The attachment of the synaptonemal complex to the nuclear envelope

An ultrastructural and cytochemical analysis of the attachment sites of the synaptonemal complex to the nuclear envelope in rat spermatocytes is presented. The association is formed by the ends of the lateral elements in the form of a plate associated with the inner membrane, as well as by formations of fibrillar character found in the perinuclear space and in the cytoplasm adjacent to the outer membrane. By means of uranyl-EDTA-lead staining, both the area of association between the lateral elements and the fibrillar formations can be strongly stained, while the same areas appear unstained when the tissue has been previously treated with RNase. Staining with alcoholic PTA also produces a strong staining effect at the level of the attachment plates of the lateral elements, and very weak staining of the perinuclear space and of the formations in the adjacent cytoplasm. On the basis of this analysis of both the associations of autosomes and of the XY pair, we suggest that this associating structure consists partly of RNA and partly proteins, presumably histones.     

The pachytene synaptonemal complex complement of the cat

Surface spreads of pachytene spermatocyte nuclei from two cats were used to construct a synaptonemal complex karyotype for the cat. It was possible to recognise the 18 autosomal synaptonemal complexes by reference to a published light microscopic banded somatic karyotype. Some variation from the somatic karyotype was noted, presumably as a result of differential contraction during prophase I. The X and Y chromosome axes were joined by a synaptonemal complex in many of the nuclei, but the structure of the unpaired portion of the X axis was quite variable. In some nuclei it was highly contracted, while in others it was extended and often was split into two or more axes. In most nuclei the autosomal synaptonemal complexes had numerous axial twists.     

Cytochemical and ultrastructural studies on the synaptonemal complex of rat spermatocytes

The effects of several dehydration treatments on the synaptonemal complex (SC), histone solubility in 2.0 M NaCl, and histone-DNA interaction in unfixed rat spermatocytes were evaluated. Freeze substitution with ethanol or dehydration with polyethylene glygol resulted in loss of the SC, preservation of histone solubility and DNA-histone salt linkages. Dehydration with ethylene glycol or hexylene glycol resulted in preservation of SC with a clear delineation of attachment of the chromatin fibrils to the lateral elements, but a loss of histone solubility and histone-DNA linkages. Dehydration to a fifty percent concentration with glycerol with completion of dehydration with ethylene glycol had the same effect but also resulted in an even distribution of chromatin fibrils. Dehydration with glycerol alone resulted in clumping of chromatin and loss of SC structure, histone solubility and histone-DNA linkages. Partial dehydration to a fifty percent concentration with these three solvents followed by freeze substitution with ethanol resulted in the loss of SC structure and histone solubility but the preservation of histone-DNA linkages. It is likely that these nonaqueous solvents affected the histone hydrophobic groups and thereby altered histone conformation and interactions. These alterations, depending on the treatment used, resulted in the loss or preservation of SC, histone solubility and histone-DNA interactions thereby indicating that the hydrophobic interactions of the histones are crucial for the preservation of these feature of meiotic chromosomes. These results also demonstrate that neither does the preservation of the histone-DNA salt linkages suffice for the preservation of the SC nor does their disruption necessarily result in its loss. The lysine-rich histones, particularly that one unique to meiotic cells, may through their interactions play a crucial role in SC structure.     

Differential distribution and association of repeat DNA sequences in the lateral element of the synaptonemal complex in rat spermatocytes

The synaptonemal complex (SC) is an evolutionarily conserved structure that mediates synapsis of homologous chromosomes during meiotic prophase I. Previous studies have established that the chromatin of homologous chromosomes is organized in loops that are attached to the lateral elements (LEs) of the SC. The characterization of the genomic sequences associated with LEs of the SC represents an important step toward understanding meiotic chromosome organization and function. To isolate these genomic sequences, we performed chromatin immunoprecipitation assays in rat spermatocytes using an antibody against SYCP3, a major structural component of the LEs of the SC. Our results demonstrated the reproducible and exclusive isolation of repeat deoxyribonucleic acid (DNA) sequences, in particular long interspersed elements, short interspersed elements, long terminal direct repeats, satellite, and simple repeats. The association of these repeat sequences to the LEs of the SC was confirmed by in situ hybridization of meiotic nuclei shown by both light and electron microscopy. Signals were also detected over the chromatin surrounding SCs and in small loops protruding from the lateral elements into the SC central region. We propose that genomic repeat DNA sequences play a key role in anchoring the chromosome to the protein scaffold of the SC. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Evolution of the synaptonemal complex in Helix aspersa spermatocytes

