Specific cytogenetic labeling of bovine spermatozoa bearing X or Y chromosomes using fluorescent in situ hybridization (FISH)

X and Y specific probes were identified in order to apply the fluorescent in situ hybridization (FISH) technique to bovine spermatozoa. For Y chromosome detection, the BRY4a repetitive probe, covering three quarters of the chromosome, was used. For X chromosome detection, a goat Bacterial Artificial Chromosome (BAC) specific to the X chromosome of bovine and goats and giving a strong FISH signal was used. Each probe labeled roughly 45% of sperm cells. The hybridization method will be useful for evaluating the ratio of X- and Y- bearing spermatozoa in a sperm sample and consequently can be used to evaluate the efficiency of sperm sorting by different techniques such as flow cytometry.     

Molecular cytogenetic techniques in detecting subtle chromosomal imbalances

Diagnostic possibilities of CGH and M-FISH techniques for detection of submicroscopic chromosomal imbalancies were compared on the basis of two cases of t(X;Y) and one case of marker chromosome. In cases with t(X;Y), the sequences specific for chromosome Y were detected by PCR and CGH, but the localisation of these sequences on the short arm of chromosome X was confirmed by the FISH technique, employing two Yp-specific probes for SRY and TSPY genes. Significant differences between above cases were revealed in the size of Yp chromosome fragments translocated on chromosome X. An extra material of chromosome marker could not be identified by classical banding and FISH techniques and it was only CGH and M-FISH techniques that enabled detecting the chromosomal origin of the marker. The applied CGH technique enabled finding subtle chromosomal imbalancies in the presented cases with a resolution of approximately 3 Mbp.     

Use of the primed in situ labelling (PRINS) technique for a rapid detection of chromosomes 13, 16, 18, 21, X and Y

The primed in situ labelling (PRINS) technique is an alternative to in situ hybridization for chromosomal screening. We have developed a semi-automatic PRINS protocol, using a programmable thermocycler. The method has been successfully tested with specific primers for chromosomes, 13, 16, 18, 21, X and Y. Specific chromosome detection has been obtained on both metaphases and interphase nuclei. This suggests that PRINS may be a reliable technique for detecting aneuploidies and some chromosomal aberrations.     

Quinacrine Mustard—a Selective Fluorescent Stain for the Y Chromosome in Human Tissues for Routine Cytogenetic Screening

A simple sensitive and specific cytochemical method for detection of the Y chromosome in man (George 1970, Pearson et al. 1970) is based on staining by alkylating fluorochromes. The dye binds strongly to the heterochromatic regions of the Y chromosome, thus causing these regions to fluoresce very brightly under ultraviolet (uv) light. The Y chromosome could be identified easily at metaphase and also as a highly fluorescent body in interphase cells. Two bodies were seen by Pearson et at. in a subject having the XYY anomaly. In prophase cells, it appears as a fluorescing chromatin thread. Its presence has been demonstrable only in males; hence the usefulness of this technique for rapid cytogenetic screening is obvious. The constancy and reproducibility of the technique seems to rule out the possibility of unreliability caused by nonspecific binding of the dye. The present report gives techniques applied to tissues usually studied in cytogenetic screening     

An Improved Diamond Knife Holder for Ultramicrotomy

A simple sensitive and specific cytochemical method for detection of the Y chromosome in man (George 1970, Pearson et al. 1970) is based on staining by alkylating fluorochromes. The dye binds strongly to the heterochromatic regions of the Y chromosome, thus causing these regions to fluoresce very brightly under ultraviolet (uv) light. The Y chromosome could be identified easily at metaphase and also as a highly fluorescent body in interphase cells. Two bodies were seen by Pearson et at. in a subject having the XYY anomaly. In prophase cells, it appears as a fluorescing chromatin thread. Its presence has been demonstrable only in males; hence the usefulness of this technique for rapid cytogenetic screening is obvious. The constancy and reproducibility of the technique seems to rule out the possibility of unreliability caused by nonspecific binding of the dye. The present report gives techniques applied to tissues usually studied in cytogenetic screening     

