Y-specific DNA Probes Research Articles

Role of microchimerism in the pathogenesis of oral lichen planus

Microchimerism of persistent fetal cells has been implicated in some cell-mediated autoimmune diseases. This study examines the hypothesis that fetal microchimerism plays a role in the pathogenesis of lichen planus (LP) affecting the oral cavity. Mucosal biopsies of 12 women with oral LP (OLP) were tested for the presence of both male cells and male DNA originating from prior pregnancies or prior blood transfusions. Six male patients with OLP served as a control group. Biopsies were analyzed for the presence of Y-chromosome-positive cells by fluorescence in situ hybridization (FISH) with X- and Y-specific DNA probes. To confirm the FISH findings, we used fluorescent polymerase chain reaction (PCR) to identify Y-chromosome sequences in DNA extracted from mucosal lesions. Using FISH technology, all the 18 biopsy samples showed good hybridization results. In females, Y-chromosome-specific signals were not detected by FISH at any site of the lesions. PCR amplification demonstrated a single peak at the locus specific for the X-chromosome. Male DNA microchimerism was not present in any of the investigated lesions, suggesting that microchimerism because of persisting male fetal cells is unlikely to play a major role in the pathogenesis of OLP.

Use of the Kleihauer test to detect fetal erythroblasts in the maternal circulation

Non-invasive prenatal diagnosis of aneuploidies on fetal nucleated erythrocytes present in the maternal circulation is hampered by the extremely small cell number of uncertain origin (70% of erythroblasts circulating during pregnancy have a maternal origin). Therefore, a method allowing selection of the fetal cells among the maternal cells is indispensable after the erythroblast enrichment step. In the present study, after an erythroblast enrichment step on a ficoll gradient followed by a positive immuno-magnetic selection with anti-CD71 or anti-GPA antibodies, a rapid, simple and direct chemical staining method adapted from the classical Kleihauer test was developed to select fetal cells. Precise differentiation between fetal and maternal erythroblasts is based on the constitutional difference between fetal and adult haemoglobin (Hb). The fetal cells appear with an intense pink cytoplasmic staining while maternal cells with adult haemoglobin are colourless. Preservation of the cytoplasmic integrity allows one to distinguish morphological characteristics and to visualize simultaneously nuclear hybridization signal by FISH (fluorescent in situ hybridization). This approach was tested by FISH analysis using dual-colour X- and Y-specific DNA probes on blood samples from 15 pregnant women, with the results being compared to cytogenetic or sonographic sex determination. For 12 pregnancies fetal sex was determined successfully (5 XY/7 XX), in two cases in situ hybridization failed, and in one case no fetal erythroblast was observed after the Kleihauer test. The selection method was applied to a pregnancy at risk for cystic fibrosis (CF). After a Kleihauer test, fetal erythroblasts were collected by microdissection, whole genomic DNA was amplified by primer extension pre-amplification (PEP) followed by a nested CF PCR. The fetal genotype was successfully characterized and confirmed by conventional prenatal diagnosis.

Use of the Kleihauer test to detect fetal erythroblasts in the maternal circulation

Non-invasive prenatal diagnosis of aneuploidies on fetal nucleated erythrocytes present in the maternal circulation is hampered by the extremely small cell number of uncertain origin (70% of erythroblasts circulating during pregnancy have a maternal origin). Therefore, a method allowing selection of the fetal cells among the maternal cells is indispensable after the erythroblast enrichment step. In the present study, after an erythroblast enrichment step on a ficoll gradient followed by a positive immuno-magnetic selection with anti-CD71 or anti-GPA antibodies, a rapid, simple and direct chemical staining method adapted from the classical Kleihauer test was developed to select fetal cells. Precise differentiation between fetal and maternal erythroblasts is based on the constitutional difference between fetal and adult haemoglobin (Hb). The fetal cells appear with an intense pink cytoplasmic staining while maternal cells with adult haemoglobin are colourless. Preservation of the cytoplasmic integrity allows one to distinguish morphological characteristics and to visualize simultaneously nuclear hybridization signal by FISH (fluorescent in situ hybridization). This approach was tested by FISH analysis using dual-colour X- and Y-specific DNA probes on blood samples from 15 pregnant women, with the results being compared to cytogenetic or sonographic sex determination. For 12 pregnancies fetal sex was determined successfully (5 XY/7 XX), in two cases in situ hybridization failed, and in one case no fetal erythroblast was observed after the Kleihauer test. The selection method was applied to a pregnancy at risk for cystic fibrosis (CF). After a Kleihauer test, fetal erythroblasts were collected by microdissection, whole genomic DNA was amplified by primer extension pre-amplification (PEP) followed by a nested CF PCR. The fetal genotype was successfully characterized and confirmed by conventional prenatal diagnosis.

