RESUMO
The protein kinase p90(Rsk) has previously been implicated as a key target of the MAPK pathway during M phase of meiosis II in Xenopus oocytes. To determine whether Rsk is a mediator of MAPK for stimulation of the G(2)/M transition early in meiosis I, we sought to generate a form of Rsk that would be constitutively active in resting, G(2) phase oocytes. Initial studies revealed that an N-terminal truncation of 43 amino acids conferred enhanced specific activity on the enzyme in G(2) phase, and stability was highest if the C terminus was not truncated. The full-length enzyme is known to be activated by phosphorylation at five sites. Two of these sites and flanking residues were replaced with either aspartic or glutamic acid, and Tyr(699) was mutated to alanine. The resulting construct, termed fully activated (FA) Rsk, had constitutive activity in G(2) phase, with a specific activity equivalent to that of wild type Rsk in M phase. In eight independent experiments approximately 45% of oocytes expressing FA-Rsk underwent germinal vesicle breakdown (GVBD, the G(2)/M transition) in the absence of progesterone, and this effect could be observed even in the presence of the MAPK kinase inhibitor U0126. Moreover, the specific activity of FA-Rsk in vivo was unaffected by U0126. In oocytes that did not undergo GVBD with FA-Rsk expression, subsequent treatment with progesterone resulted in a very rapid rate of GVBD even in the presence of U0126 to inhibit the endogenous MAPK/Rsk pathway. These results indicate that Rsk is the mediator of MAPK effects for the G(2)/M transition in meiosis I and in a subpopulation of oocytes Rsk is sufficient to trigger the G(2)/M transition.
Assuntos
Fase G2 , Mitose , Proteínas Quinases S6 Ribossômicas/metabolismo , Sequência de Aminoácidos , Animais , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Quinases S6 Ribossômicas/química , Proteínas Quinases S6 Ribossômicas/genética , Homologia de Sequência de Aminoácidos , XenopusRESUMO
Sister chromatid separation and cyclin degradation in mitosis depend on the association of the anaphase-promoting complex (APC) with the Fizzy protein (Cdc20), leading to the metaphase/anaphase transition and exit from mitosis [1--3]. In Xenopus, after metaphase of the first meiotic division, only partial cyclin degradation occurs, and chromosome segregation during anaphase I proceeds without sister chromatid separation [4--7]. We investigated the role of xFizzy during meiosis using an antisense depletion approach. xFizzy accumulates to high levels in Meiosis I, and injection of antisense oligonucleotides to xFizzy blocks nearly all APC-mediated cyclin B degradation and Cdc2/cyclin B (MPF) inactivation between Meiosis I and II. However, even without APC activation, xFizzy-ablated oocytes progress to Meiosis II as shown by cyclin E synthesis, further accumulation of cyclin B, and evolution of the metaphase I spindle to a metaphase II spindle via a disc-shaped aggregate of microtubules known to follow anaphase I [8]. Inhibition of the MAPK pathway by U0126 in antisense-injected oocytes prevents cyclin B accumulation beyond the level that is present at metaphase I. Full synthesis and accumulation can be restored in the presence of U0126 by the expression of a constitutively active form of the MAPK target, p90(Rsk). Thus, p90(Rsk) is sufficient not only to partially inhibit APC activity [7], but also to stimulate cyclin B synthesis in Meiosis II.
