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Characterization and expression of a maternal axolotl Cyclin B1 during oogenesis and early development
Abstract
The M phase promoting factor (MPF) is a dimer composed of a catalytic Cdk1 subunit and a Cyclin B regulatory subunit. We have characterized a cDNA containing the entire coding sequence of an axolotl Cyclin B1 protein that is able to promote MPF activity when added to a fraction from prophase I oocytes that contains monomeric Cdk1. The axolotl cyclin B1 gene is expressed as a maternal mRNA in oocytes and early embryos. Its poly(A) tail length increases in metaphase II oocytes and then decreases regularly during the first embryonic cell cycles. Endogenous Cyclin B1 protein is first expressed during oocyte meiotic maturation. Its level oscillates after fertilization and is coordinated to the phosphorylation level of tyrosine 15 residue of Cdk1 (pTyr15), with both maxima preceding each cell division. As expected, when translated into microinjected oocytes, axolotl Cyclin B1 induces the resumption of meiosis. In electrically activated unfertilized eggs (UFE), Cyclin B1 and pTyr15 cyclic accumulations are observed with kinetics different from those of the early embryonic cycles. The axolotl embryo and UFE provide interesting in vivo comparative models for studying events controlling Cyclin B1 regulation during development.
... M phase-promoting factor (MPF; cdc2 kinase (cdk1), and cyclin B) maintained by c-Mos (Iwao & Masui 1995, Sakamoto et al. 1998, Vaur et al. 2004, Pelczar et al. 2007). The amount of cyclin B in the unfertilized egg is approximately one-fourth in cleaving eggs but is primarily distributed in the cortex of the animal hemisphere and chromosomes (Sakamoto et al. 1998, Iwao et al. 2002. ...
... The amount of cyclin B in the unfertilized egg is approximately one-fourth in cleaving eggs but is primarily distributed in the cortex of the animal hemisphere and chromosomes (Sakamoto et al. 1998, Iwao et al. 2002. Cyclin B as well as c-Mos disappeared soon after fertilization when the sperm asters expand through the egg cytoplasm (Yamamoto et al. 2001, Iwao et al. 2002, Pelczar et al. 2007) and then the activity of both cdc2 kinase and MAPK decreases (Iwao et al. 1993, Sakamoto et al. 1998, Pelczar et al. 2007). Degradation of MPF might occur downstream of the Ca 2C rises at fertilization through a calcineurin/CaMKII/APC cascade alike in Xenopus eggs (Nishiyama et al. 2007). ...
... The amount of cyclin B in the unfertilized egg is approximately one-fourth in cleaving eggs but is primarily distributed in the cortex of the animal hemisphere and chromosomes (Sakamoto et al. 1998, Iwao et al. 2002. Cyclin B as well as c-Mos disappeared soon after fertilization when the sperm asters expand through the egg cytoplasm (Yamamoto et al. 2001, Iwao et al. 2002, Pelczar et al. 2007) and then the activity of both cdc2 kinase and MAPK decreases (Iwao et al. 1993, Sakamoto et al. 1998, Pelczar et al. 2007). Degradation of MPF might occur downstream of the Ca 2C rises at fertilization through a calcineurin/CaMKII/APC cascade alike in Xenopus eggs (Nishiyama et al. 2007). ...
Fertilization is indispensable not only for restoring diploid genomes but also for the initiation of early embryonic cell cycles in sexual reproduction. While most animals exhibit monospermy, which is ensured by polyspermy blocks to prevent the entry of extra sperm into the egg at fertilization, several animals exhibit physiological polyspermy, in which the entry of several sperm is permitted but only one sperm nucleus participates in the formation of a zygote nucleus. Polyspermy requires that the sperm transmit the egg activation signal more slowly, thus allowing the egg to accept several sperm. An increase in intracellular Ca(2+) concentration induced by the fertilizing sperm is both necessary and sufficient for egg activation in polyspermy. Multiple small Ca(2+) waves induced by several fertilizing sperm result in a long-lasting Ca(2+) rise, which is a characteristic of polyspermic amphibian eggs. We introduced a novel soluble sperm factor for egg activation, sperm-specific citrate synthase, into polyspermic newt eggs to cause Ca(2+) waves. Citrate synthase may perform dual functions: as an enzyme in mitochondria and as a Ca(2+)-inducing factor in egg cytoplasm. We also discuss the close relationship between the mode of fertilization and the Ca(2+) rise at egg activation and consider changes in this process through evolution in vertebrates.
... The third situation covers many vertebrates, including fish and amphibian species as well as nearly all mammals examined except small rodents [193][194][195][196][197][198][199]. In oocytes from these animals, new proteins must be synthesized to initiate MPF activation, despite the presence of a stockpile of inactive Cdk1-Cyclin B. A lot of studies have been dedicated to the identification of these newly synthesized proteins and have focused on two main candidates: Mos and Cyclin B. Synthesis of either of these two proteins is sufficient to promote meiosis resumption and MPF activation in Xenopus oocyte [200,201]. ...
During oocyte development, meiosis arrests in prophase of the first division for a remarkably prolonged period firstly during oocyte growth, and then when awaiting the appropriate hormonal signals for egg release. This prophase arrest is finally unlocked when locally produced maturation initiation hormones (MIHs) trigger entry into M-phase. Here, we assess the current knowledge of the successive cellular and molecular mechanisms responsible for keeping meiotic progression on hold. We focus on two model organisms, the amphibian Xenopus laevis, and the hydrozoan jellyfish Clytia hemisphaerica. Conserved mechanisms govern the initial meiotic programme of the oocyte prior to oocyte growth and also, much later, the onset of mitotic divisions, via activation of two key kinase systems: Cdk1-Cyclin B/Gwl (MPF) for M-phase activation and Mos-MAPkinase to orchestrate polar body formation and cytostatic (CSF) arrest. In contrast, maintenance of the prophase state of the fully-grown oocyte is assured by highly specific mechanisms, reflecting enormous variation between species in MIHs, MIH receptors and their immediate downstream signalling response. Convergence of multiple signalling pathway components to promote MPF activation in some oocytes, including Xenopus, is likely a heritage of the complex evolutionary history of spawning regulation, but also helps ensure a robust and reliable mechanism for gamete production.
... Exactly how such citrate-derived breakdown products might facilitate Ca 2þ -wave production during egg activation has yet to be determined, although acetyl-CoA has been shown to sensitize IP 3 receptors in the endoplasmic reticulum of other cell types, and the Ca 2þ wave observed following injection of oxaloacetate into unfertilized Cynops oocytes could arise from an oxaloacetate-induced release of Ca 2þ from mitochondria, as noted for hepatocytes (Harada et al., 2011). In any case, regardless of the precise signaling pathways triggered by citrate synthase, the fertilization-induced Ca 2þ response of Cynops and related amphibians is capable of driving MPF/ MAPK inactivation and cell-cycle progression (Sakamoto et al., 1998;Yamamoto et al., 1999;Pelczar et al., 2007;Iwao, 2012). ...
