Etoposide induces chromosomal abnormalities in mouse spermatocytes and stem cell spermatogonia

BACKGROUND: Etoposide (ET) is a chemotherapeutic agent widely used in the treatment of leukaemia, lymphomas and many solid tumours such as testicular and ovarian cancers, all of which are common in patients of reproductive age. The purpose of the study was to characterize the long-term effects of ET on male germ cells using sperm fluorescence in situ hybridization (FISH) analyses. METHODS: Chromosomal aberrations (partial duplications and deletions) and whole chromosomal aneuploidies were detected in sperm of mice treated with a clinical dose of ET. Semen samples were collected at 25 and 49 days after dosing to investigate the effects of ET on meiotic pachytene cells and spermatogonial stem-cells, respectively. RESULTS: ET treatment resulted in major increases in the frequencies of sperm-carrying chromosomal aberrations in both meiotic pachytene (27- to 578-fold) and spermatogonial stem-cells (8- to 16-fold), but aneuploid sperm were induced only after treatment of meiotic cells (27-fold) with no persistent effects in stem cells. CONCLUSION: These results show that ET may have long-lasting effects on the frequencies of sperm with structural aberrations. This has important implications for cancer patients undergoing chemotherapy with ET because they may remain at higher risk for abnormal reproductive outcomes long after the end of chemotherapy.     

Analysis using dual-colour fluorescencein situ hybridization of meiotic chromosome segregation in male mice heterozygous for a reciprocal translocation

Meiotic chromosome behaviour was investigated in male mice heterozygous for the translocation T(7;16)67H. At metaphase I, chain-of-four quadrivalents were present in approximately 80% of the spermatocytes; the bulk of remaining cells contained a ring quadrivalent, with only a few having either a trivalent plus univalent configuration or two bivalents. A low rate of non-disjunction, approximately 5%, was found through analysis of C-banded metaphase II spermatocytes. Using fluorescencein situ hybridization with differentially labelled whole chromosome paints, a wide array of segregation products were observed at metaphase II, depending on whether they arose from alternate, adjacent I, adjacent II orientation at metaphase I or were uninformative for alternative/adjacent I because of the presence of a chiasma in an interstitial pairing segment. Some 62% of the cells fell into this latter category, with only small proportions clearly arising through alternate (1.8%) or adjacent I (0.7%) orientations. Approximately 30% of the cells contained the products of adjacent II orientation. Consideration of the data suggested that most of these cells arose from metaphase I cells that contained a chain quadrivalent. Ring quadrivalents appeared predominantly to orientate in an alternate/adjacent I manner.for publication by J. S. (Pat) Heslop-Harrison     

Cot-1 banding of human chromosomes using fluorescencein situ hybridization with Cy3 labeling

Summary We developed a new chromosome banding method byin situ hybridization of human Cot-1 DNA as a probe. Clear banding was produced on metaphase chromosomes of lymphoblastoid cells after probe detection with a fluorescent dye Cy3. Comparison with the known banding patterns revelaed a similarity to the R-banding with some significant differences: some centromeric heterochromatin regions show Cot-1 positive bands. This suggests that some repetitive sequences from the heterochromatin regions constitute a major component of Cot-1 DNA. This unique chromosome banding method, Cot-1 banding, may be used as a supplement to the conventional karyotype analysis. Scanning analysis of the fluorescence intensities of Cot-1 banding and Q-banding are useful for objectively analyzing the banding pattern including a detection of chromosome aberrations. The Cot-1 banding with Cy3 is particularly powerful when applied for the gene mapping by fluorescencein situ hybridization (FISH) because red fluorescence of Cy3 for chromosome staining can be readily distinguished from green fluorescence of fluorescein isothiocyanate (FITC) for probe labeling. Using this novel method, we mapped a 4 kb-DNA fragment from myelin protein zero (MPZ) gene on the chromosome 1q22 to q23.     

