RESUMO
A cytoplasmic component(s), previously shown to rescue the 'blocked' 2-cell mouse embryo in vitro, has been demonstrated to peak in activity during the transition between G2 and M phase and decline thereafter. The possible significance of this component(s) in the regulation of cleavage of the cultured mouse embryo is discussed.
Assuntos
Fase de Clivagem do Zigoto , Citoplasma/fisiologia , Embrião de Mamíferos/fisiologia , Mitose , Animais , Células Cultivadas , Embrião de Mamíferos/ultraestrutura , Camundongos , Microscopia EletrônicaRESUMO
Mouse embryos with their zonae pellucidae intact are more resistant to suboptimal culture conditions than are zona-free embryos. This observation questions the general practice of using zona-intact preimplantation mouse embryos to monitor the quality of media and reagents used in human IVF programs.
Assuntos
Meios de Cultura/normas , Embrião de Mamíferos , Fertilização in vitro , Indicadores e Reagentes/normas , Camundongos , Animais , Feminino , Camundongos Endogâmicos , Controle de Qualidade , Zona PelúcidaAssuntos
Plaquetas/fisiologia , Prenhez/sangue , Animais , Estro , Feminino , Fertilização , Masculino , Camundongos , GravidezRESUMO
The preimplantation mouse blastocyst consists of two differentiated tissues, the trophectoderm (a structurally and functionally polarized epithelium) and the inner cell mass. The divergence of these two cell types can be traced back to a contact dependent polarization of the surface and cytoplasm at the 8-cell stage. Membrane/cytocortical organization during this preimplantation period has been studied using freeze fracture in conjunction with the sterol-binding antibiotic filipin in an attempt to discern the molecular basis and origin of these surface asymmetries. The distribution of filipin reactivity within the different membrane domains showed that the surface polarity exhibited by trophectoderm and by blastomeres of the 8-cell stage is underlain by a heterogeneity in molecular organization of the membrane/cytocortex which may originate prior to the appearance of any overt surface polarity. The results are discussed in terms of the likely basis of this membrane/cytocortical asymmetry, its probable origins and the use of the preimplantation mouse embryo as a model system for studying the assembly of a polarized epithelium.
Assuntos
Blastocisto/ultraestrutura , Animais , Blastocisto/metabolismo , Membrana Celular/metabolismo , Colesterol/metabolismo , Feminino , Filipina/metabolismo , Técnica de Fratura por Congelamento , Camundongos , Microscopia Eletrônica , Óvulo/ultraestruturaRESUMO
Embryos from certain strains of mice do not develop into blastocysts when cultured in vitro from the 1- or 2-cell stages but arrest development as 2-cell embryos--a phenomenon referred to as the '2-cell block'. Reciprocal crosses between eggs and sperm of a 'blocking' (CFLP) and 'non-blocking' (F1) strain show that in this combination the genotype of the egg alone determines whether the embryo 'blocks' at the 2-cell stage (or continues retarded development to the 4- to 6-cell stage in a minority of cases). A comparison of molecular and cellular development in normal and 'blocked 2-cell' embryos was therefore undertaken to investigate the influence of these maternal components on early mouse development. The results show that the majority of 'blocked 2-cells' arrest development at a stage equivalent to the late 2-cell stage in terms of cellular and nuclear division, DNA synthesis, activation of the embryonic genome, qualitative and quantitative changes in amino acid uptake, polypeptide synthesis and morphological maturation of organelles. These observations are compatible with the notion that maternally inherited developmental information plays an important role in controlling early cleavage of the mouse embryo.
Assuntos
Camundongos Endogâmicos/embriologia , Animais , Blastocisto/metabolismo , Contagem de Células , Divisão Celular , Núcleo Celular/análise , Células Cultivadas , Cruzamentos Genéticos , DNA/análise , Eletroforese em Gel de Poliacrilamida , Feminino , Metionina/metabolismo , Camundongos , Microscopia Eletrônica de Varredura , Biossíntese PeptídicaRESUMO
Mouse embryogenesis from ovulation to the early two-cell stage is controlled at a post-transcriptional level by components of the egg assembled during oogenesis and, in some cases, sequestered in an inactive form. These molecular changes appear to be under dual control. The terminal stages of oogenesis initiate a programme of sequential activation of previously untranslated mRNAs and post-translational modifications of pre-existing and newly synthesized polypeptides. Superimposed upon this programme is a sequence of events set in train by fertilization. The earliest of these events that we have detected is a post-translational modification(s). This is followed closely by the activation and translation of new species of mRNA(s). We suggest that the oocyte programme controls the general 'housekeeping' functions of the cell and that the transition to the fertilized state may be initiated at the post-translational level and lead to other 'fertilization-specific' changes that influence processes later in development. The transition from maternal (post-transcriptional) to embryonic (transcriptional) control of development occurs at the early two-cell stage and involves two periods of transcriptional activity closely coupled to translation. In contrast, the maternal mRNAs show an abrupt decline in stability during the two-cell stage. However, many of their polypeptide products appear to persist undegraded until at least the morula stage.
