Multicolor karyotype analyses of mouse embryonic stem cells

Summary The manipulation of embryonic stem (ES) cells to introduce directional genetic changes into the genome of mice has become an important tool in biomedical research. Monitoring of cell morphology before and after DNA manipulation and special culture conditions are a prerequisite to preserve the pluripotent properties of ES cells and thus their ability to generate chimera and effective germline transmission (GLT). It has been reported that prolonged cell culturing may affect the diploid chromosomal composition of cells and therefore the percentage of chimerism and GLT. Herein, we report multicolor-fluorescence in situ hybridization (M-FISH) analysis of four different ES cell lines/clones. Although the morphology of all four ES cell lines/clones appeared normal and all four expressed the early markers Oct-3/4 and Nanog, two cell lines presented consistent numerical and structural chromosome aberrations. We demonstrate that M-FISH is a sensitive and accurate method for a comprehensive karyotype analysis of ES cells and may minimize time, costs, and disappointment due to inadequate ES cell sources. Both authors contributed equally to this work.     

Conventional and ultrastructural analyses of the chromosomes of Discocyrtus pectinifemur (Opiliones, Laniatores, Gonyleptidae)

Among the Opiliones, species of the suborders Cyphophthalmi, Eupnoi, Dyspnoi and Laniatores have shown very diverse diploid chromosome numbers. However, only certain Eupnoi species exhibit XY/XX and ZZ/ZW sex chromosome systems. Considering the scarcity of karyotypical information and the absence of structurally identifiable sex chromosomes in the suborder Laniatores, we decided to analyse the chromosomes and bivalents of Discocyrtus pectinifemur (Gonyleptidae) to identify possible sex differences. Testicular cells examined under light microscopy showed a high diploid number, 2 n  = 88, meta/submetacentric chromosome morphology and a nucleolar organizer region on pair 35. Prophase I microspreading observed in transmission electron microscopy exhibited 44 synaptonemal complexes with similar electron density and thickness. The total and regular synapsis between the chromosomes of the bivalents was also noted in pachytene nuclei. Male mitotic and meiotic chromosomes revealed no distinct characteristic that could be related to the occurrence of heteromorphic sex chromosomes. Evolutionary trends of chromosome differentiation in the four suborders of Opiliones are discussed here.     

Preimplantation embryonic development of spontaneous mouse parthenotes after oocyte meiotic maturation in vitro

Of eggs ovulated in LT/Sv mice, 10–20% undergo spontaneous parthenogenetic activation, and 40–50% of the parthenotes develop to blastocysts when cultured in simple defined medium from the one-cell stage. Similar percentages of oocytes isolated from Graafian follicles undergo parthenogenetic activation after spontaneous maturation in simple defined medium, but embryonic development proceeds no further than the two-cell stage. The simple defined medium that supported preimplantation development of ovulated eggs and spontaneous maturation of extrafollicular oocytes contained no serum, free amino acids, or vitamins. The present experiments were conducted to determine what conditions during spontaneous maturation of extrafollicular oocytes could promote the ability of oocytes to develop to blastocysts after parthenogenetic activation and mimic the environment of preovulatory follicles. Cumulus-enclosed oocytes that were matured in simple medium supplemented with fetal bovine serum (FBS) developed to blastocysts after spontaneous parthenogenetic activation. Furthermore, minimum essential medium (MEM), a complex medium containing free amino acids and vitamins, could substitute completely for FBS for maturing oocytes from (C57BL/6J × LT/Sv)F₁ mice, and to a lesser extent for maturing LT/Sv oocytes. Therefore, even though germinal vesicle breakdown in mouse oocytes and preimplantation development of mouse eggs can occur in the absence of an exogenous supply of free amino acids and vitamins, a complete, or normal, mouse oocyte maturation cannot. These results also demonstrated that gonadotropins are not necessary during oocyte meiotic maturation for parthenogenetically activated eggs to develop through the preimplantation stages. Luteinizing hormone or 17β-estradiol in MEM during oocyte maturation had no effect on the subsequent development of parthenotes. In contrast, follicle stimulating hormone (FSH) and progesterone in the maturation medium decreased the number of ova that subsequently cleaved, and FSH decreased the number of cleaved eggs that developed to blastocysts.     

核型分析在细胞分类学中的应用

细胞学的核型分析资料作为分类学的证据,结合形态学、解剖学、胚胎学和遗传学等方面的证据,在解决分类上的疑难问题,起过不少的作用,而且是建立生物学种和建立新的自然分类系统所必不可少的资料。     

The karyotype of the dab (Limanda limanda L.)

