Translocation breakpoint possibly predisposes to nonrandom X-chromosome inactivation in mouse embryos bearing Searle''s T(X;16)16H translocation

To clarify the sequence of events that ultimately achieves the nonrandom inactivation of the paternally inherited X chromosome in postpartum female mice heterozygous for T(X;16)16H, we set out to examine the expression of Xist alleles and the X-linked HMG-lacZ transgene in embryos recovered at the egg cylinder stage. Lack of expression of the Xist(b) allele on the 16X translocation chromosome in the embryonic region of 7.5 d postcoitum (dpc) X16/X(n)Xist(a);16(X)Xist(b)/16 embryos strongly suggested the occurrence of nonrandom inactivation in favor of the normal X chromosome. The simplest explanation would be biased choice, followed by postinactivation selection against genetically unbalanced cells. However, the frequency and distribution of beta-galactosidase-positive cells in X16/X(n)lacZ;16X/16 embryos at 6.5 and 7.5 dpc, together with earlier cytogenetic data, raised an intriguing possibility that the majority of 16X chromosomes were prevented from completing the inactivation process, when they had been chosen to be silenced. Phenotypes of female mice carrying a spontaneous recombination between Xn and 16X in the segment defined by the T16H breakpoint and the X-linked Ta locus suggested that the nonrandomness was brought about by disruption of an X-chromosomal sequence or structure at the translocation breakpoint.     

Mosaic methylation of Xist gene before chromosome inactivation in undifferentiated female mouse embryonic stem and embryonic germ cells

Epigenetic modification is implicated in the choice of the X chromosome to be inactivated in the mouse. In order to gain more insight into the nature of such modification, we carried out a series of experiments using undifferentiated mouse cell lines as a model system. Not only the paternally derived X (XP) chromosome, but the maternally derived one (XM) was inactivated in the outer layer of the balloon-like cystic embryoid body probably corresponding to the yolk sac endoderm of the post-implantation embryo in which XP is preferentially inactivated. Hence, it is likely that the imprint responsible for the nonrandom XP inactivation in early mouse development has been erased or masked in female ES cells. CpG sites in the 5' region of the Xist gene were partially methylated in female ES and EG and parthenogenetic ES cell lines as in the female somatic cell in which the silent Xist allele on the active X is fully methylated, whereas the expressed allele on the inactive X is completely unmethylated. In the case of undifferentiated ES cells, however, methylation was not differential between two Xist alleles. This observation was supported by the demonstration that single-cell clones derived from female ES cell lines were not characterized by either allele specific Xist methylation or nonrandom X inactivation upon cell differentiation. Apparently these findings are at variance with the view that Xist expression and X inactivation are controlled by preemptive methylation in undifferentiated ES cells and probably in epiblast.     

Mouse choroideremia gene mutation causes photoreceptor cell degeneration and is not transmitted through the female germline

Choroideremia (CHM) is an X-linked progressive eye disorder which results from defects in the human Rab escort protein-1 (REP-1) gene. A gene targeting approach was used to disrupt the mouse chm/rep-1 gene. Chimeric males transmitted the mutated gene to their carrier daughters but, surprisingly, these heterozygous females had neither affected male nor carrier female offspring. The targeted rep-1 allele was detectable, however, in male as well as female blastocyst stage embryos isolated from a heterozygous mother. Thus, disruption of the rep-1 gene gives rise to lethality in male embryos; in female embryos it is only lethal if the mutation is of maternal origin. This observation can be explained by preferential inactivation of the paternal X chromosome in murine extraembryonic membranes suggesting that expression of the rep-1 gene is essential in these tissues. In both heterozygous females and chimeras the rep-1 mutation causes photoreceptor cell degeneration. Consequently, conditional rescue of the embryonic lethal phenotype of the rep-1 mutation may provide a faithful mouse model for choroideremia.     

