Unusual nuclear structures in meiotic prophase of fission yeast: a cytological analysis

Earlier results from sectioned nuclei indicating that Schizosaccharomyces pombe does not develop a classical tripartite synaptonemal complex (SC) during meiotic prophase are confirmed by spreading of whole nuclei. The linear elements appearing during prophase I resemble the axial cores (SC precursors) of other organisms. The number of linear elements in haploid, diploid, and tetraploid strains is always higher than the chromosome number, implying that they are not formed continuously along the chromosomes. Time course experiments reveal that the elements appear after DNA replication and form networks and bundles. Later they separate and approximately 24 individual elements with a total length of 34 microns are observed before degradation and meiotic divisions. Parallel staining of DNA reveals changes in nuclear shape during meiotic prophase. Strains with a mei4 mutation are blocked at a late prophase stage. In serial sections we additionally observed a constant arrangement of the spindle pole body, the nucleolus, and the presumptive centromere cluster. Thus, S. pombe manages to recombine and segregate its chromosomes without SC. This might correlate with the absence of crossover interference. We propose a mechanism for chromosome pairing with initial recognition of the homologs at the centromeres and suggest functions of the linear elements in preparation of the chromosomes for meiosis I disjunction. With the spreading technique combined genetic, molecular, and cytological approaches become feasible in S. pombe. This provides an opportunity to study essential meiotic functions in the absence of SCs which may help to clarify the significance of the SC and its components for meiotic chromosome structure and function.     

Dynamics of chromosome organization and pairing during meiotic prophase in fission yeast

Interactions between homologous chromosomes (pairing, recombination) are of central importance for meiosis. We studied entire chromosomes and defined chromosomal subregions in synchronous meiotic cultures of Schizosaccharomyces pombe by fluorescence in situ hybridization. Probes of different complexity were applied to spread nuclei, to delineate whole chromosomes, to visualize repeated sequences of centromeres, telomeres, and ribosomal DNA, and to study unique sequences of different chromosomal regions. In diploid nuclei, homologous chromosomes share a joint territory even before entry into meiosis. The centromeres of all chromosomes are clustered in vegetative and meiotic prophase cells, whereas the telomeres cluster near the nucleolus early in meiosis and maintain this configuration throughout meiotic prophase. Telomeres and centromeres appear to play crucial roles for chromosome organization and pairing, both in vegetative cells and during meiosis. Homologous pairing of unique sequences shows regional differences and is most frequent near centromeres and telomeres. Multiple homologous interactions are formed independently of each other. Pairing increases during meiosis, but not all chromosomal regions become closely paired in every meiosis. There is no detectable axial compaction of chromosomes in meiotic prophase. S. pombe does not form mature synaptonemal complexes, but axial element-like structures (linear elements), which were analyzed in parallel. Their appearance coincides with pairing of interstitial chromosomal regions. Axial elements may define minimal structures required for efficient pairing and recombination of meiotic chromosomes.     

Centromere and telomere redistribution precedes homologue pairing and terminal synapsis initiation during prophase I of cattle spermatogenesis

Alterations in nuclear topology associated with meiotic chromosome pairing were studied in premeiotic cells and spermatocytes I of adult bovine males. To this end, we performed FISH with chromosome, pericentromeric satellite-DNA and telomere-specific probes in combination with immunostaining of synaptonemal complex proteins (SCP3, SCP1) on testis tissue sections. Nuclei of premeiotic cells (spermatogonia) exhibited a scattered telomere distribution while pericentromeres formed a few intranuclear clusters. We observed that the chromosome pairing process in cattle prophase I is preceded by repositioning of centromeres and telomeres to the nuclear periphery during preleptotene. Clustering of chromosome ends (bouquet formation) was observed during the transition from leptonema to zygonema and coincided with pairing of a sub-centromeric marker of bovine chromosomes 7. Dissolution of bouquet topology during zygonema left perinuclear telomeres scattered over the nuclear periphery at pachynema. SCP3 staining in frozen tissue sections revealed the appearance of this axial element protein in intranuclear aggregates during preleptotene, followed by extensive axial element formation during leptotene. Synapsis as revealed by SCP1 staining initiated peripherally at earliest zygotene, at this stage nuclei still contained numerous SCP3 clusters. Our observations reveal prominent non-homologous satellite-DNA associations in spermatogonia and indicate the conservation of topological features of the meiotic chromosome pairing process among mammals. The comparison of telomere dynamics in mouse and cattle prophase I suggests that a larger number of chromosomes prolongs the duration of the bouquet stage.     

