A low-level X chromosome mosaicism in mares, detected by chromosome painting

Fluorescence in situ hybridization with the use of the equine X whole chromosome painting probe was carried out on chromosome spreads originating from three mares with poor reproductive performance (infertility, miscarriage or stillbirth). The numbers of analysed spreads were high (105, 300 and 480) and in all three mares a low frequency of mosaicism was identified. The mares had the following karyotypes: 64,XX/63,X/65,XXX (93.6%/5.7%/0.7%), 64,XX/63,X (98.9%/1.1%) and 64,XX/63,X (94.3%/5.7%). The incidence and importance of the low percentage X chromosome mosaicism are discussed.     

Cytogenetic observations on XX/XY chimaeras and a reassessement of the evidence for germ cell chimaerism in heterosexual twin cattle and marmosets.

Testicular preparations were obtained from 7 bulls, twins of freemartins, and 1 male marmoset, all proved XX/XY chimaeras. X and Y sex chromosomes were confidently identified in nearly all the 87 spermatogonia at mitotic metaphase and 1052 primary spermatocytes at diakinesis-metaphase examined: no cell was identified as containing two X chromosomes. The germ cell chimaerism previously reported in these species is therefore not confirmed. Cultures grown from presumptive somatic these species is therefore not confirmed. Cultures grown from presumptive somatic cells in the testes of two of the bulls yielded 248 identifiable mitotic spreads, all XY-type; cultures from the gonads of their freemartin twins yielded 442 mitotic spreads, all XX-type. Direct preparations from one freemartin gonad, however, yielded 3 XY mitotic spreads out of 18 examined. The conflicting evidence concerning germ cell chimaerism in cattle and marmosets is discussed, particularly in relation to reports of XX/XY bulls that have sired a great excess of daughters. The possibility that XX germ cells contributed to the functional spermatozoa of these bulls is not favoured by present information, but is not excluded.     

Differential loss of histone H3 isoforms mono-, di- and tri-methylated at lysine 4 during X-inactivation in female embryonic stem cells

Silencing of genes on one of the two female X chromosomes early in development helps balance expression of X-linked genes between XX females and XY males and involves chromosome-wide changes in histone variants and modifications. Mouse female embryonic stem (ES) cells have two active Xs, one of which is silenced on differentiation, and provide a powerful model for studying the dynamics of X inactivation. Here, we use immunofluorescence microscopy of metaphase chromosomes to study changes in H3 mono-, di- or tri-methylated at lysine 4 (H3K4mel, -2 or -3) on the inactivating X (Xi) in female ES cells. H3K4me3 is absent from Xi in approximately 25% of chromosome spreads by day 2 of differentiation and in 40-50% of spreads by days 4-6, making it one of the earliest detectable changes on Xi. In contrast, loss of H3K4me2 occurs 1-2 days later, when histone acetylation also diminishes. Remarkably, H3K4mel is depleted on both (active) X chromosomes in undifferentiated female ES cells, and on the single X in males, and remains depleted on Xi. Consistent with this, chromatin immunoprecipitation reveals differentiation-related reductions in H3K4me2 and H3K4me3 at the promoter regions of genes undergoing X-inactivation in female ES cells, but no comparable change in H3K4me1.     

X inactivation in a mammal species with three sex chromosomes

X inactivation is a fundamental mechanism in eutherian mammals to restore a balance of X-linked gene products between XY males and XX females. However, it has never been extensively studied in a eutherian species with a sex determination system that deviates from the ubiquitous XX/XY. In this study, we explore the X inactivation process in the African pygmy mouse Mus minutoides, that harbours a polygenic sex determination with three sex chromosomes: Y, X, and a feminizing mutant X, named X*; females can thus be XX, XX*, or X*Y, and all males are XY. Using immunofluorescence, we investigated histone modification patterns between the two X chromosome types. We found that the X and X* chromosomes are randomly inactivated in XX* females, while no histone modifications were detected in X*Y females. Furthermore, in M. minutoides, X and X* chromosomes are fused to different autosomes, and we were able to show that the X inactivation never spreads into the autosomal segments. Evaluation of X inactivation by immunofluorescence is an excellent quantitative procedure, but it is only applicable when there is a structural difference between the two chromosomes that allows them to be distinguished.     

