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.     

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.     

Identification of two major components of the lateral elements of synaptonemal complexes of the rat

This paper describes the identification of two major components of the lateral elements of synaptonemal complexes of the rat by immunocytochemical techniques. We prepared monoclonal antibodies against synaptonemal complexes (SCs) by immunization of mice with purified SCs. One of these antibodies, II52F10, reacts with a 30 and a 33 kDa polypeptide, which are major components of purified SCs. Using this antibody, we studied the localization of its antigens light microscopically, by means of the indirect immunoperoxidase technique, as well as ultrastructurally, by means of the immunogold labeling technique. The immunolocalization was carried out on whole-mount preparations of lysed spermatocytes. The antibody reacts with paired as well as unpaired segments of zygotene, pachytene and diplotene SCs. In light microscopic preparations, the attachment plaques, particularly those of late pachytene and diplotene SCs, also appear to react strongly. In electron micrographs the lateral elements in paired as well as unpaired segments could be seen to react. No reaction was observed in the attachment plaques; however, in late pachytene and diplotene SCs the swollen terminal segments of the lateral elements did react with the antibody. Thus, we conclude that a 30 and a 33 kDa polypeptide make part of the lateral elements of synaptonemal complexes of the rat.     

A method for preparing two-dimensional surface-spreads of synaptonemal complexes from plant meiocytes for light and electron microscopy

A method has been developed for preparing two-dimensional surface spreads of synaptonemal complexes (SCs) from plant meiocytes for examination by light and/or electron microscopy. Clear, well-spread preparations of SCs and unpaired axial cores have been obtained from a range of meiotic prophase I stages (leptotene to pachytene) from Allium and Secale meiocytes.     

Synaptonemal complexes of the A- and B-chromosomes of the spermatocytes from the East Asian mouse Apodemus peninsulae

The analysis of whole-mount preparations of synaptonemal complexes (SCs) from surface-spread spermatocytes of A. peninsulae (2n = 48A + 1, 2, ... 12 B) had revealed SCs of 23 autosomal bivalents, sex bivalent XY, axial cores and SCs of the B-chromosomes. The intercellular and interindividual variability of the number of B-chromosomes varied from 1 to 12 per cell. The SCs of autosomal bivalents were shown to have a typical structure. The structure and behaviour of SCs of sex bivalent throughout meiotic prophase I appeared to be similar to those observed in other species of this order. Mainly B-univalents and less frequently B-bivalents containing SCs were found to be formed in meiotic prophase I. The full homologues appear to be rarely seen among B-chromosomes of the East-Asiatic mouse. A tendency of forming clusters of B-univalents near the sex bivalent was found, in addition to B-bivalents with lateral elements, having the form of bi- and tri-stranded elements with rare synaptic fragments. Besides this, the SCs of the autosomes of pachytene cells were found to contain structures resembling the recombination nodules.     

Effects of the male contraceptive agent gossypol on meiotic chromosomes of the male rat

Synaptonemal complexes (SCs) of rat spermatocytes were analyzed in silver-stained meiotic preparations 10-24 days after treatment with gossypol acetic acid, 30 mg/kg/day, for 70 days. Gossypol did not affect SC formation or function, as judged by the absence of pairing anomalies, SC fragmentation, or presynaptic arrest. The unpaired lateral axes could be seen at zygotene, and at pachytene normal SCs could be observed. The behavior of the XY axes also appeared to be normal.     

Control of genetic consequences of vaccinations: electron microscopic analysis of murine synaptonemal complexes]

The ability of two inactivated bacterial vaccines, from Proteus vulgaris and Klebsiella pneumoniae, to injure synaptonemal complexes (SCs) was studied by means of electron microscopy. The preparations were given intraperitoneally to C57BL/6J male mice during five successive days and testes were fixed 24 h after the last injection. Cyclophosphamide was used for positive control. The vaccine from Klebsiella given at subtoxic dose, which was about 200 times higher that that given to people during vaccinations, induced the 10-fold rise in the frequency of SC abnormalities in murine 1-storger spermatocytes. Breaks of SCs and of single lateral elements of SCs predominated over other types of vaccine-induced anomalies. According to the preliminary data, vaccine from Proteus at subtoxic dose showed no damaging SC activity. The results of the given study are discussed in connection with the negative data obtained earlier when genotoxicity of these two vaccines had been studied in the Ames test, in routine investigations of bone marrow metaphases of vaccinated mice as well as under light microscopy of their SCs.     

