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.     

The use of gene conversion to study synaptinemal complex structure and molecular details of chromatid pairing in meiosis

Summary Gene conversion can be used to study: the topography and pairing relationships of the four chromatids of a bivalent at the time of crossing over and hybrid DNA formation, the lengths of intimately paired segments and the frequency of intimate pairing at particular sites. Conversion ratios of different types, corresponding-site interference, co-conversion, and the range and distribution of conversion frequencies are discussed in relation to DNA and chromatid pairing, and synaptinemal complex organisation. Conversion data from Ascobolus immersus and other fungi are compared with electron microscope data from various organisms and with models of the synaptinemal complex.     

The synaptinemal complex in yeast

The occurrence of a synaptinemal complex in yeast was demonstrated by an electron-microscopical examination of meiotic prophase I. The complex is formed during the interval between the maximum of meiotic DNA synthesis and the first division.     

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.     

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.     

Karyosphere of the oocytes of the common frog, Rana temporaria]

Karyospheres of ca. 200 mcm in diameter were isolated from the common frog oocytes of definitive size. An electron microscope study has revealed in the karyosphere fibrillar nucleoli and micronucleoli, modified synaptinemal complexes sometimes connected with chromatin and fibrillar material containing a great number of, mostly atypical, pore complexes resembling those of nuclear membrane and forming "pseudomembranes". An electrophoresis of the isolated karyosphere has revealed 12 distinct protein bands, of 3 which correspond to the protein triplet characteristic of the nuclear matrix and the rest 9 represent high molecular weight components with the molecular weight from 130 to 200,000 D.     

STUDIES ON THE FIXATION OF ARTIFICIAL AND BACTERIAL DNA PLASMS FOR THE ELECTRON MICROSCOPY OF THIN SECTIONS

The process of fixation of DNA-containing plasms is investigated by macroscopical and electron microscopical observations on solutions of DNA, nucleohistones, as well as on bacterial nuclei. The following treatments were found to produce a gelation of a solution of DNA or nucleohistones: (a) OsO₄ fixation at pH 6 in the presence of amino acids (tryptone) and Ca⁺⁺. (b) Exposure to aqueous solutions of uranyl acetate. (c) Exposure to aqueous solutions of indium chloride. Observed in the electron microscope, these gels show a fine fibrillar material. From experiments in which solutions of DNA or nucleohistones are mixed with bacteria and treated together, it is concluded that the behavior of the bacterial nucleoplasm is similar to that of the DNA solutions. The appearance of birefringence indicates that uranyl acetate and indium chloride produce an orientation of the molecules of a DNA solution during gelation. Bacterial chromosomes fixed by these agents also show a certain order, while those fixed by the OsO₄-amino acid-Ca⁺⁺ formula do not. Whether or not the order can be considered to be artificial is discussed, and a tentative conclusion is presented: (a) Uranyl acetate may induce artificial order. (b) Fixatives which do not gel DNA probably result in the grossest artifacts. (c) OsO₄ fixation at pH 6 in the presence of amino acids (tryptone) and Ca⁺⁺ may give the most accurate preservation of the in vivo disposition of DNA (RK⁺ fixation).     

DNA-DEPENDENT FORMATION OF THE SYNAPTINEMAL COMPLEX AT MEIOTIC PROPHASE

Evalaution of microsporocytes cultured during discrete periods of meiotic prophase in the presence of deoxyadenosine, an inhibitor of DNA synthesis, indicate that: (1) late leptonema or early zygonema DNA synthesis is required to initiate the formation of the synaptinemal complex; (2) DNA synthesized during late zygonema is necessary for the disjunction of the paired homologs at diplonema; and (3) DNA synthesis in pachynema is a requisite for normal anaphase II separation of sister chromatids.     