Chromosome systems in the eriococcidae (Coccoidea Homoptera)

Spermatogenesis is described in two eriococcid species and the observations are compared to those previously reported. In Gossyparia spuria the diploid chromosome number is 28 in both males and females. In the female all the chromosomes are euchromatic. In most male tissues 14 of the chromosomes are euchromatic (E) and 14 are heterochromatic (H). Prior to the first meiotic division in males the number of H chromosomes was reduced. During prophase I all the cells showed 14 E chromosomes and from 1 to over 9 H chromosomes. The range of chromosome numbers in metaphase I was similar to that in prophase I. All the chromosomes divided in anaphase I, and, following differential uncoiling at interkinesis, the E and H groups of chromosomes segregated from each other at anaphase II. Only the E groups formed sperm. The presence of a variable number of H chromosomes and a haploid number of E chromosomes in spermatogenesis suggested the presence of the multiple-D variant of the Comstockiella chromosome system. In this system some of the H chromosomes become euchromatic prior to prophase I of spermatogenesis and pair with their E homologues. All the remaining H chromosomes are thus univalents, while among the E elements, some are univalents and the rest are bivalents. The observed reduction in the number of H chromosomes in the first meiotic division which was previously attributed to pairing among the H chromosomes, is now interpreted to be the result of the return of some of the H chromosomes to a euchromatic state and to their subsequent pairing with their E homologues. Spermatogenesis in Eriococcus araucariae was similar to that of G. spuria except that the reduction in the number of H chromosomes was not as extensive. The chromosome systems of the two species are compared to those of other eriococcids and the differences are briefly discussed.Supported by grant GB1585 from the National Science Foundation, Washington, D. C.     

A Meiotic Chromosomal Core Consisting of Cohesin Complex Proteins Recruits DNA Recombination Proteins and Promotes Synapsis in the Absence of an Axial Element in Mammalian Meiotic Cells

The behavior of meiotic chromosomes differs in several respects from that of their mitotic counterparts, resulting in the generation of genetically distinct haploid cells. This has been attributed in part to a meiosis-specific chromatin-associated protein structure, the synaptonemal complex. This complex consist of two parallel axial elements, each one associated with a pair of sister chromatids, and a transverse filament located between the synapsed homologous chromosomes. Recently, a different protein structure, the cohesin complex, was shown to be associated with meiotic chromosomes and to be required for chromosome segregation. To explore the functions of the two different protein structures, the synaptonemal complex and the cohesin complex, in mammalian male meiotic cells, we have analyzed how absence of the axial element affects early meiotic chromosome behavior. We find that the synaptonemal complex protein 3 (SCP3) is a main determinant of axial-element assembly and is required for attachment of this structure to meiotic chromosomes, whereas SCP2 helps shape the in vivo structure of the axial element. We also show that formation of a cohesin-containing chromosomal core in meiotic nuclei does not require SCP3 or SCP2. Our results also suggest that the cohesin core recruits recombination proteins and promotes synapsis between homologous chromosomes in the absence of an axial element. A model for early meiotic chromosome pairing and synapsis is proposed.     

Nonrandom chromosome arrangements in germ line nuclei of Sciara coprophila males: the basis for nonrandom chromosome segregation on the meiosis I spindle

Meiosis I in males of the Dipteran Sciara coprophila results in the nonrandom distribution of maternally and paternally derived chromosome sets to the two division products. Based on an earlier study (Kubai, D.F. 1982. J. Cell Biol. 93:655-669), I suggested that the meiosis I spindle does not play a direct role in the nonrandom sorting of chromosomes but that, instead, haploid sets are already separated in prophase nuclei well before the onset of spindle formation. Here I report more direct evidence that this hypothesis is true; this evidence was gained from ultrastructural reconstruction analyses of the arrangement of chromosomes in germ line nuclei (prophase nuclei in spermatogonia and spermatocytes) of males heterozygous for an X- autosome chromosome translocation. Because of this translocation, the maternal and paternal chromosome sets are distinguishable, so it is possible to demonstrate that (a) the two haploid chromosome sets occupy distinct maternal and paternal nuclear compartments and that (b) nuclei are oriented so that the two haploid chromosome sets have consistent relationships to a well-defined cellular axis. The consequences of such nonrandom aspects of nuclear structure for chromosome behavior on premeiotic and meiotic spindles are discussed.     

