Intranuclear fibrillar material in cereal pollen mother cells

Ultrastructural studies of cereal anthers found intranuclear bundles of microfilaments in pollen mother cells (PMCs) but not elsewhere. The ultrastructure, distribution, and behaviour of this fibrillar material (FM) are described. FM was seen in all 19 genotypes studied comprising Aegilops, Triticum, Secale, Hordeum and Avena species, which together included haploid, diploid and allo-and autopolyploid, and natural and synthetic polyploid examples. Detailed studies in diploid S. cereale, and hexaploid T. aestivum and Triticale showed that FM was present in PMC nuclei during premeiotic interphase, leptotene and zygotene but not at pachytene and later meiotic stages. Moreover, it was most abundant at late premeiotic interphase in T. aestivum, and at leptotene in S. cereale and Triticale, when it occurred in up to 100% of sampled PMC nuclei in an anther. Although FM and synaptonemal complex (SC) occurred together in some PMC nuclei at later stages, FM was present long before SC, and reached its peak of abundance before SC did. Bundles of FM often formed links at their ends between either two masses of chromatin, or more rarely, between chromatin and the nuclear membrane. Individual bundles of FM varied in length but showed roughly similar ranges of lengths and widths in these three species. They were up to about 0.2 m in diameter and about 3 m in length, equivalent to about 20% of the maximum diameter of the nuclei containing them. Reconstructions of PMC nuclei indicated that FM was never associated with centromeres but was sometimes, and perhaps usually, associated with telomeric or sub-telomeric chromosome segments.The function of FM is unknown but its possible role is discussed in relation to (1) previously described intranuclear inclusions in meiocytes and (2) the cytogenetics and developmental behaviour of meiotic nuclei in the wheat comparium. As FM was a constant and characteristic structural component of PMC nuclei, its presence is probably of functional significance to the meiotic process. If so, it may function before, and over greater distances, than SC in establishing or maintaining the coorientation of chromosomes prerequisite for normal chromosome pairing. As FM was most abundant at stages when major chromosome movements occur, yet its distribution was non-centromeric, it is suggested that it may function in the attachment and movement of telomeres at the nuclear membrane formed after premeiotic mitosis. The possibility that a bundle of FM normally links corresponding sites on two homologues is considered.     

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.     

Gene amplification in oocytes with 8 germinal vesicles from the tailed frog Ascaphus truei Stejneger

In the last 3 oogonial mitoses in Ascaphus truei all daughter nuclei remain in the same cell. The oocyte is 8-nucleate at the start of meiotic prophase and remains so until late in oogenesis when 7 of the nuclei disappear. All 8 nuclei in a single oocyte resemble one another with respect to size and chromatin distribution at all stages of meiotic prophase. Much of the Feulgen-positive material in pachytene nuclei is concentrated into one region of the nucleus. — All of the 8 germinal vesicles of yolky oocytes have a full set of lampbrush diplotene bivalents. Germinal vesicles from oocytes of up to 0.8 mm diameter have less than 100 nucleoli, some of which are multiple nucleoli in the sense that they have more than one core region. Each of the 8 nuclei in oocytes from one animal had about the same volume of nucleolar material. — Two values have been obtained for the amount of DNA in a diploid nucleus from Ascaphus. A biochemical estimate utilizing erythrocyte nuclei and the diphenylamine reaction yielded a value of 7.1 pg per nucleus. Microphotometry of erythrocyte nuclei stained with Feulgen's reagent gave a value of 8.2 pg per nucleus. — Microphotometric measurements of Feulgen-stained nuclei at various stages of meiotic prophase up to diplotene indicate that each nucleus synthesizes up to 5 pg of extrachromosomal DNA during and immediately after pachytene. This DNA is considered to be nucleolar. Autoradiography of nuclei from oocytes which had been incubated for 6h in ³H thymidine showed silver grains over pachytene and early diplotene nuclei only. In pachytene nuclei the silver grains overlaid that part of the nucleus where Feulgen-positive material was most concentrated. Most of the chromosomal material was unlabelled. — The significance of the 8-nucleate condition in Ascaphus oocytes is discussed, and the amount of nucleolar DNA synthesized at pachytene and of nucleolar material present in germinal vesicles is compared with corresponding situations in other amphibians.     

