Proteomic screen for potential regulators of M-phase entry and quality of meiotic resumption in Xenopus laevis oocytes

The quality of oocytes depends largely on the capacity to resume meiotic maturation. In Xenopus laevis, only fully grown oocytes react to progesterone stimulation by resumption of meiotic maturation associated with the entry into the meiotic M-phase. Proteins involved in this process are poorly known. To identify novel proteins regulating M-phase entry, we performed a differential proteomic screen. We compared proteomes of fully grown stage VI oocytes characterized as poorly or highly responsive to progesterone treatment. The comparison of 2-D gels allowed us to identify several spots including two specifically present in highly responsive oocytes and two specifically present in poorly responsive ones. By mass spectrometry we identified the two proteins specifically present in highly responsive oocytes as inosine 5′monophosphate cyclohydrolase and YjgF homologues, and the two specifically present in poorly responsive oocytes as elongation factor 2 (EF2) and S-adenosyl-l-homocysteine hydrolase (SAHH). The proteins specifically expressed in highly responsive oocytes may participate in the stimulation of meiotic maturation and M-phase entry, while the proteins specifically present in poorly maturing oocytes may participate in the inhibition of meiotic resumption.     

Temporal regulation of embryonic M-phases

Temporal regulation of M-phases of the cell cycle requires precise molecular mechanisms that differ among different cells. This variable regulation is particularly clear during embryonic divisions. The first embryonic mitosis in the mouse lasts twice as long as the second one. In other species studied so far (C. elegans, Sphaerechinus granularis, Xenopus laevis), the first mitosis is also longer than the second, yet the prolongation is less pronounced than in the mouse. We have found recently that the mechanisms prolonging the first embryonic M-phase differ in the mouse and in Xenopus embryos. In the mouse, the metaphase of the first mitosis is specifically prolonged by the unknown mechanism acting similarly to the CSF present in oocytes arrested in the second meiotic division. In Xenopus, higher levels of cyclins B participate in the M-phase prolongation, however, without any cell cycle arrest. In Xenopus embryo cell-free extracts, the inactivation of the major M-phase factor, MPF, depends directly on dissociation of cyclin B from CDK1 subunit and not on cyclin B degradation as was thought before. In search for other mitotic proteins behaving in a similar way as cyclins B we made two complementary proteomic screens dedicated to identifying proteins ubiquitinated and degraded by the proteasome upon the first embryonic mitosis in Xenopus laevis. The first screen yielded 175 proteins. To validate our strategy we are verifying now which of them are really ubiquitinated. In the second one, we identified 9 novel proteins potentially degraded via the proteasome. Among them, TCTP (Translationally Controlled Tumor Protein), a 23-kDa protein, was shown to be partially degraded during mitosis (as well as during meiotic exit). We characterized the expression and the role of this protein in Xenopus, mouse and human somatic cells, Xenopus and mouse oocytes and embryos. TCTP is a mitotic spindle protein positively regulating cellular proliferation. Analysis of other candidates is in progress.     

Regulation of Cdc25C Activity During the Meiotic G2/M Transition

The Cdc25C phosphatase is a key activator of Cdc2/cyclin B that controls M-phase entry in eukaryotic cells. Here we discuss the regulation of Cdc25C by phosphorylation during the meiotic maturation of Xenopus oocytes. In G2 arrested oocytes, Cdc25C is phosphorylated on Ser287 and associated with 14-3-3 proteins. Entry of the oocytes into M-phase of meiosis is triggered by progesterone, which activates a signaling pathway leading to the dephosphorylation of Ser287, probably mediated by the PP1 phosphatase. The activation of Cdc25C during oocyte maturation correlates also with its phosphorylation on multiple sites. These phosphorylations involve several signaling pathways, including Polo kinases and MAP kinases, and might require also the inhibition of the PP2A phosphatase. Finally, Cdc25C is further phosphorylated by its substrate Cdc2/cyclin B, as part of an auto-amplification loop that ensures the high Cdc2/cyclin B activity level required to drive the oocyte through the meiotic cell cycle.     

