Meiotic Maturation Of Xenopus Oocytes Research Articles

RSK promotes G2/M transition through activating phosphorylation of Cdc25A and Cdc25B

Activation of the mitogen-activated protein kinase (MAPK) cascade in mammalian cell lines positively regulates the G2/M transition. The molecular mechanism underlying this biological phenomenon remains poorly understood. Ribosomal S6 kinase (RSK) is a key downstream element of the MAPK cascade. Our previous studies established roles of RSK2 in Cdc25C activation during progesterone-induced meiotic maturation of Xenopus oocytes. In this study we demonstrate that both recombinant RSK and endogenous RSK in Xenopus egg extracts phosphorylate all three isoforms of human Cdc25 at a conserved motif near the catalytic domain. In human HEK293 and PC-3mm2 cell lines, RSK preferentially phosphorylates Cdc25A and Cdc25B in mitotic cells. Phosphorylation of the RSK sites in these Cdc25 isoforms increases their M-phase-inducing activities. Inhibition of RSK-mediated phosphorylation of Cdc25 inhibits G2/M transition. Moreover, RSK is likely to be more active in mitotic cells than in interphase cells, as evidenced by the phosphorylation status of T359/S363 in RSK. Together, these findings indicate that RSK promotes G2/M transition in mammalian cells through activating phosphorylation of Cdc25A and Cdc25B.

Porcine SPDYA2 (RINGO A2) Stimulates CDC2 Activity and Accelerates Meiotic Maturation of Porcine Oocytes1

RINGO, a protein with no homology to cyclin B, has been reported to be involved in activation of CDC2 and regulation of meiotic maturation in Xenopus oocytes. Although the presence of homologues of RINGO families, which are known as SPDY families, has been reported in mammals, their roles in meiotic maturation of mammalian oocytes have never been examined. In the present study, the effects of SPDY on meiotic maturation of porcine oocytes were examined. At first, Xenopus RINGO (xRINGO) mRNA was injected into immature porcine oocytes and found to significantly accelerate CDC2 activation and meiotic resumption. The CCNB (also known as cyclin B) synthesis was prematurely started at 12 h of culture, whereas it started at 18 h in normal oocytes. We next cloned RINGO A2 homologue in pig (pigSPDYA2) from total RNA of immature porcine oocytes by RT-PCR and obtained full-length cDNA that was more than 85% and 40% homologous with mammalian SPDYA2 and xRINGO, respectively. Acceleration effects similar to those by xRINGO were observed in CDC2 activation, meiotic resumption, and the start of CCNB synthesis in pigSPDYA2 mRNA-injected porcine oocytes. In clear contrast with the effects of xRINGO, which was accumulated abnormally in porcine oocytes and arrested them in the first meiotic metaphase (M1), pigSPDYA2 accelerated the meiotic progression, with about half of pigSPDYA2 mRNA-injected oocytes completing meiotic maturation within 30 h. These results suggest that pigSPDYA2 has important roles on meiotic maturation of porcine oocytes and that the rapid degradation of SPDY was necessary for the normal maturation of oocytes.

Xp38gamma/SAPK3 promotes meiotic G(2)/M transition in Xenopus oocytes and activates Cdc25C.

We have studied the role of p38 mitogen-activated protein kinases (MAPKs) in the meiotic maturation of Xenopus oocytes. Overexpression of a constitutively active mutant of the p38 activator MKK6 accelerates progesterone-induced maturation. Immunoprecipit ation experiments indicate that p38gamma/SAPK3 is the major p38 activated by MKK6 in the oocytes. We have cloned Xenopus p38gamma (Xp38gamma) and show that co-expression of active MKK6 with Xp38gamma induces oocyte maturation in the absence of progesterone. The maturation induced by Xp38gamma requires neither protein synthesis nor activation of the p42 MAPK-p90Rsk pathway, but it is blocked by cAMP-dependent protein kinase. A role for the endogenous Xp38gamma in progesterone-induced maturation is supported by the inhibitory effect of kinase-dead mutants of MKK6 and Xp38gamma. Furthermore, MKK6 can rescue the inhibition of oocyte maturation by anthrax lethal factor, a protease that inactivates MAPK kinases. We also show that Xp38gamma can activate the phosphatase XCdc25C, and we identified Ser205 of XCdc25C as a major phosphorylation site for Xp38gamma. Our results indicate that phosphorylation of XCdc25C by Xp38gamma/SAPK3 is important for the meiotic G(2)/M progression of Xenopus oocytes.

