A high degree of aneuploidy in frozen-thawed human preimplantation embryos

We have studied the chromosomal content in 68 normally fertilised freeze-thawed human embryos of good morphology from 34 patients with an average maternal age of 32,6 years. Forty embryos showed post-thaw cellular division and twenty-eight post-thaw cleavage arrest. After spreading of the embryos on microscope slides, analysis of chromosomes X, Y, 15, 16, 17 and 18 was performed using two rounds of fluorescent in situ hybridisation (FISH). According to the results, the embryos were divided into four groups: (I) normal, all nuclei uniformly diploid, (II) diploid mosaics, normal diploid blastomeres in combination with abnormal blastomeres, (III) abnormal, all nuclei abnormal, (IV) chaotic, the chromosome constitution varies randomly from cell to cell. Approximately 25% of the embryos had normal number of the chromosomes tested, while the majority of the embryos were abnormal. Most of the abnormal embryos were diploid mosaics (57%). This was true for the embryos showing cleavage division as well as the embryos showing cleavage arrest. Our data show a slightly higher incidence of abnormal embryos compared to those obtained with FISH in non-cryopreserved embryos and confirm that the majority of preimplantation embryos fertilised in vitro contain abnormal blastomeres. The results, mechanisms, significance and implications are discussed. Received: 19 November 1998 / Accepted: 4 March 1999     

Reliability of aneuploidy estimates in human sperm: Results of fluorescence in situ hybridization studies using two different scoring criteria

Aneuploidy estimates for chromosomes 1, 12, X, and Y were obtained in human sperm from five donors using multicolor fluorescence in situ hybridization (FISH) analysis. Disomy frequencies were obtained by scoring a minimum of 10,000 sperm for each chromosome probe per donor. This analysis was replicated for two scoring criteria: one used one half of a signal domain as the minimum distance between two signals to be counted as two and thus disomic; the other set one signal domain as the minimum distance between two signals. A total of 120,870 sperm were assessed using one half of a domain as the criterion, and 113,478 were scored using one domain as the criterion. The percentage of disomy for chromosomes 1, 12, X, Y, and XY was 0.18, 0.16, 0.15, 0.19, and 0.25, respectively, using the one-half-domain criterion, and 0.08, 0.17, 0.07, 0.12, and 0.16, respectively, using the one-domain criterion. The percentage of disomy decreased significantly with use of one domain as the minimum distance for signal separation for all chromosomes except for chromosome number 12. These lower disomy frequencies correlated well with frequencies derived from human sperm karyotypes analyzed in our laboratory. This suggests that the fluorescent signals for chromosomes 1, X, and Y split into more than one domain in decondensed interphase sperm, and that the use of the one-half-domain criterion would lead to an overestimate of aneuploidy frequencies. The factors known to affect aneuploidy estimates derived from FISH studies are discussed, and recommendations for stringent scoring criteria are proposed. © 1995 wiley-Liss, Inc.     

Aneuploidy in late-step spermatids of mice detected by two-chromosome fluorescence in situ hybridization

A multicolor procedure employing fluorescence in situ hybridization is described for detecting chromosomal domains and germinal aneuploidy in late-step spermatids in mice using DNA probes specific for repetitive sequences near the centromeres of chromosomes 8 and X. These probes were nick-translated with biotin- or digoxigenin-labeled nucleotides, and were detected with FITC or rhodamine. Probe and hybridization specificities were confirmed using metaphase chromosomes from spleen and bone marrow cells as well as from primary and secondary spermatocytes. Late-step spermatids, identified in testicular preparations by their hooked shape, yielded compact fluorescence domains in ～ 50% and > 99% of cells when hybridized with probes for chromosomes X and 8, respectively. In a survey of > 80,000 late-step spermatids from 8 healthy young adult C57BL/6 or B6C3F1 mice, ～ 3/10,000 spermatids had fluorescence phenotypes indicative of X-X or 8–8 hyperhaploidy. These frequencies are consistent with published frequencies of aneuploidy in meiotic metaphase II and first cleavage metaphases of the mouse, providing preliminary validation of sperm hybridization for the detection of aneuploidy. No significant animal or strain differences were observed. In addition, the hyperhaploidy frequencies for murine spermatids were indistinguishable for those for sperm from healthy men obtained by a similar hybridization procedure. These procedures for detecting aneuploid male gametes are examples of “bridging biomarkers” between human and animal studies. They have promising applications for investigations of the genetic, reproductive, and toxicological factors leading to abnormal reproductive outcomes of paternal origin. © 1995 Wiley-Liss, Inc.     