Gender distinctions and lateral asymmetry in the low-level auditory brainstem response of the human neonate

Given the reported long-range cis-inactivating effect of the XIST gene in early embryonic development and the lack of requirement of X-chromosome-specific elements for propagating the inactive state, there exists the possibility of cis inactivation of autosomal material after de novo translocation to an inactive X chromosome (Xi) in differentiated cells. We have analyzed de novo radiation-induced translocations between the Xi and autosomes to study the maintenance and spreading of X-chromosome inactivation (X inactivation) in relation to the position of the X-inactivation center (XIC)/XIST in differentiated cells. Autosome/Xi translocations were detected by fluorescence in situ hybridization (FISH). The activation status of the chromosomes involved in the translocation was determined by simultaneous immunocytogenetic studies using antibodies against either BrdU incorporated at late S phase or acetylated histone H4. The position of XIC/XIST in the reciprocal products of the translocation was determined by XIST-specific FISH and computer enhancement. In other experiments, the Xq13 region carrying XIC/XIST was localized by computer enhancement of the DAPI banding pattern. Our study in differentiated cells provides a visual demonstration that physical separation from XIC/XIST does not result in reactivation of inactive X-chromosome material and that X inactivation is not spread to the translocated autosomes irrespective of the position of XIC/XIST. This observation suggests that physical linkage to XIC/XIST does not lead to de novo inactivation of autosomal material.     

X chromosome inactivation and the Xist gene

X chromosome inactivation in mammals was first described over 30 years ago. The biological problem is how to achieve gene dosage equivalence between XX females and XY males; the solution is to genetically silence one whole X chromosome in each cell of the early developing female embryo. The molecular mechanism by which this is achieved, however, remains a mystery. Recently, through the discovery of the Xist gene, it appears that we may be on the brink of learning how this unique phenomenon is mediated. Here, I discuss the developmental regulation of X inactivation and the candidacy of Xist as the X chromosome inactivation centre, with particular reference to its possible role in the initiation, spread and maintenance of X inactivation.     

The effect of multiple cryopreservation procedures and blastomere biopsy on the in-vitro development of mouse embryos

Preimplantation genetic diagnosis is currently used in clinical practice. In experienced hands the biopsy procedure alone does not affect the further in-vitro and in-vivo developmental potential of animal and human embryos. No data exist on the combination of cryopreservation of embryos at the pronuclear and/or 8-cell stage and/or biopsy at the 8-cell stage. Pronuclear stages of mouse F1 hybrids (C57Bl/jxCBA) were harvested and divided into several experimental groups. The developmental rates of zygotes, which were neither biopsied nor cryopreserved were used as data control. Others were only cryopreserved at the pronuclear stage (C-PN), or at the cleavage stage (C-CS), or both. Each of these groups was also combined with or without a biopsy. Only the hatched blastocyst rate (HBR), but not the 'simple' blastocyst rate, showed significant differences between groups. Neither C-PN (HBR = 60.42%), nor C-CS (63.16%), nor a combination of both (59.46%) had an impact on the hatched blastocyst rate when compared with that of the control group (67.46%). The biopsy procedure (55.93%) also proved not to be harmful for the embryos. The embryos, which were C-PN and C-CS, and subsequently biopsied, showed a significantly lower hatched blastocyst rate (39.62%) than that of the control, C-PN, C-CS, and C-PN/C-CS groups (P < 0.05). The combination of C-PN and cleavage-stage biopsy also lead to a lower hatched blastocyst rate (42.22%), compared with that of the control group (P < 0.05). It was concluded that couples must be advised that an effect on embryos which have undergone a combined cryopreservation and micromanipulation procedure cannot be ruled out. However, cryopreservation at the pronuclear or at the 8-cell stage alone, or in combination with a biopsy procedure, does not influence the further development of the embryo.     

Corticosteroid prescribing in rheumatoid arthritis and psoriatic arthritis

Embryos from certain mouse strains are arrested at the 2-cell stage in cell culture ('2-cell block'), whereas those from other strains develop to the blastocyst stage under the same conditions. It was previously shown that blocking embryos can be rescued in culture by aggregation with an excess of 2-cell stages of a non-blocking strain such as CBA x C57BL/6 F2. Here we have employed a LacZ transgene in a blocking strain (NMRI) to follow the fate of rescued blastomeres up to the blastocyst stage. We found that rescued blastomeres can participate in both inner cell mass and trophoblast formation, thus completely overcoming the 2-cell block.     

