Xenopus oocyte maturation does not require new cyclin synthesis

Progesterone induces fully grown, stage VI, Xenopus oocytes to pass through meiosis I and arrest in metaphase of meiosis II. Protein synthesis is required twice in this process: in order to activate maturation promoting factor (MPF) which induces meiosis I, and then again after the completion of meiosis I to reactivate MPF in order to induce meiosis II. We have used antisense oligonucleotides to destroy maternal stores of cyclin mRNAs, and demonstrate that new cyclin synthesis is not required for entry into either meiosis I or II. This finding is consistent with the demonstration that stage VI oocytes contain a store of B-type cyclin polypeptides (Kobayashi, H., J. Minshull, C. Ford, R. Golsteyn, R. Poon, and T. Hunt. 1991. J. Cell Biol. 114:755-765). Although approximately 70% of cyclin B2 is destroyed at first meiosis, the surviving fraction, together with a larger pool of surviving cyclin B1, must be sufficient to allow the reactivation of MPF and induce entry into second meiotic metaphase. Since stage VI oocytes do not contain any cyclin A, our results show that cyclin A is not required for meiosis in Xenopus. We discuss the possible nature of the proteins whose synthesis is required to induce meiosis I and II.     

Activation of maturation promoting factor in Bufo arenarum oocytes: injection of mature cytoplasm and germinal vesicle contents

Although progesterone is the established maturation inducer in amphibians, Bufo arenarum oocytes obtained during the reproductive period (spring-summer) resume meiosis with no need of an exogenous hormonal stimulus if deprived of their enveloping follicle cells, a phenomenon called spontaneous maturation. In this species it is possible to obtain oocytes competent and incompetent to undergo spontaneous maturation according to the seasonal period in which animals are captured. Reinitiation of meiosis is regulated by maturation promoting factor (MPF), a complex of the cyclin-dependent kinase p34cdc2 and cyclin B. Although the function and molecule of MPF are common among species, the formation and activation mechanisms of MPF differ according to species. This study was undertaken to evaluate the presence of pre-MPF in Bufo arenarum oocytes incompetent to mature spontaneously and the effect of the injection of mature cytoplasm or germinal vesicle contents on the resumption of meiosis. The results of our treatment of Bufo arenarum immature oocytes incompetent to mature spontaneously with sodium metavanadate (NaVO3) and dexamethasone (DEX) indicates that these oocytes have a pre-MPF, which activates and induces germinal vesicle breakdown (GVBD) by dephosphorylation on Thr-14/Tyr-15 by cdc25 phosphatase and without cyclin B synthesis. The injection of cytoplasm containing active MPF is sufficient to activate an amplification loop that requires the activation of cdc25 and protein kinase C, the decrease in cAMP levels, and is independent of protein synthesis. However, the injection of germinal vesicle content also induces GVBD in the immature receptor oocyte, a process dependent on protein synthesis but not on cdc25 phosphatase or PKC activity.     

Mitogen-activated protein kinase (MAP kinase) activation in Xenopus oocytes: Roles of MPF and protein synthesis

Mitogen-activated protein kinase (MAP kinase) is a serine/threonine kinase whose enzymatic activity is thought to play a crucial role in mitogenic signal transduction and also in the progesterone-induced meiotic maturation of Xenopus oocytes. We have purified MAP kinase from Xenopus oocytes and have shown that the protein is present in metaphase ll oocytes under two different forms: an inactive 41-kD protein able to autoactivate and to autophosphorylate in vitro, and an active 42-kD kinase resolved into two tyrosine phosphorylated isoforms on 2D gels. During meiotic maturation, MAP kinase becomes tyrosine phosphorylated and activated following the activation of the M-phase promoting factor (MPF), a complex between the p34^cdc2 kinase and cyclin B. In vivo, MAP kinase activity displays a different stability in metaphase l and in metaphase II: protein synthesis is required to maintain MAP kinase activity in metaphase I but not in metaphase II oocytes. Injection of either MPF or cyclin B into prophase oocytes promotes tyrosine phosphorylation of MAP kinase, indicating that its activation is a downstream event of MPF activation. In contrast, injection of okadaic acid, which induces in vivo MPF activation, promotes only a very weak tyrosine phosphorylation of MAP kinase, suggesting that effectors other than MPF are required for the MAP kinase activation. Moreover, in the absence of protein synthesis, cyclin B and MPF are unable to promote in vivo activation of MAP kinase, indicating that this activation requires the synthesis of new protein(s). © 1993 Wiley-Liss, Inc.     