Histone H3 phosphorylation during Xenopus oocyte maturation: regulation by the MAP kinase/p90Rsk pathway and uncoupling from DNA condensation

Here we show that during the meiotic maturation of Xenopus oocytes, histone H3 becomes phosphorylated on serine-10 at about the time of maturation promoting factor activation and meiosis I entry. However, overexpression of cAMP-dependent protein kinase that blocks entry into M phase, also leads to massive serine-10 phosphorylation of histone H3 in intact Xenopus oocytes but does not cause chromosome condensation. We also show that the phosphorylation of histone H3 during oocyte maturation requires the activation of the mitogen-activated protein kinase/p90Rsk pathway. Our results indicate that in G2-arrested oocytes, which are about to enter M phase, histone H3 phosphorylation is not sufficient for chromosome condensation.

The activation of MAP kinase and p34cdc2/cyclin B during the meiotic maturation of Xenopus oocytes.

G2-arrested Xenopus oocytes are induced to enter M-phase of meiosis by progesterone stimulation. This process, known as meiotic maturation, requires the activation of p34cdc2/cyclin B complexes (pre-MPF) which is brought about by the prior translation of specific maternal mRNAs stored in the oocyte. One of these mRNAs encodes for the protein kinase Mos which has an essential role in oocyte maturation, most likely due to its ability to activate MAP kinase (MAPK). Here we review our current knowledge on the Mos/MAPK signalling pathway and a recently found connection between MAPK-activated p90rsk and the p34cdc2 inhibitory kinase Myt1. We also discuss a pathway that involves the protein kinase Plx1 and leads to the activation of the phosphatase Cdc25, as well as other regulators of p34cdc2/cyclin B activity which may have a role in oocyte maturation.

Dissociation of MAP kinase activation and MPF activation in hormone-stimulated maturation of Xenopus oocytes.

MAP kinase activation occurs during meiotic maturation of oocytes from all animals, but the requirement for MAP kinase activation in reinitiation of meiosis appears to vary between different classes. In particular, it has become accepted that MAP kinase activation is necessary for progesterone-stimulated meiotic maturation of Xenopus oocytes, while this is clearly not the case in other systems. In this paper, we demonstrate that MAP kinase activation in Xenopus oocytes is an early response to progesterone and can be temporally dissociated from MPF activation. We show that MAP kinase activation can be suppressed by treatment with geldanamycin or by overexpression of the MAP kinase phosphatase Pyst1. A transient and low-level early activation of MAP kinase increases the efficiency of cell cycle activation later on, when MAP kinase activity is no longer essential. Many oocytes can still undergo reinitiation of meiosis in the absence of active MAP kinase. Suppression of MAP kinase activation does not affect the formation or activation of Cdc2-cyclin B complexes, but reduces the level of active Cdc2 kinase. We discuss these findings in the context of a universal mechanism for meiotic maturation in oocytes throughout the animal kingdom.

Two distinct mechanisms control the accumulation of cyclin B1 and Mos in Xenopus oocytes in response to progesterone.

Progesterone-induced meiotic maturation of Xenopus oocytes requires the synthesis of new proteins, such as Mos and cyclin B. Synthesis of Mos is thought to be necessary and sufficient for meiotic maturation; however, it has recently been proposed that newly synthesized proteins binding to p34(cdc2) could be involved in a signaling pathway that triggers the activation of maturation-promoting factor. We focused our attention on cyclin B proteins because they are synthesized in response to progesterone, they bind to p34(cdc2), and their microinjection into resting oocytes induces meiotic maturation. We investigated cyclin B accumulation in response to progesterone in the absence of maturation-promoting factor-induced feedback. We report here that the cdk inhibitor p21(cip1), when microinjected into immature Xenopus oocytes, blocks germinal vesicle breakdown induced by progesterone, by maturation-promoting factor transfer, or by injection of okadaic acid. After microinjection of p21(cip1), progesterone fails to induce the activation of MAPK or p34(cdc2), and Mos does not accumulate. In contrast, the level of cyclin B1 increases normally in a manner dependent on down-regulation of cAMP-dependent protein kinase but independent of cap-ribose methylation of mRNA.