In the dab ( Limanda limanda L.), the diploid number is 46 and the karyotype consists of 23 pairs of chromosomes, all of which are telocentric or subtelocentric, so that the total number of chromosome arms is 46 (AN-46).     

Ribosomal, telomeric and heterochromatin sequences localization in the karyotype of Anemone hortensis

The karyotype of the Mediterranean species Anemone hortensis L. (Ranunculaceae) was characterized with emphasis on heterochromatin distribution and localization of ribosomal (18S−5.8S−26S and 5S rDNA) and telomeric repeats (TTTAGGG). Diploid chromosome complement, 2 n  = 2 x  = 16, common to all investigated populations, consisted of three acrocentric, one meta-submetacentric and four metacentric chromosomes ranging in size from 6.34 to 10.47 µm. Fluorescence in situ hybridization (FISH) with 18S and 5S rDNA probes revealed two 18S−5.8S−26S rDNA loci on a satellite and secondary constriction of acrocentric chromosome pair 2 and terminally on acrocentric chromosome pair 3, and two 5S rDNA loci in the pericentromeric region of meta-submetacentric chromosome pair 4 and in the proximity of the 18S−5.8S−26S rDNA locus on chromosome pair 2. The only GC-rich heterochromatin, as revealed by fluorochrome Chromomycin A3 staining, was that associated with nucleolar organizer regions, whereas AT-rich heterochromatin, stained with 4,6-diamino-2-phenylindole (DAPI), was distributed intercalarly and terminally on the long arm of all three acrocentric chromosomes, and terminally on chromosomes 4 and 5. FISH with Arabidopsis -type telomeric repeats (TTTAGGG) as a probe revealed two classes of signals, small dot-like and large bands, at chromosome termini exclusively, where they corresponded to terminal DAPI-stained heterochromatin. Heteromorphism of chromosome pair 4, which refers to terminal DAPI bands and FISH signals, was observed in populations of Anemone hortensis . Chromosome pairing during meiosis was regular with formation of localized chiasmata proximal to the centromere.  © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society , 2006, 150 , 177–186.     

The origin of the nascent blastocoele in preimplantation mouse embryos ultrastructural cytochemistry and effect of chloroquine

Summary Mouse morulae are known to undergo cavitation as soon as some external cells have entered the sixth cell cycle (Garbutt et al. 1987). Since the early cytological features of cavitation are still unclear, we undertook a careful ultrastructural analysis of late morulae-nascent blastocysts. In addition, since maturation of lysosomes might be involved in the first step of cavity formation, we focused our attention on these organelles by means of the cytochemical localization of trimetaphosphatase activity and by the study of the effects of chloroquine on precavitation embryos. Our results suggest that cavitation starts in a few external cells (presumably competent cells entering the sixth cell cycle), by the chloroquine-sensitive formation of degradative autophagic vacuoles engulfing lipid droplets and vacuoles containing osmiophilic material. These complex structures enlarge (as a result of lipid metabolism?) and so transform into intrablastomeric cavities which, by means of a membrane fusion process, very rapidly become extracellular cavities that coalesce. The abembryonic pole of the blastocyst is determined in this way. Moreover, we suggest that the juxtacoelic cytoplasmic processes covering the inner cell mass (ICM) cells, which are known to restrict the expression of their totipotency during early cavitation (Fleming et al. 1984), are the latest remnants of the walls of the growing intrablastomeric cavities.     

Regulation of cap-dependent translation initiation in the early stage porcine parthenotes

The binding of mRNAs to ribosomes is mediated by the protein complex eIF4F in conjunction with eIF4B (eukaryotic initiation factor 4F and 4B). EIF4F is a three subunit complex consisting of eIF4A (RNA helicase), eIF4E (mRNA cap binding protein), and eIF4G (bridging protein). The crucial role is played by eIF4E, which directly binds the 5'-cap structure of the mRNA and facilitates the recruitment to the mRNA of other translation factors and the 40S ribosomal subunit. EIF4E binding to mRNA and to other initiation factors is regulated on several levels, including its phosphorylation on Ser-209, and association with its regulatory protein 4E-binding protein (4E-BP1). In this study we document that both the translation initiation factor eIF4E and its regulator 4E-BP1 become dephosphorylated in the early stage porcine zygotes already 8 hr post-activation. Similarly, the activities of ERK1/2 MAP and Mnk1 kinases, which are both involved in eIF4E phosphorylation, gradually decrease during this period with the timing similar to that of eIF4E dephosphorylation. The formation of an active eIF4F complex is also diminished after 9-15 hr post-activation, although substantial amounts of this complex have been detected also 24 hr post-activation (2-cell stage). The overall protein synthesis in the parthenotes decreases gradually from 12 hr post-activation reaching a minimum after 48 hr (4-cell stage). Although the translation is gradually decreasing during early preimplantation development, the eIF4F complex, which is temporarily formed, might be a premise for the translation of a small subset of mRNAs at this period of development.     