The Rec8 Gene of Schizosaccharomyces Pombe Is Involved in Linear Element Formation, Chromosome Pairing and Sister-Chromatid Cohesion during Meiosis

The fission yeast Schizosaccharomyces pombe does not form tripartite synaptonemal complexes during meiotic prophase, but axial core-like structures (linear elements). To probe the relationship between meiotic recombination and the structure, pairing, and segregation of meiotic chromosomes, we genetically and cytologically characterized the rec8-110 mutant, which is partially deficient in meiotic recombination. The pattern of spore viability indicates that chromosome segregation is affected in the mutant. A detailed segregational analysis in the rec8-110 mutant revealed more spores disomic for chromosome III than in a wild-type strain. Aberrant segregations are caused by precocious segregation of sister chromatids at meiosis I, rather than by nondisjunction as a consequence of lack of crossovers. In situ hybridization further showed that the sister chromatids are separated prematurely during meiotic prophase. Moreover, the mutant forms aberrant linear elements and shows a shortened meiotic prophase. Meiotic chromosome pairing in interstitial and centromeric regions is strongly impaired in rec8-110, whereas the chromosome ends are less deficient in pairing. We propose that the rec8 gene encodes a protein required for linear element formation and that the different phenotypes of rec8-110 reflect direct and indirect consequences of the absence of regular linear elements.     

The nature and extent of synaptonemal complex formation in haploid barley

Normal synaptonemal complexes have been found in haploid barley meiotic prophase at stages equivalent to pachytene in diploids. Reconstructions of serially sectioned nuclei have shown that up to 60% of the haploid chromosomes may pair in either intra- or interchromosomal associations. The extent and nature of the synaptonemal complex formation suggest that the chromosome pairing is non-homologous. From the virtual absence of chiasmata in metaphase I stages of the haploids it is inferred that crossing over requires a more precise DNA alignment than is provided by synaptonemal complex formation alone.     

Genetic collection of meiotic mutants of rye Secale cereale L

Genetic collection of meiotic mutants of winter rye Secale cereale L. (2n = 14) was created. Mutations were detected in inbred F2 generations after self-fertilization of the F1 hybrids, obtained by individual crossing of rye plants (cultivar Vyatka) or weedy rye with plants from autofertile lines. The mutations cause partial or complete plant sterility and are maintained in collection in a heterozygous state. Genetic analysis accompanied by cytogenetic study of meiosis has revealed six mutation types. (1) Nonallelic asynaptic mutations sy1 and sy9 caused the formation of only axial chromosome elements in prophase and anaphase. The synaptonemal complexes (SCs) were absent, the formation of the chromosome "bouquet" was impaired, and all chromosomes were univalent in meiotic metaphase I in 96% (sy1) and 67% (sy2) of cells. (2) Weak asynaptic mutation sy3, which hindered complete termination of synapsis in prophase II. Subterminal asynaptic segments were always observed in the SC, and at least one pair of univalents was present in metaphase I, but the number of cells with univalents did not exceed 2%. (3) Mutations sy2, sy6, sy7, sy8, sy10, and sy19, which caused partially nonhomologous synapsis: change in pairing partners and fold-back chromosome synapsis in prophase I. In metaphase I, the number of univalents varied and multivalents were observed. (4) Mutation mei6, which causes the formation of ultrastructural protrusions on the lateral SC elements, gaps and branching of these elements. (5) Allelic mutations mei8 and mei10, which caused irregular chromatin condensation along chromosomes in prophase I, sticking and fragmentation of chromosomes in metaphase I. (6) Allelic mutations mei5 and mei10, which caused chromosome hypercondensation, defects of the division spindle formation, and random arrest of cells at different meiotic stages. However, these mutations did not affect the formation of microspore envelopes even around the cells, whose development was blocked at prophase I. Analysis of cytological pictures of meiosis in double rye mutants reveled epistatic interaction in the mutation series sy9 > sy1 > sy3 > sy19, which reflects the order of switching these genes in the course of meiosis. The expression of genes sy2 and sy19 was shown to be controlled by modifier genes. Most meiotic mutations found in rye have analogs in other plant species.     

