Localization of single- and low-copy sequences on tomato synaptonemal complex spreads using fluorescence in situ hybridization (FISH)

Fluorescence in situ hybridization (FISH) is a powerful means by which single- and low-copy DNA sequences can be localized on chromosomes. Compared to the mitotic metaphase chromosomes that are normally used in FISH, synaptonemal complex (SC) spreads (hypotonically spread pachytene chromosomes) have several advantages. SC spreads (1) are comparatively free of debris that can interfere with probe penetration, (2) have relatively decondensed chromatin that is highly accessible to probes, and (3) are about ten times longer than their metaphase counterparts, which permits FISH mapping at higher resolution. To investigate the use of plant SC spreads as substrates for single-copy FISH, we probed spreads of tomato SCs with two single-copy sequences and one low-copy sequence (ca. 14 kb each) that are associated with restriction fragment length polymorphism (RFLP) markers on SC 11. Individual SCs were identified on the basis of relative length, arm ratio, and differential staining patterns after combined propidium iodide (PI) and 4', 6-diamidino-2-phenylindole (DAPI) staining. In this first report of single-copy FISH to SC spreads, the probe sequences were unambiguously mapped on the long arm of tomato SC 11. Coupled with data from earlier studies, we determined the distance in micrometers, the number of base pairs, and the rates of crossing over between these three FISH markers. We also observed that the order of two of the FISH markers is reversed in relation to their order on the molecular linkage map. SC-FISH mapping permits superimposition of markers from molecular linkage maps directly on pachytene chromosomes and thereby contributes to our understanding of the relationship between chromosome structure, gene activity, and recombination.     

Identification of aberrant chromosomes based on the morphometry of the synaptonemal complexes in a sterile male mouse

The synaptonemal complexes (SCs) of surface-spread spermatocytes of male mouse from the F1 progeny of a male exposed to a mutagen have been examined by electron microscopy. Nonreciprocal translocation was recognised in analysing configuration of SC. Electron microscope analysis revealed translocation in 100% pachytene spermatocytes and light microscope analysis of air-dried metaphase spermatocytes demonstrated this in 58% cells. Different types of association of X-chromosome with aberrant chromosomes were discovered in pachytene spermatocytes. Computer analysis of relative length of SCs permits to detect a nonreciprocal translocation from chromosome 4 to chromosome 16. The length of the translocated fragment was determined to be from 66 to 75% of the length of chromosome 4. It has been impossible to discover a telomere fragment of chromosome 16, because the break point of chromosome 16 is too close to the distal end.     

Spreading the synaptonemal complex of Neurospora crassa

A protocol was developed to spread the synaptonemal complex (SC) of the fungus Neurospora crassa. It involves direct mechanical breakage of meiotic cells before spreading. This technique makes it possible to examine the SC of the same nucleus with both light and electron microscopy. This protocol is potentially applicable for other Pyrenomycetes. The SCs were examined at zygotene, pachytene and diplotene. The central elements and the recombination nodules (RN) were well revealed by silver staining. Ten to 13 RNs were counted at pachytene. The total genomic SC length varied with the stage. This whole mount electron microscopy of the SC is particularly useful for studying chromosomal rearrangements.     

Recombination nodules in coleopteran species: Palembus dermestoides (Tenebrionidae) and Epicauta atomaria (Meloidae)

This work describes the first report about the occurrence of recombination nodules (RNs) in spread pachytene cells of two species of Coleoptera: Palembus dermestoides (Tenebrionidae) and Epicauta atomaria (Meloidae). The RNs were observed in preparations contrasted with phosphotungstic acid. Considering RN morphology and its occurrence in pachytene bivalents (one per autosome bivalent) these structures were interpreted to be late RNs. P. dermestoides and E. atomaria have 2n = 20 chromosomes including an Xy(p) sex determination system. In spite of most frequently subtelocentric morphology observed in the autosomes of both species, the occurrence of RNs is limited only to the synaptonemal complex (SC) structure of the long arms. These findings are in agreement with those obtained using light microscopy analysis in which only one chiasma or terminalization event is observed per autosomal bivalent in early or late metaphase I cells. The RNs have the same average width of the SC of each analyzed species, a circular shape, strong electron density, and are observed mainly between the lateral elements of the SC. The RNs of P. dermestoides and E. atomaria have approximately the same average size (width), 180 +/- 20 nm and 160 +/- 80 nm, respectively. The absence of RNs in the short arms and its occurrence in the long arms are discussed considering the short arm pericentromeric and pro-centric heterochromatin.     