Interphase chromosome arrangement in Sertoli cells of adult mice.

Sertoli cells of adult male laboratory mice were examined with a number of banding techniques and by nonradioactive in situ hybridization applying different repetitive DNA probes. All banding methods revealed the typical features of mouse Sertoli cells, i.e., a central nucleolus, usually with two chromocenters associated at diametrically opposed sides in which the centromeric regions of the chromosomes are clustered. Silver staining as well as in situ hydridization with rDNA labeled part of the chromocenters and the nucleolus, indicating transcriptional activity of at least some of the nucleolus organizer regions. In situ hybridization with X- and Y-specific DNA probes showed both sex chromosomes to be undercondensed in Sertoli cells This decondensation suggests expression of sex chromosomal genes in Sertoli cells. While the X chromosome appeared to occupy a central position near one of the chromocenters, the Y chromosome was found at the periphery of the nucleus in the majority of cells. Hybridization with telomeric sequences resulted in strong labeling of the chromocenters and dispersed signals at the nuclear periphery.

Application of reproductive technology to buffaloes

This paper focuses on recent advances in the application of embryo transfer technologies to buffalo. The early recovery rate of 0.15 transferable embryos has now increased to 2.0, with altered superovulation protocols. Lower follicular population and poor follicular development, with adverse seasonal influences explain the lower ovulatory responses. Unovulated follicles at superovulation contribute high quantities of oestrogen, altering the uterine milieu. The birth of calves using in vitro fertilisation technology and frozen-thawed embryos and determination of embryonic sex using a Y-specific DNA probe, are recent milestones achieved using these technologies.

Cytogenetic heterogeneity and histologic tumor growth patterns in prostatic cancer.

Twenty-five prostatic adenocarcinomas were studied for the presence of intratumoral cytogenetic heterogeneity by interphase in situ hybridization (ISH) to routinely processed tissue sections. ISH with a chromosome Y-specific repetitive DNA probe provided a model to investigate patterns of chromosomal heterogeneity within and between different pathological grades. The Gleason grading system was used, since it is based on a detailed classification of growth patterns. Heterogeneity with respect to ploidy of the tumor was examined by ISH with a repetitive DNA probe specific for chromosome 1. The ploidy status of these cancers was confirmed by DNA flow cytometry (P < 0.001). Cytogenetic heterogeneity at the (Y) chromosomal level was observed between Gleason areas, within one area, and even within single tumor glands. The different patterns of chromosomal heterogeneity were seen in all tumor grades and stages. Differences in ploidy status were also found following the aforementioned histological patterns, again, in all grades and stages. Intraglandular heterogeneity was most frequently seen. No correlation was found between cytogenetic heterogeneity and proliferative activity (Ki-67 immunostaining). In contrast to current views on clonality, suggesting regional separation of subclones with different DNA content, this study demonstrates that these subclones can be interspersed.

Non-syncytial sources of fetal DNA in transcervically recovered cell populations.

We have previously shown that fetal DNA can be detected in swabs and flushings obtained from the lower uterine pole prior to the termination of pregnancy. The presence of syncytiotrophoblast vesicles in transcervically retrieved samples suggested that this distinctive placental tissue was an abundant source of fetal DNA and a valuable resource in prenatal diagnosis strategies. In a more extensive study involving 150 terminations of pregnancy between 7 and 17 weeks gestational age, 29% of transcervically retrieved samples contained visible syncytial vesicles. Flushing of the uterine pole more frequently contained syncytia than direct aspiration (39% compared with 26% of samples) but this difference was not statistically significant. No samples > 14 weeks gestational age contained syncytia. Polymerase chain reaction analysis using Y-sequence specific-nested primers indicated the presence of fetal DNA in the absence of intact syncytial vesicles. We therefore examined samples by in-situ hybridization using Y-specific DNA probes. Positive labelling was observed in syncytial vesicles where present and in clumps of unidentified fetal cells. In addition, high numbers of naked nuclei were labelled in samples devoid of syncytia. These isolated nuclei are possibly derived from disrupted syncytia, and may be an important and hitherto overlooked contributory factor in fetal material which collects at the lower uterine pole.