Fertilization causes mature oocytes or eggs to increase their concentrations of intracellular calcium ions (Ca(2+) ) in all animals that have been examined, and such Ca(2+) elevations, in turn, provide key activating signals that are required for non-parthenogenetic development. Several lines of evidence indicate that the Ca(2+) transients produced during fertilization in mammals and other taxa are triggered by soluble factors that sperm deliver into oocytes after gamete fusion. Thus, for a broad-based analysis of Ca(2+) dynamics during fertilization in animals, this article begins by summarizing data on soluble sperm factors in non-mammalian species, and subsequently reviews various topics related to a sperm-specific phospholipase C, called PLCζ, which is believed to be the predominant activator of mammalian oocytes. After characterizing initiation processes that involve sperm factors or alternative triggering mechanisms, the spatiotemporal patterns of Ca(2+) signals in fertilized oocytes or eggs are compared in a taxon-by-taxon manner, and broadly classified as either a single major transient or a series of repetitive oscillations. Both solitary and oscillatory types of fertilization-induced Ca(2+) signals are typically propagated as global waves that depend on Ca(2+) release from the endoplasmic reticulum in response to increased concentrations of inositol 1,4,5-trisphosphate (IP3 ). Thus, for taxa where relevant data are available, upstream pathways that elevate intraoocytic IP3 levels during fertilization are described, while other less-common modes of producing Ca(2+) transients are also examined. In addition, the importance of fertilization-induced Ca(2+) signals for activating development is underscored by noting some major downstream effects of these signals in various animals. Mol. Reprod. Dev. © 2013 Wiley Periodicals, Inc.
... The mammalian fully-grown or ovulated oocyte is the largest single cell in which large amounts of maternal mRNAs and proteins are stored. Degradation of enormous amounts of maternal mRNA and proteins after fertilization contributes to dynamic changes from the oogenic program to the embryonic program Solter et al., 2004;Pelczar et al., 2007;Li et al., 2010). A lack of this degradation and regulation would be harmful to embryonic development (Stitzel and Seydoux, 2007). ...
During the maternal-to-zygotic transition (MZT), maternal proteins in oocytes are degraded by the ubiquitin-proteasome system (UPS), and new proteins are synthesized from the zygotic genome. However, the specific mechanisms underlying the UPS at the MZT are not well understood. We identified a molecule named zygote-specific proteasome assembly chaperone (ZPAC) that is specifically expressed in mouse gonads, and expression of ZPAC was transiently increased at the mouse MZT. ZPAC formed a complex with Ump1 and associated with precursor forms of 20S proteasomes. Transcription of ZPAC genes was also under the control of an autoregulatory feedback mechanism for the compensation of reduced proteasome activity similar to Ump1 and 20S proteasome subunit gene expression. Knockdown of ZPAC in early embryos caused a significant reduction of proteasome activity and decrease in Ump1 and mature proteasomes, leading to accumulation of proteins that need to be degraded at the MZT and early developmental arrest. Therefore, a unique proteasome assembly pathway mediated by ZPAC is important for progression of the mouse MZT.
... In a Xenopus LSE, incubation of demembranated sperm nuclei leads to the loading of prereplication complexes on DNA and Ca ++ addition allows the Cdk (cell-cycle dependent kinase) dependent initiation of DNA replication through activation of the licensing factor [37,38]. Preliminary experiments indicated that axolotl LSE were indeed not responsive to Ca ++ addition : both the levels of the phosphorylated forms of MAP kinases and Cdk1 (on tyrosine 15) that accumulate in axolotl UFE [39] remained unchanged in axolotl extracts following Ca ++ addition (data not shown), although they are modulated when intact axolotl UFE are activated [40]. This observation therefore raises the possibility that at least one pathway necessary to the coordinated regulation of Cdk was disrupted during axolotl LSE preparation. ...
We have previously reported a post-transcriptional RNA amplification observed in vivo following injection of in vitro synthesized transcripts into axolotl oocytes, unfertilized (UFE) or fertilized eggs. To further characterize this phenomenon, low speed extracts (LSE) from axolotl and Xenopus UFE were prepared and tested in an RNA polymerization assay. The major conclusions are: i) the amphibian extracts catalyze the incorporation of radioactive ribonucleotide in RNase but not DNase sensitive products showing that these products correspond to RNA; ii) the phenomenon is resistant to α-amanitin, an inhibitor of RNA polymerases II and III and to cordycepin (3'dAMP), but sensitive to cordycepin 5'-triphosphate, an RNA elongation inhibitor, which supports the existence of an RNA polymerase activity different from polymerases II and III; the detection of radiolabelled RNA comigrating at the same length as the exogenous transcript added to the extracts allowed us to show that iii) the RNA polymerization is not a 3' end labelling and that iv) the radiolabelled RNA is single rather than double stranded. In vitro cell-free systems derived from amphibian UFE therefore validate our previous in vivo results hypothesizing the existence of an evolutionary conserved enzymatic activity with the properties of an RNA dependent RNA polymerase (RdRp).
Trichomonas vaginalis is an early divergent protozoan parasite that causes trichomoniasis, the most common non-viral sexually transmitted infection. In metazoans, there is abundant and detailed research on the cell cycle and the components involved in the regulation mechanisms. Regulators such as the cyclin-dependent kinases (CDKs) and cyclins activate the highly regulated processes of cell division. While CDKs have important roles in the phosphorylation of specific substrates, cyclins are important activating-components of CDKs that allow orderly passage through the different stages of the cell cycle. Cell cycle cyclins are characterized by showing drastic changes in their concentration during the cell cycle progression. However, in protists such as T. vaginalis, some biological processes such as cell cycle regulation remain less well studied. In an attempt to gain insight into cell cycle regulation in T. vaginalis, as an initial approach we characterized four proteins with features of cyclins. The genes encoding these putative cyclins were cloned to produce the recombinant proteins TvCYC1, TvCYC2, TvCYC3, and TvCYC4. The functional activity of TvCYC2, TvCYC3, and TvCYC4 was assessed through their complementation of a yeast cln1,2,3Δ mutant strain; TvCYC1 was not able to complement this mutant. Furthermore, our results suggest that TvCYC1, TvCYC2, and TvCYC3, are able to interact with and activate the kinase activity of TvCRK1, a kinase previously characterized by our group. The present study represents the first characterization of cyclins potentially involved in cell cycle regulation in T. vaginalis.
Cyclin B functions as a regulatory protein through association with its catalytic partner Cdc2 kinase forming M-phase promoting factor (MPF), which plays a central role in the meiotic maturation of oocyte. To gain insight into the molecular events, we here cloned a cyclin B cDNA from the ovary of the prawn Macrobrachium rosenbergii and compared its spatial-temporal expression patterns during oocyte maturation with those of crab Eriocheir sinensis. The prawn cyclin B cDNA encodes a 398 amino acid protein with predicted molecular weight of 45.16 kDa. Immunodetection of cyclin B protein by Western blot showed that a target band of approximately 53 kDa protein in the prawn ovaries at both late vitellogenesis (lVt) and germinal vesicle breakdown (GVBD) stages, whereas a 41 kDa band was present in the crab ovaries. Cyclin B protein expression changes indicating that the newly synthesis of cyclin B proteins could be required for GVBD in both prawn and crab. Immunohistochemical analysis revealed that both the prawn and crab cyclin B proteins, were localized in the ooplasm of previtellogenic oocytes, then relocated into germinal vesicle at vitellogenesis stage and localized on meiotic spindle at M phase. These similar behaviors suggested that the prawn and the crab cyclin B proteins associated with Cdc2 kinase have conserved roles in inducing GVBD and regulating the formation of meiotic spindle. The similar expression patterns of the cyclin B proteins during oocyte maturation implicated that the molecular mechanisms for MPF activation could be identical between the prawn and the crab.