Assuntos
Ciclina B/metabolismo , Meiose/fisiologia , Oócitos/citologia , Proteínas de Saccharomyces cerevisiae , Proteínas de Xenopus , Anáfase , Animais , Elementos Antissenso (Genética) , Proteínas Cdc20 , Proteínas Cdh1 , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiologia , Metáfase , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oócitos/química , Oócitos/fisiologia , Proteínas Quinases S6 Ribossômicas/metabolismo , XenopusRESUMO
BACKGROUND: The kinetochore attachment (spindle assembly) checkpoint arrests cells in metaphase to prevent exit from mitosis until all the chromosomes are aligned properly at the metaphase plate. The checkpoint operates by preventing activation of the anaphase-promoting complex (APC), which triggers anaphase by degrading mitotic cyclins and other proteins. This checkpoint is active during normal mitosis and upon experimental disruption of the mitotic spindle. In yeast, the serine/threonine protein kinase Bub1 and the WD-repeat protein Bub3 are elements of a signal transduction cascade that regulates the kinetochore attachment checkpoint. In mammalian cells, activated MAPK is present on kinetochores during mitosis and activity is upregulated by the spindle assembly checkpoint. In vertebrate unfertilized eggs, a special form of meiotic metaphase arrest by cytostatic factor (CSF) is mediated by MAPK activation of the protein kinase p90(Rsk), which leads to inhibition of the APC. However, it is not known whether CSF-dependent metaphase arrest caused by p90(Rsk) involves components of the spindle assembly checkpoint. RESULTS: xBub1 is present in resting oocytes and its protein level increases slightly during oocyte maturation and early embryogenesis. In Xenopus oocytes, Bub1 is localized to kinetochores during both meiosis I and meiosis II, and the electrophoretic mobility of Bub1 upon SDS-PAGE decreases during meiosis I, reflecting phosphorylation and activation of the enzyme. The activation of Bub1 can be induced in interphase egg extracts by selective stimulation of the MAPK pathway by c-Mos, a MAPKKK. In oocytes treated with the MEK1 inhibitor U0126, the MAPK pathway does not become activated, and Bub1 remains in its low-activity, unshifted form. Injection of a constitutively active target of MAPK, the protein kinase p90(Rsk), restores the activation of Bub1 in the presence of U0126. Moreover, purified p90(Rsk) phosphorylates Bub1 in vitro and increases its protein kinase activity. CONCLUSIONS: Bub1, an upstream component of the kinetochore attachment checkpoint, is activated during meiosis in Xenopus in a MAPK-dependent manner. Moreover, a single substrate of MAPK, p90(Rsk), is sufficient to activate Bub1 in vitro and in vivo. These results indicate that in vertebrate eggs, kinetochore attachment/spindle assembly checkpoint proteins, including Bub1, are downstream of p90(Rsk) and may be effectors of APC inhibition and CSF-dependent metaphase arrest by p90(Rsk).
Assuntos
Oócitos/fisiologia , Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Primers do DNA , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Fases de Leitura Aberta , Fosforilação , Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases , Proteínas Quinases S6 Ribossômicas , Homologia de Sequência de Aminoácidos , XenopusRESUMO
Apoptosis is controlled by a complex interplay between regulatory proteins. Previous work has shown that Xenopus embryos remove damaged cells by apoptosis when irradiated before, but not after, the midblastula transition (MBT). Here we demonstrate that Akt/protein kinase B is activated and mediates an antiapoptotic signal only in embryos irradiated after the MBT. In addition, an increase in xBcl-2/xBax oligomerization and a decrease in xBax homodimerization promote a protective effect against apoptosis only after the MBT. The post-MBT survival mechanism arrests cells in G(1) phase by increasing expression of the cyclin-dependent kinase inhibitor p27(Xic1). p27(Xic1) associates with cyclin D/Cdk4 and cyclin A/Cdk2 complexes to cause G(1)/S arrest, perhaps allowing more time for DNA repair. Taken together, the results define the DNA damage response as an element of the MBT and indicate that multiple mechanisms prevent apoptosis after the MBT.