The program of sex chromosome pairing in meiosis is highly conserved across marsupial species: implications for sex chromosome evolution

Marsupials present a series of genetic and chromosomal features that are highly conserved in very distant species. One of these features is the absence of a homologous region between X and Y chromosomes. According to this genetic differentiation, sex chromosomes do not synapse during the first meiotic prophase in males, and a special structure, the dense plate, maintains sex chromosome association. In this report we present results on the process of meiotic sex chromosome pairing obtained from three different species, Thylamys elegans, Dromiciops gliroides, and Rhyncholestes raphanurus, representing the three orders of American marsupials. We have investigated the relationships between the axial structures organized along sex chromosomes and the formation of the dense plate. We found that in the three species the dense plate arises as a modification of sex chromosomal axial elements, but without the involvement of other meiotic axial structures, such as the cohesin axes. Considering the phylogenetic relationships among the marsupials studied here, our data reinforce the idea that the dense plate emerged early in marsupial evolution as an efficient mechanism to ensure the association of the nonhomologous sex chromosomes. This situation could have influenced the further evolution of sex chromosomes in marsupials.     

Asymmetrically distributed oligonucleotide repeats in the Caenorhabditis elegans genome sequence that map to regions important for meiotic chromosome segregation

The roundworm Caenorhabditis elegans has a haploid karyotype containing six linear chromosomes. The termini of worm chromosomes have been proposed to play an important role in meiotic prophase, either when homologs are participating in a genome-wide search for their proper partners or in the initiation of synapsis. For each chromosome one end appears to stimulate crossing-over with the correct homolog; the other end lacks this property. We have used a bioinformatics approach to identify six repetitive sequence elements in the sequenced C.elegans genome whose distribution closely parallels these putative meiotic pairing centers (MPC) or homolog recognition regions (HRR). We propose that these six DNA sequence elements, which are largely chromosome specific, may correspond to the genetically defined HRR/MPC elements.     

Immunofluorescent synaptonemal complex analysis in azoospermic men

The molecular cause of germ cell meiotic defects in azoospermic men is rarely known. During meiotic prophase I, a proteinaceous structure called the synaptonemal complex (SC) appears along the pairing axis of homologous chromosomes and meiotic recombination takes place. Newly-developed immunofluorescence techniques for SC proteins (SCP1 and SCP3) and for a DNA mismatch repair protein (MLH1) present in late recombination nodules allow simultaneous analysis of synapsis, and of meiotic recombination, during the first meiotic prophase in spermatocytes. This immunofluorescent SC analysis enables accurate meiotic prophase substaging and the identification of asynaptic pachytene spermatocytes. Spermatogenic defects were examined in azoospermic men using immunofluorescent SC and MLH1 analysis. Five males with obstructive azoospermia, 18 males with nonobstructive azoospermia and 11 control males with normal spermatogenesis were recruited for the study. In males with obstructive azoospermia, the fidelity of chromosome pairing (determined by the percentage of cells with gaps [discontinuities]/splits [unpaired chromosome regions] in the SCs, and nonexchange SCs [bivalents with 0 MLH1 foci]) was similar to those in normal males. The recombination frequencies (determined by the mean number of MLH1 foci per cell at the pachytene stage) were significantly reduced in obstructive azoospermia compared to that in controls. In men with nonobstructive azoospermia, a marked heterogeneity in spermatogenesis was found: 45% had a complete absence of meiotic cells; 5% had germ cells arrested at the zygotene stage of meiotic prophase; the rest had impaired fidelity of chromosome synapsis and significantly reduced recombination in pachytene. In addition, significantly more cells were in the leptotene and zygotene meiotic prophase stages in nonobstructive azoospermic patients, compared to controls. Defects in chromosome pairing and decreased recombination during meiotic prophase may have led to spermatogenesis arrest and contributed in part to this unexplained infertility.     