The deadenylating nuclease (DAN) is involved in poly(A) tail removal during the meiotic maturation of Xenopus oocytes.

Exonucleolytic degradation of the poly(A) tail is often the first step in the decay of eukaryotic mRNAs and is also used to silence certain maternal mRNAs translationally during oocyte maturation and early embryonic development. We previously described the purification of a poly(A)-specific 3'-exoribonuclease (deadenylating nuclease, DAN) from mammalian tissue. Here, the isolation and functional characterization of cDNA clones encoding human DAN is reported. Recombinant DAN overexpressed in Escherichia coli has properties similar to those of the authentic protein. The amino acid sequence of DAN shows homology to the RNase D family of 3'-exonucleases. DAN appears to be localized in both the nucleus and the cytoplasm. It is not stably associated with polysomes or ribosomal subunits. Xenopus oocytes contain nuclear and cytoplasmic DAN isoforms, both of which are closely related to the human DAN. Anti-DAN antibody microinjected into oocytes inhibits default deadenylation during progesterone-induced maturation. Ectopic expression of human DAN in enucleated oocytes rescues maturation-specific deadenylation, indicating that amphibian and mammalian DANs are functionally equivalent.

Inactivation of protein kinase A is not required for c-mos translation during meiotic maturation of Xenopus oocytes.

It has been shown previously that protein kinase A (PKA) maintains Xenopus oocytes arrested at G2, at least in part by preventing c-mos translation, but how PKA controls c-mos translation is not known. Using microinjection of recombinant c-mos, which still activates MAP kinase in the presence of active PKA, we have found that PKA does not exert any effect on translation of endogenous c-mos if MAP kinase is first activated. Even though they accumulate c-mos and contain MAP kinase activity as high as control oocytes, oocytes do not exit G2 in the presence of active PKA. These results are discussed in connection with recent findings on regulation of c-raf activity.

Meiotic maturation in Xenopus requires polyadenylation of multiple mRNAs.

Cytoplasmic polyadenylation of specific mRNAs commonly is correlated with their translational activation during development. Here, we focus on links between cytoplasmic polyadenylation, translational activation and the control of meiotic maturation in Xenopus oocytes. We manipulate endogenous c-mos mRNA, which encodes a protein kinase that regulates meiotic maturation. We determined that translational activation of endogenous c-mos mRNA requires a long poly(A) tail per se, rather than the process of polyadenylation. For this, we injected 'prosthetic' poly(A)_synthetic poly(A) tails designed to attach by base pairing to endogenous c-mos mRNA that has had its own polyadenylation signals removed. This prosthetic poly(A) tail activates c-mos translation and restores meiotic maturation in response to progesterone. Thus the role of polyadenylation in activating c-mos mRNA differs from its role in activating certain other mRNAs, for which the act of polyadenylation is required. In the absence of progesterone, prosthetic poly(A) does not stimulate c-mos expression, implying that progesterone acts at additional steps to elevate c-Mos protein. By using a general inhibitor of polyadenylation together with prosthetic poly(A), we demonstrate that these additional steps include polyadenylation of at least one other mRNA, in addition to that of c-mos mRNA. These other mRNAs, encoding regulators of meiotic maturation, act upstream of c-Mos in the meiotic maturation pathway.

Inhibition of poly(A) polymerase requires p34cdc2/cyclin B phosphorylation of multiple consensus and non-consensus sites.

