The Des-1 protein, required for central spindle assembly and cytokinesis, is associated with mitochondria along the meiotic spindle apparatus and with the contractile ring during male meiosis in Drosophila melanogaster

Spermatogenesis in Drosophila melanogaster serves as an excellent model system for the isolation and analysis of genes required in the control of chromosome segregation and cytokinesis. We report here the isolation and molecular characterization of a novel P-element induced allele of the des-1 gene, which leads to male sterility as a consequence of the failure of central spindle assembly in meiotic spermatocytes and the formation of aberrant meiotic end products characteristic of cytokinesis failure. We have raised affinity-purified antibodies against a Des-1 fusion protein, and localized the Des-1 protein in Drosophila spermatocytes. We show that the Des-1 protein is colocalized with mitochondria throughout male meiosis, becoming intimately associated with mitochondria along the spindle apparatus during anaphase and telophase, and with the Nebenkern, or mitochondrial derivative, of the meiotic end products. In addition, a significant association of Des-1 with the contractile ring is observed during anaphase and telophase of meiosis. These observations, together with the presence of six potential transmembrane domains in the Des-1 protein, raise the possibility that Des-1 may act as part of an anchoring mechanism that links membrane-bounded cellular compartments to components of the cytoskeleton.     

A Drosophila orthologue of larp protein family is required for multiple processes in male meiosis

It is important for the proper execution of cell division in both mitosis and meiosis that the chromosome segregation, cytokinesis, and partition of cell organelles progress in smooth coordination. We show here that the mitochondria inheritance is closely linked with microtubules during meiotic divisions in Drosophila males. They are first clustered in a cell equator at metaphase associated with astral microtubules and then distributed along central spindle microtubules after anaphase. The molecular mechanism for the microtubule-dependent inheritance of mitochondria in male meiosis has not been demonstrated yet. We first isolated mutations for a larp gene that is highly conserved among eukaryotes and showed that these mutant males exhibited multiple meiotic phenotypes such as a failure of chromosome segregation, cytokinesis, and mitochondrial partition. Our cytological examination revealed that the mutants showed defects in spindle pole organization and spindle formation. The larp encodes a Drosophila orthologue of a La-related protein containing a domain exhibiting an outstanding homology with a La type RNA-binding protein. Surprisingly, the dLarp protein is localized in the cytoplasm of the male germ line cells, as observed by its distinct co-localization with mitochondria in early spermatocytes and during meiotic divisions. We discuss here the essential role that dLarp plays in multiple processes in Drosophila male meiosis.     

Spermatogenesis in Tenebrio molitor (Tenebrionidae,Coleoptera): A Fine Structure and Anti-tubulin Immunofluorescence Study

Spermatogonia and both generations of spermatocytes of Tenebrio molitor possess conventional bipolar spindles with only few aster MTs. Spindles in metaphase spermatogonia are surrounded by fenestrated two-layered cisternae and do not contain intraspindle membranes. In metaphase spermatocytes, a spindle envelope is missing, but intraspindle membranes are abundant. Mitochondria form long threads lateral to the nucleus in prophase I of meiosis. The elongated mitochondria also align parallel to the spindle apparatus in prometaphase I. As a consequence, the spindles reside in a cage formed of mitochondria. This arrangement may guarantee proper bisection of the chondriome during division. Cells are tightly packed during spermatogonial divisions and in prophase I, but large intercellular spaces develop when the first meiotic spindle assembles. Then, cytoplasmic bridges which persist between the cells as a result of incomplete cytokinesis appear as slender tubes. Anti-tubulin immunofluorescence using an antibody against acetylated α-tubulin revealed intense acetylation throughout spermatogonial mitosis but a low degree of α-tubulin acetylation in meiotic spindles prior to telophase. This may indicate a high microtubule turnover in meiosis.     

