Abnormal meiosis in bisporic mutants of white button mushroom Agaricus bisporus (Lange) Imbach

A formerly developed method of obtaining spread preparations of mushroom basidial nuclei was applied to study of meiotic prophase I in bisporic white button mushroom (Agaricus bisporus) strains. Meiotic recombination and assemblage of axial structures (axial elements and synaptonemal complexes) of chromosomes in meiotic prophase I are interrelated. It is known that the frequency of meiotic recombination is reduced in the bisporic A. bisporus variety. We showed that formation of axial structures of meiotic chromosomes in bisporic strains of this mushroom was disrupted. The phenotypes of disruptions in spread prophase nuclei are diverse. In leptotene and early zygotene, many nuclei contain abnormal, often short, and, as a rule, few chromosomal axial elements. The abnormalities in the formation of synaptonemal complexes at the zygotene-diplotene stage are of the same kind and even more pronounced. We discovered an important feature of meiosis in A. bisporus associated with fruit-body morphogenesis. Meiosis starting in basidia (meiocytes) of young closed fruit bodies is accompanied by disruption of chromatin condensation in prophase I and, probably, is arrested. After indusium breakage, the course of meiosis normalizes. Preparations with clearly observable chromosomal axial structures can be obtained only at this stage of fruit-body development.     

Abnormal meiosis in bisporic strains of white button mushroom Agaricus bisporus (Lange) imbach

A formerly developed method of microspreading of mushroom basidial nuclei was applied to study meiotic prophase I in bisporic white button mushroom (Agaricus bisporus) strains. Meiotic recombination and assemblage of axial structures (axial elements and synaptonemal complexes) of chromosomes in meiotic prophase I are interrelated. It is known that the frequency of meiotic recombination is reduced in the bisporic A. bisporus variety. We showed that formation of axial structures of meiotic chromosomes in bisporic strains of this mushroom was disrupted. The anomalous phenotypes in spread prophase nuclei are diverse. In leptotene and early zygotene, many nuclei contain abnormal, often short, and, as a rule, few chromosomal axial elements. The abnormalities in the formation of synaptonemal complexes at the zygotene-diplotene stage are of the same kind and even more pronounced. We discovered an important feature of meiosis in A. bisporus associated with fruit-body morphogenesis. Meiosis starting in basidia (meiocytes) of young closed fruit bodies is accompanied by disruption of chromatin condensation in prophase I and, probably, is arrested. After partial veil breakage, the course of meiosis normalizes. Preparations with clearly observable chromosomal axial structures can be obtained only at this stage of fruit-body development.     

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.     

Neurospora spore killers Sk-2 and Sk-3 suppress meiotic silencing by unpaired DNA

In Neurospora crassa, pairing of homologous DNA segments is monitored during meiotic prophase I. Any genes not paired with a homolog, as well as any paired homologs of that gene, are silenced during the sexual phase by a mechanism known as meiotic silencing by unpaired DNA (MSUD). Two genes required for MSUD have been described previously: sad-1 (suppressor of ascus dominance), encoding an RNA-directed RNA polymerase, and sad-2, encoding a protein that controls the perinuclear localization of SAD-1. Inactivation of either sad-1 or sad-2 suppresses MSUD. We have now shown that MSUD is also suppressed by either of two Spore killer strains, Sk-2 and Sk-3. These were both known to contain a haplotype segment that behaves as a meiotic drive element in heterozygous crosses of killer x sensitive. Progeny ascospores not carrying the killer element fail to mature and are inviable. Crosses homozygous for either of the killer haplotypes suppress MSUD even though ascospores are not killed. The killer activity maps to the same 30-unit-long region within which recombination is suppressed in killer x sensitive crosses. We suggest that the region contains a suppressor of MSUD.     

