Orientation of nonrandomly segregating sex chromosomes in spermatocytes of the flea beetle, Alagoasa bicolor L.

In males of the flea beetle, Alagoasa bicolor L., spermatocytes have two achiasmate sex chromosomes, X and Y, each of which is approximately five times larger than the ten pairs of chiasmate autosomes. At metaphase I, these univalent sex chromosomes are located on a spindle domain separated from the autosomal spindle domain by a sheath of mitochondria. A single centriole pair is located at each pole of the spindle. In prometaphase I, each sex chromosome appears to maintain an attachment to both spindle poles via kinetochore microtubules (i.e., amphitelic orientation). Before anaphase I, this orientation changes to the syntelic orientation (both sister kinetochores connected to the same pole), perhaps by the release of microtubule attachments from the more distant pole by each of the chromosomes. The syntelic orientation just prior to anaphase I leaves each sex chromosome attached to the nearest pole via kinetochore microtubules, ensuring nonrandom segregation. As the sex chromosomes reorient, the autosomes follow in a sequential manner, starting with the bivalent closest to the sex spindle domain. We report here data that shed new light on the mechanism of this exceptional meiotic chromosome behavior.     

Mechanism of haploidy-dependent unreductional meiotic cell division in polyploid wheat

Unreductional meiotic cell division (UMCD) generates unreduced gametes and leads to polyploidy. The tetraploid wheat “Langdon” (LDN) undergoes normal meiosis, but its polyhaploid undergoes UMCD. Here, we found that sister kinetochores oriented syntelically at meiosis I in LDN, but amphitelically in LDN polyhaploid and the interspecific hybrid of LDN with Aegilops tauschii. We also observed that sister centromere cohesion persisted until anaphase II in LDN, LDN polyhaploid, and the interspecific hybrid. Meiocytes with all chromosomes oriented amphitelically underwent UMCD in LDN polyhaploid, and the interspecific hybrid, suggesting the tension created by the amphitelic orientation of sister kinetochores and persistence of centromeric cohesion between sister chromatids at meiosis I contribute to the onset of UMCD. Most likely, some ploidy-regulated genes were responsible for kinetochore orientation at meiosis I in LDN and LDN-derived polyhaploids. In addition, we found sister kinetochores of synapsed chromosomes oriented syntelically, whereas asynapsed chromosomes oriented either amphitelically or syntelically. Thus, synapsis probably is another factor for the coordination of kinetochore orientation in LDN.     

Light and electron microscopy of rat kangaroo cells in mitosis

Chromosome orientation and behavior during prometaphase of mitosis in PtK₁ rat kangaroo cells were investigated by cinémicrography and electron microscopy. The first chromosome movements occur soon after the nuclear envelope begins to break down in the region near each pole. Initial chromosome behavior is primarily determined by the distance from the kinetochore region to the spindle poles. The predominant pattern is a movement to and/or association with the proximal pole. Movement to and association with the more distant pole, or direct alignment at or near the spindle equator (direct congression) are less frequent patterns. Except for rare cases, pole-associated chromosomes congress sooner or later and most congressed chromosomes oscillate about the equator. — Ultrastructural observations suggest that pole-associated chromosomes are oriented only to the proximal pole (monotelic or syntelic orientation) and they demonstrate that the sister-kinetochores of congressing or oscillating chromosomes are oriented to opposite poles (amphitelic orientation). — Based on the structure of the early prometaphase spindle and four assumptions concerning the formation of kinetochore fibers and their force-producing interaction with complementary elements, the different patterns of chromosome behavior observed can be explained as a result of synchronous or asynchronous formation of sister-kinetochore fibers. The few chromosomes whose kinetochore region is approximately equidistant from the poles amphi-orient immediately because their sister-kinetochores form fibers synchronously and they congress directly because of the bidirectional forces to which they are subjected. The kinetochore region of most chromosomes is not equidistant from the poles. Therefore, they form a functional fiber first to the nearer pole and move to, or associate with, it because of the unidirectional force. Eventually, however, these chromosomes achieve amphitelic orientation and congress. Once established, amphitelic orientation is stable. Re-orientations do not occur during congression or oscillatory movements.     

