Molecular mechanisms of the chromosome condensation and decondensation cycle in mammalian cells

The chromosomes undergo a condensation-decondensation cycle within the life cycle of mammalian cells. Chromosome condensation is a complex and critical event that is necessary for the equal distribution of genetic material between the two daughter cells. Although chromosome condensation-decondensation and segregation is mechanistically complex, it proceeds with high fidelity during the eukaryotic cell division cycle. Cell fusion studies have indicated the presence of chromosome condensation factors in mammalian cells during mitosis. If extracts from mitotic cells are injected into immature oocytes of Xenopus laevis, they induce meiotic maturation (i.e. germinal vesicle breakdown and chromosome condensation) within 2–3 hours. Recently, we showed that the maturation-promoting activity of the mitotic cell extracts is inactivated by certain protein factors present in cells during the G₁ period. The activity of the G₁ factors coincides with the process of chromosome decondensation that begins at telophase and continues throughout the G₁ period. These studies have revealed that the mitotic factors and the G₁ factors play a pivotal role in the regulation of condensation and decondensation of chromosomes. Furthermore, our studies strongly suggest that nonhistone protein phosphorylation and dephosphorylation may mediate chromosome condensation and decondensation, respectively.     

Meiotic maturation of mouse oocytes in vitro: association of newly synthesized proteins with condensing chromosomes.

The nature, intracellular distribution, and role of proteins synthesized during meiotic maturation of mouse oocytes in vitro have been examined. Proteins synthesized during the initial stages of maturation are concentrated within the nucleus (germinal vesicle) and become intimately associated with the condensing chromosomes. Inhibition of protein synthesis during this period does not prevent germinal vesicle dissolution or chromosome condensation, but meiotic progression is blocked reversibly at the circular bivalent stage. A protein is synthesized during meiotic maturation of the mouse oocyte which exhibits several of the characteristics of the very lysine-rich histone, FI; this and other histones are phosphorylated during the initial stages of maturation. These results are discussed in relation to studies of meiotic maturation of oocytes from non-mammalian species and chromosome condensation in both oocytes and mitotic cells.     

Control of chromosome behavior in amphibian oocytes. I. The activity of maturing oocytes inducing chromosome condensation in transplanted brain nuclei

The capability of oocyte cytoplasm to induce chromosome condensation was studied by transplantation of isolated brain nuclei into Rana pipiens oocytes induced to undergo maturation in vitro by progesterone treatment. It was found that the chromosome condensation activity (CCA) first appeared in the cytoplasm of maturing oocytes shortly after germinal vesicle breakdown (GVBD), persisted in fully mature oocytes, but rapidly disappeared when the oocytes were artificially activated. A comparison of the time course of the oocyte chromosome condensation cycle and of brain chromosome condensation in maturing and activated oocytes revealed a close temporal correlation between the two, suggesting that both are under the control of the same cytoplasmic factor(s). Oocytes enucleated before GVBD always failed to develop CCA. The CCA could be restored in enucleated oocytes by injecting nucleoplasm obtained from oocytes that had not yet undergone GVBD although this same nucleoplasm was incapable of producing CCA when mixed with the cytoplasm of oocytes that had not reached the stage of GVBD. It was therefore suggested that the CCA had a dual origin involving both cytoplasmic maturation and GV materials.     

Effects of the v-mos oncogene on Xenopus development: meiotic induction in oocytes and mitotic arrest in cleaving embryos

