XGef influences XRINGO/CDK1 signaling and CPEB activation during Xenopus oocyte maturation

CPEB-mediated polyadenylation-induced translation of several developmentally important mRNAs drives Xenopus laevis oocyte meiotic progression and production of fertilizable eggs. To date, the signal transduction events that induce CPEB activation remain somewhat unclear, however, XGef has been shown to be involved in this process. P42 MAPK (ERK2) activity and XRINGO accumulation are also required for the activating phosphorylation of CPEB. We show here that XGef activity influences the early meiotic function of XRINGO/CDK1, a novel component of the progesterone signaling pathway. An XGef-specific antibody depresses XRINGO-induced GVBD, whereas XGef overexpression accelerates this process. XGef and CPEB interact with XRINGO in immature and maturing oocyte extracts and XGef, XRINGO and ERK2 interact directly in vitro. These data suggest that an XGef/XRINGO/ERK2/CPEB complex forms in ovo during early meiotic resumption. Notably, specific inhibition of XRINGO/CDK1 activity in CPEB phosphorylation-competent extracts completely blocks phosphorylation of CPEB, which suggests that XRINGO/CDK1 directly phosphorylates CPEB. Finally, overexpression of XGef (65-360), which cannot bind CPEB or ERK2, but is capable of XRINGO association, blocks XRINGO-induced meiotic progression potentially through titration of endogenous XRINGO. Combined, our results suggest that XGef is involved in XRINGO/CDK1 mediated activation of CPEB and that an XGef/XRINGO/ERK2/CPEB complex forms in ovo to facilitate this process.     

XGef is a CPEB-interacting protein involved in Xenopus oocyte maturation

XGef was isolated in a screen for proteins interacting with CPEB, a regulator of mRNA translation in early Xenopus development. XGef is a Rho-family guanine nucleotide exchange factor and activates Cdc42 in mammalian cells. Endogenous XGef (58 kDa) interacts with recombinant CPEB, and recombinant XGef interacts with endogenous CPEB in Xenopus oocytes. Injection of XGef antibodies into stage VI Xenopus oocytes blocks progesterone-induced oocyte maturation and prevents the polyadenylation and translation of c-mos mRNA; injection of XGef rescues these events. Overexpression of XGef in oocytes accelerates progesterone-induced oocyte maturation and the polyadenylation and translation of c-mos mRNA. Overexpression of a nucleotide exchange deficient version of XGef, which retains the ability to interact with CPEB, no longer accelerates oocyte maturation or Mos synthesis, suggesting that XGef exchange factor activity is required for the influence of overexpressed XGef on oocyte maturation. XGef overexpression continues to accelerate c-mos polyadenylation in the absence of Mos protein, but does not stimulate MAPK phosphorylation, MPF activation, or oocyte maturation, indicating that XGef may function through the Mos pathway to influence oocyte maturation. These results suggest that XGef may be an early acting component of the progesterone-induced oocyte maturation pathway.     

CPEB degradation during Xenopus oocyte maturation requires a PEST domain and the 26S proteasome

Cytoplasmic poly(A) elongation is widely utilized during the early development of many organisms as a mechanism for translational activation. Targeting of mRNAs for this mechanism requires the presence of a U-rich element, the cytoplasmic polyadenylation element (CPE), and its binding protein, CPEB. Blocking cytoplasmic polyadenylation by interfering with the CPE or CPEB prevents the translational activation of mRNAs that are crucial for oocyte maturation. The CPE sequence and CPEB are also important for translational repression of mRNAs stored in the Xenopus oocyte during oogenesis. To understand the contribution of protein metabolism to these two roles for CPEB, we have examined the mechanisms influencing the expression of CPEB during oogenesis and oocyte maturation. Through a comparison of CPEB mRNA levels, protein synthesis, and accumulation, we find that CPEB is synthesized during oogenesis and stockpiled in the oocyte. Minimal synthesis of CPEB, <3.6%, occurs during oocyte maturation. In late oocyte maturation, 75% of CPEB is degraded coincident with germinal vesicle breakdown. Using proteasome and ubiquitination inhibitors, we demonstrate that CPEB degradation occurs via the proteasome pathway, most likely through ubiquitin-conjugated intermediates. In addition, we demonstrate that degradation requires a 14 amino acid PEST domain.     