体外受精和孤雌活化过程中小鼠胚胎细胞骨架的动态变化

为研究微管在体外受精与孤雌活化过程中的动态变化,本实验比较了体外受精胚胎、SrCl2激活的孤雌胚胎和体内受精的原核期胚胎在体外发育的情况,采用免疫荧光化学与激光共聚焦显微术检测卵母细胞孤雌活化过程中及体外受精后微管及核的动态变化,以分析微管在减数分裂过程中的作用及其对早期发育的影响.结果显示,体内受精胚胎的发育率显著高于体外受精和孤雌激活胚胎体外发育率(P<0.05),而体外受精与孤雌激活胚胎在各阶段发育率差异均不显著.在体外受精中,精子入卵,激活卵母细胞,减数分裂恢复,纺锤丝牵拉赤道板卜致密排列的母源染色体向纺锤体两侧迁移;后期将染色体拉向两极;末期时,微管分布于两组已去凝集的母源染色体之间,卵母细胞排出第二极体(the second polarbody,Pb2),解聚的母源染色体形成雌原核.同时,在受精后5～8 h精子染色质发生去浓缩与再浓缩,形成雄原核.在原核形成的同时,胞质星体在雌、雄原核的周围重组形成长的微管,负责雌、雄原核的迁移靠近.孤雌活化过程中,卵母细胞恢复减数分裂,姐妹染色单体分离,被拉向两极,经细胞松弛素B处理后,活化4～6 h,卵周隙中未见Pb2,而在胞质中出现两个混合的单倍体原核,之间由微管相连接,负责两个单倍体原核的迁移靠近.与体外受精相比较,孤雌活化时卵母细胞更容易被激活,减数分裂期间微管的发育早且更完善.     

The karyotype of the mouse

A denaturating and renaturating technique, applied to mouse chromosomes, makes visible characteristic banding patterns by which all elements of the karyotype can be individually distinguished. The Y chromosome as a whole appears darkly stained. The X chromosome comprises 6.33% of the homogametic haploid set. The banding pattern of the chromosomes is compared with that obtained by aid of the quinacrine dihydrochloride fluorescence technique. After its use a banding pattern results which is similar to, but less distinct than, that found after the renaturation procedure.     

NEK5 regulates cell cycle progression during mouse oocyte maturation and preimplantation embryonic development

NEK5, a member of never in mitosis‐gene A‐related protein kinase, is involved in the regulation of centrosome integrity and centrosome cohesion at mitosis in somatic cells. In this study, we investigated the expression and function of NEK5 during mouse oocyte maturation and preimplantation embryonic development. The results showed that NEK5 was expressed from germinal vesicle (GV) to metaphase II (MII) stages during oocyte maturation with the highest level of expression at the GV stage. It was shown that NEK5 localized in the cytoplasm of oocytes at GV stage, concentrated around chromosomes at germinal vesicle breakdown (GVBD) stage, and localized to the entire spindle at prometaphase I, MI and MII stages. The small interfering RNA‐mediated depletion of Nek5 significantly increased the phosphorylation level of cyclin‐dependent kinase 1 in oocytes, resulting in a decrease of maturation‐promoting factor activity, and severely impaired GVBD. The failure of meiotic resumption caused by Nek5 depletion could be rescued by the depletion of Wee1B. We found that Nek5 depletion did not affect CDC25B translocation into the GV. We also found that NEK5 was expressed from 1‐cell to blastocyst stages with the highest expression at the blastocyst stage, and Nek5 depletion severely impaired preimplantation embryonic development. This study demonstrated for the first time that NEK5 plays important roles during meiotic G2/M transition and preimplantation embryonic development.     