In code biology we seek a presumably arbitrary, and thus symbolic relationship between two or more entities, such as the relationship of the DNA triplet code to amino acids. Here we review the differentiation code from the code biology point of view. We observe that lineage trees of mosaic organisms can be subsumed as special cases of differentiation trees of regulating embryos. The latter can be empirically discovered as a bifurcating tree of contraction and expansion differentiation waves that recursively divide the embryo into its cell types. A binary digit, 0 or 1, assigned to each wave results in a binary number corresponding to each cell type, and may be called the differentiation code for that cell type. The differentiation tree has a correspondence in the genome, in terms of the genome's logical structure. For a given cell type, the path to it from the zygote is marked epigenetically on the genome. Thus the differentiation code symbolically maps an epigenetically marked subset of the logical structure of the genome to the phenotype of a particular cell type. The waves involved and signal transduction from the cell state splitter to the genome are intermediaries in this relationship, and may also be arbitrary choices, and thus part of the code. In full, differentiation code ⇔ history along the differentiation tree of the differentiation wave types leading to a given cell type ⇔ contraction or expansion of the cell's cell state splitter ⇔ activation of one of two signal transduction pathways from the cell state splitter to the nucleus ⇔ activation of one of two readied gene cascades (the "nuclear state splitter") ⇔ epigenetic marking of the selected portion of the logical path. Each wave is in effect a cybernetic control system that results in differentiation of a set of cells and initiation of two new waves (cybernetic systems) as its goals. The differentiation code forms a basis for open evolution and its appearance was one of the major evolutionary transitions.
It has been known for more than one hundred years that cells multiply through division. It is however only during the last three decades that it has become possible to identify the molecular mechanisms that regulate cell division, especially the enzyme which literally catalyses mitotic division, MPF (M-phase promoting factor). This chapter reviews the advances in understanding MPF activation during the meiotic cell cycle. Oocytes are arrested at prophase of meiosis I. Resumption of meiosis is called meiotic maturation and is comprised of two consecutive M-phases with no intervening S-phase. Oocyte meiosis then arrests again to await fertilization. We discuss the meiosis-specific modulations in the regulation of MPF related to the release of the prophase meiotic arrest and entry into meiosis I.
We have measured the levels of cyclin mRNAs and polypeptides during oogenesis, progesterone-induced oocyte maturation, and immediately after egg activation in the frog, Xenopus laevis. The mRNA for each cyclin is present at a constant level of approximately 5 x 10(7) molecules per oocyte from the earliest stages of oogenesis until after fertilization. The levels of polypeptides show more complex patterns of accumulation. The B-type cyclins are first detectable in stage IV and V oocytes. Cyclin B2 polypeptide is present at approximately 2 x 10(9) molecules (150 pg) per oocyte by stage VI. The amount increases after progesterone treatment, but returns to its previous level after GVBD and undergoes no further change until it is destroyed at fertilization. Cyclin B1 is present at 4 x 10(8) molecules per oocyte in stage VI oocytes, and rises steadily during maturation, ultimately reaching similar levels to cyclin B2 in unfertilized eggs. Unlike the B-type cyclins, cyclin A is barely detectable in stage VI oocytes, and only starts to be made in significant amounts after oocytes are exposed to progesterone. A portion of all the cyclins are destroyed after germinal vesicle breakdown (GVBD), and cyclins B1 and B2 also experience posttranslational modifications during oocyte maturation. Progesterone strongly stimulates both cyclin and p34cdc2 synthesis in these oocytes, but whereas cyclin synthesis continues in eggs and after fertilization, synthesis of p34cdc2 declines strongly after GVBD. The significance of these results is discussed in terms of the activation and inactivation of maturation-promoting factor.
We have examined the time course of protein tyrosine phosphorylation in the meiotic cell cycles of Xenopus laevis oocytes and the mitotic cell cycles of Xenopus eggs. We have identified two proteins that undergo marked changes in tyrosine phosphorylation during these processes: a 42-kDa protein related to mitogen-activated protein kinase or microtubule-associated protein-2 kinase (MAP kinase) and a 34-kDa protein identical or related to p34cdc2. p42 undergoes an abrupt increase in its tyrosine phosphorylation at the onset of meiosis 1 and remains tyrosine phosphorylated until 30 min after fertilization, at which point it is dephosphorylated. p42 also becomes tyrosine phosphorylated after microinjection of oocytes with partially purified M-phase-promoting factor, even in the presence of cycloheximide. These findings suggest that MAP kinase, previously implicated in the early responses of somatic cells to mitogens, is also activated at the onset of meiotic M phase and that MAP kinase can become tyrosine phosphorylated downstream from M-phase-promoting factor activation. We have also found that p34 goes through a cycle of tyrosine phosphorylation and dephosphorylation prior to meiosis 1 and mitosis 1 but is not detectable as a phosphotyrosyl protein during the 2nd through 12th mitotic cell cycles. It may be that the delay between assembly and activation of the cyclin-p34cdc2 complex that p34cdc2 tyrosine phosphorylation provides is not needed in cell cycles that lack G2 phases. Finally, an unidentified protein or group of proteins migrating at 100 to 116 kDa increase in tyrosine phosphorylation throughout maturation, are dephosphorylated or degraded within 10 min of fertilization, and appear to cycle between low-molecular-weight forms and high-molecular-weight forms during early embryogenesis.
We have purified to near homogeneity the M-phase-specific protein kinase from starfish oocytes at first meiotic metaphase, using an improved procedure based on affinity chromatography on the immobilized yeast protein suc1. As already reported, this is identical to MPF, the cytoplasmic factor that controls entry of eukaryotic cells into M-phase. MPF is a complex formed by the stoichiometric association of a 34-kd polypeptide previously identified as cdc2 with a polypeptide that migrates with the same mobility as starfish cyclin in SDS-PAGE (apparent mol. wt 47 kd). A cDNA clone encoding starfish cyclin B has been isolated and its sequence determined. It contains a single open reading frame encoding a predicted 43 729-dalton protein. Partial microsequencing of the 47-kd polypeptide component of MPF allowed its identification as the starfish cyclin. Since the apparent mol. wt of native starfish MPF was found to be less than 100 kd, it is a heterodimer comprising one molecule of cdc2 and one molecule of cyclin B.
Early in the development of many animals, before transcription begins, any change in the pattern of protein synthesis is attributable to a change in the translational activity or stability of an mRNA in the egg. As a result, translational control is critical for a variety of developmental decisions, including axis formation in Drosophila and sex determination in Caenorhabditis elegans. Previous work demonstrated that increases in poly(A) length can activate translation, whereas removal of poly(A) can prevent it. In this report we focus on the control of c-mos and cyclin A1, B1, and B2 mRNAs during meiotic maturation and after fertilization of frog eggs. We show that addition and removal of poly(A) from these mRNAs is extensively regulated: The time at which each mRNA receives or loses poly(A), as well as the number of adenosines it gains or loses, differ substantially. Signals in the 3'-untranslated region (UTR) of each mRNA are sufficient to reconstitute both the temporal and quantitative control of poly(A) addition: Chimeric mRNAs in which a luciferase-coding region is joined to the 3' UTRs of cyclin A1, cyclin B1, or c-mos mRNA, receive poly(A) of the same length and at the same time as do the endogenous mRNAs. Moreover, each 3' UTR also regulates translation of the chimeric mRNAs, determining when and how much translation of the luciferase reporter is stimulated during maturation. The magnitude of stimulation in luciferase activity varies from 5- to 100-fold, depending on the 3' UTR. Translational stimulation by each 3' UTR requires poly(A) lengthening, as it is prevented by mutations that prevent that process. These results suggest that the 3' UTRs of cyclin and c-mos mRNAs control not only whether or not an mRNA is turned on during maturation, but when that activation occurs and to what extent. Translational control of c-mos mRNA, which may be achieved through regulation of poly(A) length, may be critical in the activation of maturation, and in the onset of cleavage divisions. Our findings, as well as those of others, suggest that even quite complex patterns of translational activation in the early embryo can be attained through the differential control of a common mechanism.
