The meiotic prophase in Bombyx mori females analyzed by three-dimensional reconstructions of synaptonemal complexes

Serial sectioning followed by three dimensional reconstruction of lateral components of the synaptonemal complex have been used to follow chromosome pairing during the prophase of the achiasmatic meiotic division in the silkworm, Bombyx mori. During leptotene and early zygotene, the lateral components become attached to the nuclear envelope at a specific region, thus forming a chromosome bouquet. The attachment of lateral components to the nuclear envelope precedes the completion of the components between their attachment points. Synapsis and synaptonemal complex formation start during the period of lateral component organization in the individual nucleus. Telomeric movements on the nuclear envelope occur at two stages of the prophase: the chromosome pairing appears to be initiated by an association of unpaired ends of homologous chromosomes, the nature of this primary attraction and recognition being unknown. Secondly, the paired chromosomes become dispersed in the nucleus by shifting of attachment sites of completed synaptonemal complexes at the end of zygotene. This movement is possibly related to a membrane flow occurring during this stage. Membrane material is synthesized at the region of synaptonemal complex attachment. Later, the excess membrane material is shifted to the opposite pole where it protrudes into the lumen of the nuclei thus forming vacuoles. — Two previously undescribed features of chromosome pairing were revealed. In late zygotene, chromosome pairing and synaptonemal complex formation were frequently observed to be delayed or even prevented over a short distance by interlocking of two bivalents, both being attached to the nuclear envelope. Such interlocking of bivalents was not found in pachytene. Secondly, one nucleus was found in which two homologous chromosomes were totally unpaired while the remaining 27 bivalents were completed or in a progressed state of pairing. The lateral components of the two unpaired chromosomes had the same length and were located several microns apart, thus eliminating the possibility of a permanent association of homologous chromosomes before the onset of meiosis in Bombyx mori females. — During pachytene, one of the 8 cells belonging to the syncytial cell cluster characteristic of oogenesis continues the meiotic prophase whereas the remaining 7 cells, the nurse cells, enter a different developmental sequence, finally resulting in their degeneration. The synaptonemal complex of the oocyte develops into a sausage-like structure after pachytene by a deposition of dense material onto the lateral components, thus filling out most of the central region. The diameter of this modified synaptonemal complex reaches at least 300 nm, as compaired to a pachytene width of approximately 130 nm. Also, the length of synaptonemal complexes increases from 212 at zygotene/pachytene to at least 300 at the modified pachytene stage. In nurse cells, synaptonemal complexes are shed from the bivalents shortly after pachytene simultaneously with a condensation of the chromatin. These free synaptonemal complex fragments associate and form various aggregates, either more or less normal looking polycomplexes or various complex figures formed by reorganized synaptonemal complex subunits. Later stages have not been included in the present investigation.     

The Effect of Bovine Serum Albumin and Cytohelicase on Surface-Spread Synaptonemal Complexes of Rye (Secale Cereale)

Synaptonemal complexes of rye meiocytes were spread on plastic coated slides for electron microscopic observation. Two proteins generally used in synaptonemal complex spreading techniques, bovine serum albumin and cytobelicase, were applied separately or in combination in an isotonic protoplast medium at concentrations of 0.1-5%. At high concentrations these proteins proved to enhance notably the ultimate number of cells with synaptonemal complexes in the preparations. Also under this condition, centromere structures became stainable with silver nitrate in both the synaptonemal complexes of pollen mother cells and in interphase nuclei of other cell types. Since the true action of cytohelicase under appropriate spreading conditions was uncertain, the putative enzymatic digestion of cell walls was determined in a series of experiments using the fluorochrome calcofluor white as a stain of callose walls. Obvious breakdown of the cell walls was not observed before 8 min of treatment under standard conditions. This made it plausible that the prime effect of cytohelicase is that of a nonspecific protein interacting with the chromatin and improving the adhesion of synaptonemal complexes to the hydrophobic plastic film. The differential staining of the centromere structures in the presence of bovine serum albumin and cytohelicase probably reflects a reduced spreading of these structures due to preferential binding between these proteins and centromeric proteins.     

