Nodules associated with axial cores and synaptonemal complexes during zygotene in Psilotum nudum

Two-dimensional spreads of synaptonemal complexes (SCs) from the lower vascular plant Psilotum nudum were examined after staining with uranyl acetate-lead citrate (UP). Staining with UP allows visualization of lateral elements/axial cores (ACs), central elements, kinetochores, and nodules. Numerous darkly stained nodules were associated with forming SCs. In addition to nodules found on the central element of SC segments, other nodules were found at points of convergence between two adjacent ACs. Of these latter nodules, some were obviously associated with a fiber that connected adjacent ACs. No central element material was visible between the ACs, and the nodule complex appeared to be the only structure holding the ACs together. Although the function(s) of nodules during zygotene is unknown, the presence of a nodule-fiber complex that connects adjacent ACs before central element formation suggests that at least some of the nodules may be involved in synaptic initiation.     

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

Synaptonemal complexes (SCs), X and Y axes, and various nucleolar structures stain preferentially with silver in surface microspread preparations and are analyzable by both light and electron microscopy. Central elements, kinetochore region material and nuclear annuli which stain with ethanolic phosphotungstic acid are seldom visible after silver staining. SCs can be characterized by length measurements equally well in light and electron micrographs, from which stages of pachytene can also be determined by differentiation of the axes of the XY pair. By electron microscopy, the lateral elements appear as single strands at zygotene and early pachytene, then become double in a plane perpendicular to that of the SC and appear denser and thicker until late pachytene when they become progressively more attenuated and again appear single. These transitions are difficult to explain in terms of separation of associated chromatids. Identification of various silver stained bodies as nucleoli is supported by their orange-red fluorescence with acridine orange. SCs, X and Y axes and associated sex body material are, with a few exceptions, virtually indistinguishable from the background yellow-green fluorescence of the chromatin. Comet-shaped nucleolar bodies are regularly associated with five (in one animal) or six (in two animals) SCs; their positions along particular SCs identifiable by relative lengths indicate these bodies to be expressions of nucleolus organizer regions. They first appear at leptotene in association with unpaired axes and undergo progressive changes through late pachytene, at which time they redistribute their contents coincident with disappearance of the SCs. A characteristic nucleolar double dense body appears at zygotene; unlike the comet-shaped nucleoli, it is unassociated with other nuclear structures, and is assumed to arise from coalescence of previously existing smaller dense bodies. — The silver staining method described is remarkable for the speed and simplicity with which large numbers of spermatocyte nuclei are obtainable for light and electron microscopy. The fidelity of the light microscopic counterpart of the electron microscopic image has been directly assessed at different stages of pachytene. For cytogenetic analysis, critical information often lies beyond the limits of light optical resolution; the correlated electron microscopy required for verification is easily obtained with this method.This paper is warmly dedicated to Professor Hans Bauer on the occasion of his seventy-fifth birthday and as our expression of gratitude and admiration for his lasting contributions to chromosome biology     

The distribution of early recombination nodules on zygotene bivalents from plants.

Early recombination nodules (ENs) are protein complexes approximately 100 nm in diameter that are associated with forming synaptonemal complexes (SCs) during leptotene and zygotene of meiosis. Although their functions are not yet clear, ENs may have roles in synapsis and recombination. Here we report on the frequency and distribution of ENs in zygotene SC spreads from six plant species that include one lower vascular plant, two dicots, and three monocots. For each species, the number of ENs per unit length is higher for SC segments than for (asynapsed) axial elements (AEs). In addition, EN number is strongly correlated with SC segment length. There are statistically significant differences in EN frequencies on SCs between species, but these differences are not related to genome size, number of chromosomes, or phylogenetic class. There is no difference in the frequency of ENs per unit length of SC from early to late zygotene. The distribution of distances between adjacent ENs on SC segments is random for all six species, but ENs are found at synaptic forks more often than expected for a random distribution of ENs on SCs. From these observations, we conclude that in plants: (1) some ENs bind to AEs prior to synapsis, (2) most ENs bind to forming SCs at synaptic forks, and (3) ENs do not bind to already formed SCs.     