Morphological studies on the structure of univalent sex chromosomes during anaphase movement in spermatocytes of the crane fly Pales ferruginea

The ultrastructure of moving amphitelic sex chromosomes during anaphase of the first meiotic division of spermatocytes was studied by means of serial sections. The chromosomes have radial lamellae-like projections at their surface running in the direction of the spindle axis. Parallel spindle microtubules lie between these lamellae. The kinetochoral region pointing to the interzone (K_IZ) is stretched, while the kinetochore pointing polewards (K_p) is flat. It is suggested that the importance of the poleward kinetochore as an application site for pulling forces during anaphase movement is possibly reduced in favour of the lamellaemicrotubule association at the periphery of the chromosome. Distal parts of kinetochoral tubules of K_IZ may be associated with the lamellae of the partner sex chromosome. This arrangement may have some additional importance for the synchronization of sex chromosome migration in anaphase.     

MAPK-Activated Protein Kinase 2 Is Required for Mouse Meiotic Spindle Assembly and Kinetochore-Microtubule Attachment

MAPK-activated protein kinase 2 (MK2), a direct substrate of p38 MAPK, plays key roles in multiple physiological functions in mitosis. Here, we show for the first time the unique distribution pattern of MK2 in meiosis. Phospho-MK2 was localized on bipolar spindle minus ends and along the interstitial axes of homologous chromosomes extending over centromere regions and arm regions at metaphase of first meiosis (MI stage) in mouse oocytes. At metaphase of second meiosis (MII stage), p-MK2 was localized on the bipolar spindle minus ends and at the inner centromere region of sister chromatids as dots. Knockdown or inhibition of MK2 resulted in spindle defects. Spindles were surrounded by irregular nondisjunction chromosomes, which were arranged in an amphitelic or syntelic/monotelic manner, or chromosomes detached from the spindles. Kinetochore–microtubule attachments were impaired in MK2-deficient oocytes because spindle microtubules became unstable in response to cold treatment. In addition, homologous chromosome segregation and meiosis progression were inhibited in these oocytes. Our data suggest that MK2 may be essential for functional meiotic bipolar spindle formation, chromosome segregation and proper kinetochore–microtubule attachments.     

The role of sister chromatid cohesiveness and structure in meiotic behaviour

Sister chromatid cores, kinetochores and the connecting strand between sister kinetochores were differentially silver stained to analyse the behaviour of these structures during meiosis in normal and two spontaneous desynaptic individuals of Chorthippus jucundus (Orthoptera). In these desynaptic individuals most of the chromosomes appear as univalents and orient equationally in the first meiotic division. Despite this abnormal segregation pattern, the changes in chromosome structure follow the same timing as in normal individuals and seem to be strictly phase dependent. Chromosomes in the first prometaphase have associated sister kinetochores and sister chromatid cores that lie in the chromosome midline; we propose that this promotes the initial monopolar orientation of chromosomes. However, the requirements of tension for stable attachment to the spindle force the autosomal univalents to acquire amphitelic orientation. Sister kinetochores behave in a chromosome orientation-dependent manner and, in the first metaphase, they appear to be interconnected by a strand that can be detected by silver impregnation, as seen in the second metaphase of wild-type individuals. The disappearance of the sister kinetochore-connecting strand, needed for equational chromatid segregation, however, can only take place in the second meiotic division. This connecting strand is ultimately responsible for the inability of chromosomes to segregate sister chromatids in the first anaphase. Received: 25 March 1997; in revised form: 14 July 1997 / Accepted: 22 August 1997     

The spatial arrangement of chromosomes during prometaphase facilitates spindle assembly

Error-free chromosome segregation requires stable attachment of sister kinetochores to the opposite spindle poles (amphitelic attachment). Exactly how amphitelic attachments are achieved during spindle assembly remains elusive. We employed photoactivatable GFP and high-resolution live-cell confocal microscopy to visualize complete 3D movements of individual kinetochores throughout mitosis in nontransformed human cells. Combined with electron microscopy, molecular perturbations, and immunofluorescence analyses, this approach reveals unexpected details of chromosome behavior. Our data demonstrate that unstable lateral interactions between kinetochores and microtubules dominate during early prometaphase. These transient interactions lead to the reproducible arrangement of chromosomes in an equatorial ring on the surface of the nascent spindle. A computational model predicts that this toroidal distribution of chromosomes exposes kinetochores to a high density of microtubules which facilitates subsequent formation of amphitelic attachments. Thus, spindle formation involves a previously overlooked stage of chromosome prepositioning which promotes formation of amphitelic attachments.     

