Mechanisms of chromosome orientation revealed by two meiotic mutants in Drosophila melanogaster

Two disjunction defective meiotic mutants, ord and mei-S332, each of which disrupts meiosis in both male and female Drosophila melanogaster, were analyzed cytologically and genetically in the male germ-line. It was observed that sister-chromatids are frequently associated abnormally during prophase I and metaphase I in ord. Sister chromatid associations in mei-S332 are generally normal during prophase I and metaphase I. By telophase I, sister chromatids have frequently precociously separated in both mutants. During the first division sister chromatids disjoin from one another frequently in ord and rarely in mei-S332. It is argued that the simplest interpretation of the observations is that each mutant is defective in sister chromatid cohesiveness and that the defect in ord manifests itself earlier than does the defect in mei-S332. In addition, based on these mutant effects, several conclusions regarding normal meiotic processes are drawn. (1) The phenotype of these mutants support the proposition that the second meiotic metaphase (mitotic-type) position of chromosomes and their equational orientation is a consequence of the equilibrium, at the metaphase plate, of pulling forces acting at the kinetochores and directed towards the poles. (2) Chromosomes which lag during the second meiotic division tend to be lost. (3) Sister chromatid cohesiveness, or some function necessary for sister chromatid cohesiveness, is required for the normal reductional orientation of sister kinetochores during the first meiotic division. (4) The kinetochores of a half-bivalent are double at the time of chromosome orientation during the first meiotic division. Finally, functions which are required throughout meiosis in both sexes must be considered in the pathways of meiotic control.     

Cytological evidences of SDR-FDR mixture in the formation of 2n eggs in a potato diploid clone

Summary Macrosporogenesis and microsporogenesis were investigated in a diploid S. tuberosum x S. chacoense potato hybrid, characterized by more than 50% 2n egg formation. Fifty-five percent of dyad formation of 2n macrospores is ascribed to two meiotic abnormalities: omission of the second meiotic division, occurring at a frequency of 38%, and irregular spindle axis orientation at metaphase I at a frequency of 16%: These abnormalities give origin to a mixture of 2n eggs, composed of mostly second division restitution (SDR) and a small portion of first division restitution (FDR). Microsporogenesis showed rare dyads of 2n microspores depending on parallel spindles observed in anaphase II.Contribution no. 53 from the Center of Vegetable Breeding, CNR, Portici, Italy     

Chromosome pairing in meiosis of partially fertile wheat/rye hybrids

The normal course of meiosis depends on regular pairing of homologous chromosomes. In intergeneric hybrids, including those of wheat, there is no chromosome pairing because there are no homologs. In F₁ wheat/rye hybrids, pairing is largely prevented by the pairing homoeologous1 (Ph1) gene. In its presence, there are only rare instances of pairing; most chromosomes are univalent, and their orientation at metaphase I initiates different pathways of the meiotic cycle. The meiotic-like pathway includes a combination of the reductional and the equational + reductional steps at AI followed by the second division. The resulting gametes are mostly non-functional. The mitotic-like pathway involves equational division of univalents at AI and the absence of the second division. Any fertility of wheat/rye hybrids depends on the production of unreduced gametes arising from meiotic restitution (mitotic-like division). We examined the meiotic pairing in wheat/rye hybrids created from wheat lines with single rye chromosome substitutions and Ph1 present. This guaranteed F₁ meiosis with one pair of rye homologs. All hybrids formed bivalents, but proportions of meiocytes with bivalents varied. In the meiocytes where bivalents were present, there was a higher tendency for the meiotic-like pathway, while in meiocytes where bivalent pairing failed, the tendency was stronger for the mitotic-like pathway. Among the equationally dividing cells, we observed more than 90 % of meiocytes without bivalents, where rye homologs did not form bivalents, too. The data indicate a potential application of wheat/rye lines in producing genetic stocks of amphidiploids with designated genomic constitutions.     