Redundant pathways for Cdc2 activation in Xenopus oocyte: either cyclin B or Mos synthesis

Xenopus oocytes are arrested in meiotic prophase I. Progesterone induces the resumption of meiotic maturation, which requires continuous protein synthesis to bring about Cdc2 activation. The identification of the newly synthesized proteins has long been a goal. Two plausible candidates have received extensive study. The synthesis of cyclin B and of c-Mos, a kinase that activates the mitogen-activated protein kinase pathway in oocytes, is clearly upregulated by translational control in response to progesterone. Recent studies suggest that ablation of either c-Mos or cyclin B synthesis by antisense oligonucleotides does not block meiotic maturation. Here, however, we show that when both pathways are simultaneously inhibited, progesterone no longer triggers maturation; adding back either c-Mos or cyclin B restores meiotic maturation. We conclude that the specific synthesis of either B-type cyclins or c-Mos, induced by progesterone, is required to induce meiotic maturation. The two pathways seem to be functionally redundant.     

Activation of Cdc2 kinase during meiotic maturation of axolotl oocyte

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

The role of cyclin B in meiosis I

In clams, fertilization is followed by the prominent synthesis of two cyclins, A and B. During the mitotic cell cycles, the two cyclins are accumulated and then destroyed near the end of each metaphase. Newly synthesized cyclin B is complexed with a small set of other proteins, including a kinase that phosphorylates cyclin B in vitro. While both cyclins can act as general inducers of entry into M phase, the two are clearly distinguished by their amino acid sequences (70% nonidentity) and by their different modes of expression in oocytes and during meiosis. In contrast to cyclin A, which is stored solely as maternal mRNA, oocytes contain a stockpile of cyclin B protein, which is stored in large, rapidly sedimenting aggregates. Fertilization results in the release of cyclin B to a more disperse, soluble form. Since the first meiotic division in clams can proceed even when new protein synthesis is blocked, these results strongly suggest it is the fertilization-triggered unmasking of cyclin B protein that drives cells into meiosis I. We propose that the unmasking of maternal cyclin B protein allows it to interact with cdc2 protein kinase, which is also stored in oocytes, and that the formation of this cyclin B/cdc2 complex generates active M phase-promoting factor.     

Function of the Mos/MAPK pathway during oocyte maturation in the Japanese brown frog Rana japonica

Fully grown immature oocytes acquire the ability to be fertilized with sperm after meiotic maturation, which is finally accomplished by the formation and activation of the maturation-promoting factor (MPF). MPF is the complex of Cdc2 and cyclin B, and its function in promoting metaphase is common among species. The Mos/mitogen-activated protein kinase (MAPK) pathway is also commonly activated during vertebrate oocyte maturation, but its function seems to be different among species. We investigated the function of the Mos/MAPK pathway during oocyte maturation of the frog Rana japonica. Although MAPK was activated in accordance with MPF activation during oocyte maturation, MPF activation and germinal vesicle breakdown (GVBD) was not initiated when the Mos/MAPK pathway was activated in immature oocytes by the injection of c-mos mRNA. Inhibition of Mos synthesis by c-mos antisense RNA and inactivation of MAPK by CL100 phosphatase did not prevent progesterone-induced MPF activation and GVBD. However, continuous MAPK activation and MAPK inhibition through oocyte maturation accelerated and delayed MPF activation, respectively. Furthermore, Mos induced a low level of cyclin B protein synthesis in immature oocytes without the aid of MAPK. These results suggest that the general function of the Mos/MAPK pathway, which is not essential for MPF activation and GVBD in Rana oocytes, is to enhance cyclin B translation by Mos itself and to stabilize cyclin B protein by MAPK during oocyte maturation.     