Effect of encapsulation on development of mouse pronuclear stage embryos in vitro

Pronuclear stage embryos with intact (ZI), slit (ZS) or completely removed (ZF) zona pellucida were encapsulated with an artificial zona pellucida (AZP) made of 1.5% sodium alginate. Embryos were cultured in KSOM medium with or without protein and their development in vitro to the blastocyst stage was recorded. AZP significantly (P < 0.05) improved the development of embryos to the blastocyst stage regardless of the presence of the natural zona pellucida. The encapsulated embryos developed at a higher rate (P < 0.05) in the absence of protein as compared with non-encapsulated embryos. Furthermore, the cell contacts at the 4-cell stage were significantly improved (P < 0.05) with encapsulation. AZP improved (P < 0.05) the development of pronuclear stage embryos with a slit zona pellucida to morula and blastocyst stages as compared with ZS embryos. It is concluded that AZP improves the in vitro development of pronuclear stage embryos with intact or completely removed zona pellucida as well as micromanipulated embryos to the blastocyst stage.     

Comparison of monolayer and bilayer plates used in antibiotic assay

Development of the rat embryo is arrested at the 2-cell stage in vitro in the presence of inorganic phosphate (Pi). Rat embryos were affected by exposure to 1.19 mM KH2PO4 in modified hamster embryo culture medium-1 at the late 2-cell stage only. When exposure durations were 6 h, embryos whose exposure timings were prior to cleavage had a reduced rate of development to the blastocyst stage (2-8%) when compared with embryos with no exposure to Pi (97%, P < 0.05). When exposure durations were 18 h, all embryos were arrested at the 2- to 4-cell stage. These timings would correspond to the G2 to M phase of the second cell cycle. Maturation-promoting factor (MPF), which is regulated by a phosphorylation cascade, controls cell division, and its kinase activity is necessary in order for the cell to enter the M phase. However, the histone H1 kinase activity levels and the patterns of the state of phosphorylation of cdc2 were the same in blocked and non-blocked embryos. Because MPF was active in blocked embryos, the developmental block in rat 2-cell embryos caused by phosphate was not due to MPF activity or its phosphorylation cascade.     

Skewed X-chromosome inactivation is common in fetuses or newborns associated with confined placental mosaicism

The inactivation of one X chromosome in females is normally random with regard to which X is inactivated. However, exclusive or almost-exclusive inactivation of one X may be observed in association with some X-autosomal rearrangements, mutations of the XIST gene, certain X-linked diseases, and MZ twinning. In the present study, a methylation difference near a polymorphism in the X-linked androgen-receptor gene was used to investigate the possibility that nonrandom X inactivation is increases in fetuses and newborns that are associated with confined placental mosaicism (CPM) involving an autosomal trisomy. Extreme skewing was observed in 7 (58%) of 12 cases with a meiotic origin of the trisomy, but in none of 10 cases examined with a somatic origin of the trisomy, and in only 1 (4%) of 27 control adult females. In addition, an extremely skewed X-inactivation pattern was observed in 3 of 10 informative cases of female uniparental disomy (UPD) of chromosome 15. This may reflect the fact that a proportion of UPD cases arise by "rescue" of a chromosomally abnormal conceptus and are therefore associated with CPM. A skewed pattern of X inactivation in CPM cases is hypothesized to result from a reduction in the size of the early-embryonic cell pool, because of either poor early growth or subsequent selection against the trisomic cells. Since approximately 2% of pregnancies detected by chorionic villus sampling are associated with CPM, this is likely a significant contributor to both skewed X inactivation observed in the newborn population and the expression of recessive X-linked diseases in females.     

Physical mapping of 2000 kb of the mouse X chromosome in the vicinity of the Xist locus

A physical map encompassing approximately 2.0 megabases (Mb) in the region of the mouse X-inactivation center has been constructed. The map extends from the Gjb-1 locus to the Xist locus and demonstrates the order of probes inseparable by genetic analysis. The deduced locus order is as follows: Gjb-1, Ccg-1, DXCrc171, Rps4, Phka, DXCrc177, DXCrc318, Xist. Detailed physical mapping in the region between the Phka and Xist loci indicates the position of CpG-rich islands associated with the 5' end of genes. The DXCrc177 and DXCrc318 loci, both defined by probes derived from linking clones, are associated with CpG-rich islands. The map provides a framework for the isolation of underlying sequences in the mouse X-inactivation center region.     

