The fine structure of meiotic chromosome polarization and pairing in Locusta migratoria spermatocytes

At the leptotene stage of meiotic prophase in Locusta spermatocytes (2n=22 telocentric autosomes + X-chromosome), each chromosome forms an axial core. The 44 ends of the autosomal cores are all attached to the nuclear membrane in a small region opposite the two pairs of centrioles of the juxtanuclear mitochondrial mass. At later stages of meiotic prophase, the cores of homologous chromosomes synapse into synaptinemal complexes. Synapsis is initiated near the nuclear membrane, in the centromeric and the non-centromeric ends of the chromosomes. Homologous cores have their attachment points close together and some cores are co-aligned prior to synapsis. At subsequent stages of zygotene, the number of synaptinemal complexes at the membrane increases, while the number of unpaired axial cores diminishes. At pachytene, all 11 bivalents are attached to the membrane at both ends, so that there are 22 synaptinemal complexes at the membrane near the centrioles. Because each bivalent makes a complete loop, the configuration of the classic Bouquet stage is produced. The X-chromosome has a poorly defined single core at pachytene which also attaches to the nuclear membrane. These observations are based on consecutive serial sections (50 to 100) through the centriolar zone of the spermatocytes. Labeling experiments demonstrated that tritiated thymidine was incorporated in the chromatin of young spermatocytes prior to the formation of the axial cores at leptotene. It is concluded that premeiotic DNA synthesis is completed well in advance of pairing of homologous chromosomes, as marked by the formation of synaptinemal complexes.     

The ultrastructure of the human sex vesicle

A correlated light and electron microscopical study has been made on the sex vesicle of human spermatocytes. The human sex vesicle contains no RNA and no ultrastructural granular element. The sex vesicle is formed at zygotene by the two heteropycnotic sex chromosomes that come into end-to-end contact at this stage. Neither the main nor secondary nucleoli are formed in connection with the human sex vesicle. The ultrastructure of the sex vesicle shows two main components: chromatin threads 250 of Å in diameter, separated by interthread spaces, and the filament or core, 700 Å wide and smoothly curved. No synaptinemal complexes have been observed in the human sex vesicle, although the filaments can become parallel for short segments. The absence of synaptinemal complexes is discussed in relation to the observations on other mammals.     

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.     

FINE STRUCTURE OF SYNAPSED CHOMOSOMES IN F1 LYCOPERSICON ESCULENTUM-SOLANUM LYCOPERSICOIDES AND ITS PARENTS

Typical synaptinemal complexes consisting of electron-dense central and lateral elements and much less dense outer fibrillar material are formed at meiotic prophase in Lycopersicon esculentum (tomato), Solanum lycopersicoides, their diploid hybrid, and occasionally in tomato haploids (in which an average of about one chromosome region per cell is synapsed nonhomologously). Complexes in the hybrid (in which the chromosomes synapse completely but often fail to form chiasmata) are similar to those in the parents. Complexes in the haploid are similar to those of diploid tomato. The data suggest that synaptinemal complexes form whenever chromosomes undergo meiotic synapsis, regardless of whether synapsis leads to chiasmata.     

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.     

The Relationship between Synaptinemal Complexes, Recombination Nodules and Crossing over in NEUROSPORA CRASSA Bivalents and Translocation Quadrivalents

Reconstruction of serially sectioned zygotene and pachytene nuclei has allowed the estimation of both the number and position of central component recombination nodules in the synaptinemal complexes of two chromosomally different strains of Neurospora crassa. In both strains the number of nodules is that expected if each nodule represents one crossover event (50 map units). The distribution of nodules within the arms of bivalents shows evidence of centromeric repulsion and telomeric localization. Nodules appear quite early in the zygotene before pairing of chromosomes is complete. Evidence was found of size differences in nodules, and multiple nodules were occasionally seen. Chromosome lengths and nuclear sizes increased from early zygotene to late pachytene. The three quadrivalents present in the alcoy translocation heterozygotes were readily distinguishable in reconstructions, and their cytological dimensions were in agreement with predictions from linkage map distances.     

