been tested directly. In the study reported here, biochemical and immunofluorescent techniques were used to test Xenopus ovarian follicle cells for the presence of hormone-sensitive IGF-1 receptor. Anti-xIGF-1 receptor beta-subunit antibodies detected a 90- and 98-kDa protein doublet in manually dissected oocyte cortices (plasma membrane-vitelline envelope complexes) by protein immunoblotting both before and after removal of follicle cells from oocytes by sandpaper rolling. The 90-kDa IGF-1 receptor beta-subunit was also detected in follicle cell pellets. Tyrosine phosphorylation of the receptor beta-subunits was increased by treatment of cortices with 10 nM IGF-1 both in the presence and absence of associated follicle cells, was reduced by removal of follicle cells, and was detected in follicle cell pellets. Treatment of follicle cell pellets with nanomolar concentrations of IGF-1 stimulated receptor tyrosine phosphorylation in a dose-dependent fashion that correlated with dose-dependent stimulation of oocyte maturation. IGF-1 receptor was also detected in cultured follicle cells by immunofluorescence. Removal of follicle cells significantly reduced the IGF-1-stimulated oocyte maturation response. These results offer the first direct evidence for hormone-responsive IGF-1 receptors in Xenopus laevis ovarian follicle cells and demonstrate that follicle cells somehow support IGF-1-stimulated oocyte maturation.

Involvement of the dehydroleucodine alpha-methylene-gamma-lactone function in GVBD inhibition inBufo arenarumoocytes

Dehydroleucodine (DhL), a sesquiterpenic lactone, was isolated and purified from aerial parts of Artemisia douglasiana Besser, a medicinal herb used in Argentina. DhL is an alpha-methylene butyro-gamma-lactone ring connected to a seven-membered ring fused to an exocyclic alpha,beta-unsaturated cyclopentenone ring. It has been previously shown that DhL selectively induces a dose-dependent transient arrest in G2 of both meristematic cells and vascular smooth muscle cells. Treatment with DhL induces an inhibition of spontaneous and progesterone-induced maturation in a dose-dependent manner in Bufo arenarum fully grown oocytes arrested at G2, at the beginning of meiosis I. However, the nature of the mechanisms involved in the process is still unknown. The aim of this work was to analyse whether DhL's alpha-methylene-gamma-lactone function is responsible for the inhibition effect on meiosis reinitiation of Bufo arenarum oocytes as well as some of the transduction pathways that could be involved in this effect using a derivative of DhL inactivated for alpha-methylenelactone, the 11,13-dihydro-dehydroleucodine (2H-DhL). The use of 2H-DhL in the maturation promoting factor (MPF) amplification experiments by injection of both cytoplasm with active MPF and of germinal vesicle content showed results similar to the ones obtained with DhL, suggesting that the hydrogenated derivative would act in a similar way to DhL. Pretreatment with DhL or 2H-DhL did not affect the percentage of germinal vesicle breakdown (GVBD) induced by H89, a protein kinase A (PKA) inhibitor, which suggests that these lactones would act on another step of the signalling pathway that induces MPF activation. The fact that both DhL and 2H-Dhl inhibit GVBD induced by okadaic acid microinjection suggests that they could act on the activity of the Myt1 kinase. This idea is supported by the experiments of injection of GV contents in which an inhibitory effect of these lactones on GVBD was also observed. Our results indicate that the inhibitory effect on meiosis progression of DhL does not depend only on the activity of the alpha-methylenelactone function, as its hydrogenated derivative, 2H-DhL, in which this function has been inactivated, causes similar effects on amphibian oocytes. However, 2H-DhL was less active than DhL as higher doses were required to obtain a significant inhibition. On the other hand, the analysis of the participation of certain mediators in some of the signalling pathways leading to MPF activation suggests that the Myt1 kinase could be a target of these lactones, while cdc25 phosphatase would not be affected. Besides, the PKA inhibition assays indicate that these lactones would act earlier in the signalling pathways.