Assuntos
Apoptose/fisiologia , Blastocisto/citologia , Blastocisto/fisiologia , Quinases relacionadas a CDC2 e CDC28 , Proteínas de Ciclo Celular , Ciclo Celular/fisiologia , Dano ao DNA , Proteínas Supressoras de Tumor , Xenopus/embriologia , Animais , Blastocisto/efeitos da radiação , Ciclo Celular/efeitos da radiação , Ciclina D , Quinase 2 Dependente de Ciclina , Quinase 4 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/fisiologia , Fase G1 , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Morfogênese , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fase S , Proteínas de XenopusRESUMO
BACKGROUND: During oocyte maturation in Xenopus, progesterone induces entry into meiosis I, and the M phases of meiosis I and II occur consecutively without an intervening S phase. The mitogen-activated protein (MAP) kinase is activated during meiotic entry, and it has been suggested that the linkage of M phases reflects activation of the MAP kinase pathway and the failure to fully degrade cyclin B during anaphase I. To analyze the function of the MAP kinase pathway in oocyte maturation, we used U0126, a potent inhibitor of MAP kinase kinase, and a constitutively active mutant of the protein kinase p90(Rsk), a MAP kinase target. RESULTS: Even with complete inhibition of the MAP kinase pathway by U0126, up to 90% of oocytes were able to enter meiosis I after progesterone treatment, most likely through activation of the phosphatase Cdc25C by the polo-like kinase Plx1. Subsequently, however, U0126-treated oocytes failed to form metaphase I spindles, failed to reaccumulate cyclin B to a high level and failed to hyperphosphorylate Cdc27, a component of the anaphase-promoting complex (APC) that controls cyclin B degradation. Such oocytes entered S phase rather than meiosis II. U0126-treated oocytes expressing a constitutively active form of p90(Rsk) were able to reaccumulate cyclin B, hyperphosphorylate Cdc27 and form metaphase spindles in the absence of detectable MAP kinase activity. CONCLUSIONS: The MAP kinase pathway is not essential for entry into meiosis I in Xenopus but is required during the onset of meiosis II to suppress entry into S phase, to regulate the APC so as to support cyclin B accumulation, and to support spindle formation. Moreover, one substrate of MAP kinase, p90(Rsk), is sufficient to mediate these effects during oocyte maturation.
Assuntos
Meiose , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Oócitos/enzimologia , Proteínas Quinases S6 Ribossômicas/metabolismo , Proteínas de Xenopus , Animais , Butadienos/farmacologia , Proteínas de Ciclo Celular/metabolismo , Ciclina B/metabolismo , Replicação do DNA/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Feminino , Immunoblotting , Meiose/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Mutação , Nitrilas/farmacologia , Oócitos/citologia , Oócitos/efeitos dos fármacos , Reação em Cadeia da Polimerase , Progesterona/farmacologia , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/farmacologia , Proteínas Quinases S6 Ribossômicas/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas/genética , Xenopus , Fosfatases cdc25/metabolismoRESUMO
Before fertilization, vertebrate eggs are arrested in metaphase of meiosis II by cytostatic factor (CSF), an activity that requires activation of the mitogen-activated protein kinase (MAPK) pathway. To investigate whether CSF arrest is mediated by the protein kinase p90Rsk, which is phosphorylated and activated by MAPK, a constitutively activated (CA) form of Rsk was expressed in Xenopus embryos. Expression of CA Rsk resulted in cleavage arrest, and cytological analysis showed that arrested blastomeres were in M phase with prominent spindles characteristic of meiotic metaphase. Thus, Rsk appears to be the mediator of MAPK-dependent CSF arrest in vertebrate unfertilized eggs.
Assuntos
Blastômeros/citologia , Sistema de Sinalização das MAP Quinases , Metáfase , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Animais , Blastômeros/enzimologia , Ativação Enzimática , Meiose , Oócitos/citologia , Oócitos/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-mos/genética , Proteínas Proto-Oncogênicas c-mos/metabolismo , RNA Mensageiro/genética , Proteínas Recombinantes/metabolismo , Proteínas Quinases S6 Ribossômicas/genética , Fuso Acromático/ultraestrutura , XenopusRESUMO
At the midblastula transition during Xenopus development, the cell cycle is remodeled, and zygotic transcription is initiated. Additionally, cyclin E1 is degraded at the midblastula transition independently of protein synthesis, the number of cell cycles, and the nuclear-to-cytoplasmic ratio. In the studies reported here, cell cycles were delayed by transient inhibition of protein synthesis with cycloheximide (100 microg/ml) prior to the midblastula transition. Even after reaccumulation of mitotic cyclins and resumption of cell divisions, cycloheximide-treated embryos did not resume DNA synthesis, failed to initiate transcription, and synchronously became apoptotic before the gastrula stage. These results were independent of the stage at which embryos were treated or the duration of treatment. Inhibition of zygotic transcription with alpha-amanitin also induced apoptosis. These data suggest that a developmental checkpoint at the midblastula transition is maternally regulated and can trigger apoptosis. Apoptosis induced by cycloheximide or alpha-amanitin was blocked by injection of RNA encoding Xenopus Bcl-2, suggesting that this maternal program is normally blocked by expression of an apoptotic inhibitor. Embryos pulsed with lower doses of cycloheximide (10 microg/ml) delayed development prior to the midblastula transition but resumed DNA synthesis, initiated transcription, and gastrulated normally. This indicates that the apoptotic response is initiated only when delayed embryos are unable to support initiation of zygotic transcription.
