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.     

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.     

Differential regulation of cyclin B1 degradation between the first and second meiotic divisions of bovine oocytes

During mammalian oocyte maturation, two consecutive meiotic divisions are required to form a haploid gamete. For each meiotic division, oocytes must transfer from metaphase to anaphase, but maturation promoting factor (cyclin-dependent kinase 1/cyclin B1) activity would keep the oocytes at metaphase. Therefore, inactivation of maturation promoting factor is needed to finish the transition and complete both these divisions; this is provided through anaphase-promoting complex/cyclosome-dependent degradation of cyclin B1. The objective of this study was to examine meiotic divisions in bovine oocytes after expression of a full length cyclin B1 and a nondegradable N-terminal 87 amino acid deletion, coupled with the fluorochrome Venus, by microinjecting their complementary RNA (cRNA). Overexpression of full-length cyclin B1-Venus inhibited homologue disjunction and first polar body formation in maturing oocytes (control 70% vs. overexpression 16%; P < 0.05). However at the same levels of expression, it did not block second meiotic metaphase and cleavage of eggs after parthenogenetic activation (control: 82% pronuclei and 79% cleaved; overexpression: 91% pronuclei and 89% cleaved). The full length cyclin B1 and a nondegradable N-terminal 87 amino acid deletion caused metaphase arrest in both meiotic divisions, whereas degradation of securin was unaffected. Roscovitine, a potent cyclin-dependent kinase 1 (CDK1) inhibitor, overcame this metaphase arrest in maturing oocytes at 140 μM, but higher doses (200 μM) were needed to overcome arrest in eggs. In conclusion, because metaphase I (MI) blocked by nondegradable cyclin B1 was distinct from metaphase II (MII) in their different sensitivities to trigger CDK1 inactivation, we concluded that mechanisms of MI arrest differed from MII arrest.     

Regulation of ubiquitin-proteasome pathway on pig oocyte meiotic maturation and fertilization

Degradation of proteins mediated by the ubiquitin-proteasome pathway (UPP) plays essential roles in the eukaryotic cell cycle. The main aim of the present study was to analyze the functional roles and regulatory mechanisms of the UPP in pig oocyte meiotic maturation, activation, and early embryo mitosis by drug treatment, Western blot analysis, and confocal microscopy. By using the hypoxanthine-maintained meiotic arrest model, we showed that the meiotic resumption of both cumulus-enclosed oocytes and denuded oocytes was stimulated in a dose- and time-dependent manner by two potent and cell-permeable proteasome inhibitors. Both the mitogen-activated protein kinase (MAPK) kinase inhibitor U0126 and the maturation-promoting factor inhibitor roscovitine overcame the stimulation of germinal vesicle breakdown induced by proteasome inhibitors. The phosphorylation of MAPK and p90rsk and the expression of cyclin B1 increased in a dose- and time-dependent manner when treated with proteasome inhibitors during oocyte in vitro-maturation culture. Both U0126 and roscovitine inhibited the phosphorylation of MAPK and p90rsk, and the synthesis of cyclin B1 stimulated by proteasome inhibitors. When matured oocytes were pretreated with proteasome inhibitors and then fertilized or artificially activated, the second polar body emission and the pronuclear formation were inhibited, and the dephosphorylation of MAPK and p90rsk as well as the degradation of cyclin B1 that should occur after oocyte activation were also inhibited. We also investigated, to our knowledge for the first time, the subcellular localization of 20S proteasome alpha subunits at different stages of oocyte and early embryo development. The 20S proteasome alpha subunits were accumulated in the germinal vesicle, around the condensed chromosomes at prometaphase, with spindle at metaphase I and II, the region between the separating chromosomes, and especially the midbody at anaphase I and telophase I, the pronucleus, and the nucleus in early embryonic cells. In conclusion, our results suggest that the UPP is important at multiple steps of pig oocyte meiosis, fertilization, and early embryonic mitosis and that it may play its roles by regulating cyclin B1 degradation and MAPK/p90rsk phosphorylation.     