The effect of bovine serum albumin and cytohelicase on surface-spread synaptonemal complexes of rye (Secale cereale)

Synaptonemal complexes of rye meiocytes were spread on plastic coated slides for electron microscopic observation. Two proteins generally used in synaptonemal complex spreading techniques, bovine serum albumin and cytohelicase, were applied separately or in combination in an isotonic protoplast medium at concentrations of 0.1-5%. At high concentrations these proteins proved to enhance notably the ultimate number of cells with synaptonemal complexes in the preparations. Also under this condition, centromere structures became stainable with silver nitrate in both the synaptonemal complexes of pollen mother cells and in interphase nuclei of other cell types. Since the true action of cytohelicase under appropriate spreading conditions was uncertain, the putative enzymatic digestion of cell walls was determined in a series of experiments using the fluorochrome calcofluor white as a stain of callose walls. Obvious breakdown of the cell walls was not observed before 8 min of treatment under standard conditions. This made it plausible that the prime effect of cytohelicase is that of a nonspecific protein interacting with the chromatin and improving the adhesion of synaptonemal complexes to the hydrophobic plastic film. The differential staining of the centromere structures in the presence of bovine serum albumin and cytohelicase probably reflects a reduced spreading of these structures due to preferential binding between these proteins and centromeric proteins.     

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.     

Germ cell differentiation and synaptonemal complex formation are disrupted in CPEB knockout mice.

CPEB is a sequence-specific RNA binding protein that regulates translation during vertebrate oocyte maturation. Adult female CPEB knockout mice contained vestigial ovaries that were devoid of oocytes; ovaries from mid-gestation embryos contained oocytes that were arrested at the pachytene stage. Male CPEB null mice also contained germ cells arrested at pachytene. The germ cells from the knockout mice harbored fragmented chromatin, suggesting a possible defect in homologous chromosome adhesion or synapsis. Two CPE-containing synaptonemal complex protein mRNAs, which interact with CPEB in vitro and in vivo, contained shortened poly(A) tails and mostly failed to sediment with polysomes in the null mice. Synaptonemal complexes were not detected in these animals. CPEB therefore controls germ cell differentiation by regulating the formation of the synaptonemal complex.     

Nuclear proteins : III. The fibrillar nature of the nuclear matrix

The nuclear matrix of mouse liver nuclei was examined after extraction of the chromatin with high salt, deoxyribonuclease and Triton X-100. The residual nuclear matrix is composed of a nuclear pore-lamina complex, fibrillar nucleoli, and intranuclear matrix. Whole mount electron microscopy shows that a portion of the nuclear matrix is composed of 20–30 Å protein fibers which we call matrixin. The fibers may associate to form larger 100–300 Å fibers. When mouse testicular cells were used, intact synaptonemal complexes and the sex vesicle were intimately associated with the matrix and we suggest these structures may be composed of matrixin. SDS gel electrophoresis of the matrix shows three major polypeptides of 65 000, 67 000 and 68 000 D. Several observations suggest DNA is attached to the matrix at many sites throughout the nucleus. The matrix may play a role in the arrangement of chromatin into the chromomeres of meiotic and mitotic chromosomes.     