Assuntos
Apoptose/genética , Transcrição Gênica , Zigoto/citologia , Amanitinas/farmacologia , Animais , Ciclinas/metabolismo , Cicloeximida/farmacologia , Replicação do DNA/efeitos dos fármacos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Xenopus laevis , Zigoto/efeitos dos fármacos , Zigoto/metabolismoRESUMO
During Xenopus development, the early cell cycles consist of rapid oscillations between DNA synthesis and mitosis until completion of the 12th mitotic division. Then the cycle lengthens and becomes asynchronous, zygotic transcription begins, and G phases are established, a period known as the midblastula transition (MBT). Some aspects of the MBT, such as zygotic transcription, depend on acquisition of a threshold nuclear to cytoplasmic (N/C) ratio, whereas others, such as maternal cyclin E degradation, are independent of nuclear events and appear to be controlled by an autonomous maternal timer. To investigate the function of cyclin E during the early cycles, cyclin E/Cdk2 kinase activity was specifically inhibited in fertilized eggs by a truncated form of the Xenopus Cdk inhibitor, Xic1 (Delta34Xic1). Delta34Xic1 caused lengthening of the embryonic cell cycles that correlated with increased levels of mitotic cyclins. However, DNA synthesis was not inhibited. Several hallmarks of the MBT were delayed for several hours in Delta34Xic1-injected embryos, including the disappearance of cyclins E and A, the initiation of zygotic transcription, and the reappearance of phosphotyrosine on Cdc2. In both control and Delta34Xic1-injected embryos, cyclin E was degraded after the 12th mitotic division as zygotic transcription began, but experiments with alpha-amanitin show that cyclin E degradation is not dependent on zygotic transcription. Thus, the length of the early cycles and the timing of maternal cyclin degradation depend upon cyclin E/Cdk2 activity. Neither oscillations in cyclin E/Cdk2 activity during the early cycles nor the disappearance of cyclin E at the MBT were dependent on protein synthesis. These data suggest that cyclin E/Cdk2 is directly linked to an autonomous maternal timer that drives the early embryonic cell cycles until the MBT.
Assuntos
Blastocisto/fisiologia , Quinases relacionadas a CDC2 e CDC28 , Ciclo Celular , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Embrião não Mamífero/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae , Xenopus/embriologia , Animais , Blastocisto/citologia , Divisão Celular , Quinase 2 Dependente de Ciclina , Proteínas Inibidoras de Quinase Dependente de Ciclina , Quinases Ciclina-Dependentes/antagonistas & inibidores , Proteínas Fúngicas/biossíntese , Mitose , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Recombinantes de Fusão/biossíntese , Proteínas de XenopusRESUMO
After the twelfth cell division in Xenopus embryos, zygotic gene transcription is activated, cells become motile, and cell division becomes asynchronous. This developmental change is termed the midblastula transition. High doses of gamma-irradiation (gamma-IR) before the midblastula transition induced apoptotic cell death and increased the levels of cyclin A1 and cyclin A1-Cdk2 activity. The addition of recombinant cyclin A1-Cdk2 induced the formation of apoptotic nuclei in Xenopus egg extracts, suggesting a role for cyclin A1-Cdk2 in apoptosis. Hallmarks of apoptosis, such as internucleosomal DNA fragmentation, pyknotic and uniformly condensed nuclei, and loss of intercellular attachments, were evident in embryos exposed to gamma-IR before the midblastula transition. Apoptotic cells accumulated in the blastocoel, suggesting that before the midblastula transition Xenopus embryos use apoptosis to eliminate cells containing damaged DNA. However, embryos treated with the same dose of gamma-IR after the midblastula transition developed normally and exhibited no signs of apoptosis, no change in cyclin A1 level, and no increase in cyclin A1-Cdk2 activity. These results indicate that there is a change in the response to DNA damage at the midblastula transition in Xenopus embryos.