We showed previously that p34(cdc2)/cyclin B (MPF) hyperphosphorylates poly(A) polymerase (PAP) during M-phase of the cell cycle, causing repression of its enzymatic activity. Mutation of three cyclin-dependent kinase (cdk) consensus sites in the PAP C-terminal regulatory domain prevented complete phosphorylation and MPF-mediated repression. Here we show that PAP also contains four nearby non-consensus cdk sites that are phosphorylated by MPF. Remarkably, full phosphorylation of all these cdk sites was required for repression of PAP activity, and partial phosphorylation had no detectable effect. The consensus sites were phosphorylated in vitro at a 10-fold lower concentration of MPF than the non-consensus sites. Consistent with this, during meiotic maturation of Xenopus oocytes, consensus sites were phosphorylated prior to the non-consensus sites at metaphase of meiosis I, and remained so throughout maturation, while the non-consensus sites did not become fully phosphorylated until after 12 h of metaphase II arrest. We propose that PAP's multiple cdk sites, and their differential sensitivity to MPF, provide a mechanism to link repression specifically to late M-phase. We discuss the possibility that this reflects a general means to control the timing of cdk-dependent regulatory events during the cell cycle.

V‐erbA oncogene initiates ultrastructural changes characteristic of early and intermediate events of meiotic maturation in Xenopus oocytes

The growth-promoting properties of the retroviral v-erbA oncogene, a highly mutated version of the chicken thyroid hormone receptor (TR) α, have so far exclusively been linked to dominant repression of the antimitogenic roles of TR and retinoic acid receptors. Here we show that when expressed in Xenopus oocytes v-ErbA induced ultrastructural changes characteristic of early and intermediate events of meiotic maturation by activating gene transcription. v-ErbA–induced maturation events occurred without activation of the cAMP/maturation-promoting factor signal pathway and were arrested prior to meiotic spindle formation. The effects of v-ErbA were not mimicked by a dominant negative in vitro–generated mutant of human TR, suggesting that v-ErbA can contribute to cell cycle reentry by interference with regulatory pathways distinct from those involving TR. Interestingly, a portion of v-ErbA expressed in oocytes was present at the cytoplasmic fibrils of the nuclear pore complexes, suggesting that in addition to its intranuclear function v-ErbA may modulate nucleocytoplasmic transport. J. Cell. Biochem. 67:184–200, 1997. © 1997 Wiley-Liss, Inc.

V-erbA oncogene initiates ultrastructural changes characteristic of early and intermediate events of meiotic maturation inXenopus oocytes

The growth-promoting properties of the retroviral v-erbA oncogene, a highly mutated version of the chicken thyroid hormone receptor (TR) alpha, have so far exclusively been linked to dominant repression of the antimitogenic roles of TR and retinoic acid receptors. Here we show that when expressed in Xenopus oocytes v-ErbA induced ultrastructural changes characteristic of early and intermediate events of meiotic maturation by activating gene transcription. v-ErbA-induced maturation events occurred without activation of the cAMP/maturation-promoting factor signal pathway and were arrested prior to meiotic spindle formation. The effects of v-ErbA were not mimicked by a dominant negative in vitro-generated mutant of human TR, suggesting that v-ErbA can contribute to cell cycle reentry by interference with regulatory pathways distinct from those involving TR. Interestingly, a portion of v-ErbA expressed in oocytes was present at the cytoplasmic fibrils of the nuclear pore complexes, suggesting that in addition to its intranuclear function v-ErbA may modulate nucleocytoplasmic transport.

Remodeling of regulatory nucleoprotein complexes on the Xenopus hsp70 promoter during meiotic maturation of the Xenopus oocyte.

Transcriptional repression occurs during meiotic maturation of Xenopus oocytes. Injection of a DNA template containing an hsp70 promoter into Xenopus oocytes, followed by progesterone-induced maturation has been used to demonstrate a dynamic competition between the assembly of transcription factor-containing nucleoprotein complexes and repressive nucleosomal arrays during the maturation process. In particular, it is shown that increased levels of injected heat shock protein, the transcriptional activator Gal4-VP16 or the DNA template itself all lead to reduced repression of transcription on maturation. Conversely, injection of additional histone increases repression. Repression of transcription is shown to be accompanied by the formation of a more regular array of nucleosomes and by an increase in the efficiency of nucleosome assembly on the injected plasmid. Meiotic maturation is therefore accompanied by replacement of transcription factor complexes by a repressive chromatin environment.

Meiotic cell cycle in Xenopus oocytes is independent of cdk2 kinase.