Regulatory roles of ubiquitin-proteasome pathway in pig oocyte meiotic maturation and fertilization

The ubiquitin-proteasome pathway is involved in the degradation of proteins related to cell cycle progression including cyclins. The present study, using two specific proteasome inhibitors, for the first time investigated the roles of ubiquitin-proteasome in cell cycle progression during pig oocyte meiotic maturation and after fertilization. In contrast to its effect in rodent oocytes, proteasome inhibition strongly prevented germinal vesicle breakdown (GVBD). After GVBD, proteasome inhibition disrupted meiotic apparatus organization, cell cycle progression, and first polar body (PB1) extrusion. Sperm penetration into the oocytes was completely inhibited when proteasome inhibitors were added at the beginning of insemination. However, sperm chromatin decondensation and metaphase-interphase transition were not affected when inhibitors were added once sperm penetrated. The results suggest that ubiquin-proteasome complex is one of the critical regulators of meiotic cell cycle, but proteasome inhibitors do not affect major fertilization events when added after sperm penetration into the oocytes in the pig.     

Meiotic maturation of the mouse oocyte requires an equilibrium between cyclin B synthesis and degradation

Among the proteins whose synthesis and/or degradation is necessary for a proper progression through meiotic maturation, cyclin B appears to be one of the most important. Here, we attempted to modulate the level of cyclin B1 and B2 synthesis during meiotic maturation of the mouse oocyte. We used cyclin B1 or B2 mRNAs with poly(A) tails of different sizes and cyclin B1 or B2 antisense RNAs. Oocytes microinjected with cyclin B1 mRNA showed two phenotypes: most were blocked in MI, while the others extruded the first polar body in advance when compared to controls. Moreover, these effects were correlated with the length of the poly(A) tail. Thus it seems that the rate of cyclin B1 translation controls the timing of the first meiotic M phase and the transition to anaphase I. Moreover, overexpression of cyclin B1 or B2 was able to bypass the dbcAMP-induced germinal vesicle block, but only the cyclin B1 mRNA-microinjected oocytes did not extrude their first polar body. Oocytes injected with the cyclin B1 antisense progressed through the first meiotic M phase but extruded the first polar body in advance and were unable to enter metaphase II. This suggested that inhibition of cyclin B1 synthesis only took place at the end of the first meiotic M phase, most likely because the cyclin B1 mRNA was protected. The injection of cyclin B2 antisense RNA had no effect. The life observation of the synthesis and degradation of a cyclin B1-GFP chimera during meiotic maturation of the mouse oocyte demonstrated that degradation can only occur during a given period of time once it has started. Taken together, our data demonstrate that the rates of cyclin B synthesis and degradation determine the timing of the major events taking place during meiotic maturation of the mouse oocyte.     

Securin and not CDK1/cyclin B1 regulates sister chromatid disjunction during meiosis II in mouse eggs

Mammalian eggs remain arrested at metaphase of the second meiotic division (metII) for an indeterminate time before fertilization. During this period, which can last several hours, the continued attachment of sister chromatids is thought to be achieved by inhibition of the protease separase. Separase is known to be inhibited by binding either securin or Maturation (M-Phase)-Promoting Factor, a heterodimer of CDK1/cyclin B1. However, the relative contribution of securin and CDK/cyclin B1 to sister chromatid attachment during metII arrest has not been assessed. Although there are conditions in which either CDK1/cyclinB1 activity or securin can prevent sister chromatid disjunction, principally by overexpression of non-degradable cyclin B1 or securin, we find here that separase activity is primarily regulated by securin and not CDK1/cyclin B1. Thus the CDK1 inhibitor roscovitine and an antibody we designed to block the interaction of CDK1/cyclin B1 with separase, both failed to induce sister disjunction. In contrast, securin morpholino knockdown specifically induced loss of sister attachment, that could be restored by securin cRNA rescue. During metII arrest separase appears primarily regulated by securin binding, not CDK1/cyclin B1.     