Quantitative immunofluorescence and immunoelectron microscopy of the topoisomerase IIα associated with nuclear matrices from wild-type and drug-resistant Chinese hamster ovary cell lines

Topo IIα is considered an important constituent of the nuclear matrix, serving as a fastener of DNA loops to the underlying filamentous scaffolding network. To further define a mechanism of drug resistance to topo II poisons, we studied the quantity of topo IIα associated with the nuclear matrix in drug-resistant SMR₁₆ and parental cells in the presence and absence of VP-16. Nuclear matrices were prepared from nuclei isolated in EDTA buffer, followed by nuclease digestion with DNase II in the absence of RNase treatment and extraction with 2 M NaCl. Whole-mount spreading of residual structures permits, by means of isoform-specific antibody and colloidal-gold secondary antibodies, an estimate of the amount of topo IIα in individual nuclear matrices. There are significant variations in topo IIα amounts between individual nuclear matrices due to the cell cycle distribution. The parental cell line contained eight to ten times more nuclear matrix–associated topo IIα than the resistant cell line matrices. Nuclear matrix–associated topo IIα from wild-type and resistant cell lines correlated well with the immunofluorescent staining of the enzyme in nuclei of intact cells. The amount of DNA associated with residual nuclear structures was five times greater in the resistant cell line. This quantity of DNA was not proportional to the quantity of topo IIα in the same matrix; in fact they were inversely related. In situ whole-mount nuclear matrix preparations were obtained from cells grown on grids and confirmed the results from labeling of isolated residual structures. J. Cell. Biochem. 67:112–130, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Nuclear matrix-like filaments and fibrogranular complexes form through the rearrangement of specific nuclear ribonucleoproteins

The behavior of nuclear pre-mRNA-binding proteins after their nuclease and/or salt-induced release from RNA was investigated. After RNase digestion or salt extraction, two proteins that initially exist as tetramers (A2)(3)B1 in isolated heterogeneous nuclear ribonucleoprotein (hnRNP) complexes quantitatively reassociated to form regular helical filaments ranging in length from 100 nm to >10 microm. In highly magnified preparations prepared for scanning transmission electron microscopy, single filaments have diameters near 18 nm. In conventional negatively stained preparations viewed at low magnification, the diameters of the thinnest filaments range from 7 to 10 nm. At protein concentrations of >0.1 mg/ml, the filaments rapidly aggregated to form thicker filamentous networks that look like the fibrogranular structures termed the "nuclear matrix." Like the residual material seen in nuclear matrix preparations, the hnRNP filaments were insoluble in 2 M NaCl. Filament formation is associated with, and may be dependent on, disulfide bridge formation between the hnRNP proteins. The reducing agent 2-mercaptoethanol significantly attenuates filament assembly, and the residual material that forms is ultrastructurally distinct from the 7- to 10-nm fibers. In addition to the protein rearrangement leading to filament formation, nearly one-third of the protein present in chromatin-clarified nuclear extracts was converted to salt-insoluble material within 1 min of digestion with RNase. These observations are consistent with the possibility that the residual material termed the nuclear matrix may be enriched in, if not formed by, denatured proteins that function in pre-mRNA packaging, processing, and transport.     

Genetic control of chromosome synapsis in yeast meiosis

Both meiosis-specific and general recombination functions, recruited from the mitotic cell cycle, are required for elevated levels of recombination and for chromosome synapsis (assembly of the synaptonemal complex) during yeast meiosis. The meiosis-specific SPO11 gene (previously shown to be required for meiotic recombination) has been isolated and shown to be essential for synaptonemal complex formation but not for DNA metabolism during the vegetative cell cycle. In contrast, the RAD52 gene is required for mitotic and meiotic recombination but not for synaptonemal complex assembly. These data suggest that the synaptonemal complex may be necessary but is clearly not sufficient for meiotic recombination. Cytological analysis of spread meiotic nuclei demonstrates that chromosome behavior in yeast is comparable with that observed in larger eukaryotes. These spread preparations support the immunocytological localization of specific proteins in meiotic nuclei. This combination of genetic, molecular cloning, and cytological approaches in a single experimental system provides a means of addressing the role of specific gene products and nuclear structures in meiotic chromosome behavior.     

