The late-replicating X chromosome in digynous mouse triploid embryos

Using BrdU-labeling and acridine orange staining, the behavior of X-chromosome replication was studied in 28 XXX and 19 XXY digynous mouse triploids. In some of these the paternal and maternal X chromosome could by cytologically distinguished. Such embryos were obtained by mating chromosomally normal females with males carrying Cattanach's X chromosome which contains an autosomal insertion that substantially increases the length of this chromosome. In the XXX triploids there were two distinct cell lines, one with two late-replicating X chromosomes, and the other with only one late-replicating X. The XXY triploids were also composed of two cell populations, one with a single late-replicating X and the other with no late replicating X chromosome. Assuming that the late-replicating X is genetically inactive, in both XXX and XXY triploids, cells from the embryonic region tended to have only one active X chromosome, whereas those from the extra-embryonic membranes tended to have two active X chromosomes. The single active X chromosome was either paternal or maternal in origin, but two active X chromosomes were overwhelmingly maternal in origin, suggesting paternal X-inactivation in extra-embryonic tissues.     

A liveborn 69,XXX triploid. Origin, X chromosome activity and gene dosage

A 69,XXX female liveborn triploid survived 45 days. The phenotype was consistent with the average clinical picture of liveborn triploids. Autopsy revealed slight atrophy of cerebral cortex and corpus callosum and severe adrenal hypoplasia. Chromosome polymorphisms indicated that the origin of this triploid was dispermy. Replication studies of the X chromosome performed on lymphocytes and fibroblasts showed that the majority of cells had two late replicating X chromosomes. X chromosome inactivation in spontaneous abortuses and liveborn triploids is discussed. Nine enzymes encoded by autosomal genes were tested, five had normal, three increased, and one reduced levels of activity. The reduced activity of alpha-galactosidase, an X-linked enzyme, is in agreement with cytogenetic findings and demonstrated a gene dosage effect.     

The pattern of X-chromosome inactivation in the embryonic and extra-embryonic tissues of post-implantation digynic triploid LT/Sv strain mouse embryos

Spontaneously cycling LT/Sv strain female mice were mated to hemizygous Rb(X.2)2Ad males in order to facilitate the distinction of the paternal X chromosome, and the pregnant females were autopsied at about midday on the tenth day of gestation. Out of a total of 222 analysable embryos recovered, 165 (74.3%) were diploid and 57 (25.7%) were triploid. Of the triploids, 26 had an XXY and 31 an XXX sex chromosome constitution. Both embryonic and extra-embryonic tissue samples from the triploids were analysed cytogenetically by G-banding and by the Kanda technique to investigate their X-inactivation pattern. The yolk sac samples were separated enzymatically into their endodermally-derived and mesodermally-derived components, and these were similarly analysed, as were similar samples from a selection of control XmXp diploid embryos. In the case of the XmXmY digynic triploid embryos, a single darkly-staining Xm chromosome was observed in 485 (82.9%) out of 585, 304 (73.3%) out of 415, and 165 (44.7%) out of 369 metaphases from the embryonic, yolk sac mesodermally-derived and yolk sac endodermally-derived tissues, respectively. The absence of a darkly staining X-chromosome in the other metaphase spreads could either indicate that both X-chromosomes present were active, or that the Kanda technique had failed to differentially stain the inactive X-chromosome(s) present. In the case of the XmXmXp digynic triploid embryos, virtually all of the tissues analysed comprised two distinct cell lineages, namely those with two darkly-staining X-chromosomes, and those with a single darkly staining X-chromosome.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human X chromosomes: synchrony of DNA replication in diploid and triploid fibroblasts with multiple active or inactive X chromosomes