Preferential Occurrence of Nonsister Spores in Two-Spored Asci of SACCHAROMYCES CEREVISIAE: Evidence for Regulation of Spore-Wall Formation by the Spindle Pole Body

Yeast cells subjected to a reversible thermal arrest of meiosis yielded progressively fewer spores per ascus as the arrest was extended. Dissection of two-spored asci by a newly developed method that prevents selection of false asci revealed that the spores were not a random sample of the haploid meiotic products. Most, if not all, pairs of spores contain nonsister products of the reductional division. Electron microscopic examination of the meiotic cells revealed the cytological basis for this bias. All four spindle pole bodies (SPBs) present at the second meiotic division normally gain a structural modification (the outer plaque) upon which the initiation of the prospore wall occurs. In the formation of a two-spored ascus, only one spindle pole body on each meiosis II spindle was so modified. These observations suggest that the morphogenesis of spores is regulated at meiosis II by limiting the number of SPBs gaining the outer plaque. The enhancement of spore yield upon addition of fresh medium suggests that this morphogenetic regulation responds more directly to nutrient deprivation arising during the thermal arrest, rather than to elevated temperature per se.     

Meiotic division and fusion of nuclei in multinucleate cells from the induced fusion of meiotic protoplasts of liliaceous plants

Multinucleate protoplasts were produced from meiotic cells at the zygotene and pachytene stages in a lily andTrillium, and their meiotic divisions were followed during subsequent culture. In each multinucleate, a complete synchrony of nuclear division was maintained throughout the meiotic process, and chromosome behavior appeared normal up to the metaphase stage. In most dinucleates, chromosome segregation movement was organized in a common spindle, and the daughter nuclei at the telophase appeared to envelope each other in the newly formed nuclear membrane. The cell was divided into two daughter cells by a common cell plate. Trinucleates were similarly converted to two cells with a hexaploid number of chromosomes. Some of the di- and trinucleates subsequently completed the second meiotic division with the formation of typical tetrad configurations. In giant cells with more than several nuclei, chromosomes separated at random but reaggregated into one giant resting nucleus, with no later cytokinesis. The rate of meiotic development in multinucleates was relatively slower in cells which contained greater numbers of nuclei.     

Formation of flagella during interphase in secondary spermatocytes from Xenopus laevis in vitro

In cell culture, single motile flagella, 1 micron in length, were observed to grow from secondary spermatocytes of Xenopus laevis within 2-3 hours after telophase I, at 22 degrees C. About 90% of the secondary spermatocytes formed flagella as observed by phase-contrast microscopy. The flagella grew up to 2-6 microns in length during interphase II, which lasted about 18 hours. The presence of the "9 + 2" microtubular structure of the flagellar axonemes of secondary spermatocytes was confirmed by electron microscopy. When chromosomal condensation began (prophase II), the flagella were resorbed into the cells and, after the second meiotic division, a flagellum was formed again by each of the round spermatids. Thus, there appears to be a close relationship between the meiotic division cycle and the formation of flagella. The possible contribution of Sertoli cells to the formation of flagella in secondary spermatocytes was examined by reducing the number of Sertoli cells to less than ten per culture. Under these conditions, flagella formed in secondary spermatocytes with very high efficiency. It is very likely that secondary spermatocytes form flagella in vivo, since the secondary spermatocytes were observed to have flagella immediately after dissociation of the testes.     

Sisters Unbound Is Required for Meiotic Centromeric Cohesion in Drosophila melanogaster

Regular meiotic chromosome segregation requires sister centromeres to mono-orient (orient to the same pole) during the first meiotic division (meiosis I) when homologous chromosomes segregate, and to bi-orient (orient to opposite poles) during the second meiotic division (meiosis II) when sister chromatids segregate. Both orientation patterns require cohesion between sister centromeres, which is established during meiotic DNA replication and persists until anaphase of meiosis II. Meiotic cohesion is mediated by a conserved four-protein complex called cohesin that includes two structural maintenance of chromosomes (SMC) subunits (SMC1 and SMC3) and two non-SMC subunits. In Drosophila melanogaster, however, the meiotic cohesion apparatus has not been fully characterized and the non-SMC subunits have not been identified. We have identified a novel Drosophila gene called sisters unbound (sunn), which is required for stable sister chromatid cohesion throughout meiosis. sunn mutations disrupt centromere cohesion during prophase I and cause high frequencies of non-disjunction (NDJ) at both meiotic divisions in both sexes. SUNN co-localizes at centromeres with the cohesion proteins SMC1 and SOLO in both sexes and is necessary for the recruitment of both proteins to centromeres. Although SUNN lacks sequence homology to cohesins, bioinformatic analysis indicates that SUNN may be a structural homolog of the non-SMC cohesin subunit stromalin (SA), suggesting that SUNN may serve as a meiosis-specific cohesin subunit. In conclusion, our data show that SUNN is an essential meiosis-specific Drosophila cohesion protein.     

