The induction of orientational instability and bivalent interlocking at meiosis

The administration of 40° C heat-treatments was found to induce bivalent orientational instability and interlocking at male meiosis in the locust Locusta migratoria. Only the longest members of the complement showed orientational instability and these usually possessed single distally sited chiasmata, with near-maximal intercentromeric distances. An effect on the stability of spindle fibre microtubule association, or attachment to the chromosome, is considered to be a possible explanation of the behaviour found. Bipolar orientation was generally achieved prior to anaphase I so that chromosome segregation was usually normal. Diamphitelic bivalents provided the most common exception to this rule. They sometimes lagged at anaphase, with the separation of half-bivalents and the production of structures indistinguishable from lagging univalents. The bivalent interlocking also involved the longest members of the complement. Most combinations of rod/rod, rod/ring and ring/ring types of interlocking were found. Usually only two bivalents were interlocked in any one cell, although occasionally three were found interlocked. All types appeared to involve an effect on the regulation of chromosome pairing, although at least one of the cells found showed interlocking caused by the metaphase orientational instability. In most cells, interlocked bivalents showed stable orientation and this usually involved the unipolar orientation of each bivalent's two centromeres. Such configurations provide concrete support for the importance of physical tension in the maintenance of metaphase orientational stability. They lead to double non-disjunction at anaphase I. Interlocked bivalents showed normal congression to a mid-equatorial position with no tendency for the re-adjustment of arm ratios to equalise centromere distances from the poles. This behaviour is discussed in relation to spindle fibre dynamics and it is concluded that no hypothesis of congression currently available can satisfactorily explain all that we know of the behaviour of univalents, bivalents, multivalents and interlocked bivalents.     

Ultrastructure of meiosis in the hollyhock rust fungus,Puccinia malvacearum II. Metaphase I—Telophase I

Summary The three-dimensional structure of the spindle pole body (SPB) and meiotic spindle during early metaphase I through telophase I inPuccinia malvacearum is analyzed ultrastructurally from serial sections. During early metaphase I the spindle rotates from the perpendicular to a position oblique to the longitudinal axis and parallel to the sagittal plane of the cell. Tubular cisternae are present within the spindle at this stage. The half middle piece (MP) subtends a collateral disc (co-disc) which is inserted eccentrically within each SPB. The SPB, co-disc and half MP at opposite poles are in mirror image. During the transition from early metaphase I to full metaphase I, the spindle orients parallel to the lateral wall of the promycelium and the half MPs are lost. The co-discs partially detach from each discoid SPB and maintain this relation until the end of interphase I. Co-discs become further differentiated as they attach to the subtending sheath-like extension of the nuclear envelope previously occupied by the half MPs. Microvesicles within the nucleoplasm are specific to mid metaphase I. A metaphase plate is absent. The 14 bivalents, which are directly connected to each polar SPB by 2 to 3 kinetochore MTs, are spread over nearly the entire length of the central spindle. The first anaphasic movement involves asynchronous shortening of the kinetochore MTs while the second consists of extensive pole-to-pole elongation. Astral MTs first appear at early metaphase I and become most numerous at anaphase I. An intact nuclear envelope constricts against the central spindle at either end of the interzonal region. Concurrently, centripetal growth of the nuclear envelope under each SPB results in their gradual externalization by the end of telophase I. The sibling nuclei are cut off by constriction of the nuclear envelope at either end of the interzonal region. These meiotic stages inP. malvacearum are compared with those in other basidiomycetes and ascomycetes.     

Meiotic behaviour of chromosomes 1R, 2R and 5R in autotetraploid rye

The meiotic behaviour of chromosomes 1R, 2R and 5R was studied in C-banded preparations of autotetraploid rye. Analysis of pairing and chiasma formation was based on metaphase I configurations, using the model designed by Sybenga, with slight modifications. Frequencies of two modes of pairing (one quadrivalent or two bivalents) differed from those expected for random pairing. Although preferential pairing for some arm pairs of chromosome 2R was detected, this did not seem to be the cause of the increased bivalent pairing. This increase was attributed to either the spatial separation of the four homologous chromosomes in some premeiotic cells into two groups of two, or a correction of the synaptonemal complex, or both. The number of chiasmate associations showed variation between chromosomes and between arms within the same chromosome. It was closely related to arm length, but different after quadrivalent and bivalent pairing. This is suggested to be a consequence of partner exchange interfering with pairing and, consequently, with chiasma formation, and a different chiasma distribution after quadrivalent pairing. Variation between chromosomes in the frequencies of alternate and adjacent co-orientation in metaphase I quadrivalents without interstitial chiasmata suggests that the relative positions of the centromeres in the quadrivalent influence their co-orientation.     