In spermatocytes of Helix aspersa, the structure of the synaptonemal complexes undergoes changes in the course of the pachytene, the lateral elements being transformed into wide bands of lesser density than the chromatin. By using the uranyl-EDTA-lead sequence, which preferentially stains RNA, the lateral elements can be made to appear positive in the early pachytene while the corresponding areas, which become wider and more diffuse, are positive during late pachytene. — Apparently, the lateral elements do not persist in the diplotene and remnants of the central element can occasionally be observed. Using the uranyl-EDTA-lead method reveals some positively stained material surrounding the chromatin, mostly granular in appearance, which is observed in late pachytene and attains its maximum amount during diplotene. Several aspects of these observations are here discussed.     

Changes of the synaptonemal complex at the end of pachytene

Summary Observations on the changes of the synaptonemal complex at the end of pachytene and in diplotene are reported. The synaptonemal complex disintegrates in diplotene by progressive disjoining of the lateral arms. The structures of the pairing space disappear. A residual piece of the synaptonemal complex is interpreted as a transitional stage of the process of disintegration. The lateral arms form single threads which disappear at the end of diplotene. No doubleness of the lateral arms can be detected, except a splitting in 3–4 laminae in the region near the fixation points at the nuclear envelope. The lateral arms do not separate in subunits at the end of the meiotic prophase. Chiasmata can not be recognized.

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Zusammenfassung Beobachtungen über die Veränderungen des synaptonemalen Komplexes am Ende des Pachytäus und im Diplotän werden mitgeteilt. Im Diplotän löst sich der synaptonemale Komplex durch fortschreitende Trennung der lateralen Arme. Die Strukturen des Paarungs-Raumes verschwinden. Ein Rest des synaptonemalen Komplexes wird als Übergangsstadium im Laufe des Desintegrations-Prozesses gedeutet. Die lateralen Arme bilden Einzelstränge, die am Ende des Diplotäns verschwinden. Eine Verdopplung des lateralen Armes kann nicht entdeckt werden; lediglich in der Nähe des Fixationspunktes an der Nuclearmembran findet sich eine Aufspaltung in 3–4 Lamellen. Am Ende der meiotischen Prophase trenne sich demnach die lateralen Arme nicht in Untereinheiten. Chiasmata sind nicht erkennbar.

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This work was supported by the Deutsche Forschungsgemeinschaft.     

Immunochemical detection of Z-DNA in rat pachytene spermatocytes

Rat testicular nuclei have been probed for the presence of Z-DNA conformation by employing indirect immunofluorescence technique using anti-Z-DNA antibodies. Pachytene nuclei, in which meiotic recombination takes place, showed brighter fluorescence than the premeiotic and postmeiotic spermatogenic nuclei. Moreover, utilizing a novel chromatin immunoblotting technique, Z-DNA conformation was found to be enriched in the poly(ADP-ribosyl)ated chromatin domains of the pachytene nucleus.     

XY pair associates with the synaptonemal complex of autosomal male-sterile translocations in pachytene spermatocytes of the mouse (Mus musculus)

Analysis of the chromosome behaviour at pachytene has been performed by means of the silver staining technique visualizing the synaptonemal complexes (SCs) in male mice heterozygous for the male-sterile translocations T(5;12)31H, T(16;17)43H and T(7;19)145H, respectively. The T(9;17)138Ca male heterozygotes and T43H/T43H homozygous males were used as fertile controls. The sterile mice displayed a high frequency (about 60%) of pachytene spermatocytes with autosomal translocation configuration located in close vicinity of the XY pair. The dense round body (XAB), normally located near the X-chromosome axis in fertile males, exhibited abnormal affinity to translocation configuration in the sterile translocation heterozygotes. The incomplete synapsis of autosomes involved in translocation configuration was observed in more than 70% of the pachytene spermatocytes with the male-sterile translocations but in less than 20% of the cells from T138Ca fertile male.s. A hypothesis relating the spermatogenic arrest of carriers of male-sterile rearrangements to the presumed interference with X chromosome inactivation in male meiosis is discussed.     