Aneugenic Effects of Epirubicin in Somatic and Germinal Cells of Male Mice

The ability of the antineoplastic agent epirubicin to induce aneuploidy and meiotic delay in the somatic and germinal cells of male mice was investigated by fluorescence in situ hybridization assay using labeled DNA probes and BrdU-incorporation assay. Mitomycin C and colchicine were used as positive controls for clastogen and aneugen, respectively, and these compounds produced the expected responses. The fluorescence in situ hybridization assay with a centromeric DNA probe for erythrocyte micronuclei showed that epirubicin is not only clastogenic but also aneugenic in somatic cells in vivo. By using the BrdU-incorporation assay, it could be shown that the meiotic delay caused by epirubicin in germ cells was approximately 48 h. Disomic and diploid sperm were shown in epididymal sperm hybridized with DNA probes specific for chromosomes 8, X and Y after epirubicin treatment. The observation that XX- and YY-sperm significantly prevailed over XY-sperm indicates missegregation during the second meiotic division. The results also suggest that earlier prophase stages contribute less to epirubicin-induced aneuploidy. Both the clastogenic and aneugenic potential of epirubicin can give rise to the development of secondary tumors and abnormal reproductive outcomes in cured cancer patients and medical personnel exposed to epirubicin.     

DNA-Binding Activities of Hop1 Protein, a Synaptonemal Complex Component from Saccharomyces cerevisiae

The meiosis-specific HOP1 gene is important both for crossing over between homologs and for production of viable spores. hop1 diploids fail to assemble synaptonemal complex (SC), which normally provides the framework for meiotic synapsis. Immunochemical methods have shown that the 70-kDa HOP1 product is a component of the SC. To assess its molecular function, we have purified Hop1 protein to homogeneity and shown that it forms dimers and higher oligomers in solution. Consistent with the zinc-finger motif in its sequence, the purified protein contained about 1 mol equivalent of zinc whereas mutant protein lacking a conserved cysteine within this motif did not. Electrophoretic gel mobility shift assays with different forms of M13 DNA showed that Hop1 binds more readily to linear duplex DNA and negatively superhelical DNA than to nicked circular duplex DNA and even more weakly to single-stranded DNA. Linear duplex DNA binding was enhanced by the addition of Zn²⁺, was stronger for longer DNA fragments, and was saturable to about 55 bp/protein monomer. Competitive inhibition of this binding by added oligonucleotides suggests preferential affinity for G-rich sequences and weaker binding to poly(dA-dT). Nuclear extracts of meiotic cells caused exonucleolytic degradation of linear duplex DNA if the extracts were prepared from hop1 mutants; addition of purified Hop1 conferred protection against this degradation. These findings suggest that Hop1 acts in meiotic synapsis by binding to sites of double-strand break formation and helping to mediate their processing in the pathway to meiotic recombination.     

THE QUADRIPOLAR MICROTUBULE SYSTEM AND MEIOTIC SPINDLE ONTOGENY IN HORNWORTS (BRYOPHYTA: ANTHOCEROTAE)

Ontogeny of the meiotic spindle in hornworts was studied by light microscopy of live materials, transmission electron microscopy, and indirect immunofluorescence microscopy. As in monoplastidic meiosis of mosses and Isoetes, the single plastid divides twice, and the four resultant plastids migrate into the future spore domains where they organize a quadripolar microtubule system (QMS). Additionally, a unique axial microtubule system (AMS) was found to parallel the plastid isthmus at each division in meiosis, much as in the single plastid division of mitosis. This finding is used to make a novel comparison of mitotic and meiotic spindle development. The AMS contributes directly to development of the mitotic spindle, whereas ontogeny of the meiotic spindle is more complex. Nuclear division in meiosis is delayed until after the second plastid division; the first AMS disappears without spindle formation, and the two AMSs of the second plastid division contribute to development of the QMS. Proliferation of microtubules at each plastid results in the QMS consisting of four cones of microtubules interconnecting the plastids and surrounding the nucleus. The QMS contributes to the development of a functionally bipolar spindle. The meiotic spindle is comparable to a merger of two mitotic spindles. However, the first division spindle does not terminate in what would be the poles of mitosis; instead the poles converge to orient the spindle axis midway between pairs of non-sister plastids.     