The metaphase II arrest in mouse oocytes is controlled through microtubule-dependent destruction of cyclin B in the presence of CSF.

In unfertilized eggs from vertebrates, the cell cycle is arrested in metaphase of the second meiotic division (metaphase II) until fertilization or activation. Maintenance of the long-term meiotic metaphase arrest requires mechanisms preventing the destruction of the maturation promoting factor (MPF) and the migration of the chromosomes. In frog oocytes, arrest in metaphase II (M II) is achieved by cytostatic factor (CSF) that stabilizes MPF, a heterodimer formed of cdc2 kinase and cyclin. At the metaphase/anaphase transition, a rapid proteolysis of cyclin is associated with MPF inactivation. In Drosophila, oocytes are arrested in metaphase I (M I); however, only mechanical forces generated by the chiasmata seem to prevent chromosome separation. Thus, entirely different mechanisms may be involved in the meiotic arrests in various species. We report here that in mouse oocytes a CSF-like activity is involved in the M II arrest (as observed in hybrids composed of fragments of metaphase II-arrested oocytes and activated mitotic mouse oocytes) and that the high activity of MPF is maintained through a continuous equilibrium between cyclin B synthesis and degradation. In addition, the presence of an intact metaphase spindle is required for cyclin B degradation. Finally, MPF activity is preferentially associated with the spindle after bisection of the oocyte. Taken together, these observations suggest that the mechanism maintaining the metaphase arrest in mouse oocytes involves an equilibrium between cyclin synthesis and degradation, probably controlled by CSF, and which is also dependent upon the three-dimensional organization of the spindle.     

The proteasome is involved in the first metaphase-to-anaphase transition of meiosis in rat oocytes

The proteasome engages in protein degradation as a regulatory process in biological transactions. Among other cellular processes, the proteasome participates in degradation of ubiquinated cyclins in mitosis. However, its role in meiosis has not been established. Resumption of meiosis in the oocyte involves the activation of maturation promoting factor (MPF), a complex of p34cdc2 and cyclin B. Inactivation of this factor, occurring between the two meiotic divisions, is associated with degradation of cyclin B. In this study, we examined the possible involvement of the proteasome in regulation of the exit from metaphase I in spontaneously maturing rat oocytes. We found that upon resumption of meiosis, proteasomes translocate to the spindle apparatus. We further demonstrated that specific inhibitors of proteasome catalytic activity, MG132 and lactacystin, blocked polar body extrusion. Chromosome and microtubule fluorescent staining verified that MG132-treated oocytes were arrested at metaphase I. Intervention of proteasomal action with this inhibitor also resulted in accumulation of cyclin B and elevated activity of MPF. These data demonstrate that proteasomal catalytic activity is absolutely essential for the decrease in MPF activity and completion of the first meiotic division. Its translocation to the spindle apparatus may facilitate the timely degradation of cyclin B.     

Discrimination of the roles of MPF and MAP kinase in morphological changes that occur during oocyte maturation

Maturing amphibian oocytes undergo drastic morphological changes, including germinal vesicle breakdown (GVBD), chromosome condensation, and spindle formation in response to progesterone. Two kinases, maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK), are involved in these changes, but their precise roles are unknown. Unlike in Xenopus oocytes, discrimination of the functions of MAPK and MPF in Rana oocytes is easy owing to the lack of pre-MPF. We investigated the roles of these kinases by careful observations of chromosomes and microtubules in Rana oocytes. MPF and MAPK activities were manipulated by treatment with progesterone, c-mos mRNA, or cyclin B mRNA in combination with MAPK kinase inhibitors. Activation of one kinase without activation of the other induced only limited events; GVBD was induced by MPF without MAPK, and reorganization of microtubules at GVBD was induced by MAPK without MPF, but other events were not induced. In contrast, coactivation of MPF and MAPK by injection of c-mos and cyclin B mRNA promoted almost all of the morphological changes that occur during maturation without progesterone, indicating that these are controlled by cooperation of MPF and MAPK. The results revealed the functions of MAPK and MPF in each process of sequential morphological changes during oocyte maturation.     