Non-syncytial sources of fetal DNA in transcervically recovered cell populations

We have previously shown that fetal DNA can be detected in swabs and flushings obtained from the lower uterine pole prior to the termination of pregnancy. The presence of syncytiotrophoblast vesicles in transcervically retrieved samples suggested that this distinctive placental tissue was an abundant source of fetal DNA and a valuable resource in prenatal diagnosis strategies. In a more extensive study involving 150 terminations of pregnancy between 7 and 17 weeks gestational age, 29% of transcervically retrieved samples contained visible syncytial vesicles. Flushing of the uterine pole more frequently contained syncytia than direct aspiration (39% compared with 26% of samples) but this difference was not statistically significant. No samples >14 weeks gestational age contained syncytia. Polymerase chain reaction analysis using Y-sequence specific-nested primers indicated the presence of fetal DNA in the absence of intact syncytial vesicles. We therefore examined samples by in-situ hybridization using Y-specific DNA probes. Positive labelling was observed in syncytial vesicles where present and in clumps of unidentified fetal cells. In addition, high numbers of naked nuclei were labelled in samples devoid of syncytia. These isolated nuclei are possibly derived from disrupted syncytia, and may be an important and hitherto overlooked contributory factor in fetal material which collects at the lower uterine pole.

In situ hybridization utilizing a Y chromosome DNA probe. Use as a cell marker for hepatocellular transplantation.

Research in hepatocellular gene therapy requires a consistently reproducible cell marker to detect transplanted hepatocytes. We have used a Y-specific genomic DNA probe to accomplish this goal. This technique enables the identification of transplanted male cells in recipient female tissues. Donor hepatocytes from male mice were transplanted into female mice via splenic injection. Recipient mouse livers were harvested 1, 24, and 48 hr after transplant. Transplanted (male) hepatocytes were detected in liver biopsy sections using in situ hybridization with the Y chromosome probe.

Aneuploidy and ageing: sex chromosome exclusion into micronuclei

In lymphocyte cultures, the number of aneuploid cell nuclei increases with proband age mainly because of the loss of sex chromosomes. Since one possible cause of aneuploidy in cell nuclei is chromosomal lag at anaphase, with subsequent chromosome loss via micronucleus formation, we scored 5000 interphase nuclei from ten female and ten male probands for associated micronuclei. Whereas, in young (< 10 years) probands, an average of 0.15% interphase nuclei exhibited micronuclei, the frequency rose to 0.46% in older probands (> 70 years). In situ hybridizations with X-specific and Y-specific DNA probes were carried out, and the signal distribution in ten nuclei with associated micronuclei was documented for each donor. Our results indicate that the exclusion of sex chromosomes into micronuclei doubles during a human life, from 11% in young probands to 20% in old donors.

Sephadex filtration and human serum albumin gradients do not select spermatozoa by sex chromosome: a fluorescent in-situ hybridization study.

Fluorescent in-situ hybridization (FISH) of decondensed sperm nuclei has been used directly to evaluate the enrichment efficiency of human sperm separation using Sephadex gel filtration and human serum albumin (HSA) gradients. Control and processed spermatozoa were fixed and their nuclei decondensed. In-situ hybridization was carried out with a Y-specific DNA probe (DYZ1). Sephadex filtration yielded 52.5% Y-chromosome-bearing spermatozoa, HSA separation resulted in 49.4% Y-chromosome-bearing spermatozoa and in the untreated control sample the percentage of Y spermatozoa was 49.3%. Statistical analysis revealed no significant differences between the selection methods employed and the controls, and no real enrichment for X- or Y-bearing spermatozoa was detected for any of the selection methods assayed. The usefulness of the protocols reported for selection of spermatozoa by sex chromosome in couples at risk for X-linked diseases is discussed.