Proteomics reveals a switch in CDK1-associated proteins upon M-phase exit during the Xenopus laevis oocyte to embryo transition

Cyclin-dependent kinase 1 (CDK1) is a major M-phase kinase which requires the binding to a regulatory protein, Cyclin B, to be active. CDK1/Cyclin B complex is called M-phase promoting factor (MPF) for its key role in controlling both meiotic and mitotic M-phase of the cell cycle. CDK1 inactivation is necessary for oocyte activation and initiation of embryo development. This complex process requires both Cyclin B polyubiquitination and proteosomal degradation via the ubiquitin-conjugation pathway, followed by the dephosphorylation of the monomeric CDK1 on Thr161. Previous proteomic analyses revealed a number of CDK1-associated proteins in human HeLa cells. It is, however, unknown whether specific partners are involved in CDK1 inactivation upon M-phase exit. To better understand CDK1 regulation during MII-arrest and oocyte activation, we immunoprecipitated (IPed) CDK1 together with its associated proteins from M-phase-arrested and M-phase-exiting Xenopus laevis oocytes. A mass spectrometry (MS) analysis revealed a number of new putative CDK1 partners. Most importantly, the composition of the CDK1-associated complex changed rapidly during M-phase exit. Additionally, an analysis of CDK1 complexes precipitated with beads covered with p9 protein, a fission yeast suc1 homologue well known for its high affinity for CDKs, was performed to identify the most abundant proteins associated with CDK1. The screen was auto-validated by identification of: (i) two forms of CDK1: Cdc2A and B, (ii) a set of Cyclins B with clearly diminishing number of peptides identified upon M-phase exit, (iii) a number of known CDK1 substrates (e.g. peroxiredoxine) and partners (e.g. HSPA8, a member of the HSP70 family) both in IP and in p9 precipitated pellets. In IP samples we also identified chaperones, which can modulate CDK1 three-dimensional structure, as well as calcineurin, a protein necessary for successful oocyte activation. These results shed a new light on CDK1 regulation via a dynamic change in the composition of the protein complex upon M-phase exit and the oocyte to embryo transition.     

Transient reactivation of CSF in parthenogenetic one-cell mouse embryos

During meiosis, the cytostatic factor (CSF) activity stabilizes the activity of the M-phase promoting factor (MPF) in metaphase II arrested vertebrate oocytes. Upon oocyte activation, the inactivation of both MPF and CSF enables the entry into the first embryonic mitotic cell cycle. Using a biological assay based on cell-fusion (hybrid between a parthenogenetically activated egg entering the first mitotic division and an activated oocyte), we observed that in activated mouse oocytes a first drop in CSF activity is detectable as early as 20 min post-activation. This suggests that CSF is inactivated upon MPF inactivation. However, CSF activity increases again to reach a maximum 60 min post-activation and gradually disappears during the following 40 min. Thus, in activated mouse oocytes (undergoing the transition to interphase) CSF activity fluctuates before definitive inactivation. We found that hybrids arrested in M-phase, thus containing CSF activity after oocyte activation, have activated forms of MAP kinases while hybrids in interphase have inactive forms of these enzymes. We postulate that CSF inactivation in mouse oocytes proceeds in two steps. The initial inactivation of CSF, required for MPF inactivation, is transient and does not require MAP kinase inactivation. The final inactivation of CSF, required for normal embryonic cell cycle progression, is dependent upon the inactivation of MAP kinases.     

MPF components and meiotic competence in growing pig oocytes

Growing pig oocytes (≤90 μm in diameter) are unable to resume meiosis in vitro. The objective of our present experiments has been to identify the reasons for meiotic competence in these cells. By comparing histone H1 kinase activity in growing and fully grown oocytes we demonstrate that incompetence is associated with an inability to activate H1 kinase in growing oocytes. Immunoblotting was used to determine whether this kinase inactivity resulted from a lack of either p34^cdc2 protein or B-type cyclin. The results established that each of these cell cycle molecules are present in comparable amounts in both growing and fully grown oocytes. In the third series of studies experiments were carried out in an attempt to induce p34^cdc2 activation during growth. Treatment with okadaic acid, an inhibitor of phosphatase 1 and 2A known to stimulate and accelerate the transition into M-phase of the meiotic cycle in a number of different species, was able to induce p34^cdc2 kinase activity and facilitated the G₂- to M-phase in growing oocytes. We conclude that although growing oocytes in pigs have sufficient key cell cycle components for the G₂ to M transition, they remain incapable of converting these components to active maturation-promoting factor (MPF) until growth is virtually completed. Inhibition of phosphatase 1 or 2A induces the formation of active MPF during growth by an as yet unidentified pathway. © 1994 Wiley-Liss, Inc.     