Mitotic cyclins are abruptly degraded at the end of mitosis by a cell-cycle-regulated ubiquitin-dependent proteolytic system. To understand how cyclin is recognized for ubiquitin conjugation, we have performed a mutagenic analysis of the destruction signal of mitotic cyclins. We demonstrate that an N-terminal cyclin B segment as short as 27 residues, containing the 9-amino-acid destruction box, is sufficient to destabilize a heterologous protein in mitotic Xenopus extracts. Each of the three highly conserved residues of the cyclin B destruction box is essential for ubiquitination and subsequent degradation. Although an intact destruction box is essential for the degradation of both A- and B-type cyclins, we find that the Xenopus cyclin A1 destruction box cannot functionally substitute for its B-type counterpart, because it does not contain the highly conserved asparagine necessary for cyclin B proteolysis. Physical analysis of ubiquitinated cyclin B intermediates demonstrates that multiple lysine residues function as ubiquitin acceptor sites, and mutagenic studies indicate that no single lysine residue is essential for cyclin B degradation. This study defines the key residues of the destruction box that target cyclin for ubiquitination and suggests there are important differences in the way in which A- and B-type cyclins are recognized by the cyclin ubiquitination machinery.
M-phase promoting factor or maturation promoting factor, a key regulator of the G2-->M transition of the cell cycle, is a complex of cdc2 and a B-type cyclin. We have previously shown that Xenopus cyclin B1 has five sites of Ser phosphorylation, four of which map to a recently identified cytoplasmic retention signal (CRS). The CRS appears to be responsible for the cytoplasmic localization of B-type cyclins, although the underlying mechanism is still unclear. Phosphorylation of cyclin B1 is not required for cdc2 binding or cdc2 kinase activity. However, when all of the Ser phosphorylation sites in the CRS are mutated to Ala to abolish phosphorylation, the mutant cyclin B1Ala is inactivated; activity can be enhanced by mutation of these residues to Glu to mimic phosphoserine, suggesting that phosphorylation of cyclin B1 is required for its biological activity. Here we show that biological activity can be restored to cyclin B1Ala by appending either a nuclear localization signal (NLS), or a second CRS domain with the Ser phosphorylation sites mutated to Glu, while fusion of a second CRS domain with the Ser phosphorylation sites mutated to Ala inactivates wild-type cyclin B1. Nuclear histone H1 kinase activity was detected in association with cyclin B1Ala targeted to the nucleus by a wild-type NLS, but not by a mutant NLS. These results demonstrate that nuclear translocation mediates the biological activity of cyclin B1 and suggest that phosphorylation within the CRS domain of cyclin B1 plays a regulatory role in this process. Furthermore, given the similar in vitro substrate specificity of cyclin-dependent kinases, this investigation provides direct evidence for the hypothesis that the control of subcellular localization of cyclins plays a key role in regulating the biological activity of cyclin-dependent kinase-cyclin complexes.
Cytoplasmic polyadenylation controls the translation of several maternal mRNAs during Xenopus oocyte maturation and requires two sequences in the 3' untranslated region (UTR), the U-rich cytoplasmic polyadenylation element (CPE), and the hexanucleotide AAUAAA. c-mos mRNA is polyadenylated and translated soon after the induction of maturation, and this protein kinase is necessary for a kinase cascade culminating in cdc2 kinase (MPF) activation. Other mRNAs are polyadenylated later, around the time of cdc2 kinase activation. To determine whether there is a hierarchy in the cytoplasmic polyadenylation of maternal mRNAs, we ablated c-mos mRNA with an antisense oligonucleotide. This prevented histone B4 and cyclin A1 and B1 mRNA polyadenylation, indicating that the polyadenylation of these mRNAs is Mos dependent. To investigate a possible role of cdc2 kinase in this process, cyclin B was injected into oocytes lacking c-mos mRNA. cdc2 kinase was activated, but mitogen-activated protein kinase was not. However, polyadenylation of cyclin B1 and histone B4 mRNA was still observed. This demonstrates that cdc2 kinase can induce cytoplasmic polyadenylation in the absence of Mos. Our data further indicate that although phosphorylation of the CPE binding protein may be involved in the induction of Mos-dependent polyadenylation, it is not required for Mos-independent polyadenylation. We characterized the elements conferring Mos dependence (Mos response elements) in the histone B4 and cyclin B1 mRNAs by mutational analysis. For histone B4 mRNA, the Mos response elements were in the coding region or 5' UTR. For cyclin B1 mRNA, the main Mos response element was a CPE that overlaps with the AAUAAA hexanucleotide. This indicates that the position of the CPE can have a profound influence on the timing of cytoplasmic polyadenylation.
In Xenopus, cdc2 tyrosine phosphorylation is detected in the first 60-75 minute cell cycle but not in the next eleven cell cycles (cycles 2-12) which are only 30 minutes long. Here we report that the wee1/cdc25 ratio increases before the first mitotic interphase. We show that the Xe-wee1 protein is absent in stage VI oocytes and is expressed from meiosis II until gastrulation. A dominant negative form of Xe-wee1 (KM wee1) reduced the level cdc2 tyrosine phosphorylation and length of the first cycle. However, the ratio of wee1/cdc25 did not decrease after the first cycle and therefore did not explain the lack of cdc2 tyrosine phosphorylation in, nor the rapidity of, cycles 2-12. Furthermore, there was no evidence for a wee1/myt1 inhibitor in cycles 2-12. We examined the role of Mos in the first cycle because it is present during the first 20 minutes of this cycle. We arrested the rapid embryonic cell cycle (cycle 2 or 3) with Mos and restarted the cell cycle with calcium ionophore; the 30 minute cycle was converted into a 60 minute cycle, with cdc2 tyrosine phosphorylation. In addition, the injection of a non-degradable Mos (MBP-Mos) into the first cycle resulted in a dramatic elongation of this cycle (to 140 minutes). MBP-Mos did not delay DNA replication or the translation of cyclins A or B; it did, however, result in the marked accumulation of tyrosine phosphorylated cdc2. Thus, while the wee1/cdc25 ratio changes during development, these changes may not be responsible for the variety of cell cycles observed during early Xenopus embryogenesis. Our experiments indicate that Mos/MAPK can also contribute to cell cycle length.
Activation of the Cyclin B/Cdc2 kinase complex triggers entry into mitosis in all eukaryotic cells. Cyclin B1 localization changes dramatically during the cell cycle, precipitously transiting from the cytoplasm to the nucleus at the beginning of mitosis. Presumably, this relocalization promotes the phosphorylation of nuclear targets critical for chromatin condensation and nuclear envelope breakdown. We show here that the previously characterized cytoplasmic retention sequence of Cyclin B1, responsible for its interphase cytoplasmic localization, is actually an autonomous nuclear export sequence, capable of directing nuclear export of a heterologous protein, and able to bind specifically to the recently identified export mediator, CRM1. We propose that the observed cytoplasmic localization of Cyclin B1 during interphase reflects the equilibrium between ongoing nuclear import and rapid CRM1-mediated export. In support of this hypothesis, we found that treatment of cells with leptomycin B, which disrupted Cyclin B1-CRM1 interactions, led to a marked nuclear accumulation of Cyclin B1. In mitosis, Cyclin B1 undergoes phosphorylation at several sites, a subset of which have been proposed to play a role in Cyclin B1 accumulation in the nucleus. Both CRM1 binding and the ability to direct nuclear export were affected by mutation of these phosphorylation sites; thus, we propose that Cyclin B1 phosphorylation at the G2/M transition prevents its interaction with CRM1, thereby reducing nuclear export and facilitating nuclear accumulation.