用C-带和涂染技术检测棕色田鼠Y染色体

采用染色体C 带技术和小鼠整条Y染色体特异探针检测棕色田鼠的Y染色体 ,结果如下 :棕色田鼠雄性个体C 带中期分裂相中 ,X性染色体是亚中部着丝粒染色体 ,在着丝粒处存在着强烈的C阳性带 ,而且在短臂的中间也有一条C阳性带 ,但是没有发现深染的Y染色体。用小鼠整条Y染色体特异探针涂染棕色田鼠的骨髓细胞中期分裂相和间期核 ,以小鼠骨髓细胞中期分裂相和间期核作为对照。涂染结果表明 :棕色田鼠骨髓细胞中期分裂相和间期核涂染信号检出率分别为 0 - 2 %和 3% - 5 % ,两者均呈阴性反应 ,而对照都呈阳性反应。根据实验结果 ,作者认为在棕色田鼠的Y染色体上及整个基因组DNA中不存在小鼠整条Y染色体特异DNA的同源序列 ,其Y染色体上可能没有决定雄性性别的重要基因     

Dual Y chromosome painting and in situ cell-specific immunofluorescence staining in lung tissue: an improved method of identifying donor marrow cells in lung following bone marrow transplantation

Several recent studies have demonstrated localization of donor bone marrow-derived cells in recipient lungs following transplantation from male to female mice or patients. Donor cells are identified by detection of the Y chromosome by fluorescence in situ hybridization (FISH). However, protein digestion pretreatments usually required for tissue FISH significantly limit the ability to detect cell type-specific markers by immunohistochemistry. We have used an alternative protein digest approach that entails heating the slides in 10 mM sodium citrate rather than utilizing a protease digestion. This approach preserves cell proteins following FISH, and allows lung tissue to remain intact for subsequent detection of cell-specific markers by immunohistochemistry. We have examined this technique in mouse lungs using a Y chromosome paint and three cell-specific markers, a pan-cytokeratin for epithelial cells, PECAM-1 for endothelial cells, and CD45 for leukocytes. Excellent visualization of both the Y chromosome and cell-specific surface protein markers was obtained on a single slide. This approach will significantly enhance the ability to detect and identify donor bone marrow cells in recipient mouse lungs following male to female transplantation.     

Flow cytometric analysis of DNA content differences in blood samples obtained by leucoconcentration

The leucoconcentration technique allows rapid obtainment of cellular suspensions from total blood or bone marrow for flow cytometric analysis. The technique is based on picric acid in ethyl alcohol fixation and saponin red cell lysis, followed by mithramycin staining for DNA. It gives a good resolution of DNA distributions that allow detection of slight variations in DNA content. These results were obtained with cellular suspensions differing only in one X or Y chromosome (male, female, Klinefelter and Turner syndromes). In these studies the ratio of the DNA content of X and Y chromosomes agrees with the chromosomal mass ratio already reported by other authors, but the "absolute values" are 10-fold more compared to these same works. Our conclusion is that leucoconcentration technique followed by DNA staining with mithramycin increases the difference in the dye's penetration and binding between X and Y chromosomes.     

Reassessment of presumed Y/22 and Y/15 translocations in man using a new technique.

A new chromosome banding technique, distamycin A plus DAPI, has been used to reexamine cases of presumed Y/autosome translocations. In contrast with the results obtained with quinacrine fluorescence (Q-banding), the satellites of acrocentric chromosomes do not fluoresce brightly with this new (DA-DAPI) method, making it more specific for the long arm of the Y chromosome. Previous cases with intensely Q-fluorescent and abnormally long short arms on a chromosome 22 were considered as presumptive 22/Y translocations: The new technique clearly shows that, in these cases, the additional material on 22p is not derived from Yq. In contrast, in other cases the Yq nature of additional material on 15p, in conjunction with the presence of an extra Y-body in interphase nuclei and the presence of a male-specific DNA, supports the previous diagnosis of a presumptive 15/Y translocation.     

The karyotype of the mouse

A denaturating and renaturating technique, applied to mouse chromosomes, makes visible characteristic banding patterns by which all elements of the karyotype can be individually distinguished. The Y chromosome as a whole appears darkly stained. The X chromosome comprises 6.33% of the homogametic haploid set. The banding pattern of the chromosomes is compared with that obtained by aid of the quinacrine dihydrochloride fluorescence technique. After its use a banding pattern results which is similar to, but less distinct than, that found after the renaturation procedure.     