Aurora-A kinase Ser349 phosphorylation is required during Xenopus laevis oocyte maturation

Xenopus laevis Aurora-A is phosphorylated in vivo onto three amino acids: Ser53, Thr295 and Ser349. The activation of the kinase depends on its autophosphorylation on Thr295 within the T-loop. The phosphorylation of Ser53 by still unknown kinase(s) prevents its degradation. The present work focused on the regulation of Aurora-A function via Ser349 phosphorylation. Mutagenesis of Ser349 to alanine (S349A) had few impact in vitro on the capability of the kinase to autophosphorylate as well as on its activity. These data in addition to in gel kinase assays and site-specific proteolytic digestion experiments prove that Ser349 is clearly neither a primary autophosphorylation site, nor an autophosphorylation site depending on the priming phosphorylation of Thr295. Using specific antibodies, we also show that the phosphorylation of Aurora-A Ser349 is a physiological event during Xenopus oocyte maturation triggered by progesterone. A peak of phosphorylation paralleled the decrease of Aurora activity observed between meiosis I and II. In response to progesterone, X. laevis stage VI oocytes microinjected with the Aurora-A S349A mutant proceeded normally to germinal vesicle breakdown (GVBD), but degenerated rapidly soon after. Since phosphorylation of Ser349 is responsible for a decrease in kinase activity, our results suggest that a down-regulation of Aurora-A activity involving Ser349 phosphorylation is required in the process of maturation.     

A predominant basic alpha-tubulin isoform present in prophase Xenopus oocyte decreases during meiotic maturation.

Xenopus oocytes are blocked in prophase of the first meiotic division. During the G2/M transition drastic changes occur both in the cytoskeletal organization and in the capacity of tubulin to polymerize. Posttranslational modification of tubulin isoforms might be one of the factors that control the dynamic properties of microtubules. We have therefore analysed, by two-dimensional polyacrylamide gel electrophoresis, the isotubulins purified from Xenopus oocytes, and we show that tubulin is resolved into at least four alpha-isoforms and four beta-isoforms. We have identified a basic alpha (alpha b)-tubulin isoform which is specific to prophase arrested oocyte and that progressively disappears during meiotic maturation; its decrease is initiated when the nuclear envelope breaks down and is controlled by the nucleus. Using 35S methionine labelled oocytes we demonstrate that the disappearance of the alpha b isotubulin results from both an arrest of its biosynthesis after maturation, and from posttranslational modification which induces a shift of this alpha-isoform to a more acidic pI. Moreover, in vitro experiments using 35S prelabelled tubulin purified from prophase oocytes show that metaphase extracts containing MPF activity are able to induce the acidification of the alpha b-isoform, suggesting that the observed posttranslational modification might be regulated by p34cdc2. However, the nature of this modification remains to be elucidated.     

Calcium signaling differentiation during Xenopus oocyte maturation

Ca(2+) is the universal signal for egg activation at fertilization in all sexually reproducing species. The Ca(2+) signal at fertilization is necessary for egg activation and exhibits specialized spatial and temporal dynamics. Eggs acquire the ability to produce the fertilization-specific Ca(2+) signal during oocyte maturation. However, the mechanisms regulating Ca(2+) signaling differentiation during oocyte maturation remain largely unknown. At fertilization, Xenopus eggs produce a cytoplasmic Ca(2+) (Ca(2+)(cyt)) rise that lasts for several minutes, and is required for egg activation. Here, we show that during oocyte maturation Ca(2+) transport effectors are tightly modulated. The plasma membrane Ca(2+) ATPase (PMCA) is completely internalized during maturation, and is therefore unable to extrude Ca(2+) out of the cell. Furthermore, IP(3)-dependent Ca(2+) release is required for the sustained Ca(2+)(cyt) rise in eggs, showing that Ca(2+) that is pumped into the ER leaks back out through IP(3) receptors. This apparent futile cycle allows eggs to maintain elevated cytoplasmic Ca(2+) despite the limited available Ca(2+) in intracellular stores. Therefore, Ca(2+) signaling differentiates in a highly orchestrated fashion during Xenopus oocyte maturation endowing the egg with the capacity to produce a sustained Ca(2+)(cyt) transient at fertilization, which defines the egg's competence to activate and initiate embryonic development.     

