Dispersion of cyclin B mRNA aggregation is coupled with translational activation of the mRNA during zebrafish oocyte maturation

Cyclin B mRNA stored in immature zebrafish oocytes is translationally activated upon the stimulation of 17alpha,20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-DP), an event prerequisite for initiating oocyte maturation in this species. We investigated localization of cyclin B mRNA in zebrafish oocytes. Cyclin B mRNA was found to be exclusively localized as an aggregation along the cytoplasm at the animal pole of full-grown immature oocytes. When oocytes were treated with 17alpha,20beta-DP, a meshwork of microfilaments in the oocyte cortex disappeared and the aggregation of cyclin B mRNA dispersed just prior to the initiation of cyclin B synthesis and germinal vesicle breakdown (GVBD). Cytochalasin B, but not nocodazole or taxol, deformed the aggregation of cyclin B mRNA, indicating the involvement of microfilaments in organizing this form. Like 17alpha,20beta-DP, cytochalasin B (10 microg/ml) induced both complete dispersion of the aggregation and translational activation of cyclin B mRNA, forcing the oocytes to undergo GVBD without 17alpha,20beta-DP. Conversely, disturbance of the aggregation of cyclin B mRNA with a low concentration (1 microg/ml) of cytochalasin B inhibited 17alpha,20beta-DP-induced GVBD. These results suggest that the direct change in cyclin B mRNA from the aggregated form to the dispersed form is responsible for translational activation of the mRNA during zebrafish oocyte maturation.     

Xtr, a plural tudor domain-containing protein, is involved in the translational regulation of maternal mRNA during oocyte maturation in Xenopus laevis

Xtr in the fertilized eggs of Xenopus has been demonstrated to be a member of a messenger ribonucleoprotein (mRNP) complex that plays a crucial role in karyokinesis during cleavage. Since the Xtr is also present both in oocytes and spermatocytes and its amount increases immediately after spematogenic cells enter into the meiotic phase, this protein was also predicted to act during meiotic progression. Taking advantage of Xenopus oocytes' large size to microinject anti-Xtr antibody into them for inhibition of Xtr function, we examined the role of Xtr in meiotic progression of oocytes. Microinjection of anti-Xtr antibody into immature oocytes followed by reinitiation of oocyte maturation did not affect germinal vesicle break down and the oscillation of Cdc2/cyclin B activity during meiotic progression but caused abnormal spindle formation and chromosomal alignment at meiotic metaphase I and II. Immunoprecipitation of Xtr showed the association of Xtr with FRGY2 and mRNAs such as RCC1 and XL-INCENP mRNAs, which are involved in the progression of karyokinesis. When anti-Xtr antibody was injected into oocytes, translation of XL-INCENP mRNA, which is known to be repressed in immature oocytes and induced after reinitiation of oocyte maturation, was inhibited even if the oocytes were treated with progesterone. A similar translational regulation was observed in oocytes injected with a reporter mRNA, which was composed of an enhanced green fluorescent protein open reading frame followed by the 3' untranslational region (3'UTR) of XL-INCENP mRNA. These results indicate that Xtr regulates the translation of XL-INCENP mRNA through its 3'UTR during meiotic progression of oocyte.     

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.     

Autoregulation of Musashi1 mRNA translation during Xenopus oocyte maturation

The mRNA translational control protein, Musashi, plays a critical role in cell fate determination through sequence‐specific interactions with select target mRNAs. In proliferating stem cells, Musashi exerts repression of target mRNAs to promote cell cycle progression. During stem cell differentiation, Musashi target mRNAs are de‐repressed and translated. Recently, we have reported an obligatory requirement for Musashi to direct translational activation of target mRNAs during Xenopus oocyte meiotic cell cycle progression. Despite the importance of Musashi in cell cycle regulation, only a few target mRNAs have been fully characterized. In this study, we report the identification and characterization of a new Musashi target mRNA in Xenopus oocytes. We demonstrate that progesterone‐stimulated translational activation of the Xenopus Musashi1 mRNA is regulated through a functional Musashi binding element (MBE) in the Musashi1 mRNA 3′ untranslated region (3′ UTR). Mutational disruption of the MBE prevented translational activation of Musashi1 mRNA and its interaction with Musashi protein. Further, elimination of Musashi function through microinjection of inhibitory antisense oligonucleotides prevented progesterone‐induced polyadenylation and translation of the endogenous Musashi1 mRNA. Thus, Xenopus Musashi proteins regulate translation of the Musashi1 mRNA during oocyte maturation. Our results indicate that the hierarchy of sequential and dependent mRNA translational control programs involved in directing progression through meiosis are reinforced by an intricate series of nested, positive feedback loops, including Musashi mRNA translational autoregulation. These autoregulatory positive feedback loops serve to amplify a weak initiating signal into a robust commitment for the oocyte to progress through the cell cycle and become competent for fertilization.Mol. Reprod. Dev. 79: 553‐563, 2012. © 2012 Wiley Periodicals, Inc.     

c-Mos and cyclin B/cdc2 connections during Xenopus oocyte maturation.

