Chromosome micromanipulation

The relationship of kinetochore orientation and reorientation to orderly chromosome distribution in anaphase has been studied experimentally by micromanipulation of living grasshopper spermatocytes. Bivalents or the X chromosome at prometaphase or metaphase I can be detached from the spindle with a microneedle and moved to any desired location within the cell. Following a pause of variable duration the detached chromosome invariably moved, kinetochores foremost, back to the spindle, reassumed its characteristic metaphase position, and, with one exception, segregated normally at anaphase I. Detachment from the spindle is demonstrated unequivocally (1) by manipulation evidence for the absence of the firm spindle connections seen both before detachment and after reattachment and (2) by a functional criterion: a given kinetochore, oriented to one pole before detachment, often orients to the opposite pole after detachment. The segregation in anaphase was always as expected from the final, post-operation, orientation. Reorientation and prometaphase and anaphase movement after detachment cannot be distinguished from their counterparts in control cells. Kinetochore position after detachment is the primary determinant of the pole to which that kinetochore will orient. Therefore, since the experimenter determines kinetochore position, he can cause any given half-bivalent to segregate to a predetermined pole at anaphase I. Similarly, orientation of both half-bivalents to the same pole can be induced. These mal-oriented bivalents invariably reorient and normal anaphase segregation ensues. Non-disjunction can, however, be produced directly in late anaphase. These experiments are based upon current views of orderly chromosome distribution; their success confirms our understanding of the fundamental orientation process.     

Influence of autosome movements and of sex-chromosome movements on sex-chromosome segregation in crane fly spermatocytes

In crane fly spermatocyte meiosis 3 autosome half-bivalents normally move to each spindle pole in anaphase while the 2 amphitelic sex-chromosome univalents remain at the equator. The sex-chromosome univalents move to opposite poles after the autosomes reach the poles. — We used micromanipulation to detach half-bivalents in anaphase. When re-attached half-bivalents were syntelically oriented to the original pole, sex-chromosome segregation was usually not altered. When re-attached half-bivalents were amphitelically oriented, sex-chromosome segregation was usually altered: usually the amphitelic autosome segregated against one sex-chromosome while the other sex-chromosome remained at the equator. When re-attached half-bivalents were syntelically oriented to the opposite pole, sex-chromosome segregation was often altered: often one sex-chromosome moved normally to the spindle pole with 2 autosomal half-bivalents, while the other sex-chromosome did not move to the spindle pole with 4 autosomal half-bivalents, but remained at the equator. — The direction of motion of a sex-chromosome could be altered even after sex-chromosome segregation had begun, by suitable micromanipulation of the other sex-chromosome. — Amphitelic chromosomes that were not on the equator at the start of anaphase segregated predominantly to the closer spindle pole. Detached half-bivalents showed no preference for the closer pole when they re-attached with syntelic orientation. — We discuss some possible hypotheses for non-independent movements, and some implications of the results.     

Micromanipulation studies of chromosome movement. I. Chromosome-spindle attachment and the mechanical properties of chromosomal spindle fibers

We have used micromanipulation to study the attachment of chromosomes to the spindle and the mechanical properties of the chromosomal spindle fibers. Individual chromosomes can be displaced about the periphery of the spindle, in the plane of the metaphase plate, without altering the structure of the spindle or the positions of the nonmanipulated chromosomes. From mid-prometaphase through the onset of anaphase, chromosomes resist displacement toward either spindle pole, or beyond the spindle periphery. In anaphase a chromosome can be displaced either toward its spindle pole or laterally, beyond the periphery of the spindle; however, the chromosome resists displacement away from the spindle pole. When an anaphase half-bivalent is displaced toward its spindle pole, it stops migrating until the nonmanipulated half-bivalents reach a similar distance from the pole. The manipulated half-bivalent then resumes its poleward migration at the normal anaphase rate. No evidence was found for mechanical attachments between separating half-bivalents in anaphase. Our observations demonstrate that chromosomes are individually anchored to the spindle by fibers which connect the kinetochores of the chromosomes to the spindle poles. These fibers are flexible, much less extensible than the chromosomes, and are to pivot about their attachment points. While the fibers are able to support a tensile force sufficient to stretch a chromosome, they buckle when subjected to a compressive force. Preliminary evidence suggests that the mechanical attachment fibers detected with micromanipulation correspond to the birefringent chromosomal spindle fibers observed with polarization microscopy.     