雷公藤多甙对小鼠卵母细胞成熟和体外受精的影响

采用超排卵技术研究雷公藤多甙（GTW）对小鼠卵母细胞的成熟和体外受精以及脏器等的影响,GTW对小鼠卵母细胞生发泡破裂没有影响,但可以抑制卵母细胞第一极体的释放,影响卵母细胞的存活率并可降低体外受精率和超排卵的卵母细胞数量。GTW可以破坏卵母细胞成熟,降低卵母细胞的体外受精能力,影响小鼠的正常生殖功能。     

Characterization of polo-like kinase 1 during meiotic maturation of the mouse oocyte

We have characterized plk1 in mouse oocytes during meiotic maturation and after parthenogenetic activation until entry into the first mitotic division. Plk1 protein expression remains unchanged during maturation. However, two different isoforms can be identified by SDS-PAGE. A fast migrating form, present in the germinal vesicle, seems characteristic of interphase. A slower form appears as early as 30 min before germinal vesicle breakdown (GVBD), is maximal at GVBD, and is maintained throughout meiotic maturation. This form gradually disappears after exit from meiosis. The slow form corresponds to a phosphorylation since it disappears after alkaline phosphatase treatment. Plk1 activation, therefore, takes place before GVBD and MAPK activation since plk1 kinase activity correlates with its slow migrating phosphorylated form. However, plk1 phosphorylation is inhibited after treatment with two specific p34(cdc2) inhibitors, roscovitine and butyrolactone, suggesting plk1 involvement in the MPF autoamplification loop. During meiosis plk1 undergoes a cellular redistribution consistent with its putative targets. At the germinal vesicle stage, plk1 is found diffusely distributed in the cytoplasm and enriched in the nucleus and during prometaphase is localized to the spindle poles. At anaphase it relocates to the equatorial plate and is restricted to the postmitotic bridge at telophase. After parthenogenetic activation, plk1 becomes dephosphorylated and its activity drops progressively. Upon entry into the first mitotic M-phase at nuclear envelope breakdown plk1 is phosphorylated and there is an increase in its kinase activity. At the two-cell stage, the fast migrating form with weak kinase activity is present. In this work we show that plk1 is present in mouse oocytes during meiotic maturation and the first mitotic division. The variation of plk1 activity and subcellular localization during this period suggest its implication in the organization and progression of M-phase.     

Involvement of the pentose phosphate pathway and redox regulation in fertilization in the mouse

Glucose metabolism is necessary for successful fertilization in the mouse. Both spermatozoa and oocytes metabolize glucose through the pentose phosphate pathway (PPP), and NADPH appears required for gamete fusion. The aims of this study were to further characterize the utilization of glucose by the fertilizing spermatozoon and the fertilized oocyte, to demonstrate the importance of the PPP in different steps of fertilization, and to examine whether the beneficial effect of glucose could be mediated by a NADPH-dependent enzyme involved in redox regulation. By using a fluorescent analog of 2-deoxyglucose, glucose uptake was evidenced in both the head and flagellum of motile spermatozoa. After sperm-oocyte fusion, an increase in glucose uptake by the fertilized oocyte was observed but not before the formation of the male and female pronuclei. By using a microphotometric technique, activity of glucose 6-phosphate dehydrogenase (G6PDH), the key enzyme of the PPP, was localized to the sperm head and midpiece. When epididymal spermatozoa were released into a glucose-containing medium, the NADPH/NADP ratio increased with capacitation. Sperm-oocyte fusion and meiosis reinitiation of the fertilized oocyte was inhibited by the PPP inhibitor 6-aminonicotinamide (6-AN); inhibition of sperm-oocyte fusion was relieved by NADPH. Sperm-oocyte fusion and meiosis reinitiation were also inhibited by diphenylamine iodonium, which is a flavoenzyme inhibitor reported to prevent reactive oxygen species (ROS) generation in mouse spermatozoa and embryos. These findings indicate that the PPP is involved in different steps of fertilization. Subsequent regulation of a NADPH-dependent flavoenzyme responsible of ROS production is envisaged.     