Xenopus oocytes and eggs provide a dramatic example of how the consequences of p42 mitogen-activated protein kinase (p42 MAPK) activation depend on the particular context in which the activation occurs. In oocytes, the activation of Mos, MEK, and p42 MAPK is required for progesterone-induced Cdc2 activation, and activated forms of any of these proteins can bring about Cdc2 activation in the absence of progesterone. However, in fertilized eggs, activation of the Mos/MEK/p42 MAPK pathway has the opposite effect, inhibiting Cdc2 activation and causing a G2 phase delay or arrest. In the present study, we have investigated the mechanism and physiological significance of the p42 MAPK-induced G2 phase arrest, using Xenopus egg extracts as a model system. We found that Wee1-depleted extracts were unable to arrest in G2 phase in response to Mos, and adding back Wee1 to the extracts restored their ability to arrest. This finding formally places Wee1 downstream of Mos/MEK/p42 MAPK. Purified recombinant p42 MAPK was found to phosphorylate recombinant Wee1 in vitro at sites that are phosphorylated in extracts. Phosphorylation by p42 MAPK resulted in a modest ( approximately 2-fold) increase in the kinase activity of Wee1 toward Cdc2. Titration experiments in extracts demonstrated that a twofold increase in Wee1 activity is sufficient to cause the delay in mitotic entry seen in Mos-treated extracts. Finally, we present evidence that the negative regulation of Cdc2 by Mos/MEK/p42 MAPK contributes to the presence of an unusually long G2 phase in the first mitotic cell cycle. Prematurely inactivating p42 MAPK in egg extracts resulted in a corresponding hastening of the first mitosis. The negative effect of p42 MAPK on Cdc2 activation may help ensure that the first mitotic cell cycle is long enough to allow karyogamy to be accomplished successfully.
Mitotic cyclins A and B contain a conserved N-terminal helix upstream of the cyclin box fold that contributes to a significant
interface between cyclin and cyclin-dependent kinase (CDK). To address its contribution on cyclin-CDK interaction, we have
constructed mutants in conserved residues of the N-terminal helix of Xenopus cyclins B2 and A1. The mutants showed altered binding affinities to Cdc2 and/or Cdk2. We also screened for mutations in the
C-terminal lobe of CDK that exhibited different binding affinities for the cyclin-CDK complex. These mutations were at residues
that interact with the cyclin N-terminal helix motif. The cyclin N-terminal helix mutations have a significant effect on the
interaction between the cyclin-CDK complex and specific substrates, Xenopus Cdc6 and Cdc25C. These results suggest that the N-terminal helix of mitotic cyclins is required for specific interactions
with CDKs and that to interact with CDK, specific substrates Cdc6 and Cdc25C require the CDK to be associated with a cyclin.
The interaction between the cyclin N-terminal helix and the CDK C-terminal lobe may contribute to binding specificity of the
cyclin-CDK complex.
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.
Fully grown Xenopus oocyte is arrested at prophase I of meiosis. Re-entry into meiosis depends on the activation of MPF (M-phase promoting factor or cyclin B.Cdc2 complex), triggered by progesterone. The prophase-arrested oocyte contains a store of Cdc2. Most of the protein is present as a monomer whereas a minor fraction, called pre-MPF, is found to be associated with cyclin B. Activation of Cdc2 depends on two key events: cyclin binding and an activating phosphorylation on Thr-161 residue located in the T-loop. To get new insights into the regulation of Thr-161 phosphorylation of Cdc2, monomeric Cdc2 was isolated from prophase oocytes. Based on its activation upon cyclin addition and detection by an antibody directed specifically against Cdc2 phosphorylated on Thr-161, we show for the first time that the prophase oocyte contains a significant amount of monomeric Cdc2 phosphorylated on Thr-161. PP2C, a Mg2+-dependent phosphatase, negatively controls Thr-161 phosphorylation of Cdc2. The unexpected presence of a population of free Cdc2 already phosphorylated on Thr-161 could contribute to the generation of the Cdc2 kinase activity threshold required to initiate MPF amplification.
Cyclin B is a regulatory subunit of CDK1 within MPF complex. Degradation of cyclin B via ubiquitin-proteasome pathway seemed to be absolutely required for the M-phase exit. However, inhibition of the proteasome proteolytic activity upon the exit from the meiotic metaphase II-arrest in Xenopus cell-free extract revealed that the proteasome-dependent dissociation of cyclin B from CDK1 is sufficient to inactivate MPF without cyclin B degradation. In this study we analyze whether the same mechanism operates during the exit from mitotic M-phase. We show in Xenopus cell-free extract undergoing the first or the second embryonic mitosis that CDK1 oscillations are not affected by proteasome inhibition with MG132 or ALLN despite effective inhibition of cyclins B degradation. The majority of cyclins B1 and B2 surviving CDK1 inactivation is CDK-free and cyclin B2 becomes resistant to phosphatase lambda dephosphorylation. The pool of cyclins B remaining after CDK1 inactivation in the presence of MG132 is mitotically inert, while exogenous or newly synthesized cyclin B activates CDK1. This suggests that cyclins B remain sequestered within the proteasome upon MPF inactivation in the presence of MG132. Comparison of the dynamics of the decline of total and CDK-bound pools of cyclins B1, B2 and B4 upon mitotic exit in absence of protein synthesis reveals that CDK-bound cyclins B diminish clearly faster. Our results thus show that cyclin B dissociation from CDK1 precedes cyclins B degradation upon CDK1 inactivation in mitotic embryo extracts and that proteasome proteolytic activity is dispensable for both activation and inactivation of CDK1 in such extracts.
Inhibition of cyclin-dependent kinase 1 (CDK1) activity by Tyr-15 phosphorylation directly regulates entry into mitosis and
is an important element in the control of the unperturbed cell cycle. Active site phosphorylation of other members of the
CDK family that regulate cell cycle progression instates checkpoints that are fundamental to eukaryotic cell cycle regulation.
Kinetic and crystallographic analyses of CDK2-cyclin A complexes reveal that this inhibitory mechanism operates through steric
blockade of peptide substrate binding and through the creation of an environment that favors a non-productive conformation
of the terminal group of ATP. By contrast, tyrosine phosphorylation of CDK2 alters neither its Km for ATP nor its significant intrinsic ATPase activity. Tyr-15-phosphorylated CDK2 retains trace protein phosphorylation activity
that should be considered in quantitative and qualitative cell cycle models.
The present work is based on the study of individual cell cycle times for a given category of cells. The material considered in detail is the Axolotl embryo during the 11th cleavage cycle. In spite of the exceptional homogeneity of this population, individual cycle times show a remarkable variation from cell to cell, coinciding with a characteristic statistical distribution. To describe the kinetics of cell proliferation, we propose a model for which the theoretical distribution of cycle times fits with the distribution observed in our material. Numerous observations allow us to generalize the model to other types of populations. According to this concept the notion of cell cycle time disappears in favour of the notion of a statistical distribution of individual cycle times. This variability is integral to the cell division process itself. We suggest that in the cycle there is a particular event the probability of occurrence of which is constant beginning with a critical state. The cells would therefore remain a certain time in this state overcoming it at a characteristic rate. To this exponential distribution variability would be added the variability of other events whose cumulative effects would result in a normal distribution. The resultant of both factors conforms to the frequency distribution implicated in our kinetic model. Discussing in a more general way the distributions of the cycle times of each cell division during cleavage, we propose the following interpretation of this development. The introduction of new events in the cyclic process would imply the switching on of certain essential genetic activities. The final consequences would be the desynchronization and the lengthening of the cycles observed at the blastula stage. Thus considered, this period of embryonic development would be eminently suitable for the study of the factors of cell division control.
