Some facts concerning the nature and formation of axial core structures in spermatids of Gryllus domesticus

Examination of living spermatid nuclei of Gryllus domesticus has revealed the presence of the same structures, the X chromosome, the round body and the axial core structures, which have been described from electron microscopic observations. The outer ribbons of the axial core structures and the round body are composed of 100 Å fibres indiscernible from and often continuous with the fibres composing the X chromosome. That the outer ribbons of the axial core structures and the round body are chromosomal is further substantiated by the results of cytochemical examinations of formaldehyde fixed material which show that the axial core structures and the round body contain RNA, DNA and basic protein. Neither acetic acid-ethanol nor cold ethanol fixation preserve the round body and the axial core structures suggesting that a protein may be responsible for maintenance of the central core structure. The central core structures are always found in close association with condensed chromatin in regions where the chromosome elements are about 1000 Å apart, suggesting that the relative state of condensation of the chromatin and the spacial relationship between condensed regions may be two of the chief factors concerned in central core formation. Maintainance of the condensed state of the chromatin, however, may in turn depend upon central core integrity.Herrn Prof. J. Seiler zu seinem 80. Geburtstag gewidmet.     

The structure and function of the synaptinemal complex inLilium longiflorum sporocytes

The development of meiotic prophase in pollen mother cells ofLilium longiflorum is presented through photomicrographs of squashes and sections and through electron micrographs of thick and thin sections. Emphasis is placed on the first appearance of axial cores, the participation of axial cores in the formation of synaptinemal complexes, the fine structure of the complex and the fate of the complex at the end of pachytene. It is shown that axial cores are formed in early meiotic prophase chromosomes and that the two axial cores of a set of homologous chromosomes participate in the formation of a synaptinemal complex. It is proposed that the transverse filaments of each axial core meet and interdigitate and so produce the transverse filaments of the complex. It is shown that the complex is axial to the pachytene bivalent and that the association of the complex with chromosomal material is terminated at the end of pachytene. The pairing affinity of the cores in homologous and non-homologous chromosome associations is discussed. The zygotene stage is defined in terms of the occurrence of synaptinemal complexes and the attachment of the nucleolus to the nuclear membrane during this stage is noted.     

Abnormal meiosis in bisporic strains of white button mushroom Agaricus bisporus (Lange) imbach

A formerly developed method of microspreading of mushroom basidial nuclei was applied to study meiotic prophase I in bisporic white button mushroom (Agaricus bisporus) strains. Meiotic recombination and assemblage of axial structures (axial elements and synaptonemal complexes) of chromosomes in meiotic prophase I are interrelated. It is known that the frequency of meiotic recombination is reduced in the bisporic A. bisporus variety. We showed that formation of axial structures of meiotic chromosomes in bisporic strains of this mushroom was disrupted. The anomalous phenotypes in spread prophase nuclei are diverse. In leptotene and early zygotene, many nuclei contain abnormal, often short, and, as a rule, few chromosomal axial elements. The abnormalities in the formation of synaptonemal complexes at the zygotene-diplotene stage are of the same kind and even more pronounced. We discovered an important feature of meiosis in A. bisporus associated with fruit-body morphogenesis. Meiosis starting in basidia (meiocytes) of young closed fruit bodies is accompanied by disruption of chromatin condensation in prophase I and, probably, is arrested. After partial veil breakage, the course of meiosis normalizes. Preparations with clearly observable chromosomal axial structures can be obtained only at this stage of fruit-body development.     

B-Chromosomen beiChironomus

In the population “Herrenmühle” ofChironomus plumosus 11% of the individuals contain one supernumerary chromosome. This B-chromosome is present both in germ-line and somatic cells. — InChironomus melanotus 6% of the larvae of the population “Falkau” carry supernumerary chromosomes. These B-chromosomes cannot be found in all nuclei of testis and soma, their number varies between cells within the individual. In both species the B-chromosomes represent centromeric fragments of chromosome IV as can be shown by their structure and pairing behaviour. — The polytene B-chromosome ofCh. plumosus exhibits a banding pattern in the salivary gland nuclei. Furthermore it is able to form an additional nucleolus in the nuclei of the malpighian tubules. InCh. melanotus band structures can be seen only in the B-chromosome of malpighian tubules. The larvae ofCh. melanotus, carrying B-chromosomes, show heterochromatic bodies in the salivary gland nuclei, varying in number and size in the nuclei of the same gland. These bodies are interpreted to be polytenic B-chromosomes divided into subunits.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Untersuchungen über die Anordnung der menschlichen Metaphasechromsomen

