X-chromosome inactivation in XX androgenetic mouse embryos surviving implantation

Using genetic and cytogenetic markers, we assessed early development and X-chromosome inactivation (X-inactivation) in XX mouse androgenones produced by pronuclear transfer. Contrary to the current view, XX androgenones are capable of surviving to embryonic day 7.5, achieving basically random X-inactivation in all tissues including those derived from the trophectoderm and primitive endoderm that are characterized by paternal X-activation in fertilized embryos. This finding supports the hypothesis that in fertilized female embryos, the maternal X chromosome remains active until the blastocyst stage because of a rigid imprint that prevents inactivation, whereas the paternal X chromosome is preferentially inactivated in extra-embryonic tissues owing to lack of such imprint. In spite of random X-inactivation in XX androgenones, FISH analyses revealed expression of stable Xist RNA from every X chromosome in XX and XY androgenonetic embryos from the four-cell to morula stage. Although the occurrence of inappropriate X-inactivation was further suggested by the finding that Xist continues ectopic expression in a proportion of cells from XX and XY androgenones at the blastocyst and the early egg cylinder stage, a replication banding study failed to provide positive evidence for inappropriate X-inactivation at E6. 5.     

Early development and X-chromosome inactivation in mouse parthenogenetic embryos.

Early development and X-chromosome inactivation were studied in ethanol-induced mouse parthenogenones. About 24% of oocytes transferred to 0.5-day pseudopregnant recipients successfully implanted. However, only 49%, 20%, and 16% of implanted parthenogenones survived 5, 6, and 7 days later, respectively. Abnormal development was evident in every parthenogenone as early as 5 days after activation with the degenerating polar trophectoderm. These embryos were destined to become either small disorganized embryos or embryonic ectoderm vesicles bounded by the visceral endoderm. Only 2 of 51 representative 6- to 8-day parthenogenones sectioned had morphology of the normal egg cylinder, although growth retardation was evident. Spontaneous LT/Sv parthenogenones shared similar morphological features. In late blastocysts, the frequency of cells with an apparently inactivated X chromosome was lower in parthenogenones than in fertilized embryos. The failure of X-inactivation in the trophectoderm seems to contribute to the defective development of parthenogenones.     

Phorbol ester-mediated modulation of cell proliferation and primary differentiation of mouse preimplantation embryos

The tumour promoter, phorbol myristate acetate (PMA) at concentrations of 5–50 ng/ml substantially affected 2-, 4-, 8-cell and morula mouse embryos cultured in vitro. PMA evoked a delay of cell growth and caused premature cell differentiation. In the former there was a formation of binuclear blastomeres, in the latter of giant cell formation in trophectoderm of blastocyst and premature cavitation. PMA-mediated delay of growth rate was completely reversible in 8-cell embryos, partially reversible in 4-cell embryos and poorly reversible, if at all, in 2-cell embryos. In the presence of PMA, nuclear DNA synthesis proceeded although the rate of nuclear labelling with [³H]thymidine was lower than in the control. Blastomeres of some 2-cell embryos treated with PMA fused, resulting in the formation of 1-cell embryos.     

X-chromosome inactivation in monkey embryos and pluripotent stem cells

Inactivation of one X chromosome in female mammals (XX) compensates for the reduced dosage of X-linked gene expression in males (XY). However, the inner cell mass (ICM) of mouse preimplantation blastocysts and their in vitro counterparts, pluripotent embryonic stem cells (ESCs), initially maintain two active X chromosomes (XaXa). Random X chromosome inactivation (XCI) takes place in the ICM lineage after implantation or upon differentiation of ESCs, resulting in mosaic tissues composed of two cell types carrying either maternal or paternal active X chromosomes. While the status of XCI in human embryos and ICMs remains unknown, majority of human female ESCs show non-random XCI. We demonstrate here that rhesus monkey ESCs also display monoallelic expression and methylation of X-linked genes in agreement with non-random XCI. However, XIST and other X-linked genes were expressed from both chromosomes in isolated female monkey ICMs indicating that ex vivo pluripotent cells retain XaXa. Intriguingly, the trophectoderm (TE) in preimplantation monkey blastocysts also expressed X-linked genes from both alleles suggesting that, unlike the mouse, primate TE lineage does not support imprinted paternal XCI. Our results provide insights into the species-specific nature of XCI in the primate system and reveal fundamental epigenetic differences between in vitro and ex vivo primate pluripotent cells.     

Sequential X-chromosome reactivation and inactivation in cell hybrids between murine embryonal carcinoma cells and female rat thymocytes

By means of a 5-bromodeoxyuridine (BrdU) incorporation and an acridine orange fluorescence staining method together with [3H]thymidine ([3H]TdR) autoradiography, we studied the chronology of X-chromosome replication in newly formed cell hybrids between the hypoxanthine phosphoribosyl transferase (HPRT)-deficient OTF9-63 murine embryonal carcinoma (EC) cell with 43 +/- chromosomes and the female rat thymocyte having 42 chromosomes. Most near-tetraploid hybrid cells retained all chromosomes from both parents including one mouse X (XM) and two rat X (XR) chromosomes throughout the period of this study. Data showing changes in the chronology of X-chromosome replication obtained here were indicative of reactivation of the inactive X chromosome from the rat thymocyte, and de novo X-inactivation of one or two chromosomes. The extinction of lymphocyte phenotypes from the hybrids and their subsequent differentiation to the cell type resembling endoderm found in the peri-implantation mouse embryo apparently occurred in parallel with the above changes. These hybrids also showed an interesting possibility of preferential reinactivation of the reactivated XR chromosome in the early stages after cell fusion.     