Detection of chromosomal abnormalities of chromosome 12 in uterine leiomyoma using fluorescencein situ hybridization

Summary Fifty uterine leiomyomas were examined using conventional cytogenetic method and fluorescencein situ hybridization (FISH) for detection of chromosomal, abnormalities of chromosome 12. Of the 50 tumors, nine were examined using FISH on the non-cultured samples. Two (4.0%) of 50 tumor samples examined showed chromosomal abnormalities of chromosome 12 by the conventional cytogenetic analysis. For FISH, the whole-chromosome painting probe and D12Z3 probe specific for the centromeric region were used. Of the 50 cultured samples, 10 showed structural aberrations and four showed numerical aberrations of chromosome 12 by FISH analysis. Of the nine non-cultured samples, four showed structural abnormalities of chromosome 12, all of which also showed structural abnormalities of chromosome 12 on the cultured samples. These results indicate that chromosomal abnormalities of chromosome 12 are important in the biology of at least some types of uterine leiomyoma, and that FISH is a useful complement to the conventional cytogenetic analysis in the study of solid tumors.     

Detection of numerical chromosomal abnormalities by fluorescence in situ hybridization of interphase cell nuclei with chromosome-specific probes on archival cytologic samples

Fluorescence in situ hybridization (FISH) is rapidly emerging as a tool for analyzing numerical and structural chromosomal abnormalities in both liquid and solid tumors. Most studies make use of fresh samples. To determine the feasibility of detecting numerical chromosomal abnormalities (NCA) by FISH using chromosome-specific probes 8, 12, 17, and X (Vysis, Inc., Downers Grove, IL) on archival cytologic preparations, we studied 23 patient samples, one Papanicolaou- and one Diff-Quik-stained slide per case (46 slides), and two additional unstained slides (fresh ascitic fluids) as controls. Included in this study were nine ascitic fluids (four benign and five malignant), four malignant pleural fluids, three benign bladder washes, and seven malignant fine-needle aspirates (FNA) from various sites. The slides ranged from 1–94 days old. After removal of coverslips using xylene, all slides were destained in a series of alcohol and water washes. Pretreatment of slides with pepsin was followed by the in situ hybridization procedure. Two hundred cells per slide were evaluated for distinct separate signals. Results showed the following: 1) all slides were evaluable except for eight (8/46) which had either too few cells or enough cells but with faint signals, 2) the oldest sample showed distinct signals, 3) previously Diff-Quik-stained slides showed relatively better signals than Papanicolaou-stained slides, 4) samples less than a month old showed relatively better signals, and 5) malignant samples showed various NCA, but not the benign samples. We conclude that FISH on archival cytologic preparation 1) is feasible, although age of the slide is a factor since better signals were seen in those less than a month old, 2) shows better results in previously Diff-Quik-stained slides, and 3) is a tool that can be used in the retrospective study of various liquid and solid neoplasms. Diagn Cytopathol 1996;14:178–181. © 1996 Wiley-Liss, Inc.     

Reexamination of chromosomal loci of human muscle actin genes by fluorescencein situ hybridization

Summary Human genes for cardiac (ACTC), skeletal (ACTA), and vascular type (aortic type or -) smooth (ACTSA) muscle actins have been localized to chromosomes 15q14, 1q42.1, and 10q23.3, respectively, by fluorescencein situ hybridization.     