We have analyzed patterns of DNA replication in X chromosomes from diploid cultured human fibroblasts and from three triploid 69,XXY fibroblast strains, using BrdU--33258 Hoechst--Giemsa techniques. Both X chromosomes in each of these Barr body-negative triploid strains were early-replicating. The results of gene dosage studies using (1) a histochemical stain to measure X-linked glucose-6-phosphate dehydrogenase (G6PD) activity in single cells and (2) cellulose acetate electrophoresis of G6PD activity in cell extracts also indicated that both Xs in these strains were genetically active. When we compared the synchrony of X chromosome DNA replication kinetics both between cells and within cells containing multiple inactive Xs, a marked variability and asynchrony was observed for late-replicating X chromosomes. In a culture of 47,XXX fibroblasts administered an 8-h terminal pulse of dT after growth in BrdU-containing medium, asynchrony was detected between the two late-replicating Xs in approximately 70% of cells examined. No such asynchrony was observed between the two early-replicating Xs in similarly cultured 69,XXY cells; in the triploid strains, the two Xs were distinguished by asynchronous replication in only approximately 15% of cells. The striking variability in late X chromosome replication kinetics appears, then, to be a property unique to inactive Xs and is not inherent to all X chromosomes.     

Novel cytogenetic finding: an unusual X;X-translocation in Mehsana buffalo (Bubalus bubalis)

Cytogenetic investigations of a phenotypically normal Mehsana river buffalo calf (Bubalusbubalis) revealed an XXY chromosome complement due to X;X-translocation in all screened metaphase plates. The chromosomal anomaly was identified by GTG-banding while CBG- and RBG-banding revealed two heterochromatin blocks and that one of the two X chromosomes was late replicating, respectively. The normal cytogenetic profiles of sire, dam and relatives of the calf suggest that the anomaly could have arisen spontaneously during oogenesis. This is the first report on a male river buffalo calf having an XXY chromosome complement with translocation between the two X chromosomes.     

Replication pattern of the X and Y chromosomes in partially synchronized human lymphocyte cultures

The replication pattern of the X and Y chromosomes at the beginning of the synthetic phase was studied in human lymphocyte cultures partially synchronized by the addition of 5-fluoro-2-deoxyuridine (FUdR). The data were evaluated statistically by an analysis of the distribution of silver grain counts over the X and Y chromosomes. —In cells from normal females, one of the X chromosomes began replication later than any other chromosomes of the complement. The short arm of the late replicating X chromosome started replication earlier than the long arm. The telomeric region of the short arm was a preferential site of DNA synthesis at the beginning of replication. —In partially synchronized lymphocyte cultures from a patient with the XXY syndrome, the Y chromosome started replication together with the late replicating X chromosome. The Y chromosome most frequently replicated synchronously with the short arm of the X. The centromeric region of the Y chromosome initiated synthesis before the telomeric region and appeared to replicate synchronously with the telomeric region of the short arm of the X. These findings are discussed with reference to the pairing of the X and Y chromosomes at meiosis.Supported in part by the National Institute of Health Research Grant HD-01979 and National Foundation Birth Defects Research Grant CRCS-40. Dr. Knight was a predoctoral fellow under National Institute of Health Training Program HD-00049-09.     

Correlation between X-chromosome inactivation and cell differentiation in female preimplantation mouse embryos

By means of a cytological method involving BrdU incorporation and acridine orange fluorescence staining in combination with embryo manipulation, we studied X-chromosome activity in female preimplantation mouse embryos with special reference to the correlation between X-chromosome inactivation and cell differentiation. There was no sign of asynchronous replication between the two X chromosomes from the one-cell to intermediate blastocyst stage. The allocyclic X chromosome, first detected in late blastocysts, was paternal in origin, mostly replicating early in the S phase and limited to the trophectoderm. Subsequent X-chromosome inactivation occurring in the primary endoderm was also characterized by the involvement of the paternal X and early replication. Both X chromosomes continued to replicate synchronously in the embryonic ectoderm or epiblast at this stage. It was evident that overt cell differentiation preceded the appearance of the asynchronously replicating X chromosome in the trophectoderm and primary endoderm. This finding seems to support the view that cell differentiation is an important correlate of X-chromosome inactivation.     