Spindle formation and dynamics of gamma-tubulin and nuclear mitotic apparatus protein distribution during meiosis in pig and mouse oocytes

This work focuses on the assembly and transformation of the spindle during the progression through the meiotic cell cycle. For this purpose, immunofluorescent confocal microscopy was used in comparative studies to determine the spatial distribution of alpha- and gamma-tubulin and nuclear mitotic apparatus protein (NuMA) from late G2 to the end of M phase in both meiosis and mitosis. In pig endothelial cells, consistent with previous reports, gamma-tubulin was localized at the centrosomes in both interphase and M phase, and NuMA was localized in the interphase nucleus and at mitotic spindle poles. During meiotic progression in pig oocytes, gamma-tubulin and NuMA were initially detected in a uniform distribution across the nucleus. In early diakinesis and just before germinal vesicle breakdown, microtubules were first detected around the periphery of the germinal vesicle and cell cortex. At late diakinesis, a mass of multi-arrayed microtubules was formed around chromosomes. In parallel, NuMA localization changed from an amorphous to a highly aggregated form in the vicinity of the chromosomes, but gamma-tubulin localization remained in an amorphous form surrounding the chromosomes. Then the NuMA foci moved away from the condensed chromosomes and aligned at both poles of a barrel-shaped metaphase I spindle while gamma-tubulin was localized along the spindle microtubules, suggesting that pig meiotic spindle poles are formed by the bundling of microtubules at the minus ends by NuMA. Interestingly, in mouse oocytes, the meiotic spindle pole was composed of several gamma-tubulin foci rather than NuMA. Further, nocodazole, an inhibitor of microtubule polymerization, induced disappearance of the pole staining of NuMA in pig metaphase II oocytes, whereas the mouse meiotic spindle pole has been reported to be resistant to the treatment. These results suggest that the nature of the meiotic spindle differs between species. The axis of the pig meiotic spindle rotated from a perpendicular to a parallel position relative to the cell surface during telophase I. Further, in contrast to the stable localization of NuMA and gamma-tubulin at the spindle poles in mitosis, NuMA and gamma-tubulin became relocalized to the spindle midzone during anaphase I and telophase I in pig oocytes. We postulate that in the centrosome-free meiotic spindle, NuMA aggregates the spindle microtubules at the midzone during anaphase and telophase and that the polarity of meiotic spindle microtubules might become inverted during spindle elongation.     

Monastral bipolar spindles in meiosis II of male Trichosia pubescens (Sciaridae): early stages of spindle formation and chromosome orientation.

The metaphase spindle of male meiosis II in fungus gnats (Sciaridae) is one example of naturally occurring monastral bipolar spindles. To gain further insights into how the bipolar spindle is formed in the presence of only one polar center, prometaphase of male meiosis II was investigated in the sciarid Trichosia pubescens by means of anti-tubulin immunofluorescence, DAPI chromosome staining, and electron microscopy of ultrathin serial sections. The first step in spindle formation after interkinesis seems to be the organization of an astral half-spindle, probably by MTOC activity of the astral region. With the exception of the non-disjunctional X chromosome, which always lies close to the aster, the chromosomes are found to occupy various positions with respect to the astral region, revealing different orientations of their chromatid kinetochores. It was observed that some of the mal-oriented kinetochores are associated with microtubules that, due to their orientation perpendicular to the spindle axis, are unlikely to originate from the astral region. Therefore, these mal-oriented microtubules are taken as an indication of a dispersed MTOC activity near the chromosomes or at kinetochores. According to recent models of chromosome-induced spindle self-organization [e.g., Merdes et al., 1997: J. Cell Biol. 138:953-956], they could be responsible for the formation of the other (anastral) half-spindle and for amphitelic (bipolar) orientation of the chromosomes.     

Chromosomal influence on meiotic spindle assembly: abnormal meiosis I in female Mlh1 mutant mice.