多重荧光原位杂交技术检测多发性骨髓瘤复杂核型异常

目的 探讨多重荧光原位杂交(multiplex fluorescence in situ hybridization,M-FISH)技术在多发性骨髓瘤(multiple myeloma,MM)复杂核型异常(complex chromosomal aberrations,CCAs)检测中的价值.方法 对10例常规细胞遗传学(conventional cytogenetics,CC)方法检测具有复杂核型的MM患者应用M-FISH技术确定复杂染色体的重排及标记染色体的组成.结果 M-FISH证实了CC显示的29种结构异常,并进一步明确了1p-、6q-、9q-、9q+、+8q+×2、14q+、?14q、der(4)、der(22)、der(1)t(1;?)(q10;?)、der(3)、del(7)的具体来源;同时也发现CC分析没有发现或不能识别的21种异常,其中t(2;15)(q33;q22)、t(6;7)(q23;q34)、t(8;11)(q24;q23)、t(1;14)(q10;q32)和t(X;1)(q26;q25)是新发现的核型异常.这10例随访的MM患者病例资料中9例已死亡,中位生存期仅为23个月,较公认的MM患者的平均生存期34个月明显缩短.结论 对伴有CCAs的MM患者,M-FISH技术可以明确CC分析中复杂染色体异常,并发现和纠正CC分析中漏检及误检的异常,为MM的染色体异常研究提供了一种重要的方法,已经成为精确染色体核型分析所不可缺少的手段之一.     

Detection of variation in the ribosomal RNA gene clusters by a modified fluorescencein situ hybridization method

Summary Physical mapping of genes by fluorescencein situ hybridization (FISH) has become routine using fluorescein isothiocyanate (FITC) for probe detection and propidium iodide (PI) for chromosome staining. We have modified this conventional FISH method in a way that utilizes Texas red (TR) for signal detection and quinacrine mustard (QM) for chromosome banding. Using this Texas red and quinacrine (TRQ) method, we were able to identify individual acrocentric chromosomes with varying degrees of ribosomal RNA gene clusters. Two acrocentric chromosomes were found to carry extremely small number of rRNA gene copies as compared to the other eight counterparts in human diploid lymphoblastoid cell line GM00130B. Thus, the TRQ method allows one to probe for a specific sequence while identifying individual chromosomes and will be powerful for the chromosomal localization of various genes.     

Condensation behaviour of the human X chromosome in male germ cells and Sertoli cells examined by fluorescencein situ hybridization

The chromatin condensation behaviour of the human X chromosome has been studied by fluorescencein situ hybridization (FISH) analysis in germ cells and Sertoli cells of the adult testis, and comparisons are made with previous findings for the human Y chromosome and for chromosome 7. In meiotic prophase, the X chromosome can be seen to extend greatly at zygotene and to contract through pachytene into the sex vesicle. Such extension, which has also been noted for the human Y chromosome at this stage of meiosis, could be a prerequisite for XY pairing and crossing-over. Byin situ hybridization analysis, the sex chromosomes of patients with Sertoli-cell-only syndrome appear extremely contracted compared with the normally extended state of those in adult Sertoli cells of fertile men. By contrast, the state of expansion for chromosome 7 in Sertoli cells appears identical for sterile and fertile testes. This could suggest an association between gene-controlled germ cell losses and failure of expansion of the sex chromosome axes. The variable patterns of extension and contraction for the X and Y chromosome axes in germ cells and Sertoli cells might provide underlying clues to patterns of expression noted for sex-linked genes in the human testis.     

Comparative fluorescencein situ hybridization mapping of primate chromosomes withAlu polymerase chain reaction generated probes from human/rodent somatic cell hybrids

We have usedAlu polymerase chain reaction generated probes from rearranged human/rodent somatic cell hybrids for fluorescencein situ hybridization and comparative mapping of some intrachromosomal changes in the karyotypes of great apes (Pan troglodytes, P. paniscus, Gorilla gorilla Pongo pygmaeus), a gibbon (Hylobates lar), and an Old World monkey (Macaca fuscata). Probes containing chromosomes 2 and 18 fragments confirmed inversions already suggested by the banding pattern of great ape homologues. However, a chromosome 3 fragment showed complex rearrangements in the gibbon and macaque karyotype which were previously not well defined from banding. Subchromosomal painting will allow the identification of intrachromosomal changes on the basis of DNA homology and provides a powerful method to study karyological and genomic evolution.accepted for publication by M. SchmidInstitut für Anthropologie und Humangenetik, Universität München     

Chromosomal translocations in human soft tissue sarcomas by interphase fluorescence in situ hybridization

