A method for producing synaptonemal complex complements in lily and mouse

A whole-mount procedure for producing pachytene synaptonemal complex complements of Lilium longiflorum was developed. The method involves swelling of the meiotic nuclei followed by nonionic detergent lysis of the nuclear envelope. This technique adequately spreads out the long lily chromosomes while producing only minimal distortion of the chromosomal axes. The ultrastructure of the synaptonemal complex is normal, and the chromatin remains closely associated with the synaptonemal complex. The procedure also was used successfully to produce pachytene synaptonemal complex preparations of mouse chromosomes. In the mouse, the centromeric heterochromatin remains associated with the synaptonemal complex, but the euchromatin is more widely dispersed.     

Solving a meiotic LEGO puzzle: transverse filaments and the assembly of the synaptonemal complex in Caenorhabditis elegans

The structure of the meiosis-specific synaptonemal complex, which is perhaps the central visible characteristic of meiotic prophase, has been a matter of intense interest for decades. Although a general picture of the interactions between the transverse filament proteins that create this structure has emerged from studies in a variety of organisms, a recent analysis of synaptonemal complex structure in Caenorhabditis elegans by Schild-Prüfert et al. (2011) has provided the clearest picture of the structure of the architecture of a synaptonemal complex to date. Although the transverse filaments of the worm synaptonemal complex are assembled differently then those observed in yeast, mammalian, and Drosophila synaptonemal complexes, a comparison of the four assemblies shows that achieving the overall basic structure of the synaptonemal complex is far more crucial than conserving the structures of the individual transverse filaments.     

The central region of the synaptonemal complex revealed in three dimensions

The synaptonemal complex plays a key role in pairing of homologous chromosomes during meiosis. Its gross structure was already known by conventional electron microscopy, but only recently has it been possible to reveal the synaptonemal complex in three dimensions at higher resolution by electron microscope tomography. As the molecular analysis of meiosis is developing rapidly, a more thorough understanding of the principal organization of the synaptonemal complex is essential.     

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.     

The synaptonemal complex as part of the nuclear matrix of the flour moth, Ephestia kuehniella

A nuclear matrix fraction was prepared from ovaries of the achiasmatic flour moth, Ephestia kuehniella, by removal of the chromatin, using detergent treatment of homogenized ovaries or dissected ovary tips followed by DNase digestion and high salt extraction. Removal of DNA and histones from the nuclei was demonstrated by Feulgen staining and polyacrylamide gel electrophoresis (PAGE), respectively. By light microscopy, ribbon-like structures similar in dimension to the synaptonemal complex were observed in the oocyte after digestion of the chromosomes. Electron microscopic examination of matrix preparations of pachytene cells showed a defined synaptonemal complex structure with both lateral and central elements. Such structures were not found in either the fully differentiated nurse cells or in follicle cells which were exposed to the same preparative technique concurrently. However, in early post-pachytene nurse cells the typical polycomplex structures, formed in these cells from the synaptonemal complex, were found in nuclear matrix preparations. The results suggest an association of synaptonemal complexes with the nuclear matrix.     

Distribution of MLH1 foci on the synaptonemal complexes of chicken oocytes

The frequency and distribution of the mismatch repair protein MLH1 was analyzed on synaptonemal complex spreads of chicken oocytes using indirect immunofluorescence. MLH1 foci appeared in late zygotene and their number remains constant throughout pachytene. The average number of foci on autosomal synaptonemal complexes (65.02 +/- 4.02) is in agreement with the number of chiasmata estimated from lampbrush chromosomes. The distribution of foci along the synaptonemal complexes is shown to be nonrandom and nonuniform in terms of the distances between them. It is concluded that MLH1 foci are good markers of crossing over in bird (chicken) meiocytes.     