Flow cytometric quantification of human chromosome specific repetitive DNA sequences by single and bicolor fluorescent in situ hybridization to lymphocyte interphase nuclei

Fluorescent in situ hybridization allows for rapid and precise detection of specific nucleic acid sequences in interphase and metaphase cells. We applied fluorescent in situ hybridization to human lymphocyte interphase nuclei in suspension to determine differences in amounts of chromosome specific target sequences amongst individuals by dual beam flow cytometry. Biotinylated chromosome 1 and Y specific repetitive satellite DNA probes were used to measure chromosome 1 and Y polymorphism amongst eight healthy volunteers. The Y probe fluorescence was found to vary considerably in male volunteers (mean fluorescence 169, S.D. 35.6). It was also detectable in female volunteers (mean fluorescence 81, S.D. 10.7), because 5-10% of this repetitive sequence is located on autosomes. The Y probe fluorescence in males was correlated with the position of the Y chromosome cluster in bivariate flow karyotypes. When chromosome 1 polymorphism was studied, one person out of the group of eight appeared to be highly polymorphic, with a probe fluorescence 26% below the average. By means of fluorescent in situ hybridization on a glass slide and bivariate flow karyotyping, this 26% difference was found to be caused by a reduction of the centromere associated satellite DNA on one of the homologues of chromosome 1. The simultaneous hybridization to human lymphocyte interphase nuclei of biotinylated chromosome 1 specific repetitive DNA plus AAF-modified chromosome Y specific DNA was detected by triple beam flow cytometry. The bicolor double hybridized nuclei could be easily distinguished from the controls. When the sensitivity of this bicolor hybridization is improved, this approach could be useful for automatic detection of numerical chromosome aberrations, using one of the two probes as an internal control.     

Chromosome sorting of primates: Isolation and identification of Y chromosome in green monkeys

Single laser flow cytometry was applied to the karyotype analysis of green monkeys. Clear sex difference in flow karyotype was recognized in this monkey, because Y chromosome could be identified as a single peak in the histogram of male specimens. We could isolate Y chromosome of this species by the use of a cell sorter, and demonstrate by polymerase chain reaction that the sorted-out chromosomes contained the Y chromosome specific nucleotide sequence (SRY). This chromosome sorting technique provides a powerful strategy for constructing the DNA library specific to Y chromosome in this species.     

Giemsa-11 technique elucidating three structurally altered nonfluorescent Y chromosomes: r (Y), idic (Yp), dir tan dup (Yp)

Three patients are presented in whom a structurally altered Y chromosome was finally diagnosed using Giemsa-11 technique. The first patient, a 19-year-old woman with primary amenorrhea and some features of Turner syndrome had ring (Y). The second patient, a 2-year-old boy with small stature and incomplete masculinization was found to have an isodicentric (Yp). In the third patient who was examined because of ambigous genitalia the chromosome abnormality, a nonfluorescent pseudodicentric (Y) was interpreted as a direct tandem duplication of the short arm, centromere, and a piece of the long arm, a rearrangement not described before. In each of the patients Q-, G-, and C-bandings failed to elucidate the kind of chromosome abnormality. Since clarification of a given Y structural rearrangement by cytogenetic methods cannot be avoided even in the era of molecular genetics, Giemsa-11 technique should be applied in the analysis of every dubious small sex chromosome.     

Demonstration of fluorescent X and Y bodies after antibody- and complement-mediated cytotoxicity

A technique for fluorescence staining of X and Y bodies (sex chromatin) after antibody- and complement-mediated cytotoxicity test has been developed. Cytotoxicity was quantitated by staining the dead cells with trypan blue (dye exclusion test). X bodies (Barr bodies) of human female fibroblast (stained with acridine orange) were observed in about 40 percent of the cells which survived cytotoxicity. Y bodies were studied on human male fibroblasts and in a hamster/human hybrid line which retained the human Y chromosome only. Fluorescent Y body was detectable in from 50 to 60 percent of the cells which survived the serological test. The double staining procedure did not significantly affect the proportion of killed (trypan blue-positive) cells. We suggest that this is a useful method for the detection of cytotoxic antibodies against the products of X and Y chromosomes, especially when mixed cell populations-such as tumor, sex chromosome mosaics, sperm, and artificially mixed human male and female cell lines-are tested.     

The Y chromosome of the mouse is decondensed in Sertoli cells

The condensation of the Y chromosome in mouse cells was studied with two repetitive DNA probes, pY353/B and M34. Both DNA probes are specific to the Y chromosome and hybridize in situ along the whole chromosome. Due to the high resolution of the in situ hybridization technique with non-radioactive labeled DNA probes it was possible to observe the degree of condensation of the Y chromosome in the interphase cell nuclei of various somatic tissues and on testes preparations. The Sertoli cells were the only cell type in which the Y chromosome was always observed to be in a highly decondensed state. The decondensation of the Y chromosome in the Sertoli cells supports the view that the genetic activity of the Y chromosome is cell autonomous and opens the way to its molecular analysis.     