Mouse polo-like kinase 1 associates with the acentriolar spindle poles, meiotic chromosomes and spindle midzone during oocyte maturation

We have examined the dynamics of the localisation of the polo-like kinase 1 (Plk1) during maturation of the mouse oocyte. Levels of Plk1 protein increase following germinal vesicle breakdown, at which time the enzyme begins to accumulate at discrete positions on the condensing chromosomes and, subsequently, at the poles of the meiotic spindle, which moves towards the cortex of the egg. Interestingly, at metaphase in both meiotic divisions, Plk1 shows a punctate localisation along the broad spindle poles. Moreover, the punctate distribution of Plk1 on the meiotic chromosomes appears at early anaphase to correspond to the centromeric regions. The protein relocates to the spindle midzone during late anaphase and then associates with the midbody at telophase. We have confirmed the specific pattern of immuno-localisation seen in fixed preparations by observing the distribution of Plk1 tagged with green fluorescent protein in living oocytes. We discuss the localisation of the enzyme in light of the structure of the spindle poles, which are known to lack centrioles, and the highly asymmetric nature of the meiotic divisions. Received: 8 August 1998 / Accepted: 13 September 1998     

The Drosophila kinesin-like protein KLP3A is a midbody component required for central spindle assembly and initiation of cytokinesis

We describe here a new member of the kinesin superfamily in Drosophila, KLP3A (Kinesin-Like-Protein-at-3A). The KLP3A protein localizes to the equator of the central spindle during late anaphase and telophase of male meiosis. Mutations in the KLP3A gene disrupt the interdigitation of microtubules in spermatocyte central spindles. Despite this defect, anaphase B spindle elongation is not obviously aberrant. However, cytokinesis frequently fails after both meiotic divisions in mutant testes. Together, these findings strongly suggest that the KLP3A presumptive motor protein is a critical component in the establishment or stabilization of the central spindle. Furthermore, these results imply that the central spindle is the source of signals that initiate the cleavage furrow in higher cells.     

Visualizing the spindle checkpoint in Drosophila spermatocytes

The spindle assembly checkpoint detects defects in spindle structure or in the alignment of the chromosomes on the metaphase plate and delays the onset of anaphase until defects are corrected. Thus far, the evidence regarding the presence of a spindle checkpoint during meiosis in male Drosophila has been indirect and contradictory. On the one hand, chromosomes without pairing partners do not prevent meiosis progression. On the other hand, some conserved components of the spindle checkpoint machinery are expressed in these cells and behave as their homologue proteins do in systems with an active spindle checkpoint. To establish whether the spindle checkpoint is active in Drosophila spermatocytes we have followed meiosis progression by time-lapse microscopy under conditions where the checkpoint is likely to be activated. We have found that the presence of a relatively high number of misaligned chromosomes or a severe disruption of the meiotic spindle results in a significant delay in the time of entry into anaphase. These observations provide the first direct evidence substantiating the activity of a meiotic spindle checkpoint in male Drosophila.     

The kinesinlike protein Subito contributes to central spindle assembly and organization of the meiotic spindle in Drosophila oocytes

In the oocytes of many species, bipolar spindles form in the absence of centrosomes. Drosophila melanogaster oocyte chromosomes have a major role in nucleating microtubules, which precedes the bundling and assembly of these microtubules into a bipolar spindle. Here we present evidence that a region similar to the anaphase central spindle functions to organize acentrosomal spindles. Subito mutants are characterized by the formation of tripolar or monopolar spindles and nondisjunction of homologous chromosomes at meiosis I. Subito encodes a kinesinlike protein and associates with the meiotic central spindle, consistent with its classification in the Kinesin 6/MKLP1 family. This class of proteins is known to be required for cytokinesis, but our results suggest a new function in spindle formation. The meiotic central spindle appears during prometaphase and includes passenger complex proteins such as AurB and Incenp. Unlike mitotic cells, the passenger proteins do not associate with centromeres before anaphase. In the absence of Subito, central spindle formation is defective and AurB and Incenp fail to properly localize. We propose that Subito is required for establishing and/or maintaining the central spindle in Drosophila oocytes, and this substitutes for the role of centrosomes in organizing the bipolar spindle.     

Caenorhabditis elegans Aurora A kinase is required for the formation of spindle microtubules in female meiosis

In many animals, female meiotic spindles are assembled in the absence of centrosomes, the major microtubule (MT)-organizing centers. How MTs are formed and organized into meiotic spindles is poorly understood. Here we report that, in Caenorhabditis elegans, Aurora A kinase/AIR-1 is required for the formation of spindle microtubules during female meiosis. When AIR-1 was depleted or its kinase activity was inhibited in C. elegans oocytes, although MTs were formed around chromosomes at germinal vesicle breakdown (GVBD), they were decreased during meiotic prometaphase and failed to form a bipolar spindle, and chromosomes were not separated into two masses. Whereas AIR-1 protein was detected on and around meiotic spindles, its kinase-active form was concentrated on chromosomes at prometaphase and on interchromosomal MTs during late anaphase and telophase. We also found that AIR-1 is involved in the assembly of short, dynamic MTs in the meiotic cytoplasm, and these short MTs were actively incorporated into meiotic spindles. Collectively our results suggest that, after GVBD, the kinase activity of AIR-1 is continuously required for the assembly and/or stabilization of female meiotic spindle MTs.     