Effects of trehalose vitrification and artificial oocyte activation on the development competence of human immature oocytes

Sucrose and trehalose are conventional cryoprotectant additives for oocytes and embryos. Ethanol can artificially enhance activation of inseminated mature oocytes. This study aims to investigate whether artificial oocyte activation (AOA) with ethanol can promote the development competence of in vitro matured oocytes. A total of 810 human immature oocytes, obtained from 325 patients undergoing normal stimulated oocyte retrieval cycles, were in vitro maturated (IVM) either immediately after collection (Fresh group n = 291)) or after being vitrified as immature oocytes (Vitrified group n = 519). These groups were arbitrarily assigned. All fresh and vitrified oocytes which matured after a period of IVM then underwent intra-cytoplasmic sperm injection (ICSI). Half an hour following ICSI, they were either activated by 7% ethanol (AOA group) or left untreated (Non-AOA group). Fertilization, cleavage rate, blastocyst quality and aneuploidy rate were then evaluated. High-quality blastocysts were only obtained in both the fresh and vitrified groups which had undergone AOA after ICSI. Trehalose vitrification slightly, but not significantly, increased the formation rates of high-quality embryos (21.7% VS 15.4%, P > 0.05) and blastocysts (15.7% VS 7.69%, P > 0.05)) when compared with sucrose vitrification. Aneuploidy was observed in 12 of 24 (50%) of the AOA derived high quality blastocysts. High-quality blastocysts only developed from fresh or vitrified immature oocytes if the ICSI was followed by AOA. This information may be important for human immature oocytes commonly retrieved in normal stimulation cycles and may be particularly important for certain patient groups, such as cancer patients. AOA with an appropriate concentration of ethanol can enhance the developmental competence of embryos.     

Molecular cytogenetic characterisation and phylogenetic analysis of the seven cultivated Vigna species (Fabaceae)

The genomic organisation of the seven cultivated Vigna species, V. unguiculata, V. subterranea, V. angularis, V. umbellata, V. radiata, V. mungo and V. aconitifolia, was determined using sequential combined PI and DAPI (CPD) staining and dual‐colour fluorescence in situ hybridisation (FISH) with 5S and 45S rDNA probes. For phylogenetic analyses, comparative genomic in situ hybridisation (cGISH) onto somatic chromosomes and sequence analysis of the internal transcribed spacer (ITS) of 45S rDNA were used. Quantitative karyotypes were established using chromosome measurements, fluorochrome bands and rDNA FISH signals. All species had symmetrical karyotypes composed of only metacentric or metacentric and submetacentric chromosomes. Distinct heterochromatin differentiation was revealed by CPD staining and DAPI counterstaining after FISH. The rDNA sites among all species differed in their number, location and size. cGISH of V. umbellata genomic DNA to the chromosomes of all species produced strong signals in all centromeric regions of V. umbellata and V. angularis, weak signals in all pericentromeric regions of V. aconitifolia, and CPD‐banded proximal regions of V. mungo var. mungo. Molecular phylogenetic trees showed that V. angularis and V. umbellata were the closest relatives, and V. mungo and V. aconitifolia were relatively closely related; these species formed a group that was separated from another group comprising V. radiata, V. unguiculata ssp. sesquipedalis and V. subterranea. This result was consistent with the phylogenetic relationships inferred from the heterochromatin and cGISH patterns; thus, fluorochrome banding and cGISH are efficient tools for the phylogenetic analysis of Vigna species.     