Germinal vesicle material drives meiotic cell cycle of mouse oocyte through the 3'UTR-dependent control of cyclin B1 synthesis

We compared the profile of histone H1 kinase activity, reflecting Maturation Promoting Factor (MPF) activity in oocytes bisected at the germinal vesicle (GV) stage and allowed to mature as separate oocyte halves in vitro. Whereas the oocyte halves containing the nucleus exhibited the same profile of increased kinase activity as that typical for intact oocytes, the anuclear halves revealed strong inhibition of the increase in this activity soon after germinal vesicle breakdown (GVBD). In contrast, the profile of MAP kinase activity did not differ significantly between anuclear and nucleus-containing oocyte halves throughout maturation. Of the two MPF components, CDK1 and cyclin B1, the amount of the latter was significantly reduced in anuclear halves, a reduction due to low-level synthesis and not to enhanced degradation. Expression of three reporter luciferase RNAs constructed, respectively, to contain cyclin B1-specific 3'UTR, the globin-specific 3'UTR, or no 3'UTR sequence was enhanced in nuclear halves, with significantly greater enhancement for the construct containing cyclin B1-specific 3'UTR as compared to the two other RNAs. We conclude that the profile of activity of MPF during mouse oocyte maturation is controlled by an unknown GV-associated factor(s) acting via 3'UTR-dependent control of cyclin B1 synthesis. These results require the revision of the hitherto prevailing view that the control of MPF activity during mouse oocyte maturation is independent of GV-derived material.     

Polo-like kinase-1 is a pivotal regulator of microtubule assembly during mouse oocyte meiotic maturation,fertilization, and early embryonic mitosis

Polo-like kinases (Plks) are a family of serine/threonine protein kinases that have been activated through phosphorylation. The activity of these kinases has been shown to be required for regulating multiple stages of mitotic progression in somatic cells. In this experiment, the changes in Plk1 expression were detected in mouse oocytes through Western blotting. The subcellular localization of Plk1 during oocyte meiotic maturation, fertilization, and early cleavage as well as after antibody microinjection or microtubule assembly disturbance was studied by confocal microscopy. The quantity of Plk1 protein remained stable during meiotic maturation and decreased gradually after fertilization. Plk1 was localized to the spindle poles of both meiotic and mitotic spindles at the early M phase and then translocated to the middle region. At anaphase and telophase, Plk1 was concentrated at the midbody of cytoplasmic cleavages. Plk1 was concentrated between the male and female pronuclei after fertilization. Plk1 disappeared at the spindle region when microtubule formation was inhibited by colchicine or staurosporine, while it was concentrated as several dots in the cytoplasm after taxol treatment. Plk1 antibody injection decreased the germinal vesicle breakdown rate and distorted MI spindle organization. Our results indicate that Plk1 is a pivotal regulator of microtubule organization during mouse oocyte meiosis, fertilization, and cleavage and that its functions may be regulated by other kinases, such as staurosporine-sensitive kinases.     

Localization and function of the Ska complex during mouse oocyte meiotic maturation

The Ska (spindle and kinetochore-associated) complex is composed of three proteins: Ska1, Ska2 and Ska3. It is required for stabilizing kinetochore-microtubule (KT-MT) interactions and silencing spindle checkpoint during mitosis. However, its roles in meiosis remain unclear. The present study was designed to investigate the localization and function of the Ska complex during mouse oocyte meiotic maturation. Our results showed that the localization and function of Ska complex in mouse oocyte meiosis differ in part from those in mitosis. Injection of low dose exogenous Myc-Ska mRNA showed that, instead of localizing to the kinetochores (KTs) and mediating KT-MT interactions from pro-metaphase to mid-anaphase stages as in mitosis, the members of the Ska complex were only localized on spindle microtubules from the Pro-MI to MII stages in mouse oocyte meiosis. Time-lapse live imaging analysis showed that knockdown of any member of the Ska complex by Morpholino injection into mouse oocytes resulted in spindle movement defects and enlarged polar bodies. Depletion of the whole Ska complex disrupted the stability of the anaphase spindle and influenced the extrusion of the first polar body. Taken together, these results show that the Ska complex plays an important role in meiotic spindle migration and anaphase spindle stability during mouse oocyte maturation.     