In soft tissue sarcomes, clonal rearrangement of chromosomes has been shown by cytogenetic analysls to be unique and specific for tumor types. The development of fluorescence in situ hybridbation (FISH) has allowed detection of chromosomal rearrangements In the interphase nuclel isolated from paraffin-embedded tissues. Three kinds of trans-locations in the interphase nuclel that were isolated from 47 cases of soft tissue sarcomas ware examined by FISH with chromosome-speclfic DNA probes of centromeric and total probes. of 47 soft tissue sarcomas 42 (89.4%) revealed tumor-specitic transiocations by retrospective cytogenetic analysis. Transiocation t(X;18) was detected In 25/28 synovial sarcomas; translocation t(11;22) In 5/6 Ewlng's sarcomas and primitive neuroectodermal tumors (PNET); and translocation t(12;16) was found in 12/13 liposarcomas, including 10 myxold and two round cell lypes as clonal chromosomal aberrations specific for both subtypes. Based on the cytogenetic analysis, Ewing's sarcoma is related closely with PNET as shown by MIC2–protein reactivity. Other cytogenetic findings of translocation t(12;16) Indicate that round cell liposarcomas share chromosomal changes with myxoid lipo sarcomas, and further suggest that both tumor subtypes of liposarcoma may possess common precursor cells. FISH is a useful aid in determining the tumor type of soft tissue sarcomas with regard to histogenetic origin.     

Preparation of tomato meiotic pachytene and mitotic metaphase chromosomes suitable for fluorescencein situ hybridization (FISH)

Fluorescencein situ hybridization (FISH) is an increasingly powerful tool with a variety of applications in both basic and applied research. With excellent genetic, cytogenetic and molecular maps available, the tomato genome provides a good model to benefit from the full potential of FISH. Tomato chromosomes at mitotic metaphase are small and not particularly suitable for high-resolution FISH. In contrast, chromosomes at meiotic pachytene are about 15 times longer, and easier to identify by their differences in chromosome arm lengths and chromomere pattern. We have developed a technique for preparing chromosomal spreads of young pollen mother cells at midprophase I which is suitable for FISH. In a first series of experiments, the hybridization patterns of three classes of repetitive DNA sequences were studied in single and multicolour FISH.accepted for publication by J. S. (Pat) Heslop-Harrison     

Variation of mouse oocyte sensitivity to griseofulvin-induced aneuploidy and meiotic delay during the first meiotic division

The effects of varying the time of chemical treatment on the induction of areuploidy and meiotic delay in metaphase II (Mll) oocytes were studied by administering 1,500 mg/kg griseofulvin (GF) at 0, 2, 4, 6, or 8 hr after on injection of human chorionic gonadotrophin (HCG). The results show that the oocytes have a different sensitivity to GF-induced aneuploidy and meiotic delay during the course of meiotic maturation. Although not restricted to a particular period of meiotic maturation, the frequency of aneuploidy was highest (P < 0.05) when GF was given at 2, 4, or 6 hr after HCG. The maximum frequency of hyperploidy (42.4%) occurred at the 4-hr treatment time. Also, GF treatment resulted in the induction of meiotic delay as demonstrated by ovulated metaphase I (Ml) and polyploid Mll oocytes. The meiotic delay data depict a period of relative resistance between two periods of sensitivity in that the percentages of ovulated Ml oocytes were 53.3, 21.3, 3.5, 6.7, and 25.7 when GF was given at 0, 2, 4, 6, and 8 hr after HCG, respectively. Also, at these treatment times the percentages of polyploid oocytes were 0.6, 1.7, 7.7, 20.1, and 15.4, respectively. Therefore, the oocytes seem to be more sensitive to GF-induced meiotic delay during the periods preceding and following meiotic spindle assembly. In conclusion, the results demonstrate that the time of chemical treatment influences the frequency of aneuploidy and the degree of meiotic delay. Also, the results emphasize that to thoroughly characterize the aneugenic potential of a specific chemical several treatment times may be needed. © 1994 Wiley-Liss, Inc.     