Summary The association pattern of the acrocentric chromosomes shows no significant difference between a population of mothers of mongoloid children and male and female controls of the same age-group. It could only be demonstrated that the associations of the mothers were interconnected by thread-like structures in a higher percentage. However, no significance could be deduced from for this phenomenon (P 0.1).

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Mit Unterstützung durch die Fritz-Thyssen-Stiftung und die Deutsche Forschungs-gemeinschaft

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Mit technischer Assistenz von Regine Heiland     

Lampenbürstenchromosomen und Amphinukleolen in Oocytenkernen der Schnecke Bithynia tentaculata L.

The oocyte nuclei of Bithynia tentaculata have been investigated by light and electron microscopy. The small oocyte nuclei of the pachytene stage are characterised by a well developed bouquet arrangement of the chromosomes, a small nucleolus located at the nuclear envelope and lamellar inclusions of unknown function. During previtellogenesis, oocytes contain typical molluscan amphinucleoli consisting of the nucleolus and the so called Proteinkörper. It is suggested, that the Proteinkörper is identical with the Binnenkörper of insects. Living oocyte nuclei of Bithynia have been isolated and investigated by phase contrast microscopy. They contain lampbrush chromosomes bearing chromomeres, pairs of lateral loops and some spheres. Maximal loop formation occurs early in oogenesis whereas chromosomes of later developmental stages show contraction of the loops and formation of many spheres. Their histochemistry and ultrastructure is described.

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Mit Unterstützung durch die Stiftung Volkswagenwerk.     

Nuclear activity during oogenesis in sea urchins

Summary Early meiotic stages of Arbacia punctulata oocytes have revealed the presence of synaptinemal complexes in the chromosomes, which persist through zygotene-pachytene. The synaptinemal complexes conform broadly to the usual tripartite structures found in other higher forms. In addition, nuclei at these stages consist of a small nucleolus and dense bodies of varying sizes. The nucleolus is fibrillar in texture throughout and does not seem to incorporate Uridine-5-³H after pulse labeling, whereas the chromosomes are labeled. The nucleolar label is visualized at diplotene stages and onwards. The nuclear envelope differentiates by the appearance of numerous nuclear pore complexes with dense material in the annuli, and the chromosomes become markedly diffused. At vitellogenesis stage the nucleolus and chromatin become highly labeled after pulse incorporation of Uridine, indicating synthesis of ribosomal and chromosomal RNAs.This investigation was supported by grants No. A-5049, A-3624 and D-17 from National Research Council, Canada, grant No. DRB-9340-05 (U6) from Defense Research Board, Canada, and grant No. DRG-918 AT from Damon Runyon Memorial Fund for Cancer Research.     

Specific Staining of Nucleolar Substance with Aceto-Carmine

A method is described for staining nucleoli intensely by treating tissues with formaldehyde, hydrolysing in normal HC1 at 60°C. and staining with aceto-carmine. With correct hydrolysis time, chromosomes and cytoplasm are almost colorless.

Formaldehyde increases the acidity of cell parts, especially the nucleolus, presumably by neutralizing the basic protein groups, and increases the resistance to hydrolysis, perhaps by protecting the phospholipoprotein complexes which are most abundant in the nucleolus.

Hydrolysis reduces the acidity of cell parts, chiefly by removal of nucleic acids.

Aceto-carmine stains cell structures which are weakly acid in character (about pH 4-5) probably by precipitating as large dye aggregates.

The technic appears to be highly specific for nucleoli and related cell bodies.     