CDC6 controls dynamics of the first embryonic M-phase entry and progression via CDK1 inhibition

CDC6 is essential for S-phase to initiate DNA replication. It also regulates M-phase exit by inhibiting the activity of the major M-phase protein kinase CDK1. Here we show that addition of recombinant CDC6 to Xenopus embryo cycling extract delays the M-phase entry and inhibits CDK1 during the whole M-phase. Down regulation of endogenous CDC6 accelerates the M-phase entry, abolishes the initial slow and progressive phase of histone H1 kinase activation and increases the level of CDK1 activity during the M-phase. All these effects are fully rescued by the addition of recombinant CDC6 to the extracts. Diminution of CDC6 level in mouse zygotes by two different methods results in accelerated entry into the first cell division showing physiological relevance of CDC6 in intact cells. Thus, CDC6 behaves as CDK1 inhibitor regulating not only the M-phase exit, but also the M-phase entry and progression via limiting the level of CDK1 activity. We propose a novel mechanism of M-phase entry controlled by CDC6 and counterbalancing cyclin B-mediated CDK1 activation. Thus, CDK1 activation proceeds with concomitant inhibition by CDC6, which tunes the timing of the M-phase entry during the embryonic cell cycle.     

Proteomic Profiling Reveals the Molecular Control of Oocyte Maturation

Meiotic maturation is an intricate and precisely regulated process orchestrated by various pathways and numerous proteins. However, little is known about the proteome landscape during oocytes maturation. Here, we obtained the temporal proteomic profiles of mouse oocytes during in vivo maturation. We successfully quantified 4694 proteins from 4500 oocytes in three key stages (germinal vesicle, germinal vesicle breakdown, and metaphase II). In particular, we discovered the novel proteomic features during oocyte maturation, such as the active Skp1–Cullin–Fbox pathway and an increase in mRNA decay–related proteins. Using functional approaches, we further identified the key factors controlling the histone acetylation state in oocytes and the vital proteins modulating meiotic cell cycle. Taken together, our data serve as a broad resource on the dynamics occurring in oocyte proteome and provide important knowledge to better understand the molecular mechanisms during germ cell development.     

An M-phase-specific protein kinase of Xenopus oocytes: partial purification and possible mechanism of its periodic activation

The activity of a Ca2+- and cyclic nucleotide-independent protein kinase(s) which catalyzes hyperphosphorylation of a set of endogenous proteins, including a 95-kDa soluble phosphoprotein, is found to fluctuate in both the meiotic and mitotic cell cycles of Xenopus oocytes and activated eggs. The activity is high in M-phase and hardly detectable in interphase. The activity copurifies with a major histone kinase(s) throughout four purification steps: ammonium sulfate precipitation, DEAE-cellulose chromatography, high-performance liquid chromatography on TSK G3000, and CM-Sepharose chromatography. This suggests that a single enzyme shares activity against endogenous proteins and added histones. Changes in the activity of the M-phase-specific protein kinase(s) as assayed in vitro correlate with changes in the extent of protein phosphorylation in oocytes pulse-labeled with 32P-phosphate by microinjection during meiotic maturation and the early embryonic cell cycle. This suggests that the kinase(s) has a broad specificity and plays a key role in the increased protein phosphorylation which occurs at the transition to M-phase. Microinjection of the maturation-promoting factor (MPF) into immature oocytes triggers, after a 10-min lag period, the activation of the M-phase specific kinase(s), even in the absence of protein synthesis. In contrast MPF microinjection does not induce kinase activation in cycloheximide-treated oocytes arrested after completion of the first meiotic cell cycle or in activated eggs arrested in S-phase by incubation in cycloheximide. This suggests that immature oocytes contain an inactive kinase precursor (prokinase) which is synthesized at each of the following cell cycles. In the absence of MPF addition, the prokinase to kinase transition occurs "spontaneously" after a 2-hr lag period in high-speed supernatants prepared from prophase-arrested oocytes if low-molecular-weight metabolites are eliminated by gel filtration. Addition of ATP, but not of AMP-PNP (adenylyl-imidodiphosphate), prevents spontaneous kinase activation in gel-filtered extracts. We propose that MPF activates the M-phase-specific protein kinase in the intact cell by inactivating a factor which requires phosphorylation conditions to inhibit the prokinase to kinase transition.     