In mouse oocytes, the first meiotic spindle is formed through the action of multiple microtubule organizing centers rather than a pair of centrosomes. Although the chromosomes are thought to play a major role in organizing the meiotic spindle, it remains unclear how a stable bipolar spindle is established. We have studied the formation of the first meiotic spindle in murine oocytes from mice homozygous for a targeted disruption of the DNA mismatch repair gene, Mlh1. In the absence of the MLH1 protein meiotic recombination is dramatically reduced and, as a result, the vast majority of chromosomes are present as unpaired univalents at the first meiotic division. The orientation of these univalent chromosomes at prometaphase suggests that they are unable to establish stable bipolar spindle attachments, presumably due to the inability to differentiate functional kinetochore domains on individual sister chromatids. In the presence of this aberrant chromosome behavior a stable first meiotic spindle is not formed, the spindle poles continue to elongate, and the vast majority of cells never initiate anaphase. These results suggest that, in female meiotic systems in which spindle formation is based on the action of multiple microtubule organizing centers, the chromosomes not only promote microtubule polymerization and organization but their attachment to opposite spindle poles acts to stabilize the forming spindle poles.     

Segregation of the amphitelically attached univalent X chromosome in the spittlebug <Emphasis Type="Italic">Philaenus spumarius</Emphasis>

In meiosis I, homologous chromosomes combine to form bivalents, which align on the metaphase plate. Homologous chromosomes then separate in anaphase I. Univalent sex chromosomes, on the other hand, are unable to segregate in the same way as homologous chromosomes of bivalents due to their lack of a homologous pairing partner in meiosis I. Here, we studied univalent segregation in a Hemipteran insect: the spittlebug Philaenus spumarius. We determined the chromosome number and sex determination mechanism in our population of P. spumarius and showed that, in male meiosis I, there is a univalent X chromosome. We discovered that the univalent X chromosome in primary spermatocytes forms an amphitelic attachment to the spindle and aligns on the metaphase plate with the autosomes. Interestingly, the X chromosome remains at spindle midzone long after the autosomes have separated. In late anaphase I, the X chromosome initiates movement towards one spindle pole. This movement appears to be correlated with a loss of microtubule connections between the kinetochore of one chromatid and its associated spindle pole.     

Mitosis in the cellular slime mold Polysphondylium violaceum

Myxamebas of Polysphondylium violaceum were grown in liquid medium and processed for electron microscopy. Mitosis is characterized by a persistent nuclear envelope, ring-shaped extranuclear spindle pole bodies (SPBs), a central spindle spatially separated from the chromosomal microtubules, well-differentiated kinetochores, and dispersion of the nucleoli. SPBs originate from the division, during prophase, of an electron-opaque body associated with the interphase nucleus. The nuclear nevelope becomes fenestrated in their vicinity, allowing the build-up of the intranuclear, central spindle and chromosomal microtubules as the SPBs migrate to opposite poles. At metaphase the chromosomes are in amphitelic orientation, each sister chromatid being directly connected to the corresponding SPB by a single microtubule. During ana- and telophase the central spindle elongates, the daughter chromosomes approach the SPBs, and the nucleus constricts in the equatorial region. The cytoplasm cleaves by furrowing in late telophase, which is in other respects characterized by a re- establishment of the interphase condition. Spindle elongation and poleward movement of chromosomes are discussed in relation to hypotheses of the mechanism of mitosis.     

How meiotic cells deal with non-exchange chromosomes

The chromosomes which segregate in anaphase I of meiosis are usually physically bound together through chiasmata. This association is necessary for proper segregation, since univalents sort independently from one another in the first meiotic division and this frequently leads to genetically unbalanced offspring. There are, however, a number of species where genetic exchanges in the form of meiotic cross-overs, the prerequisite of the formation of chiasmata, are routinely missing in one sex or between specific chromosomes. These species nevertheless manage to segregate these non-exchange chromosomes. There are four direct modes for associating achiasmatic chromosomes: (a) modified SC, (b) adhesion of chromatids comparable to somatic pairing, (c) ‘stickiness’ of heterochromatin or (d) specific ‘segregation bodies’, consisting of material structurally different from chromatin. There is also the possibility that the spindlepossibly joining forces with the kinetochores-carries out the faithful segregation of univalents which are not directly physically attached to one another. Finally, amphitelic orientation of univalents in metaphase I and pairing of the chromatids in meiosis II appear to ensure correct segregation as well.     