Meiotic Recombination in Arabidopsis Is Catalysed by DMC1, with RAD51 Playing a Supporting Role

Recombination establishes the chiasmata that physically link pairs of homologous chromosomes in meiosis, ensuring their balanced segregation at the first meiotic division and generating genetic variation. The visible manifestation of genetic crossing-overs, chiasmata are the result of an intricate and tightly regulated process involving induction of DNA double-strand breaks and their repair through invasion of a homologous template DNA duplex, catalysed by RAD51 and DMC1 in most eukaryotes. We describe here a RAD51-GFP fusion protein that retains the ability to assemble at DNA breaks but has lost its DNA break repair capacity. This protein fully complements the meiotic chromosomal fragmentation and sterility of Arabidopsis rad51, but not rad51 dmc1 mutants. Even though DMC1 is the only active meiotic strand transfer protein in the absence of RAD51 catalytic activity, no effect on genetic map distance was observed in complemented rad51 plants. The presence of inactive RAD51 nucleofilaments is thus able to fully support meiotic DSB repair and normal levels of crossing-over by DMC1. Our data demonstrate that RAD51 plays a supporting role for DMC1 in meiotic recombination in the flowering plant, Arabidopsis.     

Commitment of Mitotic Cells to Meiosis during the G2 Phase of Premeiosis

In the developing anther, archesporial cells that proliferateby mitotic division are converted into meiotic cells duringthe premeiotic interphase. Experiments with explanted microsporocytesof Lilium and Trillium were made to obtain evidence for theconversion of mitotic to meiotic cells during the premeioticperiod. Explanted premeiotic cells were cultured through thedivision cycle at relatively high division frequencies and showeda variety of division types with respect to chromosomal events.The type of division depended on the premeiotic stage at whichthe cells were explanted. Cells in the G₁, S and early G² phasesunderwent mitotic division and formed a diad or binucleate monad.Cells explanted at the late G₂ phase were cultured throughoutthe normal meiotic cycle, which resulted in typical tetrad configuration. In microsporocytes explanted during the main part of the G₂interval, centromere behavior was meiotic, but chromosome pairingand chiasma formation were disturbed. Thus, she G₂ intervalwas shown to be critical for the commitment of mitotic cellsto meiotic division. Detailed analysis showed that the intracellularchanges that commit the cells to meiosis begin shortly aftercompletion of premeiotic DNA synthesis and that these changesare progressive and cumulative. (Received February 2, 1982; Accepted May 24, 1982)     

The Arabidopsis KNOLLE Protein Is a Cytokinesis-specific Syntaxin

In higher plant cytokinesis, plasma membrane and cell wall originate by vesicle fusion in the plane of cell division. The Arabidopsis KNOLLE gene, which is required for cytokinesis, encodes a protein related to vesicle-docking syntaxins. We have raised specific rabbit antiserum against purified recombinant KNOLLE protein to show biochemically and by immunoelectron microscopy that KNOLLE protein is membrane associated. Using immunofluorescence microscopy, KNOLLE protein was found to be specifically expressed during mitosis and, unlike the plasma membrane H⁺-ATPase, to localize to the plane of division during cytokinesis. Arabidopsis dynamin-like protein ADL1 accumulates at the plane of cell plate formation in knolle mutant cells as in wild-type cells, suggesting that cytokinetic vesicle traffic is not affected. Furthermore, electron microscopic analysis indicates that vesicle fusion is impaired. KNOLLE protein was detected in mitotically dividing cells of various parts of the developing plant, including seedling root, inflorescence meristem, floral meristems and ovules, and the cellularizing endosperm, but not during cytokinesis after the male second meiotic division. Thus, KNOLLE is the first syntaxin-like protein that appears to be involved specifically in cytokinetic vesicle fusion.     