CDC6 requirement for spindle formation during maturation of mouse oocytes

A master regulator of DNA replication, CDC6 also functions in the DNA-replication checkpoint by preventing DNA rereplication. Cyclin-dependent kinases (CDKs) regulate the amount and localization of CDC6 throughout the cell cycle; CDC6 phosphorylation after DNA replication initiation leads to its proteolysis in yeast or translocation to the cytoplasm in mammals. Overexpression of CDC6 during the late S phase prevents entry into the M phase by activating CHEK1 kinase that then inactivates CDK1/cyclin B, which is essential for the G2/M-phase transition. We analyzed the role of CDC6 during resumption of meiosis in mouse oocytes, which are arrested in the first meiotic prophase with low CDK1/cyclin B activity; this is similar to somatic cells at the G2/M-phase border. Overexpression of CDC6 in mouse oocytes does not prevent resumption of meiosis. The RNA interference-mediated knockdown of CDC6, however, reveals a new and unexpected function for CDC6; namely, it is essential for spindle formation in mouse oocytes.     

Phosphorylation changes associated with the early cell cycle in Xenopus eggs

Enucleated and nondividing amphibian eggs undergo cyclic changes in cell morphology and in the level of maturation promoting factor (MPF) with a period similar to the early cleavage cycle. We show here that there is a corresponding phosphorylation and dephosphorylation of specific proteins associated with this fundamental cell cycle. M-phase is associated with a general increase in phosphatase activity and specific phosphorylation of a small set of M-phase proteins, reflected in an increased stochiometry of phosphate and increased turnover. At the end of metaphase and correlated with a drop in MPF the phosphoproteins are rapidly lost. By microinjecting M-phase phosphoproteins into arrested interphase and metaphase eggs we could show that the specific M-phase phosphorylation was not due to specificity in phosphatase action. The ability to segregate synthesis from phosphorylation demonstrates that regulation is not on the level of synthesis of the M-phase proteins. Taken together these data suggest that regulation of kinase activity in M-phase in the face of general rapid phosphate turnover in the egg plays an important role in the regulation of the fundamental mitotic cycle.     

Cucurbitacin B induces rapid depletion of the G-actin pool through reactive oxygen species-dependent actin aggregation in melanoma cells

Cucurbitacin B (CuB), a triterpenoid compound isolated from Cucurbitaceae plants, has been reported as a promising anti-cancer agent, yet its action mechanism is still controversial. In this study, we explored the potential mechanism of CuB in murine B16F10 melanoma cells. Anti-proliferation and anti-invasion effects were assessed in cultured cells, and in vivo anti-tumor activity was evaluated in a murine subcutaneous melanoma model. Flow cytometry was adopted to analyze cell cycle distribution and reactive oxygen species (ROS) levels. Actin levels were determined by western blot analysis, and the profiles of differential expressed proteins were identified by a quantitative proteomic approach. The results showed that CuB exerted inhibitory effects on cell proliferation, colony formation, as well as migration and invasion potential of the melanoma cells. The growth of subcutaneous melanoma was significantly inhibited in mice treated with CuB when compared with control group. Furthermore, CuB treatment caused rapid cell membrane blebbing and deformation, and induced G(2)/M-phase arrest and formation of multiploid cells. Notably, the G-actin pool was rapidly depleted and actin aggregates were formed quickly after CuB treatment. A number of cytoskeleton-regulatory proteins were differentially regulated. Blockage of ROS production significantly reduced the G-actin depletion ability and the anti-tumor activity of CuB. These findings indicate that CuB induces rapid depletion of the G-actin pool through ROS-dependent actin aggregation in melanoma cells, which may at least partly account for its anti-tumor activity.     

Activation of p42 MAP kinase and the release of oocytes from cell cycle arrest.