In vertebrates, M phase-promoting factor (MPF), a universal G2/M regulator in eukaryotic cells, drives meiotic maturation of oocytes, while cytostatic factor (CSF) arrests mature oocytes at metaphase II until fertilization. Cdk2 kinase, a G1/S regulator in higher eukaryotic cells, is activated during meiotic maturation of Xenopus oocytes and, like Mos (an essential component of CSF), is proposed to be involved in metaphase II arrest in mature oocytes. In addition, cdk2 kinase has been shown recently to be essential for MPF activation in Xenopus embryonic mitosis. Here we report injection of Xenopus oocytes with the cdk2 kinase inhibitor p21Cip in order to (re)evaluate the role of cdk2 kinase in oocyte meiosis. Immature oocytes injected with p21Cip can enter both meiosis I and meiosis II normally, as evidenced by the typical fluctuations in MPF activity. Moreover, mature oocytes injected with p21Cip are retained normally in metaphase II for a prolonged period, whereas those injected with neutralizing anti-Mos antibody are released readily from metaphase II arrest. These results argue strongly against a role for cdk2 kinase in MPF activation and its proposed role in metaphase II arrest, in Xenopus oocyte meiosis. We discuss the possibility that cdk2 kinase stored in oocytes may function, as a maternal protein, solely for early embryonic cell cycles.

Palmitoylation of Ha-Ras Facilitates Membrane Binding, Activation of Downstream Effectors, and Meiotic Maturation in Xenopus Oocytes

Ras proteins serve as critical relays in signal transduction pathways that control growth and differentiation and must undergo posttranslational modifications before they become functional. While it is established that farnesylation is necessary for membrane binding and cellular functions of all Ras proteins, the significance of palmitoylation is unclear. We have studied the contribution of Ha-Ras palmitoylation for biological activity in Xenopus oocytes. In contrast to wild-type Ha-Ras, which binds to membranes and induces meiosis when microinjected into oocytes, a nonpalmitoylated but farnesylated and methylated mutant mislocalizes to the cytosol and fails to promote maturation. This lack of responsiveness correlates with the inability of the mutant to induce phosphorylation and activation of mitogen-activated protein kinase and maturation promoting factor, which are both strongly activated by wild-type Ha-Ras. Costimulation of oocytes with insulin increases their responsiveness to Ras and partially rescues the biological activity of the palmitoylation-resistant mutant. However, 25-50 times higher doses of mutant were required to elicit responses equivalent to wild-type Ha-Ras. These results suggest that palmitoylation and membrane association of Ha-Ras is necessary for efficient activation of the mitogen-activated protein kinase cascade in vivo and are consistent with a biochemical function for Ras as a membrane targeting signal for downstream effectors in this pathway.

Newly synthesized protein(s) must associate with p34cdc2 to activate MAP kinase and MPF during progesterone-induced maturation of Xenopus oocytes.

The meiotic maturation of Xenopus oocytes triggered by progesterone requires new protein synthesis to activate both maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAP kinase). Injection of mRNA encoding mutant p34cdc2 (K33R) that can bind cyclins but lacks protein kinase activity strongly inhibited progesterone-induced activation of both MPF and MAP kinase in Xenopus oocytes. Similar results were obtained by injection of GST-p34cdc2 K33R protein or by injection of a monoclonal antibody (A17) against p34cdc2 that blocks its activation by cyclins. Both the dominant-negative p34cdc2 and monoclonal antibody A17 blocked the accumulation of p39mos and activation of MAP kinase in response to progesterone, as well as blocking the appearance of MPF, although they did not inhibit the translation of p39mos mRNA. These results suggest that: (i) activation of free p34cdc2 by newly made proteins, probably cyclin(s), is normally required for the activation of both MPF and MAP kinase by progesterone in Xenopus oocytes; (ii) the activation of translation of cyclin mRNA normally precedes, and does not require either MPF or MAP kinase activity; and (iii) de novo synthesis and accumulation of p39mos is probably both necessary and sufficient for the activation of MAP kinase in response to progesterone.