Bivalent orientation and behavior in crane-fly spermatocytes recovering from cold exposure

At metaphase in crane-fly primary spermatocytes, the two sister kinetochores at the centromere of each homologue in a bivalent normally are adjacent and face the same pole; one homologue has all its kinetochore microtubules (kMTs) extending toward one pole and its partner has all its kMTs extending toward the opposite pole. In contrast, during recovery from exposure to 2 degrees C, one or both homologues in many metaphase bivalents had bipolar malorientations: all kMTs of one kinetochore extended toward one pole and some or all those of its sister extended toward the other. Metaphase sister kinetochores that had most of their kMTs extending toward the same pole were adjacent, and those with most extending toward opposite poles were separated from each other. Distances between homologous centromeres were similar to those in properly oriented bivalents. Maloriented bivalents were tilted relative to the spindle axis, and analysis of living cells showed that tilted configurations were rare during prometaphase in untreated cells but frequently arose in cold-recovering cells as initial configurations, then persisted through metaphase. This was in contrast to unipolar configurations of bivalents (configurations suggesting orientation of both homologous centromeres toward the same pole), which always reoriented shortly after the configuration arose. We conclude that in cold-recovering cells, bipolar malorientations are more stable than unipolar malorientations, and the orientation process is affected such that bipolar malorientations arise in bivalents upon initial interaction with the spindle and persist through metaphase.     

Centromere co-orientation in a spontaneous translocation heterozygote of Euchorthippus pulvinatus gallicus (Acrididae,Orthoptera)

The co-orientation of a quadrivalent in an individual of E. pulvinatus gallicus heterozygous for a reciprocal translocation is studied. The data agree with the hypothesis that the co-orientation behaviour of two adjacent centromeres in a quadrivalent depends on the relative distance between them at metaphase I, this distance being determined by chiasmata.     

Chromosomal interchange inEleutherine plicata Herb

Chromosome behaviour at metaphase I and anaphase I of meiosis inEleutherine plicata Herb. (2n=14) is studied. Cells with chromosome associations comprising an association of four long chromosomes, in addition to five bivalents were observed more frequently than those with seven bivalents. it is concluded that the ring of four is due to a segmental interchange between the two long non-homologous chromosome pairs. The ring of four at anaphase I showed delayed disjunction, bridge formation and irregular separation of chromosomes in a number of cells while the behaviour of the other bivalents was normal.     

The quantitative analysis of chromosome pairing and chiasma formation based on the relative frequencies of M I configurations

In autotetraploids, chromosome pairing may be in the form of quadrivalents or bivalent pairs. Whether or not the quadrivalents are maintained until first meiotic metaphase depends on the formation of chiasmata. The relative frequencies of M I configurations thus contain information both on pairing and on chiasma formation. With distal chiasma localisation six configurations can be recognised and their relative frequencies determined: ring quadrivalents, chain quadrivalents, trivalents (with univalent), ring bivalents, open (rod) bivalents, univalent pairs. These represent five degrees of freedom permitting five parameters to be estimated: the frequency (f) of quadrivalent pairing; the frequencies of chiasmate association of the two ends (arms in metacentrics), a′, b′, after quadrivalent pairing, and a, b after bivalent pairing. — The appropriate formulae have been derived and applied to observations on Tradescantia virginiana (4n=24) which has pronounced distal chiasma localisation. Slight modifications make the model applicable to autotetraploids with interstitial in addition to distal chiasmata. In T. virginiana, chromosome pairing appeared to be random between homologues (65.8% quadrivalent pairing; 55.4% observed at M I). After quadrivalent pairing chiasmate association is frequent in the “average long” arm (95.0%) and much less so in the other arm (60.5%). This is attributed to partner exchange. After bivalent pairing chiasma frequencies are still different for the two arms (93.8% and 83.5% association respectively) but much less pronounced. Various complications are discussed.     

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.     