Specific nuclear envelope transmembrane proteins can promote the location of chromosomes to and from the nuclear periphery

Background

Different cell types have distinctive patterns of chromosome positioning in the nucleus. Although ectopic affinity-tethering of specific loci can be used to relocate chromosomes to the nuclear periphery, endogenous nuclear envelope proteins that control such a mechanism in mammalian cells have yet to be widely identified.

Results

To search for such proteins, 23 nuclear envelope transmembrane proteins were screened for their ability to promote peripheral localization of human chromosomes in HT1080 fibroblasts. Five of these proteins had strong effects on chromosome 5, but individual proteins affected different subsets of chromosomes. The repositioning effects were reversible and the proteins with effects all exhibited highly tissue-restricted patterns of expression. Depletion of two nuclear envelope transmembrane proteins that were preferentially expressed in liver each reduced the normal peripheral positioning of chromosome 5 in liver cells.

Conclusions

The discovery of nuclear envelope transmembrane proteins that can modulate chromosome position and have restricted patterns of expression may enable dissection of the functional relevance of tissue-specific patterns of radial chromosome positioning.     

Development of the synaptonemal complex of two types of pachytene in oocytes and spermatocytes of Carausius morosus Br. (Phasmatodea)

During oogenesis of the parthenogenetic stick insect Carausius morosus (2n =61+XXX) pachytene is followed by a duplication of the desynapsed chromosomes, which results in a second type of pachytene (tetrapachytene) consisting of paired sister chromosomes (autobivalents). Electron microscopic studies on sections revealed that synaptonemal complexes (SCs) are formed during tetrapachytene only. This means that the parthenogenetically produced progeny have the genetic constitution of the mother. During spermatogenesis of rare fatherless males (2n=61 + XX) and intersexes (2n=61 +XXX) either an incomplete chromosome doubling (demonstrated by up to 10% additional DNA synthesis) or a complete chromosome doubling takes place during zygotene. EM studies on sections and spreads of germ cells of the first type of meiosis showed that unpaired lateral components (LCs), pieces of SCs and complete SCs are formed during pachytene only, the sex chromosomes being represented by unpaired thickened LCs. The incomplete SC formation reflects the complex heterozygosity of the chromosome complement. In the duplicated type SCs are found in tetrapachytene nuclei only; they are wider than the SCs in oocytes. The sex chromosome bivalents are represented by unpaired thickened LCs or partially paired LCs, in which localized chiasma formation was found. The idea is discussed that formation of SCs does not take place as long as a germ cell has been programmed either to replicate or to be able to replicate its chromosomes and that consequently SCs can be formed only once per meiosis.     

Biochemical and ultrastructural analyses of the synaptonemal complex in mammalian spermatocytes

In order to study the molecular organization of synaptonemal complex (SC), a preparative method for isolation of relatively purified SC from rat, mouse and hamster testes was elaborated which involves isolation of SC-containing (pachytene) nuclei, their lysis, DNAase digestion of DNA and fractionation of nuclear elements by the discontinuous sucrose density gradient centrifugation. Electron microscopy revealed a rather good preservation of the SC structure after the isolation procedure. Effects of the dissociating agents on the SC structural integrity were studied. It has been demonstrated that the treatment with 2M NaCl, Triton X-100, sodium deoxycholate, 6M urea, and with a buffer containing 2% SDS and 5% mercaptoetthanol does not lead to a complete SC dissociation, though it results in some structural chanes. Possible reasons of the high resistance of SC to dissociating treatment are discussed.     