家鸡联会复合体的亚显微结构分析

本文以表面铺展——硝酸银染色技术,对家鸡的联会复合体(Syneptonemal Complex,SC)作亚显微结构分析。根据对10个精母细胞和10个卵母细胞SC的测量结果,绘制组型图。发现雌雄家鸡的常染色体的SC组型相同。在精母细胞中,性染色体(ZZ)的行为与常染色体相似。在卵母细胞中,性染色体ZW的长度不同,长轴为Z,短轴为W,两者之间只有部分配对,形成SC。从早粗线期到晚粗线期,由同源配对调整为非同源配对。另外,在一只雌鸡中,第一次观察到,有些细胞的常染色体能正常配对,而性染色体完全不配对的现象。     

The influence of the Robertsonian translocation Rb(X.2)2Ad on anaphase I non-disjunction in male laboratory mice

A Robertsonian translocation in the mouse between the X chromosome and chromosome 2 is described. The male and female carriers of the Rb(X.2)2Ad were fertile. A homozygous/hemizygous line was maintained. The influence of the X-autosomal Robertsonian translocation on anaphase I non-disjunction in male mice was studied by chromosome counts in cells at metaphase II of meiosis and by assessment of aneuploid progeny. The results conclusively show that the inclusion of Rb2Ad in the male genome induces non-disjunction at the first meoitic division. In second metaphase cells the frequency of sex-chromosomal aneuploidy was 10.8%, and secondary spermatocytes containing two or no sex chromosome were equally frequent. The Rb2Ad males sired 3.9% sex-chromosome aneuploid progeny. The difference in aneuploidy frequencies in the germ cells and among the progeny suggests that the viability of XO and XXY individuals is reduced. The pairing configurations of chromosomes 2, Rb2Ad and Y were studied during meiotic prophase by light and electron microscopy. Trivalent pairing was seen in all well spread nuclei. Complete pairing of the acrocentric autosome 2 with the corresponding segment of the Rb2Ad chromosome was only seen in 3.2% of the cells analysed in the electron microscope. The pairing between the X and Y chromosome in the Rb2Ad males corresponded to that in males with normal karyotype. Reasons for sex-chromosomal non-disjunction despite the normal pairing pattern between the sex chromosomes may be seen in the terminal chiasma location coupled with the asynchronous separation of the sex chromosomes and the autosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

A large-scale screen in S. pombe identifies seven novel genes required for critical meiotic events

Meiosis is a specialized form of cell division by which sexually reproducing diploid organisms generate haploid gametes. During a long prophase, telomeres cluster into the bouquet configuration to aid chromosome pairing, and DNA replication is followed by high levels of recombination between homologous chromosomes (homologs). This recombination is important for the reductional segregation of homologs at the first meiotic division; without further replication, a second meiotic division yields haploid nuclei. In the fission yeast Schizosaccharomyces pombe, we have deleted 175 meiotically upregulated genes and found seven genes not previously reported to be critical for meiotic events. Three mutants (rec24, rec25, and rec27) had strongly reduced meiosis-specific DNA double-strand breakage and recombination. One mutant (tht2) was deficient in karyogamy, and two (bqt1 and bqt2) were deficient in telomere clustering, explaining their defects in recombination and segregation. The moa1 mutant was delayed in premeiotic S phase progression and nuclear divisions. Further analysis of these mutants will help elucidate the complex machinery governing the special behavior of meiotic chromosomes.     