Clam oocytes are arrested naturally at the G2/M border in meiosis and contain an inactive 42 kDa ERK/MAP kinase, p42MAPK. Following fertilization, p42MAPK is rapidly phosphorylated on tyrosine residues and concomitantly activated. Both tyrosine phosphorylation and activation of p42MAPK begin within 2-3 min of fertilization, peak at approximately 15 min, then rapidly decline and disappear around the end of meiosis I. Neither the tyrosine phosphorylated form of p42MAPK nor p42MAPK activity reappears during meiosis II or the succeeding mitotic cell cycles. High doses of molybdate, a potent PTPase inhibitor, block the phosphorylation of p42MAPK and entry into the cell cycle. Lower doses of molybdate delay both p42MAPK phosphorylation and the release from cell cycle arrest, but once cells have re-entered the cell cycle, they continue with near-normal timing. These results argue that the transient activation of p42MAPK at fertilization is a one-time event linked to release from cell cycle arrest. In trying to reconcile this one-time activation of p42MAPK in clam embryos with the recurring, M-phase specific activation of MBP/MAP kinases reported in other systems, we show that cdc2 kinase contributes a major portion of the MBP kinase activity in mitotic extracts. Furthermore, a small fraction of p42MAPK and other related kinases are present in p13suc1-bound material, cautioning against the use of p13suc1 beads for experiments where, in addition to cdc2, the unaccounted presence of other kinase activities could be misleading.     

Induction of reinitiation of meiosis in amphibian Bufo and Xenopus oocytes by injection of M-phase extracts of ciliate Tetrahymena needs the recipient protein synthesis

We show here that germinal vesicle breakdown of amphibian Bufo and Xenopus oocytes can be induced if ciliate Tetrahymena extracts are injected into them. The activity of meiosis-reinitiation-inducing factor (MRIF) appeared only a M-phase of a synchronously dividing culture, indicating that this MRIF has an important function for induction of M-phase in the mitotic cell cycle. MRIF of Tetrahymena differed from MPF (M-phase-promoting factor), because its action on the induction of GVBD was inhibited by cycloheximide and it could not induce GVBD in starfish oocytes by microinjection. MPF activity was not detected in extracts of vegetatively growing Tetrahymena. Preliminary experiments showed that MRIF was a heat-labile, Ca2(+)-sensitive, and trypsin-sensitive soluble protein.     

Proteomic analysis of mouse oocytes reveals 28 candidate factors of the "reprogrammome"

The oocyte is the only cell of the body that can reprogram transplanted somatic nuclei and sets the gold standard for all reprogramming methods. Therefore, an in-depth characterization of its proteome holds promise to advance our understanding of reprogramming and germ cell biology. To date, limitations on oocyte numbers and proteomic technology have impeded this task, and the search for reprogramming factors has been conducted in embryonic stem (ES) cells instead. Here, we present the proteome of metaphase II mouse oocytes to a depth of 3699 proteins, which substantially extends the number of proteins identified until now in mouse oocytes and is comparable by size to the proteome of undifferentiated mouse ES cells. Twenty-eight oocyte proteins, also detected in ES cells, match the criteria of our multilevel approach to screen for reprogramming factors, namely nuclear localization, chromatin modification, and catalytic activity. Our oocyte proteome catalog thus advances the definition of the "reprogrammome", the set of molecules--proteins, RNAs, lipids, and small molecules--that enable reprogramming.     

Activation of extracellular signal-regulated kinase (ERK) in G2 phase delays mitotic entry through p21CIP1