Karyogamy and meiosis in an Amblyospora sp. (Microspora) in the mosquito Culex salinarius

Stages of merogony and sporogony are illustrated in photomicrographs of lacto-aecto-orcein stained smears of an undescribed species of Amblyospora in male Culex salinarius larvae. Karyogamy was observed to take place in diplokaryotic meronts followed by an unusual meiosis-like process in young sporonts. Karyogamy was confirmed by DNA measurements in the nuclei of meronts. DNA measurements at mingling indicate that tetrads may not be formed during “pachytene.” Therefore, structures previously reported to be synaptonemal complexes and meiotic configurations of chromosomes may not actually represent true pachytene chromosomes. It is further shown that karyogamy has been either misplaced or overlooked by numerous investigators and that the behavior of chromosomes during the meiosis-like process was misinterpreted by previous researchers studying chromosomes in microsporidian sporonts.     

Morphologische Variabilität der chromosomalen Funktionsstrukturen in den Spermatocytenkernen von Drosophila-Arten

Zusammenfassung In der Gattung Drosophila treten in den Zellkernen von primären Spermatocyten chromosomale Funktionsstrukturen auf, die im Prinzip wie die lateralen Schleifenpaare von Lampenbürstenchromosomen organisiert sind. Die Form der Schleifen ist bei jeder Art in artspezifischer Weise abgewandelt. Die morphologische Variabilität der Spermatocytenstrukturen von 54 Drosophila-Arten wird beschrieben. Das genetische Material in den Schleifen spielt möglicherweise eine entscheidende Rolle bei der Vorfertigung, Stabilisierung und programmierten Verpackung von Genprodukten, die erst in späteren Stadien während der Spermiohistogenese in den Zellen verbraucht werden.

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In the genus Drosophila the nuclei of primary spermatocytes contain special chromosomal functional structures which are organized as the pairs of lateral loops in lampbrush chromosomes. In each species the loops have their own species specific morphology. The morphological variability of the spermatocyte structures in 54 Drosophila species is described. Although the nuclei of spermatids are synthetically inactive, the cells are able to synthesize proteins which are essential for the differentiation of sperm. Therefore, special mechanisms are necessary for the preformation, stabilization, and programmed packaging of gene products during the spermatocyte stage. This may be the function of the spermatocyte loops.

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Verfasser dankt der Deutschen Forschungsgemeinschaft für eine Reisebeihilfe. Die Untersuchungen werden jetzt ebenfalls mit Unterstützung durch die Deutsche Forschungsgemeinschaft fortgesetzt. Mein besonderer Dank gilt Herrn Prof. Dr. W. S. Stone und seinen Mitarbeitern von der Genetics Foundation an der Universität von Texas in Austin. Die Zeichnungen wurden von Herrn E. Freiberg angefertigt.

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Herrn Prof. Dr. W. E. Ankel, meinem verehrten Lehrer, zum 70. Geburtstag gewidmet.     

Electron microscope study on meiosis

Summary The cycle of the nucleolus and sex chromosome was studied with the electron microscope during the following stages of spermatogenesis and spermiogenesis of Gryllus argentinus: (1) spermatogonia; (2) prophase cyte I, leptotene, part of pachytene, and end of diplotene till breakdown of the nuclear envelope; (3) division I, metaphase and anaphase; (4) cyte II, prometaphase; (5) division II, metaphase, anaphase and telophase; (6) early and late spermatids. Some observations were also carried out in the primary oocyte until beginning of the growth period.It was found that nucleolus and sex chromosome are associated, at first without mixture of their components (leptotene) and afterwards interchanging components (pachytene). The interchange takes place by the passage from one element to another of filamentous units ot low electron density, similar in appearance to those existing in the medial plane of tripartite groups (synaptinemal complexes).At pachytene the primary results of interchange are: (1) the nucleolus contains filaments of chromosomal nature; (2) the nucleolus emits a long rod-like prolongation containing a cylindrical bundle of filaments, and an axial unit of the same nature; two equidistant clear spaces separate the axial unit from the cylindrical bundle and the latter from the dark wall of nucleolar material. At the end of diplotene these components are found organized in two bodies and a prolongation. One of the bodies is formed by a number of alternatively dark and light bands, the other by a pack of tubular units each showing the structure of the former nucleolar prolongation. The prolongation is either formed as in the preceding stage or it is composed of five ribbons, two dark ones outside and three light ones between them. It is supposed that both bodies are united by the prolongation but no definite proof was obtained. It is assumed that the complex thus constituted represents the sex chromosome.The sex chromosome was found at any phase of both divisions as well as at the intermediate stages between them; at the division phase the chromosome is separated from the autosomes and moves independently of them.The element could not be traced at telophase II but it reappears within the reorganized nuclei of spermatids. Amorphous nucleolar-like material and chromosome-like material are found associated at this stage with banded complexes like those seen at the end of prophase I. All these components undergo involution during spermatid maturation. At the final step of maturation no traces of them are found.A similar association of nucleolus and chromosome was found at prophase of primary oocytes of the same species. The associated body is of the same structure as that described for primary spermatocytes. The structures existing in the primary oocytes disorganize at the beginning of growth. At this time the nucleolus has developed into a large body containing masses of chromatin-like material.This investigation was supported in part by U.S. Public Health Service, Research Grant No. GM-08337 from the National Institute of General Medical Sciences.     