Case report: a successful pregnancy outcome in a patient with non-mosaic Turner syndrome (45, X) via in vitro fertilization

We describe a successful pregnancy outcome in a patient with non-mosaic Turner syndrome (45, X) via in vitro fertilization. The patient achieved a second pregnancy at 35 years of age. The her blood lymphocyte karyotype was examined by G-band and FISH. Furthermore, cumulus cells and her elbow skin cells were evaluated via FISH. Non-mosaic Turner syndrome was determined by G-banding [100 % (50/50) 45, X]. Lymphocytes were shown as 478/500 (95.6 %) cells of X sex chromosome signal, 15/500 (3.0 %) cells of XXX signal, and 7/500 (1.4 %) cells of XX signal. The cumulus cells were mosaic: 152/260 (58.5 %) were X; 84/260 (32.3 %) were XXX, 20/260 (7.7 %) were XX, and 4/260 (1.5 %) were XY. Moreover, skin cells included a mosaic karyotype [47, XXX(29)/46, XX(1)]. We conclude that the collection of a large number of blood lymphocytes can reveal different mosaic patterns (X, XX and XXX) by FISH in spite of non-mosaic Turner syndrome.     

Diagnosis of bovine freemartinism by fluorescence in situ hybridization on interphase nuclei using a bovine Y chromosome-specific DNA probe

A heifer co-twin to a bull, in most cases, is a sterile freemartin which needs to be identified and culled from replacement stock. Various methods are available for the diagnosis of freemartinism, but none is ideal in terms of speed, sensitivity, or specificity. The present study was thus conducted to develop and validate a satisfactory fluorescence in situ hybridization procedure on interphase nuclei (I-FISH) for identifying the bovine XX/XY-karyotypic chimerism, the hallmark of freemartinism. A 190-bp DNA FISH probe containing the bovine male-specific BC1.2 DNA sequence was synthesized and labeled with digoxigenin by PCR. The FISH was performed on metaphase spreads and interphase nuclei of blood lymphocytes. Upon FISH, the probe expectedly bound to the nucleus of the male cell or to a region of the p12 locus of the Y chromosome. Twenty-four young heterosexual twins (Holstein-Friesian and Korean Cattle breeds; 10 pairs and 4 singletons) were analyzed in the present study; all but three exhibited the XX/XY-karyotypic chimerism to varying extents in both I-FISH and karyotyping. One heifer was identified to have 100% XX cells by both analyses, whereas two bulls were judged as 100% XY- and XX/XY-chimeric karyotypes by karyotyping and I-FISH, respectively. Nevertheless, the ratios of the XY to XX cells in these animals were very similar between the two analyses. In conclusion, the present I-FISH was a rapid and reliable procedure that can be used for early-life diagnosis of bovine freemartinism.     

Chromosomal in situ suppression hybridization of human gonosomes and autosomes and its use in clinical cytogenetics

Summary DNA libraries from sorted human gonosomes were used selectively to stain the X and Y chromosomes in normal and aberrant cultured human cells by chromosomal in situ suppression (CISS-) hybridization. The entire X chromosome was stained in metaphase spreads. Interphase chromosome domains of both the active and inactive X were clearly delineated. CISS-hybridization of the Y chromosome resulted in the specific decoration of the euchromatic part (Ypter-q11), whereas the heterochromatic part (Yq12) remained unlabeled. The stained part of the Y chromosome formed a compact domain in interphase nuclei. This approach was applied to amniotic fluid cells containing a ring chromosome of unknown origin (47,XY; +r). The ring chromosome was not stained by library probes from the gonosomes, thereby suggesting its autosomal origin. The sensitivity of CISS-hybridization was demonstrated by the detection of small translocations and fragments in human lymphocyte metaphase spreads after irradiation with ⁶⁰Co-gamma-rays. Lymphocyte cultures from two XX-males were investigated by CISS-hybridization with Y-library probes. In both cases, metaphase spreads demonstrated a translocation of Yp-material to the short arm of an X chromosome. The translocated Y-material could also be demonstrated directly in interphase nuclei. CISS-hybridization of autosomes 7 and 13 was used for prenatal diagnosis in a case with a known balanced translocation t(7;13) in the father. The same translocation was observed in amniotic fluid cells from the fetus. Specific staining of the chromosomes involved in such translocations will be particularly important, in the future, in cases that cannot be solved reliably by conventional chromosome banding alone.Dedicated to Professor Friedrich Vogel on the occasion of his 65th birthday     