Analysis of chromosome aberrations on the basis of synaptonemal complexes in the offspring of mice irradiated with gamma-rays

Electron-microscopic analysis of synaptonemal complexes (SC) spread on the surface of hypophase was carried out to study chromosome rearrangements in sterile and semisterile F1 offsprings of mice exposed to gamma-radiation at a dose of 5 Gy. Chromosome rearrangements were microscopically scored at diakinesis - metaphase I in the same animals. SC analysis at pachytene revealed chromosome rearrangements in 63% spermatocytes. Analysis of chromosomes at diakinesis - metaphase I in the same animals only revealed chromosome rearrangements in 32% cells. SC analysis permits detecting chromosome rearrangements undetectable at diakinesis - metaphase I.     

Meiosis in Mesostoma ehrenbergii ehrenbergii (Turbellaria,Rhabdocoela)

At metaphase I during spermatogenesis in Mesostoma ehrenbergii ehrenbergii (2 × = 10), 3 bivalents and 4 univalents form. The bivalents each have a single distally localised chiasma. Electron microscope serial section reconstructions have shown that 3 short pieces of synaptonemal complex (SC) are present in pachytene nuclei in the testis. All the SC is contained in a lobe which is separated by an invagination from the main body of the nucleus. The SC pieces vary in length from approximately 2 m to 5 m, and the greatest amount found in one nucleus was approximately 13 m. It is proposed that the pieces of SC correspond to the paired regions of the 3 bivalents present at metaphase I. This system is therefore an example of localised pairing leading to localised chiasmata. Later prophase stage nuclei have also been reconstructed, in which stacks of polycomplex are present but no SC has been found. The polycomplex is thought to be an association of discarded SC sub-units.     

Synaptonemal complex karyotyping in spermatocytes of the Chinese hamster (Cricetulus griseus)

Relative length is a constant and distinctive characteristic for each autosomal SC, despite variations in absolute length from cell to cell. Arm ratio is distinctive for each SC except for two of the three sub-acrocentrics, and serves, together with relative length, for identification. The constancy of relative length and arm ratios indicates biological stability and lack of physical distortion in these spread preparations. There is a 11 relationship between relative lengths of autosomal SCs and mitotic autosomes; their arm ratios are also similar. These close parallels provide strikingly similar SC and somatic karyotypes. Variability was observed in sub-acrocentric arm ratios and in lengths of unpaired X and Y axes, correlated with the presence of constitutive heterochromatin. — Utilizing progressive differentiations of the X and Y chromosomes for staging, it is demonstrated that autosomal SCs decrease in length from late zygotene to mid-pachytene, and then increase at late pachytene. Within a nucleus, synchrony of length changes is maintained. It is concluded that the factors governing autosomal SC length are regular for any given bivalent from cell to cell, and may be related to those that control somatic autosome length relationships. — The X and Y axes differ quantitatively as well as qualitatively from autosomal SCs. The SC portion of the X and Y is constant in length through most of pachytene; the unpaired axes shorten and lengthen, but not in proportion to autosomal SCs. X and Y relative lengths and arm ratios vary throughout pachytene and do not maintain proportionality with somatic values. The evidence suggests, but does not prove, that the long arm of the X is paired with the short arm of the Y. — Twists occur in autosomal SCs at increasing frequencies throughout pachytene but cannot account for length changes. The number of twists per SC is directly proportional to SC length. Intertwining of SCs is random and proportional to SC length. End-to-end associations of autosomal SCs appear to be random; however, the ends of the X and Y are less often involved in such connections. — The length of axial material in all chromosomes at pachytene, expressed as an equivalent length of DNA double helix, represents 0.013% of the diploid DNA complement.     

Metaphase and meiotic chromosomes,synaptonemal complexes (SC) of the lizard <Emphasis Type="Italic">Zootoca vivipara</Emphasis>

Somatic mitotic and meiotic chromosomes at the pachytene and at the metaphase I of the males of the viviparous lizard, Zootoca vivipara (Lichtenstein, 1823), from northwestern Russia, belonging to the Russian form of Z. v. vivipara, are examined. The spreading of synaptonemal complexes (SC) of their chromosomes are obtained and analyzed for the first time. Eighteen SC are observed, including SC of the Z₁Z₁ (pairs 5 or 6) and the Z₂Z₂ (pair 13) sex chromosomes. Characteristics of SC are compared with the number and the shape of bivalents and with those of the karyotype structure. In the studied Russian form of Z. v. vivipara, the length ratios of bivalents correlate with that of mitotic chromosomes (2n = 36); however, some specificity in the morphology of SC of the Z₁Z₁ sex chromosomes is reported in this article.     