CDK-1 inhibits meiotic spindle shortening and dynein-dependent spindle rotation in C. elegans

In animals, the female meiotic spindle is positioned at the egg cortex in a perpendicular orientation to facilitate the disposal of half of the chromosomes into a polar body. In Caenorhabditis elegans, the metaphase spindle lies parallel to the cortex, dynein is dispersed on the spindle, and the dynein activators ASPM-1 and LIN-5 are concentrated at spindle poles. Anaphase-promoting complex (APC) activation results in dynein accumulation at spindle poles and dynein-dependent rotation of one spindle pole to the cortex, resulting in perpendicular orientation. To test whether the APC initiates spindle rotation through cyclin B-CDK-1 inactivation, separase activation, or degradation of an unknown dynein inhibitor, CDK-1 was inhibited with purvalanol A in metaphase-I-arrested, APC-depleted embryos. CDK-1 inhibition resulted in the accumulation of dynein at spindle poles and dynein-dependent spindle rotation without chromosome separation. These results suggest that CDK-1 blocks rotation by inhibiting dynein association with microtubules and with LIN-5-ASPM-1 at meiotic spindle poles and that the APC promotes spindle rotation by inhibiting CDK-1.     

Meiotic regulation of TPX2 protein levels governs cell cycle progression in mouse oocytes

Formation of female gametes requires acentriolar spindle assembly during meiosis. Mitotic spindles organize from centrosomes and via local activation of the RanGTPase on chromosomes. Vertebrate oocytes present a RanGTP gradient centred on chromatin at all stages of meiotic maturation. However, this gradient is dispensable for assembly of the first meiotic spindle. To understand this meiosis I peculiarity, we studied TPX2, a Ran target, in mouse oocytes. Strikingly, TPX2 activity is controlled at the protein level through its accumulation from meiosis I to II. By RNAi depletion and live imaging, we show that TPX2 is required for spindle assembly via two distinct functions. It controls microtubule assembly and spindle pole integrity via the phosphorylation of TACC3, a regulator of MTOCs activity. We show that meiotic spindle formation in vivo depends on the regulation of at least a target of Ran, TPX2, rather than on the regulation of the RanGTP gradient itself.     

Meiotic segregation of a homeologous chromosome pair

During meiosis, the alignment of homologous chromosomes facilitates their subsequent migration away from one another to opposite spindle poles at anaphase I. Recombination is part of the mechanism by which chromosomes identify their homologous partners, and serves to link the homologs in a way that, in some organisms, has been shown to promote proper attachment to the meiotic spindle. We have built a diploid strain that contains a pair of homeologous chromosomes V': one is derived from Saccharomyces cerevisiae and one originates from S. carlsbergensis. Sequence analysis reveals that these chromosomes share 71% sequence identity. The homeologs experience high levels of meiotic double-stranded breaks. Despite their relatedness and their competence to initiate recombination, the meiotic segregation behavior of the homeologous chromosomes suggests that, in most meioses, they are partitioned by a meiotic segregation system that has been shown previously to partition non-exchange chromosomes and pairs with no homology. Though the homeologous chromosomes show a degree of meiotic segregation fidelity similar to that of other non-exchange pairs, our data provide evidence that their limited sequence homology may provide some bias in meiotic partner choice.     

Astrin regulates meiotic spindle organization,spindle pole tethering and cell cycle progression in mouse oocytes

Astrin has been described as a microtubule and kinetochore protein required for the maintenance of sister chromatid cohesion and centrosome integrity in human mitosis. However, its role in mammalian oocyte meiosis is unclear. In this study, we find that Astrin is mainly associated with the meiotic spindle microtubules and concentrated on spindle poles at metaphase I and metaphase II stages. Taxol treatment and immunoprecipitation show that Astrin may interact with the centrosomal proteins Aurora-A or Plk1 to regulate microtubule organization and spindle pole integrity. Loss-of-function of Astrin by RNAi and overexpression of Tof the coiled-coil domain results in spindle disorganization, chromosome misalignment and meiosis progression arrestT. Thr24, Ser66 or Ser447 may be the potential phosphorylated sites of Astrin by Plk1, as site-directed mutation of these sites causes oocyte meiotic arrest at HTmetaphaseTH I with highly disordered spindles and disorganized chromosomes, although mutant Astrin localizes to the spindle apparatus. Taken together, these data strongly suggest that Astrin is critical for meiotic spindle assembly and maturation in mouse oocytes.     