The process of segmentation was investigated in a comparative manner on axolotl embryos possessing 3 types of cytoplasm (large eggs obtained spontaneously; small eggs obtained spontaneously; 'small eggs' obtained artificially), and 2 types of nuclei (diploid; haploid). The following results were obtained: the rhythm of cleavage during synchronous phase is a specific characteristic of the cytoplasm, independent of quantitative modifications of either the nucleus or the cytoplasm; the precise time that the blastula transition appears depends on a qualitative cytoplasmic factor and a quantitative nuclear factor; and the time of occurrence of gastrulation seems to be a characteristic depending on the cytoplasm as does the rhythm of cleavage.
Study of the incorporation of 3H-uridine in cleaving embryo of Axolotl has shown a nuclear RNA synthesis during the period of synchronous cleavage (6th cycle) as well as after the onset of asynchronous divisions (9th or 10th cycle). In the early development of the Axolotl, the extent of the transcription phase looks to be an essential element of the quantitative control of gene activity.
We have measured the levels of cyclin mRNAs and polypeptides during oogenesis, progesterone-induced oocyte maturation, and immediately after egg activation in the frog, Xenopus laevis. The mRNA for each cyclin is present at a constant level of approximately 5 x 10(7) molecules per oocyte from the earliest stages of oogenesis until after fertilization. The levels of polypeptides show more complex patterns of accumulation. The B-type cyclins are first detectable in stage IV and V oocytes. Cyclin B2 polypeptide is present at approximately 2 x 10(9) molecules (150 pg) per oocyte by stage VI. The amount increases after progesterone treatment, but returns to its previous level after GVBD and undergoes no further change until it is destroyed at fertilization. Cyclin B1 is present at 4 x 10(8) molecules per oocyte in stage VI oocytes, and rises steadily during maturation, ultimately reaching similar levels to cyclin B2 in unfertilized eggs. Unlike the B-type cyclins, cyclin A is barely detectable in stage VI oocytes, and only starts to be made in significant amounts after oocytes are exposed to progesterone. A portion of all the cyclins are destroyed after germinal vesicle breakdown (GVBD), and cyclins B1 and B2 also experience posttranslational modifications during oocyte maturation. Progesterone strongly stimulates both cyclin and p34cdc2 synthesis in these oocytes, but whereas cyclin synthesis continues in eggs and after fertilization, synthesis of p34cdc2 declines strongly after GVBD. The significance of these results is discussed in terms of the activation and inactivation of maturation-promoting factor.
After fertilization in axolotl, the synchronous cell cleavages are triphasic (S, G2 and M phases). Midblastula transition (MBT) begins at the ninth cleavage and is the consequence of lengthening of cell cycles. By spectro-fluorometry and incorporation of 3H thymidine into the nuclear DNA followed by autoradiography on individual cells, the time at which a G1 phase appears during early development was investigated. The present results show that the G1 phase was introduced for the first time at MBT and its duration was variable from one blastomere to another. This variability could account for lengthening of cell cycles and be required for zygotic transcriptions necessary for DNA replication. From this point of view, axolotl represents an interesting alternative amphibian model to identify regulators involved in the G1–S transition at MBT during early development.
Fully grown oocytes of the frog (Rana pipiens) undergo cytoplasmic and nuclear maturation when treated with progesterone after the follicular envelopes have been removed. The mechanism of this maturation was investigated by injection of cytoplasm from progesterone-treated oocytes at various stages of maturation into fully grown but immature oocytes. The injected cytoplasm becomes effective in inducing maturation by 12 hours after progesterone administration, reaches a maximum effectiveness around 20 hours, and then declines after the donor oocytes complete maturation. However, even cytoplasm from early embryos retains some capacity to induce oocyte maturation. The frequency with which maturation is induced is proportional to the volume of the injected cytoplasm. Progesterone itself is not directly responsible for the maturation-producing effect of injected cytoplasm since injected progesterone does not promote maturation. However, externally applied progesterone does induce the completion of the first meiotic division, presumably by releasing a cytoplasmic “maturation promoting factor.” The production of this cytoplasmic factor was not affected by removal of the nucleus.
When full-grown oocytes of the newt Cynops pyrrhogaster were treated with progesterone in O-R2 solution containing antibiotics, approximately 85% of the oocytes completed meiosis synchronously. Maturation-promoting factor (MPF) activity appeared just before germinal vesicle breakdown (GVBD) and the oocytes maintained high MPF activity throughout metaphase I and metaphase II of meiosis. A slight decrease of MPF activity was observed at the first polar body emission. The distribution of cyclin B1 was investigated with anti-cyclin B1 antibody. No cyclin B1 was found in the oocytes before progesterone treatment. Cyclin B1 appeared in the cortex of animal hemispheres, especially around and inside germinal vesicle just before GVBD. A large amount of cyclin B1 accumulated at metaphase I, approximately half disappeared at the first polar body emission, and then cyclin B1 accumulated again at metaphase II. An inactive form of cdc2 kinase was observed in both the germinal vesicles and the oocyte cytoplasm, while an active form appeared at the M phase. No MPF was observed in the oocytes from which the germinal vesicle had been removed. A cdk7-like molecule was localized in the germinal vesicle, but not in oocyte cytoplasm, indicating that inactive cdc2 kinase associated with cyclin B1 derived from cytoplasm is activated by phosphorylation in the germinal vesicle. The changes in the amount of cyclin B1 were synchronous with the first cell cycle after fertilization. Cyclin B1 was primarily localized in the cortex of the animal hemisphere. A shift in band mobility upon electrophoresis of cyclin B1 was observed from samples taken during the cell cycle; this shift was probably due to the protein's phosphorylation state.
Study of the incorporation of 3H-uridine in cleaving embryo of Axolotl has shown a nuclear RNA synthesis during the period of synchronous cleavage (6th cycle) as well as after the onset of asynchronous divisions (9th or 10th cycle). In the early development of the Axolotl, the extent of the transcription phase looks to be an essential element of the quantitative control of gene activity.
Illustrations and descriptions of normal stages of the axolotl Ambystoma mexicanum are given. The time to reach each morphological stage is compared with similar stages in two other species, Ambystoma maculatum and Taricha torosa.
The cyclins are a family of proteins encoded by maternal mRNA. Cyclin polypeptides accumulate during interphase and are destroyed during mitosis at about the time of entry into anaphase. We show here that Xenopus oocytes contain mRNAs encoding two cyclins that are major translation products in a cell-free extract from activated eggs. Cutting these mRNAs with antisense oligonucleotides and endogenous RNAase H blocks entry into mitosis in a cell-free egg extract. The extracts can enter mitosis if either of the cyclin mRNAs is left intact. We conclude that the synthesis of these cyclins is necessary for mitotic cell cycles in cleaving Xenopus embryos.
When an M-phase promoting factor (MPF) is injected into Xenopus oocytes, which are naturally arrested at the G2/prophase boundary, it induces rapid entry of the cells into M-phase. MPF is present in late G2 and in M-phase of a variety of cell types, such as Xenopus eggs (naturally arrested in M), cleaving embryos, yeast, HeLa, and CHO cultures. MPF has been purified approximately 50-fold from eggs. It is stabilized by gamma-thio-ATP and by phosphoprotein phosphatase inhibitors. It runs as a protein of approximately 100 kd size on gel filtration. Oocytes contain a precursor of MPF, which is activated by post-translational means when a small amount of purified MPF is injected into the cell. Thus, MPF appears to be an auto-activating cytoplasmic trigger of M-phase. At anaphase of the cell cycle, MPF is inactivated due to the appearance of an 'anti-MPF' activity. Monoclonal antibodies have been prepared to partially purified MPF stabilized by gamma-thio-ATP, and several preparations which inactivate MPF were obtained. The antibodies are directed against thio-phosphate groups carried by a set of proteins including MPF. This indicates that MPF is present in our active preparations as a thio-phosphoprotein. These and other data suggest that MPF is normally activated in the cell cycle by a phosphorylation reaction.