Lateral elements inside synaptonemal complex-like polycomplexes in ndt80 mutants of yeast bind DNA

The synaptonemal complex (SC) keeps the synapsed homologous chromosomes together during pachytene in meiotic prophase I. Structures that resemble stacks of SCs, polycomplexes, are sometimes found before or after pachytene. We have investigated ndt80 mutants of yeast, which arrest in pachytene. SCs appear normal in spread chromosome preparations, but are only occasionally found in intact nuclei examined in the electron microscope. Instead, large polycomplexes occur in almost every ndt80 mutant nucleus. Immunoelectron microscopy using DNA antibodies show strong preferential labeling to the lateral element parts of the polycomplexes. In situ hybridization using chromosome-specific probes confirms that the chromosomes in ndt80 mutants are paired and attached to the SCs. Our results suggest that polycomplexes can be involved in binding of chromosomes and possibly also in synapsis.     

昆虫的联合复合体微铺展技术

研究昆虫的ＳＣ结构对促进细胞遗传学研究富有重要意义。但由于早期制备ＳＣ的技术方法复杂繁锁制约了ＳＣ研究的深入。近年来,随着制备ＳＣ标本技术方法的完善,促使ＳＣ研究的更深入发展。本文以昆虫的ＳＣ为研究对象讨论微铺展技术方法。     

Cultured Schwann cells assemble normal-appearing basal lamina only when they ensheathe axons

Previous work demonstrated that Schwann cells (SCs) must interact with nerve cells (NCs) in order to generate their basal lamina (BL) in culture (M. B. Bunge, A. K. Williams, and P. M. Wood, 1982, Dev. Biol. 92, 449-460). The present study was undertaken to determine if this interaction requires proximity of NCs to SCs. Coverslips carrying isolated SCs were placed into culture dishes containing normally contacting SCs + NCs, NCs alone, or SCs alone and were maintained in these dishes for 3-4 weeks in medium known to foster the differentiation of axon-related SCs (BL formation, myelination). The SCs on the coverslip were not allowed to contact the cells in the culture dish. In other experiments, SCs isolated on coverslips were simply cultured in medium conditioned by contacting SCs + NCs, NCs alone, or SCs alone. The accumulation of BL components was monitored by light microscopic immunocytochemistry and the assembly of BL structure assessed by electron microscopy. When SCs were cocultured with but not contacted by neurons, immunostaining for BL constituents revealed a patchy deposition of material in sharp contrast to the linear deposition observed on axon-related SCs. Electron microscopy of these isolated SCs revealed short segments of BL, strands or clumps of BL-like material extending away from the cell surface, and accumulation of this material between cells. A greater number of isolated SCs were immunostained when grown with contacting SCs + NCs than with NCs or SCs. The conditioned medium experiments yielded similar results; only patchy BL was observed and more immunostaining was detected on isolated SCs when the medium had been conditioned by contacting SCs + NCs than by NCs alone or SCs alone. Immunostaining was less overall in the conditioned medium experiments than in the cell coculture work. In addition, standard SC + NC cultures grown in differentiation-supporting medium were studied by electron microscopy. SCs that were not contacted by axons but were positioned between fascicles of normally contacting SCs + NCs were identified under phase microscopy and then examined for the presence of BL. These SCs exhibited only occasional segments of BL or detached BL-like material. Lastly, within differentiated fascicles, nonensheathing SCs were compared with neighboring myelinating SCs that were in substantial contact with axons. BL-deficient nonensheathing SCs were found directly adjacent to axons and BL-coated myelinating SCs.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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     

Silver staining of synaptonemal complexes in surface spreads for light and electron microscopy

Synaptonemal complexes (SCs), axes of the X and Y chromosomes, and nucleoli in surface-spread preparations of spermatocytes are selectively stained for both light and electron microscopy by ammoniacal silver. Combined with a simple technique for transferring material from glass slides to grids, sequential light and electron microscopic analysis of full SC complements is possible with no further preparation. This new method has potential for both basic and clinical cytogenetic research.     