Inhibition of Aurora kinases perturbs chromosome alignment and spindle checkpoint signaling in rat spermatocytes

In somatic cells, integrity of cell division is safeguarded by the spindle checkpoint, a signaling cascade that delays the separation of sister chromatids in the presence of misaligned chromosomes. Aurora kinases play important roles in this process by promoting centrosome maturation, chromosome bi-orientation, spindle checkpoint signaling, and cytokinesis. To investigate the functions of Aurora kinases in male meiosis, we applied a small molecule Aurora inhibitor, ZM447439, to seminiferous tubules in vitro. Primary and secondary spermatocytes exposed to ZM447439 exhibit defects in the spindle morphology and fail to align their chromosomes at the metaphase plate. Moreover, the treated spermatocytes undergo a forced exit from the meiotic M-phase without cytokinesis. These results suggest that the activities of Aurora kinases are required for normal spindle assembly as well as for establishment and maintenance of proper microtubule-kinetochore attachments and spindle checkpoint signaling in male mammalian meiosis.     

Centriolin,a centriole-appendage protein,regulates peripheral spindle migration and asymmetric division in mouse meiotic oocytes

Unlike somatic cells mitosis, germ cell meiosis consists of 2 consecutive rounds of division that segregate homologous chromosomes and sister chromatids, respectively. The meiotic oocyte is characterized by an absence of centrioles and asymmetric division. Centriolin is a relatively novel centriolar protein that functions in mitotic cell cycle progression and cytokinesis. Here, we explored the function of centriolin in meiosis and showed that it is localized to meiotic spindles and concentrated at the spindle poles and midbody during oocyte meiotic maturation. Unexpectedly, knockdown of centriolin in oocytes with either siRNA or Morpholino micro-injection, did not affect meiotic spindle organization, cell cycle progression, or cytokinesis (as indicated by polar body emission), but led to a failure of peripheral meiotic spindle migration, large polar body emission, and 2-cell like oocytes. These data suggest that, unlike in mitotic cells, the centriolar protein centriolin does not regulate cytokinesis, but plays an important role in regulating asymmetric division of meiotic oocytes.     

Unusual cytological patterns of microsporogenesis in Brachiaria decumbens: abnormalities in spindle and defective cytokinesis causing precocious cellularization

Cytogenetic studies carried out in the tetraploid accession BRA001068 of Brachiaria decumbens, also known as cv. Basilisk, revealed an unusual pattern of microsporogenesis. The spindle in metaphase I and anaphase I became heavily stained with propionic carmine. In telophase I, the interzonal microtubules continued to be intensely stained, and during the phragmoplast formation the fibers were pushed to the cell wall, persisting until prophase II, even after cytokinesis. Due to its tetraploid condition, the accession presented many cells with precocious chromosome migration to the poles in metaphase I and laggards in anaphase I that gave rise to micronuclei in telophase I. While in other polyploid accessions of Brachiaria micronuclei remained in this condition until the second cytokinesis, the micronuclei in this accession organized their own spindle in the second division. In several microsporocytes, the micronuclei with their minispindle were divided further into microcytes by additional cytokinesis. Some curious planes of cytokinesis were found in some cells, with partitioning of cytoplasm into cells of irregular shape. The result consisted of a high frequency of abnormal products of meiosis. Quadrivalents were observed in diakinesis at low frequency, which suggests a segmental allotetraploid and the inability of both genomes to co-ordinate their activities, leading to multiple spindle and precocious cellularization. In spite of abnormal meiotic products reducing pollen fertility, seed production was normal. Enough normal pollen was available to fertilize the central-cell nucleus of the embryo sac and produce normal endosperm in this pseudogamous aposporous apomictic accession.     

“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.     