Chromatin organization in relation to the nuclear periphery

In the limited space of the nucleus, chromatin is organized in a dynamic and non-random manner. Three ways of chromatin organization are compaction, formation of loops and localization within the nucleus. To study chromatin localization it is most convenient to use the nuclear envelope as a fixed viewpoint. Peripheral chromatin has both been described as silent chromatin, interacting with the nuclear lamina, and active chromatin, interacting with nuclear pore proteins. Current data indicate that the nuclear envelope is a reader as well as a writer of chromatin state, and that its influence is not limited to the nuclear periphery.     

Observations on the synaptonemal complex in Armenian hamster spermatocytes by light microscopy

Using the silver staining technique, in somatic and meiotic chromosomes of the Armenian hamster (Cricetulus migratorius), it is possible to stain synaptonemal complexes (SCs) and the nucleolus organizer regions (NORs) in early spermatocytes. There are five pairs of autosomes (Nos. 2, 4, 6, 7, and 8) which have terminally located NORs. Synaptonemal complexes and accessory structures present in the sex chromosomes within the sex vesicle can be easily observed using light microscopy.     

The effect of tequila in the synaptonemal complex structure of mouse spermatocytes.

The effect of tequila in the synaptonemal complex (SC) of mouse spermatocytes was determined. We tested 3 dosages (2.1, 4.2 and 8.4 g/kg) administered in a single intraperitoneal inoculation. The frequency of SC alterations was established in pachytenic nuclei 5 days after the administration using a silver impregnation technique. Three types of alterations were observed (desynapses, breaks and multiaxials) and the rate of each alteration was compared with that obtained with appropriate controls, including cyclophosphamide (CP) (150 mg/kg). The results showed a significant increase induced by tequila only in the frequency of desynapses. This damage began at the second highest dose (4.2 g/kg). The other SC alterations were in the control range. CP, however, induced a significant increase in all 3 types of SC alterations.     

Analysis of pore-rich areas on the nuclear envelope of spermatocytes

The S-antigen is a protein of photoreceptors, mainly known for its autoantigenic properties in mammals, which is widely distributed in the retina of vertebrates and in photoreceptor organs of invertebrates. Using three monoclonal antibodies specific for different epitopes of S-antigen, this study complements our previous data on retinal rods and cones and presents new results on the photosensory cells of the pineal complex. Immunoreactivity was found in (i) retinal rods and cones, (ii) cone-like and modified photoreceptor cells, and pinealocytes of the pineal organ of vertebrates, (iii) cone-like photoreceptors of the frontal organ of the frog and of the third eye of the lizard. According to the species and the antibody used, some differences were found at the level of the cellular compartments of the pineal photoreceptor cells.     

Immunocytogenetics. III. Analysis of trivalent and multivalent configurations in mouse pachytene spermatocytes by human autoantibodies to synaptonemal complexes and kinetochores

Mice heterozygous for one or more Robertsonian (Rb) translocation chromosomes have been used to analyze synaptonemal complex (SC) configurations and kinetochore arrangements in trivalents and multivalents. Rb heterozygosity without arm homologies leads to the formation of heteromorphic trivalents in meiosis I; alternating homology of the chromosome arms produces ringlike or chainlike multivalents. Immunofluorescence double-labeling with human antibodies to SCs and kinetochores was performed on surface-spread pachytene spermatocytes. Both Rb bivalents and Rb trivalents clearly showed that metacentrics possess only one centromere. In heteromorphic trivalent SCs, the nonhomologous kinetochores of the two acrocentrics were closely paired in a cis-configuration and juxtaposed opposite the kinetochore of the metacentric; the latter appeared to be an integral part of the longitudinal SC axis. Meiotic multivalents of interpopulation hybrids included up to 36 chromosome arms. In multivalent SCs, the kinetochores always lay together, with the SC arms arranged away from the central centromere cluster. The paracentromeric regions of the Rb chromosomes appeared to remain unsynapsed on both sides of the centromeres. The SC arms were often linked by end-to-end associations. Following desynapsis of the multivalent SC, the kinetochores of the Rb metacentrics showed a highly nonrandom topologic distribution within the nucleus, reminiscent of their arrangement during synapsis.