黑麂Y染色体的鉴别和Sry基因的克隆及定位

以流式细胞仪分离小麂(Muntiacus reevesi)Y染色体和黑麂(Muntiacus crinifrons)Y1,Y2,X+4和1号染色体,利用DOP-PCR技术富集了分离的各单条染色体。然后,将小麂的Y染色体的DOP-PCR产物经Cy3标记后直接作为涂染探针,应用染色体涂染技术与雌雄黑麂的核型标本进行杂交,确认了黑麂真正的Y染色体为Y2染色体。再以黑麂的Y1,Y2,X+4和1号染色体的DOP-PCR产物为模板,用人的特异性的SRY（sex determining region of the Y chromosome）基因引物对其进行扩增,结果表明黑麂只有Y2染色体出现了SRY扩增片段。然后扩增产物克隆和测序,比较它与人的同源性,初步把黑麂的Sry基因定位在Y2染色体上。最后提取雄性黑麂的基因组DNA,并用同一对引物对其进行扩增,亦得到Sry基因的片段,对此扩增片段进行克隆,测序,结果表明其与Y2染色体得到的Sry基因片段完全一样,与人SRY基因的同源性均为83%。 Abstract：The single Y chromosome of Muntiacus reevesi and Y1,Y2 ,X+4,1 chromosome of Muntiacus crinifrons were obtained by flow-sorting ,then they were amplified through DOP-PCR . After that, the metaphase karyotype of Muntiacus crinifrons were painted by using the product of the DOP-PCR of the Y chromosome of Muntiacus reevesi as a special probe and the result showed that Y2 chromosome was the real Y chromosome of Muntiacus crinifrons. Secondly the product of the DOP-PCR of Y1,Y2,X+4,1 chromosome of Muntiacus crinifrons were used as the templates of the next amplification using the special primer devised according to the human SRY gene .One band was obtained only from Y2 chromosome, then it was cloned to the T-vector and sequenced. The Sry gene sequence of Muntiacus crinifrons was acquired and the conclution was that there are 83% homology between the human and Muntiacus crinifrons. It was testified that in all mammal Sry gene is consertive. On the other side the Sry gene was located to the Y2 chromosome of the Muntiacus crinifrons.     

New technique for processing paraffin-embedded tissue for Y chromosome fluorescence identification of trophoblastic disease

A new reprocessing technique for Y chromosome fluorescent body (q 12 region) detection of trophoblastic disease in previously paraffin-embedded tissues is described. Deparaffinized sections were treated with pronase and trypsin for digestion, followed by hydrolysis with HCl and acetic acid, staining with quinacrine hydrochloride fluorochrome and mounting in S?rensen's phosphate buffer (pH 5.5). Use of the technique resulted in sufficient fluorescence quality and better accuracy for Y and X heterochromatin scoring. The technique yielded the same results in retrospective formalin-fixed, paraffin-embedded trophoblastic specimens as in fresh tissues. The combinations of enzymes and acids and the dosages necessary for optimal results are discussed.     

The TX;Y test for the detection of nondisjunction and chromosome breakage in Drosophila melanogaster. II. Results of female exposures

The TX; Y test is a short-term assay for the detection of sex-chromosome nondisjunction and chromosome breakage in Drosophila melanogaster. It has been used in previous work following the exposure of males. In this work, females are exposed. When females are the exposed parent, only chromosome gain can be detected. Positive results for the induction of aneuploidy were obtained following exposures of females to X-rays, 10 degrees C cold shock, and colchicine. No increase in aneuploidy was obtained following exposures of females to DMSO and trifluralin. Comparison with similar work in males reveals no consistent pattern concerning the more appropriate sex to use for aneuploidy testing in Drosophila, as colchicine was found to be positive in females only and DMSO and trifluralin were effective in males only. Further work is necessary to validate the TX; Y test and to understand the relative efficacy of female and male exposures to aneuploidy inducing agents in Drosophila.     

Analysis of complex Y chromosome aberrations using a single DNA probe (Y-367)

A specific cloned DNA sequence (Y-367) detects at least four loci in the euchromatic long arm and in the short arm of the human Y chromosome. Deletion mapping assigns one locus to the distal euchromatic long arm, another to a region close to the centromere on either Yq or Yp, and two additional loci to the Y short arm. Y-367 may thus be used for the rapid screening of even complex Y chromosome aberrations. This is exemplified in a 45,X male with Y chromosome material on the long arm of chromosome 10 by the detection of an inversion of a portion of Yp and by the confirmation of duplications and deletions in two individuals with duplications of part of the Y chromosome.