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．     

Indeterminate meiotic restitution (IMR): a novel type of meiotic nuclear restitution mechanism detected in interspecific lily hybrids by GISH

A detailed analysis of microsporogenesis was carried out in three diploid lily cultivars (2n=2x=24) and three diploid interspecific hybrids (2n=2x=24) using DNA in situ hybridisation methods (GISH and FISH). In cvs. Gelria (Lilium longiflorum; L genome), Connecticut King and Mont Blanc (both Asiatic hybrids; Agenome) meiosis was regular and only haploid gametes were formed while the three interspecific hybrids between L. longiflorum×Asiatic hybrid (LA) showed a variable frequency of meiotic nuclear restitution and stainable 2n-pollen formation ranging from 3% to 30%. An analysis of meiotic chromosome behaviour of the LA hybrids through GISH and FISH revealed that: (1) the parental chromosomes could be clearly discriminated into univalents, half-bivalents and bivalents in the PMCs; (2) in some of the PMCs the entire complement was present either as univalents or half-bivalents which had the potential to divide equationally (following centromere division) during the first division leading to first division restitution (FDR) gametes; (3) more frequently, however, in one and the same PMC the univalents and half-bivalents divided equationally whereas the bivalents disjoined reductionally at the same time giving rise to 2n-gametes that could vary from the well-known FDR or SDR 2n-gametes. We indicate this novel type of restitution mechanism as Indeterminate Meiotic Restitution (IMR). In order to confirm the occurrence of IMR gametes, the chromosome constitutions of eight triploid BC₁ progenies derived from backcrossing the 2n-gamete producing the LAhybrids to the Asiatic hybrid parents were analysed through in situ hybridisation. The results indicated that there were seven BC₁ plants in which FDR 2n-gametes, with or without homoeologous recombinations, were functional, whereas in one case the 2n-gamete resulting from IMR was functional. In the latter, there was evidence for the occurrence of genetic recombination through homoeologous crossing-over as well as through the assortment of homoeologous chromosomes. A singular feature of the IMR 2n-gamete was that although it transmitted a euploid number of 24 chromosomes to the BC₁ progeny, the number of chromosomes transmitted from the two parental species was dissimilar: 9 L-genome chromosomes and 15 A-genome chromosomes instead of 12 of each. Received: 15 May 2000 / Accepted: 4 December 2000     

Interphase fluorescence in situ hybridization mapping: a physical mapping strategy for plant species with large complex genomes

The chromatin in interphase nuclei is much less condensed than are metaphase chromosomes, making the resolving power of fluorescence in situ hybridization (FISH) two orders of magnitude higher in interphase nuclei than on metaphase chromosomes. In mammalian species it has been demonstrated that within a certain range the interphase distance between two FISH sites can be used to estimate the linear DNA distance between the two probes. The intephase mapping strategy has never been applied in plant species, mainly because of the low sensitivity of the FISH technique on plant chromosomes. Using a CCD (charge-coupled device) camera system, we demonstrate that DNA probes in the 4 to 8 kb range can be detected on both metaphase and interphase chromosomes in maize. DNA probes pA1-Lc and pSh2.5·SstISalI, which contain the maize locia1 andsh2, respectively, and are separated by 140 kb, completely overlapped on metaphase chromosomes. However, when the two probes were mapped in interphase nuclei, the FISH signals were well separated from each other in 86% of the FISH sites analyzed. The average interphase distance between the two probes was 0.50 µm. This result suggests that the resolving power of interphase FISH mapping in plant species can be as little as 100 kb. We also mapped the interphase locations of another pair of probes, ksu3/4 and ksu16, which span theRp1 complex controlling rust resistance of maize. Probes ksu3/4 and ksu16 were mapped genetically approximately 4 cM apart and their FISH signals were also overlapped on metaphase chromosomes. These two probes were separated by an average of 2.32 µm in interphase nuclei. The possibility of estimating the linear DNA distance between ksu3/4 and ksu16 is discussed.     