“Bouquet arrest”, monopolar chromosomes segregation, and correction of the abnormal spindle

According to our data, the arrest of univalents in bouquet arrangement is a widespread meiotic feature in cereal haploids and allohaploids (wide hybrids F₁). We have analyzed 83 different genotypes of cereal haploids and allohaploids with visualization of the cytoskeleton and found a bouquet arrest in 45 of them (in 30% to 100% pollen mother cells (PMCs)). The meiotic plant cell division in 26 various genotypes with a zygotene bouquet arrest was analyzed in detail. In three of them in PMCs, a very specific monopolar conic-shaped figure at early prometaphase is formed. This monopolar figure consists of mono-oriented univalents and their kinetochore fibers converging in pointed pole. Such figures are never observed at wild-type prometaphase or in asynaptic meiosis in the variants without a bouquet arrest. Later at prometaphase, the bipolar central spindle fibers join in this monopolar figure, and a bipolar spindle with all univalents connected to one pole is formed. As a result of monopolar chromosome segregation at anaphase and normal cytokinesis at telophase, a dyad with one member carrying a restitution nucleus and the other enucleated is formed. However, such phenotype has only three genotypes among 26 analyzed with a bouquet arrest. In the remaining 23 haploids and allohaploids, the course of prometaphase was altered after the conic monopolar figure formation. In these variants, the completely formed conic monopolar figure was disintegrated into a chaotic network of spindle fibers and univalents acquired a random orientation. This arrangement looks like a mid-prometaphase in the wild-type meiosis. At late prometaphase, a bipolar spindle is formed with the univalents distributed more or less equally between two poles, similar to the phenotypes without a bouquet arrest. The product of cell division is a dyad with aneuploid members. Thus, the spindle abnormality—monopolar chromosome orientation—is corrected. In some cells the correction of the prometaphase monopolus occurs by means of its splitting into two half-spindles and their rotation along the future division axis.     

Chromosome movement in mitosis requires microtubule anchorage at spindle poles

Anchorage of microtubule minus ends at spindle poles has been proposed to bear the load of poleward forces exerted by kinetochore-associated motors so that chromosomes move toward the poles rather than the poles toward the chromosomes. To test this hypothesis, we monitored chromosome movement during mitosis after perturbation of nuclear mitotic apparatus protein (NuMA) and the human homologue of the KIN C motor family (HSET), two noncentrosomal proteins involved in spindle pole organization in animal cells. Perturbation of NuMA alone disrupts spindle pole organization and delays anaphase onset, but does not alter the velocity of oscillatory chromosome movement in prometaphase. Perturbation of HSET alone increases the duration of prometaphase, but does not alter the velocity of chromosome movement in prometaphase or anaphase. In contrast, simultaneous perturbation of both HSET and NuMA severely suppresses directed chromosome movement in prometaphase. Chromosomes coalesce near the center of these cells on bi-oriented spindles that lack organized poles. Immunofluorescence and electron microscopy verify microtubule attachment to sister kinetochores, but this attachment fails to generate proper tension across sister kinetochores. These results demonstrate that anchorage of microtubule minus ends at spindle poles mediated by overlapping mechanisms involving both NuMA and HSET is essential for chromosome movement during mitosis.     

Chromosome micromanipulation

The attachment of individual chromosomes to the spindle has been studied by micromanipulation in functionally normal grasshopper spermatocytes. Prometaphase to anaphase I chromosomes can be repeatedly stretched with a microneedle without much increase in the distance between the kinetochores and the poles. Individual chromosomes can, however, be displaced laterally (prometaphase-anaphase) or toward the pole (anaphase) without loss of spindle attachment and without greatly disturbing other chromosomes. It is concluded that chromosomes are firmly and individually attached to the spindle by chromosomal spindle fibers which are capable of bearing any normal mitotic load, including the stretching of dikinetic (dicentric) chromosomes in anaphase. Prolonged or severe manipulation can produce a small — three or four micron — increase in the kinetochore-to-pole distance. Anaphase motion continues normally in spite of lateral or poleward displacements or of small increases in the kinetochore-to-pole distance. In late anaphase, chromosomes can be displaced to the opposite pole. An unusual, rapid motion back toward the original pole follows such displacements, but repeated displacements eventually result in non-disjunction. No evidence for firm interzonal connections between anaphase chromosomes was obtained. Prometaphase and metaphase bivalents can be detached from the spindle by manipulations other than bivalent stretching, but half-bivalents in anaphase are never detached by these manipulations.This investigation was supported in part by research grants GM-8480 and GM-13745 from the Division of General Medical Sciences, United States Public Health Service.     