Metaphase I arrest upon activation of the Mad2-dependent spindle checkpoint in mouse oocytes

BACKGROUND: The importance of mitotic spindle checkpoint control has been well established during somatic cell divisions. The metaphase-to-anaphase transition takes place only when all sister chromatids have been properly attached to the bipolar spindle and are aligned at the metaphase plate. Failure of this checkpoint may lead to unequal separation of sister chromatids. On the contrary, the existence of such a checkpoint during the first meiotic division in mammalian oocytes when homologous chromosomes are segregated has remained controversial. RESULTS: Here, we show that mouse oocytes respond to spindle damage by a transient and reversible cell cycle arrest in metaphase I with high Maturation Promoting Factor (MPF) activity. Furthermore, the mitotic checkpoint protein Mad2 is present throughout meiotic maturation and is recruited to unattached kinetochores. Overexpression of Mad2 in meiosis I leads to a cell cycle arrest in metaphase I. Expression of a dominant-negative Mad2 protein interferes with proper spindle checkpoint arrest. CONCLUSIONS: Errors in meiosis I cause missegregation of chromosomes and can result in the generation of aneuploid embryos with severe birth defects. In human oocytes, failures in spindle checkpoint control may be responsible for the generation of trisomies (e.g., Down Syndrome) due to chromosome missegregation in meiosis I. Up to now, the mechanisms ensuring correct separation of chromosomes in meiosis I remained unknown. Our study shows for the first time that a functional Mad2-dependent spindle checkpoint exists during the first meiotic division in mammalian oocytes.     

The Cdc14 phosphatase and the FEAR network control meiotic spindle disassembly and chromosome segregation

During meiosis, DNA replication is followed by two consecutive rounds of chromosome segregation. Cells lacking the protein phosphatase CDC14 or its regulators, SPO12 and SLK19, undergo only a single meiotic division, with some chromosomes segregating reductionally and others equationally. We find that this abnormal chromosome behavior is due to an uncoupling of meiotic events. Anaphase I spindle disassembly is delayed in cdc14-1, slk19Delta, or spo12Delta mutants, but the chromosome segregation cycle continues, so that both meiotic chromosome segregation phases take place on the persisting meiosis I spindle. Our results show that Cdc14, Slk19, and Spo12 are not only required for meiosis I spindle disassembly but also play a pivotal role in establishing two consecutive chromosome segregation phases, a key feature of the meiotic cell cycle.     

Septin2 is modified by SUMOylation and required for chromosome congression in mouse oocytes

In mitosis, centrosomes nucleate microtubules that capture the sister kinetochores of each chromosome to facilitate chromosome congression. In contrast, during meiosis chromosome congression on the acentrosomal spindle is driven primarily by movement of chromosomes along laterally associated microtubule bundles. Previous studies have indicated that septin2 is required for chromosome congression and cytokinesis in mitosis, we therefore asked whether perturbation of septin2 would impair chromosome congression and cytokinesis in meiosis. We have investigated its expression, localization and function during mouse oocyte meiotic maturation. Septin2 was modified by SUMO-1 and its levels remained constant from GVBD to metaphase II stages. Septin2 was localized along the entire spindle at metaphase and at the midbody in cytokinesis. Disruption of septins function with an inhibitor and siRNA caused failure of the metaphase I /anaphase I transition and chromosome misalignment but inhibition of septins after the metaphase I stage did not affect cytokinesis. BubR1, a core component of the spindle checkpoint, was labeled on misaligned chromosomes and on chromosomes aligned at the metaphase plate in inhibitor-treated oocytes that were arrested in prometaphase I/metaphase I, suggesting activation of the spindle assembly checkpoint. Taken together, our results demonstrate that septin2 plays an important role in chromosome congression and meiotic cell cycle progression but not cytokinesis in mouse oocytes.