Ribosomal DNAs: an exception to the conservation of gene order in rice genomes

rDNA (18S-5.8S-25S rDNA) and 5S rDNA loci were visualized on the chromosomes of six species of the genus Oryza by fluorescence in situ hybridization (FISH) and the labeled rice chromosomes were identified based on their condensation patterns. As a result, the chromosomes harboring rDNA and/or 5S rDNA loci were determined in the complement for all the known rice genomes. Variation in the location of the rDNA loci indicated the transpositional nature of the rDNAs in the genus Oryza, as also suggested in Triticeae and Allium. Comparative analysis of the locations of rDNA loci among rice, maize and wheat revealed that variability in the physical location of the rDNA loci was characteristic of the genus Oryza and also of the genera of Gramineae. This variability in the location of the rDNA loci between evolutionarily related species is in sharp contrast to the conservation of the general order of genes in their genomes.     

Chromosome rearrangements in Pectinidae (Bivalvia: Pteriomorphia) implied based on chromosomal localization of histone H3 gene in four scallops

Chromosomal structural rearrangement in four scallops, Chlamys farreri (n = 19), Patinopecten yessoensis (n = 19), Chlamys nobilis (n = 16) and Argopecten irradians (n = 16), was studied by fluorescence in situ hybridization using histone H3 gene probes. The results show that histone H3 gene sites differ strikingly with regard to number, location, and intensity among, or even within these species. For example, two histone H3 gene loci were detected on the metaphase chromosomes of P. yessoensis, while one locus was found in the others. In P. yessoensis, differing intensities of hybridization signals were detected between homologues 5 and 11, and within homologue 11. These data suggest that the histone H3 gene is a qualified chromosome marker for the preliminary understanding of the historical chromosomal reconstructing of the Pectinidae family. The variable distribution patterns of the histone H3 gene suggest that gene duplication/diminution as well as chromosome rearrangements by inversion and translocation may have played important roles in the genomic evolution of Pectinidae. We also compiled our present results with former published data regarding the chromosome mapping of rDNAs in species of the Pectinidae family. Such comparative chromosomal mapping should improve our understanding of historical chromosomal reconstructions of modern-day scallops.     

Structural homology in the Solanaceae: analysis of genomic regions in support of synteny studies in tomato, potato and pepper

We have analysed the structural homology in euchromatin regions of tomato, potato and pepper with special attention for the long arm of chromosome 2 (2L). Molecular organization and colinear junctions were delineated using multi-color BAC FISH analysis and comparative sequence alignment. We found large-scale rearrangements including inversions and segmental translocations that were not reported in previous comparative studies. Some of the structural rearrangements are specific for the tomato clade, and differentiate tomato from potato, pepper and other Solanaceous species. Although local gene vicinity is largely preserved, there are many small-scale synteny perturbations. Gene adjacency in the aligned segments was frequently disrupted for 47% of the ortholog pairs as a result of gene and LTR retrotransposon insertions, and occasionally by single gene inversions and translocations. Our data also suggests that long distance intra-chromosomal rearrangements and local gene rearrangements have evolved frequently during speciation in the Solanum genus, and that small changes are more prevalent than large-scale differences. The occurrence of sonata and harbinger transposable elements and other repeats near or at junction breaks is considered in the light of repeat-mediated rearrangements and a reconstruction scenario for an ancestral 2L topology is discussed.     

LNA flow–FISH: A flow cytometry–fluorescence in situ hybridization method to detect messenger RNA using locked nucleic acid probes

We present a novel method using flow cytometry–fluorescence in situ hybridization (flow–FISH) to detect specific messenger RNA (mRNA) in suspended cells using locked nucleic acid (LNA)-modified oligonucleotide probes. β-Actin mRNA was targeted in whole A549 epithelial cells by hybridization with a biotinylated, LNA-modified probe. The LNA bound to β-actin was then stained using phycoerythrin-conjugated streptavidin and detected by flow cytometry. Shifts in fluorescence signal intensity between the β-actin LNA probe and a biotinylated, nonspecific control LNA were used to determine optimal conditions for this type of flow–FISH. Multiple conditions for permeabilization and hybridization were tested, and it was found that conditions using 3 μg/ml of proteinase K for permeabilization and 90 min hybridization at 60 °C with buffer containing 50% formamide allow cells containing the LNA-bound mRNA to be detected and differentiated from the control LNA with high confidence (< 14% overlap between curves). This combined method, called LNA flow–FISH, can be used for detection and quantification of other RNA species as well as for telomerase measurement and detection.     