铺道蚁的染色体核型分析

以商丘地区的铺道蚁Tetramorium caespitum(L.)早期胚胎为材料,采用低渗处理—烘干法制备染色体标本,对其染色体组型进行研究。结果表明,铺道蚁染色体组型为n=22,2n=44,是由10条中央着丝粒染色体,3条亚中着丝粒染色体和9条亚端着丝粒染色体组成。     

中国二种癞蝗染色体C带核型

染色体C带核型在物种鉴定、分类阶元间的比较及其系统演化关系的推断中是一个有用的指标,染色体组内C带分布位置、大小、数量及异染色质含量可以反映出属、种及种下阶元的细胞学异同。文章报道中国2种癞蝗——红缘疙蝗Pseudotmethis rubimarginis Li和准噶尔贝蝗Beybienkia songorica Tzyplenkov的染色体C带核型,结果表明:2种癞蝗均为XO(♂)型性别决定机制。染色体组成均为2n♂=19,染色体为端着丝粒染色体;在各染色体相对长度,C带的大小,位置和着色程度上又存在不同程度的差异,可以作为区分种的依据。     

Bimodal karyotype in Cynomorium coccineum L. and its systematic implications

The presence of a bimodal karyotype in Cynomorium coccineum (2n = 28) is used to support its separation from Balanophoraceae and the maintenance of Cynomoriaceae as a separate family.     

The karyotype of threeCryptochironomus species of West Siberia,USSR

Karyotype characteristics C. ussouriensis and two species of C. gr. defectus from the main and special lobe cells of the salivary gland are presented in detail in this paper for the first time. C. ussouriensis has two main chromosome markers, nucleolus in chromosome II and large Balbiani ring adjacent to the centromeric band in chromosome I. Balbiani ring is developed only in the main lobe cells and is absent in the special lobe cells. Each of two chromosomes of C. gr. defectus (C. obreptans) has a nucleolus and a Balbiani ring. They are well developed in the main lobe cells but in the special lobe cells the only nucleolus in chromosome II are functioning. The karyotype of C. gr. defectus (karyotype 1) is characterized with a large number of functionally active regions: nucleoli, BRs and puffs. Two BRs (in region 10 of chromosome I and in region 8 of chromosome III) functioning in the main lobe cells are absent in the special lobe cells. Since the pattern of nucleoli, BRs and puffs are of great importance in the karyotype characteristic of the different Cryptochironomus species, a comparative study of chromosomes from the different salivary gland lobes is needed.     

凤凰蜘蛛抱蛋(铃兰科)的补充描述及核型

通过野外和标本室观察,发现凤凰蜘蛛抱蛋的原始描述存在一些错误。重新详细描述了该种的花部形态特征,补充了果实形态特征的描述,并首次报道了其染色体数目(2n=38)和核型(2n=22m+6sm+10st)。根据核型特征,认为该种的近缘种应为四川蜘蛛抱蛋和乐山蜘蛛抱蛋,而非黄花蜘蛛抱蛋。     

淡黄蚊蝎蛉染色体特征及其系统发育意义

【目的】染色体特征在昆虫系统发育研究中起着重要作用。然而,长翅目(Mecoptera)蚊蝎蛉科(Bittacidae)昆虫的染色体研究却比较匮乏。【方法】通过室内饲养获得淡黄蚊蝎Bittacus flavidus Huang & Hua各龄幼虫、蛹和成虫。取淡黄蚊蝎雄性末龄(4龄)幼虫、蛹和新羽化成虫精巢细胞进行染色体制片,通过4′,6-二脒基-2-苯基吲哚(4′,6-diamidino-2-phenylindole, DAPI)荧光染色,研究染色体核型、减数分裂行为和性别决定机制。【结果】结果表明,淡黄蚊蝎蛉的染色体数目为2n=26+X0,染色体均具有中着丝粒,核型高度对称。二价体绝对长度为(3.20±0.07)~(1.53±0.19) μm,相对长度为(5.31±0.29)~(2.73±0.24),均呈梯度变化。淡黄蚊蝎蛉的减数分裂为交叉型,其中核的平均交叉频率为11.5,二价体的平均交叉频率为0.88。性别决定机制为XX/X0型。DAPI荧光带型显示,粗线期二价体一端呈现高亮的AT富集区块。【结论】染色体特征(包括染色体数目、染色体臂基本数和核型公式等)在蚊蝎蛉科中表现出显著变异,表明染色体重排(尤其是融合、断裂和倒位)在蚊蝎蛉科昆虫的谱系分化和染色体演化中起着重要作用。