Staufen1 is expressed in preimplantation mouse embryos and is required for embryonic stem cell differentiation

Pluripotent mouse embryonic stem (mES) cells derived from the blastocyst of the preimplantation embryo can be induced to differentiate in vitro along different cell lineages. However the molecular and cellular factors that signal and/or determine the expression of key genes, and the localisation of the encoded proteins, during the differentiation events are poorly understood. One common mechanism by which proteins can be targeted to specific regions of the cell is through the asymmetric localisation of mRNAs and Staufen, a double-stranded RNA binding protein, is known to play a direct role in mRNA transport and localisation. The aims of the present study were to describe the expression of Staufen in preimplantation embryos and mES cells and to use RNA interference (RNAi) to investigate the roles of Staufen1 in mES cell lineage differentiation. Western blotting and immunocytochemistry demonstrated that Staufen is present in the preimplantation mouse embryo, pluripotent mES cells and mES cells stimulated to differentiate into embryoid bodies, but the Staufen staining patterns did not support asymmetric distribution of the protein. Knockdown of Staufen1 gene expression in differentiating mES cells reduced the synthesis of lineage-specific markers including Brachyury, alpha-fetoprotein (AFP), PAX-6, and Vasa. There was however no significant change in either the gene expression of Nanog and Oct4, or in the synthesis of SSEA-1, all of which are key markers of pluripotency. These data indicate that inhibition of Staufen1 gene expression by RNAi affects an early step in mES cell differentiation and suggest a key role for Staufen in the cell lineage differentiation of mES cells.     

Metabolism in preimplantation mouse embryos

The ability of preimplantation mouse embryos to utilize glucose oxidatively is controlled, in part at least, at the level of glycolysis. Various experimental observations are reviewed that indicate the regulatory mechanism in delayed implanting blastocysts involves the classic negative allosteric feedback of high levels of ATP on phosphofructokinase while the situation in 2-cell embryos appears to be more complicated. That is, in addition to the usual negative effect of ATP and citrate on phosphofructokinase, there appears to be a modification of hexokinase that prevents phosphorylation of adequate amounts of glucose and results in low levels of fructose-6-phosphate at the 2-cell stage and consequently there is a failure to release the inhibition of phosphofructokinase even if ATP and citrate levels decrease. Although both types of embryos have limited glycolytic activity, they do have adequate capacity for citric acid cycle activity and oxidative phosphorylation, and are able to maintain a high ATP : ADP. It is argued, therefore, that the reduced levels of macromolecular synthesis characteristic of 2-cell and delayed implanting blastocysts are not due to restricted energy substrates or regulatory controls on glycolysis and a subsequent low energy state. On the contrary, it seems that the reduction in oxidative utilization of glucose in these situations is a result of diminished energy demand because of the low level of synthetic activity. The potential significance of this relationship between energy production and utilization in terms of potential regulatory mechanisms in preimplantation embryos is discussed.     

Imprinted X-chromosome inactivation: enlightenment from embryos in vivo

There are two forms of X chromosome inactivation (XCI) in the laboratory mouse, random XCI in the fetus and imprinted paternal XCI limited to the extraembryonic tissues supporting the fetal life in utero. Imprinted XCI has been studied extensively because it takes place first in embryogenesis and it may hold clues to the mechanism of control of XCI in general and to the evolution of random' XCI. Classical microscopic and biochemical studies of embryos in vivo provide a basis for interpreting the multifaceted information yielded by various inventive approaches and for planning further experiments.     

Immunological detection of cell surface galactosyltransferase in preimplantation mouse embryos

Presence of cell surface galactosyltransferase was surveyed in preimplantation mouse embryos by indirect immunofluorescence staining using an affinity-purified antibody against galactosyltransferase from human milk. Distinct fluorescence staining was observed in embryos ranging from late 8-cell stage to early blastocysts, while the embryos at other stages were stained only weakly. The cell surface enzyme was also present in F9 embryonal carcinoma cells, in a fraction of bone marrow cells of the mouse, and in a few percent of testicular sperm.     

X-chromosome inactivation and cell memory.

Mammalian X-chromosome inactivation is an excellent example of the faithful maintenance of a determined chromosomal state. As such, it may provide insight into the mechanisms for cell memory, defined as the faithful maintenance of a determined state in clonally derived progeny cells. We review here the aspects of X-chromosome inactivation that are relevant to cell memory and discuss the various molecular mechanisms that have been proposed to explain its occurrence, with emphasis on DNA methylation and a recently proposed mechanism that depends on the timing of replication.     