Histone H4 acetylation analyses in patients with polysomy X: implications for the mechanism of X inactivation

In humans, it is thought that the X-inactivation phenomenon occurs no matter how many X chromosomes are present, and that only one of them remains active. Nevertheless, individuals who have an abnormal number of X chromosomes show a wide spectrum of abnormalities, which increase with the number of X chromosomes present in a given individual. It has been shown that the inactive X chromosome in female mammals is distinguished by a lack of histone H4 acetylation, and that this could be used as an accessible marker for distinguishing between Xi and Xa in spreads of metaphase chromosomes. We studied three X-polysomic patients for the presence of active chromatin by analysis of histone H4 acetylation on unfixed metaphase spreads. Using antisera to H4 acetylated at lysines 16, 8 and 5, respectively, we observed frequencies different from those expected from cells with only one underacetylated X chromosome. In particular, when antiserum to H4 acetylated at lysine 16 was used about 90% of the cells showed acetylation of all X chromosomes. This suggests a possible disturbance in the deacetylation process, probably due to the presence of multiple Xs. Received: 25 April 1997 / Accepted: 15 March 1998     

Investigation of telomere length dynamics in induced pluripotent stem cells using quantitative fluorescence in situ hybridization

Here we attempted to clarify telomere metabolism in parental cells and their derived clonal human induced pluripotent stem cells (iPSCs) at different passages using quantitative fluorescence in situ hybridization (Q-FISH). Our methodology involved estimation of the individual telomere lengths of chromosomal arms in individual cells within each clone in relation to telomere fluorescence units (TFUs) determined by Q-FISH. TFUs were very variable within the same metaphase spread and within the same cell. TFUs of the established iPSCs derived from human amnion (hAM933 iPSCs), expressed as mean values of the median TFUs of 20 karyotypes, were significantly longer than those of the parental cells, although the telomere extension rates varied quite significantly among the clones. Twenty metaphase spreads from hAM933 iPSCs demonstrated no chromosomal instability. The iPSCs established from fetal lung fibroblasts (MRC-5) did not exhibit telomere shortening and chromosomal instability as the number of passages increased. However, the telomeres of other iPSCs derived from MRC-5 became shorter as the number of passages increased, and one (5%) of 20 metaphase spreads showed chromosomal abnormalities including X trisomy at an early stage and all 20 showed abnormalities including X and 12 trisomies at the late stage.     

Aberrant chromosomal sex-determining mechanisms in mammals, with special reference to species with XY females

Both mouse and man have the common XX/XY sex chromosome mechanism. The X chromosome is of original size (5-6% of female haploid set) and the Y is one of the smallest chromosomes of the complement. But there are species, belonging to a variety of orders, with composite sex chromosomes and multiple sex chromosome systems: XX/XY1Y2 and X1X1X2X2/X1X2Y. The original X or the Y, respectively, have been translocated on to an autosome. The sex chromosomes of these species segregate regularly at meiosis; two kinds of sperm and one kind of egg are produced and the sex ratio is the normal 1:1. Individuals with deviating sex chromosome constitutions (XXY, XYY, XO or XXX) have been found in at least 16 mammalian species other than man. The phenotypic manifestations of these deviating constitutions are briefly discussed. In the dog, pig, goat and mouse exceptional XX males and in the horse XY females attract attention. Certain rodents have complicated mechanisms for sex determination: Ellobius lutescens and Tokudaia osimensis have XO males and females. Both sexes of Microtus oregoni are gonosomic mosaics (male OY/XY, female XX/XO). The wood lemming, Myopus schisticolor, the collared lemming, Dirostonyx torquatus, and perhaps also one or two species of the genus Akodon have XX and XY females and XY males. The XX, X*X and X*Y females of Myopus and Dicrostonyx are discussed in some detail. The wood lemming has proved to be a favourable natural model for studies in sex determination, because a large variety of sex chromosome aneuploids are born relatively frequently. The dosage model for sex determination is not supported by the wood lemming data. For male development, genes on both the X and the Y chromosomes are necessary.     