Comparative study of the molecular mechanisms of oocyte maturation in amphibians

Maturation-promoting factor (MPF), a complex of Cdc2 and cyclin B, is the final inducer of oocyte maturation. Its activity is controlled by inhibitory phosphorylation of Cdc2 on Tyr15/Thr14 and activating phosphorylation on Thr161. Full-grown immature oocytes of the African clawed frog Xenopus laevis contain inactive MPF (pre-MPF) that comprises cyclin B-bound Cdc2 phosphorylated on Tyr15/Thr14 and Thr161. The synthesis of Mos, but not cyclin B, after stimulation by the maturation-inducing steroid progesterone, is believed to be necessary for initiating Xenopus oocyte maturation through Tyr15/Thr14 dephosphorylation of pre-MPF. In contrast, amphibians other than Xenopus (and also fishes) employ a different mechanism. Full-grown immature oocytes of these species contain monomeric Cdc2 but not cyclin B. MPF is formed after hormonal stimulation by binding of the newly produced cyclin B to the pre-existing Cdc2 and is immediately activated through Thr161 phosphorylation. Mos/MAP kinase is neither necessary nor sufficient for initiating maturation in fishes and amphibians except for Xenopus. We propose a new model of MPF formation and activation during oocyte maturation that is applicable to all amphibians (as well as fishes), based on a novel concept that pre-MPF is an artificial molecule that is not essential for inducing oocyte maturation.     

Suppression of DNA replication via Mos function during meiotic divisions in Xenopus oocytes.

Meiosis is characterized by the absence of DNA replication between the two successive divisions. In Xenopus eggs, the ability to replicate DNA develops during meiotic maturation, but is normally suppressed until fertilization. Here we show that development of the DNA-replicating ability depends on new protein synthesis during meiosis I, and that mere ablation of the endogenous c-mos product Mos allows maturing oocytes to enter interphase and replicate DNA just after meiosis I. Moreover, we demonstrate that during normal maturation cdc2 kinase undergoes precocious inactivation in meiosis I and then premature reactivation before meiosis II; importantly, this premature cdc2 reactivation absolutely requires Mos function and its direct inhibition by a dominant-negative cdc2 mutant also results in nuclear reformation and DNA replication immediately after meiosis I. These findings indicate that suppression of DNA replication during meiotic divisions in Xenopus oocytes is accomplished by the Mos-mediated premature reactivation of cdc2 kinase. We suggest that these mechanisms for suppressing DNA replication may be specific for meiosis in animal oocytes, and that the ultimate biological function, including the well known cytostatic factor activity, of Mos during meiotic maturation may be to prevent undesirable DNA replication or parthenogenetic activation before fertilization.     

Mitogen-activated protein kinase activation down-regulates a mechanism that inactivates cyclin B-cdc2 kinase in G2-arrested oocytes.

The G2 arrest of oocytes from frogs, clams, and starfish requires that preformed cyclin B-cdc2 complexes [prematuration-promoting factor (MPF)] be kept in an inactive form that is largely due to inhibitory phosphorylation of this pre-MPF. We have investigated the role of mitogen-activated protein (MAP) kinase in the activation of this pre-MPF. The cytoplasm of both frog and starfish oocytes contains an activity that can rapidly inactivate injected MPF. When the MAP kinase of G2-arrested starfish or Xenopus oocytes was prematurely activated by microinjection of c-mos or Ste-11 delta N fusion proteins, the rate and extent of MPF inactivation was much reduced. Both effects were suppressed by expression of the specific MAP kinase phosphatase Pyst 1. These results show that MAP kinase down-regulates a mechanism that inactivates cyclin B-cdc2 kinase in Xenopus oocytes. In starfish oocytes, however, MAP kinase activation occurs only after germinal vesicle breakdown, much after MPF activation. In this case, down-regulation of the cyclin B-cdc2 inhibiting pathway is a sensitive response to hormonal stimulation that does not require MAP kinase activation.     