Previous work has demonstrated that the Xenopus protooncogene mosxe can induce the maturation of prophase-arrested Xenopus oocytes. Recently, we showed that mosxe can transform murine NIH3T3 fibroblasts, although it exhibited only 1-2% of the transforming activity of the v-mos oncogene. In this study we have investigated the ability of the v-mos protein to substitute for the mosxe protein in stimulating Xenopus oocytes to complete meiosis. Microinjection of in vitro synthesized RNAs encoding either the mosxe or v-mos proteins stimulates resting oocytes to undergo germinal vesicle breakdown. Microinjection of an antisense oligonucleotide spanning the initiation codon of the mosxe gene blocked progesterone-induced oocyte maturation. When oocytes were microinjected first with the mosxe antisense oligonucleotide, and subsequently with in vitro synthesized v-mos RNA, meiotic maturation was rescued as evidenced by germinal vesicle breakdown. The v-mos protein exhibited in vitro kinase activity when recovered by immunoprecipitation from either microinjected Xenopus oocytes or transfected monkey COS-1 cells; however, in parallel experiments, we were unable to detect in vitro kinase activity associated with the mosxe protein. Microinjection of in vitro synthesized v-mos RNA into cleaving Xenopus embryos resulted in mitotic arrest, demonstrating that the v-mos protein can function like the mosxe protein as a component of cytostatic factor. These results exemplify the apparently conflicting effects of the v-mos protein, namely, its ability to induce maturation of oocytes, its ability to arrest mitotic cleavage of Xenopus embryo, and its ability to transform mammalian fibroblasts.     

mos gene transforming efficiencies correlate with oocyte maturation and cytostatic factor activities.

The mos proto-oncogenes from different vertebrate species transform mouse NIH 3T3 cells with markedly different efficiencies. v-mos, mouse (c-mosmu), and chicken (c-mosch) mos transform NIH 3T3 cells 10- to 100-fold more efficiently than do human (c-moshu) and Xenopus (c-mosxc) mos. The mos genes with the highest transforming activity efficiently induce maturation in Xenopus oocytes and mimic cytostatic factor (CSF) by causing mitotic cleavage arrest in embryos. Chimeric v-mos/c-moshu proteins that had high transforming efficiencies in NIH 3T3 cells were also effective in the induction of oocyte maturation and CSF cleavage arrest. We measured the in vitro autophosphorylation activities of the different mos proteins and found that the levels of kinase activity of v-mos, c-mosmu, and c-mosch were much higher than that of c-mosxc. These data indicate that mos gene transforming efficiency and the ability to induce oocyte maturation or mimic CSF activity are correlated with in vitro autophosphorylation activity and suggest that the mos protein plays a similar role in transformed cells and normal oocytes.     

Characterization of protein kinases in mitotic and meiotic cell extracts

A number of protein kinases have been separated and identified in extracts from mitotic and interphase culture cells and from mature and immature amphibian oocytes using nondenaturing polyacrylamide gel electrophoresis followed by in situ phosphorylation assays. Certain of these protein kinase activities appear to correlate with the biological activity of extracts, assayed by their ability to induce meiotic maturation following injection into Xenopus oocytes. These results are consistent with the notion that protein phosphorylation/dephosphorylation may be integral to the mechanisms of both nuclear membrane breakdown and chromosome condensation, events common and distinctive to mitosis and meiosis.     

Chromosome condensation activity in Rana pipiens eggs matured in vivo and in blastomeres arrested by cytostatic factor (CSF)

The ability of brain nuclei to give rise to condensed chromosomes was studied inRana pipiens eggs which had undergone meiotic maturation in vivo, in blastomeres of two-cell embryos which had been arrested at metaphase by the injection of cytostatic factor (CSF) from mature eggs, and in immature fully grown ovarian oocytes with and without prior CSF injection. Chromosomes from brain nuclei were found to condense within 4 h in mature eggs and this chromosome condensation activity was enhanced by the chelation of free Ca²⁺ in the nuclear isolation medium. Chromosomes also condensed in CSF-arrested blastomeres whether they were placed in the blastomere 30 min before the CSF injection or as long as 22 h after the CSF. Both the Ca²⁺-sensitive CSF, 1CSF, and the Ca²⁺-insensitive CSF, 2CSF, resulted in chromosome condensation within arrested blastomeres. The condensation was accompanied by the formation of multipolar spindles and asters. However, it was found that cytoplasm in CSF-arrested blastomeres does not arrest mitosis at metaphase when transferred into a cleaving blastomere. Other experiments demonstrated that chromosome condensation does not occur in ovarian oocytes even when supplied with CSF. The results are interpreted as indicating that CSF does not directly bring about chromosome condensation, but arrests the cell cycle at metaphase and stabilizes the cytoplasmic conditions of metaphase which, in turn, induce chromosome condensation in foreign nuclei as well as spindle and aster formation.     