Two kinds of "recombination nodules" in Neurospora crassa

Two morphological types of recombination nodules, termed early and late, are recognized in Neurospora crassa. Eighty nuclei at different substages were used to determine numbers of nodules per nucleus, distribution of nodules along the nucleolus-organizing chromosome, and distribution of nodules among the two largest chromosomes. Early nodules appear at the synaptonemal complex at early zygotene and increase in number during zygotene until a dramatic reduction occurs at zygotene-pachytene transition. Thereafter early nodules are steadily eliminated until they disappear by diplotene. Late nodules are also present during zygotene. Their number doubles at the zygotene-pachytene transition and stays at this level until diplotene. The total number of nodules is rather constant through zygotene and pachytene. Distribution of bivalents with 0, 1, 2, etc. nodules follows a Poisson distribution at zygotene, but not at pachytene, where variance is less than the mean, indicating positive interference. Nodules are distributed nonrandomly along the nucleolus-organizer bivalent. The pattern differs slightly in nuclei of different origin. Nuclei with unusual synaptonemal complexes sustain normal levels of recombination by having the same amount of nodules as normal nuclei. In abnormal nuclei nodules are preferentially associated with normal segments. It is proposed that early nodules do not participate in any form of recombination but have a role in finding an appropriate site for a crossing-over event. Morphological change to the late type indicates that the site has been reached and the exchange event can be mediated by the late nodule.     

Nuclear aberrations and loss of synaptonemal complexes in response to diethylstilbestrol (DES) in Caenorhabditis elegans hermaphrodites

In Caenorhabditis elegans, loss of viability and fertility is observed after treatment with DES. The decrease in life span is associated with senescent morphology of meiotic prophase nuclei, such that nuclei from young and old specimens cannot be differentiated. Aging in oocytes at the pachytene stage of meiotic prophase is manifested by nucleo-cytoplasmic aberrations, increased density of the nucleoplasm and cytoplasm and decrease in numbers of mitochondria. Increasing concentrations of DES are characterized by concomitant decrease in fertility and increased production of abnormal gametes. At DES concentrations higher than 1.25 micrograms/ml, synaptonemal complexes (SC) are absent from the nuclei, thus, effective pairing and segregation of homologous chromosomes is not possible. The absence of SCs may be the result of: a premeiotic colchicine-like effect that influences pairing of chromosomes; changes in the structure of the DNA due to DES binding that results in changes in expression of the DNA; and changes in temporal DNA synthesis in response to DES. Since the SC is essential for regulating pairing and subsequent separation of bivalents, the lack of an SC explains the loss of fertility, due to the production of unbalanced gametes, observed in DES-treated specimens.     

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.     

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.     

Synaptonemal complex components persist at centromeres and are required for homologous centromere pairing in mouse spermatocytes

Recent studies in simple model organisms have shown that centromere pairing is important for ensuring high-fidelity meiotic chromosome segregation. However, this process and the mechanisms regulating it in higher eukaryotes are unknown. Here we present the first detailed study of meiotic centromere pairing in mouse spermatogenesis and link it with key events of the G2/metaphase I transition. In mouse we observed no evidence of the persistent coupling of centromeres that has been observed in several model organisms. We do however find that telomeres associate in non-homologous pairs or small groups in B type spermatogonia and pre-leptotene spermatocytes, and this association is disrupted by deletion of the synaptonemal complex component SYCP3. Intriguingly, we found that, in mid prophase, chromosome synapsis is not initiated at centromeres, and centromeric regions are the last to pair in the zygotene-pachytene transition. In late prophase, we first identified the proteins that reside at paired centromeres. We found that components of the central and lateral element and transverse filaments of the synaptonemal complex are retained at paired centromeres after disassembly of the synaptonemal complex along diplotene chromosome arms. The absence of SYCP1 prevents centromere pairing in knockout mouse spermatocytes. The localization dynamics of SYCP1 and SYCP3 suggest that they play different roles in promoting homologous centromere pairing. SYCP1 remains only at paired centromeres coincident with the time at which some kinetochore proteins begin loading at centromeres, consistent with a role in assembly of meiosis-specific kinetochores. After removal of SYCP1 from centromeres, SYCP3 then accumulates at paired centromeres where it may promote bi-orientation of homologous centromeres. We propose that, in addition to their roles as synaptonemal complex components, SYCP1 and SYCP3 act at the centromeres to promote the establishment and/or maintenance of centromere pairing and, by doing so, improve the segregation fidelity of mammalian meiotic chromosomes.     