Assuntos
Apoptose/efeitos da radiação , Blastocisto/efeitos da radiação , Quinases relacionadas a CDC2 e CDC28 , Ciclina A , Quinases Ciclina-Dependentes/efeitos da radiação , Ciclinas/efeitos da radiação , Raios gama , Proteínas Serina-Treonina Quinases/efeitos da radiação , Animais , Blastocisto/metabolismo , Núcleo Celular/efeitos dos fármacos , Quinase 2 Dependente de Ciclina , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Embrião não Mamífero/metabolismo , Embrião não Mamífero/efeitos da radiação , Óvulo/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transcrição Gênica/efeitos da radiação , Xenopus , Proteínas de Xenopus , Zigoto/metabolismo , Zigoto/efeitos da radiaçãoRESUMO
The function of the Xenopus c-mos proto-oncogene product (Mos(xe)) has been investigated during oocyte maturation. Experiments with a new antibody able to immunoblot Mos(xe) demonstrated the time course of MAP kinase (MAP K) activation in oocytes paralleled Mos(xe) accumulation, and in activated eggs the deactivation of MAP K paralleled the degradation of Mos(xe). Ablation of Mos synthesis by microinjection of antisense oligodeoxynucleotides abolished activation of MAP K by progesterone, but microinjection of GST-Mos fully restored both MAP K activation and germinal vesicle breakdown (GVBD). The Mos(xe) level at metaphase of Meiosis I (MI) was 2 - 3-fold less than that at metaphase of Meiosis II (MII), but MAP K activation was maximal at metaphase in both MI and MII. In the transition between MI and MII, both cyclin B and Mos(xe) levels rapidly declined in the presence of cycloheximide and injection of exogenous GST-Mos(xe) did not prevent degradation of either protein, although MAP K was activated. Microinjection of GST-Mos(xe) into oocytes was able to activate MAP K before GVBD and H1 kinase activation, and microinjection of constitutively-activated thiophosphorylated MAP K induced de novo synthesis of Mos(xe) before H1 kinase activation, suggesting the existence of a positive feedback loop between MAP K and Mos(xe) accumulation.
Assuntos
Proteínas Oncogênicas v-mos/fisiologia , Oócitos/fisiologia , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Ativação Enzimática , Feminino , Genes mos , Meiose/fisiologia , Metáfase/fisiologia , Proteínas Oncogênicas v-mos/genética , Proteínas Oncogênicas v-mos/metabolismo , Oócitos/citologia , Oócitos/enzimologia , Xenopus laevisRESUMO
MAP kinase (MAPK) is activated in animal cap explants from Xenopus embryos in response to mesoderm induction by FGF. This activation is rapid, appearing within 1 min of treatment with FGF, and prolonged, lasting for at least 2 hr. By immunoblot analysis, this activation of MAPK is coupled with an electrophoretic shift to the slowly migrating, phosphorylated form of MAPK. Activin-stimulated mesoderm induction also results in the activation of MAPK, but only upon prolonged exposure. However, activin can potentiate the activation of MAPK by FGF as early as 1 min after administration. These findings suggest that MAPK is involved in the early signaling events of FGF-mediated mesoderm induction, and this involvement is modulated by other mesoderm inducers such as activin.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Xenopus laevis/embriologia , Sequência de Aminoácidos , Animais , Indução Embrionária , Ativação Enzimática , Fertilização , Fator 2 de Crescimento de Fibroblastos/farmacologia , Proteína Quinase 1 Ativada por Mitógeno , Dados de Sequência Molecular , Óvulo/enzimologia , Peptídeos/química , Peptídeos/imunologiaRESUMO
The growth and differentiation of the epithelium in many tissues is mediated by interactions with the adjacent mesenchyme, but the mechanisms responsible remain undefined. To identify the factors involved in the growth and branching morphogenesis of ureteric bud, which is the collecting duct anlagen, buds from 13-gestation-day rat embryos were separated from the metanephrogenic mesenchyme and explanted to culture dishes coated with gelled type I collagen in a defined medium. Under these conditions buds attached to the substrate and grew out without indication of cell senescence. When buds were instead suspended in gelled type I collagen, branching morphogenesis was observed despite the absence of mesenchyme although it was not as extensive as in vivo. Since growth occurred much more slowly in culture than expected, culture conditions were varied in attempts to accelerate the process. Despite extensive screening of matrices and growth factors, only epidermal and endothelial cell growth factors stimulated growth to a significant extent. Transforming growth factor-beta, on the other hand, was a potent inhibitor of growth. Homogenates from tumors that caricature metanephrogenic mesenchyme were highly mitogenic for bud cells and, thus, will be a source of material for characterizing regulatory factors involved in renal growth. These studies show that growth and branching morphogenesis of the ureteric bud can occur without direct cell-cell interactions with the metanephrogenic mesenchyme and that matrices and factors secreted by the mesenchyme may mediated these activities in vivo.