The Xenopus embryo undergoes 12 rapid synchronous cleavages followed by a period of slower asynchronous divisions more typical of somatic cells. This change in cell cleavage has been termed the midblastula transition (MBT). We show that at the MBT the blastomeres become motile and transcriptionally active for the first time. We have investigated the timing of the MBT and found that it does not depend on cell division, on time since fertilization or on a counting mechanism involving the sequential modification of DNA. Rather, the timing of the MBT depends on reaching a critical ratio of nucleus to cytoplasm. We view the MBT as a consequence of the titration of some substance, originally present in the egg, by the exponentially increasing nuclear material. When this substance is exhausted a new cell program is engaged, leading to the acquisition of several new cell properties.
The injection of amphibian oocytes was one of the first systems in which purified DNA was correctly transcribed and expressed as protein. Amphibian oocytes have three characteristics. First, a very small amount of DNA needs to be injected into a single oocyte to obtain recognizable transcription and expression. Second, the expression of DNA can be monitored within a few hours of injection, during which time it is not replicated or integrated into host cell chromosomes, but is assembled with nucleosomes into an apparently normal chromatin structure. In most other expression systems, DNA is integrated and replicated, as a result of which it may undergo genetic changes and its expression may be influenced by the properties of adjacent host DNA. Third, oocyte injection makes it possible to introduce any cell components, such as RNA, chromatin, or nuclear proteins; this is likely to be particularly valuable for analyzing the regulation of gene expression.
A procedure is described for the large-scale purification of light (L) and heavy (H) chain mRNAs from plasmacytomas produced in mice. Intact RNA is selectively precipitated in high yield from frozen tumors homogenized in 3 M LiCl and 6 M urea. L and H-chain mRNAs were purified by oligo(dT)-cellulose chromatography and either sucrose gradient centrifugation in conditions preventing aggregation or by means of high-resolution preparative gel electrophoresis under non-denaturing conditions. gamma 2a and alpha H-chain mRNAs sedimented as major components at 15.5 S and 16.5 S respectively, when L-chain mRNAs sedimented as 12-S species. H-chain mRNAs isolated by continuous elution during preparative gel electrophoresis were completely separated from both L-chain mRNA and residual 18-S rRNA, and migrated as single components of 1900 +/- 50 nucleotides on analytical denaturing gels. The partially purified H-chain mRNAs were translated into major components of molecular weights of 56,000 (gamma 2a) and 60,000 (alpha) in an mRNA-dependent rabbit reticulocyte lysate, whereas L-chain mRNAs yielded polypeptides of molecular weights of 25,000 (gamma) and 27,000 (chi). Up to 95% of the translation products directed by the purified mRNAs were immunoprecipitated using specific antisera. The purity of L and H-chain mRNAs was assessed by hybridization of corresponding cDNAs with excess recombinant plasmid DNA. The results indicated a minimum purity of 47% (gamma 2a), 62% (alpha), for H-chain mRNAs and 60% (chi), for L-chain mRNAs.
Early development of an amphibian egg does not depend on the genetic activity of its nuclei. Rather, early development is guided by a stockpile of gene products accumulated during oogenesis. Later, the zygotic genome participates in development and it is only then that one can detect, in a progressive manner, the intervention of the individual’s own genome for any particular developmental event.
Oocyte maturation is triggered by the activation in the oocyte cytoplasm of maturation-promoting factor (MPF), which consists of cdc2 (a catalytic subunit) and cyclin B (a regulatory subunit). Immature goldfish oocytes contain only inactive monomeric 35-kDa cdc2 and do not stockpile cyclin B. In maturing oocytes, activation of cdc2 is associated with its Thr161 phosphorylation and mobility shift on SDS-PAGE from 35 to 34 kDa after binding to cyclin B. Using mutant cdc2, we show that Thr161 phosphorylation is required for both the downward shift and the kinase activation. Since cdc2 Tyr15 is not phosphorylated after binding to cyclin B, it does not require dephosphorylation. This situation is obviously different from that in immature Xenopus oocytes, in which the cdc2-cyclin B complex preexists with cdc2 phosphorylated on both Tyr15 and Thr161, thereby requiring Tyr15 dephosphorylation catalyzed by cdc25 phosphatase for MPF activation. These results indicate that these species employ different mechanisms of MPF activation during oocyte maturation, although the final molecular structure of the active MPF (cdc2 bound to cyclin B and phosphorylated on Thr161) is identical.
We report here the first extensive in vivo study of cell cycle regulation in the Xenopus embryo. Cyclin A1, B1, B2, and E1 levels, Cdc2 and Cdk2 kinase activity, and Cdc25C phosphorylation states were monitored during early Xenopus embryonic cell cycles. Cyclin B1 and B2 protein levels were high in the unfertilized egg, declined upon fertilization, and reaccumulated to the same level during the first cell cycle, a pattern repeated during each of the following 11 divisions. Cyclin A1 showed a similar pattern, except that its level was lower in the egg than in the cell cycles after fertilization. Cyclin B1/Cdc2 kinase activity oscillated, peaking before each cleavage, and Cdc25C alternated between a highly phosphorylated and a less phosphorylated form that correlated with high and low cyclin B1/Cdc2 kinase activity, respectively. Unlike the mitotic cyclins, the level of cyclin E1 did not oscillate during embryogenesis, although its associated Cdk2 kinase activity cycled twice for each oscillation of cyclin B1/Cdc2 activity, consistent with a role for cyclin E1 in both S-phase and mitosis. Although the length of the first embryonic cycle is regulated by both the level of cyclin B and the phosphorylation state of Cdc2, cyclin accumulation alone was rate-limiting for later cycles, since overexpression of a mitotic cyclin after the first cycle caused cell cycle acceleration. The activity of Cdc2 closely paralleled the accumulation of cyclin B2, but cell cycle acceleration caused by cyclin B overexpression was not associated with elevation of Cdc2 activity to higher than metaphase levels. Tyrosine phosphorylation of Cdc2, absent during cycles 2-12, reappeared at the midblastula transition coincident with the disappearance of cyclin E1. Cyclin A1 disappeared later, at the beginning of gastrulation. Our results suggest that the timing of the cell cycle in the Xenopus embryo evolves from regulation by accumulation of mitotic cyclins to mechanisms involving periodic G1 cyclin expression and inhibitory tyrosine phosphorylation of Cdc2.
Regulation of both the cell cycle and gene transcription is essential for orderly progression of cell growth and division. Recent results on the structures of two cyclins, cyclin A and cyclin H, and two transcription factor mediator proteins, TFIIB and the A pocket region of the retinoblastoma tumour suppressor protein (Rb), show that they share domains with a strikingly similar alpha-helical topology, despite remote sequence identity.
Oocyte maturation is finally triggered by the maturation-promoting factor (MPF), which consists of Cdc2 and cyclin B. We have cloned cDNAs encoding frog (Rana japonica) cyclins B1 and B2 and produced antibodies against their products. Using the antibodies, we investigated changes in protein states and levels of Cdc2 and cyclins B1 and B2 during oocyte maturation. In immature oocytes, all Cdc2 was a monomeric unphosphorylated inactive 35 kDa form and neither cyclin B1 nor cyclin B2 was present. Mature oocytes contained the MPF complex consisting of an active 34 kDa Cdc2 phosphorylated on threonine161 and a 49 kDa cyclin B1 or a 51 kDa cyclin B2. After progesterone stimulation, both cyclins B1 and B2 were synthesized from their stored mRNAs and bound to the preexisting 35 kDa Cdc2. The binding of Cdc2 with cyclin B and its activation probably through the phosphorylation on threonine161 occurred at almost the same time, in accordance with an electrophoretic mobility shift of Cdc2 from 35 to 34 kDa. Microinjection into immature oocytes of cyclin B1 or B2 mRNA alone, or a mixture of them, induced germinal vesicle breakdown (GVBD) with similar dose-dependence. When the translation of endogenous mRNAs of both cyclins B1 and B2 was inhibited with antisense RNAs, progesterone failed to induce GVBD in the oocytes, but the inhibition of only one of the two was unable to inhibit the progesterone-induced GVBD. These results indicate that either cyclin B1 or B2 is necessary and sufficient for inducing GVBD during Rana oocyte maturation.