Comparison of nerve cell and nerve cell plus Schwann cell cultures, with particular emphasis on basal lamina and collagen formation

The availability of cultures of normal cells (NCs) and Schwann cells (SCs) with and without fibroblasts has allowed us to investigate the sources of endoneurial and perineurial constituents of peripheral nerve. NCs cultured alone, devoid of ensheathment but healthy in appearance, lack basal lamina and extracellular fibrils. In contrast, when SCs accompany NCs, basal lamina and extracellular fibrils are consistently visible around SCs in outgrowth areas formed de novo in culture. These fibrils average 18 nm in diameter, exhibit a repeating banding pattern, and are trypsin-resistant and collagenase-sensitive. Collagen synthesis is also indicated by the incorporation of [14C]proline into peptide-bound hydroxy-proline in NC + SC or SC cultures. That the [14C]hydroxyproline polypeptides formed in NC + SC cultures are collagenous was determined in part by pepsin digestion- ammonium sulfate precipitation-polyacrylamide gel electrophoresis techniques; the 14C-polypeptides migrate to the positions of alpha 1 (I), alpha 2, alpha 1 (III), and alpha B chains of type I, type III, and A-B collagens. Also formed are thin, ruthenium red-preserved strands interconnecting basal laminae. SC ensheathment of axons is similar to that found in the animal; one SC is related to a number of unmyelinated axons or a single myelinated axon. This proclivity to ensheathe and myelinate axons indicates that SC function is not lost during the preparative procedures or after lengthy isolation in culture and provides the most reliable means for SC identification. Perineurial ensheathment and macrophages are lacking in NC + SC culture preparations divested of fibroblasts. We conclude that SCs do not form perineurium or the larger diameter collagen fibrils typical of endoneurium but that in combination with neurons they generate biochemically detectable collagens and morphologically visible basal lamina and thin collagenous fibrils.     

Spatial constraints dictate glial territories at murine neuromuscular junctions

Schwann cells (SCs), the glial cells of the peripheral nervous system, cover synaptic terminals, allowing them to monitor and modulate neurotransmission. Disruption of glial coverage leads to axon degeneration and synapse loss. The cellular mechanisms that establish and maintain this coverage remain largely unknown. To address this, we labeled single SCs and performed time-lapse imaging experiments. Adult terminal SCs are arranged in static tile patterns, whereas young SCs dynamically intermingle. The mechanism of developmental glial segregation appears to be spatial competition, in which glial-glial and axonal-glial contacts constrain the territory of single SCs, as shown by four types of experiments: (1) laser ablation of single SCs, which led to immediate territory expansion of neighboring SCs; (2) axon removal by transection, resulting in adult SCs intermingling dynamically; (3) axotomy in mutant mice with blocked axon fragmentation in which intermingling was delayed; and (4) activity blockade, which had no immediate effects. In summary, we conclude that glial cells partition synapses by competing for perisynaptic space.     

Stem-cell approaches for kidney repair: choosing the right cells

With the increasing rate of end-stage renal failure and limited alternatives for its treatment, stem cell (SC) therapy for kidney injury is urgently needed. Choosing the right SC type is the critical step in realizing the potential of this therapeutic approach. Four possible sources of SCs are envisioned for the development of this type of treatment: (i) bone-marrow-derived SCs (BMSCs), (ii) renal adult SCs, (iii) embryonic SCs and (iv) fetal renal SCs. We suggest that resident SCs recently identified in the Bowman's capsule of adult human kidneys might prospectively be the ideal cell type for treatment of both acute and chronic renal injury because they display the potential to differentiate into multiple types of renal cells. However, BMSCs also represent an attractive alternative, especially for the treatment of patients affected by acute renal failure.     