A CORRELATED HISTOCHEMICAL AND ELECTRON MICROSCOPIC STUDY OF THE INTRANUCLEAR CRYSTALLINE AGGREGATES OF ADENOVIRUS (RI-APC VIRUS) IN HELA CELLS

HeLa cells in tissue cultures infected with types 3, 4, or 7 of adenovirus (RI-APC virus) were studied in order to correlate certain histochemical and electron microscopic findings. Adjacent thin (ca. 0.05 µ) and thick (2–4 µ) sections of osmium-fixed, methacrylate-embedded cells were cut; by mapping the sections the same cells could be identified with both the electron and the light microscope. Intranuclear crystalline aggregates seen with the electron microscope to be composed of ordered arrays of viral particles were found by means of the Feulgen reaction to contain DNA. DNA is therefore assumed to be a constituent of the viral particle. The virus appeared to develop from an osmiophilic Feulgen-negative matrix. Displacement of nuclear chromatin occurred during this process. A Feulgen-azure staining method was found to permit clear distinction between viral and nuclear (host) DNA in thick sections.     

Nuclear structures in the telotrophic ovarioles of nocturnal ground beetles (Tenebrionidae, Polyphaga). III. The oocyte nucleus. Electron microscopic data]

The fine structural organization of nuclei was studied in the growing oocytes of Blaps lethifera, B. mortisaga and Gnaptor spinimanus. In the beginning of diplotene the nuclei contain primary fibrillar nucleoli and numerous electron dense globules dispersed all over the nucleus; the loose chromosome material (lampbrush chromosomes) is distributed all over the nucleus. With the oocyte growth the chromosomes are spiralized and join into the karyosphere. A capsule of fibrous material forms around the karyosphere. The karyosphere nucleoli appear on the chromosomes and, then, move to the capsule region and outside its limits, to the nuclear envelope. They are fibrillar and non-active with respect to RNA synthesis. The fibrous material of the capsule is represented by strands which consist of bundles of cross-striated filaments. These latter contact directly with the chromosomes in the karyosphere and with the surface of the karyosphere nucleoli. The fibrillar-granular bodies are distributed along the strands in the capsule; they contain both RNA and DNA. The nature of extrachromosomal DNA in the karyosphere capsule and its participation in the formation of the capsule material are discussed. A suggestion is put forward on the similarity of the capsule strands with the modified central elements of synaptinemal complex.     

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.     

THE USE OF SPECIFIC ANTIBODY IN ELECTRON MICROSCOPY : III. Localization of Antigens by the Use of Unmodified Antibody

Antibody staining was observed in the electron microscope by means of untagged antibody and osmium fixation. The antibody was visualized as a change in morphology due to its deposition on the antigenic structures. Glycerinated chicken breast muscle was stained with antimyosin, anti-H-meromyosin, and antiactin. The staining patterns obtained by electron microscopy were consistent with those previously demonstrated by fluorescence microscopy. A second method was used for confirmation of antibody staining. This consisted of extraction of unstained portions of the sarcomere with 0.6 M potassium iodide, 10^-4 M adenosine triphosphate solution. Stained regions of the sarcomere remained intact because of insolubility of the combined antigen and antibody.     

Application of automatic image analysis for morphometric studies of peroxisomes stained cytochemically for catalase

Summary The feasibility of the application of a television-based image analyzer, the Texture Analysis System (TAS, Leitz Wetzlar, FRG) in conjunction with a light microscope for morphometric studies of hepatic peroxisomes has been investigated. Rat liver peroxisomes were stained with the alkaline-DAB method for localization of catalase and semi-thin (0.25 and 1 m) sections of plastic-embedded material were examined under an oil immersion objective. The TAS detected the peroxisomal profiles selectively and determined their morphometric parameters automatically. The same parameters were obtained also by morphometric analysis of electron micrographs from the same material. The volume density of peroxisomes determined by TAS in semithin sections of normal liver, after correction for section thickness, is quite close to the corresponding value obtained by morphometry of electron micrographs. The difference is approximately 20%. In animals treated with the hypolipidemic drug bezafibrate, which causes proliferation of peroxisomes, TAS detected readily the increase in volume density of peroxisomes in semithin sections. In comparison with electron microscopy, however, the light-microscopic approach seems to underestimate the proliferation. The lower resolution of the light microscope and overlapping of neighbouring particles in relatively thick sections used for lightmicroscopic analysis may account for the differences.The present study has demonstrated the usefulness of automatic image analysis in conjunction with selective cytochemical staining of peroxisomes for morphometry of this organelle in rat liver. The light-microscopic approach is not only faster but is also extremely economical by obviating the use of an electron microscope.     