Fully-grown G2 arrested Xenopus oocytes can be induced to enter and progress into meiotic cell cycle by progesterone stimulation. This process is termed oocyte maturation. An early response to progesterone is the synthesis of the onco-protein c-Mos, defined as the candidate initiator of Xenopus oocyte maturation, which triggers the MAPK cascade, MPF activation and promotes CSF activity. Here we review our current knowledge on the synthesis, activation and functions of c-Mos in connection with MPF activation during maturation. We also discuss our recent results concerning the dispensability of cyclin B degradation in meiosis I-meiosis II transition and the stabilization of c-Mos through its direct phosphorylation by cyclin B/cdc2.     

Analyses of zebrafish and Xenopus oocyte maturation reveal conserved and diverged features of translational regulation of maternal cyclin B1 mRNA

Background  

Vertebrate development relies on the regulated translation of stored maternal mRNAs, but how these regulatory mechanisms may have evolved to control translational efficiency of individual mRNAs is poorly understood. We compared the translational regulation and polyadenylation of the cyclin B1 mRNA during zebrafish and Xenopus oocyte maturation. Polyadenylation and translational activation of cyclin B1 mRNA is well characterized during Xenopus oocyte maturation. Specifically, Xenopus cyclin B1 mRNA is polyadenylated and translationally activated during oocyte maturation by proteins that recognize the conserved AAUAAA hexanucleotide and U-rich Cytoplasmic Polyadenylation Elements (CPEs) within cyclin B1 mRNA's 3'UnTranslated Region (3'UTR).     

The subcellular localization of cyclin B2 is required for bipolar spindle formation during Xenopus oocyte maturation

Cyclins B1 and B2 are subtypes of cyclin B, a regulatory subunit of a maturation/M-phase promoting factor, and they are also highly conserved in many vertebrate species. Cyclin B1 is essential for mitosis, whereas cyclin B2 is regarded as dispensable. However, the overexpression of the cyclin B2 N-terminus containing the cytoplasmic retention signal, but not cyclin B1, inhibits bipolar spindle formation in Xenopus oocytes and embryos. Here we show that endogenous cyclin B2 was localized in and around the germinal vesicle. The perinuclear localization of cyclin B2 was perturbed by the overexpression of its N-terminus containing the cytoplasmic retention signal, which resulted in a spindle defect. This spindle defect was rescued by the overexpression of bipolar kinesin Eg5, which is located at the perinuclear region in the proximity of endogenous cyclin B2. These results demonstrate that the proper localization of cyclin B2 is essential for bipolar spindle formation in Xenopus oocytes.     

New B-type cyclin synthesis is required between meiosis I and II during Xenopus oocyte maturation

Progression through meiosis requires two waves of maturation promoting factor (MPF) activity corresponding to meiosis I and meiosis II. Frog oocytes contain a pool of inactive "pre-MPF" consisting of cyclin-dependent kinase 1 bound to B-type cyclins, of which we now find three previously unsuspected members, cyclins B3, B4 and B5. Protein synthesis is required to activate pre-MPF, and we show here that this does not require new B-type cyclin synthesis, probably because of a large maternal stockpile of cyclins B2 and B5. This stockpile is degraded after meiosis I and consequently, the activation of MPF for meiosis II requires new cyclin synthesis, principally of cyclins B1 and B4, whose translation is strongly activated after meiosis I. If this wave of new cyclin synthesis is ablated by antisense oligonucleotides, the oocytes degenerate and fail to form a second meiotic spindle. The effects on meiotic progression are even more severe when all new protein synthesis is blocked by cycloheximide added after meiosis I, but can be rescued by injection of indestructible B-type cyclins. B-type cyclins and MPF activity are required to maintain c-mos and MAP kinase activity during meiosis II, and to establish the metaphase arrest at the end of meiotic maturation. We discuss the interdependence of c-mos and MPF, and reveal an important role for translational control of cyclin synthesis between the two meiotic divisions.     

Musashi regulates the temporal order of mRNA translation during Xenopus oocyte maturation

A strict temporal order of maternal mRNA translation is essential for meiotic cell cycle progression in oocytes of the frog Xenopus laevis. The molecular mechanisms controlling the ordered pattern of mRNA translational activation have not been elucidated. We report a novel role for the neural stem cell regulatory protein, Musashi, in controlling the translational activation of the mRNA encoding the Mos proto-oncogene during meiotic cell cycle progression. We demonstrate that Musashi interacts specifically with the polyadenylation response element in the 3' untranslated region of the Mos mRNA and that this interaction is necessary for early Mos mRNA translational activation. A dominant inhibitory form of Musashi blocks maternal mRNA cytoplasmic polyadenylation and meiotic cell cycle progression. Our data suggest that Musashi is a target of the initiating progesterone signaling pathway and reveal that late cytoplasmic polyadenylation element-directed mRNA translation requires early, Musashi-dependent mRNA translation. These findings indicate that Musashi function is necessary to establish the temporal order of maternal mRNA translation during Xenopus meiotic cell cycle progression.     