Protein composition of the chromosomal scaffold and interphase nuclear matrix

Residual protein structures were prepared from isolated chromosomes and interphase nuclei of in vitro cultured bovine liver cells and the protein compositions were analysed. Chromosomes with minimal cytoplasmic contamination were obtained by a simple procedure using a pH 8 isolation medium containing Triton X-100 and polyamines, and residual protein-DNA complexes were prepared by extraction with 2 M NaCl. Residual protein structures were also obtained by digesting isolated chromosomes with staphylococcal nuclease. Protein compositions of both structures as obtained by SDS-polyacrylamide gel electrophoresis were essentially the same. Residual protein structures were prepared from isolated nuclei by the same procedures. The major nuclear matrix proteins, i.e., the lamins A, B, and C, were not found in the chromosomes and chromosome scaffolds. On the other hand, the residual chromosome structures contained two major polypeptides of 37 and 83 kilodalton relative molecular weights that were absent from the nuclear matrix preparations. A few polypeptides with the same or very similar electrophoretic mobilities were found in the residual structures of both the nuclei and the chromosomes.     

Isolation of synaptonemal complexes from hamster spermatocytes

Nuclei from Chinese hamster testicular cells in suspension were prepared in a sucrose gradient. Following the basic procedure of Blobel and co-workers for separating a fibrous lamina-nuclear pore complex, synaptonemal complexes (SCs) from spermatocytes were isolated free of other nuclear structures, except for fibrillar tufts at the attachment plaques in which annuli were observed. All the major morphological components of the SC appeared to be intact, showing that the structure could survive the procedure and was not dispersed by the removal of DNA with DNase and solubilization of membranes and some proteins with Triton X-100. Isolated sex bodies were also well preserved, as were various structures from other cell types in the mixed cell suspension, such as spermatid manchettes, acrosomal ‘ghosts’, axonemes, etc. While no nuclear matrix was found associated with autosomal SCs, a residual material was present in the sex body, in which the X and Y axes were embedded. The results indicate the feasibility of isolating and fractionating SCs from testicular cell suspensions enriched for pachytene spermatocytes. The association between SC attachment plaques and annuli that is seen in spreads of whole nuclei persists through the isolation procedure and implies an integrated structural relationship.     

Whole mount electron microscopy of meiotic chromosomes and the synaptonemal complex

The mechanism by which homologous chromosomes pair and crossover has been a major unsolved problem in genetics. Thin section electron microscopy of the synaptonemal complex has not provided enough details to allow any significant insight into this problem. Whole mount preparations of the testis of mice, quail, crayfish, and frogs provided a striking improvement in visualization of the morphological features of meiotic chromosomes. These studies, when combined with the use of deoxyribonuclease and trypsin allowed the following conclusions. 1. The synaptonemal complex (lateral and central elements with connecting L-C fibers) is composed of protein. 2. Contrary to common speculation the central element is not the pairing surface of homologous chromosomes. 3. The L-C fibers, averaging 75–100 Å in width, extend from the lateral elements and meet to form the central element which is usually composed of four fibers. 4. During leptotene, homologous axial elements, although unpaired for most of their length, attach next to each other at the nuclear membrane. 5. Short segments of the chromatin fibers attach to the lateral elements. These points of attachment are clustered, producing the chromomeres seen by light microscopy. 6. The chromatin fibers extend out from the lateral element as loops. Lampbrush chromosomes are thus not restricted to oogenesis but are common to all meiotic chromosomes.Since the morphological features of the central element of the synaptonemal complex persist despite extensive deoxyribonuclease digestion, pairing is perhaps best visualized as a two-step process consisting of a) chromosomal pairing during which the proteinaceous synaptonemal complex pulls homologous chromosomes into approximate association with each other, and b) molecular pairing, which probably takes place in the area around the synaptonemal complex.Supported by NIH Grants GM-15886 and C-2568, and The Charles and Henrietta Detoy Research Fellowship.     

In vitro survival and amethopterin effects on the structure of ovarian tissues of the sawfly, Acantholyda nemoralis Thoms.