Chromosome segregation in crane-fly spermatocytes: cold treatment and cold recovery induce anaphase lag

Anaphase lagging of autosomes was observed in 6.1±5.4% of the primary spermatocytes in untreated larvae of the crane fly, Nephrotoma suturalis. Lagging was induced by exposure of larvae to 6 ° C and during recovery at 22 ° C from exposure to 0.2, 2, and 6 ° C. The incidence of anaphase lag was maximal at 80 to 90 min of recovery. Induced lagging was observed at that recovery time after exposures of only 2.5 h to 2 or 0.2 ° C, but its incidence increased with longer exposures. As many as 85% of the cells in anaphase contained autosomal laggards after 61 h at 2 ° C and 80 to 90 min of recovery. At 2 ° C, cells reached the prophase-prometaphase transition, but spindles did not appear to form. Those cells proceeded through prometaphase during recovery, reaching mid-anaphase after 80 to 90 min of recovery. Chromosomes that lagged at anaphase during recovery from 2 ° C were observed in living cells to be half-bivalents derived from bivalents that congressed to the metaphase plate. One or both half-bivalents of any bivalent could lag. In some cells, one half-spindle had more half-bivalents than the other. Cells with autosomal laggards often did not cleave, and in uncleaved cells the second division employed spindles having two, three, or four poles. The basis of induced lagging might be a lapse in spindle attachment or motive force application at the start of anaphase or a failure of chromosomes to achieve proper orientation before the onset of anaphase.     

Functional autonomy of monopolar spindle and evidence for oscillatory movement in mitosis

The oscillations of chromosomes associated with a single spindle pole in monocentric and bipolar spindles were analysed by time-lapse cinematography in mitosis of primary cultures of lung epithelium from the newt Taricha granulosa. Chromosomes oscillate toward and away from the pole in all stages of mitosis including anaphase. The duration, velocity, and amplitude of such oscillations are the same in all stages of mitosis. The movement away from the pole in monocentric spindle is rapid enough to suggest the existence of a previously unrecognized active component in chromosome movement, presumably resulting from a pushing action of the kinetochore fiber. During prometaphase oscillations, chromosomes may approach the pole even more closely than at the end of anaphase. Together, these observations demonstrate that a monopolar spindle is sufficient to generate the forces for chromosome transport, both toward and away from the pole. The coordination of the aster/centrosome migration in prophase with the development of the kinetochore fibers determines the course of mitosis. After the breaking of the nuclear envelope in normal mitosis, aster/centrosome separation is normally followed by the rapid formation of bipolar chromosomal fibers. There are two aberrant extremes that may result from a failure in coordination between these processes: (a) A monocentric spindle will arise when aster separation does not occur, and (b) an anaphaselike prometaphase will result if the aster/centrosomal complexes are already well-separated and bipolar chromosomal fibers do not form. In the latter case, the two monopolar prometaphase half-spindles migrate apart, each containing a random number of two chromatid (metaphase) monopolar-oriented chromosomes. This random segregation of prometaphase chromosome displays many features of a standard anaphase and may be followed by a false cleavage. The process of polar separation during prometaphase occurs without any visible interzonal structures. Aster/centrosomes and monopolar spindles migrate autonomously by an unknown mechanism. There are, however, firm but transitory connections between the aster center and the kinetochores as demonstrated by the occasional synchrony of centrosome-kinetochore movement. The data suggest that aster motility is important in the progress of both prometaphase and anaphase in normal mitosis.     