Meiotic segregation of chromosomes in Drosophila melanogaster oocytes

A new technique was developed for a light microscopic analysis of meiosis in Drosophila oocytes. — When the nuclear envelope breaks down the bivalents, till then compressed into a karyosome, separate in early prometaphase. The homologues remain associated by chiasmata except for the fourth chromosomes which are no longer associated. Non-homologous chromosomes regularly segregating from each other in genetic experiments are also unconnected after karyosome disintegration but during metaphase I the fourth chromosomes and the heterologous pairs coorient on the same arc of the spindle and move precociously towards opposite poles. Nondisjunction and other irregularities are not infrequent in oocytes having an uneven number of achiasmatic elements. The fourth chromosomes and the Xs or the large autosomes, when lacking chiasmata, may be involved in non-homologous segregation. In c3G homozygotes all chromosomes appear as univalents in prometaphase. Segregation is variable but the observations suggest the polar distribution of equal numbers of chromosomes in variable combinations irrespective of the size. — Coorientation of univalents may be accounted for if the centromeres, whether homologous or non-homologous, are associated in pairs during early meiotic prophase, and that in the karyosome these pairing relationships are preserved until spindle organization at the onset of prometaphase.     

Cytology of egg maturation in the silkworm (Bombyx mori L.) in artificial parthenogenesis

The orientation of bivalentss in the first meiotic metaphase was studied on the squash preparations and sections of eggs in Bombyx mori L., Antheraea pernyi Guèr and Carpocapsa pomonella L. The reduction fissure of bivalents separating the homologous chromosomes lies in the plane of spindle equator; the equation fissure separating the sister chromatids is parallel to the long spindle axis. Complicated rearrangements are observed in the nuclei of the silkworm eggs stimulated to ameiotic parthenogenesis by the high temperature dissociation of bivalents, full destruction of spindle, formation of a new spindle and metaphase plate. The latter includes univalent chromosomes involved in the equation division. As a result, two genetically identical pronuclei with the somatic set of chromosomes are formed.     

Meiosis in Mesostoma ehrenbergii ehrenbergii (Turbellaria,Rhabdocoela)

At metaphase I of meiosis in spermatocytes of Mesostoma ehrenbergii ehrenbergii [2n=10] three bivalents and four univalents form. The same two chromosome pairs always form the univalents. Analysis of metaphase I, anaphase I and metaphase II configurations in fixed testis material suggested that the distribution of the four univalents is not a random process but the correct segregation of one member of each pair to each pole is actively achieved before the end of metaphase I. In live preparations of testis material univalents were observed to move between the poles of metaphase I cells, eventually reaching the correct segregation. All cells observed to enter anaphase I had the correct segregation of univalents. It is proposed that the univalent movement during metaphase I is directed towards obtaining the correct segregation of univalents before the cells enter anaphase.     

Meiosis in a triploid hybrid of <Emphasis Type="Italic">Gossypium</Emphasis>: high frequency of secondary bipolar spindles at metaphase II

Studies on meiosis in pollen mother cells (PMCs) of a triploid interspecific hybrid (3x = 39 chromosomes, AAD) between tetraploid Gossypium hirsutum (4n = 2x = 52,AADD) and diploid G. arboreum (2n = 2x = 26,AA) are reported. During meiotic metaphase I, 13 AA bivalents and 13 D univalents are expected in the hybrid. However, only 28% of the PMCs had this expected configuration. The rest of the PMCs had between 8 and 12 bivalents and between 12 and 17 univalents. Univalents lagged at anaphase I, and at metaphase II one or a group of univalents remained scattered in the cytoplasm and failed to assemble at a single metaphase plate. Primary bipolar spindles organized around the bivalents and multivalents. In addition to the primary spindle, several secondary and smaller bipolar spindles organized themselves around individual univalents and groups of univalents. Almost all (97%) of the PMCs showed secondary spindles. Each spindle functioned independently and despite their multiple numbers in a cell, meiosis I proceeded normally, with polyad formation. These observations strongly support the view that in plant meiocytes bilateral kinetochore symmetry is not required for establishing a bipolar spindle and that single unpaired chromosomes can initiate and stabilize the formation of a functional bipolar spindle.     