Changes in cyclin B localisation during pig oocyte in vitro maturation

The localisation of cyclin B throughout in vitro maturation of pig oocytes was determined by indirect immunofluorescence using a monoclonal antibody specific for an epitope of the human cyclin B. Maturation of pig oocytes was induced by addition of Pergonal (2 UI/ml of FSH/LH) and beta-oestradiol to the medium where isolated ovarian follicles were cultured for up to 72 h. Immature gametes with an intact germinal vesicle were observed during the first 30 h of culture. Only 10% were competent to reinitiate meiosis and showed germinal vesicle breakdown (GVBD) after 36 h. However, after 48-72 h, 60% of the oocytes accomplished their maturation and showed metaphase chromosomes. Immature oocytes showed cyclin B immunofluorescent staining in the cytoplasm, whereas mature oocytes showed the immunofluorescent label concentrated in the nucleus. Metaphase chromosomes showed an intense immunofluorescence. The migration of cyclin B to the nucleus and its association with metaphase chromosomes in pig oocytes able to progress through meiosis resembled the subcellular localisation of cyclin B and the distribution of maturation promoting factor (MPF) in mitotic dividing cells.     

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.     

Aven is dynamically regulated during Xenopus oocyte maturation and is required for oocyte survival

We have analyzed the expression and function of the cell death and cell cycle regulator Aven in Xenopus. Analysis of Xenopus Aven expression in oocytes and embryos revealed a band close to the predicted molecular weight of the protein (36 kDa) in addition to two bands of higher molecular weight (46 and 49 kDa), one of which was determined to be due to phosphorylation of the protein. The protein is primarily detected in the cytoplasm of oocytes and is tightly regulated during meiotic and mitotic cell cycles. Progesterone stimulation of oocytes resulted in a rapid loss of Aven expression with the protein levels recovering before germinal vesicle breakdown (GVBD). This loss of Aven is required for the G2–M1 cell cycle transition. Aven morpholino knockdown experiments revealed that early depletion of the protein increases progesterone sensitivity and facilitates GVBD, but prolonged depletion of Aven results in caspase-3 activation and oocyte death by apoptosis. Phosphorylated Aven (46 kDa) was found to bind Bcl-x_L in oocytes, but this interaction was lost in apoptotic oocytes. Thus, Aven alters progesterone sensitivity in oocytes and is critical for oocyte survival.     

c-mos and cdc2 cooperate in the translational activation of fibroblast growth factor receptor-1 during Xenopus oocyte maturation

During oocyte maturation in Xenopus, previously quiescent maternal mRNAs are translationally activated at specific times. We hypothesized that the translational recruitment of individual messages is triggered by particular cellular events and investigated the potential for known effectors of the meiotic cell cycle to activate the translation of the FGF receptor-1 (XFGFR) maternal mRNA. We found that both c-mos and cdc2 activate the translation of XFGFR. However, although oocytes matured by injection of recombinant cdc2/cyclin B translate normal levels of XFGFR protein, c-mos depletion reduces the level of XFGFR protein induced by cdc2/cyclin B injection. In oocytes blocked for cdc2 activity, injection of mos RNA induced low levels of XFGFR protein, independent of MAPK activity. Through the use of injected reporter RNAs, we show that the XFGFR 3' untranslated region inhibitory element is completely derepressed by cdc2 alone. In addition, we identified a new inhibitory element through which both mos and cdc2 activate translation. We found that cdc2 derepresses translation in the absence of polyadenylation, whereas mos requires poly(A) extension to activate XFGFR translation. Our results demonstrate that mos and cdc2, in addition to functioning as key regulators of the meiotic cell cycle, cooperate in the translational activation of a specific maternal mRNA during oocyte maturation.     

CDK1/cyclin B regulation during oocyte maturation in two closely related lugworm species, Arenicola marina and Arenicola defodiens

The molecular mechanisms underlying oocyte maturation in the annelid polychaetes Arenicola marina and Arenicola defodiens were investigated. In both species, a hitherto unidentified hormone triggers synchronous and rapid transition from prophase to metaphase, a maturation process which can be easily reproduced in vitro. Activation of a roscovitine- and olomoucine-sensitive M-phase-specific histone, H1 kinase, occurs during oocyte maturation. Using affinity chromatography on immobilized p9CKShs1, we purified CDK1 and cyclin B from oocyte extracts prepared from both phases and both species. In prophase, CDK1 is present both as an inactive, but Thr161-phosphorylated monomer, and as an inactive (Tyr15-phosphorylated) heterodimer with cyclin B. Prophase to metaphase transition is associated with complete tyrosine dephosphorylation of the cyclin B-associated CDK1, with phosphorylation of cyclin B, and with dramatic activation of the kinase activity of the CDK1/cyclin B complex. We propose that Arenicola oocytes may provide an ideal model system to investigate the acquisition of the ability of oocytes to be fertilized that occurs as oocyte shift from prophase to metaphase, an important physiological event, probably regulated by active CDK1/cyclin B.     