CORTICAL GRANULES PERSISTING IN GERM CELLS OF XENOPUS LAEVIS AFTER THE CLEAVAGE STAGES

Cortical granules were demonstrated, in two successive Epon sections (0.7 μm thick) stained with PAS reagent and the triple staining method respectively, to persist beyond the cleavage stages of development to the tadpole stages in Xenopus laevis. They were also examined by electron microscope. The granules which are similar both cytochemically and ultrastructurally to the cortical granules of the unfertilized eggs were observed not only in germ cells, pPGCs and PGCs, but also in somatic cells at all the stages examined. An ultrastructural similarity between the granules found in the PGCs at the tadpole stages and chromatoid body was discussed.     

DNA measurements of cervical epithelial cells in combination with scanning electron microscopy

A method is described in which the surface morphology of benign and malignant cervical cells is investigated with a combined light microscope-scanning electron microscope, after the measurement of the DNA content of each individual cell in the same instrument. The suspect cells can thus be identified by an increased aneuploid DNA content (greater than 5C) and not primarily by morphology. The DNA content was measured, after a quantitative acriflavine-Feulgen staining, by using a microphotometer attached to the combined microscope. It was found that the suspect cells show a different surface morphology compared to normal cells from a benign specimen.     

KERATIN SYNTHESIS DURING DEVELOPMENT OF THE EMBRYONIC CHICK FEATHER

The synthesis of keratin proteins during development of the embryonic chick feather was studied by quantitative gel electrophoresis of the reduced and carboxymethylated proteins. The results demonstrated a coordinated synthesis of the major keratin proteins, during and after the onset of keratin synthesis. The results from gel electrophoresis correlated well with electron microscope visualization or keratin fibrils in the developing feathers. Autoradiography at the electron microscope level indicated that the feather cells lose the ability to synthesize DNA before keratin synthesis begins, but retain the ability to synthesize RNA after keratin synthesis begins.     

THE USE OF BISMUTH AS AN ELECTRON STAIN FOR NUCLEIC ACIDS

Evidence is presented to show that bismuth combines in vitro with the phosphate of nucleic acids in a manner similar to its reaction with inorganic phosphate. When tested under similar conditions, protein exhibited no attraction for bismuth. The results of the in vitro experiments, which are of interest within themselves, may be indirectly applicable to in vivo staining. Dividing cells of onion root tips were fixed in OsO₄, stained with bismuth, and examined in the electron microscope. The electron opacity of cell structures known to contain nucleic acids was enhanced by bismuth, while organelles known to lack appreciable quantities of DNA or RNA showed little, if any, change. Bismuth is particularly effective as a stain for the chromatin material during interphase and for the chromosomes during division.     

DNA measurements of cervical epithelial cells in combination with scanning electron microscopy

Summary A method is described in which the surface morphology of benign and malignant cervical cells is investigated with a comined light microscope-scanning electron microscope, after the measurement of the DNA content of each individual cell in the same instrument. The suspect cells can thus be identified by an increased aneuploid DNA content (>5C) and not primarily by morphology. The DNA content was measured, after a quantitative acriflavine-Feulgen staining, by using a microphotometer attached to the combined microscope. It was found that the suspect cells show a different surface morphology compared to normal cells from a benign specimen.In honour of Prof. P. van Duijn