用三色荧光原位杂交(Three-Color FISH)检测小鼠精子中染色体数目异常的研究

用小鼠X、Y和8号染色体特异的DNA探针,与经DTT（dithiotreitol）和LIS（lithium-3,5-diiodosalicylicacid）解聚的小鼠附单精子进行三色荧光原位杂交（fluoresceoceinsituhybridization,FISH）,以检测精子中的染色体数目异常,并与MMⅡ染色体分析比较．结果表明精子三色FISH具有以下优点和特点：（1）方法敏感稳定,且简便快速;（2）在每一个体至少分析10000尾精子的基础上计算非整倍体单,因此结果更为准确;（3）能检测多倍体即减数分裂停止的发生率及停止的时期;（4）不仅能测定发生于试数分裂Ⅰ（MI）的染色体分离异常,还能检测发生于减数分裂Ⅱ（MII）的不分离和丢失．并对探针的选用、分析标准的建立以及三色FISH用于精于染色体分析的必要性等进行了讨论．     

多色荧光原位杂交检测苯系物接触工人精子1,18号染色体畸变率

为研究苯系物暴露对工人精子染色体的损伤 ,用 4条DNA探针与间期精子核染色体进行多色荧光原位杂交 ,同时检测精子 1号、18号染色体数目畸变和 1号染色体结构畸变 (末端缺失与重复 )。作业车间空气中苯的时间加权浓度 (TWA)为 4 2 2 9mg m3,高于国家卫生标准 (6mg m3)。暴露组工人尿粘糠酸 (ttMA)高于对照组。共计数15例暴露组工人 14 4 2 82条精子 ,14例对照组工人 135 937条精子 ,杂交效率为 99 85 %。非整倍体测定结果 :暴露组精子 1号、18号染色体双体率 (分别为 0 0 88%± 0 0 4 1% ,0 0 87%± 0 0 4 9% )显著高于对照组 1号、18号双体率(0 0 4 5 %± 0 0 2 4 % ,0 0 5 3%± 0 0 2 8% ) ;暴露组 1号、18号染色体缺体率分别为 (0 11%± 0 0 5 9% ,0 0 75 %±0 0 35 % )显著高于对照组相应数值 (0 0 4 8%± 0 0 18% ,0 0 4 5 %± 0 0 2 4 % ) ;而二倍体精子率 ,两组差别无显著性。结构畸变测定结果 :暴露组 1号染色体的末端重复率、末端缺失率 (分别为 0 16 %± 0 0 37% ,0 14 %± 0 0 5 3% )显著高于对照组数值 (分别为 0 0 82 %± 0 0 2 3% ,0 0 6 9%± 0 0 2 8% ) ;暴露组 1号染色体着丝粒重复率及着丝粒缺失率(0 10 %± 0 0 35 % ,0 10 %± 0 0 4 1% )显著性高于对     

三色荧光原位杂交技术（Triple—FISH）检测男性不育患者精子染色体的非整倍体性

目的：研究精子非整倍体染色体与男性不育之间的关系。方法：用荧光原位杂交（FISH）技术,对12个不育患精子的染色体进行了研究,其中,10例少,弱,畸精症患为A组,2例正常或接近正常的样品为B组,另有2例正常健康献精为对照组,原位杂交采用染色体XY和18号探针,每个个体记数1000个精子,结果：杂交总有效率为98％,在B组男性不育XY和18号的二体频率为0．30％和0．30％,与对照组相比（0．15％,0％）无显差别,XY和18号失体的精子为0．15％和0％,与对照组（0％,0．15％）也无显差别,而A组的XY和18号染色体二体率为1．13％和0．96％,失体为1．13％和1．6％,与前两组相比,差异有显性,估算总的非整倍体率：A组为42．44％,B组为6．05％,对照组为2．59％,前与后两组相比,差异有显性,结论：不育患精子的染色体具有显的非整倍体性。     