In vivo activation of a microtubule-associated protein kinase during meiotic maturation of the Xenopus oocyte

We have characterized a serine/threonine protein kinase from Xenopus metaphase-II-blocked oocytes, which phosphorylates in vitro the microtubule-associated protein 2 (MAP2). The MAP2 kinase activity, undetectable in prophase oocytes, is activated during the progesterone-induced meiotic maturation (G2-M transition of the cell cycle). p-Nitrophenyl phosphate, a phosphatase inhibitor, is required to prevent spontaneous deactivation of the MAP2 kinase in crude preparations; conversely, the partially purified enzyme can be in vitro deactivated by the low-Mr polycation-stimulated (PCSL) phosphatase (also termed protein phosphatase 2A2), working as a phosphoserine/phosphothreonine-specific phosphatase and not as a phosphotyrosyl phosphatase indicating that phosphorylation of serine/threonine is necessary for its activity. S6 kinase, a protein kinase activated during oocyte maturation which phosphorylates in vitro ribosomal protein S6 and lamin C, can be deactivated in vitro by PCSL phosphatase. S6 kinase from prophase oocytes can also be activated in vitro in fractions known to contain all the factors necessary to convert pre-M-phase-promoting factor (pre-MPF) to MPF. Active MAP2 kinase can activate in vitro the inactive S6 kinase present in prophase oocytes or reactivate S6 kinase previously inactivated in vitro by PCSL phosphatase. These data are consistent with the hypothesis that the MAP2 kinase is a link of the meiosis signalling pathway and is activated by a serine/threonine kinase. This will lead to the regulation of further steps in the cell cycle, such as microtubular reorganisation and S6 kinase activation.     

Protein phosphorylation during meiotic maturation of Xenopus oocytes: cdc2 protein kinase targets

M-Phase specific protein kinase or cdc2 protein kinase is a component of MPF (M-Phase promoting factor). During meiotic maturation of Xenopus oocytes, cdc2 protein kinase is activated in correlation with MPF activity. A protein phosphorylation cascade takes place involving several protein kinases, among which casein kinase II, and different changes associated with meiosis occur such as germinal vesicle breakdown, chromosome condensation, cytoskeletal reorganization and increase in protein synthesis. Our results provide a biochemical link between cdc2 protein kinase and protein synthesis since they show that the kinase phosphorylates in vitro a p47 protein identified as elongation factor EF1 (gamma subunit) and that the in vitro site of p47 corresponds to the site phosphorylated in vivo. Immunofluorescence showed that the elongation factor (EF1-beta gamma) is localized in the oocyte cortex. Furthermore, they show that cdc2 kinase phosphorylates and activates casein kinase II in vitro, strongly supporting the view that casein kinase II is involved in the phosphorylation cascade originated by cdc2 kinase.     

Regulation of Greatwall kinase during Xenopus oocyte maturation

Greatwall kinase has been identified as a key element in M phase initiation and maintenance in Drosophila, Xenopus oocytes/eggs, and mammalian cells. In M phase, Greatwall phosphorylates endosulfine and related proteins that bind to and inhibit protein phosphatase 2A/B55, the principal phosphatase for Cdk-phosphorylated substrates. We show that Greatwall binds active PP2A/B55 in G2 phase oocytes but dissociates from it when progesterone-treated oocytes reach M phase. This dissociation does not require Greatwall kinase activity or phosphorylation at T748 in the presumptive T loop of the kinase. A mutant K71M Greatwall, also known as Scant in Drosophila, induces M phase in the absence of progesterone when expressed in oocytes, despite its reduced stability and elevated degradation by the proteasome. M phase induction by Scant Greatwall requires protein synthesis but is not associated with altered binding or release of PP2A/B55 as compared to wild-type Greatwall. However, in vitro studies with Greatwall proteins purified from interphase cells indicate that Scant, but not wild-type Greatwall, has low but detectable activity against endosulfine. These results demonstrate progesterone-dependent regulation of the PP2A/B55-Greatwall interaction during oocyte maturation and suggest that the cognate Scant Greatwall mutation has sufficient constitutive kinase activity to promote M phase in Xenopus oocytes.     