Involvement of catecholamines in the regulation of oocyte maturation in frogs

The present study investigates the role of catecholamines in the regulation of Bufo arenarum oocyte maturation. The metabolic changes in the oxidation of carbohydrates and the meiotic resumption evinced by the germinal vesicle breakdown were used as indicators of cytoplasmic and nuclear maturation, respectively. The results obtained suggest that noradrenaline (norepinephrine) could be one of the factors responsible for the metabolic behaviour that characterises cytoplasmically immature oocytes. The use of adrenaline (epinephrine), on the other hand, induced a metabolic change which made oocytes cytoplasmically mature. The effect of both catecholamines, which was dose-dependent, was observed in ovarian oocytes (surrounded by follicle cells) as well as in coelomic oocytes (free from follicle cells), suggesting the presence of adrenergic receptors in the gamete. The results obtained using adrenergic agonists and antagonists suggest that the effect of adrenaline would be due to an interaction with beta2-receptors. Although catecholamines have an influence on the determination of the stage of cytoplasmic maturation of the oocytes, they do not affect nuclear maturation by themselves. Nevertheless, pretreatment of follicles with adrenaline caused a significant inhibition in progesterone-induced nuclear maturation even though this effect was markedly weaker when using noradrenaline.     

Cell-cycle-dependent subcellular localization of cyclin B1, phosphorylated cyclin B1 and p34cdc2 during oocyte meiotic maturation and fertilization in mouse

M phase or maturation promoting factor (MPF), a kinase complex composed of the regulatory cyclin B and the catalytic p34cdc2 kinase, plays important roles in meiosis and mitosis. This study was designed to detect and compare the subcellular localization of cyclin B1, phosphorylated cyclin B1 and p34cdc2 during oocyte meiotic maturation and fertilization in mouse. We found that all these proteins were concentrated in the germinal vesicle of oocytes. Shortly after germinal vesicle breakdown, all these proteins were accumulated around the condensed chromosomes. With spindle formation at metaphase I, cyclin B1 and phosphorylated cyclin B1 were localized around the condensed chromosomes and concentrated at the spindle poles, while p34cdc2 was localized in the spindle region. At the anaphase/telophase transition, phosphorylated cyclin B1 was accumulated in the midbody between the separating chromosomes/chromatids, while p34cdc2 was accumulated in the entire spindle except for the midbody region. At metaphase II, both cyclin B1 and p34cdc2 were horizontally localized in the region with the aligned chromosomes and the two poles of the spindle, while phosphorylated cyclin B1 was localized in the two poles of spindle and the chromosomes. We could not detect a particular distribution of cyclin B1 in fertilized eggs when the pronuclei were initially formed, but in late pronuclei cyclin B1 was accumulated in the pronuclei. p34cdc2 and phosphorylated cyclin B1 were always concentrated in one pronucleus after parthenogenetic activation or in two pronuclei after fertilization. At metaphase of 1-cell embryos, cyclin B1 was accumulated around the condensed chromosomes. Cyclin B1 was accumulated in the nucleus of late 2-cell embryos but not in early 2-cell embryos. Furthermore, we also detected the accumulation of p34cdc2 in the nucleus of 2- and 4-cell embryos. All these results show that cyclin B1, phosphorylated cyclin B1 and p34cdc2 have similar distributions at some stages but different localizations at other stages during oocyte meiotic maturation and fertilization, suggesting that they may play a common role in some events but different roles in other events during oocyte maturation and fertilization.     

A novel regulatory element determines the timing of Mos mRNA translation during Xenopus oocyte maturation

Progression through vertebrate oocyte maturation requires that pre-existing, maternally derived mRNAs be translated in a strict temporal order. The mechanism that controls the timing of oocyte mRNA translation is unknown. In this study we show that the early translational induction of the mRNA encoding the Mos proto-oncogene is mediated through a novel regulatory element within the 3' untranslated region of the Mos mRNA. This novel element is responsive to the MAP kinase signaling pathway and is distinct from the late acting, cdc2-responsive, cytoplasmic polyadenylation element. Our findings suggest that the timing of maternal mRNA translation is controlled through signal transduction pathways targeting distinct 3' UTR mRNA elements.     

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.     

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.