Autosomal synaptonemal complexes and sex chromosomes without axes in Triatoma infestans (Reduviidae; Hemiptera)

Meiotic and somatic cells at interphase in Triatoma infestans are characterized by the formation of a large chromocenter, which was assumed to contain the whole of the three large pairs of autosomes and the sex chromosomes. Observations with C-banding techniques show that the chromocenter is formed only by the terminal and subterminal heterochromatic blocks of the three large pairs of autosomes and the sex chromosomes. During pachytene the two largest autosomal pairs loop on themselves and their condensed ends form the chromocenter, together with the single heterochromatic end of the third autosomal pair. The X and Y chromosomes seem to associate with these condensed ends by their affinity for C-heterochromatin. During a very short pachytene stage, bivalents and synaptonemal complexes (SCs) are observed. Pachytene is followed by a very long diffuse stage, during which SCs are disassembled, multiple complexes aggregate on the inner face of the chromocenter and finally all complexes disappear and a dense material is extruded to the cytoplasm through the annuli. The 3-dimensional reconstruction of early pachytene chromocenters show 3 SCs entering and tunnelling the chromocenter, while during mid-pachytene 4 SCs enter this mass and a 5th SC is in a separate small mass. The looping of a whole SC which has both ends in the chromocenter was shown by the reconstructions. These data are interpreted as the progressive looping of the two largest bivalents during pachytene, forming finally the association of 5 bivalent ends corresponding to the 5 C-banding blocks of the large autosomal pairs. No single axis or SC that could be ascribed to the sex chromosomes was found. This agrees with the pachytene microspreads, which show only 10 SCs corresponding to the autosomal bivalents. The X and Y chromosomes are enclosed in the chromocenter, as shown by the unravelling chromocenters at diplotene-diakinesis. Thus the sex chromosomes do not form axial condensations, and this fact may be related to the ability of the X and Y chromosomes to divide equationally at metaphase I. SCsThis paper is dedicated to the memory of the late Professor Francisco A. Saez     

Meiosis in Mesostoma ehrenbergii ehrenbergii (Turbellaria,Rhabdocoela)

Female meiosis in Mesostoma ehrenbergii ehrenbergii is achiasmate. Electron microscope serial section reconstructions of nuclei from the germarium region of the ovary have shown that synaptonemal complex (SC) is present during the early prophase stages. The greatest amounts present were in two nuclei containing 389 and 401 respectively. The reconstructions showed that discontinuities in the SC existed along the lengths of the bivalents in these two nuclei which were taken to represent the maximally paired stage, i.e., pachytene. — Reconstructions of post pachytene nuclei showed that SC was absent from the bivalents and therefore retention of SC until metaphase I is not the mechanism used by this achiasmate species to ensure homologous chromosome segregation. An alternative mechanism for this function is proposed based on observations made on the later stages of meiosis in maturing oocytes.     

Pachytene karyotype analysis of tetraploid Meloidogyne hapla females by electron microscopy

Pairing of homologous chromosomes results in the formation of 34 synaptonemal complexes (SC) at pachytene, corresponding to the 34 bivalents at metaphase I. No multivalent associations were observed and pairing occurs two-by-two. The modified SC, which lacks a central element, does not affect the pairing process. Only one end of the SC is attached to the nuclear envelope, although either end can attach. Total SC length and the number of recombination nodules in the tetraploid were about 1.5 times greater than in the diploid.     

Synaptonemal complex karyotyping in spermatocytes of the Chinese hamster (Cricetulus griseus)