Fetal granulocytes in maternal venous blood detected by in situ hybridization.

Fetal male cells from maternal venous blood were detected by a non-radioactive in situ hybridization method using the biotinylated Y-specific DNA probe pY431. The hybridizations were performed on Ficoll-Paque-isolated nucleated blood cells obtained from 11 pregnant women in the seventh to 31st week of gestation. A Y-specific signal was detected in both granulocytes and lymphocyte-like cells in seven of the 11 women studied. These women gave birth to boys. In one of the four remaining cases, a Y-specific signal was detected in the lymphocyte-like cells but not in the granulocytes. This woman gave birth to a girl. The other three women had no cells with a Y-specific signal and all three gave birth to girls. Altogether, 83,500 nucleated cells were analysed. One hundred and three cells showed a Y-specific signal. Of these Y-specific cells, 62 per cent were granulocytes and 38 per cent lymphocyte-like cells. Our results suggest that fetomaternal transfer of granulocytes is common and that it occurs as early as in the seventh week of gestation. None of the ten non-pregnant female control samples showed positive cells with the Y-chromosome-specific probe; approximately 97 per cent of the cells from the five adult male controls showed a Y-specific signal. Our results indicate that in situ hybridization using a Y-specific DNA probe performed on granulocytes in maternal blood can be used for fetal male sex determination.

Marker chromosomes in gonadal dysgenesis: Avoiding unnecessary surgery

Women with partial or intact Y chromosomes are at increased risk for developing gonadoblastoma or dysgerminoma; prophylactic gonadectomy is usually recommended. However, some women with gonadal dysgenesis have marker chromosomes which can complicate assessment and management. To evaluate a young woman with gonadal dysgenesis and a 45,X/46,X,+ mar karyotype, we used a direct assay for H-Y antigen and, in addition, polymerase chain reaction (PCR) and in situ hybridization with X- and Y-specific DNA probes. The marker chromosome was found to be an X chromosome derivative, indicating that the patient was at low risk for tumor formation. The technology presented is useful for assessing the risk for tumor formation in women with marker sex chromosomes.

Embryo Sexing: A Review of Current Techniques and Their Potential for Commercial Amdication in Livestock Production

The development of embryo transfer procedures has created the potential for embryo sexing as a method of controlling the sex of offspring. Five techniques have been developed for this purpose: cytogenetic analysis, assay of X-linked enzymes, analysis of differential development rates, detection of male-specific antigens, and the use of Y-specific DNA probes. These methods are reviewed and their potential for commercial application in domestic animal production is discussed. The use of Y-specific DNA probes, particularly those that employ DNA amplification techniques, has resulted in the greatest potential for commercial application. An assay based on a bovine Y-chromosome-specific DNA sequence is described. This assay has a determination rate greater than 90% with samples as small as two cells and accurately determines sex 94% of the time. For economic and technical reasons, embryo sexing has not been widely used in conventional embryo transfer, but these procedures, particularly Y-chromosome-specific DNA amplification techniques, are quite applicable to embryo cloning and will be vital when cloning becomes commercially viable.

Cytogenetic and molecular characterization of a small ring chromosome in the complex karyotype of a girl with Turner syndrome

Blood samples of an 8-year-old girl with Turner syndrome were examined using cytogenetic and molecular methods. Chromosomal analyses revealed a mosaic karyotype consisting of 25% 47,X,der(X), +r(X) and 75% 46,X,der(X) cells. Southern blot hybridizations with Y-specific DNA probes excluded a Y chromosomal origin of the small ring chromosome. In situ hybridization using DNA probe pXBR showed it to be X-derived. Examination of C-, Q-, and R-banding patterns indicated that the der(X) chromosome probably arose by a translocation event.

DNA analysis of a patient with two different marker chromosomes using Y-specific DNA probes

A female patient with unilateral gonadal dysgenesis was a mosaic for three cell lines, 45,X/46,X, + marI/46,X, + marII, including two different marker chromosomes. DNA analysis using 17 Y-specific DNA probes revealed that each marker consists of different segments of the Y chromosome.