Evidence for the presence of inhibitors of mitotic factors during G1 period in mammalian cells

Our earlier studies indicated that the mitotic factors, which induce germinal vesicle breakdown and chromosome condensation when injected into fully grown Xenopus oocytes, are preferentially associated with metaphase chromosomes and that they bind to chromatin as soon as they are synthesized during the G2 phase. In this study, we attempted to determine the fate of these factors as the cell completes mitosis and enters G1. Extracts from HeLa cells at different points during G1, S, and G2 periods were mixed with mitotic extracts in various proportions, incubated, and then injected into Xenopus oocytes to determine their maturation-promoting activity. The maturation-promoting activity of the mitotic extracts was neutralized by extracts of G1 cells during all stages of G1 but not by those of late S and G2 phase cells. Extracts of quiescent (G0) human diploid fibroblasts exhibited very little inhibitory activity. However, UV irradiation of G0 cells, which is known to cause decondensation of chromatin, significantly enhanced the inhibitory activity of extracts of these cells. These factors are termed inhibitors of mitotic factors (IMF). They seem to be activated, rather than newly synthesized, as the cell enters telophase when chromosomes begin to decondense. The IMF are nondialyzable, nonhistone proteins with a molecular weight of greater than 12,000. Since mitotic factors are known to induce chromosome condensation, it is possible that IMF, which are antagonistic to mitotic factors, may serve the reverse function of the mitotic factors, i.e., regulation of chromosome decondensation.     

Generality of the action of various maturation-promoting factors

It has been known in amphibians and starfishes that a cytoplasmic factor called maturation-promoting factor (MPF), produced in maturing oocytes under the influence of the maturation-inducing hormones, can induce germinal vesicle breakdown (GVBD) and the subsequent process of meiotic maturation. The present study revealed that injection of cytoplasm of maturing starfish oocytes (starfish MPF) into immature sea cucumber oocytes brought about maturation of the recipients. Amphibian MPF obtained from mature oocytes of Xenopus laevis or Bufo bufo was found to induce maturation of starfish oocytes following injection. Cytoplasm taken from cleaving starfish blastomeres induced maturation when injected into immature starfish oocytes. The maturation-inducing activity of cytoplasm of starfish blastomeres changed along with the mitotic cell cycle during 1- to 4-cell stages so far tested and reached a peak just before cleaving. Furthermore, an extract of mammalian cultured cells, CHO or V-79, synchronized in M phase, induced GVBD in starfish oocytes following injection, whereas S phase extract had little activity. These facts suggest that MPF generally brings about nuclear membrane breakdown in both meiosis and mitosis, and that the nature of MPF is very similar among vertebrates and invertebrates.     

Oncogenic ras protein induces meiotic maturation of amphibian oocytes in the presence of protein synthesis inhibitors

Microinjection of the activated ras oncogenic protein can induce the meiotic maturation of Xenopus laevis oocytes, a process that can also be triggered by progesterone or high concentrations of insulin. Cycloheximide and puromycin, well-known inhibitors of protein synthesis, block the maturation process induced by progesterone and insulin but do not affect the maturation caused by H-raslys12 protein microinjection. Theophylline, an inhibitor of cAMP phosphodiesterase that also affects oocyte protein synthesis, does cause a partial inhibition of ras protein-induced maturation. These findings indicate that ras protein acts on the oocyte maturation process at a point that is downstream of the protein synthesis requirement, a characteristic shared with the maturation promoting factor, an activity that appears in oocytes and mitotic cells at the onset of cell division.     

Cell coupling and maturation-promoting factor activity in in vitro-matured prepubertal and adult sheep oocytes.