The von Kossa reaction for calcium deposits: silver lactate staining increases sensitivity and reduces background

Summary The classical von Kossa method has been modified: the high silver nitrate concentration in the original was replaced by 0.05% silver lactate with hydroquinone remaining the reducing agent of choice. The present modification stained calcification nodules with a sensitivity comparable to the original von Kossa reaction, but resulted in a reduced background staining in cultured osteoblasts. The method works well also with plastic- or paraffin-embedded tissue sections.     

Localized chiasmata and meiotic nodules in the tetraploid onion Allium porrum.

Allium porrum L. (cultivated leek) (2n = 4x = 32) is a fertile tetraploid that forms bivalents with pericentric chiasmata at metaphase I. To investigate the basis of this unusual behavior for a tetraploid, we describe the karyotype, axial cores, synaptonemal complexes (SCs), and meiotic nodules of A. porrum. The karyotype appears to be autotetraploid. This conclusion is also supported by presynaptic alignment of axial cores in groups of four and partner trades between pairs of SCs. Numerous early nodules are distributed all along axial cores and SCs during zygonema, but they are lost by late zygonema - early pachynema. Late (recombination) nodules (RNs) are present on SCs near kinetochores throughout the remainder of pachynema. This pattern of RNs corresponds to the pattern of pericentric chiasmata. Pachytene quadrivalents usually are resolved into bivalents because partner trades between SC lateral elements rarely occur between RNs on the same segment of SC. Thus, the patterns of crossing-over and partner trades promote balanced disjunction and high fertility in autotetraploid A. porrum. Rare quadrivalents observed at metaphase I must be due to infrequent partner trades between RNs. Polycomplexes, unusual in their number and size, were observed during zygonema. Key words : synaptonemal complex, recombination nodules, localized chiasmata, polycomplex, Allium porrum.     

Synaptonemal complex-associated centromeres and recombination nodules in plant meiocytes prepared by an improved surface-spreading technique

An improved method of preparing two-dimensional surface spreads of plant synaptonemal complexes (SCs) is described. This technique produces clear preparations of SCs and, in addition, consistently reveals both centromeres and recombination nodules (RNs) in PTA-stained preparations viewed by electron microscopy. A preliminary study of RN number and distribution in Allium fistulosum indicates that they faithfully reflect the positions of cross-over exchange events as revealed by chiasmata.     

Spreading synaptonemal complexes from Zea mays

Four different inversion heterozygotes of maize were examined for the occurrence of synaptic adjustment. Three substages of pachytene were identified in synaptonemal complex (SC) spreads using side-by-side comparisons of chromosome squashes with two-dimensional spreads of SCs. In SC spreads, inversion loop frequency did not change substantially from early through late pachytene for any of the four inversion heterozygotes examined. In addition, the position and size of the inversion loops remained essentially constant throughout pachytene. These results indicate that synaptic adjustment of inversion loops does not occur during pachytene in Zea mays.     

褐家鼠精母细胞中联会复合体的研究Ⅳ.常染色体联会复合体的发育和偶线期节的研究

采用表面铺展-SDS处理、硝酸银和磷钨酸（Phosphotungsticacid,PTA）染色电镜技术,研究了褐家鼠精母细胞中常染色体联会复合体（Synaptonemacomplex,SC）的发育及偶线期节（Zygotenenodule,ZN）。在褐家鼠精母细胞的细线期,常染色体轴心（Axialcores,ACs）已形成,同源轴心在空间上靠近,偶线期SCs开始形成,到粗线期SCs完全形成,于双线期SCs开始解体。在双线期除了个别SCs侧生组分分开外,大多数SCs发生碎片化（fragmentation）．在偶线期未配对的ACs和SCs侧生组分及中央组分上均发现电子密度高的球形或椭圆形的节状结构──偶线期节,ZNss在同源染色体配对过程中起很重要的作用。     

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。     

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.     