Assuntos
Túbulos Renais Coletores/embriologia , Mesoderma/fisiologia , Morfogênese/fisiologia , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Colágeno , Meios de Cultura Livres de Soro/farmacologia , Fatores de Crescimento Endotelial/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Feminino , Neoplasias Renais/patologia , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/fisiologia , Masculino , Mesoderma/citologia , Morfogênese/efeitos dos fármacos , Ratos , Ratos Endogâmicos F344 , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/fisiologia , Fator de Crescimento Transformador beta/farmacologia , Células Tumorais CultivadasRESUMO
Previous work identified in blastocele fluid a soluble activity which killed embryonal carcinoma cells with trophectodermal potential but not those with embryonic potential [35]. From use of a malignant caricature of the late blastocyst, this toxic activity was postulated to be H2O2 [8]. The purpose of this paper was to determine if blastocele fluid also contained amounts of H2O2 capable of mediating the preferential killing of malignant pretrophectodermal cells (ECa 247). We not only observed that blastocele fluid is not toxic for these cells in the presence of catalase, but that malignant cells with embryonic potential (P19) that normally survive exposure to blastocele fluid become sensitive to it if their intracellular glutathione levels are lowered. Thus, it is concluded that the blastocyst contains amounts of H2O2 toxic to malignant pretrophectodermal cells and that glutathione-dependent mechanisms protect malignant inner cell mass cells with embryonic potential. Apparently, H2O2 production and glutathione-dependent protection mechanisms are developmentally regulated in the inner cell mass. These results are discussed with regards to apoptosis and the regulation of tissue mass.
Assuntos
Blastocisto/fisiologia , Peróxido de Hidrogênio/farmacologia , Animais , Antimetabólitos/farmacologia , Butionina Sulfoximina , Catalase/farmacologia , Sobrevivência Celular/fisiologia , Modelos Animais de Doenças , Glutationa/fisiologia , Técnicas In Vitro , Metionina Sulfoximina/análogos & derivados , Metionina Sulfoximina/farmacologia , Camundongos , Neoplasias Embrionárias de Células Germinativas/patologia , Células Tumorais CultivadasRESUMO
The malignant growth potential of embryonal carcinoma cells may be controlled by environmental factors. For example, embryonal carcinoma cells placed into normal blastocysts may not exhibit the continued growth expected of malignant cells but rather may lose all aspects of the malignant phenotype and become apparently normal embryonic cells. Loss of the malignant phenotype of embryonal carcinoma cells occurs early in these injected blastocysts and has been used as the basis of assays to study the mechanisms of regulation of embryonal carcinoma by the blastocyst. In this regard, P19, an embryonal carcinoma that makes midgestation chimeras, was regulated by blastocele fluid plus contact with trophectoderm but not by blastocele fluid plus contact with inner cell mass (ICM). In contrast, ECa 247, which makes trophectoderm, was regulated by exposure to blastocele fluid plus contact with trophectoderm or ICM. During the course of these experiments, dead embryonal carcinoma and ICM cells were observed, and blastocele fluid was then shown to kill ECa 247 and normal ICM cells of early blastocysts with trophectodermal potential. P19 cells and ICM cells with potential to make the embryo were not killed by blastocele fluid. Programmed cell death occurs in the ICM of the blastocyst during the transition from early (when ICM has the potential to make trophectoderm) to late (when the ICM lacks the potential to make trophectoderm). It is postulated that this programmed cell death is designed to eliminate redundant ICM cells with trophectodermal potential, and its mechanism of action is mediated by epigenetic factors in blastocele fluid.