Synaptonemal complex antigen location and conservation

The axial cores of chromosomes in the meiotic prophase nuclei of most sexually reproducing organisms play a pivotal role in the arrangement of chromatin, in the synapsis of homologous chromosomes, in the process of genetic recombination, and in the disjunction of chromosomes. We report an immunogold analysis of the axial cores and the synaptonemal complexes (SC) using two mouse monoclonal antibodies raised against isolated rat SCs. In Western blots of purified SCs, antibody II52F10 recognizes a 30- and a 33-kD peptide (Heyting, C., P. B. Moens, W. van Raamsdonk, A. J. J. Dietrich, A. C. G. Vink, and E. J. W. Redeker, 1987, Eur. J. Cell Biol., 43: 148-154). In spreads of rat spermatocyte nuclei it produces gold grains over the cores of autosomal and sex chromosomes. The cores label lightly during the chromosome pairing stage (zygotene) of early meiotic prophase and they become more intensely labeled when they are parallel aligned as the lateral elements of the SC during pachytene (55 grains/micron SC). Statistical analysis of electronically recorded gold grain positions shows that the two means of the bimodal gold grain distribution coincide with the centers of the lateral elements. At diplotene, when the cores separate, the antigen is still detected along the length of the core and the enlarged ends are heavily labeled. Shadow-cast SC preparations show that recombination nodules are not labeled. The continued presence suggests that the antigens serve a continuing function in the cores, such as chromatin binding, and/or structural integrity. Antibody III15B8, which does not recognize the 30- and 33-kD peptides, produces gold grains predominantly between the lateral elements. The grain distribution is bimodal with the mean of each peak just inside the pairing face of the lateral element. The antigen is present where and while the cores of the homologous chromosomes are paired. From the location and the timing, it is assumed that the antigen recognized by III15B8 functions in chromosome pairing at meiotic prophase. The two anti-rat SC antibodies label rat and mouse SCs but not rabbit or dog SCs. A positive control using human CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) anti-centromere serum gives equivalent labeling of SC centromeres in the rat, mouse, rabbit, and dog. It is concluded that the SC antigens recognized by II52F10 and III15B8 are not widely conserved. The two antibodies do not bind to cellular or nuclear components of somatic cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparative analysis of female and male meiosis in three meiotic mutants of tomato.

Female meiosis was analysed in squash preparations of ovules from three meiotic mutants and wild-type plants of tomato. In the completely asynaptic mutant as6, chromosome pairing and chiasma formation were virtually absent in both sexes. In the partially asynaptic mutant asb, with intermediate levels of chromosome pairing at pachytene, there were a higher number of chiasmate chromosome arms in female meiosis than in male meiosis, whereas in the desynaptic mutant as5 there were normal levels of chromosome pairing at pachytene and a similar reduction in chiasma frequency in the two sexes. In wild-type tomato, we found slightly higher numbers of chiasmate chromosome arms in female meiosis than in male meiosis. We propose that the higher female chiasma frequencies in mutant asb and wild-type tomato result from a longer duration of female meiotic prophase. This would allow chromosomes more time to pair and recombine. It is possible that a longer duration of prophase I does not affect mutants as5 and as6, either because the meiotic defect acts before the pairing process begins (in as6) or because it acts at a later stage and involves chiasma maintenance (in as5).     

Centromere pairing precedes meiotic chromosome pairing in plants

Meiosis is a specialized eukaryotic cell division, in which diploid cells undergo a single round of DNA replication and two rounds of nuclear division to produce haploid gametes. In most eukaryotes, the core events of meiotic prophase I are chromosomal pairing,synapsis and recombination. To ensure accurate chromosomal segregation, homologs have to identify and align along each other at the onset of meiosis. Although much progress has been made in elucidating meiotic processes, information on the mechanisms underlying chromosome pairing is limited in contrast to the meiotic recombination and synapsis events. Recent research in many organisms indicated that centromere interactions during early meiotic prophase facilitate homologous chromosome pairing, and functional centromere is a prerequisite for centromere pairing such as in maize. Here, we summarize the recent achievements of chromosome pairing research on plants and other organisms, and outline centromere interactions, nuclear chromosome orientation,and meiotic cohesin, as main determinants of chromosome pairing in early meiotic prophase.     

How do meiotic chromosomes meet their homologous partners?: lessons from fission yeast

Homologous chromosome pairing is required for proper chromosome segregation and recombination during meiosis. The mechanism by which a pair of homologous chromosomes contact each other to establish pairing is not fully understood. When pairing occurs during meiotic prophase in the fission yeast, Schizosaccharomyces pombe, the nucleus oscillates between the cell poles and telomeres remain clustered at the leading edge of the moving nucleus. These meiosis-specific activities produce movements of telomere-bundled chromosomes. Several lines of evidence suggest that these movements facilitate homologous chromosome pairing by aligning homologous chromosomes and promoting contact between homologous regions. Since telomere clustering and nuclear or chromosome movements in meiotic prophase have been observed in a wide range of eukaryotic organisms, it is suggested that telomere-mediated chromosome movements are general activities that facilitate homologous chromosome pairing.