Xenopusoocytes reactivate muscle gene transcription in transplanted somatic nuclei independently of myogenic factors

Transplantation into eggs or oocytes is an effective means of achieving the reprogramming of somatic cell nuclei. We ask here whether the provision of gene-specific transcription factors forms part of the mechanism by which a gene that is repressed in somatic cells is transcribed in oocytes. We find that M1 oocytes have an extremely strong transcription-inducing activity. They cause muscle genes of nuclei from non-muscle somatic cells, after injection into oocytes, to be transcribed to nearly the same extent as muscle genes in muscle cells. We show, surprisingly, that the myogenic factor MyoD and other known myogenic factors are not required to induce the transcription of muscle genes in a range of non-muscle somatic cell nuclei after transplantation to Xenopus oocytes. The overexpression of Id, a dominant-negative repressor of MyoD, prevents maternal MyoD from binding to its consensus sequences; nevertheless, muscle genes are activated in somatic nuclei to the same extent as without Id. We conclude that M1 oocytes can reprogram somatic nuclei in a different way to other experimental procedures: oocytes do not suppress the transcription of inappropriate genes and they activate a gene without the help of its known transcription factors. We suggest that these characteristics might be a special property of amphibian oocytes, and possibly of oocytes in general.

Anoctamin/TMEM16 family members are Ca2+‐activated Cl− channels

Ca(2+)-activated Cl- channels (CaCCs) perform many important functions in cell physiology including secretion of fluids from acinar cells of secretory glands, amplification of olfactory transduction, regulation of cardiac and neuronal excitability, mediation of the fast block to polyspermy in amphibian oocytes, and regulation of vascular tone. Although a number of proteins have been proposed to be responsible for CaCC currents, the anoctamin family (ANO, also known as TMEM16) exhibits characteristics most similar to those expected for the classical CaCC. Interestingly, this family of proteins has previously attracted the interest of both developmental and cancer biologists. Some members of this family are up-regulated in a number of tumours and functional deficiency in others is linked to developmental defects.

Influence of Lamin A on the Mechanical Properties of Amphibian Oocyte Nuclei Measured by Atomic Force Microscopy

The nuclear lamina is part of the nuclear envelope (NE). Lamin filaments provide the nucleus with mechanical stability and are involved in many nuclear activities. The functional importance of these proteins is highlighted by mutations in lamin genes, which cause a variety of human diseases (laminopathies). Here we describe a method that allows one to quantify the contribution of lamin A protein to the mechanical properties of the NE. Lamin A is ectopically expressed in Xenopus oocytes, where it is incorporated into the NE of the oocyte nucleus, giving rise to a prominent lamina layer at the inner nuclear membrane. Nuclei are then isolated and probed by atomic force microscopy. From the resulting force curves, stiffness values are calculated and compared with those of control nuclei. Expression of lamin A significantly increases the stiffness of oocyte nuclei in a concentration-dependent manner. Since chromatin adds negligibly to nuclear mechanics in these giant nuclei, this method allows one to measure the contribution of individual NE components to nuclear mechanics.

Epigenetic marks in somatic chromatin are remodelled to resemble pluripotent nuclei by amphibian oocyte extracts

Reprogramming pluripotency after nuclear transplantation shows that molecules in oocytes can remodel somatic chromatin to a stem cell state. Here we report on an ex-ovo system using axolotl oocyte extracts to remodel epigenetic marks of somatic chromatin. Molecules present in axolotl oocyte extracts induce the reduction of the overall levels of H3K9me3, HP1α, and DNA methylation of somatic cells, and they increase the levels of H3K9ac. The levels of signal intensity detected in treated differentiated cells resemble those detected in embryonic stem cells, which are, in contrast, unaffected by these extracts. Analysis of specific genome sequences shows that somatic cells exposed to oocyte extracts undergo demethylation of LINE-1 repeats but Major Satellite repeats and the imprinted gene H19 remain unchanged. In addition, they induce demethylation of the Oct-4 promoter. Finally, the kinetics of activation of Oct-4 and Nanog expression from MEF nuclei treated in extracts suggests that these genes are subject to different levels of epigenetic control. The results demonstrate that axolotl oocyte extracts are a useful tool for studying epigenetic remodelling of somatic cells to a stem cell configuration, and for elucidating oocyte specific mechanisms of nuclear reprogramming.