Spiral cores of synaptonemal complex lateral elements at the diplotene stage in rye include the ASY1 protein

After completing their functioning, synaptonemal complexes (SCs) degrade during the diplotene stage. In the pollen mother cells of rye Secale cereale L., this occurs through the formation of gaps in lateral elements of the SCs and the shortening of fragments of SCs until their complete disappearance. However, when contrasting SCs with silver nitrate solution at a pH 3.5–4.5, these gaps appear to be filled with threads associated with SC lateral elements. As the diplotene stage proceeds and gradual degradation of SC fragments continues, these threads turn into submicroscopic spirals. In this study, we found that the threads and spirals associated with degrading synaptonemal complexes are stained by antibodies to the ASY1 protein of Arabidopsis thaliana lateral elements and thus are degradation products of the lateral elements of SCs.     

Colchicine effects on meiosis in the male mouse

Antimitotic agents administered at the time of synapsis (leptotene/zygotene) have been shown to induce synaptic abnormalities visible during pachytene in the male mouse. The object of this study was to test the hypothesis that cells with relatively large amounts of colchicine-induced damage to the synaptonemal complex (SC) are eliminated from prophase whereas cells with relatively small amounts of SC damage proceed through to the end of prophase. Male mice were injected with tritiated thymidine to mark a cohort of spermatocytes at premeiotic S-phase for tracking through pachytene. Forty-eight hours later, when those cells were at leptotene/zygotene, colchicine was administered intratesticularly. Whole-mount SC spreads were made from animals sacrificed at various times following colchicine administration, and prepared for autoradiography. The marked cells were examined by light and electron microscopy and the kind and number of synaptic abnormalities were scored throughout pachytene. Colchicine-induced SC damage included single axial elements (univalents), together with partially synapsed and nonhomologously synapsed SCs. The amount of SC damage (amount and type per cell and frequency of cells with damage) scored at early pachytene exceeded by three- to fivefold the amount at late pachytene. This is consistent with spermatogenic cell loss from the seminiferous tubule via colchicine-induced destruction of Sertoli cell microtubules. The presence of spermatocytes with no more than four autosomal univalents at late pachytene indicates that some cells with low amounts of synaptic damage progress to the end of pachytene. The loss of the most severely damaged cells may represent a meiotic checkpoint at early pachytene in the male mouse. Received: 24 April 1996; in revised form: 29 August 1996 / Accepted: 11 March 1997     

A method for preparation of synaptonemal complexes of meiotic chromosomes from basidial protoplasts of the common mushroom Agaricus bisporus (Lange) Imbach

For the first time, preparations of synaptonemal complexes (SCs) were made from meiotic chromosomes of white button mushroom (Agaricus bisporus) basidia. It is the first experience of obtaining SC preparations of filamentous fungi from isolated meiosporangium protoplasts. Previously, only yeast SC preparations were obtained following this approach. The method includes four major stages: isolation of basidium protoplasts by treatment of basidia with lytic enzymes, spreading of protoplast nuclei on a filmy support by osmotic shock, staining the preparations with silver nitrate, and examination under light and electron microscopes. The structures of spread premeiotic nuclei, axial elements of chromosomes, SCs, chromatin, and nucleoli were studied at the leptotene-diplotene stage of meiotic prophase I.     

Behavior of silver stainable material during mitosis inVicia faba

To reveal the behavior of silver stainable material localized mainly in the nucleoli and nucleolar organizing regions (NORs), the somatic cells ofVicia faba were investigated by silver staining throughout the mitotic cell cycle. Nucleoli of interphase and early prophase nuclei were darkly stained. From late prophase to anaphase the secondary constrictions were discriminated as silver stained NORs and many silver grains appeared throughout the cytoplasm. At late prophase the NOR condensed at the same rate as the chromosome arm. Small spherical bodies and two new nucleoli appeared in telophase nuclei and at the same time the cytoplasmic grains disappeared. On the basis of the above observations on the silver stainable material during each mitotic phase, the behavior of silver stainable material is interpreted.     