Prenatal detection of aneuploidies using fluorescencein situ hybridization: A preliminary experience in an Indian set up

Fluorescencein situ hybridization (FISH) is a powerful molecular cytogenetic technique which allows rapid detection of aneuploidies on interphase cells and metaphase spreads. The aim of the present study was to evaluate FISH as a tool in prenatal diagnosis of aneuploidies in high risk pregnancies in an Indian set up. Prenatal diagnosis was carried out in 88 high-risk pregnancies using FISH and cytogenetic analysis. Multicolour commercially available FISH probes specific for chromosomes 13, 18, 21, X and Y were used. Interphase FISH was done on uncultured cells from chorionic villus and amniotic fluid samples. FISH on metaphase spreads was done from cord blood samples. The results of FISH were in conformity with the results of cytogenetic analysis in all the normal and aneuploid cases except in one case of structural chromosomal abnormality. The hybridization efficiency of the 5 probes used for the detection of aneuploidies was 100%. Using these probes FISH assay yielded discrete differences in the signal profiles between cytogenetically normal and abnormal samples. The overall mean interphase disomic signal patterns of chromosomes 13, 18, 21, X and Y were 94.45%; for interphase trisomic signal pattern of chromosome 21 was 97.3%. Interphase FISH is very useful in urgent high risk cases. The use of FISH overcomes the difficulties of conventional banding on metaphase spreads and reduces the time of reporting. However, with the limited number of probes used, the conventional cytogenetic analysis serves as a gold standard at present. It should be employed as an adjunctive tool to conventional cytogenetics     

Y chromosome length variation and its significance in the horse

The results of Y chromosome measurements in 31 horses are presented. The Y chromosome was identified using G-, R-, and C-banding techniques. From G-banded metaphase spreads, total X and Y chromosome and separate proximal (P) and distal (D) Y-band measurements were made. Within this group, the Y/X ratio (%) for each animal varied from 18.93 to 43.95, with an overall mean of 34.85 and a coefficient of variation (CV) of 16.12. The overall mean P/X ratio (%) was 23.57 with a CV of 20.57, compared with an overall mean D/X ratio (%) of 11.26 with a CV of 15.18. The group studied included 27 Thoroughbred and 4-non-Thoroughbred animals, of which 20 were clinically normal controls and 11 presented with various clinical abnormalities. By comparison with data from other species, the possible breed association and clinical significance of the observed heteromorphism for the Y chromosome in this species is discussed.     

Meiotic analysis of two human reciprocal X-autosome translocations

Two cases of human reciprocal X-autosome translocation, t(X;12) and t(X;2), are described in sterile males, along with meiotic findings. Each carrier had inherited the translocation from his mother. Both showed azoospermia and germ-cell maturation arrest at the primary spermatocyte level, with most cells being arrested at the pachytene stage. A few metaphase I (MI) divisions were found, with occasional metaphase II cells being seen in the t(X;2) carrier. MI air-dried preparations gave clear evidence of chain quadrivalent formation. In the t(X;2) heterozygote, the pairing characteristics of the quadrivalent at pachytene were also analyzed in electron microscopic spreads. Disturbance of pairing around the breakpoints characterized most quadrivalents, and there was evidence in about 20% of the cells that nonhomologous pairing had taken place between the translocated chromosomes and the normal chromosome 2. Comparisons are made with similar nonhomologous pairing configurations seen at pachytene in quadrivalents of male reciprocal X-autosome translocations of the mouse.     