Model Progesterone Response

Interacting negative feedback loops control the sensitivity of amphibian oocytes to the hormone progesterone.

Tuning the Activation Threshold of a Kinase Network by Nested Feedback Loops

Determining proper responsiveness to incoming signals is fundamental to all biological systems. We demonstrate that intracellular signaling nodes can tune a signaling network's response threshold away from the basal median effective concentration established by ligand-receptor interactions. Focusing on the bistable kinase network that governs progesterone-induced meiotic entry in Xenopus oocytes, we characterized glycogen synthase kinase-3beta (GSK-3beta) as a dampener of progesterone responsiveness. GSK-3beta engages the meiotic kinase network through a double-negative feedback loop; this specific feedback architecture raises the progesterone threshold in correspondence with the strength of double-negative signaling. We also identified a marker of nutritional status, l-leucine, which lowers the progesterone threshold, indicating that oocytes integrate additional signals into their cell-fate decisions by modulating progesterone responsiveness.

Lampbrush chromosomes enable study of cohesin dynamics

The lampbrush chromosomes present in the nuclei of amphibian oocytes offer unique biological approaches for study of the mechanisms that regulate chromatin structure with high spatial resolution. We discuss fundamental aspects of the remarkable organization and plasticity exhibited by lampbrush chromosomes. We then utilize lampbrush chromosomes to characterize the chromosomal distribution and dynamics of cohesin, the four-protein complex (RAD21/MCD1/SCC1, SMC1, SMC3, SCC3/SA2) responsible for sister chromatid cohesion. We find that endogenous SMC3 and newly expressed hRAD21 co-localize on chromosomal axes, sites where sister chromatids are tightly paired. We present evidence suggesting that hRAD21 recruitment to lampbrush chromosomes is modulated by chromosomal SMC1 and SMC3. Notably, using a technique for de novo chromosome assembly, we demonstrate that both SMC3 and hRAD21 are recruited to single, unreplicated lampbrush chromatids. Finally, we used our novel method of analyzing the oocyte nucleus under oil combined with fluorescence recovery after photobleaching, to provide direct evidence that cohesin is highly dynamic at discrete, condensed chromosomal regions. Collectively, these data demonstrate that lampbrush chromosomes provide a unique and powerful tool for combining biochemical and cytological analyses for dissection of complex chromosomal processes.

Fragile X Mental Retardation Protein FMRP Binds mRNAs in the Nucleus

The fragile X mental retardation protein FMRP is an RNA binding protein that associates with a large collection of mRNAs. Since FMRP was previously shown to be a nucleocytoplasmic shuttling protein, we examined the hypothesis that FMRP binds its cargo mRNAs in the nucleus. The enhanced green fluorescent protein-tagged FMRP construct (EGFP-FMRP) expressed in Cos-7 cells was efficiently exported from the nucleus in the absence of its nuclear export sequence and in the presence of a strong nuclear localization sequence (the simian virus 40 [SV40] NLS), suggesting an efficient mechanism for nuclear export. We hypothesized that nuclear FMRP exits the nucleus through its bound mRNAs. Using silencing RNAs to the bulk mRNA exporter Tap/NXF1, we observed a significantly increased number of cells containing EGFP-FMRP in the nucleus, which was further augmented by removal of FMRP's nuclear export sequence. Nuclear-retained SV40-FMRP could be released upon treatment with RNase. Further, Tap/NXF1 coimmunoprecipitated with EGFP-FMRP in an RNA-dependent manner and contained the FMR1 mRNA. To determine whether FMRP binds pre-mRNAs cotranscriptionally, we expressed hemagglutinin-SV40 FMRP in amphibian oocytes and found it, as well as endogenous Xenopus FMRP, on the active transcription units of lampbrush chromosomes. Collectively, our data provide the first lines of evidence that FMRP binds mRNA in the nucleus.