双色荧光原位杂交检测苯系物暴露工人精子9、18号染色体数目畸变

为研究苯系物暴露工人精子常染色体数目畸变.用地高辛标记的9号染色体探针(D9Zl)和生物素标记的18号染色体探针(D18Z1)进行双色荧光原位杂交,测定精子9、18号染色体非整倍体率.车间空气苯时间加权平均浓度(TWA)为86.49mg/m3,高于国家卫生标准1倍,苯暴露工人尿粘糠酸(ttMA)显著高于对照组.共计数14名暴露工人136401条精子,16名对照工人156955条精子.暴露组9、18染色体双体精子率(分别为0.168%±0.063%、0.055%±0.031%)和二倍体精子率(0.073%±0.045%)均高于对照组相应数值(分别为0.050%±0.030%、0.033%±0.025%和0.040%±0.036%);暴露组9、18号染色体缺体率(分别为0.206%±0.047%,0.068%±0.044%)高于对照组值(0.067%±0.037%、0.048%±0.034%).总数目畸变率(0.570%±0.144%)亦高于对照组值(0.218%±0.071%).实验表明接触较高浓度苯可引起长期暴露者精子常染色体非整倍体率增高.     

植物非整倍体及其遗传

生物物种在长期的进化过程中形成了稳定的遗传体系,都具有整倍的染色体数。但整倍体生物能够自发地或人为地产生非整倍体。植物非整倍体(三体)是在曼陀罗中首先发现的,并且在育种的过程中陆续培育出12种可能的三体。此后,又在烟草和燕麦中发现单体等非整倍体。     

丙烯酰胺非整倍体诱发效应的荧光原位杂交和CREST染色的研究

本文以小鼠着丝粒次要卫星DNA探针FISH和抗着丝粒CREST染色,研究了可疑的非整倍体毒剂丙烯酰胺(AA)诱导的小鼠NIH3T3细胞微核(MN)的着丝粒组成情况和小鼠骨髓染色体畸变(CA)情况。结果发现AA在100—400μg/ml诱导的MN约52.7％—71.6％为FISH阳性,60.5％—68.2％的MN为CR-EST阳性,两种结果均显示AA具有较强的非整倍体诱发效应。小鼠骨髓CA的FISH表明,AA既能诱导染色体结构畸变,又能诱导非整倍体形成,而以非整倍体诱发效应更为明显。     

贝类非整倍体研究进展

近年来,在贝类多倍体育种研究中,人们发现了成活的非整倍体胚胎和成体,并就此开展了相关的研究.对此研究进行了综述,对非整倍体的类型、产生原因、生长发育、鉴定方法等进行探讨,并展望了非整倍体研究的前景和应用价值.     

秋水仙胺(colcemid)诱发雄性小鼠生殖细胞减数分裂延迟和非整倍体的研究

本文以101/E1和C3 H/E1的杂种第一代雄性小鼠为材料,一次性腹腔注射秋水仙胺(COM)(1mg/kg体重)后,分别于第2,6,10,14和18h取材,进行减数分裂延迟和中期Ⅱ(MMII)染色体分析。结果表明,COM可诱发小鼠减数分裂延迟,并对其可能的生物学机制进行了讨论。在本实验条件下,COM未能导致MMII非整倍体率显著升高,并对可能的原因进行了分析。     

非整倍体专用术语

简要回顾了非整倍体专用术语研究和使用的历史概况，详细介绍了三体、四体，单体及缺体植物缺失和补偿染色体的３３种类型，并对各类型予以准确的定名和解释。     

Simultaneous detection of X- and Y-bearing human sperm by double fluorescence in situ hybridization