Involvement of Xenopus Pumilio in the translational regulation that is specific to cyclin B1 mRNA during oocyte maturation

Protein synthesis of cyclin B by translational activation of the dormant mRNA stored in oocytes is required for normal progression of maturation. In this study, we investigated the involvement of Xenopus Pumilio (XPum), a cyclin B1 mRNA-binding protein, in the mRNA-specific translational activation. XPum exhibits high homology to mammalian counterparts, with amino acid identity close to 90%, even if the conserved RNA-binding domain is excluded. XPum is bound to cytoplasmic polyadenylation element (CPE)-binding protein (CPEB) through the RNA-binding domain but not to its phosphorylated form in mature oocytes. In addition to the CPE, the XPum-binding sequence of cyclin B1 mRNA acts as a cis-element for translational repression. Injection of anti-XPum antibody accelerated oocyte maturation and synthesis of cyclin B1, and, conversely, over-expression of XPum retarded oocyte maturation and translation of cyclin B1 mRNA, which was accompanied by inhibition of poly(A) tail elongation. The injection of antibody and the over-expression of XPum, however, had no effect on translation of Mos mRNA, which also contains the CPE. These findings provide the first evidence that XPum is a translational repressor specific to cyclin B1 in vertebrates. We propose that in cooperation with the CPEB-maskin complex, the master regulator common to the CPE-containing mRNAs, XPum acts as a specific regulator that determines the timing of translational activation of cyclin B1 mRNA by its release from phosphorylated CPEB during oocyte maturation.     

Chronic restraint stress disturbs meiotic resumption through APC/C-mediated cyclin B1 excessive degradation in mouse oocytes

Psychological stress, which exerts detrimental effects on human reproduction, may compromise the meiotic competence of oocytes. Meiotic resumption, germinal vesicle breakdown (GVBD), is the first milestone to confer meiotic competence to oocytes. In the practice of assisted reproductive technology (ART), the timing for GVBD is associated with the rates of cleavage and blastocyst formation. However, whether chronic stress compromises oocyte competence by influencing GVBD and the underlying mechanisms are unclear. In the present study, a chronic restraint stress (CRS) mouse model was used to investigate the effects of stress on oocyte meiotic resumption, as well as the mechanisms. Following a 4-week chronic restraint stress in female mice, the percentage of abnormal bipolar spindles increased and indicated compromised oocyte competence in the CRS group. Furthermore, we identified a decreased percentage of GVBD and prolonged time of GVBD in the CRS mouse oocytes compared with the control group. CRS simultaneously reduced the expression of cyclin B1 (CCNB1), which represents a regulatory subunit of M-phase/mature promoting factor (MPF). However, MG132, an inhibitor of anaphase-promoting complex/cyclosome (APC/C), could rescue the prolonged time of GVBD and increase the expression level of CCNB1 of oocytes from the CRS mice. Collectively, our results demonstrated that stress disturbed meiotic resumption through APC/C-mediated CCNB1 degradation, thus providing a novel understanding for stress-related oocyte quality decline; moreover, it may provide a non-invasive approach to select high-quality gametes and novel targets for molecular therapy to treat stress-related female infertility.     