X chromosome inactivation in female embryos of a marsupial mouse (Antechinus stuartii)

Blastocysts and late gestation stages of the marsupial mouse, Antechinus stuartii, were examined cytologically and electrophoretically to investigate X chromosome activity during embryogenesis. A late replicating X chromosome was identified in the protoderm cells of female unilaminar blastocysts and in the cells of embryonic and extra-embryonic regions of older blastocysts. Sex chromatin bodies were also observed in female bilaminar and trilaminar blastocysts. The X linked enzyme -galactosidase showed no evidence of paternal allele expression in the extra-embryonic region of bilaminar blastocysts or in the yolk sac and embryonic tissue of known heterozygotes. It is concluded that the late replicating X chromosome is paternal in origin and that unlike the laboratory mouse, X inactivation is not correlated with cell differentiation in Antechinus.     

Presence of an Allocyclic Early Replicating X Chromosome in Female Embryos of the Rat, Chinese Hamster and Rabbit

Previous studies on early female mouse embryos revealed the presence of two kinds of inactive X chromosomes, one replicating late and the other early in the DNA synthetic period. The X chromosome that replicates early is of special interest because of its paternal origin, preferential occurrence in trophectoderm and primitive endoderm derivatives, and programmed shift to the late replicator. This study by BrdU labeling and acridine orange fluorescence staining was undertaken to examine whether the inactive X chromosome behaves in a similar manner in other laboratory mammals. In rat embryos the paternal X chromosome was found to show the same behavior in extraembryonic tissues. Early replicating chromosomes were also found in the extraembryonic regions of Chinese hamster and rabbit embryos, although their parental origin could not be determined due to the absent of X chromosome polymorphism in these species. Probably the early replicating X chromosome occurs commonly in mammals. Its functional significance is unknown.     

Cytogenetic and biochemical investigations on fibroblast cultures and clones with one and two active X chromosomes of a 69,XXY triploidy

Bromodeoxyuridine replication patterns showed that fibroblasts from a 69,XXY triploidy carried either one or two early replicating X chromosomes. The activity of alpha-galactosidase A measured in single cells fell into two classes with a ratio of 1:2. Dilute plating produced clones of both types with the activity of alpha-galactosidase A corresponding to the number of active X chromosomes. To our knowledge, this is the first report on clones of a triploidy with different numbers of active X chromosomes, and on a gene-dosage effect of an X-linked gene using triploid cells with one active X as control.     

Untersuchungen zur armverhältnisbestimmung am spätreplizierenden X-Chromosom des Menschen

Zusammenfassung Das Armverhältnis des spätreplizierenden X-Chromosoms wurde bei 5 Kontrollpersonen, 5 47,XXY Klinefelter-Patienten, 1 46/47,XX/XXX-Mosaik und 1 45/46,X/XX-Mosaik bestimmt. Die statistische Analyse der erhaltenen Werte zeigt, daß die Methode der Armverhältnisbestimmung durch Messung der Armlängen an vergrößert projizierten Chromosomen als geeignet erscheint. Die Mittelwerte der Armverhältnisse der 5 Kontroll-personen können nicht als gleich angesehen werden. Der gesamte Mittelwert der Armverhältnisse ist signifikant kleiner (1,487) für die Klinefelter-Patienten als für die Kontrollpersonen (1,546). Die Werte für die Mosaike XX/XXX (1,557) und XO/XX (1,528) liegen im Bereich der Normalwerte.

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Investigations of defining the arm ratio of the late replicating X-chromosome in man

Summary The arm ratio of the late replicating X-chromosome was determined in 5 control persons, 5 47,XXY Klinefelter patients, 1 46/47,XX/XXX mosaic and 1 45/46,X/XX mosaic. The analysis of the values obtained shows that the method of defining the arm ratio by means of measuring arm lengths on drawings of highly magnified projected chromosomes proves practicable. The mean values of the arm ratios of the 5 control persons cannot be valued as equal. The total mean value of the arm ratios is significantly smaller (1.487) for the Klinefelter patients than for the normal control persons (1.546). The values of the mosaics XX/XXX (1.557) and XO/XX (1.528) lie in the range of the control values.