Ultrastructural analysis of maize pachytene karyotypes by three dimensional reconstruction of the synaptonemal complexes

Aldehyde fixation followed by staining with phosphotungstic acid produces differential contrast between the synaptonemal complex and the chromatin of maize pachytene bivalents. Centromeres, heterochromatic knobs and large chromomeres are easily recognised. With this and other staining techniques the nucleolus organizer region can be differentiated into two components. — Microsporocyte nuclei at pachytene were serially sectioned and all ten bivalents reconstructed in five nuclei. An idiogram was derived from the mean chromosome (= synaptonemal complex) lengths, the arm ratios, positions of knobs and the nucleolus organizer region. The idiogram agrees well with that published from light microscopic analyses. However, bivalent lengths are only two thirds of those observed by light microscopy of squash preparations. Many telomeres of the bivalents are connected via chromatin to the nuclear envelope, but a varying number of free bivalent ends are observed in all five reconstructed nuclei. — Bivalents heterozygous for inversion 3b were reconstructed. In the presence of abnormal chromosome 10 (K10) the lateral components of the synaptonemal complex of chromosome 3 formed a typical inversion loop, while in one of the nuclei having no K10 the two lateral components of the long arms of chromosome 3 remained unpaired in the region of inversion heterozygosity. The presence of K10, which increases crossing-over frequencies and promotes intimate pairing at the light microscopic level, was thus found to permit formation of complete synaptonemal complexes in the inverted region. The extra terminal portion of the K10 chromosome folded back on itself and formed a morphologically normal synaptonemal complex in this — possibly non-homologously paired — region. The chromatin of centromeres and knobs from different bivalents were sometimes found to fuse, but the synaptonemal complexes transversing the fused centromeres or knobs retained their individuality.     

Alteration of meiotic chromosomal pairing and synaptonemal complexes by cycloheximide

When cells are exposed to cycloheximide during the synaptic period of meiotic prophase, the structure of the synaptonemal complex is markedly altered. The bulk of the lateral component is removed. When lily zygotene microsporocytes are subsequently transferred into a culture medium free from cycloheximide, normal synaptonemal complexes are again seen. Modification of the structure of the synaptonemal complex by treatment with cycloheximide for 4 days has little permanent effect on meiosis except at late zygonema or early pachynema. Treatment at this time produces meiocytes in which no synaptonemal complexes reform. When these cells proceed into diplotene and diakinesis they are devoid of chiasmatic chromosomes. The data suggest that the synaptonemal complex is essential if chiasmata are to be formed, and that a unique period exists when the formation can be interrupted.This work was supported by grants from the National Science Foundation (GB 5173X and GB 6476) and the National Institutes of Health (GM 16882).     

Synaptonemal complex analysis of mole rats (Spalax ehrenbergi): unusual polymorphisms of chromosome 1

Two unusual structural polymorphisms in the largest chromosomal pair of the Israeli mole rat, Spalax ehrenbergi, were analyzed from surface-spread and silver-stained preparations of synaptonemal complexes. A C-band negative polymorphism for the length of the 1p arm was visible as axial length differences during late zygonema and early pachynema. This region underwent synaptic adjustment resulting in a fully paired, mid-pachytene synaptonemal complex with equalized axial lengths. The somatically variable and nonargentophilic secondary constriction in the 1q arm was evident as a distinct silver-stained thickening along the synaptonemal complex. Presence of this structure on the synaptonemal complex varied both among individuals and among cells within individuals. The intraindividual variation of this region is hypothesized to represent differential biochemical activity with its cellular visualization being regulated in a manner similar to that of nucleolus organizer regions.     

Expression of the Meiosis-Specific Synaptonemal Complex Protein 1 in a Heterologous System Results in the Formation of Large Protein Structures

The synaptonemal complex is a meiosis-specific structure essential for synapsis of homologous chromosomes. The synaptonemal complex protein 1 (SCP1) is a major constituent of the transversal filament, a fibrous structure that connects the central element of the synaptonemal complex with the two lateral elements. The SCP1 protein forms filamentous dimers with the two molecules that have the same polarity, with the C-termini being anchored in the lateral elements and the N-termini reaching into the central element. We investigated whether the SCP1 protein can take part in the formation of higher order protein structures by expressing it in a heterologous system. We find that expression of SCP1 in Swiss-3T3 fibroblast cells results in the formation of large protein structures. These protein structures resemble a higher order protein structure produced by overexpression of a yeast transversal filament protein in meiotic cells. Our results show that SCP1 is a structural protein and that it most likely is directly involved in the assembly of the synaptonemal complex.     