Modulation of O-GlcNAc glycosylation during Xenopus oocyte maturation

O-linked N-acetylglucosamine (O-GlcNAc) glycosylation is a post-translational modification, which is believed antagonises phosphorylation. We have studied the O-GlcNAc level during Xenopus oocyte meiotic resumption, taking advantage of the high synchrony of this model which is dependent upon a burst of phosphorylation. Stimulation of immature stage VI oocytes using progesterone was followed by a 4.51 +/- 0.32 fold increase in the GlcNAc content, concomitantly to an increase in phosphorylation, notably on two cytoplasmic proteins of 66 and 97 kDa. The increase of O-GlcNAc for the 97 kDa protein, which we identified as beta-catenin was partly related to its accumulation during maturation, as was demonstrated by the use of the protein synthesis inhibitor--cycloheximide. Microinjection of free GlcNAc, which inhibits O-glycosylated proteins-lectins interactions, delayed the progesterone-induced maturation without affecting the O-GlcNAc content. Our results suggest that O-GlcNAc glycosylation could regulate protein-protein interactions required for the cell cycle kinetic.     

In vivo phosphorylation and activation of ribosomal protein S6 kinases during Xenopus oocyte maturation

Previous studies have shown that increased ribosomal protein S6 kinase activity in unfertilized Xenopus eggs can be resolved by DEAE-Sephacel chromatography into two peaks, designated S6 kinase I and S6 kinase II. We show here that antibody against bacterially expressed S6 kinase II cross-reacts with S6 kinase I. Both S6 kinases undergo marked phosphorylation when they are activated during oocyte maturation, and both become deactivated and dephosphorylated upon activation of eggs. Immunoblotting of extracts of oocytes reveals that all S6 kinase molecules undergo a decrease and increase in electrophoretic mobility upon activation and deactivation, respectively. The increase in electrophoretic mobility can be produced in vitro by incubation of activated S6 kinase with purified phosphatases. Phosphoamino acid analysis of S6 kinase II labeled in vivo during maturation reveals both phosphoserine and phosphothreonine, and phosphopeptide maps suggest that several kinases may phosphorylate and activate S6 kinase II in vivo. These results demonstrate that, during oocyte maturation and early development, S6 kinase activation and deactivation are regulated by phosphorylation and dephosphorylation, suggesting a probable mechanism for S6 kinase regulation in other mitogenically stimulated cells.     

Localization and function of mSpindly during mouse oocyte meiotic maturation

Spindly was first identified in Drosophila; its homologues are termed SPDL-1 in Caenorhabditis elegans and Hs Spindly/hSpindly in humans. In all species, Spindly and its homologues function by recruiting dynein to kinetochores and silencing SAC in mitosis of somatic cells. Depletion of Spindly causes an extensive metaphase arrest during somatic mitoses in Drosophila, C. elegans and humans. In Drosophila, Spindly is required for shedding of Rod and Mad2 from the kinetochores in metaphase; in C. elegans, SPDL-1 presides over the recruitment of dynein and MDF-1 to the kinetochores; in humans, Hs Spindly is required for recruiting both dynein and dynactin to kinetochores but it is dispensable for removal of checkpoint proteins from kinetochores. The present study was designed to investigate the localization and function of the Spindly homologue (mSpindly) during mouse oocyte meiotic maturation by immunofluorescent analysis, and by overexpression and knockdown of mSpindly. We found that mSpindly was typically localized to kinetochores when chromatin condensed into chromosomes after GVBD. In metaphase of both first meiosis and second meiosis, mSpindly was localized not only to kinetochores but also to the spindle poles. Overexpression of mSpindly did not affect meiotic progression, but its depletion resulted in an arrest of the pro-MI/MI stage, failure of anaphase entry and subsequent polar body emission, and in abnormal spindle morphology and misaligned chromosomes. Our data suggest that mSpindly participates in SAC silencing and in spindle formation as a recruiter and/or a transporter of kinetochore proteins in mouse oocytes, but that it needs to cooperate with other factors to fulfill its function.     

Requirement for phosphorylation of cyclin B1 for Xenopus oocyte maturation.