Stability of cyclin B protein during meiotic maturation and the first mitotic cell division in mouse oocytes

This report examines in detail the metabolism of the cyclin protein B1 during meiotic maturation and following the activation of mature mouse oocytes using immunoprecipitation of the radiolabelled protein. The net synthesis of cyclin B increases progressively during meiotic maturation, reaching its maximum levels at least 1 h before oocytes exit into metaphase of meiosis II (MII). This increase correlates with the rise in cdc2 kinase activity reported previously and suggests an association between the length of the first meiotic M phase (MI) and the net synthesis of cyclin B, that seems to regulate the time required for the cdc2 kinase to reach its maximum activity. Moreover, no marked degradation of cyclin B was observed before the MI to MII transition and that which occurs does so independently of the presence of microtubules, which are essential for cyclin degradation during metaphase II arrest and exit of oocytes into interphase of the first mitotic cell cycle. Cyclin B is degraded rapidly during the transitions MI to MII, MII to the first mitotic interphase and MII to an abortive third metaphase state (MIII). However, whilst its degradation was incomplete during the MI to MII transition, virtually no cyclin B protein was detected following both the MII to interphase and MII to MIII transitions. Thus, the decision of oocytes to exit into MIII, or interphase is not controlled at the level of cyclin B degradation. Lastly, in aging, non-activated oocytes, the net synthesis of cyclin B declines. Whereas, in activated eggs cultured in parallel although the rate of net synthesis declines initially, it is effectively ‘rescued’ being two-fold greater than in non-activated oocytes of an equivalent age. This gradual fall in the net synthesis of cyclin B observed in aging oocytes may contribute to the increasing ease with which they become activated, compared to recently ovulated oocytes.     

Overexpression of cyclin A1 promotes meiotic resumption but induces premature chromosome separation in mouse oocyte

Mammalian cyclin A1 is prominently expressed in testis and essential for meiosis in the male mouse, however, it shows weak expression in ovary, especially during oocyte maturation. To understand why cyclin A1 behaves in this way in the oocyte, we investigated the effect of cyclin A1 overexpression on mouse oocyte meiotic maturation. Our results revealed that cyclin A1 overexpression triggered meiotic resumption even in the presence of germinal vesicle breakdown inhibitor, milrinone. Nevertheless, the cyclin A1-overexpressed oocytes failed to extrude the first polar body but were completely arrested at metaphase I. Consequently, cyclin A1 overexpression destroyed the spindle morphology and chromosome alignment by inducing premature separation of chromosomes and sister chromatids. Therefore, cyclin A1 overexpression will prevent oocyte maturation although it can promote meiotic resumption. All these results show that decreased expression of cyclin A1 in oocytes may have an evolutional significance to keep long-lasting prophase arrest and orderly chromosome separation during oocyte meiotic maturation.     

Translational control of cyclin B1 mRNA during meiotic maturation: coordinated repression and cytoplasmic polyadenylation

Translational control is prominent during meiotic maturation and early development. In this report, we investigate a mode of translational repression in Xenopus laevis oocytes, focusing on the mRNA encoding cyclin B1. Translation of cyclin B1 mRNA is relatively inactive in the oocyte and increases dramatically during meiotic maturation. We show, by injection of synthetic mRNAs, that the cis-acting sequences responsible for repression of cyclin B1 mRNA reside within its 3'UTR. Repression can be saturated by increasing the concentration of reporter mRNA injected, suggesting that the cyclin B1 3'UTR sequences provide a binding site for a trans-acting repressor. The sequences that direct repression overlap and include cytoplasmic polyadenylation elements (CPEs), sequences known to promote cytoplasmic polyadenylation. However, the presence of a CPE per se appears insufficient to cause repression, as other mRNAs that contain CPEs are not translationally repressed. We demonstrate that relief of repression and cytoplasmic polyadenylation are intimately linked. Repressing elements do not override the stimulatory effect of a long poly(A) tail, and polyadenylation of cyclin B1 mRNA is required for its translational recruitment. Our results suggest that translational recruitment of endogenous cyclin B1 mRNA is a collaborative effect of derepression and poly(A) addition. We discuss several molecular mechanisms that might underlie this collaboration.