An Improved Method for Chromosome Preparations from Preimplantation Mammalian Embryos, Oocytes or Isolated Blastomeres

A method is described for making chromosome preparations from mammalian oocytes or preimplantation embryos, with or without the zona pellucida, or from isolated blastomeres. It is more robust and requires less skill and experience than previous techniques, yet chromosome structure is well preserved and very high quality preparations can be made. The method, which involves use of cold hypotonic solution and very cold fixative, reduces turbulence and allows even single blastomeres to be located and handled with relative ease, while the duration of hypotonic treatment becomes noncritical. The softening solution recommended contains no lactic acid and hence does not harm the chromosomes.     

大鳞副泥鳅粗线期二价染色体分带研究

报道了一种鱼类二价体显带的新,采用ＰＨ值在７．２－７．４之间的低涌液对大鳞副泥鳅精巢进行整体长低渗、卡诺固定液处理方法得到粗线期二阶染色体的高分辨Ｇ带,其带纹特征和数目相对稳定且清晰可辨。与胰酶显和ＥＤＴＡ显相比具有操作简单,耗时短以及二价体分散相对较好等特点,这一方法在所有显带方法中最为简便。     

Formation of the enveloping layer of the chum salmon egg in isotonic salt solutions

After stimulation in a hypotonic solution (9.4 mOsm kg⁻¹), inseminated eggs of the chum salmon Oncorhynchus keta initiate cleavages in isotonic salmon Ringer's solution (267.3 mOsm kg⁻¹) containing 3.2 mM Ca²⁺ ions. Blastomeres of these eggs, however, separate from each other and the enveloping layer is not observed at the blastula stage. An increase in external divalent cations rescues the separation; the concentration of CaCl₂ in the external medium should be 25 mM or more to induce close contact of blastomeres and the formation of an enveloping layer in isotonic salt solutions. The effectiveness of Ca²⁺ ions can be substituted by Mg²⁺, Sr²⁺ and Zn²⁺ ions; the same results are obtained in isotonic MgCl₂ and SrCl₂ solutions (100 mM) or in isotonic salmon Ringer's solution containing Zn ions (6.2 mM). The close contact of blastomeres and the formation of an enveloping layer are also observed in a low Ca²⁺ concentration (< 0.1 mM) in a hypotonic salt solution (9.4 mOsm kg⁻¹). The Ca²⁺ level in the external medium to induce the enveloping layer formation seems to be correlated with the salinity of the incubation medium. It is suggested that adhesion molecules on the surface of blastomeres in the chum salmon eggs are different in properties from those found in sea urchin and other fish species.     

一种野外制备染色体的新方法-骨髓整体低渗法

本文以整体低渗的方法进行哺乳与鸟类的染色体制备,以探讨一种在野外条件下,无法进行离心时,适合细胞遗传学研究的染色体制备新方法。应用该方法可在野外考察中,同时开展细胞遗传学方面的工作在某些特殊研究课题中,该方法有着广阔的应用前景。     

Chromosome analysis of blastomeres from human embryos by using comparative genomic hybridization

Karyotypic studies of aborted fetuses have been used to draw the inference that the proportion of conceptuses with chromosome abnormalities is very high. Fluorescent in situ hybridization (FISH) studies of blastomeres from early cleavage embryos have provided some support for this inference but they are limited to the study of a few chromosomes. We describe the novel application of comparative genomic hybridization (CGH) to the study of numerical and structural abnormalities of single blastomeres from disaggregated 3-day-old human embryos. CGH results were obtained for 63 blastomeres from 12 embryos. Identification of all chromosomes with the exception of chromosomes 17, 19, 20 and 22 was possible. The embryos divided into four groups: (1) embryos with a normal CGH karyotype seen in all blastomeres; (2) embryos with consistent aneuploidy suggesting meiotic non-disjunction had occurred; (3) embryos that were mosaic generally with one or more cells showing aneuploidy for one or two chromosomes but some with cells showing extensive aneuploidy; and (4) one embryo with extensive aneuploidy in all blastomeres. The extensive aneuploidy in group 4 is interpreted as corresponding to the random aneuploidy seen in "chaotic" embryos reported by using interphase FISH. Partial chromosome loss and gain following chromosome breakage was observed in one embryo. Our analysis provides basic biological information on the occurrence of constitutional and post-zygotic chromosome abnormalities in early human embryos. Used in conjunction with embryo biopsy, diagnostic CGH should allow the exclusion of a proportion of embryos that appear normal but that have a poor probability of survival and, therefore, may improve the implantation rate after in vitro fertilization.     