Hanging on to your homolog: the roles of pairing, synapsis and recombination in the maintenance of homolog adhesion

Homologous chromosomes initially undergo weak alignments that bring homologous sequences into register during meiosis. These alignments can be facilitated by two types of mechanisms: interstitial homology searches and telomere-telomere alignments. As prophase (and chromatin compaction) proceeds, these initial pairings or alignments need to be stabilized. In at least some organisms, such as Saccharomyces cerevisiae and S. pombe, these pairings can apparently be maintained by the creation of recombination intermediates. In contrast, synapsis during zygotene may be able to facilitate and/or maintain chromosome pairing even in the absence of exchange in several higher organisms. It thus seems possible that the synaptonemal complex plays a role both in maintaining homolog adhesion during meiotic prophase and, more speculatively, in facilitating meiotic exchange. Received: 15 November 1999; in revised form: 17 January 2000 / Accepted: 18 January 2000     

Robertsonian translocations modify genomic distribution of γH2AFX and H3.3 in mouse germ cells

Heterozygosity for Robertsonian translocations hampers pairing and synapsis between the translocated chromosome and its normal homologs during meiotic prophase I. This causes meiotic silencing of unsynapsed chromatin in pericentromeric regions. Several lines of evidence suggest that autosomal asynapsis leads to meiotic arrest in males and two underlying mechanisms have been proposed: (1) reactivation of the X and Y chromosomes due to competition for silencing factors and (2) meiotic silencing of genes that are located in the unsynapsed regions and are essential for meiotic progression. The latter mechanism requires that asynapsis and meiotic silencing spread beyond the p-arms of the normal homologs into gene-rich regions. We used chromatin immunoprecipitation assays to determine whether histones γH2AFX and H3.3, both marks of asynapsis and meiotic silencing, are enriched in gene-rich regions of the translocated chromosomes and their homologs in the spermatocytes of heterozygous carriers of Robertsonian translocations. We also asked if γH2AFX and H3.3 enrichment was reduced at the X chromosome and if γH2AFX and H3.3 enrichment was higher on the normal homolog. Our data show that γH2AFX enrichment extends as far as 9–15 Mb of the annotated genomic sequence of the q-arms of the translocated chromosomal trivalents and that both γH2AFX and H3.3 levels are reduced over the X chromosome. Our data are also suggestive of an asymmetry in γH2AFX and H3.3 enrichment with a bias toward the non-translocated homolog.     

The nuclear envelope protein Matefin/SUN-1 is required for homologous pairing in C. elegans meiosis

We identify a highly specific mutation (jf18) in the Caenorhabditis elegans nuclear envelope protein matefin MTF-1/SUN-1 that provides direct evidence for active involvement of the nuclear envelope in homologous chromosome pairing in C. elegans meiosis. The reorganization of chromatin in early meiosis is disrupted in mtf-1/sun-1(jf18) gonads, concomitant with the absence of presynaptic homolog alignment. Synapsis is established precociously and nonhomologously. Wild-type leptotene/zygotene nuclei show patch-like aggregations of the ZYG-12 protein, which fail to develop in mtf-1/sun-1(jf18) mutants. These patches remarkably colocalize with a component of the cis-acting chromosomal pairing center (HIM-8) rather than the centrosome. Our data on this mtf-1/sun-1 allele challenge the previously postulated role of the centrosome/spindle organizing center in chromosome pairing, and clearly support a role for MTF-1/SUN-1 in meiotic chromosome reorganization and in homolog recognition, possibly by mediating local aggregation of the ZYG-12 protein in meiotic nuclei.     

Meiotic cytology of Saccharomyces cerevisiae in protoplast lysates

Summary This report describes cytological features of meiosis in Saccharomyces cerevisiae prepared for electron microscopy by lysis of protoplasts or nuclei on an aqueous surface. Whereas the chromatin of cells lysed before or after meiotic prophase was widely dispersed, pachytene bivalents appeared as discrete, elongate masses of compact chromatin. These bivalents were of nearly uniform thickness; they ranged in length from about 0.6 m to 4.0 m, with a median of 1.6–1.8 m. Enzymatic digestion of chromosomal DNA removed the chromatin to reveal the underlying synaptonemal complex. The lysis of partially purified nuclei was less disruptive and thereby revealed the regular association of the telomeres with fragments of the nuclear envelope. In tetraploid cells, pachytene lysates contained quadrivalents characterized by the close apposition of chromatin masses of similar length. One or more points of intimate association appear to represent sites of exchange between pairing partners. The departure of the diploid cells from pachytene was accompanied by the renewed association of spindle microtubules with the chromosomes shortly before the diplotene chromosomes decondensed. Later, the successive meiotic divisions were identified by the appearance of a single spindle for meiosis I and of two spindles for meiosis II.     