雪旺氏细胞与同种异体骨支架的体外共培养研究

目的:探讨雪旺细胞(Schwann’s cells,SCs)在同种异体骨支架上的生物相容性,体外构建组织工程骨神经化模型。方法:利用新鲜人体骨骼制备同种异体骨支架材料,检测其物理性能;采用优化方法提取新生SD大鼠坐骨、臂丛神经培养SCs,实验分为三维培养实验组(SCs+同种异体骨)、二维培养对照组(SCs+胶原玻片),S-100抗体免疫荧光染色鉴定SCs纯度;细胞计数法检测两组细胞增殖特点;细胞接种后第3、7天取样,扫描电镜观察。结果:同种异体骨支架具有良好的三维孔隙结构,适宜细胞贴附生长;S-100免疫荧光染色证实SCs纯度95%;扫描电镜检测显示两组SCs均可正常粘附增殖,细胞间排布规律相似,培养早期实验组SCs胞体更加细长,伪足更加明显,随着培养时间的延长表现出较强的迁移能力;细胞增殖检测:两组SCs生长曲线特征基本一致,支架材料对SCs无毒性作用。结论:同种异体骨支架SCs具有良好的生物相容性,其三维立体多孔结构有利于SCs的粘附与迁移,初步构建了体外组织工程骨神经化模型。     

Cytological study on Sertoli cells and their interactions with germ cells during annual reproductive cycle in turtle

Sertoli cells (SCs) play a central role in the development of germ cells within functional testes and exhibit varying morphology during spermatogenesis. This present study investigated the seasonal morphological changes in SCs in the reproductive cycle of Pelodiscus sinensis by light microscopy, transmission electron microscopy (TEM), and immunohistochemistry. During hibernation period with the quiescent of spermatogenesis, several autophagosomes were observed inside the SCs, the processes of which retracted. In early spermatogenesis, when the germ cells started to proliferate, the SCs contained numerous lipid droplets instead of autophagosomes. In late spermatogenesis, the SCs processes became very thin and contacted several round/elongated spermatids in pockets. At this time, abundant endoplasmic reticulum and numerous mitochondria were present in the SCs. The organization of the tight junctions and the adherens junctions between the SCs and germ cells also changed during the reproductive cycle. Moreover, SCs were involved in the formation of cytoplasmic bridges, phagophores, and exosome secretions during spermatogenesis. Tubulobulbar complexes (TBC) were also developed by SCs around the nucleus of the spermatid at the time of spermiation. Strong, positive expression of vimentin was noted on the SCs during late spermatogenesis compared with the hibernation stage and the early stage of spermatogenesis. These data provide clear cytological evidence about the seasonal changes in SCs, corresponding with their different roles in germ cells within the Chinese soft‐shelled turtle Pelodiscus sinensis.     

The protective effects of resveratrol on Schwann cells with toxicity induced by ethanol in vitro

Schwann cells (SCs) are the myelin forming cells in the peripheral nervous system, they play a key role in the pathology of various polyneuropathies and provide trophic support to axons via expression of various neurotrophic factors, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). Ethanol (EtOH) adversely affected both SCs proliferation and myelin formation in culture. Resveratrol (Res) has been shown to regulate many cellular processes and to display multiple protective and therapeutic effects. Whether Res has protective effects on SCs with EtOH-induced toxicity is still unclear. The protective efficacy of Res on EtOH-treated SCs in vitro was investigated in the present study. Res improved cell viability of the EtOH-treated SCs. Hoechst 33342 staining and terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate nick-end labeling analysis showed that the EtOH-induced apoptosis was inhibited by Res. The effects of Res were blocked by the 5′-adenosine monophosphate-activated protein kinase inhibitor Compound C and the silencing information regulator T1 inhibitor nicotinamide. Res could increase the mRNA and protein levels of BDNF and GDNF in the EtOH-treated SCs. However, the EtOH-induced increase of NGF in the SCs is inhibited by Res. The data from the present study indicate that Res protects SCs from EtOH-induced cell death and regulates the expression of neurotrophic factors. Res and its derivative may be effective for the treatment of neuropathic diseases induced by EtOH.