We examined some differences between prepubertal and adult ovine oocytes; in particular we analyzed the functional status of the cumulus-oocyte complex, protein synthesis during in vitro maturation, and because no information is available on prepubertal and adult sheep, maturation-promoting factor (MPF) fluctuations throughout meiotic progression both in prepubertal and adult sheep oocytes. After 24 h of maturation, percentages of MII oocytes were similar between prepubertal and adult animals. Electron microscopy examinations showed that prepubertal oocytes had fewer transzonal projections than adult oocytes. Methionine uptake was significantly lower in prepubertal cumulus-enclosed oocytes examined through meiotic progression. On the contrary, denuded prepubertal oocytes showed a higher methionine incorporation in the first 4 h of incubation compared with adult oocytes. We also found some differences in MPF activity between prepubertal and adult oocytes at MII stage. In fact, prepubertal MII oocytes had a significantly lower level of MPF activity than adult oocytes did and, after fusion with germinal vesicle oocytes, they were unable to induce nuclear breakdown and chromosome condensation 1-2 h post-fusion, whereas adult MII oocytes could induce these processes. Our findings show that the lesser competence of prepubertal oocytes could be due to morphological anomalies and alterations in physiological activity and that oocytes do not reach full developmental competence until puberty.     

Injected mitotic extracts induce condensation of interphase chromatin

Although extracts from mitotic cells have been shown to induce chromosome condensation when injected into amphibian oocytes, they have not as yet been shown to induce this response in somatic interphase cells. In the experiments reported here, when mitotic extracts were injected into syncytial frog embryos, whose somatic nuclei were arrested in interphase, chromosome condensation was observed. The inability of interphase extracts, injected at similar concentrations, to induce this event demonstrates the cell cycle-specific accumulation of the factors responsible.     

Winter Hibernation and UCHL1-p34cdc2 Association in Toad Oocyte Maturation Competence

Currently, it is believed that toad oocyte maturation is dependent on the physiological conditions of winter hibernation. Previous antibody-blocking experiments have demonstrated that toad ubiquitin carboxyl-terminal hydrolase L1 (tUCHL1) is necessary for germinal vesicle breakdown during toad oocyte maturation. In this paper, we first supply evidence that tUCHL1 is highly evolutionarily conserved. Then, we exclude protein availability and ubiquitin carboxyl-terminal hydrolase enzyme activity as factors in the response of oocytes to winter hibernation. In the context of MPF (maturation promoting factor) controlling oocyte maturation and to further understand the role of UCHL1 in oocyte maturation, we performed adsorption and co-immunoprecipitation experiments using toad oocyte protein extracts and determined that tUCHL1 is associated with MPF in toad oocytes. Recombinant tUCHL1 absorbed p34^cdc2, a component of MPF, in obviously larger quantities from mature oocytes than from immature oocytes, and p13^suc1 was isolated from tUCHL1 with a dependence on the ATP regeneration system, suggesting that still other functions may be involved in their association that require phosphorylation. In oocytes from hibernation-interrupted toads, the p34^cdc2 protein level was significantly lower than in oocytes from toads in artificial hibernation, providing an explanation for the different quantities isolated by recombinant tUCHL1 pull-down and, more importantly, identifying a mechanism involved in the toad oocyte’s dependence on a low environmental temperature during winter hibernation. Therefore, in toads, tUCHL1 binds p34^cdc2 and plays a role in oocyte maturation. However, neither tUCHL1 nor cyclin B1 respond to low temperatures to facilitate oocyte maturation competence during winter hibernation.     