The association of ATR protein with mouse meiotic chromosome cores

The ATR (ataxia telangiectasia- and RAD3-related) protein is present on meiotic prophase chromosome cores and paired cores (synaptonemal complexes, SCs). Its striking characteristic is that the protein forms dense aggregates on the cores and SCs of the last chromosomes to pair at the zygotene-pachytene transition. It would appear that the ATR protein either signals delays in pairing or it is directly involved in the completion of the pairing phase. Atm-deficient spermatocytes, which are defective in the chromosome pairing phase, accumulate large amounts of ATR. The behaviour of ATR at meiotic prophase sets it apart from the distribution of the RAD51/DMC1 recombinase complex and our electron microscope observations confirm that they do not co-localize. We failed to detect ATM in association with cores/SCs and we have reported elsewhere that RAD1 protein does not co-localize with DMC1 foci. The expectation that putative DNA-damage checkpoint proteins, ATR, ATM and RAD1, are associated with RAD51/DMC1 recombination sites where DNA breaks are expected to be present, is therefore not supported by our observations. Received: 23 November 1998 / Accepted: 3 January 1999     

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 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.     

Synaptonemal complex damage induced by clastogenic and anti-mitotic chemicals: implications for non-disjunction and aneuploidy

Mice were treated with mitomycin C, cyclophosphamide, amsacrine, colchicine, or vinblastine sulfate, and meiotic prophase cells analyzed for synaptonemal complex (SC) damage. All test agents caused synaptonemal complex breakage and synapsis irregularities, although propensities for inducing specific types of damage at S-phase or prophase stages varied among the chemicals. The data indicate that SC analysis can reveal chemical-specific alterations to meiotic homologue pairing/synapsis which have not generally been recognized, and which theoretically may be implicated in non-disjunction.     

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.     

Effects of dimethyl sulphoxide on early gametogenesis in Caenorhabditis elegans: ultrastructural aberrations and loss of synaptonemal complexes from pachytene nuclei

In Caenorhabditis elegans, loss of viability and fertility was observed after treatment with dimethyl sulphoxide (DMSO). The decrease in life span is associated with senescent morphology of meiotic prophase nuclei, such that nuclei from young and old specimens cannot be differentiated. Aging in oocytes at the pachytene stage of meiotic prophase is characterized by nucleo-cytoplasmic aberrations, increased density of the nucleoplasm and cytoplasm and decrease in numbers of mitochondria (Goldstein and Curis, 1987). Increasing concentrations of DMSO result in decrease in fertility and increased production of abnormal gametes. At DMSO concentrations higher than 5.0%, synaptonemal comlexes (SC) are absent from the nuclei, thus, effective pairing and segregation of homologous chromosomes is not possible. The absence of SCs may be the result of: (1) a premeiotic colchicine-like effect which influences pairing of chromosomes; (2) changes in the structure of the DNA due to DMSO binding that results in changes in expression of the DNA; and (3) changes in temporal DNA synthesis in response to DMSO. Since the SC is essential for regulating pairing and subsequent separation of bivalents, the lack of an SC explains the loss of fertility, due to the production of unbalanced gametes, observed in DMSO treated specimens.     