Maturation-promoting factor, consisting of cdc2 protein kinase and a regulatory B-type cyclin, is a universal regulator of meiosis and mitosis in eukaryotes. In Xenopus, there are two subtypes of B-type cyclins, designated B1 and B2, both of which are phosphorylated. In this study, we have investigated the biological significance of this phosphorylation for Xenopus cyclin B1 during meiotic maturation. We have used a combination of site-directed mutagenesis and phosphopeptide-mapping to identify serine residues 2, 94, 96, 101, and 113 as presumptive phosphorylation sites, and together these sites account for all cyclin B1 phosphorylation in oocytes before germinal vesicle breakdown (GVBD). Single Ser-->Ala mutants as well as multiple site mutants have been constructed and characterized. Phosphorylation of cyclin B1 appears to be required for Xenopus oocyte maturation, based on the significantly diminished ability of the quintuple Ala mutant to induce oocyte maturation. Furthermore, partial phosphorylation of these five sites is sufficient to meet this requirement. Phosphorylation of cyclin B1 is not required for cdc2 kinase activity, for binding to cdc2 protein, for stability of cyclin B1 before GVBD, or for destruction of cyclin B1 after GVBD or after egg activation. A quintuple Glu mutant was also constructed, with serine residues 2, 94, 96, 101, and 113 mutated to Glu. In contrast to the quintuple Ala mutant, the quintuple Glu mutant was able to induce oocyte maturation efficiently, and with more rapid kinetics than wild-type cyclin B1. These data confirm that phosphorylation, as mimicked by Ser-->Glu mutations, confers enhanced biological activity to cyclin B1. Possible roles of cyclin B1 phosphorylation are discussed that might account for the increased biological activity of the quintuple Glu mutant.     

Regulation of Src kinase activity during Xenopus oocyte maturation

Expression of constitutively active Src protein tyrosine kinase in Xenopus oocytes has been shown to accelerate oocyte maturation suggesting that Src may be involved in meiotic progression. However, meiotic regulation of endogenous Src kinase in oocytes has not been investigated in detail. To address this problem, we measured the activity, expression level, and phosphorylation state of the endogenous Xenopus Src (xSrc) and overexpressed xSrc mutants in the process of progesterone-induced oocyte maturation. We found that the enzyme is first transiently activated in the plasma membrane-containing fraction of oocytes within 3 min of progesterone administration. This event represents one of the earliest responses of oocytes to the hormone and should be related to triggering some early signaling pathways of maturation. Thereafter, xSrc activity increases again at the time of germinal vesicle breakdown (GVBD) and remains elevated till the completion of maturation. This elevation of xSrc activity is associated with a 2-fold increase of xSrc protein content in the absence of change in its specific activity and xSrc mRNA content. No significant changes in the phosphorylation state of C-terminal regulatory phosphotyrosine can be registered either in endogenous xSrc or in overexpressed kinase-negative and wild-type xSrc proteins during maturation. Altogether, these results indicate that upregulation of xSrc in the meiotic metaphase occurs at the translation level. We also demonstrate here that the expression of constitutively active xSrc in Xenopus oocytes is accompanied by the activation of mitogen-activated protein kinase (MAPK). Our data suggest that the Src kinase acts through the MAPK pathway to accelerate oocyte maturation.     

Nitric oxide inhibits oocyte meiotic maturation

Recently, we have found that the nitrate/nitrite concentrations in preovulatory follicles significantly decrease after hCG injection and that inducible nitric oxide synthase (iNOS) plays a main role in the decrease of the intrafollicular nitric oxide (NO) concentration. The purpose of the present study was to investigate the role of NO on oocyte meiotic maturation and to consider the physiological means of the decrease in intrafollicular NO concentration. Immature rats received 15 IU of eCG, and ovaries were removed under ether anesthesia 48 h later. Each ovary was bluntly divided into five or six pieces containing from four to seven preovulatory follicles under the microscope and then incubated with hCG, aminoguanidine (AG; an iNOS inhibitor), or S-nitroso-L-acetyl penicillamine (SNAP; an NO donor) for 5 h. After incubation, preovulatory follicles were punctured, and germinal vesicle breakdown (GVBD) was observed. Also, cGMP concentrations in these follicles were measured. Next, denuded oocytes were recovered from preovulatory follicles at 48 h after injection of 15 IU of eCG and incubated with SNAP with or without ferrous hemoglobin. Every 30 min up to 12 h, GVBD was observed. Both AG and hCG promoted GVBD, and SNAP prevented this effect. In addition, AG decreased intrafollicular cGMP levels, and the concomitant addition of SNAP prevented this decrease. Finally, SNAP dose-dependently inhibited GVBD in denuded oocyte, and this effect of SNAP was reversed by the addition of hemoglobin. We conclude that the iNOS-NO-(cGMP) axis may play an important role in oocyte meiotic maturation.     