Response of preimplantation murine embryos to heat shock as modified by developmental stage and glutathione status

Objectives were to characterize developmental changes in response to heat shock in the preimplantation mouse embryo and to evaluate whether ability to synthesize glutathione is important for thermal resistance in mouse embryos. Heat shock (41 degrees C for 1 or 2 h) was most effective at disrupting development to the blastocyst stage when applied to embryos at the 2-cell stage that were delayed in development. Effects of heat shock on ability of embryos to undergo hatching were similar for 2-cell, 4-cell, and morula stage embryos. The phenomenon of induced thermotolerance, for which exposure to a mild heat shock increases resistance to a more severe heat shock, depended upon stage of development and whether embryos developed in vitro or in vivo. In particular, induced thermotolerance was observed for morulae derived from development in vivo but not for 2-cell embryos or morulae that developed in culture. Administration of buthionine sulfoximine to inhibit glutathione synthesis did not increase thermal sensitivity of 2-cell embryos or morulae but did reduce subsequent development of 2-cell embryos at both 37 degrees and 41 degrees C. In summary, changes in the ability of 2-cell through morula stages to continue to develop following a single heat shock were generally minimal. However, 2-cell embryos delayed in development had reduced thermal resistance, and therefore, maternal heat stress may be more likely to cause mortality of embryos that are already compromised in development. There were also developmental changes in the capacity of embryos to undergo induced thermotolerance. Glutathione synthesis was important for development of embryos but inhibition of glutathione synthesis did not make embryos more susceptible to heat shock.     

Hypersensitivity of ataxia telangiectasia fibroblasts to ionizing radiation is associated with a repair deficiency of DNA double-strand breaks

Amphiregulin (Ar) is an EGF receptor ligand that functions to modulate the growth of both normal and malignant epithelial cells. We asked whether mouse preimplantation embryos express Ar, and if so, what the function of Ar is during preimplantation development. We used RT-PCR to show expression of Ar mRNA in mouse blastocysts, and using a polyclonal anti-Ar antibody and indirect immunofluorescence, we detected the presence of Ar protein in morula- and blastocyst-stage embryos. Ar protein was present in both the cytoplasm and nucleus in both morulae- and blastocyst-stage embryos, which is similar to Ar distribution in other cell types. Embryos cultured in Ar developed into blastocysts more quickly and also exhibited increased cell numbers compared to control embryos. In addition, 4-cell stage embryos cultured in an antisense Ar phosphorothioate-modified oligodeoxynucleotide (S-oligo) for 48 hr exhibited slower rates of blastocyst formation and reduced embryo cell numbers compared to embryos exposed to a random control S-oligo. TGF-alpha significantly improved blastocyst formation, but not cell numbers, for embryos cultured in the antisense Ar S-oligo. From these observations, we propose that Ar may function as an autocrine growth factor for mouse preimplantation embryos by promoting blastocyst formation and embryo cell number. We also propose that blastocyst formation is stimulated by Ar and TGF-alpha, while Ar appears to exert a greater stimulatory effect on cell proliferation than does TGF-alpha in these embryos.     

Development, glycolytic activity, and viability of preimplantation mouse embryos subjected to different periods of glucose starvation

After compaction, the preimplantation mouse embryo switches to a glucose-based metabolism, whereas for the 2- to 4-cell stage embryo, glucose can be inhibitory. In this study, we investigated the adaptability of preimplantation embryos to different periods of glucose starvation by culturing in vitro fertilized (IVF) and in vivo-fertilized 1-cell OF1 mouse embryos. Blastocysts obtained from exposure to glucose starvation for different periods of time were examined for the number of cells in the trophectoderm and inner cell mass, and for glycolytic activity and viability. A high percentage of blastocysts was obtained when 1-cell embryos fertilized in vitro or in vivo were cultured in M16 until the 2-cell stage, were transferred to M16 without glucose (M16-G) until the 4- or 8-cell stage, and then were transferred to fresh M16-G. When in vivo-fertilized 1-cell embryos were cultured to the 2-cell stage and then left in M16, less than 5% formed blastocysts compared to 26% of those transferred into M16-G. Blastocysts obtained when in vivo-fertilized 1-cell embryos were left in M16-G after the 2-cell stage, however, showed a significantly elevated glycolytic activity compared to those transferred to fresh M16 or M16-G medium at the 4- or 8-cell stage. Interestingly, even though these embryos displayed elevated glycolytic activity, they did not exhibit differences in the numbers of inner cell mass and trophectoderm cells or in viability compared to embryos cultured according to other protocols. Blastocysts from all cultured protocols had a significantly lower total cell number and a lower trophectoderm, but not inner cell mass, cell number compared to blastocysts developed in vivo. This study documents the metabolic adaptability of the preimplantation embryo by highlighting its ability to proceed with development and retain viability when challenged with glucose starvation at different periods.