Meiotic association and segregation of the achiasmatic giant sex chromosomes in the male field vole (Microtus agrestis)

Controversy exists regarding the meiotic behaviour of the giant sex chromosomes during spermatogenesis in the field vole, Microtus agrestis. Both univalents and bivalents have been observed between diakinesis and metaphase I. These differences seem to be dependent on the technique used. The present study employs electron microscopy of serially sectioned testes tubules and light microscopy of microspread preparations to re-examine the behaviour of sex chromosomes during meiosis. In microspreads, about one-third of the early pachytene nuclei examined showed end joining of the X and Y axes. The longitudinal heterogeneity of the chromosomes in the form of axial thickenings allowed the detection of two different end-joining patterns. In the remaining early pachytene cells as well as in all mid to late pachytene cells seen, the X and Y axes had, though near to each other, no contact in the form of a synaptonemal complex. If a synaptonemal complex is a prerequisite for genetic exchange, the sex chromosomes in M. agrestis males must be achiasmatic. The analysis of serial sections through an early pachytene and a late prophase I nucleus with the electron microscope revealed that the sex chromosomes occupied a common area. By metaphase I, the centromeres of the X and Y were oriented towards opposite spindle poles while the chromosomes remained attached to one another by their distal segments at the level of the metaphase I plate. As a consequence of the large size of the sex chromosomes their centromeres lay close to the spindle poles. In anaphase I the sex chromosomes maintained their metaphase position until the autosomes approached the spindle poles. During autosomal migration a medial constriction developed where the sex chromosomes were mutually associated, the X and Y became separated, and joined the autosomes. In metaphase II the chromatids of the sex chromosomes lay side by side and exhibited a delayed separation in the subsequent anaphase. It is suggested that heterochromatin, which represents a major part of both sex chromosomes, plays a role in the association of the two achiasmatic sex chromosomes in metaphase I and in the delayed separation of the chromatids of the sex chromosomes in anaphase II.Dedicated to Prof. C.-G. Arnold (Erlangen) on the occasion of his 60th birthday     

Chiasma interference and centromere co-orientation in a spontaneous translocation heterozygote of Euchorthippus pulvinatus gallicus (Acrididae; Orthoptera)

The meiosis of an individual of the species Euchorthippus pulvinatus gallicus heterozygous for a reciprocal translocation involving chromosomes 3 and 6 has been analysed using the Giemsa C-banding technique. It is concluded that: (i) Chiasma interference in the quadrivalent seems to act only at the arm level. There is no interference across the translocation break point. No interchromosomal chiasma interference could be demonstrated, (ii) The results concerning the co-orientation of the quadrivalent suggest that the length of the chromosomal segments between two adjacent centromeres at metaphase I is related with their orientation behaviour.     

Prometaphase I and anaphase I in an interchange heterozygote of Allium triquetrum (Liliaceae)

Four unstable malorientations in a chain quadrivalent, as well as maloriented bivalents, were studied in fixed prometaphase I pollen-mothercells from an interchange heterozygote of Allium triquetrum. The relative frequencies of these malorientations, in cells from pollen sacs of different developmental ages, suggest that maloriented bivalents, on the average, reorient before maloriented quadrivalents; and that, similarly, there are differences in the timing of reorientation amongst the four types of maloriented quadrivalents. — Further, the proportion of anaphase I cells derived from alternate orientation in the interchange quadrivalent is lower than expected in pollen sacs with a high percentage of cells in mid-anaphase; but higher than expected in pollen sacs in which relatively few cells have as yet proceeded into anaphase, and also in those in which most cells have already passed through anaphase. Arguably, these data are a direct outcome of the differential behavior of unstable maloriented quadrivalents in the preceeding prometaphase.     

Ultrastructure of meiosis in the hollyhock rust fungus,Puccinia malvacearum I. Prophase I — Prometaphase I

Summary A thoroughly documented account of the ultrastructure of the meiotic spindle pole body (SPB) cycle in a rust (Basidiomycota, Uredinales) is presented for the first time. The three-dimensional structure of the SPB and spindle during meiosis in the hollyhock rust fungusPuccinia malvacearum is analyzed from serial sections of preselected stages. This paper covers prophase I to prometaphase I. At late prophase I, the nucleolus disperses and does not reappear until the end of meiosis. The SPB at late prophase I consists of two, 4-layered discs, 0.8–1.0 m in diameter, connected by a middle piece (MP). The SPB is associated with a differentiated region of the nuclear envelope and nucleoplasm. At late diplotene to diakinesis, each disc generates a half spindle as it inserts into an otherwise intact nuclear envelope. The MP connecting the interdigitating half spindles elongates and eventually splits transversely during subsequent spindle elongation. Each half MP, which is attached to a SPB disc, becomes inserted in a sheath-like extension of the nuclear envelope. The intranuclear late prometaphase I spindle always becomes oriented perpendicularly to the longitudinal axis and sagittal plane of the metabasidium. There are 200–290 spindle microtubules (MTs) at each SPB at late prometaphase. The nonkinetochore MTs form a coherent central spindle around which the kinetochore MTs and bivalents are spread. A metaphase plate is absent. The results are compared with SPB behavior and spindle structure in early meiosis of other basidiomycetes and ascomycetes.     