Possible involvement of Nemo-like kinase 1 in Xenopus oocyte maturation as a kinase responsible for Pumilio1, Pumilio2, and CPEB phosphorylation

Members of the mitogen-activated protein kinase (MAPK) family play important roles in Xenopus oocyte maturation. Nemo-like kinase (NLK), an atypical MAPK, is known to function in multiple developmental processes in vertebrates and invertebrates, but its involvement in gametogenesis and gamete maturation is unknown. In this study, we biochemically examined NLK1 during Xenopus oocyte maturation. NLK1 is expressed in immature oocytes, and its protein level remains constant during maturation. NLK1 is inactive in immature oocytes but is activated during maturation, depending on Mos protein synthesis but not on p42 MAPK activation. Overexpression of NLK1 by injection of 5 ng of mRNA accelerates progesterone-induced oocyte maturation by enhancing Cyclin B1 protein synthesis through the translational activation of its mRNA, in accordance with precocious phosphorylation of Pumilio1 (Pum1), Pumilio2 (Pum2), and cytoplasmic polyadenylation element-binding protein (CPEB), key regulators of the translational control of mRNAs stored in oocytes. A higher level of NLK1 expression by injection of 50 ng of mRNA induces Pum1/Pum2/CPEB phosphorylation, CPEB degradation, Cyclin B1 protein synthesis, and oocyte maturation in the absence of progesterone. NLK1 phosphorylates Pum1, Pum2, and CPEB in vitro. These findings provide the first evidence for the involvement of NLK1 in Xenopus oocyte maturation. We suggest that NLK1 acts as a kinase downstream of Mos and catalyzes phosphorylation of Pum1, Pum2, and CPEB to regulate the translation of mRNAs, including Cyclin B1 mRNA, stored in oocytes.     

Heat-shock response in Xenopus oocytes during meiotic maturation and activation

After a 60 min heat-shock at 36 degrees C, Xenopus oocytes are still able to accomplish a complete meiotic maturation in response to a progesterone treatment. The 36 degrees C heat-shock applied to maturing oocytes strongly enhances the synthesis of a single heat-shock protein of approx. 70 000 molecular weight (hsp70); after activation with the Ca2+-ionophore A 23187, matured oocytes still display the ability to synthesize hsp70 and to survive a heat-shock. A cycloheximide treatment combined with a heat-shock induces, during the recovery period, the synthesis of two heat-shock proteins, of approx. 70 000 and 83 000 molecular weight.     

Activation of Cdc2 kinase during meiotic maturation of axolotl oocyte

Activity of Cdc2, the universal inducer of mitosis, is regulated by phosphorylation and binding to cyclin B. Comparative studies using oocytes from several amphibian species have shown that different mechanisms allow Cdc2 activation and entry into first meiotic division. In Xenopus, immature oocytes stockpile pre-M-phase promoting factor (MPF) composed of Cdc2-cyclin B complexes maintained inactive by Thr14 and Tyr15 phosphorylation of Cdc2. Activation of MPF relies on the conversion of pre-MPF into MPF by Cdc2 dephosphorylation, implying a positive feedback loop known as MPF auto-amplification. On the contrary, it has been proposed that pre-MPF is absent in immature oocyte and that MPF activation depends on cyclin synthesis in some fishes and other amphibians. We demonstrate here that MPF activation in the axolotl oocyte, an urodele amphibian, is achieved through mechanisms resembling partly those found in Xenopus oocyte. Pre-MPF is present in axolotl immature oocyte and is activated during meiotic maturation. However, monomeric Cdc2 is expressed in large excess over pre-MPF, and pre-MPF activation by Cdc2 dephosphorylation takes place progressively and not abruptly as in Xenopus oocyte. The intracellular compartmentalization as well as the low level of pre-MPF in axolotl oocyte could account for the differences in oocyte MPF activation in both species.