Multiple synaptinemal complexes at the region of gene amplification in Acheta

Ovaries of Acheta domesticus (house cricket) were fixed for electron microscopy at two stages of development: (1) ovaries containing mainly oocytes at interphase and early prophase of meiosis, and (2) ovaries with oocytes mainly at pachytene and diplotene. The E.M. study was accompanied by three types of light microscopy controls consisting mainly of cytochemical tests. Every oocyte contains a DNA body which at pachytene and diplotene acquires the appearance of a puff. In the light microscope two zones can be distinguished inside the body: (1) an inner core of DNA and (2) and outer shell of RNA. In the E. M. the inner core consists of a fibrillar material and the outer shell is composed of areas of high electron opacity consisting mainly of tightly packed particles and fibrils. At these stages synaptinemal complexes are seldom seen associated with the DNA body but are present throughout the nucleus as part of the paired chromosomes. The complexes are present as single units. — In the oocytes at interphase and early prophase of meiosis, where the DNA body is active in DNA replication, the body appears in the light microscope as a large Feulgen positive sphere containing Feulgen negative areas. In the E. M. at these stages the DNA body consists of: (a) the two components found at pachytene, (b) a third electron dense component which is more homogeneous than the other two, and (c) of large assemblies of synaptinemal complexes originating from several centers. The most significant features of the axial complexes are: (1) the circular packing of the complexes, (2) their occurrence in packages of 300 to 400 units and (3) the fact that not all of the DNA body forms complexes but only a part of it.Biochemical experiments (Lima-de-Faria, Birnstiel and Jaworska, 1969) have demonstrated the amplification of ribosomal cistrons in the DNA body of Acheta. The simplest explanation is that the multiple complexes are formed either between the extra gene copies of the two homologues, or between the extra copies of each chromosome as well. There seems to be a correlation between the presence of multiple axial complex formation and gene amplification in Acheta but the exact relation between the two phenomena demands further study.Dedicated to Dr. Sally Hughes-Schrader on the occasion of the seventyfifth birthday on the twentyfifth of January 1970.     

The Relation between the Axial Complex of Meiotic Prophase Chromosomes and Chromosome Pairing in a Salamander (Plethodon cinereus)

An investigation of the structure of meiotic chromosomes from primary spermatocytes of two salamanders, Plethodon cinereus and Desmognathus fusca, has been made using correlated light and electron microscopy. Feulgen squashes were compared with stained sections and these related to adjacent thin sections in the electron microscope. A transition from the familiar cytological preparation to the electron image was thus effected. A linear complex consisting of three parallel strands has been observed with the electron microscope, passing along the central axis of primary spermatocyte chromosomes. The complex is similar to that found in comparable chromosomes from at least a dozen animal species. The structure in Plethodon is described in detail. Synapsis has been positively identified as the stage of meiotic prophase at which the complex occurs. Thus the complex is a part of bivalent chromosomes. It has not been seen in other stages or other divisions and is thus thought to be exclusively of synaptic occurrence. The term synaptinemal complex is suggested for the entire structure. By virtue of the material condensed around it, the complex is also seen in the light microscope where it appears as a fine, densely Feulgen-positive central core along the chromosome. The complex is thus closely associated with DNA, if not at least in part, composed of it. In the stages studied, homologous chromosomes are not always completely paired. The lateral elements of the complex separate and follow the single chromosome axes at these points. The central element disappears and thus may be a phenomenon of pairing. It is concluded that the lateral elements of the synaptinemal complex may more correctly be a "core" of the single meiotic prophase chromosome, possibly being concerned with its linear organization.     

Meiotic prophase in anthers of asynaptic wheat

The light microscope showed that zygotene and pachytene were completely suppressed in pollen mother cells of an asynaptic mutant of Triticum durum; the chromosomes passed through a normal chromomeral leptotene condition and remained unpaired throughout prophase. The electron microscope confirmed the absence of synaptinemal complexes, as would be expected with no pairing. Prominent opaque axial cores were present in the chromatin from the onset of leptotene up to an indeterminate stage during prophase condensation. At an early time during condensation 150 Å particles appeared between chromatin masses. Coincident to the disappearance of axial cores from the chromatin, polycomplexes consisting of linearly associated core fragments arrayed in single layer sheets appeared between chromatin masses. The aligned fragments were invariably spaced about 625 Å from centre to centre; this is approximately half the distance between centres of the lateral elements (axial cores) of the synaptinemal complex of pachytene of synaptic sister seedlings. There was no central element between the associated fragments. The significance of these observations is discussed, as is also the essential difference between asynapsis and desynapsia.     