Extracellular signal-regulated kinase activity is essential for mediating cell cycle progression from G(1) phase to S phase (DNA synthesis). In contrast, the role of extracellular signal-regulated kinase during G(2) phase and mitosis (M phase) is largely undefined. Previous studies have suggested that inhibition of basal extracellular signal-regulated kinase activity delays G(2)- and M-phase progression. In the current investigation, we have examined the consequence of activating the extracellular signal-regulated kinase pathway during G(2) phase on subsequent progression through mitosis. Using synchronized HeLa cells, we show that activation of the extracellular signal-regulated kinase pathway with phorbol 12-myristate 13-acetate or epidermal growth factor during G(2) phase causes a rapid cell cycle arrest in G(2) as measured by flow cytometry, mitotic indices and cyclin B1 expression. This G(2)-phase arrest was reversed by pre-treatment with bisindolylmaleimide or U0126, which are selective inhibitors of protein kinase C proteins or the extracellular signal-regulated kinase activators, MEK1/2, respectively. The extracellular signal-regulated kinase-mediated delay in M-phase entry appeared to involve de novo synthesis of the cyclin-dependent kinase inhibitor, p21(CIP1), during G(2) through a p53-independent mechanism. To establish a function for the increased expression of p21(CIP1) and delayed cell cycle progression, we show that extracellular signal-regulated kinase activation in G(2)-phase cells results in an increased number of cells containing chromosome aberrations characteristic of genomic instability. The presence of chromosome aberrations following extracellular signal-regulated kinase activation during G(2)-phase was further augmented in cells lacking p21(CIP1). These findings suggest that p21(CIP1) mediated inhibition of cell cycle progression during G(2)/M phase protects against inappropriate activation of signalling pathways, which may cause excessive chromosome damage and be detrimental to cell survival.     

chk-1 is an Essential Gene and is Required for an S-M Checkpoint During Early Embryogenesis

The chk1 gene was first discovered in screens for radiation sensitive mutants in S. pombe [1]. Genetic analysis revealed that chk1 is involved in a DNA damage G2-M checkpoint. Chk1 becomes activated in response to DNA damage and prevents entry into mitosis by inhibiting the cell cycle machinery. This checkpoint decreases the risk of defective DNA being inherited by daughter cells, therefore reducing the risk of genetic instability. In higher eukaryotes, chk1 homologues have similar checkpoint functions. For example, an avian B-lymphoma cell line that is defective for Chk1 fails to arrest in G2-M after DNA damage. Nonetheless, these Chk1 defective cells are viable indicating that Chk1 is not essential for normal somatic cells to divide [2]. In spite of this, mouse and Drosophila homozygous Chk1 mutants die during embryogenesis suggesting that this is an essential gene for embryonic cell cycles [3, 4]. What particular role does Chk1 have in directing embryonic cell divisions? Here we used the model organism, C. elegans, to address the role of chk-1 during development. As expected, disruption of chk-1 by RNAi eliminated the DNA damage checkpoint response in C. elegans. In addition, we revealed that chk-1 was predominantly expressed during embryogenesis and in the postembryonic germline. Indeed, we found that chk-1 had an essential role in embryo and germline development. More specifically, disruption of chk-1 expression resulted in embryo lethality, which was attributed to a defect in an intrinsic S-M hence causing premature entry into M-phase.     

Amphibian oocyte maturation induced by extracts of Physarum polycephalum in mitosis

The orderly progression of eukaryotic cells from interphase to mitosis requires the close coordination of various nuclear and cytoplasmic events. Studies from our laboratory and others on animal cells indicate that two activities, one present mainly in mitotic cells and the other exclusively in G1-phase cells, play a pivotal role in the regulation of initiation and completion of mitosis, respectively. The purpose of this study was to investigate whether these activities are expressed in the slime mold Physarum polycephalum in which all the nuclei traverse the cell cycle in natural synchrony. Extracts were prepared from plasmodia in various phases of the cell cycle and tested for their ability to induce germinal vesicle breakdown and chromosome condensation after microinjection into Xenopus laevis oocytes. We found that extract of cells at 10-20 min before metaphase consistently induced germinal vesicle breakdown in oocytes. Preliminary characterization, including purification on a DNA-cellulose affinity column, indicated that the mitotic factors from Physarum were functionally very similar to HeLa mitotic factors. We also identified a number of mitosis-specific antigens in extracts from Physarum plasmodia, similar to those of HeLa cells, using the mitosis-specific monoclonal antibodies MPM-2 and MPM-7. Interestingly, we also observed an activity in Physarum at 45 min after metaphase (i.e., in early S phase since it has no G1) that is usually present in HeLa cells only during the G1 phase of the cell cycle. These are the first studies to show that maturation-promoting factor activity is present in Physarum during mitosis and is replaced by the G1 factor (or anti-maturation-promoting factor) activity in a postmitotic stage. A comparative study of these factors in this slime mold and in mammalian cells would be extremely valuable in further understanding their function in the regulation of eukaryotic cell cycle and their evolutionary relationship to one another.