Using the Counce-Meyer spreading technique, in over 70 spermatocytes it was possible consistently to obtain whole, flattened nuclei containing complete sets of pachytene SCs. The SCs are visible in both the phase and electron microscopes. Each SC is morphologically intact, preferentially stained, and attached to the nuclear envelope by a dense, terminal plaque. It is thus possible to trace each SC for its entire length. Also, a structure representing the kinetochore is clearly visible in each autosomal SC. Karyotypes comparable to the somatic karyotype can be constructed by arranging SCs according to length and kinetochore position. The observed regularity of SC morphology implies structural stability sufficient to withstand the stresses imposed by the procedure.— A coarse network of closely packed nuclear annuli connecting SC attachment plaques often provides end-to-end associations and may tend to immobilize SCs during processing.— Three kinds of perturbation of SC structure are encountered. Twists in the SC frequently occur, but no regular pattern or correspondence with chiasma distribution is observed. SCs occasionally hook around each other without disruption, but in two instances the unpaired axis of the X apparently was interlocked within an autosomal SC. Stretching of the SC is infrequent; it is conspicuous when it occurs and is usually associated with other obvious distortions of the nucleus.— Distinctive morphologies of the X and Y chromosomes facilitate their identification in all preparations. — During zygotene, autosomal synapsis, i.e., the formation of SCs from the pairing of single axial elements, initiates at distal ends and terminates at the kinetochore region; neither initiation nor termination is synchronous among all autosomes.     

Ultrastructure of meiotic pairing in B chromosomes of Crepis capillaris

The meiotic pairing behaviour of four B isochromosomes of Crepis capillaris was studied by synaptonemal complex (SC) surface spreading of pollen mother cells. The four B chromosomes form a tightly associated group, separate from the standard chromosomes, throughout zygotene and pachytene. All four B chromosomes are also folded around their axis of symmetry, the centromere, and the eight homologous arms are closely aligned from the earliest prophase I stages. A high frequency of multivalent pairing of the four B chromosomes is observed at pachytene, in excess of 90%, mirroring the situation observed at metaphase I but exceeding the frequency expected (76.2%) on the assumption of random pairing among the eight B isochromosome arms with a single distal pairing initiation site per arm. The higher than expected frequency of multivalents is due to the occurrence of multiple pairing initiations along the B isochromosome arms, resulting in high frequencies of pairing partner switches. Pairing of the standard chromosome set is frequently incomplete in the presence of four B chromosomes, and abnormalities of SC structure such as thickening and splitting of axes and lateral elements are also frequently seen. Similarly, B chromosomes show partial pairing failure, the extent of which is correlated with pairing failure in the standard chromosome set. The B chromosomes themselves also show abnormalities of SC structure. Both standard and B chromosomes show non-homologous foldback pairing of regions that have failed to pair homologously.by D. Schweizer     

Synaptonemal complex analysis of mouse chromosomal rearrangements

Electron microscopy of surface-spread spermatocytes from mice heterozygous for a tandem duplication shows the heteromorphic synaptonemal complex (SC) to comprise two lateral elements of unequal length, the longer of which is buckled out in a characteristic loop, representing the unsynapsed portion of the duplication. The loop is a regular feature of late zygotene-early pachytene nuclei; it is longest at these early stages, but, through equalization of the two axes as a consequence of synaptic adjustment, it is replaced by a normal appearing SC at late pachytene. Because equalization, as indicated by a decrease in the percent difference between axes, may begin shortly after completion of synapsis, estimates of duplication segment length are restricted to a sample selected for least adjustment. — Although the mean position of the loop is constant at various pachytene substages, individual positions vary widely from cell to cell, consistent with the behavior expected of a duplication, but not of a deletion or an inversion. The length of the segment that is duplicated is estimated to be 22% of the normal chromosome, the midpoint of the segment is mapped at 0.61 of the chromosome distal to the kinetochore, and the ends of the segment are mapped at 0.50 to 0.72. Measurements of G-banded mitotic chromosomes give comparable values: duplication length, 24%; midpoint, 0.60, and segment ends, 0.48 and 0.71. This agreement constitutes further validation of the SC/spreading method for detecting and analyzing chromosomal rearrangements at pachytene and substantiates the fidelity with which the axes and SCs represent the behavior of chromosomes in synapsis.     

黄鳝减数分裂和联会复合体组型分析

联会复合体(Synaptonemal Complex,SC)是减数分裂前期同源染色体配对形成的一种非永久性核内细胞器,同染色体配对、遗传交换以及染色体的分离有着密切的关系。自Moses和Fawcett的早期工作以来,围绕着SC的结构、行为及化学组成等开展了大量的工作,积累了丰富的资料。近年来,由于界面铺张技术的发展,进一步推动了     

Delayed Formation of Chromosome Aberrations in Mouse Pachytene Spermatocytes Treated with Triethylenemelamine (Tem)