Physical mapping of three fruit ripening genes：Endopolygalacturonase,ACC oxidase and ACC synthase from apple（Malus x domestica） in an apple rootstock A106（Malus sieboldii

The apple rootstock,A106(Malus sieboldii),had 17 bivalents in pollen mother cells at meiotic metaphase 1,and 17 chromosomes in a haploid pollen cell.Karyotypes were prepared from root-tip cells with 2n=34 chromosomes,Seven out of 82 karyotypes(8.5%) showed one pari of satellites at the end of the short arm of chromosome 3.C-bands were shown on 6 pairs of chromosomes 2,4,6,8,14,and 16 near the telomeric regions of short arms.Probes for three ripening-related genes from Malus x domestica:endopolygalacturonase(EPG,0.6kb),ACC oxidase(1.2kb),and ACC synthase(2kb)were hybridized in situ to metaphase chromosomes of A106.Hybridization sites for the EPG gene were observed on the long arm of chromosome 14 in 15 out of 16 replicate spreads and proximal to the centromere of chromosomes 6 and 11.For the ACC oxidase gene,hylridization sites were observed in the telomeric region of the short arm of chromosomes 5 and 11 in 87% and 81% of 16 spreads respectively,proxiaml to the centromere of chromosome 1 in 81% of the spreads,and on the long arm of chromosome 13 in 50% of the spreads. Physical mapping of three fruit ripening genes in an apple rootstock A106.Twenty five spreads were studied for the ACC synthase gene and hybridization sites were observed in the telomeric region of the short arm of chromosome 12 in 96% of the spreads.chromosomes 9 and 10 in 76% of the spreads,and chromosome 17 in 56% of the spreads.     

Analysis of repetitive DNA sequences in the sex chromosomes of Oreochromis niloticus

In the Nile tilapia, Oreochromis niloticus, sex determination is primarily genetic, with XX females and XY males. While the X and Y chromosomes (the largest pair) cannot be distinguished in mitotic chromosome spreads, analysis of comparative hybridization of X and Y chromosome derived probes (produced, by microdissection and DOP-PCR, from XX and YY genotypes, respectively) to different genotypes (XX, XY and YY) has demonstrated that sequence differences exist between the sex chromosomes. Here we report the characterization of these probes, showing that a significant proportion of the amplified sequences represent various transposable elements. We further demonstrate that concentrations of a number of these individual elements are found on the sex chromosomes and that the distribution of two such elements differs between the X and Y chromosomes. These findings are discussed in relation to sex chromosome differentiation in O. niloticus and to the changes expected during the early stages of sex chromosome evolution.     

Visualization of pig sperm chromosomes by in-vitro penetration of zona-free hamster ova

Zona-free hamster eggs were penetrated by pig spermatozoa capacitated using bovine follicular fluid and Percoll gradients. A mean penetration rate of 80.1% was obtained from 5 ejaculates from 2 boars. Penetrated eggs were cultured and analysable metaphase chromosome spreads were obtained from 16.8%. The analysis of 20 pig sperm complements indicated that 9 were 19, Y, 10 were 19, X and 1 appeared to have an XY sex chromosome constitution.     

Carrier detection of deletions in female relatives of X-linked disorders by non-isotopic in situ hybridisation.

Recent studies suggest that a non-isotopic in situ hybridisation (NISH) approach can be successfully employed to investigate the carrier status of female relatives in families of selected patients with Duchenne muscular dystrophy (DMD) or Hunter syndrome, whose diseases are due to a specific X chromosome deletion. Whilst the majority of metaphase spreads from normal females show specific hybridisation signals on both X chromosomes when tested with either dystrophin or Hunter gene-derived probes, only one X chromosome in each metaphase spread will show the relevant hybridisation complex in female carriers of deletions involving the dystrophin or Hunter gene. Thus, the NISH method can be a valuable diagnostic tool for the detection of the carrier status of female relatives of patients with X chromosome deletions.     

Cytogenetic analysis of 34 early stage bovine embryos from superovulated Hereford donors

One hundred and thirty-four bovine embryos were collected from 17 superovulated Hereford cows and processed for metaphase chromosome spreads. Mitotic figures were obtained from 54 embryos (40%), but only 34 embryos (25%) provided analyzable chromosomes spreads. Thirty embryos were 2n = 60, one was a possible 2n/4n mosaic and three were hypodiploid. The hypodiploid cases and the 2n/4n mosaic could be due to technical artifacts.