An Air-Drying Technique for Flattening Chromosomes in Mammalian Cells Grown In Vitro

The method described was developed to facilitate the analysis of chromosome complements in cells freshly isolated from monkey kidney cortex and grown on glass, and in “altered” monkey cells grown on glass or in suspension. Cells were treated with hypotonic solution (quarter-strength Tyrode or diluted medium) for 30 min, or with colchicine in a final concentration of 25 μg/ml (.0025%) for 12-18 hr followed by hypotonic salt solution for 5 min, then fixed in acetic alcohol (1:3) for 5 min. With cells centrifuged from suspended cultures, addition of fixative had to be gradual. Directly after fixation, films of cells on slides were air dried completely. This produces a more uniform and complete flattening of cells than can be achieved by manual pressure; yet, fragmentation of chromosome complements does not occur. Fixed and air dried slides may be stored for days without deterioration or they may be stained immediately in 2% natural orcein (G. T. Gurr, London) in 50% acetic acid. Preparations can be made permanent by a dry ice schedule, without loss, shrinkage, or distortion of cells.     

Chromosome preparation from human bone marrow--technique modified for immediate clinical investigation.

A modified method for chromosome preparation from bone marrow aspirates of normal individuals and leukaemic patients is described for immediate clinical investigation. Hanks' balanced salt solution at different concentrations was used for in vitro incubation and hypotonic treatment. The method yields quite a high mitotic index with good metaphase spreads.     

Male chromosomes of sea urchin hybrid andromerogones created with cryopreserved sperm

We developed a method for preparing male chromosomes from sea urchin hybrid andromerogones created with cryopreserved sperm. We obtained hybrid andromerogones by heterospermic insemination of Hemicentrotus pulcherrimus non-nucleate egg fragments produced by centrifuging unfertilized eggs in a stepwise saccharose density gradient. The hybrid andromerogones showed cleavage rates of 1%-93%, cleaved successively into two- and four- blastomeres and developed to early blastulae. The morulae or early blastulae were treated with colchicine (0.1-1.0 mg/ml), dissociated into single blastomeres by pippeting, swollen with 7%-10% sodium citrate for 10 min and fixed with methanol:acetic acid (3:1). The fixed cells were dropped on slides and air-dried. The andromerogones for 5 sperm species showed a half of their respective diploid chromosome numbers without chromosome elimination. This method is applicable for analysis of the haploid male chromosome complement in sea urchin species for which only sperm can be obtained.     

Optimization of osmotic shock process variables for enhancement of the release of periplasmic interferon-α2b from Escherichia coli using response surface method

The osmotic shock process for the release of periplasmic recombinant human interferon-α2b from Escherichia coli was optimized using response surface method (RSM). The process parameters such as pH, buffer concentration and sucrose concentration in hypertonic solution, cell concentration to hypertonic solution, contact time of cells with hypertonic solution, temperature of hypertonic solution, cell concentration to hypotonic solution, contact time of cells with hypotonic solution and temperature of hypotonic solution were initially screened using Plackett Burman design. Further optimization was carried out using central composite design (one of the design in RSM) for sucrose concentration in hypertonic solution as well as cell concentration to hypertonic and hypotonic solutions. The optimal cell concentration was 0.05 g/mL in hypertonic solution and 0.2 g/mL in hypotonic solution. The use of hypertonic solution containing 18% sucrose with a combination of 100 mM Tris and 2.5 mM EDTA buffer (pH 8.0 and 25 °C) and cold water (4 °C) as a hypotonic solution gave the optimum release of interferon-α2b. Increased product concentration in the final solution resulted from the optimized process would reduce the downstream steps during purification. The concept of reuse of hypertonic solution was also demonstrated.     

Mesoderm formation by isolated and cultivated 8-cell stage blastomeres of the teleost, Leucopsarion ptersii (shiro-uo).

Isolation of cleavage-stage blastomeres and the study of their developmental potential has been used extensively for analyzing the mechanisms of embryogenesis in vertebrates, including amphibians and echinoderms. We devised a method to isolate 8-cell stage blastomeres in the teleost, shiro-uo, by utilizing its unique cleavage pattern of the horizontal 3rd cleavage plane. Removal of all the upper blastomeres at the 8-cell stage allowed almost normal embryogenesis from the remaining lower blastomeres and yolk cell mass. Isolated upper or lower blastomeres formed vesicles and spherical bodies, which later showed morphological changes during cultivation. Mesoderm formation was detected not only in the cultivated lower blastomeres or whole blastomeres but also in the upper blastomeres isolated from the yolk cell mass at the 8-cell stage, although at a lower frequency than the lower blastomeres. These results indicated the presence of very early signaling for mesoderm induction, which is independent from the currently postulated signals from the yolk syncytial layer at later stages. This also indicated non-equivalence or differentiation of the blastomeres from the very early cleavage stage in teleost embryos.     