After fertilization in axolotl, the synchronous cell cleavages are triphasic (S, G2 and M phases). Midblastula transition (MBT) begins at the ninth cleavage and is the consequence of lengthening of cell cycles. By spectrofluorometry and incorporation of 3H thymidine into the nuclear DNA followed by autoradiography on individual cells, the time at which a G1 phase appears during early development was investigated. The present results show that the G1 phase was introduced for the first time at MBT and its duration was variable from one blastomere to another. This variability could account for lengthening of cell cycles and be required for zygotic transcriptions necessary for DNA replication. From this point of view, axolotl represents an interesting alternative amphibian model to identify regulators involved in the G1-S transition at MBT during early development.
The poly(A) tail present at the 3' end of most eukaryotic mRNAs can play a critical role in message translation and stability. Therefore, identifying alterations in poly(A) tail length can yield important insights into an mRNA's function and subsequent physiological impact. Here, we present three methods for assaying polyadenylation of a specific mRNA in the context of total cellular RNA. The first method described, oligo(dT)/RNase H-Northern analysis, is the classic labor-intensive assay for polyadenylation and is included for historical reference and as a potential experimental control for the poly(A) test (PAT) assays described subsequently. The PAT methods-rapid amplification of cDNA ends-PAT (RACE-PAT), and ligase-mediated PAT (LM-PAT)-are polymerase chain reaction-driven assays that allow speed, sensitivity, and length quantitation. The PAT assays can be conducted in a single day and can readily detect the poly(A) status of an mRNA present in subnanogram quantities of total cellular RNA.
During oocyte maturation, cyclin B1 mRNA is translationally activated by cytoplasmic polyadenylation. This process is dependent on cytoplasmic polyadenylation elements (CPEs) in the 3' untranslated region (UTR) of the mRNA. To determine whether a titratable factor might be involved in the initial translational repression (masking) of this mRNA, high levels of cyclin B1 3' UTR were injected into oocytes. While this treatment had no effect on the poly(A) tail length of endogenous cyclin B1 mRNA, it induced cyclin B1 synthesis. A mutational analysis revealed that the most efficient unmasking element in the cyclin 3' UTR was the CPE. However, other U-rich sequences that resemble the CPE in structure, but which do not bind the CPE-binding polyadenylation factor CPEB, failed to induce unmasking. When fused to the chloramphenical acetyl transferase (CAT) coding region, the cyclin B1 3' UTR inhibited CAT translation in injected oocytes. In addition, a synthetic 3' UTR containing multiple copies of the CPE also inhibited translation, and did so in a dose-dependent manner. Furthermore, efficient CPE-mediated masking required cap-dependent translation. During the normal course of progesterone-induced maturation, cytoplasmic polyadenylation was necessary for mRNA unmasking. A model to explain how cyclin B1 mRNA masking and unmasking could be regulated by the CPE is presented.
In mammalian cells the Cdc25 family of dual-specificity phosphatases has three distinct isoforms, termed A, B, and C, which are thought to play discrete roles in cell-cycle control. In this paper we report the cloning of Xenopus Cdc25A and demonstrate its developmental regulation and key role in embryonic cell-cycle control. Northern and Western blot analyses show that Cdc25A is absent in oocytes, and synthesis begins within 30 min after fertilization. The protein product is localized in the nucleus in interphase and accumulates continuously until the midblastula transition (MBT), after which it is degraded. Upon injection into newly fertilized eggs, wild-type Cdc25A shortened the cell cycle and accelerated the timing of cleavage, whereas embryos injected with phosphatase-dead Cdc25A displayed a dose-dependent increase in the length of the cell cycle and a slower rate of cleavage. In contrast, injection of the phosphatase-dead Cdc25C isoform had no effect. Western blotting with an antibody specific for phosphorylated tyr15 in Cdc2/Cdk2 revealed a cycle of phosphorylation/dephosphorylation in each cell cycle in control embryos, and in embryos injected with phosphatase-dead Cdc25A there was a twofold increase in the level of p-tyr in Cdc2/Cdk2. Consistent with this, the levels of cyclin B/Cdc2 and cyclin E/Cdk2 histone H1 kinase activity were both reduced by approximately 50% after phosphatase-dead Cdc25A injection. The phosphatase-dead Cdc25A could be recovered in a complex with both Cdks, suggesting that it acts in a dominant-negative fashion. These results indicate that periodic phosphorylation of Cdc2/Cdk2 on tyr15 occurs in each pre-MBT cell cycle, and dephosphorylation of Cdc2/Cdk2 by Cdc25A controls at least in part the length of the cell cycle and the timing of cleavage in pre-MBT embryos. The disappearance of Cdc25A after the MBT may underlie in part the lengthening of the cell cycle at that time.
In Xenopus development, the expression of several maternal mRNAs is regulated by cytoplasmic polyadenylation. CPEB and maskin, two factors that control polyadenylation-induced translation are present on the mitotic apparatus of animal pole blastomeres in embryos. Cyclin B1 protein and mRNA, whose translation is regulated by polyadenylation, are colocalized with CPEB and maskin. CPEB interacts with microtubules and is involved in the localization of cyclin B1 mRNA to the mitotic apparatus. Agents that disrupt polyadenylation-induced translation inhibit cell division and promote spindle and centrosome defects in injected embryos. Two of these agents inhibit the synthesis of cyclin B1 protein and one, which has little effect on this process, disrupts the localization of cyclin B1 mRNA and protein. These data suggest that CPEB-regulated mRNA translation is important for the integrity of the mitotic apparatus and for cell division.
Substrates for mitotic proteolysis such as cyclin B have a 9 residue destruction motif, the destruction box (D-box). To identify the receptor that specifically binds the D-box, we used affinity chromatography with immobilized D-box matrices. We find that the APC/C from Xenopus egg extracts binds to the D-box of cyclin B, whereas Fizzy (Cdc20) does not. Mutations in the D-box abolished this interaction. We show that this binding is regulated in the cell cycle, such that the APC/C from egg extracts in interphase does not bind to the D-box matrix. Our results suggest that the APC/C forms a stable interaction with the D-box of its substrates in a cell cycle-dependent manner.
There are two major problems for the cell to solve in mitosis: how to ensure that each daughter cell receives an equal and identical complement of the genome, and how to prevent cell separation before chromosome segregation. Both these problems are solved by controlling when two specific proteins are destroyed: securin, an inhibitor of chromosome segregation, and cyclin B, which inhibits cell separation (cytokinesis). It has recently become clear that several other proteins are degraded at specific points in mitosis. This review (which is part of the Chromosome Segregation and Aneuploidy series) focuses on how specific proteins are selected for proteolysis at defined points in mitosis and how this contributes to the proper coordination of chromosome segregation and cytokinesis.
Exp. 1.—Mortality curves for (1) haploid hybrid controls, pipiens (♀) × catesbeiana ♂; (2) enucleated pipiens eggs injected with haploid catesbeiana nuclei; and (3) control diploid hybrids, pipiens ♀ × catesbeiana ♂.
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.
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.
Contribution to the study of axolotl segmentation, I: definition of the blastulean transition
- J. Signoret
- J. Lefresne
- J. Signoret
- J. Lefresne