Orbital marginal zone lymphomas: an immunohistochemical, polymerase chain reaction, and fluorescence in situ hybridization study

Many studies have been performed on chromosomal aberrations of extranodal marginal zone lymphomas. However, only a few have been published so far on ocular adnexal marginal zone lymphomas. We studied 18 cases of orbital lymphoid cell infiltrates. Using fluorescence in situ hybridization (FISH), we studied some of the most common chromosomal aberrations found in extranodal marginal zone lymphomas as: trisomies 3, and rearrangements of the 18q21 MALTI gene to detect the translocations t(11;18)(q21;q21) and t(14;18)(q32;q21)MALT1. Our goals were as follows: (1) study those aberrations in our material and compare them with the literature, (2) check their prognostic significance, and (3) check whether studying those aberrations with FISH can be used as a diagnostic tool to differentiate reactive from neoplastic infiltrates, in addition to immunohistochemistry and polymerase chain reaction. We found a high frequency of trisomies 3 (68%) and 18 (56.6%), the highest published so far in orbital lymphomas. On the other hand, no rearrangement was seen in any of our cases. The histologic picture and the clinical course were the same when there was one or more aberrations. As for the diagnostic significance, the presence of a prior, concurrent, or subsequent lymphoma in almost all the positive for aberrations cases suggests that either the orbital infiltrates in these cases are lymphomas, or they have, at least, a malignant potential or a genetic instability. Therefore, the demonstration of these numerical aberrations by FISH may be an additional sensitive, reliable, and relatively simple tool to differentiate reactive from neoplastic orbital lymphoid cell infiltrates when the immunohistochemistry and polymerase chain reaction, performed in a busy and routine-based histopathology laboratory, are unsatisfactory.     

Analysis of rye B-chromosome structure using fluorescencein situ hybridization (FISH)

Fluorescencein situ hybridization (FISH) has been used to analyse the structure of the rye B chromosome. Genomicin situ hybridization (GISH) demonstrates the high level of overall similarity between A and B chromosomes of rye, as well as the presence of a number of specific sequences. The B-specific repeat families D1100 and E3900 have been analysed in terms of their physical location and possible contiguity. Rye Bs contain members of the rye-specific dispersed repetitive family R173, as well as centromeric regions similar to those of the As. The B chromosomes analysed in our study lack detectable rDNA sequences. Anomalous results have been obtained with a number of subtelomeric repetitive probes from rye. Bs usually lack these sequences, but evidence is presented that in some cases A–B translocation events may relocate such sequences from the As to the Bs. These data are discussed in the context of current models for the origin of the B chromosome.     

Detection of aneuploidy in human spermatozoa using fluorescencein situ hybridization (FISH)

Summary Fluorescencein situ hybridization (FISH) with chromosome specific alpha-satellite DNA probes was used to estimate the rates of aneuploidy of chromosomes 1, 17, 18, X and Y in human ejaculated sperm. Sperm samples were collected from six donors, and biotinylated DNA probes, D1Z5, D17Z1, D18Z1, DXZ1 and DYZ3 were hybridized to interphase sperm which had been pretreated with dithiothreitol to expand their nuclei. A minimum of 3,000 sperm per donor were analyzed. The hybridization efficiency was 99.68% for all the five probes. The frequencies of aneuploidy for chromosomes 1, 17 and 18 were 0.65%, 0.66% and 0.61% respectively. For XX-and YY-sperm the frequencies were 0.28% and 0.27% respectively. To estimate the diploidy and disomy rates, a mixture of D17Z1 and D18Z1 were used as probes, and the frequency of diploid sperm was calculated to be 0.27%. After subtraction of the diploidy rate, the disomy rates for chromosomes 1, 17, and 18 were estimated to be 0.38%, 0.39% and 0.33%, respectively. The proportion of X- and Y-bearing sperm were 49.9% and 49.66%, consistent with an expected 1: 1 ratio.     