小鼠单卵裂球体外培养及染色体制备

为了探索研究了植入前胚胎染色体病（包括平衡易位）诊断的可行性方法,作者进行了单卵裂球体外培养、染色体制备及显带技术研究,在Ｂ２培养基加血清进行培养的基础上,比较了在两种没处理因素进行体外培养时小鼠单卵裂球增殖情况,Ｂ２培养基加血清再加输卵管包埋进行培养,其单卵裂球体外增殖率为５０％（８７／１７４）,单卵裂球染色体制备成功率为２７．６％;Ｂ２培养基加腹水过滤液进行培养,其单卵裂球体外增殖率为３３％（     

Intravital selection of early mouse embryos by sex and karyotypic characteristics

The possibility of live karyotyping by a single blastomere isolated at the 2-, 4-, and 8-cell stage has been investigated. It is expedient to use to this end a single blastomere isolated from a 4-cell embryo. Three rest blastomeres formed normal morulae or blastocysts upon cultivation during 44 or 68 hrs. When the isolated blastomeres were cultivated for 14-16 hrs in a medium 0.5 micrograms/ml colcemide, 97% of blastomeres were at the metaphase stage and 72% of chromosome plates were suitable for karyotyping. The prospects of the method proposed in experimental embryology and for selection of the early embryos of farm animals by sex are discussed.     

Identification of the facultative heterochromatic X chromosome in females of 25 rodent species.

Treatment of the chromosomes of 25 rodent species with a 50 degrees C hypotonic solution and Giemsa staining permitted identification of the heterochromatic X chromosome in 24 species. With this technique, the facultative of the heterochromatic X chromosome or the facultative portion of large, composite-type X chromosoms is stained darker than the other chromosomes, allowing it to be distinguished from the homologous euchromatic X chromosome in female metaphase cells. Intense staining of the single X chromosome was not observed in male metaphase cells. It is suggested that this differential staining of one of the two X chromosomes might be due to qualitative differences in chromosomal proteins rather than to differences in the degree of chromosomal condensation or in DNA base sequence.     

Chromosome breakage in human preimplantation embryos from carriers of structural chromosomal abnormalities in relation to fragile sites, maternal age, and poor sperm factors

Chromosome breakage is a fairly widespread phenomenon in preimplantation embryos affecting at least 10% of day 3 cleavage stage embryos. It may be detected during preimplantation genetic diagnosis (PGD). For carriers of structural chromosomal abnormalities, PGD involves the removal and testing of single blastomeres from cleavage stage embryos, aiming towards an unaffected pregnancy. Twenty-two such couples were referred for PGD, and biopsied blastomeres on day 3 and untransferred embryos (day 5/6) were tested using fluorescence in situ hybridisation (FISH) with appropriate probes. This study investigated whether chromosome breakage (a) was detected more frequently in cases where the breakpoint of the aberration was in the same chromosomal band as a fragile site and (b) was influenced by maternal age, sperm parameters, reproductive history, or the sex of the carrier parent. The frequency of breakage seemed to be independent of fragile sites, maternal age, reproductive history, and sex of the carrier parent. However, chromosome breakage was very significantly higher in embryos from male carriers with poor sperm parameters versus embryos from male carriers with normal sperm parameters. Consequently, embryos from certain couples were more prone to chromosome breakage, fragment loss, and hence chromosomally unbalanced embryos, independently of meiotic segregation.     

Chromosome abnormalities in human arrested preimplantation embryos: a multiple-probe FISH study.

Numerical chromosome abnormalities were studied in single blastomeres from arrested or otherwise morphologically abnormal human preimplantation embryos. A 6-h FISH procedure with fluorochrome-labeled DNA probes was developed to determine numerical abnormalities of chromosomes X, Y, and 18. The three chromosomes were stained and detected simultaneously in 571 blastomeres from 131 embryos. Successful analysis including biopsy, fixation, and FISH analysis was achieved in 86.5% of all blastomeres. The procedure described here offers a reliable alternative to sexing of embryos by PCR and allows simultaneous ploidy assessment. For the three chromosomes tested, numerical aberrations were found in 56.5% of the embryos. Most abnormal embryos were polyploid or mosaics, and 6.1% were aneuploid for gonosomes or chromosome 18. Extrapolation of these results to all human chromosomes suggests that the majority of abnormally developing and arrested human embryos carry numerical chromosome abnormalities.     