A simple and economical modification of the Masson-Fontana method for staining melanin granules and enterochromaffin cells

Enterochromaffin cells from the small intestine of man, guinea pig, dog, chicken, rabbit, cat and rat were stained using the Masson-Fontana ammoniacal silver method with varying dilutions of silver nitrate solution (0.25 to 5 g per 100 ml of distilled water) and incubation temperatures (60 C and 75 C). The 0.5% solution of silver nitrate gave an argentaffin pattern similar to that of the 5% solution and had two major advantages: economically, since much less silver nitrate is used, and methodologically, since low background resulted with tissue of those species (rat, cat and rabbit) that required unusually long incubation. The staining of melanocytes was similar for all dilutions at the usual staining time (15-30 min).     

A Simple and Economical Modification of the Masson-Fontana Method for Staining Melanin Granules and Enterochromaffin Cells

Enterochromaffin cells from the small intestine of man, guinea pig, dog, chicken, rabbit, cat and rat were stained using the Masson-Fontana ammoniacal silver method with varying dilutions of silver nitrate solution (0.25 to 5 g per 100 ml of distilled water) and incubation temperatures (60 C and 75 C). The 0.5% solution of silver nitrate gave an argentaffin pattern similar to that of the 5% solution and had two major advantages: economically, since much less silver nitrate is used, and methodologically, since low background resulted with tissue of those species (rat, cat and rabbit) that required unusually long incubation. The staining of melanocytes was similar for all dilutions at the usual staining time (15-30 min).     

Heterochromatin and silver banding of rye (Secale cereale,Gramineae) chromosomes

Air-dried chromosomes of rye when stained with aqueous silver nitrate show differential banding patterns. In addition to staining the NOR sites, the silver nitrate stains all regions of constitutive heterochromatin, as identified by Giemsa C-banding, as well as a number of small interstitial regions. However, the heterochromatin on the B chromosome is not stained by the silver method. This is proposed as a rapid and reliable banding method.     

A Method for Preparation of Synaptonemal Complexes of Meiotic Chromosomes from Basidial Protoplasts of the White Button Mushroom Agaricus bisporus (Lange) Imbach.

For the first time, preparations of synaptonemal complexes (SCs) were made from meiotic chromosomes of white button mushroom (Agaricus bisporus) basidia. It is the first experience of obtaining SC preparations of filamentous fungi from isolated meiosporangium protoplasts. Previously, only yeast SC preparations were obtained following this approach. The method includes four major stages: isolation of basidium protoplasts by treatment of basidia with lytic enzymes, spreading of protoplast nuclei on a filmy support by osmotic shock, staining the preparations with silver nitrate, and examination under light and electron microscopes. The structures of spread premeiotic nuclei, axial elements of chromosomes, SCs, chromatin, and nucleoli were studied at the leptotene–diplotene stage of meiotic prophase I.     

Rapid Bielschowsky silver impregnation method using microwave heating.

The Bielschowsky silver impregnation method has been used extensively to demonstrate neuronal processes including dendrites, axons and neurofibrils. In this study, we examined the differences in the time required for and the staining quality of the Bielschowsky method for neuronal processes when microwave heating was used instead of processing at room temperature. For this purpose, a control group of sections stained according to the conventional method at room temperature was compared to an experimental group stained in a microwave oven at 180 W for 2, 4 and 1 min in 2% silver nitrate, ammoniacal silver nitrate and gold chloride, respectively. Light microscopic examination demonstrated that the normal structure was preserved in both groups and that there was no difference in the staining quality between the control and the microwave groups. In addition, staining time for this procedure was reduced to 8 min by using the microwave oven. Our study revealed that microwave irradiation can be used safely for Bielschowsky silver impregnation of neuronal tissues.