Couples,pairs, and clusters: mechanisms and implications of centromere associations in meiosis

Observations of a wide range of organisms show that the centromeres form associations of pairs or small groups at different stages of meiotic prophase. Little is known about the functions or mechanisms of these associations, but in many cases, synaptonemal complex elements seem to play a fundamental role. Two main associations are observed: homology-independent associations very early in the meiotic program—sometimes referred to as centromere coupling—and a later association of homologous centromeres, referred to as centromere pairing or tethering. The later centromere pairing initiates during synaptonemal complex assembly, then persists after the dissolution of the synaptonemal complex. While the function of the homology-independent centromere coupling remains a mystery, centromere pairing appears to have a direct impact on the chromosome segregation fidelity of achiasmatic chromosomes. Recent work in yeast, Drosophila, and mice suggest that centromere pairing is a previously unappreciated, general meiotic feature that may promote meiotic segregation fidelity of the exchange and non-exchange chromosomes.     

The fate of multivalents during meiotic prophase in the hybrid Gibasis consobrina x G. karwinskyana Rafin. (Commelinaceae)

The chromosomes of the two closely related diploid species, Gibasis consobrina and G. karwinskyana (Commelinaceae; 2n=2x=10), are morphologically alike, yet form few chiasmate associations at metaphase I in the f₁ hybrid. During meiotic prophase, however, synaptonemal complexes join the majority of the chromosomes of the complement in complex multiple pairing configurations. The F₁ hybrid between different tetraploid genotypes of the same two species similarly forms multivalents during meiotic prophase, which are subsequently eliminated in favour of strictly homologous bivalents before metaphase I. One quadrivalent comprising interchange chromosomes inherited from one of the parents, usually persists to first metaphase. Evidently the resolution of multivalents to bivalents at first metaphase, which accounts for diploidisation, is not attributable to the elimination of multivalents per se, but of multivalents comprising chromosomes of limited homology.     

Stage-specific damage to synaptonemal complexes and metaphase chromosomes induced by X rays in male mouse germ cells

Synaptonemal complexes reveal mutagen-induced effects in germ cell meiotic chromosomes. This study was aimed at characterizing relationships between damage to synaptonemal complexes and metaphase I chromosomes following radiation exposure at various stages of spermatogenesis. Male mice were irradiated with doses of 0, 2, or 4 Gy, and spermatocytes were harvested at times consistent with earlier exposures as spermatogonial stem cells, preleptotene cells (premeiotic DNA synthesis), or meiotic prophase cells. After stem-cell exposure, twice as many rearrangements were observed in synaptonemal complexes as in metaphase I chromosomes. Irradiation during premeiotic DNA synthesis resulted in dose-related increases in synaptonemal complex breakage and rearrangements (including novel forms) and in metaphase chromosomal aberrations. Following prophase exposure, various types and levels of damage to synaptonemal complexes and metaphase chromosomes were observed. Irradiation of zygotene cells led to high frequencies of chromosome multivalents in metaphase I without a correspondingly high level of damage in preceding prophase synaptonemal complexes. Thus irradiation of premeiotic and meiotic cells results in variable relationships between damage to synaptonemal complexes and metaphase chromosomes. Interpretations of these relationships are based upon what is known about both radiation clastogenesis and the structural/temporal relationships between synaptonemal complexes at prophase and chromosomes at metaphase I of meiosis.     

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).     

Genetic and cytological characterization of the recombination protein RAD-51 in<Emphasis Type="Italic"> Caenorhabditis elegans</Emphasis>

We investigated the role of Caenorhabditis elegans rad-51 during meiotic prophase. We showed that rad-51 mutant worms are viable, have no defects in meiotic homology recognition and synapsis but exhibit abnormal chromosomal morphology and univalent formation at diakinesis. During meiosis RAD-51 becomes localized to distinct foci in nuclei of the transition zone of the gonad and is most abundant in nuclei at late zygotene/early pachytene. Foci then gradually disappear from chromosomes and no foci are observed in late pachytene. RAD-51 localization requires the recombination genes spo-11 and mre-11 as well as chk-2, which is necessary for homology recognition and presynaptic alignment. Mutational analysis with synapsis- and recombination-defective strains, as well as the analysis of strains bearing heterozygous translocation chromosomes, suggests that presynaptic alignment may be required for RAD-51 focus formation, whereas homologous synaptonemal complex formation is required to remove RAD-51 foci.