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Studie im Rahmen des Assoziations-Vertrages Hämatologie EURATOM-GSF Nr.031-64-1 BIAD.     

A comparative study on steroid sulfatase and arylsulfatase C in fibroblast clones from 45,X/47,XXX and 69,XXY

Summary Steroid sulfatase (STS) and arylsulfatase C (ARSC) were studied in fibroblast clones from a 45,X/47,XXX mosaic and from a 69,XXY triploidy with one or two active X chromosomes. The comparison of the 47,XXX with 45,X clones showed an incomplete gene dosage effect (1.8 for STS and 2.0 for ARSC). This was not the case for the triploid clones with different X-inactivation patterns. These results confirm previous reports on the non-inactivation of the STS gene, and establish X linkage and non-inactivation for the ARSC gene as well.     

Q-banding of chromosomes in human spontaneous abortions

Chromosome studies of 242 spontaneous abortions were carried out by Q-banding technique. The abortuses were selected for study because they were phenotypically abnormal, had not progressed beyond 12 weeks development or were from women with repeated abortions. Chromosome anomalies were found in 126 (52%) of the abortuses. Of these, 71 (56%) were trisomies. Trisomies were found for all the autosomes except Nos. 1, 3, 5, 11, 17 and 18. Triploidy was the second commonest anomaly in this series, making up 26 (21%) of the total anomalies. About 70% of these had an XXY sex chromosome complement. Only 16 (13%) of the abortuses had X monosomy, a lower frequency than would be expected in an unselected study. Tetraploidy was found in 8 abortuses and the 5 remaining specimens had various anomalies. These included 3 translocations, a trisomy 21,X monosomy and a ring chromosome 13. Except for the greater frequency of XXY than XXX sex chromosomes in the triploids, there was no evidence of a distortion of the sex ratio, either among the trisomic or among the chromosomally normal abortuses.     

X-chromosome inactivation and enzymatic activities in diploid and triploid fibroblasts

Diploid and triploid rabbit embryos obtained by artificial fertilization were cultured. No Barr's body in XXY and only one Barr's body in XXX triploid fibroblasts was observed. Six enzymatic activities were also determined. Two X-chromosome-bound enzymatic activities, glucose-6-phosphate dehydrogenase and phosphoglycerate kinase, were significantly increased in triploid fibroblasts, while another X-chromosome-bound activity (hypoxanthine phosphoribosyl transferase) was not modified. Among the autosome-bound activities studied, pyruvate kinase and adenine phosphoribosyl transferase were not modified, whereas in the triploid cells the 6-phosphogluconate dehydrogenase activity was decreased. The relationship between these modifications of enzymatic activities in triploids and the X-chromosome inactivation is discussed.     

Prenatal diagnosis of a 46,XX,inv(12)pat/47,XX,i(Xq),inv(12)pat

Summary A 46,XX,inv(12)pat/47,XX,i(Xq),inv(12)pat was diagnosed prenatally in a 36-year-old woman whose husband was a known carrier of a pericentric inversion of chromosome 12. The diagnosis was confirmed in fetal tissue. Terminal bromodeoxyuridine (BrdU) labelling demonstrated that in the line with 46 chromosomes one X was late replicating, while one X and the i(Xq) were late replicating in 100% of the cells with 47 chromosomes. We present the first case of this type of sex chromosome mosaicism. Genetic counseling presented difficulties since it was not possible to predict the fetal phenotype.     

Immunoglobulins and the X-chromosome

Serum levels of immunoglobulins (Ig) G, M, and A were determined in 28 women with an additional X-chromosome (XXX), and in equal numbers of age-matched normal women and men. Mean IgM levels were found to be highest in the XXX group, intermediate in normal women, and lowest in men; these differences were statistically significant. Mean IgM values obtained from seven XXY and three XXXY cases were almost identical with those of normal women and XXX women respectively. No such sex linkage was observed for IgA and IgG levels. These results support the suggestion that the serum level of IgM is related to the number of X chromosomes present.     