Chromosome behavior after laser microirradiation of a single kinetochore in mitotic PtK2 cells

The role of the kinetochore in chromosome movement was studied by 532- nm wavelength laser microirradiation of mitotic PtK2 cells. When the kinetochore of a single chromatid is irradiated at mitotic prometaphase or metaphase, the whole chromosome moves towards the pole to which the unirradiated kinetochore is oriented, while the remaining chromosomes congregate on the metaphase plate. The chromatids of the irradiated chromosome remain attached to one another until anaphase, at which time they separate by a distance of 1 or 2 micrometers and remain parallel to each other, not undergoing any poleward separation. Electron microscopy shows that irradiated chromatids exhibit either no recognizable kinetochore structure or a typical inactive kinetochore in which the tri-layer structure is present but has no microtubules associated with it. Graphical analysis of the movement of the irradiated chromosome shows that the chromosome moves to the pole rapidly with a velocity of approximately 3 micrometers/min. If the chromosome is close to one pole at irradiation, and the kinetochore oriented towards that pole is irradiated, the chromosome moves across the spindle to the opposite pole. The chromosome is slowed down as it traverses the equatorial region, but the velocity in both half-spindles is approximately the same as the anaphase velocity of a single chromatid. Thus a single kinetochore moves twice the normal mass of chromatin (two chromatids) at the same velocity with which it moves a single chromatid, showing that the velocity with which a kinetochore moves is independent, within limits, of the mass associated with it.     

Involvement of chromatid cohesiveness at the centromere and chromosome arms in meiotic chromosome segregation: A cytological approach

Kinetochores and chromatid cores of meiotic chromosomes of the grasshopper species Arcyptera fusca and Eyprepocnemis plorans were differentially silver stained to analyse the possible involvement of both structures in chromatid cohesiveness and meiotic chromosome segregation. Special attention was paid to the behaviour of these structures in the univalent sex chromosome, and in B univalents with different orientations during the first meiotic division. It was observed that while sister chromatid of univalents are associated at metaphase I, chromatid cores are individualised independently of their orientation. We think that cohesive proteins on the inner surface of sister chromatids, and not the chromatid cores, are involved in the chromatid cohesiveness that maintains associated sister chromatids of bivalents and univalents until anaphase I. At anaphase I sister chromatids of amphitelically oriented B univalents or spontaneous autosomal univalents separate but do not reach the poles because they remain connected at the centromere by a long strand which can be visualized by silver staining, that joins stretched sister kinetochores. This strand is normally observed between sister kinetochores of half-bivalents at metaphase II and early anaphase II. We suggest that certain centromere proteins that form the silver-stainable strand assure chromosome integrity until metaphase II. These cohesive centromere proteins would be released or modified during anaphase II to allow normal chromatid segregation. Failure of this process during the first meiotic division could lead to the lagging of amphitelically oriented univalents. Based on our results we propose a model of meiotic chromosome segregation. During mitosis the cohesive proteins located at the centromere and chromosome arms are released during the same cellular division. During meiosis those proteins must be sequentially inactivated, i.e. those situated on the inner surface of the chromatids must be eliminated during the first meiotic division while those located at the centromere must be released during the second meiotic division.by D.P. Bazett-Jones     

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.     

The kinetochore fiber structure in the acentric spindles of the green algaOedogonium

Summary The microtubule (MT) arrangement in three kinetochore fibers in the acentric spindles of the green algaOedogonium cardiacum were reconstructed from serial sections of prometaphase and metaphase cells. The majority of the MTs attached to the kinetochore (kMTs) are relatively short, extending less than a third of the distance to the putative spindle pole region, and none extended the full distance. Fine filaments and a matrix described earlier (Schibler andPickett-Heaps 1980) were associated with the MTs all along the fibers. Live cells ofOedogonium were also studied by time lapse cinematography for correlation with the ultrastructural observations. Late prometaphase and metaphase kinetochore fibers appear to move independently as if unattached at their poleward ends. These observations suggest that kinetochore fibers inOedogonium are not attached to a specific pole structure from late prometaphase until the inception of anaphase. The results are discussed with reference to spindle structure and function in general.     