Confirmation of the copy number of chromosome 1 in interphase nuclei from paraffin sections of breast tumours by fluorescencein situ hybridization

Fluorescencein situ hybridization (FISH) was used to establish the copy number of chromosome 1 in a set of nine breast tumours in which the chromosome had previously been shown to have undergone a variety of rearrangements by loss of heterozygosity studies. In each case, FISH with satellite III DNA from chromosome 1q12 confirmed the results obtained by Southern hybridization. Importantly, in all five cases with rearrangements thought not to involve the centromeric region, FISH showed that the events had not disrupted the gross chromosome structure. This study highlights the potential of using the two techniques together to obtain a clearer picture of both large- and small-scale alterations to chromosomes in solid tumours.     

Characterization of somatic cell hybrids by bivariate flow karyotyping and fluorescencein situ hybridization

We report on the use of flow karyotyping and fluorescence in situ hybridization (FISH) to characterize the human chromosomes in somatic cell hybrids. The identity, DNA content, and relative frequency of human chromosomes are derived from flow karyotypes, i.e., measurements of Hoechst and chromomycin fluorescence intensities of chromosomes by dual beam flow cytometry. Chromosome integrity is assessed by comparing the peak position of a human chromosome in the flow karyotypes of a hybrid cell line and its human donor. When human donor cells are unavailable, the peak position of a human chromosome in a hybrid line is compared to the range of peak positions among normal individuals. The relative frequency of human chromosomes in subclones or hybrids grown in culture is monitored using the volumes of peaks in flow karyotypes. FISH with biotinylated human genomic DNA or chromosome-specific repeat sequences as probe is used in conjunction with flow karyotyping to confirm the number of human chromosomes in hybrids. Some small rearrangements are detected by flow karyotyping and not by FISH. On the other hand, translocations between human and rodent chromosomes are detected by FISH and not always by flow karyotyping. Flow karyotyping and FISH were used to characterize over 100 hybrid lines donated by other laboratories. A hybrid set useful for the construction of chromosome-enriched gene libraries is presented. In this set, each of the 24 human chromosome types is present and intact, as judged by these techniques, in a line containing little or no other human material.     

Acrylamide: Induction of heritable translocations in male mice

Acrylamide (AA), known to induce dominant lethals in male rodents, was studied in the mouse heritable translocation test by using intraperitoneal injections on 5 consecutive days. Matings on days 7-10 following the last injection yielded a high frequency of translocation carriers in the F₁ male population, which demonstrated that acrylamide is an effective inducer of translocations in postmeiotic germ cells. As an inducer of both dominant lethals and heritable translocations in late spermatids and early spermatozoa, AA is similar to alkylating agents such as ethylmethanesulfonate and ethylene oxide. However, AA′s chemical structure, the nature of adducts formed with DNA, and its lack of mutagenicity in bacteria suggest a different mechanism as the basis for AA′s germ cell mutagenicity.     

多重荧光原位杂交结合染色体涂抹技术检测骨髓增生异常综合征复杂核型异常

目的探讨多重荧光原位杂交(multiplex fluorescence in situ hybridization,M-FISH)及全染色体涂抹(whole chromosome painting,WCP)技术在骨髓增生异常综合征(myelodysplastic syndromes,MDS)复杂核型异常检测中的价值。方法对7例常规R显带具有复杂染色体异常的MDS患者应用M-FISH技术确定复杂染色体的重排及标记染色体的组成,识别微小易位。并进一步采用双色WCP技术验证M-FISH检测的结果。结果M-FISH不仅证实了R显带的结果,而且确定了R带核型分析没有确定的6种标记染色体、9种有不明来源的额外物质增加的染色体、5种衍生染色体的组成和来源及4种被忽略的微小易位。涉及17号染色体的异常及-5/5q-是MDS最为常见的两种染色体异常。WCP技术纠正了一些M-FISH漏检及误检的异常。结论M-FISH是明确复杂染色体异常的很有用的分子生物学工具,WCP是M-FISH技术的重要补充,R带核型分析结合分子细胞遗传学工具M-FISH和WCP可以更加准确地描述复杂染色体异常。