Detection of Pseudo-nitzschia (Bacillariophyceae) species and amnesic shellfish toxins in Scottish coastal waters using oligonucleotide probes and the Jellet Rapid Test™

Species specific LSU rRNA targeted fluorescent oligonucleotide probes, designed by researchers at the Monterey Bay Aquarium Research Institute (USA) for a limited range of Pseudo-nitzschia species, were applied to unialgal cultures and Scottish field samples, to investigate possible applications in Scottish phytoplankton monitoring programmes to detect potential amnesic shellfish poisoning (ASP) toxin producing species. The existing available probe for Pseudo-nitzschia australis gave good results, positively labelling cells from cultures and field samples. However, application of the P. pungens, P. delicatissima and P. fraudulenta probes gave poor results, with little or no fluorescence label observed in field samples, while transmission electron microscopy (TEM) showed these species to be present. Comparison of the same region of the LSU sequence from cultures of P. delicatissima, isolated from Scottish waters, with the probe designed for detection of P. delicatissima isolated from Monterey Bay revealed the presence of a single base difference between the two sequences, which may have prevented the probe from hybridising to Scottish isolates and cells from field samples. In an attempt to assess the potential ASP toxin production by field populations of Pseudo-nitzschia a rapid immunodiagnostic test (the Jellet Rapid Test, JRT) for ASP toxins was examined. Results indicate that additional development of molecular probes for the detection of a range of Pseudo-nitzschia species detected in Scottish coastal waters and the use of JRT for toxin detection could conceivably provide an effective tool for broad-scale mapping of toxin events and management of coastal zone activities.     

Heterometallic coordination compounds of dipicolinic acid with Ce(III, IV) and Cu(II): Synthesis, crystal structure and spectral studies

Reaction of pyridine-2,6-dicarboxylic acid (dipicH₂) with ammonium ceric nitrate and Cu(II) salts yielded three heterometallic compounds all of which contain [Ce(dipic)₃]²⁻ linked to aquo-Cu(II) complex units. Part of the Ce(IV) gets reduced by solvent during the reaction leading to [(Ce(dipic)₃Ce(H₂O)₈)₂Cu(H₂O)₄][Ce(dipic)₃]₂·12H₂O (1). Other lanthanide(III) ions could take the place of Ce(III) as demonstrated by the preparation of [(Ce(dipic)₃La(H₂O)₈)₂Cu(H₂O)₄][Ce(dipic)₃]₂·12H₂O (4), which is isomorphous with compound 1. [Ce(dipic)₃Cu(H₂O)₄]·8H₂O (2) is a one-dimensional coordination polymer in which two types of aquo-Cu(II) complex units which differ in the orientation of the tetragonal axis alternate along the chain. The central Cu(H₂O)₂²⁺ unit in the trinuclear anion of [Cu(H₂O)₆][Ce(dipic)₃Cu(H₂O)₂Ce(dipic)₃]·8H₂O (3) is chelated by two carboxylate groups in trans positions in off-axis mode. In all the four complexes, the Cu(II) centres are magnetically isolated leading well-resolved EPR spectra in polycrystalline samples.     

中间偃麦草(Thinopyrum intermedium)18S-5.8S-26S rDNA位点在染色体上的分布及对其核ITS序列异质性的分析(英文)