Assuntos
Fase de Clivagem do Zigoto/fisiologia , Camundongos/embriologia , Oócitos/fisiologia , Animais , Gonadotropina Coriônica/farmacologia , Proteínas do Ovo/biossíntese , Feminino , Regulação da Expressão Gênica , Ponto Isoelétrico , Peso Molecular , RNA Mensageiro/genética , Fatores de TempoRESUMO
A variety of Agents (viz : cytochalasin D, colcemid, cytochalasin D+ colcemid, Ca2+-free medium, 7-ketocholesterol, cholesterol, concanavalin A, anti-embryonal carcinoma antiserum and tunicamycin) which modify the cell membrane and/or cytoskeleton were used to investigate the molecular and cellular basis of the intercellular and intracellular components of compaction and analyse the relationships between them. It was found that the individual components could be selectively dissociated from one another. Cell flattening and close intercellular apposition were the most sensitive features and affected by the majority of agents. Tight junctions did not form in the absence of intercellular apposition, however an apparently normal degree of intercellular apposition did not necessarily lead to the assembly of these junctions. Polarization of individual blastomeres, as assessed by the reorganization of the cell surface, was the component most resistant to experimental intervention since it occurred in the presence of all agents used, though it was modified by some of them. The result are discussed in terms of the molecular and cellular events underlying polarization, intercellular apposition and tight junction formation as well as the significance of these events for normal blastocyst formation.
Assuntos
Blastômeros/efeitos dos fármacos , Animais , Blastocisto/citologia , Blastômeros/citologia , Cálcio , Colesterol/farmacologia , Concanavalina A/farmacologia , Citocalasina D , Citocalasinas/farmacologia , Demecolcina/farmacologia , Soros Imunes/farmacologia , Junções Intercelulares , Cetocolesteróis/farmacologia , Camundongos , Mórula/citologia , Tunicamicina/farmacologiaRESUMO
Between the 1-cell zygote and the early blastocyst stage of mouse embryos the net rate of uptake of methionine increased, the internal pool became progressively more expanded and less easily reached steady state, and the specificity of competitor amino acids changed. Sodium-dependent transport was first detected in compacted morulae (16--32-cell stage). Uptake of [14C]methylaminoisobutyric acid was detectable in blastocysts but not in unfertilized eggs. Efflux of methionine by an exchange transport system was detectable at all stages, but in intact blastocysts much higher external concentrations were required to activate exchange transport. An exchange system with properties similar to that operating at cleavage stages was exposed when blastocysts were collapsed with cytochalasin D. Since this exchange system was not detectable in isolated inner cell masses, it may be confined to the juxtacoelic surface of trophectoderm cells.
Assuntos
Blastocisto/metabolismo , Desenvolvimento Embrionário , Metionina/metabolismo , Aminoácidos/metabolismo , Ácidos Aminoisobutíricos/análogos & derivados , Ácidos Aminoisobutíricos/metabolismo , Animais , Transporte Biológico , Feminino , Camundongos , Gravidez , Sódio/metabolismoAssuntos
Blastocisto/metabolismo , Membrana Celular/metabolismo , Colesterol/biossíntese , Lanosterol/biossíntese , Lipídeos de Membrana/biossíntese , Animais , Ésteres do Colesterol/biossíntese , Cromatografia em Camada Fina , Feminino , Hidroximetilglutaril-CoA Redutases/metabolismo , Ácido Mevalônico/metabolismo , Camundongos , Esqualeno/biossínteseRESUMO
Cleavage stage mouse embryos were incubated with cytochalasin D, which inhibited cytokinesis, intercellular adhesion and junction formation but not aspects of the morphological polarization associated with compaction. If the drug was removed when controls had formed blastocysts, the embryos compacted within 1 hr, assembled junctional complexes at the apices of their cells and accumulated fluid to form "blastocyst-like vesicles" within the next 8 to 12 hr. The molecular and morphological development of these blastocyst-like vesicles and embryos cultured continuously in cytochalasin D followed the same temporal sequence as untreated controls, indicating that they are similar to normal intact blastocysts despite their decreased number of cells, altered ploidy and the absence of an overt inner cell mass. Since maturation of the embryo can take place in the absence of the intercellular features of compaction, the cellular polarization of blastomeres may be important for initiating the differentiation of inner cell mass and trophectoderm in the normal compacting morula.