Ady4p and Spo74p are components of the meiotic spindle pole body that promote growth of the prospore membrane in Saccharomyces cerevisiae

Spore formation in Saccharomyces cerevisiae occurs via the de novo synthesis of the prospore membrane during the second meiotic division. Prospore membrane formation is triggered by assembly of a membrane-organizing center, the meiotic outer plaque (MOP), on the cytoplasmic face of the spindle pole body (SPB) during meiosis. We report here the identification of two new components of the MOP, Ady4p and Spo74p. Ady4p and Spo74p interact with known proteins of the MOP and are localized to the outer plaque of the SPB during meiosis II. MOP assembly and prospore membrane formation are abolished in spo74Δ/spo74Δ cells and occur aberrantly in ady4Δ/ady4Δ cells. Spo74p and the MOP component Mpc70p are mutually dependent for recruitment to SPBs during meiosis. In contrast, both Ady4p and Spo74p are present at SPBs, albeit at reduced levels, in cells that lack the MOP component Mpc54p. Our findings suggest a model for the assembled MOP in which Mpc54p, Mpc70p, and Spo74p make up a core structural unit of the scaffold that initiates synthesis of the prospore membrane, and Ady4p is an auxiliary component that stabilizes the plaque.     

The Arabidopsis BLAP75/Rmi1 Homologue Plays Crucial Roles in Meiotic Double-Strand Break Repair

In human cells and in Saccharomyces cerevisiae, BLAP75/Rmi1 acts together with BLM/Sgs1 and TopoIIIα/Top3 to maintain genome stability by limiting crossover (CO) formation in favour of NCO events, probably through the dissolution of double Holliday junction intermediates (dHJ). So far, very limited data is available on the involvement of these complexes in meiotic DNA repair. In this paper, we present the first meiotic study of a member of the BLAP75 family through characterisation of the Arabidopsis thaliana homologue. In A. thaliana blap75 mutants, meiotic recombination is initiated, and recombination progresses until the formation of bivalent-like structures, even in the absence of ZMM proteins. However, chromosome fragmentation can be detected as soon as metaphase I and is drastic at anaphase I, while no second meiotic division is observed. Using genetic and imunolocalisation studies, we showed that these defects reflect a role of A. thaliana BLAP75 in meiotic double-strand break (DSB) repair—that it acts after the invasion step mediated by RAD51 and associated proteins and that it is necessary to repair meiotic DSBs onto sister chromatids as well as onto the homologous chromosome. In conclusion, our results show for the first time that BLAP75/Rmi1 is a key protein of the meiotic homologous recombination machinery. In A. thaliana, we found that this protein is dispensable for homologous chromosome recognition and synapsis but necessary for the repair of meiotic DSBs. Furthermore, in the absence of BLAP75, bivalent formation can happen even in the absence of ZMM proteins, showing that in blap75 mutants, recombination intermediates exist that are stable enough to form bivalent structures, even when ZMM are absent.     

PLK1 regulates centrosome migration and spindle dynamics in male mouse meiosis

Cell division requires the regulation of karyokinesis and cytokinesis, which includes an essential role of the achromatic spindle. Although the functions of centrosomes are well characterised in somatic cells, their role during vertebrate spermatogenesis remains elusive. We have studied the dynamics of the meiotic centrosomes in male mouse during both meiotic divisions. Results show that meiotic centrosomes duplicate twice: first duplication occurs in the leptotene/zygotene transition, while the second occurs in interkinesis. The maturation of duplicated centrosomes during the early stages of prophase I and II are followed by their separation and migration to opposite poles to form bipolar spindles I and II. The study of the genetic mouse model Plk1(Δ/Δ) indicates a central role of Polo‐like kinase 1 in pericentriolar matrix assembly, in centrosome maturation and migration, and in the formation of the bipolar spindles during spermatogenesis. In addition, in vitro inhibition of Polo‐like kinase 1 and Aurora A in organotypic cultures of seminiferous tubules points out to a prominent role of both kinases in the regulation of the formation of meiotic bipolar spindles.     