中间偃麦草(Thinopyrum intermedium(Host)Barkworth et Dewey)是禾本科小麦族植物中的一个异源六倍体物种,是重要的牧草植物,在小麦的抗病育种中发挥了重要作用。利用荧光原位杂交(FISH)技术,在体细胞中期染色体上,对18S-5.8S-26S rDNA位点进行了物理定位,发现该物种有3～4对染色体携带18S-5.8S-26S rDNA主位点。结合基因组原位杂交(GISH)分析,证明中间偃麦草的St基因组中有一对同源染色体短臂末端携带一个主位点,其余2～3对主位点位于E基因组染色体上。对不同来源的材料研究表明:18S-5.8S-26S rDNA位点的数目(包括主位点和小位点)、位置、拷贝数在不同收集材料之间的差异较大,甚至在同一个体的不同细胞中也存在差异。讨论了rDNA物理作图数据在分析系统发育问题中的局限性。结合中间偃麦草的三个可能的二倍体基因组供体(Th.bessarabicum、Th. elongatum和Pseudoroegneria stipifolia)rDNA位点分析的结果,对中间偃麦草进化过程中rDNA位点的变化进行了分析,同时,对其中一份材料的核ITS序列进行了克隆、测序和系统发育分析,发现在中间偃麦草中,ITS序列具有很高的异质性。     

植物45S rDNA的染色体位置的CPD染色和FISH分析

采用PI和DAPI组合(CPD)染色结合45SrDNA探针的荧光原位杂交(FISH)对分属6个科的16种植物的45S rDNA的染色体位置进行了分析。在所有供试植物中,共检测到53个45S rDNA位点。大多数45S rDNA位点分布在染色体的短臂;位于染色体臂内和染色体末端的位点的比例大体相当;多数位于染色体臂内的45S rDNA位点有次缢痕形成,但rDNA重复单位簇所处的位置存在差异。根据45S rDNA所处的染色体臂的不同、距着丝粒远近的差异、形成次缢痕与否以及rDNA重复单位簇相对于次缢痕的位置等特征,将植物的45S rDNA位点划分为12种染色体分布类型。基于我们的结果和其他的报道对45S rDNA位点、核仁组织区(NOR)、次缢痕和随体相互之间的关系进行了分析。     

用FISH技术研究人类体外未受精卵的21号染色体非整倍体

采用荧光原位杂交技术,选用人类21号染色体端粒探针（21qter）,检测人类体外未受精卵的21号染色体非整倍体发生率,并比较非整倍体率与25－30岁和31－35岁这两个女性年龄组、IVF指征、超排方案之间的关系,在54个未受精卵中,正常21号单体30枚,二体16枚,三体4枚,缺体4枚,非整倍体率为44.4％（24／54）;25－30岁和31－35岁这两个年龄组、IVF指征、超排方案的患者的21号染色体非整倍体率之间的差异无显著性,卵母细胞21号染色体的非整倍性是造成体外受精失败的重要原因之一。     

Active site-specifically reconstituted nickel(II) horse liver alcohol dehydrogenase: Optical spectra of binary and ternary complexes with coenzymes,coenzyme analogues,substrates, and inhibitors

Insertion of nickel ions into the empty catalytic site of horse liver alcohol dehydrogenase yields an active enzyme with 65% metal substitution and about 12% intrinsic activity. The electronic absorption spectrum is characterized by bands at 357 nm (2900 M^?1 cm^?1, 407 nm (3500 M^?1 cm^?1), 505 nm (300 M^?1 cm^?1), 570 nm (?130 M^?1 cm^?1), and 680 nm (?80 M^?1 cm^?1). The absorption and CD spectra are similar to those of nickel(II) azurin and nickel(II) aspartate transcarbamoylase and prove coordination of the nickel(II) ions to sulfur in a distorted tetrahedral coordination geometry. Changes of the spectra upon ligand binding at the metal or conformation changes of the protein induced by coenzyme, or both, indicate alterations of the metal geometry.The chromophoric substrate trans-4-(N, N-dimethylamino)-cinnamaldehyde forms a ternary complex with Ni(II) liver alcohol dehydrogenase and the coenzyme analogue 1,4,5,6-tetrahydronicotinamide-adenine-dinucleotide, stable between pH 6 and 10. The corresponding ternary complex with NADH is only stable at pH > 9.0. The spectral redshifts induced in the substrate are 11 nm larger than those found in the zinc enzyme. We suggest direct coordination of the substrate to the catalytic metal ion which acts as a Lewis acid in both substrate coordination and catalysis.