Functional implications of cold-stable microtubules in kinetochore fibers of insect spermatocytes during anaphase

In normal anaphase of crane fly spermatocytes, the autosomes traverse most of the distance to the poles at a constant, temperature-dependent velocity. Concurrently, the birefringent kinetochore fibers shorten while retaining a constant birefringent retardation (BR) and width over most of the fiber length as the autosomes approach the centrosome region. To test the dynamic equilibrium model of chromosome poleward movement, we abruptly cooled or heated primary spermatocytes of the crane fly Nephrotoma ferruginea (and the grasshopper Trimerotropis maritima) during early anaphase. According to this model, abrupt cooling should induce transient depolymerization of the kinetochore fiber microtubules, thus producing a transient acceleration in the poleward movement of the autosomal chromosomes, provided the poles remain separated. Abrupt changes in temperature from 22 degrees C to as low as 4 degrees C or as high as 31 degrees C in fact produced immediate changes in chromosome velocity to new constant velocities. No transient changes in velocity were observed. At 4 degrees C (10 degrees C for grasshopper cells), chromosome movement ceased. Although no nonkinetochore fiber BR remained at these low temperatures, kinetochore fiber BR had changed very little. The cold stability of the kinetochore fiber microtubules, the constant velocity character of chromosome movement, and the observed Arrhenius relationship between temperature and chromosome velocity indicate that a rate-limiting catalyzed process is involved in the normal anaphase depolymerization of the spindle fiber microtubules. On the basis of our birefringence observations, the kinetochore fiber microtubules appear to exist in a steady-state balance between comparatively irreversible, and probably different, physiological pathways of polymerization and depolymerization.     

Backward chromosome movement in anaphase, after irradiation of kinetochores or kinetochore fibres

Summary We used an ultraviolet microbeam to irradiate kinetochores, or to irradiate kinetochore fibres just in front of kinetochores, in anaphase crane-fly spermatocytes. Forward movements were blocked after most irradiations, but in 7 cells the associated anaphase half-bivalents moved backward, toward their partner half-bivalents, with speeds faster than poleward movements. The occurrence of backward movement suggests that there may be mechanical connections between separating half-bivalents. We have been unable to find conditions to obtain these results reproducibly.     

Malorientation and abnormal segregation of chromosomes during recovery from colcemid and nocodazole

Reversal of meiotic arrest in crane-fly spermatocytes by U. V. irradiation of Colcemid-arrested cells or by rinsing Nocodazole-arrested cells in fresh buffer results in the induction of chromosome malorientation. Malorientations observed among Colcemid-recovering and Nocodazole-recovering spermatocytes at frequencies higher than normally observed in untreated cells included associations of sister kinetochores of half-bivalents with both spindle poles (amphitely), in contrast with associations of sisters with only one pole (syntely) as is usually found during the first meiotic division. In several cases, prior to anaphase onset, maloriented bivalents appeared unusually tilted with respect to the spindle axis, and during anaphase they gave rise to laggard half-bivalents that did not segregate during anaphase along with half-bivalents having proper syntelic orientation. The results parallel previous findings obtained during cold recovery, and the properties of the drugs used here suggest that their action on microtubules, although reversible, induces malorientation during recovery from meiotic arrest.     

Distance segregation of sex chromosomes in crane-fly spermatocytes studied using laser microbeam irradiations

Univalent sex chromosomes in crane-fly spermatocytes have kinetochore spindle fibres to each spindle pole (amphitelic orientation) from metaphase throughout anaphase. The univalents segregate in anaphase only after the autosomes approach the poles. As each univalent moves in anaphase, one spindle fibre shortens and the other spindle fibre elongates. To test whether the directionality of force production is fixed at anaphase, that is, whether one spindle fibre can only elongate and the other only shorten, we cut univalents in half with a laser microbeam, to create two chromatids. In both sex-chromosome metaphase and sex-chromosome anaphase, the two chromatids that were formed moved to opposite poles (to the poles to which their fibre was attached) at speeds about the same as autosomes, much faster than the usual speeds of univalent movements. Since the chromatids moved to the pole to which they were attached, independent of the direction to which the univalent as a whole was moving, the spindle fibre that normally elongates in anaphase still is able to shorten and produce force towards the pole when allowed (or caused) to do so.     