SEXUAL REPRODUCTION AND MEIOSIS IN PERIDINIUM INCONSPICUUM LEMMERMANN (DINOPHYCEAE)

In Peridinium inconspicuum Lemmermann, sexual reproduction occurs in both nitrogen-enriched and nitrogen-deficient media. In this homothallic strain, protoplasmic fusion begins between two thecate gametes; but zygote formation is completed in a space outside the fusing pair. This diploid cell can form a plated theca which is shed as the cell enlarges. This spherical zygote then forms a new non-plated theca. The process of ecdysis and the formation of a new non-plated theca is repeated several times. During this process the zygote gradually elongates and by cytoplasmic infurrowing becomes peanut-shaped. Eventually two cells are formed. The first and second meiotic divisions are greatly separated in time. The first meiotic division occurs in the spherical non-thecate zygote. The second meiotic division can occur in the peanut-shaped zygote before it completes cytokinesis. This meiotic division may not be synchronous, occasionally resulting in a trinucleate stage. Eventually four flagellated, haploid products are produced.     

Inhibition of the Smc5/6 Complex during Meiosis Perturbs Joint Molecule Formation and Resolution without Significantly Changing Crossover or Non-crossover Levels

Meiosis is a specialized cell division used by diploid organisms to form haploid gametes for sexual reproduction. Central to this reductive division is repair of endogenous DNA double-strand breaks (DSBs) induced by the meiosis-specific enzyme Spo11. These DSBs are repaired in a process called homologous recombination using the sister chromatid or the homologous chromosome as a repair template, with the homolog being the preferred substrate during meiosis. Specific products of inter-homolog recombination, called crossovers, are essential for proper homolog segregation at the first meiotic nuclear division in budding yeast and mice. This study identifies an essential role for the conserved Structural Maintenance of Chromosomes (SMC) 5/6 protein complex during meiotic recombination in budding yeast. Meiosis-specific smc5/6 mutants experience a block in DNA segregation without hindering meiotic progression. Establishment and removal of meiotic sister chromatid cohesin are independent of functional Smc6 protein. smc6 mutants also have normal levels of DSB formation and repair. Eliminating DSBs rescues the segregation block in smc5/6 mutants, suggesting that the complex has a function during meiotic recombination. Accordingly, smc6 mutants accumulate high levels of recombination intermediates in the form of joint molecules. Many of these joint molecules are formed between sister chromatids, which is not normally observed in wild-type cells. The normal formation of crossovers in smc6 mutants supports the notion that mainly inter-sister joint molecule resolution is impaired. In addition, return-to-function studies indicate that the Smc5/6 complex performs its most important functions during joint molecule resolution without influencing crossover formation. These results suggest that the Smc5/6 complex aids primarily in the resolution of joint molecules formed outside of canonical inter-homolog pathways.     

Effects of several meiotic mutations on female meiosis in maize

A modified enzyme digestion technique of ovary isolation followed by staining and squash preparation has allowed us to observe female meiosis in normal maize meiotically dividing megaspore mother cells (MMCs). The first meiotic division in megasporogenesis of maize is not distinguishable from that in mi-crosporogenesis. The second female meiotic division is characterized as follows: (1) the two products of the first meiotic division do not simultaneously enter into the second meiotic division; as a rule, the chalazal-most cell enters division earlier than the micropylar one, (2) often the second of the two products does not proceed with meiosis, but degenerates, and (3) only a single haploid meiotic product of the tetrad remains alive, and this cell proceeds with three rounds of mitoses without any intervening cell wall formation to produce the eight-nucleate embryo sac. This technique has allowed us to study the effects of five meiotic mutations (aml, aml-pral, afdl, dsy *-9101, and dvl) on female meiosis in maize. The effects of the two alleles of the aml gene (aml and aml-pral) and of the afdl and dsy *-9101mutations are the same in both male and female meiosis. The aml allele prevents the entrance of MMCs into meiosis and meiosis is replaced by mitosis; the aml-pral permits MMCs to enter into meiosis, but their progress is stopped at early prophase I stages. The afdl gene is responsible for substitution of the first meiotic (reductional) division by an equational division including the segregation of sister chromatid centromeres at anaphase I. The dsy * -9101 gene exhibits abnormal chromosome pairing; paired homologous chromosomes are visible at pachytene, but only univalents are observed at diakinesis and metaphase I stages. These mutation specific patterns of abnormal meiosis are responsible for the bisexual sterility of these meiotic mutants. The abnormal divergent shape of the spindle apparatus and the resulting abnormal segregation of homologous chromosomes observed in micro-sporogenesis in plants homozygous for the dv1 mutation have not been found in meiosis of megasporogenesis. Only male sterility is induced by the dv1 gene in the homozygous condition. © 1993 Wiley-Liss, Inc.