X chromosome imprinting and inactivation in preimplantation mammalian embryos

Dosage compensation for the mammalian X chromosome involves the silencing of one X chromosome to achieve equal X-linked gene expression between males and females. X chromosome inactivation (XCI) is controlled by a complex set of genetic elements located in a region known as the X chromosome inactivation center, and is regulated by a combination of genomic imprinting, cell lineage-dependent erasure of imprinting, an unidentified mechanism of X chromosome counting, an incompletely understood means of selection of one X chromosome for inactivation and developmentally regulated changes in X chromosome chromatin. A detailed understanding of when and how these components of XCI occur is essential for elucidating the operative mechanisms. A model accounting for early events related to XCI, including observations in uniparental and aneuploid embryos, is presented.     

Fucosylated glycoconjugates in mouse preimplantation embryos

Preimplantation mouse embryos were metabolically labelled with 3H or 14C-fucose to investigate the synthesis of fucosylated macromolecules. Scintillation counting revealed that there was a progressive increase in both total fucose taken up by the embryo and incorporation of fucose into TCA-precipitable material as embryos developed from the 4-cell to the blastocyst stage. This was reflected in the increasing intensity of bands on autoradiographs of radioactive fucose labelled proteins separated on 10% SDS-PAGs between the 4-cell embryo (at which stage bands were first detectable) and the blastocyst. Minor qualitative changes in fucoproteins were detected at the time of compaction and additional bands appeared at the blastocyst stage. Preliminary analysis of fucolipids in 6- to 8-cell embryos indicated that an approximately equal amount of fucose was incorporated into lipid and protein. Autoradiographs of semi-thin sections of 3H-fucose-labelled embryos showed substantial amounts of radioactive material in the vicinity of the plasma membrane both adjacent to other cells and facing the zona pellucida. These data would support a predominant role for fucoconjugates in cell surface events in the preimplantation embryo from the 8-cell stage.     

Alkaline phosphatase in preimplantation mouse embryos

This report deals with alkaline phosphatase in preimplantation mouse embryos. The enzyme activity is cytochemically demonstrated by an azo dye coupling method and biochemically determined by measuring phosphate liberated from β-glycerophosphate. The cytochemical procedure reveals alkaline phosphatase beginning suddenly in late 4-cell embryos. With the biochemical procedure, in spite of the large samples used, no activity is detected until the 8-cell stage when the activity rises abruptly, though less abruptly than the cell number. These results, which suggest the initiation of enzyme activity, are discussed and compared with those obtained by the Gomori-Takamatsu method on the same material.     

Skewed X-chromosome inactivation in human embryos with mosaic trisomy 16

The sex ratio and X-chromosome inactivation were analyzed in placental tissues of human spontaneous abortuses with pure and mosaic forms of chromosome 16 trisomy. The sex ratio value was found to decrease with an increase in the share of cells with the trisomic karyotype, which suggests differential survival of embryos belonging to different sexes. The pattern of X-chromosome inactivation in cells of extraembryonic mesoderm in the control group of embryos and in spontaneous abortuses with the level of trisomy 16 below 80% corresponded to random X-inactivation, whereas in most embryos with a frequency of trisomy 16 exceeding 80% skewed inactivation was observed. Our results support the hypothesis about the existence of an autosomal transfactor influencing the initiation of X-chromosome inactivation and suggest its possible localization on chromosome 16.     

Ontogeny of X-chromosome inactivation in the female germ line

Data are presented on G6PD electrophoretic patterns in fetal ovarian preparations of G6PD heterozygotes. The results indicate that in the early or mitotic period of female germ cell development, only a single X chromosome is active in each oogonium just as is the case for X inactivated somatic tissue. However, in the later or meiotic stage, reactivation of the inactive X chromosome in each oocyte occurs so that two functional X chromosomes are present in each oocyte.     

Expression and distribution of cell adhesion molecule uvomorulin in mouse preimplantation embryos

We have examined the synthesis and distribution of the cell adhesion molecule uvomorulin in mouse preimplantation embryos. Uvomorulin can already be detected on the cell surface of unfertilized and fertilized eggs but is not synthesized in these cells. Uvomorulin synthesis starts in late two-cell embryos and seems not to be correlated with the onset of compaction. The first signs of compaction are accompanied by a redistribution of uvomorulin on the surface of blastomeres. During compaction uvomorulin is progressively removed from the apical membrane domains of peripheral blastomeres. In compact morulae uvomorulin is no longer present on the outer surface of the embryo but is localized predominantly in membrane domains involved in cell-cell contacts of adjacent outer blastomeres. On inner blastomeres of compact morulae uvomorulin remains evenly distributed. This uvomorulin distribution once established during compaction is maintained and also found in the blastocyst: on trophectodermal cells uvomorulin localization is very similar to that in adult intestinal epithelial cells while uvomorulin remains evenly distributed on the surface of inner cell mass cells. The possible role of the redistribution of uvomorulin for the generation of trophectoderm and inner cell mass in early mouse embryos is discussed.