Meiotic chromosome missegregation during apyrene meiosis in the gypsy moth,Lymantria dispar,is preceded by an aberrant prophase I

The gypsy moth, Lymantria dispar, produces two structurally and genetically distinct types of spermatozoa. The eupyrene spermatozoa are genetically haploid and structurally typical. The apyrene spermatozoa are anucleate and structurally different from eupyrene spermatozoa. To understand further the events contributing to meiotic chromosome missegregation in apyrene spermatocytes, we examined the progression of meiosis in these cells with respect to their eupyrene counterparts. Chromosomal bouquet formation and fusion of nucleolar organizing regions are disrupted in apyrene nuclei. In addition, the chromatin of apyrene nuclei is prematurely and extremely condensed compared with that of eupyrene nuclei. An antibody to the conserved synaptonemal complex protein 3 (SCP3) labeled eupyrene pachytene chromosomes, but not apyrene pachytene chromosomes. In addition, apyrene meiotic spindles are missing a subset of microtubules, which likely include kinetochore microtubules. Because the condensation behavior of meiotic chromatin in apyrene spermatocytes deviates from that of eupyrene spermatocytes, we examined the appearance and distribution of the phosphorylated form of histone H3, but no significant differences in histone H3 phosphorylation were found between apyrene and eupyrene spermatocytes. We argue that because a pachytene checkpoint is not initiated in apyrene spermatocytes, this system may provide a way to understand better the underlying biochemical connections between pairing, recombination, synapsis, kinetochore assembly and segregation of chromosomes during meiosis in a higher eukaryote.     

HIM-8 binds to the X chromosome pairing center and mediates chromosome-specific meiotic synapsis

The him-8 gene is essential for proper meiotic segregation of the X chromosomes in C. elegans. Here we show that loss of him-8 function causes profound X chromosome-specific defects in homolog pairing and synapsis. him-8 encodes a C2H2 zinc-finger protein that is expressed during meiosis and concentrates at a site on the X chromosome known as the meiotic pairing center (PC). A role for HIM-8 in PC function is supported by genetic interactions between PC lesions and him-8 mutations. HIM-8 bound chromosome sites associate with the nuclear envelope (NE) throughout meiotic prophase. Surprisingly, a point mutation in him-8 that retains both chromosome binding and NE localization fails to stabilize pairing or promote synapsis. These observations indicate that stabilization of homolog pairing is an active process in which the tethering of chromosome sites to the NE may be necessary but is not sufficient.     

Distribution of Atr protein in primary spermatocytes of a mouse chromosomal mutant: a comparison of preparation techniques

In this study, we examined the suitability of a three dimensional preparation technique for studying chromosome behaviour in the first meiotic prophase in the mouse chromosomal mutant T(1;13)H/T(1;13)Wa. To preserve cellular shape, primary spermatocytes were encapsulated in a fibrin clot. Conventionally sedimented prophase nuclei served as controls. Axial elements and lateral synaptonemal complex components were subsequently stained by immunofluorescence and the presence of axial elements at the pachytene stage was highlighted with indirect immunofluorescence against the Atr protein. We compared the distribution of Atr signal in the fibrin-embedded spermatocytes with surface-spread preparations and immunohistochemically stained histological sections of seminiferous tubules. Furthermore, fluorescence in situ hybridisation of the mouse minor satellite DNA was done on fibrin-embedded spermatocytes. The Atr signal is most conspicuous in fibrin-embedded nuclei on unpaired axial elements during pachytene, both for sex chromosomal and for autosomal segments, and expanding from these elements into the surrounding chromatin. Both spread and encapsulated zygotene nuclei with extended axial element formation proved to be positive for Atr. Mid- to late zygotene nuclei were devoid of 3,3′-diaminodibenzene deposition in the histological sections. Highlighting the unpaired axial elements in the small heteromorphic 1¹³H;1¹³Wa bivalent with an Atr signal enabled meiotic analysis of this bivalent to be carried out in a three-dimensional context. Thus, proximity of this bivalent with the sex chromosomes is found more often in three-dimensional preparations than in spread preparations. Furthermore, the development of the Atr signal over the sex chromosomes as pachytene proceeds helps in substaging of this long and heterogeneous meiotic phase, in sedimented but especially in fibrin-encapsulated nuclei. Received: 22 September 1999; in revised form: 20 December 1999 / Accepted: 21 December 1999     