Analysis of sex-chromosome aneuploidy in interspecific backcross progeny between the laboratory mouse strain C57BL/6 and Mus spretus.

Sex-chromosomal aneuploidy was identified in four female progeny of 200 interspecific backcrosses between laboratory mice (C57BL/6Ros) and Mus spretus. The progeny included two 39,XO monosomy mice resulting from a backcross with M. spretus, as well as a 41,XXX trisomic mouse and a 40,XX/41,XXX mosaic mouse resulting from two separate backcrosses with C57BL/6 mice. The parental origin and meiotic stage of the aneuploidies was determined for each of the mice using a series of markers that identified allelic differences in the parental X-chromosome genes present in the hybrid female. Two of the probes identified differences in repeated elements between the M. spretus and laboratory mouse X chromosomes, whereas the remaining sites involved restriction fragment length differences of single-copy genes detectable by Southern analysis. These markers indicated that the aneuploidies were most likely of maternal origin and that the trisomy resulted from a nondisjunction at the second meiotic division. In contrast, the mosaic female could have originated either from a trisomic embryo that had lost a single X in a portion of its cells or from a mitotic nondisjunction during early embryogenesis that resulted in XXX and XO daughter cells, with subsequent loss of the XO cells.     

Aging and aneuploidy: evidence for the preferential involvement of the inactive X chromosome

It has been known for some time that there is an association between chronological aging and X-chromosome aneuploidy in peripheral blood lymphocyte cultures from females. In an attempt to elucidate the mechanism of X-chromosome aneuploidy in aging females, we used a BrdU late-labeling technique to determine the X-inactivation pattern in 45,X and 47,XXX lymphocytes of older women. In 50 of 58 X-aneuploid cells the inactive X chromosome was missing or extra. This implies that either the inactive X has a special propensity for mitotic errors or mitotic errors occur at random but subsequent selection is less stringent against cells with a missing or additional inactive X chromosome than against aneuploid cells involving the active X chromosome. Evidence is presented in favor of the former hypothesis.     

Cell fusion-induced quick change in replication time of the inactive mouse X chromosome: an implication for the maintenance mechanism of late replication.

It is unknown how and why the genetically inactivated mammalian X chromosome replicates late in S phase. There are also occasional inactive X chromosomes characterized by an opposite behavior replicating early in S phase. Two clonal cell lines, MTLB3 and MTLH8, isolated from a cultured murine T-cell lymphoma have an allocyclic X chromosome of the latter type. This precociously replicating X chromosome was judged to be genetically inactive as the late replicating one. Immediately after fusion with another cell line, the precociously replicating X chromosome from these cells starts to replicate late in S phase. This finding seems to suggest that late replication characterizing the inactive X chromosome is actively maintained by a trans-acting factor in female somatic cells, and that its lack entails a switch from late replication to precocious replication. It remains unknown whether this presumptive factor also modifies the autosomal replication pattern.     

Comparative characteristics of phenotypes in numerical anomalies of human X-chromosomes (morphologic and psychological features)]

On the basis of data on antropological and psychological investigation of 30 XXY males, 30 X0 females and 10 XXX females comparative characteristics of phenotypes of patients are given. The persons with X monosomy (the absence X- or Y-chromosomes) are observed to have decreased total sizes of body, to retain the ability to compensate congenital mental defects, to use standard methods of the adaptation to social environment and to assimilate standard norms of social behaviour. The persons with trisomies XXY and XXX (the presence of the additional heterochromatic X-chromosome) have the tendency to an increase of the longitudinal body sizes independently on sex; they have a delayed development of mental functions and reduced possibilities to compensation of these functions; they also have different degrees of disorganization of the social behaviour and the disadaptation to social environment. It is found that the patients with numerical anomalies of human X-chromosome (both the lack and the excess of chromosome material) have a disturbance of development of the sex dimorphism and sex dipsychism.