WHAMM is required for meiotic spindle migration and asymmetric cytokinesis in mouse oocytes

WASP homolog associated with actin, membranes and microtubules (WHAMM) is a newly discovered nucleation-promoting factor that links actin and microtubule cytoskeleton and regulates transport from the endoplasmic reticulum to the Golgi apparatus. However, knowledge of WHAMM is limited to interphase somatic cells. In this study, we examined its localization and function in mouse oocytes during meiosis. Immunostaining showed that in the germinal vesicle (GV) stage, there was no WHAMM signal; after meiosis resumption, WHAMM was associated with the spindle at prometaphase I (Pro MI), metaphase I (MI), telophase I (TI) and metaphase II (MII) stages. Nocodazole and taxol treatments showed that WHAMM was localized around the MI spindle. Depletion of WHAMM by microinjection of specific short interfering (si)RNA into the oocyte cytoplasm resulted in failure of spindle migration, disruption of asymmetric cytokinesis and a decrease in the first polar body extrusion rate during meiotic maturation. Moreover, actin cap formation was also disrupted after WHAMM depletion, confirming the failure of spindle migration. Taken together, our data suggest that WHAMM is required for peripheral spindle migration and asymmetric cytokinesis during mouse oocyte maturation.     

An original meiotic mutation in Paspalum regnellii

 Cytogenetic studies carried out over a period of 2 consecutive years on a native Brazilian accession of Paspalum regnellii (2n=40) revealed a meiotic mutation that has not been previously reported for any other species. Among 13 inflorescences investigated during the first collection year, three presented anomalous meiotic behavior starting from metaphase I. At the beginning of this phase, the chromosomes occupied the entire equatorial plate in a membrane-to-membrane arrangement, and the spindle fibers, which were clearly visible, did not converge towards the poles. Degeneration of spindle fibers occurred at the end of metaphase I. Chromosome segregation did not occur and the bivalents were left scattered at random in the cytoplasm. Remnants of chromosome fibers could be seen close to the centromere during this stage. The bivalents gave origin to micronuclei in telophase I, with extremely wide variations in number and size among cells. With the absence of spindle formation during meiosis II, metaphase and anaphase II were not observed. Second cytokinesis occurred in prophase II cells after the occurrence of first cytokinesis. The final product of meiosis was completely abnormal, with a predominance of polyads with microspores of different sizes that resulted in abortive pollen grains. In the affected inflorescences, all microsporocytes presented this anomaly, which caused total sterility. Received: 27 March 1997 / Revision accepted: 7 July 1997     

Dynamic localization and functional implications of Aurora-C kinase during male mouse meiosis

Aurora-C was first identified during screening for kinases expressed in mouse sperm and eggs. Herein, we report for the first time the precise subcellular localization of endogenous Aurora-C during male meiotic division. The localization of Aurora-C was analyzed by immunofluorescence staining on chromosome spreads of mouse spermatocytes or in squashed seminiferous tubules. Aurora-C was first detected at clusters of chromocenters in diplotene spermatocytes and was concentrated at centromeres in metaphase I and II. Interestingly, Aurora-C was also found along the chromosome axes, including both the regions of centromeres and the chromosome arms in diakinesis. During the anaphase I/telophase I and anaphase II/telophase II transitions, Aurora-C was relocalized to the spindle midzone and midbody. A similar distribution pattern was also observed for Aurora-B during male meiotic divisions. Surprisingly, we detected no Aurora-C in mitotic spermatogonia. Furthermore, immunoprecipitation analyses revealed that INCENP associated with Aurora-C in the male testis. We propose that INCENP recruits Aurora-C (or some other factor(s) recruit INCENP and Aurora-C) to meiotic chromosomes, while Aurora-C may either work alone or cooperate with Aurora-B to regulate chromosome segregation during male meiosis.     

Microtubule organization during successive microsporogenesis in Allium cepa and simultaneous cytokinesis in Nicotiana tabacum

Microtubule cytoskeleton organization during microspore mother cell (MMC) meiosis in Allium cepa L. and microsporogenesis in Nicotiana tabacum L. was examined. The MMC microtubules (MTs) were short and well dispersed in the cytoplasm of both taxa. As the MMCs of both species entered metaphase of meiosis I, the MTs constructed a spindle that facilitated the chromosomes to orient in the meridian plane. At anaphase of meiosis I, the spindle MTs differentiated into two types: one MT type became short, pulled the chromosomes toward the two poles, and was designated as centromere MTs; the second type of MT connected the two poles, and was designated as pole MTs. In A. cepa, where successive cytokinesis was observed, pole MTs assumed a tubbish shape. Some new short MTs aggregated in the meridian plane and constricted to form a phragmoplast, which developed into a cell plate, divided the cytoplasm into two parts and produced a dyad. However, in tobacco, a phragmoplast was not generated in anaphase of meiosis I and II and cytokinesis did not occur. The spindle MTs depolymerized and reorganized the radial arrangement of MTs from the nucleate surface to the periplasm during anaphase. Following telophase of meiosis II, the cytoplasm produced centripetal furrows, which met in the center of the cell and divided it into four parts, serving as a form of cytokinesis. In this process, MTs appeared to bear no relationship to cytokinesis.     