The effect of the wheat Ph1 locus on chromatin organisation and meiotic chromosome pairing analysed by genome painting

The Ph1 locus in wheat influences homo(eo)logous chromosome pairing. We have analysed its effect on the behaviour and morphology of two 5RL rye telosomes in a wheat background, by genomic in situ hybridisation (GISH), using rye genomic DNA as a probe. Our main objective was to study the effect of different alleles of the Ph1 locus on the morphology and behaviour of the rye telosomes in interphase nuclei of tapetal cells and in pollen mother cells at early stages of meiosis. The telosomes, easily detectable at all stages, showed a brightly fluorescing chromomere in the distal region and a constriction in the proximal part. These diagnostic markers enabled us to define the centromere and telomere regions of the rye telosomes. In the presence of functional copies of Ph1, the rye telosomes associated at pre-leptotene, disjoined and reorganised their shape at leptotene, and became fully homologously paired at zygotene – pachytene. In plants without functional alleles (ph1bph1b), the rye telosomes displayed an aberrant morphology, their premeiotic associations were clearly disturbed and their pairing during zygotene and pachytene was reduced and irregular. The Ph1 locus also influenced the behaviour of rye telosomes in the interphase nuclei of tapetal cells: in Ph1Ph1 plants, the rye telosomes occupied distinct, parallel-oriented domains, whereas in tapetal nuclei of ph1bph1b plants they were intermingled with wheat chromosomes and showed a heavily distorted morphology. The results shed new light on the effect of Ph1, and suggest that this locus is involved in chromosome condensation and/or scaffold organisation. Our explanation might account for various apparently contradictory and pleiotropic effects of this locus on both premeiotic associations of homologues, the regulation of meiotic homo(eo)logous chromosome pairing and synapsis, the resolution of bivalent interlockings and centromere behaviour. Received: 27 April 1998; in revised form: 5 August 1998 / Accepted: 11 August 1998     

Synaptonemal complex-dependent centromeric clustering and the initiation of synapsis in Drosophila oocytes

The pairing of homologous chromosomes and the intimate synapsis of the paired homologs by the synaptonemal complex (SC) are essential for subsequent meiotic processes including recombination and chromosome segregation. Here we show that the centromere clustering plays an important role in initiating homolog synapsis during meiosis in Drosophila females. Although centromeres are not clustered prior to the onset of meiosis, all four pairs of centromeres are actively clustered into one or two masses during early meiotic prophase. Within the 16-cell cyst, centromeric clustering appears to define the first step in the initiation of synapsis. Clustering is restricted to the nuclei that form the SC and is dependent on all known SC proteins. Surprisingly, both centromeric clusters and the SC components associated with them persist long after the disassembly of the euchromatic SC at the end of pachytene. The initiation of homologous recombination through the formation of programmed double-strand breaks (DSBs) is not required for either the formation or the maintenance of the centromeric clusters. Our data support a view in which the SC-mediated clustering at the centromeres is the initiating event for meiotic synapsis.     

The control of meiosis progression in the fungus Coprinus cinereus by light/dark cycles

Meiosis progression in Coprinus cinereus is controlled by light/dark cycles. Light is essential to propel basidia into karyogamy and light intensity determines the timing of meiotic events. The higher the light intensities, the faster the fruiting bodies enter karyogamy. The critical period when light has this influence is between 16 and 6 h before karyogamy. The control is highly stage specific. A 3-h dark period is essential for a Java dikaryon and the Japanese A(mut)B(mut) homokaryon to enter meiotic metaphase; without it the fruit body is permanently arrested at diffused diplotene. This arrest is light intensity-dependent (>20 hlx) and temperature-dependent (e.g., 27 degrees C). The placement of the dark period is very stage specific; it has no effect when placed before karyogamy stage. A dikaryon of London origin is light blind and able to complete meiosis under continuous high light regime. Fruiting bodies arrested under a continuous high light can be rescued by a 3-h dark treatment, but there is always an 8-h lag time to enter meiotic metaphase. It is possible that the dark effect signals cellular processes leading to division events. Cytological studies of arrested fruiting bodies showed that chromosomes are normal in meiotic prophase through pachytene and diplotene, but are unable to undergo chromosome condensation. Genetic crosses between a monokaryon of Java stock J6;5.4 and a monokaryon BL55 or H5 of London stock showed that light-blindness is dominant, and is controlled by a single Mendelian gene.     