Synaptonemal complex karyotype of Eimeria tenella

In most organisms, biological variability rests on the behaviour of the chromosomes in the meiotic context. Despite the importance of meiosis, very little is known about the meiotic behaviour of the Eimeria chromosomes. The aim of the present study is to describe the standard synaptonemal complex karyotype from Eimeria tenella oocyst spreads by electron microscopy. For that purpose, complete sets of pachytene synaptonemal complexes were obtained and the morphological pachytene karyotype was determined. The authors used a previously reported method that overcomes the difficulty of the extreme resistance of protozoan oocysts to disruption and permits the release of intact meiotic chromosomes. The chromosomes were selected under a light microscope and those selected were stained with phosphotungtic acid and studied by transmission electron microscopy. The authors confirmed 14 chromosomes, which were observed as synaptonemal complexes, and the karyotype was constructed by arranging synaptonemal complexes according to their relative lengths and kinetochore position. Components of the synaptonemal complex, lateral elements, central element, recombination nodules and kinetochore were observed. Measures of the kynetochore, width of the synaptonemal complex, diameter of the recombination nodule and length of the telomeres are given. Minimal and no significant differences were found between measures of chromosomes isolated from different Eimeria tenella strains. To the best of our knowledge, the present investigation for the first time identifies and describes the morphological characteristics of the synaptonemal complex of Eimeria tenella during the meiosis that occurs within the oocysts. In addition, the authors provide evidence of the presence of recombination nodules, suggesting that the recombination process may play an important role in the molecular evolution of this parasite.     

Synaptonemal complex formation: where does it start?

The synaptonemal complex is a prominent, evolutionarily conserved feature of meiotic prophase. The assembly of this structure is closely linked to meiotic recombination. A recent study in budding yeast reveals an unexpected role in centromere pairing for a protein component of the synaptonemal complex, Zip1. These findings have implications for synaptonemal complex formation.     

The staining of the synaptonemal complex for light microscopic study in the mouse

Demonstration of the synaptonemal complex for light microscopy has until now been based on staining with silver. After fixation at pH 9-10 it is also possible to visualize synaptonemal complexes with several nonspecific protein stains such as Coomassie brilliant blue, Giemsa, fast green, light green and Stains All. Although staining with silver gives the best contrast between synaptonemal complexes and the background, the other dyes have a number of advantages, such as more even staining, easy extractability, and lower cost than silver.     

The Staining of the Synaptonemal Complex for Light Microscopic Study in the Mouse

Demonstration of the synaptonemal complex for light microscopy has until now been based on staining with silver. After fixation at pH 9-10 it is also possible to visualize synaptonemal complexes with several nonspecific protein stains such as Coomassie brilliant blue, Giemsa, fast green, light green and Stains All. Although staining with silver gives the best contrast between synaptonemal complexes and the background, the other dyes have a number of advantages, such as more even staining, easy extractability, and lower cost than silver.     

Meiosis in Bombyx mori females.

Crossing over is absent in oocytes of the silkworm, Bombyx mori. Synaptonemal complexes are present during pachytene between the paired chromosomes. At leptotene, lateral components of the synaptonemal complex are attached in a bouquet to a limited region of the nuclear envelope. Before completion of lateral components, synaptonemal complex formation begins at the nuclear envelope. With synaptonemal complex formation proceeding from both ends bivalents occasionally become interlocked. After pairing is completed, the bouquet arrangement is dissolved possibly as a result of a flow of the inner membrane of the nuclear envelope thereby separating the telomeres. After the telomeres are released from the nuclear envelope, material is deposited onto the lateral components of the synaptonemal complex. The modified synaptonemal complexes are retained by the bivalents until metaphase I. It is suggested that these modified synaptonemal complexes substitute for chiasmata in order to ensure regular disjunction of homologous chromosomes in the absence of crossing over.     