The role of cAMP in oocyte maturation and the role of the germinal vesicle contents in mediating maturation and subsequent developmental events in hydrozoans

Summary Externally applied membrane permeable cAMP derivatives and the injection of cAMP induce oocyte maturation in several species of hydrozoans. This technique for inducing oocyte maturation has been used to study ion permeability changes, maturation promoting factor activity and surface tension changes during maturation. Oocyte membrane potential remains constant during maturation. Cyclic AMP induced maturation proceeds in the absence of external Ca²⁺, K⁻, Mg²⁺ or Na⁺. Cytoplasm from maturing oocytes that induces oocyte maturation when it is injected into untreated oocytes is produced during cAMP induced maturation. Surface tension, as measured by the application of a standardized force that mechanically deforms individual oocytes, declines during the first part of maturation. This is followed by a sharp rise and fall of surface tension at first and second polar body formation that accompanies a slow rise in the resistance of oocytes to deformation during the last part of maturation. The production of maturation promoting factor activity and some of the changes in surface tension during maturation can occur in the absence of germinal vesicle material. Two early developmental events that follow oocyte maturation are the production of sperm chemoattractant and calcium channel function. Neither of these events occurs in eggs that have undergone maturation in the absence of germinal vesicle material. The addition of germinal vesicle contents from oocytes to eggs that have undergone maturation in the absence of germinal vesicle material initiates calcium channel function. This experiment indicates that the germinal vesicle contains factors that are necessary for post-maturation developmental events.     

The molecular basis of drug-induced G2 arrest in mammalian cells

Summary The purpose of this review was to focus mainly on the molecular events related to the progression of cells through the G2 period to examine the cause for G2-arrest in mammalian cells after exposure to various anticancer drugs. With few exceptions, most of the eukaryotic cells exhibit a G2 period in their life cycles. The G2 period, which separates S phase from mitosis, represents the time necessary for the synthesis of the various components related to the condensation of chromosomes, assembly of the mitotic spindle, and cytokinesis. Continued synthesis of RNA and protein is necessary for the successful completion of G2 and the initiation of mitosis. Inhibition of RNA and protein synthesis, replacement of phenylalanine by its analog parafluorophenylalanine, or the elevation of intracellular cAMP concentrations, induce reversible G2 arrest in cultured cells. Exposure of cells to certain antineoplastic drugs also blocks cells preferentially in G2. This irreversible drug-induced G2 arrest is associated with extensive chromosome damage. The G2-arrested cells were found to be deficient in certain proteins that may be specific for the G2-mitotic transition. These mitotic or chromosome condensation factors synthesized during the G2 period, reach their maximum levels at mitosis. A preliminary characterization of the chromosome condensation factor revealed that it is a heat labile, Ca²⁺-sensitive, nondialyzable protein with a sedimentation value of 4–5S.     

Involvement of GABAA receptor in Bufo arenarum oocyte maturation

Amphibian oocytes meiotic arrest is released under the stimulus of progesterone; this hormone interacts with the oocyte surface and starts a cascade of events leading to the activation of a cytoplasmic maturation promoting factor (MPF) that induces germinal vesicle breakdown (GVBD), chromosome condensation and extrusion of the first polar body.The aim of this work was to determine whether the activation of a GABAA receptor is able to induce GVBD in fully grown denuded oocytes of Bufo arenarum and to analyse its possible participation in progesterone-induced maturation. We also evaluated the role of purines and phospholipids in the maturation process induced by a GABAA receptor agonist such as muscimol.Our results indicated that the activation of the GABAA receptor by muscimol induces maturation in a dose- and time-dependent manner and that this activation is a genuine maturation that enables oocytes to form pronuclei. Assays with a receptor antagonist, picrotoxine, showed that the maturation induced by muscimol was inhibited. Treatment with picrotoxine, however, shows that the participation of GABAA receptor in progesterone-induced maturation is not significant.In addition, our results indicate that high intracellular levels of purines obtained by the use of db-AMPc and theophylline or the inhibition of the phosphatidylinositol 4,5-bisphosphate (PIP2 hydrolysis by neomycin and PIP2 turn over by LiCl, respectively, inhibited the maturation induced by muscimol. Treatment with H-7 indicated, however, that PKC activation is not necessary for GVBD induced by the GABAA receptor agonist. Results suggest that the transduction pathway used by the GABAA receptor to induce maturation is different from those used by progesterone.     