Newly synthesized protein(s) must associate with p34cdc2 to activate MAP kinase and MPF during progesterone-induced maturation of Xenopus oocytes.

The meiotic maturation of Xenopus oocytes triggered by progesterone requires new protein synthesis to activate both maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAP kinase). Injection of mRNA encoding mutant p34cdc2 (K33R) that can bind cyclins but lacks protein kinase activity strongly inhibited progesterone-induced activation of both MPF and MAP kinase in Xenopus oocytes. Similar results were obtained by injection of GST-p34cdc2 K33R protein or by injection of a monoclonal antibody (A17) against p34cdc2 that blocks its activation by cyclins. Both the dominant-negative p34cdc2 and monoclonal antibody A17 blocked the accumulation of p39mos and activation of MAP kinase in response to progesterone, as well as blocking the appearance of MPF, although they did not inhibit the translation of p39mos mRNA. These results suggest that: (i) activation of free p34cdc2 by newly made proteins, probably cyclin(s), is normally required for the activation of both MPF and MAP kinase by progesterone in Xenopus oocytes; (ii) the activation of translation of cyclin mRNA normally precedes, and does not require either MPF or MAP kinase activity; and (iii) de novo synthesis and accumulation of p39mos is probably both necessary and sufficient for the activation of MAP kinase in response to progesterone.     

Interaction of cell cycle kinases, microtubules, and chromatin in ascidian oocytes during meiosis

We used kinase assays and confocal microscopy to study the interaction of cell cycle proteins with microtubule organising centres (MTOC) and chromatin in ascidian oocytes during meiosis. The activity of maturation promoting factor (MPF) and mitogen activated protein kinase (MAPK) appear not to be correlated in control oocytes. MPF activity peaks during metaphase I and II of the meiotic cell cycle whereas the activity of MAPK peaks at telophase I and is subsequently degraded to remain at low levels for the remainder of meiosis. The protein synthesis inhibitor emetine induces the degradation in MPF activity in unfertilized metaphase-I (M-I) oocytes, while MAPK is unaffected. Emetine does not alter the activities of these cell cycle kinases in fertilized oocytes during meiosis I but MPF activity remains low while MAPK activity is high for an elongated time period and oocytes do not complete meiosis I. Emetine induces maternal MTOC duplication in unfertilized M-I oocytes and prevents sperm aster growth in fertilized oocytes, but it does not alter the M-I meiotic apparatus in unfertilized oocytes. These experiments suggest that neither MPF alone nor emetine-sensitive proteins are responsible for M-I arrest in ascidian oocytes, MAPK may ensure this stability. In addition, we showed that the maternal MTOC is present at M-I but suppressed from duplicating in an emetine-sensitive manner.     

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

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

Phosphorylation of Xenopus cyclins B1 and B2 is not required for cell cycle transitions.

The cdc2 kinase and B-type cyclins are known to be components of maturation- or M-phase-promoting factor (MPF). Phosphorylation of cyclin B has been reported previously and may regulate entry into and exit from mitosis and meiosis. To investigate the role of cyclin B phosphorylation, we replaced putative cdc2 kinase phosphorylation sites in Xenopus cyclins B1 and B2 by using oligonucleotide site-directed mutagenesis. We found that Ser-90 of cyclin B2 and Ser-94 or Ser-96 of cyclin B1 are the main phosphorylation sites both in functional Xenopus egg extracts and after phosphorylation with purified MPF in vitro. Microtubule-associated protein (MAP) kinase from Xenopus eggs phosphorylated cyclin B1 significantly at Ser-94 or Ser-96, whereas it was largely inactive against cyclin B2. The substitutions that ablated phosphorylation at these sites, however, resulted in no functional differences between mutant and wild-type cyclin, as judged by the kinetics of M-phase degradation, induction of mitosis in egg extracts, or induction of oocyte maturation. These results indicate that the phosphorylation of Xenopus B-type cyclins by cdc2 kinase or MAP kinase is not required for the hallmark functions of cyclin.     