Ultrastructure of the micronucleus of the infusorian Didinium nasutum during meiosis]

Six stages can be distinguished in the micronuclear first maturation division prophase of D. nasutum. Nucleolus-like structures of fibrillar nature, connected with micronuclear chromosomes seem to develop at the late leptotene. At zygotene-pachytene, the chromosomes condense, forming irregular loops. This coincides with formation of classically structured synaptinemal complexes in the micronuclei. At diplotene-diakinesis, chromosomal bivalents are uniformly scattered throughout the micronucleus. They aggregate into a net equatorial plate in the first division metaphase; chromosomes show prominent kinetochores with attached chromosomal microtubule bundles. The second maturation division starts immediately after the completion of the first division and is morphologically similar to agamic mitosis of the micronuclei of D. nasutum. During the 2th maturation division prophase, the compact chromosomes form a dense group and show no spreading inside the nucleus. They are interspaced by an amorphous material being possibly involved in the formation of spindle microtubules. The telophase spindle of the 2nd division likely as that of the Ist division divides into three parts, the two daughter nuclei and the separation spindle containing a material of depolymerized microtubules. Only one of the 2nd division derivatives enters the third maturation division. A short telophasic third division spindle is perpendicular to the surface of the contact between the conjugants and produces two pronuclei. The envelopes of the daughter micronuclei are formed from parts of the original nuclear envelope surrounding the entire spindle.     

Synaptinemal complexes in the achiasmatic spermatogenesis of Bolbe nigra Giglio-Tos (Mantoidea)

In chiasmatic meiosis of mosquitoes, ascomycetes and lilies the synaptinemal complex (SC) disassociates from the bivalent before metaphase I. Conversely, in the achiasmatic meiosis of Bolbe nigra, the SC remains associated with the bivalent during first metaphase. Light microscopy reveals mid-bodies between disjoining half-bivalents during early first anaphase in Bolbe. Optically controlled serial sections for electron microscopy show that the mid-bodies seen in light micrographs and synaptinemal complexes seen in electron micrographs are the same structure. Electron micrographs indicate that the SC breaks transversely at a point corresponding to the chromosomal kinetochore during anaphase I as the chromatin and the SC begin to separate. During telophase I, SC remnants are at the poles with the chromosomes or between poles. Presently, the evidence is inadequate to state whether the SC serves alternately or simultaneously as a biological contrivance for conjunction and crossing-over or singly as a device for one of these phenomena.Supported by a University of Melbourne Research Fellowship.     

Core-Strukturen in den meiotischen und post-meiotischen Kernen der Spermatogenese von Gryllus domesticus

Summary electron microscope study of spermatogenesis and spermiogenesis in Gryllus domesticus has revealed the existence of peculiar lamellate bodies which occur both in spermatocyte and spermatid nuclei. These bodies must be considered as multiple complexes of the axial core structures which are regularly found in paired pachytene chromosomes. Their shape is irregular, and their constituent structural elements, although having dimensions and a fine structure identical to those of regular axial complexes, may assemble in sheets rather than ribbons, often in a concentric rather than planparallel multiple layer system.Spcrmatocyte nuclei may either contain just one or two large bodies of this type, often but not always in close association with the nucleolus and/or the X chromosome, or they may show several such structures of smaller dimensions which have some connection to chromosome fibrils. None of the two types of nuclei simultaneously contains regular axial core complexes. In spermatid nuclei one or two such multiple structures are usually found, again often in association to the X and/or (in O-spermatids) to what appears to be a nucleolus.It is considered likely that the multiple core complexes are due to the self-assembly of those protein molecules which typically assemble only under the control of and in close association with the pachytene chromosomes to form ordered axial complexes. Their occurrence in spermatids shows that the constituent molecular material may not be decomposed during the meiotic divisions after it is dissociated from the chromosomes.

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Mit Unterstützung durch die Göttinger Akademie der Wissenschaften.

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Herrn Prof. Dr. H. Bauer zu seinem 60. Geburtstag gewidmet.     