Regulation of the meiosis-inhibited protein kinase, a p38(MAPK) isoform, during meiosis and following fertilization of seastar oocytes

A p38(MAPK) homolog Mipk (meiosis-inhibited protein kinase) was cloned from seastar oocytes. This 40-kDa protein shares approximately 65% amino acid identity with mammalian p38-alpha isoforms. Mipk was one of the major tyrosine-phosphorylated proteins in immature oocytes arrested at the G(2)/M transition of meiosis I. The tyrosine phosphorylation of Mipk was increased in response to anisomycin, heat, and osmotic shock of oocytes. During 1-methyladenine-induced oocyte maturation, Mipk underwent tyrosine dephosphorylation and remained dephosphorylated in mature oocytes and during the early mitotic cell divisions until approximately 12 h after fertilization. At the time of differentiation and acquisition of G phases in the developing embryos, Mipk was rephosphorylated on tyrosine. In oocytes that were microinjected with Mipk antisense oligonucleotides and subsequently were allowed to mature and become fertilized, differentiation was blocked. Because MipK antisense oligonucleotides and a dominant-negative (K62R)Mipk when microinjected into immature oocytes failed to induce germinal vesicle breakdown, inhibition of Mipk function was not sufficient by itself to cause oocyte maturation. These findings point to a putative role for Mipk in cell cycle control as a G-phase-promoting factor.     

Protein synthesis requirement for maturation promoting factor (MPF) initiation of meiotic maturation in Rana oocytes.

Mechanisms controlling disintegration or breakdown of the germinal vesicle (GVBD) in Rana oocytes were investigated. A secondary cytoplasmic maturation promoting factor (MPF), produced in response to steroid stimulation, was shown to induce maturation when injected into immature recipient oocytes. Exposure of immature Rana oocytes to cycloheximide following injection of MPF or steroid treatment completely inhibited such maturation. Results indicate that injected MPF required protein synthesis for germinal vesicle breakdown and thus acted at some translational level. These results contrast with data obtained in Xenopus oocytes where injected MPF induced maturation in the presence of cycloheximide. Cytoplasmic MPF was also produced in Rana oocytes following treatment with lanthanum salts. This activity was similarly inhibited by cycloheximide. Time course studies conducted to compare the onset of cycloheximide insensitivity in steroid-treated and MPF-injected oocytes demonstrated that MPF-injected oocytes become insensitive to cycloheximide prior to steroid-treated germ cells. These results suggest that MPF acts as an intermediary in progesterone-induced maturation. Insensitivity to cycloheximide occurred several hours prior to the onset of germinal vesicle breakdown in both MPF-injected and steroid-treated oocytes. The data indicate that injected MPF in Rana does not induce nuclear disintegration directly, but rather requires amplification and/or autocatalytic synthesis of additional MPF or other factors for maturation to be induced. Molecular mechanisms involved in nuclear disintegration are discussed in relation to these species differences.     

Cytoplasmic factors that control nuclear behavior in mammalian oocytes: a re-evaluation of studies performed as a student of Yoshio Masui

Studies performed by the author in the laboratory of Dr Yoshio Masui are reviewed and interpreted in the light of subsequent findings. The first series of studies indicated that that chromosome condensation during meiotic maturation of mouse oocytes is controlled initially by a stable protein that decays during maturation and subsequently by an unstable protein synthesized after germinal vesicle breakdown. Cyclin B is present in immature oocytes, becomes partially degraded near metaphase I and then re-accumulates, suggesting that this may be protein whose activity was inferred from the original results. The second series of experiments indicated that factors which appear in the oocyte cytoplasm during maturation are able to remodel the sperm into metaphase-like chromosomes, and that the supply of these factors is limited. Recent work indicates that these factors are required for the assembly of histones onto the sperm DNA, and has identified two molecular species, mNAP-1 and NPM-3, known to promote replication-independent chromatin assembly in somatic cells, that are expressed in oocytes.