14-3-3 Is Not Essential for Raf-1 Function: Identification of Raf-1 Proteins That Are Biologically Activated in a 14-3-3- and Ras-Independent Manner

Recent reports have demonstrated the in vivo association of Raf-1 with members of the 14-3-3 protein family. To address the significance of the Raf-1-14-3-3 interaction, we investigated the enzymatic activity and biological function of Raf-1 in the presence and absence of associated 14-3-3. The interaction between these two molecules was disrupted in vivo and in vitro with a combination of molecular and biochemical techniques. Biochemical studies demonstrated that the enzymatic activities of Raf-1 were equivalent in the presence and absence of 14-3-3. Furthermore, mixing of purified Raf-1 and 14-3-3 in vitro was not sufficient to activate Raf-1. With a molecular approach, Cys-165 and Cys-168 as well as Ser-259 were identified as residues of Raf-1 required for the interaction with 14-3-3. Cys-165 and Cys-168 are located within the conserved cysteine-rich region of the CR1 domain, and Ser-259 is a conserved site of serine phosphorylation found within the CR2 domain. Mutation of either Cys-165 and Cys-168 or Ser-259 prevented the stable interaction of Raf-1 with 14-3-3 in vivo. Consistent with the model in which a site of serine phosphorylation is involved in the Raf-1-14-3-3 interaction, dephosphorylated Raf-1 was unable to associate with 14-3-3 in vitro. Phosphorylation may represent a general mechanism mediating 14-3-3 binding, because dephosphorylation of the Bcr kinase (known to interact with 14-3-3) also eliminated its association with 14-3-3. Finally, mutant Raf-1 proteins unable to stably interact with 14-3-3 exhibited enhanced enzymatic activity in human 293 cells and Xenopus oocytes and were biologically activated, as demonstrated by their ability to induced meiotic maturation of Xenopus oocytes. However, in contrast to wild-type Raf-1, activation of these mutants was independent of Ras. Our results therefore indicate that interaction with 14-3-3 is not essential for Raf-1 function.

Human Dual Specificity Phosphatase VHR Activates Maturation Promotion Factor and Triggers Meiotic Maturation in Xenopus Oocytes

Bacterially expressed, dual specificity phosphatase VHR protein induced germinal vesicle breakdown (GVBD) when microinjected into Xenopus oocytes, albeit with slower kinetics than that observed in progesterone- or insulin-induced maturation. A mutant VHR protein missing an essential cysteine residue for its in vitro phosphatase activity completely lacked activity in injected oocytes. VHR injection done in conjunction with progesterone or insulin treatment resulted in highly synergized GVBD responses showing much faster kinetics than that produced by VHR or either hormone alone. The delayed kinetics of VHR-induced GVBD and the synergistic responses obtained in the presence of hormones suggested that this protein may be promoting G2/M transition by weakly mimicking the action of cdc25, the dual specificity phosphatase that physiologically activates the maturation promotion factor. Various experimental observations are consistent with such a role for the injected VHR in oocytes: 1) as opposed to hormone-treated oocytes, histone H1 kinase activation is not preceded by MAPK activation in the process of GVBD in VHR-injected oocytes; 2) incubation of purified VHR with highly concentrated cell-free extracts of untreated oocytes resulted in activation of histone H1 kinase activity in the lysates; 3) coinjection of VHR with activated Ras proteins resulted in synergized responses, faster than those produced by either protein alone; 4) coinjection of VHR with the purified amino-terminal SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase (which blocks insulin-induced GVBD) does not affect VHR-induced maturation. The biological actions of VHR in oocytes clearly distinguish it from other dual specificity phosphatases, which have shown inhibitory effects when tested in oocytes. We speculate that VHR may represent a dual specificity phosphatase responsible for activation of cdk-cyclin complex(es) at a still undetermined stage of the cell cycle.

Intracellular Expression of Anti-p21ras Single Chain Fv Fragments Inhibits Meiotic Maturation of Xenopus Oocytes

The recombinant variable regions of the monoclonal antibody Y13-259, directed against the p21ras protein, have been engineered for expression as intracellular single chain Fv fragments. The activity of the plasmid was confirmed by in vitro and in vivo translation of mRNA showing that the intracellularly expressed single chain fragments are stably and efficiently expressed as cytosolic proteins. The expression of the anti-p21ras single chain antibodies in the cytoplasm of Xenopus laevis oocytes leads to the inhibition of the insulin-induced meiotic maturation. This finding represents the first successful application of the strategy of intracellular antibodies to block a complex biological process in the cytosol of vertebrate cells.