Micromanipulation of Chromosomes in Mitotic Vertebrate Tissue Cells: Tension Controls the State of Kinetochore Movement

In mitotic vertebrate tissue cells, chromosome congression to the spindle equator in prometaphase and segregation to the poles in anaphase depend on the movements of kinetochores at their kinetochore microtubule attachment sites. To test if kinetochores sense tension to control their states of movement poleward (P) and away from the pole (AP), we applied an external force to the spindle in preanaphase newt epithelial cells by stretching chromosome arms with microneedles. For monooriented chromosomes (only one kinetochore fiber), an abrupt stretch of an arm away from the attached pole induced the single attached kinetochore to persist in AP movement at about 2 μm/min velocity, resulting in chromosome movement away from the pole. When the stretch was reduced or the needle removed, the kinetochore switched to P movement at about 2 μm/min and pulled the chromosome back to near the premanipulation position within the spindle. For bioriented chromosomes (sister kinetochores attached to opposite poles) near the spindle equator, stretching one arm toward a pole placed the kinetochore facing away from the direction of stretch under tension and the sister facing toward the stretch under reduced tension or compression. Kinetochores under increased tension exhibited prolonged AP movement while kinetochores under reduced tension or compression exhibited prolonged P movement, moving the centromeres at about 2 μm/min velocities off the metaphase plate in the direction of stretch. Removing the needle resulted in centromere movement back to near the spindle equator at similar velocities. These results show that tension controls the direction of kinetochore movement and associated kinetochore microtubule assembly/disassembly to position centromeres within the spindle of vertebrate tissue cells. High tension induces persistent AP movement while low tension induces persistent P movement. The velocity of P and AP movement appears to be load independent and governed by the molecular mechanisms which attach kinetochores to the dynamic ends of kinetochore microtubules.     

Antikinetochore antibodies interfere with prometaphase but not anaphase chromosome movement in living PtK2 cells.

Injection of CREST antikinetochore antiserum (AKA) containing antibodies to the kinetochore into living prometaphase PtK2 cells decreased chromosome velocity to near zero. Injection of either phosphate-buffered saline or CREST antiserum without antikinetochore antibodies (antikinetochore negative: AKN) had no effect on prometaphase oscillations. AKA antiserum injected into anaphase cells at the beginning of chromatid separation had no effect on anaphase chromosome velocity, spindle elongation, or cytokinesis. Visible binding of antikinetochore antibodies in prometaphase cells at room temperature occurred between 5 and 15 minutes after injection. Anaphase cells injected at the beginning of chromatid separation had bound antibody at the end of anaphase. AKA antiserum recognizes in Western blots proteins associated with the primary constriction: CENP-B, -C, and -D, as reported by other workers. The control antiserum, AKN, does not recognize these proteins. These results imply that the antigens recognized by CREST antibodies are important for chromosome movement. Whether or not these antigens are themselves motor molecules cannot be addressed by the present data. In addition, the results suggest that these antigens are not involved in an important way in anaphase movement.     

The relationship between nuclear envelope-derived annulate lamellae and the asymmetric division of primary spermatocytes in aphids

The structure of dividing primary spermatocytes of Amphorophora tuberculata (Aphididae, Hemiptera) as determined by electron microscopy and serial sectioning is described. The developmental stages examined extend from late prophase I to late telophase I. We looked for any asymmetric organization that could be causally linked to the differences in chromatin behaviour between the two daughter nuclei towards the end of meiosis I of this species. In late prophase I, evaginations of the nuclear envelope in the vicinity of two neigh-bouring centrosomes develop into closed cytoplasmic compartments with a dense content. The compartments open in prometaphase I and come to lie together with fragments of the nuclear envelope within the spindle area. Since nuclear pores are preserved in the membranes, intraspindle annulate lamellae have formed. These and material of presumed nuclear origin associated with them are asymmetrically distributed within the cell. Although dispersed at stages beyond prometaphase I, the material may be largely incorporated into one of the two daughter cells and thus be decisive for further development. Some annulate lamellae form a cap at the chromosome surface opposite to the neighbouring centrosomes in prometaphase I. These membranes may prevent interaction between spindle microtubules and chromosomes until a bipolar spindle forms in metaphase I. At this stage, both the banana-shaped autosomal bivalent and the X univalent occupy the equatorial plane. This is strange, because the X univalent has microtubular connections with one spindle pole and would be expected to migrate towards that pole. Possibly, the kinetochore of the X chromosome is inactive, and remains so in anaphase I, when the X univalent remains located between the two autosomal half-bivalents.M.F. Trendelenburg