Whole ovaries of the sawfly, Acantholyda nemoralis Thoms., Tenthredinidae, Hymenoptera, were cultivated by the organ culture technique of Fell in the medium of Jones and Cunningham modified by doubling the contents of salts, sugars and lactalbumin hydrolysate and supplemented with an addition of folate. In the experimental conditions the sawfly ovary survived for several days showing mitoses in the follicular epithelium. In some of the nurse cell nuclei of the cultured ovaries nucleolus-like structures developed which showed a positive reaction to the Hamm test for viral polyhedra. Formation of these structures was stimulated by amethopterin. Electron microscopical examination revealed that these structures were not viral polyhedra but enlarged nucleoli whose modified ultrastructure differed considerably from that of the nucleoli of cells of trophic ovaries in normally developing insects. Amethopterin appears to induce enlargement of the granules of the granular component of nucleolonema, probably due to disturbance in RNA biosynthesis. The positive Hamm test obtained in the nucleoli shows that this test cannot be regarded as specific for nuclear polyhedra.     

Micrococcal nuclease digestion of nuclei reveals extended nucleosome ladders having anomalous DNA lengths for chromatin assembled on non-replicating plasmids in transfected cells.

The chromatin structures of a variety of plasmids and plasmid constructions, transiently transfected into mouse Ltk- cells using the DEAE-dextran procedure, were studied by micrococcal nuclease digestion of nuclei and Southern hybridization. Although regularly arranged nucleosome-like particles clearly were formed on the transfected DNA, the nucleosome ladders, in some cases with 13-14 bands, were anomalous. Most often, a ladder of DNA fragments with lengths of approximately 300, 500, 700, 900 bp, etc. was generated. In contrast, typical 180-190 bp multiples were generated from bulk cellular or endogenous beta-actin gene chromatin. Very similar results were obtained with all DNA's transfected, and in a variety of cell lines, provided that plasmid replication did not occur. Additionally, after digestion of nuclei, about 90% of the chromatin fragments that contained transfected DNA sequences could not be solubilized at low ionic strength, in contrast with bulk cellular chromatin, suggesting association with nuclear structures or nuclear matrix. The remaining 10% of transfected DNA sequences, arising from soluble chromatin fragments, generated a typical nucleosome ladder. These results are consistent with the idea that assembly of atypical chromatin structures might be induced by proximity to elements of the nuclear pore complex or by nuclear compartmentalization.     

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.     

Characterization of a novel Dp71 dystrophin-associated protein complex (DAPC) present in the nucleus of HeLa cells: members of the nuclear DAPC associate with the nuclear matrix

Dystrophin is an essential component in the assembly and maintenance of the dystrophin-associated protein complex (DAPC), which includes members of the dystroglycan, syntrophin, sarcoglycan and dystrobrevin protein families. Distinctive complexes have been described in the cell membrane of different tissues and cultured cells. In this work, we report the identification and characterization of a novel DAPC present in the nuclei of HeLa cells, which contains dystrophin Dp71 as a key component. Using confocal microscopy and cell fractionation analyses, we found the presence of Dp71, beta-sarcoglycan, beta-dystroglycan, alpha- and beta-syntrophin, alpha1- and beta-dystrobrevin and nNOS in the nuclei of HeLa cells. Furthermore, we demonstrated by co-immunoprecipitation experiments that most of these proteins form a complex in the nuclear compartment. Next, we analyze the possible association of the nuclear DAPC with the nuclear matrix. We found the presence of Dp71, beta-dystroglycan, nNOS, beta-sarcoglycan, alpha/beta syntrophin, alpha1-dystrobrevin and beta-dystrobrevin in the nuclear matrix protein fractions and in situ nuclear matrix preparations from HeLa cells. Moreover, we found that Dp71, beta-dystroglycan and beta-dystrobrevin co-immunoprecipitated with the nuclear matrix proteins lamin B1 and actin. The association of members of the nuclear DAPC with the nuclear matrix indicates that they may work as scaffolding proteins involved in nuclear architecture.