Cyclin is a component of maturation-promoting factor from Xenopus

Highly purified maturation-promoting factor (MPF) from Xenopus eggs contains both cyclin B1 and cyclin B2 as shown by Western blotting and immunoprecipitation using Xenopus anti-B-type cyclin antibodies. Immunoprecipitates with these antibodies display the histone H1 kinase activity characteristic of MPF, for which exogenously added B1 and B2 cyclins are both substrates. Protein kinase activity against cyclin oscillates in maturing oocytes and activated eggs with the same kinetics as p34cdc2 kinase activity. These data indicate that B-type cyclin is the other component of MPF besides p34cdc2.     

Overexpression of the cytoplasmic retention signal region of cyclin B2, but not of cyclin B1, inhibits bipolar spindle formation in Xenopus oocytes

Cyclin B, a regulatory subunit of maturation/M-phase promoting factor (MPF), has several subtypes in many vertebrate species. However, it is not known whether the different B-type cyclins have any different functions in vertebrate cells, although their subcellular localizations seem to differ largely from each other. To examine the roles of two major B-type cyclins, B1 and B2, in spindle formation in M phase, we overexpressed their N-termini in Xenopus oocytes; the N-termini of cyclins B1 and B2 contained a cytoplasmic retention signal (CRS), and hence their overexpressions were expected to competitively inhibit the subcellular localizations of the endogenous cyclins B1 and B2, respectively. Upon entry into meiosis I, oocytes overexpressing the cyclin B1 N-terminus formed an apparently normal bipolar spindle, but those oocytes overexpressing the cyclin B1 N-terminus formed a monopolar (or monoastral) spindle. This defect in bipolar spindle formation was observed only when the cyclin B2 N-terminus contained its own CRS sequence, and was able to be rescued by overexpression of full-length cyclin B2. These results suggest, for the first time, that the correct subcellular localization of cyclin B2, but not of cyclin B1, is essential for (the initiation of) bipolar spindle formation in Xenopus oocytes.     

Meiotic cell cycle in Xenopus oocytes is independent of cdk2 kinase.

In vertebrates, M phase-promoting factor (MPF), a universal G2/M regulator in eukaryotic cells, drives meiotic maturation of oocytes, while cytostatic factor (CSF) arrests mature oocytes at metaphase II until fertilization. Cdk2 kinase, a G1/S regulator in higher eukaryotic cells, is activated during meiotic maturation of Xenopus oocytes and, like Mos (an essential component of CSF), is proposed to be involved in metaphase II arrest in mature oocytes. In addition, cdk2 kinase has been shown recently to be essential for MPF activation in Xenopus embryonic mitosis. Here we report injection of Xenopus oocytes with the cdk2 kinase inhibitor p21Cip in order to (re)evaluate the role of cdk2 kinase in oocyte meiosis. Immature oocytes injected with p21Cip can enter both meiosis I and meiosis II normally, as evidenced by the typical fluctuations in MPF activity. Moreover, mature oocytes injected with p21Cip are retained normally in metaphase II for a prolonged period, whereas those injected with neutralizing anti-Mos antibody are released readily from metaphase II arrest. These results argue strongly against a role for cdk2 kinase in MPF activation and its proposed role in metaphase II arrest, in Xenopus oocyte meiosis. We discuss the possibility that cdk2 kinase stored in oocytes may function, as a maternal protein, solely for early embryonic cell cycles.