Development of the synaptonemal complex and the “recombination nodules” during meiotic prophase in the seven bivalents of the fungus Sordaria macrospora Auersw

Complete reconstruction of seven leptotene, six zygotene, three pachytene and three diplotene nuclei has permitted to follow the pairing process in the Ascomycete Sordaria macrospora. The seven bivalents in Sordaria can be identified by their length. The lateral components of the synaptonemal complexes (SC) are formed just after karyogamy but are discontinuous at early leptotene. Their ends are evenly distributed on the nuclear envelope. The homologous chromosomes alignment occurs at late leptotene before SC formation. The precise pairing starts when a distance of 200–300 nm is reached. Each bivalent has several independent central component initiation sites with preferentially pairing starting near the nuclear envelope. These sites are located in a constant position along the different bivalents in the 6 observed nuclei. The seven bivalents are not synchronous either in the process of alignment or in SC formation: the small chromosomes are paired first. At pachytene the SC is completed in each of the 7 bivalents. Six bivalents have one fixed and one randomly attached telomeres. The fixed end of the nucleolar organizer is the nucleolus anchored end. At diffuse stage and diplotene, only small stretches of the SC are preserved. The lateral components increase in length is approximately 34% between leptotene and pachytene. Their lengths remain constant during pachytene. From zygotene to diplotene the central components contain local thickenings (nodules). At late zygotene and pachytene each bivalent has 1 to 4 nodules and the location of at least one is constant. The total number of nodules remains constant from pachytene to diplotene and is equal to the mean total number of chiasmata. The observations provide additional insight into meiotic processes such as chromosome movements, initiation and development of the pairing sites during zygotene, the existence of fixed telomeres, the variations in SC length. The correspondence between nodules and chiasmata are discussed.     

Nuclear structure and chromosome segregation in Drosophila male meiosis depend on the ubiquitin ligase dTopors

In many organisms, homolog pairing and synapsis at meiotic prophase depend on interactions between chromosomes and the nuclear membrane. Male Drosophila lack synapsis, but nonetheless, their chromosomes closely associate with the nuclear periphery at prophase I. To explore the functional significance of this association, we characterize mutations in nuclear blebber (nbl), a gene required for both spermatocyte nuclear shape and meiotic chromosome transmission. We demonstrate that nbl corresponds to dtopors, the Drosophila homolog of the mammalian dual ubiquitin/small ubiquitin-related modifier (SUMO) ligase Topors. We show that mutations in dtopors cause abnormalities in lamin localizations, centriole separation, and prophase I chromatin condensation and also cause anaphase I bridges that likely result from unresolved homolog connections. Bridge formation does not require mod(mdg4) in meiosis, suggesting that bridges do not result from misregulation of the male homolog conjunction complex. At the ultrastructural level, we observe disruption of nuclear shape, an uneven perinuclear space, and excess membranous structures. We show that dTopors localizes to the nuclear lamina at prophase, and also transiently to intranuclear foci. As a role of dtopors at gypsy insulator has been reported, we also asked whether these new alleles affected expression of the gypsy-induced mutation ct(6) and found that it was unaltered in dtopors homozygotes. Our results indicate that dTopors is required for germline nuclear structure and meiotic chromosome segregation, but in contrast, is not necessary for gypsy insulator function. We suggest that dtopors plays a structural role in spermatocyte lamina that is critical for multiple aspects of meiotic chromosome transmission.     

Synaptonemal complex and recombination nodules in rye (Secale cereale)

Meiotic prophase in rye was investigated by serial-section reconstruction of pollen mother cell nuclei. In the mid-late zygotene nucleus, all lateral elements were continuous from telomere to telomere, and 9–20 pairing initiation sites per bivalent were observed. Chromosome and bivalent interlockings detected during zygotene were resolved at early pachytene when pairing was completed. In the three pachytene nuclei, the relative synaptonemal complex (SC) lengths and arm ratios were found to be in good correlation with light microscopic data of pachytene bivalents. Spatial tracing of the bivalents showed that they occupy separate areas in the nucleus. Three types of recombination nodules were observed: large, ellipsoïdal and small nodules at early pachytene and irregularly shaped nodules mainly associated with chromatin at late pachytene. Their number and position along the bivalents correlated well with the number and distribution of chiasmata. The classification of the seven bivalents was based on arm ratio and heterochromatic knob distribution.