Meiotic self-pairing of the Psalidodon (Characiformes,Characidae) iso-B chromosome: A successful perpetuation mechanism

B chromosomes are non-essential additional genomic elements present in several animal and plant species. In fishes, species of the genus Psalidodon (Characiformes, Characidae) harbor great karyotype diversity, and multiple populations carry different types of non-essential B chromosomes. This study analyzed how the dispensable supernumerary B chromosome of Psalidodon paranae behaves during meiosis to overcome checkpoints and express its own meiosis-specific genes. We visualized the synaptonemal complexes of P. paranae individuals with zero, one, or two B chromosomes using immunodetection with anti-medaka SYCP3 antibody and fluorescence in situ hybridization with a (CA)₁₅ microsatellite probe. Our results showed that B chromosomes self-pair in cells containing only one B chromosome. In cells with two identical B chromosomes, these elements remain as separate synaptonemal complexes or close self-paired elements in the nucleus territory. Overall, we reveal that B chromosomes can escape meiotic silencing of unsynapsed chromatin through a self-pairing process, allowing expression of their own genes to facilitate regular meiosis resulting in fertile individuals. This behavior, also seen in other congeneric species, might be related to their maintenance throughout the evolutionary history of Psalidodon.     

PRODUCTION AND RELEASE OF A LOCUS-SPECIFIC RIBONUCLEOPROTEIN PRODUCT IN POLYTENE NUCLEI OF DROSOPHILA HYDEI

A specific, 0.1–0.3-µm large ribonucleoprotein complex consisting of a central core with stalklike extensions on top of which 280–320-Å ribonucleoprotein particles are situated is found in an experimentally activated chromosome region, 2–48C, of the polytene chromosomes of Drosophila hydei. Alkaline hydrolysis, RNAse digestion, and uranyl-EDTA-lead staining indicated the ribonucleoprotein character of the 280–320-Å particles, whereas the central core seems to be devoid of RNA. The characteristic complexes are present in the nucleoplasm and at the nuclear membrane, but absent from the cytoplasm. It is suggested that the large RNP complexes are the specific products of the puff at 2–48C. Complexes similar to the ones described have not been observed in any other region of the polytene salivary gland chromosomes of this species.     

Meiosis in Microsporidia: effects on biological cycles]

Synaptinemal complexes have been demonstrated in 7 microsporidian species belonging to 6 different genera (Gurleya, Thelohania, Pleistophora, Tuzetia, Baculea, Glugea). Thus, it can be presumed that a meiosis and consequently a karyogamy occur during their life cycle. Meisis occurs at the beginning of sporogony; therefore, karyogamy, must occur between spore and merogany, i.e. during the poorly known part of the life cycle. In the microsporidian species studied, with uninucleate spores and diplokaryotic merogony (Thelohania for instance), the 2 joined nuclei, each of them containing meiotic chromosomes, not only fail to fuse, but actually separate at the beginning of sporogony; afterwards, each of them undergoes meiosis. Their separation is accompanied by the appearance of an organelle whose structure and function are poorly understood. However, its structure resembles that of the kinetic center. The Nosema species studied do not have synaptinemal complexes; thus, their life cycle is difficult to understand: either karyogamy and meiosis occur during the unobserved part of the life-cycle, or sexual phanomena are absent altogether. In the latter case, the Nosema-type life cycle might be limited to vegetative multiplication which could be explained by the dimorphism theory of Microsporidia. It is shown also in the present study that the life cycle of Microsporidia does not involve haploid organisms which it might be thought to contain by comparing it with the cycles of sporozoa.     