Partial complementation by murinethaplotypes: deficit of males amongt6/tw5double heterozygotes and correlation with transmission-ratio distortion

To evaluate whether sex reversal contributes to sex-ratio imbalance among t6/tw5 double heterozygotes, the cross performed by K. B. Bechtol (Genetical Research 39, 1982, 79-84), T/t6 x T/tw5, was repeated. Significantly more normal-tailed (t6/tw5) females than males were recovered. By contrast, sex ratios were normal among tailless progeny resulting from this cross and among all classes produced by control crosses. Hybridization of a Y-specific DNA probe with genomic DNA from phenotypic females revealed no XY, sex-reversed males. On the genetic backgrounds that generated only moderate transmission distortion of tw5 (81-85%), the overall viability of the doubly heterozygous progeny was only 50% and the sex-ratio skew among this class was strong. However, on a genetic background that displayed extreme tw5 transmission (99%), embryonic viability was more than 80% and the sex-ratio imbalance was weak.

Determination of sex chromosomal constitution and chromosomal origin of drumsticks, drumstick-like structures, and other nuclear bodies in human blood cells at interphase by fluorescence in situ hybridization

The sex chromosomal constitution has been determined in various types of human leukocytes at interphase by use of fluorescence in situ hybridization with X- and/or Y-specific DNA probes. It is found that during aging and differentiation of myelocytes into polymorphs there is no significant change in the relative frequency of various types of male and female cells with a specific type of sex chromosomal constitution. Non-random variability of the relative proximity between the X chromosomes within the nuclei is also observed in female cells. Moreover, we are the first to determine that sex-specific "drumsticks" and "sessile nodules" in female polymorphs originate from the X chromosomes and that non-sex-specific "drumstick-like" bodies in male polymorphs are of Y chromosomal origin.

Determining the origins and the structural aberrations of small marker chromosomes in two cases of 45,X/46,X, +mar by use of chromosome‐specific DNA probes

A 17-year-old girl (S.M.) and a 13-year-old girl (C.L.) both with Ullrich-Turner syndrome (UTS) were found to have 45,X/46,X, + mar mosaicism. The marker chromosomes in both patients were very small in size. In S.M. the marker chromosome was present in 80% of phytohemagglutinin-stimulated lymphocytes, 28% of skin fibroblasts, and 11-20% of gonadal fibroblasts. In C.L., the small marker chromosome was found in 50% of stimulated lymphocytes. S.M. is of normal height, but C.L. is short. Molecular hybridization with a number of Y-specific DNA probes demonstrated their presence in S.M. but absence in C.L. In situ hybridization with Y-specific and X-centromere-specific DNA probes confirmed the Y origin of the marker chromosome in S.M. and the X origin of the minute chromosome in C.L. Biotinylated centromere and telomere probes were also used for in situ hybridization to show the presence of centromeric and telomeric sequences in the Y-marker chromosome, suggesting that the deletion of this marker chromosome is interstitial.

XY female mice resulting from a heritable mutation in the primary testis determining gene, Tdy

Chimeric mice constructed with XY embryonic stem (ES) cells that had been multiply infected with a retroviral vector were used in a genetic screen to look for mutations affecting the sex determination pathway in mice. From a small number of chimeras screened one was identified that gave rise to a low proportion of XY females amongst his offspring. Analysis of the segregating patterns of retroviral insertions demonstrated that the mutation was found in a subset of the offspring derived from one originally infected ES cell. However, the mutation appeared to have occurred subsequent to the infection. Some of the XY females proved to be fertile, and the mutant phenotype was found to segregate exclusively with the Y chromosome. Analysis of the offspring also confirmed the absence of any retroviral insertion that could be correlated with the mutation. Further characterisation of the Y chromosome carrying the mutation by karyotypic analysis, and by Southern blotting with a range of Y-specific DNA probes suggested that there has been no gross deletion or rearrangement of the Y carrying the mutation. There also appeared to be no loss of Y-specific gene functions apart from that of testis determination. Moreover, the mutation is complemented by Sxr', the minimum portion of the mouse Y known to carry Tdy. From the phenotype and deduced location of the mutation, we conclude that it is within the Tdy locus. This is the first such mutation to be described in mice.