Co-orientation stability by physical tension: a demonstration with experimentally interlocked bivalents

Using living spermatocyte cultures of the grasshopper Melanoplus differentialis three experiments were successfully carried out in which bivalents were interlocked at metaphase I, using a micromanipulator. Two rod-shaped bivalents, with terminal kinetochores, were each made unipolar to opposite poles and placed together in such a way that each put tension upon the other when pulled by its sets of spindle fibers. The experiments unambiguously demonstrated and supported the importance of physical tension in maintaining metaphase I coorientation stability. In two of these experiments the interlocked structures remained stable for 168 and 100 minutes prior to anaphase I separation. In the third experiment, following 25 minutes stability under tension the two bivalents pulled apart. No longer under tension, each bivalent reoriented within 15–30 min, became bipolar, and congressed.     

Studies on the Sporogenous Lineage in the Moss Timmiella barbuloides

This correlated immunofluorescence and electron microscope study reveals that the microtubule arrays during meiosis in Timmiella barbuloides mirror those in other mosses but the organization of the metaphase I spindle is quite different. In other mosses the sagittiform metaphase I spindle initially contains four bands of microtubules derived from the tetrahedral system present at prophase. These bands converge towards the division axis and each half spindle contains two focal points of microtubules straddling a cleavage furrow. In Timmiella the sagittiform spindle also contains four microtubular foci derived from the preprophasic tetrahedron. However, one of these contributes to one half spindle, the other half deriving from the three remaining foci orientated at approximately 120° to each other. In contrast to other mosses the sporocytes in Timmiella are hardly lobed, the cleavage-furrows ill-defined, the prophasic plastid positioning in the lobes is also more variable and the organelle band in meiocytes comprises mitochondria alone.     

THE QUESTION OF POST-REDUCTION IN SPHAGNUM

The 19 spatially distinct chromosomal units at first meiotic metaphase in sporophytically diploid species of Sphagnum have usually been considered to be bivalents, but one investigator (Sorsa, 1956) has interpreted them as chromosomes from dissociated bivalents and meiosis as post-reductional. The present studies on diploid S. squarrosum (Pers.) Crome establish the chromosome number on the basis of the following evidence: there are in addition to m-chromosomes, 19 pairs of chromosomes in early prophase, 19 bivalents at diakinesis, 19 chromosomes in each of the two sets at second metaphase, 19 daughter chromosomes in each of the four sets at late second anaphase, and 19 chromosomes in gametophytic mitoses. The 19 bodies at first meiotic metaphase in diploid species are true bivalents in loose secondary association, which has led to their erroneous interpretation as chromosomes of dissociated bivalents. The gametic chromosome number in sporophytically diploid Sphagnum is therefore, without doubt, n = 19, and this evidence negates the claim for post-reduction in Sphagnum.     

The relation between cell size,chromosome length and the orientation of chromosomes in dividing root cortex cells

Cells in the root meristem are organised in longitudinal files. Repeated transverse cell divisions in these files are the prime cause of root growth. Because of the orientation of the cell divisions, we expected to find mitoses with an spindle axis parallel to the file axis. However, we observed in the root cortex ofVicia faba large number of oblique chromosome orientations. From metaphase to telophase there was a dramatic increase of the rotation of the spindle axis. Measurements of both the size of the cortex cells and the chromosome configurations indicated that most cells were too small for an orientation of the spindle parallel to the file axis. Space limitation force the spindle into an oblique position. Despite this spindle axis rotation, most daughter cells remained within the original cell file. Only in extremely flat cells did the position of the daughter nuclei forced the cell to set a plane of division parallel to the file axis, which result in side-by-side orientation of the daughter cells. Telophase spindle axis rotations are also observed inCrepis capillaris andPetunia hybrida.. These species have respectively medium and small sized chromosomes compared toVicia. Since space limitation, which causes the rotation, depends both on cell and chromosome size, the frequency and extent of the phenomenon in former two species is comparatively low.