ULTRASTRUCTURE AND CYTOCHEMISTRY OF METABOLIC DNA IN TIPULA

A DNA body is present in the females of the fly Tipula oleracea and is formed in contact with the sex chromosomes in the oogonial interphases. At each oogonial mitosis, the DNA body follows the chromosomes to one anaphase group and is included in one of the telophase nuclei. The body increases appreciably in size during the interphase of meiosis. All oocytes have the body, but only a few nurse cells possess it. The DNA body synthesizes its DNA at a different time than the chromosomes, as is shown by incorporation of tritiated thymidine, and contains 59% of the DNA of the nucleus, as is disclosed by spectrophotometric measurements. At late diplotene the DNA body disintegrates, releasing its DNA into either the nucleus or the cytoplasm. When studied in the electron microscope, the DNA body appears composed of a tight mass of intertwined fibrils. Demonstration that the main mass of the body is composed of DNA is obtained from cytochemical tests which reveal that the DNA body is Feulgen positive, stains green with azure B, incorporates H³-thymidine, and after digestion with DNase is Feulgen negative. The DNA of the body is complexed with histone, like the DNA of the chromosomes, as is revealed by an intense alkaline fast green staining. Electron microscope examination of oocytes reveals that one side of the DNA body is in close contact with the nuclear envelope and that the other side possesses an outer shell composed mainly of particles 150 to 250 A in diameter. Between the outer shell and the chromosomes there is a band of low electron opacity, 4000 to 7000 A thick. In the light microscope, this light band together with the outer shell is Feulgen negative and stains violet with azure B; this is confirmation of the presence of RNA. In the oocytes the nucleoli are found inside the DNA body. These nucleoli have a nucleolonema composed mainly of particles 150 to 250 A. The nucleoli are Feulgen negative, alkaline fast green negative, stain violet with azure B, and do not stain with azure B after RNase digestion, thus confirming their RNA content. The presence of the nucleoli inside the DNA body and of a band of RNA between the body and the chromosomes is indicative of a high RNA synthetic activity. Since the DNA of the body is complexed with histone, as in the chromosomes, and the nucleoli are located inside the body, the simplest interpretation of the DNA body is that it represents hundreds of copies of the operons of the nucleolar organizing region or neighboring regions. The situation found in Tipula has several basic features in common with the polytene chromosomes of other Diptera and with the hundreds of nucleoli present in Triturus oocytes. In all three cases, genes seem to be copied hundreds of times but are kept in different types of packages. A DNA body like the one in Tipula oleracea is found in other species of Diptera and in the Coleoptera. There is no indication, from the present investigation, that the DNA body is in any way associated with a virus.     

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to assemble. Therefore, to identify these functional protein complexes, it is important to stabilize them in vivo before cell lysis and subsequent purification. Here we describe a method used to isolate large bona fide protein complexes from Drosophila embryos. This method is based on embryo permeabilization and stabilization of the complexes inside the embryos by in vivo crosslinking using a low concentration of formaldehyde, which can easily cross the cell membrane. Subsequently, the protein complex of interest is immunopurified followed by gel purification and analyzed by mass spectrometry. We illustrate this method using purification of a Tudor protein complex, which is essential for germline development. Tudor is a large protein, which contains multiple Tudor domains - small modules that interact with methylated arginines or lysines of target proteins. This method can be adapted for isolation of native protein complexes from different organisms and tissues.     

Meiotic Exchange without the Synaptinemal Complex

THE synaptinemal complex is a ribbonlike tripartite structure, normally restricted to the nucleus of meiocytes and located along the longitudinal axis of bivalent chromosomes¹. It consists of two dense lateral elements (about 400 Å in width) separated from the central element (about 250 Å in width) by spaces of about 400 Å (Fig. 1). Various functions have been proposed for it, primarily that it is intimately involved in meiotic exchange, either directly by promoting effective pairing between complementary nucleotide strands of homologous chromosomes^1–3 or indirectly by providing for the rough alignment of homologues before their more intimate association^4,5. In this view, the complex is an essential feature of the regular and extensive exchange process typical of meiosis. The firmest evidence in support of its role in exchange has been its concomitant presence in primary meiocytes whenever genetic or cytological evidence indicates that crossing-over is occurring¹. The presence of the complex in meiocytes which lack exchange and/or chiasmata, for example, the female of Bombyx mori⁶, intergeneric hybrids, haploid genomes and certain achiasmate male insects, as well as its presence in univalent X chromosomes of primary spermatocytes and in postmeiotic spermatids, has been interpreted as “the exceptions that prove, or at least refine, the rule¹.” Here, for the first time, we describe the converse situation, one in which high frequencies of meiotic exchange, as determined by genetic tests, are not accompanied by a detectable synaptinemal complex. This means that both pairing of homologues, which must precede exchange and the exchange process itself can occur in the meiocyte without the aid of the synaptinemal complex.     

The assembly of the synaptinemal complex.

The assembly of the synaptinemal complex in the ascomycete Neottiella was studied by three-dimensional reconstruction of a late zygotene nucleus. A single banded lateral component is formed between the two sister chromatids of each homologous chromosome prior to their pairing. The central regions are pre-assembled in organized form in folds of the granular part of the nucleolus and then converted into an amorphous transport form. The latter appears to move through the nucleoplasm to sites between the lateral components of synapsing homologous chromosomes. The central region material is reorganized into blocks with a recognizable central component and attached to one lateral component. The last step in the completion of the synaptinemal complex is the association of the free surface of the organized central region with the corresponding segment of the homologous lateral component. The findings are discussed in relation to mechanisms of chromosome pairing and chiasma formation.