Autoradiographische Untersuchungen zur funktionellen und pathologischen Kernschwellung in der Rattenleber nach Fütterung von Thioacetamid

Summary In the liver parenchyma of rats a markedly increase of the nuclear volume occurs after the application of thioacetamide. According to autoradiographic and morphological criterions this nuclear enlargement can be separated into a functional and a pathological swelling. In case of the functional swelling the rate of incorporation for H³-phenylalanine and H³-cytidine in the karyoplasm — as a measurement for protein and ribonucleic acid metabolism — is proportional to the nuclear volume. But in case of the pathological swelling the incorporation of these precursors is very much lower. In the functional swollen as well as in normal nuclei the volumetrical nucleolus/karyoplasm ratio can serve as an expression of the synthetic activity of both nuclear components. Yet this relation does not exist in pathological swollen nuclei. Therefore the functional swelling of the nucleus in comparison with the normal synthetic activities of the nucleus (karyoplasm + nucleolus) is an increased stage of function with completely preserved nuclear productivity. The pathological swelling however is a decreased state of function with a decompensated nuclear capacity.

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Die autoradiographischen Arbeiten wurden im Institut für Medizinische Isotopenforschung der Universität Köln (Leiter: Prof. Dr. W. Maurer) durchgeführt, für die Synthese der tritiierten Aminosäure danken wir Herrn Dr. Dr. K. Hempel.

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Die Arbeit lag 1963 der Medizinischen Fakultät der Universität Würzburg als Teil einer Habilitationsschrift vor. Sie wurde durch Mittel des Bundesministeriums für Wissenschaftliche Forschung ermöglicht.     

Über die Struktur und Teilung der Kerne von Geotrichum candidum und Geotrichum magnusii

Summary The nuclei in germinating spores and growing hyphae ofGeotrichum magnusii andG. candidum have been examined during life and in fixed and stained preparations.The spores and the cells of the hyphae are multinucleate. The nuclei consist of a dense Feulgen-negative nucleolus surrounded by a less dense shell of Feulgen-positive particles. No membrane was seen at the margin of either living or fixed and stained nuclei. The mass of chromatin and the nucleolus divide at the same time by elongation followed by constriction. Chromosomes could not be detected in either resting or dividing nuclei.

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Mit 5 Textabbildungen     

Lamelläre Einschlußkörper in den Zellkernen vonRhinanthus serotinus Oborny

Zusammenfassung In den Zellkernen der Antherenwand (mit Ausnahme des Tapetums) vonRhinanthus serotinus Oborny treten bis zu vier Einschlußkörper auf, die als Kristalloide vorliegen. Jedes Kristalloid besteht aus einem Stapel ± gleichartig ausgebildeter Lamellen, deren Periode etwa 180 Å beträgt (die Lamellendicke ist etwa 70 Å, der Lamellenabstand rund 110 Å). Die enge Nachbarschaft zwischen Nukleolus und Einschlußkörper scheint nicht zufällig zu sein: vielleicht dienen gewisse Orte am Nukleolus als Kristallisationszentren bei der Stapelbildung.