Activation of amphibian oocytes by sperm extracts

In the fertilization of most animals, egg activation is accompanied by an increase in cytoplasmatic Ca2+; however, the mechanism through which the fertilizing sperm induce this phenomenon is still controversial. An increase in intracellular free Ca2+ is required to trigger egg activation events, a process that includes cortical granule exocytosis, resumption and completion of meiosis and DNA replication, and culminates in the first mitotic cleavage. In this work, we investigated the effect of microinjection and incubation of different fractions of homologous sperm extract on the activation of Bufo arenarum oocytes matured in vitro. Two heat treatment-sensitive fractions obtained by chromatography were able to induce oocyte activation. The sperm fraction, which contained a 24 kDa protein, induced 90% activation when it was microinjected into the oocytes. Whilst the sperm fraction, which contained a 36 kDa protein, was able to induce about 70% activation only when it was applied on the oocyte surface.

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.

Activation of the progesterone-signaling pathway by methyl-β-cyclodextrin or steroid in Xenopus laevis oocytes involves release of 45-kDa Gα s

Treatment of Xenopus laevis oocytes with cholesterol-depleting methyl-β-cyclodextrin (MeβCD) stimulates phosphorylation of mitogen-activated protein kinase (MAPK) and oocyte maturation, as reported previously [Sadler, S.E., Jacobs, N.D., 2004. Stimulation of Xenopus laevis oocyte maturation by methyl-β-cyclodextrin. Biol. Reprod. 70, 1685–1692.]. Here we report that treatment of oocytes with MeβCD increased levels of immunodetectable 39-kDa mos protein. The protein synthesis inhibitor, cycloheximide, blocked the appearance of Mos, blocked MeβCD-stimulated phosphorylation of MAPK, and inhibited MeβCD-induced oocyte maturation. These observations suggest that MeβCD activates the progesterone-signaling pathway. Chemical inhibition of steroid synthesis and mechanical removal of follicle cells were used to verify that MeβCD acts at the level of the oocyte and does not require production of steroid by surrounding follicle cells. Cortical Gα s is contained in low-density membrane; and treatment of oocytes with progesterone or MeβCD reduced immunodetectable levels of Gα s protein in cortices and increased internal levels of 45-kDa Gα s in cortical-free extracts. Dose-dependent increases in internal Gα s after treatment of oocytes with progesterone correlated with the steroid-induced maturation response, and the increase in internal Gα s after hormone treatment was comparable to the decrease in cortical Gα s. These results are consistent with a model in which release of Gα s from the plasma membrane is involved in activation of the progesterone-signaling pathway that leads to amphibian oocyte maturation.

Possible Involvement of Phosphatidylinositol 3-Kinase, but Not Protein Kinase B or Glycogen Synthase Kinase 3β, in Progesterone-Induced Oocyte Maturation in the Japanese Brown Frog, Rana japonica

It is known that amphibian oocytes undergo maturation through the formation and activation of maturation-promoting factor (MPF) in response to stimulation by the maturation-inducing hormone progesterone; however, the signal transduction pathway that links the hormonal stimulation on the oocyte surface to the activation of MPF in the oocyte cytoplasm remains a mystery. The aim of this study was to investigate whether the signal transduction mediated by phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB), and glycogen synthase kinase 3beta (GSK3beta) is involved in progesterone-induced oocyte maturation in the Japanese brown frog, Rana japonica. Inhibitors of PI3K, wortmannin and LY294002, inhibited progesterone-stimulated germinal vesicle breakdown (GVBD) only when the oocytes were treated at the initial phase of maturation, suggesting that PI3K is involved in the progesterone-induced maturation of Rana oocytes. However, we also obtained results suggesting that PKB and GSK3beta are not involved in Rana oocyte maturation. A constitutively active PKB expressed in the oocytes failed to induce GVBD in the absence of progesterone despite its high level of kinase activity. A Myc-tagged PKB expressed in the oocytes (used to monitor endogenous PKB activity) was not activated in the process of progesterone-induced oocyte maturation. Overexpression of GSK3beta, which is reported to retard the progress of Xenopus oocyte maturation, had no effect on Rana oocyte maturation. On the basis of these results, we propose that PI3K is involved in the initiation of Rana oocyte maturation, but that neither PKB nor GSK3beta is a component of the PI3K signal transduction pathway.