Meiosis spindle formation and chromosome behavior in diploid potatoes with 'fused spindles' mutation

Chromosomal behaviour and spindle morphology were studied in microsporogenesis of two kinds of diploid potato clones: with normal meiosis, and with "fused spindles" (fs) occurring during the second meiotic division from prometaphase II (proMII) to telophase II (TII). For the first time, morphological effect of fs was found at the late proMII stage to be expressed as two interrelated processes: 1) abnormal chromosome movement, which resulted in joining two groups of chromosomes in the central zone of meiocytes, and 2) abnormal formation of two spindles in the direction to two division poles instead of four poles that actually led to the formation of a united bipolar spindle. Thus, it is not the fusion of two parallel spindles but the formation of united bipolar spindle that constitutes fs abnormality, while the parallel co-orientation of two spatially separated meiotic spindles is a norm in diploid potato. These primary abnormalities detected at proMII resulted in abnormalities at its subsequent meiotic stages: formation of fused spindle and united metaphase plate at MII, bipolar chromosome segration at anaphase II, formation of two telophase nuclei at TII and dyads at the tetrad stage. The results obtained evidence the polar division disturbance in diploid potato clones with fs abnormality.     

Cytological studies on meiosis and male gametophyte development in autotetraploid cucumber

With improved staining and chromosome preparation techniques, meiosis of pollen mother cells (PMCs) and male gametophyte development in autotetraploid cucumber (Cucumis sativus L.) was studied to understand the correlation between chromosomes behaviour and fertility. Various chromosome configurations, e.g. multivalent, quadrivalents, trivalents, bivalents and univalents were observed in most PMCs at metaphase I. Lagging chromosomes were frequently observed at anaphase in both meiotic divisions. In addition, chromosomes segregations were not synchronous and equal in some PMCs during anaphase II and telophase II. Dyads, triads, tetrads with micronuclei and polyads were observed at tetrad stage, and the frequencies of normal tetrad with four microcytes were only 55.4 %. The frequency of abnormal behaviour in each stage of meiosis was counted, and the average value was 37.2 %. The normal meiotic process could be accomplished to form the microspore tetrads via simultaneous cytokinesis. Most microspores could develop into fertile gametophytes with 2 cells and 3 germ pores through the following stages: single-nucleus early stage, single-nucleus late stage and 2-celled stage. The frequency of abnormalities was low during the process of male gametophyte development. The germination rate of pollen grains was 46.9 %. These results suggested that abnormal meiosis in PMCs was the reason for low pollen fertility in the autotetraploid cucumber.     

Mutations in twinstar, a Drosophila gene encoding a cofilin/ADF homologue, result in defects in centrosome migration and cytokinesis

We describe the phenotypic and molecular characterization of twinstar (tsr), an essential gene in Drosophila melanogaster. Two P-element induced alleles of tsr (tsr1 and tsr2) result in late larval or pupal lethality. Cytological examination of actively dividing tissues in these mutants reveals defects in cytokinesis in both mitotic (larval neuroblast) and meiotic (larval testis) cells. In addition, mutant spermatocytes show defects in aster migration and separation during prophase/prometaphase of both meiotic divisions. We have cloned the gene affected by these mutations and shown that it codes for a 17-kD protein in the cofilin/ADF family of small actin severing proteins. A cDNA for this gene has previously been described by Edwards et al. (1994). Northern analysis shows that the tsr gene is expressed throughout development, and that the tsr1 and tsr2 alleles are hypomorphs that accumulate decreased levels of tsr mRNA. These findings prompted us to examine actin behavior during male meiosis to visualize the effects of decreased twinstar protein activity on actin dynamics in vivo. Strikingly, both mutants exhibit abnormal accumulations of F- actin. Large actin aggregates are seen in association with centrosomes in mature primary spermatocytes. Later, during ana/telophase of both meiotic divisions, aberrantly large and misshaped structures appear at the site of contractile ring formation and fail to disassemble at the end of telophase, in contrast with wild-type. We discuss these results in terms of possible roles of the actin-based cytoskeleton in centrosome movement and in cytokinesis.