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Lamellar inclusions in the nuclei ofRhinanthus serotinus oborny

Summary The nuclei of the anthere wall (with the exception of the tapetum) contain inclusions of stacks of lamellae. The thickness of one lamella amounts to 70 Å, the distance from one lamella to the next one measures about 110 Å; the periodicity amounts to 180 Å. The close contact of stack and nucleolus in most nuclei seems to be not accidentically: perhaps locations around the nucleolus are places of cristallization of the stack.

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Herrn Prof. Dr.Karl Carniel danke ich für fruchtbringende Diskussion und FrauChrista Grubmann für technische Mithilfe.     

A LIGHT AND ELECTRON MICROSCOPE STUDY OF SMALL, SPHERICAL NUCLEAR BODIES IN MERISTEMATIC CELLS OF ALLIUM CEPA, VICIA FABA, AND RAPHANUS SATIVUS

Interphase, preprophase, and prophase nuclei of meristematic cells of Allium cepa, Vicia faba, and Raphanus sativus are characterized by the presence of spherical bodies approximately 1 µ in diameter. These structures are Feulgen-negative but stain metachromatically with azure B, as the nucleolus, following fixation with glutaraldehyde. At the ultrastructural level, they consist predominantly of fibrils estimated to be between 70 and 100 A in diameter which greatly resemble those found within certain zones of the nucleolus in these plant species. Moreover, in Allium cepa, these spherules often exhibit dense particles which are also found within the fibrillar zones of the nucleolus in this species. The observations that the bodies in question are frequently located at the surface of the nucleolus and moreover show cytochemical and ultrastructural similarities with this organelle suggest that they may originate from the nucleolus. However, the common association of the spherules with chromosome strands may indicate instead that these bodies represent extranucleolar ribonucleoprotein materials synthesized by specific chromosomal loci.     

Distribution of Atr protein in primary spermatocytes of a mouse chromosomal mutant: a comparison of preparation techniques

In this study, we examined the suitability of a three dimensional preparation technique for studying chromosome behaviour in the first meiotic prophase in the mouse chromosomal mutant T(1;13)H/T(1;13)Wa. To preserve cellular shape, primary spermatocytes were encapsulated in a fibrin clot. Conventionally sedimented prophase nuclei served as controls. Axial elements and lateral synaptonemal complex components were subsequently stained by immunofluorescence and the presence of axial elements at the pachytene stage was highlighted with indirect immunofluorescence against the Atr protein. We compared the distribution of Atr signal in the fibrin-embedded spermatocytes with surface-spread preparations and immunohistochemically stained histological sections of seminiferous tubules. Furthermore, fluorescence in situ hybridisation of the mouse minor satellite DNA was done on fibrin-embedded spermatocytes. The Atr signal is most conspicuous in fibrin-embedded nuclei on unpaired axial elements during pachytene, both for sex chromosomal and for autosomal segments, and expanding from these elements into the surrounding chromatin. Both spread and encapsulated zygotene nuclei with extended axial element formation proved to be positive for Atr. Mid- to late zygotene nuclei were devoid of 3,3′-diaminodibenzene deposition in the histological sections. Highlighting the unpaired axial elements in the small heteromorphic 1¹³H;1¹³Wa bivalent with an Atr signal enabled meiotic analysis of this bivalent to be carried out in a three-dimensional context. Thus, proximity of this bivalent with the sex chromosomes is found more often in three-dimensional preparations than in spread preparations. Furthermore, the development of the Atr signal over the sex chromosomes as pachytene proceeds helps in substaging of this long and heterogeneous meiotic phase, in sedimented but especially in fibrin-encapsulated nuclei. Received: 22 September 1999; in revised form: 20 December 1999 / Accepted: 21 December 1999     

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     

Pachytene variations in Ricinus

The pachytene complement in microsporocytes was compared among ten races of Ricinus communis L., the castor plant (2n=20). Four kinds of pachytene variations were observed: (1) variation in degree of spreading of the chromosomes; (2) variation in degree of attenuation of heterochromatin; (3) variation in morphology, which appeared to be restricted to the two nucleolar organizing bivalents; (4) variation in frequency of association of each of these two bivalents with the nucleolus. It is suggested that variations in the pachytene chromosomes occur ubiquitously in this species.