Induction of chromosome aberrations in pachytene spermatocytes of mice by 2 mg/kg TEM was compared with induction by 400 R X rays. These doses induced comparably high dominant lethal effects in pachytene spermatocytes of mice. Cytological analysis at diakinesis–metaphase I stage showed that whereas 76.4% of the cells treated with X rays at pachytene stage had aberrations, the frequencies observed in two TEM experiments were only 0.8 and 2.2%. On the other hand, 5% of the progeny from TEM-treated pachytene spermatocytes were found to be translocation heterozygotes. This is the first report on the recovery of heritable translocations from treated spermatocytes of mice. The aberration frequencies observed for TEM in diakinesis–metaphase I were much too low to account for all the lethal mutations and heritable translocations. Thus, the formation of the bulk of aberrations induced by TEM in pachytene spermatocytes was delayed—a marked contrast to the more immediate formation of X-ray-induced aberrations. It is postulated that the formation of the bulk of TEM-induced aberrations in pachytene spermatocytes and in certain postmeiotic stages occurs sometime during spermiogenesis, and not through the operation of postfertilization pronuclear DNA synthesis.     

六种鱼的精母细胞联会复合体的电镜观察

我们以界面铺张——硝酸银染色技术,对鲈形目三种鱼(尼罗罗非鱼、莫桑比克罗非鱼、刺鳅)和鲤形目(鱼句)亚科三种鱼(花(鱼骨)、黑鳍鳈、麦穗鱼)的精母细胞联会复合体进行了电镜观察研究。系统考察了鱼类常染色体SC的亚显微结构、形成过程和配对行为,比较分析了刺鳅的性染色体SC的异配形态和行为,并绘制了鲈形目三种鱼的SC组型模式图。     

A human 9;20 reciprocal translocation associated with male infertility analyzed at prophase and metaphase I of meiosis

Details are given of a meiotic prophase analysis, carried out by spreading, of a human 9;20 reciprocal translocation ascertained in a subfertile, oligospermic male. Air-dried meiotic preparations revealed the presence of translocation quadrivalents at metaphase I. Germ-cell degeneration was evident from the early prophase of meiosis onward. Associations between the translocation quadrivalent and XY bivalent at pachytene were seen in only 20% of the cells and seemed not to be the prime cause of germ-cell failure. Pairing disruption around the breakpoints of the translocation at pachytene and/or pairing failure in one arm of the pachytene cross was observed in a total of 87% of all cells analyzed. This could have contributed significantly to germ-cell atresia.     

Synaptonemal complex analysis of mouse chromosomal rearrangements

Synaptonemal complex (SC) analysis by electron microscopy of spermatocytes in surface microspreads was carried out in mice heterozygous for two paracentric inversions: either In(1)1RK or In(2)5Rk. Characteristic SC inversion loops are formed at synapsis in bivalents carrying the rearrangements. Although all loops were observed to be eliminated by late pachytene through synaptic adjustment, every spermatocyte at early pachytene contained a fully synapsed loop. Cells in the earliest stage of pachytene contained the longest loops and thus had undergone minimal adjustment. The SC estimates of inversion lengths and breakpoint positions in such cells corresponded well with those from mitotic chromosome banding and could be correlated with genetic maps of chromosomes # 1 and # 2, thus demonstrating the basis for the mapping of pachytene chromosomes. The regularity of loop formation and reproducibility of the SC analysis are reflected in the constant relative positions of the estimated breakpoints. The method is sensitive enough to reflect small, real, interstitial length differences between meiotic and mitotic chromosomes. The results demonstrate the feasibility and precision of detection and quantitative characterization of inversions at early meiotic prophase by SC analysis.This paper is warmly dedicated to Prof. Dr. Wolfgang Beermann, on the occasion of his 60th birthday     

Mitotic and meiotic analysis in Arctocephalus australis (Otariidae)

The karyotype with C-, G- and NOR-banding of Arctocephalus australis is reported for the first time. The chromosomal number is 2n = 36. The X chromosome, identified in G-banded metaphases from males, is metacentric and the Y chromosome is a minute chromosome, also metacentric. Pachytene spermatocytes were used for synaptonemal complexes analysis with a surface spreading technique. A total of 17 autosomal synaptonemal complexes are observed plus the XY pair. During early pachytene, the X and Y axes are thickened and remain unpaired. As pachytene advances, a short SC is formed between the gonosomes, as it is common among eutherian mammals. The particular asymmetrical appearance of the synaptonemal complex in the sex pair is described and compared to other cases among mammals.