Sequential analysis of synaptonemal complexes in the repopulating spermatocytes of Rattus norvegicus after restricting the germ cell population to spermatogonia by gossypol treatment

Synaptonemal complexes of the repopulating spermatocytes of male rats were analyzed day by day using silver-stained surface spread nuclei between 8 and 25 days after restricting the germ cell population to spermatogonia by treatment of gossypol acetic acid at 30 mg/kg body weight/day for 70 days. The method allowed sequential analysis of male meiotic prophase on successive days after the last day of treatment. The leptotene cells appeared on day 11 and were characterized by a network of lateral elements and large nucleolar bodies in a diffuse mass. On day 13 the unpaired lateral elements and short stretches of synaptonemal complexes characteristic for zygotene could be seen. Pachytene nuclei showing 20 autosomal synaptonemal complexes and XY axes appeared on day 15. The diplotene cells were defined on day 22 by the loss of a complete synaptonemal complex set and by the appearance of disjoined lateral elements and persistent segments of synaptonemal complexes.     

Synaptonemal complex stability depends on repressive histone marks of the lateral element-associated repeat sequences

The synaptonemal complex (SC) is the central key structure for meiosis in organisms undergoing sexual reproduction. During meiotic prophase I, homologous chromosomes exchange genetic information at the time they are attached to the lateral elements by specific DNA sequences. Most of these sequences, so far identified, consist of repeat DNA, which are subject to chromatin structural changes during meiotic prophase I. In this work, we addressed the effect of altering the chromatin structure of repeat DNA sequences mediating anchorage to the lateral elements of the SC. Administration of the histone deacetylase inhibitor trichostatin A into live rats caused death of cells in the pachytene stage as well as changes in histone marks along the synaptonemal complex. The most notable effect was partial loss of histone H3 lysine 27 trimethylation. Our work describes the epigenetic landscape of lateral element-associated chromatin and reveals a critical role of histone marks in synaptonemal complex integrity.     

Developing and testing a method of in silico identification and characterization of meiotic DNA

A method of in silico search for specific repetitive DNA sequences related to the synaptonemal complex (meiDNA) in mammalian genomes was developed. A study of the distribution of these repeats over chromosomes revealed their scarcity on the Y chromosome and a decrease in recombination frequency in regions enriched in meiDNA. The results are discussed in context of the model of the looplike meiotic chromosome organization during the formation of the synaptonemal complex.     

SCF-Fbxo42 promotes synaptonemal complex assembly by downregulating PP2A-B56

Meiosis creates genetic diversity by recombination and segregation of chromosomes. The synaptonemal complex assembles during meiotic prophase I and assists faithful exchanges between homologous chromosomes, but how its assembly/disassembly is regulated remains to be understood. Here, we report how two major posttranslational modifications, phosphorylation and ubiquitination, cooperate to promote synaptonemal complex assembly. We found that the ubiquitin ligase complex SCF is important for assembly and maintenance of the synaptonemal complex in Drosophila female meiosis. This function of SCF is mediated by two substrate-recognizing F-box proteins, Slmb/βTrcp and Fbxo42. SCF-Fbxo42 down-regulates the phosphatase subunit PP2A-B56, which is important for synaptonemal complex assembly and maintenance.     

Histone electrophoretic pattern in the characterization of synaptonemal complexes

The presence of a stoechiometric electrophoresis pattern of histones in carefully isolated synaptonemal complexes is reported. The use of this pattern is suggested as an internal standard of synaptonemal complex purification, in addition to the more generally used electron micrographs. This is especially useful in experiments leading to the characterization of the protein components of SCs. The use of mice of an age at which pachytenes predominate (90%) in the prophase-cell population is also advantageous to improve the final yield in synaptonemal complexes.