Double fluorescence in situ hybridization (FISH) was used to detect sex chromosomes in decondensed human sperm nuclei. Biotinylated X chromosome specific (TRX) and digoxigenin-labeled Y chromosome specific (HRY) probes were simultaneously hybridized to sperm preparations from 12 normal healthy donors. After the hybridization, the probes were detected immuno-cytochemically, using two different and independent affinity systems. Ninety-six percent of the 12,636 sperm showed fluorescent labeling, of which 47.4% were haploid X and 46.8% were haploid Y. A frequency of 0.46% of XX-bearing sperm (0.28% disomic, 0.18% diploid) and 0.38% YY-bearing sperm (0.21% disomic, 0.17% diploid) was found. The overall proportions of X- and Y-bearing sperm in the ejaculates were 47.9% and 47.2%, respectively, which was not significantly different from the expected 50:50 ratio. In addition 0.21% of cells appeared to be haploid XY-bearing sperm, 0.62% were diploid XY-bearing cells, and 0.05% of cells were considered to be tetraploid cells. The application of double FISH to human sperm using X-chro-mosome and Y-chromosome probes has allowed a more accurate assessment of the sex chromosal complements in sperm than single FISH method and quinacrine staining for Y-bodies. © 1993 Wiley-Liss, Inc.     

应用荧光原位杂交检测EB病毒BNLF-1基因在转基因小鼠子代染色体上的整合及其定位

应用荧光原位杂交技术研究了EB病毒潜伏膜蛋白基因(BNLF-1)在转基因小鼠子二代染色体上的整合及其定位。结果在两只子二代转基因小鼠中,分别观察80个和60个分裂相,出现杂交信号的核型分别为27和18个,检出率为33.8％和30％。转基因分别整合在14号染色体和10号染色体上。提示转基因BNLF-1已稳定整合到转基因小鼠的染色体上,并通过生殖细胞遗传给子代;推测转基因原代鼠的转基因整合可能是随机的多位点整合。     

Acrocentric centromere organization within the chromocenter of the human sperm nucleus.

It has recently been reported that in human sperm cells, the centromeres are clustered in a chromocenter in the interior region of the nucleus. The aim of the present study was to determine the intra-chromocenter organization of the five centromeres of the acrocentric chromosomes responsible for the biosynthesis of rRNA. The acrocentric centromeres were labeled by fluorescence in situ hybridization (FISH) after mild decondensation of the sperm nuclei to preserve the tail structure. The tail was used as a topographical marker for the orientation of the nucleus. The following results were obtained: (a) the association among the five centromeres was higher than expected from random distribution; (b) all the centromeres observed were randomly located within the chromocenter, occupying about 87% of the total area of the internal nucleus; (c) a major subpopulation of centromeres was located in a preferred area occupying 8.3% of the total nuclear area, with a peak 0.6 microm on the lateral axis and 1.0 microm on the apical side of the longitudinal axis; and (d) The dispersion of the centromeres was not influenced by the degree of the nuclear decondensation. We conclude that in human sperm nuclei, the acrocentric centromeres are organized within a nonlocalized structural element in the chromocenter. The chromocenter can range from an expanded size of 87% of the whole nucleus to a preferred size of 8.3% independent of the degree of nuclear decondensation. These findings have important implications for nuclear function (rRNA) that is not directly related to sperm cell function or early embryo development.     

Optimization of PRINS and C-PRINS for detection of telomeric sequences in Vicia faba

Primed in situ labelling (PRINS) of nucleic acids was developed as an alternative to traditionally used fluorescence in situ hybridization (FISH). Compared to FISH, PRINS is faster and does not require preparation of labelled probes. Nevertheless, the number of applications for physical mapping of DNA sequences on plant chromosomes remains low. This is due to the fact that there are a number of factors which influence the specificity and sensitivity of the reaction. The purpose of this work was to analyse the effect of some of them, including the age of slides, type of Taq DNA polymerase, number and concentration of primers, the presence and concentration of bovine serum albumine and MgCl₂ in the reaction mixture. Furthermore, the effect of various pre-treatments on signal intensity and non-specific fluorescence was studied. A consensus Arabidopsis-type telomeric sequence and Vicia faba mitotic chromosomes were used as a model system. We have found that the age of slides was critical and that under optimal conditions it was possible to achieve relatively high signal to noise ratio. This revised version was published online in July 2006 with corrections to the Cover Date.     