Assuntos
Blastocisto/citologia , Diferenciação Celular/efeitos dos fármacos , Citocalasinas/farmacologia , Animais , Blastocisto/ultraestrutura , Citocalasina D , Relação Dose-Resposta a Droga , Feminino , Técnicas In Vitro , Camundongos , Microscopia Eletrônica , Mórula/citologia , Mórula/ultraestrutura , Gravidez , Fatores de TempoRESUMO
The role of membrane sterols in the compaction and subsequent development of the preimplantation mouse embryo was studied by incubating embryos in 7-ketocholesterol and other oxygenated sterols. These sterols have been shown to inhibit sterol synthesis and deplete membranes of cholesterol in a variety of ther cell types. Compaction and subsequent blastocyst formation were normal when embryos were incubated in physiological sterols but were inhibited by oxygenated sterols to a degree which depended upon the concentration of sterol, duration of incubation and developmental age of the embryos. Precompaction 8-cell embryos were most susceptible to the action of these sterols and failed to compact (as assessed by cell flattening and increased intercellular adhesion) but continued to divide, whilst later stage embryos developed normally, 7-ketocholesterol had a specific effect on the ultrastructure of the smooth endoplasmic reticulum of treated embryos. The developmental and ultrastructural effects induced by the oxygenated sterols could be reversed or prevented by the use of products of the blocked reaction (i.e. mevalonate, desmosterol or cholesterol). These results substantiate the evidence that preimplantation mammalian embryos are capable of synthesizing membrane sterols from the 8-cell stage onwards and emphasize the importance of the sterol composition of membranes for normal cytokinesis and compaction of the mouse embryo.
Assuntos
Blastocisto/fisiologia , Esteróis/metabolismo , Animais , Blastocisto/efeitos dos fármacos , Blastocisto/ultraestrutura , Contagem de Células , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Técnicas de Cultura , Cetocolesteróis/farmacologia , Camundongos , Microscopia Eletrônica , Microscopia de Contraste de FaseRESUMO
Pregnancy block caused by exposure of mated female mice to a strange male was significantly reduced by bilateral destruction of the vomeronasal organ. Treatment of newly mated females with alpha-bromocriptine also produced pregnancy block. Pregnancy block also occurred in mated females exposed to strange male odours, but the blastocysts which had failed to implant were still present in the uterus and were viable for up to 15 days after mating. Implantation was induced in such mice by administration of exogenous progesterone and oestradiol.
Assuntos
Implantação Tardia do Embrião , Implantação do Embrião , Septo Nasal/fisiologia , Feromônios/fisiologia , Prolactina/fisiologia , Animais , Bromocriptina/farmacologia , Implantação do Embrião/efeitos dos fármacos , Implantação Tardia do Embrião/efeitos dos fármacos , Feminino , CamundongosRESUMO
Synthesis of phospholipid during cleavage, compaction and blastocyst formation of the preimplantation mouse embryo was investigated using [methyl-3H]-choline as a specific precursor. The only choline-containing lipids found to incorporate label were phosphatidylcholine and lysolecithin. [Methyl-3H]choline incorporation into lipid was detectable at the 2-cell stage and increased 9-13-fold (on a per embryo basis) during the 8-cell stage and subsequent compaction of the morula. Incorporation of choline was also elevated in the blastocyst but could not be compared accurately with the rates observed in earlier embryos due to uncertainty about the size of the endogenous choline pool at this stage. Choline kinase (assayed in vitro) was detectable at every stage, its activity increased during development and paralleled (qualitatively) the extent of phosphocholine formation in intact embryos. Phospholipid turnover and choline base exchange did not contribute significantly to [methyl-3H]choline incorporation into lipid, which is hence judged to represent denovo synthesis of phospholipid via the Kennedy pathway. Mouse embryo lipids exhibit several features which may be characteristic of immature cells and which could influence the properties of their membranes. These include the absence of detectable sphingomyelin synthesis and the presence of demonstrable deacylation and turnover of phosphatidylcholine.
Assuntos
Blastocisto/metabolismo , Fosfolipídeos/biossíntese , Animais , Blastocisto/enzimologia , Células Cultivadas , Colina/metabolismo , Colina Quinase/metabolismo , Cromatografia em Camada Fina , Feminino , Cinética , CamundongosRESUMO
Ovariectomy-induced delay of implantation was used to study the role of the uterine environment in controlling implantation in mice. Labelling studies in vivo showed that uterine protein synthesis and secretion is maximal 2-5 h and 24-30 h after the oestradiol injection which initiates implantation. Embryos removed from uteri 5,12 or 30 h after oestradiol injection were able to transport and utilize precursors of nucleic acids and proteins in short-term cultures at the same rate as normal embryos, although "delayed" embryos had low levels of activity. These results suggest that "delayed" embryos are metabolically activated within 5 h of release from delay, perhaps because of the hormonally-induced changes in uterine proteins which occur at this time.