制备绒毛细胞染色体的研究

在以往工作的基础上,建立了制备绒毛细胞染色体的24小时培养并结合酶解的方法。主要特点是将解离细胞、低渗处理与秋水仙素处理合并为一个步骤。用这种方法能使绒毛细胞染色体的制作成功率明显提高,并对研究内容进行了讨论。 Abstract:A study of enzymolysis for chromosome preparation from chorionic villus was established upon previous work .Essential feature of this technique was to merge cell separation from the cytotrophoblastic layer,treatment with hypotonic solution and colchicines treatment into one step.This technique significantly improved the success rate of chromosome analysis from CVS.The results of the study were discussed.     

Stabilization of chromonema--one of the highest compactization levels--by visible light in the presence of ethidium bromide

This paper deals with the ultrastructure and behavior of interphase chromatin and metaphase chromosomes of L-197 culture cells under experimental conditions, which help to reveal the chromonemal level of chromosomal structure after the treatment of living cells with 0.1% Triton X-100 and 3 mM CaCl2. In these conditions, the chromonemata can be seen as dense chromatin fibers with thickness about 100 nm. Such chromosomes, whose chromonemal substructure after the treatment with hypotonic solution (10 mM Tris-HCl), look like loose chromosomal bodies composed of elementary 30 nm DNP fibrils. On the other hand, if chromosomes, in which chromonemal levels were revealed by 3 mM CaCl2, were treated with etidium bromide and then illuminated by light with length wave about 460 nm, no chromosomal decondensation in hypotonic conditions is observed. Chromonemata in chromosomes stabilized by light retain their density and dimensions. It is very important that chromonemata in stabilizated chromatin of metaphase chromosome keep specific connections between themselves and also general trend in their composition inside the chromosome. Thus, we have found conditions for observation of chromonemal elements in metaphase chromosome, providing the possibility for future three-dimensional investigation of chromonema packing in mitotic chromosomes.     

Chromosome core structure revealed by silver staining

Chromosomes were subjected to either prolonged hypotonic solution pretreatment or aging. Both conditions greatly loosened and dispersed the overlying epichrormatin from the central chromosome core structure. This was followed by silver staining and examination with bright-field microscopy. The chromosome core selectively reduced the silver and stained black while the surrounding epichrormatin stained yellow. A single core was seen extending the length of each chromatid. Nucleolus organizer regions appeared to be attached to the core, while kinetochores seemed to be specialized regions of the core itself. Cytochemical tests indicated that the core component(s) responsible for silver staining was non-histone protein(s).     

Oocyte Maturation and Furrow Formation in an Unfertilized Egg by Fusion with a Fertilized Egg or Blastomeres in the Ascidian, Ascidia sydneiensis divisa

A technique for fusing an ascidian egg with blastomeres using a chemical fusiogen was established and then used to identify cytoplasmic factors that regulate the process of oocyte maturation in ascidian eggs. Unfertilized eggs fused with fertilized eggs or blastomeres in hypotonic artificial sea water containing 20% polyvinyl alcohol within 10 min. After fusion polar bodies were extruded from the unfertilized portion of the fused eggs. Furrows were formed not only in the fertilized portion but also in the unfertilized portion in the fused eggs. No polar body extrusion and furrow formation occur in either portion of fused unfertilized eggs. These results suggest that fertilized eggs and blastomeres contain a factor that induces oocyte maturation. Polar body extrusion and furrow formation were not suppressed in the fertilized portion of fused eggs, suggesting that unfertilized eggs do not contain a factor that inhibits oocyte maturation.     

Sexing of mouse preimplantation embryos by detection of Y chromosome-specific sequences using polymerase chain reaction.

Detection of genes known to be present on the mammalian Y chromosome was adapted for sexing mouse early embryos using the polymerase chain reaction (PCR) method. Sry and Zfy genes located in the sex-determining region of the Y chromosome were chosen for Y-specific target sequences, and DXNds3 sequence on the X chromosome was chosen for control. The two-step PCR method using two pairs of primers for each of the target sequences was employed for detecting the sequences. When DNAs of male and female mice were amplified with these primers, male-specific fragments were detected even in DNAs that were equivalent in amount to two cells. Mouse embryos at the two-cell stage were separated into two individual blastomeres, and one blastomere was karyotyped at the second cleavage. The remaining blastomere was subjected to PCR amplification immediately or after having been cultured for 48 h up to the morula stage. The Sry and Zfy sequences were detected in about half the embryos; detection of the Sry and Zfy sequences corresponded exactly to the presence of the Y chromosome, except in one sample of male morula in which embryos may have been lost before the PCR amplification. It is concluded that the sex of mouse preimplantation embryos can be accurately determined through detection of the Y-specific sequences using the two-step PCR method, even with the single blastomeres separated at the two-cell stage.