A new FISH assay to simultaneously detect structural and numerical chromosomal abnormalities in mouse sperm

De novo aberrations in chromosome structure represent important categories of paternally transmitted genetic damage. Unlike numerical abnormalities, the majority of de novo structural aberrations among human offspring are of paternal origin. We report the development of a three-color fluorescence in situ hybridization (FISH) assay (CT8) to detect mouse sperm carrying structural and numerical chromosomal abnormalities. The CT8 assay uses DNA probes for the centromeric and telomeric regions of chromosome 2, and a probe for the subcentromeric region of chromosome 8. The CT8 assay was used to measure the frequencies of sperm carrying certain structural aberrations involving chromosome 2 (del2ter, dup2ter, del2cen, dup2cen), disomy 2, disomy 8, and sperm diploidy. Analysis of approximately 80,000 sperm from eight B6C3F1 mice revealed an average baseline frequency of 2.5 per 10,000 sperm carrying partial duplications and deletions of chromosome 2. Extrapolated to the entire haploid genome, approximately 0.4% of mouse sperm are estimated to carry structural chromosomal aberrations, which is more than fivefold lower than the spontaneous frequencies of sperm with chromosome structural aberrations in man. We validated the CT8 assay by comparing the frequencies of abnormal segregants in sperm of T(2;14) translocation carriers detected by this assay against those detected by chromosome painting cytogenetic analysis of meiosis II spermatocytes. The CT8 sperm FISH assay is a promising method for detecting structural chromosome aberrations in mouse sperm with widespread applications in genetics, physiology, and genetic toxicology.     

Sperm allocation in the three-spined stickleback

Male three-spined stickleback Gasterosteus aculeatus have a fixed amount of sperm during the breeding season because spermatogenesis is inhibited at this time. A method was developed to estimate ejaculate size in situ by removing the sperm from the male's nest. The reliability of the method was tested using known numbers of sperm. In their first mating, males ejaculated 11·64 × 10⁶ sperm (median), representing c. 5% of the male's sperm store (median 27·88 × 10⁷ sperm). The amount of sperm in the testes was significantly reduced in males that had mated several times (median 8·09 × 10⁷). Additionally, ejaculate size was smaller in these experienced males (median 8·79 × 10⁵). Heavier and larger fish invested absolutely and relatively more sperm in a mating than did lighter and smaller fish. Ejaculate size did not correlate with the mass of the egg clutch.     

Rapid and simultaneous detection of chromosome Y- and 1-bearing porcine spermatozoa by fluorescence in situ hybridization

This study was carried out to develop a rapid and simultaneous detection system of chromosome Y- and 1-bearing porcine spermatozoa by fluorescence in situ hybridization (FISH). Chromosome Y- and 1-specific DNA probes were produced by polymerase chain reaction with digoxigenin (Dig)- or biotin-dUTP. The hybridization probe mixture of labeled Y-chromosome and chromosome 1-specific DNA was applied to the preparation, immediately denatured at 75°C for 8 min, hybridized for 5 min at 37°C and overall FISH steps were done within a few hours. When double FISH with Dig-labeled chromosome Y-specific and biotin-labeled chromosome 1-specific probes was applied to sperm nuclei pretreated with dithiothreitol, the average of 50.9% of sperm nuclei had the Dig-signal, 99.2% of the sperm nuclei had the biotin-signal and the average of 0.3% of sperm nuclei showed no signal. The putative rate of Y-bearing spermatozoa ranged from 49.8% to 52.8% among 5 boars and the average putative rate of Y-bearing spermatozoa was 51.0%. The results indicated that a rapid and simultaneous FISH with chromosome Y- and 1-specific porcine DNA probes produced by PCR made possible more accurate assessment of Y-bearing porcine spermatozoa. © 1996 Wiley-Liss, Inc.